TY - JOUR
T1 - Identification of ERK and JNK as signaling mediators on protein kinase C activation in cultured granulosa cells
AU - Sriraman, Venkataraman
AU - Modi, Swati R.
AU - Bodenburg, Yvonne
AU - Denner, Larry A.
AU - Urban, Randall J.
N1 - Funding Information:
This research was supported by NIH grant R01 HD044566-01 (R.J.U. and L.A.D.). V.S. was supported in part by the NICHD, NIAID, and ORWH as a BIRCWH scholar (HD052023) and ASRM/Organon grant.
PY - 2008/11/6
Y1 - 2008/11/6
N2 - 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 2 kb 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 4 h. 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 15 min. 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.
AB - 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 2 kb 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 4 h. 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 15 min. 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.
KW - ERK
KW - Granulosa cells
KW - JNK
KW - Protein kinase C signaling
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U2 - 10.1016/j.mce.2008.07.011
DO - 10.1016/j.mce.2008.07.011
M3 - Article
C2 - 18694803
AN - SCOPUS:53649110719
SN - 0303-7207
VL - 294
SP - 52
EP - 60
JO - Molecular and Cellular Endocrinology
JF - Molecular and Cellular Endocrinology
IS - 1-2
ER -