Michael John Garabedian, Ph.D.

PEST-domain-enriched tyrosine phosphatase and glucocorticoids as regulators of anaphylaxis in mice
Obiri, D D; Flink, N; Maier, J V; Neeb, A; Maddalo, D; Thiele, W; Menon, A; Stassen, M; Kulkarni, R A; Garabedian, M J; Barrios, A M; Cato, A C B
2012 Feb;67(2):175-182, Allergy
To cite this article: Obiri DD, Flink N, Maier JV, Neeb A, Maddalo D, Thiele W, Menon A, Stassen M, Kulkarni RA, Garabedian MJ, Barrios AM, Cato ACB. PEST-domain-enriched tyrosine phosphatase and glucocorticoids as regulators of anaphylaxis in mice. Allergy 2012; 67: 175-182. ABSTRACT: Background: PEST-domain-enriched tyrosine phosphatase (PEP) is a protein tyrosine phosphatase exclusively expressed in hematopoietic cells. It is a potent negative regulator of T-cell receptor signalling that acts on receptor-coupled protein tyrosine kinases. PEST-domain-enriched tyrosine phosphatase is also expressed in mast cell and is positively regulated by glucocorticoids, but its function is unknown. In this communication, the function of PEP is analysed in mast cells. Methods: Signal transduction cascades following IgE receptor cross-linking were compared in bone marrow-derived mast cells (BMMC) from PEP(-/-) and PEP(+/+) mice. Furthermore, antigen-induced passive systemic anaphylaxis (PSA) was analysed in PEP(+/+) and PEP(-/-) mice. Results: Bone marrow-derived mast cells from PEP(-/-) mice showed impaired PLCgamma1 phosphorylation and Ca(2+) mobilization. Additionally, mice deficient in PEP showed impaired mast cell degranulation and were less susceptible to PSA. Treatment of wild-type BMMC or mice with an Au(I)-phosphine complex that selectively inhibits PEP activity produced defects in Ca(2+) signalling pathway and reduced anaphylaxis similar to that caused by the deletion of the PEP gene. Glucocorticoid that negatively regulates a wide range of mast cell action increased PEP expression and only partially inhibited anaphylaxis. However, glucocorticoid potently inhibited anaphylaxis when combined with the PEP inhibitor. Conclusions: PEST-domain-enriched tyrosine phosphatase is an important positive regulator of anaphylaxis. Pharmacological inhibition of its activity together with glucocorticoid administration provide an effective rescue for PSA in mice
— id: 149793, year: 2012, vol: 67, page: 175, stat: Journal Article,

Research Resource: Enhanced Genome-Wide Occupancy of Estrogen Receptor alpha by the Cochaperone p23 in Breast Cancer Cells
Simpson, Natalie E; Gertz, Jason; Imberg, Keren; Myers, Richard M; Garabedian, Michael J
2012 Jan;26(1):194-202, Molecular endocrinology
p23 is a chaperone with multiple heat shock protein 90 dependent and independent cellular functions, including stabilizing unliganded steroid receptors and modulating receptor-DNA dynamics. p23 protein is also up-regulated in several cancers, notably breast cancer. We previously demonstrated that higher expression of p23 in the estrogen-dependent breast cancer line MCF-7 (MCF-7+p23) selectively increased estrogen receptor (ER) target gene transcription and ER recruitment to regulatory elements, promoted cell invasion, and predicted a poor prognosis in breast cancer patients. To probe the impact of p23 on ER binding throughout the human genome, we compared ER occupancy in MCF-7+p23 cells relative to MCF-7-control cells by using chromatin immunoprecipitation followed by ultrahigh-throughput DNA sequencing in the absence and presence of 17beta-estradiol (E2) treatment. We found that increased expression of p23 resulted in a 230% increase in the number of E2-induced ER-binding sites throughout the genome compared with control cells and also increased ER binding under basal conditions. Motif analysis indicated that ER binds to a similar DNA sequence regardless of p23 status. We also observed that ER tends to bind closer to genes that were induced, rather than repressed by either E2 treatment or p23 overexpression. Interestingly, we also found that the increased invasion of MCF-7+p23 cells was not only p23 dependent but also ER dependent. Thus, a small increase in the expression of p23 amplifies ER-binding genome wide and, in combination with ER, elicits an invasive phenotype. This makes p23 an attractive target for combating tumor cell metastasis in breast cancer patients
— id: 148730, year: 2012, vol: 26, page: 194, stat: Journal Article,

Antidepressants increase human hippocampal neurogenesis by activating the glucocorticoid receptor
Anacker, C.; Zunszain, P. A.; Cattaneo, A.; Carvalho, L. A.; Garabedian, M. J.; Thuret, S.; Price, J.; Pariante, C. M.
2011 JUL ;16(7):738-750, Molecular psychiatry
Antidepressants increase adult hippocampal neurogenesis in animal models, but the underlying molecular mechanisms are unknown. In this study, we used human hippocampal progenitor cells to investigate the molecular pathways involved in the antidepressant-induced modulation of neurogenesis. Because our previous studies have shown that antidepressants regulate glucocorticoid receptor (GR) function, we specifically tested whether the GR may be involved in the effects of these drugs on neurogenesis. We found that treatment (for 3-10 days) with the antidepressant, sertraline, increased neuronal differentiation via a GR-dependent mechanism. Specifically, sertraline increased both immature, doublecortin (Dcx)-positive neuroblasts (+16%) and mature, microtubulin-associated protein-2 (MAP2)-positive neurons (+26%). This effect was abolished by the GR-antagonist, RU486. Interestingly, progenitor cell proliferation, as investigated by 5'-bromodeoxyuridine (BrdU) incorporation, was only increased when cells were co-treated with sertraline and the GR-agonist, dexamethasone, (+14%) an effect which was also abolished by RU486. Furthermore, the phosphodiesterase type 4 (PDE4)-inhibitor, rolipram, enhanced the effects of sertraline, whereas the protein kinase A (PKA)-inhibitor, H89, suppressed the effects of sertraline. Indeed, sertraline increased GR transactivation, modified GR phosphorylation and increased expression of the GR-regulated cyclin-dependent kinase-2 (CDK2) inhibitors, p27(Kip1) and p57(Kip2). In conclusion, our data suggest that the antidepressant, sertraline, increases human hippocampal neurogenesis via a GR-dependent mechanism that requires PKA signaling, GR phosphorylation and activation of a specific set of genes. Our data point toward an important role for the GR in the antidepressant-induced modulation of neurogenesis in humans. Molecular Psychiatry (2011) 16, 738-750; doi:10.1038/mp.2011.26; published online 12 April 2011
— id: 134895, year: 2011, vol: 16, page: 738, stat: Journal Article,

Global Functional Map of the p23 Molecular Chaperone Reveals an Extensive Cellular Network
Echtenkamp, Frank J; Zelin, Elena; Oxelmark, Ellinor; Woo, Joyce I; Andrews, Brenda J; Garabedian, Michael; Freeman, Brian C
2011 Jul 22;43(2):229-241, Molecular cell
In parallel with evolutionary developments, the Hsp90 molecular chaperone system shifted from a simple prokaryotic factor into an expansive network that includes a variety of cochaperones. We have taken high-throughput genomic and proteomic approaches to better understand the abundant yeast p23 cochaperone Sba1. Our work revealed an unexpected p23 network that displayed considerable independence from known Hsp90 clients. Additionally, our data uncovered a broad nuclear role for p23, contrasting with the historical dogma of restricted cytosolic activities for molecular chaperones. Validation studies demonstrated that yeast p23 was required for proper Golgi function and ribosome biogenesis, and was necessary for efficient DNA repair from a wide range of mutagens. Notably, mammalian p23 had conserved roles in these pathways as well as being necessary for proper cell mobility. Taken together, our work demonstrates that the p23 chaperone serves a broad physiological network and functions both in conjunction with and sovereign to Hsp90
— id: 136494, year: 2011, vol: 43, page: 229, stat: Journal Article,

HDL promotes rapid atherosclerosis regression in mice and alters inflammatory properties of plaque monocyte-derived cells
Feig, Jonathan E; Rong, James X; Shamir, Raanan; Sanson, Marie; Vengrenyuk, Yuliya; Liu, Jianhua; Rayner, Katey; Moore, Kathryn; Garabedian, Michael; Fisher, Edward A
2011 Apr 26;108(17):7166-7171, Proceedings of the National Academy of Sciences of the United States of America
HDL cholesterol (HDL-C) plasma levels are inversely related to cardiovascular disease risk. Previous studies have shown in animals and humans that HDL promotes regression of atherosclerosis. We hypothesized that this was related to an ability to promote the loss of monocyte-derived cells (CD68(+), primarily macrophages and macrophage foam cells) from plaques. To test this hypothesis, we used an established model of atherosclerosis regression in which plaque-bearing aortic arches from apolipoprotein E-deficient (apoE(-/-)) mice (low HDL-C, high non-HDL-C) were transplanted into recipient mice with differing levels of HDL-C and non-HDL-C: C57BL6 mice (normal HDL-C, low non-HDL-C), apoAI(-/-) mice (low HDL-C, low non-HDL-C), or apoE(-/-) mice transgenic for human apoAI (hAI/apoE(-/-); normal HDL-C, high non-HDL-C). Remarkably, despite persistent elevated non-HDL-C in hAI/apoE(-/-) recipients, plaque CD68(+) cell content decreased by >50% by 1 wk after transplantation, whereas there was little change in apoAI(-/-) recipient mice despite hypolipidemia. The decreased content of plaque CD68(+) cells after HDL-C normalization was associated with their emigration and induction of their chemokine receptor CCR7. Furthermore, in CD68(+) cells laser-captured from the plaques, normalization of HDL-C led to decreased expression of inflammatory factors and enrichment of markers of the M2 (tissue repair) macrophage state. Again, none of these beneficial changes were observed in the apoAI(-/-) recipients, suggesting a major requirement for reverse cholesterol transport for the beneficial effects of HDL. Overall, these results establish HDL as a regulator in vivo of the migratory and inflammatory properties of monocyte-derived cells in mouse atherosclerotic plaques, and highlight the phenotypic plasticity of these cells
— id: 131816, year: 2011, vol: 108, page: 7166, stat: Journal Article,

Statins Promote the Regression of Atherosclerosis via Activation of the CCR7-Dependent Emigration Pathway in Macrophages
Feig, Jonathan E; Shang, Yueting; Rotllan, Noemi; Vengrenyuk, Yuliya; Wu, Chaowei; Shamir, Raanan; Torra, Ines Pineda; Fernandez-Hernando, Carlos; Fisher, Edward A; Garabedian, Michael J
2011 ;6(12):e28534-e28534, PLoS ONE
HMG-CoA reductase inhibitors (statins) decrease atherosclerosis by lowering low-density-lipoprotein cholesterol. Statins are also thought to have additional anti-atherogenic properties, yet defining these non-conventional modes of statin action remains incomplete. We have previously developed a novel mouse transplant model of atherosclerosis regression in which aortic segments from diseased donors are placed into normolipidemic recipients. With this model, we demonstrated the rapid loss of CD68+ cells (mainly macrophages) in plaques through the induction of a chemokine receptor CCR7-dependent emigration process. Because the human and mouse CCR7 promoter contain Sterol Response Elements (SREs), we hypothesized that Sterol Regulatory Element Binding Proteins (SREBPs) are involved in increasing CCR7 expression and through this mechanism, statins would promote CD68+ cell emigration from plaques. We examined whether statin activation of the SREBP pathway in vivo would induce CCR7 expression and promote macrophage emigration from plaques. We found that western diet-fed apoE(-/-) mice treated with either atorvastatin or rosuvastatin led to a substantial reduction in the CD68+ cell content in the plaques despite continued hyperlipidemia. We also observed a significant increase in CCR7 mRNA in CD68+ cells from both the atorvastatin and rosuvastatin treated mice associated with emigration of CD68+ cells from plaques. Importantly, CCR7(-/-)/apoE(-/-) double knockout mice failed to display a reduction in CD68+ cell content upon statin treatment. Statins also affected the recruitment of transcriptional regulatory proteins and the organization of the chromatin at the CCR7 promoter to increase the transcriptional activity. Statins promote the beneficial remodeling of plaques in diseased mouse arteries through the stimulation of the CCR7 emigration pathway in macrophages. Therefore, statins may exhibit some of their clinical benefits by not only retarding the progression of atherosclerosis, but also accelerating its regression
— id: 146266, year: 2011, vol: 6, page: e28534, stat: Journal Article,

Modulation of human estrogen receptor alpha activity by multivalent estradiol-peptidomimetic conjugates
Holub, Justin M; Garabedian, Michael J; Kirshenbaum, Kent
2011 Feb 1;7(2):337-345, Molecular bioSystems
Estradiol-peptidomimetic conjugates (EPCs) are linear, sequence-specific peptoid oligomers that site-specifically display multiple copies of 17beta-estradiol (E2), a ligand for the human estrogen receptor alpha (hERalpha). We evaluate the ability of multivalent EPCs to activate hERalpha-mediated transcription. EPCs activated the hERalpha in both a length- and valence-dependent manner, with the highest levels of activation generated by divalent peptoid 6-mers, divalent 18-mers, and trivalent 9-mers. Hexavalent EPCs did not activate hERalpha, but instead blocked E2-mediated hERalpha activation. The physicochemical features of EPCs can be precisely tuned, which may allow the generation of a library of chemical tools for modulating specific effects of estrogens
— id: 120740, year: 2011, vol: 7, page: 337, stat: Journal Article,

Regulation of Androgen Receptor-Mediated Transcription by RPB5 Binding Protein URI/RMP
Mita, Paolo; Savas, Jeffrey N; Djouder, Nabil; Yates, John R 3rd; Ha, Susan; Ruoff, Rachel; Schafler, Eric D; Nwachukwu, Jerome C; Tanese, Naoko; Cowan, Nicholas J; Zavadil, Jiri; Garabedian, Michael J; Logan, Susan K
2011 Sep;31(17):3639-3652, Molecular & cellular biology
Androgen receptor (AR)-mediated transcription is modulated by interaction with coregulatory proteins. We demonstrate that the unconventional prefoldin RPB5 interactor (URI) is a new regulator of AR transcription and is critical for antagonist (bicalutamide) action. URI is phosphorylated upon androgen treatment, suggesting communication between the URI and AR signaling pathways. Whereas depletion of URI enhances AR-mediated gene transcription, overexpression of URI suppresses AR transcriptional activation and anchorage-independent prostate cancer cell growth. Repression of AR-mediated transcription is achieved, in part, by URI binding and regulation of androgen receptor trapped clone 27 (Art-27), a previously characterized AR corepressor. Consistent with this idea, genome-wide expression profiling in prostate cancer cells upon depletion of URI or Art-27 reveals substantially overlapping patterns of gene expression. Further, depletion of URI increases the expression of the AR target gene NKX-3.1, decreases the recruitment of Art-27, and increases AR occupancy at the NKX-3.1 promoter. While Art-27 can bind AR directly, URI is bound to chromatin prior to hormone-dependent recruitment of AR, suggesting a role for URI in modulating AR recruitment to target genes
— id: 136514, year: 2011, vol: 31, page: 3639, stat: Journal Article,

LXR{alpha} Regulates Macrophage Arginase 1 Through PU.1 and Interferon Regulatory Factor 8
Pourcet, Benoit; Feig, Jonathan E; Vengrenyuk, Yuliya; Hobbs, Adrian J; Kepka-Lenhart, Diane; Garabedian, Michael J; Morris, Sidney M Jr; Fisher, Edward A; Pineda-Torra, Ines
2011 Aug 19;109(5):492-501, Circulation research
Rationale: Activation of liver X receptors (LXRs) inhibits the progression of atherosclerosis and promotes regression of existing lesions. In addition, LXRalpha levels are high in regressive plaques. Macrophage arginase 1 (Arg1) expression is inversely correlated with atherosclerosis progression and is markedly decreased in foam cells within the lesion. Objective: To investigate LXRalpha regulation of Arg1 expression in cultured macrophages and atherosclerotic regressive lesions. Methods and Results: We found that Arg1 expression is enhanced in CD68+ cells from regressive versus progressive lesions in a murine aortic arch transplant model. In cultured macrophages, ligand-activated LXRalpha markedly enhances basal and interleukin-4-induced Arg1 mRNA and protein expression as well as promoter activity. This LXRalpha-enhanced Arg1 expression correlates with a reduction in nitric oxide levels. Moreover, Arg1 expression within regressive atherosclerotic plaques is LXRalpha-dependent, as enhanced expression of Arg1 in regressive lesions is impaired in LXRalpha-deficient CD68+ cells. LXRalpha does not bind to the Arg1 promoter but instead promotes the interaction between PU.1 and interferon regulatory factor (IRF)8 transcription factors and induces their binding of a novel composite element. Accordingly, knockdown of either IRF8 or PU.1 strongly impairs LXRalpha regulation of Arg1 expression in macrophage cells. Finally, we demonstrate that LXRalpha binds the IRF8 locus and its activation increases IRF8 mRNA and protein levels in these cells. Conclusions: This work implicates Arg1 in atherosclerosis regression and identifies LXRalpha as a novel regulator of Arg1 and IRF8 in macrophages. Furthermore, it provides a unique molecular mechanism by which LXRalpha regulates macrophage target gene expression through PU.1 and IRF8
— id: 137018, year: 2011, vol: 109, page: 492, stat: Journal Article,

