Manna P R, Pakarinen P, El-Hefnawy T, Huhtaniemi I T
Department of Physiology, Institute of Biomedicine, University of Turku, Finland.
Endocrinology. 1999 Apr;140(4):1739-51. doi: 10.1210/endo.140.4.6650.
The steroidogenic acute regulatory (StAR) protein, a 30-kDa mitochondrial factor, is a key regulator of steroid hormone biosynthesis, facilitating the transfer of cholesterol from the outer to the inner mitochondrial membrane. StAR protein expression is restricted to steroidogenic tissues, and it responds to hormonal stimulation through different second messenger pathways. The present study was designed to explore the mechanisms of extracellular calcium (Ca2+) involved in the hCG-stimulated expression of StAR protein and steroidogenesis in a mouse Leydig tumor cell line (mLTC-1). Extracellular Ca2+ (1.5 mmol/liter) enhanced the hCG (50 microg/liter)-induced increases in StAR messenger RNA (mRNA) and protein levels (1.7 +/- 0.3-fold; 4 h), as monitored by quantitative RT-PCR and immunoblotting. The potentiating effect of Ca2+ on the hCG-stimulated StAR response correlated with the acute progesterone (P) response. In accordance, omission of Ca2+ from the extracellular medium by specific Ca2+ chelators, EDTA or EGTA (4 mmol/liter each), markedly diminished the hCG-stimulated P production. The Ca2+ effect on hCG-induced StAR mRNA expression was dramatically suppressed by 10 micromol/liter verapamil, a Ca2+ channel blocker. The Ca2+-mobilizing agonist, potassium (K+; 4 mmol/liter), greatly increased the hCG responses of StAR expression and P production, which conversely were attenuated by Ca2+ antagonists, further supporting the involvement of intracellular free Ca2+ ([Ca2+]i) in these responses. The interaction of Ca2+ or K+ with hCG accounted for a clear increase in the StAR protein level (1.4-1.8-fold; 4 h) compared with that after hCG stimulation. Inhibition of protein synthesis by cycloheximide (CHX) drastically diminished the hCG-induced StAR protein content, indicating the requirement for on-going protein synthesis for hCG action. The transmembrane uptake of 45Ca2+ was increased by 26% with hCG and was strongly inhibited by verapamil. [Ca2+]i moderately augmented the response to hCG in fura-2/AM-loaded mLTC-1 cells within 30-40 sec, reaching a plateau within 1-3 min. Interestingly, the calcium ionophore (A23187) clearly increased (P < 0.01) StAR mRNA expression, in additive fashion with hCG. Northern hybridization analysis revealed four StAR transcripts at 3.4, 2.7, 1.6, and 1.4 kb, with the 1.6-kb band corresponding to the functional StAR protein; all of them were up-regulated 3- to 5-fold upon hCG stimulation, with a further increase in the presence of Ca2+. The mechanism of the Ca2+ effect on hCG-stimulated StAR expression and P production was evaluated by assessing the involvement of the nuclear orphan receptor, steroidogenic factor 1 (SF-1). Stimulation of hCG significantly elevated (2.1 +/- 0.3-fold) the SF-1 mRNA level, which was further augmented in the presence of Ca2+, whereas EGTA and verapamil completely abolished the increase caused by Ca2+. Cells expressing SF-1 marginally increased StAR expression, but coordinately elevated StAR mRNA levels in response to hCG and hCG plus Ca2+ compared with those in mock-transfected cells. On the other hand, overexpression of the nuclear receptor DAX-1 remarkably diminished (P < 0.0001) the endogenous SF-1 mRNA level as well as hCG-induced StAR mRNA expression. In summary, our results provide evidence that extracellular Ca2+ rapidly increases [Ca2+]i after hCG stimulation, presumably through opening of the transmembrane Ca2+ channel. Neither extracellular Ca2+ nor K+ alone has a noticeable effect on StAR expression and steroidogenesis, whereas they clearly potentiate hCG induction. The Ca2+-mediated increase in hCG involved in StAR expression and P production is well correlated to the levels of SF-1 expression. The stimulatory effect of hCG that rapidly increases [Ca2+]i is responsible at least in part for the regulation of SF-1-mediated StAR expression that consequently regulates steroidogenesis in mouse Leydig tumor cells.
