Miroslav Blumenberg, Ph.D.

The p38 mitogen-activated protein kinase isoforms p38 beta and p38 delta are essential in vivo regulators of mouse skin carcinogenesis
Koppel, A. C.; Lin, C.; Burns, C.; Gribben, E.; Anders, J.; Cataisson, C.; Yuspa, S. H.; Kissling, G. E.; Blumenberg, M.; Arthur, J. C.; Efimova, T.
2011 APR ;131(5):S21-S21, Journal of investigative dermatology
— id: 131836, year: 2011, vol: 131, page: S21, stat: Journal Article,

Specific and shared targets of ephrin A signaling in epidermal keratinocytes
Walsh, Rebecca; Blumenberg, Miroslav
2011 Mar 18;286(11):9419-9428, Journal of biological chemistry
Both ephrins (EFNs) and their receptors (Ephs) are membrane-bound, restricting their interactions to the sites of direct cell-to-cell interfaces. They are widely expressed, often co-expressed, and regulate developmental processes, cell adhesion, motility, survival, proliferation, and differentiation. Both tumor suppressor and oncogene activities are ascribed to EFNs and Ephs in various contexts. A major conundrum regarding the EFN/Eph system concerns their large number and functional redundancy given the promiscuous cross-activation of ligands and receptors and the overlapping intracellular signaling pathways. To address this issue, we treated human epidermal keratinocytes with five EFNAs individually and defined the transcriptional responses in the cells. We found that a large set of genes is coregulated by all EFNAs. However, although the responses to EFNA3, EFNA4, and EFNA5 are identical, the responses to EFNA1 and EFNA2 are characteristic and distinctive. All EFNAs induce epidermal differentiation markers and suppress cell adhesion genes, especially integrins. Ontological analysis showed that all EFNAs induce cornification and keratin genes while suppressing wound healing-associated, signaling, receptor, and extracellular matrix-associated genes. Transcriptional targets of AP1 are selectively suppressed by EFNAs. EFNA1 and EFNA2, but not the EFNA3, EFNA4, EFNA5 cluster, regulate the members of the ubiquitin-associated proteolysis genes. EFNA1 specifically induces collagen production. Our results demonstrate that the EFN-Eph interactions in the epidermis, although promiscuous, are not redundant but specific. This suggests that different members of the EFN/Eph system have specific, clearly demarcated functions
— id: 138317, year: 2011, vol: 286, page: 9419, stat: Journal Article,

Comprehensive transcriptional profiling of human epidermis, reconstituted epidermal equivalents, and cultured keratinocytes using DNA microarray chips
Lee, Ding-Dar; Zavadil, Jiri; Tomic-Canic, Marjana; Blumenberg, Miroslav
2010 ;585:193-223, Methods in molecular biology
Because of its accessibility, skin has been among the first organs analyzed using DNA microarrays; psoriasis, melanomas, carcinomas, chronic wound biopsies, and epidermal keratinocytes in culture have been intensely investigated. Skin has everything: stem cells, differentiation, signaling, inflammation, diseases, cancer, etc. Here we provide step-by-step instructions for bioinformatics analysis of transcriptional profiling of skin. Specifically, we describe the use of GCOS and RMA programs for initial normalization and selection of differentially expressed genes, DAVID and LOLA programs for annotation of genes, and statistically relevant identification of over- and under-represented functional and biological categories in identified gene sets, L2L and Venn diagrams for comparing multiple lists of genes, and oPOSSUM for identification of statistically over-represented transcription factor binding sites in the promoter regions of gene sets. The work can be a primer for researchers embarking on skinomics, the comprehensive analysis of transcriptional changes in the skin
— id: 105252, year: 2010, vol: 585, page: 193, stat: Journal Article,

Attenuation of TGFbeta Signaling Pathway in Chronic Venous Ulcers
Pastar, Irena; Stojadinovic, Olivera; Krzyzanowska, Agata; Barrientos, Stephan; Stuelten, Christina; Zimmerman, Karen; Blumenberg, Miroslav; Brem, Harold; Tomic-Canic, Marjana
2010 Mar;16(3-4):92-101, Molecular medicine
TGFbeta is important in inflammation, angiogenesis, re-epithelialization and connective tissue regeneration during wound healing. We analyzed components of TGFbeta signaling pathway in biopsies from ten patients with non-healing venous ulcers (VUs). Using comparative genomics of transcriptional profiles of VUs and TGFbeta treated keratinocytes, we found deregulation of TGFbeta target genes in VUs. Using qPCR and immunohistochemistry, we found suppression of TGFbetaRI, RII and RIII and complete absence of phosphorylated Smad2 (pSmad2) in VUs epidermis. In contrast, pSmad2 was induced in the cells of migrating epithelial tongue of acute wounds. TGFbeta inducible transcription factors (GADD45beta, ATF3 and ZFP36L1) were suppressed in VUs. Likewise, genes suppressed by TGFbeta (FABP5, CSTA and S100A8) were induced in non-healing VUs. An inhibitor of Smad signaling, Smad7 was also down-regulated in VUs. We conclude that TGFbeta signaling is functionally blocked in VUs by down-regulation of TGFbeta receptors and attenuation of Smad signaling resulting in deregulation of TGFbeta target genes and consequent hyperproliferation. These data suggest that application of exogenous TGFbeta may not be beneficial treatment for VUs
— id: 107002, year: 2010, vol: 16, page: 92, stat: Journal Article,

Retinoid-responsive transcriptional changes in epidermal keratinocytes
Lee, Ding-Dar; Stojadinovic, Olivera; Krzyzanowska, Agata; Vouthounis, Constantinos; Blumenberg, Miroslav; Tomic-Canic, Marjana
2009 Aug;220(2):427-439, Journal of cellular physiology
Retinoids (RA) have been used as therapeutic agents for numerous skin diseases, from psoriasis to acne and wrinkles. While RA is known to inhibit keratinocyte differentiation, the molecular effects of RA in epidermis have not been comprehensively defined. To identify the transcriptional targets of RA in primary human epidermal keratinocytes, we compared the transcriptional profiles of cells grown in the presence or absence of all-trans retinoic acid for 1, 4, 24, 48, and 72 h, using large DNA microarrays. As expected, RA suppresses the protein markers of cornification; however the genes responsible for biosynthesis of epidermal lipids, long-chain fatty acids, cholesterol, and sphingolipids, are also suppressed. Importantly, the pathways of RA synthesis, esterification and metabolism are activated by RA; therefore, RA regulates its own bioavailability. Unexpectedly, RA regulates many genes associated with the cell cycle and programmed cell death. This led us to reveal novel effects of RA on keratinocyte proliferation and apoptosis. The response to RA is very fast: 315 genes were regulated already after 1 h. More than one-third of RA-regulated genes function in signal transduction and regulation of transcription. Using in silico analysis, we identified a set of over-represented transcription factor binding sites in the RA-regulated genes. Many psoriasis-related genes are regulated by RA, some induced, others suppressed. These results comprehensively document the transcriptional changes caused by RA in keratinocytes, add new insights into the molecular mechanism influenced by RA in the epidermis and demonstrate the hypothesis-generating power of DNA microarray analysis
— id: 99235, year: 2009, vol: 220, page: 427, stat: Journal Article,

MOLECULAR MECHANISM OF ATTENUATION OF TGF beta SIGNALING PATHWAY IN CHRONIC ULCERS
Pastar, I; Barrientos, S; Krzyzanowska, A; Stojadinovic, O; Zimmerman, K; Blumenberg, M; Vukelic, S; Brem, H; Tomic-Canic, M
2009 MAR-APR ;17(2):A48-A48, Wound repair & regeneration
— id: 97665, year: 2009, vol: 17, page: A48, stat: Journal Article,

Induction of gene encoding FABP4 in Pten-null keratinocytes
Tsuda, Masaaki; Inoue-Narita, Tae; Suzuki, Akira; Itami, Satoshi; Blumenberg, Miroslav; Manabe, Motomu
2009 Apr 17;583(8):1319-1322, FEBS letters
Keratinocyte-specific Pten-null mice revealed distinct phenotypes, including epidermal and sebaceous gland hyperplasia. To determine the candidate genes that contribute to their phenotypes, we analyzed a comprehensive gene expression of Pten-null keratinocytes using microarray technology. Consequently, it was demonstrated that the most induced gene was adipocyte-specific fatty acid-binding protein (FABP4). Collectively, it is conceivable that the FABP4 pathway mediates the sebaceous gland hyperplasia in keratinocyte-specific Pten-null mice
— id: 115708, year: 2009, vol: 583, page: 1319, stat: Journal Article,

Transcriptional profiling defines the roles of ERK and p38 kinases in epidermal keratinocytes
Gazel, Alix; Nijhawan, Rajiv I; Walsh, Rebecca; Blumenberg, Miroslav
2008 May;215(2):292-308, Journal of cellular physiology
Epidermal keratinocytes respond to extracellular influences by activating cytoplasmic signal transduction pathways that change gene expression. Using pathway-specific transcriptional profiling, we identified the genes regulated by two such pathways, p38 and ERK. These pathways are at the fulcrum of epidermal differentiation, proliferative and inflammatory skin diseases. We used SB203580 and PD98059 as specific inhibitors and Affymetrix Hu133Av2 microarrays, to identify the genes regulated after 1, 4, 24, and 48 h and compared them to genes regulated by JNK. Unexpectedly, inhibition of MAPK pathways is compensated by activation of the NFkappaB pathway and suppression of the DUSP enzymes. Both pathways promote epidermal differentiation; however, there is a surprising disconnect between the expression of steroid synthesis enzymes and differentiation markers. The p38 pathway induces the expression of extracellular matrix and proliferation-associated genes, while suppressing microtubule-associated genes. The ERK pathway induces nuclear envelope and mRNA splicing proteins, while suppressing steroid synthesis and mitochondrial energy production enzymes. Transcription factors SRY, c-FOS, and N-Myc are the principal targets of the p38 pathway, Elk-1 SAP1 and HLH2 of ERK, while FREAC-4, ARNT and USF are shared. The results suggest a list of targets potentially useful in therapeutic interventions in cutaneous diseases and wound healing
— id: 76465, year: 2008, vol: 215, page: 292, stat: Journal Article,

Transcriptional profiling defines the effects of nickel in human epidermal keratinocytes
Gazel, Alix; Rosdy, Martin; Tornier, Carine; De Fraissinette, Anne De Brugerolle; Blumenberg, Miroslav
2008 Dec;217(3):686-692, Journal of cellular physiology
Nickel is a ubiquitous and virtually unavoidable environmental pollutant and occupational hazard, but its molecular and cellular effects are not well understood. Human epidermal keratinocytes are the sentinel and the primary target for nickel. We treated with nickel salts skin equivalents containing differentiating epidermal keratinocytes grown on air-liquid interface in standard cell culture conditions. We identified the transcriptional profiles affected by nickel in reconstructed human epidermis (RHE) using DNA microarrays. The Ni-regulated genes were determined at two time points, immediate-early, 30 min after treatment, and late, at 6 h. Using in silico data analysis, we determined that 134 genes are regulated by nickel; of these, 97 are induced and 37 suppressed. Functional categories of regulated genes suggest that Ni inhibits apoptosis, promotes cell cycle and induces synthesis of extracellular matrix proteins and extracellular proteases. Importantly, Ni also regulates a set of secreted signaling proteins, inducing VEGF, amphiregulin, PGF, GDF15, and BST2, while suppressing IL-18, galectin-3, and LITAF. These secreted proteins may be important in Ni-caused allergic reactions. Ni induced inhibitors of the NFkappaB signaling pathway, and suppressed its activators. Correspondingly, NFkappaB binding sites were found to be overrepresented in the Ni-suppressed genes, whereas cFOS/AP1 binding sites were common in the Ni-induced genes. Significant parallels were found between the Ni-regulated genes and the genes regulated by TGFbeta, EGF, glucocorticoids, or Oncostatin-M. The comprehensive identification of Ni-regulated genes in human epidermal equivalents significantly advances our understanding of the molecular effects of nickel in skin
— id: 93342, year: 2008, vol: 217, page: 686, stat: Journal Article,

Cellular genomic maps help dissect pathology in human skin disease
Haider, Asifa S; Lowes, Michelle A; Suarez-Farinas, Mayte; Zaba, Lisa C; Cardinale, Irma; Blumenberg, Miroslav; Krueger, James G
2008 Mar;128(3):606-615, Journal of investigative dermatology
Genomic signature maps of different cell types can aid in the interpretation of genomic data of specimens collected during disease states. We have defined 'lineage-specific' genes, as well as 'activation' genes, for cellular components of the skin: keratinocytes, fibroblasts, macrophages, monocytes, T cells, immature, and mature dendritic cells (DCs). Re-analysis of a previously published gene set of psoriasis then provided a model for assessing the usefulness of these maps. We were able to ascribe over 90% of these genes to specific cell types, and there was a surprisingly large contribution from DCs. This shows the utility of such cellular gene maps
— id: 95166, year: 2008, vol: 128, page: 606, stat: Journal Article,

