Forkhead Box O1 Is a Repressor of Basal and GnRH-Induced Fshb Transcription in Gonadotropes

Abstract

Synthesis of the gonadotropin β-subunits is tightly controlled by a complex network of hormonal signaling pathways that may be modulated by metabolic cues. Recently, we reported that insulin regulates FOXO1 phosphorylation and cellular localization in pituitary gonadotropes and that FOXO1 overexpression inhibits Lhb transcription. In the current study, we investigated whether FOXO1 modulates Fshb synthesis. Here, we demonstrate that FOXO1 represses basal and GnRH-induced Fshb transcription in LβT2 cells. In addition, we show that PI3K inhibition, which increases FOXO1 nuclear localization, results in decreased Fshb mRNA levels in murine primary pituitary cells. FOXO1 also decreases transcription from the human FSHB promoter, suggesting that FOXO1 regulation of FSHB transcription may be conserved between rodents and humans. Although the FOXO1 DNA-binding domain is necessary for suppression of Fshb, we do not observe direct binding of FOXO1 to the Fshb promoter, suggesting that FOXO1 exerts its effect through protein-protein interactions with transcription factors required for Fshb synthesis. FOXO1 suppression of basal Fshb transcription may involve PITX1 because PITX1 interacts with FOXO1, FOXO1 repression maps to the proximal Fshb promoter containing a PITX1-binding site, PITX1 induction of Fshb or a PITX1 binding element in CV-1 cells is decreased by FOXO1, and FOXO1 suppresses Pitx1 mRNA and protein levels. GnRH induction of an Fshb promoter containing a deletion at -50/-41 or -30/-21 is not repressed by FOXO1, suggesting that these two regions may be involved in FOXO1 suppression of GnRH-induced Fshb synthesis. In summary, our data demonstrate that FOXO1 can negatively regulate Fshb transcription and suggest that FOXO1 may relay metabolic hormonal signals to modulate gonadotropin production.

Homeodomain Proteins SIX3 and SIX6 Regulate Gonadotrope-specific Genes During Pituitary Development

“…Mouse Six6 and Six3 expression vectors (1), mouse FSHb (18) and GnRHR (19), human Cga (20), and rat LHb (21) luciferase reporter constructs, the HD-binding element (HDBE) multimer (22) and ATTA multimer (1) have been previously described. The Six6 eh1 mutant and the Ptx1 promoter element mutations were previously described or constructed by QuikChange Site-Directed Mutagenesis kit from Agilent Technologies (20).…”

GnRH Transactivates Human AMH Receptor Gene via Egr1 and FOXO1 in Gonadotrope Cells

“…As shown in Figure 5c, GnRHa treatment still increased the mutated h AMHR2 promoter to a similar extent as that observed with the wild-type promoter. This suggests that although FOXO1 decreases GnRH induction of h AMHR2 activity , it may not act through direct binding to the h AMHR2 promoter as already reported for murine Fshb and Lhb promoters [13, 14, 52]. …”

“…In this study, we also uncovered the functional importance of the transcription factor FOXO1 in the regulation of the h AMHR2 promoter. FOXO1 was recently detected in native gonadotrope cells as well as in the LβT2 cell line and identified as a negative regulator of FSHβ and LHβ gene expression [13-15, 52]. Here, we demonstrate using a constitutively active FOXO1 mutant that FOXO1 is a negative regulator of basal and GnRH-dependent h AMHR2 promoter activity.…”

“…Egr1 and FOXO1. FOXO1 was shown to act as a repressor of AMHR2 expression as previously reported for gonadotropin β-subunit genes [13-15, 52]. In murine granulosa cells, FOXO1 represses the expression of the LH/choriogonadotropin receptor gene [60], as well as of several genes involved in the steroidogenic biosynthesis pathways [61].…”

“…However, mutations of the identified FOXO binding site did not affect GnRH-stimulated promoter activity. Although this may indicate the use of alternative yet unidentified motifs within the promoter, several studies reported that the FOXO1 suppressive effect on Fshb and Lhb expression in LβT2 cells occurs without direct binding of FOXO1 to their respective promoters [13, 14, 52]. The authors of those studies postulated that FOXO1 acts through protein-protein interactions with other transcription factors/cofactors.…”