Dihydrotestosterone Inhibits Insulin-Stimulated Cyclin D2 Messenger Ribonucleic Acid Expression in Rat Ovarian Granulosa Cells by
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《内分泌学杂志》
Departments of Obstetrics and Gynecology and Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109
Abstract
The effect of 5-dihydrotestosterone (DHT) on insulin-stimulated granulosa cell proliferation was examined using cyclin D2 mRNA as a marker. Granulosa cells from 3-d estradiol-treated immature rats showed a concentration-dependent increase in cyclin D2 mRNA expression in response to insulin. Exposure to DHT reduced the insulin-stimulated cyclin D2 mRNA expression. Inhibition of the two insulin-signaling pathways, ERK and phosphatidylinositol 3 kinase (PI3 kinase), by using specific inhibitors, also reduced this insulin-stimulated response. These results suggest that both ERK and PI3 kinase signaling are involved in insulin stimulated granulosa cell proliferation. DHT exposure resulted in reduced insulin-stimulated ERK phosphorylation. DHT treatment also reduced the insulin mediated insulin receptor substrate-1 and Raf-1 phosphorylation, the upstream molecules of ERK in insulin signaling pathway. Additionally, inhibition of insulin stimulated PI3 kinase activation reduced ERK phosphorylation. The present study therefore shows that the inhibitory effect of DHT on insulin-stimulated granulosa cell proliferation occurs early in the signaling pathway at the level of insulin receptor substrate-1 phosphorylation, leading to reduced ERK phosphorylation and subsequent inhibition of cyclin D2 mRNA expression.
Introduction
THE DEVELOPMENT OF mammalian follicle is regulated by gonadotrophins and steroids as well as various growth factors. The primordial follicle in the developing mammalian ovary consists of an immature oocyte surrounded by a single layer of granulosa cells, and follicular growth is initiated as the granulosa cells begin to proliferate, when they acquire responsiveness to FSH, resulting in the formation of large preantral follicle (1, 2, 3). The proliferation of granulosa cells is crucial for the formation of preovulatory follicle. Disturbance in this orderly progression of follicle growth could result in anovulation. It is now well recognized that, in the human, under pathophysiological conditions often related to polycystic ovarian syndrome, anovulation is associated with excess androgen production (4, 5, 6). It has been reported that in hyperandrogenic ovaries, granulosa cells proliferate at a subnormal rate (7), and follicular development is arrested at a stage at which selection of dominant follicle usually occurs (8). Furthermore, the level of 5-reductase, the enzyme that converts androgens to their 5-reduced metabolites, has been shown to be high in hyperandrogenic states (9, 10).
Before proliferation, granulosa cells undergo a well-coordinated series of events that are controlled by regulatory proteins such as cyclins and cyclin-dependent kinases. Progression of cell cycle from G1/S phase is regulated by D-type cyclins, which are regulated by extracellular signals (11). The D-type cyclins bind to cyclin-dependent kinases (cdk) 4 and 6, which activate a cascade of events that eventually leads to the progression from G1 to S phase (12, 13). Conversely, the kinase-inhibitory protein, p27kip1, binds to the cyclin D-cdk4/6 complex and inhibits their activation and blocks the progression of cell cycle from G1 to S phase, arresting the cells at G1 phase (14, 15).
We have previously shown that 5-dihydrotestosterone (DHT), the 5-reduced metabolite of testosterone, inhibits FSH-mediated granulosa cell proliferation by reducing cyclin D2 mRNA expression and subsequent cell cycle arrest at G1/S interphase in cultured rat granulosa cells (16). We have also reported that DHT exerts its inhibitory effect on cyclin D2 mRNA expression by reducing FSH-mediated phosphorylation of the ERK or MAPK by inhibiting protein kinase A (17).
The mitogenic and metabolic pathways in granulosa cells of preantral follicles are also known to be regulated by insulin (18, 19). Insulin acts through its own receptor to modulate the response of granulosa cells to gonadotropins (20, 21). Because insulin is an important mitogenic agent of proliferating granulosa cells, we examined whether DHT exerts any inhibitory effect on insulin-mediated mitogenic signaling pathway.
Upon interaction with insulin, the receptor becomes activated through an autophosphorylation reaction (22). Cellular scaffold proteins bind to the autophosphorylation sites that are then phosphorylated on the tyrosine residues. Most of the intracellular signals are generated through signaling complexes that are assembled around the tyrosine phosphorylated scaffold proteins including insulin receptor substrates (IRS), which are largely responsible for insulin-mediated functions. IRS proteins couple insulin receptors to the phosphatidylinositol 3-kinase (PI3 kinase) and ERK cascades (23). In the present studies, we examined the effect of insulin in cyclin D2 mRNA expression because cyclin D2 is a crucial molecule in FSH-mediated granulosa cell proliferation by regulating cell cycle progression at G1/S phase (13, 16, 24).
Our studies show that in cultured rat granulosa cells, insulin treatment stimulates cyclin D2 mRNA expression in a dose-dependent manner. DHT inhibits insulin mediated IRS-1 phosphorylation which leads to the inhibition of ERK phosphorylation. The reduced ERK activation in turn reduces cyclin D2 mRNA expression. Our results also show that inhibition of PI3 kinase reduces insulin-mediated cyclin D2 mRNA expression and ERK phosphorylation, suggesting a cross-talk between these two pathways.
