Functional Assessment of the Calcium Messenger System in Cultured Mouse Leydig Tumor Cells: Regulation of Human Chorionic Gonadotropin-Induced Expression of the Steroidogenic Acute Regulatory Protein
The steroidogenic acute regulatory (StAR) protein, a 30-kDa mi- tochondrial 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 (Ca21) involved in the hCG-stimulated expression of StAR protein and ste- roidogenesis in a mouse Leydig tumor cell line (mLTC-1). Extracel- lular Ca21 (1.5 mmol/liter) enhanced the hCG (50 mg/liter)-induced increases in StAR messenger RNA (mRNA) and protein levels (1.7 6 0.3-fold; 4 h), as monitored by quantitative RT-PCR and immuno- blotting. The potentiating effect of Ca21 on the hCG-stimulated StAR response correlated with the acute progesterone (P) response. In ac- cordance, omission of Ca21 from the extracellular medium by specific Ca21 chelators, EDTA or EGTA (4 mmol/liter each), markedly di- minished the hCG-stimulated P production. The Ca21 effect on hCG- induced StAR mRNA expression was dramatically suppressed by 10 mmol/liter verapamil, a Ca21 channel blocker. The Ca21-mobilizing agonist, potassium (K1; 4 mmol/liter), greatly increased the hCG responses of StAR expression and P production, which conversely were attenuated by Ca21 antagonists, further supporting the involve- ment of intracellular free Ca21 ((Ca21)i) in these responses. The in- teraction of Ca21 or K1 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, indi- cating the requirement for on-going protein synthesis for hCG action. The transmembrane uptake of 45Ca21 was increased by 26% with hCG and was strongly inhibited by verapamil. (Ca21)i moderately aug- mented 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 (A 23187) 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 Ca21. The mechanism of the Ca21 effect on hCG-stimulated StAR expression and P production was evaluated by assessing the involvement of the nuclear orphan recep- tor, steroidogenic factor 1 (SF-1). Stimulation of hCG significantly elevated (2.1 6 0.3-fold) the SF-1 mRNA level, which was further augmented in the presence of Ca21, whereas EGTA and verapamil completely abolished the increase caused by Ca21. Cells expressing SF-1 marginally increased StAR expression, but coordinately ele- vated StAR mRNA levels in response to hCG and hCG plus Ca21 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 Ca21 rapidly increases (Ca21)i after hCG stimula- tion, presumably through opening of the transmembrane Ca21 chan- nel. Neither extracellular Ca21 nor K1 alone has a noticeable effect on StAR expression and steroidogenesis, whereas they clearly poten- tiate hCG induction. The Ca21-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 (Ca21)i is responsible at least in part for the regulation of SF-1- mediated StAR expression that consequently regulates steroidogen- esis in mouse Leydig tumor cells. (Endocrinology 140: 1739 -1751, 1999)