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BOR - Papers in Press, published online ahead of print February 15, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.049908
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BIOLOGY OF REPRODUCTION 74, 1034–1040 (2006)
DOI: 10.1095/biolreprod.105.049908
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Insulin and Oxidative Stress Modulate Proliferation of Rat Ovarian Theca-Interstitial Cells Through Diverse Signal Transduction Pathways1

Jakub Kwintkiewicz 3, Robert Z. Spaczynski 4, Nastaran Foyouzi 3, Tugce Pehlivan 5, and Antoni J. Duleba 2 3

Department of Obstetrics and Gynecology,3 Yale University School of Medicine, New Haven, Connecticut 06510 Department of Gynecology and Obstetrics,4 Poznan University of Medical Sciences, 60-535 Poznan, Poland Instituto Valenciano de Infertilidad,5 46015 Valencia, Spain

ABSTRACT

Insulin and moderate oxidative stress stimulate proliferation of ovarian theca-interstitial cells. The effects of these agents on selected signal transduction pathways were examined. PD98059 (inhibitor of MAP2K1, also known as MEK-1, upstream of extracellular signal-regulated protein kinases MAPK3/1, also known as ERK1/2), wortmannin (inhibitor of PIK3C2A, also known as PI3K), and rapamycin (inhibitor of FRAP1, also known as mTOR, upstream of RPS6KB1) each significantly decreased insulin and oxidative stress-induced proliferation of theca-interstitial cells. The greatest inhibition was observed in the presence of rapamycin; this effect occurred without a significant change in cell viability. Phosphorylation of AKT was stimulated by insulin only, while phosphorylation of MAPK3/1 and RPS6KB1 was increased by insulin and oxidative stress. Insulin-induced and oxidative stress-induced phosphorylation of RPS6KB1 was partly inhibited by wortmannin and partly by PD98059; the greatest inhibition was observed in the presence of a combination of wortmannin plus PD98059. Effects of insulin and oxidative stress on phosphorylation of RPS6KB1 were confirmed by kinase activity assays. These findings indicate that actions of insulin and oxidative stress converge on MAPK3/1 and RPS6KB1. Furthermore, we speculate that activation of RPS6KB1 may be in part induced via the MAPK3/1 pathway.

IGF1, insulin, oxidative stress, signal transduction, theca cells

INTRODUCTION

Ovarian thecal and interstitial tissues provide structural support and play an important paracrine and endocrine role, especially as an ovarian source of androgens. Appropriate regulation of proliferation of these tissues is essential for normal ovarian development and follicular function. We have previously demonstrated that insulin stimulates proliferation of both rat and human ovarian theca-interstitial cells in a concentration-dependent fashion [1, 2]. These actions of insulin and IGF1 may be mediated by activation of insulin receptors as well as IGF1 receptors, both of which have been identified in theca and interstitial cells [3–5]. IGF1 receptors possess the greatest affinity to IGF1 but also bind insulin. In rat theca-interstitial cells, IGF1 has a greater potency than insulin with respect to stimulation of proliferation; furthermore, IGF1 mutants with decreased affinity to IGF binding proteins have an even greater proliferative potency [1]. These observations indicate that the proliferative effects of insulin and IGF1 may be primarily due to activation of IGF1 receptors. Activation of insulin receptors and IGF1 receptors may induce several signal transduction pathways including phosphatidylinositol 3-kinase (PIK3C2A), AKT, and mitogen-activated protein kinase 1 (MAPK1) pathways [6–10]. PIK3C2A, AKT, and MAPK1 pathways have been implicated in stimulation of proliferation; however, the actual role and contribution to proliferation vary depending on cell type [9, 11–13]. Activation of PIK3C2A, AKT, and MAPK1 may lead to downstream signaling via RPS6KB1 [14–16].

Recently, we found that moderate oxidative stress also stimulates thecal-stromal proliferation [17]. These observations are consistent with growing evidence that reactive oxygen species (ROS) play a role as signaling molecules modulating growth and function of various cell types including fibroblasts and aortic endothelial cells [18–22]. The mode of signal transduction by ROS may include MAPK1 pathways, especially extracellular signal-regulated protein kinases (MAPK3/1) and/or RPS6KB1 [23–26].

This study was designed to compare the effects of insulin and oxidative stress on selected steps of the relevant signal transduction pathways including AKT, MAPK3/1, and RPS6KB1. Our findings indicate that in ovarian theca-interstitial cells, insulin stimulates AKT, MAPK3/1, and RPS6KB1, while oxidative stress activates MAPK3/1 and RPS6KB1. We propose that the proliferative effects of insulin/IGF and oxidative stress converge on RPS6KB1.

