Research Interests
Mechanism of Nuclear Hormone Receptor Action in Cancer and Heart Disease
The overall goal of my laboratory is to define the mechanism of signal transduction and transcriptional regulation by nuclear hormone receptors, including the estrogen, androgen, glucocorticoid and liver X receptors (ER, AR, GR and LXR). Our approach combines genetic, molecular and biochemical strategies to identify and characterize molecules that affect the ER, AR, GR and LXR functions.
Phosphorylation of LXR alpha reprograms target gene expression in macrophages
Genetic, molecular and biochemical strategies are being used to define the effects of phosphorylation on LXR alpha function. LXR alpha regulates cholesterol metabolism as well as the inflammatory and immune responses in macrophages. We have identified by mass spectrometry a novel phosphorylation site in LXR alpha at serine 198 (S198). To elucidate the specific signal transduction pathways that lead to LXR alpha phosphorylation and to determine whether this modification affects the transcriptional activity of the receptor, RAW 267.4 macrophages expressing either wild-type LXR (RAW-LXR alpha) or its non-phosphorylatable serine to alanine S198A mutant (RAW-S198A) were generated by retroviral infection. In RAW macrophages phosphorylation of LXR alpha was induced by ligand (T0901317), by lipopolysacharide (LPS) as well as by cholesterol loading as detected with an antibody that specifically recognizes the phosphorylated S198 site. Wild-type LXRa expression enhanced the basal and ligand-induced levels of the LXR target genes ABCA1, ABCG1, PLTP, AIM, LPL and SREBP. Interestingly, only the expression of AIM and LPL was increased in the RAW-S198A cells compared to RAW-LXRa wild-type macrophages. This reflects an ability of the non-phosphorylated receptor to specifically modulate the expression of AIM and LPL, and was not due to different expression levels or subcellular localization of wild-type and S198A LXRa proteins. Intriguingly, basal expression of the chemokine receptor CCR7, which promotes macrophage emigration from atherosclerotic lesions in a mouse model of atherosclerotic plaque regression, was over 15-fold higher in the LXRa phospho-mutant expressing macrophages compared to the wild-type cells. Upon ligand stimulation, however, CCR7 levels were reduced up to 50% in RAW-S198A, but not in RAW-LXRa wild-type cells. Therefore, LXRa phosphorylation at S198 plays an inhibitory role in the transcription of a subset of known LXR responsive genes (AIM and LPL), and induces ligand-independent activation of a novel target CCR7. This may reflect differences in the phospho-S198-LXRa binding to the LXRE within their promoter or from distinct cofactor recruitment by LXR alpha. Energy minimization simulations of LXR alpha peptides, one with S198 phosphorylated and one without, indicates that LXR alpha phosphorylation at S198 promotes a conformational change, which exposes a novel surface, likely facilitating the recruitment of corepressors and/or release of coactivators. Thus, LXR alpha phosphorylation can selectively affect the transcription and expand the repertoire of LXR alpha responsive genes in macrophages. LXR ligands that activate CCR7 by mimicking the non-phosphorylatable LXR alpha conformation could represent an important new class of therapeutics that facilitate atherosclerotic plaque regression and decrease heart failure. We are currently screening for compounds that induce LXR-dependent CCR7 expression and elucidating the mechanism of LXR alpha phosphorylation-dependent gene expression by chromatin immunoprecipitation.
In a separate a genetic screen in yeast, we identified RDI1, a Rho guanine nucleotide dissociation inhibitor (Rho GDI), as a positive regulator of ER transactivation. Our findings indicate that mammalian Rho GDIa specifically increases the transcriptional activity of ER a and b as well as the glucocorticoid receptor (GR) and androgen receptor (AR), but not of unrelated transcription factors serum response factor (SRF) and Sp1, and that this activation is mediated via repression of Rho GTPases. More recent data suggest that Rho GDI increases ER transactivation by regulating the ER coactivators GRIP1 and CBP/p300. We are currently examining the effect of Rho signaling pathway on endogenous genes regulated by ER.
