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BOR - Papers in Press, published online ahead of print November 27, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.009266
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BIOLOGY OF REPRODUCTION 68, 1054–1063 (2003)
DOI: 10.1095/biolreprod.102.009266
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

Stimulatory Effect of Progesterone on the Expression of Steroidogenic Acute Regulatory Protein in MA-10 Leydig Cells1

Heidi Schwarzenbacha, Pulak R. Mannab, Douglas M. Stoccob, Gopa Chakrabartia, and Amal K. Mukhopadhyay2,a

a Institute for Hormone and Fertility Research, University of Hamburg, D-22529 Hamburg, Germany b Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The steroidogenic acute regulatory protein (StAR), by virtue of its ability to facilitate the intramitochondrial transport of cholesterol, plays an important role in regulating steroid hormone biosynthesis in steroidogenic cells. In agreement with published data, both StAR expression and progesterone production in MA-10 mouse Leydig tumor cells could be stimulated with hCG and 8Br-cAMP. Addition of aminoglutethimide, an inhibitor of cholesterol side chain cleavage (P450scc) enzyme, not only resulted in a drastic inhibition of progesterone production but also in an attenuation of StAR expression in response to either hCG or 8-Br-cAMP. Therefore, we addressed the question of whether progesterone, the end product of the steroidogenic cascade in these cells, could be in a position to regulate the StAR gene expression. In MA-10 cells, we report here that progesterone in microgram amounts can induce StAR gene expression in a time- and dose-dependent manner. StAR expression in response to a maximally effective concentration of progesterone of 10 µg/ml was highest at 6 h and started decreasing thereafter. The effect of progesterone on StAR protein and StAR mRNA induction was mimicked by its synthetic analog, progestin R5020, but not by other steroids, including dexamethasone, estradiol, testosterone, and dihydrotestosterone. Dexamethasone, in contrast, was able to inhibit StAR expression in MA-10 cells. Surprisingly, RU486, a potent antagonist of progesterone and glucocorticoid action, had a stimulatory effect on StAR mRNA levels. Reverse transcription-polymerase chain reaction analysis demonstrated the absence of the classical form of progesterone receptor in MA-10 cells. Thus, for the first time, a direct stimulatory effect of a steroid on StAR gene expression has been demonstrated. Furthermore, these results provide a new insight, indicating that progesterone mediates the activation of StAR expression exerted presumably through a novel, nonclassical progesterone receptor in mouse Leydig cells.

Leydig cells, progesterone, progesterone receptor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The steroidogenic acute regulatory protein (StAR) plays a crucial role in the regulation of steroid hormone biosynthesis in the gonads and adrenals. StAR protein mediates the rate-limiting step in steroidogenesis, i.e., translocation of cholesterol from the outer to the inner mitochondrial membrane, the site of side chain cleavage cytochrome P450 enzyme (P450scc), which converts cholesterol into pregnenolone. Pregnenolone is then further metabolized to progesterone or testosterone in Leydig cells [1–4]. StAR was identified and cloned from the murine Leydig tumor cell line MA-10 [5]. StAR mRNA has been shown to be specifically expressed in adrenal, testis, and ovary, the sites of steroid biosynthesis [6], indicating its specific role in steroidogenesis. Importantly, mutations in the StAR gene cause congenital lipoid adrenal hyperplasia, a lethal disease that is characterized by marked impairment of adrenal and gonadal steroidogenesis [7, 8]. The important role of StAR in regulating steroidogenesis has been further demonstrated in StAR knockout mice that lack steroid synthesis [9]. The inner mitochondrial 30-kDa mature StAR protein is proteolytically processed from a short-lived 37-kDa precursor protein [10]. Northern blot analyses revealed three StAR transcripts in the mouse (3.4, 2.7, and 1.6 kb) [5]. MA-10 cells stimulated with gonadotropins, LH or hCG, produces increased amounts of progesterone, a major end product of the steroidogenic cascade, by a mechanism that depends on a cAMP-mediated increase in the expression of StAR protein [5, 11].

Although steroidogenesis in Leydig cells is primarily regulated by LH/hCG in a cAMP-dependent manner [12], a number of autocrine and paracrine factors are known to influence this process [12, 13]. Similarly, for StAR gene expression, in addition to the cAMP-protein kinase A (PKA)-dependent pathway, several other signal transduction pathways can also play a regulatory role. For example, StAR expression is induced by angiotensin II, a phorbol ester (phorbol 12-myristate 12-acetate [PMA]), and a calcium agonist BAY K8644 via protein kinase C- and Ca2+-dependent signal transduction pathways [14–16]. Conversely, the prostaglandin F2{alpha} can repress StAR transcription via the protein kinase C-dependent pathway [17]. Thyroid hormone has also been shown to be a potent stimulator of StAR expression in a mouse Leydig tumor cell line [18]. In Leydig cells, the acute inhibition of steroid production was shown to be correlated with the inhibition of StAR protein expression [19, 20]. However, whether the gonadal steroids have any direct effect on StAR expression has yet to be investigated. Based on experiments involving estradiol administration in vivo to pseudopregnant rabbits, Townson et al. [21] proposed a role for StAR protein in the luteotrophic effect of estrogen to promote progesterone synthesis by corpora lutea. Although in these experiments it was clearly shown that StAR expression is responsive to estrogen in vivo, it is not apparent whether estrogen has a direct effect on StAR expression in this tissue or whether estrogen's action involves possible mediation by another factor, e.g., pituitary hormones, including prolactin or a uterine product. The only steroid so far known to have a direct effect on StAR expression is dexamethasone, which was shown to suppress LH-mediated stimulation of StAR protein in preovulatory follicles of rat cultured in vitro [22].

