Differential Roles of MAPK-Erk 1 2 and MAPK-p 38

Differential Roles of MAPK-Erk 1/2 and MAPK-p 38 in Insulin or IGF-I Signaling Pathways for Progesterone Production in Human Ovarian Cells Grishma Parikh, Dimiter Avtanski, Miroslava Varadinova, Alice Park, Pauline Suwandhi, Aliza Leiser, Leonid Poretsky, Donna Seto-Young G. J. Friedman Diabetes Institute and Division of Endocrinology, Department of Medicine, Beth Israel Medical Center, New York Summary of article from Horm Metab Res 2011; 43: 386 -390

Background l l Insulin and IGF-I participate in the regulation of ovarian function and steroidogenesis Insulin can bind to and up regulate IGF-I receptor and activate PI-3 kinase independent of insulin signaling pathways (1)

Progesterone Synthesis in Human Granulosa-Lutein Cells and Thecal Cells l l l c. AMP-dependent activation of MAPK-erk 1/2 by forskolin/LH increases progesterone production and steroid acute regulatory protein (St. AR) expression But, in the presence of a potent MAPK-Erk 1/2 inhibitor PD 98059, LH induced progesterone production or St. AR expression is not affected. The requirement for MAPK-Erk 1/2 activation in regulation of progesterone production in the ovary is stimulus-specific (2 -4)

Objectives l l Study the role of MAPK in progesterone production in mixed ovarian cells Examine the effect of MAPK inhibitors, PD 98059 - specific inhibitor of MAPK-Erk 1/2 SB 203580 -specific inhibitor of MAPK-p 38 LY 294002 - specific inhibitor of PI-3 -kinase

Methods l Cell cultures: mixed ovarian cell culture contains granulosa, thecal and stromal cells and is responsive to stimulation by gonadotropins, insulin and IGF-I (5) l Cells were incubated in tissue culture medium with or without 10, 102, 103, or 104 ng/ml insulin or 1, 2. 5, 5, or 10 ng/ml IGF-1, with or without 25 -50 m. M PD 98059, with or without 2. 5 -5 m. M LY 294002 and with or without 10 -25 m. M SB 203580 l For the studies of IGF-induced progesterone production, cells were pre-incubated with 10 ng/ml of insulin for 2 hours

Statistical analysis l 2 -way analysis of variance (ANOVA) to compare mean values according to insulin or IGF-I concentrations in the presence or absence of PD 98059, LY 294002 or SB 203580 were calculated l Pairwise Bonferroni-adjusted contrasts were analyzed to determine statistical significance l Adjustments were made for initial inhibition or stimulation of progesterone production induced by the MAPK inhibitors in the absence of insulin or IGF-I

Effects of PD 98059 on Phospho-MAKPErk 1/2 Activity Fig. 1 l l A representative immuno-blot of the effect of 25 -50µM PD 98059 in the absence or in the presence of insulin (0 -103 ng/ml) (A) and IGF-1 (0 -5 ng/ml) (B) PD 98059 completely inhibited both insulin-induced and IGF-I-induced phospho-MAPK-Erk 1/2 activity

Effects of PD 98059 on Progesterone Production Fig. 2

Effects of PD 98059 on Progesterone Production Cont. . l PD 98059 alone stimulated progesterone production in a dose-dependent manner by up to 65% (p<0. 001) (C) l Insulin alone stimulated progesterone production in a dose-dependent manner by 50% (p<0. 001) (D) l In the presence of PD 98059, insulin-induced progesterone production was stimulated by 80%- 100% (p<0. 001) (D) l The effect of PD 98059 on insulin-induced stimulation of progesterone production was not significant when the adjustments were made for initial stimulation of progesterone production induced by PD 98059 alone (D) l IGF-I alone stimulated progesterone production by 60% (p<0. 001) (E). In the presence of PD 98059 (25 m. M-50 m. M), IGF-I had no additional stimulatory effect on progesterone production