LXR promotes the maximal egress of monocyte-derived cells from mouse aortic plaques during atherosclerosis regression
Feig, Jonathan E; Pineda-Torra, Ines; Sanson, Marie; Bradley, Michelle N; Vengrenyuk, Yuliya; Bogunovic, Dusan; Gautier, Emmanuel L; Rubinstein, Daniel; Hong, Cynthia; Liu, Jianhua; Wu, Chaowei; van Rooijen, Nico; Bhardwaj, Nina; Garabedian, Michael; Tontonoz, Peter; Fisher, Edward A
2010 Dec 1;120(12):4415-4424, Journal of clinical investigation
We have previously shown that mouse atherosclerosis regression involves monocyte-derived (CD68+) cell emigration from plaques and is dependent on the chemokine receptor CCR7. Concurrent with regression, mRNA levels of the gene encoding LXRalpha are increased in plaque CD68+ cells, suggestive of a functional relationship between LXR and CCR7. To extend these results, atherosclerotic Apoe-/- mice sufficient or deficient in CCR7 were treated with an LXR agonist, resulting in a CCR7-dependent decrease in plaque CD68+ cells. To test the requirement for LXR for CCR7-dependent regression, we transplanted aortic arches from atherosclerotic Apoe-/- mice, or from Apoe-/- mice with BM deficiency of LXRalpha or LXRbeta, into WT recipients. Plaques from both LXRalpha and LXRbeta-deficient Apoe-/- mice exhibited impaired regression. In addition, the CD68+ cells displayed reduced emigration and CCR7 expression. Using an immature DC line, we found that LXR agonist treatment increased Ccr7 mRNA levels. This increase was blunted when LXRalpha and LXRbeta levels were reduced by siRNAs. Moreover, LXR agonist treatment of primary human immature DCs resulted in functionally significant upregulation of CCR7. We conclude that LXR is required for maximal effects on plaque CD68+ cell expression of CCR7 and monocyte-derived cell egress during atherosclerosis regression in mice
— id: 119227, year: 2010, vol: 120, page: 4415, stat: Journal Article,

Glucocorticoid receptor DNA binding decoy is a gas
Garabedian, Michael J; Logan, Susan K
2010 ;3(108):pe5-pe5, Science signaling
The glucocorticoid receptor (GR) is a paradigmatic DNA binding transcription factor and was described over 20 years ago as one of the first proteins identified to bind the enhancer region of genes called 'response elements.' Since that time, an immense amount of work has revealed that GR transcriptional regulation is controlled at virtually every step of its activity: ligand binding, nuclear translocation, transcriptional cofactor binding, and DNA binding. Just when the major modes of GR regulation appear known, a new study provides yet another mechanism whereby GR transcriptional activity is controlled under conditions of cell growth arrest. In this case, GR activity is repressed by a small noncoding RNA (ncRNA) from the growth arrest-specific transcript 5 gene that folds into a soluble glucocorticoid response element-like sequence and serves as a decoy for GR DNA binding. This unexpected mode of regulation by nucleic acid molecular mimicry is probably not confined to GR and should spark interest in the hunt for other ncRNAs that regulate transcription factor binding to DNA
— id: 106599, year: 2010, vol: 3, page: pe5, stat: Journal Article,

Tumor Suppressor Function of Androgen Receptor Coactivator ARA70{alpha} in Prostate Cancer
Ligr, Martin; Li, Yirong; Zou, Xuanyi; Daniels, Garrett; Melamed, Jonathan; Peng, Yi; Wang, Wei; Wang, Jinhua; Ostrer, Harry; Pagano, Michele; Wang, Zhengxin; Garabedian, Michael J; Lee, Peng
2010 Apr;176(4):1891-1900, American journal of pathology
Androgen receptor (AR), a member of the steroid receptor family, is a transcription factor that has an important role in the regulation of both prostate cell proliferation and growth suppression. AR coactivators may influence the transition between cell growth and growth suppression. We have shown previously that the internally spliced ARA70 isoform, ARA70beta, promotes prostate cancer cell growth and invasion. Here we report that the full length ARA70alpha, in contrast, represses prostate cancer cell proliferation and anchorage-independent growth in vitro and inhibits tumor growth in nude mice xenograft experiments in vivo. Further, the growth inhibition by ARA70alpha is AR-dependent and mediated through induction of apoptosis rather than cell cycle arrest. Interestingly, AR with T877A mutation in LNCaP cells decreased its physical and functional interaction with ARA70alpha, facilitating the growth of LNCaP cells. This is consistent with our previous findings that ARA70alpha expression is decreased in prostate cancer cells compared with benign prostate. ARA70alpha also reduced the invasion ability of LNCaP cells. Although growth inhibition by ARA70alpha is AR-dependent, the inhibition of cell invasion is an androgen-independent process. These results strongly suggest that ARA70alpha functions as a tumor suppressor gene
— id: 107298, year: 2010, vol: 176, page: 1891, stat: Journal Article,

Androgen receptor coactivator p44/Mep50 in breast cancer growth and invasion
Peng, Yi; Li, Yirong; Gellert, Lan Lin; Zou, Xuanyi; Wang, Jun; Singh, Baljit; Xu, Ruliang; Chiriboga, Luis; Daniels, Garrett; Pan, Ruimin; Zhang, David Y; Garabedian, Michael J; Schneider, Robert J; Wang, Zhengxin; Lee, Peng
2010 Dec;14(12):2780-2789, Journal of cellular & molecular medicine
Hormones and their receptors play an important role in the development and progression of breast carcinoma. Although the primary focus has been on oestrogen and oestrogen receptor (ER), androgen, androgen receptor (AR) and its coactivator(s) have been implicated in tumorigenesis of breast carcinoma and warrant further investigation. AR coactivator p44/Mep50 is identified as a subunit of methylosome complex and lately characterized as an AR coactivator that enhances AR mediated transcription activity in a ligand dependent manner. In prostate cancer, p44 is expressed in the nucleus of benign epithelia and translocated into the cytoplasm in cancer cells. Furthermore, nuclear expression of p44 inhibits prostate cancer growth. In this report, we examined the expression and function of p44 in breast cancer. In addition to being an AR coactivator, p44 also functions as an ER coactivator. In contrast to findings in prostate cancer, the expression of p44 shows strong cytoplasmic expression in morphologically normal terminal ductal lobular units, while nuclear p44 is observed in both ductal carcinoma in situ and invasive carcinoma. Further, overexpression of nuclear-localized p44 stimulates proliferation and invasion in MCF7 breast cancer cells in the presence of oestrogen and the process is ERalpha dependent. These findings strongly suggest that p44 plays a role in mediating the effects of hormones during tumorigenesis in breast
— id: 138376, year: 2010, vol: 14, page: 2780, stat: Journal Article,

High levels of Hsp90 cochaperone p23 promote tumor progression and poor prognosis in breast cancer by increasing lymph node metastases and drug resistance
Simpson, Natalie E; Lambert, W Marcus; Watkins, Renecia; Giashuddin, Shah; Huang, S Joseph; Oxelmark, Ellinor; Arju, Rezina; Hochman, Tsivia; Goldberg, Judith D; Schneider, Robert J; Reiz, Luiz Fernando Lima; Soares, Fernando Augusto; Logan, Susan K; Garabedian, Michael J
2010 Nov 1;70(21):8446-8456, Cancer research
p23 is a heat shock protein 90 (Hsp90) cochaperone located in both the cytoplasm and nucleus that stabilizes unliganded steroid receptors, controls the catalytic activity of certain kinases, regulates protein-DNA dynamics, and is upregulated in several cancers. We had previously shown that p23-overexpressing MCF-7 cells (MCF-7+p23) exhibit increased invasion without affecting the estrogen-dependent proliferative response, which suggests that p23 differentially regulates genes controlling processes linked to breast tumor metastasis. To gain a comprehensive view of the effects of p23 on estrogen receptor (ER)-dependent and -independent gene expression, we profiled mRNA expression from control versus MCF-7+p23 cells in the absence and presence of estrogen. A number of p23-sensitive target genes involved in metastasis and drug resistance were identified. Most striking is that many of these genes are also misregulated in invasive breast cancers, including PMP22, ABCC3, AGR2, Sox3, TM4SF1, and p8 (NUPR1). Upregulation of the ATP-dependent transporter ABCC3 by p23 conferred resistance to the chemotherapeutic agents etoposide and doxorubicin in MCF-7+p23 cells. MCF-7+p23 cells also displayed higher levels of activated Akt and an expanded phosphoproteome relative to control cells, suggesting that elevated p23 also enhances cytoplasmic signaling pathways. For breast cancer patients, tumor stage together with high cytoplasmic p23 expression more accurately predicted disease recurrence and mortality than did stage alone. High nuclear p23 was found to be associated with high cytoplasmic p23, therefore both may promote tumor progression and poor prognosis by increasing metastatic potential and drug resistance in breast cancer patients
— id: 114177, year: 2010, vol: 70, page: 8446, stat: Journal Article,

Atherosclerosis Regression Promoted by an LXR Agonist is Dependent on the Chemokine Receptor CCR7 and Requires Both LXR alpha and LXR beta: Insights into Reducing Stroke Incidence
Feig, JE; Pineda-Torra, I; Garabedian, MJ; Tontonoz, P; Fisher, EA
2009 APR ;40(4):E149-E149, Stroke
— id: 97792, year: 2009, vol: 40, page: E149, stat: Journal Article,

LEF1 in androgen-independent prostate cancer: regulation of androgen receptor expression, prostate cancer growth, and invasion
Li, Yirong; Wang, Longgui; Zhang, Miao; Melamed, Jonathan; Liu, Xiaomei; Reiter, Robert; Wei, Jianjun; Peng, Yi; Zou, Xuanyi; Pellicer, Angel; Garabedian, Michael J; Ferrari, Anna; Lee, Peng
2009 Apr 15;69(8):3332-3338, Cancer research
A major obstacle in treating prostate cancer is the development of androgen-independent disease. In this study, we examined LEF1 expression in androgen-independent cancer as well as its regulation of androgen receptor (AR) expression, prostate cancer growth, and invasion in androgen-independent prostate cancer cells. Affymetrix microarray analysis of LNCaP and LNCaP-AI (androgen-independent variant LNCaP) cells revealed 100-fold increases in LEF1 expression in LNCaP-AI cells. We showed that LEF1 overexpression in LNCaP cells resulted in increased AR expression and consequently enhanced growth and invasion ability, whereas LEF1 knockdown in LNCaP-AI cells decreased AR expression and, subsequently, growth and invasion capacity. Chromatin immunoprecipitation, gel shift, and luciferase assays confirmed LEF1 occupancy and regulation of the AR promoter. Thus, we identified LEF1 as a potential marker for androgen-independent disease and as a key regulator of AR expression and prostate cancer growth and invasion. LEF1 is highly expressed in androgen-independent prostate cancer, potentially serving as a marker for androgen-independent disease
— id: 99128, year: 2009, vol: 69, page: 3332, stat: Journal Article,

Genome-wide impact of androgen receptor trapped clone-27 loss on androgen-regulated transcription in prostate cancer cells
Nwachukwu, Jerome C; Mita, Paolo; Ruoff, Rachel; Ha, Susan; Wang, Qianben; Huang, S Joseph; Taneja, Samir S; Brown, Myles; Gerald, William L; Garabedian, Michael J; Logan, Susan K
2009 Apr 1;69(7):3140-3147, Cancer research
The androgen receptor (AR) directs diverse biological processes through interaction with coregulators such as AR trapped clone-27 (ART-27). Our results show that ART-27 is recruited to AR-binding sites by chromatin immunoprecipitation analysis. In addition, the effect of ART-27 on genome-wide transcription was examined. The studies indicate that loss of ART-27 enhances expression of many androgen-regulated genes, suggesting that ART-27 inhibits gene expression. Surprisingly, classes of genes that are up-regulated upon ART-27 depletion include regulators of DNA damage checkpoint and cell cycle progression, suggesting that ART-27 functions to keep expression levels of these genes low. Consistent with this idea, stable reduction of ART-27 by short-hairpin RNA enhances LNCaP cell proliferation compared with control cells. The effect of ART-27 loss was also examined in response to the antiandrogen bicalutamide. Unexpectedly, cells treated with ART-27 siRNA no longer exhibited gene repression in response to bicalutamide. To examine ART-27 loss in prostate cancer progression, immunohistochemistry was conducted on a tissue array containing samples from primary tumors of individuals who were clinically followed and later shown to have either recurrent or nonrecurrent disease. Comparison of ART-27 and AR staining indicated that nuclear ART-27 expression was lost in the majority of AR-positive recurrent prostate cancers. Our studies show that reduction of ART-27 protein levels in prostate cancer may facilitate antiandrogen-resistant disease
— id: 99292, year: 2009, vol: 69, page: 3140, stat: Journal Article,

Genomic determination of the glucocorticoid response reveals unexpected mechanisms of gene regulation
Reddy, Timothy E; Pauli, Florencia; Sprouse, Rebekka O; Neff, Norma F; Newberry, Kimberly M; Garabedian, Michael J; Myers, Richard M
2009 Dec;19(12):2163-2171, Genome research
The glucocorticoid steroid hormone cortisol is released by the adrenal glands in response to stress and serves as a messenger in circadian rhythms. Transcriptional responses to this hormonal signal are mediated by the glucocorticoid receptor (GR). We determined GR binding throughout the human genome by using chromatin immunoprecipitation followed by next-generation DNA sequencing, and measured related changes in gene expression with mRNA sequencing in response to the glucocorticoid dexamethasone (DEX). We identified 4392 genomic positions occupied by the GR and 234 genes with significant changes in expression in response to DEX. This genomic census revealed striking differences between gene activation and repression by the GR. While genes activated with DEX treatment have GR bound within a median distance of 11 kb from the transcriptional start site (TSS), the nearest GR binding for genes repressed with DEX treatment is a median of 146 kb from the TSS, suggesting that DEX-mediated repression occurs independently of promoter-proximal GR binding. In addition to the dramatic differences in proximity of GR binding, we found differences in the kinetics of gene expression response for induced and repressed genes, with repression occurring substantially after induction. We also found that the GR can respond to different levels of corticosteroids in a gene-specific manner. For example, low doses of DEX selectively induced PER1, a transcription factor involved in regulating circadian rhythms. Overall, the genome-wide determination and analysis of GR:DNA binding and transcriptional response to hormone reveals new insights into the complexities of gene regulatory activities managed by GR
— id: 120741, year: 2009, vol: 19, page: 2163, stat: Journal Article,

Development of phosphorylation site-specific antibodies to nuclear receptors
Torra, Ines Pineda; Staverosky, Julia A; Ha, Susan; Logan, Susan K; Garabedian, Michael J
2009 ;505:221-235, Methods in molecular biology
Protein phosphorylation is a versatile posttranslational modification that can regulate nuclear receptor function. Although the precise role of receptor phosphorylation is not fully understood, it appears that it functions to direct or refine receptor activity in response to particular physiological requirements. Identifying and characterizing specific nuclear receptor phosphorylation sites is an important step in elucidating the role(s) receptor phosphorylation plays in function. Although traditional methods of metabolic labeling and in vitro protein phosphorylation have been informative, receptor phosphorylation site-specific antibodies are simple and reliable tools to study receptor phosphorylation. This chapter will discuss how to develop nuclear receptor phosphorylation site-specific antibodies to elucidate function
— id: 92774, year: 2009, vol: 505, page: 221, stat: Journal Article,

Lef1 Expression in Androgen-Independent Prostate Cancer
Zhang, M; Li, YR; Wang, LG; Melamed, J; Liu, XM; Wei, JJ; Peng, Y; Pellicer, A; Garabedian, MJ; Ferrari, A; Lee, P
2009 ;89:921-921, Laboratory investigation
— id: 104576, year: 2009, vol: 89, page: 921, stat: Journal Article,

Lef1 Expression in Androgen-Independent Prostate Cancer
Zhang, M; Li, YR; Wang, LG; Melamed, J; Liu, XM; Wei, JJ; Peng, Y; Pellicer, A; Garabedian, MJ; Ferrari, A; Lee, P
2009 ;22(Suppl 1):203A-203A 921, Modern pathology
— id: 104577, year: 2009, vol: 22, page: 203A, stat: Journal Article,

