Regulation of steroidogenesis and the steroidogenic acute regulatory protein by a member of the cAMP response-element binding protein family.
The mitochondrial phosphoprotein, the steroidogenic acute regulatory (StAR) protein, is an essential component in the regulation of steroid biosynthesis in adrenal and gonadal cells through cAMP-dependent pathways. In many cases transcriptional induction by cAMP is mediated through the interaction of a cAMP response-element binding protein (CREB) family member with a consensus cAMP response element (CRE; 5'-TGACGTCA-3') found in the promoter of target genes. The present investigation was carried out to determine whether a CRE-binding protein (CREB) family member [CREB/CRE modulator (CREM) family] was involved in the regulation of steroidogenesis and StAR protein expression. Transient expression of wild- type CREB in MA-10 mouse Leydig tumor cells further increased the levels of (Bu)2cAMP-induced progesterone synthesis, StAR promoter activity, StAR mRNA, and StAR protein. These responses were significantly inhibited by transfection with a dominant-negative CREB (A-CREB), or with a CREB mutant that cannot be phosphorylated (CREB-M1), the latter observation indicating the importance of phosphorylation of a CREB/CREM family member in steroidogenesis and StAR expression. The CREB/CREM-responsive region in the mouse StAR gene was located between -110 and -67 bp upstream of the transcriptional start site. An oligonucleotide probe (-96/-67 bp) containing three putative half-sites for 5'-canonical CRE sequences (TGAC) demonstrated the formation of protein-DNA complexes in EMSAs with recombinant CREB protein as well as with nuclear extracts from MA-10 or Y-1 mouse adrenal tumor cells. The predominant binding factor observed with EMSA was found to be the CREM protein as demonstrated using specific antibodies and RT-PCR analyses. The CRE elements identified within the -96/-67 bp region were tested for cAMP responsiveness by generating mutations in each of the CRE half-sites either alone or in combination. Although each of the CRE sites contribute in part to the CREM response, the CRE2 appears to be the most important site as determined by EMSA and by reporter gene analyses. Binding specificity was further assessed using specific antibodies to CREB/CREM family members, cold competitors, and mutations in the target sites that resulted in either supershift and/or inhibition of these complexes. We also demonstrate that the inducible cAMP early repressor markedly diminished the endogenous effects of CREM on cAMP-induced StAR promoter activity and on StAR mRNA expression. These are the first observations to provide evidence for the functional involvement of a CREB/CREM family member in the acute regulation of trophic hormone-stimulated steroidogenesis and StAR gene expression.
Manna
Pulak R
PR
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.
Dyson
Matthew T
MT
Eubank
Darrell W
DW
Clark
Barbara J
BJ
Lalli
Enzo
E
Sassone-Corsi
Paolo
P
Zeleznik
Anthony J
AJ
Stocco
Douglas M
DM
D024506:5' Flanking Region; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D051786:Cyclic AMP Response Element Modulator; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D007985:Leydig Cells; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins; D020218:Response Elements / Q000235:genetics; D013256:Steroids / Q000378:metabolism*; D014158:Transcription, Genetic; D000098682:Steroidogenic Acute Regulatory Protein
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D012097:Repressor Proteins; D013256:Steroids; D000098682:Steroidogenic Acute Regulatory Protein; D051786:Cyclic AMP Response Element Modulator; D000242:Cyclic AMP