As discussed above, StAR protein is essential for regulating steroidogenesis, and therefore, it is relevant to ask whether steroids may have a feedback effect on the regulation of StAR expression. In ovarian cells as well as in tumor Leydig cell lines, the major end product of steroid biosynthesis is progesterone. Previous studies demonstrated that progesterone action can be mediated in the gonads by two forms of receptors, i.e., the classical nuclear receptor and the nonconventional membrane receptor [23, 24]. In a recent report, a novel nonclassical type of progesterone receptor (PR) has been identified and characterized in mLTC-1 mouse Leydig tumor cells [25]. In the present study, we have evaluated the effect of progesterone and its possible mode of action on the expression of the StAR protein in MA-10 cells. The findings reported here provide the first evidence that, in MA-10 cells, progesterone is capable of stimulating StAR expression and does so through a novel type of receptor that is distinct from the conventional nuclear receptor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

MA-10 mouse Leydig tumor cells were a gift from Dr. M. Ascoli (Department of Pharmacology, University of Iowa, Iowa City, IA). The StAR antisera used in these studies were from two sources: one was kindly provided by Dr. D.B. Hales (Department of Physiology and Biophysics, Chicago, IL) and the other was produced by the laboratory of D.M.S.. Both these antisera produced similar results. Dulbecco minimum essential medium was obtained from Gibco (Life Technologies Inc., Gaithersburg, MD). R5020 (promegestone); (NEN Life Science Products, Inc., Boston, MA), 8-Bromo adenosine 3',5'-cAMP (8-Br-cAMP); (Boehringer Mannheim, Mannheim, Germany), PeqGold RNA Pure (Peq Lab Biotechnologie, Erlangen, Germany), and all other hormones and cell culture reagents were obtained from Sigma (Deisenhofen, Germany). The reagents for Northern blot analyses were purchased from Ambion (Austin, TX). The positively charged nylon membranes (Schleicher & Schuell, Dassel, Germany), the polyvinylidene fluoride membranes (Millipore, Bedford, MA), and the peroxidase-conjugated affinity purified goat anti-rabbit IgG were purchased from Jackson Immuno Research Laboratories, Inc. (Hamburg, Germany). Reverse transcription-polymerase chain reaction (RT-PCR) reagents were obtained from Gene Craft (Münster, Germany). All other chemicals were commercially obtained.

Cell Culture, Incubations, and Determination of Progesterone Levels

MA-10 mouse Leydig tumor cells [26] and uterine smooth muscle cells (A10) were maintained in culture medium (a 1:1 mixture of Dulbecco minimum essential medium and Ham F-12 nutrient mixture supplemented with 7.5% horse serum, 2.5% fetal calf serum, 2 mM L-glutamine, and 200 IU:200 µg/ml penicillin:streptomycin sulfate). Cells were plated ~24 h preceding experiments, washed with 0.01 mol/L PBS, and incubated in serum-free media. The additives (stimulators, inhibitors, etc.) were freshly prepared and added to the incubations as specified in the figure captions. Progesterone production in the culture media was determined by a competitive double-antibody enzyme immunoassay using a solid-phase technique (IHF, Hamburg, Germany) as previously described [27].

Extraction of Total RNA and Northern Blot Analysis

Total RNA was isolated from MA-10 cells using PeqGold RNA Pure containing guanidinium thiocyanate and phenol. Twenty micrograms of RNA per lane was separated in a formaldehyde agarose (1.3%) gel and subsequently transferred to a positively charged nylon membrane. Briefly, cDNA probes, a 339–601 base pair (bp) fragment of mouse StAR and a 341–539 bp fragment of glyceraldehyde phosphate dehydrogenase (GAPDH), were labeled with a psoralen-biotin kit according to the manufacturer's instructions (Ambion). Hybridization of the cDNA probes was carried out overnight at 42°C in 5 ml ULTRAhyb solution, and the signals were detected using a streptavidin-alkaline phosphatase conjugate and the substrate CDP-Star (alkaline phosphate-reactive 1,2-dioxetane chemiluminescent substrate). The membranes were incubated with Fuji film (Noroldeutscher Roentgenvertrieb GmbH, Oststeinbek, Germany) for 3.5 h at RT. The relative mRNA levels of different signals were evaluated by densitometry using NIH Image 1.62 software (by Wayne Rasband, National Institute of Health, Bethesda, MD).

