Androgen and glucocorticoid regulation of androgen receptor cDNA expression

Androgen receptor (AR) levels are regulated by androgens, other steroids and non-steroidal hormones via complex, tissue-specific processes. Since alterations in receptor levels may influence cellular sensitivity to androgens, understanding AR regulation is of fundamental and potentially therapeutic significance. In most target tissues and AR-containing cell lines, AR mRNA is down-regulated in response to androgens. We have reconstituted this androgen-mediated down-regulation of AR mRNA in COS 1 cells transfected with a human AR cDNA under the control of the cytomegalovirus (CMV) promoter. The sequences mediating receptor mRNA down-regulation are represented within the AR cDNA and not within the CMV promoter. Androgenic down-regulation of AR cDNA expression was time- and dose-dependent, resembling native AR mRNA down-regulation. In addition, androgenic regulation of the receptor cDNA was not dependent on protein synthesis suggesting that AR and/or another pre-existing protein(s) is involved in this process. In COS 1 cells co-transfected with androgen and glucocorticoid receptor cDNAs, dexamethasone mimicked the action of androgen in down-regulating AR mRNA. This response depended on glucocorticoid receptors. Androgen had little effect on steady-state levels of AR protein consistent with reports that androgen down-regulates AR mRNA but increases AR protein half-life (Kemppainen et al. (1992) J. Biol. Chem. 267, 968–974; Zhou et al. (1995) Mol. Endocrinol. 9, 208–218). However, glucocorticoids decreased AR protein levels in cells that co-expressed androgen and glucocorticoid receptors. These results indicate that sequences represented in the AR cDNA mediate AR mRNA down-regulation by both androgens and glucocorticoids. Inhibition of AR mRNA and protein by glucocorticoids suggests that these steroids may modulate androgen action in tissues, such as mammary gland and prostate, which express both androgen and glucocorticoid receptors

Vitamin D receptor content and transcriptional activity do not fully predict antiproliferative effects of vitamin D in human prostate cancer cell lines

Prostate cancer cell lines exhibit variable growth suppression by the hormonal form of vitamin D 3, 1,25-Dihydroxyvitamin D 3 [1,25 (OH) 2D] (1,25 D 3). To understand the molecular basis for this differential sensitivity to 1,25 D 3, we compared growth response to 1,25 D 3, vitamin D receptor (VDR) content and VDR transcriptional activity in four well-characterized human prostate cancer cell lines: LNCaP, DU145, PC-3 and ALVA-31. In PC-3 and DU145 cells, relative lack of growth inhibition by 1,25 D 3 (<10% inhibition) correlates with very low levels of VDR (9–15 fmol/mg protein) compared to classical vitamin D 3 target tissues (∼75–200 fmol/mg protein). Transfection of DU145 and PC-3 cells with a VDR cDNA expression vector is sufficient to establish growth sensitivity to 1,25 D 3, suggesting that low VDR levels are responsible for the failure of these cell lines to respond to 1,25 D 3. LNCaP cells are highly sensitive to growth inhibition by 1,25 D 3 (∼55% inhibition) and contain ∼2–3-fold more VDR (25 fmol/mg) than the relatively 1,25 D 3-insensitive PC-3 and DU145 cell lines. However, ALVA-31 cells display less than 20% growth inhibition to 1,25 D 3 although they contain the highest levels of VDR (45 fmol/mg) of the four cell lines. Thus, sensitivity to growth inhibition by 1,25 D 3 does not correlate with VDR content in ALVA-31 and LNCaP cells. This lack of correlation between VDR density and growth responses to 1,25 D 3 led us to investigate VDR-mediated gene transcription in these cell lines. We employed two different naturally-occurring vitamin D response elements (VDREs) linked to a reporter gene. Reporter gene activation by 1,25 D 3 correlated well with VDR content in all four cell lines. Therefore, compared to LNCaP cells, decreased sensitivity of ALVA-31 to growth inhibition by 1,25 D 3 is not due to a decrease in the general transcriptional activity of VDR. We conclude that growth inhibition by 1,25 D 3 in prostate cancer cells requires VDR but that this response is modulated by non-receptor factors that are cell line-specific

Absence of androgen-mediated transcriptional effects in osteoblastic cells despite presence of androgen receptors

Androgen excess and deficiency affect skeletal maturation and bone cell function. Understanding the molecular basis for these androgen effects could improve therapy/prevention of short stature and osteoporosis. Androgens act through binding to androgen receptors (ARs), which modulate gene transcription via interactions with DNA response elements on target genes. Because osteoblasts contain ARs at levels just below certain androgen-sensitive tissues, we sought to define the function of AR in a number of commonly used osteoblastic cell lines. Presence and quantification of AR protein and mRNA were evaluated by ligand binding assay, western blotting, and RNAse protection assay. AR-containing osteoblastic cell lines were exposed to nonaromatizable androgens and effects on gene expression were assessed. We found no evidence for direct effects of androgen on endogenous genes nor was androgen involved in modulation of parathyroid hormone effects on early gene activation. Androgen-sensitive reporter gene constructs were stimulated by androgen only when AR cDNA expression vectors were introduced into cells by cotransfection. We conclude that, in commonly used osteoblastic cell lines, the presence of AR at the levels described here does not guarantee androgen transcriptional activity. The effects of androgen on bone in vivo may involve direct stimulation of osteoblastic cells in a different setting or stage of differentiation. Alternatively, androgen may act on bone cells other than osteoblasts, or through metabolic conversion to estrogens

In vivo 17β-estradiol treatment contributes to podocyte actin stabilization in female db/db mice.

We recently showed that 17β-estradiol (E(2)) treatment ameliorated type 2 diabetic glomerulosclerosis in mice in part by protecting podocyte structure and function. Progressive podocyte damage is characterized by foot process effacement, vacuolization, detachment of podocytes from the glomerular basement membrane, and apoptosis. In addition, podocytes are highly dependent on the preservation of their actin cytoskeleton to ensure proper function and survival. Because E(2) administration prevented podocyte damage in our study on diabetic db/db mice and has been shown to regulate both actin cytoskeleton and apoptosis in other cell types and tissues, we investigated whether actin remodeling and apoptosis were prevented in podocytes isolated from E(2)-treated diabetic db/db mice. We performed G-actin/F-actin assays, Western analysis for Hsp25 expression, Ras-related C(3) botulinum toxin substrate 1 (Rac1) activity, and apoptosis assays on previously characterized podocytes isolated from both in vivo-treated placebo and E(2) female db/db mice. We found that in vivo E(2) protects against a phenotype change in the cultured podocytes characterized by a percent increase of F-actin vs. G-actin, suppression of Hsp25 expression and transcriptional activation, increase of Rac1 activity, and decreased apoptotic intermediates. We conclude from these studies that E(2) treatment protects against podocyte damage and may prevent/reduce diabetes-induced kidney disease