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Department of Woman and Child Health, Pediatric Endocrinology Unit, Karolinska Institute and University Hospital, SE-17176 Stockholm, Sweden
Address all correspondence and requests for reprints to: Dr. Konstantin Svechnikov, Department of Woman and Child Health, Pediatric Endocrinology Unit, Q2:08, Karolinska Institute and Hospital, Astrid Lindgren Children’s Hospital, S-17176 Stockholm, Sweden. E-mail: konstantin.svechnikov{at}kbh.ki.se.
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Introduction |
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In connection with the signaling cascade of events triggered by gonadotropins and resulting in activation of steroidogenesis, the cAMP-dependent protein kinase [protein kinase A (PKA)] plays a key role (2). PKA activation leads to up-regulation of the expression of the steroidogenic acute regulatory (StAR) protein, the function of which is to translocate cholesterol from the outer to the inner mitochondrial membrane, which constitutes the rate-limiting step in steroid hormone synthesis (3). After this translocation, cholesterol is converted to pregnenolone by the cytochrome P450 side-chain cleavage complex (4).
ERKs (1/2), belonging to the family of signaling MAPKs, are involved in the regulation of a number of important biological functions, including cell proliferation, differentiation, and apoptosis as well as carcinogenesis (5). Several recent reports (6, 7) have indicated that ERK1/2 participate in the regulation of steroidogenesis in steroid-producing cells, but to our knowledge virtually nothing is known concerning their possible functions with regard to the Leydig cell. In the present investigation, we demonstrate that the steroidogenesis induced in immature rat Leydig cells by hCG is dependent on activation of the ERK cascade.
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Materials and Methods |
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Animals
Forty-d-old male Sprague Dawley rats (B&K Laboratories, Sollentuna, Sweden) were used as the source of immature Leydig cells (8). These animals were fed a standard pellet diet and water ad libitum. These experiments were approved by the Northern Stockholm Animal Ethics Committee (registration no. N192/03).
Isolation and culture of Leydig cells
Leydig cells were prepared from the immature rats by treatment of testes with collagenase as described earlier (9). Briefly, decapsulated testes were incubated with collagenase (0.25 mg/ml) for 20 min at 37 C, after which the crude mixture of interstitial cells was collected by centrifugation at 300 x g for 10 min, following by washing in HBSS containing 0.1% (wt/vol) BSA. To obtain purified Leydig cells, this crude cell suspension was loaded on top of a discontinuous Percoll gradient (consisting of layers of 20, 40, 60, and 90% Percoll in HBSS) and subsequently centrifuged at 800 x g for 20 min. The fractions enriched in Leydig cells thus obtained were then centrifuged in a continuous, self-generating density gradient starting with 60% Percoll at 20,000 x g for 30 min at 4 C.
The purity of the Leydig cells was shown to be 90%, as determined by histochemical staining for 3ß-hydroxysteroid dehydrogenase (10). The cell viability, as assessed by Trypan blue exclusion, was greater than 90%. These purified Leydig cells were washed twice in DMEM-F12 and thereafter resuspended in DMEM-F12 supplemented with 15 mM HEPES (pH 7.4), 1 mg/ml BSA, 365 mg/liter glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin.
For culturing, 100 µl of a suspension containing 1.5 x 105 cells/ml were plated into each well of a 96-well Falcon plate (Falcon, Franklin Lake, NJ) and incubated for 24 h at 34 C. At this time point, fresh culture medium was added and the cells preincubated with the specific inhibitor of ERK, Uo126 (0.1–10 µM) for 30 min, before incubation with hCG (10 ng/ml), (Bu)2cAMP (1 mM), and/or 22R-OHC (10 µM) for 3 h. This range of concentrations of Uo126 was shown earlier to inhibit ERK activities without affecting a number of other protein kinases (11).
In other experiments, 0.5 x 106 Leydig cells in a total volume of 2 ml were plated onto a culture dish (35 x 10 mm) (Falcon) and incubated for 24 h at 34 C. Thereafter, fresh medium was added and the cells pretreated with Uo126 (10 µM), H-89 (10 µM), and Calphostin C (1 µM), followed by incubation with hCG (10 ng/ml) for 60 min. This treatment was terminated by removing the culture medium by aspiration and subsequently rinsing the cells twice with PBS.
Testosterone determination
Samples of culture medium were stored at –20 C until being assayed for testosterone employing a Coat-a-Count RIA kit (Diagnostic Products Corp., Los Angeles, CA), according to the manufacturer’s instructions.