MATERIALS AND METHODS

Animals

Sprague-Dawley female rats were obtained on Day 25 of age from Taconic Farms (Germantown, NY) and housed with a 12L:12D photoperiod in an air-conditioned environment. Standard rat chow and water were given ad libitum. Starting on Day 28 of age, the animals were injected with 17ß-estradiol (1 mg/0.3 ml of sesame oil s.c.) daily for 3 days in order to stimulate ovarian development and growth of antral follicles. Approximately 24 h after the last injection (Day 31 of age), the animals were anesthetized with ketamine and xylazine (i.p.) and killed by intracardiac perfusion using 0.9% saline. All treatments and procedures were carried out in accordance with accepted standards of humane animal care as outlined in the NIH Guide for the Care and Use of Laboratory Animals and a protocol approved by the Yale University Animal Care Committee.

Cell Culture and Reagents

Ovarian theca-interstitial cells were obtained as described previously [1, 27]. Ovaries were dissected, and theca-interstitial cells were purified using discontinuous Percoll gradient centrifugation. The cells were counted and viability was routinely in the 85%–95% range. Theca-interstitial cells were incubated in 96-well plates (35 000 cells/well) for proliferation assays and in 100-mm plates (1.5 million cells/plate) for phosphorylation and kinase activity assays. The cultures were carried out for up to 48 h at 37°C in an atmosphere of 5% CO2 in humidified air, in serum-free McCoys 5a medium (supplemented with antibiotics, 0.1% bovine serum albumin, and 2 mM L-glutamine). The cells were incubated without (control) or with insulin (30–100 nM), and oxidative stress was induced by hypoxanthine and xanthine oxidase (HX/XO; 1 mM/30 µU/ml) or hydrogen peroxide (100 nM), wortmannin (100 nM), PD98059 (10 µM), and rapamycin (5 nM). All above chemicals were purchased form Sigma Chemical Co. (St. Louis, MO).

Thymidine Incorporation Assay

Theca-interstitial cells were incubated for 48 h in 96-well culture plates with or without individual additives. DNA synthesis was quantified using thymidine incorporation assay. Radiolabeled [3H] thymidine (1 µCi/well) was added to the cells during the last 24 h of culture. At the end of the culture period, the cells were harvested using a multiwell cell harvester (PHD Harvester, Model 290; Cambridge Technology, Inc., Watertown, MA). Radioactivity was measured in a liquid scintillation counter, SL 4000 (Intertechnique, Fairfield, NJ). Each treatment was carried out in at least eight replicates.

Variability in the DNA synthesis was noted between experiments; however, within each individual cell preparation (each 96-well plate), the variability was low. Proliferation was assessed in approximately 60%–70% confluent cells, and the proliferation was rapid. Variability in the interexperimental proliferative effects is likely related to changes in proliferation among nearly confluent cells. However, the relative effects of all used agents were consistent.

Western Blot Analysis

Following incubation with individual treatments, culture dishes with attached cells were transferred to an ice bath and washed twice with ice-cold PBS. Afterward, lysis buffer was added (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM PMSF, protease inhibitor cocktail). Lysed cells were transferred to Eppendorf tubes and incubated on ice for 30 min with vigorous vortexing at 5-min intervals. Lysates were centrifuged for 20 min at 20 000 x g. Protein concentration was determined by using standard Bradford colorimetric assay. Supernatants were collected and stored at –80°C or directly subjected to electrophoresis. Protein preparations were mixed with the sample buffer (125 mM Tris-HCl, 4% SDS, 21% glycerol, 10% ß-mercaptoethanol, 0.04% bromophenol blue) and loaded to 10% acrylamide SDS-PAGE gel. Gel separation was carried out for 200 min at 100 volts at room temperature. After separation, protein was transferred to a PVDF membrane that was pre-equilibrated in transfer buffer (25 mM Tris, 192 mM glycine, 20% v/v methanol, pH 8.3). Transfer was carried out overnight (30 volts at 4°C). The next day the PVDF membrane was first blocked with 5% nonfat dry milk in TBST-T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) at room temperature for 1 h and subsequently incubated with primary antibody: anti-RPS6KB1 pTpS 421/424 and anti-total actin (both from Santa Cruz Biotechnology Inc., Santa Cruz, CA) diluted 1:2500 in 5% BSA in TBS-T for 2 h at room temperature. Membranes were washed six times for 15 min in TBS-T, and subsequently a secondary, horseradish peroxidase-labeled anti-IgG antibody incubation was performed. This step was followed by six 15-min washes in TBS-T. The membrane was wetted in ECL solution for 1–5 min, exposed to x-ray film, and developed. For sequential immunoblot analysis, the membranes were stripped in Restore Western Blot Stripping Buffer (Pierce Biotechnology, Rockford, IL) at 37°C for 30 min, washed extensively with TBS-T, and reused. Westerns were quantified using Image J NIH software by measuring the intensity of individual bands.

