Activation of protein kinase Cζ mediates luteinizing hormone- or forskolin-induced NGFI-B expression in preovulatory granulosa cells of rat ovary

We have previously demonstrated that luteinizing hormone (LH) induces a rapid and transient expression of NGFI-B in the ovary. In this report, we investigated the signaling pathway for LH- and forskolin-induced NGFI-B expression in cultured rat granulosa cells of preovulatory follicles. LH- or forskolin-induced NGFI-B expression was suppressed by high dose of protein kinase C (PKC) inhibitor RO 31-8220 (10 μM), but not by low doses RO 31-8220 (0.1–1.0 μM) or adenylate cyclase inhibitor MDL-12,300A, implicating the involvement of atypical PKCs. Kinase assay revealed that LH treatment of granulosa cells resulted in a rapid stimulation of atypical PKCζ activity. Interestingly, like LH, forskolin was also able to activate PKCζ. Treatment with the cell-permeable PKCζ-specific inhibitor pseudosubstrate peptide inhibited LH-or forskolin-induced NGFI-B expression, indicating the essential role of PKCζ. Consistent with this promise, in granulosa cells depleted of diacylglycerol sensitive PKCs by prolonged treatment with tetradecanoylphobol-13-acetate, LH or forskolin could still induce NGFI-B expression, and RO 31-8220 or the PKCζ pseudosubstrate peptide inhibited LH- or forskolin-induced NGFI-B expression. Furthermore, overexpression of dominant-negative PKCζ in primary granulosa cells using a replication-defective adenovirus vector resulted in the suppression of LH- or forskolin-induced NGFI-B expression. Our findings demonstrate that PKCζ, which is activated by LH or forskolin, contributes to the induction of NGFI-B in granulosa cells of preovulatory follicles.