Modulation of androgen receptor (AR)-mediated transcriptional activity by EGF in the developing mouse reproductive...

Recently, a role for epidermal growth factor in male sexual differentiation was reported from different laboratories. We demonstrated that androgen receptor (AR) mediates the EGF-induced effects. The mechanism, by which EGF modulates AR mediated activity, is not known and the current studies were designed to investigate the role of AR. Using mesenchymal cell preparation from the 18-day fetal reproductive tract, first, we determined whether EGF induced sexual differentiation by enhancing AR gene expression. Thus, AR mRNA and AR protein levels were measured in response to EGF-treatment using RT-PCR based analysis of AR mRNA and Western blot analysis of AR protein level respectively. Both of these analysis detected presence of AR gene expression in this cell preparation, however no effect of EGF was observed in AR protein or AR mRNA expression. Next, we examined whether EGF enhanced AR mediated transcriptional activity in the developing tract. Using the mesenchymal cells, co-transfected with AR expression vector (pSV-AR) and androgen response element linked to luciferase reporter vector (pMAMneoLUC) we assessed AR-mediated transcriptional activity in response to EGF treatment in the presence and absence of testosterone. The results showed that androgen stimulated the luciferase activity in a dose dependent manner, as expected. EGF also enhanced such activity; however, the response was significantly lower than that by androgen. EGF, however, produced striking enhancement of the androgen-induced transcriptional activity when used with androgen. EGF and testosterone produced no stimulation of the luciferase activity either alone or in combination in the cells lacking AR expression vector, suggesting a role for AR in the effect of EGF and testosterone. Flutamide, an AR antagonist, also blocked the enhancement of luciferase activity induced by EGF and testosterone, further confirming the role of AR in the effect of EGF and testosterone. Thus, it appears that EGF-modulation of sexual differentiation involves enhancement of AR-mediated transcriptional activity and not enhancement of AR gene expression. Additionally, it appears that EGF modulates sexual differentiation in the presence of testosterone possibly by potentiating the testosterone-effect.