Regulation of Steroidogenic Genes by Trophic Hormones

营养激素对类固醇基因的调节

• 批准号: 6826636
• 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/6826636
• 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）：通过CAMP/PKA依赖性途径介导了肾上腺皮质中转录的类固醇生成基因作用的刺激性肾上腺皮质激素（ACTH）。我们已经证明，人CYP17基因（HCYP17）的ACTH/CAMP刺激的转录需要有丝分裂原激活的蛋白激酶磷酸酶1（MKP-1），一种灭活细胞外信号相关的激酶1/2（ERK1/2）的灭活剂。 。在人肾上腺皮质H295R细胞中，MKP-1迅速由ACTH/CAMP诱导，PKA可以在体外磷酸化MKP-1。我们还表明，抑制ERK1/2激活模拟ACTH/CAMP对HCYP17基因表达的刺激作用。我们假设ERK1/2组成型磷酸化类固醇生成因子1（SF-1），并且在响应ACTH/CAMP时，MKP-1起作用，可用于去磷酸化ERK1/2，从而增加HCYP17转录。我们先前的研究还表明，SF-1，p54NRB和多吡啶氨酸链结合蛋白相关的剪接因子（PSF）形成了一种复合物，这对于HCYP17的CAMP依赖性转录至关重要。此外，我们已经证明了Corepressor MSIN3A和组蛋白脱乙酰基酶（HDAC）与这种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从复合和募集共激活剂中解离。该建议旨在表征ACTH/CAMP刺激的HCYP17基因转录中MKP-1激活和SF-1去磷酸化的功能意义。此外，我们将确定SF-1由组成型磷酸化的机制，并检查SF-1的磷酸化如何抑制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Corepressor/共激活器相互作用，以允许HCYP17的抑制/激活。使用生化和分子技术，包括报告基因转染测定法，染色质免疫沉淀，磷酸酶/激酶活性测定和质谱法，我们的发现将提供有关ACTH/CAMP途径和MAPK Signaling Cascade相互作用的机制的见解。肾上腺激素以满足人类的生理需求。