Nexus between epidermolysis bullosa and transcriptional regulation by thyroid hormone in epidermal keratinocytes
Tomic-Canic, Marjana; Stojadinovic, Olivera; Lee, Brian; Walsh, Rebecca; Blumenberg, Miroslav
2008 May;1(1):45-49, Clinical & Translational Science
Abstract Thyroid hormone, T3, through the interaction of its receptor with the recognition sequences in the DNA, regulates gene expression. This regulation includes the promoter activity of keratin genes. The receptor shares coregulators with other members of the nuclear receptor family, including RXR. Intending to define the transcriptional effects of thyroid hormones in keratinocytes, we used Affymetrix microarrays to comprehensively compare the genes expressed in T3-treated and untreated human epidermal keratinocytes. The transcriptomes were compared at 1, 4, 24, 48, and 72 hours. Surprisingly, T3 induced only 9 and suppressed 28 genes, much fewer than expected. Significantly, genes associated with epidermolysis bullosa, a set of inherited blistering skin diseases, were found statistically highly overrepresented among the suppressed genes. These genes include Integrin beta4, Plectin, Collagen XVII, MMP1, MMP3, and MMP14. The data imply that in keratinocytes T3 could suppresses the remodeling by, attachment to, and production of extracellular matrix. The results suggest that topical treatment with T3 may be effective for alleviation of symptoms in patients with epidermolysis bullosa
— id: 109574, year: 2008, vol: 1, page: 45, stat: Journal Article,

Transcriptional responses of human epidermal keratinocytes to cytokine interleukin-1
Yano, Shoichiro; Banno, Tomohiro; Walsh, Rebecca; Blumenberg, Miroslav
2008 Jan;214(1):1-13, Journal of cellular physiology
Interleukin-1 is a proinflammatory and immunomodulatory cytokine that plays a crucial role in inflammatory diseases of the skin, including bacterial infections, bullous diseases, UV damage, and especially psoriasis. To characterize the molecular effects of IL-1 in epidermis, we defined the transcriptional changes in human epidermal keratinocytes 1, 4, 24, and 48 h after treatment with IL-1alpha. IL-1 significantly regulated 388 genes, including genes associated with proteolysis, adhesion, signal transduction, proliferation, and epidermal differentiation. IL-1 induces many genes that have antimicrobial function. Secreted cytokines, chemokines, growth factors, and their receptors are the prominent targets of IL-1 regulation, including IL-8, IL-19, elafin, C3, and S100A proteins, which implicate IL-1 in the pathogenesis of inflammatory diseases. IL-1 induced not only proliferation-associated genes but also differentiation marker genes such as transglutaminase-1 and involucrin, which suggests that IL-1 plays an important role in the aberrant proliferation and differentiation seen in psoriasis. Correlation of IL-1 regulated genes with the TNFalpha and IFNgamma regulated ones showed more similarities between IL-1 and TNFalpha than IL-1 and IFNgamma, whereas Oncostatin-M (OsM) affected a largely unrelated set of genes. IL-1 regulates many genes previously shown to be specifically over-expressed in psoriasis. In summary, IL-1 regulates a characteristic set of genes that define its specific contribution to inflammation and aberrant differentiation in skin diseases
— id: 75390, year: 2008, vol: 214, page: 1, stat: Journal Article,

Gene profiling: Implications in dermatology
Blumenberg M.; Tomic-Canic M.
2007 ;2(6):763-768, Expert Review of Dermatology
DNA microarrays are capable of following the level of expression of virtually all genes in a human tissue. This has been employed to determine aberrant gene-expression profiles in many skin diseases, including UV-light damage, inflammatory processes and cancers. Owing to its accessibility, skin served as one of the initial targets of basic research using DNA microarrays; both the epidermis and dermis have been extensively investigated. Development of bedside uses of DNA arrays, coupled with the concomitant price reduction of the materials and methods of microarray analyses, holds great promise for improved diagnosis, treatment and prevention of dermatologic disorders. copyright 2007 Future Drugs Ltd
— id: 75469, year: 2007, vol: 2, page: 763, stat: Journal Article,

Chromatin structure regulation in transforming growth factor-beta-directed epithelial-mesenchymal transition
Blumenberg, Miroslav; Gao, Shujuan; Dickman, Kathleen; Grollman, Arthur P; Bottinger, Erwin P; Zavadil, Jiri
2007 ;185(1-3):162-174, Cells tissues organs
Epithelial-mesenchymal transitions (EMTs) occur in organogenesis throughout embryonic development and are recapitulated during epithelial tissue injury and in carcinoma progression. EMTs are regulated by complex, precisely orchestrated cell signaling and gene expression networks, with the participation of key developmental pathways. Here we review context-dependent modules of gene regulation by hairy/enhancer-of-split-related (H/E(spl)) repressors downstream of transforming growth factor-beta (TGF-beta)/Smad and Notch signals in EMT and in other phenotype transitions such as differentiation and cancer. Based on multiple models of disease-related EMT, we propose that Polycomb group epigenetic silencers and histone-lysine methyl-transferases EZH1 and EZH2 are candidate targets of H/E(spl)-mediated transcriptional repression, in a process accompanied by replacement of modified core histone H3 with de novo synthesized histone variant H3.3B. Finally, we discuss the potential significance of this scenario for EMT in the light of recent findings on gene regulation by histone modifications and chromatin structure changes.
— id: 73002, year: 2007, vol: 185, page: 162, stat: Journal Article,

Novel genomic effects of glucocorticoids in epidermal keratinocytes: inhibition of apoptosis, interferon-gamma pathway, and wound healing along with promotion of terminal differentiation
Stojadinovic, Olivera; Lee, Brian; Vouthounis, Constantinos; Vukelic, Sasa; Pastar, Irena; Blumenberg, Miroslav; Brem, Harold; Tomic-Canic, Marjana
2007 Feb 9;282(6):4021-4034, Journal of biological chemistry
Glucocorticoids (GCs) have a long history of use as therapeutic agents for numerous skin diseases. Surprisingly, their specific molecular effects are largely unknown. To characterize GC action in epidermis, we compared the transcriptional profiles of primary human keratinocytes untreated and treated with dexamethasone (DEX) for 1, 4, 24, 48, and 72 h using large scale microarray analyses. The majority of genes were found to be regulated only after 24 h and remained regulated throughout treatment. In addition to regulation of the expected pro-inflammatory genes, we found that GCs regulate cell fate, tissue remodeling, cell motility, differentiation, and metabolism. GCs suppress the expression of essentially all IFNgamma-regulated genes, including IFNgamma receptor and STAT-1, an effect that was previously unknown. GCs also block STAT-1 activation and nuclear translocation. Unexpectedly, GCs induce the expression of anti-apoptotic genes and repress pro-apoptotic ones, preventing UV-induced keratinocyte apoptosis. Consequently, treatment with GCs blocked UV-induced apoptosis of keratinocytes. GCs have profound effect on wound healing by inhibiting cell motility and the expression of the proangiogenic factor, vascular endothelial growth factor. They play an important role in tissue remodeling and scar formation by suppressing the expression of TGFbeta1 and -2 and MMP1, -2, -9, and -10 and inducing TIMP-2. Finally, GCs promote terminal epidermal differentiation while simultaneously inhibiting early stage differentiation. These results provide new insights into the beneficial and adverse effects of GCs in the epidermis, defining the participating genes and mechanisms that coordinate the cellular responses important for GC-based therapies
— id: 79480, year: 2007, vol: 282, page: 4021, stat: Journal Article,

Transforming growth factor-beta and microRNA:mRNA regulatory networks in epithelial plasticity
Zavadil, Jiri; Narasimhan, Manisha; Blumenberg, Miroslav; Schneider, Robert J
2007 ;185(1-3):157-161, Cells tissues organs
Noncoding microRNAs act as posttranscriptional repressors of gene function and are often deregulated in cancers and other diseases. Here we review recent findings on microRNA roles in tumorigenesis and report a microRNA profiling screen in transforming growth factor-beta1 (TGF-beta)-induced epithelial-mesenchymal transition (EMT) in human keratinocytes, a model of epithelial cell plasticity underlying epidermal injury and skin carcinogenesis. We describe a novel EMT-specific microRNA signature that includes induction of miR-21, a candidate oncogenic microRNA associated with carcinogenesis. By integrating the microRNA screen results with target prediction algorithms and gene expression profiling data, we outline a framework for TGF-beta-directed microRNA:messenger RNA (mRNA) regulatory circuitry and discuss its biological relevance for tumor progression.
— id: 73003, year: 2007, vol: 185, page: 157, stat: Journal Article,

Overview of the skin barrier
Blumenberg, M
2006 JUL ;126(7):1670-1670, Journal of investigative dermatology
— id: 66451, year: 2006, vol: 126, page: 1670, stat: Journal Article,

DNA microarrays in dermatology and skin biology
Blumenberg, Miroslav
2006 Fall;10(3):243-260, Omics : a journal of integrative biology
Because of its accessibility, skin has been among the first organs analyzed using DNA microarrays. Skin cancers, melanomas, and basal and squamous cell carcinomas have been intensely investigated because they are very frequent and can be fatal. Psoriasis, one of the most common human inflammatory diseases, has been studied comprehensively using DNA microarrays. In addition, epidermal keratinocytes have been the target of many studies because they respond to a rich variety of inflammatory and immunomodulating cytokines, hormones, vitamins, ultraviolet (UV) light, toxins, and physical injury. Because of the ethical considerations, the effects of harmful or dangerous agents on skin have been studied using artificial skin substitutes. Transcriptional mechanisms that regulate epidermal differentiation and cornification have begun to yield their mysteries, and very exciting recent studies identified the genes specifically expressed in epidermal stem cells. Thus, skin has everything: stem cells, differentiation, signaling, inflammation, diseases, and cancer. All these exciting facets of skin have been explored using DNA microarrays. Researchers in skin biology and dermatology were among the first to implement this technology and we expect that they will continue to generate exciting and useful new knowledge
— id: 69598, year: 2006, vol: 10, page: 243, stat: Journal Article,

Inhibition of JNK promotes differentiation of epidermal keratinocytes
Gazel, Alix; Banno, Tomohiro; Walsh, Rebecca; Blumenberg, Miroslav
2006 Jul 21;281(29):20530-20541, Journal of biological chemistry
In inflamed tissue, normal signal transduction pathways are altered by extracellular signals. For example, the JNK pathway is activated in psoriatic skin, which makes it an attractive target for treatment. To define comprehensively the JNK-regulated genes in human epidermal keratinocytes, we compared the transcriptional profiles of control and JNK inhibitor-treated keratinocytes, using DNA microarrays. We identified the differentially expressed genes 1, 4, 24, and 48 h after the treatment with SP600125. Surprisingly, the inhibition of JNK in keratinocyte cultures in vitro induces virtually all aspects of epidermal differentiation in vivo: transcription of cornification markers, inhibition of motility, withdrawal from the cell cycle, stratification, and even production of cornified envelopes. The inhibition of JNK also induces the production of enzymes of lipid and steroid metabolism, proteins of the diacylglycerol and inositol phosphate pathways, mitochondrial proteins, histones, and DNA repair enzymes, which have not been associated with differentiation previously. Simultaneously, basal cell markers, including integrins, hemidesmosome and extracellular matrix components, are suppressed. Promoter analysis of regulated genes finds that the binding sites for the forkhead family of transcription factors are over-represented in the SP600125-induced genes and c-Fos sites in the suppressed genes. The JNK-induced proliferation appears to be secondary to inhibition of differentiation. The results indicate that the inhibition of JNK in epidermal keratinocytes is sufficient to initiate their differentiation program and suggest that augmenting JNK activity could be used to delay cornification and enhance wound healing, whereas attenuating it could be a differentiation therapy-based approach for treating psoriasis
— id: 67550, year: 2006, vol: 281, page: 20530, stat: Journal Article,

A Characteristic Subset of Psoriasis-Associated Genes Is Induced by Oncostatin-M in Reconstituted Epidermis
Gazel, Alix; Rosdy, Martin; Bertino, Beatrice; Tornier, Carine; Sahuc, Florent; Blumenberg, Miroslav
2006 Dec;126(12):2647-2657, Journal of investigative dermatology
The pathological manifestations of psoriasis are orchestrated by many secreted proteins, but only a handful, tumor necrosis factor-alpha, IFN-gamma and IL-1, have been studied in great detail. Oncostatin-M (OsM) has also been found in psoriatic skin and we hypothesized that it makes a unique and characteristic contribution to the psoriatic processes. To define in-depth the molecular effects of OsM in epidermis, we used high-density DNA microarrays for transcriptional profiling of OsM-treated human skin equivalents. We identified 374 unambiguously OsM-regulated genes, out of 22,000 probed. OsM suppressed the expression of the 'classical' epidermal differentiation markers, but strongly and specifically induced the S100A proteins. Cytoskeletal and complement proteins, proteases, and their inhibitors were also induced by OsM. Interestingly, a large set of genes was induced by OsM at early time points but suppressed later; these genes are known regulatory targets of IFN and thus provide a nexus between the OsM and IFN pathways. OsM induces IL-4 and suppresses the T-helper 1-type and IL-1-responsive signals, potentially attenuating the psoriatic pathology. The data suggest that OsM plays a unique role in psoriasis, different from all other, more thoroughly studied cytokines.Journal of Investigative Dermatology advance online publication, 17 August 2006; doi:10.1038/sj.jid.5700461
— id: 67548, year: 2006, vol: 126, page: 2647, stat: Journal Article,

