Faculty Bibliography

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

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

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

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

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

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