Preparation of Mitochondrial, Cytosolic, and Nuclear Fractions and Immunoblotting

For preparation of the mitochondrial fraction, cells were resuspended in Tris-sucrose buffer containing 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 0.25 mM sucrose, 1 mM dithiothreitol (DTT), 1 mM PMSF, and protease inhibitors. Mitochondrial preparation was performed using differential centrifugation as described previously [10, 16]. Preparation of the cytosolic fraction was carried out in buffer A (25 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 0.5 mM DTT) containing protease inhibitors. Cells were homogenized in a glass dounce homogenizer, the homogenate centrifuged at 300 x g for 15 min at 4°C, and the supernatant collected and subjected to ultracentrifugation at 100 000 x g for 40 min at 4°C. The supernatant was used as the cytosolic fraction.

Nuclear preparation was carried out in buffer B (10 mM Tris-HCl, pH 7.5, 2.5 mM EGTA, 1 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT, 0.1% triton X-100, and 10% glycerol). Cells were sonicated for 15 sec and the homogenate subjected to ultracentrifugation at 100 000 x g for 40 min at 4°C. The supernatant was used as the nuclear fraction. The protein content of the mitochondrial, cytosolic, and nuclear fractions was determined according to the method of Bradford using the BioRad protein dye assay [28]. Immunodetection of StAR and PR was performed by SDS-PAGE analysis. Briefly, 25–100 µg of protein fractions from different experiments were separated by SDS-PAGE using standard procedures and were electrophoretically transferred to polyvinylidene fluoride membranes. The membranes were incubated with antisera against mouse StAR or specific polyclonal antisera against PR and glucocorticoid receptor (GR) and subsequently treated with goat anti-rabbit IgG antibodies linked to peroxidase. Immunoactivity was detected using chemiluminescent reagents, and their levels were quantified as above.

Reverse Transcription and Polymerase Chain Reaction

Isolation and amplification of PR, GAPDH, StAR, and L19 cDNAs were carried out using the following primer pairs. The progesterone sense primer, 5'-CCCACAGGAGTTTGTCAAACTC-3', and the progesterone antisense primer, 5'-TAACTTCAGACATCATTTCCGG-3', were used to amplify a 325-bp DNA fragment encoding the conserved steroid binding domain of the classical PR [29]. A 198-bp DNA fragment of GAPDH was generated using specific primers and served as a control. The StAR sense primer, 5'-GACCTTGAAAGGCTCAGGAAGAAC-3', and the StAR antisense primer, 5'-TAGCTGAAGATGGACAGACTTGC-3', were designed from the mouse cDNA sequences [5]. The variation in RT-PCR efficiency for StAR expression was assessed with the L19 ribosomal protein gene using the sense primer 5'-GAAATCGCCAATGCCAACTC-3' and the antisense primer 5'-TCTTAGACCTGCGAGCCTCA-3' [30].

Three micrograms of total RNA from MA-10 and smooth muscle cells (A10) were incubated with 500 µg/ml oligo (dT) as primers at 70°C for 10 min. After chilling on ice, the cDNA synthesis was performed at 42°C for 1.5 h using first-strand buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, and 3 mM MgCl2), 10 mM dithiothreitol, 500 µM dNTP mixtures, and 10 U of Superscript reverse transcriptase. Fifty nanograms of cDNA samples were amplified in a 50-µl reaction volume containing polymerase buffer (16 mM (NH4)2SO4, 67 mM Tris-HCl, pH 8.8, 1.5 mM MgCl2, 0.01% Tween 20), 200 µM dNTPs, 1 µM sense and antisense progesterone or 0.1 µM GAPDH primers, and 5 U polymerase using a programmable Hybaid thermal cycler. The reaction was started with denaturation for 3 min at 94°C, followed by PCR amplification for 0.5 min at 94°C, 1 min at 60°C, and 1 min at 72°C for 34 cycles. A final cycle of extension for 5 min at 72°C was included. A quantitative approach was employed for StAR and L19 by amplifying 980- and 395-bp fragments, respectively [16, 18]. Briefly, RT and PCR of the target genes were run sequentially in the same assay tube using 2 µg of total RNA from different treatment groups under optimized conditions as described previously [18]. The molecular sizes of the PCR products (PR, GAPDH, StAR, and L19) were determined in 1% agarose gels by comparing with molecular weight markers run in parallel. For StAR and L19, the gels were vacuum dried, exposed to Hyperfilm (Amersham International PLC, Buckinghamshire, England) at 4°C between 1 and 3 h, and the relative levels of different signals were quantified by densitometry (Molecular Dynamics Inc., Sunnyvale, CA).

Presentation and Analysis of Data

The results are presented as the mean ± SEM of data obtained from at least three separate experiments. ELISA for progesterone was performed in triplicate within any single assay and were repeated at least three times using different batches of cells. The Student unpaired t-test has been applied to determine the significance of the difference between untreated control and agonist-treated experimental groups.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Aminoglutethimide on Progesterone Production and StAR mRNA Expression

Aminoglutethimide (AGT), an inhibitor of steroidogenesis that acts at the level of the cholesterol side chain cleavage enzyme (P450scc), was used to inhibit the steroidogenic cascade in MA-10 cells from the mitochondrial step downward to determine if cAMP-stimulated StAR expression was affected. MA-10 cells stimulated for 5 h with either hCG (5, 25, and 50 ng/ml) or 8-Br-cAMP (1 mM) produced significantly greater amounts of progesterone (Fig. 1A) compared with the unstimulated cells (basal). Addition of AGT (5 µg/ml) decreased both hCG- and 8-Br-cAMP-stimulated progesterone production by 60–80% and 90%, respectively. However, AGT had no effect on basal progesterone production. These findings demonstrate that AGT is a potent inhibitor of progesterone synthesis in MA-10 cells.