A new approach to dissecting skin disease pathogenesis through cellular genomics
Haider, AS; Cardinale, I; Blumenberg, M; Ott, J; Giuli, L; Lowes, MA; Carucci, JC; Krueger, JG
2006 ;126(2):457-457, Journal of investigative dermatology
— id: 114999, year: 2006, vol: 126, page: 457, stat: Journal Article,

"Genomic analysis defines a cancer specific gene expression signature for human squamous cell carcinoma and identifies potential roles for WNT, FZD and PTN in the pathogenesis of SCC"
Haider, AS; Kaporis, HG; Ott, J; Blumenberg, M; Krueger, JG; Carucci, J
2006 ;126(2):104-104, Journal of investigative dermatology
— id: 114998, year: 2006, vol: 126, page: 104, stat: Journal Article,

Genomic analysis defines a cancer-specific gene expression signature for human squamous cell carcinoma and distinguishes malignant hyperproliferation from benign hyperplasia
Haider, Asifa S; Peters, Sara B; Kaporis, Helen; Cardinale, Irma; Fei, Ji; Ott, Jurg; Blumenberg, Miki; Bowcock, Ann M; Krueger, James G; Carucci, John A
2006 Apr;126(4):869-881, Journal of investigative dermatology
Using high-density oligonucleotide arrays, we measured expression of >12,000 genes in surgical excisions of invasive human squamous cell carcinomas (SCCs) versus site-matched control skin. This analysis defined >1,900 genes with altered expression in SCCs that were statistically different from controls. As SCCs are composed of epithelial cells, which are both hyperplastic and invasive, we sought to define gene sets associated with these biologic processes by comparing gene expression to psoriasis vulgaris, which is a condition of benign keratinocyte hyperplasia without invasiveness or pre-malignant potential. Through this analysis, we found genes that were commonly upregulated in both conditions and unique genes with increased expression in SCCs. Differential gene regulation in these two conditions was confirmed by real-time reverse transcription-PCR and immunohistochemistry. We found that benign hyperplasia is associated with upregulation of genes including DEFB4 (defensin B4), SERPINB3 (serine proteinase inhibitor, member 3), STAT1 (signal transducer and activator of transcription 1), K16 (keratin 16), CEACAMs (carcinoembryonic antigen-related cell adhesion molecules), and WNT 5A (wingless-type MMTV integration site family, member 5A). WNT receptor frizzled homolog 6 (FZD6) and prostaglandin-metabolizing enzyme hydroxyprostaglandin dehydrogenase were increased in SCC alone. Growth factor pleiotrophin (PTN) was expressed at higher levels in non-tumor-bearing skin adjacent to excised SCC. SCC was further characterized by upregulation of matrix metalloproteinases 1, 10, and 13, cathepsin L2, cystatin E/M as well as STAT3 and microseminoprotein, beta (MSMB), and downregulation of inducible nitric oxide synthase, granzyme B, CD8, and CD83. The current study defines a unique gene expression signature for cutaneous SCC in humans and suggests potential roles for WNT, FZD, and PTN in the pathogenesis of SCC
— id: 114941, year: 2006, vol: 126, page: 869, stat: Journal Article,

Mechanical stretch induces extracellular matrix proteins, adhesion molecules, and actin-related cytoskeleton proteins, by regulating Rho, cdc42 and Rac family gene expression in normal human keratinocytes
Komine, M; Yano, S; Okochi, H; Blumenberg, M; Tamaki, K
2006 APR ;126(1):33-33, Journal of investigative dermatology
— id: 70332, year: 2006, vol: 126, page: 33, stat: Journal Article,

Interleukin IL-12 blocks a specific subset of the transcriptional profile responsive to UVB in epidermal keratinocytes
Molenda, Matthew; Mukkamala, Lakshmi; Blumenberg, Miroslav
2006 May;43(12):1933-1940, Molecular immunology
Interleukin-12 (IL-12) is a proinflammatory and immunomodulatory cytokine that plays a critical role it in innate and adaptive immunity by inducing production of interferon-gamma and other cytokines. IL-12 was shown to block the ultraviolet light-induced immunosuppression, important in cancer immunosurveillance, cutaneous allergies and inflammation. To characterize the molecular effects of IL-12 in epidermis we used large DNA microarrays and defined the transcriptional changes in human epidermal keratinocytes 1 h, 4 h, 24 h, and 48 h after treatment with IL-12, as well as in cells treated with both IL-12 and UV light. In keratinocytes, IL-12 activates STAT3 and STAT4; surprisingly, despite activating these transcription factors, the transcriptional effects of IL-12 did not rise above background levels. However, pre-treatment of keratinocytes with IL-12 strongly modulated the transcriptional effects of UV. Pre-treatment with IL-12 enhanced the UV-mediated regulation of 20 and antagonized the regulation of 263 genes. IL-12 enhanced the induction of cytokines by UV. IL-12 antagonized the suppression of cytoskeletal, junctional, metabolic, mitochondrial, and extracellular matrix proteins, while antagonizing the induction of certain signaling proteins and RNA processing enzymes. We conclude that in the epidermis, IL-12 interferes with a specific subset of transcriptional effects of UV irradiation
— id: 67551, year: 2006, vol: 43, page: 1933, stat: Journal Article,

TRANSCRIPTIONAL PROFILING OF EPIDERMAL DIFFERENTIATION
Radoja, Nada; Gazel, Alix; Banno, Tomohiro; Yano, Shoichiro; Blumenberg, Miroslav
2006 Oct 3;27(1):65-78, Physiological genomics
In epidermal differentiation basal keratinocytes detach from the basement membrane, stop proliferating, and express a new set of structural proteins and enzymes, which results in an impermeable protein/lipid barrier that protects us. To define the transcriptional changes essential for this process, we purified large quantities of basal and suprabasal cells from human epidermis, using the expression of beta4 integrin as the discriminating factor. The expected expression differences in cytoskeletal, cell cycle and adhesion genes confirmed the effective separation of the cell populations. Using DNA microarray chips, we comprehensively identify the differences in genes expressed in basal and differentiating layers of the epidermis, including the ECM components produced by the basal cells, the proteases in both the basal and suprabasal cells, and the lipid and steroid metabolism enzymes in suprabasal cells responsible for the permeability barrier. We identified the signaling pathways specific for the two populations, and found two previously unknown paracrine and one juxtacrine signaling pathway operating between the basal and suprabasal cells. Furthermore, using specific expression signatures, we identified a new set of late differentiation markers and mapped their chromosomal loci, as well as a new set of melanocyte-specific markers. The data represent a quantum jump in understanding the mechanisms of epidermal differentiation
— id: 67549, year: 2006, vol: 27, page: 65, stat: Journal Article,

Global transcriptional profiling of human epidermal keratinocytes to interleukin 1-alpha
Yano, S; Walsh, R; Banno, T; Blumenberg, M
2006 APR ;126(1):86-86, Journal of investigative dermatology
— id: 70333, year: 2006, vol: 126, page: 86, stat: Journal Article,

Pathway-specific profiling identifies the NFkB-dependent TNFa-regulated genes in epidermal keratinocytes
Banno, Tomohiro; Gazel, Alix; Blumenberg, Miroslav
2005 May;280(19):18973-18980, Journal of biological chemistry
Identification of TNFa as the key agent in inflammatory disorders led to new therapies specifically targeting TNFa and avoiding many side-effects of earlier anti-inflammatory drugs. However, because of the wide spectrum of systems affected by TNFa, drugs targeting TNFa have a potential risk of delaying wound healing, secondary infections and cancer. TNFa regulates many processes, e.g., immune response, cell-cycle and apoptosis, through several signal transduction pathways that convey the TNFa signals to the nucleus. Hypothesizing that specific TNFa-dependent pathways control specific processes and that inhibition of a specific pathway may yield even more precisely targeted therapies, we used oligonucleotide microarrays and parthenolide, an NFkB-specific inhibitor, to identify the NFkB-dependent set of the TNFa-regulated genes in human epidermal keratinocytes. Expression of approximately 40% of all TNFa-regulated genes depends on NFkB, 17% are regulated early (1-4 hrs post-treatment) and 23% late (24-48 hrs). Cytokines, apoptosis-related and cornification proteins belong to the 'early' NFkB-dependent group, antigen presentation proteins to the 'late', while most cell-cycle, RNA-processing and metabolic enzymes are not NFkB-dependent. Therefore, inflammation, immunomodulation, apoptosis and differentiation are on the NFkB pathway, cell-cycle, metabolism and RNA processing are not. Most early genes contain consensus NFkB binding sites in their promoter DNA and are, presumably, directly regulated by NFkB, except, curiously, the cornification markers. Using siRNA-silencing, we identified cFLIP/CFLAR as an essential NFkB-dependent anti-apoptotic gene. The results confirm our hypothesis, suggesting that inhibiting a specific TNFa-dependent signaling pathway may inhibit a specific TNFa-regulated process, leaving others unaffected, which could lead to more specific anti-inflammatory agents that are both more effective and safer
— id: 49566, year: 2005, vol: 280, page: 18973, stat: Journal Article,

Skinomics
Blumenberg, Miroslav
2005 Apr;124(4):viii-viix, Journal of investigative dermatology
— id: 54110, year: 2005, vol: 124, page: viii, stat: Journal Article,

Transcriptional responses of human epidermal keratinocytes to Oncostatin-M
Finelt, Nika; Gazel, Alix; Gorelick, Steven; Blumenberg, Miroslav
2005 Aug 21;31(4):305-313, Cytokine
Oncostatin-M (OsM) plays an important role in inflammatory and oncogenic processes in skin, including psoriasis and Kaposi sarcoma. However, the molecular responses to OsM in keratinocytes have not been explored in depth. Here we show the results of transcriptional profiling in OsM-treated primary human epidermal keratinocytes, using high-density DNA microarrays. We find that OsM strongly and specifically affects the expression of many genes, in particular those involved with innate immunity, angiogenesis, adhesion, motility, tissue remodeling, cell cycle and transcription. The timing of the responses to OsM comprises two waves, early at 1h, and late at 48 h, with much fewer genes regulated in the intervening time points. Secreted cytokines and growth factors and their receptors, as well as nuclear transcription factors, are primary targets of OsM regulation, and these, in turn, effect the secondary changes
— id: 58897, year: 2005, vol: 31, page: 305, stat: Journal Article,

Interleukin (IL12) blocks a specific subset of transcriptional responses to UVB in epidermal keratinocytes (vol 124, pg A75, 2005)
Molenda, M; Mukkamala, L; Blumenberg, M
2005 JUL ;125(1):A8-A8, Journal of investigative dermatology
— id: 56288, year: 2005, vol: 125, page: A8, stat: Journal Article,

Effects of tumor necrosis factor-alpha (TNF alpha) in epidermal keratinocytes revealed using global transcriptional profiling
Banno, Tomohiro; Gazel, Alix; Blumenberg, Miroslav
2004 Aug 30;279(31):32633-32642, Journal of biological chemistry
Identification of tumor necrosis factor-alpha (TNF alpha) as the key agent in inflammatory disorders, e.g. rheumatoid arthritis, Crohn's disease, and psoriasis, led to TNF alpha-targeting therapies, which, although avoiding many of the side-effects of previous drugs, nonetheless causes other side-effects, including secondary infections and cancer. By controlling gene expression, TNF alpha orchestrates the cutaneous responses to environmental damage and inflammation. To define TNF alpha action in epidermis, we compared the transcriptional profiles of normal human keratinocytes untreated and treated with TNF alpha for 1, 4, 24, and 48 h by using oligonucleotide microarrays. We found that TNF alpha regulates not only immune and inflammatory responses but also tissue remodeling, cell motility, cell cycle, and apoptosis. Specifically, TNF alpha regulates innate immunity and inflammation by inducing a characteristic large set of chemokines, including newly identified TNF alpha targets, that attract neutrophils, macrophages, and skin-specific memory T-cells. This implicates TNF alpha in the pathogenesis of psoriasis, fixed drug eruption, atopic and allergic contact dermatitis. TNF alpha promotes tissue repair by inducing basement membrane components and collagen-degrading proteases. Unexpectedly, TNF alpha induces actin cytoskeleton regulators and integrins, enhancing keratinocyte motility and attachment, effects not previously associated with TNF alpha. Also unanticipated was the influence of TNF alpha upon keratinocyte cell fate by regulating cell-cycle and apoptosis-associated genes. Therefore, TNF alpha initiates not only the initiation of inflammation and responses to injury, but also the subsequent epidermal repair. The results provide new insights into the harmful and beneficial TNF alpha effects and define the mechanisms and genes that achieve these outcomes, both of which are important for TNF alpha-targeted therapies
— id: 45940, year: 2004, vol: 279, page: 32633, stat: Journal Article,

