David E Levy, Ph.D.

TLR4 Engagement during TLR3-Induced Proinflammatory Signaling in Dendritic Cells Promotes IL-10-Mediated Suppression of Antitumor Immunity
Bogunovic, Dusan; Manches, Olivier; Godefroy, Emmanuelle; Yewdall, Alice; Gallois, Anne; Salazar, Andres M; Marie, Isabelle; Levy, David E; Bhardwaj, Nina
2011 Aug 15;71(16):5467-5476, Cancer research
Toll-like receptor (TLR) agonists are promising adjuvants for immune therapy of cancer, but their potential efficacy as single or combinatorial agents has yet to be fully evaluated. Here, we report that among all TLR agonists tested, dendritic cells (DC) stimulated with the TLR3 agonist polyI:C displayed the strongest activity in stimulating proinflammatory responses and the production of melanoma antigen-specific CD8(+) T cells. Simultaneous treatment with TLR7/8 agonists further improved these responses, but the inclusion of bacterial lipopolysaccharide (LPS), a TLR4 agonist, suppressed proinflammatory cytokine production. This inhibition was contingent upon rapid induction of the suppressive cytokine interleukin (IL)-10 by LPS, leading to dysregulated immune responses and it could be reversed by signal transducers and activators of transcription 3 knockdown, p38 blockade or antibodies to IL-10 and its receptor. Our findings show how certain TLR agonist combinations can enhance or limit DC responses associated with antitumor immunity, through their relative ability to induce IL-10 pathways that are immune suppressive. Cancer Res; 71(16); 5467-76. (c)2011 AACR
— id: 136637, year: 2011, vol: 71, page: 5467, stat: Journal Article,

STAT3 Plays a Critical Role in KRAS-Induced Pancreatic Tumorigenesis
Corcoran, Ryan B; Contino, Gianmarco; Deshpande, Vikram; Tzatsos, Alexandros; Conrad, Claudius; Benes, Cyril H; Levy, David E; Settleman, Jeffrey; Engelman, Jeffrey A; Bardeesy, Nabeel
2011 Jul 15;71(14):5020-5029, Cancer research
The STAT3 transcription factor is an important regulator of stem cell self-renewal, cancer cell survival, and inflammation. In the pancreas, STAT3 is dispensable for normal development, whereas the majority of pancreatic ductal adenocarcinomas (PDAC) show constitutive activation of STAT3, suggesting its potential as a therapeutic target in this cancer. Here, we sought to define the mechanisms of STAT3 activation and its functional importance in PDAC pathogenesis. Large-scale screening of cancer cell lines with a JAK2 inhibitor that blocks STAT3 function revealed a more than 30-fold range in sensitivity in PDAC, and showed a close correlation of sensitivity with levels of tyrosine-phosphorylated STAT3 and of the gp130 receptor, an upstream signaling component. Correspondingly, upregulation of the IL6/LIF-gp130 pathway accounted for the strong STAT3 activation in PDAC subsets. To define functions of STAT3 in vivo, we developed mouse models that test the impact of conditional inactivation of STAT3 in KRAS-driven PDAC. We showed that STAT3 is required for the development of the earliest premalignant pancreatic lesions, acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN). Moreover, acute STAT3 inactivation blocked PDAC initiation in a second in vivo model. Our results show that STAT3 has critical roles throughout the course of PDAC pathogenesis, supporting the development of therapeutic approaches targeting this pathway. Moreover, our work suggests that gp130 and phospho-STAT3 expression may be effective biomarkers for predicting response to JAK2 inhibitors. Cancer Res; 71(14); 5020-9. (c)2011 AACR
— id: 135540, year: 2011, vol: 71, page: 5020, stat: Journal Article,

STAT3-dependent IL-21 production from T helper cells regulates hematopoietic progenitor cell homeostasis
Kaplan, Mark H; Glosson, Nicole L; Stritesky, Gretta L; Yeh, Norman; Kinzfogl, John; Rohrabaugh, Sara L; Goswami, Ritobrata; Pham, Duy; Levy, David E; Brutkiewicz, Randy R; Blum, Janice S; Cooper, Scott; Hangoc, Giao; Broxmeyer, Hal E
2011 Jun 9;117(23):6198-6201, Blood
The contribution of specific cell types to the production of cytokines that regulate hematopoiesis is still not well defined. We have previously identified T cell-dependent regulation of hematopoietic progenitor cell (HPC) numbers and cycling. In this report, we demonstrated that HPC activity is decreased in mice with STAT3-deficient T cells, a phenotype that is not because of decreased expression of IL-17 or RORgammat. STAT3 expression in T cells was required for IL-21 production by multiple T helper subsets, and neutralization of IL-21 resulted in decreased HPC activity identical to that in mice with STAT3-deficient T cells. Importantly, injection of IL-21 rescued HPC activity in mice with STAT3-deficient T cells. Thus, STAT3-dependent IL-21 production in T cells is required for HPC homeostasis
— id: 141123, year: 2011, vol: 117, page: 6198, stat: Journal Article,

Distinct inflammatory signals have physiologically divergent effects on epigenetic regulation of foxp3 expression and treg function
Lal, G; Yin, N; Xu, J; Lin, M; Schroppel, S; Ding, Y; Marie, I; Levy, D E; Bromberg, J S
2011 Feb;11(2):203-214, American journal of transplantation
Foxp3 expression in regulatory T cells (Treg) is required for their development and suppressive function. How different inflammatory signals affect Foxp3 chromatin structure, expression and Tregs plasticity are not completely known. In the present study, the Toll-like receptor 2 (TLR2) ligand peptidoglycan inhibited Foxp3 expression in both natural Treg (nTreg) and TGFbeta-driven adaptive Treg (aTreg). Inhibition was independent of paracrine Th1, Th2 and Th17 cytokines. PGN-induced T cell-intrinsic TLR2-Myd88-dependent IFR1 expression and induced IRF1 bound to IRF1 response elements (IRF-E) in the Foxp3 promoter and intronic enhancers, and negatively regulated Foxp3 expression. Inflammatory IL-6 and TLR2 signals induced divergent chromatin changes at the Foxp3 locus and regulated Treg suppressor function, and in an islet transplant model resulted in differences in their ability to prolong graft survival. These findings are important for understanding how different inflammatory signals can affect the transplantation tolerance and immunity
— id: 122535, year: 2011, vol: 11, page: 203, stat: Journal Article,

Induction and function of type i and III interferon in response to viral infection
Levy D.E.; Marie I.J.; Durbin J.E.
2011 ;1(6):476-486, Current Opinion in Virology
The type I and III interferon (IFN) families consist of cytokines rapidly induced during viral infection that confer antiviral protection on target cells and are critical components of innate immune responses and the transition to effective adaptive immunity. The regulation of their expression involves an intricate and stringently regulated signaling cascade, initiated by recognition most often of viral nucleic acid in cytoplasmic and endosomal compartments and involving a series of protein conformational rearrangements and interactions regulated by helicase action, ubiquitin modification, and protein aggregation, culminating in kinase activation and phosphorylation of critical transcription factors and their regulators. The many IFN subtypes induced by viruses confer amplification, diversification, and cell-type specificity to the host response to infection, providing fertile ground for development of antiviral therapeutics and vaccines
— id: 147760, year: 2011, vol: 1, page: 476, stat: Journal Article,

High frequencies of de novo cnvs in bipolar disorder and schizophrenia
Malhotra D.; McCarthy S.; Michaelson J.J.; Vacic V.; Burdick K.E.; Yoon S.; Cichon S.; Corvin A.; Gary S.; Gershon E.S.; Gill M.; Karayiorgou M.; Kelsoe J.R.; Krastoshevsky O.; Krause V.; Leibenluft E.; Levy D.L.; Makarov V.; Bhandari A.; Malhotra A.K.; McMahon F.J.; Nothen M.M.; Potash J.B.; Rietschel M.; Schulze T.G.; Sebat J.
2011 ;72(6):951-963, Neuron
While it is known that rare copy-number variants (CNVs) contribute to risk for some neuropsychiatric disorders, the role of CNVs in bipolar disorder is unclear. Here, we reasoned that a contribution of CNVs to mood disorders might be most evident for de novo mutations. We performed a genome-wide analysis of de novo CNVs in a cohort of 788 trios. Diagnoses of offspring included bipolar disorder (n = 185), schizophrenia (n = 177), and healthy controls (n = 426). Frequencies of de novo CNVs were significantly higher in bipolar disorder as compared with controls (OR = 4.8 [1.4,16.0], p = 0.009). De novo CNVs were particularly enriched among cases with an age at onset younger than 18 (OR = 6.3 [1.7,22.6], p = 0.006). We also confirmed a significant enrichment of de novo CNVs in schizophrenia (OR = 5.0 [1.5,16.8], p = 0.007). Our results suggest that rare spontaneous mutations are an important contributor to risk for bipolar disorder and other major neuropsychiatric diseases. 2011 Elsevier Inc
— id: 150876, year: 2011, vol: 72, page: 951, stat: Journal Article,

Spatiotemporal trafficking of HIV in human plasmacytoid dendritic cells defines a persistently IFN-alpha-producing and partially matured phenotype
O'Brien, Meagan; Manches, Olivier; Sabado, Rachel Lubong; Baranda, Sonia Jimenez; Wang, Yaming; Marie, Isabelle; Rolnitzky, Linda; Markowitz, Martin; Margolis, David M; Levy, David; Bhardwaj, Nina
2011 Mar 1;121(3):1088-1101, Journal of clinical investigation
Plasmacytoid DCs (pDCs) are innate immune cells that are specialized to produce IFN-alpha and to activate adaptive immune responses. Although IFN-alpha inhibits HIV-1 replication in vitro, the production of IFN-alpha by HIV-activated pDCs in vivo may contribute more to HIV pathogenesis than to protection. We have now shown that HIV-stimulated human pDCs allow for persistent IFN-alpha production upon repeated stimulation, express low levels of maturation molecules, and stimulate weak T cell responses. Persistent IFN-alpha production by HIV-stimulated pDCs correlated with increased levels of IRF7 and was dependent upon the autocrine IFN-alpha/beta receptor feedback loop. Because it has been shown that early endosomal trafficking of TLR9 agonists causes strong activation of the IFN-alpha pathway but weak activation of the NF-kappaB pathway, we sought to investigate whether early endosomal trafficking of HIV, a TLR7 agonist, leads to the IFN-alpha-producing phenotype we observed. We demonstrated that HIV preferentially traffics to the early endosome in human pDCs and therefore skews pDCs toward a partially matured, persistently IFN-alpha-secreting phenotype
— id: 130297, year: 2011, vol: 121, page: 1088, stat: Journal Article,

The transcription factor STAT3 is required for T helper 2 cell development
Stritesky, Gretta L; Muthukrishnan, Rajarajeswari; Sehra, Sarita; Goswami, Ritobrata; Pham, Duy; Travers, Jared; Nguyen, Evelyn T; Levy, David E; Kaplan, Mark H
2011 Jan 28;34(1):39-49, Immunity
Signal transducer and activator of transcription (STAT) family members direct the differentiation of T helper cells, with specific STAT proteins promoting distinct effector subsets. STAT6 is required for the development of T helper 2 (Th2) cells, whereas STAT3 promotes differentiation of Th17 and follicular helper T cell subsets. We demonstrated that STAT3 was also activated during Th2 cell development and was required for the expression of Th2 cell-associated cytokines and transcription factors. STAT3 bound directly to Th2 cell-associated gene loci and was required for the ability of STAT6 to bind target genes. In vivo, STAT3 deficiency in T cells eliminated the allergic inflammation in mice sensitized and challenged with ovalbumin or transgenic for constitutively active STAT6. Thus, STAT3 cooperates with STAT6 in promoting Th2 cell development. These results demonstrate that differentiating T helper cells integrate multiple STAT protein signals during Th2 cell development
— id: 132218, year: 2011, vol: 34, page: 39, stat: Journal Article,

Inhibition of nonsense-mediated RNA decay by the tumor microenvironment promotes tumorigenesis
Wang, Ding; Zavadil, Jiri; Martin, Leenus; Parisi, Fabio; Friedman, Eugene; Levy, David; Harding, Heather; Ron, David; Gardner, Lawrence B
2011 Sep;31(17):3670-3680, Molecular & cellular biology
While nonsense-mediated RNA decay (NMD) is an established mechanism to rapidly degrade select transcripts, the physiological regulation and biological significance of NMD are not well characterized. We previously demonstrated that NMD is inhibited in hypoxic cells. Here we show that the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) translation initiation factor by a variety of cellular stresses leads to the inhibition of NMD and that eIF2alpha phosphorylation and NMD inhibition occur in tumors. To explore the significance of this NMD regulation, we used an unbiased approach to identify approximately 750 NMD-targeted mRNAs and found that these mRNAs are overrepresented in stress response and tumor-promoting pathways. Consistent with these findings, the inhibition of NMD promotes cellular resistance to endoplasmic reticulum stress and encourages tumor formation. The transcriptional and translational regulations of gene expression by the microenvironment are established mechanisms by which tumor cells adapt to stress. These data indicate that NMD inhibition by the tumor microenvironment is also an important mechanism to dynamically regulate genes critical for the response to cellular stress and tumorigenesis
— id: 136513, year: 2011, vol: 31, page: 3670, stat: Journal Article,

STAT3 negatively regulates type I IFN-mediated antiviral response
Wang, Wei-Bei; Levy, David E; Lee, Chien-Kuo
2011 Sep 1;187(5):2578-2585, Journal of immunology
Type I IFNs are crucial cytokines of innate immunity for combating viral infections. Signaling through type I IFN receptors triggers the activation of STAT proteins, including STAT1, STAT2, and STAT3. Although an essential role of STAT1 and STAT2 for type I IFN-induced antiviral response has been well established by studies of gene-targeted mice and human mutations, the role of STAT3 for this response remains unclear. Using gain-of-function and loss-of-function approaches, we demonstrated that STAT3 negatively regulates type I IFN-mediated response. STAT3 knockdown or knockout cells displayed enhanced gene expression and antiviral activity in response to IFN-alpha/beta. Restoration of STAT3 to STAT3KO cells resulted in attenuation of the response. Upon viral infection, increased type I IFN production in STAT3KO cells resulted in enhanced STAT activation and ISG expression. One mechanism for the enhanced IFN production and response in the absence of STAT3 might operate through an MDA5-dependent manner. STAT3 also appeared to suppress IFN response directly in a manner dependent on its N-terminal domain and independent of its function as a transcriptional factor. Taken together, these results define STAT3 as a negative regulator of type I IFN response and provide a therapeutic target for viral infections
— id: 138428, year: 2011, vol: 187, page: 2578, stat: Journal Article,

A STAT3-mediated metabolic switch is involved in tumour transformation and STAT3 addiction
Demaria, Marco; Giorgi, Carlotta; Lebiedzinska, Magdalena; Esposito, Giovanna; D'Angeli, Luca; Bartoli, Antonietta; Gough, Daniel J; Turkson, James; Levy, David E; Watson, Christine J; Wieckowski, Mariusz R; Provero, Paolo; Pinton, Paolo; Poli, Valeria
2010 Nov;2(11):823-842, Aging
The pro-oncogenic transcription factor STAT3 is constitutively activated in a wide variety of tumours that often become addicted to its activity, but no unifying view of a core function determining this widespread STAT3-dependence has yet emerged. We show here that constitutively active STAT3 acts as a master regulator of cell metabolism, inducing aerobic glycolysis and down-regulating mitochondrial activity both in primary fibroblasts and in STAT3-dependent tumour cell lines. As a result, cells are protected from apoptosis and senescence while becoming highly sensitive to glucose deprivation. We show that enhanced glycolysis is dependent on HIF-1alpha up-regulation, while reduced mitochondrial activity is HIF-1alpha-independent and likely caused by STAT3-mediated down-regulation of mitochondrial proteins. The induction of aerobic glycolysis is an important component of STAT3 pro-oncogenic activities, since inhibition of STAT3 tyrosine phosphorylation in the tumour cell lines down-regulates glycolysis prior to leading to growth arrest and cell death, both in vitro and in vivo. We propose that this novel, central metabolic role is at the core of the addiction for STAT3 shown by so many biologically different tumours
— id: 134403, year: 2010, vol: 2, page: 823, stat: Journal Article,

Functional crosstalk between type I and II interferon through the regulated expression of STAT1
Gough, Daniel J; Messina, Nicole L; Hii, Linda; Gould, Jodee A; Sabapathy, Kanaga; Robertson, Ashley P S; Trapani, Joseph A; Levy, David E; Hertzog, Paul J; Clarke, Christopher J P; Johnstone, Ricky W
2010 ;8(4):e1000361-e1000361, PLoS biology
Autocrine priming of cells by small quantities of constitutively produced type I interferon (IFN) is a well-known phenomenon. In the absence of type I IFN priming, cells display attenuated responses to other cytokines, such as anti-viral protection in response to IFNgamma. This phenomenon was proposed to be because IFNalpha/beta receptor1 (IFNAR1) is a component of the IFNgamma receptor (IFNGR), but our new data are more consistent with a previously proposed model indicating that regulated expression of STAT1 may also play a critical role in the priming process. Initially, we noticed that DNA binding activity of STAT1 was attenuated in c-Jun(-/-) fibroblasts because they expressed lower levels of STAT1 than wild-type cells. However, expression of STAT1 was rescued by culturing c-Jun(-/-) fibroblasts in media conditioned by wild-type fibroblasts suggesting they secreted a STAT1-inducing factor. The STAT1-inducing factor in fibroblast-conditioned media was IFNbeta, as it was inhibited by antibodies to IFNAR1, or when IFNbeta expression was knocked down in wild-type cells. IFNAR1(-/-) fibroblasts, which cannot respond to this priming, also expressed reduced levels of STAT1, which correlated with their poor responses to IFNgamma. The lack of priming in IFNAR1(-/-) fibroblasts was compensated by over-expression of STAT1, which rescued molecular responses to IFNgamma and restored the ability of IFNgamma to induce protective anti-viral immunity. This study provides a comprehensive description of the molecular events involved in priming by type I IFN. Adding to the previous working model that proposed an interaction between type I and II IFN receptors, our work and that of others demonstrates that type I IFN primes IFNgamma-mediated immune responses by regulating expression of STAT1. This may also explain how type I IFN can additionally prime cells to respond to a range of other cytokines that use STAT1 (e.g., IL-6, M-CSF, IL-10) and suggests a potential mechanism for the changing levels of STAT1 expression observed during viral infection
— id: 138944, year: 2010, vol: 8, page: e1000361, stat: Journal Article,

Non-canonical functions of STAT3 in growth and tumorigenesis
Gough, Daniel; Jones, Courtney; Wang, Yaming; Marie, Isabelle; Levy, David E.
2010 OCT-NOV ;52(1-2):36-36, Cytokine
— id: 113933, year: 2010, vol: 52, page: 36, stat: Journal Article,

NF-kappaB-ISGF3 transcription factor cooperation: coincidence detector or memory chip?
Levy, David E
2010 Jul 23;33(1):1-2, Immunity
Induction of gene expression involves deployment of transcription factors. In this issue of Immunity, Farlik et al. (2010) provide a view in which cooperation between transcription factors NF-kappaB and ISGF3 divides the task of transcription by recruiting and activating distinct components of the transcriptional machinery
— id: 111362, year: 2010, vol: 33, page: 1, stat: Journal Article,

A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells
Manel, Nicolas; Hogstad, Brandon; Wang, Yaming; Levy, David E; Unutmaz, Derya; Littman, Dan R
2010 Sep 9;467(7312):214-217, Nature
Dendritic cells serve a key function in host defence, linking innate detection of microbes to activation of pathogen-specific adaptive immune responses. Whether there is cell-intrinsic recognition of human immunodeficiency virus (HIV) by host innate pattern-recognition receptors and subsequent coupling to antiviral T-cell responses is not yet known. Dendritic cells are largely resistant to infection with HIV-1, but facilitate infection of co-cultured T-helper cells through a process of trans-enhancement. Here we show that, when dendritic cell resistance to infection is circumvented, HIV-1 induces dendritic cell maturation, an antiviral type I interferon response and activation of T cells. This innate response is dependent on the interaction of newly synthesized HIV-1 capsid with cellular cyclophilin A (CYPA) and the subsequent activation of the transcription factor IRF3. Because the peptidylprolyl isomerase CYPA also interacts with HIV-1 capsid to promote infectivity, our results indicate that capsid conformation has evolved under opposing selective pressures for infectivity versus furtiveness. Thus, a cell-intrinsic sensor for HIV-1 exists in dendritic cells and mediates an antiviral immune response, but it is not typically engaged owing to the absence of dendritic cell infection. The virulence of HIV-1 may be related to evasion of this response, the manipulation of which may be necessary to generate an effective HIV-1 vaccine
— id: 112431, year: 2010, vol: 467, page: 214, stat: Journal Article,

STAT3 negatively regulates type I IFN-mediated signaling and functions
Wang, Wei-Bei; Den, Hao-Kang; Levy, David E.; Lee, Chien-Kuo
2010 OCT-NOV ;52(1-2):65-65, Cytokine
— id: 113935, year: 2010, vol: 52, page: 65, stat: Journal Article,

Mitochondrial STAT3 supports Ras-dependent oncogenic transformation
Gough, Daniel J; Corlett, Alicia; Schlessinger, Karni; Wegrzyn, Joanna; Larner, Andrew C; Levy, David E
2009 Jun 26;324(5935):1713-1716, Science
Signal transducer and activator of transcription 3 (STAT3) is a latent cytoplasmic transcription factor responsive to cytokine signaling and tyrosine kinase oncoproteins by nuclear translocation when it is tyrosine-phosphorylated. We report that malignant transformation by activated Ras is impaired without STAT3, in spite of the inability of Ras to drive STAT3 tyrosine phosphorylation or nuclear translocation. Moreover, STAT3 mutants that cannot be tyrosine-phosphorylated, that are retained in the cytoplasm, or that cannot bind DNA nonetheless supported Ras-mediated transformation. Unexpectedly, STAT3 was detected within mitochondria, and exclusive targeting of STAT3 to mitochondria without nuclear accumulation facilitated Ras transformation. Mitochondrial STAT3 sustained altered glycolytic and oxidative phosphorylation activities characteristic of cancer cells. Thus, in addition to its nuclear transcriptional role, STAT3 regulates a metabolic function in mitochondria, supporting Ras-dependent malignant transformation
— id: 100615, year: 2009, vol: 324, page: 1713, stat: Journal Article,

Activation of Stat3 in renal tumors
Guo, Charles; Yang, Guanyu; Khun, Kyle; Kong, Xiantian; Levy, David; Lee, Peng; Melamed, Jonathan
2009 ;1(3):283-290, American Journal of Translational Research
Signal transducer and activator of transcription 3 (Stat3) plays a vital role in signal transduction pathways that mediate transformation and inhibit apoptosis. Oncogenic Stat3 is persistently activated in several human cancers and transformed cell lines. Previous studies indicate activation of Stat3 in renal cell carcinoma (RCC). However, the detailed characterization of the Stat3 expression pattern in different histologic types of RCC is lacking. We have analyzed the immunoprofile of activated or phosphorylated Stat3 (pStat3) in a tissue microarray of renal tumors of different histologic types, including 42 cases of conventional clear cell type, 24 chromophobe, and 7 papillary, 15 oncocytoma, 7 urothelial carcinoma and 21 normal kidney tissues using an anti-pStat3 antibody (recognizes only activated STAT3). pStat3 nuclear staining was observed in 25 of 42 conventional clear cell RCC (59.5 %), 8 of 24 chromophobe RCC (33.3%), 4 of 7 papillary RCC (57.1%). In the other tumor groups, 4 of 15 oncocytomas (26.7%) and 6 of 7 urothelial carcinomas (85.7%) showed positive nuclear staining. Weak nuclear immunoreactivity for pStat3 was seen in 4 of 21 cases of non-neoplastic kidney tissue (19.0%). The extent of Stat3 activation as determined by nuclear expression of its phosphorylated form is increased in histologic types of renal tumors with greater malignant potential, specifically conventional clear cell RCC, papillary RCC and urothelial carcinoma, only slightly increased in chromophobe RCC, and not increased in oncocytoma. These results suggest a role of Stat3 activation in different types of renal neoplasia, possibly serving as a prognostic marker or therapeutic target
— id: 105529, year: 2009, vol: 1, page: 283, stat: Journal Article,

Analysis of interleukin-21-induced Prdm1 gene regulation reveals functional cooperation of STAT3 and IRF4 transcription factors
Kwon, Hyokjoon; Thierry-Mieg, Danielle; Thierry-Mieg, Jean; Kim, Hyoung-Pyo; Oh, Jangsuk; Tunyaplin, Chainarong; Carotta, Sebastian; Donovan, Colleen E; Goldman, Matthew L; Tailor, Prafullakumar; Ozato, Keiko; Levy, David E; Nutt, Stephen L; Calame, Kathryn; Leonard, Warren J
2009 Dec 18;31(6):941-952, Immunity
Interleukin-21 (IL-21) is a pleiotropic cytokine that induces expression of transcription factor BLIMP1 (encoded by Prdm1), which regulates plasma cell differentiation and T cell homeostasis. We identified an IL-21 response element downstream of Prdm1 that binds the transcription factors STAT3 and IRF4, which are required for optimal Prdm1 expression. Genome-wide ChIP-Seq mapping of STAT3- and IRF4-binding sites showed that most regions with IL-21-induced STAT3 binding also bound IRF4 in vivo and furthermore revealed that the noncanonical TTCnnnTAA GAS motif critical in Prdm1 was broadly used for STAT3 binding. Comparing genome-wide expression array data to binding sites revealed that most IL-21-regulated genes were associated with combined STAT3-IRF4 sites rather than pure STAT3 sites. Correspondingly, ChIP-Seq analysis of Irf4(-/-) T cells showed greatly diminished STAT3 binding after IL-21 treatment, and Irf4(-/-) mice showed impaired IL-21-induced Tfh cell differentiation in vivo. These results reveal broad cooperative gene regulation by STAT3 and IRF4
— id: 138943, year: 2009, vol: 31, page: 941, stat: Journal Article,

