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 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-alpha mRNA levels. This study examines whether the cAMP-induced increase in inhibin-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. 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 alpha 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 alpha 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 alpha (2.2 kb):luciferase chimeric construct (containing 2.2 kb of the alpha 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 alpha mRNA stability, cells were pretreated with medium or 50 micrograms/ml 8-Br-cAMP for 24 h before addition of 5 microM actinomycin D to arrest new RNA synthesis, and the decay of alpha mRNA transcripts was assessed over 24 h by Northern analysis and nonlinear regression.(ABSTRACT TRUNCATED AT 250 WORDS)
Najmabadi
H
H
Department of Medicine, Harbor-University of California, Los Angeles Medical Center, Torrance 90509.
Rosenberg
L A
LA
Yuan
Q X
QX
Bhatia
N
N
Albiston
A L
AL
Burger
H
H
Bhasin
S
S
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D000818:Animals; D015152:Blotting, Northern; D002822:Choriocarcinoma; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007265:Inhibins / Q000235:genetics*; D007700:Kinetics; D008156:Luciferases / Q000235:genetics; D008297:Male; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins; D015290:Second Messenger Systems; D012708:Sertoli Cells / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D007265:Inhibins; D008156:Luciferases