Molecular mechanism of suppression of testicular steroidogenesis by proinflammatory cytokine tumor necrosis factor alpha

Tumor necrosis factor alpha (TNF-alpha) has been demonstrated to inhibit steroidogenesis in Leydig cells at the transcriptional level of steroidogenic enzymes. However, the molecular mechanism of this observed gene repression is not well understood. We now demonstrate that nuclear factor kappaB (NF-kappaB) activated by TNF-alpha inhibits the transactivation of orphan nuclear receptors, which regulate the expression of steroidogenic-enzyme genes. TNF-a treatment suppressed the luteinizing-hormone-induced or Nur77/SF-1-stimulated promoter activity of steroidogenic-enzyme genes in Leydig cells. The TNF-alpha-mediated gene suppression was blocked by treatment with an inhibitor of NF-kappaB. In addition, overexpression of the p65 (Re1A) subunit of NF-kappaB showed the same effect as TNF-alpha and inhibited Nur77 transactivation, suggesting the involvement of NF-kappaB activation in the observed gene repression. Physical association of Nur77 with p65 was revealed by mammalian twohybrid, GST pull-down, and coimmunoprecipitation analyses. The NF-kappaB inhibition of Nur77 transactivation was likely due to the competition of p65 for Nur77 binding with coactivators. Finally, chromatin immunoprecipitation assays revealed that TNF-alpha treatment caused the recruitment of NF-kappaB to the promoter of the steroidogenic-enzyme P450c17 gene, supporting the hypothesis that the TNF-alpha-mediated gene repression involves NF-kappaB inhibition of the transcriptional activity of Nur77 and other orphan nuclear receptors. These findings provide a molecular mechanism underlying the inhibition of testicular steroidogenesis by proinflammatory cytokines.