Materials and Methods
Materials
Insulin (bovine), DHT (5-androstan-17-ol-3-one), 17-estradiol (1,3,5 [10]-estratriene-3, 17-diol), and PI3 kinase inhibitor, wortmannin, were purchased from Sigma Chemical Co. (St. Louis, MO). Antibodies for total phosphorylated ERK and total ERK-2 were obtained form Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against phosphorylated Raf (Ser 338) as well as p27kip were obtained from Cell Signaling Technology Inc. (Beverly, MA). Antiphosphotyrosine antibody (clone 4G10) was from Upstate Cell Signaling Solutions (Lake Placid, NY), and IRS-1 antibody was from BD Transduction Laboratories (San Diego, CA). MAPK kinase (MEK) inhibitor PD 98059 was from Promega (Madison, WI). Antimouse and antirabbit IgG horseradish peroxidase conjugates and enhanced chemiluminescent Western blotting detection reagents were purchased from Amersham Pharmacia Biotech. (Piscataway, NJ). Phenol red-free DMEM-F12 medium, Trizol reagent, and the RadPrime DNA labeling system were products of Life Technologies Inc. (Gaithersburg, MD). [32-P]Deoxy-CTP 3000 Ci/mmol) was purchased from MP Biomedicals (Irvine, CA). DHT was dissolved in ethanol, whereas wortmannin and PD 98059 were dissolved in dimethylsulfoxide. A dilute solution of acid water (HCl, pH 2–3) was used to dissolve insulin to make required concentrations. The total concentrations of diluents in the culture medium did not exceed 0.15%.
Animals and treatment
Female Sprague Dawley rats (22 d old) were housed in a temperature-controlled room with proper dark-light cycles and were under the care of University of Michigan Unit of Laboratory Animal Medicine. The animals were injected with estradiol (1.5 mg/d) for 3 d to stimulate the development of preantral follicles (25). Twenty-four hours after the last estradiol administration, animals were killed by CO2 asphyxiation. All the experiments conducted were approved by the University Committee on Use and Care of Animal, University of Michigan (approval no. 8360).
Granulosa cell isolation and culture
The ovaries from rats were collected and granulosa cells were isolated as previously described (16, 26, 27). In brief, ovaries were cleared from the surrounding fat and punctured with 25-gauge needles. Cells were collected in phenol red-free DMEM-F12 containing 0.2% BSA, 10 mM HEPES, and 6.8 mM EGTA, incubated for 15 min at 37 C under 95% O2-5% CO2, and centrifuged for 5 min at 250 x g. The pellets were suspended in a solution containing 0.5 M sucrose, 0.2% BSA, and 1.8 mM EGTA in DMEM-F12 and incubated for 5 min. After incubation, the suspension was diluted with 3 vol DMEM-F12, centrifuged at 250 x g, and treated sequentially with trypsin (20 μg/ml) for 1 min, 300 μg/ml soybean trypsin inhibitor for 5 min, and deoxyribonuclease (DNase I) (100 μg /ml) for 5 min at 37 C to remove dead cells. The cells were then rinsed twice with serum-free media, suspended in DMEM-F12, and the cell number determined. Cell viability was examined by trypan blue exclusion. Cells were cultured in serum-free DMEM-F12 supplemented with 20 mM HEPES (pH 7.4), 4 mM glutamine, 100 IU penicillin/ml, and 100 μg/ml streptomycin. Before plating, the culture dishes were coated with 10% fetal calf serum for 2 h at 37 C and washed with DMEM-F12. After allowing cells to attach overnight, they were treated with DHT (90 ng/ml) or vehicle alone for 24 h.
Northern blot analysis
Granulosa cells, after overnight attachment, were treated with 0, 10, 25, 50, and 100 ng/ml insulin for 2 h to examine the dose-dependent effect on cyclin D2 mRNA expression. In experiments in which the effect of the MEK inhibitor PD 98059 and the PI3 kinase inhibitor wortmannin on insulin-stimulated cyclin D2 mRNA expression was tested, one set of cultures was pretreated with the inhibitors (20 μM PD 98059 for 1 h and 100 nM wortmannin for 30 min) followed by incubation with insulin (100 ng/ml) for 2 h. To test the effect of DHT on insulin-mediated cyclin D2 mRNA expression, cells were pretreated with DHT (90 ng/ml) for 24 h followed by insulin (100 ng/ml) for 2 h. Control groups received equal amounts of respective vehicles. At the end of incubation, the medium was decanted, and the cells were washed once with PBS. Total RNA was extracted using TRIzol reagent according to the manufacturer’s instructions (Life Technologies). The cyclin D2 cDNA (1.1 kb) probe was radiolabeled using [32-P]deoxy-CTP and the RadPrime DNA labeling system and was hybridized to blots overnight at 42 C using 2 x 107 cpm of labeled probe. The hybridized blots were washed and exposed at –70 C to XAR film (Eastman Kodak Co., Rochester, NY). The blots were stripped and rehybridized with a radiolabeled cDNA probe corresponding to 18 S rRNA to monitor total RNA loading.
Western blot analysis
In experiments in which the specific inhibitors (20 μM PD 98059 and 100 nM wortmannin) were used, cells were pretreated with these agents before the incubation with insulin as described above. To examine the effect of DHT on insulin-mediated phosphorylation of ERK, Raf, and IRS-1, granulosa cells, after 24 h exposure to DHT (90 ng/ml) or vehicle, were stimulated with insulin (100 ng/ml) for time intervals described for each experiment. Because the expression of phosphorylated ERK-2 was considerably higher than ERK-1 in granulosa cells, we specifically looked at phosphorylated ERK-2 expression. The effect of insulin on p27kip was examined by treating the cells with insulin for 4 h. The reaction was stopped by removing the medium. In all the experiments, control sets were run simultaneously with respective vehicles. Cells after treatments were washed with PBS, lysed in modified radioimmunoprecipitation assay buffer. Total protein was subjected to Western blot analysis, and protein loading was normalized by reprobing the same blots with antibodies against the nonphosphorylated form of each protein probe or -tubulin. Detection was performed with an enhanced chemiluminescent Western blotting detection system (Amersham Pharmacia Biotech).
