title: Induction of cyclin D2 in rat granulosa cells requires FSH-dependent relief from FOXO1 repression coupled with positive signals from Smad.
abstract: Ovarian follicles undergo exponential growth in response to follicle-stimulating hormone (FSH), largely as a result of the proliferation of granulosa cells (GCs). In vitro under serum-free conditions, rat GCs differentiate in response to FSH but do not proliferate unless activin is also present. In the presence of FSH plus activin, GCs exhibit enhanced expression of cyclin D2 as well as inhibin-alpha, aromatase, steroidogenic factor-1 (SF-1), cholesterol side chain (SCC), and epiregulin. In this report we sought to identify the signaling pathways by which FSH and activin promote GC proliferation and differentiation. Our results show that these responses are associated with prolonged Akt phosphorylation relative to time-matched controls and are dependent on phosphatidylinositol 3-kinase (PI 3-kinase) and Smad2/3 signaling, based on the ability of the PI 3-kinase inhibitor LY294002 or infection with adenoviral dominant negative Smad3 (DN-Smad3) mutant to attenuate induction of cyclin D2, inhibin-alpha, aromatase, SCC, SF-1, and epiregulin. The DN-Smad3 mutant also abolished prolonged Akt phosphorylation stimulated by FSH plus activin 24 h post-treatment. Infection with the adenoviral constitutively active forkhead box-containing protein, O subfamily (FOXO)1 mutant suppressed induction of cyclin D2, aromatase, inhibin-alpha, SF-1, and epiregulin. Transient transfections of GCs with constitutively active FOXO1 mutant also suppressed cyclin D2, inhibin-alpha, and epiregulin promoter-reporter activities. Chromatin immunoprecipitation results demonstrate in vivo the association of FOXO1 with the cyclin D2 promoter in untreated GCs and release of FOXO1 from the cyclin D2 promoter upon addition of FSH plus activin. These results suggest that proliferation and differentiation of GCs in response to FSH plus activin requires both removal of FOXO1-dependent repression and positive signaling from Smad2/3.
authors.affiliation: Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA.
authors.forename: Youngkyu
authors.initials: Y
authors.lastname: Park
authors.forename: Evelyn T
authors.initials: ET
authors.lastname: Maizels
authors.forename: Zachary J
authors.initials: ZJ
authors.lastname: Feiger
authors.forename: Hena
authors.initials: H
authors.lastname: Alam
authors.forename: Carl A
authors.initials: CA
authors.lastname: Peters
authors.forename: Teresa K
authors.initials: TK
authors.lastname: Woodruff
authors.forename: Terry G
authors.initials: TG
authors.lastname: Unterman
authors.forename: Eun Jig
authors.initials: EJ
authors.lastname: Lee
authors.forename: J Larry
authors.initials: JL
authors.lastname: Jameson
authors.forename: Mary
authors.initials: M
authors.lastname: Hunzicker-Dunn
mesh_terms: D028341:Activins / Q000378:metabolism; D000818:Animals; D056742:Cyclin D2; D016213:Cyclins / Q000235:genetics*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000235:genetics; D066260:Epiregulin; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D051858:Forkhead Transcription Factors; D006107:Granulosa Cells / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D028322:Inhibin-beta Subunits / Q000378:metabolism; D007265:Inhibins / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D051785:Smad Proteins; D054339:Steroidogenic Factor 1; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D000071161:Forkhead Box Protein O1
chemical_list: D028341:Activins; D056742:Cyclin D2; D016213:Cyclins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D004815:Epidermal Growth Factor; D066260:Epiregulin; D005640:Follicle Stimulating Hormone; D051858:Forkhead Transcription Factors; D028322:Inhibin-beta Subunits; D007265:Inhibins; D009419:Nerve Tissue Proteins; D019869:Phosphatidylinositol 3-Kinases; D011994:Recombinant Proteins; D051785:Smad Proteins; D054339:Steroidogenic Factor 1; D015534:Trans-Activators; D014157:Transcription Factors; C486197:Ccnd2 protein, rat; C587880:EREG protein, human; C587882:Ereg protein, rat; C515057:NR5A1 protein, human; C422405:activin A; C094377:inhibin-alpha subunit; C515060:steroidogenic factor 1, rat; C074639:Foxo1 protein, rat; D000071161:Forkhead Box Protein O1