TRANSCRIPTIONAL AND POSTTRANSCRIPTIONAL REGULATION OF INHIBIN ALPHA-SUBUNIT GENE-EXPRESSION IN RAT SERTOLI CELLS BY 8-BROMO-3',5'-CYCLIC-ADENOSINE MONOPHOSPHATE

FSH is a major regulator of inhibin production in the testis. FSH effe cts on Sertoli cell inhibin production are believed to be mediated, at least in part, via the cAMP second messenger system. Previously, it h as been shown that 8-bromo-cAMP (8-Br-cAMP) stimulates inhibin-alpha m RNA levels. This study examines whether the cAMP-induced increase in i nhibin-alpha mRNA levels results from increased alpha mRNA synthesis, decreased degradation of mRNA, or both. The effects of cAMP on inhibin -alpha gene transcription were examined using nuclear run-on assays. F urthermore, the ability of 8-Br-cAMP to drive the transcription of chi meric constructs containing a 2.2-kilobase (kb) segment of the 5'-regu latory region of the alpha gene placed upstream of the coding region o f the luciferase reporter gene was also examined. Data from nuclear ru n-on assays demonstrated rapid induction of alpha gene transcription b y cAMP within 2 h and maximal 4- to 5-fold increase within 4-8 h in pr imary Sertoli cells. Transfection of TM.4 and JEG.3 cells with an alph a (2.2 kb):luciferase chimeric construct (containing 2.2 kb of the alp ha gene 5'-flanking DNA) revealed rapid time-dependent induction of lu ciferase activity by 8-Br-cAMP in these cell types. To examine the eff ects of 8-Br-cAMP on alpha mRNA stability, cells were pretreated with medium or 50 mug/ml 8-Br-cAMP for 24 h before addition of 5 mum actino mycin D to arrest new RNA synthesis, and the decay of alpha mRNA trans cripts was assessed over 24 h by Northern analysis and nonlinear regre ssion. 8-Br-cAMP significantly altered the slope of the alpha mRNA dec ay curve and increased the half-life of the 1.5-kb alpha mRNA transcri pt. Collectively, these data provide evidence that cAMP increases alph a mRNA levels both by stimulating transcription rates and by stabilizi ng the alpha mRNA transcripts.