Lhcgr Expression in Granulosa Cells: Roles for PKA-Phosphorylated β-Catenin, TCF3, and FOXO1

Abstract

Ovarian follicles lacking FSH or FSH receptors fail to progress to a preovulatory stage, resulting in infertility. One hallmark of the preovulatory follicle is the presence of luteinizing hormone/choriogonadotropin receptors (LHCGR) on granulosa cells (GCs). However, the mechanisms by which FSH induces Lhcgr gene expression are poorly understood. Our results show that protein kinase A (PKA) and phosphoinositide 3-kinase (PI3K)/AKT pathways are required for FSH to activate both the murine Lhcgr-luciferase reporter and expression of Lhcgr mRNA in rat GCs. Based on results showing that an adenovirus (Ad) expressing a steroidogenic factor 1 (SF1) mutant that cannot bind β-catenin abolished FSH-induced Lhcgr mRNA, we evaluated the role of β-catenin in the regulation of Lhcgr gene expression. FSH promoted the PKA-dependent, PI3K-independent phosphorylation of β-catenin on Ser552 and Ser665. FSH activated the β-catenin/T-cell factor (TCF) artificial promoter-reporter TOPFlash via a PKA-dependent, PI3K-independent pathway, and dominant-negative (DN) TCF abolished FSH-activated Lhcgr-luciferase reporter and induction of Lhcgr mRNA. Microarray analysis of GCs treated with Ad-DN-TCF and FSH identified the Lhcgr as the most down-regulated gene. Chromatin immunoprecipitation results placed β-catenin phosphorylated on Ser552 and Ser675 and SF1 on the Lhcgr promoter in FSH-treated GCs; TCF3 was constitutively associated with the Lhcgr promoter. Transduction with an Ad-phospho-β-catenin mutant (Ser552/665/Asp) enhanced Lhcgr mRNA expression in FSH-treated cells greater than 3-fold. Finally, we identified a recognized PI3K/AKT target, forkhead box O1, as a negative regulator of Lhcgr mRNA expression. These results provide new understanding of the complex regulation of Lhcgr gene expression in GCs.

Extracellular Signal-regulated Kinase (ERK)-dependent Phosphorylation of Y-Box-binding Protein 1 (YB-1) Enhances Gene Expression in Granulosa Cells in Response to Follicle-stimulating Hormone (FSH)

“…CREB, SF-1(Nr5a1)/LRH-1(Nr5a2), and GATA-4/6 enhance expression of both Cyp19a1 and Inha (46,(75)(76)(77), and FOXO1 (15) and T-cell factor 3 (9) repress expression of both genes. Many transcriptional proteins that regulate expression of the Lhcgr (9,78,79) and Cyp11a1 (76,80,81) have been elucidated, although the mechanisms by which FSH enhances Egfr and Pappa expression have not been reported. However, a role for YB-1 and indeed for the ERK signaling pathway in the transcriptional activation of FSH target genes is novel.…”

“…However, the mechanisms by which these signaling pathways regulate gene expression are less well understood. One of the more important pathways activated by FSH is the PI3K/ AKT pathway, based on evidence that this pathway is necessary for GC differentiation (9,15,17,18 …”

“…PKA then either directly phosphorylates proteins that regulate transcription or indirectly activates signaling cascades whose targets regulate primarily transcription and translation. Direct PKA targets in GCs include cAMP-response element binding protein (CREB) (Ser 133 ) (7), histone H3 (Ser 10 ) (8), and ␤-catenin (Ser 552 and Ser 675 ) (9). Upon phosphorylation, these direct PKA target proteins participate in the regulation of gene expression though interactions with DNA and additional transcription factors.…”

“…However, the mechanisms by which these signaling pathways regulate gene expression are less well understood. One of the more important pathways activated by FSH is the PI3K/ AKT pathway, based on evidence that this pathway is necessary for GC differentiation (9,15,17,18). Yet the only transcriptional factor regulated by this pathway that has been shown to modulate gene expression in GCs is FOXO1 (15,18).…”

“…Adenoviral Transduction of GCs-GCs were plated on fibronectin-coated 6-well plates at a density of 1.5 ϫ 10 6 cells/ well in DMEM/F12 ϩ P/S ϩ E2. After allowing the cells to adhere to the plate for ϳ4 h, the indicated adenoviruses were added to the media (9). After an overnight treatment, cells were allowed ϳ1 h recovery and then treated as indicated.…”