Stocco D M, Ascoli M
Department of Biochemistry and Molecular Biology, Texas Tech University Health Sciences Center, Lubbock 79430.
Endocrinology. 1993 Mar;132(3):959-67. doi: 10.1210/endo.132.3.8382603.
The true rate-limiting step in steroidogenesis is the delivery of cholesterol to the inner mitochondrial membrane where it is converted to pregnenolone by the cholesterol side-chain cleavage complex. This process is known to require de novo protein synthesis. We have previously described the synthesis of a family of 37, 32, and 30 kilodalton mitochondrial proteins in response to hormone stimulation in MA-10 mouse Leydig tumor cells and have proposed that these proteins are involved in the acute regulation of steroidogenesis. In this study we have used two subclones of MA-10 cells to further demonstrate the correlation between the quantity of these proteins and the production of steroids in response to hormone treatment. One of these, designated MA-10(K3), has been transfected with a mutant gene of the type 1 regulatory subunit of the cAMP-dependent protein kinase under the control of a metallothionein promoter, whereas the other, designated MA-10(P+29), is a constitutive overproducer of a cAMP-phosphodiesterase (PDE). MA-10 parent cells designated (P), produce large amounts of progesterone in response to LH, human CG, and (Bu)2cAMP. The MA-10(K3) cells, on the other hand, whereas significantly higher than controls, produce much less steroid than the parent cells in response to hormone stimulation. Activation of the mutant gene with Zn+2 results in yet a further decrease in the amount of steroid produced. The MA-10(P+29) cells display greatly reduced progesterone production when stimulated with LH, because of the presence of high amounts of PDE, but return to maximally stimulated levels when a PDE inhibitor is present. Quantitation of the synthesis of the mitochondrial proteins described above in MA-10(K3) cells in the presence and absence of Zn+2 and in MA-10(P+29) cells in the presence and absence of PDE inhibitor clearly demonstrate that the amount of the 30 kilodalton mitochondrial proteins present in these cells closely parallels that of progesterone production. The high degree of correlation between the appearance and quantity of these mitochondrial proteins and the production of steroids make them strong candidates for the putative proteins involved in acute regulation of steroidogenesis.
类固醇生成过程中真正的限速步骤是胆固醇转运至线粒体内膜，在那里胆固醇侧链裂解复合物将其转化为孕烯醇酮。已知这一过程需要从头合成蛋白质。我们之前曾描述过，在MA - 10小鼠睾丸间质细胞瘤细胞中，响应激素刺激会合成一族分子量分别为37、32和30千道尔顿的线粒体蛋白，并提出这些蛋白参与类固醇生成的急性调节。在本研究中，我们使用了MA - 10细胞的两个亚克隆，以进一步证明这些蛋白的量与激素处理后类固醇生成之间的相关性。其中一个亚克隆命名为MA - 10(K3)，已用金属硫蛋白启动子控制下的cAMP依赖性蛋白激酶1型调节亚基的突变基因进行转染；另一个亚克隆命名为MA - 10(P + 29)，是cAMP磷酸二酯酶（PDE）的组成型过量生产者。命名为(P)的MA - 10亲本细胞，响应促黄体生成素（LH）、人绒毛膜促性腺激素（hCG）和双丁酰环磷腺苷（(Bu)2cAMP）会产生大量孕酮。另一方面，MA - 10(K3)细胞虽然显著高于对照组，但在激素刺激下产生的类固醇比亲本细胞少得多。用Zn + 2激活突变基因会导致产生的类固醇量进一步减少。MA - 10(P + 29)细胞在受到LH刺激时，由于存在大量PDE，孕酮生成大幅减少，但当存在PDE抑制剂时会恢复到最大刺激水平。对MA - 10(K3)细胞在有无Zn + 2情况下以及MA - 10(P + 29)细胞在有无PDE抑制剂情况下上述线粒体蛋白合成的定量分析清楚地表明，这些细胞中存在的30千道尔顿线粒体蛋白的量与孕酮生成量密切平行。这些线粒体蛋白的出现和量与类固醇生成之间的高度相关性，使它们成为参与类固醇生成急性调节的假定蛋白的有力候选者。
