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Garabedian Lab
Research Interests

Mechanism of Nuclear Hormone Receptor Action in Cancer, Heart Disease and Neuroendocrine Function

My laboratory is interested in understanding how nuclear hormone receptors –which transduce signaling information by binding to small molecules and assembling into transcriptional regulatory complexes at target genes -regulate cell physiology and pathophysiology in areas such as breast and prostate cancer, cardiovascular disease and neuroendocrine function. Our approach combines genetic, molecular and biochemical strategies to identify and characterize pathways and molecules that affect the ER, AR, GR and LXR functions.

Regulation of GR transcriptional activity in the brain by phosphorylation

Our group has been elucidating the mechanism of transcriptional regulation by the glucocorticoid receptor (GR). We were the first to identify components of the Mediator complex, and in particular MED14 (DRIP150), as a protein that interacted with the GR N-terminal transcriptional activation domain to selectively regulate GR target gene expression [1, 2]. We have subsequently shown that the interaction between GR and MED14 is modulated by GR phosphorylation [3]. We have also demonstrated that GR phosphorylation selectively affected target gene expression and have proposed a model whereby the gene specific effect of GR phosphorylation is dependent upon the amount of activated receptor in the cell [3]. We were the first to develop GR phosphorylation site-specific antibodies and have used them to elucidate interacting cellular signaling pathways, and to identify kinases and phosphatases that modulate GR phosphorylation and transcriptional regulatory function [4-10]. More recently in collaboration with Moses Chao’s laboratory [11], we have identified new phosphorylation sites on GR in response to neurotrophin (BDNF and NGF) signaling. Our hypothesis is that by altering phosphorylation, neurotrophins modulate GR gene regulatory function, which in turn affects the hippocampal-pituitary-adrenal (HPA) axis activity. This represents an exciting new area of research for my lab.

Role of LXR alpha in atherosclerosis

We have been also been involved in examining the role of phosphorylation in LXR alpha function [12]. Again, we were the first to identify the site of phosphorylation in LXR, to develop LXR alpha phosphorylation site-specific antibodies, and to demonstrate changes in LXR phosphorylation in macrophages loaded with cholesterol in vitro and in atherosclerotic plaques in vivo. We have also shown that a change in LXR alpha phosphorylation restricted the repertoire of LXR-responsive genes. The mechanism is in part through changes in corepressor recruitment by the phosphorylated form of LXR. More recently, in collaboration with Ed Fisher’s lab, we have shown a requirement for LXR in the emigration of macrophages out of atherosclerotic plaques through LXR’s ability to regulate the expression of CCR7, a chemokine receptor involved in cell movement. We are currently pursuing the mechanism whereby LXR alpha regulates CCR7 gene expression by changes in LXR alpha phosphorylation mediated through alterations in the cellular lipid environment. We have also begun a screen for small molecules that activate CCR7 expression in macrophages to identify compounds that would facilitate “regression” of atherosclerotic plaques.

Regulation of AR transcriptional activity in prostate cancer

We are also pursing the mechanism of androgen receptor (AR) regulation by the cofactor ART-27. ART-27 was cloned in my laboratory as a factor that interacted with the AR N-terminal activation domain [13]. We have proceeded to show in collaboration with the Logan lab that ART-27 expression in the prostate is restricted to epithelial cells. ART-27 expression is often reduced in prostate cancer, and ART-27 overexpression suppresses cell proliferation in AR-dependent prostate cancer (LNCaP) cells suggesting that ART-27 plays a tumor suppressor role in the prostate [14]. We further showed that germline and somatic mutations in AR associated with androgen insensitivity syndrome and prostate cancer no longer interact with ART-27[15]. Recently, we have examined the genome-wide impact of ART-27 on AR-target gene expression and found that the loss of ART-27 enhanced the expression of many androgen-regulated genes, suggesting that ART-27 plays a repressive role in AR-mediated gene expression. We also found that a reduction of ART-27 protein levels in prostate cancer facilitates anti-androgen resistance. Thus, loss of ART-27 expression in prostate cancer cells enhances AR activity and may also help cells evade anti-androgen therapy. Future directions include testing ART-27’s tumor suppressor function in vivo using ART-27 overexpression and ablation in the prostate.