Statistical analysis
Statistical analysis was carried out using unpaired t test (GraphPad Prism, version 3.0; GraphPad Inc., San Diego, CA). Each experiment was repeated at least three times with similar results. Blots are representative of one experiment, and graphs represent the mean ± SE of three replicates.
Results
Insulin increases cyclin D2 mRNA expression in granulosa cells
Initial experiments examined the effect of insulin on cyclin D2 mRNA expression. The cultured granulosa cells from 3-d estradiol primed rats were treated with increasing concentration (0, 10, 25, 50, and 100 ng/ml) of insulin for 2 h, and cyclin D2 expression was measured using Northern blot analysis. Insulin treatment increased cyclin D2 in a dose-dependent manner (Fig. 1). The maximum expression was observed with 100 ng/ml insulin and therefore selected for all subsequent studies. Insulin treatment also resulted in a significant reduction (P < 0.05) of p27kip (cdk inhibitor) an inhibitor of cell proliferation (Fig. 2), compared with control.
DHT treatment reduces insulin mediated cyclin D2 mRNA expression
To examine whether DHT affects insulin-mediated stimulation of cyclin D2 mRNA expression, granulosa cells were pretreated with DHT (90 ng/ml) for 24 h followed by stimulation with 100 ng/ml insulin for 2 h. Northern blot analysis shown in Fig. 3 indicates an inhibition in cyclin D2 expression in response to pretreatment with DHT (P < 0.05). This result demonstrates that at a dose of 90 ng/ml, DHT inhibits insulin-stimulated cyclin D2 mRNA expression.
Inhibition of ERK and PI3 kinase activation inhibits insulin mediated cyclin D2 mRNA expression
The signaling pathways through which insulin stimulates cyclin D2 expression were then examined. ERK and PI3 kinase activation, representing the two well-established insulin signaling pathways, were selected as targets for the study. Cells were pretreated with specific inhibitors against ERK (PD 98059) and PI3 kinase (wortmannin), followed by stimulation with insulin. The results presented in Fig. 4 show that inhibition of ERK activation by pretreatment with PD 98059 (20 μM) completely abolished insulin’s stimulatory effect on cyclin D2 mRNA (P < 0.05). Blocking PI3 kinase activation using wortmannin (100 nM), on the other hand, evoked partial but significant (P < 0.05) inhibition of this stimulation (Fig. 5). These results indicate that ERK is a crucial molecule in insulin stimulated cyclin D2 mRNA expression and that both ERK and PI3 kinase pathways participate in insulin-stimulated cyclin D2 mRNA expression.
DHT treatment inhibits insulin stimulated ERK phosphorylation
Experiments were then designed to examine the effect of DHT on insulin mediated ERK-2 phosphorylation. Granulosa cells were pretreated with DHT or vehicle for 24 h followed by stimulation with insulin for 5 min. Phosphorylation of ERK-2 was examined by Western blot analysis using specific antibody. As expected, pretreatment with DHT significantly reduced insulin-stimulated ERK-2 phosphorylation (P < 0.05) (Fig. 6), compared with control cells subjected to similar treatments, suggesting that DHT inhibits cyclin D2 mRNA expression by reducing ERK phosphorylation.
DHT treatment reduces phosphorylation of Raf-1 and IRS-1
After establishing that DHT exerts its inhibitory effect by blocking insulin-dependent ERK activation, the effect of DHT on upstream molecules of ERK in the Ras/Raf/MEK signaling cascade was examined. Incubation with DHT for 24 h followed by insulin stimulation for 3 min resulted in reduced Raf-1 phosphorylation (P < 0.05), compared with control (Fig 7). For IRS-1 phosphorylation, which is a very rapid process, exposure time of insulin was reduced to 1 min. Control cells showed a significantly higher response in IRS-1 phosphorylation (P < 0.05) with insulin treatment, whereas in DHT-exposed cells, insulin stimulation of IRS-1 phosphorylation is completely abrogated (Fig. 8). The antiphosphotyrosine antibody used for the detection of IRS-1 phosphorylation recognizes all tyrosine phosphorylated sites. Because IRS-1 is phosphorylated at multiple sites, this antibody rather than those against specific sites, allowed us to identify the changes in the extent of phosphorylation with improved signal intensity. The identity of IRS-1 was established by stripping and reprobing the blots with antibody specific for total IRS-1. Together these results show that DHT inhibits the activation of upstream molecules of ERK in insulin signaling.
Inhibition of PI3 kinase reduces insulin stimulated ERK phosphorylation
A reduction in insulin-mediated cyclin D2 mRNA expression by PI3 kinase inhibition (Fig. 5) suggests that PI3 kinase also plays (at least partially) a role in insulin’s mitogenic effect. Because ERK activation is critical for cyclin D2 mRNA expression, a possible cross-talk between PI3 kinase and ERK activation was examined. Pretreatment of granulosa cells with 100 nM wortmannin, an inhibitor of PI3 kinase, significantly reduced insulin-mediated ERK-2 phosphorylation (P < 0.05) (Fig. 9). This result suggests that insulin may also increase cyclin D2 mRNA expression by stimulating ERK activation through PI3 kinase.