Differential recruitment of glucocorticoid receptor phospho-isoforms to glucocorticoid-induced genes
Blind, Raymond D; Garabedian, Michael J
2008 Mar;109(1-2):150-157, Journal of steroid biochemistry & molecular biology
The human glucocorticoid receptor (GR) is phosphorylated on its N-terminus at three major sites (S203, S211 and S226) within activation function 1 (AF1). Although GR has been shown to assemble at glucocorticoid responsive elements (GREs) in the presence of hormone, the timing and specificity of GR phospho-isoform recruitment to receptor target genes has not been established. Using chromatin immunoprecipitation (ChIP) and GR phosphorylation site-specific antibodies, we examined GR phospho-isoform recruitment to several glucocorticoid-induced genes including tyrosine aminotransferase (tat) and sulfonyltransferase-1A1 (sult) in rat hepatoma cells, and the glucocorticoid-induced leucine zipper (gilz) gene in human U2OS cells. GR P-S211 and GR P-S226 isoforms were efficiently recruited to the tat, sult and gilz GREs in a hormone-dependent manner. In contrast, the GR P-S203 isoform displayed no significant recruitment to any GREs of the genes analyzed, consistent with its lack of nuclear accumulation. Interestingly, the kinetics of GR P-S211 and GR P-S226 recruitment differed among genes. Our findings indicate that GR phospho-isoforms selectively occupy GR target genes, and suggests gene specific requirements for GR phosphorylation in receptor-dependent transcriptional activation
— id: 79137, year: 2008, vol: 109, page: 150, stat: Journal Article,

Glucocorticoid receptor phosphorylation differentially affects target gene expression
Chen, Weiwei; Dang, Thoa; Blind, Raymond D; Wang, Zhen; Cavasotto, Claudio N; Hittelman, Adam B; Rogatsky, Inez; Logan, Susan K; Garabedian, Michael J
2008 Aug;22(8):1754-1766, Molecular endocrinology
The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor
— id: 80349, year: 2008, vol: 22, page: 1754, stat: Journal Article,

Atherosclerosis regression promoted by an LXR agonist is dependent on the chemokine receptor CCR7 and requires both LXR alpha and LXR beta
Feig, JE; Bradley, MN; Pineda-Torra, I; Randolph, GJ; Garabedian, MJ; Tontonoz, P; Fisher, EA
2008 JUN ;28(6):E43-E43, Arteriosclerosis, thrombosis, & vascular biology
— id: 86977, year: 2008, vol: 28, page: E43, stat: Journal Article,

Atherosclerosis regression promoted by an LXR agonist is dependent on the chemokine receptor CCR7 and requires both LXR and LXR
Feig, JE; Pineda-Torra, I; Garabedian, MJ; Fisher, EA
2008 MAR 11 ;51(10):A374-A375, Journal of the American College of Cardiology
— id: 78390, year: 2008, vol: 51, page: A374, stat: Journal Article,

Atypical regulation of SRC-3
Garabedian, Michael J; Logan, Susan K
2008 Jul;33(7):301-304, Trends in biochemical sciences
Overexpression of steroid receptor coactivator 3 (SRC-3) is associated with an increased incidence of breast cancer. A recent study shows that SRC-3 is protected from proteasomal degradation by atypical protein kinase C (aPKC)-mediated phosphorylation in an estrogen receptor alpha (ERalpha)-dependent manner. This finding provides a novel mechanism for coupling increased SRC-3 expression with enhanced estrogen-dependent cellular proliferation
— id: 80347, year: 2008, vol: 33, page: 301, stat: Journal Article,

Activation of Trk neurotrophin receptors by glucocorticoids provides a neuroprotective effect
Jeanneteau, Freddy; Garabedian, Michael J; Chao, Moses V
2008 Mar 25;105(12):4862-4867, Proceedings of the National Academy of Sciences of the United States of America
Glucocorticoids (GCs) display both protective and destructive effects in the nervous system. In excess, GCs produce neuronal damage after stress or brain injury; however, the neuroprotective effects of adrenal steroids also have been reported. The mechanisms that account for the positive actions are not well understood. Here we report that GCs can selectively activate Trk receptor tyrosine kinases after in vivo administration in the brain and in cultures of hippocampal and cortical neurons. Trk receptors are normally activated by neurotrophins, such as NGF and brain-derived neurotrophic factor, but the activation of Trk receptors by GCs does not depend on increased production of neurotrophins. Other tyrosine kinase receptors, such as EGF and FGF receptors, were not activated by GCs. The ability of GCs to increase Trk receptor activity resulted in the neuroprotection of neurons deprived of trophic support and could be modulated by steroid-converting enzymes. Pharmacological and shRNA experiments indicate that Trk receptor activation by GCs depends on a genomic action of the GC receptor. The ability of GCs to promote Trk receptor activity represents a molecular mechanism that integrates the actions of GCs and neurotrophins
— id: 77790, year: 2008, vol: 105, page: 4862, stat: Journal Article,

Stromal AR inhibition of prostate cancer growth and invasion by stromal AR and association with androgen independent disease
Li, Y; Li, CX; Melamed, J; Walden, P; Peng, Y; Lepor, H; Garabedian, MJ; Lee, P
2008 ;179(4):187-187, Journal of urology
— id: 104578, year: 2008, vol: 179, page: 187, stat: Journal Article,

Decrease in stromal androgen receptor associates with androgen-independent disease and promotes prostate cancer cell proliferation and invasion
Li, Yirong; Li, Caihong X; Ye, Huihui; Chen, Fei; Melamed, Jonathan; Peng, Yi; Liu, Jinsong; Wang, Zhengxin; Tsou, Hui C; Wei, Jianjun; Walden, Paul; Garabedian, Michael J; Lee, Peng
2008 Dec;12(6B):2790-2798, Journal of cellular & molecular medicine
Androgen receptor (AR) is expressed in both stromal and epithelial cells of the prostate. The majority of studies on AR expression and function in prostate cancer is focused on malignant epithelial cells rather than stromal cells. In this study, we examined the levels of stromal AR in androgen-dependent and -independent prostate cancer and the function of stromal AR in prostate cancer growth and invasion. We showed that stromal AR levels were decreased in the areas surrounding cancerous tissue, especially in androgen-independent cancer. Using two telomerase-immortalized human stromal cell lines, one AR-positive and the other AR-negative, we demonstrated that stromal cells lacking AR stimulated cell proliferation of co-cultured prostate cancer cells in vitro and enhanced tumor growth in vivo when co-injected with PC3 epithelial cells in nude mice. In contrast, stromal cells expressing AR suppressed prostate cancer growth in vitro and in vivo. In parallel with cancer growth, in vitro invasion assays revealed that stromal cells lacking AR increased the invasion ability of PC3 cell by one order of magnitude, while stromal cells expressing AR reduced this effect. These results indicate a negative regulation of prostate cancer growth and invasion by stromal AR. This provides potentially new mechanistic insights into the failure of androgen ablation therapy, and the reactivation of stromal AR could be a novel therapeutic approach for treating hormone refractory prostate cancer
— id: 76448, year: 2008, vol: 12, page: 2790, stat: Journal Article,

Regulation of prostate cell growth through androgen receptor cofactors
Logan, SK; Nwachukwu, JC; Mita, P; Taneja, SS; Garabedian, MJ
2008 ;179(4):190-190, Journal of urology
— id: 104579, year: 2008, vol: 179, page: 190, stat: Journal Article,

Distinct nuclear and cytoplasmic functions of androgen receptor cofactor p44 and association with androgen-independent prostate cancer
Peng, Yi; Chen, Fei; Melamed, Jonathan; Chiriboga, Luis; Wei, Jianjun; Kong, Xiangtian; McLeod, Maureen; Li, Yirong; Li, Caihong X; Feng, Alice; Garabedian, Michael J; Wang, Zhengxin; Roeder, Robert G; Lee, Peng
2008 Apr 1;105(13):5236-5241, Proceedings of the National Academy of Sciences of the United States of America
Androgen receptor (AR) mediates transcriptional activation of diverse target genes through interactions with various coactivators that may alter its function and help mediate the switch between prostate cell proliferation and differentiation. We recently identified p44/MEP50 as an AR coactivator and further showed that it is expressed primarily in the nucleus and cytoplasm of benign prostate epithelial and prostate cancer cells, respectively. We also showed that haploinsufficiency in p44(+/-) mice causes prostate epithelial cell proliferation. To establish direct cause-and-effect relationships, we have used p44 fusion proteins that are selectively expressed in the nucleus or cytoplasm of prostate cancer cells (LNCaP), along with RNAi analyses, to examine effects of p44 both in vitro and in vivo (in tumor xenografts). We show that preferential expression of p44 in the nucleus inhibits proliferation of LNCaP cells in an AR-dependent manner, whereas preferential expression of p44 in the cytoplasm enhances cell proliferation. These effects appear to be mediated, at least in part, through the regulation of distinct cell-cycle regulatory genes that include p21 (up-regulated by nuclear p44) and cyclin D2 and CDK6 (up-regulated by cytoplasmic p44). Importantly, we also demonstrate that altered p44 expression is associated with androgen-independent prostate cancer. Our results indicate that nuclear p44 and cytoplasmic p44 have distinct and opposing functions in the regulation of prostate cancer cell proliferation
— id: 76450, year: 2008, vol: 105, page: 5236, stat: Journal Article,

Stimulation of prostate cancer cellular proliferation and invasion by the androgen receptor co-activator ARA70
Peng, Yi; Li, Caihong X; Chen, Fei; Wang, Zhengxin; Ligr, Martin; Melamed, Jonathan; Wei, Jianjun; Gerald, William; Pagano, Michele; Garabedian, Michael J; Lee, Peng
2008 Jan;172(1):225-235, American journal of pathology
ARA70 was first identified as a gene fused to the ret oncogene in thyroid carcinoma and subsequently as a co-activator for androgen receptor (AR). Two isoforms of ARA70 have been identified: a 70-kDa version called ARA70 alpha and an internally spliced 35-kDa variant termed ARA70 beta. We have previously reported that ARA70 alpha expression is reduced in prostate cancer, and its overexpression inhibits proliferation of LNCaP prostate cancer cells. However, the function of the ARA70 beta isoform in prostate cancer is not understood. In this report we examined the effects of ARA70 beta on AR transcriptional regulation as well as prostate cancer cellular proliferation and invasion. Although both ARA70 alpha and ARA70 beta functioned as transcriptional co-activators of AR in cell-based reporter assays, ARA70 beta overexpression, in contrast to ARA70 alpha, promoted prostate cancer cellular proliferation and invasion through Matrigel. Interestingly, genome-wide expression profiling of cells expressing ARA70 beta revealed an increase in the expression of genes involved in the control of cell division and adhesion, compatible with a role for ARA70 beta in proliferation and invasion. Consistent with its function in promoting cell growth and invasion, ARA70 beta expression was increased in prostate cancer. Our findings implicate ARA70 beta as a regulator of tumor cell growth and metastasis by affecting gene expression
— id: 76451, year: 2008, vol: 172, page: 225, stat: Journal Article,

Phosphorylation of liver X receptor alpha selectively regulates target gene expression in macrophages
Torra, Ines Pineda; Ismaili, Naima; Feig, Jonathan E; Xu, Chong-Feng; Cavasotto, Claudio; Pancratov, Raluca; Rogatsky, Inez; Neubert, Thomas A; Fisher, Edward A; Garabedian, Michael J
2008 Apr;28(8):2626-2636, Molecular & cellular biology
Dysregulation of liver X receptor alpha (LXRalpha) activity has been linked to cardiovascular and metabolic diseases. Here, we show that LXRalpha target gene selectivity is achieved by modulation of LXRalpha phosphorylation. Under basal conditions, LXRalpha is phosphorylated at S198; phosphorylation is enhanced by LXR ligands and reduced both by casein kinase 2 (CK2) inhibitors and by activation of its heterodimeric partner RXR with 9-cis-retinoic acid (9cRA). Expression of some (AIM and LPL), but not other (ABCA1 or SREBPc1) established LXR target genes is increased in RAW 264.7 cells expressing the LXRalpha S198A phosphorylation-deficient mutant compared to those with WT receptors. Surprisingly, a gene normally not expressed in macrophages, the chemokine CCL24, is activated specifically in cells expressing LXRalpha S198A. Furthermore, inhibition of S198 phosphorylation by 9cRA or by a CK2 inhibitor similarly promotes CCL24 expression, thereby phenocopying the S198A mutation. Thus, our findings reveal a previously unrecognized role for phosphorylation in restricting the repertoire of LXRalpha-responsive genes
— id: 76646, year: 2008, vol: 28, page: 2626, stat: Journal Article,

CCR7 is functionally required for atherosclerosis regression and is activated in vivo by LXR
Feig, JE; Hoffman, JR; Torra, IP; Garabedian, MJ; Fisher, EA
2007 ;27(6):E95-E95, Arteriosclerosis, thrombosis, & vascular biology
— id: 104580, year: 2007, vol: 27, page: E95, stat: Journal Article,

Atherosclerosis regression promoted by an LXR agonist is dependent on the chemokine receptor CCR7
Feig, JE; Pmeda-Torra, I; Shamir, R; Joaquin, VA; Grauer, LS; Garabedian, MJ; Fisher, EA
2007 OCT 16 ;116(16):146-146, Circulation
— id: 75965, year: 2007, vol: 116, page: 146, stat: Journal Article,

CCR7 is functionally required for atherosclerosis regression and is activated by LXR
Feig, Jonathan E; Randolph, Gwendalyn J; Garabedian, Michael J; Fisher, Edward A
2007 ;1:30-30, Probe: the publication of research on biomedical endeavors
— id: 75322, year: 2007, vol: 1, page: 30, stat: Journal Article,

Peptoids on steroids: Precise multivalent estradiol-peptidomimetic conjugates generated via azide-alkyne [3+2] cycloaddition reactions
Holub, JM; Garabedian, MJ; Kirshenbaum, K
2007 DEC ;26(11-12):1175-1180, QSAR & Combinatorial Science
We have developed a family of functionalized peptidomimetic oligomers for the multivalent display of bioactive ligands in a site-directed manner. Sequence-specific N-substituted glycine peptoid oligomer scaffolds were synthesized on solid phase to include up to six azidoalkyl sidechains. These constructs were used as substrates for Cu(I)-catalyzed azide-alkyne [3+2] cycloaddition reactions. 17 alpha-ethynylestradiol was conjugated at up to six positions along the peptoid backbone, generating estradiol-peptidomimetic conjugates in good yield. We evaluate how the binding avidities of these compounds to the estrogen receptor are enhanced when the valency of hormone ligand presentation is increased
— id: 75630, year: 2007, vol: 26, page: 1175, stat: Journal Article,

Cyclin-dependent kinase 5 differentially regulates the transcriptional activity of the glucocorticoid receptor through phosphorylation: clinical implications for the nervous system response to glucocorticoids and stress
Kino, Tomoshige; Ichijo, Takamasa; Amin, Niranjana D; Kesavapany, Sashi; Wang, Yonghong; Kim, Nancy; Rao, Sandesh; Player, Audrey; Zheng, Ya-Li; Garabedian, Michael J; Kawasaki, Ernest; Pant, Harish C; Chrousos, George P
2007 Jul;21(7):1552-1568, Molecular endocrinology
Glucocorticoids, major end effectors of the stress response, play an essential role in the homeostasis of the central nervous system and influence diverse functions of neuronal cells. We found that cyclin-dependent kinase 5 (CDK5), which plays important roles in the morphogenesis and functions of the nervous system and whose aberrant activation is associated with development of neurodegenerative disorders, interacted with the ligand-binding domain of the glucocorticoid receptor (GR) through its activator p35 or its active proteolytic fragment p25. CDK5 phosphorylated GR at multiple serines, including Ser203 and Ser211 of its N-terminal domain, and suppressed the transcriptional activity of this receptor on glucocorticoid-responsive promoters by attenuating attraction of transcriptional cofactors to DNA. In microarray analyses using rat cortical neuronal cells, the CDK5 inhibitor roscovitine differentially regulated the transcriptional activity of the GR on more than 90% of the endogenous glucocorticoid-responsive genes tested. Thus, CDK5 exerts some of its biological activities in neuronal cells through the GR, dynamically modulating GR transcriptional activity in a target promoter-dependent fashion
— id: 96450, year: 2007, vol: 21, page: 1552, stat: Journal Article,

The expression and function of androgen receptor coactivator p44 and protein arginine methyltransferase 5 in the developing testis and testicular tumors
Liang, John J; Wang, Zhengxin; Chiriboga, Luis; Greco, M Alba; Shapiro, Ellen; Huang, Hongying; Yang, Ximing J; Huang, Jiaoti; Peng, Yi; Melamed, Jonathan; Garabedian, Michael J; Lee, Peng
2007 May;177(5):1918-1922, Journal of urology
PURPOSE: The role of androgen receptor coactivators in testicular development and cancer formation is unclear. p44/Mep50 was identified as an androgen receptor coactivator that functions in a complex with protein arginine methyltransferase 5. We studied the expression of p44 and protein arginine methyltransferase 5 in developing fetal testis and adult testicular tumors, including seminomas and Leydig cell tumors. MATERIALS AND METHODS: A total of 30 human fetal testes from abortuses at a gestational age of 10 to 40 weeks, 33 human seminomas and 11 human Leydig cell tumors were retrieved from the archives of the departments of pathology. Immunohistochemistry was performed with affinity purified p44 and IgG purified protein arginine methyltransferase 5 polyclonal antibodies. RESULTS: Protein arginine methyltransferase 5 and p44 were expressed predominantly as nuclear proteins in fetal Leydig cells and human adult nonneoplastic testes, including germ cells and Leydig cells, while they were expressed in the cytoplasm of germ cells of the fetal testis. Expression was strongest in the fetal testis during the second trimester. Compared to adult nonneoplastic testes, human seminoma and Leydig tumor cells showed a marked decrease in nuclear expression of p44 and protein arginine methyltransferase 5 with a concomitant marked increase in cytoplasmic expression of these proteins. Furthermore, average testicular size was increased by 29% in p44(+/-) heterzygotic mice. CONCLUSIONS: These results suggest distinct functions of the nuclear and the p44/protein arginine methyltransferase 5 complexes in the developing fetal testis and in the oncogenesis of testicular tumors. Further studies are needed to confirm the functional relevance of these findings
— id: 72417, year: 2007, vol: 177, page: 1918, stat: Journal Article,