Epigenetic regulation of Foxp3 expression in regulatory T cells by DNA methylation
Lal, Girdhari; Zhang, Nan; van der Touw, William; Ding, Yaozhong; Ju, Wenjun; Bottinger, Erwin P; Reid, St Patrick; Levy, David E; Bromberg, Jonathan S
2009 Jan 1;182(1):259-273, Journal of immunology
Foxp3, a winged-helix family transcription factor, serves as the master switch for CD4(+) regulatory T cells (Treg). We identified a unique and evolutionarily conserved CpG-rich island of the Foxp3 nonintronic upstream enhancer and discovered that a specific site within it was unmethylated in natural Treg (nTreg) but heavily methylated in naive CD4(+) T cells, activated CD4(+) T cells, and peripheral TGFbeta-induced Treg in which it was bound by DNMT1, DNMT3b, MeCP2, and MBD2. Demethylation of this CpG site using the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (Aza) induced acetylation of histone 3, interaction with TIEG1 and Sp1, and resulted in strong and stable induction of Foxp3. Conversely, IL-6 resulted in methylation of this site and repression of Foxp3 expression. Aza plus TGFbeta-induced Treg resembled nTreg, expressing similar receptors, cytokines, and stable suppressive activity. Strong Foxp3 expression and suppressor activity could be induced in a variety of T cells, including human CD4(+)CD25(-) T cells. Epigenetic regulation of Foxp3 can be predictably controlled with DNMT inhibitors to generate functional, stable, and specific Treg
— id: 93449, year: 2009, vol: 182, page: 259, stat: Journal Article,

Ancrod for acute ischemic stroke: a new dosing regimen derived from analysis of prior ancrod stroke studies
Levy, David E; Trammel, James; Wasiewski, Warren W
2009 Jan;18(1):23-27, Journal of stroke & cerebrovascular diseases
BACKGROUND: Ancrod, a fibrinogen-reducing agent, has been evaluated as treatment beginning within 3 or 6 hours of onset of acute ischemic stroke with inconsistent results. The data sets from these studies provide an opportunity to determine whether ancrod-related variables are associated with efficacy and safety. OBJECTIVE: This post hoc analysis of data from the Stroke Treatment with Ancrod Trial (STAT) analyzed ancrod-related variables as potential determinants of efficacy or safety. The resulting hypotheses were then tested in the European STAT (ESTAT) database. METHODS: The relationships between ancrod-related variables and the outcomes of efficacy and symptomatic intracranial hemorrhage (ICH) were analyzed using a 3-stage multivariate process. RESULTS: Good clinical outcome at 3 months based on the Barthel Index occurred almost twice as often in rapid defibrinogenators (>or=30 mg/dL/h) (52%) as in slow defibrinogenators (26%), with no increase in mortality or symptomatic ICH. Compared with a 20.7% incidence of symptomatic ICH in patients with mean post-9-hour fibrinogen levels less than or equal to 60 mg/dL, symptomatic ICH incidence was 0.8% in those with mean levels greater than 60 mg/dL (with no loss of efficacy). There were no symptomatic ICHs among 220 North American patients with mean levels greater than 70 mg/dL. It was hypothesized that an initial controlled rapid ancrod infusion with mean post-9-hour fibrinogen levels greater than 70 mg/dL would yield superior efficacy and safety. Such ESTAT patients had statistically significant efficacy versus placebo and a marked reduction in the incidence of symptomatic ICH versus patients taking ancrod with lower maintenance fibrinogen levels. CONCLUSION: Modifications to ancrod dosing may substantially improve efficacy while reducing the rate of symptomatic ICH
— id: 93448, year: 2009, vol: 18, page: 23, stat: Journal Article,

Knockdown of Stat3 activity in vivo prevents diabetic glomerulopathy
Lu, Ting-Chi; Wang, Zhao-Hui; Feng, Xiaobei; Chuang, Peter Y; Fang, Wei; Shen, Yuhong; Levy, David E; Xiong, Huabao; Chen, Nan; He, John Cijiang
2009 Jul;76(1):63-71, Kidney international
Recent studies suggest that Stat3, a transcription factor that mediates cytokine signaling, plays a critical role in the pathogenesis of diabetic nephropathy. Complete Stat3 gene knockout is embryonic lethal; therefore, we crossed Stat3+/- mice with Stat3 mutant mice (SA/SA) that lack full Stat3 activity. This strategy generated Stat3SA/- mice (25% activity) and Stat3SA/+ mice (75% activity), which were made diabetic using streptozotocin in order to define the role of Stat3 in diabetic kidney disease. While the glomerular number was not different between these two groups of mice, the diabetic SA/- mice had significantly less proteinuria, mesangial expansion, glomerular cell proliferation, and macrophage infiltration than the diabetic SA/+ mice. The reduction in Stat3 activity did not affect glomerular hyperfiltration seen after the induction of diabetes, as it was increased to the same degree in both groups of mice. Phosphorylation of Stat3 was markedly increased in the glomeruli of diabetic SA/+ mice compared to diabetic SA/- mice. The expression of inflammatory markers, IL-6, MCP-1, and activated NF-kappaB; type IV collagen, TGF-beta, and ICAM-1 mRNA; or type IV collagen and TGF-beta protein, were all found to be significantly less in glomeruli isolated from diabetic SA/- mice, as compared with diabetic SA/+ mice. Our study shows that Stat3 plays a critical role in the regulation of inflammation and abnormal matrix synthesis at an early stage of DN
— id: 138970, year: 2009, vol: 76, page: 63, stat: Journal Article,

Signal transducer and activator of transcription 4 limits the development of adaptive regulatory T cells
O'Malley, John T; Sehra, Sarita; Thieu, Vivian T; Yu, Qing; Chang, Hua-Chen; Stritesky, Gretta L; Nguyen, Evelyn T; Mathur, Anubhav N; Levy, David E; Kaplan, Mark H
2009 Aug;127(4):587-595, Immunology
T-cell responses to a cytokine milieu instruct the development of multiple effector phenotypes. While transforming growth factor-beta(1) (TGF-beta(1)) inhibits the development of T helper type 1 (Th1) and Th2 cells, we demonstrate that like interleukin-6 (IL-6) and IL-4, IL-12 can inhibit the development of TGF-beta(1)-induced Foxp3-expressing adaptive T regulatory (aTreg) cells. Signal transducer and activator of transcription 4 (STAT4) is critical for the response to IL-12, although there is a parallel pathway involving T box expressed in T cells (T-bet), and cells from mice double-deficient in STAT4 and T-bet are refractory to the inhibition of aTreg-cell development by IL-12. While the ability of these cytokines to promote Th differentiation may contribute to this effect, we observe that culture with IL-12, or other instructive cytokines, results in an increase in repressive chromatin modifications at the Foxp3 locus that limit STAT5 binding to Foxp3, without observed effects on IL-2 signalling pathways. In a model of allergic lung inflammation there are increased percentages of Treg cells in the lungs of Stat4(-/-) mice, compared with wild-type mice, and increases in Treg cells correlate with decreased allergic inflammation. Overall, these results suggest an important role for STAT4 in regulating Treg-cell development
— id: 132219, year: 2009, vol: 127, page: 587, stat: Journal Article,

Identification of a Stat3-dependent transcription regulatory network involved in metastatic progression
Ranger, Jill J; Levy, David E; Shahalizadeh, Solmaz; Hallett, Michael; Muller, William J
2009 Sep 1;69(17):6823-6830, Cancer research
High levels of activated Stat3 are often found in human breast cancers and can correlate with poor patient outcome. We employed an activated ErbB2 mouse model of breast cancer to investigate the in vivo role of Stat3 in mammary tumor progression and found that Stat3 does not alter mammary tumor initiation but dramatically affects metastatic progression. Four-fold fewer animals exhibited lung metastases in the absence of Stat3 and a 12-fold reduction in the number of lung lesions was observed in animals bearing Stat3-null tumors when compared with the wild-type cohort. The decreased malignancy in Stat3-deficient tumors is attributed to a reduction in both angiogenic and inflammatory responses associated with a Stat3-dependent transcriptional cascade involving CCAAT/enhancer binding protein delta
— id: 138942, year: 2009, vol: 69, page: 6823, stat: Journal Article,

IL-21 mediates suppressive effects via its induction of IL-10
Spolski, Rosanne; Kim, Hyoung-Pyo; Zhu, Wei; Levy, David E; Leonard, Warren J
2009 Mar 1;182(5):2859-2867, Journal of immunology
IL-21 is a pleiotropic cytokine that is required for normal Ig production. We previously showed that IL-21 was elevated in BXSB-Yaa mice with systemic lupus erythematosus. These mice also had elevated IL-10 levels, and we now show that IL-21 induces IL-10 mRNA and protein, suggesting unexpected immunosuppressive activities for IL-21. Indeed, Th1 priming with IL-21 leads to accumulation of cells with immunosuppressive activity, and IL-21 overexpression decreases specific Ab production after immunization in an IL-10-dependent fashion. Moreover, we show that IL-21 signaling is required for maximal induction of IL-10 by IL-6 or IL-27. Overall, our data indicate that IL-21 regulates immune responses at least in part by inducing IL-10 and reveal unanticipated immunosuppressive actions for this cytokine
— id: 93447, year: 2009, vol: 182, page: 2859, stat: Journal Article,

Analysis of chikungunya viral protein interactions with the interferon response pathway
Tangeman, M; Briese, T; Lipkin, WI; Levy, DE
2009 OCT-NOV ;48(1-2):60-60, Cytokine
— id: 105955, year: 2009, vol: 48, page: 60, stat: Journal Article,

c-Maf regulates IL-10 expression during Th17 polarization
Xu, Jiangnan; Yang, Yu; Qiu, Guixing; Lal, Girdhari; Wu, Zhihong; Levy, David E; Ochando, Jordi C; Bromberg, Jonathan S; Ding, Yaozhong
2009 May 15;182(10):6226-6236, Journal of immunology
IL-10 production by Th17 cells is critical for limiting autoimmunity and inflammatory responses. Gene array analysis on Stat6 and T-bet double-deficient Th17 cells identified the Th2 transcription factor c-Maf to be synergistically up-regulated by IL-6 plus TGFbeta and associated with Th17 IL-10 production. Both c-Maf and IL-10 induction during Th17 polarization depended on Stat3, but not Stat6 or Stat1, and mechanistically differed from IL-10 regulation by Th2 or IL-27 signals. TGFbeta was also synergistic with IL-27 to induce c-Maf, and it induced Stat1-independent IL-10 expression in contrast to IL-27 alone. Retroviral transduction of c-Maf was able to induce IL-10 expression in Stat6-deficient CD4 and CD8 T cells, and c-Maf directly transactivated IL-10 gene expression through binding to a MARE (Maf recognition element) motif in the IL-10 promoter. Taken together, these data reveal a novel role for c-Maf in regulating T effector development, and they suggest that TGFbeta may antagonize Th17 immunity by IL-10 production through c-Maf induction
— id: 138941, year: 2009, vol: 182, page: 6226, stat: Journal Article,

Identification of a PTEN-regulated STAT3 brain tumor suppressor pathway
Bonni, Azad; Depinho, Ronald A; Levy, David E; You, Mingjian J; Bachoo, Robert M; Chan, Jennifer A; Puram, Sidharth V; Konopka, Genevieve; de la Iglesia, Nuria
2008 Feb 15;22(4):449-462, Genes & development
Activation of the transcription factor STAT3 is thought to potently promote oncogenesis in a variety of tissues, leading to intense efforts to develop STAT3 inhibitors for many tumors, including the highly malignant brain tumor glioblastoma. However, the function of STAT3 in glioblastoma pathogenesis has remained unknown. Here, we report that STAT3 plays a pro-oncogenic or tumor-suppressive role depending on the mutational profile of the tumor. Deficiency of the tumor suppressor PTEN triggers a cascade that inhibits STAT3 signaling in murine astrocytes and human glioblastoma tumors. Specifically, we forge a direct link between the PTEN-Akt-FOXO axis and the leukemia inhibitory factor receptor beta (LIFRbeta)-STAT3 signaling pathway. Accordingly, PTEN knockdown induces efficient malignant transformation of astrocytes upon knockout of the STAT3 gene. Remarkably, in contrast to the tumor-suppressive function of STAT3 in the PTEN pathway, STAT3 forms a complex with the oncoprotein epidermal growth factor receptor type III variant (EGFRvIII) in the nucleus and thereby mediates EGFRvIII-induced glial transformation. These findings indicate that STAT3 plays opposing roles in glial transformation depending on the genetic background of the tumor, providing the rationale for tailored therapeutic intervention in glioblastoma
— id: 93452, year: 2008, vol: 22, page: 449, stat: Journal Article,

Nucleoporin levels regulate cell cycle progression and phase-specific gene expression
Chakraborty, Papia; Wang, Yaming; Wei, Jen-Hsuan; van Deursen, Jan; Yu, Hongtao; Malureanu, Liviu; Dasso, Mary; Forbes, Douglass J; Levy, David E; Seemann, Joachim; Fontoura, Beatriz M A
2008 Nov;15(5):657-667, Developmental cell
The Nup107-160 complex, the largest subunit of the nuclear pore, is multifunctional. It mediates mRNA export in interphase, and has roles in kinetochore function, spindle assembly, and postmitotic nuclear pore assembly. We report here that the levels of constituents of the Nup107-160 complex are coordinately cell cycle-regulated. At mitosis, however, a member of the complex, Nup96, is preferentially downregulated. This occurs via the ubiquitin-proteasome pathway. When the levels of Nup96 are kept high, a significant delay in G1/S progression occurs. Conversely, in cells of Nup96(+/-) mice, which express low levels of Nup96, cell cycle progression is accelerated. These lowered levels of Nup96 yield specific defects in nuclear export of certain mRNAs and protein expression, among which are key cell cycle regulators. Thus, Nup96 levels regulate differential gene expression in a phase-specific manner, setting the stage for proper cell cycle progression
— id: 93450, year: 2008, vol: 15, page: 657, stat: Journal Article,

Vaccinia virus protein E3L inhibits type I interferon induction by the cytoplasmic signaling pathway
Friedman, E; Marie, IJ; Levy, DE
2008 SEP ;43(3):300-301, Cytokine
— id: 91472, year: 2008, vol: 43, page: 300, stat: Journal Article,

IFNgamma signaling-does it mean JAK-STAT?
Gough, Daniel J; Levy, David E; Johnstone, Ricky W; Clarke, Christopher J
2008 Oct-Dec;19(5-6):383-394, Cytokine & growth factor reviews
The molecular pathways involved in the cellular response to interferon (IFN)gamma have been the focus of much research effort due to their importance in host defense against infection and disease, as well as its potential as a therapeutic agent. The discovery of the JAK-STAT signaling pathway greatly enhanced our understanding of the mechanism of IFNgamma-mediated gene transcription. However, in recent years it has become apparent that other pathways, including MAP kinase, PI3-K, CaMKII and NF-kappaB, either co-operate with or act in parallel to JAK-STAT signaling to regulate the many facets of IFNgamma biology in a gene- and cell type-specific manner. The complex interactions between JAK/STAT and alternate pathways and the impact of these signaling networks on the biological responses to IFNgamma are beginning to be understood. This review summarizes and appraises current advances in our understanding of these complex interactions, their specificity and proposed biological outcomes
— id: 93451, year: 2008, vol: 19, page: 383, stat: Journal Article,

Monocytes in the urine of children with lupus: A potential marker of active nephritis
Kahn, PJ; Zhang, HZ; Levy, D; Imundo, L; Eichenfield, A; Winchestet, R
2008 ;58(9):S252-S252, Arthritis & rheumatism
— id: 90034, year: 2008, vol: 58, page: S252, stat: Journal Article,

Inhibition of interferon regulatory factor 7 (IRF7)-mediated interferon signal transduction by the Kaposi's sarcoma-associated herpesvirus viral IRF homolog vIRF3
Joo, Chul Hyun; Shin, Young C; Gack, Michaela; Wu, Liguo; Levy, David; Jung, Jae U
2007 Aug;81(15):8282-8292, Journal of virology
Upon viral infection, the major defense mounted by the host immune system is activation of the interferon (IFN)-mediated antiviral pathway that is mediated by IFN regulatory factors (IRFs). In order to complete their life cycle, viruses must modulate the host IFN-mediated immune response. Kaposi's sarcoma-associated herpesvirus (KSHV), a human tumor-inducing herpesvirus, has developed a unique mechanism for antagonizing cellular IFN-mediated antiviral activity by incorporating viral homologs of the cellular IRFs, called vIRFs. Here, we report a novel immune evasion mechanism of KSHV vIRF3 to block cellular IRF7-mediated innate immunity in response to viral infection. KSHV vIRF3 specifically interacts with either the DNA binding domain or the central IRF association domain of IRF7, and this interaction leads to the inhibition of IRF7 DNA binding activity and, therefore, suppression of alpha interferon (IFN-alpha) production and IFN-mediated immunity. Remarkably, the central 40 amino acids of vIRF3, containing the double alpha helix motifs, are sufficient not only for binding to IRF7, but also for inhibiting IRF7 DNA binding activity. Consequently, the expression of the double alpha helix motif-containing peptide effectively suppresses IRF7-mediated IFN-alpha production. This demonstrates a remarkably efficient means of viral avoidance of host antiviral activity
— id: 138972, year: 2007, vol: 81, page: 8282, stat: Journal Article,

STAT3 signaling and the hyper-IgE syndrome
Levy, David E; Loomis, Cynthia A
2007 Oct 18;357(16):1655-1658, New England journal of medicine
— id: 93454, year: 2007, vol: 357, page: 1655, stat: Journal Article,

The zinc finger antiviral protein acts synergistically with an interferon-induced factor for maximal activity against alphaviruses
MacDonald, Margaret R; Machlin, Erica S; Albin, Owen R; Levy, David E
2007 Dec;81(24):13509-13518, Journal of virology
Type I interferons (IFNs) signal through specific receptors to mediate expression of genes, which together confer a cellular antiviral state. Overexpression of the zinc finger antiviral protein (ZAP) imparts a cellular antiviral state against Retroviridae, Togaviridae, and Filoviridae virus family members. Since ZAP expression is induced by IFN, we utilized Sindbis virus (SINV) to investigate the role of other IFN-induced factors in ZAP's inhibitory potential. Overexpressed ZAP did not inhibit virion production or SINV-induced cell death in BHK cells deficient in IFN production (and thus IFN signaling), suggesting a role for an IFN-induced factor in ZAP's activity. IFN pretreatment in the presence of ZAP resulted in greater inhibition than IFN alone. Using mouse embryo fibroblast (MEF) cells deficient in Stat1, we showed that signaling through the IFN receptor is necessary for IFN's enhancement of ZAP activity. Unlike in BHK cells, however, overexpressed ZAP exhibited antiviral activity in the absence of IFN. In wild-type MEFs with an intact Stat1 gene, IFN pretreatment synergized with ZAP to generate a potent antiviral response. Despite failing to inhibit SINV virion production and virus-induced cell death in BHK cells, ZAP inhibited translation of the incoming viral RNA. IFN pretreatment synergized with ZAP to further block protein expression from the incoming viral genome. We further show that silencing of IFN-induced ZAP reduces IFN efficacy. Our findings demonstrate that ZAP can synergize with another IFN-induced factor(s) for maximal antiviral activity and that ZAP's intrinsic antiviral activity on virion production and cell survival can have cell-type-specific outcomes
— id: 93453, year: 2007, vol: 81, page: 13509, stat: Journal Article,

Nuclear receptor coregulator (NRC): mapping of the dimerization domain, activation of p53 and STAT-2, and identification of the activation domain AD2 necessary for nuclear receptor signaling
Mahajan, Muktar A; Murray, Audrey; Levy, David; Samuels, Herbert H
2007 Aug;21(8):1822-1834, Molecular endocrinology
Nuclear receptor coregulator (NRC) is a 250-kDa nuclear protein involved in transcriptional activation of nuclear hormone receptors, nuclear factor-kappaB, c-Jun, c-Fos, and cAMP response element-binding protein. NRC is organized into a modular structure consisting of two activation domains (AD1 and AD2), two nuclear hormone receptor-interacting motifs, LxxLL-1 and LxxLL-2, and a C-terminal regulatory region rich in serines, threonines, and leucines. The LxxLL-1 motif of NRC binds to a broad spectrum of nuclear hormone receptors with high affinity whereas LxxLL-2 interacts with a very limited number of receptors. In this study we present further evidence that NRC can act as a dimer and have identified a dimerization region of 146 amino acids including LxxLL-1. Mutation of the core LxxLL-1 motif, however, indicates that it is not involved in the dimerization of NRC. AD2, just C-terminal of LxxLL-1, was found to play a central role in ligand-dependent activation by nuclear receptors even though AD1 exhibits more potent intrinsic activity. Thus, a short region of approximately 300 amino acids including and flanking LxxLL-1 plays an important role in NRC dimerization and nuclear receptor binding and transcriptional activation. In addition, consistent with its role as a cointegrator for transcriptional activation, NRC also functions as a coactivator for signal transducer and activator of transcription 2 (STAT-2) and p53. Activation of p53 by NRC appears to involve a novel mechanism where NRC interacts indirectly with p53 through Trap80, a member of the mediator complex, which binds NRC interacting factor-1 (NIF-1), which interacts with and potentiates the effect of NRC
— id: 73864, year: 2007, vol: 21, page: 1822, stat: Journal Article,

Stat3 and Stat4 direct development of IL-17-secreting Th cells
Mathur, Anubhav N; Chang, Hua-Chen; Zisoulis, Dimitrios G; Stritesky, Gretta L; Yu, Qing; O'Malley, John T; Kapur, Reuben; Levy, David E; Kansas, Geoffrey S; Kaplan, Mark H
2007 Apr 15;178(8):4901-4907, Journal of immunology
IL-17-secreting CD4(+) T cells are critically involved in inflammatory immune responses. Development of these cells is promoted in vivo and in vitro by IL-23 or TGFbeta1 plus IL-6. Despite growing interest in this inflammatory Th subset, little is known about the transcription factors that are required for their development. We demonstrate that Stat3 is required for programming the TGFbeta1 plus IL-6 and IL-23-stimulated IL-17-secreting phenotype, as well as for RORgammat expression in TGFbeta1 plus IL-6-primed cells. Moreover, retroviral transduction of a constitutively active Stat3 into differentiating T cell cultures enhances IL-17 production from these cells. We further show that Stat4 is partially required for the development of IL-23-, but not TGFbeta1 plus IL-6-primed IL-17-secreting cells, and is absolutely required for IL-17 production in response to IL-23 plus IL-18. The requirements for Stat3 and Stat4 in the development of these IL-17-secreting subsets reveal additional mechanisms in Th cell fate decisions during the generation of proinflammatory cell types
— id: 93456, year: 2007, vol: 178, page: 4901, stat: Journal Article,

Influenza virus targets the mRNA export machinery and the nuclear pore complex
Satterly, Neal; Tsai, Pei-Ling; van Deursen, Jan; Nussenzveig, Daniel R; Wang, Yaming; Faria, Paula A; Levay, Agata; Levy, David E; Fontoura, Beatriz M A
2007 Feb 6;104(6):1853-1858, Proceedings of the National Academy of Sciences of the United States of America
The NS1 protein of influenza A virus is a major virulence factor that is essential for pathogenesis. NS1 functions to impair innate and adaptive immunity by inhibiting host signal transduction and gene expression, but its mechanisms of action remain to be fully elucidated. We show here that NS1 forms an inhibitory complex with NXF1/TAP, p15/NXT, Rae1/mrnp41, and E1B-AP5, which are key constituents of the mRNA export machinery that interact with both mRNAs and nucleoporins to direct mRNAs through the nuclear pore complex. Increased levels of NXF1, p15, or Rae1 revert the mRNA export blockage induced by NS1. Furthermore, influenza virus down-regulates Nup98, a nucleoporin that is a docking site for mRNA export factors. Reduced expression of these mRNA export factors renders cells highly permissive to influenza virus replication, demonstrating that proper levels of key constituents of the mRNA export machinery protect against influenza virus replication. Because Nup98 and Rae1 are induced by interferons, down-regulation of this pathway is likely a viral strategy to promote viral replication. These findings demonstrate previously undescribed influenza-mediated viral-host interactions and provide insights into potential molecular therapies that may interfere with influenza infection
— id: 93457, year: 2007, vol: 104, page: 1853, stat: Journal Article,

JAK-STAT signaling: from interferons to cytokines
Schindler, Christian; Levy, David E; Decker, Thomas
2007 Jul 13;282(28):20059-20063, Journal of biological chemistry
— id: 93455, year: 2007, vol: 282, page: 20059, stat: Journal Article,

Restricted tissue tropism and acquired resistance to Sindbis viral vector expression in the absence of innate and adaptive immunity
Tseng, J-C; Zheng, Y; Yee, H; Levy, D E; Meruelo, D
2007 Aug;14(15):1166-1174, Gene therapy
Our previous studies suggest that replication-defective Sindbis vectors might be promising agents for specific tumor targeting and detection. However, the effects of innate and/or adaptive anti-viral immunity, in particular, the IFN-I/STAT1 signaling pathway, may impact their therapeutic potential. Using a bioluminescent imaging system, we demonstrate that although most normal cells are not permissively transduced by replication-defective Sindbis vector, transduction of liver non-sinusoidal endothelial occurs the first time IFN-I/STAT1 signaling deficient mice are inoculated with the vector. Transduction of some cells is not surprising since STAT1 knockout animals show significant delay in IFN responses such as the production of IFN-alpha/beta and transcriptional activation of several anti-viral genes (IRF7, RIG-I, PKR, TLR3, USP18, ISG15). However, beyond the initial vector transduction, which resolves rapidly, secondary inoculums of Sindbis vectors do not transduce any liver cells, suggesting that an alternative antiviral pathway may protect against further transduction. Other known signaling pathways were examined using mice lacking functional TLR3, tumor necrosis factoralphaR or nuclear factor-kappa B (p50). Surprisingly, none of those pathways seem to play a significant role in anti-Sindbis responses. Thus it appears that in vivo, in contrast to the ready transduction of tumor cells, transduction of normal cells by replication-defective Sindbis vector is limited, possibly by a novel mechanism
— id: 74661, year: 2007, vol: 14, page: 1166, stat: Journal Article,

Multiple signaling pathways are involved in IL-21-mediated proliferation
Zeng, R; Spolski, R; Casas, E; Zhu, W; Levy, DE; Leonard, WJ
2007 JUL ;39(1):48-48, Cytokine
— id: 87204, year: 2007, vol: 39, page: 48, stat: Journal Article,

