Regulation of steroidogenesis and the steroidogenic acute regulatory protein by a member of the cAMP response-element binding protein family
Regulation of steroidogenesis and the steroidogenic acute regulatory protein by a member of the cAMP response-element binding protein family
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DOI：
10.1210/me.16.1.184
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发表时间：
2002-01-01
影响因子：
--
通讯作者：
Stocco, DM
中科院分区：
文献类型：
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作者：
Manna, PR;Dyson, MT;Stocco, DM
线粒体磷蛋白是类固醇合成急性调节蛋白(STAR)，是肾上腺和性腺细胞通过cAMP依赖途径调节类固醇生物合成的重要组成部分。在许多情况下，cAMP的转录诱导是通过cAMP反应元件结合蛋白(CREB)家族成员与靶基因启动子中的共同cAMP反应元件(Cre；5‘-TGACGTCA-3’)相互作用而介导的。本研究旨在确定CRE结合蛋白(CREB)家族成员[CREB/CRE调节器(CREM)家族]是否参与类固醇合成和STAR蛋白表达的调节。野生型CREB在MA-10小鼠间质瘤细胞中的瞬时表达进一步增加了Bu、cAMP诱导的孕酮合成、STAR启动子活性、STAR mRNA和STAR蛋白的水平。这些反应被显性负性CREB(ACREB)或不能磷酸化的CREB突变体(CREB-M1)显著抑制，后者表明CREB/CREM家族成员的磷酸化在类固醇合成和STAR表达中的重要性。小鼠STAR基因的CREB/CREM响应区位于转录起始点上游-110~-67bp之间。含有3个假定的5‘-规范CRE序列(TGAC)半位点的寡核苷酸探针(-96/-67bp)证实了EMSA与重组CREB蛋白以及MA-10或Y-1小鼠肾上腺肿瘤细胞的核提取物形成蛋白质-DNA复合体。通过特异性抗体和RT-PCR分析，发现EMSA观察到的主要结合因子是CREM蛋白。在-96/-67bp区域内确定的Cre元件通过单独或组合在每个Cre半位点上产生突变来测试cAMP反应性。根据EMSA和报告基因分析，虽然每个Cre位点都参与了CREM反应，但CRE2似乎是最重要的位点。进一步使用针对CREB/CREM家族成员、冷竞争对手的特异性抗体以及导致这些复合体超位和/或抑制的靶点突变来进一步评估结合特异性。我们还证明，可诱导的cAMP早期抑制物显著减弱了CREM对cAMP诱导的STAR启动子活性和对STAR mRNA表达的内源性影响。这是第一次为CREB/CREM家族成员参与营养激素刺激的类固醇合成和STAR基因表达的急性调节提供证据。
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),cAMP-induced progesterone synthesis, StAR promoter activity, StAR mRNA, and StAR protein. These responses were significantly inhibited by transfection with a dominant-negative CREB (ACREB), 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.