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  		FIG. 1. Effects of Aminoglutethimide (AGT) on hCG and 8-Br-cAMP-stimulated progesterone (Prog) synthesis and StAR mRNA expression. A) MA-10 cells were incubated with either hCG5 (5 ng/ml), hCG25 (25 ng/ml), hCG50 (50 ng/ml), and 8-Br-cAMP (1 mM) in the absence (-) or presence (+) of AGT (5 µg/ml) for 5 h. The amount of progesterone produced was determined with an ELISA immunoassay. Values are the mean ± SEM (n = 3). ***P < 0.001 for AGT treatment in comparison with the data obtained with corresponding hCG/8-Br-cAMP concentrations. B) Cells were incubated with 8-Br-cAMP (1mM) in the absence (-) or presence (+) of 5 µg/ml AGT for 6 h. Total RNA was isolated and subjected to Northern blot analysis. Twenty micrograms of RNA used per lane were hybridized with a psoralen-biotin-labeled StAR cDNA probe as described in the Materials and Methods. The membranes were rehybridized with a GAPDH cDNA probe to control RNA loading. C) The integrated optical density (IOD) of the StAR transcripts (all three transcripts taken together) was quantified and normalized with the corresponding GAPDH signals (the 1.4-kb transcript). Values presented are the mean ± SEM of three independent experiments. *P < 0.05 in comparison with the 8-Br-cAMP value.

Using similar experimental paradigms, Northern blot analysis revealed that AGT-induced inhibition of progesterone synthesis caused a parallel inhibition of 8-Br-cAMP-mediated induction of StAR mRNA expression in different transcripts (Fig. 1, B and C). In agreement with previous reports, two major (3.4- and 1.6-kb) and a minor (2.7-kb) StAR transcripts were found [5, 11]. These results demonstrate that inhibition of progesterone synthesis is paralleled by an inhibition of StAR expression.

Involvement of Progesterone on StAR mRNA and StAR Protein Expression in MA-10 Cells

The fact that AGT-mediated inhibition of hCG/cAMP-stimulated steroidogenic activity led to an attenuation of StAR expression prompted us to investigate whether progesterone may play a role in regulating the expression of StAR protein in MA-10 cells. To examine this, optimal conditions for progesterone effects were first determined. Cells stimulated for 6 h with varying concentrations of progesterone (0–20 µg/ml) demonstrated a dose-dependent increase in StAR mRNA expression. Concentrations of progesterone of less than 2.5 µg/ml were ineffective (Fig. 2). As determined by quantitative RT-PCR analysis, the magnitude of the StAR response was highest with 10 µg/ml of progesterone, being 3-fold over control, and decreased thereafter. At a concentration of 20 µg/ml, progesterone produced a drastic inhibitory effect; the reason for this is not yet understood.



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  		FIG. 2. Dose-response pattern of progesterone-stimulated StAR mRNA expression in MA-10 cells. A) Cells were treated with serial dilutions of progesterone (0–20 µg/ml) for 6 h. Total RNA was extracted and subjected to quantitative RT-PCR analysis of StAR mRNA expression as described in Materials and Methods. The variation in RT-PCR efficiency was determined by L19 ribosomal protein gene coamplified in each sample. The RT-PCR products were resolved in 1% agarose gels, and the gels were dried and exposed to X-ray films. B) The IOD values of each band were quantified and normalized with the corresponding L19 bands. Data represent the mean ± SEM of four independent experiments. *P < 0.05; ***P < 0.001, in comparison with control

Figure 3 illustrates the time-course of the effect of progesterone on StAR expression. Northern blot analysis revealed a time-dependent increase in StAR mRNA expression in response to progesterone treatment that was significant (P < 0.05) at 3 h. The maximal increase was observed at 6 h and started decreasing between 20 and 24 h. However, a 2- to 2.5-fold increase in StAR mRNA expression was evident at all time periods investigated. A control group was subsequently maintained without progesterone (data not shown) and demonstrated that StAR mRNA expression decreased at 24 h to levels below that of control, presumably due to the limited half-life of StAR mRNA. Interestingly, induction of the 2.7-kb StAR transcript at 6 h and beyond was noted in response to progesterone. This transcript was barely detectable in unstimulated cells and cells stimulated with hCG or 8Br-cAMP. Because StAR gene expression was maximally observed using 10-µg/ml progesterone for 6 h, these parameters were employed in subsequent experiments.