The molecular basis of IL-21-mediated proliferation
Zeng, Rong; Spolski, Rosanne; Casas, Esther; Zhu, Wei; Levy, David E; Leonard, Warren J
2007 May 15;109(10):4135-4142, Blood
Interleukin-21 (IL-21) is a type I cytokine that modulates functions of T, B, natural killer (NK), and myeloid cells. The IL-21 receptor (IL-21R) is closely related to the IL-2 receptor beta chain and is capable of transducing signals through its dimerization with the common cytokine receptor gamma chain (gamma(c)), the protein whose expression is defective in humans with X-linked severe combined immunodeficiency. To clarify the molecular basis of IL-21 actions, we investigated the role of tyrosine residues in the IL-21R cytoplasmic domain. Simultaneous mutation of all 6 tyrosines greatly diminished IL-21-mediated proliferation, whereas retention of tyrosine 510 (Y510) allowed full proliferation. Y510 efficiently mediated IL-21-induced phosphorylation of Stat1 and Stat3, but not of Stat5, and CD8(+) T cells from Stat1/Stat3 double knock-out mice exhibited decreased proliferation in response to IL-21 + IL-15. In addition, IL-21 weakly induced phosphorylation of Shc and Akt, and consistent with this, specific inhibitors of the MAPK and PI3K pathways inhibited IL-21-mediated proliferation. Collectively, these data indicate the involvement of the Jak-STAT, MAPK, and PI3K pathways in IL-21 signaling
— id: 93458, year: 2007, vol: 109, page: 4135, stat: Journal Article,

IL-6 programs TH-17 cell differentiation by promoting the sequential engagement of the IL-21 and IL-23 pathways
Zhou, L; Ivanov, II; Spolski, R; Min, R; Shenderov, K; Egawa, T; Levy, DE; Leonard, WJ; Littman, DR
2007 JUL ;39(1):49-49, Cytokine
— id: 87205, year: 2007, vol: 39, page: 49, stat: Journal Article,

IL-6 programs T(H)-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways
Zhou, Liang; Ivanov, Ivaylo I; Spolski, Rosanne; Min, Roy; Shenderov, Kevin; Egawa, Takeshi; Levy, David E; Leonard, Warren J; Littman, Dan R
2007 Sep;8(9):967-974, Nature immunology
T helper cells that produce interleukin 17 (IL-17; 'T(H)-17 cells') are a distinct subset of proinflammatory cells whose in vivo function requires IL-23 but whose in vitro differentiation requires only IL-6 and transforming growth factor-beta (TGF-beta). We demonstrate here that IL-6 induced expression of IL-21 that amplified an autocrine loop to induce more IL-21 and IL-23 receptor in naive CD4(+) T cells. Both IL-21 and IL-23, along with TGF-beta, induced IL-17 expression independently of IL-6. The effects of IL-6 and IL-21 depended on STAT3, a transcription factor required for the differentiation of T(H)-17 cells in vivo. IL-21 and IL-23 induced the orphan nuclear receptor RORgammat, which in synergy with STAT3 promoted IL-17 expression. IL-6 therefore orchestrates a series of 'downstream' cytokine-dependent signaling pathways that, in concert with TGF-beta, amplify RORgammat-dependent differentiation of T(H)-17 cells
— id: 74681, year: 2007, vol: 8, page: 967, stat: Journal Article,

STAT3 positively regulates an early step in B-cell development
Chou, Wei-Chun; Levy, David E; Lee, Chien-Kuo
2006 Nov 1;108(9):3005-3011, Blood
Transcription factors are critical for instructing the development of B lymphocytes from multipotential progenitor cells in the bone marrow (BM). Here, we show that the absence of STAT3 impaired B-cell development. Mice selectively lacking STAT3 in BM progenitor cells displayed reduced numbers of mature B cells, both in the BM and in the periphery. The reduction in the B-cell compartment included reduced percentages and numbers of pro-B, pre-B, and immature B cells in the absence of STAT3, whereas the number of pre-pro-B cells was increased. We found that pro-B and pre-B-cell populations lacking STAT3 were hyporesponsive to IL-7 because of a decreased number of IL-7-responsive cells rather than decreased expression or signaling of IL-7Ralpha. Moreover, STAT3-deficient mice displayed enhanced apoptosis in the pro-B population when deprived of survival factors, suggesting that at least 2 mechanisms (impaired differentiation and enhanced apoptosis) are involved in the mutant phenotype. Last, BM transplantation confirmed that impaired B lymphopoiesis in the absence of STAT3 was caused by a cell autonomous defect. In sum, these studies defined a specific role for STAT3 in early B-cell development, probably acting at the pre-pro-B transition by contributing to the survival of IL-7-responsive progenitors
— id: 93462, year: 2006, vol: 108, page: 3005, stat: Journal Article,

The nucleoporin Nup96 is required for proper expression of interferon-regulated proteins and functions
Faria, Ana M C; Levay, Agata; Wang, Yaming; Kamphorst, Alice O; Rosa, Magda L P; Nussenzveig, Daniel R; Balkan, Wayne; Chook, Yuh Min; Levy, David E; Fontoura, Beatriz M A
2006 Mar;24(3):295-304, Immunity
Nup98 and Nup96 are components of the nuclear transport machinery and are induced by interferons (IFN). Nup98 is a constituent of an mRNA export pathway that is targeted by viruses and regulated by IFN. However, the role of Nup96 in IFN-related mechanisms has not been established. To investigate the function of Nup96 in vivo, we generated Nup96(+/-) mice that express low levels of Nup96, as Nup96(-/-) mice are lethal. The Nup96(+/-) mice presented selective alterations of the immune system, which resulted in downregulation and impaired IFN alpha- and gamma-mediated induction of MHC I and IFNgamma induction of MHC II, ICAM-1, and other proteins. Frequency of TCRalphabeta+ and CD4+ T cells, which depends on MHC function, is reduced in NUP96(+/-) mice. Upon immunization, Nup96(+/-) mice showed impaired antigen presentation and T cell proliferation. Nup96(+/-) cells and mice were highly susceptible to viral infection, demonstrating a role for Nup96 in innate and adaptive immunity
— id: 93464, year: 2006, vol: 24, page: 295, stat: Journal Article,

Gr-1+CD115+ immature myeloid suppressor cells mediate the development of tumor-induced T regulatory cells and T-cell anergy in tumor-bearing host
Huang, Bo; Pan, Ping-Ying; Li, Qingsheng; Sato, Alice I; Levy, David E; Bromberg, Jonathan; Divino, Celia M; Chen, Shu-Hsia
2006 Jan 15;66(2):1123-1131, Cancer research
The accumulation of myeloid suppressor cells (MSCs) is associated with immune suppression in tumor-bearing mice and in cancer patients. The suppressive activity of MSC correlates with the expression of the myeloid markers Gr-1, CD115 (macrophage colony-stimulating factor receptor), and F4/80. Gr-1(+)CD115(+) MSCs, in addition to being able to suppress T-cell proliferation in vitro, can induce the development of Foxp3(+) T regulatory cells (Treg) in vivo, which are anergic and suppressive. Furthermore, the secretion of interleukin (IL)-10 and transforming growth factor-beta by Gr-1(+)CD115(+) MSCs was induced and enhanced, respectively, on IFN-gamma stimulation. The development of Treg requires antigen-associated activation of tumor-specific T cells, depends on the presence of IFN-gamma and IL-10, and is independent of the nitric oxide-mediated suppressive mechanism by MSC. Our data provide evidence that Gr-1(+)CD115(+) MSC can mediate the development of Treg in tumor-bearing mice and show a novel immune suppressive mechanism by which MSCs can suppress antitumor responses
— id: 93465, year: 2006, vol: 66, page: 1123, stat: Journal Article,

STAT1 acts as a tumor promoter for leukemia development
Kovacic, Boris; Stoiber, Dagmar; Moriggl, Richard; Weisz, Eva; Ott, Rene G; Kreibich, Rita; Levy, David E; Beug, Hartmut; Freissmuth, Michael; Sexl, Veronika
2006 Jul;10(1):77-87, Cancer cell
The tumor suppressor STAT1 is considered a key regulator of the surveillance of developing tumors. Here, we describe an unexpected tumor-promoting role for STAT1 in leukemia. STAT1(-/-) mice are partially protected from leukemia development, and STAT1(-/-) tumor cells induce leukemia in RAG2(-/-) and immunocompetent mice with increased latency. The low MHC class I protein levels of STAT1(-/-) tumor cells enable efficient NK cell lysis and account for the enhanced tumor clearance. Strikingly, STAT1(-/-) tumor cells acquire increased MHC class I expression upon leukemia progression. These findings define STAT1 as a tumor promoter in leukemia development. Furthermore, we describe the upregulation of MHC class I expression as a general mechanism that allows for the escape of hematopoietic malignancies from immune surveillance
— id: 93461, year: 2006, vol: 10, page: 77, stat: Journal Article,

STAT3: a multifaceted oncogene
Levy, David E; Inghirami, Giorgio
2006 Jul 5;103(27):10151-10152, Proceedings of the National Academy of Sciences of the United States of America
— id: 67388, year: 2006, vol: 103, page: 10151, stat: Journal Article,

Gemcitabine (G) plamsa and intracellular pharmacokinetics in E6201: Greater metabolite levels using fixed dosing rate (FDR) delivery
Liebes, L; Levy, DE; Poplin, E; Mendoza, S; Fry, D; Buckley, M; Zoloratov, A; Benson, A; Hochster, H
2006 JUN 20 ;24(18):85S-85S, Journal of clinical oncology
— id: 69295, year: 2006, vol: 24, page: 85S, stat: Journal Article,

Regulation of IRF7 through cell type-specific protein stability
Prakash, Arun; Levy, David E
2006 Mar 31;342(1):50-56, Biochemical & biophysical research communications
Interferon regulatory factor 7 (IRF7) is a key component of the cellular response to virus infection that culminates in physiologically relevant IFNalpha production. We studied molecular mechanisms governing responses to respiratory viral infection that are characterized by transient induction and subsequent shut-off of interferon (IFN) gene expression. We asked whether alterations in IRF7 protein stability occurred during virus infection that might contribute to this regulation. To this end, we measured IRF7 half-life in various cell types and found it to be short-lived, in marked contrast to the pronounced stability of the related transcription factor, IRF3. Furthermore, virus infection accelerated IRF7 degradation in a proteosome-dependent manner in most cell types. However, plasmacytoid dendritic cells (pDC), which constitute the major circulating IFN producing cell type, displayed a distinct pattern of regulation. Infection of lymphoid tissues, where the majority of IRF7 is expressed in pDC, attenuated the normal proteosome-mediated degradation of IRF7, resulting in a long-lived protein. Stabilization was partially stimulated by autocrine IFN as a positive feedback mechanism, but was partially IFN independent. Thus, two distinct posttranslational mechanisms regulate IRF7 activity in response to viral infection, with protein turnover attenuating responses postinfection in most cell types while infection-induced protein stabilization contributes to the heightened IFN production characteristic of pDC
— id: 63740, year: 2006, vol: 342, page: 50, stat: Journal Article,

Regulation of Stat3 transcriptional activity by the conserved LPMSP motif for OSM and IL-6 signaling
Sun, Wei; Snyder, Marylynn; Levy, David E; Zhang, J Jillian
2006 Oct 30;580(25):5880-5884, FEBS letters
To achieve maximal transcriptional activity in response to gp130 cytokines, Serine-727 (Ser-727) of Stat3 is phosphorylated. Ser-727 resides in the LPMSP motif, the only conserved sequence among the transcription activation domains of several STATs. We show here that in addition to Ser-727, other residues in this LPMSP motif are also required for Stat3 activity in response to cytokine signaling through regulation of Ser-727 phosphorylation and recruitment of the transcription co-activator CBP/p300 to the promoters of Stat3-target genes for transcription activation. Hence, we have demonstrated a critical role for the whole conserved LPMSP motif in JAK-STAT signaling
— id: 93459, year: 2006, vol: 580, page: 5880, stat: Journal Article,

C. elegans STAT cooperates with DAF-7/TGF-beta signaling to repress dauer formation
Wang, Yaming; Levy, David E
2006 Jan 10;16(1):89-94, Current biology. CB
The DAF-7/TGF-beta pathway in C. elegans interprets environmental signals relayed through amphid neurons and actively inhibits dauer formation during reproductive developmental growth . In metazoans, the STAT pathway interprets external stimuli through regulated tyrosine phosphorylation, nuclear translocation, and gene expression , but its importance for developmental commitment, particularly in conjunction with TGF-beta, remains largely unknown. Here, we report that the nematode STAT ortholog STA-1 accumulated in the nuclei of five head neuron pairs, three of which are amphid neurons involved in dauer formation . Moreover, sta-1 mutants showed a synthetic dauer phenotype with selected TGF-beta mutations. sta-1 deficiency was complemented by reconstitution with wild-type protein, but not with a tyrosine mutant. Canonical TGF-beta signaling involves the DAF-7/TGF-beta ligand activating the DAF-1/DAF-4 receptor pair to regulate the DAF-8/DAF-14 Smads . Interestingly, STA-1 functioned in the absence of DAF-7, DAF-4, and DAF-14, but it required DAF-1 and DAF-8. Additionally, STA-1 expression was induced by TGF-beta in a DAF-3-dependent manner, demonstrating a homeostatic negative feedback loop. These results highlight a role for activated STAT proteins in repression of dauer formation. They also raise the possibility of an unexpected function for DAF-1 and DAF-8 that is independent of their normal upstream activator, DAF-7
— id: 62749, year: 2006, vol: 16, page: 89, stat: Journal Article,

C. elegans STAT: evolution of a regulatory switch
Wang, Yaming; Levy, David E
2006 Aug;20(10):1641-1652, FASEB journal
STAT transcription factors have been implicated in many biological processes, particularly host immune defense and development. Here we characterize a STAT orthologue from the nematode, C. elegans. We show that this protein, termed STA-1, is structurally and functionally related to other vertebrate and invertebrate STAT proteins, recognizing a cis DNA element conserved through phylogeny. Unexpectedly, STA-1 lacks the conserved amino-terminal oligomerization domain found in vertebrate and other invertebrate STAT proteins, a feature also lacking in orthologues from a distantly related nematode species and from the slime mold, Dictyostelium discoideum. This absence suggests that a primordial STAT protein lacked this domain, which was accreted later in evolution to provide further regulatory control of STAT signaling. Derivation of null mutants demonstrated that STA-1 is not required for nematode viability, despite its widespread expression in multiple tissues of the worm. However, mutant STA-1 proteins that lack functional coiled-coil and DNA binding domains could still be activated and accumulated in the nucleus, suggesting that DNA binding is not a necessary prerequisite for nuclear retention of activated STAT proteins. Our results shed new light on the evolution and function of the STAT signaling pathway and on the structural requirements for STAT activation
— id: 68775, year: 2006, vol: 20, page: 1641, stat: Journal Article,

Nuclear and cytoplasmic mRNA quantification by SYBR green based real-time RT-PCR
Wang, Yaming; Zhu, Wei; Levy, David E
2006 Aug;39(4):356-362, Methods
Measurement of the steady-state abundance of nuclear and cytoplasmic RNA requires efficient subcellular fractionation and RNA recovery coupled with accurate quantification of individual RNA species. Detergent lysis of tissue culture cells provides a simple fractionation procedure that can be optimized to individual cell lines. The large dynamic range, extreme sensitivity, high sequence-specificity, and fast turn-around time has allowed real-time reverse transcription polymerase chain reaction (real-time RT-PCR) to become a standard tool for mRNA quantification. Among the different chemistries used for PCR product detection during amplification, DNA binding dyes such as SYBR Green I are simple, versatile, and yet highly reliable and least expensive. With attention to primer design and cycling conditions, virtually any mRNA species can be accurately quantified from even minute quantities of starting RNA. This method provides an accurate and efficient procedure for estimating the relative ratios of nuclear and cytoplasmic RNA concentrations
— id: 93460, year: 2006, vol: 39, page: 356, stat: Journal Article,

An IL-21 receptor tyrosine is critical for IL-21-induced proliferation and the activation of Stat1 and Stat3
Zeng, R; Spolski, R; Casas, E; Levy, DE; Leonard, WJ
2006 APR 1 ;176(4):S14-S14, Journal of immunology
— id: 68834, year: 2006, vol: 176, page: S14, stat: Journal Article,

IL-6 signaling via the STAT3/SOCS3 pathway: functional analysis of the conserved STAT3 N-domain
Zhang, Ling; Badgwell, Donna B; Bevers, Jack J 3rd; Schlessinger, Karni; Murray, Peter J; Levy, David E; Watowich, Stephanie S
2006 Aug;288(1-2):179-189, Molecular & cellular biochemistry
The conserved N-domain of the STAT proteins has been implicated in several activities crucial to cytokine signaling including receptor recruitment and STAT activation, cooperative DNA binding and STAT-dependent gene expression. We evaluated the role of the STAT3 N-domain in the IL-6 signal transduction pathway leading to Socs3 gene expression, an essential mechanism that controls the quality and magnitude of IL-6-dependent transcriptional responses. Based on the model for STAT N-domain function in cooperative gene expression and the presence of tandem STAT binding motifs in the murine Socs3 promoter, we anticipated that stabilizing interactions between adjacent STAT3 dimers via N-domain sequences might be essential for Socs3 gene expression. This was underscored by the tight conservation in the location and sequence of the tandem STAT binding sites between the murine and human Socs3 promoters. Using reconstitution into Stat3-/- mouse embryonic fibroblasts (Stat3-/- MEFs), we find that a STAT3 N-domain deletion mutant (Delta 133STAT3) is activated by tyrosine phosphorylation in response to IL-6 and then undergoes dephosphorylation with kinetics similar to full-length STAT3. These results highlight important differences compared to other STATs where the N-domain has been shown to mediate activation (STAT4) or dephosphorylation (STAT1). STAT3 binds predominantly to a single STAT consensus site in the Socs3 promoter, despite the presence of an adjacent STAT motif. Significantly, Delta 133STAT3 stimulates expression of the endogenous Socs3 gene in Stat3-/- MEFs upon IL-6 treatment with an activity similar to reconstituted STAT3, demonstrating that the N-domain is dispensable for Socs3 gene expression. We propose that the Socs3 gene in its chromosomal context is activated by the IL-6/STAT3 pathway independent of STAT3 N-domain sequences
— id: 93463, year: 2006, vol: 288, page: 179, stat: Journal Article,

Regulatory serine residues mediate phosphorylation-dependent and phosphorylation-independent activation of interferon regulatory factor 7
Caillaud, Alexandre; Hovanessian, Ara G; Levy, David E; Marie, Isabelle J
2005 May 6;280(18):17671-17677, Journal of biological chemistry
Interferon regulatory factor (IRF)7 is a key transcription factor required for establishment of antiviral resistance. In response to infection, IRF7 is activated by phosphorylation through the action of the non-canonical IkappaB kinases, IkappaB kinase-epsilon and TANK-binding kinase 1. Activation leads to nuclear retention, DNA binding, and derepression of transactivation ability. Clusters of serine residues located in the carboxyl-terminal regulatory domain of IRF7 are putative targets of virus-activated kinases. However, the exact sites of phosphorylation have not yet been established. Here, we report a comprehensive structure-activity examination of potential IRF7 phosphorylation sites through analysis of mutant proteins in which specific serine residues were altered to alanine or aspartate. Phosphorylation patterns of these mutants were analyzed by two-dimensional gel electrophoresis, and their transcriptional activity was monitored by reporter assays. Essential phosphorylation events were mapped to amino acids 437-438 and a redundant set of sites at either amino acids 429-431 or 441. IRF7 recovered from infected cells was heterogeneously phosphorylated at these sites, and greater phosphorylation correlated with increased transactivation. Interestingly, a distinct serine cluster conserved in the related protein IRF3 was also essential for IRF7 activation and distal phosphorylation. However, the essential role of this motif did not appear to be fulfilled by phosphorylation. Rather, these serine residues and an adjacent leucine were required for phosphorylation at distal sites and may determine a conformational element required for function
— id: 93468, year: 2005, vol: 280, page: 17671, stat: Journal Article,

Disruption of the gamma-interferon signaling pathway at the level of signal transducer and activator of transcription-1 prevents immune destruction of beta-cells
Gysemans, Conny A; Ladriere, Laurence; Callewaert, Hanne; Rasschaert, Joanne; Flamez, Daisy; Levy, David E; Matthys, Patrick; Eizirik, Decio L; Mathieu, Chantal
2005 Aug;54(8):2396-2403, Diabetes
beta-cells under immune attack are destroyed by the aberrant activation of key intracellular signaling cascades. The aim of the present study was to evaluate the contribution of the signal transducer and activator of transcription (STAT)-1 pathway for beta-cell apoptosis by studying the sensitivity of beta-cells from STAT-1 knockout (-/-) mice to immune-mediated cell death in vitro and in vivo. Whole islets from STAT-1-/- mice were completely resistant to interferon (IFN)-gamma (studied in combination with interleukin [IL]-1beta)-mediated cell death (92 +/- 4% viable cells in STAT-1-/- mice vs. 56 +/- 3% viable cells in wild-type controls, P < or = 0.001) and had preserved insulin release after exposure to IL-1beta and IFN-gamma. Moreover, analysis of cell death in cytokine-exposed purified beta-cells confirmed that protection was due to absence of STAT-1 in the beta-cells themselves. Deficiency of STAT-1 in islets completely prevented cytokine-induced upregulation of IL-15, interferon inducible protein 10, and inducible nitric oxide synthase transcription but did not interfere with monocyte chemoattractant protein 1 and macrophage inflammatory protein 3alpha expression. In vivo, STAT-1-/- mice were partially resistant to development of diabetes after multiple low-dose streptozotocin injections as reflected by mean blood glucose at 12 days after first injection (159 +/- 28 vs. 283 +/- 81 mg/dl in wild-type controls, P < or = 0.05) and diabetes incidence at the end of the follow-up period (39 vs. 73% in wild-type controls, P < or = 0.05). In conclusion, the present results indicate that STAT-1 is a crucial transcription factor in the process of IFN-gamma-mediated beta-cell death and the subsequent development of immune-mediated diabetes
— id: 93466, year: 2005, vol: 54, page: 2396, stat: Journal Article,

New and old functions of STAT3: a pivotal target for individualized treatment of cancer
Inghirami, Giorgio; Chiarle, Roberto; Simmons, William J; Piva, Roberto; Schlessinger, Karni; Levy, David E
2005 Sep;4(9):1131-1133, Cell cycle
Signal transducers and activators of transcription (STAT) regulate a plethora of cytokine responses. Recently, aberrant signaling by STAT proteins has been demonstrated to play important roles in the pathogenesis of many neoplasms, by promoting cell cycle progression and survival, stimulating angiogenesis, and impairing immunological responses and tumor surveillance. We have developed genetic tools to evaluate STAT-dependent malignancy and showed that survival and growth of lymphoid malignancies requires expression of STAT3. In contrast, loss of STAT3 in normal cells does not impair their growth or survival; but in spite of this apparent dispensability of STAT3, STAT3-null fibroblasts are resistant to transformation by a variety of oncogenes. The precise molecular mechanisms responsible for the tumorigenic activity of STAT3 have been only partially elucidated. While the tyrosine phosphorylation of STAT3, which is indicative of its signal-dependent activation, is a common occurrence in tumors, and appears to play a crucial role in some malignancies, a variety of new data suggest that it can be dispensable under some circumstances and STAT3 can participate in transformation through novel and non-canonical mechanisms. The discovery and dissection of non-canonical modes of STAT3 action will open new avenues for the design of effective therapeutics capable of neutralizing the tumorigenic properties of this molecule
— id: 64467, year: 2005, vol: 4, page: 1131, stat: Journal Article,

Activation of mitogen-activated protein kinase kinase (MKK) 3 and MKK6 by type I interferons
Li, Yongzhong; Batra, Sandeep; Sassano, Antonella; Majchrzak, Beata; Levy, David E; Gaestel, Matthias; Fish, Eleanor N; Davis, Roger J; Platanias, Leonidas C
2005 Mar 18;280(11):10001-10010, Journal of biological chemistry
There is accumulating evidence that the p38 MAP kinase pathway plays important roles in Type I interferon (IFN) signaling, but the mechanisms regulating p38 activation during engagement of the Type I IFN receptor remain to be defined. We sought to identify the events that lead to activation of the p38 MAP kinase in response to Type I IFNs. Our data demonstrate that treatment of sensitive cell lines with IFNalpha results in activation of both MAP kinase kinase 3 (MKK3) and MAP kinase kinase 6 (MKK6). Such IFN-inducible activation of MKK3 and MKK6 is essential for downstream phosphorylation and activation of the p38 MAP kinase, as shown by studies using mouse embryonic fibroblasts (MEFs) with targeted disruption of the Mkk3 and Mkk6 genes (MKK3-/- MKK6-/-). Similarly, IFN-dependent activation of the downstream effectors of p38, MAPKAPK-2 and MAPKAPK-3, is not detectable in cells lacking Mkk3 and Mkk6, demonstrating that the function of these MAP kinase kinases is required for full activation of the p38 pathway. To define the functional relevance of MKK3/6 engagement in Type I IFN signaling, IFN-inducible gene transcription was evaluated in the MKK3/MKK6 double knock-out cells. IFNalpha- and IFNbeta-dependent transcription via either interferon-stimulated response element or IFNgamma activated site elements was defective in MKK3 -/-/MKK6 -/- MEFs in luciferase reporter assays. In addition, IFN-dependent induction of two genes known to be of importance in the generation of IFN responses, Isg15 and Irf-9, was diminished in the absence of Mkk3 and Mkk6. The effects of Mkk3 and Mkk6 on IFN-dependent transcription were unrelated to any effects on the phosphorylation and activation of STAT proteins, indicating the presence of a STAT-independent mechanism. Altogether, our findings demonstrate that MKK3 and MKK6 are rapidly activated during engagement of the Type I IFN receptor and play important roles in Type I IFN signaling and the generation of IFN responses
— id: 93471, year: 2005, vol: 280, page: 10001, stat: Journal Article,

Cell signaling. Stat acetylation--a key facet of cytokine signaling?
O'Shea, John J; Kanno, Yuka; Chen, Xiaomin; Levy, David E
2005 Jan 14;307(5707):217-218, Science
— id: 93470, year: 2005, vol: 307, page: 217, stat: Journal Article,