We have also recently initiated an unbiased genetic screen to identify new factors that influence AR function. Toward this end, and again in collaboration with the Logan lab, we have completed a genome wide siRNA screen for factors affecting AR transcriptional activity using AR-dependent transcription of a reporter gene as the readout. Out of the ~14,000 siRNAs screened, we found ~350 hits, which either decrease or increase AR transcription activity. Factors deleted by the siRNA that result in lower AR transcriptional activity -especially potentially “drugable” candidates such as kinases and GCPR- could represent new therapeutic targets that may lower AR activity in prostate cancers that are refractory to hormone ablation therapy. In contrast, deleted genes that increase AR activity may represent new tumor suppressor genes. In fact, an ART-27 homologue and an ART-27 associated protein were identified in the screen as factors that increase AR activity when deleted, further supporting a role for ART-27 as a tumor suppressor gene.

Role of molecular chaperones in breast cancer

My laboratory has also had a long-standing interest in the regulation of estrogen receptor alpha (ER) signaling by the Hsp90 cochaperone p23. We were the first to link p23 to ER signaling using a reconstituted steroid receptor signaling system in yeast [16]. A structure-function analysis of p23 was also performed that pinpointed specific residues on p23 that were required for Hsp90 binding [17]. This genetic approach was substantiated by the recent crystal structure of the yeast p23-hsp90 complex. We also found that p23 overexpression in the ER-dependent breast cancer cell line MCF7 selectively affected ER target gene expression, including genes associated with advanced stages of breast cancers [18]. We are currently examining how p23 might increase the transcriptional activity of these genes by analyzing the chromatin marks associated with p23 overexpression at the promoters of these genes by ChIP. We are also examining the effect of ER activation and Hsp90 inhibition on the expression of these genes. Interestingly we found that ER activation repressed, whereas Hsp90 inhibition differentially affected the expression of the p23 target genes. This suggests that p23 might be acting without Hsp90 to affect gene expression. We are testing this idea by generating a MCF7 cell line expressing a p23 mutant that we showed previously cannot associate with Hsp90.

In normal mammary development p23 protein is found largely in the nucleus, preliminary analysis of p23 expression from breast cancer tissue arrays linked to outcome data reveals that the cytoplasmic localization of p23 is associated with shorter disease free survival as compared to those tumors where p23 is largely confined to the nucleus. Given this information and as a long-term goal, we have generated p23 proteins with nuclear and cytoplasmic localization signals to restrict p23 to one subcellular compartment or another. Human breast cancer cell lines [e.g. MCF7 (ER+) and MDAMB231 (ER-)] and transgenic mice will ultimately be generated to determine the consequences of p23 nuclear and cytoplasmic localization on cell invasion in vitro and mammary development in vivo.