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  		FIG. 3. Temporal pattern of progesterone-induced StAR mRNA expression. A) MA-10 cells were stimulated with progesterone (10 µg/ml) for different time periods as indicated. Twenty micrograms of total RNA were analyzed for StAR mRNA expression by Northern blot analysis, as described in the Materials and Methods and in the legends of Figure 1. B) The IOD of the StAR (± SEM) in different transcripts was quantified and normalized with corresponding GAPDH signals (correspond to 1.4-kb band) from four experiments. *P < 0.05; ***P < 0.001, in comparison with basal values

In the next experiment, the potential influence of a combination of progesterone and hCG or cAMP analog on StAR mRNA and StAR protein expression was examined. MA-10 cells incubated without any addition or with progesterone, (Bu)2cAMP, (Bu)2cAMP plus progesterone, hCG, and hCG plus progesterone and StAR expression were assessed by quantitative RT-PCR analyses (Fig. 4). The data demonstrate that treatment with progesterone resulted in a 2- to 3-fold induction of StAR mRNA over the basal level. Stimulation with cAMP or hCG resulted in a robust induction of StAR expression. A combination of progesterone and cAMP did not produce any additional effect over what was achieved with cAMP alone. However, a combination of hCG plus progesterone produced an additive effect on the induction of StAR mRNA expression, suggesting that progesterone and the gonadotropin may be following separate pathways employing two different receptor systems. That progesterone did not have any additional effect when added together with the cAMP analog may be explained by the fact that cAMP appears to have already stimulated StAR expression maximally. The addition of progesterone together with submaximal concentrations of cAMP has not yet been investigated. Therefore, the question of whether progesterone could have a synergistic or additive effect together with cAMP remains unanswered at this time.



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  		FIG. 4. RT-PCR analysis of the effects of progesterone, hCG, (Bu)2cAMP, and their combinations on StAR mRNA expression. A) MA-10 cells were treated with progesterone (10 µg/ml), hCG (50 ng/ml), (Bu)2cAMP (1 mM), or a combination of progesterone and hCG or (Bu)2cAMP for 6 h. Total RNA was subjected to RT-PCR analysis of StAR mRNA expression, as described in Materials and Methods and in the legends of Figure 2. The variation in RT-PCR efficiency was assessed by L19 gene expression. B) The IOD values of each band were quantified and normalized with the corresponding L19 bands. Data represent the mean ± SEM of four independent experiments. Different letters above the bars indicate that these groups differ significantly from each other at least at P < 0.05

Identification of the Classical Progesterone Receptor in MA-10 and Uterine Smooth Muscle Cells

Because progesterone action clearly increased StAR expression, we next attempted to identify the PR in MA-10 cells by RT-PCR analysis. The response to progesterone has been proposed to be mediated by the classical nuclear as well as nonclassical plasma membrane-associated receptors [23, 24]. The classical receptor is expressed as two protein isoforms, type A (PRA) and B (PRB), which are functionally distinct with respect to progesterone's action [31]. Using specific primers that amplified a 325-bp-long DNA fragment encoding the conserved steroid-binding domain of the classical PR, we observed a complete absence of the classical PR mRNA in MA-10 cells (Fig. 5A). In contrast, smooth muscle cells of the uterus (A10) showed the presence of the classical PR mRNA and served as a positive control. Moreover, this form of the PR was also undetectable in primary mouse Leydig cells (data not shown).



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  		FIG. 5. Identification of the classical progesterone receptor (PR) and glucocorticoid receptor (GR). A) For PCR analysis, 50 ng of cDNA from smooth muscle cells (lane 2) and from two different passages of MA-10 cells (lanes 3 and 4) were amplified with specific progesterone and GAPDH primers in 34 cycles. The amplification with H20 served as a negative control (lane 1). Marker (lane M). B) Cytosolic (lanes 1, 3, and 4) and nuclear (lanes 2, 5, and 6) preparations of MA-10 and smooth muscle cells (A10) were analyzed for PR and GR by Western blot analysis. One hundred micrograms of protein per lane were incubated with specific polyclonal antisera against the PR and GR. The experiments were repeated three times, and a representative experiment is illustrated

The absence of PR protein in MA-10 cells was also confirmed by Western blot analysis using specific antiserum recognizing the C-terminus of the classical PR (Fig. 5B). However, using specific antibodies to the glucocorticoid receptor (GR), a double band of 95 kDa and 90 kDa, corresponding to the {alpha}- and ß-isoforms of the receptor, respectively, could be detected in MA-10 cells (Fig. 5B). These isoforms of the GR are likely to be due to alternative splicing. In addition, double bands corresponding to the PRB and PRA receptors, 110 and 79 kDa, respectively, were observed in smooth muscle cells (A10) when a specific antibody against the PR was used as a positive control. However, these bands were not detected in MA-10 cells (Fig. 6B), indicating that the classical form of the PR is indeed absent from MA-10 cells.



