A novel mechanism of FSH regulation of DNA synthesis in the granulosa cells of hamster preantral follicles: involvement of a protein kinase C-mediated MAP kinase 3/1 self-activation loop

Abstract

The objective was to reveal whether a protein kinase C (PKC [all isozymes])-mediated self-sustaining MAPK3/1 (3/1 extracellular signal regulated kinase 2/1, also known as ERK2/1) activation loop was necessary for FSH- or epidermal growth factor (EGF)-induced DNA synthesis in the granulosa cells of intact preantral follicles. For this purpose, hamster preantral follicles were cultured with FSH or EGF in the presence of selective kinase inhibitors FSH or EGF phosphorylated RAF1, MAP2K1, and MAPK3/1. However, a relatively higher dose of EGF was necessary to sustain the MAPK3/1 activity, which was essential for cyclin-dependent kinase 4 (CDK4) activation and DNA synthesis. In intact preantral follicles, FSH or EGF stimulated DNA synthesis only in the granulosa cells. Sustained activation of MAPK3/1 beyond 3 h was independent of EGFR kinase activity but dependent on PKC activity, which appeared to form a self-sustaining MAPK3/1 activation loop by activating RAF1, MAP2K1, and PLA2G4 (phospholipase A2 [all cytosolic isozymes]). Inhibition of PKC activity as late as 4 h after the administration of FSH or EGF arrested DNA synthesis, which corresponded with attenuated phosphorylation of RAF1 and MAPK3/1, thus suggesting an essential role of PKC in MAPK3/1 activation. Collectively, these data present a novel self-sustaining mechanism comprised of MAPK3/1, PLA2G4, PKC, and RAF1 for CDK4 activation leading to DNA synthesis in granulosa cells. Either FSH or EGF can activate the loop to activate CDK4 and initiate DNA synthesis; however, consistent with our previous findings, FSH effect seems to be mediated by EGF, which initiates the event by stimulating EGFR kinase.

Fig. 1

Effect of 25 ng/ml ovine-FSH-20 or 50 ng/ml EGF on kinase and CDK4 activation, and DNA synthesis. (A) Follicles were cultured with FSH for indicated times. CCND2/CDK4 complex was immunoprecipitated from follicular homogenate and used for CDK4 assay. The top panel represents a phosphorimage of [³²P]RB1, and the bottom panel represents an immunoblot of RB1 protein added to each sample. (B) Immunoblots of phosphorylated signaling intermediaries in preantral follicles. Follicles were cultured for 6h with or without FSH or EGF. (C) Effect of an EGFR-kinase inhibitor on FSH stimulated follicular CDK4 activity and DNA synthesis. Follicles were cultured for 6h with FSH and [³H]thymidine, both of which were added 1h after the administration of 100 nM AG1478. The top panel represents a phosphorimage of [³²P]RB1, middle panel is an immunoblot of RB1 added to each sample and the bottom panel reflects [³H]thymidine incorporation. (D) Effect of FSH or EGF on granulosa cell and thecal DNA synthesis. Follicles were cultured with FSH or EGF for 6h, granulosa cells separated from the theca and the incorporation of [³H]thymidine in both cell types determined. (E) Effect of EGF on EGFR and MAPK3/1 phosphorylation in granulosa and theca cells. Follicles were cultured with FSH or EGF for 2h, granulosa cells separated from the theca and EGFR and MAPK3/1 phosphorylation in both cell types determined by immunoblotting. (F) Autoradiographic analysis of FSH- or EGF-induced EGFR autophosphorylation in granulosa cells. Follicles were cultured with AG1478 1h before the administration of FSH or EGF. Granulosa cells were isolated 1h later, and the crude membrane pellet mixed with [³²P]-γ-ATP. After electrophoretic separation of the EGFR, gels were fixed, dried and exposed overnight to an x-ray film. Each bar represents a mean ± SEM of three separate values. P > 0.05: bars with a same letter; P < 0.05: bars with a different letter.

Fig. 2

Effects of PKC inhibitor on FSH- or EGF stimulated follicular CDK4 activity, DNA synthesis and MAPK3/1 phosphorylation. (A) Follicles were cultured for 6h without or with 100 nM GFXI, and FSH, EGF or 20 nM PMA, and [³H]-thymidine. The top panel depicts a phosphorimage of [³²P]RB1 and immunoblot of RB1 protein. The bottom panel indicates DNA synthesis. Each bar represents a mean ± SEM of three separate values. P > 0.05: bars with a same letter; P < 0.05: bars with a different letter. (B) Immunoblots of dual phosphorylated and total MAPK3/1 in follicles cultured with EGF with or without GFXI. (C) Immunoblots of phosphorylated and total RAF1, MAP2K1 and MAPK3/1 in follicles cultured with 20 nM PMA for 6h.