Discussion
Ovarian granulosa cell proliferation, a prerequisite for normal ovulation, is under the control of multiple regulatory molecules including FSH, insulin/IGF system, and activin (28, 29, 30, 31, 32, 33, 34). Signals from these mitogens flow through different signaling cascades and activate proteins that govern cell proliferation. Cyclin D2, one such regulatory protein, is critical for granulosa cell proliferation. We have previously shown that increased cyclin D2 mRNA expression correlates with increased thymidine incorporation to DNA and proliferation in granulosa cells in response to FSH-mediated mitogenesis (16, 17). The importance of cyclin D2 in granulosa cell proliferation is further substantiated by the observation of impaired proliferation and ovulation in cyclin D2 knockout mice (24).
Because insulin is a known mitogen of granulosa cells (18, 21), the present studies were carried out to determine whether its mitogenic actions are mediated through cyclin D2. We have shown that in cultured rat granulosa cells, insulin stimulates cyclin D2 mRNA expression in a dose-dependent manner (Fig. 1) and reduces cyclin-dependent kinase inhibitor, p27kip (Fig. 2). Together these data demonstrate that insulin regulates mitogenesis by up-regulating cyclin D2 expression, a positive regulator of cell cycle and by reducing the negative regulator, p27kip1.
As stated in the introductory text, elevated levels of androgens and their 5-reduced metabolites have been shown to disrupt the mitogenic signaling in granulosa cells. Because insulin stimulates granulosa cell proliferation by up-regulating cyclin D2 mRNA expression, we have examined whether this process is inhibited by DHT. Exposure of granulosa cells to DHT for 24 h reduced insulin-mediated cyclin D2 mRNA expression (Fig. 3), suggesting that elevated levels of DHT could block insulin mediated mitogenesis. Because the signaling pathways of insulin have been well characterized, we specifically examined the site of DHT’s inhibitory action. Insulin has been known to act both as mitogenic agent through the ERK pathway and metabolic regulator through the PI3 kinase pathway (23, 35). However, the use of specific inhibitors of both pathways reduced cyclin D2 mRNA expression. ERK inhibition by PD 98059 completely abolished insulin’s stimulatory effect (Fig. 4), whereas PI3 kinase inhibition by wortmannin partially reduced this stimulation (Fig. 5). Although inhibition of ERK produced a more pronounced inhibitory effect than that of PI3 kinase, it would appear that activation of both signaling pathways is involved in insulin-mediated cyclin D2 mRNA expression. The complete abolishment of insulin-mediated cyclin D2 mRNA expression by ERK inhibition makes this molecule a critical component of insulin’s mitogenic machinery. The observed reduction in insulin-stimulated ERK-2 phosphorylation in DHT-treated cells (Fig. 6) indicates that DHT-mediated inhibition of cyclin D2 mRNA is indeed due to interference at this step.
Our results show that DHT exert its inhibitory effect on insulin action in the granulosa cell at an early step in its signaling pathway. It is now well established that phosphorylation of IRS at tyrosine residues is the first post receptor step in insulin signaling, and this pathway has been implicated as a modulator of follicular growth (18). Although insulin receptor activation leads to both IRS-1 and IRS-2 phosphorylation, previous gene knockout studies have shown that IRS-1 mainly regulates mitogenic pathways, whereas IRS-2 mediates metabolic pathways (36, 37). Because cyclin D2 is a positive regulator of mitogenesis, it is conceivable that insulin stimulates cyclin D2 expression through IRS-1 rather than IRS-2. This notion is consistent with our results, in which DHT treatment inhibited insulin-stimulated phosphorylation of IRS-1 as well as its downstream target molecule, Raf-1 (Figs. 7 and 8). Phosphorylation of Raf-1 at multiple phosphorylation sites has been shown to activate ERK pathway (38). Together these observations demonstrate that DHT reduces the IRS-1 phosphorylation leading to the inhibition of Ras/Raf/ERK pathway.
The finding that inhibition of PI3 kinase activation reduced insulin-stimulated ERK-2 phosphorylation (Fig. 9) points to a possible cross-talk between the PI3 kinase and ERK pathways in insulin-stimulated cyclin D2 mRNA expression. Similar cross-talk between insulin signaling pathways has also been reported recently in other cell types (39, 40). One explanation for the existence of such a cross-talk in granulosa cells is that converging stimulatory signals from PI3 kinase to ERK could synergize insulin’s mitogenic potential in growing follicles.
In summary, inhibition of IRS-1 phosphorylation by DHT leads to inhibition of the Ras/Raf/MEK signaling cascade, resulting in reduced ERK activation and subsequent decrease in cyclin D2 mRNA expression. The present studies along with our previous reports (17), which demonstrated that DHT inhibited protein kinase A activation without affecting cAMP production, suggest that DHT can act at multiple sites in granulosa cells (17). The inhibitory effect of DHT at multiple sites in FSH and insulin-mediated ERK activation indicates that impairment of granulosa cell proliferation by androgens is a complex process and that the inhibitory effects culminate at the level of ERK phosphorylation. A recent independent study, which implicates alteration in ERK pathway in the pathogenesis of hyperandrogenism, is supportive of this notion (41). We conclude that the defective growth characteristics of granulosa cells seen under hyperandrogenic conditions might be mediated by inhibition of FSH and insulin signaling at multiple steps, but they converge to inhibit ERK activation and cyclin D2 mRNA expression.
Acknowledgments
We express our appreciation to Dr. Anil Nair, Dr. Roberto Towns, Helle Peegel, and Christine Clouser for critical reading of the manuscript.
Footnotes
This work was supported by National Institutes of Health Grant HD-38424.
First Published Online October 6, 2005
Abbreviations: cdk, Cyclin-dependent kinase; DHT, 5-dihydrotestosterone; IRS, insulin receptor substrate; MEK, MAPK kinase; PI3 kinase, phosphatidylinositol 3 kinase.
