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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

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

	Introduction

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THE ENDOCRINE regulation of Leydig cells occurs predominantly through the LH/hCG receptor after coupling to the adenylate cyclase signal transduction system (1, 2). The role of Ca²⁺ as a second messenger has been extensively studied in the regulation of diverse cellular functions (3, 4, 5). Specific extracellular stimuli increase the intracellular free Ca²⁺ levels, which, in turn, stimulate various functions of the target cells (6, 7). The cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) of Leydig cells increases in parallel with their LH-stimulated testosterone production (8). In mouse Leydig tumor cells, LH-stimulated testosterone production is inhibited in the absence of extracellular Ca²⁺ (9). It has been suggested that exogenous arachidonic acid can induce Ca²⁺ mobilization from intracellular stores, resulting in increased [Ca²⁺]_i that modulates steroidogenesis in a variety of cells (10, 11, 12). This process is well developed in rat granulosa cells, where GnRH increases [Ca²⁺]_i mobilization. However, the precise source of the [Ca²⁺]_i that participates in the stimulatory function of LH/hCG has yet to be determined.

The rate-limiting, committed, and regulatable step in steroid hormone biosynthesis is the transport of cholesterol from the outer to the inner mitochondrial membrane, and it is dependent on de novo protein synthesis (13). Recently, a novel LH-induced 30-kDa mitochondrial factor was purified and cloned from MA-10 mouse Leydig tumor cells, named the steroidogenic acute regulatory (StAR) protein (14). The mature StAR protein is synthesized from a larger 37-kDa precursor protein whose expression is restricted to steroidogenic cells and is acutely involved in the regulation of steroidogenesis (15, 16). In MA-10 mouse Leydig tumor cells, (Bu)₂cAMP coordinately stimulates StAR protein and StAR messenger RNA (mRNA) expression, which were consistent with stimulated progesterone (P) production (17). Inhibition of protein synthesis by actinomycin D in hormone-stimulated MA-10 cells has been shown to inhibit StAR gene transcription, whereas CHX has no effect on the steady state level of StAR mRNA (18).

Several lines of evidence propose that multiple second messenger systems, including cAMP, protein kinase A (PKA), protein kinase C (PKC), and Ca²⁺, are involved in the induction of StAR gene expression in adrenal and gonadal cells (19, 20, 21, 22). However, clear-cut data on involvement of the Ca²⁺ messenger system in hormone-stimulated StAR expression in Leydig cells are still lacking. Ramnath et al. (23) demonstrated in MA-10 cells that the modulation of steroidogenesis is affected by the omission of Ca²⁺ from the incubation medium in the presence or absence of chloride ions. To gain molecular insight into the mechanisms, the crucial role of Ca²⁺ messenger system in StAR protein expression and steroidogenesis has yet to be investigated in mouse Leydig tumor cells (mLTC-1) (24).

The physiological action of K⁺, especially in adrenal cells, is thought to involve the opening of plasma membrane Ca²⁺ channels, facilitating the influx of Ca²⁺ for activation of steroid hormone biosynthesis (25). In adrenal glomerulosa cells, ample evidence exists for the requirement of Ca²⁺ for the steroidogenic action of K⁺ (26, 27, 28). It has also been reported that K⁺ opens voltage-operated T- and L-type Ca²⁺ channels, where the low threshold T-type channels greatly accelerate this process (29, 30). In human H295R adrenocortical choriocarcinoma cells, StAR protein expression and aldosterone synthesis have been shown to be induced by angiotensin II (Ang-II)-, K⁺-, and PKC-dependent pathways (19, 31, 32). Much progress has been made in understanding these mechanisms in adrenal cells, but definitive evidence for the role of K⁺ in hCG-stimulated StAR expression in Leydig cells is lacking.

The mechanism of the Ca²⁺ effect that modulates hCG action in steroidogenesis needs further clarification. Studies indicate that members of the orphan nuclear receptor family are involved in the development and differentiation of endocrine functions at multiple levels, including steroid hormone biosynthesis (33, 34). In accordance, SF-1 and DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome) play pivotal roles in regulation of the cytochrome P450 steroid hydroxylase genes during development and the differentiated function of the adrenal glands and gonads (35, 36). In addition, mutations of these two receptors cause multiple abnormalities of adrenal development and function associated with hypogonadotropic hypogonadism, strongly pointing out their interactions in a common developmental pathway, with activation of target genes in cooperative fashion (37, 38). It has also been demonstrated that the 5'-flanking region of the mouse StAR gene exhibits two SF-1-binding sites at positions -135 and -42 (18), and one DAX-1-binding site at position -24, whereas the human StAR promoter possesses two putative DAX-1-binding sites at positions -27 and -61 (39), but their importance has yet to be confirmed. It is noteworthy that DAX-1-binding sites in the human promoter are conserved and form hairpin loops, whereas these regions in the mouse are ineffective to form a potential hairpin loops. The interaction of these factors with StAR in regulating steroidogenesis presents the opportunity to understand in more depth the development of the steroidogenic machinery.

In light of the above observations, the present investigation was aimed to evaluate the mechanisms of Ca²⁺ messenger system in the hCG-mediated StAR mRNA, StAR protein expression, and P synthesis. The involvement of extracellular Ca²⁺ and K⁺ potentially augmented the hCG responses without altering the basal level of steroidogenesis. The use of Ca²⁺ chelators and transmembrane channel blocker allowed assessment of the role of Ca²⁺ mobilization and the rapid increase in [Ca²⁺]_i after hCG stimulation. Our data clearly show that alterations of [Ca²⁺]_i in response to hCG stimulation are highly correlated with the expression level of SF-1, a key component in the coordinate regulation of StAR expression and steroidogenesis.

	Materials and Methods

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Hormones and reagents
Purified hCG (CR-127; biological potency, 14,900 IU/mg) was a generous gift from the National Hormone and Pituitary Program (NIH, Bethesda, MD). Verapamil (-isopropyl- [(N-methyl-N-homoveratryl)--aminopropyl]3,4-dimethoxyphenylacetonitrile hydrochloride), Ca²⁺ ionophore (A 23187), fura-2/AM, 3-isobutyl-1-methylxanthine, sodium lauorylsarcosine, guanidine thiocyanate, 2-mercaptoethanol, antifoam A, EGTA, and EDTA were purchased from Sigma Chemical Co. (St. Louis, MO). ⁴⁵CaCl₂ (37 megabecquerels/1 mCi) was purchased from DuPont-New England Nuclear (Boston, MA), [-³²P]UTP (800 Ci/mmol) and [-³²P]deoxy (d)-CTP (1800 Ci/mmol) were obtained from Amersham (Aylesbury, UK). Gentamycin was purchased from Biological Industries (Bet-Haemek, Israel), HEPES-buffered Waymouth’s medium was obtained from Life Technologies (Paisley, Scotland), and cell culture plasticware was obtained from Greiner Labortechnik (Frickenhausen, Germany). All other chemicals used in this study were of analytical grade and obtained from commercial sources.

