This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Martinelle, N. Articles by Svechnikov, K. Search for Related Content PubMed PubMed Citation Articles by Martinelle, N. Articles by Svechnikov, K.

Extracellular Signal-Regulated Kinases Are Involved in the Acute Activation of Steroidogenesis in Immature Rat Leydig Cells by Human Chorionic Gonadotropin

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.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We studied the involvement of the ERK cascade in human chorionic gonadotropin (hCG)-induced steroidogenesis by primary cultures of immature rat Leydig cells. Our findings indicate that protein kinase A and protein kinase C function as upstream kinases in connection with transduction of the signal from the gonadotropin receptor to the ERK cascade. These MAPKs enhance the stimulatory effects of hCG on the de novo synthesis of the steroidogenic acute regulatory protein and the activity of protein phosphatase 2A, which are associated with increased androgen production by the Leydig cell. Specific inhibition of ERK1/2 by Uo126 suppressed all of these cellular responses to hCG. In contrast, steroidogenesis from 22OHC (a cell-permeable form of cholesterol) is not inhibited by Uo126, suggesting that cholesterol delivery to mitochondria is being affected by this compound. We propose that the ERK cascade is an important part of the signal transduction pathway involved in the rapid hormonal responses of Leydig cells to trophic hormones. In hCG-activated Leydig cells, these MAPKs may play a role in controlling the biosynthesis of the steroidogenic acute regulatory protein as well as regulating protein phosphatase 2A activity, thereby governing cholesterol transport across the mitochondrial membrane.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
LH SECRETED BY THE pituitary gland plays a central role in regulation of reproductive function in both male and female mammals. In the case of the testis, LH regulates the expression of receptors on the surface of Leydig cells and maintains the local and peripheral concentrations of androgens required for hormonal and reproductive development (1). Human chorionic gonadotropin (hCG) exhibits pronounced structural similarity to LH and binds to the same receptors.

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.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
DMEM-Ham’s nutrient mixture F-12, MEM, Hanks’ balanced salt solution (HBSS) without Ca²⁺ and Mg²⁺, and penicillin-streptomycin were obtained from Life Technologies, Inc./BRL (Paisley, Scotland, UK). BSA (fraction V), Percoll, HEPES, hCG, (Bu)₂cAMP (dibutyryl cAMP or dbcAMP), collagenase type I, and 22R-hydroxycholesterol (22R-OHC) (Sigma Chemical Co., St. Louis, MO), Uo126, Calphostin C, and H-89 hydrochloride (Calbiochem, La Jolla, CA), specific phospho-p44/42 MAPK antibodies (mouse monoclonal antibody IgG, affinity purified), and p44/42 MAPK antibodies (rabbit polyclonal IgG, affinity purified) (Cell Signaling Technology, Inc., Beverly, MA), and ³⁵S-methionine (Amersham Pharmacia Biotech, Buckinghamshire, UK) were purchased from the sources indicated.

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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transient activation of ERK1/2 in immature Leydig cells by hCG
Treatment of immature Leydig cells with hCG resulted in transient activation of ERK1/2 (Fig. 1). 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.

View larger version (28K): [in this window] [in a new window]   FIG. 1. Time course of activation of ERK1/2 in immature Leydig cells by hCG. After incubation with hCG (10 ng/ml) for different periods of time, the cultured cells were lysed, and 40 µg total protein were used to analyze phosphorylated (P-ERK1/2, upper panel) and total (lower panel) ERK1/2 by Western blotting. Similar results were obtained in two other experiments.

View larger version (29K): [in this window] [in a new window]   FIG. 2. PKA and PKC are upstream modulators of the activation of MAPKs in Leydig cells by hCG. Effect of PKA (A) and PKC (B) inhibition on the activation of ERK1/2 in Leydig cells by hCG. The cells were pretreated with 10 µM H-89 (A), 1 µM Calphostin C (Calph) (B), or vehicle alone for 30 min and thereafter stimulated with hCG (10 ng/ml) for an additional 60 min. Whole-cell lysates (40 µg protein) were used to analyze phosphorylated and total ERK1/2 by Western blotting. Each phosphorylated ERK1/2 (P-ERK1/2) band was quantitated by densitometric scanning using NIH Image software and expressed in relative densitometric units. Mean values ± SE from three independent Leydig cell preparations are presented. , P < 0.01, compared with untreated cells; *, P < 0.05 and **, P < 0.01, compared with cells treated with hCG alone.

