StAR expression: Integration of Transcription with regulation via the mRNA 3'UTR

描述（由申请人提供）：肾上腺皮质的皮质醇合成在应激反应、饮食控制和心血管调节中起核心作用。这种下丘脑-垂体-肾上腺轴控制的异常在代谢综合征中起重要作用，代谢综合征通常与2型糖尿病相关。ACTH刺激皮质醇合成始于线粒体细胞色素P450 11A1将胆固醇转化为孕烯醇酮。这一步骤的激活取决于类固醇生成急性调节因子（StAR）的新合成及其被蛋白激酶A （PKA）磷酸化。缺乏症引起高脂血症肾上腺功能不全。本研究的重点是发现PKA和蛋白激酶C （PKC）以非常不同的方式刺激StAR表达。我们将展示这些过程是如何通过组蛋白去乙酰化酶和Erk激酶的重要贡献来明确区分的。我们将描述这些过程的不同贡献的核因素，包括分化调节剂，SF-1。这包括转录过程中上下循环的时间依赖性修饰（磷酸化/去磷酸化和乙酰化/去乙酰化）。将会有证据表明PKA也可以增强PKC过程的后期，从而在StAR转录中产生强大的协同作用。StAR的转录后调控提供了PKA和PKC调控的另一个不同点。一种新的调控因子，锌指蛋白TIS11b，靶向3.5 kb StAR mRNA延伸的3'非翻译区中的特定序列。TIS11b被PKA快速刺激，但被PKC抑制。我们将在StAR 3.5 kb mRNA末端确定特定的TIS11b识别位点，并证明这种相互作用可以增强StAR蛋白的翻译，同时增加mRNA的降解。我们将测试TIS11b是否由于共享转录因子（包括SF-1）而与StAR共转录。我们将确定这些机制在ACTH刺激过程中如何相互作用，包括在原发性肾上腺细胞系和体内肾上腺中。从小鼠到人类，在StAR表达过程中指定SF-1和TIS11b相互作用的启动子和mRNA序列基本上是保守的。我们将测试这些小鼠机制是否在人类肾上腺H295细胞中保留。我们的研究表明，TIS11b与StAR的相互作用和一个扩展的StAR mRNA的优势在原代牛肾上腺细胞中是保守的，并对ACTH有反应。这项工作将汇集几个实验室，首次研究PKA、SF-1和TIS11b（或与它们相关的因素）之间的相互作用，这种相互作用可能发生在多种类固醇组织中。肾上腺皮质的皮质醇合成在应激反应、饮食控制和心血管调节中起着核心作用。促肾上腺皮质激素在压力下升高，通过增强胆固醇向孕烯醇酮的转化来刺激皮质醇的合成。这一步取决于类固醇生成急性调节因子（StAR）的新合成。这项研究提出了一个新的过程，即ACTH不仅刺激StAR的转录，还刺激一种名为TIS11b的蛋白质的转录，这种蛋白质分别调节StAR蛋白的翻译和mRNA的降解。我们详细分析了控制StAR和TIS11b转录的调控过程，以了解它们的潜在协调。TIS11b可能会加快应激反应时间，同时在去除ACTH刺激时也有助于去除StAR。TIS11b调节存在于其他制造类固醇的组织中（睾丸）。这种mRNA调节因子的缺乏可能导致对ACTH和应激的反应更加迟缓，以及皮质醇输出异常。

DESCRIPTION (provided by applicant): Cortisol synthesis in the adrenal cortex plays a central role in stress responses, in dietary control, and in cardiovascular regulation. Aberrations in this control of the hypothalamic pituitary adrenal axis play an important part in the Metabolic Syndrome that is often associated with Type 2 diabetes. ACTH stimulation of cortisol synthesis starts with the conversion of cholesterol to pregnenolone by mitochondrial cytochrome P450 11A1. Activation of this step depends on new synthesis of the steroidogenesis acute regulator (StAR) and its phosphorylation by protein kinase A (PKA). StAR deficiency causes hyperlipidemic adrenal insufficiency. This research focuses on the finding that StAR expression is stimulated in very different ways by PKA and by protein kinase C (PKC). We will show how these processes are clearly distinguished by essential contributions from, respectively, histone de-acetylatases and Erk kinase. We will characterize these processes for different contributions from nuclear factors, including the differentiation regulator, SF-1. This includes time-dependent modifications that cycle up and down during transcription (phosphorylation/de-phosphorylation and acetylation/de- acetylation). Evidence will be developed that PKA can also enhance a late stage in the PKC process, thus producing strong synergy in StAR transcription. Post transcriptional regulation of StAR provides another point of distinction between PKA and PKC regulation. A novel regulator, the Zn finger protein, TIS11b, targets specific sequences in the extended 3'untranslated region of the 3.5 kb StAR mRNA. TIS11b is rapidly stimulated by PKA, but suppressed by PKC. We will identify specific TIS11b recognition sites at the end of the StAR 3.5 kb mRNA and show that this interaction can enhance StAR protein translation, while increasing mRNA degradation. We will test whether TIS11b is co-transcribed with StAR due to shared transcription factors, including SF-1. We will determine how these mechanisms interplay during ACTH stimulation, including in primary adrenal cell lines and in adrenals in vivo. The promoter and mRNA sequences that specify SF-1 and TIS11b interactions during StAR expression are substantially conserved from mouse to humans. We will test whether these mouse mechanisms are retained in human adrenal H295 cells. Our research shows that TIS11b interaction with StAR and predominance of an extended StAR mRNA are conserved in primary bovine adrenal cells and respond to ACTH. This work will bring together several laboratories to provide a first look at the interplay between PKA, SF-1, and TIS11b (or factors related to each), which is likely to occur in multiple steroidogenic tissues. Cortisol synthesis in the adrenal cortex plays a central role in stress responses, in dietary control, and in cardiovascular regulation. ACTH, which is elevated by stress, stimulates cortisol synthesis through enhanced conversion of cholesterol to pregnenolone. This step depends on new synthesis of the steroidogenesis acute regulator (StAR). The proposed research addresses a novel process, whereby ACTH stimulates not only transcription of StAR, but also a protein called TIS11b, which separately regulates StAR protein translation and mRNA degradation. We make a detailed analysis of regulatory processes that control StAR and TIS11b transcription in order to understand their potential coordination. TIS11b may accelerate the response time to stress, while also aiding in the removal of StAR when the ACTH stimulus is removed. TIS11b regulation is present in other tissues that make steroids (testis). Deficiency in this mRNA regulator may lead to more sluggish responses to ACTH and stress as well as abnormal cortisol output.