Western blot analysis
The effects of hCG on ERK phosphorylation were analyzed by PAGE/Western blots. The cells were washed twice with PBS and then lysed and sonicated in a lysis buffer containing 62.5 mM Tris-Cl (pH 6.8), 2% sodium dodecyl sulfate, 50 mM dithiothreitol, and 10% glycerol. Subsequently, the fraction thus solubilized was collected by centrifugation at 10,000 x g for 6 min. These proteins solubilized from whole Leydig cells (30 µg from each sample) were resolved by electrophoresis on SDS-PAGE 10% gels and then transferred electrophoretically to Hybond-P polyvinylidene difluoride membranes (Amersham Pharmacia Biotech), using 25 mM Tris-Cl, 185 mM glycine (pH 8.3), containing 20% methanol.
After this transfer, the membrane was incubated in a blocking buffer (Tris-buffered saline buffer containing 5% nonfat dry milk) for 1 h, followed by three washes with Tris-buffered saline/Tween 0.1% (3 x 10 min). Subsequently, these membranes were incubated with antibodies directed toward phospho-ERK1/2 and total ERK1/2, according to the manufacturer’s specifications (Cell Signaling Technology) and, after washing with donkey antirabbit or sheep antimouse IgG, secondary antibodies conjugated with horseradish peroxidase (Amersham Pharmacia Biotech). Finally, for detection these blots were incubated with ECL Plus Western blotting agent (Amersham Pharmacia Biotech) and then exposed to Hyperfilm ECL (Amersham Pharmacia Biotech).
Immunoprecipitation of StAR
Leydig cells first labeled with 35S-methionine (80 µCi/ml) in methionine-free DMEM were then preincubated with Uo126 (10 µM) for 30 min, followed by incubation with hCG (10 ng/ml) for an additional 2 h. These cells were then rinsed with PBS and scraped off the plates into 200 µl ice-cold immunoprecipitation buffer containing 30 mM Tris-Cl (pH 7.4), 150 mM NaCl, 2 mM Na3VO4, 10 mM NaF, 1 mM EDTA, 0.8% Nonidet P-40, 0.4% deoxycholate, and a protease inhibitor cocktail (Roche, Mannheim, Germany). The resulting cell lysates were passed repeatedly through a 29-gauge needle, followed by centrifugation at 12,000 x g for 10 min. There were no significant changes in total protein synthesis between the treatment groups (control, 41,357 ± 15,600; 70,401 ± 21,700 with hCG; 62,740 ± 16,300 with hCG + Uo126; 56,217 ± 14,350 cpm/µg with Uo126). Equal amounts of protein from each supernatant were incubated with 2 µl polyclonal StAR antiserum [kindly provided by Dr. D. M. Stocco (12)] overnight at 4 C and capture and isolation of immune complexes then performed using the Seize Classic immunoprecipitation kit (Pierce, Rockford, IL) according to the manufacturer’s instructions.
Briefly, after incubation of the immune complex with immobilized Protein A for 30 min at room temperature, the immunoprecipitated protein was recovered using an elution buffer and centrifugation.
These eluates were resuspended in sample buffer and equal amounts of radioactivity from each sample loaded on SDS-PAGE 15% gels. After electrophoresis, the gels were fixed for 30 min in 50% methanol-10% acetic acid, dried for 90 min at 65 C, and finally exposed to Hyperfilm MP (Amersham).
Assay of phosphatase activity
The activities of the two serine/threonine phosphatases, protein phosphatase (PP)1 and PP2A were assayed with 6,8-difluoro-4-methylumbelliferyl phosphate as substrate (EnzChek serine/threonine phosphatase assay kit, Molecular Probes, Eugene, OR). After treatment as described above, the cells were rinsed with cold saline, scraped into ice-cold buffer [10 mM Tris-Cl buffer (pH 7.0), 0.25 M sucrose, and a protease inhibitor cocktail (Roche)] and homogenized as also described above. The homogenates thus obtained were centrifuged at 14,000 x g for 20 min and the resulting supernatant stored at –20 C until assay of phosphatase activity.
In the pilot trial, serially diluted samples were assayed to determine the optimal amount of sample for use in measuring the activities of this distinct family of phosphatases. Consequently, samples diluted 1:20 in the reaction buffer were added to an equal volume of reaction buffer containing cofactors at twice the final concentrations i.e. (2 mM dithiothreitol and 200 µM MnCl2 in the case of PP1 or 1 mM NiCl2 for PP2A) in substrate-coated assay wells. After incubation for 60 min at room temperature, phosphatase activity was quantitated fluorometrically employing excitation/emission wavelengths of 355/460 nm, respectively, and a Wallac1420 microplate spectrofluorometer (PerkinElmer, Norwalk, CT) and the MultiCalc computer program (PerkinElmer) for calculations. Preliminary experiments were performed with selective inhibitors for each type of phosphatases to validate the assay.
