PARENT SESSION
PLATFORM SESSION 21. GENE REGULATION AND FUNCTION II

Tuesday, August 3, 2004
4:30 PM–6:30 PM
Buchanan A100
Chair: Ruth Keri Co-Chair: Chuck Greenfeld

(602) FSH AND ACTIVIN STIMULATES PROLIFERATION OF GRANULOSA CELLS BY INACTIVATING FOXO1 AND ACTIVATING SMAD ACTIVITY.

Park, Youngkyu ¹, Maizels, Evelyn¹, Lee, Eun Jig², Feiger, Zackery¹, Alam, Hena¹, Peters, Carl¹, Woodruff, Teressa³, Jameson, Larry², Hunzicker-Dunn, Mary¹, ¹ Northwestern University Medical School, Chicago, IL² Northwestern University Medical School, Chicago, IL³ Northwestern University, Evanston, IL

ABSTRACT- It has been reported that granulosa cells (GC) from rat ovarian follicles undergo differentiation and proliferation in response to combined treatment with FSH and activin in vitro. In agreement with other groups, we have found that cyclin D2 (an index of proliferation) was increased in response to cotreatment with FSH and activin within two hours. Activin synergized with FSH to increase gene expression of several markers of GC diffferentiation, including inhibin alpha, aromatase, cytochrome P450scc, and type 1 gonadal 3-beta-hydroxysteroid dehydrogenase. In this report we investigate the cross talk between FSH- and activin-signaling pathways which might contribute to the synergistic effect of these reagents on GC differentiation and proliferation. In addition to the FSH-stimulated PKA pathway, FSH signals in GC to activate the phosphatidylinositol-3 kinase(PI3K)/Akt pathway. Akt inactivates the forkhead transcription factor FOXO1 by phosphorylation and nuclear exclusion. FSH/activin-stimulated induction of cyclin D2 was blocked by the PI3K inhibitor LY292004, indicating that PI3K signaling is required for the mitogenic response to the cotreatment regimen. Synergistic induction of differentiation marker genes by cotreatment with FSH/activin was reduced by treatment with LY292004, indicating that PI3K signaling is required for differentiation responses as well. Activin, a member of the TGF beta superfamily, stimulates the phosphorylation of Smad2/3 proteins to activate target genes. Immunofluorescence showed that cotreatment of GC with FSH and activin elicited the export of FOXO1 protein from the nucleus and the transport of Smad 3 protein into the nucleus by 1 h. We constructed adenoviruses containing phosphorylation-defective (PD) mutant FOXO1 sequences (unable to respond to PI3K/Akt regulation), as well as a dominant negative (DN) mutant of smad3 (unable to respond to activin) to assess the respective roles of FOXO1 and Smad3 transcription factors in GC. Infection of adeno-FOXO1 PD mutant or adeno-smad3 DN mutant each inhibited the induction of cyclin D2 as well as differentation markers respectively. These results suggest that FOXO1 and Smad are each important regulators of GC differentiation and proliferation. Supported by HD21921.

KEY WORDS: smad, activin, foxo1, fsh