Cell culture, steroid incubation, and transfection experiments
The mouse tumor Leydig cell line (mLTC-1) was maintained in HEPES-buffered Waymouth’s medium supplemented with 9% heat-inactivated horse serum (Life Technologies) and 4.5% FCS (Bioclear, Devizes, Wilts, UK) containing 0.1 µg/liter gentamycin. The cells were cultured on 9-cm diameter plates in regular medium at 37 C under a humidified atmosphere of 95% air and 5% CO₂. During splitting, the viability of the cells was examined with 0.4% trypan blue dye exclusion, and cells were plated at a density of 6 x 10⁴ cells/well in 24-well plates for determining P production. A total of 5 x 10⁵ cells/well in 6-well plates were used for ⁴⁵Ca²⁺ incorporation studies and total RNA extractions. The cells were plated about 24 h before carrying out the stimulations.

On the day preceding an experiment, the cells were washed twice with 0.01 mol/liter PBS, and the medium was replaced with serum-free Waymouth’s medium supplemented with 0.1% BSA (fraction V, Sigma Chemical Co.) containing 0.25 mmol/liter 3-isobutyl-1-methylxanthine. The influence of Ca²⁺ on hCG-induced StAR mRNA, StAR protein, and P levels was determined in Ca²⁺-free medium. The additives (stimulators, inhibitors, etc.) were constituted fresh, diluted, applied to the cells in serum-free medium, and incubated for 4 h under a controlled atmosphere. At the end of stimulations, P content was measured from the diethyl ether-extracted incubation medium by a specific RIA, as described previously (40).

The mLTC-1 cells were subcultured at a density of 1.5 x 10⁶ cells/6-cm diameter plate in regular medium. Transfection studies were conducted by FuGENE 6 transfection reagent (Boehringer Mannheim GmbH, Mannheim, Germany) at a 60–70% confluence of cells under optimized conditions, according to the instructions of the manufacturer. Briefly, the FuGENE 6 transfection reagent was diluted in serum-free medium and incubated at room temperature in a final volume of 300 µl. Two micrograms of the pCMV119⁺-SF-1 construct (obtained from Dr. K. L. Parker, Durham, NC) and 2 µg pBKCMV-hDAX-1 construct (obtained from Dr. R. Yu, Chicago, IL) were used for transfections. To control the transfection efficiency, 2 µg of a ß-galactosidase expression vector, pRSV-ß-galactosidase (Promega Corp., Madison, WI), were used. After 15-min incubation of the FuGENE 6-DNA complex at room temperature, it was distributed dropwise to the plate containing 3 ml regular medium. Stimulation studies were conducted between 40–48 h of transfection.

RNA extraction, and quantitative RT-PCR
Total RNA was extracted from control and stimulated cells by the single step acid guanidinium thiocyanate-phenol-chloroform extraction method of Chomczynski and Sacchi (41). The purity of the extracted RNA was determined by spectrophotometric scanning at a wave length 220–320 nm.

The isolation and amplification of the mouse (mLTC-1) StAR complementary DNA (cDNA) were carried out by designing primers from the mouse StAR cDNA sequence (14). The sense primer, 5'-GACCTTGAAAGGCTCAGGAAGAAC-3', and the antisense primer, 5'-TAGCTGAAGATGGACAGACTTGC-3', spanned bases -51 to -27 and 931–908, respectively. To evaluate the potential variation in RT-PCR efficiency, an internal control, a 395-bp fragment of the L19 ribosomal protein gene was coamplified in each sample, using as the sense primer 5'-GAAATCGCCAATGCCAACTC-3' and as the antisense primer 5'-TCTTAGACCTGCGAGCCTCA-3'.

The target genes were amplified by RT and PCR, and run sequentially in the same assay tube, as described previously (42). Briefly, equal amounts of total RNA from the different experimental groups (2 µg/sample) were reverse transcribed using avian myeloblastosis virus reverse transcriptase (Finnzymes, Espoo, Finland) and the antisense primers. The cDNAs generated were further amplified by PCR using the primer pairs mentioned above. The total reaction volume was 50 µl. Each reaction contained 1 nmol/liter of each oligo primer, 200 mmol/liter of dNTP mixtures containing [³²P]dCTP, 20 U RNasin (Promega Corp.), 12.5 U avian myeloblastosis virus reverse transcriptase, and 2.5 U Dynazyme-DNA polymerase in 1 x PCR buffer (10 mmol/liter Tris-HCl, 50 mmol/liter KCl, 1.5 mmol/liter MgCl₂, and 0.1% Triton X-100, pH 8.8; Finnzymes, Espoo, Finland). The reaction was initiated at 50 C for 15 min (RT), followed by denaturation at 97 C for 5 min. Then the PCR was run with the steps of amplification defined by denaturation at 96 C for 1.5 min, annealing at 55 C for 1.5 min, and extension at 72 C for 3 min (PTC-200, Peltier Thermal Cycler, MJ Research, Inc., Cambridge, MA). Sixteen to 40 PCR cycles were examined, and 16 cycles were chosen for further analysis (data not shown). A final cycle of extension at 72 C for 15 min was included. To examine the PCR products, a 25-µl aliquot of each reaction was analyzed by gel electrophoresis on a 1.2% agarose gel. The molecular sizes of the amplified products (StAR and L19) were determined by comparison with the mol wt markers run in parallel with the RT-PCR products. The gels were then vacuum dried for 45–60 min and exposed to Kodak x-ray films (XAR-5, Eastman Kodak Co., Rochester, NY) at 4 C for 1–3 h, and the autoradiograms were analyzed for StAR mRNA expression after correcting for the variation in RT-PCR efficiency with the corresponding L19 bands. The relative levels of different signals were evaluated by densitometry (Tina 2.0 Package, Straubenhardt, Germany).

Isolation of mitochondria, SDS-PAGE, and immunodetection of StAR protein
After stimulation of the mLTC-1 cells, they were washed with 0.01 mol/liter PBS and scraped off using a rubber policeman into a buffer containing 10 mmol/liter Tris-Cl (pH 7.2), 250 mmol/liter sucrose, 0.1 mmol/liter EDTA, and 1.0 mmol/liter phenylmethylsulfonylfluoride. The cells were homogenized at 4 C (1200 rpm, 30 strokes) with a Potter-Elvehjem homogenizer fitted with a serrated pestle. Mitochondrial preparations were obtained by differential centrifugation. The homogenate was centrifuged at 500 x g for 25 min to remove broken cell debris and nuclei, and the resulting supernatant was further centrifuged at 1000 x g for 25 min. The pellet containing mitochondria was washed twice at 9000 x g for 15 min each time in the same buffer.

