Recent studies in non-human primates demonstrate that granulosa cell synthesis of the steroid progesterone increases significantly within 30 min of an ovulatory gonadotropin bolus. Concurrent with this increase, gene expression of several key steroidogenic enzymes also increases, notably the steroidogenic acute regulatory protein (STAR) and 3beta-hydroxysteroid dehydrogenase (3bHSD). While the regulation of both StAR and 3bHSD is due in part to transcription factors such as steroidogenic factor-1 (SF-1), GATA 4/6, and CAAT enhancer binding protein beta (CEBPb), we have recently discovered that the initial increase in steroid synthesis by primate granulosa cells depends upon the tumor suppressor p53. Inhibition of p53 with pharmacologic agents or viral oncogenes attenuates hCG stimulation of StAR and 3bHSD (but not SF-1), indicating that p53 plays a key role in this process, p53 in granulosa cell extracts does not bind to putative p53 sites from the 3bHSD promoter, thus it is hypothesized that p53 works as a transcriptional co-factor for SF-1, GATA, and/or CEBPb.
Specific aim 1 will determine if a hCG administration to luteinized human granulosa cells results in post-translational changes to p53, and the kinases responsible for these changes identified and blocked using RNA interference technology. The goal of aim 1 is to demonstrate that post-translational regulation of p53 is a key factor in steroidogenesis.
Specific aim 2 will test the hypothesis that nuclear p53 is essential for maximal StAR promoter activity by directly interacting with SF-1, GATA, and/or CEBPb. A non-steroidogenic, p53 deficient cell line (H1299) will be used in which various combinations of expression plasmids for p53, SF-1, GATA4/6, or CEBPb can be manipulated to demonstrate the necessity of each protein. Luteinized granulosa cells will be transfected with SF-1, GATA4/6, or CEBPb and p53 to test the hypothesis that these proteins make direct physical contact. The proposed studies are expected to demonstrate a novel role for the tumor suppressor p53 in the onset of primate and human steroidogenesis.
