Regulation of Steroidogenic Genes by Trophic Hormones

描述（由申请人提供）：促肾上腺皮质激素（ACTH）对肾上腺皮质激素基因转录作用的刺激是通过cAMP/ pka依赖途径介导的。我们已经证明，ACTH/ camp刺激的人类CYP17基因（hCYP17）的转录需要有丝分裂原激活的蛋白激酶磷酸酶1 (MKP-1)，这是一种细胞外信号相关激酶1/2 （ERK1/2）的失活因子。在人肾上腺皮质H295R细胞中，ACTH/cAMP可快速诱导MKP-1， PKA可使MKP-1在体外磷酸化。我们还发现，抑制ERK1/2的激活可以模拟ACTH/cAMP对hCYP17基因表达的刺激作用。我们假设ERK1/2构成性地磷酸化甾体生成因子1 (SF-1)，并且在ACTH/cAMP的反应中，MKP-1作用于ERK1/2去磷酸化，从而增加hCYP17的转录。我们之前的研究也表明，SF-1、p54nrb和聚嘧啶束结合蛋白相关剪接因子（PSF）形成一个复合物，对camp依赖性的hCYP17转录至关重要。此外，我们已经证明了协同抑制因子mSin3A和组蛋白去乙酰化酶（HDAC）与该复合物相互作用并抑制hCYP17的转录。我们假设SF-1的去磷酸化导致mSin3A和HDAC从复合体中分离并募集共激活因子。本提案旨在表征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组成性磷酸化的机制，并研究SF-1磷酸化如何抑制hCYP17的表达。最后，我们将确定SF-1/p54nrb/PSF复合体如何相互作用，以及该复合体如何与共抑制因子/共激活因子相互作用，从而抑制/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，我们的发现将深入了解ACTH/cAMP途径和MAPK信号级联相互作用的机制，以确保肾上腺激素的生物合成，以满足人类的生理需求。

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.