The mitochondrial protein (12 µg/lane) was solubilized in sample buffer [25 mmol/liter Tris-Cl (pH 6.8), 1% SDS, 5% ß-mercaptoethanol, 10% glycerol, and 0.01% bromophenol blue] and loaded onto a 12% SDS-polyacrylamide gel (Mini Protean II System, Bio-Rad), as described by Laemmli (43), with minor modifications. Electrophoresis was performed at 200 V for 1 h, and the proteins were electrophoretically transferred onto a nitrocellulose membrane (Hybond, Amersham). The membranes were incubated in a blocking buffer (Tris-buffered saline containing 0.1% Tween-20 and 5% nonfat dry milk) for 2 h at room temperature, followed by incubation with antipeptide antibodies of the 30-kDa StAR protein generated in rabbit against amino acids 88–98 (obtained from Dr. D. M. Stocco, Lubbock, TX) (14). The incubation with the antibodies was carried out overnight at 4 C. The membranes were washed three times (10 min each time) in Tris-buffered saline buffer and incubated for 1 h at room temperature with horseradish peroxidase-labeled donkey antirabbit IgG (Amersham). The membranes were washed as stated above, the immunodetection of StAR protein was revealed by the ECL Western blotting detection kit (Amersham), and the membranes were exposed for 1–3 min to Fuji Photo Film Co. Ltd. x-ray films (Fuji Photo Film Co., Ltd., Tokyo, Japan). The immunospecific bands were quantified by phosphorimager and densitometry.

Northern hybridization analysis of StAR mRNA expression
Twenty micrograms of the total RNA obtained from control and stimulated samples were resolved on 1.2% formaldehyde denaturing agarose gel and transferred onto Hybond-N⁺ nylon membranes (Amersham) by employing the capillary transfer method. An antisense complementary RNA probe corresponding to bases -27 to 931, a NotI fragment of the StAR cDNA, was produced by in vitro transcription (Promega Corp.) with T7 RNA polymerase, dNTPs, and [-³²P]UTP (Amersham). For preparing an SF-1 cDNA probe, an EcoRI-PstI fragment of the SF-1 cDNA was labeled with [-³²P]dCTP using the Prime-a-Gene labeling method (Promega Corp.). The labeled probes were purified using Sephadex G-50 nick columns (Pharmacia, Uppsala, Sweden). Prehybridization and hybridization were carried out under stringent conditions, as previously described (44). In brief, prehybridization was performed for at least 6 h at 65 C in a solution containing 50% formamide, 3 x SSC (150 mmol/liter sodium chloride and 50 mmol/liter sodium citrate, pH 7.0), 5 x Denhardt’s solution, 1% SDS, 0.1 µg/liter heat-denatured calf thymus DNA, and 100 mg/liter yeast transfer RNA. Hybridization was performed at 66 C in the same solution after addition of the ³²P-labeled probe and was continued for an additional 16 h. In the case of the cDNA probes SF-1 and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), prehybridization and hybridization were carried out at 42 C. The membranes were washed twice at room temperature for 20 min each time with 2 x SSC containing 0.1% SDS, followed by 2 h at 66 C with 0.1 x SSC and 0.1% SDS until removal of the background counts. Membranes were then exposed to x-ray films (Kodak XAR-5) for 36–48 h at -80 C. To normalize the variation in StAR and SF-1 mRNA levels, the membranes were subjected to rehybridization with a cDNA probe of GAPDH. The relative mRNA levels of StAR in the different transcripts and SF-1 and GAPDH expression were quantitated as described above.

Determination of ⁴⁵Ca²⁺ uptake
Radiolabeled calcium was used to determine the dependency of the hCG effects on extracellular Ca²⁺, as described previously (45) with minor modifications. Briefly, before stimulation, the cells were washed twice with 0.01 mol/liter PBS. At time zero, ⁴⁵Ca²⁺ (4 µCi/well) was added in Ca²⁺-free medium (final concentration of Ca²⁺, 1.5 mmol/liter) to each well for 2 h at 37 C in the absence or presence of hCG, K⁺, and hCG or K⁺ in the presence of verapamil (10 µmol/liter). Two hours later, the cells were washed, collected in 0.5 ml 0.1 N NaOH, vigorously mixed with the scintillation liquid, and finally measured for radioactivity by liquid scintillation spectrometer (1215 RACKBETA II, Wallac Oy, Turku, Finland).

Determination of intracellular [Ca²⁺]_i
The [Ca²⁺]_i was monitored in mLTC-1 cells with the fluorescent probe for Ca²⁺, fura-2/AM. After recovery from trypsinization shock, the cells were washed and incubated at a concentration of 1 x 10⁶/ml in Ca²⁺-free medium containing 4 µmol/liter fura-2/AM for 45 min at 37 C. The unincorporated fura-2/AM was washed away after 45 min, and the cells were sedimented and resuspended in Ca²⁺-free medium. The fluorescence intensity of the fura-2/AM was determined in a Hitachi F 2000 spectrophotometer (Tokyo, Japan) by employing excitation at 340 nm (5-nm bandpass) and emission wavelength at 505 nm. The effects of digitonin (50 µmol/liter) and EGTA (4 mmol/liter) were carried out for maximum and minimum fluorescence intensities.

Statistical analysis
The data were analyzed by one-way ANOVA, followed by Fisher’s least significant difference tests, using the StatView 4.5 program (Abacus Concepts, Inc., Berkeley, CA) fitted for the Macintosh computer. The results were expressed as the mean ± SEM, and P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Requirement of Ca²⁺ for hCG-stimulated StAR mRNA expression and P production in mLTC-1 cells
The results presented in Fig. 1 show that increasing concentrations of hCG (0–1000 µg/liter) significantly elevated the StAR mRNA levels in a concentration-dependent manner, with a maximum 2.8 ± 0.4-fold increase at 4 h, in Ca²⁺-free medium. Significant changes in StAR message were detected at concentrations of 1 µg/liter hCG or more, with half-maximal stimulation at 22 µg/liter and a maximum increase at 50 µg/liter or more. Interestingly, an increase in the extracellular Ca²⁺ concentration to 1.5 mmol/liter in the presence of hCG markedly augmented (1.7 ± 0.3-fold) the hCG-stimulated StAR mRNA levels, indicating that there is a component dependent on Ca²⁺ in hCG action. A 4-h stimulation with hCG exhibited a quantitatively similar dose-dependent increase in P production at doses of 1 µg/liter or more, with maximum stimulation of 5.6 ± 0.4 over the value in nonstimulated cells. Likewise, the presence of 1.5 mmol/liter Ca²⁺ additively increased the hCG effect (P < 0.0001) on P production, resulting in an approximately 2-fold higher response than with saturating dose of hCG (50 µg/liter) alone (Fig. 1C). Cells stimulated with varying concentrations of Ca²⁺ (0.1–4 mmol/liter) and a fixed concentration of hCG (50 µg/liter) displayed a dose response to Ca²⁺ in P production, reaching a maximum at 1–2 mmol/liter of Ca²⁺, whereas the basal level of P was not altered (data not shown). The stimulatory effect of Ca²⁺ on hCG-induced P production was completely abolished (P < 0.0001) by the Ca²⁺-chelating agents EGTA and EDTA (4 mmol/liter each; Fig. 2). The diminished effect on P production was correlated with the concentration of Ca²⁺ in the extracellular medium, further suggesting the requirement for extracellular Ca²⁺ in potentiation of hCG action (Fig. 2).

