Role of mitogen-activated protein kinase in prostaglandin F-2 alpha actionin human granulosa-luteal cells

In the ovary it has been demonstrated that PGF(2 alpha) activates the phosp holipase C (PLC)/diacylglycerol/protein kinase C pathway. However, little i s known about the downstream signaling events that mediate subsequent cellu lar responses such as steroidogenesis. The present study was designed to ex amine the effect of PGF(2 alpha) on activation of the mitogen-activated pro tein kinase (MAPK) signaling pathway and its physiological role in human gr anulosa-luteal cells (hGLCs). Human GLCs, obtained from women undergoing in vitro fertilization-embryo transfer, were treated with increasing concentr ations of PGF(2 alpha) (10 nmol/L to 10 mu mol/L) for 5 min. For time-cours e experiments, hGLCs were treated with 1 mu mol/L PGF(2 alpha) for 1, 5, 10 , or 20 min. Western blot analysis, using a monoclonal antibody that detect ed the phosphorylated forms of extracellular signal-regulated kinases 1 and 2 (p42(mapk) and p44(mapk), respectively), demonstrated that PGF(2 alpha) 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 mu mol/L; a PKC inhibitor), or PD98059 (50 mu mol/L; a MEK inhibitor an d a MAPK kinase inhibitor) significantly attenuated the PGF(2 alpha)-induce d activation of MAPK. In contrast, MAPK activation was not significantly af fected by pertussis toxin (200 ng/mL; a G(i) inhibitor) pretreatment. To de termine the role of MAPK in steroidogenesis, hGLCs were treated with PGF(2 alpha) (1 mu mol/L), hCG (1 IU/mL), or PGF(2 alpha) plus hCG in the presenc e or absence of PD98059. Progesterone levels in the culture medium were exa mined by RIA. Treatment of hGLCs with PGF(2 alpha) significantly inhibited hCG-induced progesterone production. The presence of the MEK inhibitor, PD9 8059, reversed the inhibitory effect of PGF(2 alpha) on hCG-induced progest erone production. To our knowledge, it is the first demonstration of PGF(2 alpha)-induced activation of the MAPK signaling pathway in the human ovary. These results indicated that PGF(2 alpha) activated MAPK subsequent to PLC and PKC activation through pertussis toxin-insensitive G protein in hGLCs. Further, we demonstrated that PGF(2 alpha)-induced MAPK activation is asso ciated with modulation of progesterone production. These results support th e idea that the MAPK signaling pathway is involved in mediating PGF(2 alpha ) actions in the human ovary.