View larger version (36K): [in this window] [in a new window]   FIG. 3. Uo126 inhibits elevated phosphorylation of ERK1/2 in immature Leydig cells exposed to hCG. The cultured cells were pretreated with 10 µM Uo126 or vehicle alone for 30 min and thereafter stimulated with hCG (10 ng/ml) for 60 min. The cells were subsequently lysed and 40 µg total lysate protein used to determine phosphorylated and total ERK1/2 by Western blotting. The data were quantitated and are expressed as described in the legend to Fig. 2. Mean values ± SE from three independent Leydig cell preparations are presented. , P < 0.05 and , P < 0.01, compared with untreated cells; **, P < 0.01, compared with cells treated with hCG alone.

View larger version (19K): [in this window] [in a new window]   FIG. 4. Effects of Uo126 on the activation of steroidogenesis in immature Leydig cells by hCG, dbcAMP, or 22R-OHC. Cultured Leydig cells were pretreated with different concentrations of Uo126 for 30 min and thereafter incubated with hCG (10 ng/ml), dbcAMP (1 mM), or 22R-OHC (10 µM) or vehicle alone for an additional 3 h. The testosterone secreted into the medium was then measured by RIA. Each experiment was performed independently four times with similar results. **, P < 0.01, compared with treatment with hCG and dbcAMP.

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).

View larger version (16K): [in this window] [in a new window]   FIG. 5. Time course of the inhibitory effect of Uo126 on hCG-stimulated testosterone production by immature rat Leydig cells. The Leydig cells were preincubated with or without 10 µM Uo126 for 30 min and thereafter stimulated with hCG (10 ng/ml) for various periods of time. Each experiment was performed independently three times with similar results.

View larger version (20K): [in this window] [in a new window]   FIG. 6. Effects of Uo126 on de novo synthesis of the StAR protein and on testosterone production by immature Leydig cells stimulated with hCG. The Leydig cells (0.5 x 106) were labeled with 35S-methionine (80 µCi/ml) and then preincubated in the presence or absence of 10 µM Uo126 for 30 min, followed by incubation with hCG (10 ng/ml) or vehicle alone for an additional 2 h. A, The cells were subsequently lysed and StAR protein isolated by immunoprecipitation and visualized as described in Material and Methods. The data were quantitated and are expressed as described in the legend to Fig. 2. The position of the band containing the 35S-StAR protein is indicated by an arrow. B, The testosterone secreted into the medium was assayed by RIA. In both A and B, mean values ± SE for three independent Leydig cell preparations are presented. , P < 0.01, compared with untreated cells; *, P < 0.05 and **, P < 0.01, compared with cells treated with hCG alone.

View larger version (14K): [in this window] [in a new window]   FIG. 7. Effects of Uo126 on PP2A activity in hCG-treated immature Leydig cells. The cells were preincubated in the presence or absence of Uo126 (10 µM) for 30 min and thereafter incubated with hCG (10 ng/ml) or vehicle alone for an additional 2 h. Phosphatase activity was assayed using 6,8-difluoro-4-methylumbelliferyl phosphate as substrate as described in Material and Methods. Mean values ± SE for three independent Leydig cell preparations are presented. , P < 0.05, compared with untreated cells; *, P < 0.05, compared with cells treated with hCG.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

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.

	Footnotes

 
This work was supported by grants from the Swedish Research Council (project 8282), the Frimurare Barnhuset Foundation in Stockholm, the Swedish Children’s Cancer Fund, the Swedish Environment Protection Agency, HRH Crown Princess Lovisa’s Society for Pediatric Health Care, and the Karolinska Institute.