ELISA Analysis

AKT TOTAL and pS473 as well as MAPK3/1 TOTAL and pTpY 185/187 kits were purchased in Biosource International (Camarillo, CA). Protein for ELISA tests was prepared as for Western blot using the same lysis buffer. ELISA tests were performed according to manufacturer's instructions. Briefly, protein lysates were added to 96-well plates precoated with capture antibody and incubated for 2 h at room temperature. Subsequently, detecting antibody was added and incubation continued for 1 h. Next, secondary antibody conjugated with horseradish peroxidase was added, and the mixture was incubated for an additional 30 min. The wells were rinsed four times using washing buffer. The reaction was visualized using stabilized chromogen, which was added for 30 min. Finally, a stop solution was added, and absorbance was read at 405 nm in a spectrophotometer (Biorad model 680).

Immunoprecipitation

Cells were washed twice with ice-cold PBS and lysed in lysis buffer A (50mM Tris/HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate [activated], 10 mM Na B-glycerolphosphate, 50 mM NaF, 5 mM Na pyrophosphate, 1% w/v Triton X-100, 0.1% w/v 2-mercaptoethanol, 1 mM PMSF). Lysates were snap frozen. Prior to immunoprecipitation, samples were thawed and spun at 15 000 x g for 10 min, and the supernatants were incubated with anti-RPS6KB1 antibody (Upstate Biotechnology, Lake Placid, NY) and protein G-agarose at 4°C overnight. The samples of immunoprecipitate-agarose bead complex were washed three times with lysis buffer and once with 1 M NaCl in lysis buffer (buffer B). These precipitates were subsequently used in a kinase activity assay.

RPS6KB1 Assay

Kits for assessment of enzymatic activity of RPS6KB1 were purchased from Upstate Biotechnology. Immunoprecipitates were processed according to the manufacturer's instructions. Briefly, immunoprecipitates were combined with 10 µl of dilution buffer, 10 µl of substrate cocktail (50 µM final concentration of AKRRRLSSLRA), 10 µl of inhibitor cocktail, and 10 µl of the diluted [{gamma}-32P]ATP mixture. The mechanism of the reaction is presented in the following diagram:

Formula

After 10 min of incubation at 30°C, the suspension was transferred onto P81 Phosphocellulose Squares, washed three times in 0.75% phosphoric acid and once in acetone, and subsequently placed in scintillation vials. Scintillation liquid was added, and radioactivity was determined using a scintillation counter.

Statistical Analysis

Values represent means ± SEM. Statistical analysis was performed by analysis of variance followed by pairwise comparisons using Bonferroni correction.

RESULTS

The effects of insulin (30 nM) and moderate oxidative stress on DNA synthesis by ovarian theca-interstitial cells are summarized in Figure 1. Moderate oxidative stress was induced by 1 mM hypoxanthine combined with 30 µU/ml xanthine oxidase (HX/XO) [17]. The above concentrations of insulin and HX/XO were previously shown to induce maximal proliferative effects; concentration-dependent effects of these agents were evaluated in an identical cell culture system [1, 17]. In this study, insulin increased proliferation by 61 ± 24% above control level (P < 0.001), whereas HX/XO increased proliferation by 120 ± 7% above control level (P < 0.001).


Figure 1
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  		FIG. 1. Proliferation of rat theca-interstitial cells assessed by incorporation of [3H] thymidine after 48 h of incubation without additives (control) or with insulin (30 nM) (A), hypoxanthine/xanthine oxidase (HX/XO; 1 mM/30 µU/ml) (B), and/or specific inhibitors of kinases: PD98059 (10 µM), wortmannin (100 nM), and rapamycin (5 nM) (C). Each bar represents mean ± SEM (n = 8); * denotes means significantly different from control (P < 0.001); {dagger} denotes means significantly different from insulin and/or HX/XO (P < 0.001).