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  		FIG. 6. Effects of progesterone, its agonist R5020, and antagonist RU486 on StAR mRNA expression. A) MA-10 cells were incubated with progesterone (10 µg/ml), the progesterone agonist R5020 (10 µg/ml), and the progesterone antagonist RU486 (10 µg/ml) for 6 h. Total RNA was isolated and subjected to StAR mRNA expression by Northern blot analysis, as described in the Materials and Methods and in the legends of Figure 1. B) The IOD values of StAR transcripts were quantified and normalized with the corresponding GAPDH bands (the 1.4-kb transcript). The experiments were repeated three times. ***P < 0.001, in comparison with basal values

Specificity of the Effects of Progesterone and Its Agonist on StAR mRNA Expression in MA-10 Cells

The induction of StAR expression in MA-10 cells was mimicked by the progesterone agonist R5020 (Fig. 6). In contrast, other steroids like estradiol (10 µg/ml), testosterone (10-7 M), and dihydrotestosterone (10-7 M) were ineffective (data not shown). These data demonstrate the specificity of progesterone action on StAR protein induction in MA-10 cells. Surprisingly, RU486 (10 µg/ml), a potent antagonist of progesterone and glucocorticoid action that is also known to bind with high affinity to either nuclear PR and also to the GR [32], exhibited a stimulatory effect on StAR expression (Fig. 6). The stimulatory effect of RU486 was comparable or even slightly higher than that achieved with progesterone.

Because progesterone or its agonist R5020 and RU486 can induce StAR expression in the absence of the classical PR, it is conceivable that progesterone may be acting via the GR that is present in MA-10 cells. To test this hypothesis, the role of a potent synthetic glucocorticoid, dexamethasone (Dex) was examined with respect to StAR expression. Measuring StAR protein by Western blot analysis revealed that addition of Dex (10-7 mol/L) resulted not in stimulation but rather in an inhibition of StAR protein expression in both basal and hCG-stimulated cells. The magnitude of inhibition was about 20% and 28% on basal and hCG-mediated StAR expression, respectively. On the other hand, Dex did not affect 8-Br-cAMP-stimulated StAR protein expression (Fig. 7), possibly because the inhibitory effect of Dex is exerted at loci prior to cAMP generation. Figure 7A shows the precursor (37-kDa) and mature (30-kDa) protein bands. In additional studies, Northern blot analysis demonstrated qualitatively similar effects of Dex on basal, hCG, and 8-Br-cAMP (data not shown).



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  		FIG. 7. Effect of dexamethasone on basal and hCG-stimulated StAR protein expression. A) MA-10 cells were incubated with hCG (50 ng/ml) or 8-Br-cAMP (1 mmol/L) in the absence (-) or presence (+) of 10-7 mol/L dexamethasone (Dex) for 6 h. Mitochondrial protein (100 µg) was analyzed for StAR protein expression by Western blot analysis, as described in the Materials and Methods. B) The IOD values of StAR protein expression (± SEM) are shown from three independent experiments. **P < 0.01, in comparison with the hCG value


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Transport of cholesterol from the outer to the inner mitochondrial membrane, the site of the P450scc that converts cholesterol to pregnenolone, is the rate-limiting step in steroid hormone biosynthesis and is dependent on the expression of the StAR protein. The studies described here indicated that inhibition of P450scc activity was associated with the inhibition of the steroidogenic response. Consequently, it was determined that an inhibitor of P450scc, aminoglutethimide (AGT), not only blocked hCG/cAMP-stimulated steroid synthesis but also reduced the expression of StAR protein. In light of these observations, it was hypothesized that StAR expression might be regulated by progesterone, the major end product of the steroid biosynthetic pathway in the MA-10 mouse Leydig tumor cell line. Indeed, the evidence obtained in this study shows for the first time that progesterone in microgram concentrations is able to increase the steady-state level of StAR transcripts and protein in MA-10 cells in a time- and dose-dependent manner. However, the possibility that the increase in StAR transcripts could be due to an increased half-life of the mRNA cannot be ruled out yet based on our data. Furthermore, it was striking to observe that this effect of progesterone was exerted in a cell that lacked the classical form of the PR. The notion that the action of progesterone in MA-10 cells proceeds via a novel signaling pathway is strengthened by the observation that RU486 acts antagonistically at the level of the classical progesterone/glucocorticoid receptor and produced an agonistic effect on StAR expression in MA-10 cells.

Regulation of LH/hCG-mediated induction of steroid production correlates with StAR protein expression, a well-documented phenomenon in MA-10 and other steroidogenic cells [5, 11]. It has been demonstrated that steroid biosynthesis in Leydig cells is also influenced by locally produced factors through endocrine, autocrine, and paracrine regulation [12, 13]. Northern analysis revealed the presence of three StAR transcripts, two of which are major (3.4 and 1.6 kb) and one of which is minor (2.7 kb), an observation in general agreement with previous findings [10, 11]. Although all three transcripts were coordinately increased in response to progesterone, the expression of the 2.7-kb transcript appears to be differentially regulated by hCG/cAMP and progesterone. In addition, while hCG/cAMP markedly increased this particular transcript, it was barely detectable in response to progesterone. It appears that, at the molecular level, the mechanism of action of progesterone and hCG/cAMP on StAR expression may differ, a mechanism that will require further research.