View larger version (40K): [in this window] [in a new window]   Figure 1. The dose-response pattern of hCG-induced StAR mRNA expression in the absence or presence of extracellular Ca2+ (1.5 mmol/liter) in mLTC-1 cells. The cells were stimulated for 4 h with increasing concentrations of hCG (0–1000 µg/liter), and total RNA was extracted from control and stimulated cells and subjected to RT-PCR analysis using 2 µg RNA as described (see Materials and Methods). An L19 fragment of the ribosomal protein gene (395 bp) was coamplified with each sample to normalize for the variation in RT-PCR efficiency. The RT-PCR products were resolved on 1.2% agarose gels, which were subsequently exposed to x-ray films. A, A representative autoradiogram showing the StAR mRNA expression in the absence (upper part) or presence (lower part) of Ca2+ in response to hCG (0–1000 µg/liter) stimulation. B, The arbitrary densitometric units (A.D.U.) of the StAR mRNA expression exhibited in each band after correction for intensities of the corresponding L19 bands. The results are the mean ± SEM of four independent experiments. C, The determination of hCG-stimulated P production in the absence (-Ca2+) or presence (+Ca2+) of extracellular Ca2+ (1.5 mmol/liter). The cells were cultured in 24-well plates at a density of 6 x 104 cells/well, and stimulated for 4 h with varying doses of hCG (0–1000 µg/liter). Tha data represent the mean ± SEM of six experiments performed in triplicate. The asterisk with arrow represents the first significant differences compared to respective controls (0): *, P 0.05.

View larger version (59K): [in this window] [in a new window]   Figure 2. Effect of Ca2+ chelators on hCG-induced P production. The mLTC-1 cells were stimulated in medium without (CON) or with Ca2+ (1.5 mmol/liter), hCG (50 µg/liter), hCG plus Ca2+, and hCG, Ca2+, and EDTA or EGTA (4 mmol/liter each). The data represent the mean ± SEM of five separate experiments performed in triplicate. Different letters above the bars indicate that those groups differ significantly from each other at P < 0.0001.

Effect of Ca²⁺ channel blocker verapamil on hCG-induced StAR mRNA expression
To corroborate the observed effect of extracellular Ca²⁺, we next examined the effect of verapamil, a voltage-sensitive transmembrane Ca²⁺ channel blocker. The results summarized in Fig. 3 demonstrate that this substance, when added at concentrations of 0.1–100 µmol/liter to the hCG and Ca²⁺ incubations inhibited StAR mRNA expression in a dose-dependent fashion, indicating the importance of extracellular Ca²⁺ mobilization through the transmembrane channels for the modulation of hCG action. The ED₅₀ of the verapamil effect was 4 µmol/liter (Fig. 3). The blocking of extracellular Ca²⁺ entry on hCG-mediated P production followed a qualitatively similar pattern as that observed for StAR mRNA expression (data not shown). These data imply that Ca²⁺ entry from the extracellular space is responsible for modulation of the hCG action on StAR mRNA expression and steroidogenesis.

View larger version (44K): [in this window] [in a new window]   Figure 3. Effects of increasing concentrations of verapamil (0–100 µmol/liter) on StAR mRNA expression. The mLTC-1 cells were stimulated with fixed concentrations of hCG (50 µg/liter) and Ca2+ (1.5 mmol/liter) in the presence of varying doses of verapamil. After a 4-h stimulation, the cells were harvested for RNA extraction, and 2 µg total RNA were examined by RT-PCR (see Materials and Methods and Fig. 1). A, A representative autoradiogram showing the effects of increasing doses of verapamil on StAR mRNA expression. The cells stimulated in Ca2+-free medium without (CON) or with hCG (50 µg/liter) are shown for comparison. B, The arbitrary densitometric unit (A.D.U.) values of each band quantified and normalized for the intensity of the corresponding L19 bands. The data represent the mean ± SEM of five independent determinations performed in duplicate.

View larger version (46K): [in this window] [in a new window]   Figure 4. Interaction of K+ with hCG-stimulated StAR mRNA expression and P production in mLTC-1 cells. The cells were stimulated for 4 h without (CON) or with K+ (4 mmol/liter); hCG (50 µg/liter); hCG plus K+; hCG, K+, and EGTA (4 mmol/liter); or verapamil (10 µmol/liter). Two micrograms of total RNA from each group were analyzed by RT-PCR (see Materials and Methods and Fig. 1). A shows a representative autoradigram of StAR mRNA expression in response to the different treatment groups. B, The A.D.U. values of each band as quantified and corrected for the corresponding L19 bands (±SEM; n = 4). C, P levels in media of the corresponding incubations. Different letters above the bars indicate that those groups are significantly different from each other at P < 0.0001.

View larger version (53K): [in this window] [in a new window]   Figure 5. Effect of A 23187 (CaI) on hCG-induced StAR mRNA expression. The mLTC-1 cells were stimulated without (CON) or with CaI (1 µmol/liter) for 4 h in the presence or absence of hCG (50 µg/liter); hCG, calcium ionophore A 23187 (CaI), and EGTA (4 mmol/liter); and verapamil (10 µmol/liter). Two micrograms of the total RNA from different treatment groups were used for RT-PCR (see Materials and Methods and Fig. 1). A, A representative autoradiogram showing StAR mRNA expression in the different stimulation groups. B, The A.D.U. values of the bands as normalized for the corresponding L19 bands (±SEM; n = 3). Different letters above the bars indicate that they differ significantly from each other at P < 0.01.