Transcriptional regulation of the androgen receptor cofactor androgen receptor trapped clone-27
Nwachukwu, Jerome C; Li, Wenhui; Pineda-Torra, Ines; Huang, Hong Ying; Ruoff, Rachel; Shapiro, Ellen; Taneja, Samir S; Logan, Susan K; Garabedian, Michael J
2007 Dec;21(12):2864-2876, Molecular endocrinology
Cofactors modulate nuclear receptor activity and impact human health and disease, yet surprisingly little is known about their transcriptional regulation. Androgen receptor trapped clone-27 (ART-27) is a cofactor that binds to androgen receptor (AR) amino terminus and modulates AR-dependent transcription. Interestingly, ART-27 displays both a cell type- and developmental stage-specific expression pattern. However, the cis-acting elements and trans-acting factors affecting ART-27 gene expression have not been elucidated. We found that ART-27 gene expression is repressed and its promoter is histone H3-K27 tri-methylated in human embryonic kidney cells, but not prostate cells, and the histone deacetylase inhibitor, trichostatin A, relieves this inhibition. The DNA response elements that control the induction of ART-27 gene expression were also characterized. The major cis-acting element corresponds to a consensus cAMP-responsive element (CRE) and binds the CRE-binding protein (CREB) as shown by EMSA and chromatin immunoprecipitation assays. Furthermore, ART-27 promoter activity is induced upon CREB overexpression. Epidermal growth factor, which activates CREB via phosphorylation, also induces ART-27 expression, whereas a reduction in CREB phosphorylation or expression blocks this induction in prostate cells. In human prostate development, both epithelial and stromal cells express CREB; however, active phosphorylated CREB is restricted to epithelial cells where ART-27 is expressed. Based on these findings, we propose a transcriptional regulatory circuit for the developmental expression of ART-27 that includes repression by chromatin modification through a trichostatin A-sensitive factor and activation upon growth factor stimulation via CREB
— id: 94948, year: 2007, vol: 21, page: 2864, stat: Journal Article,

Modulation of glucocorticoid receptor phosphorylation and transcriptional activity by a C-terminal-associated protein phosphatase
Wang, Zhen; Chen, Weiwei; Kono, Evelyn; Dang, Thoa; Garabedian, Michael J
2007 Mar;21(3):625-634, Molecular endocrinology
The glucocorticoid receptor (GR) is phosphorylated at three major sites on its N terminus (S203, S211, and S226), and phosphorylation modulates GR-regulatory functions in vivo. We examined the phosphorylation site interdependence, the contribution of the receptor C-terminal ligand-binding domain, and the participation of protein phosphatases in GR N-terminal phosphorylation and gene expression. We found that GR phosphorylation at S203 was greater when S226 was not phosphorylated and vice versa, indicative of intersite dependency. We also observed that a GR derivative lacking the ligand-binding domain, which no longer binds the heat shock protein 90 (Hsp90) complex, exhibits increased GR phosphorylation at all three sites as compared with the full-length receptor. A GR mutation (F602S) that produces a receptor less dependent on Hsp90 for function as well as treatment with the Hsp90 inhibitor geldanamycin also increased basal GR phosphorylation at a subset of sites. Pharmacological inhibition of serine/threonine protein phosphatases increased GR basal phosphorylation. Likewise, a reduction in protein phosphatase 5 protein levels enhanced GR phosphorylation at a subset of sites and selectively reduced the induction of endogenous GR target genes. Together, our findings suggest that GR undergoes a phosphorylation/dephosphorylation cycle that maintains steady-state receptor phosphorylation at a low basal level in the absence of ligand. Our findings also suggest that the ligand-dependent increase in GR phosphorylation results, in part, from the dissociation of a ligand-binding domain-linked protein phosphatase(s), and that changes in the intracellular concentration of protein phosphatase 5 differentially affect GR target gene expression
— id: 72536, year: 2007, vol: 21, page: 625, stat: Journal Article,

MED14 and MED1 differentially regulate target-specific gene activation by the glucocorticoid receptor
Chen, Weiwei; Rogatsky, Inez; Garabedian, Michael J
2006 Mar;20(3):560-572, Molecular endocrinology
The Mediator subunits MED14 and MED1 have been implicated in transcriptional regulation by the glucocorticoid receptor (GR) by acting through its activation functions 1 and 2. To understand the contribution of these Mediator subunits to GR gene-specific regulation, we reduced the levels of MED14 and MED1 using small interfering RNAs in U2OS-hGR osteosarcoma cells and examined the mRNA induction by dexamethasone of four primary GR target genes, interferon regulatory factor 8 (IRF8), ladinin 1, IGF-binding protein 1 (IGFBP1), and glucocorticoid-inducible leucine zipper (GILZ). We found that the GR target genes differed in their requirements for MED1 and MED14. GR-dependent mRNA expression of ladinin 1 and IRF8 required both MED1 and MED14, whereas induction of IGFBP1 mRNA by the receptor was dependent upon MED14, but not MED1. In contrast, GILZ induction by GR was largely independent of MED1 and MED14, but required the p160 cofactor transcriptional intermediary factor 2. Interestingly, we observed higher GR occupancy at GILZ than at the IGFBP1 or IRF8 glucocorticoid response element (GREs). In contrast, recruitment of MED14 compared with GR at IGFBP1 and IRF8 was higher than that observed at GILZ. At GILZ, GR and RNA polymerase II were recruited to both the GRE and the promoter, whereas at IGFBP1, RNA polymerase II occupied the promoter, but not the GRE. Thus, MED14 and MED1 are used by GR in a gene-specific manner, and the requirement for the Mediator at GILZ may be bypassed by increased GR and RNA polymerase II occupancy at the GREs. Our findings suggest that modulation of the Mediator subunit activities would provide a mechanism for promoter selectivity by GR
— id: 66678, year: 2006, vol: 20, page: 560, stat: Journal Article,

CCR7 is functionally required for atherosclerosis regression and is activated by LXR
Feig, JE; Ma, YQ; Randolph, GJ; Torra, IP; Garabedian, MJ; Fisher, EA
2006 MAY ;26(5):E50-E51, Arteriosclerosis, thrombosis, & vascular biology
— id: 63866, year: 2006, vol: 26, page: E50, stat: Journal Article,

Embryonic stage-specific inactivation of glucocorticoid receptor in thymic development results in differential postnatal immune responses
Ismaili, N; Pineda-Torra, I; Shen, YL; Littman, DR; Lee, MJ; Garabedian, MJ
2006 FEB ;13(2):203A-203A, Journal of the Society for Gynecologic Investigation
— id: 62829, year: 2006, vol: 13, page: 203A, stat: Journal Article,

First-trimester trophoblast cell model gene response to hypoxia
Koklanaris, Nikki; Nwachukwu, Jerome C; Huang, S Joseph; Guller, Seth; Karpisheva, Ksenia; Garabedian, Michael; Lee, Men-Jean
2006 Mar;194(3):687-693, American journal of obstetrics & gynecology
OBJECTIVE: Trophoblast invasion, which sets the stage for placentation and pregnancy outcome, likely occurs in a hypoxic environment. We used microarray technology in a trophoblast cell line to identify hypoxia-responsive genes that may impact placentation. STUDY DESIGN: An immortalized extravillous cytotrophoblast cell line, HTR-8/SVneo, was exposed to normoxia (20% oxygen) or hypoxia (1% oxygen) for 6 hours. Total RNA was harvested and prepared for microarray study. Quantitative reverse transcriptase polymerase chain reaction was performed for array confirmation. RESULTS: We confirmed the up- and down-regulation of 10 hypoxia-responsive genes using quantitative reverse transcriptase polymerase chain reaction. Ontologic gene categories that were found to be hypoxia-responsive included motility/migration, angiogenesis, and apoptosis. CONCLUSION: Specific genes that were found to be up-regulated in this first-trimester array (such as plasminogen activator inhibitor-1 and tissue inhibitor of metalloproteinase 3) have been described in preeclampsia. The hypoxia-responsive genes that we identified may be physiologic in early pregnancy. However, up-regulation of these same genes in later pregnancy augurs poorly
— id: 64163, year: 2006, vol: 194, page: 687, stat: Journal Article,

Transcriptional regulation of chemokine receptor CCR7 by Liver X Receptor
Ma, YQ; Feig, JE; Torra, IP; Garabedian, MJ; Fisher, EA
2006 MAY ;26(5):E99-E99, Arteriosclerosis, thrombosis, & vascular biology
— id: 63871, year: 2006, vol: 26, page: E99, stat: Journal Article,

The cochaperone p23 differentially regulates estrogen receptor target genes and promotes tumor cell adhesion and invasion
Oxelmark, Ellinor; Roth, Jennifer M; Brooks, Peter C; Braunstein, Steven E; Schneider, Robert J; Garabedian, Michael J
2006 Jul;26(14):5205-5213, Molecular & cellular biology
The cochaperone p23 plays an important role in estrogen receptor alpha (ER) signal transduction. In this study, we investigated how p23 regulates ER target gene activation and affects tumor growth and progression. Remarkably, we found that changes in the expression of p23 differentially affected the activation of ER target genes in a manner dependent upon the type of DNA regulatory element. p23 overexpression enhanced the expression of the ER target genes cathepsin D and pS2, which are regulated by direct DNA binding of ER to estrogen response elements (ERE). In contrast, the expression of other target genes, including c-Myc, cyclin D1, and E2F1, to which ER is recruited indirectly through its interaction with other transcription factors remains unaffected by changes in p23 levels. The p23-induced expression of pS2 is associated with enhanced recruitment of ER to the ERE in the promoter, whereas ER recruitment to the ERE-less c-Myc promoter does not respond to p23. Intriguingly, p23-overexpressing MCF-7 cells exhibit increased adhesion and invasion in the presence of fibronectin. Our findings demonstrate that p23 differentially regulates ER target genes and is involved in the control of distinct cellular processes in breast tumor development, thus revealing novel functions of this cochaperone
— id: 67389, year: 2006, vol: 26, page: 5205, stat: Journal Article,

Stabilization of the unliganded glucocorticoid receptor by TSG101
Ismaili, Naima; Blind, Raymond; Garabedian, Michael J
2005 Mar 25;280(12):11120-11126, Journal of biological chemistry
The glucocorticoid receptor (GR) has been shown to undergo hormone-dependent down-regulation via transcriptional, post-transcriptional, and posttranslational mechanisms. However, the mechanisms involved in modulating GR levels in the absence of hormone remain enigmatic. Here we demonstrate that TSG101, a previously identified GR-interacting protein, stabilizes the hypophosphorylated form of GR in the absence of ligand. We found that a non-phosphorylated version of GR (S203A/S211A) showed enhanced interaction with TSG101 as compared with the wild type GR, suggesting that TSG101 interacts more favorably with GR when it is not phosphorylated. A significant accumulation of GR S203A/S211A protein is detected in the absence of ligand when TSG101 is overexpressed, whereas no increase in the wild type phosphorylated GR or phosphomimetic GR S203E/S211E was observed in mammalian cells. In contrast, down-regulation of TSG101 expression by siRNA renders the hypophosphorylated form of GR unstable. We further show that TSG101 stabilizes GR by impeding its degradation by the proteasome and extending receptor half-life. Thus, in absence of a ligand, TSG101 binds GR and protects the non-phosphorylated receptor from degradation
— id: 51388, year: 2005, vol: 280, page: 11120, stat: Journal Article,

EXPRESSION AND REGULATION OF GLUCOCORTICOID RECEPTOR IN HUMAN PLACENTAL VILLOUS FIBROBLASTS
Lee, Men-Jean; Wang, Zhen; Yee, Herman; Ma, Yuehong; Swenson, Nicole; Yang, Liubin; Kadner, Susan S; Baergen, Rebecca N; Logan, Susan K; Garabedian, Michael J; Guller, Seth
2005 Nov;146(11):4619-4626, Endocrinology
The human placenta is a glucocorticoid (GC)-responsive organ consisting of multiple cell types including smooth muscle cells, fibroblasts, and trophoblast that demonstrate changes in gene expression following hormone treatment. However, little is known about the relative expression or activity of the GC receptor (GR) among the various placental cell types. Normal term human placentas were examined by immunohistochemistry using either GR phosphorylation site-specific antibodies that are markers for various activation states of the GR, or a GR antibody that recognizes the receptor independent of its phosphorylation state (total GR). We found strong total GR and phospho-GR immunoreactivity in stromal fibroblasts of terminal villi, as well as perivascular fibroblasts and vascular smooth muscle cells of the stem villi. Lower levels of both total GR and phospho-GR were found within cytotrophoblast cells relative to fibroblasts, whereas syncytiotrophoblast showed very little total GR or phospho-GR immunoreactivity. This pattern holds true by immunoblot analysis of extracts from cell fractions cultured ex vivo. In cultured placental fibroblasts, phosphorylation of GR increased upon short-term GC treatment, consistent with a role for GR phosphorylation in receptor transactivation. Total GR levels were reduced by nearly 90% following long-term hormone treatment; however, this down-regulation was independent of changes in GR mRNA levels. These findings demonstrate that GR levels in fibroblasts can be modulated by changes in hormone exposure. Such cell type-specific differences in GR protein expression and phosphorylation may provide the means of differentially regulating the GC response among the cells of the human placenta
— id: 57670, year: 2005, vol: 146, page: 4619, stat: Journal Article,

Hypoxic treament supresses homologous down-regulation of glucocorticoid receptor (GR) in human placenta
Lee, MJ; Garabedian, MJ; Ma, YH; Kadner, SS; Guller, S
2005 ;12(2):672-672, Journal of the Society for Gynecologic Investigation
— id: 104582, year: 2005, vol: 12, page: 672, stat: Journal Article,

The effect of antenatal glucocorticoid exposure on glucocorticoid receptor expression in fetal non-human primate lung at 0.7 gestation
Lee, MJ; Garabedian, MJ; Sorel, MA; Guller, S; Nathanielsz, PW
2005 ;12(2):492-492, Journal of the Society for Gynecologic Investigation
— id: 104581, year: 2005, vol: 12, page: 492, stat: Journal Article,

Androgen receptor mutations identified in prostate cancer and androgen insensitivity syndrome display aberrant ART-27 coactivator function
Li, Wenhui; Cavasotto, Claudio N; Cardozo, Timothy; Ha, Susan; Dang, Thoa; Taneja, Samir S; Logan, Susan K; Garabedian, Michael J
2005 May 26;19(9):2273-2282, Molecular endocrinology
The transcriptional activity of the androgen receptor (AR) is modulated by interactions with coregulatory molecules. It has been proposed that aberrant interactions between AR and its coregulators may contribute to diseases related to AR activity, such as prostate cancer and androgen insensitivity syndrome (AIS); however, evidence linking abnormal receptor:cofactor interactions to disease is scant. The Androgen Receptor Trapped clone-27 (ART-27) is a recently identified AR N-terminal coactivator that is associated with AR-mediated growth inhibition. Here we analyze a number of naturally occurring AR mutations identified in prostate cancer and AIS for their ability to affect AR response to ART-27. Although the vast majority of AR mutations appeared capable of increased activation in response to ART-27, an AR mutation identified in prostate cancer (AR P340L) and AIS (AR E2K) show reduced transcriptional responses to ART-27, whereas their response to the p160 class of coactivators was not diminished. Relative to the wild-type receptor, less ART-27 protein associated with the AR E2K substitution, consistent with reduced transcriptional response. Surprisingly, more ART-27 associated with AR P340L, despite the fact that the mutation decreased transcriptional activation in response to ART-27. Our findings suggest that aberrant AR-coactivator association interferes with normal ART-27 coactivator function resulting in suppression of AR activity and may contribute to the pathogenesis of diseases related to alterations in AR activity, such as prostate cancer and AIS
— id: 56038, year: 2005, vol: 19, page: 2273, stat: Journal Article,

