Abstract
In the present study, we investigate whether the FOXO1 transcription factor modulates activin signaling in pituitary gonadotropes. Our studies show that overexpression of constitutively active FOXO1 decreases activin induction of murine Fshb gene expression in immortalized LβT2 cells. We demonstrate that FOXO1 suppression of activin induction maps to the −304/−95 region of the Fshb promoter containing multiple activin response elements and that the suppression requires the FOXO1 DNA-binding domain (DBD). FOXO1 binds weakly to the −125/−91 region of the Fshb promoter in a gel-shift assay. Since this region of the promoter contains a composite SMAD/FOXL2 binding element necessary for activin induction of Fshb transcription, it is possible that FOXO1 DNA binding interferes with SMAD and/or FOXL2 function. In addition, our studies demonstrate that FOXO1 directly interacts with SMAD3/4 but not SMAD2 in a FOXO1 DBD-dependent manner. Moreover, we show that SMAD3/4 induction of Fshb-luc and activin induction of a multimerized SMAD-binding element-luc are suppressed by FOXO1 in a DBD-dependent manner. These results suggest that FOXO1 binding to the proximal Fshb promoter as well as FOXO1 interaction with SMAD3/4 proteins may result in decreased activin induction of Fshb in gonadotropes.
Highlights
In mammalian reproduction, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) production from pituitary gonadotrope cells is critical for the regulation of gonadal functions such as steroidogenesis and gametogenesis [1,2].PLOS ONE | DOI:10.1371/journal.pone.0113839 November 25, 2014FOXO1 Decreases Activin Induction of FshbLH and FSH are heterodimeric glycoproteins composed of a common alpha subunit and a beta subunit which is unique to each hormone [3]
We recently published that overexpression of the FOXO1 transcription factor in immortalized LbT2 gonadotrope cells resulted in decreased basal and GnRHinduced Lhb and Fshb gene expression [35,36]
To determine whether FOXO1 can modulate activin signaling in gonadotropes, we transfected LbT2 cells with a multimer containing four repeats of a consensus Forkhead binding element (FBE) fused with a luc reporter gene (46FBE-luc) along with constitutively active FOXO1 (FOXO1-CA), which remains in the nucleus due to the inability of insulin/growth factor signaling to phosphorylate the mutated residues
Summary
In mammalian reproduction, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) production from pituitary gonadotrope cells is critical for the regulation of gonadal functions such as steroidogenesis and gametogenesis [1,2].PLOS ONE | DOI:10.1371/journal.pone.0113839 November 25, 2014FOXO1 Decreases Activin Induction of FshbLH and FSH are heterodimeric glycoproteins composed of a common alpha subunit and a beta subunit which is unique to each hormone [3]. In addition to GnRH, activin signaling via binding to activin type II serine/threonine kinase receptors, which results in the phosphorylation of activin type I receptors [8], is important for gonadotropin production. Activation of these receptors results in the phosphorylation of downstream Sma- and mothers against decapentaplegic (MAD)-related proteins, SMAD2 and SMAD3 [9,10,11]. Forkhead box L2 (FOXL2) has been reported to bind three elements at 2350, 2154 and 2113 in the murine Fshb promoter and mutation of these sites disrupt activin induction [18,19,20,21]
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