Androgen induction of cyclin-dependent kinase inhibitor p21 gene: Role of androgen receptor and transcription factor Sp1 complex

Previous studies have shown that androgen upregulates expression of the p21 (WAF1, CIP1, SDI1, CAP20) gene, which contains a canonical androgen respon se element (ARE) in its proximal promoter region. We undertook the current studies to determine whether elements in the p21 promoter other than the AR E mediate androgen action. We found that deletion of the ARE did not comple tely abolish the promoter responsiveness to androgen, suggesting that addit ional cis-regulatory elements within the p21 core promoter may also be invo lved in androgen responsiveness. The p21 core promoter is GC-rich and conta ins six binding sites for transcription factor Sp1. We determined whether o ne or more of these Sp1 sites mediate androgen responsiveness of the p21 pr omoter. To do so, we used a transient transfection assay with p21 promoter- luciferase reporter constructs. The reporter activity of a construct lackin g the ARE but containing all six Sp1 sites was induced approximately 3-fold by androgen. Mutation of Sp1-3 nearly eliminated basal promoter activity a s well as androgen responsiveness, whereas deletion of Sp1-1 and Sp1-2 site s and mutation of Sp1-4, Sp1-5, and Sp1-6 sites had relatively little effec t. We also used the mammalian one-hybrid assay and coimmunoprecipitation as say to show that androgen receptor (AR) and transcription factor Sp1 intera ct with one another. The current studies suggest a model in which AR and tr anscription factor Sp1 not only bind to their respective consensus sites wi thin the p21 promoter, but also complex with one another, thereby recruitin g coactivators and general transcription factors and inducing p21 transcrip tion.