Cell-specific regulation of androgen receptor phosphorylation in vivo
Taneja, Samir S; Ha, Susan; Swenson, Nicole K; Huang, Hong Ying; Lee, Peng; Melamed, Jonathan; Shapiro, Ellen; Garabedian, Michael J; Logan, Susan K
2005 Dec 9;280(49):40916-40924, Journal of biological chemistry
The biological ramifications of phosphorylation of the androgen receptor (AR) are largely unknown. To examine the phosphorylation of AR at serine 213, a putative substrate for Akt, a phosphorylation site-specific antibody was generated. The use of this antibody indicated that AR Ser-213 is phosphorylated in vivo and that phosphorylation is tightly regulated in a cell type-specific manner. Furthermore, Ser-213 phosphorylation took place with rapid kinetics and was inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002. Phosphorylation occurred in response to R1881 and dihydrotestosterone but weakly if at all in response to testosterone. It did not occur in response to AR antagonists or growth factor stimulation in the absence of an AR agonist. Transcription assays using an AR-responsive reporter gene construct showed that activated phosphatidylinositol 3-kinase inhibited transcription mediated by wild type AR but not that of a mutant AR variant (S213A), which could not be phosphorylated at Ser-213. By immunohistochemistry, the AR Ser(P)-213 antigen was detected in prostate epithelial but not stromal cells despite the fact that an antibody recognizing both phosphorylated and non-phosphorylated forms of AR demonstrates that AR is present in both cell types as expected. In fetal tissue the AR-Ser(P)-213 antigen was present in epithelial cells of the urogenital sinus when endogenous androgen levels were high and activated Akt was prevalent, but absent at a later stage of development when endogenous androgen levels were low and Akt activation was minimal. Immunoreactivity was evident in differentiated cells lining the lumen of the urogenital sinus but not in rapidly dividing, Ki67 positive cells within the developing prostate or stromal tissue, suggesting that site-specific phosphorylation of AR Ser-213 by cellular kinases occurs in a non-proliferating cellular milieu
— id: 61359, year: 2005, vol: 280, page: 40916, stat: Journal Article,

Modulation of glucocorticoid receptor function via phosphorylation
Ismaili, Naima; Garabedian, Michael J
2004 Jul;1024(6):86-101, Annals of the New York Academy of Sciences
The glucocorticoid receptor (GR) is phosphorylated at multiple serine residues in a hormone-dependent manner. It has been suggested that GR phosphorylation affects turnover, subcellular trafficking, or the transcriptional regulatory functions of the receptor, yet the contribution of individual GR phosphorylation sites to the modulation of GR activity remains enigmatic. This review critically evaluates the literature on GR phosphorylation and presents more recent work on the mechanism of GR phosphorylation from studies using antibodies that recognize GR only when it is phosphorylated. In addition, we present support for the notion that GR phosphorylation modifies protein-protein interactions, which can stabilize the hypophosphorylated form of the receptor in the absence of ligand, as well as facilitate transcriptional activation by the hyperphosphorylation of GR via cofactor recruitment upon ligand binding. Finally, we propose that GR phosphorylation also participates in the nongenomic activation of cytoplasmic signaling pathways evoked by GR. Thus, GR phosphorylation is a versatile mechanism for modulating and integrating multiple receptor functions
— id: 46127, year: 2004, vol: 1024, page: 86, stat: Journal Article,

Modulation of Glucocorticoid Receptor Function via Phosphorylation
Ismaili, Naima; Garabedian, Michael J
Glucocorticoid action: Basic and clinical implications New York, NY, US: New York Academy of Sciences, 2004,
(from the chapter) The glucocorticoid receptor (GR) is phosphorylated at multiple serine residues in a hormone-dependent manner. It has been suggested that GR phosphorylation affects turnover, subcellular trafficking, or the transcriptional regulatory functions of the receptor, yet the contribution of individual GR phosphorylation sites to the modulation of GR activity remains enigmatic. This review critically evaluates the literature on GR phosphorylation and presents more recent work on the mechanism of GR phosphorylation from studies using antibodies that recognize GR only when it is phosphorylated. In addition, we present support for the notion that GR phosphorylation modifies protein-protein interactions, which can stabilize the hypophosphorylated form of the receptor in the absence of ligand, as well as facilitate transcriptional activation by the hyperphosphorylation of GR via cofactor recruitment upon ligand binding. Finally, we propose that GR phosphorylation also participates in the nongenomic activation of cytoplasmic signaling pathways evoked by GR. Thus, GR phosphorylation is a versatile mechanism for modulating and integrating multiple receptor functions
— id: 4724, year: 2004, vol: , page: 86, stat: Chapter,

Regulation of glucocorticoid receptor expression in intrauterine growth restricted placentas
Lee, MJ; Garabedian, MJ; Yee, H; Ma, YH; Swenson, N; Baergen, R; Guller, S
2004 FEB ;11(2):349A-349A, Journal of the Society for Gynecologic Investigation
— id: 46695, year: 2004, vol: 11, page: 349A, stat: Journal Article,

Glucocorticoid receptor expression and function in the human placenta
Lee, MJ; Garabedian, MJ; Yee, H; Ma, YH; Yang, LB; Baergen, R; Guller, S
2004 FEB ;11(2):350A-350A, Journal of the Society for Gynecologic Investigation
— id: 46696, year: 2004, vol: 11, page: 350A, stat: Journal Article,

1,25-Dihydroxyvitamin D3 and fetal lung maturation: immunogold detection of VDR expression in pneumocytes type II cells and effect on fructose 1,6 bisphosphatase
Nguyen, M; Trubert, C L; Rizk-Rabin, M; Rehan, V K; Besancon, F; Cayre, Y E; Garabedian, M
2004 May;89-90(1-5):93-97, Journal of steroid biochemistry & molecular biology
Lung maturation before birth includes type II pneumocyte differentiation with progressive disappearance of glycogen content and onset of surfactant synthesis. We have shown previously that 1,25-(OH)2D3 increases surfactant synthesis and secretion by type II cells and decreases their glycogen content in fetal rat lung explants. Recently, the gene coding fructose 1,6 bisphosphatase (F1,6BP), a regulatory enzyme of gluconeogenesis, has been identified in type II cells and its promoter bears a Vitamin D response element. Present results show:The coexistence of type II cells at different stages of maturation. in rat fetal lung on day 21 of gestation (electron microscopy), and the association between maturation of type II cells and disappearance of their glycogen content. The immunogold labeling of all type II cells when using the 9A7g VDR-antibody, with significantly more abundant gold particles in cells exhibiting an intermediate glycogen content. The expression of F1,6BP mRNA in a human type II cell line (NCI-H441) and the increase of this expression after 18h incubation with 1,25-(OH)2D3 (10(-8)M). These results bring further evidence for a physiological role of 1,25-(OH)2D3 during type II pneumocyte maturation. Activation of F1,6BP may participate to the 1,25-(OH)2D3 action on surfactant synthesis via the gluconeogenesis pathway
— id: 44730, year: 2004, vol: 89-90, page: 93, stat: Journal Article,

Identification of DRIP205 as a coactivator for the Farnesoid X receptor
Pineda Torra, Ines; Freedman, Leonard P; Garabedian, Michael J
2004 Aug 27;279(35):36184-36191, Journal of biological chemistry
Farnesoid X receptor (FXR) is a bile acid sensor that regulates the expression of a number of genes the products of which control bile acid and cholesterol homeostasis; however, the role of DRIP205 in FXR-mediated gene regulation remains unexplored. In this study we demonstrate that DRIP205 binds FXR in a ligand-dependent manner in vitro and in vivo. Glutathione S-transferase pull-down assays showed that DRIP205 binds FXR in response to bile acid ligands in a dose-dependent fashion and that the potency of this interaction is associated with the ability of the ligand to activate FXR. In addition, the FXR-DRIP205 interaction required the presence of an intact LXXLL nuclear receptor box 1 (N-terminal) motif of DRIP205. In gel shift assays FXR was also able to recruit DRIP205 in the context of a DNA-bound FXR/RXR (retinoid X receptor) heterodimer. In transient transfection assays, DRIP205 efficiently enhanced a bile acid-activated FXRE-driven reporter gene in a dose-dependent manner in cells overexpressing FXR/RXR, demonstrating that DRIP205 enhances FXR-mediated transactivation. By contrast, an FXRW469A mutant in the activation function 2 domain that does not bind to DRIP205 was unable to activate ligand-stimulated FXR transcription, indicating that DRIP205 is recruited to activation function 2 of FXR. Requirement for the FXR/RXR heterodimer in the DRIP205-FXR interaction was evaluated using an RXR heterodimerization-deficient FXR mutant (FXRL433R). FXRL433R was not able to bind to DRIP205 and failed to enhance an FXRE-driven reporter gene. In addition, DRIP205 was unable to induce FXR-mediated transactivation in the absence of RXR overexpression, indicating that FXR heterodimerization with RXR is required for coactivation by DRIP205. Finally, in HepG2 cells, overexpression or reduction of DRIP205 levels modulated the induction of endogenous FXR target gene mRNA expression by ligand. Together, these results demonstrate that DRIP205 acts as a bona fide coactivator of FXR and underscore the importance of DRIP205 in modulating the bile acid response of FXR target genes
— id: 44731, year: 2004, vol: 279, page: 36184, stat: Journal Article,

Inhibition of glucocorticoid receptor-mediated transcriptional activation by p38 mitogen-activated protein (MAP) kinase
Szatmary, Zoltan; Garabedian, Michael J; Vilcek, Jan
2004 Oct 15;279(42):43708-43715, Journal of biological chemistry
Tumor necrosis factor (TNF) promotes certain immune and inflammatory responses, whereas glucocorticoids exert immunosuppressive and anti-inflammatory actions. We show that TNF treatment produced a modest inhibition of glucocorticoid receptor (GR)-mediated transcriptional activation of a mouse mammary tumor virus (MMTV) promoter-driven luciferase construct in HeLa cells. The mitogen-activated protein (MAP) kinases, p38 and c-Jun N-terminal kinase (JNK), are important mediators of target gene activation by TNF, and JNK activation was earlier shown to inhibit GR-mediated transcriptional activation by direct phosphorylation of GR at Ser-246. Transfection of HeLa cells with MKK6b(E), a constitutively active specific upstream activator of p38, led to a potent inhibition of GR activation of the MMTV promoter-driven luciferase construct. A similar inhibition of activation of the MMTV promoter-driven luciferase construct was seen in HeLa cells transfected with MKK7(D), a constitutively functional activator of JNK. Data from 'domain swap' experiments using GR chimeras indicated that the main target of the p38-mediated (but not JNK-mediated) inhibition is the ligand-binding domain of GR (spanning amino acids 525-795), whereas the constitutively active N-terminal AF-1 region (spanning amino acids 106-237) is dispensable for the inhibitory effect of p38. We also demonstrate that activated p38 targets the GR ligand-binding domain indirectly. Suppression of GR function by activated p38 and JNK MAP kinases may be physiologically important as a mechanism of resistance to glucocorticoids seen in many patients with chronic inflammatory conditions
— id: 48208, year: 2004, vol: 279, page: 43708, stat: Journal Article,

ART-27, an androgen receptor coactivator regulated in prostate development and cancer
Taneja, Samir S; Ha, Susan; Swenson, Nicole K; Torra, Ines Pineda; Rome, Serge; Walden, Paul D; Huang, Hong Ying; Shapiro, Ellen; Garabedian, Michael J; Logan, Susan K
2004 Apr 2;279(14):13944-13952, Journal of biological chemistry
Androgen receptor trapped clone-27 (ART-27) is a newly described transcriptional coactivator that binds to the N terminus of the androgen receptor (AR). Given the vital importance of AR signaling in prostate growth and differentiation, we investigated the role of ART-27 in these processes. Immunohistochemical studies indicate that ART-27 protein is expressed in differentiated epithelial cells of adult human prostate and breast tissue. In prostate, ART-27 is abundant in AR-positive prostate luminal epithelial cells, in contrast to the stroma, where cells express AR but not ART-27. The use of a rat model of androgen depletion/reconstitution indicates that ART-27 expression is associated with the elaboration of differentiated prostate epithelial cells. Interestingly, regulated expression of ART-27 in the androgen-sensitive LNCaP prostate cancer cell line inhibits androgen-mediated cellular proliferation and enhances androgen-mediated transcription of the prostate-specific antigen (PSA) gene. Consistent with a growth suppressive function, we show that ART-27 expression levels are negligible in human prostate cancer. Importantly, examination of ART-27 protein expression in early fetal prostate development demonstrates that ART-27 is detected only when the developing prostate gland has proceeded from a solid mass of undifferentiated cells to a stage in which differentiated luminal epithelial cells are evident. Thus, ART-27 is an AR cofactor shown to be subject to both cell type and developmental regulation in humans. Overall, the results suggest that decreased levels of ART-27 protein in prostate cancer tissue may occur as a result of de-differentiation, and indicate that ART-27 is likely to regulate a subset of AR-responsive genes important to prostate growth suppression and differentiation
— id: 44732, year: 2004, vol: 279, page: 13944, stat: Journal Article,

The liver X receptor - alpha is phosphorylated at Ser207
Torra, IP; Garabedian, MJ
2004 OCT 26 ;110(17):210-211, Circulation
— id: 55939, year: 2004, vol: 110, page: 210, stat: Journal Article,

Glucocorticoid receptor expression in fetal non-human primate lung
Lynch, D; Lee, MJ; Natha-Nielsz, P; Guller, S; Swenson, N; Garabe-Dian, M
2003 DEC ;189(6):S162-S162, American journal of obstetrics & gynecology
— id: 42535, year: 2003, vol: 189, page: S162, stat: Journal Article,

Genetic dissection of p23, an Hsp90 cochaperone, reveals a distinct surface involved in estrogen receptor signaling
Oxelmark, Ellinor; Knoblauch, Roland; Arnal, Suzzette; Su, Laura F; Schapira, Matthieu; Garabedian, Michael J
2003 Sep 19;278(38):36547-36555, Journal of biological chemistry
p23 is an Hsp90-associated protein that regulates signal transduction by the estrogen receptor alpha (ER); however, the mechanism through which p23 governs ER function remains enigmatic. To obtain a collection of p23 molecules with distinct effects on ER signaling, we screened in yeast a series of random mutations as well as specific sequence alterations based on the p23 crystal structure and further analyzed these mutations for their effect on p23-Hsp90 association in vitro and in vivo. We found that the ability of the p23 mutants to decrease or increase ER signal transduction correlated with their association with Hsp90. We also identified a mutation in the C-terminal tail of p23, which displayed a dominant inhibitory effect on ER transcriptional activation and associates more avidly with Hsp90 relative to the wild type p23. Interestingly, this mutant interacts with Hsp90 in its non-ATP-bound state, whereas the wild type p23 protein interacts exclusively with the ATP-bound form of Hsp90, which may account for its dominant phenotype. In addition, we have uncovered a novel activity of p23 that antagonizes Hsp90 action during times of cell stress. Using molecular modeling and the p23 crystal structure, we found that the p23 mutations affecting ER signaling identified in the screen localized to one face of the molecule, whereas those that had no effect mapped to other parts of the protein. Thus, our structure/function analysis has identified an important regulatory surface on p23 involved in ER signaling and p23 binding to Hsp90
— id: 44735, year: 2003, vol: 278, page: 36547, stat: Journal Article,

Target-specific utilization of transcriptional regulatory surfaces by the glucocorticoid receptor
Rogatsky, Inez; Wang, Jen-Chywan; Derynck, Mika K; Nonaka, Daisuke F; Khodabakhsh, Daniel B; Haqq, Christopher M; Darimont, Beatrice D; Garabedian, Michael J; Yamamoto, Keith R
2003 Nov 25;100(24):13845-13850, Proceedings of the National Academy of Sciences of the United States of America
The glucocorticoid receptor (GR) activates or represses transcription depending on the sequence and architecture of the glucocorticoid response elements in target genes and the availability and activity of interacting cofactors. Numerous GR cofactors have been identified, but they alone are insufficient to dictate the specificity of GR action. Furthermore, the role of different functional surfaces on the receptor itself in regulating its targets is unclear, due in part to the paucity of known target genes. Using DNA microarrays and real-time quantitative PCR, we identified genes transcriptionally activated by GR, in a translation-independent manner, in two human cell lines. We then assessed in U2OS osteosarcoma cells the consequences of individually disrupting three GR domains, the N-terminal activation function (AF) 1, the C-terminal AF2, or the dimer interface, on activation of these genes. We found that GR targets differed in their requirements for AF1 or AF2, and that the dimer interface was dispensable for activation of some genes in each class. Thus, in a single cell type, different GR surfaces were used in a gene-specific manner. These findings have strong implications for the nature of gene response element signaling, the composition and structure of regulatory complexes, and the mechanisms of context-specific transcriptional regulation
— id: 44733, year: 2003, vol: 100, page: 13845, stat: Journal Article,

