In the ovary it has been demonstrated that PGF2α activates the phospholipase C (PLC)/diacylglycerol/protein kinase C pathway. However, little is known about the downstream signaling events that mediate subsequent cellular responses such as steroidogenesis. The present study was designed to examine the effect of PGF2α on activation of the mitogen-activated protein kinase (MAPK) signaling pathway and its physiological role in human granulosa-luteal cells (hGLCs). Human GLCs, obtained from women undergoing in vitro fertilization-embryo transfer, were treated with increasing concentrations of PGF2α (10 nmol/L to 10 μmol/L) for 5 min. For time-course experiments, hGLCs were treated with 1 μmol/L PGF2α for 1, 5, 10, or 20 min. Western blot analysis, using a monoclonal antibody that detected the phosphorylated forms of extracellular signal-regulated kinases 1 and 2 (p42mapk and p44mapk, respectively), demonstrated that PGF2α activated MAPK in hGLCs in a dose- and time-dependent manner. Treatment of the cells with neomycin (10 mmol/L; a PLC inhibitor), bisindolylmaleimide I (5 μmol/L; a PKC inhibitor), or PD98059 (50 μmol/L; a MEK inhibitor and a MAPK kinase inhibitor) significantly attenuated the PGF2α-induced activation of MAPK. In contrast, MAPK activation was not significantly affected by pertussis toxin (200 ng/mL, a Gi inhibitor) pretreatment. To determine the role of MAPK in steroidogenesis, hGLCs were treated with PGF2α, (1 μmol/L), hCG (1 IU/mL), or PGF2α, plus hCG in the presence or absence of PD98059. Progesterone levels in the culture medium were examined by RIA. Treatment of hGLCs with PGF2α significantly inhibited hCG-induced progesterone production. The presence of the MEK inhibitor, PD98059, reversed the inhibitory effect of PGF2α on hCG-induced progesterone production. To our knowledge, it is the first demonstration of PGF2α,-induced activation of the MAPK signaling pathway in the human ovary. These results indicated that PGF2α activated MAPK subsequent to PLC and PKC activation through pertussis toxin-insensitive G protein in hGLCs. Further, we demonstrated that PGF2α-induced MAPK activation is associated with modulation of progesterone production. These results support the idea that the MAPK signaling pathway is involved in mediating PGF2α, actions in the human ovary.
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