In order to determine which signal transduction pathways may be involved in modulation of DNA synthesis, the effects of the following selective inhibitors were evaluated: PD98059 (10 µM; inhibitor of MAP2K1, upstream of MAPK3/1), wortmannin (100 nM; inhibitor of PIK3C2A), and rapamycin (5 nM; inhibitor of FRAP1, upstream of RPS6KB1). Inhibitors were always added to the cells 30 min before other treatments. As presented in Figure 1A, PD98059 alone, as well wortmannin alone, decreased insulin-induced proliferation from 161 ± 24% of control to 114 ± 5% (P < 0.001) and 118 ± 5% of control (P < 0.001), respectively. A combination of PD98059 and wortmannin decreased insulin-induced effect on proliferation to a greater extent than each of these inhibitors alone (to 88 ± 5% of control; P < 0.001). Rapamycin exerted the most potent effect, inhibiting insulin-induced proliferation to 11 ± 3% of control level (P < 0.001). Thus, these treatments not only entirely reversed insulin effects but actually reduced proliferation to levels below basal (control) level.

As shown in Figure 1B, PD98059 alone and wortmannin alone decreased HX/XO-induced proliferation (220 ± 7% of control) to 151 ± 5% (P < 0.001) and 130 ± 5% (P < 0.001) of control, respectively. The combination of both inhibitors had an approximately additive effect, decreasing HX/XO-induced proliferation to 61 ± 2% (P < 0.001) of control. Rapamycin decreased HX/XO-induced proliferation to 13 ± 1% (P < 0.001). PD98059, wortmannin, combination of PD98059 and wortmannin, and rapamycin also blocked basal proliferation (Fig. 1C), respectively, to 68 ± 3%, 46 ± 2%, 25 ± 1%, and 5 ± 3% of control level (all significant at P < 0.001).

The viability of cells in the absence or in the presence of inhibitors was assessed by the trypan blue exclusion test following a 48-h exposure to inhibitors in culture: the proportion of stained cells was 4.1 ± 1.1% in control cultures, 12.6 ± 2.8% in the presence of PD98059, 7.2 ± 1.3% in the presence of wortmannin, and 3.5 ± 1.3% in the presence of rapamycin. These differences in cell viability were not statistically significant.

In subsequent evaluations of signal transduction pathways of insulin and oxidative stress, we studied activation of selected steps: MAPK3/1, AKT, and RPS6KB1. To study a major MAPK1 pathway, phosphorylation of MAPK3/1 was evaluated at tyrosine/threonine 185/187; a maximum effect was observed after 10 min of exposure (Fig. 2A). Moderate oxidative stress induced by hydrogen peroxide (HP) and HX/XO stimulated MAPK3/1 phosphorylation by 70 ± 15% (P < 0.05) and 115 ± 24% (P < 0.05), respectively. Insulin increased MAPK3/1 phosphorylation by 82 ± 19% (P < 0.05). In contrast (Fig. 2B), AKT phosphorylation (at pS473) was increased by insulin by 432 ± 234% (P < 0.01), but not by oxidative stress.


Figure 2
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  		FIG. 2. Phosphorylation of MAPK3/1 (A) and AKT (B) in rat theca-interstitial cells determined by ELISA after 10 min of incubation without additives (control), with hydrogen peroxide (HP; 100 nM), with hypoxanthine/xanthine oxidase (HX/XO; 1 mM/30 µU/ml), or with insulin (100 nM). Each bar represents mean ± SEM (n = 4); * denotes means significantly different from control (P < 0.05).

Activation of RPS6KB1 (phosphorylation of tyrosine/serine at 421/424) was determined by Western blotting; the maximum effect was observed after 10 min of exposure. As presented in Figure 3A, moderate oxidative stress induced by hydrogen peroxide and HX/XO stimulated RPS6KB1 phosphorylation by 29 ± 8% (P < 0.05) and 60 ± 25% (P < 0.05), respectively. Insulin increased phosphorylation by 65 ± 13% (P < 0.01) above control levels. The stimulatory effects of insulin and moderate oxidative stress on RPS6KB1 were confirmed by the activity assay (Fig. 3B); insulin increased the activity of RPS6KB1 by 105 ± 3% and HX/XO by 177 ± 61% above control level (all significant at P < 0.05).