Reduced STAT3 activity in mice mimics clinical disease syndromes
Shen, Yuhong; La Perle, Krista M D; Levy, David E; Darnell, James E Jr
2005 Apr 29;330(1):305-309, Biochemical & biophysical research communications
Phosphorylation on Y705 is obligatory for STAT3 activation, but full transcriptional activity of this widely expressed protein also requires phosphorylation on S727. We described earlier the STAT3 SA/- mice (SA, S727A allele) on a Black 6 (Bl6) background that showed 75% perinatal lethality and early growth retardation presumably due to the decreased transcription supported by STAT3 S727A. We now report additional analyses of long-term surviving SA/- animals which show no important tissue abnormalities. However, we have found a much greater susceptibility to doxorubicin-induced heart failure in the SA/- mice. Also we introduced the SA allele into strain 129 and found the SA/- mice showed greater susceptibility to LPS-induced toxicity. These results suggest a continued need for normal STAT3 transcriptional activity to resist two different noxious challenges that mimic the conditions necessary to induce adult diseases
— id: 93467, year: 2005, vol: 330, page: 305, stat: Journal Article,

Expression of many immunologically important genes in Mycobacterium tuberculosis-infected macrophages is independent of both TLR2 and TLR4 but dependent on IFN-alphabeta receptor and STAT1
Shi, Shuangping; Blumenthal, Antje; Hickey, Christopher M; Gandotra, Sheetal; Levy, David; Ehrt, Sabine
2005 Sep 1;175(5):3318-3328, Journal of immunology
Macrophages respond to several subcellular products of Mycobacterium tuberculosis (Mtb) through TLR2 or TLR4. However, primary mouse macrophages respond to viable, virulent Mtb by pathways largely independent of MyD88, the common adaptor molecule for TLRs. Using microarrays, quantitative PCR, and ELISA with gene-disrupted macrophages and mice, we now show that viable Mtb elicits the expression of inducible NO synthase, RANTES, IFN-inducible protein 10, immune-responsive gene 1, and many other key genes in macrophages substantially independently of TLR2, TLR4, their combination, or the TLR adaptors Toll-IL-1R domain-containing adapter protein and Toll-IL-1R domain-containing adapter inducing IFN-beta. Mice deficient in both TLR2 and TLR4 handle aerosol infection with viable Mtb as well as congenic controls. Viable Mtb also up-regulates inducible NO synthase, RANTES, IFN-inducible protein 10, and IRG1 in macrophages that lack mannose receptor, complement receptors 3 and 4, type A scavenger receptor, or CD40. These MyD88, TLR2/4-independent transcriptional responses require IFN-alphabetaR and STAT1, but not IFN-gamma. Conversely, those genes whose expression is MyD88 dependent do not depend on IFN-alphabetaR or STAT1. Transcriptional induction of TNF is TLR2/4, MyD88, STAT1, and IFN-alphabetaR independent, but TNF protein release requires the TLR2/4-MyD88 pathway. Thus, macrophages respond transcriptionally to viable Mtb through at least three pathways. TLR2 mediates the responses of a numerically minor set of genes that collectively do not appear to affect the course of infection in mice; regulation of TNF requires TLR2/4 for post-transcriptional control, but not for transcriptional induction; and many responding genes are regulated through an unknown, TLR2/4-independent pathway that may involve IFN-alphabetaR and STAT1
— id: 138940, year: 2005, vol: 175, page: 3318, stat: Journal Article,

Interleukin-10 induces inhibitory C/EBPbeta through STAT-3 and represses HIV-1 transcription in macrophages
Tanaka, Naohiko; Hoshino, Yoshihiko; Gold, Jeffrey; Hoshino, Satomi; Martiniuk, Frank; Kurata, Takeshi; Pine, Richard; Levy, David; Rom, William N; Weiden, Michael
2005 Oct;33(4):406-411, American journal of respiratory cell & molecular biology
Pulmonary tuberculosis (TB) has been characterized by inflammation with increased pro- or anti-inflammatory cytokines produced by macrophages. We have reported that IFN produces inhibitory C/EBPbeta and represses transcription of the HIV-1 LTR in macrophages. STAT-1 and type I IFN receptor knockout mice have macrophages that are defective in IFN signaling, yet LPS stimulation induces inhibitory C/EBPbeta, demonstrating that other cytokines can induce this repressor. LPS or Mycobacterium tuberculosis-derived lipoarabinomannan induce the anti-inflammatory cytokine interleukin (IL)-10, which represses the HIV-1 LTR in differentiated THP-1 macrophages by inducing inhibitory C/EBPbeta. In contrast, in undifferentiated THP-1 monocytes, IL-10 did not inhibit HIV-1 replication or induce C/EBPbeta. IL-10 signal transduction uses STAT-3, and macrophages from STAT-3-/- mice fail to produce inhibitory C/EBPbeta after LPS or IL-10 stimulation. Transfection of STAT-3 into THP-1 cells enhances C/EBPbeta promoter activity. THP-1 differentiation also increases STAT-3 protein, but not STAT-3 gene transcription, and induces a translational regulator, CUG-binding protein, that was essential for production of C/EBPbeta. Differentiation induced post-transcriptional regulation is required to produce inhibitory C/EBPbeta in response to IL-10. Only macrophages are able to repress HIV-1 LTR promoter activity and inhibit viral replication in response to IL-10 or type I IFN
— id: 58745, year: 2005, vol: 33, page: 406, stat: Journal Article,

Novel roles of unphosphorylated STAT3 in oncogenesis and transcriptional regulation
Yang, Jinbo; Chatterjee-Kishore, Moitreyee; Staugaitis, Susan M; Nguyen, Hannah; Schlessinger, Karni; Levy, David E; Stark, George R
2005 Feb 1;65(3):939-947, Cancer research
Signal transducer and activator of transcription 3 (STAT3) is phosphorylated on tyrosine residue 705 in response to growth factors or cytokines to form activated homodimers that drive gene expression. Because the stat3 promoter has a binding site for STAT3 dimers, the amount of STAT3 protein increases when STAT3 is activated (e.g., in response to interleukin 6). Unphosphorylated STAT1 is known to drive the expression of certain genes. To explore the possibility of a similar role for the induced expression of unphosphorylated STAT3, we overexpressed either Y705F STAT3, which can not be phosphorylated on residue 705, or wild-type STAT3 in normal human mammary epithelial cells or STAT3-null mouse cells. The levels of many mRNAs were affected strongly by high levels of either form of STAT3. Some genes whose expression was increased by overexpressed STAT3, but not by activated STAT3 dimers, encode well-known oncoproteins (e.g., MRAS and MET). In many tumors, STAT3 is activated constitutively, and thus the unphosphorylated form is likely to be expressed highly, driving oncogene expression by a novel mechanism. In addition, expression of the stat3 gene is increased strongly in response to interleukin 6, and the high levels of unphosphorylated STAT3 that result drive a substantial late phase of gene expression in response to this cytokine. Thus, unphosphorylated STAT3, which activates gene expression by a novel mechanism distinct from that used by STAT3 dimers, is very likely to be an important transcription factor both in cancer and in responses to cytokines
— id: 93469, year: 2005, vol: 65, page: 939, stat: Journal Article,

Interferon-gamma-induced inhibition of neuronal vesicular stomatitis virus infection is STAT1 dependent
Chesler, David A; Dodard, Cindy; Lee, Grace Y; Levy, David E; Reiss, Carol Shoshkes
2004 Feb;10(1):57-63, Journal of neurovirology
In this report, the signaling pathways utilized by interferon (IFN)-gamma in neurons and their respective roles in the inhibition of vesicular stomatitis virus (VSV) replication were studied. The authors have previously shown that IFN-gamma treatment of NB41A3 neuroblastoma cells results in a 2-log inhibition of VSV production. This inhibition of VSV replication is dependent both in vitro and in vivo on nitric oxide (NO) production by NO synthase (NOS)-1. In NB41A3 neuroblastoma cells, IFN-gamma was found to induce the signal transducer and activator of transcription (STAT) STAT1 phosphorylation, interferon regulatory factor (IRF)-1 expression, and p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation; MAPK, however, was not required for inhibition of viral replication. Using olfactory bulb-enriched primary neuronal cultures, the inhibition of VSV replication was found to be STAT1 dependent, but did not require IRF-1
— id: 42151, year: 2004, vol: 10, page: 57, stat: Journal Article,

Role of interferons in an in vivo model of inhalational anthrax
Gold JA; Jones MB; Levy DE; Hoshino Y; Nolan A; Weiden MD
2004 ;169:A230-A230, American journal of respiratory & critical care medicine
— id: 101401, year: 2004, vol: 169, page: A230, stat: Journal Article,

Art and the brain: the influence of art on Roger Shepard's studies of mental rotation
Levy, Ellen K; Levy, David E; Goldberg, Michael E
2004 Mar;13(1):79-90, Journal of the history of the neurosciences
This paper explores the influence of visual sources on Roger N. Shepard's 1971 mental rotation experiments and the centrality of ambiguity as one of his experimental and artistic concerns. Sources include Shepard's statements about ambiguity as expressed in the book, Mind Sights, and a recent interview. Parallel investigations of ambiguity by the contemporary artists Al Held and Robert Smithson are considered. Shepard utilized a wide range of visual sources while formulating his experimental design, namely Necker cube illusions, hypnopompic images, Rene Magritte, and M.C. Escher. In addition, he drew upon key art historical theses of the time, such as Ernst Gombrich's theories about schemas. For Shepard as for Gombrich, the world of appearances is a world of ambiguity
— id: 93472, year: 2004, vol: 13, page: 79, stat: Journal Article,

Role of p38alpha Map kinase in Type I interferon signaling
Li, Yongzhong; Sassano, Antonella; Majchrzak, Beata; Deb, Dilip K; Levy, David E; Gaestel, Matthias; Nebreda, Angel R; Fish, Eleanor N; Platanias, Leonidas C
2004 Jan 9;279(2):970-979, Journal of biological chemistry
Multiple signaling pathways are activated during engagement of the Type I interferon (IFN) receptor to mediate biological responses, including the Jak-Stat and Rac1/p38 Map kinase signaling cascades. In the present study we sought to determine the functional relevance of the p38alpha isoform in IFN signaling, using cells from mouse embryos with targeted disruption of the p38alpha gene. Our data demonstrate that p38alpha activation is essential for Type I IFN-dependent transcriptional regulation via ISRE or GAS elements. On the other hand, the function of p38alpha is not required for IFN-dependent Ser727 or Tyr701 phosphorylation of Stat1 and does not impact on the formation of ISGF3 or SIF nuclear binding complexes. In efforts to identify downstream effectors of p38 that may mediate IFN-dependent transcriptional responses, we found that IFNalpha activates the kinase Msk1, a known regulator of histone phosphorylation and chromatin remodeling. In other studies, we demonstrate that Type I IFN-dependent activation of the kinases MapKapK-2 and MapKapK-3 is defective in the absence of p38alpha, while Type I IFN-dependent antiviral properties are decreased in cells with targeted disruption of the MapKapK-2 gene. Altogether, our data establish that the p38alpha Map kinase pathway regulates activation of downstream effectors that participate in the induction of IFN-dependent gene transcription, to mediate IFN-responses
— id: 42152, year: 2004, vol: 279, page: 970, stat: Journal Article,

Essential role of STAT3 in postnatal survival and growth revealed by mice lacking STAT3 serine 727 phosphorylation
Shen, Yuhong; Schlessinger, Karni; Zhu, Xuejun; Meffre, Eric; Quimby, Fred; Levy, David E; Darnell, J E Jr
2004 Jan;24(1):407-419, Molecular & cellular biology
A large number of extracellular polypeptides bound to their cognate receptors activate the transcription factor STAT3 by phosphorylation of tyrosine 705. Supplemental activation occurs when serine 727 is also phosphorylated. STAT3 deletion in mice leads to embryonic lethality. We have produced mice with alanine substituted for serine 727 in STAT3 (the SA allele) to examine the function of serine 727 phosphorylation in vivo. Embryonic fibroblasts from SA/SA mice had approximately 50% of the transcriptional response of wild-type cells. However, SA/SA mice were viable and grossly normal. STAT3 wild-type/null (+/-) animals were also normal and were interbred with SA/SA mice to study SA/- mice. The SA/- mice progressed through gestation, showing 10 to 15% reduced birth weight, three-fourths died soon after birth, and the SA/- survivors reached only 50 to 60% of normal size at 1 week of age. The lethality and decreased growth were accompanied by altered insulin-like growth factor 1 (IGF-1) levels in serum, establishing a role for the STAT3 serine phosphorylation acting through IGF-1 in embryonic and perinatal growth. The SA/- survivors have decreased thymocyte number associated with increased apoptosis, but unexpectedly normal STAT3-dependent liver acute phase response. These animals offer the opportunity to study defined reductions in the transcriptional capacity of a widely used signaling pathway
— id: 93473, year: 2004, vol: 24, page: 407, stat: Journal Article,

Ringing the interferon alarm: differential regulation of gene expression at the interface between innate and adaptive immunity
Levy, David E; Marie, Isabelle; Prakash, Arun
2003 Feb;15(1):52-58, Current opinion in immunology
— id: 39347, year: 2003, vol: 15, page: 52, stat: Journal Article,

An essential role of Th1 responses and interferon gamma in infection-mediated suppression of neoplastic growth
Rankin, Erinn B; Yu, Duonan; Jiang, Jiu; Shen, Hao; Pearce, Edward J; Goldschmidt, Michael H; Levy, David E; Golovkina, Tatyana V; Hunter, Christopher A; Thomas-Tikhonenko, Andrei
2003 Nov-Dec;2(6):687-693, Cancer biology & therapy
We had previously demonstrated that in mice acute toxoplasmosis leads to systemic inhibition of angiogenesis and, consequently, strong suppression of neoplastic growth. Here we investigated the role of Th1 cytokines, in particular interferon gamma (IFN-gamma), in this phenomenon. Besides toxoplasma, neoplastic growth was readily blocked during acute infection with other Th1 response-inducing pathogens such as Listeria monocytogenes and lymphocytic choriomeningitis virus (LCMV). In contrast, chronic infection with LCMV (when Th1 responses were strongly suppressed) and acute infection with Schistosoma mansoni (when Th2 responses predominated) afforded no anti-tumor protection. To corroborate the involvement of Th1 cytokines in infection-mediated suppression of neoplastic growth, we utilized mice deficient in interleukin-10 (IL10), a suppressor of Th1 responses. When challenged with B16 cells concomitantly with toxoplasma infection, both IL10-null and wild type mice exhibited resistance to neoplastic growth. However, tumors borne by IL10-null animals were even smaller than those borne by their wild type counterparts. This enhanced resistance correlated with dramatically elevated levels of circulating IFN-gamma, a principal Th1 cytokine. Furthermore, while interleukin-12 and tumor necrosis factor a were dispensable for tumor suppression, in animals deficient in IFN-gamma production or signaling, tumor growth and neovascularization were markedly enhanced. Interestingly, the enhancement was also apparent in uninfected animals suggesting that IFN-gamma and its anti-angiogenic effects underlie both infection-dependent and -independent tumor surveillance
— id: 42153, year: 2003, vol: 2, page: 687, stat: Journal Article,

The WD motif-containing protein RACK-1 functions as a scaffold protein within the type I IFN receptor-signaling complex
Usacheva, Anna; Tian, Xinyong; Sandoval, Raudel; Salvi, Debra; Levy, David; Colamonici, Oscar R
2003 Sep 15;171(6):2989-2994, Journal of immunology
The WD repeat-containing protein receptor for activated protein kinase C (RACK)-1 has been linked to a variety of signaling systems including protein kinase C, growth factors, and IFNs. In the IFN system, RACK-1 functions as an adaptor recruiting the transcription factor STAT1 to the receptor complex. However, RACK-1 should play a broader role in type I IFN signaling because mutation of the RACK-1 binding site in the IFN-alpha receptor 2/beta subunit of the type I IFN receptor abrogates not only STAT1, but also STAT2, activation. In this study, we demonstrate that RACK-1 serves as a scaffold protein for a multiprotein complex that includes the IFN-alpha receptor 2/beta-chain of the receptor, STAT1, Janus kinase 1, and tyrosine kinase 2. In vitro data further suggest that within this complex tyrosine kinase 2 is the tyrosine kinase responsible for the phosphorylation of STAT1. Finally, we provide evidence that RACK-1 may also serve as a scaffold protein in other cytokine systems such as IL-2, IL-4, and erythropoietin
— id: 138939, year: 2003, vol: 171, page: 2989, stat: Journal Article,

STAT1 and STAT3 activation during the course of experimental arthritis
De Hooge, ASK; Van De Loo, FAJ; Kolbe, T; Mueller, M; Levy, DE; Bennink, M; Arntzl, OJ; Van Den Berg, WB
2002 SEP ;46(9):S250-S250, Arthritis & rheumatism
— id: 37120, year: 2002, vol: 46, page: S250, stat: Journal Article,

Role of nucleoporin induction in releasing an mRNA nuclear export block
Enninga, Jost; Levy, David E; Blobel, Gunter; Fontoura, Beatriz M A
2002 Feb 22;295(5559):1523-1525, Science
Signal-mediated nuclear import and export proceed through the nuclear pore complex (NPC). Some NPC components, such as the nucleoporins (Nups) Nup98 and Nup96, are also associated with the nuclear interior. Nup98 is a target of the vesicular stomatitis virus (VSV) matrix (M) protein-mediated inhibition of messenger RNA (mRNA) nuclear export. Here, Nup98 and Nup96 were found to be up-regulated by interferon (IFN). M protein-mediated inhibition of mRNA nuclear export was reversed when cells were treated with IFN-gamma or transfected with a complementary DNA (cDNA) encoding Nup98 and Nup96. Thus, increased Nup98 and Nup96 expression constitutes an IFN-mediated mechanism that reverses M protein-mediated inhibition of gene expression
— id: 93477, year: 2002, vol: 295, page: 1523, stat: Journal Article,

Deletion of Stat3 blocks mammary gland involution and extends functional competence of the secretory epithelium in the absence of lactogenic stimuli
Humphreys, Robin C; Bierie, Brian; Zhao, Ling; Raz, Regina; Levy, David; Hennighausen, Lothar
2002 Sep;143(9):3641-3650, Endocrinology
The transcription factor Stat3 is activated through tyrosine phosphorylation by many cytokines and is a fundamental mediator of their signals. In the mammary gland, Stat3 activity increases sharply shortly after weaning, and involution is delayed in mice, that contain a mutant Stat3 lacking 33 amino acids including the key tyrosine residue. We have now generated a more extensive mutation of Stat3 through the deletion of exons 15-21 in mammary epithelium. This resulted in the loss of 245 amino acids including the DNA binding and SH2 domains, and Stat3 protein was undetectable. Pregnancy-mediated mammary development and lactation were normal in these mice. Involution was delayed and, remarkably, Stat3-null mammary epithelium maintained its functional integrity and competence even 6 d after weaning, whereas control mammary tissue was rendered nonfunctional within 2 d. The lack of remodeling and functional stasis of the epithelium correlated with the disruption of proteinase activity. Our data demonstrate that mammary tissue can retain its functional competence in the absence of external lactogenic stimuli and demonstrate a delay in the initiation of the irreversible stage of involution
— id: 106996, year: 2002, vol: 143, page: 3641, stat: Journal Article,

STAT3 is a negative regulator of granulopoiesis but is not required for G-CSF-dependent differentiation
Lee, Chien-kuo; Raz, Regina; Gimeno, Ramon; Gertner, Rachel; Wistinghausen, Birte; Takeshita, Kenichi; DePinho, Ronald A; Levy, David E
2002 Jul;17(1):63-72, Immunity
STAT3 has been described as an essential component of G-CSF-driven cell proliferation and granulopoiesis. This notion was tested by conditional gene ablation in transgenic mice. Contrary to expectation, granulocytes developed from STAT3 null bone marrow progenitors, and STAT3 null neutrophils displayed mature effector functions. Rather than a deficit in granulopoiesis, mice lacking STAT3 in their hematopoietic progenitors developed neutrophilia, and bone marrow cells were hyperresponsive to G-CSF stimulation. These studies provide direct evidence for STAT3-independent granulopoiesis and suggest that STAT3 directs a negative feedback loop necessary for controlling neutrophil numbers, possibly through induced expression of the signaling inhibitor, SOCS3
— id: 32457, year: 2002, vol: 17, page: 63, stat: Journal Article,

Whence interferon? Variety in the production of interferon in response to viral infection
Levy, David E
2002 Feb 18;195(4):F15-F18, Journal of experimental medicine
— id: 39705, year: 2002, vol: 195, page: F15, stat: Journal Article,

Stats: transcriptional control and biological impact
Levy, David E; Darnell, J E Jr
2002 Sep;3(9):651-662, Nature reviews. Molecular cell biology
Extracellular proteins bound to cell-surface receptors can change nuclear gene expression patterns in minutes, with far-reaching consequences for development, cell growth and homeostasis. The signal transducer and activator of transcription (STAT) proteins are among the most well studied of the latent cytoplasmic signal-dependent transcription-factor pathways. In addition to several roles in normal cell decisions, dysregulation of STAT function contributes to human disease, making the study of these proteins an important topic of current research
— id: 39597, year: 2002, vol: 3, page: 651, stat: Journal Article,

What does Stat3 do?
Levy, David E; Lee, Chien-kuo
2002 May;109(9):1143-1148, Journal of clinical investigation
— id: 39652, year: 2002, vol: 109, page: 1143, stat: Journal Article,

Enhancement and diversification of IFN induction by IRF-7-mediated positive feedback
Levy, David E; Marie, Isabelle; Smith, Eric; Prakash, Arun
2002 Jan;22(1):87-93, Journal of interferon & cytokine research
Interferons (IFN) are potent components of the innate immune response to microbial infection. The genes for type I IFN (IFN-alpha and IFN-beta) are rapidly induced in response to viral infection through a mechanism that involves latent cellular transcription factors that are activated in response to innate recognition of viral components. IFN regulatory factor (IRF) proteins are key to this regulation, and their conversion from latent to active involves virus-induced serine phosphorylation. Differential utilization of distinct IRF proteins by different members of the type I IFN gene family produces a graded induction of gene expression, resulting in tight control of these cytokines through a positive feedback mechanism. Early response to virus causes secretion of a subset of IFN genes through the action of IRF-3 in conjunction with additional transcription factors, such as NF-kappaB and activator protein-1 (AP-1) (c-jun/ATF). This early IFN acts in an autocrine manner to stimulate production of IRF-7, a transcription factor capable of activating the many additional members of the IFN-alpha gene family. The dependence of IRF-7 on virus-induced phosphorylation for its activity insures that IFN production is limited to virus-infected cells. Characterization of the cellular components involved in viral detection and IRF activation will further delineate this vital mechanism of innate immune response
— id: 39713, year: 2002, vol: 22, page: 87, stat: Journal Article,

IFN-Stimulated transcription through a TBP-free acetyltransferase complex escapes viral shutoff
Paulson, Matthew; Press, Carolyn; Smith, Eric; Tanese, Naoko; Levy, David E
2002 Feb;4(2):140-147, Nature cell biology
Type I interferon (IFN) stimulates transcription through a heteromeric transcription factor that contains tyrosine-phosphorylated STAT2. We show that STAT2 recruits histone acetyltransferases (HAT) through its transactivation domain, resulting in localized transient acetylation of histones. GCN5, but not p300/CBP or PCAF, is required for STAT2 function. However, GCN5 function is impaired by the transcriptional antagonist, adenovirus E1A oncoprotein. The TFIID component TAFII130 potentiates STAT2 function, but TAFII28 or the HAT activity of TAFII250 do not, and transcriptional induction can proceed independently of the TATA-binding protein, TBP. Moreover, IFN-stimulated transcription was resistant to poliovirus-targeted degradation by TBP, and continued despite host-cell transcriptional shutoff during poliovirus infection. We conclude that a non-classical transcriptional mechanism combats an anticellular action of poliovirus, through a TBP-free TAF-containing complex and GCN5
— id: 25648, year: 2002, vol: 4, page: 140, stat: Journal Article,

Cost-effectiveness of ancrod treatment of acute ischaemic stroke: results from the Stroke Treatment with Ancrod Trial (STAT)
Samsa, Gregory P; Matchar, David B; Williams, G Rhys; Levy, David E
2002 Feb;8(1):61-70, Journal of evaluation in clinical practice
RATIONALE, AIMS AND OBJECTIVES: This paper describes a recent randomized controlled trial in which 42% of patients receiving ancrod attained a favourable outcome in comparison with 34% of controls. Although the above effect size corresponds to a number needed to treat (to achieve a favourable outcome) of approximately 13, intuition does not necessarily suggest what would be the overall impact of a treatment with this level of efficacy. METHODS: The objective was to evaluate the cost-effectiveness of ancrod. Cost-effectiveness analysis of data from the Stroke Treatment with Ancrod Trial (STAT) trial was carried out. The participants were 495 patients with data on functional status at the conclusion of follow-up. Short-term results were based upon utilization and quality of life observed during the trial; these were merged with expected long-term results obtained through simulation using the Stroke Policy Model. The main outcome measure was incremental cost-effectiveness ratio. RESULTS: Ancrod treatment resulted in both better quality-adjusted life expectancy and lower medical costs than placebo as supported by sensitivity analysis. The cost differential was primarily attributable to the long-term implications of ancrod's role in reducing disability. CONCLUSIONS: If ancrod is even modestly effective, it will probably be cost-effective (and, indeed, cost-saving) as well. The net population-level impact of even modestly effective stroke treatments can be substantial
— id: 93476, year: 2002, vol: 8, page: 61, stat: Journal Article,

Production of type I IFN sensitizes macrophages to cell death induced by Listeria monocytogenes
Stockinger, Silvia; Materna, Tilo; Stoiber, Dagmar; Bayr, Lourdes; Steinborn, Ralf; Kolbe, Thomas; Unger, Hermann; Chakraborty, Trinad; Levy, David E; Muller, Mathias; Decker, Thomas
2002 Dec 1;169(11):6522-6529, Journal of immunology
Type I IFNs (IFN-alpha/beta) modulate innate immune responses. Here we show activation of transcription factor IFN regulatory factor 3, the synthesis of large amounts of IFN-beta mRNA, and type I IFN signal transduction in macrophages infected with Listeria monocytogenes. Expression of the bacterial virulence protein listeriolysin O was necessary, but not sufficient, for efficient IFN-beta production. Signaling through a pathway involving the type I IFN receptor and Stat1 sensitized macrophages to L. monocytogenes-induced cell death in a manner not requiring inducible NO synthase (nitric oxide synthase 2) or protein kinase R, potential effectors of type I IFN action during microbial infections. The data stress the importance of type I IFN for the course of infections with intracellular bacteria and suggest that factors other than listeriolysin O contribute to macrophage death during Listeria infection
— id: 93474, year: 2002, vol: 169, page: 6522, stat: Journal Article,

