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.

Aromatase expression in the ovary: Hormonal and molecular regulation

“…FSH stimulation of Akt seems to relieve aromatase from a repressive effect of forkhead box O1 (FOXO1) since a constitutively active FOXO1 protein prevents aromatase stimulation by FSH and activin [58]. Consistent with this hypothesis, both FSH and IGF-I, act posttranslationally to phosphorylate FOXO1 [59], resulting in cytoplasmic localization and, presumably, loss of FOXO1 repression activity.…”

“…Of these pathways, PI3K, which activates protein kinase B (PKB or viral proto-oncogene 1; Akt), also participates in the induction of aromatase expression by FSH [57,58]. For instance, aromatase stimulation by FSH is amplified by the expression of constitutively activated Akt [57], whereas treatment with an inhibitor of the PI3K [58] or overexpression of dominant negative Akt [57] prevents aromatase up-regulation.…”

“…Using a granulosa cell line, Parakh et al demonstrated for the first time that SF-1 binds to the endogenous ovarian-specific aromatase promoter and that this finding is stimulated by cAMP [122]. Moreover, overexpression of constitutively active mutant FOXO1, which prevents aromatase stimulation by FSH and activin, also decreases SF-1 expression [58]. Levels of SF-1 protein do not exhibit major changes during follicular growth or in response to low levels of FSH in culture but decrease rapidly after the LH surge along with the downregulation of aromatase [115], further supporting the idea of an important role for SF-1 in the regulation of aromatase expression.…”

Relative expression of genes encoding SMAD signal transduction factors in human granulosa cells is correlated with oocyte quality

“…Thus, understanding the mechanisms and pathways underlying fertility and fecundity remains of crucial importance. As the SMAD signal transduction proteins are known to mediate signaling by members of the TGF-β superfamily, which is involved in the control of normal folliculogenesis and ovulation [7,8], and they associate with members of the Forkhead family of transcription factors, the FOXO class [34], thought to play important roles in oocyte maturation, ovulation, and possibly luteinization [34][35][36][37][38][39], with expression in human granulosa cells [40], it is possible that SMAD expression levels may also affect ovarian folliculogenesis and fertility. Female Smad3 −/− mice exhibit impaired folliculogenesis and reduced fertility [28,30] and Smad4 ovarian-specific knockout mice exhibit multiple defects in folliculogenesis and decreased fertility over time [27].…”

La signalisation FSH a-t-elle un sexe ?

“…De plus, chez toutes les espèces de mammifères, la FSH inhibe la synthèse des protéines de liaison des IGF et stimule leur protéolyse, augmentant ainsi la biodisponibilité de l'IGF1 pour les cellules de la granulosa au cours du développement folliculaire terminal précédant l'ovulation [22]. De façon intéressante, ces deux facteurs activent des mécanismes de signalisation communs, touchant principalement la voie PI-3 kinase/Akt impliquée dans la survie et la prolifération des cellules de la granulosa et induisent, par cette voie, la phosphorylation du facteur de transcription Foxo-1, permettant sa redistribution du noyau vers le cytoplasme [23]. Pour permettre la différenciation du follicule pré-ovulatoire, la FSH lève l'inhibition transcriptionnelle exercée par Foxo-1 sur les gènes P450 aromatase, épiréguline ou inhibine a, vraisemblablement en activant de façon autonome Sgk (serum/glucocorticoid-regulated kinase) et PKB [24].…”