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The androgen receptor (AR) in Sertoli cells mediates the actions of testosterone on spermatogenesis. However, the transcription factors responsible for AR gene regulation in Sertoli cells remain unknown. In this study, we determined that nuclear factor-κB (NF-κB) regulates transcription of AR in primary cultures of Sertoli cells isolated from testes of adult rats. Electrophoretic mobility shift and antibody supershift assays with nuclear extracts prepared from Sertoli cells identified two binding sites, termed κB1 at −491/−482 bp and κB2 at −574/−565 bp, upstream of the transcription start site of the AR gene that bind the NF-κB subunits, p50 and p65. DNAse I footprint analyses showed that binding of the p50 NF-κB subunit protected the same regions on the rat AR promoter. Analyses of AR promoter-luciferase reporter gene activity after transfection of primary cultures of Sertoli cells demonstrated that mutation of the κB2 site or combined mutation of the κB1 and κB2 sites reduced activity by 40%. Preferential binding of the transcriptionally active p65/p50 heterodimer to the κB2 site rather than to the κB1 site supported these observations. Overexpression of the NF-κB p65 and p50 subunits in Sertoli cells increased activity from the wild-type AR promoter and the promoter with mutation of the κB1 site, but not the κB2 site. Activity was further stimulated by CBP (CREB binding protein), a coactivator of p65 transcriptional activity. Taken together, our data show that NF-κB is an activator of AR gene transcription in Sertoli cells and may be an important determinant of androgen activity during spermatogenesis.

Nuclear Factor-κB Activates Transcription of the Androgen Receptor Gene in Sertoli Cells Isolated from Testes of Adult Rats