ATP activates nuclear translocation of mitogen-activated protein kinases in human granulosa-luteal cells

Abstract

To investigate the specific role of adenosine triphosphate (ATP) in the subsequent MAPK-induced signaling cascade in human granulosa-luteal cells (hGLCs). We have previously demonstrated an effect of extracellular adenosine triphosphate (ATP) on ERKs and gonadotropin-induced progesterone secretion, implicating the significance of ATP in the regulation of ovarian function. The present study was designed to examine the effect of ATP on the activation of the MAPK signaling pathway, including nuclear translocation in hGLCs. Human GLCs were collected from patients undergoing in vitro fertilization programs. Human GLCs were cultured in FBS-supplemented DMEM for 3 days prior to our studies. Cells were starved in serum-free medium for 4h prior to subsequent treatments. To examine the dose-response relationship, hGLCs were treated with increasing concentrations of ATP (100nM, 1μ, 10μ, 100μ) for 5 min. For time-course experiments, hGLCs were treated with 10μM ATP for 1, 5, 10, 20 min. To determine the translocation of MAPKs, hGLCs were fixed 5 min after 10μM ATP exposure. Western blot analysis was performed using antibodies against the phosphorylated forms of ERK1 and ERK2 (P-MAPK, p42mapk and p44mapk, respectively), p42/p44 MAPK (total MAPK, T- MAPK), phospho JNK and activated P38. To examine the translocation of activated ERKs, FITC-conjugated secondary antibody was used to detect the distribution of total and phosphorylated ERKs. Western blot analysis, which detected the total and phosphorylated forms of ERK1 and ERK2, demonstrated that exogenous ATP evoked ERKs in a dose- and time-dependent manner. In contrast, p38 and JNK were not significantly activated following ATP treatment. Immunofluorescent staining revealed that phosphorylated ERKs were translocated from cytoplasm into nucleus subsequent to 10μ ATP treatment. To our knowledge, this is the first demonstration of the ATP-induced nuclear translocation of MAPKs in the human ovary. These results suggest that the MAPKs signaling pathway plays a role in mediating ATP actions in the human ovary.