A significant elevation of StAR mRNA levels was observed within 3 h of progesterone treatment and lasted for 20 h. The specificity of progesterone-stimulated StAR mRNA levels was further confirmed by RT-PCR analyses. The synthetic progesterone agonist R5020 was also able to induce StAR gene transcription with a potency similar to progesterone itself. It is noteworthy that the progesterone antagonist RU486 was also capable of stimulating StAR expression in MA-10 cells. This is consistent with previous studies documenting that some steroid antagonists can display agonistic activity depending on receptor, cell type, cofactor, or signaling pathway [33–35]. It has also been demonstrated that RU486 has a high affinity for both the PR and GR and also has a weak binding affinity to the androgen receptor [32, 36]. Recently, Beck et al. [35] reported that, in T47D human breast cancer cells, the agonistic effect of RU486 was mediated through the nuclear PR. Furthermore, an antagonist of estrogen, tamoxifen, was also found to act in a manner similar to that of RU486, and its agonistic activity through the estrogen receptor has also been demonstrated [37, 38]. However, the response of RU486 in MA-10 cells is rather difficult to explain, as these cells lack the nuclear PR. Further studies will be necessary to explore the precise mechanism involved in the antagonistic or agonistic action of RU486 in MA-10 cells.

Progesterone generally exerts its effects upon binding to the nuclear PR, resulting in an activation of target gene(s) transcription [23]. While many effects of progesterone are known to be mediated by the nuclear PR, little is known about progesterone action in the testis and particularly in Leydig cells. The presence of the PR has been detected in the cytosol of rat Leydig cells by a radioligand-binding assay employing [3H]-R5020 [39]; however, it should be noted that such a binding assay will detect both classical and nonclassical progesterone receptors. Heikinheimo et al. [40] detected PR in the nonhuman primate testis using RT-PCR analysis, although Leydig cells have not been specifically examined. In agreement with previous findings [24], the data obtained in this study allow us to conclude that the classical form of the PR is absent in MA-10 cells, as determined by Western and RT-PCR analyses. On the other hand, the presence of the GR is clearly evident in these cells. Recent studies also demonstrated that progesterone action in the rat corpus luteum could be mediated via the GR [41]. It is possible that progesterone at micromolar concentrations might exert its effect on StAR expression promiscuously via receptors for other steroids like the androgen or glucocorticoid receptors. Treatment of MA-10 cells with testosterone and dihydrotestosterone produced no effect on StAR expression, demonstrating the observed effect of progesterone in MA-10 cells is not mediated by androgen receptors, which are known to be predominant in the testis [42]. On the other hand, the synthetic glucocorticoid Dex produced an inhibitory effect on StAR expression in MA-10 cells. Such an inhibitory effect has been reported for Dex in the case of LH-stimulated StAR protein induction in rat preovulatory follicles [22]. Also, it is possible that the inhibitory effect of Dex might be due to LH receptor inhibition, as has been demonstrated with cortisol [43]. The data obtained in the present study suggest therefore that the induction of StAR expression in response to progesterone is mediated by neither the GR nor the androgen receptor (AR). As several steroids examined could not mimic the action of progesterone, its action seems to be specific. The effects of progesterone appear to be mediated presumably in an autocrine fashion in MA-10 cells using a nonclassical PR.

Disruption of the classical PR led to significant defects in reproductive function and infertility in adult female mice, whereas the adult mutant male mice seem to be normal [44]. This indicates that, unlike the female, the nuclear PR is not essential for male reproductive function and presumably a nonconventional, membrane-bound PR might play a putative role. In the present findings, the nuclear PR was not detected in MA-10 cells, an observation consistent with other studies in Leydig cells as well as in the testis [25]. A growing number of studies have demonstrated novel, nonconventional, membrane-bound forms of steroid receptors (reviewed in [45, 46]) involving various nongenomic and rapid effects in a variety of cells. Progesterone has been reported to induce a rapid increase in intracellular [Ca2+] in human sperm [47], in granulosa cells [48], and in the mLTC-1 murine Leydig tumor cell line [25]. Moreover, the existence of progesterone binding sites have been demonstrated in the plasma membrane of mouse and rat Leydig cells, in bovine follicular and luteal cell membranes, and in human spermatozoa membranes [25, 49–53]. In addition, a low copy number of nonclassical PR transcripts were detected in human spermatozoa using RT-PCR analysis [53]. Therefore, it is highly likely that the effect of progesterone on the induction of StAR protein in MA-10 cells is mediated by a novel, nonclassical PR acting at the level of the StAR gene. Currently, the localization of this novel type of PR in MA-10 cells remains obscure, and additional research will be necessary to determine if this receptor is a membrane-associated form.