Figure 3
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  		FIG. 3. Activation RPS6KB1 by oxidative stress and insulin were determined by phosphorylation assay (A) and RPS6KB1 activity assay (B). Phosphorylation of RPS6KB1 was detected by Western blot after 10 min of incubation without additives (control), with hydrogen peroxide (HP; 100 nM), with hypoxanthine/xanthine oxidase (HX/XO; 1 mM/30 µU/ml), or with insulin (100 nM). Activity of RPS6KB1 was determined following 30 min of incubation without additives (control) or with hypoxanthine/xanthine oxidase (HX/XO; 1 mM/30 µU/ml) or insulin (100 nM). Each bar represents mean ± SEM (n = 4); * denotes means significantly different from control (P < 0.05).

Role of PIK3C2A and MAPK1 in activation of RPS6KB1 was studied in experiments evaluating effects of wortmannin and PD98059 on RPS6KB1 phosphorylation (Fig. 4). Insulin induced RPS6KB1 phosphorylation to 155 ± 6% of control; this effect was inhibited by PD98059 alone (to 103 ± 6% of control; P < 0.05) and wortmannin alone (to 66 ± 5% of control; P < 0.05). A combination of PD98059 and wortmannin had the greatest inhibitory effect, decreasing insulin-induced RPS6KB1 phosphorylation to 33 ± 23% (P < 0.05); this effect was significantly greater than that of either inhibitor used alone (P < 0.05). In a similar fashion, HX/XO induced RPS6KB1 phosphorylation to 162 ± 13% of control; this effect was decreased by PD98059 (to 116 ± 13% of control; P < 0.05) and wortmannin (to 104 ± 2% of control; P < 0.05). When used in a combination, wortmannin and PD98059 reduced HX/XO-induced RPS6KB1 phosphorylation to 43 ± 8% (P < 0.05); the effect was significantly greater than that of either inhibitor alone.


Figure 4
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  		FIG. 4. Phosphorylation of RPS6KB1 of rat theca-interstitial cells assessed by Western blot after 10 min of incubation with vehicle (control), with insulin (100 nM) (A), or with hypoxanthine/xanthine oxidase (HX/XO; 1 mM/30 µU/ml) (B). Specific inhibitors of kinases—PD98059 (10 µM) and/or wortmannin (100 nM)—were added 30 min before other treatments. Each bar represents mean ± SEM (n = 4); * denotes means significantly different from control (P < 0.05); {dagger} denotes means significantly different from insulin (A; P < 0.05) and HX/XO (B; P < 0.05); § denotes means significantly different from insulin plus wortmannin (A; P < 0.05) and from HX/XO plus wortmannin (B; P < 0.05).

DISCUSSION

The present study demonstrates that in ovarian theca-interstitial cells: 1) MAPK3/1, PIK3C2A, and RPS6KB1 are all involved in modulation of proliferation; 2) insulin and oxidative stress induce increased activity of MAPK3/1 and RPS6KB1 pathways; 3) insulin stimulates AKT; and 4) PIK3C2A and MAPK3/1 may converge on RPS6KB1. A proposed model of relevant signal transduction pathways and studied inhibitors is presented in Figure 5.


Figure 5
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  		FIG. 5. Proposed interactions of insulin and oxidative stress with selected steps of signal transduction pathways.

To our knowledge, this is the first study evaluating signal transduction pathways with regard to the effects on proliferation of ovarian mesenchyme. Our experiments using inhibitors of MAPK3/1, PIK3C2A, and RPS6KB1 indicate that all these pathways are important in modulation of proliferation, both at the basal level as well as in response to insulin and oxidative stress. Reduction of basal proliferation by these inhibitors suggests that even in the absence of added insulin or ROS, the above signal transduction pathways are partly activated. Such activation may be related to factors such as endogenous generation of moderate oxidative stress or other activators. The greatest inhibitory effect of rapamycin underscores the importance of RPS6KB1. The key role of RPS6KB1 is further supported by induction of its phosphorylation and activity both by insulin and by moderate oxidative stress.

The phosphorylation assay of RPS6KB1 evaluated only phosphorylation sites at pTpS421/424. Since activation of this kinase requires multiple inputs directed at different domains [28], phosphorylation assays were validated by evaluation of kinase activity. Figure 3B presents a RPS6KB1 activity assay, confirming the stimulatory effects of insulin and oxidative stress. These findings are in accord with observations in several cell systems whereby activation of RPS6KB1 is important in induction of proliferation in response to stimuli such as various growth factors, cytokines, and agents such as angiotensin and Ca2+ [29–31].

