GnRH induces SET protein down-regulation in infantile mice pituitary.

<p>(a) <i>Left panel</i>—SET protein abundance was analyzed in infantile pituitaries (7–17 dpn) by Western blotting, with GAPDH used as a loading control. Bar graphs show the mean ± SEM of SET levels normalized to those of GAPDH (n = 3 to 8 pituitaries/age). A representative immunoblot of SET expression is shown. Data were analyzed by one-way ANOVA, followed by Tukey’s test with distinct letters indicating significant differences between ages (p<0.05). <i>Right panel</i>—The relative pituitary abundance of SET mRNA in infantile (7–17 dpn) females was determined by real-time RT-PCR and normalized to the mRNA levels of <i>Hprt</i> (n = 4 to 8 pituitaries/age). Bar graphs show the mean ± SEM of relative quantification. Data were analyzed by one-way ANOVA, followed by Tukey’s test with distinct letters indicating significant differences between ages (p<0.05). (b) SET protein abundance was analyzed in infantile pituitaries after treatment with the GnRH antagonist Ganirelix or saline vehicle at 12 and 13 dpn. A representative blot is shown. Bar graphs show the mean ± SEM of SET levels normalized to those of GAPDH (n = 3 to 5 pituitaries/condition). Data were analyzed by t test for unpaired groups, **: p<0.01, compared to 14 dpn saline.</p

Pulsatile native GnRH induces SET protein down-regulation in LβT2 cells.

<p>(a) LβT2 gonadotrope cells were treated with GnRHa (100 nM) for 2 hours and SET protein (<i>left panel</i>) and SET mRNA (<i>right panel</i>) levels were determined by Western blotting and by real-time RT-PCR, respectively. A representative immunoblot of SET expression is shown. Results are normalized by vinculin signals (SET protein) or by mRNA levels of <i>Hprt</i> (SET mRNA) and are expressed as percentage of the amount of SET in unstimulated cells. Results are expressed as mean ± SEM from 3 independent experiments. Data were analyzed by t test for unpaired groups *: p<0.05, compared to no GnRHa. (b) LβT2 gonadotrope cells were cultured in perifusion chambers as described in “Materials and methods” and challenged or not with pulsatile GnRH (10 nM) at high and low frequencies (one pulse every 0.5 hour or one pulse every 2 hours, respectively). At the end of the incubation, proteins and mRNA were extracted and SET protein and <i>Set</i>, <i>Lhb and Fshb</i> transcripts levels were determined by Western blotting and by real-time RT-PCR, respectively. A representative immunoblot of SET expression is shown. Results are normalized by vinculin signals (SET protein) or by mRNA levels of <i>Hprt</i> (<i>Set</i>, <i>Lhb and Fshb</i> mRNA) and expressed as percentage of the amount of SET in unstimulated cells. Results are expressed as mean ± SEM from 3 independent experiments. One-way ANOVA followed by Tukey’s test, ***: p<0.001, compared to no GnRH.</p

GnRHa increases SET phosphorylation–Involvement of PKC.

