Constitutively Active FOXO1 Diminishes Activin Induction of Fshb Transcription in Immortalized Gonadotropes

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.

FOXO1 Negates the Cooperative Action of FOXL2C134W and SMAD3 in CYP19 Expression in HGrC1 Cells by Sequestering SMAD3

“…Figure 6 shows the input levels of SMAD3, FOXL2 C134W , and FOXO1 3A (top three panels) and the levels of SMAD3 coprecipitated with FOXL2 C134W or FOXO1 3A (bottom panel). Interestingly, FOXO1 3A , similar to FOXL2 C134W , associates with SMAD3, consistent with the putative role of SMAD3 in regulating FOXO1 activity [37].…”

“…Interestingly, the deletion of the DNA binding domain in FOXO1 3A (FOXO1 3A-DBD ) compromised the inhibition of FOXL2 C134W by FOXO1, despite a lack of binding by FOXO1 to FBE5. Indeed, the DNA binding domain of FOXO1 is also essential for SMAD3 binding [37], further supporting a model in which FOXO1 directly competes with FOXL2 C134W for binding to SMAD3. FOXL2 C134W action at the FBE5 site on CYP19 promoter occurs with SMAD3 as a cofactor [52].…”

“…To approach the mechanism by which FOXO1 inhibits SMAD3- and FOXL2 C134W -induced CYP19 mRNA expression, we next tested the effect of the FOXO1 3A mutant that is confined to the nucleus and is thereby constitutively active [55]. As an additional control, we included the FOXO1 3A-DBD mutant in this study, which lacks the capacity to bind SMAD3 as well as DNA [37].…”

“…To extend our studies on the role of FOXL2 C134W in regulating CYP19 expression via enhanced recruitment of SMAD3 to a proximal FBE, we have now assessed whether another forkhead protein, FOXO1, is involved in the mechanism because it was demonstrated to partner with SMAD3 in the regulation of activin induction of Fshb in immortalized murine L β T2 gonadotrope cells [37].…”

Rapid Communication: A MicroRNA-132/212 Pathway Mediates GnRH Activation of FSH Expression

“…Mechanisms of the repressive effect of FOXO1 on the Fshb promoter have not been clearly established. It has been hypothesized that FOXO1, which does not appear to physically bind to the Fshb promoter, could interact with a positive effector like Forkhead box L2 (28,35) or paired-like homeodomain 1 (29), preventing them from binding to their responsive element. In our experiments, no significant modification of basal Fshb mRNA level were observed when FOXO1 acetylation was prevented by miR-132/212 LNA inhibition (Figure 1).…”