Anaplastic lymphoma kinase (ALK) activates Stat3 and protects hematopoietic cells from cell death
Zamo, Alberto; Chiarle, Roberto; Piva, Roberto; Howes, Jennifer; Fan, Yan; Chilosi, Marco; Levy, David E; Inghirami, Giorgio
2002 Feb 7;21(7):1038-1047, Oncogene
The anaplastic lymphoma kinase (ALK) gene is characteristically translocated in Anaplastic Large Cell Lymphomas (ALCL) and the juxtaposition of the ALK gene to multiple partners results in its constitutive protein tyrosine kinase activity. We show here that expression of activated ALK induces the constitutive phosphorylation of Stat3 in transfected cells as well as in primary human ALCLs. Furthermore, immunohistochemical studies demonstrate that among distinct human B and T cell lymphomas, activation of Stat3 nuclear translocation is uniquely associated with ALK expression. NPM-ALK also binds and activates Jak3; however, Jak3 is not required for Stat3 activation or for cell transformation in vitro. Moreover, src family kinases are not necessary for NPM-ALK-mediated Stat3 activation or transformation, suggesting that Stat3 may be phosphorylated directly by ALK. To evaluate relevant targets of ALK-activated Stat3, we investigated the regulation of the anti-apoptotic protein Bcl-x(L) and its role in cell survival in NPM-ALK positive cells. NPM-ALK expression caused enhanced Bcl-x(L) transcription, largely mediated by Stat3. Increased expression of Bcl-x(L) provided sufficient anti-apoptotic signals to protect cells from treatment with specific inhibitors of the Jaks/Stat pathway or the Brc-Abl kinase. These studies support a pathogenic mechanism whereby stimulation of anti-apoptotic signals through activation of Stat3 contributes to the successful outgrowth of ALK positive tumor cells
— id: 27551, year: 2002, vol: 21, page: 1038, stat: Journal Article,

A Kaposi's sarcoma-associated herpesviral protein inhibits virus-mediated induction of type I interferon by blocking IRF-7 phosphorylation and nuclear accumulation
Zhu, Fan Xiu; King, Sonya M; Smith, Eric J; Levy, David E; Yuan, Yan
2002 Apr 16;99(8):5573-5578, Proceedings of the National Academy of Sciences of the United States of America
Interferons constitute the earliest immune response against viral infection. They elicit antiviral effects as well as multiple biological responses involved in cell growth regulation and immune activation. Because the interferon-induced cellular antiviral response is the primary defense mechanism against viral infection, many viruses have evolved strategies to antagonize the inhibitory effects of interferon. Here, we demonstrate a strategy that Kaposi's sarcoma-associated herpesvirus uses to block virus-mediated induction of type I interferon. We found that a viral immediate-early protein, namely ORF45, interacts with cellular interferon-regulatory factor 7 (IRF-7). In consequence, IRF-7 phosphorylation is inhibited and the accumulation of IRF-7 in the nucleus in response to viral infection is blocked. IRF-7 is a transcription regulator that is responsible for virus-mediated activation of type I interferon genes. By blocking the phosphorylation and nuclear translocation of IRF-7, ORF45 efficiently inhibits the activation of interferon alpha and beta genes during viral infection. Inhibition of interferon gene expression through a viral protein blocking the activation and nuclear translocation of a crucial transcription factor is a novel mechanism for viral immune evasion
— id: 93475, year: 2002, vol: 99, page: 5573, stat: Journal Article,

Cooperation between STAT3 and c-jun suppresses Fas transcription
Ivanov, V N; Bhoumik, A; Krasilnikov, M; Raz, R; Owen-Schaub, L B; Levy, D; Horvath, C M; Ronai, Z
2001 Mar;7(3):517-528, Molecular cell
Decreased Fas expression during tumor progression often results in a loss of Fas-ligand (FasL)-mediated apoptosis. Human and mouse melanoma exhibit an inverse correlation between the degree of Fas cell surface expression, tumorigenicity, and metastatic capacity. The expression of dominant negative Stat3 or c-Jun in melanoma cells efficiently increased Fas expression and sensitized cells to FasL-induced apoptosis. Stat3+/- as well as c-Jun-/- cells exhibited increased Fas cell surface expression and higher sensitivity to FasL-mediated apoptosis. Suppression of Fas expression by Stat3 and c-Jun is uncoupled from Stat3-mediated transcriptional activation. Our findings indicate that Stat3 oncogenic activities could also be mediated through its cooperation with c-Jun, resulting in downregulation of Fas surface expression, which is implicated in the tumor's ability to resist therapy and metastasize
— id: 106997, year: 2001, vol: 7, page: 517, stat: Journal Article,

Specificity of signaling by STAT1 depends on SH2 and C-terminal domains that regulate Ser727 phosphorylation, differentially affecting specific target gene expression
Kovarik, P; Mangold, M; Ramsauer, K; Heidari, H; Steinborn, R; Zotter, A; Levy, D E; Muller, M; Decker, T
2001 Jan 15;20(1-2):91-100, EMBO journal
Complete activation of signal transducer and activator of transcription 1 (STAT1) requires phosphorylation at both Y701 and a conserved PMS(727)P sequence. S727 phosphorylation of STAT1 in interferon-gamma (IFN-gamma)-treated mouse fibroblasts occurred without a need for p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1 and 2 or c-Jun kinases, and required both an intact SH2 domain and phosphorylation of Y701. In contrast, UV irradiation-induced STAT1 phosphorylation on S727 required p38MAPK, but no SH2 domain- phosphotyrosine interactions. Mutation of S727 differentially affected IFN-gamma target genes, at the level of both basal and induced expression. Particularly strong effects were noted for the GBP1 and TAP1 genes. The PMS(727)P motif of STAT3 was phosphorylated by stimuli and signaling pathways different from those for STAT1 S727. Transfer of the STAT3 C-terminus to STAT1 changed the stimulus and pathway specificity of STAT1 S727 phosphorylation to that of STAT3. Our data suggest that STAT C-termini contribute to the specificity of cellular responses by linking individual STATs to different serine kinase pathways and through an intrinsically different requirement for serine phosphorylation at different target gene promoters
— id: 138937, year: 2001, vol: 20, page: 91, stat: Journal Article,

The virus battles: IFN induction of the antiviral state and mechanisms of viral evasion
Levy DE; Garcia-Sastre A
2001 Jun;12(2-3):143-156, Cytokine & growth factor reviews
Response to IFN involves a rapid and direct signal transduction mechanism that quickly reports that presence of extracellular cytokine to the cell nucleus, preserving the specificity inherent in cytokine-receptor interactions to transcriptionally induce expression of a set of genes encoding important antiviral proteins. Establishment of the resulting antiviral state provides a crucial initial line of defense against viral infection. Studies of IFN-deficient cells and animals derived by gene targeting have demonstrated the essential nature of IFN-mediated innate immunity. The long co-evolutionary history of viruses with their hosts as seen the development of a variety of evasive adaptions that allow viruses to circumvent or inactivate host antiviral mechanisms. Further understanding of both host and viral components of this battle may provide important new strategies for vaccine development and creation of novel antiviral compounds
— id: 20700, year: 2001, vol: 12, page: 143, stat: Journal Article,

Hepatitis C virus nonstructural 5A protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response
Polyak, S J; Khabar, K S; Paschal, D M; Ezelle, H J; Duverlie, G; Barber, G N; Levy, D E; Mukaida, N; Gretch, D R
2001 Jul;75(13):6095-6106, Journal of virology
Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). The HCV nonstructural 5A (NS5A) protein has been implicated in HCV antiviral resistance in many studies. NS5A antagonizes the IFN antiviral response in vitro, and one mechanism is via inhibition of a key IFN-induced enzyme, the double-stranded-RNA-activated protein kinase (PKR). In the present study we determined if NS5A uses other strategies to subvert the IFN system. Expression of full-length NS5A proteins from patients who exhibited a complete response (FL-NS5A-CR) or were nonresponsive (FL-NS5A-NR) to IFN therapy in HeLa cells had no effect on IFN induction of IFN-stimulated gene factor 3 (ISGF-3). Expression of mutant NS5A proteins lacking 110 (NS5A-DeltaN110), 222 (NS5A-DeltaN222), and 334 amino-terminal amino acids and mutants lacking 117 and 230 carboxy-terminal amino acids also had no effect on ISGF-3 induction by IFN. Expression of FL-NS5A-CR and FL-NS5A-NR did not affect IFN-induced STAT-1 tyrosine phosphorylation or upregulation of PKR and major histocompatibility complex class I antigens. However, NS5A expression in human cells induced interleukin 8 (IL-8) mRNA and protein, and this effect correlated with inhibition of the antiviral effects of IFN in an in vitro bioassay. NS5A induced transcription of a reporter gene driven by the IL-8 promoter, and the first 133 bp of the IL-8 promoter made up the minimal domain required for NS5A transactivation. NS5A-DeltaN110 and NS5A-DeltaN222 stimulated the IL-8 promoter to higher levels than did the full-length NS5A protein, and this correlated with increased nuclear localization of the proteins. Additional mutagenesis of the IL-8 promoter suggested that NF-kappaB and AP-1 were important in NS5A-DeltaN222 transactivation in the presence of tumor necrosis factor alpha and that NF-IL-6 was inhibitory to this process. This study suggests that NS5A inhibits the antiviral actions of IFN by at least two mechanisms and provides the first evidence for a biological effect of the transcriptional activity of the NS5A protein. During HCV infection, viral proteins may induce chemokines that contribute to HCV antiviral resistance and pathogenesis
— id: 138938, year: 2001, vol: 75, page: 6095, stat: Journal Article,

STAT1 mediates the increased apoptosis and reduced chondrocyte proliferation in mice overexpressing FGF2
Sahni M; Raz R; Coffin JD; Levy D; Basilico C
2001 Jun;128(11):2119-2129, Development
Unregulated FGF receptor signaling results in bone malformations that affect both endochondral and intramembranous ossification, and is the basis for several genetic forms of human dwarfism. FGF signaling inhibits chondrocyte proliferation and we have previously shown that the transcription factor STAT1 mediates the growth inhibitory effect of FGF in vitro. We provide genetic evidence that STAT1 is a modulator of the negative regulation of bone growth by FGF in vivo. We crossed Stat1(-/-) mice with a transgenic mouse line overexpressing human FGF2 (TgFGF). TgFGF mice exhibit phenotypes characterized by chondrodysplasia and macrocephaly, which affect endochondral and intramembranous ossification. We found that the chondrodysplasic phenotype of these mice results both from reduced proliferation and increased apoptosis of growth plate chondrocytes. Loss of STAT1 function in TgFGF mice led to a significant correction of the chondrodysplasic phenotype, but did not affect the skull malformations. The reduced proliferation of TgFGF growth plate chondrocytes, as well as their excessive apoptosis, were restored to near-normal levels in the absence of STAT1 function. Unregulated FGF signaling in TgFGF mice also induced apoptosis in calvarial osteoblasts that was not, however, corrected by the absence of STAT1. Detailed analysis of Stat1(-/-) growth plates uncovered a transient phenotype, characterized by an expansion of the proliferative zone and by acceleration of longitudinal bone growth, that attenuated as the animals grew older. These results document an essential role for STAT1 in FGF-mediated regulation of cell growth that is specific to the epiphyseal growth plate
— id: 26721, year: 2001, vol: 128, page: 2119, stat: Journal Article,

IRF3 and IRF7 phosphorylation in virus-infected cells does not require double-stranded RNA-dependent protein kinase R or Ikappa B kinase but is blocked by Vaccinia virus E3L protein
Smith EJ; Marie I; Prakash A; Garcia-Sastre A; Levy DE
2001 Mar 23;276(12):8951-8957, Journal of biological chemistry
Induction of interferon-alpha (IFNalpha) gene expression in virus-infected cells requires phosphorylation-induced activation of the transcription factors IRF3 and IRF7. However, the kinase(s) that targets these proteins has not been identified. Using a combined pharmacological and genetic approach, we found that none of the kinases tested was responsible for IRF phosphorylation in cells infected with Newcastle disease virus (NDV). Although the broad-spectrum kinase inhibitor staurosporine potently blocked IRF3 and -7 phosphorylation, inhibitors for protein kinase C, protein kinase A, MEK, SAPK, IKK, and protein kinase R (PKR) were without effect. Both IkappaB kinase and PKR have been implicated in IFN induction, but cells genetically deficient in IkappaB kinase, PKR, or the PKR-related genes PERK, IRE1, or GCN2 retained the ability to phosphorylate IRF7 and induce IFNalpha. Interestingly, PKR mutant cells were defective for response to double-stranded (ds) RNA but not to virus infection, suggesting that dsRNA is not the only activating viral component. Consistent with this notion, protein synthesis was required for IRF7 phosphorylation in virus-infected cells, and the kinetics of phosphorylation and viral protein production were similar. Despite evidence for a lack of involvement of dsRNA and PKR, vaccinia virus E3L protein, a dsRNA-binding protein capable of inhibiting PKR, was an effective IRF3 and -7 phosphorylation inhibitor. These results suggest that a novel cellular protein that is activated by viral products in addition to dsRNA and is sensitive to E3L inhibition is responsible for IRF activation and reveal a novel mechanism for the anti-IFN effect of E3L distinct from its inhibition of PKR
— id: 21260, year: 2001, vol: 276, page: 8951, stat: Journal Article,

Type I IFN modulates innate and specific antiviral immunity
Durbin JE; Fernandez-Sesma A; Lee CK; Rao TD; Frey AB; Moran TM; Vukmanovic S; Garcia-Sastre A; Levy DE
2000 Apr 15;164(8):4220-4228, Journal of immunology
IFNs protect from virus infection by inducing an antiviral state and by modulating the immune response. Using mice deficient in multiple aspects of IFN signaling, we found that type I and type II IFN play distinct although complementing roles in the resolution of influenza viral disease. Both types of IFN influenced the profile of cytokines produced by T lymphocytes, with a significant bias toward Th2 differentiation occurring in the absence of responsiveness to either IFN. However, although a Th1 bias produced through inhibition of Th2 differentiation by IFN-gamma was not required to resolve infection, loss of type I IFN responsiveness led to exacerbated disease pathology characterized by granulocytic pulmonary inflammatory infiltrates. Responsiveness to type I IFN did not influence the generation of virus-specific cytotoxic lymphocytes or the rate of viral clearance, but induction of IL-10 and IL-15 in infected lungs through a type I IFN-dependent pathway correlated with a protective response to virus. Combined loss of both IFN pathways led to a severely polarized proinflammatory immune response and exacerbated disease. These results reveal an unexpected role for type I IFN in coordinating the host response to viral infection and controlling inflammation in the absence of a direct effect on virus replication
— id: 11766, year: 2000, vol: 164, page: 4220, stat: Journal Article,

Differential regulation of TSG-14 expression in murine fibroblasts and peritoneal macrophages
Goodman AR; Levy DE; Reis LF; Vilcek J
2000 Mar;67(3):387-395, Journal of leukocyte biology
Tumor necrosis factor (TNF)-stimulated gene 14 (TSG-14, also termed PTX3) encodes a secreted glycoprotein whose carboxy-terminal half shares sequence similarity with the pentraxin family of acute phase proteins (C-reactive protein and serum amyloid P component). We compared TSG-14 mRNA expression in cultures of murine BALB/c 3T3 fibroblasts and thioglycollate-elicited peritoneal macrophages. TNF and interleukin-1 (IL-1) potently induced TSG-14 expression in 3T3 fibroblasts but not in peritoneal macrophages. Lipopolysaccharide (LPS) elicited TSG-14 expression in both cell types, but induction in 3T3 cells and macrophages showed several distinct characteristics. Whereas in 3T3 fibroblasts TSG-14 mRNA was rapidly up-regulated by LPS, expression in macrophages was substantially delayed. Furthermore, cycloheximide greatly reduced LPS-induced TSG-14 mRNA up-regulation in macrophages but not in 3T3 cells. Finally, interferon-gamma (IFN-gamma; but not IFN-alpha/beta) inhibited LPS-induced TSG-14 expression in macrophages and not in 3T3 fibroblasts. The antioxidant pyrrolidine dithiocarbamate inhibited LPS-induced nuclear factor-kappaB (NF-kappaB) activation and TSG-14 expression in macrophages. In contrast, IFN-gamma did not inhibit NF-kappaB function as measured by IkappaB-alpha and IkappaB-beta degradation, IkappaB-alpha resynthesis, or electrophoretic mobility shift analysis. Inhibition of LPS-induced TSG-14 mRNA expression by IFN-gamma in macrophages was also observed in the presence of cycloheximide and in cells from STAT1 null mice, suggesting that IFN-gamma inhibits TSG-14 expression through an unconventional mechanism
— id: 11789, year: 2000, vol: 67, page: 387, stat: Journal Article,

Distinct requirements for IFNs and STAT1 in NK cell function
Lee CK; Rao DT; Gertner R; Gimeno R; Frey AB; Levy DE
2000 Oct 1;165(7):3571-3577, Journal of immunology
NK cell functions were examined in mice with a targeted mutation of the STAT1 gene, an essential mediator of IFN signaling. Mice deficient in STAT1 displayed impaired basal NK cytolytic activity in vitro and were unable to reject transplanted tumors in vivo, despite the presence of normal numbers of NK cells. IL-12 enhanced NK-mediated cytolysis, but poly(I:C) did not, and a similar phenotype occurred in mice lacking IFNalpha receptors. Molecules involved in activation and lytic function of NK cells (granzyme A, granzyme B, perforin, DAP10, and DAP12) were expressed at comparable levels in both wild-type and STAT1(-/-) mice, and serine esterase activity necessary for CTL function was normal, showing that the lytic machinery was intact. NK cells with normal cytolytic activity could be derived from STAT1(-/-) bone marrow progenitors in response to IL-15 in vitro, and enhanced NK lytic activity and normal levels of IFN-gamma were produced in response to IL-12 treatment in vivo. Despite these normal responses to cytokines, STAT1(-/-) mice could not reject the NK-sensitive tumor RMA-S, even following IL-12 treatment in vivo. Whereas in vitro NK cytolysis was also reduced in mice lacking both type I and type II IFN receptors, these mice resisted tumor challenge. These results demonstrate that both IFN-alpha and IFN-gamma are required to maintain NK cell function and define a STAT1-dependent but partially IFN-independent pathway required for NK-mediated antitumor activity
— id: 20292, year: 2000, vol: 165, page: 3571, stat: Journal Article,

STAT1 affects lymphocyte survival and proliferation partially independent of its role downstream of IFN-gamma
Lee CK; Smith E; Gimeno R; Gertner R; Levy DE
2000 Feb 1;164(3):1286-1292, Journal of immunology
Lymphocytes derived from mice deficient in STAT1 showed reduced apoptosis and enhanced proliferation in vitro. To understand the involvement of STAT1 in the observed reduction in apoptosis, we examined the levels of caspase and bcl-2 family genes that are involved in cell survival and/or apoptosis. The levels of caspase 1 and 11, two enzymes involved in both cytokine protein processing and induction of apoptosis, were reduced in STAT1-/- cells compared with wild-type. However, the levels of bcl-2 genes were comparable in both mice. STAT1-/- cells also displayed an enhanced proliferation following TCR stimulation. This hyperproliferation could not be ascribed completely to the loss of IFN-gamma-mediated antiproliferation. First, similar phenotypes were also observed in fibroblasts and pre-B cells derived from STAT1-/- mice, which do not produce IFN-gamma. Second, comparisons with cells lacking the gene for IFN-gamma or with cells treated with neutralizing Abs to IFN-gamma only partially mimicked the STAT1-/- phenotype. Interestingly, the kinetics of degradation of p27kip1, a CDK inhibitor, following TCR ligation were faster, and, concomitantly, the up-regulation of CDK2 kinase activity and protein levels were increased in stimulated T cells of STAT1-/- mice relative to those of wild-type mice. Furthermore, STAT1-/- animals were more susceptible to carcinogen-induced thymic tumors, a possible consequence of altered T cell growth and/or survival. These results demonstrate an essential role for STAT1 for lymphocyte survival and proliferation that is only partially dependent on IFN-gamma signaling
— id: 8573, year: 2000, vol: 164, page: 1286, stat: Journal Article,

Divergent roles of STAT1 and STAT5 in malignancy as revealed by gene disruptions in mice
Levy DE; Gilliland DG
2000 May 15;19(21):2505-2510, Oncogene
Stat proteins are latent transcription factors activated by tyrosine phosphorylation downstream of cytokine and growth factor receptors and have been implicated in a variety of cell growth regulatory pathways. Constitutive phosphorylation has also been observed in various transformed cell line and in primary malignant tissue, suggesting that Stat protein activation may contribute to the transformed phenotype. One method to distinguish between a causative role in malignancy as opposed to bystander phosphorylation from the increased tyrosine phosphorylation that accompanies transformation is to investigate cell growth and malignancy in the absence of particular Stat proteins using targeted gene disruptions in transgenic mice. Such studies show that Stat1 primarily mediates growth inhibitory signals and contributes to the host rejection of tumors, and that its activation in transformed cells is not necessary for malignancy. Activation of Stat5 can be both necessary and sufficient for malignant transformation, and single Stat5-target genes have been identified that are critical for heightened proliferation. Nonetheless, some malignancies that are characterized by constitutively phosphorylated Stat5 are not altered by the loss of Stat5 protein. Its role in these cases may be redundant with other transforming events that are in themselves sufficient to cause disease, rendering tyrosine phosphorylation of Stat5 unnecessary in these transformed cells. Oncogene (2000)
— id: 11657, year: 2000, vol: 19, page: 2505, stat: Journal Article,

Phosphorylation-induced dimerization of interferon regulatory factor 7 unmasks DNA binding and a bipartite transactivation domain
Marie I; Smith E; Prakash A; Levy DE
2000 Dec;20(23):8803-8814, Molecular & cellular biology
Interferon regulatory factor 7 (IRF7) is an interferon (IFN)-inducible transcription factor required for activation of a subset of IFN-alpha genes that are expressed with delayed kinetics following viral infection. IRF7 is synthesized as a latent protein and is posttranslationally modified by protein phosphorylation in infected cells. Phosphorylation required a carboxyl-terminal regulatory domain that controlled the retention of the active protein exclusively in the nucleus, as well as its binding to specific DNA target sequences, multimerization, and ability to induce target gene expression. Transcriptional activation by IRF7 mapped to two distinct regions, both of which were required for full activity, while all functions were masked in latent IRF7 by an autoinhibitory domain mapping to an internal region. A conditionally active form of IRF7 was constructed by fusing IRF7 with the ligand-binding and dimerization domain of estrogen receptor (ER). Hormone-dependent dimerization of chimeric IRF7-ER stimulated DNA binding and transcriptional transactivation of endogenous target genes. These studies demonstrate the regulation of IRF7 activity by phosphorylation-dependent allosteric changes that result in dimerization and that facilitate nuclear retention, derepress transactivation, and allow specific DNA binding
— id: 39519, year: 2000, vol: 20, page: 8803, stat: Journal Article,

Stat5 is essential for the myelo- and lymphoproliferative disease induced by TEL/JAK2
Schwaller J; Parganas E; Wang D; Cain D; Aster JC; Williams IR; Lee CK; Gerthner R; Kitamura T; Frantsve J; Anastasiadou E; Loh ML; Levy DE; Ihle JN; Gilliland DG
2000 Sep;6(3):693-704, Molecular cell
STAT5 is activated in a broad spectrum of human hematologic malignancies. We addressed whether STAT5 activation is necessary for the myelo- and lymphoproliferative disease induced by TEL/JAK2 using a genetic approach. Whereas mice transplanted with bone marrow transduced with retrovirus expressing TEL/JAK2 develop a rapidly fatal myelo- and lymphoproliferative syndrome, reconstitution with bone marrow derived from Stat5ab-deficient mice expressing TEL/JAK2 did not induce disease. Disease induction in the Stat5a/b-deficient background was rescued with a bicistronic retrovirus encoding TEL/JAK2 and Stat5a. Furthermore, myeloproliferative disease was induced by reconstitution with bone marrow cells expressing a constitutively active mutant, Stat5a, or a single Stat5a target, murine oncostatin M (mOSM). These data define a critical role for Stat5a/b and mOSM in the pathogenesis of TEL/JAK2 disease
— id: 18491, year: 2000, vol: 6, page: 693, stat: Journal Article,

Differentiation of monocytes to macrophages switches the Mycobacterium tuberculosis effect on HIV-1 replication from stimulation to inhibition: modulation of interferon response and CCAAT/enhancer binding protein beta expression
Weiden M; Tanaka N; Qiao Y; Zhao BY; Honda Y; Nakata K; Canova A; Levy DE; Rom WN; Pine R
2000 Aug 15;165(4):2028-2039, Journal of immunology
HIV-1 replication is inhibited in uninflamed lung macrophages and is stimulated during tuberculosis. Attempts to recapitulate activation of HIV-1 replication in primary monocytes and macrophages ex vivo and in the untreated and PMA-treated THP-1 cell line model in vitro have produced opposite results depending on the state of differentiation of the cells. After infection with Mycobacterium tuberculosis, monocytes enhanced HIV-1 replication and produced a stimulatory 37-kDa CCAAT/enhancer binding protein beta (C/EBPbeta) transcription factor, whereas macrophages suppressed HIV-1 replication and produced an inhibitory 16-kDa C/EBPbeta transcription factor. IFN-beta induced inhibitory 16-kDa C/EBPbeta in macrophages, but had no effect on C/EBPbeta expression in monocytes. Macrophages, but not monocytes, were able to activate IFN-stimulated gene factor-3 (ISGF-3), a transcription factor composed of STAT-1, STAT-2, and IFN regulatory factor (IRF)-9, after infection with M. tuberculosis or stimulation with type I IFN. Macrophages expressed IRF-9 DNA-binding activity, but monocytes did not, and addition of the IRF-9 component reconstituted ISGF-3 in extracts of IFN-treated monocytes. Modulation of IFN responsiveness upon differentiation occurred at least in part through a post-transcriptionally regulated increase in IRF-9 expression. Both monocytes and macrophages maintained IFN responsiveness, activating STAT-1 homodimer formation and transcription of the STAT-1 gene after IFN stimulation. In addition, both monocytes and macrophages were able to activate NF-kappaB upon infection with M. tuberculosis. These results show that induction of ISGF-3, expression of the inhibitory 16-kDa C/EBPbeta, and suppression of HIV-1 replication via a transcriptional mechanism are macrophage-specific responses to infection with M. tuberculosis
— id: 11568, year: 2000, vol: 165, page: 2028, stat: Journal Article,

