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The Role of Mitogen-Activated Protein Kinase in Insulin and Insulin-Like Growth Factor I (IGF-I) Signaling Cascades for Progesterone and IGF-Binding Protein-1 Production in Human Granulosa Cells

Division of Endocrinology, Beth Israel Medical Center and Albert Einstein College of Medicine (D.S.-Y., J.Z., L.P.), New York, New York 10003; and Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University (H.-C.L., Z.R.), New York, New York 10021

Address all correspondence and requests for reprints to: Leonid Poretsky M.D., Division of Endocrinology, Beth Israel Medical Center, 317 East 17th Street, Fierman Hall, 7th Floor, New York, New York 10003. E-mail: lporetsk{at}bethisraelny.org.

Insulin and IGF-I participate in the regulation of ovulation, steroidogenesis, and IGF-binding protein (IGFBP) production in the ovary. Insulin and IGF-I actions in the ovary are closely related. For example, insulin may amplify IGF-I action in the ovary by up-regulating type I IGF receptors and inhibiting IGFBP-1 production, thus increasing the bioavailability of IGF-I. It is hypothesized that ovarian effects of insulin in insulin-resistant states are mediated via an insulin action pathway(s) distinct from those involved in glucose transport. We previously reported that insulin-induced stimulation of progesterone and inhibition of IGFBP-1 production in the human ovary are mediated by signaling pathways that are independent of phosphatidylinositol 3-kinase, the enzyme whose activation is crucial for glucose transport. We now examined whether activation of MAPK is necessary to mediate insulin-induced or IGF-I-induced stimulation of progesterone or inhibition of IGFBP-1 production in human granulosa cells.

Human granulosa cells were obtained during in vitro fertilization. Cells (0.5–1 x 10⁵) were incubated for 24 h in the presence of 0, 10, 10², or 10³ ng/ml insulin or 0, 0.5, 1, 2.5, or 5 ng/ml IGF-I and in the presence or absence of 1 µM PD98059, a specific inhibitor of ERK1/2 MAPK. The progesterone concentration in the tissue culture medium was measured by RIA (Pantex, Santa Monica, CA), and the IGFBP-1 concentration was measured by immunoradiometric assay (DSL-7800, Diagnostic Systems Laboratories, Inc., Webster, TX). MAPK activity was assessed using the MAPK IP-Kinase assay kit (Upstate Biotechnology, Inc., Lake Placid, NY). ANOVA was used to compare mean values of progesterone or IGFBP-1 concentrations.

MAPK was stimulated by insulin up to 350% of the baseline value. Progesterone production in human granulosa cells was stimulated by insulin in a dose-related manner to 123% of the control value (P < 0.001), and IGFBP-1 production was inhibited to 25% of the baseline value (P < 0.001). Despite inhibiting MAPK activity by 99%, PD98059 (1 µM) did not interfere with insulin-induced stimulation of progesterone or inhibition of IGFBP-1 production.

MAPK was stimulated by IGF-I to 730% of the baseline value, with maximal stimulation achieved at 0.5 ng/ml IGF-I. Progesterone production in granulosa cells was stimulated by IGF-I to 130% of the control value (P < 0.001), whereas IGFBP-1 production was inhibited to 44% of the control value (P < 0.001). PD98059 (1 µM) inhibited IGF-I-induced MAPK activity by 94%. In the presence of 1 µM PD98059, IGF-I-induced stimulation of progesterone production was inhibited by 96% (P < 0.001). The inhibitory effect of IGF-I on IGFBP-1 production was reduced in the presence of 1 µM PD98059 by 45% at 5 ng/ml IGF-I and was completely abolished in the presence of 1 µM PD98059 at concentrations of IGF-I ranging from 0.5–2.5 ng/ml (P < 0.001). We conclude that, under conditions of our experiments, insulin-induced stimulation of progesterone or inhibition of IGFBP-1 production in human granulosa cells does not require MAPK activation, whereas similar effects of IGF-I are largely MAPK dependent.

This work was supported by The Gerald J. Friedman Foundation (to D.S.-Y. and L.P.), the Singer/Hellman Award at Beth Israel Medical Center (to L.P. and D.S.-Y.), Amersham Pharmacia Biotech (to D.S.-Y. and L.P.), and NIH Grant RO1-MH-60563 (to L.P.).

Abbreviations: FBS, Fetal bovine serum; IGFBP, IGF-binding protein; PI-3-kinase, phosphatidylinositol 3-kinase.

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