Insulin and IGF1 have been shown to activate RPS6KB1 and induce proliferation of several cell types including smooth muscle cells [32], hepatocytes [33], pancreatic beta cells [34], and lens epithelial cells [35]. However, these effects appear to be not ubiquitous but dependent on the cell type; thus, for example, RPS6KB1 preferentially mediates IGF1-induced differentiation rather than proliferation of myoblasts [36]. Notably, relatively little is known regarding the activation of RPS6KB1 in response to oxidative stress. In previous studies, RPS6KB1 has been shown to be involved in response to oxidative stress by epidermal cells [26, 37] and cardiomyocytes [38].

Activation of RPS6KB1 is usually attributed to upstream signals mediated by PIK3C2A, possibly through the kinase AKT [39]. However, AKT-independent and even PIK3C2A-independent induction of RPS6KB1 has been demonstrated [40]. Furthermore, there is evidence indicating stimulatory involvement of MAPK1 pathway in activation of RPS6KB1 [16]. Present findings support this concept. First, oxidative stress induced phosphorylation and activity of RPS6KB1 without significant phosphorylation of AKT. Second, inhibition of MAPK3/1 pathway by PD98059 reduced both DNA synthesis and phosphorylation of RPS6KB1; these effects were observed in the presence of both insulin and oxidative stress (Figs. 1 and 4).

While the above observations support the role of MAPK3/1 in stimulating RPS6KB1, it is apparent that activation of PIK3C2A also contributes to stimulation of proliferation and activation of RPS6KB1. In support of this concept, we found that inhibition of PIK3C2A by wortmannin decreased activation of p RPS6KB1. Furthermore, a combination of wortmannin and PD98059 exerted a greater inhibitory effect than wortmannin alone or PD98059 alone on proliferation and on RPS6KB1 phosphorylation. These observations were consistent for both insulin-induced and oxidative stress-induced effects (Fig. 1).

Another related conclusion pertains to our observation that insulin and oxidative stress share common signal transduction pathways including MAPK3/1 and RPS6KB1. Indeed, it is well recognized that MAPK3/1 is a major pathway transducing insulin/IGF1 signals and stimulating proliferation in many [13, 36, 41, 42] but not all biological systems [43]. There is also growing evidence that oxidative stress induces proliferation via the MAPK3/1 pathway [44–46]. In view of the present findings, it is tempting to speculate that the proliferative actions of insulin may be mimicked and/or even mediated by generation of reactive oxygen species. Indeed, insulin and IGF1 have been shown to induce oxidative stress [47–50]. However, to date, such effects were not demonstrated in ovarian tissues.

Notably, we observed that all tested inhibitors of signal transduction pathways also decreased basal rate of DNA synthesis (Fig. 1C). This observation may reflect nonspecific effects of inhibitors; however, a significant toxic effect of these agents is not likely in view of lack of significant effects on cell viability. Another, more interesting explanation may be that, under basal conditions, signal transduction pathways are already active. Activity of these pathways may be due to putative basal level of oxidative stress and/or presence of other autocrine agents activating PIK3C2A, MAPK3/1, and RPS6KB1.

The present study may also have potential clinical relevance to a better understanding of the pathophysiology of polycystic ovary syndrome (PCOS), a condition typically associated with decreased effectiveness of insulin in regulation of glucose uptake/metabolism [51–53]. In PCOS, other actions of insulin, such as stimulation of thecal-interstitial proliferation, may be intact. Glucose uptake is stimulated primarily via PIK3C2A rather than MAPK3/1 or RPS6KB1 [54, 55]. In view of the findings of the present study, it is tempting to speculate that insulin-induced proliferation of ovarian theca-interstitial cells, mediated via MAPK3/1 and RPS6KB1, may be maintained even in the presence of dysfunction of signal transduction pathways regulating glucose uptake. However, this concept remains to be validated in studies utilizing human tissues.

In summary, our studies have identified MAPK3/1 and RPS6KB1 as important signal transduction steps involved in the modulation of theca-interstitial cell responses to insulin and oxidative stress. It is apparent that oxidative stress shares with insulin some but not all transduction pathways evoking comparable stimulatory effects on proliferation.

FOOTNOTES

1 Supported by NIH grant R01 HD40207 to A.J.D. Back

2 Correspondence: Antoni J. Duleba, Yale University School of Medicine, Department of Obstetrics and Gynecology, 333 Cedar St., New Haven, CT 06510. FAX: 203 785 7134; Back

101 antoni.duleba{at}yale.edu Back

Received: 1 December 2005.

First decision: 20 December 2005.

Accepted: 7 February 2006.

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