Effects of oxidative stress, insulin and IGF-I on p70S6 kinase in rat thecal-interstitial cells

Abstract

Appropriate regulation of ovarian thecal-interstitial proliferation and steroidogenesis is essential for normal ovarian function. Previously, we have shown that insulin, insulin-like growth factor-I (IGF-I) and moderate oxidative stress, all stimulate proliferation and steroidogenesis of theca-interstitial cells. We have also observed that the effects of the combination of these agents are not additive indicating a possibility of a common saturable step(s) of their signal transduction pathways. Recently, we have shown that oxidative stress and insulin/IGF-I stimulate the MAPK pathway. In contrast, insulin and IGF-I, but not oxidative stress, stimulate the PI3K/Akt pathway. In the present study we proposed that both oxidative stress and insulin/IGF-I signaling pathways converge at the level of p70S6 kinase (p70S6K). In vitro evaluation of human endometrial cell proliferation. Purified rat theca-interstitial cells were cultured in chemically defined media without (control) or with IGF-I (10 nM), insulin (100 nM), hypoxanthine + xanthine oxidase (X/XO; 1 mM/30 μU/mL), hydrogen peroxide (100 nM), wortmannin (100 nM) and/or PD98059 (10 μM). The above concentrations of X/XO and hydrogen peroxide have been shown previously to induce moderate oxidative stress and to promote theca-interstitial proliferation and steroidogenesis. Phosphorylation of p70S6 kinase was determined by western blot of p70S6K[pT/pS421/424]. Quantitative data was analyzed by ANOVA followed by post-hoc pairwise comparisons. In rat theca-interstitial cells, insulin and IGF-I stimulated p70S6K phosphorylation at tyrosine/serine 421/424 with a maximum effect after 10 minutes of exposure. Insulin and IGF-I increased phosphorylation by 63±13% (mean±SEM; p<0.001) and 51±13% (p<0.001), respectively, above control levels. Moderate oxidative stress induced by X/XO and hydrogen peroxide stimulated p70S6K phosphorylation by 60±25% (p<0.05) and 29±8% (p<0.01), respectively. Wortmannin, an inhibitor of PI3K, decreased insulin-induced p70S6K phosphorylation by 53±2%. Similarly, PD98059, a selective inhibitor of MAPK, decreased insulin-induced p70S6K phosphorylation by 44±3%. The present findings indicate that in thecal-interstitial cells, both PI3K and MAPK pathways activate p70S6K. A convergence of actions of insulin/IGF-I and oxidative stress at the level of p70S6K may explain the comparable effects of the above agents on proliferation and testosterone production. We propose that excessive activation of this kinase by oxidative stress and hyperinsulinemia may promote hyperplasia of ovarian mesenchyme in conditions such as polycystic ovary syndrome (PCOS).