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 androgensensitive LNCaP prostate cancer cell line inhibits androgen-mediated cellular proliferation and enhances androgen-mediated transcription of the prostatespecific 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.

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. Like other steroid hormone receptors, the androgen receptor (AR)2 is a phosphoprotein (1). Phosphorylated amino acids within the AR include serines 16, 81, 94, 256, 308, 424, and 650 (2-4). All of these sites show increased phosphorylation in the presence of androgen, with the exception of Ser-94, which is constitutively phosphorylated. Although the biological function of AR phosphorylation is relatively obscure, analysis of AR receptor mutants that are defective in nuclear export shows that they accumulate in subnuclear foci and are hyperphosphorylated at Ser-81 (5). Interestingly, phosphorylation of Ser-650 is enhanced by treatment with forskolin, epidermal growth factor (EGF), and phorbol-12-myristate-13-acetate (4), suggesting that AR phosphorylation may be intricately linked to signal transduction processes regulating tumor promotion and cell growth. In addition, it has been reported that Akt, a cytoplasmic protein kinase activated by the lipid products of PI3K, phosphorylates AR at serines 213 and 791 in vitro (6, 7) and that overexpression of activated Akt destabilizes AR (8).Multiple laboratories have investigated the functional link ...

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 after 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 for 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% after 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. (Endocrinology 146: 4619 -4626, 2005)

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