View larger version (35K): [in this window] [in a new window]   Figure 6. Effect of hCG and K+ on 45Ca2+ uptake of cultured mLTC-1 cells in the absence or presence of verapamil. A, Determination of the transmembrane Ca2+ influx in mLTC-1 cells in response to hCG and K+. The cells were incubated for 2 h with 45Ca2+ (4 µCi/ml) in the absence (CON) or presence of hCG (50 µg/liter), K+ (4 mmol/liter), hCG, or K+ and verapamil (10 µmol/liter). After washing, the cells were lysed in 0.1 N NaOH, and the uptake of Ca2+ was determined as described in Materials and Methods. The data represent the mean ± SEM of four independent experiments performed in triplicate. Different letters above the bars indicate that these groups are significantly different from each other at P < 0.001. B, Time-dependent alteration of mLTC-1 cell [Ca2+]i upon extracellular stimulation. The cells were incubated with fura-2/AM (3 µmol/liter) for 45 min at 37 C in Ca2+-free medium. After washing, the cells were incubated in Ca2+-replete medium sequentially with hCG (50 µg/liter), digitonin (50 µmol/liter), and EGTA (4 mmol/liter), as described in Materials and Methods. The arrows indicate the times of addition of the extracellular stimuli. The figure represents one of three experiments with similar results.

View larger version (56K): [in this window] [in a new window]   Figure 7. Western blot analysis of the effect of Ca2+ on hCG-induced StAR protein expression in mitochondrial preparations of mLTC-1 cells. The cells were stimulated for 4 h without (CON) or with Ca2+ (1.5 mmol/liter); hCG (50 µg/liter); hCG plus Ca2+; hCG, Ca2+, and CHX (10 mg/liter); or EGTA (4 mmol/liter). The effect of K+ (4 mmol/liter) on the hCG action was examined using the same experimental paradigm. For each case, 12 µg mitochondrial protein were resolved on SDS-PAGE as described in Materials and Methods. A, A representative experiment showing the dependence of the magnitude of hCG-induced StAR protein expression on extracellular Ca2+. B, The arbitrary densitometric unit (A.D.U.) values of the immunoreactive 30-kDa protein as quantified by densitometric scanning from three independent experiments. C, A Western blot demonstrating the effect of K+ on hCG-induced StAR protein expression in one representative experiment. D, The A.D.U. values of each immunospecific StAR protein band as quantified from three independent experiments. Different letters above the bars indicate that those groups are significantly different from each other at P < 0.0001.

View larger version (85K): [in this window] [in a new window]   Figure 8. Northern hybridization analysis of StAR mRNA expression by mLTC-1 cells and its regulation by Ca2+ (1.5 mmol/liter), hCG (50 µg/liter), EGTA (4 mmol/liter), and verapamil (10 µmol/liter). The cells were stimulated in duplicate with the different treatments for 4 h and subjected thereafter to RNA extraction. Twenty micrograms of total RNA were probed with full-length StAR complementary RNA as described in Materials and Methods. The apparent molecular sizes of the different transcripts are indicated on the right. The expression of the GAPDH mRNA level in the same lanes (lower panel) indicates equal RNA loading. One of four independent experiment with similar results is presented.

View larger version (61K): [in this window] [in a new window]   Figure 9. Effect of Ca2+ and hCG on SF-1 mRNA expression in mLTC-1 cells. The cells were stimulated in duplicate for 4 h without (CON) or with Ca2+ (1.5 mmol/liter); hCG (50 µg/liter); hCG plus Ca2+; hCG, Ca2+, and EGTA (4 mmol/liter); or verapamil (10 µmol/liter) in Ca2+-free medium. The cells were thereafter harvested for RNA extraction, and 20 µg total RNA from all groups were hybridized with an SF-1 cDNA probe. A, A representative autoradiogram showing the expression of SF-1 after the different treatments. The expression of GAPDH mRNA is shown as evidence of equal RNA loading (lower part). B, The arbitrary densitometric unit (A.D.U.) values of each band, represented by the mean ± SEM of three independent experiments, are shown. Different letters above the bars indicate that these groups are significantly different from each other at P < 0.0001.

View larger version (41K): [in this window] [in a new window]   Figure 10. The effect of constitutive overexpression of the DAX-1 cDNA on the basal levels of SF-1 in mLTC-1 cells. The cells were transiently transfected with viral promoter-driven DAX-1 cDNA (2 µg), as described in Materials and Methods. After 48 h, total RNA was extracted from mock-transfected (MT) and DAX-1-expressing cells and subjected to Northern hybridization with an SF-1 cDNA probe, using 20 µg total RNA. A shows a representative autoradiogram of the SF-1 mRNA expression. The GAPDH mRNA expression is indicated below as evidence for equal RNA loading. B, The arbitrary densitometric unit (A.D.U.) values of each band are quantified; each bar represents the mean ± SEM of four separate experiments performed in triplicate. The asterisks represent significant differences between the two groups: ****, P < 0.0001.

View larger version (46K): [in this window] [in a new window]   Figure 11. The effect of constitutive overexpression of SF-1 and DAX-1 in mLTC-1 cells on their Ca2+- and hCG-stimulated StAR mRNA expression. The cells were transiently transfected with SF-1 and DAX-1 (2 µg cDNA each), as described in Materials and Methods. After 48 h, the cells were stimulated without (CON) and with hCG (50 µg/liter) in the absence or presence of Ca2+ (1.5 mmol/liter). Total RNA was extracted from mock-transfected (MT) and SF-1- and DAX-1-expressing cells after the different stimulations and subjected to RT-PCR analysis using 2 µg total RNA. A, A representative autoradiogram showing the Ca2+ dependence of hCG action on StAR mRNA expression in mock-transfected (MT) and SF-1- and DAX-1-expressing cells. Ca2+ alone had no effect on StAR mRNA levels (not illustrated). B, The arbitray densitometric unit (A.D.U.) values of each band, as quantitated and normalized for the variation in RT-PCR efficiency with the corresponding L19 bands, are shown. The bars denote the mean ± SEM of four independent experiments. Different letters above the bars represent significance differences at P < 0.01. Comparisons were made within the different transfection groups, i.e. MT, SF-1, and DAX-1.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The role of Ca²⁺ as a second messenger of hormone action has been implicated in diverse cellular functions along with gonadal steroidogenesis (3, 6, 23). In Leydig cells, the steroid hormone biosynthesis is regulated by LH/hCG through activation of multiple second messenger systems (4, 8). The present experiments were designed to elucidate the precise involvement of extracellular Ca²⁺ in the hCG-stimulated StAR gene expression and steroidogenesis in mLTC-1 mouse Leydig tumor cells. Our data allowed us to conclude that the functional response of hCG involved in steroidogenesis is not dependent of extracellular Ca²⁺, whereas the addition of Ca²⁺ markedly enhanced hCG activity. The increase in [Ca²⁺]_i from extracellular space after hCG stimulation is responsible for inducing SF-1-dependent StAR expression that is intimately associated with increased steroidogenesis. Collectively, these results provide evidence for the molecular involvement of the Ca²⁺ messenger system in hCG-induced StAR expression and steroidogenesis in mouse Leydig cells.

