Regulation of Steroidogenic Genes by Trophic Hormones

说明书(申请人提供)：促肾上腺皮质激素(ACTH)对肾上腺皮质类固醇生成基因转录的作用是通过cAMP/PKA依赖的途径介导的。我们已经证明，ACTH/cAMP刺激的人细胞色素P17基因(HCYP17)的转录需要丝裂原激活的蛋白激酶磷酸酶1(MKP-1)，它是细胞外信号相关蛋白1/2(ERK1/2)的失活因子。在人肾上腺皮质H295R细胞中，ACTH/cAMP能快速诱导MKP-1，PKA在体外可使MKP-1磷酸化。我们还发现，抑制ERK1/2的激活类似于ACTH/cAMP对hCYP17基因表达的刺激作用。我们推测ERK1/2结构性地磷酸化类固醇生成因子1(SF-1)，并且作为对ACTH/cAMP的反应，MKP-1作用于去磷酸化ERK1/2，从而增加hCYP17的转录。我们以前的研究也证明了SF-1、p54nrb和多嘧啶结合蛋白相关剪接因子(PSF)形成了一个复合体，对cAMP依赖的hCYP17转录是必不可少的。此外，我们还证明了辅阻遏子mSin3A和组蛋白脱乙酰酶(HDAC)与这个复合体相互作用并抑制hCYP17的转录。我们假设，SF-1的去磷酸化导致mSin3A和HDAC从复合体和共激活子的招募中解离。本研究旨在研究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被结构性磷酸化的机制，并研究SF-1的磷酸化如何抑制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辅阻遏子/共激活子相互作用，从而抑制/激活hCYP17。利用生化和分子技术，包括报告基因转染试验、染色质免疫沉淀、磷酸酶/激酶活性分析和质谱分析，我们的发现将为深入了解ACTH/cAMP途径和MAPK信号级联反应确保肾上腺激素的生物合成以满足人体生理需求的机制提供依据。

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.