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.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Dufau ML 1988 Endocrine regulation and communicating functions of the Leydig cell. Annu Rev Physiol 50:483–508[CrossRef][Medline]
2. Stocco DM, Clark BJ 1996 Regulation of the acute production of steroids in steroidogenic cells. Endocr Rev 17:221–244[Abstract/Free Full Text]
3. Stocco DM 2001 StAR protein and the regulation of steroid hormone biosynthesis. Annu Rev Physiol 63:193–213[CrossRef][Medline]
4. Christenson LK, Strauss 3rd JF 2000 Steroidogenic acute regulatory protein (StAR) and the intramitochondrial translocation of cholesterol. Biochim Biophys Acta 1529:175–187[Medline]
5. Lewis TS, Shapiro PS, Ahn NG 1998 Signal transduction through MAP kinase cascades. Adv Cancer Res 74:49–139[Medline]
6. Seger R, Hanoch T, Rosenberg R, Dantes A, Merz WE, Strauss 3rd JF, Amsterdam A 2001 The ERK signaling cascade inhibits gonadotropin-stimulated steroidogenesis. J Biol Chem 276:13957–13964[Abstract/Free Full Text]
7. Gyles SL, Burns CJ, Whitehouse BJ, Sugden D, Marsh PJ, Persaud SJ, Jones PM 2001 ERKs regulate cyclic AMP-induced steroid synthesis through transcription of the steroidogenic acute regulatory (StAR) gene. J Biol Chem 276:34888–34895[Abstract/Free Full Text]
8. Ge RS, Hardy DO, Catterall JF, Hardy MP 1999 Opposing changes in 3-hydroxysteroid dehydrogenase oxidative and reductive activities in rat Leydig cells during pubertal development. Biol Reprod 60:855–860[Abstract/Free Full Text]
9. Svechnikov KV, Sultana T, Söder O 2001 Age-dependent stimulation of Leydig cell steroidogenesis by interleukin-1 isoforms. Mol Cell Endocrinol 182:193–201[CrossRef][Medline]
10. Payne AH, Downing JR, Wong KL 1980 Luteinizing hormone receptors and testosterone synthesis in two distinct populations of Leydig cells. Endocrinology 106:1424–1429[Abstract/Free Full Text]
11. Davies SP, Reddy H, Caivano M, Cohen P 2000 Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351:95–105[CrossRef][Medline]
12. Clark BJ, Wells J, King SR, Stocco DM 1994 The purification, cloning, and expression of a novel luteinizing hormone-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]
13. Jarvis WD, Turner AJ, Povirk LF, Traylor RS, Grant S 1994 Induction of apoptotic DNA fragmentation and cell death in HL-60 human promyelocytic leukemia cells by pharmacological inhibitors of protein kinase C. Cancer Res 54:1707–1714[Abstract/Free Full Text]
14. Artemenko IP, Zhao D, Hales DB, Hales KH, Jefcoate CR 2001 Mitochondrial processing of newly synthesized steroidogenic acute regulatory protein (StAR), but not total StAR, mediates cholesterol transfer to cytochrome P450 side chain cleavage enzyme in adrenal cells. J Biol Chem 276:46583–46596[Abstract/Free Full Text]
15. Jones PM, Sayed SB, Persaud SJ, Burns CJ, Gyles S, Whitehouse BJ 2000 Cyclic AMP-induced expression of steroidogenic acute regulatory protein is dependent upon phosphoprotein phosphatase activities. J Mol Endocrinol 24:233–239[Abstract]
16. Maciel FC, Poderoso C, Gorostizaga A, Paz C, Podesta EJ 2001 LH/chorionic gonadotropin signaling pathway involves protein tyrosine phosphatase activity downstream of protein kinase A activation: evidence of an obligatory step in steroid production by Leydig cells. J Endocrinol 170:403–411[Abstract]
17. Niswender GD 2002 Molecular control of luteal secretion of progesterone. Reproduction 123:333–339[Abstract]
18. Cameron MR, Foster JS, Bukovsky A, Wimalasena J 1996 Activation of mitogen-activated protein kinases by gonadotropins and cyclic adenosine 5'-monophosphates in porcine granulosa cells. Biol Reprod 55:111–119[Abstract]
19. Das S, Maizels ET, DeManno D, St. Clair E, Adam SA, Hunzicker-Dunn M 1996 A stimulatory role of cyclic adenosine 3',5'-monophosphate in follicle-stimulating hormone-activated mitogen-activated protein kinase signaling pathway in rat ovarian granulosa cells. Endocrinology 137:967–974[Abstract]
20. Tai CJ, Kang SK, Choi KC, Tzeng CR, Leung PC 2001 Role of mitogen-activated protein kinase in prostaglandin F(2) action in human granulosa-luteal cells. J Clin Endocrinol Metab 86:375–380[Abstract/Free Full Text]
21. Li X, Hales KH, Watanabe G, Lee RJ, Pestell RG, Hales DB 1997 The effect of tumor necrosis factor- and cAMP on induction of AP-1 activity in MA-10 tumor Leydig cells. Endocrine 6:317–324[Medline]
22. de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, Bos JL 1998 Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396:474–477[CrossRef][Medline]
23. McPhail LC, Clayton CC, Snyderman R 1984 A potential second messenger role for unsaturated fatty acids: activation of Ca²⁺-dependent protein kinase. Science 224:622–625[Abstract/Free Full Text]

This article has been cited by other articles:

				P. R. Manna, I. T. Huhtaniemi, and D. M. Stocco Mechanisms of Protein Kinase C Signaling in the Modulation of 3',5'-Cyclic Adenosine Monophosphate-Mediated Steroidogenesis in Mouse Gonadal Cells Endocrinology, July 1, 2009; 150(7): 3308 - 3317. [Abstract] [Full Text] [PDF]

				M. Ramanjaneya, A. C. Conner, J. Chen, P. R. Stanfield, and H. S. Randeva Orexins Stimulate Steroidogenic Acute Regulatory Protein Expression through Multiple Signaling Pathways in Human Adrenal H295R Cells Endocrinology, August 1, 2008; 149(8): 4106 - 4115. [Abstract] [Full Text] [PDF]

				K. Shiraishi and M. Ascoli Lutropin/Choriogonadotropin Stimulate the Proliferation of Primary Cultures of Rat Leydig Cells through a Pathway that Involves Activation of the Extracellularly Regulated Kinase 1/2 Cascade Endocrinology, July 1, 2007; 148(7): 3214 - 3225. [Abstract] [Full Text] [PDF]

				H. Johnston, P. J King, and P. J O'Shaughnessy Effects of ACTH and expression of the melanocortin-2 receptor in the neonatal mouse testis Reproduction, June 1, 2007; 133(6): 1181 - 1187. [Abstract] [Full Text] [PDF]

				G.-S. Hwang, S.-W. Wang, W.-M. Tseng, C.-H. Yu, and P. S. Wang Effect of hypoxia on the release of vascular endothelial growth factor and testosterone in mouse TM3 Leydig cells Am J Physiol Endocrinol Metab, June 1, 2007; 292(6): E1763 - E1769. [Abstract] [Full Text] [PDF]

				P. R Manna, Y. Jo, and D. M Stocco Regulation of Leydig cell steroidogenesis by extracellular signal-regulated kinase 1/2: role of protein kinase A and protein kinase C signaling J. Endocrinol., April 1, 2007; 193(1): 53 - 63. [Abstract] [Full Text] [PDF]

				E. Colon, F. Zaman, M. Axelson, O. Larsson, C. Carlsson-Skwirut, K. V. Svechnikov, and O. Soder Insulin-Like Growth Factor-I Is an Important Antiapoptotic Factor for Rat Leydig Cells during Postnatal Development Endocrinology, January 1, 2007; 148(1): 128 - 139. [Abstract] [Full Text] [PDF]

				P. R Manna, S. P Chandrala, Y. Jo, and D. M Stocco cAMP-independent signaling regulates steroidogenesis in mouse Leydig cells in the absence of StAR phosphorylation. J. Mol. Endocrinol., August 1, 2006; 37(1): 81 - 95. [Abstract] [Full Text] [PDF]

				K. Shiraishi and M. Ascoli Activation of the Lutropin/Choriogonadotropin Receptor in MA-10 Cells Stimulates Tyrosine Kinase Cascades that Activate Ras and the Extracellular Signal Regulated Kinases (ERK1/2) Endocrinology, July 1, 2006; 147(7): 3419 - 3427. [Abstract] [Full Text] [PDF]

				N Renlund, Y Jo, I Svechnikova, M Holst, D M Stocco, O Soder, and K Svechnikov Induction of steroidogenesis in immature rat Leydig cells by interleukin-1alpha is dependent on extracellular signal-regulated kinases. J. Mol. Endocrinol., April 1, 2006; 36(2): 327 - 336. [Abstract] [Full Text] [PDF]

				T. Mizutani, K. Shiraishi, T. Welsh, and M. Ascoli Activation of the Lutropin/Choriogonadotropin Receptor in MA-10 Cells Leads to the Tyrosine Phosphorylation of the Focal Adhesion Kinase by a Pathway that Involves Src Family Kinases Mol. Endocrinol., March 1, 2006; 20(3): 619 - 630. [Abstract] [Full Text] [PDF]

				P. R. Manna, S. P. Chandrala, S. R. King, Y. Jo, R. Counis, I. T. Huhtaniemi, and D. M. Stocco Molecular Mechanisms of Insulin-like Growth Factor-I Mediated Regulation of the Steroidogenic Acute Regulatory Protein in Mouse Leydig Cells Mol. Endocrinol., February 1, 2006; 20(2): 362 - 378. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Martinelle, N. Articles by Svechnikov, K. Search for Related Content PubMed PubMed Citation Articles by Martinelle, N. Articles by Svechnikov, K.