Accepted for publication September 28, 2005.
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Abstract
The effect of 5-dihydrotestosterone (DHT) on insulin-stimulated granulosa cell proliferation was examined using cyclin D2 mRNA as a marker. Granulosa cells from 3-d estradiol-treated immature rats showed a concentration-dependent increase in cyclin D2 mRNA expression in response to insulin. Exposure to DHT reduced the insulin-stimulated cyclin D2 mRNA expression. Inhibition of the two insulin-signaling pathways, ERK and phosphatidylinositol 3 kinase (PI3 kinase), by using specific inhibitors, also reduced this insulin-stimulated response. These results suggest that both ERK and PI3 kinase signaling are involved in insulin stimulated granulosa cell proliferation. DHT exposure resulted in reduced insulin-stimulated ERK phosphorylation. DHT treatment also reduced the insulin mediated insulin receptor substrate-1 and Raf-1 phosphorylation, the upstream molecules of ERK in insulin signaling pathway. Additionally, inhibition of insulin stimulated PI3 kinase activation reduced ERK phosphorylation. The present study therefore shows that the inhibitory effect of DHT on insulin-stimulated granulosa cell proliferation occurs early in the signaling pathway at the level of insulin receptor substrate-1 phosphorylation, leading to reduced ERK phosphorylation and subsequent inhibition of cyclin D2 mRNA expression.
Introduction
THE DEVELOPMENT OF mammalian follicle is regulated by gonadotrophins and steroids as well as various growth factors. The primordial follicle in the developing mammalian ovary consists of an immature oocyte surrounded by a single layer of granulosa cells, and follicular growth is initiated as the granulosa cells begin to proliferate, when they acquire responsiveness to FSH, resulting in the formation of large preantral follicle (1, 2, 3). The proliferation of granulosa cells is crucial for the formation of preovulatory follicle. Disturbance in this orderly progression of follicle growth could result in anovulation. It is now well recognized that, in the human, under pathophysiological conditions often related to polycystic ovarian syndrome, anovulation is associated with excess androgen production (4, 5, 6). It has been reported that in hyperandrogenic ovaries, granulosa cells proliferate at a subnormal rate (7), and follicular development is arrested at a stage at which selection of dominant follicle usually occurs (8). Furthermore, the level of 5-reductase, the enzyme that converts androgens to their 5-reduced metabolites, has been shown to be high in hyperandrogenic states (9, 10).
Before proliferation, granulosa cells undergo a well-coordinated series of events that are controlled by regulatory proteins such as cyclins and cyclin-dependent kinases. Progression of cell cycle from G1/S phase is regulated by D-type cyclins, which are regulated by extracellular signals (11). The D-type cyclins bind to cyclin-dependent kinases (cdk) 4 and 6, which activate a cascade of events that eventually leads to the progression from G1 to S phase (12, 13). Conversely, the kinase-inhibitory protein, p27kip1, binds to the cyclin D-cdk4/6 complex and inhibits their activation and blocks the progression of cell cycle from G1 to S phase, arresting the cells at G1 phase (14, 15).
We have previously shown that 5-dihydrotestosterone (DHT), the 5-reduced metabolite of testosterone, inhibits FSH-mediated granulosa cell proliferation by reducing cyclin D2 mRNA expression and subsequent cell cycle arrest at G1/S interphase in cultured rat granulosa cells (16). We have also reported that DHT exerts its inhibitory effect on cyclin D2 mRNA expression by reducing FSH-mediated phosphorylation of the ERK or MAPK by inhibiting protein kinase A (17).
The mitogenic and metabolic pathways in granulosa cells of preantral follicles are also known to be regulated by insulin (18, 19). Insulin acts through its own receptor to modulate the response of granulosa cells to gonadotropins (20, 21). Because insulin is an important mitogenic agent of proliferating granulosa cells, we examined whether DHT exerts any inhibitory effect on insulin-mediated mitogenic signaling pathway.
Upon interaction with insulin, the receptor becomes activated through an autophosphorylation reaction (22). Cellular scaffold proteins bind to the autophosphorylation sites that are then phosphorylated on the tyrosine residues. Most of the intracellular signals are generated through signaling complexes that are assembled around the tyrosine phosphorylated scaffold proteins including insulin receptor substrates (IRS), which are largely responsible for insulin-mediated functions. IRS proteins couple insulin receptors to the phosphatidylinositol 3-kinase (PI3 kinase) and ERK cascades (23). In the present studies, we examined the effect of insulin in cyclin D2 mRNA expression because cyclin D2 is a crucial molecule in FSH-mediated granulosa cell proliferation by regulating cell cycle progression at G1/S phase (13, 16, 24).
Our studies show that in cultured rat granulosa cells, insulin treatment stimulates cyclin D2 mRNA expression in a dose-dependent manner. DHT inhibits insulin mediated IRS-1 phosphorylation which leads to the inhibition of ERK phosphorylation. The reduced ERK activation in turn reduces cyclin D2 mRNA expression. Our results also show that inhibition of PI3 kinase reduces insulin-mediated cyclin D2 mRNA expression and ERK phosphorylation, suggesting a cross-talk between these two pathways.