Modulation of glucocorticoid receptor transcriptional activation, phosphorylation, and growth inhibition by p27Kip1
Wang, Zhen; Garabedian, Michael J
2003 Dec 19;278(51):50897-50901, Journal of biological chemistry
The cyclin-dependent kinase inhibitor p27Kip1 is frequently inactivated in human cancers. Glucocorticoids, acting through the glucocorticoid receptor (GR), are frequently used to treat certain malignancies and are growth inhibitive, but the relationship between GR activity and p27 status has not been explored. We have therefore examined GR-dependent transcriptional activation, receptor phosphorylation, and glucocorticoid-dependent growth inhibition in p27-deficient (p27-/-) murine embryonic fibroblasts (MEFs). We find that GR transcriptional enhancement as well as receptor phosphorylation at two putative cyclin-dependent kinase sites are elevated in p27-/- MEFs, relative to control cells. This increased GR transcriptional activation appears to be mediated through the GR N terminus, and coexpression of the GR N-terminal coactivator, DRIP150, further enhanced GR-dependent transcriptional activation. Furthermore, p27-/- MEFs are partially resistant to the growth inhibitory effects of glucocorticoids. Thus, p27 appears to be an important element in the GR transcription and growth inhibitory responses
— id: 44734, year: 2003, vol: 278, page: 50897, stat: Journal Article,

The dentino-enamel junction revisited
Goldberg, M; Septier, D; Bourd, K; Hall, R; Jeanny, J C; Jonet, L; Colin, S; Tager, F; Chaussain-Miller, C; Garabedian, M; George, A; Goldberg, H; Menashi, S
2002 ;43(2-3):482-489, Connective tissue research
The dentino-enamel junction is not an simple inert interface between two mineralized structures. A less simplistic view suggests that the dentino-enamel junctional complex should also include the inner aprismatic enamel and the mantle dentin. At early stages of enamel formation, fibroblast growth factor (FGF)-2 is stored in and released from the inner aprismatic enamel, possibly under the control of matrix metalloproteinase (MMP)-3. The concentration peak for MMP-2 and -9 observed in the mantle dentin coincided with a very low labeling for TIMP-1 and -2, favoring the cross-talk between mineralizing epithelial and connective structures, and as a consequence the translocation of enamel proteins toward odontoblasts and pulp cells, and vice versa, the translocation of dentin proteins toward secretory ameloblasts and cells of the enamel organ. Finally, in X-linked hypophosphatemic rickets, large interglobular spaces in the circumpulpal dentin were the major defect induced by the gene alteration, whereas the mantle dentin was constantly unaffected. Altogether, these data plead for the recognition of the dentino-enamel junctional complex as a specific entity bearing its own biological characteristics
— id: 44736, year: 2002, vol: 43, page: 482, stat: Journal Article,

ART-27, a novel androgen receptor (AR) interacting protein, is a potential. mediator of prostate eptihelial differentiation
Logan, S; Ha, S; Rome, S; Garabedian, MJ; Taneja, SS
2002 ;167(4):56-56, Journal of urology
— id: 104583, year: 2002, vol: 167, page: 56, stat: Journal Article,

Identification and characterization of ART-27, a novel coactivator for the androgen receptor N terminus
Markus, Steven M; Taneja, Samir S; Logan, Susan K; Li, Wenhui; Ha, Susan; Hittelman, Adam B; Rogatsky, Inez; Garabedian, Michael J
2002 Feb;13(2):670-682, Molecular biology of the cell
The androgen receptor (AR) is a ligand-regulated transcription factor that stimulates cell growth and differentiation in androgen-responsive tissues. The AR N terminus contains two activation functions (AF-1a and AF-1b) that are necessary for maximal transcriptional enhancement by the receptor; however, the mechanisms and components regulating AR transcriptional activation are not fully understood. We sought to identify novel factors that interact with the AR N terminus from an androgen-stimulated human prostate cancer cell library using a yeast two-hybrid approach designed to identify proteins that interact with transcriptional activation domains. A 157-amino acid protein termed ART-27 was cloned and shown to interact predominantly with the AR(153-336), containing AF-1a and a part of AF-1b, localize to the nucleus and increase the transcriptional activity of AR when overexpressed in cultured mammalian cells. ART-27 also enhanced the transcriptional activation by AR(153-336) fused to the LexA DNA-binding domain but not other AR N-terminal subdomains, suggesting that ART-27 exerts its effect via an interaction with a defined region of the AR N terminus. ART-27 interacts with AR in nuclear extracts from LNCaP cells in a ligand-independent manner. Interestingly, velocity gradient sedimentation of HeLa nuclear extracts suggests that native ART-27 is part of a multiprotein complex. ART-27 is expressed in a variety of human tissues, including sites of androgen action such as prostate and skeletal muscle, and is conserved throughout evolution. Thus, ART-27 is a novel cofactor that interacts with the AR N terminus and plays a role in facilitating receptor-induced transcriptional activation
— id: 39704, year: 2002, vol: 13, page: 670, stat: Journal Article,

Regulation of GRIP1 and CBP Coactivator activity by Rho GDI modulates estrogen receptor transcriptional enhancement
Su, Laura F; Wang, Zhen; Garabedian, Michael J
2002 Oct 4;277(40):37037-37044, Journal of biological chemistry
Estrogen receptor alpha (ER) coordinates gene expression with cellular physiology in part by controlling receptor- cofactor interactions in response to extracellular signals. We have previously shown that the Rho signaling pathway modulates ER transcriptional activation. We now demonstrate that Rho GDI-dependent increase in ER transactivation is dependent on the ER AF-2 coactivator binding site, prompting us to examine regulation of receptor coactivators by Rho GDI. Indeed, Rho GDI cooperates with GRIP1 to increase ER ligand-independent and ligand-dependent transactivation and also enhances GRIP1 transcriptional activity when GRIP1 is tethered to DNA. The GRIP1 activation domain 1 (AD1), which binds CBP/p300, is necessary for Rho GDI to modulate GRIP1 activity. Using E1A to inhibit the endogenous CBP/p300 and a Gal4-CBP fusion protein to assay CBP activity, we find that the effect of Rho GDI on ER transactivation is CBP/p300-dependent. Importantly, the ability of CBP/p300 to transduce the Rho GDI signal to ER occurs through both GRIP1-dependent and -independent pathways. These data suggest a complex interplay between ER transcriptional activation and the Rho signaling pathways through modulation of receptor cofactors, which may have evolved to coordinate receptor-dependent gene expression with Rho-regulated events, such as cell migration. We speculate that dysregulation of the Rho-ER axis may participate in cancer progression
— id: 39613, year: 2002, vol: 277, page: 37037, stat: Journal Article,

Deciphering the phosphorylation "code" of the glucocorticoid receptor in vivo
Wang, Zhen; Frederick, Jeremy; Garabedian, Michael J
2002 Jul 19;277(29):26573-26580, Journal of biological chemistry
The glucocorticoid receptor (GR) is phosphorylated at multiple serine residues in a hormone-dependent manner, yet progress on elucidating the function of GR phosphorylation has been hindered by the lack of a simple assay to detect receptor phosphorylation in vivo. We have produced antibodies that specifically recognize phosphorylation sites within human GR at Ser(203) and Ser(211). In the absence of hormone, the level of GR phosphorylation at Ser(211) was low compared with phosphorylation at Ser(203). Phosphorylation of both residues increased upon treatment with the GR agonist dexamethasone. Using a battery of agonists and antagonists, we found that the transcriptional activity of GR correlated with the amount of phosphorylation at Ser(211), suggesting that Ser(211) phosphorylation is a biomarker for activated GR in vivo. Mechanistically, the kinetics of Ser(203) and Ser(211) phosphorylation in response to hormone differed, with Ser(211) displaying a more robust and sustained phosphorylation relative to Ser(203). Analysis of GR immunoprecipitates with phospho-GR-specific antibodies indicated that the receptor was phosphorylated heterogeneously at Ser(203) in the absence of hormone, whereas in the presence of hormone, a subpopulation of receptors was phosphorylated at both Ser(203) and Ser(211). Interestingly, biochemical fractionation studies following hormone treatment indicated that the Ser(203)-phosphorylated form of the receptor was predominantly cytoplasmic, whereas Ser(211)-phosphorylated GR was found in the nucleus. Likewise, by immunofluorescence, Ser(203)-phosphorylated GR was located in the cytoplasm and perinuclear regions of the cell, but not in the nucleoplasm, whereas strong phospho-Ser(211) staining was evident in the nucleoplasm of hormone-treated cells. Our results suggest that differentially phosphorylated receptor species are located in unique subcellular compartments, likely modulating distinct aspects of receptor function
— id: 39648, year: 2002, vol: 277, page: 26573, stat: Journal Article,

Rho GTPases as modulators of the estrogen receptor transcriptional response
Su LF; Knoblauch R; Garabedian MJ
2001 Feb 2;276(5):3231-3237, Journal of biological chemistry
The estrogen receptor alpha (ER) is a ligand-dependent transcription factor that plays a critical role in the development and progression of breast cancer, in part, by regulating target genes involved in cellular proliferation. To identify novel components that affect the ER transcriptional response, we performed a genetic screen in yeast and identified RDI1, a Rho guanine nucleotide dissociation inhibitor (Rho GDI), as a positive regulator of ER transactivation. Overexpression of the human homologue of RDI1, Rho GDIalpha, increases ERalpha, ERbeta, androgen receptor, and glucocorticoid receptor transcriptional activation in mammalian cells but not activation by the unrelated transcription factors serum response factor and Sp1. In contrast, expression of constitutively active forms of RhoA, Rac1, and Cdc42 decrease ER transcriptional activity, suggesting that Rho GDI increases ER transactivation by antagonizing Rho function. Inhibition of RhoA by expression of either the Clostridium botulinum C3 transferase or a dominant negative RhoA resulted in enhanced ER transcriptional activation, thus phenocopying the effect of Rho GDI expression on ER transactivation. Together, these findings establish the Rho GTPases as important modulators of ER transcriptional activation. Since Rho GTPases regulate actin polymerization, our findings suggest a link between the major regulators of cellular architecture and steroid receptor transcriptional response
— id: 48157, year: 2001, vol: 276, page: 3231, stat: Journal Article,

Androgen stimulated cellular proliferation in the human prostate cancer cell line LNCaP is associated with reduced retinoblastoma protein expression
Taneja SS; Ha S; Garabedian MJ
2001 ;84(1):188-199, Journal of cellular biochemistry
To elucidate the mechanism of androgen-dependent cellular proliferation in prostate cancer, androgen-dependent alterations of individual cell cycle regulatory proteins in the androgen-sensitive prostate cancer cell line LNCaP were evaluated. LNCaP cells were deprived of androgens by culture in steroid-depleted media for 5 days, which resulted in the maximal accumulation of cells in G(0)/G(1) phase of the cell cycle. The mitogenic concentration of the synthetic androgen R1881 was established as 0.1 nM using cell proliferation assay. Protein and mRNA levels of particular cyclins, cyclin-dependent kinases (Cdks), cyclin-dependent kinase inhibitors (Ckis), and the retinoblastoma proteins (Rb) were assessed. Androgen stimulation resulted in a post-transcriptional reduction in Rb protein levels, an increase in Rb phosphorylation at serine 780 and an accumulation of high molecular weight Rb protein species. Androgen stimulation also induced the expression of the Cdk2 and Cdk1 as well as their regulatory partners, cyclin A and cyclin B, resulting in a corresponding increase in cyclin A/Cdk2 activity in vitro. Pulse-chase showed decreased Rb protein stability in androgen-treated LNCaP cells. Collectively, our findings suggest a novel mechanism of androgen-dependent prostate cancer growth in which androgen stimulation results in decreased Rb protein expression in LNCaP cells. The observation of decreased Rb protein stability in the setting of increased phosphorylation supports the concept of phosphorylation mediated protein degradation. We propose that the observed reduction in Rb protein level occurs through Rb degradation via the ubiquitin/proteasome pathway, and is preceded by selective Rb phosphorylation by cyclin A/Cdk2 and cyclin B/Cdk1
— id: 39463, year: 2001, vol: 84, page: 188, stat: Journal Article,

Selective activation of the glucocorticoid receptor by steroid antagonists in human breast cancer and osteosarcoma cells
Fryer, C J; Kinyamu, H K; Rogatsky, I; Garabedian, M J; Archer, T K
2000 Jun 9;275(23):17771-17777, Journal of biological chemistry
Steroid hormones regulate the transcription of numerous genes via high affinity receptors that act in concert with chromatin remodeling complexes, coactivators and corepressors. We have compared the activities of a variety of glucocorticoid receptor (GR) antagonists in breast cancer and osteosarcoma cell lines engineered to stably maintain the mouse mammary tumor virus promoter. In both cell types, GR activation by dexamethasone occurs via the disruption of mouse mammary tumor virus chromatin structure and the recruitment of receptor coactivator proteins. However, when challenged with a variety of antagonists the GR displays differential ability to activate transcription within the two cell types. For the breast cancer cells, the antagonists fail to activate the promoter and do not promote the association of the GR with either remodeling or coactivator proteins. In contrast, in osteosarcoma cells, the antiglucocorticoids, RU486 and RU43044, exhibit partial agonist activity. The capacity of these antagonists to stimulate transcription in the osteosarcoma cells is reflected in the ability of the RU486-bound receptor to remodel chromatin and associate with chromatin-remodeling proteins. Similarly, the observation that the RU486-bound receptor does not fully activate transcription is consistent with its inability to recruit receptor coactivator proteins
— id: 120742, year: 2000, vol: 275, page: 17771, stat: Journal Article,

Differential regulation of glucocorticoid receptor transcriptional activation via AF-1-associated proteins
Hittelman AB; Burakov D; Iniguez-Lluhi JA; Freedman LP; Garabedian MJ
1999 Oct 1;18(19):5380-5388, EMBO journal
The hormone-activated glucocorticoid receptor (GR), through its N- and C-terminal transcriptional activation functions AF-1 and AF-2, controls the transcription of target genes presumably through interaction(s) with transcriptional regulatory factors. Utilizing a modified yeast two-hybrid approach, we have identified the tumor susceptibility gene 101 (TSG101) and the vitamin D receptor-interacting protein 150 (DRIP150) as proteins that interact specifically with a functional GR AF-1 surface. In yeast and mammalian cells, TSG101 represses whereas DRIP150 enhances GR AF-1-mediated transactivation. Thus, GR AF-1 is capable of recruiting both positive and negative regulatory factors that differentially regulate GR transcriptional enhancement. In addition, we show that another member of the DRIP complex, DRIP205, interacts with the GR ligand binding domain in a hormone-dependent manner and facilitates GR transactivation in concert with DRIP150. These results suggest that DRIP150 and DRIP205 functionally link GR AF-1 and AF-2, and represent important mediators of GR transcriptional enhancement
— id: 6257, year: 1999, vol: 18, page: 5380, stat: Journal Article,

Role for Hsp90-associated cochaperone p23 in estrogen receptor signal transduction
Knoblauch R; Garabedian MJ
1999 May;19(5):3748-3759, Molecular & cellular biology
The mechanism of signal transduction by the estrogen receptor (ER) is complex and not fully understood. In addition to the ER, a number of accessory proteins are apparently required to efficiently transduce the steroid hormone signal. In the absence of estradiol, the ER, like other steroid receptors, is complexed with Hsp90 and other molecular chaperone components, including an immunophilin, and p23. This Hsp90-based chaperone complex is thought to repress the ER's transcriptional regulatory activities while maintaining the receptor in a conformation that is competent for high-affinity steroid binding. However, a role for p23 in ER signal transduction has not been demonstrated. Using a mutant ER (G400V) with decreased hormone binding capacity as a substrate in a dosage suppression screen in yeast cells (Saccharomyces cerevisiae), we identified the yeast homologue of the human p23 protein (yhp23) as a positive regulator of ER function. Overexpression of yhp23 in yeast cells increases ER transcriptional activation by increasing estradiol binding in vivo. Importantly, the magnitude of the effect of yhp23 on ER transcriptional activation is inversely proportional to the concentration of both ER and estradiol in the cell. Under conditions of high ER expression, ER transcriptional activity is largely independent of yhp23, whereas at low levels of ER expression, ER transcriptional activation is primarily dependent on yhp23. The same relationship holds for estradiol levels. We further demonstrate that yhp23 colocalizes with the ER in vivo. Using a yhp23-green fluorescent protein fusion protein, we observed a redistribution of yhp23 from the cytoplasm to the nucleus upon coexpression with ER. This nuclear localization of yhp23 was reversed by the addition of estradiol, a finding consistent with yhp23's proposed role as part of the aporeceptor complex. Expression of human p23 in yeast partially complements the loss of yhp23 function with respect to ER signaling. Finally, ectopic expression of human p23 in MCF-7 breast cancer cells increases both hormone-dependent and hormone-independent transcriptional activation by the ER. Together, these results strongly suggest that p23 plays an important role in ER signal transduction
— id: 12024, year: 1999, vol: 19, page: 3748, stat: Journal Article,