Role of the molecular chaperone p23 in estrogen receptor-dependent gene expression and tumorigenesis
p23 is a ubiquitous and evolutionarily conserved protein that functions as a component of the Hsp90-based molecular chaperone complex. We have demonstrated that this complex chaperones steroid receptors such as estrogen receptor (ER) to a mature form and plays an important role in ER signal transduction. We are currently investigating how p23 regulates ER target gene activation and affects tumor growth and progression. We have recently shown that MCF-7 cells with elevated p23 levels display an increased expression of the ER target genes Cathepsin D and pS2. In contrast, the expression of other target genes, including c-Myc, Cyclin D1 and E2F1 remains unaffected by changes in p23 levels. The p23-induced expression of pS2 is associated with enhanced recruitment of ER to the estrogen response element (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. We are currently examining the effects of p23-hsp90 pathway on breast cancer growth in vivo.
Role of the glucocorticoid receptor in the epigenetic regulation of T cell function
The role GR plays during thymocyte differentiation as well as its subsequent effects in adult T-cell lineages has not been fully elucidated. To approach this problem, we have selectively inactivated GR in CD4+CD8+ thymocytes using a Cre–Lox approach. We found that T-cell subsets in the thymus were unchanged between the GR knock out and control mice, indicating that GR is dispensable for T-cell development. However, a small decrease was observed in the number of splenic T-cells lacking GR, suggesting that GR affects T-cell movement or survival in the periphery. Surprisingly, and in contrast to a previous report that GR gene inactivation at the earlier CD4-CD8- stage of thymocyte development resulted in derepression of COX-2, intestinal inflammation and lethality upon T-cell activation, mice lacking GR at the later CD4+CD8+ stage displayed none of these phenotypes. Thus, the phenotypic consequences of the loss GR in thymocytes are stage specific, and the results suggest that GR imparts an epigenetic memory early in thymocyte development that persists in mature T-cells to limit the inflammatory response. Our findings link the pathophysiology of T-cell mediated inflammatory diseases to GR stage-specific signaling during thymocyte development. We are currently elucidating the mechanism of GR imprinting during T-cell development.
Regulation of AR transcriptional activity in prostate cancer
Activation of androgen receptor (AR) plays a critical role in the development and progression of prostate cancer. The AR transduces the signaling information conveyed by androgens and controls gene expression by associating with critical transcriptional regulatory proteins termed coactivators. This in turn allows the AR to “switch on” or “switch off” genes important for prostrate cancer growth. Therefore, modulation of AR activity via AR coactivators will provide an important new avenue of prostate cancer treatment.
We have, through a unique screening process, identified a protein that interacts with the AR, which we call androgen receptor trapped clone 27 (ART-27). In addition to playing a role in facilitating AR-dependent transcriptional activation, ART-27 displays both cell type and developmental regulation in humans, consistent with a role in AR-dependent differentiation. For example, during prostate development in humans, ART-27 protein is expressed in differentiated luminal epithelial cells but is not detected in undifferentiated epithelial cell precursors, suggesting a role for ART-27 in AR-mediated growth suppression and differentiation. Consistent with a growth suppressive function, ART-27 expression levels are negligible in human prostate cancer and regulated expression of ART-27 in the androgen sensitive LNCaP prostate cancer cell line inhibits androgen-mediated cellular proliferation. Moreover, a somatic alteration in AR (AR P340L) associated with prostate cancer shows a diminished capacity to enhance ART-27 mediated AR-transcriptional activation. Thus, loss of ART-27 is associated with cell growth and prostate cancer. ART-27 is also part of a multiprotein transcriptional regulatory complex that includes a subunit of RNA polymerase. Based on these findings, we propose that ART-27 is a novel tumor suppressor gene that plays a key role in AR-mediated growth suppression of prostate epithelial cells by regulating genes important for prostate cell growth.
To further elucidate the role of ART-27 in prostate growth and differentiation, we will create a prostate cancer cell lines and transgenic mouse that overexpresses and ablate ART-27 expression in prostate epithelial cells. These cell liens and transgenic animals will be examined for changes in AR-dependent gene expression and prostate cell proliferation and differentiation. A clear demonstration that ART-27 regulates androgen-dependent prostate cell growth suppression and differentiation will make ART-27 an attractive therapeutic target in prostate cancer treatment.