Statistical analyses
The ECL Hyperfilms were scanned using an HP ScanJet 5100C and HP PrecisionScan software (Hewlett-Packard Sverige AB, Kista, Sweden) and the extent of antibody binding quantitated using National Institutes of Health Image 1.57 software. The differences between various values were analyzed for statistical significance by the Student’s t test. In addition, the dose-response data and enzyme activities were analyzed with the one-way ANOVA test where appropriate, with supplementation by the Dunnett t test. P < 0.05 was considered as statistically significant.
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Results |
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). Phosphorylation of ERK1/2 was detectably elevated within 5 min and maximal after 180 min. These effects had disappeared almost completely 6 h after initiation of treatment.
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, A and B). The cells were also exposed to H-89 and Calphostin C, specific inhibitors of PKA and PKC activities, respectively (11, 13). Both of these compounds inhibited hCG-activated MAPK phosphorylation by 50% (P < 0.05), indicating the upstream involvement of these kinases in the pathways that communicate signals from gonadotropin receptors to the MAPK. The enhanced phosphorylation of these MAPKs induced by hCG was completely abolished by the specific ERK1/2 inhibitor Uo126 (Fig. 3).
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). Furthermore, a pronounced inhibitory effect of 10 µM Uo126 on basal Leydig cell steroidogenesis was also observed. In the presence of this inhibitor, basal testosterone secretion was reduced from 0.55 ± 0.057 to 0.27 ± 0.048 ng/ml (P < 0.05).
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) or in combination with hCG (23.3 ± 2.3 ng testosterone per milliliter with hCG + Uo + 22R-OHC vs. 26 ± 1.6 ng/ml with 22R-OHC alone, P = 0.4) suggested that trafficking of cholesterol across mitochondria might be regulated, at least in part, by ERK1/2.
Time course of the inhibition of hCG-induced androgen production by immature Leydig cells by Uo126
Inhibition of hCG-induced steroidogenesis by Uo126 was evident after as little as 15 min of incubation (1.56 ± 0.11 ng testosterone per milliliter with hCG alone vs. 0.31 ± 0.01 ng/ml with hCG + Uo126; P < 0.01) and was observed throughout the entire 6-h incubation period (Fig. 5). Pilot experiments showed that Uo126 inhibited hCG-induced androgen production by Leydig cells even after pretreatment with hCG, when steroidogenesis was well established (data not shown).
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). The increased rate of StAR protein synthesis induced by hCG was accompanied by a dramatic 38-fold stimulation (P < 0.01) of androgen production by cultured immature Leydig cells, an effect that was again significantly attenuated by Uo126 (Fig. 6B).
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). Furthermore, there were no significant changes in PP1 activity on treatment with hCG alone or in combination with Uo126 (control activity 530 ± 96; 523 ± 101 with hCG; 508 ± 163 with hCG + Uo126; 513 ± 51 pmol/µg·h with Uo126).
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Discussion |
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Together, these findings led us to propose that expression of the StAR protein [whose function is to translocate cholesterol from the outer to the inner mitochondrial membrane (3)], in response to stimulation by hCG, might be regulated by the ERK cascade. Because only newly synthesized and processed StAR is required for this transfer of cholesterol (14), we examined whether these MAPKs are involved in de novo StAR synthesis by activated Leydig cells. Inhibition of the ERK cascade was seen to attenuate de novo StAR synthesis stimulated by hCG in primary cultures of immature rat Leydig cells.
These findings are in agreement with the description by Gyles et al. (7) of stimulatory effects of ERK1/2 on steroidogenesis in the mouse adrenocortical Y1 cell line. These investigators reported that acute cAMP-induced steroid synthesis is dependent on activation of the ERK cascade, which up-regulates transcription of the StAR gene via stimulation of the phosphorylation of steroidogenic factor 1. Thus, inhibition of ERK1/2 attenuates agonist-stimulated increases in the levels of StAR mRNA and protein and in steroid production by Y1 cells. Similarly, the same authors have shown that MAPK kinase inhibitors totally inhibited forskolin-stimulated steroid production by the mouse testicular MA-10 cell line (7).
The finding that modulation of the state of phosphorylation/dephosphorylation in steroidogenic cells may influence the expression of StAR (15, 16) led us to suggest that the ERK cascade might control the activities of certain phosphoprotein phosphatases. Indeed, we found here that hCG stimulates serine/threonine phosphatase PP2A activity in immature rat Leydig cells and that Uo126 inhibits this process. These observations are in line with a previous report demonstrating that specific inhibition of two types of serine/threonine phosphatases (PP2A and PP1) in mouse Y1 adrenocortical cells suppresses both steroid production and expression of the StAR protein (15).
