Transcriptional and Post-Translational Regulation of Cyclin D2 in FSH-Induced Rat Granulosa Cell Proliferation In Vitro.

Abstract

Cyclin D2 plays an important role in the induction of early-to-mid G1 phase transition by mitogen and is believed to be essential for granulosa cell proliferation during folliculogenesis. However, mechanism of regulation of granulosa cell cyclin D2 expression by FSH is poorly understood. The objective of the present studies is to elucidate the regulatory mechanism of FSH on cyclin D2 expression in granulosa cell proliferation in vitro. We hypothesize that transcriptional and post-translational regulation of cyclin D2 are important in FSH-induced granulosa cell proliferation. Granulosa cells from diethylstilbestrol (DES, 1 mg/rat/day, for 3 days)-treated immature rats were cultured with FSH (0 – 100 ng/ml) in the absence or presence of transcriptional inhibitor (actinomycin D, 20 μg/ml) or translational inhibitor (cycloheximide 5 μg/ml), and cyclin D2 and cell proliferation will be assessed by qPCR & Western blot as well as flow cytometry & alamar blue assay, respectively. FSH significantly increased granulosa cell proliferation in 24h, a response preceded by increased cyclin D2 mRNA and protein as early as 2h and subsequent by DNA synthesis. The FSH-induced cyclin D2 expression declined rapidly and reached basal level at 12h of culture. The effects of FSH could be mimicked by forskolin (10 μM), and suppressed by the protein kinase A inhibitors H89 and PKI, and by gene silencing (siRNA). Moreover, FSH-enhanced cyclin D2 mRNA abundance was blocked by actinomycin D, indicating a regulation at the transcriptional level. The quick decline in cyclin D2 content appeared to be a result of accelerated proteasomal degradation, since the response was attenuated by proteasome inhibitors (MG132, lactacystin and epoxomicin). In summary, the present studies indicate that FSH-induced granulosa cell proliferation involves transcriptional and post-translational regulation of cyclin D2. (Supported by grants from CIHR and NSFC (BKT and GLX); YYH is a recipient of a China Scholarship Council Doctoral Scholarship).