Thyroid hormones and gamma interferon specifically increase K15 keratin gene transcription
Radoja, Nada; Stojadinovic, Olivera; Waseem, Ahmad; Tomic-Canic, Marjana; Milisavljevic, Vladana; Teebor, Susan; Blumenberg, Miroslav
2004 Apr;24(8):3168-3179, Molecular & cellular biology
Basal layers of stratified epithelia express keratins K5, K14, and K15, which assemble into intermediate filament networks. Mutations in K5 or K14 genes cause epidermolysis bullosa simplex (EBS), a disorder with blistering in the basal layer due to cell fragility. Nonkeratinizing stratified epithelia, e.g., in the esophagus, produce more keratin K15 than epidermis, which alleviates the esophageal symptoms in patients with K14 mutations. Hypothesizing that increasing the cellular content of K15 could compensate for the mutant K14 and thus ease skin blistering in K14 EBS patients, we cloned the promoter of the K15 gene and examined its transcriptional regulation. Using cotransfection, gel mobility shifts, and DNase I footprinting, we have identified the regulators of K15 promoter activity and their binding sites. We focused on those that can be manipulated with extracellular agents, transcription factors C/EBP, AP-1, and NF-kappaB, nuclear receptors for thyroid hormone, retinoic acid, and glucocorticoids, and the cytokine gamma interferon (IFN-gamma). We found that C/EBP-beta and AP-1 induced, while retinoic acid, glucocorticoid receptors, and NF-kappaB suppressed, the K15 promoter, along with other keratin gene promoters. However, the thyroid hormone and IFN-gamma uniquely and potently activated the K15 promoter. Using these agents, we could boost the amounts of K15 in human epidermis. Our findings suggest that treatments based on thyroid hormone and IFN-gamma could become effective agents in therapy for patients with EBS
— id: 44939, year: 2004, vol: 24, page: 3168, stat: Journal Article,

Mouse striatal transcriptome analysis: effects of oral self-administration of alcohol
Saito, Mariko; Szakall, Istvan; Toth, Reka; Kovacs, Krisztina M; Oros, Melinda; Prasad, Vidudala V T S; Blumenberg, Miroslav; Vadasz, Csaba
2004 May;32(3):223-241, Alcohol
Results of recent studies support the notion that substance self-administration is partially a genetically controlled component of addiction tied to habit formation and cellular modification of the striatum. Aiming to define pathways among genomic, neural, and behavioral determinants of addiction, we investigated global striatal gene expression in a paradigm of oral self-administration of alcohol by using genomically very similar alcohol-nonpreferring B6.Cb(5)i(7)-alpha 3/Vad (C5A3) and alcohol-preferring B6.Ib(5)i(7)-beta 25A/Vad (I5B25A) quasi-congenic mouse strains and their progenitors, C57BL/6By (B6By) and BALB/cJ. Expression of 12,488 genes and expressed sequence tags (ESTs) was studied by using 24 high-density oligonucleotide microarrays. Transcript signal intensity differences were analyzed with z test after iterative median normalization across groups and Hochberg step-down Bonferroni procedure. As expected, striatal transcriptome differences were far more extensive between the independently derived progenitor strains than between the quasi-congenic strains and their background partner, B6By. However, the genes, which were differentially expressed between the quasi-congenic strains and their background partner, were not subsets of the progenitorial differences and were not located on the chromosome segments introgressed into the quasi-congenic strains from the donor BALB/cJ strain that have been so far defined. Although 25 transcripts showed significantly different expression between the progenitor strains, only two transcripts, phosphatidylserine decarboxylase and a hypothetical 21.2-kDa protein, and one transcript, molybdenum co-factor synthesis 2, showed significantly different expression between C5A3 and I5B25A, and between B6By and I5B25A, respectively. The latter three transcripts are not located on previously identified chromosome segments introgressed from the donor BALB/cJ strain, supporting the suggestion of trans-acting regulatory variations among strains. Exposure to alcohol did not induce statistically significant striatal gene expression changes in any of the mouse strains. In conclusion, the results support the hypothesis that in functional genomic studies the chance of detecting function-relevant genes can be increased by the comparative analysis of quasi-congenic and background strains because the number of functionally irrelevant, differentially expressed genes between genomically similar strains is reduced. Lack of statistically significant alcohol-induced changes in transcript abundance indicated that oral self-administration had subtle effects on striatal gene expression and directed attention to important implications for the experimental design of future microarray gene expression studies on complex behaviors
— id: 45939, year: 2004, vol: 32, page: 223, stat: Journal Article,

Specificity in Stress Response: Epidermal Keratinocytes Exhibit Specialized UV-Responsive Signal Transduction Pathways
Adachi, Makoto; Gazel, Alix; Pintucci, Giuseppe; Shuck, Alyssa; Shifteh, Shiva; Ginsburg, Dov; Rao, Laxmi S; Kaneko, Takehiko; Freedberg, Irwin M; Tamaki, Kunihiko; Blumenberg, Miroslav
2003 Oct;22(10):665-677, DNA & cell biology
UV light, a paradigmatic initiator of cell stress, invokes responses that include signal transduction, activation of transcription factors, and changes in gene expression. Consequently, in epidermal keratinocytes, its principal and frequent natural target, UV regulates transcription of a distinctive set of genes. Hypothesizing that UV activates distinctive epidermal signal transduction pathways, we compared the UV-responsive activation of the JNK and NFkappaB pathways in keratinocytes, with the activation of the same pathways by other agents and in other cell types. Using of inhibitors and antisense oligonucleotides, we found that in keratinocytes only UVB/UVC activate JNK, while in other cell types UVA, heat shock, and oxidative stress do as well. Keratinocytes express JNK-1 and JNK-3, which is unexpected because JNK-3 expression is considered brain-specific. In keratinocytes, ERK1, ERK2, and p38 are activated by growth factors, but not by UV. UVB/UVC in keratinocytes activates Elk1 and AP1 exclusively through the JNK pathway. JNKK1 is essential for UVB/UVC activation of JNK in keratinocytes in vitro and in human skin in vivo. In contrast, in HeLa cells, used as a control, crosstalk among signal transduction pathways allows considerable laxity. In parallel, UVB/UVC and TNFalpha activate the NFkappaB pathway via distinct mechanisms, as shown using antisense oligonucleotides targeted against IKKbeta, the active subunit of IKK. This implies a specific UVB/UVC responsive signal transduction pathway independent from other pathways. Our results suggest that in epidermal keratinocytes specific signal transduction pathways respond to UV light. Based on these findings, we propose that the UV light is not a genetic stress response inducer in these cells, but a specific agent to which epidermis developed highly specialized responses
— id: 38998, year: 2003, vol: 22, page: 665, stat: Journal Article,

Unique keratinocyte-specific effects of interferon-gamma that protect skin from viruses, identified using transcriptional profiling
Banno, Tomohiro; Adachi, Makoto; Mukkamala, Lakshmi; Blumenberg, Miroslav
2003 Dec;8(6):541-554, Antiviral therapy
Interferon (IFN)-gamma, is a multifunctional, immunomodulatory cytokine with cell type-specific antiviral activities, particularly important in skin, where it is implicated in many diseases ranging from warts to psoriasis and cancer. Since epidermis is our first line of defence against many viruses, we investigated the molecular processes regulated by IFN-gamma in keratinocytes using DNA microarrays. We identified the IFN-gamma-regulated keratinocyte-specific genes in keratinocytes, IFN-gamma-induced tight junction proteins, presumably to deny viruses paracellular routes of infection. Furthermore, differing from published data, we find that IFN-gamma suppressed the expression of keratinocytes differentiation markers including desmosomal proteins, cornified envelope components and suprabasal cytokeratins. Inhibition of differentiation may interfere with the epidermal tropism of viruses that require differentiating cells for growth, for example, papillomaviruses. As in other cell types, IFN-gamma induced HLA, cell adhesion and proteasome proteins, facilitating leukocyte attraction and antigen-presentation by keratinocytes. IFN-gamma also induced chemokine/cytokines specific for mononuclear cells. IFN-gamma suppressed the expression of over 100 genes responsible for cell cycle, DNA replication and RNA metabolism, thereby shutting down many nuclear processes and denying viruses a healthy cell in which to replicate. Thus, uniquely in keratinocytes, IFN-gamma initiates a well-organized molecular programme boosting host antiviral defences, obstructing viral entry, suppressing cell proliferation and impeding differentiation
— id: 46265, year: 2003, vol: 8, page: 541, stat: Journal Article,

Transcriptional profiling of epidermal keratinocytes: comparison of genes expressed in skin, cultured keratinocytes, and reconstituted epidermis, using large DNA microarrays
Gazel, Alix; Ramphal, Patricia; Rosdy, Martin; De Wever, Bart; Tornier, Carine; Hosein, Nadia; Lee, Brian; Tomic-Canic, Marjana; Blumenberg, Miroslav
2003 Dec;121(6):1459-1468, Journal of investigative dermatology
Epidermal keratinocytes are complex cells that create a unique three-dimensional (3-D) structure, differentiate through a multistage process, and respond to extracellular stimuli from nearby cells. Consequently, keratinocytes express many genes, i.e., have a relatively large 'transcriptome.' To determine which of the expressed genes are innate to keratinocytes, which are specific for the differentiation and 3-D architecture, and which are induced by other cell types, we compared the transcriptomes of skin from human subjects, differentiating 3-D reconstituted epidermis, cultured keratinocytes, and nonkeratinocyte cell types. Using large oligonucleotide microarrays, we analyzed five or more replicates of each, which yielded statistically consistent data and allowed identification of the differentially expressed genes. Epidermal keratinocytes, unlike other cells, express many proteases and protease inhibitors and genes that protect from UV light. Skin specifically expresses a higher number of receptors, secreted proteins, and transcription factors, perhaps influenced by the presence of nonkeratinocyte cell types. Surprisingly, mitochondrial proteins were significantly suppressed in skin, suggesting a low metabolic rate. Three-dimensional samples, skin and reconstituted epidermis, are similar to each other, expressing epidermal differentiation markers. Cultured keratinocytes express many cell-cycle and DNA replication genes, as well as integrins and extracellular matrix proteins. These results define innate, architecture-specific, and cell-type-regulated genes in epidermis
— id: 44940, year: 2003, vol: 121, page: 1459, stat: Journal Article,

Keratins and the keratinocyte activation cycle
Freedberg IM; Tomic-Canic M; Komine M; Blumenberg M
2001 May;116(5):633-640, Journal of investigative dermatology
In wound healing and many pathologic conditions, keratinocytes become activated: they turn into migratory, hyperproliferative cells that produce and secrete extracellular matrix components and signaling polypeptides. At the same time, their cytoskeleton is also altered by the production of specific keratin proteins. These changes are orchestrated by growth factors, chemokines, and cytokines produced by keratinocytes and other cutaneous cell types. The responding intracellular signaling pathways activate transcription factors that regulate expression of keratin genes. Analysis of these processes led us to propose the existence of a keratinocyte activation cycle, in which the cells first become activated by the release of IL-1. Subsequently, they maintain the activated state by autocrine production of proinflammatory and proliferative signals. Keratins K6 and K16 are markers of the active state. Signals from the lymphocytes, in the form of Interferon-gamma, induce the expression of K17 and make keratinocytes contractile. This enables the keratinocytes to shrink the provisional fibronectin-rich basement membrane. Signals from the fibroblasts, in the form of TGF-beta, induce the expression of K5 and K14, revert the keratinocytes to the healthy basal phenotype, and thus complete the activation cycle
— id: 20670, year: 2001, vol: 116, page: 633, stat: Journal Article,

