title: 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.
abstract: 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 10(5)) were incubated for 24 h in the presence of 0, 10, 10(2), or 10(3) ng/ml insulin or 0, 0.5, 1, 2.5, or 5 ng/ml IGF-I and in the presence or absence of 1 micro M 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 micro M) 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 micro M) inhibited IGF-I-induced MAPK activity by 94%. In the presence of 1 micro M 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 micro M PD98059 by 45% at 5 ng/ml IGF-I and was completely abolished in the presence of 1 micro M 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.
authors.affiliation: Division of Endocrinology, Beth Israel Medical Center and Albert Einstein College of Medicine, New York, New York 10003, USA.
authors.forename: Donna
authors.initials: D
authors.lastname: Seto-Young
authors.forename: Jacek
authors.initials: J
authors.lastname: Zajac
authors.forename: Hung-Ching
authors.initials: HC
authors.lastname: Liu
authors.forename: Zev
authors.initials: Z
authors.lastname: Rosenwaks
authors.forename: Leonid
authors.initials: L
authors.lastname: Poretsky
mesh_terms: D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005419:Flavonoids / Q000494:pharmacology; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000096:biosynthesis*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011374:Progesterone / Q000096:biosynthesis*
chemical_list: D004791:Enzyme Inhibitors; D005419:Flavonoids; D007004:Hypoglycemic Agents; D007328:Insulin; D018970:Insulin-Like Growth Factor Binding Protein 1; D011374:Progesterone; D007334:Insulin-Like Growth Factor I; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one