Effects of growth differentiation factor 9 on cell cycle regulators and ERK42/44 in human granulosa cell proliferation.
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 [(3)H]thymidine incorporation, flow cytometry, quantitative PCR, and immunoblotting, respectively. GDF-9 stimulated [(3)H]thymidine incorporation, enhanced cell transition from G(0)/G(1) to S and G(2)/M phases (whereas both SB-431542 and PD-098059 attenuated these changes), increased mRNA and protein expression of cyclin D(1) 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 G(1) to S-phase regulator), ERK42/44, and Smad3. PD-098059 blocked Rb protein phorsphorylation and the increase in cyclin D(1) 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 D(1) and E induced by GDF-9. GDF-9 stimulates hLG cell proliferation by stimulating cyclin D(1) and E and suppressing p15(INK4B) and p16(INK4A) via both Smad-dependent and Smad-independent pathways.
Huang
Qing
Q
Dept. of Obstetrics and Gynecology, Univ. of British Columbia, Rm. 2H-30, 4500 Oak St., Vancouver, BC, Canada V6H 3V5.
Cheung
Anthony P
AP
Zhang
Yu
Y
Huang
He-Feng
HF
Auersperg
Nelly
N
Leung
Peter C K
PC
D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism; D050762:Cyclin-Dependent Kinase Inhibitor p15 / Q000235:genetics / Q000378:metabolism; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics / Q000378:metabolism; D005260:Female; D005419:Flavonoids / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D055429:Growth Differentiation Factor 9 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
C530104:CCND1 protein, human; D019927:Cyclin E; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D005419:Flavonoids; C525897:GDF9 protein, human; D055429:Growth Differentiation Factor 9; D011994:Recombinant Proteins; D019938:Cyclin D1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one