Interleukin-1 Induces Transcription of Keratin K6 in Human Epidermal Keratinocytes
Komine M; Rao LS; Freedberg IM; Simon M; Milisavljevic V; Blumenberg M
2001 Feb;116(2):330-338, Journal of investigative dermatology
Keratinocytes respond to injury by releasing the proinflammatory cytokine interleukin-1, which serves as the initial 'alarm signal' to surrounding cells. Among the consequences of interleukin-1 release is the production of additional cytokines and their receptors by keratinocytes and other cells in the skin. Here we describe an additional effect of interleukin-1 on keratinocytes, namely the alteration in the keratinocyte cytoskeleton in the form of the induction of keratin 6 expression. Keratin 6 is a marker of hyperproliferative, activated keratinocytes, found in wound healing, psoriasis, and other inflammatory disorders. Skin biopsies in organ culture treated with interleukin-1 express keratin 6 in all suprabasal layers of the epidermis, throughout the tissue. In cultured epidermal keratinocytes, the induction of keratin 6 is time and concentration dependent. Importantly, only confluent keratinocytes respond to interleukin-1, subconfluent cultures do not. In the cells starved of growth factors, epidermal growth factor or tumor necrosis factor-alpha, if added simultaneously with interleukin-1, they synergistically augment the effects of interleukin-1. Using DNA-mediated cell transfection, we analyzed the molecular mechanisms regulating the keratin 6 induction by interleukin-1, and found that the induction occurs at the transcriptional level. We used a series of deletions and point mutations to identify the interleukin-1 responsive DNA element in the keratin 6 promoter, and determined that it contains a complex of C/EBP binding sites. The transcription factor C/EBPbeta binds this element in vitro, and the binding is augmented by pretreatment of the cells with interleukin-1. The interleukin-1 responsive element is clearly distinct from the epidermal growth factor responsive one, which means that the proinflammatory and proliferative signals independently regulate the expression of keratin 6. Thus, interleukin-1 initiates keratinocyte activation not only by triggering additional signaling events, but also by inducing directly the synthesis of keratin 6 in epidermal keratinocytes, and thus changing the composition of their cytoskeleton
— id: 17561, year: 2001, vol: 116, page: 330, stat: Journal Article,

Inflammatory versus proliferative processes in epidermis. Tumor necrosis factor alpha induces K6b keratin synthesis through a transcriptional complex containing NFkappa B and C/EBPbeta
Komine M; Rao LS; Kaneko T; Tomic-Canic M; Tamaki K; Freedberg IM; Blumenberg M
2000 Oct 13;275(41):32077-32088, Journal of biological chemistry
Epidermal keratinocytes respond to injury by becoming activated, i.e. hyperproliferative, migratory, and proinflammatory. These processes are regulated by growth factors and cytokines. One of the markers of activated keratinocytes is keratin K6. We used a novel organ culture system to show that tumor necrosis factor alpha (TNFalpha) induces the expression of K6 protein and mRNA in human skin. Multiple isoforms of K6 are encoded by distinct genes and have distinct patterns of expression. By having shown previously that proliferative signals, such as epidermal growth factor (EGF), induce expression of the cytoskeletal protein keratin K6b, we here demonstrate that the same isoform, K6b, is also induced by TNFalpha, a proinflammatory cytokine. Specifically, TNFalpha induces the transcription of the K6b gene promoter. By using co-transfection, specific inhibitors, and antisense oligonucleotides, we have identified NFkappaB and C/EBPbeta as the transcription factors that convey the TNFalpha signal. Both transcription factors are necessary for the induction of K6b by TNFalpha and act as a complex, although only C/EBPbeta binds the K6b promoter DNA. By using transfection, site-directed mutagenesis, and footprinting, we have mapped the site that responds to TNFalpha, NFkappaB, and C/EBPbeta. This site is separate from the one responsive to EGF and AP1. Our results show that the proinflammatory (TNFalpha) and the proliferative (EGF) signals in epidermis separately and independently regulate the expression of the same K6b keratin isoform. Thus, the cytoskeletal responses in epidermal cells can be precisely tuned by separate proliferative and inflammatory signals to fit the nature of the injuries that caused them
— id: 16372, year: 2000, vol: 275, page: 32077, stat: Journal Article,

Novel mechanism of steroid action in skin through glucocorticoid receptor monomers
Radoja N; Komine M; Jho SH; Blumenberg M; Tomic-Canic M
2000 Jun;20(12):4328-4339, Molecular & cellular biology
Glucocorticoids (GCs), important regulators of epidermal growth, differentiation, and homeostasis, are used extensively in the treatment of skin diseases. Using keratin gene expression as a paradigm of epidermal physiology and pathology, we have developed a model system to study the molecular mechanism of GCs action in skin. Here we describe a novel mechanism of suppression of transcription by the glucocorticoid receptor (GR) that represents an example of customizing a device for transcriptional regulation to target a specific group of genes within the target tissue, in our case, epidermis. We have shown that GCs repress the expression of the basal-cell-specific keratins K5 and K14 and disease-associated keratins K6, K16, and K17 but not the differentiation-specific keratins K3 and K10 or the simple epithelium-specific keratins K8, K18, and K19. We have identified the negative recognition elements (nGREs) in all five regulated keratin gene promoters. Detailed footprinting revealed that the function of nGREs is to instruct the GR to bind as four monomers. Furthermore, using cotransfection and antisense technology we have found that, unlike SRC-1 and GRIP-1, which are not involved in the GR complex that suppresses keratin genes, histone acetyltransferase and CBP are. In addition, we have found that GR, independently from GREs, blocks the induction of keratin gene expression by AP1. We conclude that GR suppresses keratin gene expression through two independent mechanisms: directly, through interactions of keratin nGREs with four GR monomers, as well as indirectly, by blocking the AP1 induction of keratin gene expression
— id: 11686, year: 2000, vol: 20, page: 4328, stat: Journal Article,

Transcriptional control of K5, K6, K14, and K17 keratin genes by AP-1 and NF-kappaB family members
Ma S; Rao L; Freedberg IM; Blumenberg M
1997 ;6(6):361-370, Gene expression
The expression of keratins K5 and K14 is restricted to the basal layers of the healthy epidermis, whereas the expression of K6 and K17 is induced in response to proliferative and inflammatory signals, respectively. The control of keratin expression occurs primarily at the transcriptional level. We studied the effects of transcription factors of the AP-1 and NF-kappaB families on the expression of those four keratin genes. We chose AP-1 and NF-kappaB proteins because they are activated by many extracellular signals, including those in hyperproliferative and inflammatory processes. DNA constructs expressing the transcription factors were, in various combinations, cotransfected with constructs containing keratin gene promoters and the CAT reporter gene into HeLa cells or keratinocytes. We found that the K5 and K14 promoters, which are coexpressed in vivo, are regulated in parallel by the cotransfected genes. Both were activated by the c-Fos and c-Jun components of AP-1, but not by Fra1. On the other hand, the NF-kappaB proteins, especially p65, suppressed these two promoters. The K17 promoter was specifically activated by c-Jun, whereas the other transcription factors tested had no significant effect. In contrast, the K6 promoter was very strongly activated by all AP-1 proteins, especially by the c-Fos + c-Jun and Fra1 + c-Jun combinations. It was also strongly activated by the p65 NF-kappaB protein. AP-1 and NF-kappaB acted synergistically in activating the K6 promoter, although the AP-1 and the NF-kappaB responsive sites could be separated physically. These results suggest that the interplay of AP-1 and NF-kappaB proteins regulates epidermal gene expression and that the activation of these transcription factors by extracellular signaling molecules brings about the differential expression of keratin genes in epidermal differentiation, cutaneous diseases, and wound healing
— id: 8082, year: 1997, vol: 6, page: 361, stat: Journal Article,

The coupling of alpha6beta4 integrin to Ras-MAP kinase pathways mediated by Shc controls keratinocyte proliferation
Mainiero F; Murgia C; Wary KK; Curatola AM; Pepe A; Blumemberg M; Westwick JK; Der CJ; Giancotti FG
1997 May 1;16(9):2365-2375, EMBO journal
The signaling pathways linking integrins to nuclear events are incompletely understood. We have examined intracellular signaling by the alpha6beta4 integrin, a laminin receptor expressed in basal keratinocytes and other cells. Ligation of alpha6beta4 in primary human keratinocytes caused tyrosine phosphorylation of Shc, recruitment of Grb2, activation of Ras and stimulation of the MAP kinases Erk and Jnk. In contrast, ligation of the laminin- and collagen-binding integrins alpha3beta1 and alpha2beta1 did not cause these events. While the stimulation of Erk by alpha6beta4 was suppressed by dominant-negative Shc, Ras and RhoA, the activation of Jnk was inhibited by dominant-negative Ras and Rac1 and by the phosphoinositide 3-kinase inhibitor Wortmannin. Adhesion mediated by alpha6beta4 induced transcription from the Fos serum response element and promoted cell cycle progression in response to mitogens. In contrast, alpha3beta1- and alpha2beta1-dependent adhesion did not induce these events. These findings suggest that the coupling of alpha6beta4 integrin to the control of cell cycle progression mediated by Shc regulates the proliferation of basal keratinocytes and possibly other cells which are in contact with the basement membrane in vivo
— id: 12317, year: 1997, vol: 16, page: 2365, stat: Journal Article,

Specific organization of the negative response elements for retinoic acid and thyroid hormone receptors in keratin gene family
Radoja N; Diaz DV; Minars TJ; Freedberg IM; Blumenberg M; Tomic-Canic M
1997 Oct;109(4):566-572, Journal of investigative dermatology
Retinoic acid and thyroid hormone are important regulators of epidermal growth, differentiation, and homeostasis. Retinoic acid is extensively used in the treatment of many epidermal disorders ranging from wrinkles to skin cancers. Retinoic acid and thyroid hormone directly control the transcription of differentiation-specific genes including keratins. Their effect is mediated through nuclear receptors RAR and T3R. We have previously identified the response element in the K14 gene, K14RARE/TRE, to which these receptors bind, and found that it consists of a cluster of five half-sites with variable spacing and orientation. To determine whether this specific structure is found in other keratin genes, we have mapped and analyzed the RARE/TRE elements in three additional epidermal keratin genes: K5, K6, and K17. We used three different approaches to identify these elements: co-transfection of promoter deletion constructs, gel-shift assays, and site-specific mutagenesis. We localized the RARE/TRE elements relatively close to the TATA box in all three promoters. All three RARE/TRE elements have a similar structural organization: they consist of clusters of 3-6 half-sites with variable spacing and orientation. This means that the clustered structure of the RARE/TREs is a common characteristic for keratin genes. RARE and TRE in the K5 promoter are adjacent to each other whereas in the K17 promoter they overlap. All three keratin REs bind specifically both RAR and T3R in gel-shift assays. Interestingly, addition of ligand to the receptor changes the binding pattern ofthe T3R from homodimer to monomer, reflecting the change in regulation from induction to inhibition
— id: 7239, year: 1997, vol: 109, page: 566, stat: Journal Article,

Regulation of epidermal expression of keratin K17 in inflammatory skin diseases
Komine M; Freedberg IM; Blumenberg M
1996 Oct;107(4):569-575, Journal of investigative dermatology
Keratin K17, the myoepithelial keratin, is expressed in psoriasis but is not present in healthy skin. Psoriasis is associated with production of gamma interferon (IFN gamma), which induces the expression of keratin K17 by activating transcription factor STAT1. Our hypothesis states that the induction of K17 is specific for the inflammatory reactions associated with high levels of IFN gamma and activation of STAT1. One of the corollaries of the hypothesis is that the STAT1-activating cytokines should induce the expression of keratin K17, whereas those cytokines that work through other mechanisms should not. Furthermore, because the STAT activation pathway is dependent upon protein phosphorylation events, phosphorylation inhibitors should attenuate the induction of keratin K17, whereas protein phosphatase inhibitors should augment it. To test this hypothesis, we analyzed lesional samples of inflammatory diseases using immunofluorescence, transfected keratinocytes with K17 gene promoter DNAs in the presence of various cytokines, and followed nuclear translocation of STAT1 in keratinocytes using specific antibodies. Confirming the hypothesis, we found that K17 is induced in psoriasis and dermatitis caused by delayed type hypersensitivity, which are associated with high levels of IFN gamma, but not in samples of atopic dermatitis, which is not. Two cytokines, interleukin-6 and leukemia inhibitory factor, which can induce phosphorylation of STAT1, can also induce K17 expression, whereas interleukin-3, interleukin-4, interleukin-10, and granulocyte macrophage colony stimulating factor have no effect on K17 expression. As expected, staurosporine and genistein inhibited, whereas okadaic acid augmented, the induction of K17 by IFN gamma. Our data indicate that in inflammatory skin diseases, lymphocytes, through the cytokines they produce, differently regulate not only each other, but also keratin gene expression in epidermis one of their target tissues
— id: 12536, year: 1996, vol: 107, page: 569, stat: Journal Article,

Characterization of nuclear protein binding sites in the promoter of keratin K17 gene
Milisavljevic V; Freedberg IM; Blumenberg M
1996 Jan;15(1):65-74, DNA & cell biology
Keratin K17, while not present in healthy skin, is expressed under various pathological conditions, including psoriasis and cutaneous allergic reactions. The regulatory circuits involved in transcription of the human keratin K17 gene are poorly understood. To begin an analysis of the molecular mechanisms that regulate K17 gene transcription, we have studied the interactions between the nuclear proteins and the promoter region of the human K17 gene. That promoter region comprised 450 bp upstream from the translation initiation site. For these studies, we used electrophoretic mobility-shift assays, computer analysis, site-directed mutagenesis, and DNA-mediated cell transfection. In addition to the previously characterized interferon-gamma-responsive elements, we identified eight protein binding sites in the promoter. Five of them bind the known transcription factors NF1, AP2, and Sp1 and three others bind still unidentified proteins. Using site-directed mutagenesis, we have demonstrated the importance of the protein binding sites for the promoter function involved in both constitutive and interferon-induced expression of the K17 keratin gene
— id: 6937, year: 1996, vol: 15, page: 65, stat: Journal Article,

