Fig. 10. Activin Enhances the GnRH-Induced Activation of p38MAPK, JNK, and p53.

Cells were stimulated with GnRH for the indicated times and doses. A, Induction of p21Cip1/Waf1 mRNA by 100 nM GnRH by microarray and Q-PCR. Black bars (■) indicate cells pretreated with 25 ng/ml activin for 24 h; open bars (□) are control untreated cells. Q-PCR results are mean and SEM of four independent experiments. Horizontal axis shows time of GnRH treatment; vertical axis shows p21 mRNA levels (arbitrary units). B, Induction of p21Cip1/Waf1 protein by Western blot. Experiment was repeated three times with similar results. C, Phosphorylation of p53 on Ser15 and Ser46 after GnRH stimulation in the presence of activin. Experiment was repeated twice with similar results. D, Phosphorylation of ERK and p38MAPK by GnRH in the presence or absence of activin. Cells were stimulated for 10 min with 0, 10, and 100 nM GnRH. Whole-cell extracts were blotted for phospho-ERK. Blot was stripped and reblotted for phospho-p38MAPK and then for p38MAPK protein as a loading control. Experiment was repeated four times with similar results. E, Stimulation of JNK by GnRH in the presence or absence of activin. Cells were stimulated for increasing times with 100 nM GnRH. Whole-cell extracts were blotted for phospho-JNK and then stripped and blotted for JNK. Experiment was repeated twice with similar results. F, Pharmacological blockade of p38MAPK prevents the phosphorylation of p53. Cell were treated with activin for 24 h and then with 500 nM PD169316 or vehicle for 30 min before being stimulated with GnRH for 30 or 60 min. Whole-cell extracts were blotted for phospho-p53(Ser15), and then stripped and blotted for p53. Experiment was repeated twice with similar results. AU, Arbitrary units.