Transcriptional regulation of inhibin βB messenger ribonucleic acid levels in TM.4 or primary rat sertoli cells by 8-bromo-cyclic adenosine monophosphate

Abstract

FSH, a major regulator of inhibin production in the testis, is believed to exert its effects via cAMP second messenger system. Inhibin α-subunit gene appears to be regulated by cAMP and has a palindromic cAMP response element sequence TGACGTCA. However, the regulation of the inhibin βB-subunit gene by cAMP has been less clear. It has been assumed that βB may not be regulated by cAMP, based mainly on observations that FSH stimulates only α, not βB, mRNA levels, and that the 5′-up-stream regulatory region of the βB gene does not contain the classical cAMP response element. However, we have observed that 8-bromo-cAMP stimulates βB mRNA levels in both primary Sertoli (∼2-fold) and TM.4 cells (∼5 fold). We examined whether this cAMP-induced increase in βB mRNA levels is the result of increased transcription or altered mRNA stability. Data from nuclear run-on assays demonstrate about a 2-fold increase in relative mRNA synthesis rates in primary Sertoli-cells and about a 4-to 5-fold increase in TM.4 cells. Transfection studies in TM.4 and JEG.3 cell lines with βB:luciferase chimeric reporter gene constructs containing 1.5 kilobases of the βB 5′-up-stream regulatory region revealed marked cAMP induction of reporter gene activity in both cell types. To examine the effect of 8-bromo-cAMP on βB mRNA transcript stability, cells were pretreated with medium or 50 μg/ml 8-bromo-cAMP for 24 h before the addition of 5 μM actinomycin-D to arrest new RNA synthesis, and the decay of the βB mRNA transcripts was assessed over 24 h by Northern analysis and nonlinear regression. 8-Bromo-cAMP did not significantly affect either the slopes of the mRNA decay curves or the calculated half-lives of the two inhibin βB mRNA transcripts. Collectively, these data provide evidence that cAMP induces βB mRNA levels in these three cell types primarily by transcriptional mechanisms.