Close linkage of the two keratin gene clusters in the human genome
Milisavljevic V; Freedberg IM; Blumenberg M
1996 May 15;34(1):134-138, Genomics
Mapping studies of functional keratin genes in the human genome have localized most of the acidic keratin genes to chromosome 17q12-q21 and the basic keratin genes to chromosome 12q11-q13. Within the acidic keratin locus two clusters were identified, one containing the genes for K15 and K19, the other the genes for K14, K16, and K17. The relative positions and the distance between the two clusters have not been determined previously. In this paper we describe our analysis of P1 clones containing multiple acidic keratin genes, which were studied using restriction analysis and Southern blot hybridization with PCR-amplified probes specific for functional human keratin genes 15, 17, and 19. Our results show that the two clusters are very closely linked to each other, within a 55-kb region in the human genome. The genes are organized 5' to 3' in the following order: 5'-K19-K15-K17-K16-K14. Between K15 and K17 at least one additional, unidentified keratin gene is present
— id: 8263, year: 1996, vol: 34, page: 134, stat: Journal Article,

SIGNAL-TRANSDUCTION IN EPIDERMAL-KERATINOCYTES - 2 INDEPENDENT CASCADES
BLUMENBERG, M; KOMINE, M; GORELICK, S; FREEDBERG, IM
1995 APR ;104(4):591-591, Journal of investigative dermatology
— id: 87380, year: 1995, vol: 104, page: 591, stat: Journal Article,

TGF beta promotes the basal phenotype of epidermal keratinocytes: transcriptional induction of K:5 and K:14 keratin genes
Jiang CK; Tomic-Canic M; Lucas DJ; Simon M; Blumenberg M
1995 ;12(2):87-97, Growth factors
TGFbeta is an important regulator of epidermal keratinocyte function because it suppresses cell proliferation, while it induces synthesis of extracellular matrix proteins and their cells surface receptors. To examine whether TGFbeta affects synthesis of intracellular proteins as well, specifically the transcription of keratin genes, we transfected a series of DNA constructs that contain keratin gene promoters into human epidermal keratinocytes. The transfected cells were grown in the presence and absence of TGFbeta. We found that TGFbeta specifically induces transcription controlled by the promoters of K:5 and K:14 keratin genes, markers of basal cells. No other keratin gene promoters were induced. The effect of TGFbeta is concentration-dependent, can be demonstrated in HeLa cells, does not depend on keratinocyte growth conditions and can be elicited by both TGFbeta1 and TGFbeta2. We conclude that TGFbeta promotes the basal cell phenotype in stratified epithelia such as the epidermis
— id: 8052, year: 1995, vol: 12, page: 87, stat: Journal Article,

TH-1 AND TH-2 LYMPHOCYTES DIFFERENTLY REGULATE EPIDERMAL KERATIN GENE-EXPRESSION
KOMINE, M; FREEDBERG, IM; SEMAT, A; BLUMENBERG, M
1995 APR ;104(4):586-586, Journal of investigative dermatology
— id: 87379, year: 1995, vol: 104, page: 586, stat: Journal Article,

Keratinocyte growth factor and keratin gene regulation
Latkowski JM; Freedberg IM; Blumenberg M
1995 Jan;9(1):36-44, Journal of dermatological science
Keratinocyte growth factor (KGF) is a stromally derived paracrine mitogen that belongs to the fibroblast growth factor (FGF) family. It is secreted by dermal fibroblasts and specifically promotes keratinocyte proliferation. We have recently shown that epidermal growth factor (EGF) and transforming growth factor beta (TGF beta), modulators of keratinocyte proliferation, regulate expression of specific keratin genes. However KGF, unlike EGF and TGF beta, allows keratinocytes to differentiate normally. With this in mind, we sought to determine whether KGF may be involved in keratinocyte differentiation through a mechanism that does not involve regulation of keratin gene expression. We transfected human epidermal keratinocytes with ten different keratin gene promoters linked to a reporter gene, and grew the transfected cells in the presence or absence of KGF. Interestingly, no significant change in keratin gene regulation was observed in the presence of KGF relative to control. The possibility that KGF influences the induction of keratin gene expression by other keratinocyte modulators, such as EGF, TGF beta and gamma interferon (IFN gamma), was also explored. In these experiments, the transformed keratinocytes were exposed simultaneously to KGF and another modulator. KGF did not significantly change the effects of EGF, TGF beta or IFN gamma on keratin gene expression. KGF's lack of ability to directly regulate keratin gene expression suggests that KGF affects keratinocyte growth and differentiation through a pathway independent of keratin gene regulation. These results illustrate that regulation of keratinocyte proliferation can be separated from the regulation of keratin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 6668, year: 1995, vol: 9, page: 36, stat: Journal Article,

REGULATION OF KERATIN GENE-EXPRESSION BY STEROID-HORMONES
TOMICCANIC, M; KOMINE, M; SANTIAGO, C; DIAZ, D; FREEDBERG, IM; BLUMENBERG, M
1995 APR ;104(4):593-593, Journal of investigative dermatology
— id: 87381, year: 1995, vol: 104, page: 593, stat: Journal Article,

Disease-activated transcription factor: allergic reactions in human skin cause nuclear translocation of STAT-91 and induce synthesis of keratin K17
Jiang CK; Flanagan S; Ohtsuki M; Shuai K; Freedberg IM; Blumenberg M
1994 Jul;14(7):4759-4769, Molecular & cellular biology
Epidermal keratinocytes have important immunologic functions, which is apparent during wound healing, in psoriasis, and in allergic and inflammatory reactions. In these processes, keratinocytes not only produce cytokines and growth factors that attract and affect lymphocytes but also respond to the polypeptide factors produced by the lymphocytes. Gamma interferon (IFN-gamma) is one such signaling polypeptide. Its primary molecular effect is activation of specific transcription factors that regulate gene expression in target cells. In this work, we present a molecular mechanism of lymphocyte-keratinocyte signaling in the epidermis. We have induced cutaneous delayed-type hypersensitivity reactions that are associated with an accumulation of lymphocytes. These resulted in activation and nuclear translocation of STAT-91, the IFN-gamma-activated transcription factor, in keratinocytes in vivo and subsequent induction of transcription of keratin K17. Within the promoter of the K17 keratin gene, we have identified and characterized a site that confers the responsiveness to IFN-gamma and that binds the transcription factor STAT-91. Other keratin gene promoters tested were not induced by IFN-gamma. These results characterize at the molecular level a signaling pathway produced by the infiltration of lymphocytes in skin and resulting in the specific alteration of gene expression in keratinocytes
— id: 6414, year: 1994, vol: 14, page: 4759, stat: Journal Article,

Interleukin-1 alpha is released during transfection of keratinocytes
Komine M; Freedberg IM; Blumenberg M
1994 Oct;103(4):580-582, Journal of investigative dermatology
Keratinocytes are known to produce, store, and release IL-1 alpha and therefore we suspected that the DNA-mediated cell transfection procedure may release the stored IL-1 alpha from keratinocytes into the medium. Using enzyme-linked immunosorbent assay, we determined the IL-1 alpha concentration in culture supernatants during keratinocyte transfection. The following transfection methods were compared: lipofection with lipofectACE and lipofectAMINE (GIBCO), Ca3(PO4)2 co-precipitation, and polybrene-dimethylsulfoxide (DMSO). The supernatants were collected immediately prior to transfection, after 5-h incubation with lipofectin or Ca3(PO4)2, and 24 and 48 h after transfection. In the polybrene-DMSO method, the supernatant was also collected immediately before and after DMSO shock. LipofectAMINE caused the highest release of IL-1 alpha, whereas the lipofectACE and polybrene-DMSO mediated transfection with confluent cells released the least. The other two methods released intermediate levels of IL-1 alpha. Our data indicate that a substantial amount of IL-1 alpha is released during the keratinocyte transfection procedure, which can affect the results of transfection in studies of gene expression
— id: 6663, year: 1994, vol: 103, page: 580, stat: Journal Article,

Expression of the carcinoma-associated keratin K6 and the role of AP-1 proto-oncoproteins
Bernerd F; Magnaldo T; Freedberg IM; Blumenberg M
1993 ;3(2):187-199, Gene expression
The normal pattern of keratin expression in epidermis is altered in carcinomas as well as in nonmalignant diseases such as psoriasis and wound healing. Under these circumstances, the transcription of differentiation-specific keratins K1 and K10 is suppressed, whereas the activation- and hyperproliferation-associated keratins K6 and K16 are induced. Very little is known regarding transcriptional regulators involved in this switch. To investigate the nuclear factors that participate in regulation of expression of the K6 gene, we have characterized the binding sites for nuclear proteins on the promoter DNA of the K6 gene by gel retardation assays and site-specific deletion mutagenesis. We found four nuclear protein binding sites in the K6 gene promoter. Two are near the TATA box, but their ability to bind HeLa or keratinocyte nuclear extracts is independent of the TATA box-binding protein complex. The third binding site is a large palindrome. The sequences of these three sites do not correspond to any described target sequences for characterized transcriptional factors. The fourth is an AP-1 site, the target sequence for the proto-oncoproteins fos and jun. All four sites are independent of the previously characterized epidermal growth factor-responsive element, EGF-RE. These findings suggest that there may be two parallel pathways of induction of K6 transcription. One proceeds through the EGF-RE, which may be involved in nonmalignant hyperproliferation processes; the other, through the AP-1 site and the fos-jun proto-oncoproteins, may be related to induction in malignant processes
— id: 6321, year: 1993, vol: 3, page: 187, stat: Journal Article,

Epidermal growth factor and transforming growth factor alpha specifically induce the activation- and hyperproliferation-associated keratins 6 and 16
Jiang CK; Magnaldo T; Ohtsuki M; Freedberg IM; Bernerd F; Blumenberg M
1993 Jul 15;90(14):6786-6790, Proceedings of the National Academy of Sciences of the United States of America
Epidermal injury results in activation of keratinocytes which produce and respond to growth factors and cytokines and become migratory. Activated keratinocytes express a specific pair of keratin proteins, K6 and K16, distinct from the keratins in the healthy epidermis. Keratinocytes can be activated, for example, by binding of the appropriate ligands to the epidermal growth factor receptor (EGFR). We have analyzed the effects of EGFR activation on keratin gene transcription by transfecting DNAs containing keratin promoters linked to a reporter gene into primary cultures of human epidermal keratinocytes in the presence or absence of EGF or transforming growth factor alpha (TGF alpha), two growth factors that activate EGFR. The activation of EGFR had no effect on the promoters of simple epithelial, basal-layer-specific, or differentiation-specific keratins. In contrast, the expression of K6 and K16 was strongly and specifically induced. A 20-bp DNA segment of the K16 gene promoter conveyed the EGF regulation, functioned in a heterologous construct, and therefore constituted an EGF-responsive element. A nuclear protein specifically bound to this element and to the analogous sequence of the K6 promoter. Thus, EGF specifically induces K6 and K16, markers of activated keratinocytes, via nuclear proteins that bind to EGF-responsive elements in the promoters of these keratin genes
— id: 13107, year: 1993, vol: 90, page: 6786, stat: Journal Article,

Transcriptional regulators of expression of K:16, the disease-associated keratin
Magnaldo T; Bernerd F; Freedberg IM; Ohtsuki M; Blumenberg M
1993 Dec;12(10):911-923, DNA & cell biology
In most malignant and benign skin diseases, the normal pattern of keratin expression is altered. Among other phenotypic changes, the expression of hyperproliferation- and activation-associated keratins K:16 and K:6 is induced. Because the molecular mechanisms and the nuclear regulators involved in this induction are unknown, we have characterized the transcriptional regulators of expression of the keratin K:16 promoter. Our previous studies have shown that the transcription of K:16 is strongly and specifically induced in epidermal keratinocytes by epidermal growth factor (EGF), through the EGF-responsive element (RE). In the present work, using an electrophoretic mobility-shift assay, we have found several nuclear protein binding sites that have been identified as an Sp1 site, an AP2 site, the EGF-RE, and an enhancer element. The function of each site was assessed in transfection assays using specific deletions. Both the Sp1 and EGF-RE sites are essential for K:16 promoter activity. The site that functions as an independent enhancer, E, was found adjacent to and interacting with a sequence recognized by the AP2 transcription factor. This knowledge of the nuclear regulators of expression of the disease-associated K:16 keratin provides insight into the molecular parameters that might be important in skin diseases
— id: 6444, year: 1993, vol: 12, page: 911, stat: Journal Article,

On the role of AP2 in epithelial-specific gene expression
Magnaldo T; Vidal RG; Ohtsuki M; Freedberg IM; Blumenberg M
1993 ;3(3):307-315, Gene expression
Transcription factor AP2 plays an important role in transcription of keratin genes, and it has been suggested that AP2 confers epithelial specificity. Promoters of keratin genes contain AP2 sites, usually within tight clusters of binding sites for other nuclear transcription factors. The role of AP2 was examined by in vitro gel shift analysis, AP2 binding site mutagenesis, and stable and transient transfection experiments. Nonepithelial cells, such as GM10 fibroblasts and melanocytes, neither express keratin nor become phenotypically epithelial when transfected with an AP2-expressing vector. However, in 3T3 and HeLa cells, co-transfection of an AP2-expressing vector increases the level of transcription from keratin gene promoters. This increase requires an intact AP2 binding site. Thus, the role of AP2 in keratin gene expression is quantitative rather than qualitative. AP2 interacts with other transcription factors and may convey extracellular regulatory signals to the transcription complex in the promoters of keratin genes
— id: 13339, year: 1993, vol: 3, page: 307, stat: Journal Article,

A cluster of five nuclear proteins regulates keratin gene transcription
Ohtsuki M; Flanagan S; Freedberg IM; Blumenberg M
1993 ;3(2):201-213, Gene expression
A common feature of all epithelial cells is the presence of keratin proteins that assemble into an intermediate filament cytoskeletal network. Whereas other cell types often use a specific master transcription factor to coordinate cell type-specific transcription, analysis of transcriptional regulation of keratin genes suggests that specific groupings of widely expressed transcription factors, acting on clusters of recognition elements in the promoter regions, confer epithelia-specific transcription. We define such a cluster of three sites that binds five transcription factors in the human K5 keratin gene. Within this cluster, an unusual Sp1 site binds the Sp1 transcription factor and two additional proteins. Flanking the Sp1 site are an AP2 site and another sequence, Site A; each binds a transcription factor. Similar clusters of recognition sites for the same five transcription factors have been also identified in other keratin genes. Such clusters may play a role in epithelia-specific expression of keratins
— id: 6474, year: 1993, vol: 3, page: 201, stat: Journal Article,

Nuclear proteins involved in transcription of the human K5 keratin gene
Ohtsuki M; Tomic-Canic M; Freedberg IM; Blumenberg M
1992 Aug;99(2):206-215, Journal of investigative dermatology
Keratin K5 is expressed in the basal layer of stratified epithelia in mammals and its synthesis is regulated by hormones and vitamins such as retinoic acid. The molecular mechanisms that regulate K5 expression are not known. To initiate analysis of the protein factors that interact with the human K5 keratin gene upstream region, we have used gel-retardation and DNA-mediated cell-transfection assays. We found five DNA sites that specifically bind nuclear proteins. DNA-protein interactions at two of the sites apparently increase transcription levels, at one decrease it. The importance of the remaining two sites is, at present, unclear. In addition, the location of the retinoic acid and thyroid hormone nuclear receptor action site has been determined, and we suggest that it involves a cluster of five sites similar to the consensus recognition elements. The complex constellation of protein binding sites upstream from the K5 gene probably reflects the complex regulatory circuits that govern the expression of the K5 keratin in mammalian tissues
— id: 13508, year: 1992, vol: 99, page: 206, stat: Journal Article,

Vitamin D3, its receptor and regulation of epidermal keratin gene expression
Tomic M; Jiang CK; Connolly D; Freedberg IM; Blumenberg M
1992 Apr;1(2):70-75, Epithelial cell biology
Among extrinsic modulators of keratinization are certain hormones and vitamins, which makes them potentially important pharmacological tools for treatment of keratinization disorders. Vitamin D3 and vitamin A, and their metabolites, promote and inhibit keratinization, respectively. We have shown that retinoic acid, via its nuclear receptor, directly suppresses the expression of the keratin genes which are markers of keratinocyte differentiation. Here we present evidence that 1,25(OH)2 vitamin D3 and its nuclear receptor do not directly regulate keratin gene expression. Co-transfection of a vector expressing the nuclear receptor for vitamin D3 with responder DNA constructs containing keratin gene promoters had no effect on the level of activity of keratin gene promoters either in the presence or in the absence of vitamin D3. We conclude that vitamin D3, unlike retinoic acid, modifies keratin synthesis indirectly, by changing the differentiation phenotype of the keratinocyte
— id: 13656, year: 1992, vol: 1, page: 70, stat: Journal Article,

Identification of the retinoic acid and thyroid hormone receptor-responsive element in the human K14 keratin gene
Tomic-Canic M; Sunjevaric I; Freedberg IM; Blumenberg M
1992 Dec;99(6):842-847, Journal of investigative dermatology
The promoter of human K14 keratin gene, specific for the basal layer of stratified epithelia, is regulated by nuclear receptors for retinoic acid and thyroid hormone. However, the DNA sequences responsible for this regulation have not yet been identified. To identify the retinoic acid-responsive site, we have devised a simple site-specific mutagenesis method and introduced mutations into the K14 keratin gene promoter. These mutations identify the retinoic acid-responsive site. The site consists of a cluster of consensus palindrome half-sites in various orientations. As shown previously, retinoic acid and thyroid hormone receptors can recognize and bind common sequences in regulated genes. Here, we describe mutations that abolish regulation by both receptors. Interestingly, the hormone-dependent and -independent regulatory sites of the thyroid hormone nuclear receptor can be separated. Clusters of half-sites that share structural organization with the K14 regulatory site were found in the K5 and K10 keratin gene promoters. Similar clusters may be responsible for retinoic acid-mediated transcription regulation in epidermis
— id: 13365, year: 1992, vol: 99, page: 842, stat: Journal Article,

Comparison of methods for transfection of human epidermal keratinocytes
Jiang CK; Connolly D; Blumenberg M
1991 Dec;97(6):969-973, Journal of investigative dermatology
Several methods for DNA-mediated cell transfection were tested to determine the optimal conditions for transfection of human epidermal keratinocytes. The following methods were compared: electroporation, lipofection, Ca3(PO4)2 co-precipitation, DEAE-dextran, and polybrene-mediated transfection. The transfected DNA included human keratinocyte-specific promoter for keratin K14 as well as SV40 and RSV viral promoters. Enzyme assays and in situ staining were used to evaluate both quantitative and qualitative aspects of transfection, and both subconfluent and post-confluent, stratifying keratinocytes were examined. Lipofection, Ca3(PO4)2 co-precipitation, and polybrene methods transfect very efficiently, but lipofection is expensive and Ca++ in the co-precipitation procedure induces keratinocytes to differentiate. We have found that polybrene-mediated transfection followed by a 27% DMSO shock is optimal for introducing DNA into human epidermal keratinocytes
— id: 13832, year: 1991, vol: 97, page: 969, stat: Journal Article,

Functional comparison of the upstream regulatory DNA sequences of four human epidermal keratin genes
Jiang CK; Epstein HS; Tomic M; Freedberg IM; Blumenberg M
1991 Feb;96(2):162-167, Journal of investigative dermatology
The promoters of epidermal keratin genes, K5, K6, and K10 were cloned and their functions compared with that of the previously described promoter of the K14 keratin gene in non-epithelial and transformed epithelial cell lines, as well as in primary cultures of cells derived from simple and stratified epithelia. The four promoters were functional only in epithelial cells. Although the promoter for the basal cell-specific, acidic-type K14 gene was active in all epithelial cells tested, its basic-type partner, K5, and the promoter for the hyper-proliferation-associated K6 were active only in primary cultures of stratified epithelia. The promoter for the epidermal differentiation-specific K10 keratin gene was active at a low level in primary cultures of stratified epithelial cells on non-epidermal origin. Thus, the K14 gene promoter is functional in all epithelial cells, but the upstream regions of the K5 and K6 keratin genes restrict their expression to stratified epithelia, whereas the epidermal determinants of the K10 gene are not in the proximal upstream sequences
— id: 14151, year: 1991, vol: 96, page: 162, stat: Journal Article,

Epithelial-specific keratin gene expression: identification of a 300 base-pair controlling segment
Jiang CK; Epstein HS; Tomic M; Freedberg IM; Blumenberg M
1990 Jan 25;18(2):247-253, Nucleic acids research
To elucidate the elements required for regulation of keratin expression in epidermis, we have linked a short, 300 base pair segment, corresponding to the promoter region of a human K#14 gene, to the chloramphenicol-acetyl-transferase gene. This construct was introduced into various mammalian cell lines and primary cultures via Ca3(PO4)2 precipitation. The 300 base pair segment from the keratin gene promoter region was active in all epithelial cells studied including transformed, simple epithelial cells such as HeLa and ME-180, cell lines derived from stratified epithelium, such as SCC-12, as well as primary cultures of epithelial cells. The construct was inactive in all non-epithelial cells tested including fibroblasts and melanocytes. The segment does not function as a silencer in nonepithelial cells but it can function as an enhancer in epithelial cells. Using the polymerase chain reaction we have constructed a series of deletions of the promoter and have localized an essential function within a 40 bp sequence. We conclude that we have identified the keratin gene promoter that is sufficient to confer epithelial-specific expression
— id: 16374, year: 1990, vol: 18, page: 247, stat: Journal Article,

AN ENHANCER CONTROLS HUMAN K-NUMBER-14 KERATIN GENE-EXPRESSION
Jiang, CK; Tomic, M; Epstein, H; Freedberg, IM; Blumenberg, M
1990 Apr;94(4):539-539, Journal of investigative dermatology
— id: 31998, year: 1990, vol: 94, page: 539, stat: Journal Article,

Three parallel linkage groups of human acidic keratin genes
Savtchenko ES; Tomic M; Ivker R; Blumenberg M
1990 Jul;7(3):394-407, Genomics
Two regions of human genomic DNA, each containing several keratin genes, were isolated and partially sequenced. The keratin genes are inactive, having suffered deleterious mutations. Both regions contain at least four keratin genes arranged in a head-to-tail orientation including a pseudogene for keratin K#16. Within each segment there are two keratin genes in close linkage with only 1.5 kb of DNA between them. Sequence comparison of the two regions showed 98.9% identity in both the coding and the intronic segments of the pseudogenes. The pseudogenes show 94% identity to their functional counterparts. Southern hybridization analysis showed that the segments are paralogous, not allelic. The regions are products of two independent, recent duplication events. The first occurred approximately 24 million years ago, after the separation of primates from the rhesus/baboon line. The second is specific for the human lineage, having occurred approximately 3.8 million years ago. Analysis of the genomic DNAs of primates showed the presence of only one of the regions in the DNAs of gibbon and gorilla, while rhesus monkey and baboon were missing both copies. We conclude that the human keratin genes are still actively evolving, with new duplications having occurred as recently as after the separation of human and gorilla ancestors
— id: 17108, year: 1990, vol: 7, page: 394, stat: Journal Article,

Nuclear receptors for retinoic acid and thyroid hormone regulate transcription of keratin genes
Tomic M; Jiang CK; Epstein HS; Freedberg IM; Samuels HH; Blumenberg M
1990 Nov;1(12):965-973, Cell regulation
In the epidermis, retinoids regulate the expression of keratins, the intermediate filament proteins of epithelial cells. We have cloned the 5' regulatory regions of four human epidermal keratin genes, K#5, K#6, K#10, and K#14, and engineered constructs in which these regions drive the expression of the CAT reporter gene. By co-transfecting the constructs into epithelial cells along with the vectors expressing nuclear receptors for retinoic acid (RA) and thyroid hormone, we have demonstrated that the receptors can suppress the promoters of keratin genes. The suppression is ligand dependent; it is evident both in established cell lines and in primary cultures of epithelial cells. The three RA receptors have similar effects on keratin gene transcription. Our data indicate that the nuclear receptors for RA and thyroid hormone regulate keratin synthesis by binding to negative recognition elements in the upstream DNA sequences of the keratin genes. RA thus has a twofold effect on epidermal keratin expression: qualitatively, it regulates the regulators that effect the switch from basal cell-specific keratins to differentiation-specific ones; and quantitatively, it determines the level of keratin synthesis within the cell by direct interaction of its receptors with the keratin gene promoters
— id: 14309, year: 1990, vol: 1, page: 965, stat: Journal Article,

A rapid and simple method for introducing specific mutations into any position of DNA leaving all other positions unaltered
Tomic M; Sunjevaric I; Savtchenko ES; Blumenberg M
1990 Mar 25;18(6):1656-1656, Nucleic acids research
— id: 17109, year: 1990, vol: 18, page: 1656, stat: Journal Article,