1. Chen, W., I. Rogatsky, and M.J. Garabedian, 2006. MED14 and MED1 differentially regulate target-specific gene activation by the glucocorticoid receptor. Mol Endocrinol, 20(3): p. 560-72.
2. Hittelman, A.B., D. Burakov, J.A. Iniguez-Lluhi, L.P. Freedman, and M.J. Garabedian, 1999. Differential regulation of glucocorticoid receptor transcriptional activation via AF-1-associated proteins. Embo J, 18(19): p. 5380-8.
3. Chen, W., T. Dang, R.D. Blind, Z. Wang, C.N. Cavasotto, A.B. Hittelman, I. Rogatsky, S.K. Logan, and M.J. Garabedian, 2008. Glucocorticoid receptor phosphorylation differentially affects target gene expression. Mol Endocrinol, 22(8): p. 1754-66.
4. Blind, R.D. and M.J. Garabedian, 2008. Differential recruitment of glucocorticoid receptor phospho-isoforms to glucocorticoid-induced genes. J Steroid Biochem Mol Biol, 109(1-2): p. 150-7.
5. Ismaili, N. and M.J. Garabedian, 2004. Modulation of glucocorticoid receptor function via phosphorylation. Ann N Y Acad Sci, 1024: p. 86-101.
6. Wang, Z., W. Chen, E. Kono, T. Dang, and M.J. Garabedian, 2007. Modulation of glucocorticoid receptor phosphorylation and transcriptional activity by a C-terminal-associated protein phosphatase. Mol Endocrinol, 21(3): p. 625-34.
7. Wang, Z., J. Frederick, and M.J. Garabedian, 2002. Deciphering the phosphorylation "code" of the glucocorticoid receptor in vivo. J Biol Chem, 277(29): p. 26573-80.
8. Wang, Z. and M.J. Garabedian, 2003. Modulation of glucocorticoid receptor transcriptional activation, phosphorylation, and growth inhibition by p27Kip1. J Biol Chem, 278(51): p. 50897-901.
9. Krstic, M.D., I. Rogatsky, K.R. Yamamoto, and M.J. Garabedian, 1997. Mitogen-activated and cyclin-dependent protein kinases selectively and differentially modulate transcriptional enhancement by the glucocorticoid receptor. Mol Cell Biol, 17(7): p. 3947-54.
10. Rogatsky, I., S.K. Logan, and M.J. Garabedian, 1998. Antagonism of glucocorticoid receptor transcriptional activation by the c-Jun N-terminal kinase. Proc Natl Acad Sci U S A, 95(5): p. 2050-5.
11. Jeanneteau, F., M.J. Garabedian, and M.V. Chao, 2008. Activation of Trk neurotrophin receptors by glucocorticoids provides a neuroprotective effect. Proc Natl Acad Sci U S A, 105(12): p. 4862-7.
12. Torra, I.P., N. Ismaili, J.E. Feig, C.F. Xu, C. Cavasotto, R. Pancratov, I. Rogatsky, T.A. Neubert, E.A. Fisher, and M.J. Garabedian, 2008. Phosphorylation of liver X receptor alpha selectively regulates target gene expression in macrophages. Mol Cell Biol, 28(8): p. 2626-36.
13. Markus, S.M., S.S. Taneja, S.K. Logan, W. Li, S. Ha, A.B. Hittelman, I. Rogatsky, and M.J. Garabedian, 2002. Identification and characterization of ART-27, a novel coactivator for the androgen receptor N terminus. Mol Biol Cell, 13(2): p. 670-82.
14. Taneja, S.S., S. Ha, N.K. Swenson, I.P. Torra, S. Rome, P.D. Walden, H.Y. Huang, E. Shapiro, M.J. Garabedian, and S.K. Logan, 2004. ART-27, an androgen receptor coactivator regulated in prostate development and cancer. J Biol Chem, 279(14): p. 13944-52.
15. Li, W., C.N. Cavasotto, T. Cardozo, S. Ha, T. Dang, S.S. Taneja, S.K. Logan, and M.J. Garabedian, 2005. Androgen receptor mutations identified in prostate cancer and androgen insensitivity syndrome display aberrant ART-27 coactivator function. Mol Endocrinol, 19(9): p. 2273-82.
16. Knoblauch, R. and M.J. Garabedian, 1999. Role for Hsp90-associated cochaperone p23 in estrogen receptor signal transduction. Mol Cell Biol, 19(5): p. 3748-59.
17. Oxelmark, E., R. Knoblauch, S. Arnal, L.F. Su, M. Schapira, and M.J. Garabedian, 2003. Genetic dissection of p23, an Hsp90 cochaperone, reveals a distinct surface involved in estrogen receptor signaling. J Biol Chem, 278(38): p. 36547-55.
18. Oxelmark, E., J.M. Roth, P.C. Brooks, S.E. Braunstein, R.J. Schneider, and M.J. Garabedian, 2006. The cochaperone p23 differentially regulates estrogen receptor target genes and promotes tumor cell adhesion and invasion. Mol Cell Biol, 26(14): p. 5205-13.

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