Distinct glucocorticoid receptor transcriptional regulatory surfaces mediate the cytotoxic and cytostatic effects of glucocorticoids
Rogatsky I; Hittelman AB; Pearce D; Garabedian MJ
1999 Jul;19(7):5036-5049, Molecular & cellular biology
Glucocorticoids act through the glucocorticoid receptor (GR), which can function as a transcriptional activator or repressor, to elicit cytostatic and cytotoxic effects in a variety of cells. The molecular mechanisms regulating these events and the target genes affected by the activated receptor remain largely undefined. Using cultured human osteosarcoma cells as a model for the GR antiproliferative effect, we demonstrate that in U20S cells, GR activation leads to irreversible growth inhibition, apoptosis, and repression of Bcl2. This cytotoxic effect is mediated by GR's transcriptional repression function, since transactivation-deficient mutants and ligands still bring about apoptosis and Bcl2 down-regulation. In contrast, the antiproliferative effect of GR in SAOS2 cells is reversible, does not result in apoptosis or repression of Bcl2, and is a function of the receptor's ability to stimulate transcription. Thus, the cytotoxic versus cytostatic outcome of glucocorticoid treatment is cell context dependent. Interestingly, the cytostatic effect of glucocorticoids in SAOS2 cells involves multiple GR activation surfaces. GR mutants and ligands that disrupt individual transcriptional activation functions (activation function 1 [AF-1] and AF-2) or receptor dimerization fail to fully inhibit cellular proliferation and, remarkably, discriminate between the targets of GR's cytostatic action, the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1). Induction of p21(Cip1) is agonist dependent and requires AF-2 but not AF-1 or GR dimerization. In contrast, induction of p27(Kip1) is agonist independent, does not require AF-2 or AF-1, but depends on GR dimerization. Our findings indicate that multiple GR transcriptional regulatory mechanisms that employ distinct receptor surfaces are used to evoke either the cytostatic or cytotoxic response to glucocorticoids
— id: 8498, year: 1999, vol: 19, page: 5036, stat: Journal Article,

Potentiation of human estrogen receptor alpha transcriptional activation through phosphorylation of serines 104 and 106 by the cyclin A-CDK2 complex
Rogatsky I; Trowbridge JM; Garabedian MJ
1999 Aug 6;274(32):22296-22302, Journal of biological chemistry
Both estradiol binding and phosphorylation regulate transcriptional activation by the human estrogen receptor alpha (ER). We have previously shown that activation of the cyclin A-CDK2 complex by overexpression of cyclin A leads to enhanced ER-dependent transcriptional activation and that the cyclin A-CDK2 complex phosphorylates the ER N-terminal activation function-1 (AF-1) between residues 82 and 121. Within ER AF-1, serines 104, 106, and 118 represent potential CDK phosphorylation sites, and in this current study, we ascertain their importance in mediating cyclin A-CDK2-dependent enhancement of ER transcriptional activity. Cyclin A overexpression does not enhance transcriptional activation by an ER derivative bearing serine-to-alanine changes at residues 104, 106, and 118. Likewise, the cyclin A-CDK2 complex does not phosphorylate this triple-mutated derivative in vitro. Individual serine-to-alanine mutations at residues 104 and 106, but not 118, decrease ER-dependent transcriptional enhancement in response to cyclin A. The same relationship holds for ER phosphorylation by cyclin A-CDK2 in vitro. Finally, enhancement of ER transcriptional activation by cyclin A is evident in the absence and presence of estradiol, as well as in the presence of tamoxifen, suggesting that the effect of the cyclin A-CDK2 on ER transcriptional activation is AF-2-independent. These results indicate that the enhancement of ER transcriptional activation by the cyclin A-CDK2 complex is mediated via the AF-1 domain by phosphorylation of serines 104 and 106. We propose that these residues control ER AF-1 activity in response to signals that affect cyclin A-CDK2 function
— id: 8492, year: 1999, vol: 274, page: 22296, stat: Journal Article,

Antagonism of glucocorticoid receptor transcriptional activation by the c-Jun N-terminal kinase
Rogatsky I; Logan SK; Garabedian MJ
1998 Mar 3;95(5):2050-2055, Proceedings of the National Academy of Sciences of the United States of America
The mitogen-activated protein kinases ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), and p38 phosphorylate and activate transcription factors that promote proliferative and inflammatory responses, whereas glucocorticoid receptor (GR) activation inhibits cell growth and inflammation. We demonstrate that JNK and ERK but not p38 phosphorylate GR in vitro primarily at Ser-246. Selective activation of either ERK or JNK in vivo inhibits GR-mediated transcriptional activation, which depends on receptor phosphorylation at Ser-246 by JNK but not ERK. Thus, JNK inhibits GR transcriptional activation by direct receptor phosphorylation, whereas ERK does so indirectly. We propose that phosphorylation of GR by JNK or of a GR cofactor by ERK provides mechanisms to ensure the rapid inhibition of GR-dependent gene expression when it conflicts with mitogenic or proinflammatory signals
— id: 7764, year: 1998, vol: 95, page: 2050, stat: Journal Article,

Phosphorylation and inhibition of rat glucocorticoid receptor transcriptional activation by glycogen synthase kinase-3 (GSK-3). Species-specific differences between human and rat glucocorticoid receptor signaling as revealed through GSK-3 phosphorylation
Rogatsky I; Waase CL; Garabedian MJ
1998 Jun 5;273(23):14315-14321, Journal of biological chemistry
Transcriptional activation by the glucocorticoid receptor (GR) is regulated by both glucocorticoid binding and phosphorylation. The rat GR N-terminal transcriptional regulatory domain contains four major phosphorylation sites: threonine 171 (Thr171), serine 224 (Ser224), serine 232 (Ser232), and serine 246 (Ser246). We have previously demonstrated that Ser224 and Ser232 are phosphorylated by cyclin-dependent kinases, while Ser246 is phosphorylated by the c-Jun N-terminal kinase. We report here that the remaining GR phosphorylation site, Thr171, is a target for glycogen synthase kinase-3 (GSK-3) in vitro and in cultured mammalian cells. Increasing GSK-3 activity through its overexpression in cultured cells inhibits GR transcriptional enhancement, an effect dependent upon Thr171. Correspondingly, overexpression of a constitutively active form of the GSK-3 inhibitor, protein kinase B/Akt, increases GR transcriptional enhancement. Overexpression of GSK-3 had no effect on GR-mediated transcriptional repression of AP1-dependent gene expression. Importantly, transcriptional activation by the human GR (hGR), which contains an alanine (Ala150) at the position equivalent to Thr171 in rat GR, is not affected by GSK-3 overexpression. Introduction of a threonine residue at this position (A150T) establishes GSK-3-mediated inhibition of hGR transcriptional activation. These findings demonstrate species-specific differences in GR signaling, as revealed through GSK-3 phosphorylation, which suggests that GR function in rodents may not fully recapitulate receptor action in humans and that hGR is capable of adopting the GSK-3 signaling pathway through a somatic mutation
— id: 8300, year: 1998, vol: 273, page: 14315, stat: Journal Article,

Superactivation of expanded CAG repeat mutant androgen receptor by overexpression of TAF130
Taneja, SS; Kern, A; Tanese, N; Garabedian, MJ
1998 ;159(5):35-35, Journal of urology
— id: 104584, year: 1998, vol: 159, page: 35, stat: Journal Article,

GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors
Hong, H; Kohli, K; Garabedian, M J; Stallcup, M R
1997 May;17(5):2735-2744, Molecular & cellular biology
After binding to enhancer elements, transcription factors require transcriptional coactivator proteins to mediate their stimulation of transcription initiation. A search for possible coactivators for steroid hormone receptors resulted in identification of glucocorticoid receptor interacting protein 1 (GRIP1). The complete coding sequence for GRIP1, isolated from a mouse brain cDNA library, contains an open reading frame of 1,462 codons. GRIP1 is the probable ortholog of the subsequently identified human protein transcription intermediary factor 2 (TIF2) and is also partially homologous to steroid receptor coactivator 1 (SRC-1). The full-length GRIP1 interacted with the hormone binding domains (HBDs) of all five steroid receptors in a hormone-dependent manner and also with HBDs of class II nuclear receptors, including thyroid receptor alpha, vitamin D receptor, retinoic acid receptor alpha, and retinoid X receptor alpha. In contrast to agonists, glucocorticoid antagonists did not promote interaction between the glucocorticoid receptor and GRIP1. In yeast cells, GRIP1 dramatically enhanced the transcriptional activation function of proteins containing the HBDs of any of the above-named receptors fused to the GAL4 DNA binding domain and thus served as a transcriptional coactivator for them. This finding contrasts with previous reports of TIF2 and SRC-1, which in mammalian cells enhanced the transactivation activities of only a subset of the steroid and nuclear receptors that they physically interacted with. GRIP1 also enhanced the hormone-dependent transactivation activity of intact glucocorticoid receptor, estrogen receptor, and mineralocorticoid receptor. Experiments with glucocorticoid receptor truncation and point mutants indicated that GRIP1 interacted with and enhanced the activity of the C-terminal AF-2 but not the N-terminal AF-1 transactivation domain of the glucocorticoid receptor. These results demonstrate directly that AF-1 and AF-2 domains accomplish their transactivation activities through different mechanisms: AF-2 requires GRIP1 as a coactivator, but AF-1 does not
— id: 120743, year: 1997, vol: 17, page: 2735, stat: Journal Article,

Mitogen-activated and cyclin-dependent protein kinases selectively and differentially modulate transcriptional enhancement by the glucocorticoid receptor
Krstic MD; Rogatsky I; Yamamoto KR; Garabedian MJ
1997 Jul;17(7):3947-3954, Molecular & cellular biology
Cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK) phosphorylate the rat glucocorticoid receptor in vitro at distinct sites that together correspond to the major phosphorylated receptor residues observed in vivo; MAPK phosphorylates receptor residues threonine 171 and serine 246, whereas multiple CDK complexes modify serines 224 and 232. Mutations in these kinases have opposite effects on receptor transcriptional activity in vivo. Receptor-dependent transcriptional enhancement is reduced in yeast strains deficient in the catalytic (p34CDC28) or certain regulatory (cyclin) subunits of CDK complexes and is increased in a strain devoid of the mammalian MAPK homologs FUS3 and KSS1. These findings indicate that the glucocorticoid receptor is a target for multiple kinases in vivo, which either positively or negatively regulate receptor transcriptional enhancement. The control of receptor transcriptional activity via phosphorylation provides an increased array of regulatory inputs that, in addition to steroid hormones, can influence receptor function
— id: 7190, year: 1997, vol: 17, page: 3947, stat: Journal Article,

Mechanism of glucocorticoid-dependent inhibition of estradiol-induced proliferation of MCF7 breast cancer cells
Labat, M; Rogatsky, I; Trowbridge, J; Garabedian, M
1997 NOV ;8(5):149-149, Molecular biology of the cell
— id: 53157, year: 1997, vol: 8, page: 149, stat: Journal Article,

Glucocorticoid receptor-mediated cell cycle arrest is achieved through distinct cell-specific transcriptional regulatory mechanisms
Rogatsky I; Trowbridge JM; Garabedian MJ
1997 Jun;17(6):3181-3193, Molecular & cellular biology
Glucocorticoids inhibit proliferation of many cell types, but the events leading from the activated glucocorticoid receptor (GR) to growth arrest are not understood. Ectopic expression and activation of GR in human osteosarcoma cell lines U2OS and SAOS2, which lack endogenous receptors, result in a G1 cell cycle arrest. GR activation in U2OS cells represses expression of the cyclin-dependent kinases (CDKs) CDK4 and CDK6 as well as their regulatory partner, cyclin D3, leading to hypophosphorylation of the retinoblastoma protein (Rb). We also demonstrate a ligand-dependent reduction in the expression of E2F-1 and c-Myc, transcription factors involved in the G1-to-S-phase transition. Mitogen-activated protein kinase, CDK2, cyclin E, and the CDK inhibitors (CDIs) p27 and p21 are unaffected by receptor activation in U2OS cells. The receptor's N-terminal transcriptional activation domain is not required for growth arrest in U2OS cells. In Rb-deficient SAOS2 cells, however, the expression of p27 and p21 is induced upon receptor activation. Remarkably, in SAOS2 cells that express a GR deletion derivative lacking the N-terminal transcriptional activation domain, induction of CDI expression is abolished and the cells fail to undergo ligand-dependent cell cycle arrest. Similarly, murine S49 lymphoma cells, which, like SAOS2 cells, lack Rb, require the N-terminal activation domain for growth arrest and induce CDI expression upon GR activation. These cell-type-specific differences in receptor domains and cellular targets linking GR activation to cell cycle machinery suggest two distinct regulatory mechanisms of GR-mediated cell cycle arrest: one involving transcriptional repression of G1 cyclins and CDKs and the other involving enhanced transcription of CDIs by the activated receptor
— id: 7250, year: 1997, vol: 17, page: 3181, stat: Journal Article,

Regulation of estrogen receptor transcriptional enhancement by the cyclin A/Cdk2 complex
Trowbridge JM; Rogatsky I; Garabedian MJ
1997 Sep 16;94(19):10132-10137, Proceedings of the National Academy of Sciences of the United States of America
We have found that ectopic expression of cyclin A increases hormone-dependent and hormone-independent transcriptional activation by the estrogen receptor in vivo in a number of cell lines, including HeLa cells, U-2 OS osteosarcoma cells and Hs 578Bst breast epithelial cells. This effect can be further enhanced in HeLa cells by the concurrent expression of the cyclin-dependent kinase activator, cyclin H, and cdk7, and abolished by expression of the cdk inhibitor, p27(KIP1), or by the expression of a dominant negative catalytically inactive cdk2 mutant. ER is phosphorylated between amino acids 82 and 121 in vitro by the cyclin A/cdk2 complex and incorporation of phosphate into ER is stimulated by ectopic expression of cyclin A in vivo. Together, these results strongly suggest a direct role for the cyclin A/cdk2 complex in phosphorylating ER and regulating its transcriptional activity
— id: 57005, year: 1997, vol: 94, page: 10132, stat: Journal Article,

Synergistic transcriptional activation of the tissue inhibitor of metalloproteinases-1 promoter via functional interaction of AP-1 and Ets-1 transcription factors
Logan, S K; Garabedian, M J; Campbell, C E; Werb, Z
1996 Jan 12;271(2):774-782, Journal of biological chemistry
The tissue inhibitor of metalloproteinases-1 (TIMP-1) is an inhibitor of the extracellular matrix-degrading metalloproteinases. We characterized response elements that control TIMP-1 gene expression. One contains a binding site that selectively binds c-Fos and c-Jun in vitro and confers a response to multiple AP-1 family members in vivo. Adjacent to this is a binding site for Ets domain proteins. Although c-Ets-1 alone did not activate transcription from this element, it enhanced transcription synergistically with AP-1 either in the context of the natural promoter or when the sequence was linked upstream of a heterologous promoter. Furthermore, a complex of c-Jun and c-Fos interacted with c-Ets-1 in vitro. These results suggest that AP-1 tethers c-Ets-1 to the TIMP-1 promoter via protein-protein interaction to achieve Ets-dependent transcriptional regulation. Collectively, our results indicate that TIMP-1 expression is controlled by several DNA response elements that respond to variations in the level and activity of AP-1 and Ets transcriptional regulatory proteins
— id: 106246, year: 1996, vol: 271, page: 774, stat: Journal Article,

Mechanism of glucocorticoid receptor - Induced cell cycle arrest
Rogatsky, I; Trowbridge, JM; Garabedian, MJ
1996 MAR ;44(3):A240-A240, Journal of investigative medicine
— id: 52953, year: 1996, vol: 44, page: A240, stat: Journal Article,

Regulation of estrogen receptor-dependent transcription by a cyclin-dependent kinase
Trowbridge, JM; Rogatsky, I; Knoblauch, R; Garabedian, MJ
1996 MAR ;44(3):A271-A271, Journal of investigative medicine
— id: 52960, year: 1996, vol: 44, page: A271, stat: Journal Article,

Modular structure of glucocorticoid receptor domains is not equivalent to functional independence. Stability and activity of the steroid binding domain are controlled by sequences in separate domains
Xu, M; Chakraborti, P K; Garabedian, M J; Yamamoto, K R; Simons, S S
1996 Aug 30;271(35):21430-21438, Journal of biological chemistry
A long-standing conundrum of glucocorticoid receptors has been why the steroid binding domain is active in hybrid proteins but not in isolation. For this reason, the precise boundaries of the steroid binding domain have not been defined. These questions have now been systematically examined with a variety of receptor deletion constructs. Plasmids encoding amino acids 537-673 and 537-795 of the rat receptor did not yield stable proteins, while the fusion of receptor or non-receptor sequences upstream of 537-673 afforded stable proteins that did not bind steroid. Wild type steroid binding affinity could be obtained, however, when proteins such as beta-galactosidase or dihydrofolate reductase were fused upstream of receptor amino acids 537-795. Studies of a series of dhfr/receptor constructs with deletions at the amino- and carboxyl-terminal ends of the receptor sequence localized the boundaries of the steroid binding domain to 550-795. The absence of steroid binding upon deletion of sequences in the carboxyl-terminal half of this domain was consistent with improperly folded receptor sequences. This conclusion was supported by analyses of the proteolysis and thermal stability of the mutant receptors. Thus, three independent regions appear to be required for the generation of the steroid binding form of receptors: 1) a protein sequence upstream of the steroid binding domain, which conveys stability to the steroid binding domain, 2) sequences of the carboxyl-terminal amino acids (674-795), which are required for the correct folding of the steroid binding domain, and 3) amino-terminal sequences (550-673), which may be sufficient for steroid binding after the entire steroid binding domain is properly folded. These results establish that the steroid binding domain of glucocorticoid receptors is not independently functional and illustrate the importance of both protein stability and protein folding when constructing mutant proteins
— id: 120744, year: 1996, vol: 271, page: 21430, stat: Journal Article,

