Induction of Cyclin D2 in Rat Granulosa Cells Requires FSH-dependent Relief from FOXO1 Repression Coupled with Positive Signals from Smad

Youngkyu Park,Evelyn T Maizels,Zachary J Feiger,Hena Alam,Carl A Peters,Teresa K Woodruff,Terry G Unterman,Eun Jig Lee,J Larry Jameson,Mary Hunzicker-Dunn

doi:10.1074/jbc.m409486200

Youngkyu Park, Evelyn T Maizels + Show 8 more

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https://doi.org/10.1074/jbc.m409486200

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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.

Highlights

Ovarian follicles produce hormones and growth factors that regulate the hypothalamic-pituitary axis, uterine re
We evaluated the protein expression of the orphan nuclear receptor steroidogenic factor-1 (SF-1) in granulosa cell (GC) treated for 24 h with follicle-stimulating hormone (FSH) and/or activin, based on its established role in the transcriptional activation of FSH target genes [29, 78, 79]
Results (Fig. 1B) show that while FSH promotes a modest increase in SF-1 expression, consistent with an earlier report [80], expression of SF-1 was readily detected in cells treated with FSH plus activin compared with FSH alone

Summary

The abbreviations used are

TGF␤, transforming growth factor ␤; ERK, extracellular signal-regulated kinase; FSH, follicle-stimulating hormone; SF-1, steroidogenic factor-1; PCNA, proliferating cell nuclear antigen; SCC, P450 side chain cleavage enzyme; Cdk, cyclin-dependent kinase; PI 3-kinase, phosphatidylinositol 3-kinase; RT-PCR, reverse transcriptase polymerase chain reaction; DN, dominant negative; EGF, epidermal growth factor; IGF1, insulin-like growth factor 1, LRH-1, liver receptor homolog-1; mTOR, mammalian target of rapamycin; GC, granulosa cell; ChIP, chromatin immunoprecipitation; WT, wild type; pfu, plaque-forming unit; X-gal, 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside; PBS, phosphate-buffered saline; FOXO, forkhead box-containing protein, O subfamily. While FSH is well known to activate the PI 3-kinase pathway leading to Akt activation [18, 19, 23, 50, 61] and FOXO1 phosphorylation [23, 50] as well as to activate mTOR [20], cell cycle progression in cultured rat GCs requires FSH plus activin or GDF-9 (8 –11). In vivo association of constitutively active FOXO1 with cyclin D2 in GCs is confirmed by chromatin immunoprecipitation (ChIP) assay Taken together, these results suggest that FSH plus activin signal to sustain Akt phosphorylation by a pathway that requires Smad2/3 phosphorylation, thereby relieving cyclin D2 from repression, and allowing the cell cycle to progress

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION