Regulation of Steroidogenic Genes by Trophic Hormones

营养激素对类固醇基因的调节

• 批准号: 6901080
• 负责人: Marion B. Sewer
• 金额: $ 22.2万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-07 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6901080
• 关键词: corticosteroids; enzyme activity; gene expression; gene mutation; genetic regulation; hormone regulation /control mechanism; mitogen activated protein kinase; phosphatase inhibitor; phosphoprotein phosphatase; phosphorylation; protein kinase A; protein protein interaction; protein structure function; steroid biosynthesis; tissue /cell culture

DESCRIPTION (provided by applicant): The stimulatory adrenocorticotropin (ACTH) on steroidogenic gene action of transcription in the adrenal cortex is mediated through a cAMP/PKA-dependent pathway. We have demonstrated that the ACTH/cAMP-stimulated transcription of the human CYP17 gene (hCYP17) requires mitogen-activated protein kinase phosphatase 1 (MKP-1), an inactivator of extracellular-signal-related kinases 1/2 (ERK1/2). In human adrenocortical H295R cells, MKP-1 is rapidly induced by ACTH/cAMP and PKA can phosphorylate MKP-1 in vitro. We have also shown that inhibition of ERK1/2 activation mimics the stimulatory effect of ACTH/cAMP on hCYP17 gene expression. We postulate that ERK1/2 constitutively phosphorylates steroidogenic factor 1 (SF-1) and that in response to ACTH/cAMP, MKP-1 acts to dephosphorylate ERK1/2, thereby increasing hCYP17 transcription. Our previous studies have also demonstrated that SF-1, p54nrb and polypyrimidine tract binding protein-associated splicing factor (PSF) form a complex that is essential for cAMP-dependent transcription of hCYP17. Further, we have demonstrated that the corepressor mSin3A and a histone deacetylase (HDAC) interact with this complex and repress hCYP17 transcription. We hypothesize that dephosphorylation of SF-1 results in dissociation of mSin3A and the HDAC from the complex and recruitment of coactivators. This proposal aims to characterize the functional significance of MKP-1 activation and SF-1 dephosphorylation in ACTH/cAMP-stimulated hCYP17 gene transcription. Further, we will determine the mechanism by which SF-1 is constitutively phosphorylated and examine how phosphorylation of SF-1 represses hCYP17 expression. Finally, we will determine how the SF-1/p54nrb/PSF complex interacts with each other and how this complex interacts with corepressors/coactivators to allow for repression/activation of hCYP17. Using both biochemical and molecular techniques, including reporter gene transfection assays, chromatin immunoprecipitation, phosphatase/kinase activity assays, and mass spectrometry, our findings will provide insight into the mechanism by which the ACTH/cAMP pathway and the MAPK signaling cascade interact to ensure biosynthesis of adrenal hormones to meet physiological needs in humans.

描述（由申请人提供）：刺激性促肾上腺皮质激素（ACTH）对肾上腺皮质中类固醇生成基因转录的作用是通过cAMP/PKA依赖性途径介导的。我们已经证明，ACTH/cAMP 刺激的人 CYP17 基因 (hCYP17) 转录需要丝裂原激活蛋白激酶磷酸酶 1 (MKP-1)，它是细胞外信号相关激酶 1/2 (ERK1/2) 的灭活剂。在人肾上腺皮质H295R细胞中，MKP-1被ACTH/cAMP快速诱导，并且PKA可以在体外磷酸化MKP-1。我们还表明，抑制 ERK1/2 激活模拟了 ACTH/cAMP 对 hCYP17 基因表达的刺激作用。我们假设 ERK1/2 持续磷酸化类固醇生成因子 1 (SF-1)，并且响应 ACTH/cAMP，MKP-1 使 ERK1/2 去磷酸化，从而增加 hCYP17 转录。我们之前的研究还表明，SF-1、p54nrb 和多聚嘧啶束结合蛋白相关剪接因子 (PSF) 形成复合物，对于 hCYP17 的 cAMP 依赖性转录至关重要。此外，我们还证明了辅抑制子 mSin3A 和组蛋白脱乙酰酶 (HDAC) 与该复合物相互作用并抑制 hCYP17 转录。我们假设 SF-1 的去磷酸化导致 mSin3A 和 HDAC 从复合物中解离并招募共激活剂。该提案旨在表征 MKP-1 激活和 SF-1 去磷酸化在 ACTH/cAMP 刺激的 hCYP17 基因转录中的功能意义。此外，我们将确定 SF-1 组成型磷酸化的机制，并研究 SF-1 磷酸化如何抑制 hCYP17 表达。最后，我们将确定 SF-1/p54nrb/PSF 复合物如何相互作用，以及该复合物如何与辅阻遏物/共激活物相互作用以抑制/激活 hCYP17。利用生化和分子技术，包括报告基因转染测定、染色质免疫沉淀、磷酸酶/激酶活性测定和质谱分析，我们的研究结果将深入了解 ACTH/cAMP 途径和 MAPK 信号级联相互作用的机制，以确保生物合成肾上腺激素以满足人类的生理需要。