Fig. 3

Effect of AACOCF3 or PD98059 on FSH- or EGF stimulated follicular DNA synthesis, PLA2G4 and PKC activity. (A) Follicles were cultured with 60 μM AACOCF3 for 1h before the administration of FSH or EGF, and [³H]thymidine. After 6h of culture, DNA synthesis was determined. (B) Follicles were cultured with 60 μM AACOCF3 or 10 μM PD98059 for 1h before the administration of FSH or EGF. After 6h culture, PLA2G4 or PKC activity was measured. AACOCF3 was used as a positive control for PLA2G4 inhibition. (C) Cell-free determination of the specificity of kinase inhibitors at the tested dose level. Follicles were cultured without or with 25 ng/ml FSH, homogenate was mixed with an appropriate concentration of respective inhibitors for 30 min on ice and used in enzyme reaction. Each bar represents a mean ± SEM of three separate values. P > 0.05: bars with a same letter; P < 0.05: bars with a different letter.

Fig. 4

Effects of suboptimal and optimal doses of EGF on follicular DNA synthesis, CDK4 activity or MAPK3/1 phosphorylation. (A-B) Follicles were cultured with indicated doses of EGF and 1 μCI/ml [³H]thymidine. (A) DNA synthesis and (B) CDK4 activity were measured. Each bar represents a mean ± SEM of three separate values. P > 0.05: bars with a same letter; P < 0.05: bars with a different letter. (C) Follicles were cultured with 10 ng/ml EGF for indicated times, and MAPK3/1 phosphorylation was determined by immunoblotting. (D) Follicles were cultured for indicated times with a low (10 ng/ml) or an optimal (50 ng/ml) dose of EGF. Levels of phosphorylated MAPK3/1 were determined by immunoblotting. (E) Granulosa cells were separated from thecal layers and exposed to a suboptimal (10 ng/ml) dose of EGF for indicated times. One group was exposed to 50 ng/ml EGF for 6h to compare the data with those presented in 4D. Phosphorylated MAPK3/1 and TUBB were detected by immunoblotting.

Fig. 5

Dependency of FSH or EGF stimulated CDK4 activation and DNA synthesis on EGFR kinase and PKC. (A) Follicles were cultured with 25 ng/ml FSH or 50 ng/ml EGF, and [³H]thymidine for 6h. AG1478 was added at indicated times after the beginning of the culture. CDK4 activity and DNA synthesis were determined. (B) Follicles were cultured with 50 ng/ml EGF for 6h. A mouse monoclonal anti EGFR IgG was added to the culture to a final concentration 5 μg/ml at 2h or 4h after the beginning of the culture, and phosphorylated MAPK3/1 detected by immunoblotting. (C) Follicles were cultured with 25 ng/ml FSH or 50 ng/ml EGF, and [³H]thymidine for 6h. GFXI was added at 4h after the beginning of the culture, and DNA synthesis examined. The data were compared with thymidine incorporation for 4h. (D) Follicles were cultured with 100 nM GFXI or 10 nM U0126 for 1h before the administration of 50 ng/ml EGF. For some groups, 100 nM AG1478 was added at 2h or 4h after beginning of the culture. After 6h, EGFR phosphorylation was examined by immunoblotting. Lysate of activated A431 cells was used as a positive control for phosphorylated EGFR. Each bar represents a mean ± SEM of three separate values. P > 0.05: bars with a same letter; P < 0.05: bars with a different letter.

Fig. 6

Necessity of PKC to sustain MAPK3/1 phosphorylation. (A) Follicles were cultured with 50 ng/ml EGF for 6h, and 100 nM AG1478 was added at 2h or 4h after beginning of the culture. Another group of follicles received 100 nM GFXI along with AG1478 at 4h. Levels of phosphorylated RAF1 and MAPK3/1 were determined. (B) Quantitative values of the signal intensities presented in A. (C) Follicles were cultured with 50 ng/ml EGF for 6h, and 100 nM AG1478 or 10 nM U0126 was added at 2h or 4h after the beginning of the culture. Another group of follicles received 100 nM GFXI at 4h. PKC activity in follicular homogenate was determined. Each bar represents a mean ± SEM of three separate values. P > 0.05: bars with a same letter; P < 0.05: bars with a different letter.

Fig. 7

A model depicting the establishment of a self-sustaining MAPK3/1 activation loop for CDK4 activation and DNA synthesis in granulosa cells of hamster preantral follicles. FSH, likely via EGF, activates EGFR kinase that phosphorylates MAPK3/1 by sequential activation of RAF1 and MAP2K1. Active MAPK3/1 stimulates PKC via PLA2G4 and sets the loop in motion. After 2h of exposure to FSH or EGF, the activation loop becomes independent of the receptor kinase and sustains MAPK3/1 activity for at least 6h resulting in CDK4 activation and DNA synthesis.