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This Article Full Text Full Text (PDF) All Versions of this Article: 296/6/E1344    most recent 90929.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Huang, Q. Articles by Leung, P. C. K. PubMed PubMed Citation Articles by Huang, Q. Articles by Leung, P. C. K.

Effects of growth differentiation factor 9 on cell cycle regulators and ERK42/44 in human granulosa cell proliferation

¹Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada; and ²Zhejiang University School of Medicine, Hangzhou, China

Submitted 18 November 2008 ; accepted in final form 10 April 2009

GDF-9 stimulates granulosa cell proliferation and plays important roles during folliclogenesis. However, its molecular mechanisms are still far from clear, particularly its roles in human granulosa cells around the periovulatory stage. Therefore, we investigated the effects of GDF-9 on cell cycle distribution, regulatory molecules, and signaling pathways involved in human luteinized granulosa (hLG) cells in vitro. Primary cultures of hLG cells obtained from women undergoing IVF and treated with and without recombinant GDF-9 were evaluated with and without a specific inhibitor to activin receptor-like kinase 5 (ALK5; SB-431542), ERK42/44 (PD-098059), or Smad3 (SIS3). Cell proliferation, cell cycle distribution, mRNA expression, and protein expression of relevant cell cycle molecules were determined by [³H]thymidine incorporation, flow cytometry, quantitative PCR, and immunoblotting, respectively. GDF-9 stimulated [³H]thymidine incorporation, enhanced cell transition from G₀/G₁ to S and G₂/M phases (whereas both SB-431542 and PD-098059 attenuated these changes), increased mRNA and protein expression of cyclin D₁ and E, and decreased those of the cyclin-dependent kinase (CDK) inhibitors p15^INK4B and p16^INK4A. GDF-9 also activated Rb protein (a critical G₁ to S-phase regulator), ERK42/44, and Smad3. PD-098059 blocked Rb protein phorsphorylation and the increase in cyclin D₁ and E but not the decrease in p15^INK4B and p16^INK4A induced by GDF-9. In contrast, SIS3 reversed the decrease in p15^INK4B and p16^INK4A but not the increase in cyclin D₁ and E induced by GDF-9. GDF-9 stimulates hLG cell proliferation by stimulating cyclin D₁ and E and suppressing p15^INK4B and p16^INK4A via both Smad-dependent and Smad-independent pathways.

cyclin D₁; cyclin E; p15^INK4B; p16^INK4A; extracellular signal-regulated kinase 42/44