Because the effect of progesterone on StAR expression in MA-10 cells is affected only at micromolar concentrations, it may be questioned if steroidogenic cells are ever exposed to such high progesterone concentrations. That the effect of progesterone on StAR protein induction was exerted in response to high concentrations of progesterone might reflect the fact that the PR in Leydig cells requires high ligand concentrations to reach the activation threshold, as basal in vivo levels of progesterone in the testis are higher than those found in the circulation [54]. In that particular study and as quoted in El-Hefnawy and Huhtaniemi [43], it has been shown that the in vivo basal concentration of progesterone in the testis is far higher than that found in the circulation and, in response to trophic hormone, it can be further increased to a level between 0.3 and 3.0 µmol/L [54]. It is further possible that the local concentration of progesterone in the vicinity of the Leydig cells is even higher, but the specific level of progesterone in the interstitial compartment of the testis has not been measured. It is also possible that progesterone added to MA-10 cells is metabolized rapidly into other nonactive steroids, thus reducing the effective concentration of the progesterone that was added to the culture medium. In support of this contention, Rommerts et al. [55] reported that progesterone added to MA-10 cells is rapidly metabolized to 5{alpha}-pregnan-3ß-ol-20one or 5{alpha}-pregnan-3{alpha}-ol-20one and several other minor metabolites. It will be of interest in the future to determine if any of these metabolites have an effect on StAR gene regulation.

Expression of StAR has been demonstrated in ovarian steroidogenic cells [11]. In the ovary, progesterone concentrations can be very high, especially during the ovulatory and postovulatory phases as well as in the corpora lutea of pregnant and nonpregnant animals. It is conceivable that high local concentrations of progesterone in the ovary may be involved in regulating its own production, as hypothesized by Rothchild [56]. In a recent study, Swan et al. [57] demonstrated that, in porcine granulosa cells, addition of micromolar concentrations of progesterone or levonorgestrel increased the steady-state level of transcripts for P450scc, a key enzyme in the steroidogenic cascade and in the amount of progesterone produced by these cells. In addition, these authors reported that RU486 acted as a progesterone agonist in these cells, an observation in agreement with our findings. It will be interesting, therefore, to determine if such a mechanism involving the action of progesterone on StAR expression can be demonstrated for ovarian cells such as granulosa or luteal cells that also produce a large amount of progesterone.

In contrast with the postulated nongenomic rapid action by steroids acting via the membrane-associated steroid receptors [45, 46], we observed a genomic effect of progesterone that required several hours to maximally increase the steady-state level of StAR transcripts in MA-10 cells. Thus, it appears that the nonnuclear membrane-associated steroid receptors may cause both rapid nongenomic action as well as long-term effects at the level of transcription.

The promoter region of the StAR gene lacks consensus binding sites for the PR and the GR [58, 59]. While the StAR promoter is activated by the cAMP-dependent signaling pathway, it lacks complete consensus cAMP response elements. Several transcription factors have been demonstrated to play a role in the regulation of StAR gene function [60, 61] and seem to be a common phenomenon of many cAMP-regulated genes. Further studies will be required to demonstrate the precise involvement of specific transcription factor(s) or signal transduction pathway(s) on progesterone-mediated StAR gene expression. Signal transduction mechanisms coupled to nonconventional receptors remain largely obscure. One report, however, clearly demonstrated that progesterone, in micromolar concentrations, stimulated phospholipase C activity in plasma membrane-cortex preparations from Xenopus oocytes [62]. Other reports demonstrate Ca2+ signaling in response to membrane steroid receptor activation [47, 48]. Additional research will be required to clarify the details of the mechanisms involved in these signaling pathways.

Collectively, the present findings provide evidence that progesterone is capable of stimulating StAR protein expression and it is most likely that its action is mediated through the involvement of a novel, nonclassical PR. Because progesterone is the major product produced by MA-10 cells, it remains a possibility that progesterone may act in an autocrine fashion in these cells. Although more research will be necessary to determine the cellular localization of this receptor in MA-10 cells and its signaling mechanism, our results demonstrate that these cells are an excellent model system for in-depth investigation to understand the nonclassical mode of action of steroid hormones. In summary, in addition to trophic hormones, the regulation of StAR protein expression can be modulated by a number of substances ranging from angiotensin II, atrial natriuretic peptide, retinoic acid, etc. (reviewed in [11]), and now progesterone can be added to this growing list.


   ACKNOWLEDGMENTS
 
We would like to thank Prof. F.A. Leidenberger for his encouragement and support for completion of this study. The superb technical assistance of Björn Ehlers and Deborah Alberts is acknowledged.


   FOOTNOTES
 
1 We gratefully acknowledge the financial support of the Leidenberger-Müller-Foundation, Hamburg, Germany, to A.K.M. Also, financial support was obtained from NIH grant HD 17481 and from the Robert A. Welch Foundation (to D.M.S.). Back

2 Correspondence: Amal K. Mukhopadhyay, Institute for Hormone and Fertility Research, Grandweg 64, D-22529 Hamburg, Germany. FAX: 49 40 56 19 08 64; e-mail: amal{at}ihf.de Back

Received: 10 July 2002.

First decision: 3 August 2002.

Accepted: 7 October 2002.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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R.-S. Ge, Q. Dong, E.-m. Niu, C. M. Sottas, D. O. Hardy, J. F. Catterall, S. A. Latif, D. J. Morris, and M. P. Hardy
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Coexistence of Intracellular and Membrane-Bound Progesterone Receptors in Human Testis
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Gonadotropin-Mediated Regulation of the Murine VEGF Expression in MA-10 Leydig Cells
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