Transcriptional and posttranscriptional regulation of inhibin α-subunit gene expression in rat sertoli cells by 8-bromo-3′,5′-cyclic-adenosine monophosphate

Abstract

FSH is a major regulator of inhibin production in the testis. FSH effects on Sertoli cell inhibin production are believed to be mediated, at least in part, via the cAMP second messenger system. Previously, it has been shown that 8-bromo-cAMP (8-Br-cAMP) stimulates inhibin-α mRNA levels. This study examines whether the cAMP-induced increase in inhibin-α mRNA levels results from increased a mRNA synthesis, decreased degradation of mRNA, or both. The effects of cAMP on inhibin-α gene transcription were examined using nuclear run-on assays. Furthermore, the ability of 8-Br-cAMP to drive the transcription of chimeric constructs containing a 2.2-kilobase (kb) segment of the 5′-regulatory region of the α gene placed upstream of the coding region of the luciferase reporter gene was also examined. Data from nuclear run-on assays demonstrated rapid induction of α gene transcription by cAMP within 2 h and maximal 4- to 5-fold increase within 4-8 h in primary Sertoli cells. Transfection of TM.4 and JEG.3 cells with an α (2.2 kb):luciferase chimeric construct (containing 2.2 kb of the α gene 5′-flanking DNA) revealed rapid time-dependent induction of luciferase activity by 8-Br-cAMP in these cell types. To examine the effects of 8-Br-cAMP on α mRNA stability, cells were pretreated with medium or 50 μg/ ml 8-Br-cAMP for 24 h before addition of 5 μM actinomycin D to arrest new RNA synthesis, and the decay of α mRNA transcripts was assessed over 24 h by Northern analysis and nonlinear regression. 8-Br-cAMP significantly altered the slope of the α mRNA decay curve and increased the half-life of the 1.5-kb α mRNA transcript. Collectively, these data provide evidence that cAMP increases a mRNA levels both by stimulating transcription rates and by stabilizing the α mRNA transcripts.