NF-kappaB induces rat androgen receptor promoter activity in Sertoli cells

Expression of the androgen receptor (AR) gene in Sertoli cells is essential for normal spermatogenesis and regulation of the AR is key to the effects of androgens on spermatogenesis. However, transcription factors responsible for AR gene regulation in Sertoli cells from sexually mature rats are not known. In this study, we investigated the role of nuclear factor-κB (NF-κB) in the transcriptional regulation of AR in primary cultures of Sertoli cells isolated from the testes of sexually mature rats and in the TM4 mouse Sertoli cell line. Using electrophoretic mobility shift assays, we demonstrated that the NF-κB p65 and p50 subunits expressed in Sertoli cells can specifically interact with NF-κB binding sites, κB1 at −491/−482 and κB2 at −574/−565, on the AR promoter. Transient transfection analysis of AR gene promoter activity following mutagenesis of the NF-κB binding sites demonstrated that the activity of mutant promoters containing either mutation of the κB2 site or combined mutations of the κB1 and κB2 sites was significantly lower than that of the wild type promoter in cultures of mature Sertoli cells, indicating that the κB2 motif in the rat AR promoter played a primary regulatory role in AR expression. Preferential binding of the transcriptionally active p65/p50 heterodimer to the κB2 site rather than the κB1 site supported these observations. Overexpression of NF-κB subunits in Sertoli cells led to an increased activity from the wild type AR promoter and from the promoter with a mutated κB1 motif, but not from the promoter in which the κB2 motif was mutated. CBP, a coactivator of p65, further enhanced NF-κB mediated induction of activity from the wild type AR promoter and from the promoter with a mutated κB1 motif, but not from the promoter in which the κB2 motif was mutated. Taken together, we have established that NF-κB induces rat AR promoter activity in mature Sertoli cells and this effect is mainly mediated through the κB2 site. Similar studies performed in TM4 cells supported a role for both the κB1 and κB2 sites in the regulation of AR expression. Our data suggest that NF-κB activates AR gene transcription in Sertoli cells.