Role of mitogen-activated protein kinase in prostaglandin F_2α action in human granulosa-luteal cells

In the ovary it has been demonstrated that PGF_2α 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 PGF_2α 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 PGF_2α (10 nmol/L to 10 μmol/L) for 5 min. For time-course experiments, hGLCs were treated with 1 μmol/L PGF_2α 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 (p42^mapk and p44^mapk, respectively), demonstrated that PGF_2α 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 PGF_2α-induced activation of MAPK. In contrast, MAPK activation was not significantly affected by pertussis toxin (200 ng/mL, a G_i inhibitor) pretreatment. To determine the role of MAPK in steroidogenesis, hGLCs were treated with PGF_2α, (1 μmol/L), hCG (1 IU/mL), or PGF_2α, plus hCG in the presence or absence of PD98059. Progesterone levels in the culture medium were examined by RIA. Treatment of hGLCs with PGF_2α significantly inhibited hCG-induced progesterone production. The presence of the MEK inhibitor, PD98059, reversed the inhibitory effect of PGF_2α on hCG-induced progesterone production. To our knowledge, it is the first demonstration of PGF_2α,-induced activation of the MAPK signaling pathway in the human ovary. These results indicated that PGF_2α activated MAPK subsequent to PLC and PKC activation through pertussis toxin-insensitive G protein in hGLCs. Further, we demonstrated that PGF_2α-induced MAPK activation is associated with modulation of progesterone production. These results support the idea that the MAPK signaling pathway is involved in mediating PGF_2α, actions in the human ovary.