Our investigations thus suggest that the activity of PP2A is positively controlled by ERK and, furthermore, is required for the full stimulatory effect of hCG on steroidogenesis by the immature Leydig cell. It has been suggested recently that PKC can phosphorylate the mitochondrial targeting sequence of StAR and thereby decrease its bioactivity (17). If this is the case, then dephosphorylation of these sites by PP2A might activate StAR, and, vice versa, inhibition of this phosphatase may suppress StAR activity and steroidogenesis. In addition, inhibition of the ERK cascade, and thus of PP2A activity, might negatively influence the expression of StAR by maintaining dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 in an active phosphorylated state for a prolonged period of time.
Reports concerning the potential involvement of ERK1/2 in the regulation of steroidogenesis in different steroidproducing cells appear to be contradictory, some documenting stimulatory (7, 18, 19), and others inhibitory effects (6). For example, several investigations have shown that LH and FSH activate ERK1/2 and enhance steroid production in ovarian cells (18, 19), whereas stimulation of the ERK cascade by these same gonadotropins in cell lines derived from granulosa cells leads to down-regulation of steroidogenesis (6). Furthermore, the inhibitory effect of prostaglandin F2
on steroidogenesis has also been linked to activation of ERK1/2 (20).
Differences in the nature of the second-messenger systems linked to cell surface receptors as well as the variety of overlapping and interacting signal pathways that are functional in various cell lines and tissues may provide explanations for these seemingly contradictory findings. An additional factor in this context may be the different periods of time used to inhibit ERK activities in different studies. The mechanisms underlying acute and long-term inhibition of ERK in steroid-producing cells may be different, involving, for example, modulation of the expression of various transcription factors and other proteins participating in the regulation of steroidogenesis.
At present, little is known concerning the endocrine and paracrine regulators involved in activation of the ERK pathway in Leydig cells. The single report has demonstrated that TNF and cAMP can modulate ERK activities in MA-10 tumor Leydig cells (21). Our present findings clearly show that hCG activate this signaling cascade in primary culture of immature Leydig cells through PKA- and PKC-dependent pathways.
The cAMP/PKA pathway has previously been reported to be involved in gonadotropin-dependent activation of ERK in different steroidogenic cells (6, 7). One mechanism by which the ERK cascade may be activated by PKA includes activation of the cAMP-responsive guanine nucleotide exchange factors for the small GTPase Rap1, Epac1, and Epac2. Upon binding of cAMP, these components rapidly activate Rap1, which in turn promotes activation of B-Raf and the remainder of the ERK cascade (22).
Activation of the LH/hCG receptor could also stimulate release of arachidonic acid, a physiological regulator of PKC (23), thereby triggering the PKC-dependent MAPK kinase/ERK pathway in the Leydig cell. Because, as shown here, pharmacological inhibition of both PKA and PKC only partially (by 50%) suppresses phosphorylation of ERK, other, as-yet-unknown signaling mechanisms stimulated by gonadotropins might also be involved in activation of ERK1/2.
Finally, we have found that primary cultures of Leydig cells constitute a convenient model to study the regulation of steroidogenesis because the steroidogenic machinery in these cells is intact and not modified as in the MA-10 Leydig cell line. We believe that data obtained by using primary cultures of steroidogenic cells as model in vitro more precisely reflect the complicated mechanisms of regulation of steroidogenesis that take place in vivo.
In summary, our present investigation has revealed that the ERK cascade is part of the signal transduction pathway activated by hCG in immature Leydig cells. Our hypothesis is that these MAPKs thus play an important role(s) in regulation of the rapid hormonal responses of Leydig cells to gonadotropins.
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Footnotes |
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Abbreviations: dbcAMP or (Bu)2 cAMP, Dibutyryl cAMP; HBSS, Hanks’ balanced salt solution; hCG, human chorionic gonadotropin; PKA, protein kinase A; PKC, protein kinase C; PP, protein phosphatase; 22R-OHC, 22R-hydroxycholesterol; StAR, steroidogenic acute regulatory.
Received April 16, 2004.
Accepted for publication June 29, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxysteroid dehydrogenase oxidative and reductive activities in rat Leydig cells during pubertal development. Biol Reprod 60:855–860) action in human granulosa-luteal cells. J Clin Endocrinol Metab 86:375–380 and cAMP on induction of AP-1 activity in MA-10 tumor Leydig cells. Endocrine 6:317–324[Medline]
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