Regulation of the a Inhibin Gene by Cyclic Adenosine 3′,5′-Monophosphate after Transfection into Rat Granulosa Cells

Abstract

Inhibin gene expression in the ovary is stimulated by FSH, which uses cAMP as an intracellular second messenger. To examine further the transcriptional regulation of the alpha inhibin gene by FSH and cAMP, we have isolated and characterized a genomic clone that contains the entire rat alpha inhibin gene. Sequence analysis of the alpha inhibin promoter region revealed several potential cAMP response elements (CREs) and transcription factor AP2-binding sites that might mediate cAMP regulation. To determine the functional importance of these sequences, fusion genes including the alpha inhibin 5' flanking region linked to a luciferase reporter gene were transiently transfected into primary granulosa cells isolated from immature rats. These fusion genes were both expressed and regulated by the adenylyl cyclase activator forskolin in transfected granulosa cells. Analysis of a series of 5' deletion mutants indicated that a construct containing as little as 170 basepairs up-stream of the alpha inhibin start site, which includes a single imperfect CRE and no AP2 sites, was regulated by forskolin. DNAse footprinting was used to demonstrate that bacterially expressed CRE-binding protein (CREB) binds to this CRE located 122 basepairs up-stream of the alpha inhibin gene transcriptional start site. To investigate further the role of this CRE in alpha inhibin gene expression, site-specific mutagenesis of the CRE was performed. The alpha inhibin promoter containing a mutated CRE was not regulated by forskolin in granulosa cells and did not bind the CREB protein. Interestingly, mutation of the CRE also substantially reduced basal expression of the alpha inhibin promoter. Lastly, a gel mobility shift assay was used to examine CRE-binding proteins from granulosa cell extracts. Granulosa cells contain a protein that specifically interacts with CRE-containing oligonucleotides or with the alpha inhibin promoter and that is recognized by antibodies against the CREB protein. Our results suggest that CREB or related transcription factors play an important role in both basal and cAMP-regulated expression of the alpha inhibin gene in ovarian granulosa cells.

High levels of luteinizing hormone analog stimulate gonadal and adrenal tumorigenesis in mice transgenic for the mouse inhibin-α-subunit promoter/Simian virus 40 T-antigen fusion gene

“…In Inha/Tag mice, LH stimulation is likely to increase the cAMP levels of target cells and also to stimulate the inhibin-a promoter, leading to increased T-antigen expression. It has been shown that an important component of gonadotropin-regulated, cAMP-dependent, inhibin-a-subunit gene expression is the synthesis or activation of cAMP response element (CRE) binding protein (CREB) (Pei et al, 1991). Both gonadotropins can induce the phosphorylation of CREB, leading to its activation (Mukherjee et al, 1996).…”

Genomic Cloning and Sequence Analyses of the Bovine α‐, βA‐ and βB‐Inhibin/activin Genes

“…Recently, the genes of another protein family within the TGF-p superfamily have been found to be regulated differently, in that the promoters of TGF-P1 and TGF-/X2 were found to be regulated by Spl, whereas that of TGF-p3 was not (Geiser et al, 1993). S1 -nuclease-protection studies and primer-extension analyses have identified the transcriptional start sites of the rat (Feng et al, 1989;Albiston et al, 1990;Pei et al, 1991) and mouse (Su and Hsueh, 1992) a-inhibin genes to occur at -73 to -75 bp and at -71 bp, respectively, relative to the translational start site. The present study identified the start site of the bovine gene to be at position -82 (G; Fig.…”

“…Signalling mechanisms operating through the PKA and PKC pathways have been suggested to play roles in the regulation of inhibidactivin gene expression (Klaij et al, 1990(Klaij et al, , 1992Pei et al, 1991 ;Tanimoto et al, 1992;Tanimoto et al, 1993;Najmabadi et al, 1993a,b). As indicated in Figs 1-3, two predicted CRE sites are found following searches of the sequence data using SIGNAL SCAN/TFD, one each occurring in the a-and P,-inhibidactivin 5'-flanking regions.…”

“…The sequences and structural features of the inhibidactivin subunits has led to their inclusion as members of the TGF-p superfamily (for reviews see Kingsley, 1994;MassaguC et al, 1994). Genomic clones of the a-inhibin gene from man (Stewart et al, 1986;Mason et al, 1987), rat (Feng et al, 1989;Albiston et al, 1990;Pei et al, 1991) and mouse ( Su and Hsueh, 1992), the human P,-inhibin/activin gene (Stewart et al, 1986;Mason et al, 1987;Tanimoto et al, 1991) and the pBinhibidactivin gene from man (Forage et al, 1987;Mason et al, 1987;Mason et al, 1989), rat (Feng et al, 1989) and sheep (Rodgers, 1991) have been isolated and sequenced. Comparisons of the inhibidactivin genomic clones with their cDNAs reveal that the a-, PA-and P,-inhibidactivin genes are each comprised of two exons separated by an intron of approximately 1.5-2.1, 9-10 and 2.6-3.0 kb, respectively.…”

“…Although cAMP has been implicated as a signalling mechanism involved in the transcriptional regulation of inhibidactivin gene expression, the transcription factor CREB is the only trans-acting factor to date that has been established experimentally as a likely modulator of inhibidactivin gene expression by specifically inducing transcription of the a-inhibin subunit gene (Pei et al, 1991). To examine further the mechanisms controlling the transcriptional regulation of the inhibidactivin genes, we chose to characterize the 5'-flanking regions of the bovine a-, PA-and P,-subunit genes.…”