Glucocorticoid receptor phosphorylation in v-mos-transformed cells
Borror, K C; Garabedian, M J; DeFranco, D B
1995 May;60(5):375-382, Steroids
Nucleocytoplasmic shuttling of glucocorticoid receptors (GRs) is disrupted in v-mos-transformed cells leading to the redistribution of hormone-bound receptors from the nuclear to cytoplasmic compartments. We show here that GRs from v-mos-transformed cells are hyperphosphorylated on a specific peptide and maintain hormone-induced phosphorylations upon a prolonged hormone treatment that is associated with disruptions in its nucleocytoplasmic shuttling. Since similar effects on GR nucleocytoplasmic shuttling and phosphorylation were exerted upon treatment of nontransformed cells with the protein phosphatase inhibitor okadaic acid, we examined whether hyperphosphorylation of GRs in v-mos-transformed cells resulted from inhibition of receptor dephosphorylation. Protein phosphatase activity, measured using various substrates in vitro, was identical in cell-free extracts prepared from v-mos-transformed and nontransformed cells. Analysis of phosphate turnover in vivo from either the sum of all GR phosphorylation sites or from individual sites using pulse-chase analysis, did not reveal any significant difference between v-mos-transformed cells versus nontransformed cells. Thus, hyperphosphorylation of GR in v-mos-transformed cells does not appear to result from inhibition of GR dephosphorylation, but rather from stimulation of GR phosphorylation
— id: 120745, year: 1995, vol: 60, page: 375, stat: Journal Article,

Genetic approaches to mammalian nuclear receptor function in yeast
Garabedian, Michael J.
1993 ;5(2):138-146, Methods
Mammalian nuclear receptor function can be faithfully reconstituted in yeast, enabling a wide variety of genetic approaches to be taken toward defining the mechanisms of signal transduction and transcriptional regulation. This report describes vectors for the expression of mammalian receptors in yeast, reporter genes, yeast host strains, and simple assays that monitor receptor transcriptional activity. Methods for the generation of receptors with distinct defects in particular functions, such as DNA or hormone binding, that couple random mutagenesis with phenotypic screens are outlined as well. In addition, strategies for the identification of nonreceptor components whose gene products may act on receptors are discussed. The experimental advantages of yeast invite a detailed genetic analysis of mammalian nuclear receptor functions sbd hormone and DNA binding, nuclear localization, and interaction with nonreceptor factors sbd and should illuminate further the mechanisms of signal transduction and transcriptional regulation by this important class of regulatory molecules
— id: 98814, year: 1993, vol: 5, page: 138, stat: Journal Article,

Role of cysteines 640, 656, and 661 in steroid binding to rat glucocorticoid receptors
Chakraborti, P K; Garabedian, M J; Yamamoto, K R; Simons, S S Jr
1992 Jun 5;267(16):11366-11373, Journal of biological chemistry
The involvement of a vicinally spaced dithiol group in steroid binding to the glucocorticoid receptor has been deduced from experiments with the thiol-specific reagent methyl methanethiolsulfonate and the vicinal dithiol-specific reagent sodium arsenite. The vicinally spaced dithiol appears to reside in the 16-kDa trypsin fragment of the receptor, which is thought to contain 3 cysteines (Cys-640, -656, and -661 of the rat receptor) and binds hormone with an approximately 23-fold lower affinity than does the intact 98-kDa receptor. We now report that the steroid binding specificity of preparations of this 16-kDa fragment and the intact receptor are virtually identical. This finding supports our designation of the 16-kDa fragment as a steroid-binding core domain and validates our continued use of this tryptic fragment in studies of steroid binding. To identify the cysteines which comprise the vicinally spaced dithiol group, and to examine further the role of cysteines in steroid binding, a total of five point mutant receptors were prepared: cysteine-to-serine for each suspected cysteine, cysteine-to-glycine for Cys-656, and the C656,661S double mutant. Unexpectedly, each receptor with a single point mutation still bound steroid. Even the double mutant (C656,661S) bound steroid with wild type affinity. These results suggest that none of these cysteines are directly required either for steroid binding to the glucocorticoid receptor or for heat shock protein 90 association with the receptor. However, the presence of Cys-656 was obligatory for covalent labeling of the receptor by [3H]dexamethasone 21-mesylate. Studies with preparations of the 98 and 16 kDa forms of these mutant receptors revealed both that Cys-656 and -661 comprise the vicinally spaced dithiols reacting with arsenite and that any two of the three thiols could form an intramolecular disulfide after treatment with low concentrations of methyl methanethiolsulfonate. These data, in conjunction with those from experiments on the effects of steric bulk on various receptor functions, support a model for the ligand binding cavity of the receptor that involves all three thiols in a flexible cleft but where thiol-steroid interactions are not essential for binding
— id: 120747, year: 1992, vol: 267, page: 11366, stat: Journal Article,

Genetic dissection of the signaling domain of a mammalian steroid receptor in yeast
Garabedian, M J; Yamamoto, K R
1992 Nov;3(11):1245-1257, Molecular biology of the cell
The mechanism of signal transduction by steroid receptor proteins is complex and not yet understood. We describe here a facile genetic strategy for dissection of the rat glucocorticoid receptor 'signaling domain,' a region of the protein that binds and transduces the hormonal signal. We found that the characteristics of signal transduction by the receptor expressed in yeast were similar to those of endogenous receptors in mammalian cells. Interestingly, the rank order of particular ligands differed between species with respect to receptor binding and biological efficacy. This suggests that factors in addition to the receptor alone must determine or influence ligand efficacy in vivo. To obtain a collection of receptors with distinct defects in signal transduction, we screened in yeast an extensive series of random point mutations introduced in that region in vitro. Three phenotypic classes were obtained: one group failed to bind hormone, a second displayed altered ligand specificity, and a third bound hormone but lacked regulatory activity. Our results demonstrate that analysis of glucocorticoid receptor action in yeast provides a general approach for analyzing the mechanism of signaling by the nuclear receptor family and may facilitate identification of non-receptor factors that participate in this process
— id: 120746, year: 1992, vol: 3, page: 1245, stat: Journal Article,

Two point mutations in the hormone-binding domain of the mouse glucocorticoid receptor that dramatically reduce its function
Byravan, S; Milhon, J; Rabindran, S K; Olinger, B; Garabedian, M J; Danielsen, M; Stallcup, M R
1991 Jun;5(6):752-758, Molecular endocrinology
Mouse lymphoma cell line W7M320b, a mutant WEH17 line, requires higher than normal concentrations of glucocorticoid to elicit the hormone responses that are characteristic of this lineage. Complementary DNA clones representing the glucocorticoid receptor (GR) mRNA were derived from the mutant cells, and the sequences coding for the hormone-binding domain were substituted for the analogous wild-type sequences in a GR cDNA expression vector. The function of the resulting GR proteins was tested by transient expression in COS-7 cells along with a glucocorticoid-inducible reporter gene in the presence of varying concentrations of glucocorticoid. From these assays and DNA sequence analyses, two independent functionally significant point mutations in the GR hormone-binding domain were identified. A mutant GR protein containing the single amino acid substitution, Pro547 to Ala, was still functional as a transcriptional activator, but only at hormone concentrations 100 times higher than those required by the wild-type receptor. A second mutant GR protein with a Cys742 to Gly substitution was unstable and almost completely nonfunctional
— id: 120750, year: 1991, vol: 5, page: 752, stat: Journal Article,

Creation of "super" glucocorticoid receptors by point mutations in the steroid binding domain
Chakraborti, P K; Garabedian, M J; Yamamoto, K R; Simons, S S Jr
1991 Nov 25;266(33):22075-22078, Journal of biological chemistry
Almost all modifications of the steroid binding domain of glucocorticoid receptors are known to cause a reduction or loss of steroid binding activity. Nonetheless, we now report that mutations of cysteine 656 of the rat receptor, which was previously suspected to be a crucial amino acid for the binding process, have produced 'super' receptors. These receptors displayed an increased affinity for glucocorticoid steroids and a decreased relative affinity for cross-reacting steroids such as progesterone and aldosterone. The increased in vitro affinity of the super receptors was maintained in a whole cell bioassay. These results indicate that additional modifications of the glucocorticoid receptor, and probably the other steroid receptors, may further increase the binding affinity and/or specificity
— id: 120748, year: 1991, vol: 266, page: 22075, stat: Journal Article,

Protein phosphatase types 1 and/or 2A regulate nucleocytoplasmic shuttling of glucocorticoid receptors
DeFranco, D B; Qi, M; Borror, K C; Garabedian, M J; Brautigan, D L
1991 Sep;5(9):1215-1228, Molecular endocrinology
We have used okadaic acid (OA), a cell-permeable inhibitor of serine/threonine protein phosphatase types 1 (PP-1) and 2A (PP-2A), to demonstrate that the subcellular distribution of glucocorticoid receptor (GR) in rat fibroblasts is influenced by its phosphorylation state. Nuclear GRs in OA-treated cells retain transcriptional enhancement activity. Nuclear import or export of hormone agonist-bound GRs is not affected by OA. However, a dose of OA that fully inhibits PP-2A and partially inhibits PP-1, but not a lower dose that only partially inhibits PP-2A, leads to inefficient nuclear retention of agonist-bound GRs, and their redistribution into the cytoplasm. These receptors appear to be trapped in the cytoplasmic compartment and are unable to recycle (i.e. reenter the nucleus). Addition of OA during different steps of GR recycling demonstrates that OA must be present during nuclear export of GRs to block GR recycling. A direct role for PP-1 and/or PP-2A in GR recycling is suggested by site-specific hyperphosphorylation of GRs in vivo during OA inhibition of recycling. These are the same sites that undergo in vitro site-specific dephosphorylation by PP-1 and PP-2A. The block in GR recycling that results from inhibition of PP-1 and/or PP-2A resembles effects previously observed in v-mos-transformed rat fibroblasts. Interestingly, OA inhibition of PP-2A in v-mos-transformed cells leads to the reversal of oncoprotein effects on GR recycling and retention of receptors within the nuclear compartment. We propose that GR recycling is influenced by the activities of distinct protein phosphatases (PP-1 and/or PP-2A), and that the interference of this pathway observed in v-mos-transformed cells may be the result of effects of the oncoprotein on the phosphatases or a specific subset of their targets
— id: 120749, year: 1991, vol: 5, page: 1215, stat: Journal Article,

Reduced levels of hsp90 compromise steroid receptor action in vivo
Picard, D; Khursheed, B; Garabedian, M J; Fortin, M G; Lindquist, S; Yamamoto, K R
1990 Nov 8;348(6297):166-168, Nature
Signalling by steroid hormones is mediated by receptor proteins that bind hormonal ligands and regulate the transcription of specific genes. The heat-shock protein hsp90 seems to associate selectively with unliganded receptors (aporeceptors), but it has not been determined whether this interaction affects receptor function in vivo. To address the role of hsp90, we have taken advantage of the capacity of mammalian steroid receptors to function in yeast. We constructed a strain of Saccharomyces cerevisiae in which hsp90 expression was regulatable and could be reduced more than 20-fold relative to wild type. At low levels of hsp90, aporeceptors seem to be mostly hsp90-free, yet fail to enhance transcription; on hormone addition, the receptors are activated but with markedly reduced efficiency. Thus hsp90 does not inhibit receptor function solely by steric interference; rather, hsp90 seems to facilitate the subsequent response of the aporeceptor to the hormonal signal. This is the first biological evidence that hsp90 acts in the signal transduction pathway for steroid receptors
— id: 120751, year: 1990, vol: 348, page: 166, stat: Journal Article,

DNA regions that regulate the ovarian transcriptional specificity of Drosophila yolk protein genes
Logan SK; Garabedian MJ; Wensink PC
1989 Sep;3(9):1453-1461, Genes & development
Yolk protein genes 1 and 2 (yp1 and yp2) of Drosophila melanogaster are divergently transcribed neighboring genes. Both are transcribed in only two tissues, the ovarian follicle cells and the fat bodies of adult females. Previous work has identified a yolk protein enhancer between the genes that is sufficient to direct transcription in one of the tissues, female fat bodies. Using germ-line transformation methods, we identify two cis-acting regions with positive effects on transcription in ovaries. One, a 301-bp region located between the genes, influences both genes and is an enhancer determining the stage and cell type specificity of ovarian transcription. The other, a 105-bp region located in the first exon of yp2, acts across the yp2 promoter region to stimulate yp1 transcription in ovaries. Additional observations suggest how a single enhancer influences both promoters
— id: 57675, year: 1989, vol: 3, page: 1453, stat: Journal Article,

The nucleotide sequence of the gene coding for Drosophila melanogaster yolk protein 3
Garabedian, M J; Shirras, A D; Bownes, M; Wensink, P C
1987 ;55(1):1-8, Gene
The entire sequence of the Drosophila melanogaster yolk protein 3 (YP3) gene (yp3), including 1822 nucleotides (nt) of 5'- and 834 nt of 3'-flanking DNA, has been determined. In addition, the 5' and 3' ends of the mRNA and the two introns have been mapped. The predicted amino acid sequence of YP3 has considerable homology (43%) to the other two yolk proteins of D. melanogaster. The nucleotide sequence of yp3 was compared to the other two yolk protein genes which have the same developmental pattern of expression. In addition to extensive homology between the protein coding regions, we found two small regions of homology between yp3 flanking sequences and a segment of DNA required for normal expression of the yolk protein 1 gene in adult female fat bodies
— id: 120752, year: 1987, vol: 55, page: 1, stat: Journal Article,

A tissue-specific transcription enhancer from the Drosophila yolk protein 1 gene
Garabedian, M J; Shepherd, B M; Wensink, P C
1986 Jun 20;45(6):859-867, Cell
The yolk protein 1 gene (yp1) of Drosophila melanogaster is expressed only in the ovarian follicle cells and the fat bodies of adult females. We have previously shown that a different cis-acting DNA region is required for each of these tissue specificities. In this paper we use germ line transformation to localize and characterized one of these tissue-specific regulatory regions. We demonstrate that a 125 bp segment of DNA located 196 bp upstream of the yp1 cap site is sufficient to determine the sex-, stage-, and fat body-specific expression of the yp1 gene. We also find that this region can confer yp1-specific expression on a heterologous Drosophila promoter. This specificity is retained when the region is in different orientations and at different distances from the heterologous promoter. Thus a small regulatory region acts in vivo as a positive enhancer to determine the fat body expression pattern of yp1
— id: 120753, year: 1986, vol: 45, page: 859, stat: Journal Article,

Independent control elements that determine yolk protein gene expression in alternative Drosophila tissues
Garabedian, M J; Hung, M C; Wensink, P C
1985 Mar;82(5):1396-1400, Proceedings of the National Academy of Sciences of the United States of America
The adjacent and divergently transcribed yp1 and yp2 genes of Drosophila were separated at a site that is 342 base pairs upstream of yp2 and 883 base pairs upstream of yp1. Each gene was separately used to transform Drosophila germ-line-producing flies with a single copy of the introduced gene. Transcripts from the introduced genes were found only in adult females. Thus, the introduced genes maintain the sex- and time-specific expression pattern of the endogenous yp genes. However, the pattern of tissue-specific expression differed among the two introduced genes and the endogenous genes. Transcripts from both of the endogenous genes are found in fat bodies and ovaries. In contrast, transcripts from the introduced yp1 gene are found only in fat bodies, and those from the introduced yp2 gene are found only in ovaries. Thus, each introduced DNA segment lacks at least one of the cis-acting elements required for the normal pattern of tissue-specific expression. These results indicate that the expression of a yp gene in different tissues is determined by different cis-acting elements
— id: 120754, year: 1985, vol: 82, page: 1396, stat: Journal Article,

Developmental control of Drosophila yolk protein 1 gene by cis-acting DNA elements
Shepherd, B; Garabedian, M J; Hung, M C; Wensink, P C
1985 ;50:521-526, Cold Spring Harbor symposia on quantitative biology
In this paper we have demonstrated that at least two tissue-specific cis-acting DNA elements are necessary for the normal developmental pattern of Drosophila yp1 gene expression. One of these elements is necessary for expression in the adult female follicle cells and the other is necessary for expression in adult female fat bodies. We have localized the fat body expression element to a 125-bp fragment that lies between nucleotides -196 and -321 of the yp1 gene. This small fragment has at least one of the characteristics of enhancer sequences because it can direct a heterologous Drosophila promoter to be transcribed with the yp1 fat body expression pattern
— id: 120755, year: 1985, vol: 50, page: 521, stat: Journal Article,