The Effects of PD 98059 and LY 294002 on Progesterone Production Fig. 3 l l l MAPK-Erk 1/2 inhibitor PD 98059 (25 m. M) stimulated progesterone production by 13% (p<0. 001) PI-3 - Kinase inhibitor LY 294002 (2. 5 m. M or 5 m. M) stimulated progesterone production by 13. 6% and 18. 1% respectively PD 98059 (25 m. M) and LY 294002 (2. 5 m. M or 5 m. M) together inhibited progesterone production by 17% (p<0. 005) and 20% (p<0. 009), respectively

The Effect of SB 203580 (MAPK-p 38 inhibitor) on Phospho-MAKP-p 38 Activity Fig. 4 l At 0 -102 ng/ml insulin, 10µM and 25µM of SB 203580 inhibited phospho-MAPK-p 38 activity by 20% and 90 % respectively (A) l At 0 -10 ng/ml of IGF-I, 10µM and 25µM of SB 203580 inhibited phospho-MAPK-p 38 by 50 -80% (B)

The Effect of SB 203580 (MAPK-p 38 inhibitor) on Progesterone Production Fig. 5

The Effect of SB 203580 (MAPK-p 38 inhibitor) on Progesterone Production Cont… Fig 5 C l l l 25 m. M of SB 203580 inhibited progesterone production by 30%. Insulin alone stimulated progesterone production in a dose-dependent manner by 40%. 10 m. M and 25 m. M of SB 203580 completely abolished insulin-induced stimulation of progesterone production Fig 5 D l l l Both 10 m. M and 25 m. M SB 203580 alone inhibited progesterone production by 20% (p<0. 001) IGF-I alone stimulated progesterone production by 40% In the presence of SB 203850 (10 m. M-25 m. M), IGF-I induced stimulation of progesterone production was completely abolished

Discussion l l In insulin resistant hyperinsulinemic states the ovary can remain sensitive to insulin in part by activation of IGF-I and insulin signaling pathways unrelated to glucose transport (6) Activation of PI-3 -kinase is not necessary for the ovarian effects of insulin We have previously demonstrated that activation of MAPK (Erk 1/2) is not necessary for the effects of insulin in granulosa cells while IGF-I induced progesterone synthesis in these cells is MAPK-dependent (7) These findings provided initial evidence for the divergence of insulin signaling pathways and IGF-I signaling pathways for steroidogenesis in the human ovary

Discussion- Cont. . l l l activation of MAPK-Erk 1/2 is not necessary for the stimulatory effects of insulin on progesterone production IGF-I-induced progesterone synthesis is MAPK-Erk 1/2 dependent In contrast to MAPK-Erk 1/2, MAPK-p 38 is necessary for stimulation of progesterone production by both insulin and IGF-I

Discussion- Cont. . l l l LH-induced stimulation of progesterone synthesis in granulosa cells may be mediated by two signaling pathways: MAPK-Erk 1/2 or c. AMP-dependent protein -kinase A (PKA) pathway (2 -3) In these studies, LH-induced progesterone production and stimulation of St. AR m. RNA expression were preserved in the presence of specific inhibitor of MAPK-Erk 1/2 (2 -3) Thus, activation of progesterone production by PD 98059 alone, observed in our studies, may involve a MAPK-Erk 1/2 -independent signaling pathway

Discussion- Cont. . l l We confirmed findings of Lin et al (8) that 10 m. M of SB 203580 had no effect on progesterone production in human granulosa cells At higher concentration (25 m. M) SB 203580 independently inhibited progesterone production by 20 -30%

Conclusions l l l Insulin-induced progesterone production in human ovarian cells is dependent on the activation of MAPK -p 38, but not of MAPK-Erk 1/2 IGF-I induced progesterone production in human ovarian cells is both MAPK-Erk 1/2 and MAPK-p 38 dependent These data provide further evidence for the divergence of insulin and IGF-I signaling pathways in the human ovary

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Acknowledgements This work was supported in part by l Gerald J. and Dorothy Friedman New York Foundation for Medical Research, l Thanks to Scandinavia Foundation, l Empire Clinical Research Investigator Program of the New York State Department of Health, l The Chinese American Medical Society & Chinese American Independent Practice Association l Yen Family Foundation.