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. 2015 Mar;29(3):364-72.
doi: 10.1210/me.2014-1390. Epub 2015 Jan 30.

Rapid communication: A microRNA-132/212 pathway mediates GnRH activation of FSH expression

Jérôme Lannes  1 , David L'Hôte, Ghislaine Garrel, Jean-Noël Laverrière, Joëlle Cohen-Tannoudji, Bruno Quérat
Affiliations

Rapid communication: A microRNA-132/212 pathway mediates GnRH activation of FSH expression

Jérôme Lannes et al. Mol Endocrinol. 2015 Mar.

Abstract

GnRH plays a key role in the vertebrate reproductive system by stimulating biosynthesis and secretion of pituitary gonadotropins. However, the potential involvement of microRNAs (miRNAs) on this activation has still to be explored. In this study, we investigated the role of miRNA-132 and miRNA-212, two tandemly expressed miRNAs that target the same transcripts, on GnRH-induced FSH expression. We first showed that the GnRH stimulation of FSH secretion was reduced and Fshb mRNA abolished by blocking miR-132/212 action in rat pituitary cells. In mouse LβT2 gonadotrope cells, the GnRH stimulation of Fshb mRNA was also demonstrated to be dependent on miR-132/212 and reproduced by overexpressing one or both miRNAs. We then showed that the miR-132/212-mediated action of GnRH involved a posttranscriptional decrease of sirtuin 1 (SIRT1) deacetylase. The lower level of SIRT1 deacetylase correlated with an increase in the acetylated form of Forkhead Box O1 (FOXO1), a transcriptional repressor of Fshb. Interestingly, we show that the acetylated mimicking mutant of FOXO1 was localized outside the nucleus, thus alleviating its repressive effect on Fshb transcription. Overall, we demonstrate that the GnRH stimulation of Fshb expression is dependent on miR-132/212 and involves a SIRT1-FOXO1 pathway. This is the first demonstration of an obligatory microRNA pathway in the GnRH-regulated expression of a gonadotropin gene.

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Figures

Figure 1.
Figure 1.. The miR-132/212 pathway is involved in Fshb induction by GnRH.
A and B, Rat primary pituitary cultures were electroporated with an anti-miR-132/212 or a scrambled LNA and then treated with 1 nM GnRHa for 6 hours (n = 6). A, The concentration of accumulated FSH into the medium was measured by an ELISA. Blocking miR-132 and miR-212 significantly reduced the GnRH-increased FSH secretion. B, Fshb mRNA level was measured by qRT-PCR and normalized to Gapdh mRNA. Blocking miR-132 and miR-212 prevented the GnRH-induced Fshb mRNA expression in rat primary pituitary cells. C, LβT2 cells were electroporated with a control vector (control) or with miR-132 or miR-212 expression vectors for 72 hours and then harvested. Overexpression of miR-132 or miR-212 increased Fshb mRNA expression (n = 4). D, LβT2 cells were electroporated with an anti-miR-132/212 LNA or scrambled LNA and were treated with 10 nM GnRHa for 8 hours. Blocking miR-132 and miR-212 prevented the GnRH-induced Fshb mRNA expression in LβT2 cells (n = 6). *, P < .05; **, P < .01; ***, P < .001.
Figure 2.
Figure 2.. miR-132 and miR-212 down-regulate SIRT1 protein expression in gonadotrope cells.
A and B, LβT2 cells were treated for 6, 8, or 24 hours with 10 nM GnRHa (n = 4). A, Sirt1 mRNA level was quantified by qRT-PCR and normalized to Gapdh mRNA. GnRHa treatment did not induce any change in the Sirt1 mRNA steady-state level. B, SIRT1 protein level was quantified by Western blot analysis. GnRHa treatment induced a significant decrease in SIRT1 deacetylase level after 8 hours. C and D, Recruitment of endogenous miR-132 (C) and Sirt1 mRNA (D) into RISC were determined by qRT-PCR on AGO-immunoprecipitated samples and normalized to the input level. GnRHa treatment stimulated the recruitment of both miR-132 and Sirt1 mRNA (n = 4). E, LβT2 cells were electroporated with a control or with miR-132 and/or miR-212 expression vectors and harvested after 72 hours. SIRT1 deacetylase level was quantified by Western blot analysis and normalized with GAPDH. Overexpression of miR-132 or miR-212 decreased SIRT1 protein expression (n = 5). F, LβT2 cells were electroporated with anti-miR-132/212 or scrambled LNA and then treated with 10 nM GnRHa for 8 hours. Blocking miR-132 and miR-212 prevented the GnRH-induced SIRT1 protein decrease (n = 4). *, P < .05; **, P < .01; ***, P < .001.
Figure 3.
Figure 3.. The miR-132/SIRT1 pathway controls FSHβ expression through enhanced acetylation of FOXO1.
A and B, LβT2 cells were treated for 6, 8, or 24 hours with 10 nM GnRHa. A, upper panel, Representative Western blot of SIRT1, Ac-FOXO1, total FOXO1, and GAPDH at different time points after a 10 nM GnRHa treatment. A, lower panel, Ac-FOXO1 level was quantified by Western blot analysis and normalized with total FOXO1. Acetylation of FOXO1 increased in response to GnRH, whereas SIRT1 deacetylase expression was decreased (n = 4). B, LβT2 cells were electroporated with a control vector or with miR-132 and/or miR-212 expression vectors and harvested after 72 hours. Overexpression of miR-132 and/or miR-212 increased acetylation of FOXO1 (n = 4). C, LβT2 cells were electroporated with scrambled or anti-miR-132/212 LNAs and then treated for 24 hours with 10 nM GnRHa. The amount of Ac-FOXO1 over total FOXO1, estimated by Western blot analysis, was enhanced under GnRH, but blocking miR-132 and miR-212 prevented this effect (n = 6). D and E, LβT2 cells were electroporated with control vector or with FLAG-FOXO1 wild type (WT), constitutively active mutant (ADA), or acetylated mimicking mutant (6KQ) vectors. D, Fshb mRNA level was quantified by qRT-PCR and normalized to Gapdh. Fshb expression was activated in response to the acetylated FOXO1 mimic overexpression, whereas it was inhibited in response to the constitutively active mutant (n = 4). E, FLAG-FOXO1 mutants were localized by immunocytochemistry and 4′,6′-diamino-2-phenylindole (DAPI) staining of the nucleus. Acetylation of FOXO1 induced a shuttling of FOXO1 to the cytoplasm. *, P < .05; **, P < .01.
Figure 4.
Figure 4.. Proposed model for the GnRH-induced miR-132/212 mediation on FSHβ expression.
1, GnRH stimulation increases miR-132 and miR-212 expression. 2, miR-132/212 recruit Sirt1 mRNA into RISC and down-regulates SIRT1 protein level. 3, SIRT1 down-regulation reduces deacetylation of FOXO1, provoking shuttling of FOXO1 to the cytoplasm. 4, As a result, FOXO1-mediated inhibition of Fshb transcription is decreased, enhancing Fshb expression
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This work was supported by Université Paris-Diderot, CNRS and INSERM.