Allen John A, Shankara Tristan, Janus Paul, Buck Steve, Diemer Thorsten, Hales Karen Held, Hales Dale B
Department of Physiology and Biophysics (MC 901), University of Illinois at Chicago, Chicago, Illinois 60612-7342, USA.
Endocrinology. 2006 Aug;147(8):3924-35. doi: 10.1210/en.2005-1204. Epub 2006 May 11.
The first and rate-limiting step in the biosynthesis of steroid hormones is the transfer of cholesterol into mitochondria, which is facilitated by the steroidogenic acute regulatory (StAR) protein. Recent study of Leydig cell function has focused on the mechanisms regulating steroidogenesis; however, few investigations have examined the importance of mitochondria in this process. The purpose of this investigation was to determine which aspects of mitochondrial function are necessary for acute cAMP-stimulated Leydig cell steroidogenesis. MA-10 cells were treated with 8-bromoadenosine 3',5'-cyclic monophosphate (cAMP) and different site-specific agents that disrupt mitochondrial function, and the effects on acute cAMP-stimulated progesterone synthesis, StAR mRNA and protein, mitochondrial membrane potential (Deltapsim), and ATP synthesis were determined. cAMP treatment of MA-10 cells resulted in significant increases in both cellular respiration and Deltapsim. Dissipating Deltapsim with carbonyl cyanide m-chlorophenyl hydrazone resulted in a profound reduction in progesterone synthesis, even in the presence of newly synthesized StAR protein. Preventing electron transport in mitochondria with antimycin A significantly reduced cellular ATP, potently inhibited steroidogenesis, and reduced StAR protein levels. Inhibiting mitochondrial ATP synthesis with oligomycin reduced cellular ATP, inhibited progesterone synthesis and StAR protein, but had no effect on Deltapsim. Disruption of intramitochondrial pH with nigericin significantly reduced progesterone production and StAR protein but had minimal effects on Deltapsim. 22(R)-hydroxycholesterol-stimulated progesterone synthesis was not inhibited by any of the mitochondrial reagents, indicating that neither P450 side-chain cleavage nor 3beta-hydroxysteroid dehydrogenase activity was inhibited. These results indicate that Deltapsim, mitochondrial ATP synthesis, and mitochondrial pH are all required for acute steroid biosynthesis. These results suggest that mitochondria must be energized, polarized, and actively respiring to support Leydig cell steroidogenesis, and alterations in the state of mitochondria may be involved in regulating steroid biosynthesis.
类固醇激素生物合成的第一步也是限速步骤，是胆固醇转运至线粒体，这一过程由类固醇生成急性调节（StAR）蛋白介导。近期对睾丸间质细胞功能的研究聚焦于调节类固醇生成的机制；然而，很少有研究探讨线粒体在此过程中的重要性。本研究的目的是确定急性cAMP刺激的睾丸间质细胞类固醇生成所需的线粒体功能的哪些方面。用8-溴腺苷3',5'-环磷酸（cAMP）和不同的位点特异性试剂处理MA-10细胞，这些试剂会破坏线粒体功能，并测定其对急性cAMP刺激的孕酮合成、StAR mRNA和蛋白、线粒体膜电位（Δψm）以及ATP合成的影响。用cAMP处理MA-10细胞会导致细胞呼吸和Δψm显著增加。用羰基氰化物间氯苯腙消散Δψm会导致孕酮合成大幅减少，即使存在新合成的StAR蛋白也是如此。用抗霉素A阻止线粒体中的电子传递会显著降低细胞ATP水平，强力抑制类固醇生成，并降低StAR蛋白水平。用寡霉素抑制线粒体ATP合成会降低细胞ATP水平，抑制孕酮合成和StAR蛋白，但对Δψm没有影响。用尼日利亚菌素破坏线粒体内pH会显著降低孕酮生成和StAR蛋白，但对Δψm影响最小。22(R)-羟基胆固醇刺激的孕酮合成不受任何线粒体试剂的抑制，这表明细胞色素P450侧链裂解酶和3β-羟基类固醇脱氢酶活性均未受到抑制。这些结果表明，急性类固醇生物合成需要Δψm、线粒体ATP合成和线粒体内pH。这些结果提示，线粒体必须被激活、极化并积极进行呼吸作用以支持睾丸间质细胞的类固醇生成，线粒体状态的改变可能参与调节类固醇生物合成。
