Identification of ERK and JNK as signaling mediators on protein kinase C activation in cultured granulosa cells

V Sriraman, SR Modi, Y Bodenburg, LA Denner… - Molecular and cellular …, 2008 - Elsevier
V Sriraman, SR Modi, Y Bodenburg, LA Denner, RJ Urban
Molecular and cellular endocrinology, 2008Elsevier
PKC signaling is critical for follicular development and the induction of ovulatory genes
including Pgr, Prkg2, and Cyp11a1 (SCC). We investigated PKC signaling mechanisms in
the JC-410 porcine granulosa cell line stably expressing an SCC-luciferase reporter gene
containing 2kb of the porcine SCC promoter. Addition of phorbol 12-myristate 13-acetate
(PMA), which activates protein kinase C, induced the promoter∼ 6-fold over the basal levels
in 4h. This effect was predominantly mediated by the PKC β and δ isoforms. PMA-mediated …
PKC signaling is critical for follicular development and the induction of ovulatory genes including Pgr, Prkg2, and Cyp11a1 (SCC). We investigated PKC signaling mechanisms in the JC-410 porcine granulosa cell line stably expressing an SCC-luciferase reporter gene containing 2kb of the porcine SCC promoter. Addition of phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C, induced the promoter ∼6-fold over the basal levels in 4h. This effect was predominantly mediated by the PKC β and δ isoforms. PMA-mediated induction of the SCC promoter was sensitive to inhibition of ERK1/2 or JNK. Inhibition of p38 MAP kinase or Src tyrosine kinase did not alter the PMA-mediated inducibility of the promoter. SCC promoter induction in response to PMA treatment required basal EGF-receptor activity, but did not involve ectodomain shedding. Western blot analyses using phospho-specific antibodies showed that PMA treatment of JC-410 cells induced phosphorylation of MEK1/2, ERK1/2, and its downstream target p90 RSK at 15min. We also documented the rapid phosphorylation of JNK1/2 in response to PMA treatment. Phosphorylation of ERK and JNK was robust and sustained in contrast to activation of PKA and EGF-receptor signaling in these cells. Pretreatment of JC-410 granulosa cells with IGF-1 had a synergistic effect on PMA-mediated induction of the SCC promoter. We demonstrated the importance of PMA activation of ERK signaling and the synergism with IGF-1 by showing similar responses for Prkg2 expression in primary granulosa cells. In conclusion, our studies demonstrated PMA activation of ERK and JNK signaling which is relevant in the regulation of gene expression during follicular development, ovulation, and luteinization.
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