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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

Department of Physiology, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland

Address all correspondence and requests for reprints to: Prof. Ilpo T. Huhtaniemi, M.D., Ph.D., Department of Physiology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. E-mail: ilpo.huhtaniemi{at}utu.fi

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 µg/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 µmol/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 (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 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.

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