Materials and Methods
Materials
Insulin (bovine), DHT (5-androstan-17-ol-3-one), 17-estradiol (1,3,5 [10]-estratriene-3, 17-diol), and PI3 kinase inhibitor, wortmannin, were purchased from Sigma Chemical Co. (St. Louis, MO). Antibodies for total phosphorylated ERK and total ERK-2 were obtained form Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against phosphorylated Raf (Ser 338) as well as p27kip were obtained from Cell Signaling Technology Inc. (Beverly, MA). Antiphosphotyrosine antibody (clone 4G10) was from Upstate Cell Signaling Solutions (Lake Placid, NY), and IRS-1 antibody was from BD Transduction Laboratories (San Diego, CA). MAPK kinase (MEK) inhibitor PD 98059 was from Promega (Madison, WI). Antimouse and antirabbit IgG horseradish peroxidase conjugates and enhanced chemiluminescent Western blotting detection reagents were purchased from Amersham Pharmacia Biotech. (Piscataway, NJ). Phenol red-free DMEM-F12 medium, Trizol reagent, and the RadPrime DNA labeling system were products of Life Technologies Inc. (Gaithersburg, MD). [32-P]Deoxy-CTP 3000 Ci/mmol) was purchased from MP Biomedicals (Irvine, CA). DHT was dissolved in ethanol, whereas wortmannin and PD 98059 were dissolved in dimethylsulfoxide. A dilute solution of acid water (HCl, pH 2–3) was used to dissolve insulin to make required concentrations. The total concentrations of diluents in the culture medium did not exceed 0.15%.
Animals and treatment
Female Sprague Dawley rats (22 d old) were housed in a temperature-controlled room with proper dark-light cycles and were under the care of University of Michigan Unit of Laboratory Animal Medicine. The animals were injected with estradiol (1.5 mg/d) for 3 d to stimulate the development of preantral follicles (25). Twenty-four hours after the last estradiol administration, animals were killed by CO2 asphyxiation. All the experiments conducted were approved by the University Committee on Use and Care of Animal, University of Michigan (approval no. 8360).
Granulosa cell isolation and culture
The ovaries from rats were collected and granulosa cells were isolated as previously described (16, 26, 27). In brief, ovaries were cleared from the surrounding fat and punctured with 25-gauge needles. Cells were collected in phenol red-free DMEM-F12 containing 0.2% BSA, 10 mM HEPES, and 6.8 mM EGTA, incubated for 15 min at 37 C under 95% O2-5% CO2, and centrifuged for 5 min at 250 x g. The pellets were suspended in a solution containing 0.5 M sucrose, 0.2% BSA, and 1.8 mM EGTA in DMEM-F12 and incubated for 5 min. After incubation, the suspension was diluted with 3 vol DMEM-F12, centrifuged at 250 x g, and treated sequentially with trypsin (20 μg/ml) for 1 min, 300 μg/ml soybean trypsin inhibitor for 5 min, and deoxyribonuclease (DNase I) (100 μg /ml) for 5 min at 37 C to remove dead cells. The cells were then rinsed twice with serum-free media, suspended in DMEM-F12, and the cell number determined. Cell viability was examined by trypan blue exclusion. Cells were cultured in serum-free DMEM-F12 supplemented with 20 mM HEPES (pH 7.4), 4 mM glutamine, 100 IU penicillin/ml, and 100 μg/ml streptomycin. Before plating, the culture dishes were coated with 10% fetal calf serum for 2 h at 37 C and washed with DMEM-F12. After allowing cells to attach overnight, they were treated with DHT (90 ng/ml) or vehicle alone for 24 h.
Northern blot analysis
Granulosa cells, after overnight attachment, were treated with 0, 10, 25, 50, and 100 ng/ml insulin for 2 h to examine the dose-dependent effect on cyclin D2 mRNA expression. In experiments in which the effect of the MEK inhibitor PD 98059 and the PI3 kinase inhibitor wortmannin on insulin-stimulated cyclin D2 mRNA expression was tested, one set of cultures was pretreated with the inhibitors (20 μM PD 98059 for 1 h and 100 nM wortmannin for 30 min) followed by incubation with insulin (100 ng/ml) for 2 h. To test the effect of DHT on insulin-mediated cyclin D2 mRNA expression, cells were pretreated with DHT (90 ng/ml) for 24 h followed by insulin (100 ng/ml) for 2 h. Control groups received equal amounts of respective vehicles. At the end of incubation, the medium was decanted, and the cells were washed once with PBS. Total RNA was extracted using TRIzol reagent according to the manufacturer’s instructions (Life Technologies). The cyclin D2 cDNA (1.1 kb) probe was radiolabeled using [32-P]deoxy-CTP and the RadPrime DNA labeling system and was hybridized to blots overnight at 42 C using 2 x 107 cpm of labeled probe. The hybridized blots were washed and exposed at –70 C to XAR film (Eastman Kodak Co., Rochester, NY). The blots were stripped and rehybridized with a radiolabeled cDNA probe corresponding to 18 S rRNA to monitor total RNA loading.
Western blot analysis
In experiments in which the specific inhibitors (20 μM PD 98059 and 100 nM wortmannin) were used, cells were pretreated with these agents before the incubation with insulin as described above. To examine the effect of DHT on insulin-mediated phosphorylation of ERK, Raf, and IRS-1, granulosa cells, after 24 h exposure to DHT (90 ng/ml) or vehicle, were stimulated with insulin (100 ng/ml) for time intervals described for each experiment. Because the expression of phosphorylated ERK-2 was considerably higher than ERK-1 in granulosa cells, we specifically looked at phosphorylated ERK-2 expression. The effect of insulin on p27kip was examined by treating the cells with insulin for 4 h. The reaction was stopped by removing the medium. In all the experiments, control sets were run simultaneously with respective vehicles. Cells after treatments were washed with PBS, lysed in modified radioimmunoprecipitation assay buffer. Total protein was subjected to Western blot analysis, and protein loading was normalized by reprobing the same blots with antibodies against the nonphosphorylated form of each protein probe or -tubulin. Detection was performed with an enhanced chemiluminescent Western blotting detection system (Amersham Pharmacia Biotech).
