Regulation of Steroidogenic Genes by Trophic Hormones

营养激素对类固醇基因的调节

• 批准号: 7068030
• 负责人: Marion B. Sewer
• 金额: $ 21.67万
• 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/7068030
• 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（由申请方提供）：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作用的影响通过cAMP/PKA依赖性途径介导。我们已经证明，ACTH/cAMP刺激的人CYP 17基因（hCYP 17）的转录需要丝裂原活化蛋白激酶磷酸酶1（MKP-1），一种细胞外信号相关激酶1/2（ERK 1/2）的灭活剂。在人肾上腺皮质H295 R细胞中，MKP-1可被ACTH/cAMP快速诱导，PKA可使MKP-1磷酸化。我们还表明，抑制ERK 1/2激活模拟ACTH/cAMP对hCYP 17基因表达的刺激作用。我们推测ERK 1/2组成性磷酸化类固醇生成因子1（SF-1），MKP-1对ACTH/cAMP的反应是使ERK 1/2去磷酸化，从而增加hCYP 17的转录。我们以前的研究也表明，SF-1，p54 nrb和多聚嘧啶道结合蛋白相关剪接因子（PSF）形成的复合物是必不可少的cAMP依赖性转录的hCYP 17。此外，我们已经证明，辅阻遏蛋白mSin 3A和组蛋白脱乙酰酶（HDAC）与该复合物相互作用，并抑制hCYP 17转录。我们假设SF-1的去磷酸化导致mSin 3A和HDAC从复合物中解离并募集共激活因子。该提议旨在表征MKP-1激活和SF-1去磷酸化在ACTH/cAMP刺激的hCYP 17基因转录中的功能意义。此外，我们将确定SF-1组成性磷酸化的机制，并研究SF-1的磷酸化如何抑制hCYP 17表达。最后，我们将确定SF-1/p54 nrb/PSF复合物如何相互作用，以及该复合物如何与辅阻遏物/辅激活物相互作用以抑制/激活hCYP 17。使用生物化学和分子技术，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，我们的研究结果将提供深入了解ACTH/cAMP途径和MAPK信号级联相互作用的机制，以确保肾上腺激素的生物合成，以满足人类的生理需求。