Mechanism of STAT3 activation by insulin-like growth factor I receptor
Zong, C S; Chan, J; Levy, D E; Horvath, C; Sadowski, H B; Wang, L H
2000 May 19;275(20):15099-15105, Journal of biological chemistry
Recent evidence indicates that STAT proteins can be activated by a variety of receptor and non-receptor protein-tyrosine kinases. Unlike cytokine-induced activation of STATs, where JAKs are known to play a pivotal role in phosphorylating STATs, the mechanism for receptor protein-tyrosine kinase-mediated activation of STATs remains elusive. In this study, we investigated the activation of STAT proteins by the insulin-like growth factor I receptor (IGF-IR) in vitro and in vivo and assessed the role of JAKs in the process of activation. We found that STAT3, but not STAT5, was activated in response to IGF-I in 293T cells cotransfected with IGF-IR and STAT expression vectors. Moreover, tyrosine phosphorylation of STAT3, JAK1, and JAK2 was increased upon IGF-I stimulation of endogenous IGF-IR in 293T cells transfected with the respective STAT or JAK expression vector. Supporting the observation in 293T cells, endogenous STAT3 was tyrosine-phosphorylated upon IGF-I stimulation in the muscle cell line C2C12 as well as in various embryonic and adult mouse organs during different stages of development. Dominant-negative JAK1 or JAK2 was able to block the IGF-IR-mediated tyrosine phosphorylation of STAT3 in 293T cells. A newly identified family of proteins called SOCS (suppressor of cytokine signaling), including SOCS1, SOCS2, SOCS3 and CIS, was able to inhibit the IGF-I-induced STAT3 activation as well with varying degrees of potency, in which SOCS1 and SOCS3 appeared to have the higher inhibitory ability. Inhibition of STAT3 activation by SOCS could be overcome by overexpression of native JAK1 and JAK2. We conclude that IGF-I/IGF-IR is able to mediate activation of STAT3 in vitro and in vivo and that JAKs are essential for the process of activation
— id: 138936, year: 2000, vol: 275, page: 15099, stat: Journal Article,

Differential regulation of constitutive major histocompatibility complex class I expression in T and B lymphocytes
Lee CK; Gimeno R; Levy DE
1999 Nov 15;190(10):1451-1464, Journal of experimental medicine
Major histocompatibility complex (MHC) class I antigens are constitutively expressed yet highly induced by interferon (IFN) during inflammation. We found that not only IFN-induced but also normal basal expression of MHC I required IFN receptors and signal transducer and activator of transcription (STAT)1, providing genetic evidence for continuous IFN signaling. Surprisingly, an IFN-independent requirement for STAT1 was also found, specifically in T lymphocytes, where MHC class I expression was not fully accounted for by IFN signaling. This IFN-independent pathway maintained tyrosine phosphorylation of STAT1 in T but not B lymphocytes even in the absence of IFN receptors. Interestingly, interleukin (IL)-7 selectively activated STAT1 and induced MHC class I in mature T but not B cells. These loss of function studies demonstrate an essential role of endogenous IFN and activated STAT1 for constitutive MHC class I expression in normal mice and define IL-7-dependent but IFN-independent regulation of STAT1 restricted to T lymphocytes
— id: 6240, year: 1999, vol: 190, page: 1451, stat: Journal Article,

Physiological significance of STAT proteins: investigations through gene disruption in vivo
Levy DE
1999 Sep;55(12):1559-1567, Cellular & molecular life sciences: CMLS
Signal transducers and activators of transcription (STATs) were discovered as mediators of type I interferon-induced gene expression. This family of transcription factors has been found in widespread signaling pathways, especially those involving cytokines regulating the immune response. Because a plethora and often confusing set of activators for STAT proteins was observed in cell culture models, it became important to define the physiologically relevant actions of these molecules. One approach to this question has been through the targeted disruption of STAT genes in transgenic mice. Now that all seven STAT genes have been disrupted, both the high degree of STAT selectivity as well as many surprising and unexpected complexities are beginning to be characterized
— id: 11944, year: 1999, vol: 55, page: 1559, stat: Journal Article,

The proximal tyrosines of the cytoplasmic domain of the beta chain of the type I interferon receptor are essential for signal transducer and activator of transcription (Stat) 2 activation. Evidence that two Stat2 sites are required to reach a threshold of interferon alpha-induced Stat2 tyrosine phosphorylation that allows normal formation of interferon-stimulated gene factor 3
Nadeau OW; Domanski P; Usacheva A; Uddin S; Platanias LC; Pitha P; Raz R; Levy D; Majchrzak B; Fish E; Colamonici OR
1999 Feb 12;274(7):4045-4052, Journal of biological chemistry
The precise role of the different subunits (alpha/IFNAR1 and betaL/IFNAR2) of the type I interferon receptor (IFN-R) in the activation of signal transducer and activator of transcription (Stat) 1, Stat2, and Stat3 has not yet been established. In this report we demonstrate that there are functionally redundant phosphotyrosine-dependent and -independent binding sites for Stat2 in the alpha and beta subunits of the type I IFN-R. Expression of a type I IFN-R containing only the constitutive Stat2 site or the proximal tyrosines of betaL, but not the docking site on the alpha chain (Tyr466 and Tyr481), supported low levels of Stat2 activation. However, the presence of only one intact Stat2 site did not lead to induction of interferon-stimulated gene factor 3 (ISGF3) or an antiviral state. Normal levels of Stat2 tyrosine phosphorylation, induction of ISGF3, and an antiviral effect always required the proximal tyrosines of betaL and at least one of the other Stat2 sites (Tyralpha466, 481 or betaL404-462). These data suggest that a threshold of Stat2 tyrosine phosphorylation is required for complete activation of ISGF3. Interestingly, a receptor in which all tyrosines were mutated to phenylalanine shows normal Stat3 phosphorylation and low levels of activation of Stat1
— id: 7991, year: 1999, vol: 274, page: 4045, stat: Journal Article,

Stat protein transactivation domains recruit p300/CBP through widely divergent sequences
Paulson M; Pisharody S; Pan L; Guadagno S; Mui AL; Levy DE
1999 Sep 3;274(36):25343-25349, Journal of biological chemistry
The signal transduction and activator of transcription (Stat) gene family has been highly conserved throughout evolution. Gene duplication and divergence has produced 7 mammalian Stat genes, each of which mediates a distinct process. While some Stat proteins are activated by multiple cytokines, Stat2 is highly specific for responses to type I interferon. We have cloned mouse Stat2 and found that while its sequence was more divergent from its human homologue than any other mouse-human Stat pairs, it was fully functional even in human cells. Overall sequence identity was only 69%, compared with 85-99% similarity for other Stat genes, and several individual domains that still served similar or identical functions in both species were even less well conserved. The coiled-coil domain responsible for interaction with IRF9 was only 65% identical and yet mouse Stat2 interacted with either human or mouse IRF9; the carboxyl terminus was only 30% identical and yet both regions functioned as equal transactivation domains. Both mouse and human transactivation domains recruited the p300/CBP coactivator and were equally sensitive to inhibition by adenovirus E1A protein. Interestingly, the Stat3 carboxyl terminus also functioned as a transactivator capable of recruiting p300/CBP, as does the Stat1 protein, although with widely differing potencies. Yet these proteins share no sequence similarity with Stat2. These data demonstrate that highly diverged primary sequences can serve similar or identical functions, and that the minimal regions of similarity between human and mouse Stat2 may define the critical determinants for function
— id: 8480, year: 1999, vol: 274, page: 25343, stat: Journal Article,

Essential role of STAT3 for embryonic stem cell pluripotency
Raz R; Lee CK; Cannizzaro LA; d'Eustachio P; Levy DE
1999 Mar 16;96(6):2846-2851, Proceedings of the National Academy of Sciences of the United States of America
Propagation of mouse embryonic stem (ES) cells in vitro requires exogenous leukemia inhibitory factor (LIF) or related cytokines. Potential downstream effectors of the LIF signal in ES cells include kinases of the Src, Jak, and mitogen-activated protein families and the signal transducer and transcriptional activator STAT3. Activation of nuclear STAT3 and the ability of ES cells to grow as undifferentiated clones were monitored during LIF withdrawal. A correlation was found between levels of STAT3 activity and maintenance of an undifferentiated phenotype at clonal density. In contrast, variation in STAT3 activity did not affect cell proliferation. The requirement for STAT3 was analyzed by targeted mutagenesis in ES cell lines exhibiting different degrees of LIF dependency. An insertional mutation was devised that abrogated Stat3 gene expression but could be reversed by Cre recombination-mediated excision. ES cells heterozygous for the Stat3 mutation could be isolated only from E14 cells, the line least dependent on LIF for self-renewal. Targeted clones isolated from other ES cell lines were invariably trisomic for chromosome 11, which carries the Stat3 locus, and retained normal levels of activated STAT3. Cre-regulated reduction of Stat3 gene copy number in targeted, euploid E14 clones resulted in dose-dependent losses of STAT3 activity and the efficiency of self-renewal without commensurate changes in cell cycle progression. These results demonstrate an essential role for a critical amount of STAT3 in the maintenance of an undifferentiated ES cell phenotype
— id: 8219, year: 1999, vol: 96, page: 2846, stat: Journal Article,

FGF signaling inhibits chondrocyte proliferation and regulates bone development through the STAT-1 pathway
Sahni M; Ambrosetti DC; Mansukhani A; Gertner R; Levy D; Basilico C
1999 Jun 1;13(11):1361-1366, Genes & development
Several genetic forms of human dwarfism have been linked to activating mutations in FGF receptor 3, indicating that FGF signaling has a critical role in chondrocyte maturation and skeletal development. However, the mechanisms through which FGFs affect chondrocyte proliferation and differentiation remain poorly understood. We show here that activation of FGF signaling inhibits chondrocyte proliferation both in a rat chondrosarcoma (RCS) cell line and in primary murine chondrocytes. FGF treatment of RCS cells induces phosphorylation of STAT-1, its translocation to the nucleus, and an increase in the expression of the cell-cycle inhibitor p21WAF1/CIP1. We have used primary chondrocytes from STAT-1 knock-out mice to provide genetic evidence that STAT-1 function is required for the FGF mediated growth inhibition. Furthermore, FGF treatment of metatarsal rudiments from wild-type and STAT-1(-/-) murine embryos produces a drastic impairment of chondrocyte proliferation and bone development in wild-type, but not in STAT-1(-/-) rudiments. We propose that STAT-1 mediated down regulation of chondrocyte proliferation by FGF signaling is an homeostatic mechanism which ensures harmonious bone development and morphogenesis
— id: 12005, year: 1999, vol: 13, page: 1361, stat: Journal Article,

Superantigen-activated T cells redirected by a bispecific antibody inhibit vesicular stomatitis virus replication in vitro and in vivo
Fernandez-Sesma, A; Peluso, R W; Bai, X; Schulman, J L; Levy, D E; Moran, T M
1998 Feb 15;160(4):1841-1849, Journal of immunology
A bispecific Ab (BsAb) that binds the TCR on T cells and the G protein of the vesicular stomatitis virus (VSV) can redirect staphylococcal enterotoxin B (SEB)-activated T cells to kill VSV-infected cells and to inhibit VSV replication in vitro. Inhibition of virus replication in our system is dependent upon the specificity of the Ab for the viral protein. IFN-gamma does not play a very important role in this phenomenon, which is mainly mediated by the release of Pfp from CD8+ T cells. We have used a Stat1 knockout mouse model in which VSV infection is lethal. Infusion of staphylococcal enterotoxin-activated B T cells and bispecific Ab significantly slowed virus progression and prolonged the survival of VSV-infected Stat1 knockout mice in vivo
— id: 138966, year: 1998, vol: 160, page: 1841, stat: Journal Article,

The role of interferon in influenza virus tissue tropism
Garcia-Sastre A; Durbin RK; Zheng H; Palese P; Gertner R; Levy DE; Durbin JE
1998 Nov;72(11):8550-8558, Journal of virology
We have studied the pathogenesis of influenza virus infection in mice that are unable to respond to type I or II interferons due to a targeted disruption of the STAT1 gene. STAT1-/- animals are 100-fold more sensitive to lethal infection with influenza A/WSN/33 virus than are their wild-type (WT) counterparts. Virus replicated only in the lungs of WT animals following intranasal (i.n.) virus inoculation, while STAT1-/- mice developed a fulminant systemic influenza virus infection following either i.n. or intraperitoneal inoculation. We investigated the mechanism underlying this altered virus tropism by comparing levels of virus replication in fibroblast cell lines and murine embryonic fibroblasts derived from WT mice, STAT-/- mice, and mice lacking gamma interferon (IFNgamma-/- mice) or the IFN-alpha receptor (IFNalphaR-/- mice). Influenza A/WSN/33 virus replicates to high titers in STAT1-/- or IFNalphaR-/- fibroblasts, while cells derived from WT or IFNgamma-/- animals are resistant to influenza virus infection. Immunofluorescence studies using WT fibroblast cell lines demonstrated that only a small subpopulation of WT cells can be infected and that in the few infected WT cells, virus replication is aborted at an early, nuclear phase. In all organs examined except the lung, influenza A WSN/33 virus infection is apparently prevented by an intact type I interferon response. Our results demonstrate that type I interferon plays an important role in determining the pathogenicity and tissue restriction of influenza A/WSN/33 virus in vivo and in vitro
— id: 7576, year: 1998, vol: 72, page: 8550, stat: Journal Article,

Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems
Garcia-Sastre, A; Egorov, A; Matassov, D; Brandt, S; Levy, D E; Durbin, J E; Palese, P; Muster, T
1998 Dec 20;252(2):324-330, Virology
The NS1 protein is the only nonstructural protein encoded by influenza A virus. It has been proposed that the NS1 performs several regulatory functions during the viral replication cycle, including the regulation of synthesis, transport, splicing, and translation of mRNAs. Through the use of reverse genetics, a viable transfectant influenza A virus (delNS1) which lacks the NS1 gene has been generated. Our results indicate that the NS1 of influenza A virus is an auxiliary (virulence) factor which plays a crucial role in inhibiting interferon-mediated antiviral responses of the host
— id: 138967, year: 1998, vol: 252, page: 324, stat: Journal Article,

Analysis of interferon-regulated proteins binding the interferon-alpha-stimulated response element
Levy DE
1998 Jul;15(3):167-174, Methods
Interferon mediates its biological effects of antiviral, antiproliferative, and immunomodulatory activities through induction of specific gene expression. Transcription of target genes is regulated through a set of transcription factors that bind to specific cis-acting regulatory sequences in target promoters and enhancers. The activity or abundance of these transcription factors is modulated by interferon treatment. Because of their DNA binding activity, regulatory factors can be recognized and characterized using methods to detect protein-DNA complexes. The availability of antibodies directed against common interferon regulatory proteins coupled with analysis of binding-site specificity provides comprehensive analysis.
— id: 7387, year: 1998, vol: 15, page: 167, stat: Journal Article,

Differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7
Marie I; Durbin JE; Levy DE
1998 Nov 16;17(22):6660-6669, EMBO journal
Interferon (IFN) genes are among the earliest transcriptional responses to virus infection of mammalian cells. Although the regulation of the IFNbeta gene has been well characterized, the induction of the large family of IFNalpha genes has remained obscure. We report that the IFNalpha genes can be divided into two groups: an immediate-early response gene (IFNalpha4) which is induced rapidly and without the need for ongoing protein synthesis; and a set of genes that display delayed induction, consisting of at least IFNalpha2, 5, 6 and 8, which are induced more slowly and require cellular protein synthesis. One protein that must be synthesized for induction of the delayed gene set is IFN itself, presumably IFNalpha4 or IFNbeta, which stimulates the Jak-Stat pathway through the IFN receptor, resulting in activation of the transcription factor interferon-stimulated gene factor 3 (ISGF3). Among the IFN-stimulated genes induced through this positive feedback loop is the IFN regulatory factor (IRF) protein, IRF7. Induction of IRF7 protein in response to IFN and its subsequent activation by phosphorylation in response to virus-specific signals, involving two C-terminal serine residues, are required for induction of the delayed IFNalpha gene set
— id: 7964, year: 1998, vol: 17, page: 6660, stat: Journal Article,

Essential role of STAT3 for embryonic stem cell growth
Raz, R; D'Eustachio, P; Kennizzaro, L; Levy, DE
1998 SEP ;9(3):328-328, European cytokine network
— id: 53660, year: 1998, vol: 9, page: 328, stat: Journal Article,

Proto-oncogene PML controls genes devoted to MHC class I antigen presentation
Zheng P; Guo Y; Niu Q; Levy DE; Dyck JA; Lu S; Sheiman LA; Liu Y
1998 Nov 26;396(6709):373-376, Nature
Fragments of foreign antigens associated with class I molecules of the major histocompatibility complex (MHC) are presented at the cell surface to elicit an immune response. This presentation requires the coordinated expression of several genes contained in the MHC, including those encoding the MHC class I heavy chain, the proteins LMP-2 and LMP-7, which are involved in the proteasomal degradation of cytosolic antigens into peptide fragments that are destined for association with MHC class I molecules, and TAP-1 and TAP-2, which transport these fragments across the membrane of the endoplasmic reticulum at the start of their journey to the cell surface. In many virus-transformed cell lines and spontaneous tumours, these genes are simultaneously repressed. However, the key factor(s) that are essential for their expression and repression have not been identified. Here we report that the proto-oncogene product PML induces expression of LMP-2, LMP-7, TAP-1 and TAP-2 in an MHC-class I-negative, recurrent tumour, leading to the re-expression of cell-surface MHC in tumours and to rejection of the tumours. PML also regulates MHC expression in untransformed fibroblasts. We conclude that malfunction of PML may enable a tumour to evade the immune defence of its host
— id: 7473, year: 1998, vol: 396, page: 373, stat: Journal Article,

Stat2 is a transcriptional activator that requires sequence-specific contacts provided by stat1 and p48 for stable interaction with DNA
Bluyssen HA; Levy DE
1997 Feb 14;272(7):4600-4605, Journal of biological chemistry
Transcriptional responses to interferon (IFN) are mediated by tyrosine phosphorylation and nuclear translocation of transcription factors of the signal transducer and activator of transcription (Stat) family. The Stat1 protein is required for all transcriptional responses to IFN (both type I and type II). Responses to type I IFN (alpha and beta) also require Stat2 and the IFN regulatory factor family protein p48, which form a heterotrimeric transcription complex with Stat1 termed ISGF3. Stat1 homodimers formed in response to IFN-gamma treatment can also interact with p48 and function as transcriptional activators. We now show that Stat2 is capable of forming a stable homodimer that interacts with p48, can be recruited to DNA, and can activate transcription, raising a question of why Stat1 is required. Analysis of the transcriptional competence, affinity, and specificity of Stat2-p48 complexes compared with other Stat protein-containing transcription factor complexes suggests distinct roles for each component. Although Stat2 is a potent transactivator, it does not interact stably with DNA in complex with p48 alone. Adding Stat1 increases the affinity and alters the sequence selectivity of p48-DNA interactions by contacting a half-site of its palindromic recognition motif adjacent to a p48 interaction sequence. Thus, ISGF3 assembly involves p48 functioning as an adaptor protein to recruit Stat1 and Stat2 to an IFN-alpha-stimulated response element, Stat2 contributes a potent transactivation domain but is unable to directly contact DNA, while Stat1 stabilizes the heteromeric complex by contacting DNA directly
— id: 12382, year: 1997, vol: 272, page: 4600, stat: Journal Article,

Regulation of interferon-alpha responsiveness by the duration of Janus kinase activity
Lee CK; Bluyssen HA; Levy DE
1997 Aug 29;272(35):21872-21877, Journal of biological chemistry
Daudi B lymphoblastoid cells are highly sensitive to the anti-growth and anti-viral effects of interferon (IFN). Unlike many cell lines, these cells show prolonged transcription of IFN-stimulated genes following treatment with IFN-alpha. This prolonged response correlated with the continued presence of the activated transcription factor, IFN-stimulated gene factor 3 (ISGF3). Pulse-chase labeling experiments indicated that the half-life of the phosphorylation of signal transducers and activators of transcription (Stat)1 and Stat2 was short (<2 h) although the turnover of the proteins themselves was slow (>24 h), indicative of a constitutive phosphatase activity. The administration of protein-tyrosine kinase inhibitors at any time point during IFN stimulation led to rapid inhibition of the response, indicating that tyrosine kinase activity was continuously required. Catalytic activity of Jak1 and Tyk2 kinases remained elevated for prolonged periods following stimulation. Continuous presence of IFN-alpha was necessary for maintaining prolonged activation of ISGF3 and of Janus kinases, an activity that was blocked by antibodies to IFN-alpha or by cycloheximide. Conditioned medium of IFN-alpha-stimulated cells was capable of stimulating STAT activation in naive cells. Taken together, these results suggest that the response to IFN-alpha is controlled by the duration of stimulated Janus kinase activity over the background of constitutive dephosphorylation and that this response can be sustained by autocrine secretion of IFN-alpha
— id: 7193, year: 1997, vol: 272, page: 21872, stat: Journal Article,

The house that Jak/Stat built
Levy DE
1997 Mar;8(1):81-90, Cytokine & growth factor reviews
An international workshop on 'Cytokines, Signaling and the Jak/Stat Pathway' organized by Peter C. Heinrich of the Klinikum der RWTH Aachen under the auspices of DFG-Forschergruppe was held in Aachen, Germany, on 4-5 October 1996. In this review David Levy notes that even though some diagrams shown at the meeting displayed more arrows than there were meeting participants, remarkable progress has been made in the unraveling of the complexities of cytokine signal transduction. The central issue in the field of cytokine signaling is specificity, and this issue was addressed through experimental results from biochemical, molecular, and genetic studies of signal transduction pathways
— id: 7197, year: 1997, vol: 8, page: 81, stat: Journal Article,

Retinoic acid induces signal transducer and activator of transcription (STAT) 1, STAT2, and p48 expression in myeloid leukemia cells and enhances their responsiveness to interferons
Matikainen, S; Ronni, T; Lehtonen, A; Sareneva, T; Melen, K; Nordling, S; Levy, D E; Julkunen, I
1997 Jun;8(6):687-698, Cell growth & differentiation
IFNs are antiproliferative cytokines that have growth-inhibitory effects on various normal and malignant cells. Therefore, they have been used in the treatment of certain forms of cancer, such as chronic myelogenous leukemia and hairy cell leukemia. However, there is little evidence that IFNs would be effective in the treatment of acute myelogenous leukemia, and molecular mechanisms underlying IFN unresponsiveness have not been clarified. Here we have studied the activation and induction of IFN-specific transcription factors signal transducer and activator of transcription (STAT) 1, STAT2, and p48 in all-trans-retinoic acid (ATRA)-differentiated myeloid leukemia cells using promyelocytic NB4, myeloblastic HL-60, and monoblastic U937 cells as model systems. These cells respond to ATRA by growth inhibition and differentiation. We show that in undifferentiated NB4 cells, 2',5'-oligoadenylate synthetase and MxB gene expression is not activated by IFN-alpha, possibly due to a relative lack of signaling molecules, especially p48 protein. However, during ATRA-induced differentiation, steady-state STAT1, STAT2, and especially p48 mRNA and corresponding protein levels were elevated both in NB4 and U937 cells, apparently correlating to an enhanced responsiveness of these cells to IFNs. ATRA treatment of NB4 cells sensitized them to IFN action as seen by increased IFN-gamma activation site DNA-binding activity or by efficient formation of IFN-alpha-specific ISGF3 complex and subsequent oligoadenylate synthetase and MxB gene expression. Lack of p48 expression could be one of the mechanisms of promyelocytic leukemia cell escape from growth-inhibitory effects of IFN-alpha
— id: 138965, year: 1997, vol: 8, page: 687, stat: Journal Article,

ISGF3 gamma p48, a specificity switch for interferon activated transcription factors
Bluyssen AR; Durbin JE; Levy DE
1996 Jun;7(1):11-17, Cytokine & growth factor reviews
Interferon (IFN) induces gene expression by phosphorylating latent transcription factors of the STAT family. Two different STAT multimeric complexes that bind distinct enhancer elements are activated by IFN alpha and IFN gamma, dictated by the DNA-binding protein ISGF3 gamma p48. This protein, a member of the IFN regulatory factor (IFR) family, acts as an adaptor protein to redirect STAT multimers from their intrinsic palindromic sequence specificity to interactions with a composite element composed of an IRF site juxtaposed with a STAT half-site. Sequence similarity within the IRF family suggests that other members could serve as adaptor proteins for transcriptional activators. Recent evidence suggests that PIP (LSIRF) sequesters the Ets protein PU.1 at a composite DNA element lends support to this adaptor hypothesis
— id: 12594, year: 1996, vol: 7, page: 11, stat: Journal Article,

Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease
Durbin JE; Hackenmiller R; Simon MC; Levy DE
1996 Feb 9;84(3):443-450, Cell
The STAT1 transcription factor is activated in response to many cytokines and growth factors. To study the requirement for STAT1 in vivo, we disrupted the Stat1 gene in embryonic stem (ES) cells and in mice. Stat1(-1-)ES cells were unresponsive to interferon (IFN), but retained responsiveness to leukemia inhibitory factor (LIF) and remained LIF dependent for undifferentiated growth. Stat1(-1-1) animals were born at normal frequencies and displayed no gross developmental defects. However, these animals failed to thrive and were extremely susceptible to viral disease. Cells and tissues from Stat1(-1-) mice were unresponsive to IFN, but remained responsive to all other cytokines tested. Thus, STAT1 appears to be specific for IFN pathways that are essential for viability in the face of otherwise innocuous pathogens
— id: 6903, year: 1996, vol: 84, page: 443, stat: Journal Article,