Statistical analysis
Statistical analysis was carried out using unpaired t test (GraphPad Prism, version 3.0; GraphPad Inc., San Diego, CA). Each experiment was repeated at least three times with similar results. Blots are representative of one experiment, and graphs represent the mean ± SE of three replicates.
Results
Insulin increases cyclin D2 mRNA expression in granulosa cells
Initial experiments examined the effect of insulin on cyclin D2 mRNA expression. The cultured granulosa cells from 3-d estradiol primed rats were treated with increasing concentration (0, 10, 25, 50, and 100 ng/ml) of insulin for 2 h, and cyclin D2 expression was measured using Northern blot analysis. Insulin treatment increased cyclin D2 in a dose-dependent manner (Fig. 1). The maximum expression was observed with 100 ng/ml insulin and therefore selected for all subsequent studies. Insulin treatment also resulted in a significant reduction (P < 0.05) of p27kip (cdk inhibitor) an inhibitor of cell proliferation (Fig. 2), compared with control.
DHT treatment reduces insulin mediated cyclin D2 mRNA expression
To examine whether DHT affects insulin-mediated stimulation of cyclin D2 mRNA expression, granulosa cells were pretreated with DHT (90 ng/ml) for 24 h followed by stimulation with 100 ng/ml insulin for 2 h. Northern blot analysis shown in Fig. 3 indicates an inhibition in cyclin D2 expression in response to pretreatment with DHT (P < 0.05). This result demonstrates that at a dose of 90 ng/ml, DHT inhibits insulin-stimulated cyclin D2 mRNA expression.
Inhibition of ERK and PI3 kinase activation inhibits insulin mediated cyclin D2 mRNA expression
The signaling pathways through which insulin stimulates cyclin D2 expression were then examined. ERK and PI3 kinase activation, representing the two well-established insulin signaling pathways, were selected as targets for the study. Cells were pretreated with specific inhibitors against ERK (PD 98059) and PI3 kinase (wortmannin), followed by stimulation with insulin. The results presented in Fig. 4 show that inhibition of ERK activation by pretreatment with PD 98059 (20 μM) completely abolished insulin’s stimulatory effect on cyclin D2 mRNA (P < 0.05). Blocking PI3 kinase activation using wortmannin (100 nM), on the other hand, evoked partial but significant (P < 0.05) inhibition of this stimulation (Fig. 5). These results indicate that ERK is a crucial molecule in insulin stimulated cyclin D2 mRNA expression and that both ERK and PI3 kinase pathways participate in insulin-stimulated cyclin D2 mRNA expression.
DHT treatment inhibits insulin stimulated ERK phosphorylation
Experiments were then designed to examine the effect of DHT on insulin mediated ERK-2 phosphorylation. Granulosa cells were pretreated with DHT or vehicle for 24 h followed by stimulation with insulin for 5 min. Phosphorylation of ERK-2 was examined by Western blot analysis using specific antibody. As expected, pretreatment with DHT significantly reduced insulin-stimulated ERK-2 phosphorylation (P < 0.05) (Fig. 6), compared with control cells subjected to similar treatments, suggesting that DHT inhibits cyclin D2 mRNA expression by reducing ERK phosphorylation.
DHT treatment reduces phosphorylation of Raf-1 and IRS-1
After establishing that DHT exerts its inhibitory effect by blocking insulin-dependent ERK activation, the effect of DHT on upstream molecules of ERK in the Ras/Raf/MEK signaling cascade was examined. Incubation with DHT for 24 h followed by insulin stimulation for 3 min resulted in reduced Raf-1 phosphorylation (P < 0.05), compared with control (Fig 7). For IRS-1 phosphorylation, which is a very rapid process, exposure time of insulin was reduced to 1 min. Control cells showed a significantly higher response in IRS-1 phosphorylation (P < 0.05) with insulin treatment, whereas in DHT-exposed cells, insulin stimulation of IRS-1 phosphorylation is completely abrogated (Fig. 8). The antiphosphotyrosine antibody used for the detection of IRS-1 phosphorylation recognizes all tyrosine phosphorylated sites. Because IRS-1 is phosphorylated at multiple sites, this antibody rather than those against specific sites, allowed us to identify the changes in the extent of phosphorylation with improved signal intensity. The identity of IRS-1 was established by stripping and reprobing the blots with antibody specific for total IRS-1. Together these results show that DHT inhibits the activation of upstream molecules of ERK in insulin signaling.
Inhibition of PI3 kinase reduces insulin stimulated ERK phosphorylation
A reduction in insulin-mediated cyclin D2 mRNA expression by PI3 kinase inhibition (Fig. 5) suggests that PI3 kinase also plays (at least partially) a role in insulin’s mitogenic effect. Because ERK activation is critical for cyclin D2 mRNA expression, a possible cross-talk between PI3 kinase and ERK activation was examined. Pretreatment of granulosa cells with 100 nM wortmannin, an inhibitor of PI3 kinase, significantly reduced insulin-mediated ERK-2 phosphorylation (P < 0.05) (Fig. 9). This result suggests that insulin may also increase cyclin D2 mRNA expression by stimulating ERK activation through PI3 kinase.