Multiple levels of regulation impinge on StAR action, i.e. the PKA- and PKC-dependent pathways, the Ca²⁺ messenger systems, as well as other factors and processes that are responsible for the mobilization of cholesterol from the outer to the inner mitochondrial membrane. Recently, it has been demonstrated that phosphorylation of the serine residue at codon 194/195 of the StAR protein modulates the functional activity of the StAR intimately associated with steroidogenesis (52).

Regulation of StAR expression and aldosterone synthesis have also been demonstrated by the Ca²⁺-mobilizing agonists Ang-II and K⁺ in adrenal glomerulosa cells (19, 22, 23). It is noteworthy that in H295R adrenocortical cells, the inhibition of Ang-II and K⁺ functions markedly prevented aldosterone synthesis without altering the induction of StAR expression (19, 32). Our results demonstrate that extracellular Ca²⁺ significantly enhanced the stimulatory action of hCG on StAR mRNA and protein levels together with P production, without altering their basal levels. The stimulatory effect of hCG on StAR expression and steroidogenesis is independent of extracellular Ca²⁺, whereas the addition of Ca²⁺ potentially augments this action. In accordance, experiments with Ca²⁺ inhibitors exhibited modest, but consistent, attenuation of the hCG-induced responses, suggesting that [Ca²⁺]_i could be effectively affected by them. The increases in [Ca²⁺]_i and ⁴⁵Ca²⁺ uptake were primarily dependent on hCG action, as blockade of the transmembrane Ca²⁺ passage by verapamil reduced the modulatory action of hCG. The concentration of verapamil (10 µmol/liter) used in subsequent experiments showed a specific antagonistic effect without nonspecific inhibition, as determined by its involvement in basal P production during a 4-h incubation period (data not shown). These observations support the contention of a critical role of Ca²⁺ in potentiating LH/hCG action involved in steroidogenesis (4, 9).

With respect to the potential interaction with Ca²⁺ in StAR expression, the studies on K⁺ deserve a note. K⁺ has been regarded as a Ca²⁺-mobilizing agent in adrenal cells through opening of T- and L-type transmembrane Ca²⁺ channels that are progressively involved in steroid hormone biosynthesis (25, 27). Our findings in mLTC-1 cells demonstrate that K⁺ markedly increased the levels of hCG-stimulated StAR mRNA and protein, in close coordination with acute P production. Furthermore, the inducible effect of K⁺ on StAR expression was drastically suppressed when Ca²⁺-chelating agents or a specific Ca²⁺ channel blocker were included in the extracellular medium. Both agents significantly diminished the stimulatory actions of Ca²⁺ and K⁺, strongly suggesting the requirement of the extracellular Ca²⁺ for modulation of the hCG response. It is worth mentioning that neither Ca²⁺ nor K⁺ alone had a noticeable effect on StAR expression and P production, whereas they only potentiated the hCG action. This is expected, because the activation of PKC by phorbol ester (12-O-tetradecanoylphorbol 13-acetate) has been demonstrated to induce StAR expression without affecting steroidogenesis in MA-10 mouse Leydig tumor cells (32). The results of our study clearly show that the interaction of K⁺ is highly Ca²⁺ dependent and apparently facilitates the increase in [Ca²⁺]_i upon hCG stimulation that is responsible for StAR expression and acute P production.

The receptor binding of LH/hCG results in activation of the adenylate cyclase and phospholipase C signal transduction cascades (53, 54, 55). The steroidogenesis in Leydig cells is controlled by blood-borne endocrine influences, mainly from the pituitary gland, as well as by intratesticular autocrine and paracrine impulses (56). As LH/hCG plays a key role in the stimulation of Leydig cell steroidogenesis, a pertinent question is whether hCG is capable of increasing [Ca²⁺]_i, which then could amplify the gonadotropin effect on StAR expression. In adrenal glomerulosa cells, ionomycin-mediated calcium clamping has recently been shown to modulate StAR protein expression (22). Our findings document that the addition of hCG to fura-2/AM-loaded mLTC-1 cells exhibited a moderate elevation in [Ca²⁺]_i within 30–40 sec, with a plateau between 1–3 min. The marginal increase in [Ca²⁺]_i after hCG stimulation is possibly due to inefficient coupling of the LH receptor to phospholipase C, which is consistent with previous findings (7). In addition, the involvement of A 23187, primarily altering [Ca²⁺]_i, clearly augmented StAR gene expression in an additive fashion with hCG. It is important to note that hCG was unable to stimulate the inositol phosphate pathway in MA-10 mouse Leydig tumor cells (57). In contrast, in rat Leydig cells, hCG markedly increased [Ca²⁺]_i after a lag period of 2–3 min, and this was sustained for at least 15 min (9). It has also been shown in these cells that K⁺ clearly enhanced [Ca²⁺]_i through the voltage-sensitive Ca²⁺ channels in the plasma membrane. The inclusion of digitonin and EGTA in this experimental paradigm represented maximum and minimum levels of [Ca²⁺]_i, respectively, suggesting a physiological response of the cellular machinery. Tomic et al. (7) recently demonstrated that in rat Leydig cells, LH transiently mobilizes functional Ca²⁺ in an all or none manner. Our observations clearly show that the change in [Ca²⁺]_i upon hCG stimulation, originating from extracellular space, is responsible in inducing the hCG function involved in StAR expression.

The predicted role of StAR in steroidogenesis prompted us to examine the potential involvement of the orphan nuclear receptors, SF-1 and DAX-1. Recent studies demonstrated that these factors play a key role in the regulation of endocrine functions during development, at multiple levels of reproductive functions, and in the expression of steroidogenic enzymes (58, 59). In addition, DAX-1 binds to DNA hairpin structures and diminishes the transcriptional activity of SF-1 and StAR in adrenocortical cells (39, 60). Of great significance is the finding that SF-1 knockout mice present with a complete lack of adrenal glands and gonads, which is pertinent to the role of SF-1 as a key regulator in adrenal and gonadal developmental function (61). Our findings reinforce the idea that inhibition of endogenous SF-1 expression by DAX-1 remarkably attenuated StAR gene expression and P production, consistent with the previous findings. Recently, it has also been demonstrated that the expression of SF-1 was not impaired in Y1 cells that overexpress DAX-1 (62). The omission of Ca²⁺ from the extracellular medium or blocking of transmembrane Ca²⁺ uptake in relation to hCG resulted in a marked inhibition of Ca²⁺-mediated increase involved in SF-1 mRNA expression. The involvement of Ca²⁺ progressively modulated the hCG-induced SF-1 expression that consequently influences the following regulatory cascade of steroidogenesis, i.e. extracellular Ca²⁺hCG[Ca²⁺]_iSF-1StARacute steroidogenesis.

