Figure 6: miR-125b/miR-132 regulatory loop involvement in gonadotrope cell desensitization to GnRH.

(a–d) Dynamics of the GnRH-induced miR-125b/miR-132 regulatory loop. LβT2 cells were treated with 10 nM GnRHa. (a) Effect on the recruitment into RISC. miR-125b, miR-132 and NSun2 mRNA levels were determined on pan-AGO-immunoprecipitated samples and normalized to input level. Recruitment of miR-125b was quickly reduced and kept low until 8 h of treatment. Recruitment of miR-132 was delayed until 2 h and maintained for up to 8 h. NSun2 mRNA was recruited only after 8 h (n ≥ 5). (b) GnRHa treatment induces a decrease in NSun2 protein level (n ≥ 4). (c) Protein extracts were eluted from a phosphoprotein enrichment column and analysed by western blotting using an anti-NSun2 antibody. The GnRHa-treatment rapidly induced a strong increase of NSun2 in the phosphoprotein fraction, peaking at 4 h (n ≥ 4). (d–f) NSun2 is inactivated by a PP1α-dependent dephosphorylation. d; LβT2 cells were treated with 10 nM GnRHa. Ppp1ca mRNA was significantly induced after 6 h of treatment (n ≥ 5). (e) LβT2 cells were either pre-treated with the MEK1/2 inhibitors (U0126) or not before treatment with GnRHa (10 nM for 8 h). Inhibition of ERK1/2 phosphorylation prevented the GnRH-induced expression of Ppp1ca mRNA (n = 3). (f) Cells were electroporated with PP1CA or empty expression vector followed by a GnRHa treatment (10 nM for 4 h). PP1CA overexpression alone had no effect on basal phosphorylation level but prevented the GnRH-induced phosphorylation of NSun2 (n ≥ 3). (g) Cells were treated by GnRHa (10 nM for 4 or 8 h). Protein extracts were immunoprecipitated using anti-NSun2 antibody. Co-immunoprecipitated proteins were analysed by western blotting using anti-PP1CA. Representative blot of immunoprecipitated NSun2 proteins from GnRH-treated compared to control cells. The catalytic subunit of PP1α was associated with NSun2 after 8 h of GnRHa-treatment but not after 4 h. (h) Cells were treated with 10 nM GnRHa. Lhb and Fshb mRNAs were significantly induced in GnRH-treated cells relative to control cells and returned to control values after 24 h (n ≥ 5). Representative western blot images are given on Fig. S2M. *P < 0.05; **P < 0.01; ***P < 0.001.