Discussion
Ovarian granulosa cell proliferation, a prerequisite for normal ovulation, is under the control of multiple regulatory molecules including FSH, insulin/IGF system, and activin (28, 29, 30, 31, 32, 33, 34). Signals from these mitogens flow through different signaling cascades and activate proteins that govern cell proliferation. Cyclin D2, one such regulatory protein, is critical for granulosa cell proliferation. We have previously shown that increased cyclin D2 mRNA expression correlates with increased thymidine incorporation to DNA and proliferation in granulosa cells in response to FSH-mediated mitogenesis (16, 17). The importance of cyclin D2 in granulosa cell proliferation is further substantiated by the observation of impaired proliferation and ovulation in cyclin D2 knockout mice (24).
Because insulin is a known mitogen of granulosa cells (18, 21), the present studies were carried out to determine whether its mitogenic actions are mediated through cyclin D2. We have shown that in cultured rat granulosa cells, insulin stimulates cyclin D2 mRNA expression in a dose-dependent manner (Fig. 1) and reduces cyclin-dependent kinase inhibitor, p27kip (Fig. 2). Together these data demonstrate that insulin regulates mitogenesis by up-regulating cyclin D2 expression, a positive regulator of cell cycle and by reducing the negative regulator, p27kip1.
As stated in the introductory text, elevated levels of androgens and their 5-reduced metabolites have been shown to disrupt the mitogenic signaling in granulosa cells. Because insulin stimulates granulosa cell proliferation by up-regulating cyclin D2 mRNA expression, we have examined whether this process is inhibited by DHT. Exposure of granulosa cells to DHT for 24 h reduced insulin-mediated cyclin D2 mRNA expression (Fig. 3), suggesting that elevated levels of DHT could block insulin mediated mitogenesis. Because the signaling pathways of insulin have been well characterized, we specifically examined the site of DHT’s inhibitory action. Insulin has been known to act both as mitogenic agent through the ERK pathway and metabolic regulator through the PI3 kinase pathway (23, 35). However, the use of specific inhibitors of both pathways reduced cyclin D2 mRNA expression. ERK inhibition by PD 98059 completely abolished insulin’s stimulatory effect (Fig. 4), whereas PI3 kinase inhibition by wortmannin partially reduced this stimulation (Fig. 5). Although inhibition of ERK produced a more pronounced inhibitory effect than that of PI3 kinase, it would appear that activation of both signaling pathways is involved in insulin-mediated cyclin D2 mRNA expression. The complete abolishment of insulin-mediated cyclin D2 mRNA expression by ERK inhibition makes this molecule a critical component of insulin’s mitogenic machinery. The observed reduction in insulin-stimulated ERK-2 phosphorylation in DHT-treated cells (Fig. 6) indicates that DHT-mediated inhibition of cyclin D2 mRNA is indeed due to interference at this step.
Our results show that DHT exert its inhibitory effect on insulin action in the granulosa cell at an early step in its signaling pathway. It is now well established that phosphorylation of IRS at tyrosine residues is the first post receptor step in insulin signaling, and this pathway has been implicated as a modulator of follicular growth (18). Although insulin receptor activation leads to both IRS-1 and IRS-2 phosphorylation, previous gene knockout studies have shown that IRS-1 mainly regulates mitogenic pathways, whereas IRS-2 mediates metabolic pathways (36, 37). Because cyclin D2 is a positive regulator of mitogenesis, it is conceivable that insulin stimulates cyclin D2 expression through IRS-1 rather than IRS-2. This notion is consistent with our results, in which DHT treatment inhibited insulin-stimulated phosphorylation of IRS-1 as well as its downstream target molecule, Raf-1 (Figs. 7 and 8). Phosphorylation of Raf-1 at multiple phosphorylation sites has been shown to activate ERK pathway (38). Together these observations demonstrate that DHT reduces the IRS-1 phosphorylation leading to the inhibition of Ras/Raf/ERK pathway.
The finding that inhibition of PI3 kinase activation reduced insulin-stimulated ERK-2 phosphorylation (Fig. 9) points to a possible cross-talk between the PI3 kinase and ERK pathways in insulin-stimulated cyclin D2 mRNA expression. Similar cross-talk between insulin signaling pathways has also been reported recently in other cell types (39, 40). One explanation for the existence of such a cross-talk in granulosa cells is that converging stimulatory signals from PI3 kinase to ERK could synergize insulin’s mitogenic potential in growing follicles.
In summary, inhibition of IRS-1 phosphorylation by DHT leads to inhibition of the Ras/Raf/MEK signaling cascade, resulting in reduced ERK activation and subsequent decrease in cyclin D2 mRNA expression. The present studies along with our previous reports (17), which demonstrated that DHT inhibited protein kinase A activation without affecting cAMP production, suggest that DHT can act at multiple sites in granulosa cells (17). The inhibitory effect of DHT at multiple sites in FSH and insulin-mediated ERK activation indicates that impairment of granulosa cell proliferation by androgens is a complex process and that the inhibitory effects culminate at the level of ERK phosphorylation. A recent independent study, which implicates alteration in ERK pathway in the pathogenesis of hyperandrogenism, is supportive of this notion (41). We conclude that the defective growth characteristics of granulosa cells seen under hyperandrogenic conditions might be mediated by inhibition of FSH and insulin signaling at multiple steps, but they converge to inhibit ERK activation and cyclin D2 mRNA expression.
Acknowledgments
We express our appreciation to Dr. Anil Nair, Dr. Roberto Towns, Helle Peegel, and Christine Clouser for critical reading of the manuscript.
Footnotes
This work was supported by National Institutes of Health Grant HD-38424.
First Published Online October 6, 2005
Abbreviations: cdk, Cyclin-dependent kinase; DHT, 5-dihydrotestosterone; IRS, insulin receptor substrate; MEK, MAPK kinase; PI3 kinase, phosphatidylinositol 3 kinase.
Accepted for publication September 28, 2005.
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