Collectively, the present data suggest that extracellular Ca²⁺ provides the source of [Ca²⁺]_i, subsequently augmenting the hCG activity, and is involved in the regulation of StAR gene expression and P production. The interaction of Ca²⁺ with hCG presumably increases the abundance of StAR expression, primarily through elevations in transcription of the StAR gene. These findings further confirm and extend the idea of a crucial involvement of SF-1 and Ca²⁺ in the hCG-mediated StAR expression and the acute regulation of steroid hormone biosynthesis.

	Acknowledgments

 
We are grateful to Dr. D. M. Stocco (Department of Cell Biology and Biochemistry, Texas Tech University Health Science Center, Lubbock, TX) for the generous gift of mouse StAR cDNA and antiserum to StAR protein. We also thank Dr. K. L. Parker (Departments of Medicine and Pharmacology and Howard Huges Medical Institute, Durham, NC) for providing us with the pCMV111⁺-SF-1, and Dr. R. Yu (Center for Endocrinology, Metabolism and Molecular Medicine, Chicago, IL) for the pBKCMV-hDAX-1 constructs. The superb technical assistance of Ms. Tarja Laiho and Ms. Riikka Kytömaa is gratefully acknowledged.

Received October 1, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Dufau ML, Baukal AJ, Catt KJ 1980 Hormone-induced guanyl nucleotide binding and activation of adenylate cyclase in the Leydig cell. Proc Natl Acad Sci USA 77:5837–5841[Abstract/Free Full Text]
2. Huhtaniemi I, Katikineni M, Catt KJ 1981 Regulation of luteinizing hormone receptors and steroidogenesis in neonatal rat testis. Endocrinology 109:588–595[Medline]
3. Rasmussen H, Waisman D 1981 The messenger function of calcium in endocrine systems. Biochem Act Horm 8:1–9
4. Veldhuis JD, Klase PA 1982 Mechanisms by which calcium ions regulate the steroidogenic actions of luteinizing hormone in isolated ovarian cells in vitro. Endocrinology 111:1–6[Abstract]
5. Python CP, Laban OP, Rossier MF, Vallotton MB, Capponi AM 1995 The site of action of Ca²⁺ in the actvation of steroidogenesis: studies in Ca²⁺-clamped bovine adrenal zona-glomerulosa cells. Biochem J 305:569–576
6. Gorczynska E, Handelsman DJ 1991 The role of calcium in follicle-stimulating hormone signal transduction in Sertoli cells. J Biol Chem 266:23739–23744[Abstract/Free Full Text]
7. Tomic M, Dufau ML, Catt KJ, Stojilkovic SS 1995 Calcium signalling in single rat Leydig cells. Endocrinology 138:3422–3429
8. Sullivan MHF, Cooke BA 1986 The role of Ca²⁺ in steroidogenesis in Leydig cells: stimulation of intracellular free Ca²⁺ by lutropin (LH), luliberin (LHRH) agonist and cyclic AMP. Biochem J 236:45–51[Medline]
9. Kumar S, Blumberg DL, Canas JA, Maddaiah VT 1994 Human chorionic gonadotropin (hCG) increases cytosolic free calcium in adult Leydig cells. Cell Calcium 15:349–355[CrossRef][Medline]
10. Abayasekara DRE, Band AM, Cooke BA 1990 Evidence for the involvement of phospholipase A2 in the regulation of luteinizing hormone-stimulated steroidogenesis in rat testis Leydig cells. Mol Cell Endocrinol 70:147–153[CrossRef][Medline]
11. Alila HW, Corradino RA, Hansel W 1990 Arachidonic acid and its metabolites increase cytosolic free calcium in bovine luteal cells. Prostaglandins 39:481–496[CrossRef][Medline]
12. Hertelendy F, Molnár M, Jamaluddin M 1992 Dual action of arachidonic acid on calcium mobilibation in avian granulosa cells. Mol Cell Endocrinol 83:173–181[CrossRef][Medline]
13. Mendelson C, Dufau ML, Catt KJ 1975 Dependence of gonadotropin induced steroidogenesis upon RNA and protein synthesis in the intertitial cells of the rat testis. Biochim Biophys Acta 411:222–230[Medline]
14. Clark BJ, Wells J, King SR, Stocco DM 1994 The purification, cloning and expression of a novel LH-induced mitochondrial protein in MA-10 mouse Leydig tumor cells: characterization of the steroidogenic acute regulatory protein (StAR). J Biol Chem 269:28314–28322[Abstract/Free Full Text]
15. Stocco DM, Chen W 1991 Presence of identical mitochondrial proteins in unstimulated constitutive steroid-producing R2C rat Leydig tumor and stimulated nonconstitutive steroid-producing MA-10 mouse leydig tumor cells. Endocrinology 128:1918–1926[Abstract]
16. Sugawara T, Holt JA, Driscoll D, Strauss III JF, Lin D, Miller WL, Patterson D, Clancy KP, Hart IM, Clark BJ, Stocco DM 1995 Human steroidogenic acute regulatory protein: functional activity in COS-1 cells, tissue-specific expression, and mapping of the gene to 8p11.2 and a pseuodogene to chromosome 30. Proc Natl Acad Sci USA 92:4778–4782[Abstract/Free Full Text]
17. Clark BJ, Soo S-C, Caron KM, Ikeda Y, Parker KL, Stocco DM 1995 Hormonal and developmental regulation of the steroidogenic acute regulatory (StAR) protein. Mol Endocrinol 9:1346–1355[Abstract]
18. Caron KM, Ikeda Y, Soo S-C, Stocco DM, Parker KL, Clark BJ 1997 Characterization of the promoter region of the mouse gene encoding the steroidogenic acute regulatory protein. Mol Endocrinol 11:138–147[Abstract/Free Full Text]
19. Clark BJ, Pezzi V, Stocco DM, Rainey WE 1995 The steroidogenic acute regulatory protein is induced by angiotensin II and K⁺ in H295R cells. Mol Cell Endocrinol 115:215–219[CrossRef][Medline]
20. Stocco DM, Sodeman TC 1991 The 30 kDa mitochondrial proteins induced by hormone stimulation in MA-10 mouse Leydig tumor cells are processed from the larger precursors. J Biol Chem 266:19731–19738[Abstract/Free Full Text]
21. Nishikawa T, Sasano H, Omura M, Suematsu S 1996 Regulation of expression of the steroidogenic regulatory (StAR) protein by ACTH in bovine adrenal fasiculata cells. Biochem Biophys Res Commun 223:12–18[CrossRef][Medline]
