DISRUPTION OF STEROIDOGENISIS BY ARSENITE

亚砷酸钠是环境中砷的主要形式，它与邻苯二甲酰蛋白巯基交联，导致多种毒性细胞反应，包括氧化应激。我们发现，亚砷酸盐，即使在低浓度下，在大鼠体内和培养的细胞中，大大抑制肾上腺类固醇的产生。cAMP调节类固醇生成的关键步骤是cAMP转移胆固醇，将胆固醇转移到线粒体内的细胞色素P450/SCC。类固醇生成急性调节蛋白（星星）在这一过程中起着重要作用。这种蛋白质形成为37 kDa的前体，其被蛋白激酶A（PKA）磷酸化，并在线粒体中加工成更有活性的30 kDa。这一过程与胆固醇从线粒体外膜向线粒体内膜的转运增强有关。这种连续的翻译和加工是胆固醇转移所必需的。我们将表明，亚砷酸盐抑制肾上腺（原代和Y-1）和睾丸Leydig（MA-10）细胞的胆固醇代谢，同时也减弱了这一重要的星星处理。亚砷酸盐，在非常低的浓度，大大增强cAMP诱导的星星转录之前，在较高浓度的抑制。另一种氧化应激剂茴香霉素对这一过程显示出类似的双相效应，并且与亚砷酸盐一样，激活应激激活蛋白激酶（SAPK），如JNK和p38。我们认为，SAPK刺激星星转录提供了一种保护机制，以维持星星活性，因为后来的关键步骤被抑制。我们将建立，与选择性抑制剂，亚砷酸盐改变转录和稳定性的星星mRNA主要通过激活p38。涉及亚砷酸盐的转录调节将通过解剖星星上游调控区，特别是识别SF-1核调节子的位点进行分析。亚砷酸盐调节正常和突变的重组SF-1对StAR-荧光素酶报告基因的活性将用于进一步确定机制。拟议的研究将提供一种新的肾上腺对化学应激的反应机制，这种反应可能与其他类固醇生成细胞共享。砷对SF-1调节的影响与该因子调节的肾上腺/性腺发育有更广泛的相关性。

Sodium arsenite, a major form of environmental arsenic, cross-links vicinyl protein sulfhydryl groups, leading to multiple toxic cellular responses, including oxidative stress. We found that arsenite, even at low concentrations, substantially inhibits adrenal steroid production both in rats in vivo and in cultured cells. The key step in the regulation of steroidogenesis by cAMP is the transfer of cholesterol by cAMP is the transfer of cholesterol to the inner mitochondrial cytochrome P450/SCC. The Steroidogenic Acute Regulatory protein (StAR) plays a major role in this process. This protein is formed as a 37 kDa precursor that is phosphorylated by protein kinase A (PKA) and is processed to more active 30 kDa in the mitochondria. This processing is coupled to enhanced cholesterol transport from the outer to the inner mitochondrial membrane. This continuous translation and processing is necessary for cholesterol transfer. We will show that arsenite suppresses cholesterol metabolism in adrenal (primary and Y-1) and testis Leydig (MA-10) cells, while also attenuating this essential StAR processing. Arsenite, at very low concentrations, substantially potentiates cAMP-induced StAR transcription prior to suppression at higher concentrations. Another oxidative stress agent, anisomycin, shows a similar biphasic effect on this process and, like arsenite, activates stress-activated protein kinases (SAPK), such as JNK and p38. We propose that SAPK stimulation of StAR transcription provides a protective mechanism to sustain StAR activity as later key steps are inhibited. We will establish, with selective inhibitors that arsenite alters the transcription and the stability of StAR mRNA primarily via the activation of p38. The transcriptional modulation involving arsenite will be analyzed by dissecting the StAR upstream regulatory region, particularly sites recognizing the SF-1 nuclear regulator. Arsenite modulation of the activity of normal and mutated recombinant SF-1 on StAR-luciferase reporters will be used to further define the mechanism. The proposed research will provide mechanistic insight into a novel adrenal response to chemical stress that may be shared by other steroidogenic cells. Effects of arsenite on SF-1 regulation have broader relevance to adrenal/gonadal development regulated by this factor.