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170


Glucocorticoid receptor expression in fetal non-human primate lung [Meeting Abstract]

Lynch, D; Lee, MJ; Natha-Nielsz, P; Guller, S; Swenson, N; Garabe-Dian, M
ISI:000187910500368
ISSN: 0002-9378
CID: 42535

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
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
PMCID:283509
PMID: 14617768
ISSN: 0027-8424
CID: 44733

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
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
PMID: 12835317
ISSN: 0021-9258
CID: 44735

Regulation of GRIP1 and CBP Coactivator activity by Rho GDI modulates estrogen receptor transcriptional enhancement

Su, Laura F; Wang, Zhen; Garabedian, Michael J
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
PMID: 12138084
ISSN: 0021-9258
CID: 39613

Deciphering the phosphorylation "code" of the glucocorticoid receptor in vivo

Wang, Zhen; Frederick, Jeremy; Garabedian, Michael J
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
PMID: 12000743
ISSN: 0021-9258
CID: 39648

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
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
PMCID:65658
PMID: 11854421
ISSN: 1059-1524
CID: 39704

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
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
PMID: 12489202
ISSN: 0300-8207
CID: 44736

ART-27, a novel androgen receptor (AR) interacting protein, is a potential. mediator of prostate eptihelial differentiation [Meeting Abstract]

Logan, S; Ha, S; Rome, S; Garabedian, MJ; Taneja, SS
ISI:000184566700222
ISSN: 0022-5347
CID: 104583

Rho GTPases as modulators of the estrogen receptor transcriptional response

Su LF; Knoblauch R; Garabedian MJ
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
PMID: 11060289
ISSN: 0021-9258
CID: 48157

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
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
PMID: 11746527
ISSN: 0730-2312
CID: 39463