Roles of JAKs in activation of STATs and stimulation of c-fos gene expression by epidermal growth factor
Leaman, D W; Pisharody, S; Flickinger, T W; Commane, M A; Schlessinger, J; Kerr, I M; Levy, D E; Stark, G R
1996 Jan;16(1):369-375, Molecular & cellular biology
The tyrosine kinase JAK1 and the transcription factors STAT1 and STAT3 are phosphorylated in response to epidermal growth factor (EGF) and other growth factors. We have used EGF receptor-transfected cell lines defective in individual JAKs to assess the roles of these kinases in STAT activation and signal transduction in response to EGF. Although JAK1 is phosphorylated in response to EGF, it is not required for STAT activation or for induction of the c-fos gene. STAT activation in JAK2- and TYK2-defective cells is also normal, and the tyrosine phosphorylation of these two kinases does not increase upon EGF stimulation in wild-type or JAK1-negative cells. In cells transfected with a kinase-negative mutant EGF receptor, there is no STAT activation in response to EGF and c-fos is not induced, showing that the kinase activity of the receptor is required, directly or indirectly, for these two responses. The data do not support a role for any of the three JAK family members tested in STAT activation and are consistent with a JAK-independent pathway in which the intrinsic kinase domain of the EGF receptor is crucial. Furthermore, data from transient transfection experiments in HeLa cells, using c-fos promoters lacking the STAT regulatory element c-sis-inducible element, indicate that this element may play only a minor role in the induction of c-fos by EGF in these cells
— id: 138964, year: 1996, vol: 16, page: 369, stat: Journal Article,

Activation of the JAK-STAT signal transduction pathway by oncostatin-M cultured human and mouse osteoblastic cells
Levy, J B; Schindler, C; Raz, R; Levy, D E; Baron, R; Horowitz, M C
1996 Apr;137(4):1159-1165, Endocrinology
Oncastatin M (OSM) is one member of the leukemia inhibitory factor/interleukin-6 family of cytokines that has been shown to be a growth regulatory molecule. In osteoblastic cultures, OSM causes marked phenotypic changes and the enhanced secretion of interleukin-6. In this study, we have shown that stimulation of murine and human osteoblastic cultures and a human osteosarcoma cell line with OSM resulted in the tyrosine phosphorylation of a number of cellular proteins including members of both the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) family of signaling proteins. The JAKs, a family of intracellular kinases, and the STATs, a family of transcription factors, have both previously been shown to be tyrosine phosphorylated and activated in response to various cytokines, interferons, and growth factors in cells of non-skeletal origin. Using three different sources of cells of the osteoblast lineage, we demonstrate that OSM induces a rapid but transient tyrosine phosphorylation of the three JAK family members tested, JAK1, JAK2 and Tyk2. In addition, two members of the STAT family, Stat1alpha and Stat3, are tyrosine phosphorylated in osteoblastic cells in culture in response to OSM. OSM activation of this pathway in cells of the osteoblast lineage will result in the transcription of specific genes that ultimately may be associated with osteoblast function
— id: 106998, year: 1996, vol: 137, page: 1159, stat: Journal Article,

Combinatorial association and abundance of components of interferon-stimulated gene factor 3 dictate the selectivity of interferon responses
Bluyssen HA; Muzaffar R; Vlieststra RJ; van der Made AC; Leung S; Stark GR; Kerr IM; Trapman J; Levy DE
1995 Jun 6;92(12):5645-5649, Proceedings of the National Academy of Sciences of the United States of America
Genes containing the interferon-stimulated response element (ISRE) enhancer have been characterized as transcriptionally responsive primarily to type I interferons (IFN alpha/beta). Induction is due to activation of a multimeric transcription factor, interferon-stimulated gene factor 3 (ISGF3), which is activated by IFN alpha/beta but not by IFN gamma. We found that ISRE-containing genes were induced by IFN gamma as well as by IFN alpha in Vero cells. The IFN gamma response was dependent on the ISRE and was accentuated by preexposure of cells to IFN alpha, a treatment that increases the abundance of ISGF3 components. Overexpression of ISGF3 polypeptides showed that the IFN gamma response depended on the DNA-binding protein ISGF3 gamma (p48) as well as on the 91-kDa protein STAT91 (Stat1 alpha). The transcriptional response to IFN alpha required the 113-kDa protein STAT113 (Stat2) in addition to STAT91 and p48. Mutant fibrosarcoma cells deficient in each component of ISGF3 were used to confirm that IFN gamma induction of an ISRE reporter required p48 and STAT91, but not STAT113. A complex containing p48 and phosphorylated STAT91 but lacking STAT113 bound the ISRE in vitro. IFN gamma-induced activation of this complex, preferentially formed at high concentrations of p48 and STAT91, may explain some of the overlapping responses to IFN alpha and IFN gamma
— id: 12764, year: 1995, vol: 92, page: 5645, stat: Journal Article,

STRUCTURE-FUNCTION ANALYSIS OF ISGF3 COMPLEX-FORMATION
BLUYSSEN, HAR; RAZ, R; LEVY, DE
1995 JAN 5 ;336(3):30-30, Journal of cellular biochemistry
— id: 87348, year: 1995, vol: 336, page: 30, stat: Journal Article,

Activation of acute phase response factor (APRF)/Stat3 transcription factor by growth hormone
Campbell, G S; Meyer, D J; Raz, R; Levy, D E; Schwartz, J; Carter-Su, C
1995 Feb 24;270(8):3974-3979, Journal of biological chemistry
The mechanism by which the binding of growth hormone (GH) to its cell surface receptor elicits changes in gene transcription are largely unknown. The transcription factor Stat1/p91 has been shown to be activated by GH. Here we show that acute phase response factor or Stat3 f1p4an antigenically related protein), is also activated by GH. Stat3 has been implicated in the interleukin-6-dependent induction of acute phase response genes. GH promotes in 3T3-F442A fibroblasts the tyrosyl phosphorylation of a protein immunoprecipitated by antibodies to Stat3. This protein co-migrates with a tyrosyl phosphorylated protein from cells treated with leukemia inhibitory factor, a cytokine known to activate Stat3. Tyrosyl phosphorylated Stat3 is also observed in response to interferon-gamma. Stat3 is present in GH-inducible DNA-binding complexes that bind the sis-inducible element in the c-fos promoter and the acute phase response element in the alpha 2-macroglobulin promoter. The ability of GH to activate both Stat1 and Stat3 (i.e. increase their tyrosyl phosphorylation and ability to bind to DNA) suggests that gene regulation by GH involves multiple Stat proteins. Shared transcription factors among hormones and cytokines that activate JAK kinases provide an explanation for shared responses, while the ability of the different ligands to differentially recruit various Stat family members suggests mechanisms by which specificity in gene regulation could be achieved
— id: 106999, year: 1995, vol: 270, page: 3974, stat: Journal Article,

Prolactin, growth hormone, erythropoietin and granulocyte-macrophage colony stimulating factor induce MGF-Stat5 DNA binding activity
Gouilleux, F; Pallard, C; Dusanter-Fourt, I; Wakao, H; Haldosen, L A; Norstedt, G; Levy, D; Groner, B
1995 May 1;14(9):2005-2013, EMBO journal
The molecular components which mediate cytokine signaling from the cell membrane to the nucleus were studied. Upon the interaction of cytokines with their receptors, members of the janus kinase (Jak) family of cytoplasmic protein tyrosine kinases and of the signal transducers and activators of transcription (Stat) family of transcription factors are activated through tyrosine phosphorylation. It has been suggested that the Stat proteins are substrates of the Jak protein tyrosine kinases. MGF-Stat5 is a member of the Stat family which has been found to confer the prolactin response. MGF-Stat5 can be phosphorylated and activated in its DNA binding activity by Jak2. The activation of MGF-Stat5 is not restricted to prolactin. Erythropoietin (EPO) and growth hormone (GH) stimulate the DNA binding activity of MGF-Stat5 in COS cells transfected with vectors encoding EPO receptor and MGF-Stat5 or vectors encoding GH receptor and MGF-Stat5. The activation of DNA binding by prolactin, EPO and GH requires the phosphorylation of tyrosine residue 694 of MGF-Stat5. The transcriptional induction of a beta-casein promoter luciferase construct in transiently transfected COS cells is specific for the prolactin activation of MGF-Stat5; it is not observed in EPO- and GH-treated cells. In the UT7 human hematopoietic cell line, EPO and granulocyte-macrophage colony stimulating factor activate the DNA binding activity of a factor closely related to MGF-Stat5 with respect to its immunological reactivity, DNA binding specificity and molecular weight. These results suggest that MGF-Stat5 regulates physiological processes in mammary epithelial cells, as well as in hematopoietic cells.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 138960, year: 1995, vol: 14, page: 2005, stat: Journal Article,

Activation of the signal transducer and transcription (STAT) signaling pathway in a primary T cell response. Critical role for IL-6
Henttinen, T; Levy, D E; Silvennoinen, O; Hurme, M
1995 Nov 15;155(10):4582-4587, Journal of immunology
The T cell activation is initiated by interaction of specific Ags with TCR, followed by activation of intracellular biochemical events leading to activation of several genes. The activation of signal transducer and activator of transcription (STAT) proteins in a primary TCR-mediated activation of T cells have been explored. In purified human peripheral blood T cells, nuclear STAT proteins were activated approximately 3 h after activation by cross-linked anti-CD3 Abs. These STAT proteins were detected by using the IFN-gamma-activated sequence (GAS) and related oligonucleotides as probes in electrophoretic mobility shift assay. Analysis of the nuclear extracts with anti-STAT Abs indicated that they contained STAT-3 and additional proteins crossreactive with the STAT family. The induction of STAT activity was inhibited completely by pretreatment with either cycloheximide or cyclosporin A, thus indicating that the induction was due to a secondary factor produced by the activated T cells. As neutralizing anti-IL-6 Abs effectively down-regulated the early induction of STAT proteins and as exogenously added IL-6 rapidly activated DNA binding similar to TCR-mediated bindings, it can be concluded that IL-6 is the factor responsible for the activation of STAT proteins in a primary T cell response
— id: 138958, year: 1995, vol: 155, page: 4582, stat: Journal Article,

INTERFERON INDUCTION OF GENE-EXPRESSION THROUGH THE JAK-STAT PATHWAY
LEVY, DE
1995 JUN ;6(3):181-189, Seminars in virology
IFN alpha and IFN gamma induce rapid activation of gene expression following binding to cell surface receptors on target cells. Gene transcription depends on latent transcription factors that become activated in response to IFN treatment. These proteins, termed Stats for signal transducers and activators of transcription, serve as intrinsic elements of the signaling pathway. In untreated cells, they are sequestered in the cytoplasm. Upon treatment of cells with IFN, they become tyrosine phophorylated by receptor-bound protein tyrosine Kinases of the Jak family, assemble into multimeric complexes, and localise to the cell nucleus. Differential activation and combinatorial association of these transcription factors produce the biological responses characteristic of each IFN
— id: 87242, year: 1995, vol: 6, page: 181, stat: Journal Article,

Thrombopoietin activates a STAT5-like factor in hematopoietic cells
Pallard, C; Gouilleux, F; Benit, L; Cocault, L; Souyri, M; Levy, D; Groner, B; Gisselbrecht, S; Dusanter-Fourt, I
1995 Jun 15;14(12):2847-2856, EMBO journal
Thrombopoietin (TPO) is a newly cloned cytokine which is the major regulator of circulating platelet levels, acting on both proliferation and differentiation of megakaryocytes. We have investigated the ability of TPO to activate the JAK/STAT pathway in megakaryocytic cell lines. We used either the granulocyte-macrophage colony-stimulating factor (GM-CSF)- and/or erythropoietin (EPO)-dependent UT7 cell line in which the murine TPO receptor (mumpl) had been transfected (mumpl-UT7 transfectants) or the MO7E and DAMI cells which express endogenous human TPO receptors. We demonstrated that TPO activates the kinase JAK2 and a STAT5-like transcriptional factor but not STAT1, STAT2, STAT3 or STAT4, in a very rapid and transient manner. In order to better ascertain the specificity of the activation of STAT5-related factor by TPO, we investigated the effect of other cytokines/growth factors. Both GM-CSF and EPO activated the STAT5-like factor. In contrast, neither interferon (IFN)-gamma nor the mitogenic stem cell factor (SCF) activated STAT5, although IFN-gamma did activate STAT1 in those cells. The hematopoietic DNA binding activity related to STAT5 was identified as a p97 tyrosine-phosphorylated protein band which exhibited identical gel mobility to the mammary STAT5. Because v-mpl, a truncated form of the TPO receptor c-mpl, was shown to be oncogenic, we tested the activity of v-mpl on STAT5 and found STAT5 constitutively activated in two different v-mpl-expressing cells, the transiently transfected Cos7 cells and the stable v-mpl-UT7 transfectants. Overall, our data indicate that STAT5 is widely expressed in hematopoietic cells and activated by a number of cytokines, including TPO, GM-CSF and EPO, but not by IFN-gamma or SCF
— id: 138961, year: 1995, vol: 14, page: 2847, stat: Journal Article,

Three distinct loci on human chromosome 21 contribute to interferon-alpha/beta responsiveness
Raz R; Cheung K; Ling L; Levy DE
1995 Mar;21(2):139-145, Somatic cell & molecular genetics
The species specificity of interferons (IFNs) depends on restricted recognition of these ligands by multisubunit cell surface receptors. Expression of the human receptor subunit IFNAR in mouse cells conferred sensitivity only to one subtype of human IFN, IFN-alpha B. Other genes on human chromosome 21 were required for responses to other subtypes of type I IFN. In contrast, IFNAR expression in hamster cells did not confer sensitivity to any human IFN tested, including IFN-alpha B. Using human-hamster somatic cell hybrids, we mapped the Ifnabr gene, encoding a ligand-binding subunit of the IFN-alpha/beta (type I) receptor, to human chromosome 21. Ifnabr colocalized with Ifnar to the distal region of q22.1. The presence of a chromosomal fragment encoding IFNABR and IFNAR was also not sufficient to confer sensitivity to human IFN. In contrast, hybrids carrying in addition the region 21q22.2 showed a full response to human IFN-alpha B, suggesting that a gene located in this region encodes a third factor required for type I IFN receptor activity
— id: 12801, year: 1995, vol: 21, page: 139, stat: Journal Article,

Molecular interactions between interferon consensus sequence binding protein and members of the interferon regulatory factor family
Bovolenta, C; Driggers, P H; Marks, M S; Medin, J A; Politis, A D; Vogel, S N; Levy, D E; Sakaguchi, K; Appella, E; Coligan, J E
1994 May 24;91(11):5046-5050, Proceedings of the National Academy of Sciences of the United States of America
Interferon (IFN) consensus sequence binding protein (ICSBP) is a transcription factor expressed mostly in the cells of the immune system. ICSBP belongs to the IFN regulatory factor (IRF) family, which also includes IRF-1, IRF-2, and the IFN-alpha-stimulated gene factor 3 gamma (ISGF3 gamma). We show here that ICSBP forms a complex with IRF-1 or IRF-2 both in vivo and in vitro and, in the presence or absence of the target DNA, with the IFN-stimulated response element (ISRE). Further, electrophoretic mobility shift assays show that this interaction greatly enhances the otherwise very low binding affinity of ICSBP to the ISRE. We show, on the other hand, that ICSBP inhibits binding of the IFN-alpha-stimulated gene factor 3 gamma to the ISRE. Through these interactions ICSBP is likely to exert complex modulatory functions in the regulation of IFN-stimulated genes
— id: 138971, year: 1994, vol: 91, page: 5046, stat: Journal Article,

Rapid activation of proteins that interact with the interferon gamma activation site in response to multiple cytokines
Lamb, P; Kessler, L V; Suto, C; Levy, D E; Seidel, H M; Stein, R B; Rosen, J
1994 Apr 15;83(8):2063-2071, Blood
Many cytokines and growth factors trigger rapid changes in gene expression upon binding to their receptors. In many cases, the mechanism by which these changes are affected is unknown. In this report, we show that interleukin-2 (IL-2), IL-3, IL-4, IL-6, leukemia inhibitory factor (LIF), erythropoietin (Epo), and granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment of cells causes rapid activation of DNA-binding activities that recognize a DNA sequence element previously implicated in regulation of gene expression by interferon gamma (IFN gamma). The IL-4-, IL-6-, and GM-CSF-induced complexes can be distinguished from the recently characterized IFN gamma-activated protein p91 on the basis of mobility in polyacrylamide gels, sequence preferences, and lack of reactivity with an anti-p91 antiserum. The IL-4- and GM-CSF-induced complexes react with antiphosphotyrosine antibodies, demonstrating the presence of phosphotyrosine-containing proteins in these DNA-binding complexes. Transcriptional activation of a reporter gene linked to a synthetic IFN gamma-responsive promoter is observed in response to IFN gamma, IL-6, and LIF. These data suggest a pathway by which cytokines induce rapid changes in gene expression
— id: 138963, year: 1994, vol: 83, page: 2063, stat: Journal Article,

Acute phase response factor and additional members of the interferon-stimulated gene factor 3 family integrate diverse signals from cytokines, interferons, and growth factors
Raz R; Durbin JE; Levy DE
1994 Sep 30;269(39):24391-24395, Journal of biological chemistry
Cytokines and growth factors elicit responses in target cells through induction of gene expression. Signaling mechanisms leading to gene transcription from cell surface receptors often require tyrosine phosphorylation. A family of transcription factors comprising the interferon (IFN)-stimulated gene factor 3 (ISGF3) multimeric complex are phosphorylated and activated in response to interferon. We describe a protein 50% identical to the 91-kDa subunit of ISGF3 that constitutes the acute phase response factor (APRF). This protein was rapidly activated by interleukin-6 to bind an enhancer element common to genes activated in liver cells during the acute phase response to inflammation. Remarkably, APRF was also activated by IFN alpha, IFN gamma, epidermal growth factor, platelet-derived growth factor, colony stimulating factor-1, and the cytokines leukemia inhibitory factor and oncostatin M. The growth factors also activated a third, distinct but related, DNA-binding protein in addition to APRF and p91. This novel factor or a closely related one, but neither APRF nor p91, was also activated in lymphoid cells by interleukin-2, erythropoietin, and interleukin-3. Activation of APRF, p91, and additional members of the ISGF3 family is thus a general feature of a wide variety of signaling pathways, integrating diverse signals through common transcriptional regulators
— id: 12890, year: 1994, vol: 269, page: 24391, stat: Journal Article,

Cytokines and growth factors signal through tyrosine phosphorylation of a family of related transcription factors
Rothman P; Kreider B; Azam M; Levy D; Wegenka U; Eilers A; Decker T; Horn F; Kashleva H; Ihle J; et al.
1994 Sep;1(6):457-468, Immunity
The ability of cytokines to activate distinct but overlapping sets of genes defines their characteristic biological response. We now show that IFN gamma, IL-3, IL-4, IL-6, erythropoietin, EGF, and CSF-1 activate differing members of a family of latent cytoplasmic transcription factors. Although these factors have distinct physical and functional properties and exhibit different patterns of expression, they share many important features, including recognition of a related set of enhancer elements, rapid activation, tyrosine phosphorylation, and cross-reactivity to antibodies against p91, a cytoplasmic signaling protein activated by IFN alpha, IFN gamma, and IL-6. These shared features point to either parallel or common patterns of signal transduction. A general model of cytokine signal transduction is presented, in which receptor-associated tyrosine kinases activate ligand-specific members of a family of signal-transducing factors. Once activated, these factors carry their signals to the nucleus, where they bind a family of related enhancer elements
— id: 18492, year: 1994, vol: 1, page: 457, stat: Journal Article,

A novel interferon-alpha-regulated, DNA-binding protein participates in the regulation of the IFP53/tryptophanyl-tRNA synthetase gene
Seegert, D; Strehlow, I; Klose, B; Levy, D E; Schindler, C; Decker, T
1994 Mar 18;269(11):8590-8595, Journal of biological chemistry
We have investigated the transcriptional response of the IFP53/tryptophanyl-tRNA synthetase gene to interferon-alpha (IFN-alpha). A single gamma-interferon activation site (GAS) in proximity to the transcription start sites was found to mediate the response of the IFP53 gene to IFN-alpha. This DNA element bound two distinct protein factors, alpha-interferon activation factor 1 (AAF1) and AAF2, which were rapidly activated in the cytoplasm of IFN-alpha-treated HeLa cells. AAF1, like the gamma-interferon activation factor, bound to the GAS from different IFN-responsive promoters and contained the 91-kDa ISGF3 protein (p91). However, in complexes with the IFP53 or Ly6A/E GAS, p91 was the only ISGF3 protein, whereas in the case of the GBP GAS, the 48-kDa protein (p48) was also present. AAF2 was found to preferentially bind to the IFP53 GAS, but not at all to the GBP GAS, and contained no ISGF3 protein. Therefore, GAS-binding regulatory factors in the IFN-alpha response can either consist of proteins found in ISGF3 or be formed by distinct proteins that are similarly linked to IFN-alpha-induced signal transduction
— id: 138962, year: 1994, vol: 269, page: 8590, stat: Journal Article,

The interferon-stimulable response elements of two human genes detect overlapping sets of transcription factors
Parrington, J; Rogers, N C; Gewert, D R; Pine, R; Veals, S A; Levy, D E; Stark, G R; Kerr, I M
1993 Jun 15;214(3):617-626, European journal of biochemistry
We have previously reported three types of DNA-protein complexes, formed specifically with the interferon-stimulable response elements (ISRE) in the 5' flanking DNA of the interferon-inducible 6-16 and 9-27 genes, a type-I interferon-inducible early complex involving factor E (ISGF3), M and G complexes induced more slowly in response to type-I and type-II interferons, respectively and C1/C2, a constitutive complex(s). Similar complexes have been reported by others. The operationally defined band-shift complexes M, G and C1/C2 are shown here to be heterogeneous and to differ in their factor content, depending on the ISRE probe. With a 9-27 ISRE probe the M, G and C1/C2 complexes all contain the gamma subunit of ISGF3, which is present constitutively but is induced in response to IFN-alpha (to yield M) or IFN-gamma (to yield G). In contrast, a 6-16 ISRE probe forms band-shift complexes with IFN-alpha-inducible and IFN-gamma-inducible IRF1 and IRF2. With a 6-16 ISRE probe, therefore, M and G each correspond to two complexes which co-migrate in band-shift assays, one corresponding to IRF1, the other to IRF2. With this probe, the constitutive complex C1/C2 corresponds predominantly to IRF2. Consistent with this, IRF1 and IRF2 have lower affinity for the 9-27 ISRE than the 6-16 ISRE, whereas the reverse is true for E (ISGF3) and its gamma subunit. Relatively small differences in affinity appear sufficient to determine whether or not a band-shift complex is detected. In the case of IRF1 and IRF2, the different affinities for the 6-16 and 9-27 probes are dominated by a dinucleotide sequence in the centre of the 14-nucleotide 'core' ISRE. In contrast, preferential binding of E (ISGF3) by the 39-nucleotide 9-27 ISRE-containing sequence, although ISRE dependent, appears to be mediated by sequences 3' of the 'core' ISRE. Accordingly, these complexes can be simultaneously assayed using a hybrid probe consisting of the 5' flanking region and 'core' ISRE sequences from the 6-16 gene and sequences immediately 3' of the 'core' 9-27 ISRE sequence. No evidence was obtained for a modulatory role in factor binding for a pseudo-ISRE sequence close to ISRE in the 9-27 gene. The precise roles of IRF1 and IRF2 in the induction of IFN-beta and the control of interferon-inducible gene expression remain to be established.(ABSTRACT TRUNCATED AT 400 WORDS)
— id: 138959, year: 1993, vol: 214, page: 617, stat: Journal Article,

Interferon-induced nuclear signalling by Jak protein tyrosine kinases
Silvennoinen O; Ihle JN; Schlessinger J; Levy DE
1993 Dec 9;366(6455):583-585, Nature
Interferons IFN-alpha/beta and IFN-gamma act through independent cell-surface receptors, inducing gene expression through tyrosine phosphorylation of cytoplasmic transcription factors . IFN-alpha stimulates phosphorylation and nuclear localization of the 84/91K and 113K subunits of latent ISGF3 (interferon-stimulated gene factor 3), which combine with the 48K DNA-binding subunit to bind regulatory elements of IFN-alpha-responsive genes. IFN-gamma activates p91 alone, inducing IFN-gamma-responsive genes through a distinct DNA element. Genetic complementation studies implicated the tyrosine kinase Tyk2 in IFN-alpha signalling and, more recently, the related Jak2 kinase in IFN-gamma signalling. We now present biochemical evidence for Jak-family kinase involvement in IFN signal transduction. Jak1 was activated in response to IFN-alpha and IFN-gamma; Jak2 responded exclusively to IFN-gamma. Overexpression of either Jak1 or Jak2 stimulated p91 DNA-binding activity and p91-dependent transcription. Overexpression also activated endogenous Jak kinases, suggesting that interactions between Jak kinases are required during interferon signalling
— id: 18493, year: 1993, vol: 366, page: 583, stat: Journal Article,

Ras-independent growth factor signaling by transcription factor tyrosine phosphorylation [see comments]
Silvennoinen O; Schindler C; Schlessinger J; Levy DE
1993 Sep 24;261(5129):1736-1739, Science
Interferons induce transcriptional activation through tyrosine phosphorylation of the latent, cytoplasmic transcription factor interferon-stimulated gene factor-3 (ISGF-3). Growth factors and cytokines were found to use a similar pathway: The 91-kilodalton subunit of ISGF-3 was activated and tyrosine phosphorylated in response to epidermal growth factor (EGF), platelet-derived growth factor, and colony stimulating factor-1. The tyrosine phosphorylated factor acquired DNA binding activity and accumulated in nuclei. Activation required the major sites for autophosphorylation on the EGF receptor that bind Src homology region 2 domain-containing proteins implicated in Ras activation. However, activation of this factor was independent of the normal functioning of Ras
— id: 13070, year: 1993, vol: 261, page: 1736, stat: Journal Article,