Evolution of homologous domains of cytoplasmic intermediate filament proteins and lamins
Blumenberg M
1989 Jan;6(1):53-65, Molecular biology & evolution
The earliest gene duplications in the evolution of the intermediate filament proteins created the ancestors of acidic keratins, basic keratins, nonepithelial intermediate filament proteins, and lamins. Biochemistry and function of cytoplasmic intermediate filaments differ greatly from those of lamins. Cytoplasmic intermediate filament proteins have a different cellular location than lamins, form different types of supramolecular structures, and are missing a protein segment found in lamins; but the data presented here indicate that the cytoplasmic intermediate filaments do not have a common ancestor separate from the ancestor of lamins. In the non-epithelial intermediate filament branch, the ancestor of neurofilament proteins and the common ancestor of desmin, vimentin, and glial fibrillary acidic protein (GFAP) diverged first. By evolutionary criteria, the intermediate filament protein recently discovered in neuronal cells does not belong to the neurofilament family but is more closely related to desmin, vimentin, and GFAP. Sequences of different sub-domains yield different evolutionary trees, possibly indicating existence of sub-domain-specific functions
— id: 10746, year: 1989, vol: 6, page: 53, stat: Journal Article,

EMBRYONIC EXPRESSION OF THE HUMAN 40-KD KERATIN - EVIDENCE FROM A PROCESSED PSEUDOGENE SEQUENCE - REPLY
Blumenberg, M
1989 Apr;44(4):591-591, American journal of human genetics
— id: 31734, year: 1989, vol: 44, page: 591, stat: Journal Article,

Concerted gene duplications in the two keratin gene families
Blumenberg M
1988 ;27(3):203-211, Journal of molecular evolution
Evolutionary trees were derived from the keratin protein sequences using the Phylogeny Analysis Using Parsimony (PAUP) set of programs. Three major unexpected conclusions were derived from the analysis: The smallest keratin protein subunit, K#19 (Moll et al. 1982), is not the most primitive one, but has evolved to fulfill a highly specialized function, presumably to redress the unbalanced synthesis of keratin subunits. Second, the ancestors of keratins expressed in the early embryonic stages, K#8 and K#18, were the first to diverge from the ancestors of all the other keratins. The branches leading to these two keratins are relatively short, indicating a comparatively strong selection against changes in the sequences of these two proteins. Third, the two keratin families show extraordinary parallelism in their patterns of gene duplications. In both families the genes expressed in embryos diverged first, later bursts of gene duplications created the subfamilies expressed in various differentiated cells, and relatively recent gene duplications gave rise to the hair keratin genes and separated the basal cell-specific keratin from those expressed under hyperproliferative conditions. The parallelism of gene duplications in the two keratin gene families implies a mechanism in which duplications in one family influence duplication events in the other family
— id: 17110, year: 1988, vol: 27, page: 203, stat: Journal Article,

Inactivation of human keratin genes: the spectrum of mutations in the sequence of an acidic keratin pseudogene
Savtchenko ES; Freedberg IM; Choi IY; Blumenberg M
1988 Jan;5(1):97-108, Molecular biology & evolution
Keratins are cytoskeletal proteins encoded by a multigene family. We have identified the first human keratin pseudogene and determined its complete nucleotide sequence. Sequence comparisons indicate that the pseudogene arose from a very recent duplication of the 50-kd keratin (K14) gene. The coding and the intron sequences of the two genes are 95% and 93% identical, respectively. Although the sequence of the regulatory region in the pseudogene is virtually identical to that in the 50-kd functional gene, several deleterious mutations have been identified in the pseudogene. There are three frameshifts in the coding regions, one of which is a perfect 8-bp duplication. A single-base-pair deletion in the first exon and a single-base-pair insertion in the penultimate exon also result in frameshifts. The three remaining deleterious mutations interfere with the mRNA processing signals: two alter the intron/exon boundaries, and the third disrupts the polyadenylation signal. These mutations clearly identify the sequence as a human keratin pseudogene
— id: 11291, year: 1988, vol: 5, page: 97, stat: Journal Article,

Embryonic expression of the human 40-kD keratin: evidence from a processed pseudogene sequence [see comments]
Savtchenko ES; Schiff TA; Jiang CK; Freedberg IM; Blumenberg M
1988 Nov;43(5):630-637, American journal of human genetics
Analysis of the cytoskeletal components of early murine embryos has detected expression of two keratin proteins, K#8 and K#18, at the 4-8-cell stage. Comparable data for human embryos do not exist, although several processed pseudogenes corresponding to K#8 and K#18 have been discovered in the human genome. Because only genes that are expressed in pre-germ-line and germ-line cells can give rise to processed pseudogenes, the existence of human K#8 and K#18 processed pseudogenes is prima facie evidence for expression of keratins K#8 and K#18 in the early human embryo. We have cloned and determined the complete sequence of a processed pseudogene corresponding to another acidic human keratin. Comparison of its sequence with known sequences of other mammalian keratins indicates that the pseudogene arose from a reverse transcript of a correctly initiated and terminated functional human K#19 gene. This implies expression of K#19 keratin in addition to K#8 and K#18 in the early human embryo. We have proposed previously that K#19 evolved specifically to redress unbalanced production of various basic keratins, and our current evidence, that it is expressed at an early stage of development, implies that K#19 may fulfill this same role during human embryogenesis
— id: 10923, year: 1988, vol: 43, page: 630, stat: Journal Article,

Evolution of keratin genes: different protein domains evolve by different pathways
Klinge EM; Sylvestre YR; Freedberg IM; Blumenberg M
1987 ;24(4):319-329, Journal of molecular evolution
Intermediate filaments are composed of a family of proteins that evolved from a common ancestor. The proteins consist of three domains: a central, alpha-helical domain similar in all intermediate filaments, bracketed by two domains that are variable in length and structure. Within the intermediate-filament family, several subfamilies have been recognized by immunologic and nucleic acid hybridization techniques. In this paper we present the sequence of the genomic DNA coding for a 65-kilodalton human keratin and compare it with the sequences of other intermediate-filament proteins. While the central, alpha-helical domains of these proteins show homologies that indicate a common ancestor, the sequences of the variable terminal domains indicate that the variable domains evolved through a series of tandem duplications and possibly by gene-conversion mechanisms
— id: 16375, year: 1987, vol: 24, page: 319, stat: Journal Article,

Linkage of human keratin genes
Blumenberg M; Savtchenko ES
1986 ;42(1-2):65-71, Cytogenetics & cell genetics
Two families of keratins, type I and type II, can be distinguished within the intermediate filament family of proteins, and at least 20 genes in the human genome code for the 20 known keratin proteins. In epithelial intermediate filaments, keratins from both families appear to be coordinately expressed. We have screened a library of human genomic DNA and have identified several cases of linkage among homologous and heterologous pairs of keratin genes. Genes coding for type I keratins were found linked to those coding for type II keratins. Linkage was discovered also among homologous genes coding for type I keratins and among genes encoding type II keratins. In addition, we found genes coding for glycine-rich keratins linked to genes coding for those that do not contain glycine-rich regions. Our results raise the possibility that all keratin genes are linked in a single region of the human genome
— id: 17111, year: 1986, vol: 42, page: 65, stat: Journal Article,

A systematic approach for detecting high-frequency restriction fragment length polymorphisms using large genomic probes
Feder J; Yen L; Wijsman E; Wang L; Wilkins L; Schroder J; Spurr N; Cann H; Blumenberg M; Cavalli-Sforza LL
1985 Jul;37(4):635-649, American journal of human genetics
Thirteen phage clones containing low-copy sequences were isolated from a human DNA library and tested for their ability to detect restriction fragment length polymorphisms (RFLPs). Reported are the RFLPs revealed with each clone, all found in frequencies useful for linkage studies. Cytological data are available for five of the 13 clones, with regional assignments made for three of the markers by in situ hybridization. It is concluded that phage clones containing large unique DNA inserts detect multiple RFLPs with high efficiency. An analysis of the relative efficiency of 20 restriction enzymes for detecting single nucleotide changes is discussed by comparing the observed data to those expected on the basis of recognition and potential site frequencies, as computed from the dinucleotide distribution. Finally, in an effort to facilitate linkage studies using polymorphic DNA sequences, experiments were made with pools of probes from various sources
— id: 17106, year: 1985, vol: 37, page: 635, stat: Journal Article,

Evolutionary divergence of the Citrobacter freundii tryptophan operon regulatory region: comparison with other enteric bacteria
Blumenberg M; Yanofsky C
1982 Oct;152(1):57-62, Journal of bacteriology
The regulatory region of the trp operon of Citrobacter freundii was sequenced and compared with the corresponding regions of other enteric bacteria. Significant differences were noted in the promoter region. These differences are presumably responsible for the weak expression of the cloned trp operon in Escherichia coli. The presumed operator region, although nonfunctional in E. coli, has dyad symmetry, but the sequence of the symmetrical region differs appreciably from those of operators that can be regulated by the E. coli trp repressor. The sequence of the trp leader region of C. freundii resembles that of other enteric bacteria, suggesting that the C. freundii operon is also regulated by attenuation. Comparison of the sequence of the initial portion of trpE with the homologous regions of E. coli and Salmonella typhimurium indicates that the three organisms probably are evolutionary equidistant
— id: 17112, year: 1982, vol: 152, page: 57, stat: Journal Article,

Regulatory region of the Klebsiella aerogenes tryptophan operon
Blumenberg M; Yanofsky C
1982 Oct;152(1):49-56, Journal of bacteriology
The trp operon of Klebsiella aerogenes was cloned, and its regulatory region was sequenced. Comparison with previously reported trp regulatory sequences of other enteric bacteria indicates that the K. aerogenes trp promoter-operator region is most similar to the corresponding region of Salmonella typhimurium. The trp leader regions of K. aerogenes and other enteric bacteria are organized similarly, but there are significant differences in the stabilities of the predicted secondary structures in their leader transcripts. These differences should make the K. aerogenes attenuator a weaker transcription termination site than any of the other attenuator regions studied; this was confirmed in in vitro transcription experiments. The sequence of the leader transcript and the precise site of in vitro termination were determined
— id: 17113, year: 1982, vol: 152, page: 49, stat: Journal Article,

Physical maps of Klebsiella aerogenes and Salmonella typhimurium hut genes
Blumenberg M; Magasanik B
1981 Jan;145(1):664-667, Journal of bacteriology
The recognition sites for several restriction endonucleases were mapped within deoxyribonucleic acid coding for histidine utilization (hut) genes of Salmonella typhimurium and Klebsiella aerogenes. Deoxyribonucleic acid fragments containing the two hut promoters were identified by ribonucleic acid polymerase binding
— id: 17115, year: 1981, vol: 145, page: 664, stat: Journal Article,

Comparison of the nucleoside sequence of trpA and sequences immediately beyond the trp operon of Klebsiella aerogenes. Salmonella typhimurium and Escherichia coli
Nichols BP; Blumenberg M; Yanofsky C
1981 Apr 10;9(7):1743-1755, Nucleic acids research
The nucleotide sequence of trpA of Klebsiella aerogenes is presented and compared with the trpA sequences of Salmonella typhimurium and Escherichia coli. The majority of the approximately 200 differences between each pair of trpA's are single nucleotide pair changes that do not alter the amino acid sequence. Codon usage conforms to the general patterns revealed by examination of other prokaryotic gene sequences. However, codon usage in K. aerogenes trpA reflects the high G+C content of the genome of this organism. The DNA sequences just beyond trpA, the presumed transcription termination region, are also compared for the three species. Perusal of these sequences indicates that the secondary structure of the transcript segment just beyond trpA has been preserved, while the primary sequence has diverged appreciably
— id: 17114, year: 1981, vol: 9, page: 1743, stat: Journal Article,

A study in evolution: the histidine utilization genes of enteric bacteria
Blumenberg M; Magasanik B
1979 Nov 25;135(1):23-37, Journal of molecular biology
— id: 17116, year: 1979, vol: 135, page: 23, stat: Journal Article,

Biochemical parameters of glutamine synthetase from Klebsiella aerogenes
Bender RA; Janssen KA; Resnick AD; Blumenberg M; Foor F; Magasanik B
1977 Feb;129(2):1001-1009, Journal of bacteriology
The glutamine synthetase (GS) from Klebsiella aerogenes is similar to that from Escherichia coli in several respects: (i) it is repressed by high levels of ammonia in the growth medium; (ii) its biosynthetic activity is greatly reduced by adenylylation; and (iii) adenylylation lowers the pH optimum and alters the response of the enzymes to various inhibitors in the gamma-glutamyl transferase (gammaGT) assay. There are, however, several important differences: (i) the isoactivity point for the adenylylated and non-adenylylated forms in the gammaGT assay occurs at pH 7.55 in K. aerogenes and at pH 7.15 in E. coli; (ii) the non-adenylylated form of the GS from K. aerogenes is stimulated by 60 mM MgCl2 in the gammaGT assay at pH 7.15. A biosynthetic reaction assay that correlates well with number of non-adenylylated enzyme subunits, as determined by the method of Mg2+ inhibition of the gammaGT assay, is described. Finally, we have found that it is necessary to use special methods to harvest growing cells to prevent changes in the adenylylation state of GS from occurring during harvesting
— id: 17117, year: 1977, vol: 129, page: 1001, stat: Journal Article,