类固醇生成急性调节（StAR）蛋白是一种30 kDa的线粒体因子，是类固醇激素生物合成的关键调节因子，可促进胆固醇从线粒体外膜转移至内膜。StAR蛋白的表达仅限于类固醇生成组织，并通过不同的第二信使途径对激素刺激作出反应。本研究旨在探讨细胞外钙（Ca2+）参与人绒毛膜促性腺激素（hCG）刺激小鼠睾丸间质细胞瘤细胞系（mLTC-1）中StAR蛋白表达和类固醇生成的机制。细胞外Ca2+（1.5 mmol/L）增强了hCG（50 μg/L）诱导的StAR信使核糖核酸（mRNA）和蛋白水平的增加（1.7±0.3倍；4小时），通过定量逆转录聚合酶链反应（RT-PCR）和免疫印迹法监测。Ca2+对hCG刺激的StAR反应的增强作用与急性孕酮（P）反应相关。相应地，通过特异性Ca2+螯合剂乙二胺四乙酸（EDTA）或乙二醇双乙醚二胺四乙酸（EGTA）（各4 mmol/L）从细胞外培养基中去除Ca2+，显著降低了hCG刺激的P生成。10 μmol/L维拉帕米（一种Ca2+通道阻滞剂）显著抑制了Ca2+对hCG诱导的StAR mRNA表达的影响。Ca2+动员激动剂钾（K+；4 mmol/L）显著增加了StAR表达和P生成的hCG反应，相反，Ca2+拮抗剂减弱了这些反应，进一步支持细胞内游离钙（[Ca2+]i）参与这些反应。与hCG刺激后相比，Ca2+或K+与hCG的相互作用使StAR蛋白水平明显增加（1.4 - 1.8倍；4小时）。放线菌酮（CHX）抑制蛋白质合成显著降低了hCG诱导的StAR蛋白含量，表明hCG作用需要持续的蛋白质合成。hCG使45Ca2+的跨膜摄取增加26%，并被维拉帕米强烈抑制。在负载fura-2/AM的mLTC-1细胞中，[Ca2+]i在30 - 40秒内适度增强了对hCG的反应，在1 - 3分钟内达到平台期。有趣的是，钙离子载体（A23187）明显增加（P < 0.01）StAR mRNA表达，与hCG呈相加作用。Northern杂交分析显示有3.4、2.7、1.6和1.4 kb的四种StAR转录本，1.6 kb条带对应功能性StAR蛋白；hCG刺激后所有转录本均上调3至5倍，Ca2+存在时进一步增加。通过评估核孤儿受体类固醇生成因子1（SF-1）的参与情况，来评价Ca2+对hCG刺激的StAR表达和P生成的作用机制。hCG刺激显著提高（2.1±0.3倍）SF-1 mRNA水平，Ca2+存在时进一步增强，而EGTA和维拉帕米完全消除了Ca2+引起的增加。表达SF-1的细胞使StAR表达略有增加，但与mock转染细胞相比，对hCG和hCG加Ca2+的反应使StAR mRNA水平协同升高。另一方面，核受体DAX-1的过表达显著降低（P < 0.0001）内源性SF-1 mRNA水平以及hCG诱导的StAR mRNA表达。总之，我们的结果表明，细胞外Ca2+在hCG刺激后可能通过打开跨膜Ca2+通道迅速增加[Ca2+]i。单独的细胞外Ca2+或K+对StAR表达和类固醇生成没有明显影响，而它们明显增强hCG诱导作用。Ca2+介导的hCG参与StAR表达和P生成的增加与SF-1表达水平密切相关。hCG迅速增加[Ca2+]i的刺激作用至少部分负责调节SF-1介导的StAR表达，从而调节小鼠睾丸间质细胞瘤细胞中的类固醇生成。