Two domains of ISGF3 gamma that mediate protein-DNA and protein-protein interactions during transcription factor assembly contribute to DNA-binding specificity
Veals SA; Santa Maria T; Levy DE
1993 Jan;13(1):196-206, Molecular & cellular biology
Alpha interferon (IFN-alpha) induces the transcription of a large set of genes through activation of multimeric transcription factor ISGF3. This factor can be dissociated into two protein components, termed ISGF3 gamma and ISGF3 alpha. ISGF3 gamma is a 48-kDa protein related at the amino terminus to members of the IFN-regulatory factor (IRF) and Myb families of DNA-binding proteins; ISGF3 alpha consists of three polypeptides of 84, 91, and 113 kDa that self-assemble to form an activated component in response to IFN-alpha. DNA-binding studies indicated that ISGF3 gamma binds DNA alone, recognizing the IFN-stimulated response element, while the ISGF3 alpha polypeptides alone display no specific interactions with DNA. A complex between ISGF3 gamma and activated ISGF3 alpha binds the IFN-stimulated response element with much greater affinity than does the 48-kDa ISGF3 gamma protein alone. The DNA-binding domain of ISGF3 gamma and regions responsible for protein-protein interaction with ISGF3 alpha were identified by using deleted forms of ISGF3 gamma expressed in vitro. The amino-terminal region of ISGF3 gamma homologous to the IRF and Myb proteins was sufficient for interaction with DNA and displayed the binding specificity of the intact protein; phosphorylation of this region was necessary for activity. A second region of 160 amino acids separated from the DNA-binding domain by over 100 amino acids contained a domain capable of associating with ISGF3 alpha and was sufficient to confer specific ISGF3 alpha interaction to a heterologous protein. Interaction of the ISGF3 alpha component with the protein interaction domain of ISGF3 gamma altered the DNA-binding specificity of the resulting complex, suggesting that one or more of the ISGF3 alpha polypeptides make base-specific contacts with DNA. This interaction defines a mechanism through which IRF-like proteins complexed with regulatory components can display novel DNA-binding specificities
— id: 13309, year: 1993, vol: 13, page: 196, stat: Journal Article,

Subunit of an alpha-interferon-responsive transcription factor is related to interferon regulatory factor and Myb families of DNA-binding proteins
Veals SA; Schindler C; Leonard D; Fu XY; Aebersold R; Darnell JE Jr; Levy DE
1992 Aug;12(8):3315-3324, Molecular & cellular biology
Alpha interferon stimulates transcription by converting the positive transcriptional regulator ISGF3 from a latent to an active form. This receptor-mediated event occurs in the cytoplasm, with subsequent translocation of the activated factor to the nucleus. ISGF3 has two components, termed ISGF3 alpha and ISGF3 gamma. ISGF3 gamma serves as the DNA recognition subunit, while ISGF3 alpha, which appears to consist of three polypeptides, is a target for alpha interferon signaling and serves as a regulatory component whose activation is required to form ISGF3. ISGF3 gamma DNA-binding activity was identified as a 48-kDa polypeptide, and partial amino acid sequence has allowed isolation of cDNA clones. ISGF3 gamma translated in vitro from recombinant clones bound DNA with a specificity indistinguishable from that of ISGF3 gamma purified from HeLa cells. Sequencing of ISGF3 gamma cDNA clones revealed significant similarity to the interferon regulatory factor (IRF) family of DNA binding proteins in the amino-terminal 117 residues of ISGF3 gamma. The other IRF family proteins bind DNA with a specificity related to but distinct from that of ISGF3 gamma. We note sequence similarities between the related regions of IRF family proteins and the imperfect tryptophan repeats which constitute the DNA-binding domain of the c-myb oncoprotein. These sequence similarities suggest that ISGF3 gamma and IRF proteins and the c-myb oncoprotein use a common structural motif for DNA recognition. Recombinant ISGF3 gamma, like the natural protein, interacted with HeLa cell ISGF3 alpha to form the mature ISGF3 DNA-binding complex.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 13485, year: 1992, vol: 12, page: 3315, stat: Journal Article,

Protein kinase activity required for an early step in interferon-alpha signaling
Kessler, D S; Levy, D E
1991 Dec 5;266(34):23471-23476, Journal of biological chemistry
Interferon-alpha (IFN alpha) induces an immediate transcriptional response of a restricted set of genes in target cells. Specific transcription is mediated by the cytoplasmic activation of a transcription factor complex termed ISGF3. ISGF3 is a multimeric protein complex composed of a regulatory component (ISGF3 alpha), which is activated following IFN alpha treatment, and a DNA-binding component (ISGF3 gamma), which recognizes the IFN alpha-stimulated response element (ISRE). Following activation, ISGF3 alpha translocates to the nucleus where ISGF3 assembles as a high affinity complex on the ISRE. The biochemical basis for receptor-mediated activation of ISGF3 is unknown. We report that two potent protein kinase inhibitors, staurosporine and K-252a, ablated the transcriptional response to IFN alpha treatment. These inhibitors prevented the activation of the ISGF3 alpha component without affecting the ISGF3 gamma component, resulting in no accumulation of mature ISGF3 in nuclei of treated cells. Although these agents are potent inhibitors of protein kinase C (PKC), PKC does not mediate ISGF3 alpha activation. Down-regulation of PKC by chronic exposure of cells to 12-O-tetradecanoylphorbol-13-acetate, which led to complete loss of PKC-immunoreactive material, failed to ablate the transcriptional response to IFN alpha or the activation of ISGF3 alpha. The PKC-specific inhibitor calphostin C did not perturb activation or nuclear accumulation of ISGF3. We conclude that a novel, staurosporine/K-252a-sensitive kinase is required for ISGF3 activity and may participate in receptor-mediated signal transduction
— id: 138968, year: 1991, vol: 266, page: 23471, stat: Journal Article,

ISGF3, the transcriptional activator induced by interferon alpha, consists of multiple interacting polypeptide chains
Fu XY; Kessler DS; Veals SA; Levy DE; Darnell JE Jr
1990 Nov;87(21):8555-8559, Proceedings of the National Academy of Sciences of the United States of America
Interferon-stimulated gene factor 3 (ISGF3) is the ligand-dependent transcriptional activator that, in response to interferon treatment, is assembled in the cell cytoplasm, is translocated to the nucleus, and binds the consensus DNA site, the interferon-stimulated response element. We have purified ISGF3 and identified its constituent proteins: a DNA-binding protein of 48 kDa and three larger polypeptides (84, 91, and 113 kDa), which themselves do not have DNA-binding activity. The multisubunit structure of ISGF3 most likely reflects its participation in receiving a ligand-dependent signal, translocating to the nucleus, and binding to DNA to activate transcription
— id: 63335, year: 1990, vol: 87, page: 8555, stat: Journal Article,

Interferon-alpha regulates nuclear translocation and DNA-binding affinity of ISGF3, a multimeric transcriptional activator
Kessler, D S; Veals, S A; Fu, X Y; Levy, D E
1990 Oct;4(10):1753-1765, Genes & development
The interaction of interferon-alpha (IFN-alpha) with a specific cell-surface receptor elicits physiological changes that rely on rapid transcriptional activation of a group of IFN-alpha-stimulated genes (ISGs). The IFN-stimulated response element (ISRE), a conserved regulatory element of all ISGs, is the target for transcriptional activation by the positive regulator IFN-stimulated gene factor-3 (ISGF3). We reported previously that post-translational activation of ISGF3 in the cytoplasm of IFN-alpha-treated cells requires two cytoplasmic activities (ISGF3 alpha and ISGF3 gamma) to produce an ISRE-binding complex that accumulates in the nucleus. In this study, we show that these activities are actually distinct subunits of the ISGF3 complex, which associate through noncovalent interaction. Sedimentation analysis, protein renaturation, and photoaffinity cross-linking of enriched preparations of cytoplasmic ISGF3 alpha and ISGF3 gamma and of nuclear ISGF3 demonstrated that ISGF3 gamma was a 48-kD polypeptide with intrinsic, low-affinity DNA-binding activity. Four polypeptides of 48, 84, 91, and 113 kD bound to the ISRE in vitro; the larger three polypeptides most likely compose the ISGF3 alpha component. These ISGF3 alpha polypeptides were unable to bind DNA alone but formed a DNA-binding complex in conjunction with ISGF3 gamma. The resulting heteromeric complex had the same ISRE-binding specificity as the individual ISGF3 gamma polypeptide but approximately 25-fold higher affinity. Whereas ISGF3 gamma partitioned between the cytoplasm and nucleus in unstimulated cells, ISGF3 alpha was stimulated to translocate to the nucleus only following IFN-alpha treatment, resulting in preferential nuclear accumulation of both ISGF3 alpha and ISGF3 gamma as a stable ISGF3-ISRE complex. This regulated nuclear translocation of an activated transcription factor subunit maintained the specificity and rapidity of the IFN-alpha signaling pathway
— id: 138969, year: 1990, vol: 4, page: 1753, stat: Journal Article,

Synergistic interaction between interferon-alpha and interferon-gamma through induced synthesis of one subunit of the transcription factor ISGF3
Levy, D E; Lew, D J; Decker, T; Kessler, D S; Darnell, J E Jr
1990 Apr;9(4):1105-1111, EMBO journal
Interferon-alpha (IFN alpha) and interferon-gamma (IFN gamma) each induce in susceptible target cells a state of resistance to viral replication and reduced cellular proliferation, presumably through different mechanisms: these two polypeptides are unrelated by primary sequence and act through distinct cell-surface receptors to induce expression of largely non-overlapping sets of genes. However, acting in concert, they can produce synergistic interactions leading to mutual reinforcement of the physiological response. In HeLa cells, this synergistic response was initiated by cooperative induction of IFN alpha stimulated genes (ISGs). These normally quiescent genes were rapidly induced to high rates of transcription following exposure of cells to IFN alpha. Although they were only negligibly responsive to IFN gamma, combined treatment of cells with IFN gamma followed by IFN alpha resulted in an approximately 10-fold increase in ISG transcription. ISG transcription is dependent upon ISGF3, a positive transcription factor specific for a cis-acting regulatory element in ISG promoters. IFN gamma treatment induced increased synthesis of latent ISGF3, which was subsequently activated in response to IFN alpha to form approximately 10-fold higher levels than detected in cells treated with IFN alpha alone. ISGF3 is composed of two distinct polypeptide components, synthesis of one of which was induced by IFN gamma, increasing its cellular abundance from limiting concentrations to a level which allowed formation of at least 10 times as much active ISGF3. Cell lines vary in their constitutive levels of the inducible component of ISGF3 and in the ability of IFNs to increase its synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 138946, year: 1990, vol: 9, page: 1105, stat: Journal Article,

Purification and cloning of interferon-stimulated gene factor 2 (ISGF2): ISGF2 (IRF-1) can bind to the promoters of both beta interferon- and interferon-stimulated genes but is not a primary transcriptional activator of either
Pine, R; Decker, T; Kessler, D S; Levy, D E; Darnell, J E Jr
1990 Jun;10(6):2448-2457, Molecular & cellular biology
Interferon-stimulated gene factor 2 (ISGF2) was purified from HeLa cells treated with alpha interferon. The factor, a single polypeptide of 56 kilodaltons (kDa), bound both to the central 9 base pairs of the 15-base-pair interferon-stimulated response element (ISRE) that is required for transcriptional activation of interferon-stimulated genes and to the PRD-I regulatory element of the beta interferon gene. ISGF2 was a phosphoprotein, and dephosphorylation in vitro reduced its DNA-binding activity. However, conditions that changed the amount of ISGF2 did not change the phosphorylated isoforms in vivo. ISGF2 in unstimulated cells existed in trace amounts and was induced by both alpha interferon and gamma interferon as well as by virus infection. Plasmid-bearing Escherichia coli clones encoding ISGF2 were selected with antibody against purified ISGF2. Sequence analysis revealed that the ISGF2 protein was the same as that encoded by the cDNA clone IRF-1, which has been claimed to activate transcription of interferon genes. We show that transcription of the ISGF2 gene was induced by alpha interferon, gamma interferon, and double-stranded RNA. However, ISGF2 was neither necessary nor sufficient for induced transcription of the beta interferon gene, while the factor NF kappa B was clearly involved
— id: 138947, year: 1990, vol: 10, page: 2448, stat: Journal Article,

Interactions of alpha- and gamma-interferon in the transcriptional regulation of the gene encoding a guanylate-binding protein
Decker, T; Lew, D J; Cheng, Y S; Levy, D E; Darnell, J E Jr
1989 Jul;8(7):2009-2014, EMBO journal
Transcriptional regulation of the gene encoding a guanylate-binding protein (GBP) by the two interferon (IFN) types was studied. GBP gene transcription was regulated by alpha IFN in a manner identical to that of previously described IFN-stimulated genes (ISGs): rapid induction, without a need for protein synthesis, followed by a protein synthesis-dependent suppression of transcription to basal levels within 6 h. Transcriptional induction by gamma IFN was equally rapid and independent of ongoing protein synthesis but remained at elevated levels for greater than 24 h. Experiments employing combined treatments with IFNs of both types revealed that induction of the GBP gene by gamma IFN overrides the alpha IFN-induced active repression and reverses the alpha IFN-induced repressed state. Moreover, the alpha IFN-mediated repression of ISG54, a gene normally responsive to only alpha IFN, is also reversed by gamma IFN. Induction of GBP by gamma IFN is presumably mediated by a factor different from the recently described activator Interferon Stimulated Gene Factor 3 (ISGF3) because induction of this factor was not observed upon treatment of cells with gamma IFN. Finally, a complex set of reinforcing or synergistic effects were observed when induction of the GBP gene was evoked by a combined treatment with the two IFN types
— id: 138950, year: 1989, vol: 8, page: 2009, stat: Journal Article,

Cytoplasmic activation of ISGF3, the positive regulator of interferon-alpha-stimulated transcription, reconstituted in vitro
Levy DE; Kessler DS; Pine R; Darnell JE Jr
1989 Sep;3(9):1362-1371, Genes & development
The signal transduction pathway through which interferon-alpha (IFN alpha) stimulates transcription of a defined set of genes involves activation of DNA-binding factors specific for the IFN alpha-stimulated response element (ISRE). IFN-stimulated gene factor-3 (ISGF3), the positive regulator of transcription, was derived in response to IFN alpha treatment from preexisting protein components that were activated first in the cell cytoplasm prior to appearance in the nucleus. Nuclear translocation of ISGF3 required several minutes and could be inhibited by NaF. Formation of active ISGF3 was mimicked in vitro by mixing cytoplasmic extracts from IFN alpha-stimulated cells with extracts of cells treated to contain high amounts of the unactivated factor. Active ISGF3 was found to be formed from association of two latent polypeptide precursors that were distinguished biochemically by differential sensitivity to N-ethyl maleimide. One precursor was modified in response to IFN alpha occupation of its cell-surface receptor, thus enabling association with the second subunit. The resulting complex then was competent for nuclear translocation and binding to ISRE. Cytoplasmically localized transcription factor precursors thus serve as second messengers to translate directly an extracellular signal into specific transcriptional activity in the nucleus
— id: 10512, year: 1989, vol: 3, page: 1362, stat: Journal Article,

Virus infection and interferon can activate gene expression through a single synthetic element, but endogenous genes show distinct regulation
Raj, N B; Engelhardt, J; Au, W C; Levy, D E; Pitha, P M
1989 Oct 5;264(28):16658-16666, Journal of biological chemistry
Virus inducible elements (IE) in promoters of mouse alpha-interferon and human beta 1-interferon genes contain multiple copies of the hexanucleotide sequence AGT-GAA or its variants which are also found in the interferon-stimulated response element of genes transcriptionally induced by interferon. We have examined the similarities between virus and interferon induction of gene expression and the role of AGTGAA and AAT-GAA hexamers in these responses. Hybrid plasmids were constructed by inserting the IE region, the alpha 4 promoter, or the multiple copies of AGTGAA or AAT-GAA 5' to the inactive-45 human immunodeficiency-chloramphenicol acetyltransferase hybrid gene, and their inducible expression was studied in a transient expression assay. In L-cells, multiple hexamers were efficiently induced both by infection with Newcastle disease virus and by interferon treatment; while the alpha 4 promoter and the IE inducible region were induced predominantly by virus rather than by interferon. In order to dissociate the effect of virus and endogenous interferon on the induction process, we examined the gene expression in Vero cells, which have undergone homozygous deletion of type 1 interferon genes, and in VNPT-159 cells, which were derived from Vero cells by insertion of an inducible human interferon beta 1 gene. The results show that while the alpha 4 promoter was efficiently induced only by virus in both cell types, the constructs containing shorter segments of the IE were induced by both virus and interferon in Vero cells. However, the inducibility by interferon was not detected in VNPT-159 cells, suggesting that the presence of endogenous interferon suppresses interferon-induced expression of hexanucleotide repeats and the short inducible region. In contrast, virus inducibility of endogenous interferon-stimulated genes, ISG-15 and ISG-54, was about 100-fold more efficient in VNPT-159 cells than in Vero cells, suggesting that this induction is largely mediated through synthesis of endogenous interferon. Hence, endogenous interferon may play a role in the autoregulation of both interferon genes and interferon-stimulated genes
— id: 138951, year: 1989, vol: 264, page: 16658, stat: Journal Article,

Two interferon-induced nuclear factors bind a single promoter element in interferon-stimulated genes
Kessler, D S; Levy, D E; Darnell, J E Jr
1988 Nov;85(22):8521-8525, Proceedings of the National Academy of Sciences of the United States of America
Nuclear proteins induced by interferon (IFN) treatment of human cells are capable of forming two specific complexes with DNA fragments containing the IFN-stimulated response element (ISRE). These two complexes, designated B2 and B3, are distinguished by differential migration in gel retardation assays. The factor that forms the B3 complex, termed IFN-stimulated gene factor 3 (ISGF-3), preexists in cells, is activated upon IFN treatment, and appears with kinetics paralleling those for transcriptional activation of IFN-stimulated genes. The factor that forms the B2 complex (ISGF-2) appears following a time lag after IFN treatment during which protein synthesis must occur. By extensive point mutagenesis of the ISREs from two IFN-stimulated promoters (ISG54 and ISG15), we demonstrate that the B2 and B3 complexes are formed by factors binding to the same DNA sequence. Mutations at this site decrease or eliminate transcriptional activation and impair binding of both ISGF-2 and ISGF-3. This analysis has shown that the ISGF-3 binding site is slightly broader than the ISGF-2 binding site, which is completely contained within the sequence necessary both for ISGF-3 binding and for transcriptional activation. The evidence strongly implicates ISGF-3 as the positive transcriptional regulator of IFN-stimulated genes
— id: 138949, year: 1988, vol: 85, page: 8521, stat: Journal Article,

Cells resistant to interferon are defective in activation of a promoter-binding factor
Kessler, D S; Pine, R; Pfeffer, L M; Levy, D E; Darnell, J E Jr
1988 Dec 1;7(12):3779-3783, EMBO journal
Human cultured cell lines deficient in their ability to respond to type I interferon (IFN) fail to interrupt cellular proliferation or to induce an antiviral state following exposure to IFN alpha. Comparison of non-responsive Daudi and HeLa cell lines with IFN-responsive partner cell lines and examination of non-responsive Raji cells showed that the defective cell lines expressed type I IFN receptors of typical number and affinity and bound IFN equivalently compared to the normal cells. However, transcriptional induction of interferon-stimulated genes (ISGs) was greatly reduced and delayed in these cell lines, leading to reduced accumulation of ISG mRNA. Furthermore, the rapid activation of IFN-stimulated promoter binding factors whose appearance correlates with ISG transcriptional induction, did not occur in non-responsive cells. Thus, the primary defect of these cells leading to an impaired physiological response to IFN appears to be an inability to activate promoter-binding factors necessary to trigger ISG transcription, an obligate early step in antiviral and antiproliferative physiology
— id: 138952, year: 1988, vol: 7, page: 3779, stat: Journal Article,

Interferon-induced nuclear factors that bind a shared promoter element correlate with positive and negative transcriptional control
Levy, D E; Kessler, D S; Pine, R; Reich, N; Darnell, J E Jr
1988 Apr;2(4):383-393, Genes & development
Human alpha- and beta-interferons (IFNs) stimulate rapid but transient increases in transcription from a set of previously quiescent genes. Protein synthesis is not required for initial stimulation, but duration of the response is limited to a few hours by a process requiring synthesis of new proteins. An IFN-stimulated response element (ISRE) was identified 5' to an inducible gene by deletion analysis and point mutagenesis, and sequence comparisons with other promoters defined the consensus element YAGTTTC(A/T)YTTTYCC. Two classes of IFN-inducible nuclear factors were found that bind to the ISRE. The most rapidly induced factor appeared without new protein synthesis, whereas a second factor required active protein synthesis for its appearance and maintenance. The kinetics of appearance and loss of these binding activities correlate with the activation and repression of IFN-stimulated genes. These different IFN-activated or induced factors may bind sequentially to the same essential promoter element to first increase and then repress transcription
— id: 138955, year: 1988, vol: 2, page: 383, stat: Journal Article,

Transcriptional regulation of interferon-stimulated genes: a DNA response element and induced proteins that recognize it
Levy, D; Reich, N; Kessler, D; Pine, R; Darnell, J E Jr
1988 ;53 Pt 2:799-802, Cold Spring Harbor symposia on quantitative biology
— id: 138954, year: 1988, vol: 53 Pt 2, page: 799, stat: Journal Article,

Transcriptional stimulation by CaPO4-DNA precipitates
Pine, R; Levy, D E; Reich, N; Darnell, J E Jr
1988 Feb 25;16(4):1371-1378, Nucleic acids research
Genes in human chromosomes that normally require induction by alpha-interferon are activated after calcium phosphate (CaPO4) transfection, but not after DEAE-dextran transfection. The c-fos gene and genes stimulated by gamma-interferon also are affected by CaPO4-DNA precipitates, but the calcium ionophore A23187 stimulates only c-fos among this group. These results suggest caution not only in choosing gene transfer methods, but also in interpreting experiments aimed at understanding the role of second messengers in gene activation
— id: 138953, year: 1988, vol: 16, page: 1371, stat: Journal Article,

Transcription of interferon-stimulated genes is induced by adenovirus particles but is suppressed by E1A gene products
Reich, N; Pine, R; Levy, D; Darnell, J E Jr
1988 Jan;62(1):114-119, Journal of virology
Interferon treatment of cell cultures results in the rapid transcriptional induction of a specific set of genes. In this paper we explore the effect of cellular infection by several adenoviruses, both wild type and mutant, on the expression of these genes. Infection with adenovirus induces the transcription of the interferon-stimulated genes in the absence of any protein synthesis. In fact, the inhibition of protein synthesis during a wild-type infection produces enhanced stimulation of transcription of these genes. Experiments with viral mutants indicate the ability to specifically suppress this transcription maps to the E1A gene. In addition, the E1A gene products are capable of suppressing the specific transcriptional induction of interferon-stimulated promoters during cotransfection experiments and therefore presumably during viral infection. The dual effect of adenovirus on the expression of interferon-stimulated genes may represent an example of action and evolutionary reaction between virus and host
— id: 138948, year: 1988, vol: 62, page: 114, stat: Journal Article,

Differences in cerebral blood flow and glucose utilization in vegetative versus locked-in patients
Levy DE; Sidtis JJ; Rottenberg DA; Jarden JO; Strother SC; Dhawan V; Ginos JZ; Tramo MJ; Evans AC; Plum F
1987 Dec;22(6):673-682, Annals of neurology
Positron emission tomographic studies of regional cerebral metabolic rate for glucose (rCMRGlc) and cerebral blood flow were performed in 7 vegetative and 3 locked-in patients to determine objectively the level of brain function underlying these clinical states. Cortical gray rCMRGlc in the vegetative patients was 2.73 +/- 0.13 (mean +/- SEM) mg/100 gm/min, less than half the normal value of 6.82 +/- 0.23 (p less than 0.001). Cerebral blood flow exhibited similar but more variable reductions. By contrast, cortical rCMRGlc in the locked-in patients was 5.08 +/- 0.69, a 25% reduction (p less than 0.02) from normal. The massive reduction in vegetative rCMRGlc involved not only the cerebral cortex but also the basal nuclei and cerebellum. Such metabolic hypoactivity has precedent only in deep anesthesia and supports clinical evidence that cerebral cognitive function is lost in the vegetative state, leaving a body that can no longer think or experience pain
— id: 60874, year: 1987, vol: 22, page: 673, stat: Journal Article,

Interferon-induced transcription of a gene encoding a 15-kDa protein depends on an upstream enhancer element
Reich, N; Evans, B; Levy, D; Fahey, D; Knight, E Jr; Darnell, J E Jr
1987 Sep;84(18):6394-6398, Proceedings of the National Academy of Sciences of the United States of America
A human gene encoding an interferon-induced 15-kDa protein has been isolated from a genomic library. The gene appears to be single-copy and is composed of two exons, the first of which contains the ATG translation initiation codon. In vitro nuclear run-on assays showed that the transcription rate of the gene is stimulated after interferon treatment. To analyze transcriptional regulatory sequences, we constructed recombinant plasmids for use in transient transfection assays of HeLa cells. Constructs containing 115 nucleotides 5' to the transcription initiation site were found to be fully inducible by interferon. Assays of deletion mutants identified a critical element for interferon induction located between -115 and -96, just upstream of the 'CCAAT box.' Moreover, a DNA fragment including this region can confer interferon inducibility on a heterologous promoter (thymidine kinase) when cloned in either orientation upstream of the gene or downstream of the gene. These are properties characteristic of an enhancer element that is active only after treatment with interferon. This regulatory sequence may be shared by a group of interferon-induced genes, since a very similar sequence is present within the functional region near the RNA start site of another interferon-induced gene
— id: 138957, year: 1987, vol: 84, page: 6394, stat: Journal Article,

Interferon-stimulated transcription: isolation of an inducible gene and identification of its regulatory region
Levy, D; Larner, A; Chaudhuri, A; Babiss, L E; Darnell, J E Jr
1986 Dec;83(23):8929-8933, Proceedings of the National Academy of Sciences of the United States of America
A human gene (termed ISG-54K) that is induced from near undetectable levels to high transcriptional activity by alpha- and beta-interferons has been cloned. The genomic structure and nucleotide sequence of the coding region were determined and the RNA initiation site was identified. The 5' portion of the gene was fused with a heterologous gene lacking an active promoter in recombinant plasmid and adenoviral vectors. These fusion genes were used to assess the activity of the ISG-54K promoter in response to interferon. RNA was formed in HeLa cells from recombinant plasmids only in response to interferon. Furthermore, in human diploid fibroblasts, infection with the recombinant adenovirus vector resulted in a 50-fold increase in specific RNA in response to interferon, followed by a subsequent decrease, imitating the natural regulated transcriptional cycle of the endogenous gene
— id: 138956, year: 1986, vol: 83, page: 8929, stat: Journal Article,

Outcome from severe neurological illness; should it influence medical decisions?
Plum, Fred; Levy, David E
1979 ;No. 69:267-277, CIBA Foundation symposium
— id: 93478, year: 1979, vol: No. 69, page: 267, stat: Journal Article,