22. Cherradi N, Rossier MF, Vallotton MB, Timberg R, Friedberg I, Orly J, Wang XJ, Stocco DM, Capponi AM 1997 Submitochondrial distribution of three key stroidogenic proteins (steroidogenic acute regulatory protein and cytochrome P450_scc and 3ß-hydroxysteroid dehydrogenase isomerase enzymes) upon stimulation by intracellular calcium in adrenal glomerulosa cells. J Biol Chem 272:7899–7907[Abstract/Free Full Text]
23. Ramnath HI, Peterson S, Michael AE, Stocco DM, Cooke BA 1997 Modulation of steroidogenesis by choride ions in MA-10 mouse leydig tumor cells: role of calcium, protein synthesis, and the steroidogenic acute regulatory protein. Endocrinology 138:2308–2314[Abstract/Free Full Text]
24. Rebois RV 1982 Establishment of gonadotropin-responsive murine Leydig tumor cell line. J Cell Biol 94:70–76[Abstract/Free Full Text]
25. Duchatelle P, Joffre M 1990 Potassium and cloride conductances in rat Leydig cells: effects of gonadotropins and cyclic adenosine monophosphate. J Physiol 428:15–37[Abstract/Free Full Text]
26. Capponi AM, Rossier MF, Davies E, Vallotton MB 1988 Calcium stimulates steroidogenesis in permealized bovine adrenal cortical cells. J Biol Chem 263:16113–16117[Abstract/Free Full Text]
27. Cohen CJ, McCarthy RT, Barrett PQ, Rasmussen H 1988 Calcium channels in adrenal glomerulosa cells: potassium and angiotensin II increase T-type calcium current. Proc Natl Acad Sci USA 85:2412–2416[Abstract/Free Full Text]
28. Spät A, Enyedi P, Hajnoczky G, Hunyady L 1991 Generation and role of calcium signal in adrenal glomerulosa cells. Exp Physiol 76:859–885[Medline]
29. Matsunaga H, Yamashita N, Maruyama Y, Kojima I, Kurokawa 1987 Evidence for two distinct voltage-gated calcium channel currents in bovine adrenal glomerulosa cells. Biochem Biophys Res Commun 149:1049–1054[CrossRef][Medline]
30. Burnay MM, Python CP, Vallotton MB, Capponi AM, Rossier MF 1994 Role of capacitative calcium influx in the activation of steroidogenesis by angiotensin-II in adrenal glomerulosa cells. Endocrinology 135:751–758[Abstract]
31. Bird IM, Mathis JM, Mason JI, Rainey WE 1995 Ca²⁺-regulared expression of steroid hydroxylases in H295R human adrenocortical cells. Endocrinology 136:5677–5684[Abstract]
32. Chaudhary LR, Stocco DM 1991 Effect of different steroidogenic stimuli on protein phosphorylation and steroidogenesis in MA-10 mouse Leydig tumor cells. Biochim Biophys Acta 1094:175–184[Medline]
33. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schultz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835–839[CrossRef][Medline]
34. Enmark E, Gustafsson J-Å 1996 Orphan nuclear receptors–the first eight years. Mol Endocrinol 10:1293–1307[CrossRef][Medline]
35. Parker KL, Schimmer BP 1997 Steroidogenic factor 1: a key determinant of endocrine development and function. Endocr Rev 18:361–377[Abstract/Free Full Text]
36. Caron KM, Clark BJ, Ikeda Y, Parker KL 1997 Steroidogenic factor 1 acts at all levels of the reproductive axis. Steroids 62:53–56[CrossRef][Medline]
37. Ikeda Y, Swain A, Weber TJ, Hentges KE, Zanaria E, Lalli E, Tamai KT, Sassone-Corsi P, Lovell-Badge R, Camerino G, Parker KL 1996 Steroidogenic factor 1 and Dax-1 colocalize in multiple cell lineages: potential links in endocrine development. Mol Endocrinol 10:1261–1272[Abstract]
38. Lalli E, Bardoni B, Zazopoulos E, Wurtz J-M, Strom TM, Moras D, Sassone-Corsi P 1997 A transcriptional silencing domain in DAX-1 whose mutaion causes adrenal hypoplasia congenita. Mol Endocrinol 11:1950–1960[Abstract/Free Full Text]
39. Zazopoulos E, Lalli E, Stocco DM, Sassone-Corsi P 1997 DNA binding and transcriptional repression by DAX-1 blocks steroidogenesis. Nature 390:311–315[CrossRef][Medline]
40. Vuorento T, Lahti A, Hovatta O, Huhtaniemi IT 1989 Daily measurements of salivary progesterone reveals a high rate of anovulation in healthy students. Scand J Clin Lab Invest 49:395–401[Medline]
41. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:56–159
42. Tena-Sempere M, Zhang F-P, Huhtaniemi IT 1994 Persistent expression of a truncated form of the luteinizing hormone receptor messenger ribonucleic acid in the rat testis after selective Leydig cell destruction by ethylene dimethane sulfonate. Endocrinology 135:1018–1024[Abstract]
43. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
44. Zhang F-P, Rannikko AS, Manna PR, Fraser HR, Huhtaniemi IT 1997 Cloning and functional expression of the luteinizing hormone receptor complementary deoxyribonucleic acid from the marmoset monkey testis: absence of sequences encoding exon 10 in other species. Endocrinology 138:2481–2490[Abstract/Free Full Text]
45. Bhattacharya S, Manna PR, Halder S, Jamaluddin M 1990 Requirement of extracellular calcium in gonadotropin releasing hormone action. Prog Clin Biol Res 342:572–577[Medline]
46. Phillips MC, Johnson WJ, Rothblat GH 1987 Mechanisms and consequences of cellular cholesterol exchange and transfer. Biochim Biophys Acta 906:223–276[Medline]
47. Jefcoate CR, McNamara BC, Artemenko I, Yamazaki T 1992 Regulation of cholesterol movement to mitochondrial cytochrome P450scc in steroid hormone synthesis. J Steroid Biochem Mol Biol 43:315–321
48. Rennert H, Chang YJ, Strauss III JF 1993 Intracellular cholesterol dynamics in steroidogenic cells: a contemporary view. In: Adashi EY, Leung PCK (eds) The Ovary. Raven Press, New York, pp 147–164
49. Privalle CT, Crivello JF, Jefcoate CR 1983 Regulation of intramitochondrial cholesterol transfer to side-chain cleavage cytochrome P450scc in rat adrenal gland. Proc Natl Acad Sci USA 80:702–706[Abstract/Free Full Text]