<p>(a) <i>Left panel–</i>αT3-1 gonadotrope cells were plated in 6-well plates and labelled with [³²P]-orthophosphate (50 μCi/ml) as described in “Materials and methods”. Cells were stimulated (+) or not (-) with GnRHa (100 nM) for 0.5 hour followed by SET immunoprecipitation (IP SET) as described in “Materials and methods”. Immunoprecipitated SET was resolved on 10% SDS-PAGE, electrotransferred and probed with anti-SET antibody (IB SET). Phosphorylated SET (P-SET) was visualized by autoradiography using a Fuji Phosphoimager FLA7000. <i>Right panel–</i>αT3-1 gonadotrope cells were incubated (+) or not (-) with GnRHa (100 nM, 0.5 hour) and phosphoproteins were purified by chromatography as described in “Materials and methods”. Phosphorylated SET and ERK1/2 were detected by Western blotting using anti-SET and anti-total ERK1/2 antibodies, respectively. Results are representative of 4 independent experiments. (b) αT3-1 gonadotrope cells were pre-incubated or not with the PKC inhibitor GF109203X (2 μM, 1 hour) prior to GnRHa stimulation (100 nM, 0.5 hour). Phosphoproteins were purified by chromatography as described in “Materials and methods” and phosphorylated SET was detected by Western blotting using anti-SET antibody. Results are expressed as the percentage of SET protein level in absence of treatment. Results are expressed as mean ± SEM from 3 independent experiments. Data were analyzed by Two-way ANOVA followed by Tukey’s test, with distinct letters indicating significant differences between treatments (p<0.05). (c) αT3-1 gonadotrope cells were pre-incubated or not with the PKC inhibitor GF109203X (2 μM, 1 hour) prior to GnRHa stimulation (100 nM, 0.5 hour) and SET protein expression was detected by Western blotting using anti-SET antibody. Results are normalized by vinculin signals and expressed as the percentage of SET protein level in absence of treatment. Results are expressed as mean ± SEM from 3 to 4 independent experiments. Data were analyzed by Two-way ANOVA followed by Tukey’s test, with distinct letters indicating significant differences between treatments (p<0.05).</p

GnRHa down-regulates SET protein expression in αT3-1 gonadotrope.

<p>(a) <b>α</b>T3-1 gonadotrope cells were incubated during 2 hours with increasing GnRHa concentrations (10⁻⁹ to 10⁻⁶ M) and SET protein level was determined by Western blotting. A representative immunoblot of SET expression is shown. Results are normalized by vinculin signals and are expressed as the percentage of SET protein level in absence of GnRHa. Results are expressed as mean ± SEM from 3 to 4 independent experiments. Data were analyzed by One-way ANOVA followed by Dunnett’s test, *: p<0.01 and ***: p<0.001, compared to no GnRHa. (b) <b>α</b>T3-1 gonadotrope cells were pre-treated or not (control) with the proteasome inhibitors MG132 (MG132, 2 hours, 3 μM) or the clasto-lactacystin–Lactone (Lactacystin, 2 hours, 10 μM) before stimulation with GnRHa (100 nM) for the indicated times (0.5 and 2 hours). SET protein level was determined by Western blotting and normalized by vinculin signals. SET protein level in GnRHa stimulated cells was expressed as the percentage of respective basal SET expression levels at each time point in the presence or absence of MG132 or clasto-lactacystin–Lactone. Results are expressed as mean ± SEM from 3 to 5 independent experiments. Data were analyzed by Two-way ANOVA followed by Tukey’s test. Distinct letters indicate significant differences between treatments (p<0.05). (c) αT3-1 gonadotrope cells were incubated with GnRHa (100 nM) for increasing periods of time and SET mRNA levels were determined by real-time RT-PCR and normalized to the mRNA levels of <i>Hprt</i>. Results are expressed as the percentage of SET mRNA level in unstimulated cells (0 h). Results are expressed as mean ± SEM from 3 independent experiments. Data were analyzed by One-way ANOVA, no significant.</p

Impact of serine 9 phosphorylation on SET expression level.

<p><b>(</b>a) Gonadotrope cells were transfected either with A9 or E9 His-SET mutants as described in “Materials and methods”. Thirty hours after transfection, cells were pre-incubated (+) or not (-) with MG132 (10 μM, 18 hours). The relative expression levels of both mutants were assessed by Western blotting and normalized by vinculin signals. His-SET proteins migrate on SDS-PAGE at a higher molecular weight than endogenous SET allowing their specific quantification. Results are expressed as the percentage of SET A9 protein level in absence of MG132. Results are expressed as mean ± SEM from 4 to 5 independent experiments. Data were analyzed by Two-way ANOVA followed by Tukey’s test, with distinct letters indicating significant differences between treatments (p<0.05). (b) Gonadotrope cells were transfected either with the wild type His-SET or the His-SET A9 mutant and were treated or not with GnRHa (100 nM) for 0.5 or 2 hours. SET expression level was assessed by Western blotting and normalized by vinculin signals. Results are expressed as the percentage of SET WT or SET A9 protein levels in absence of GnRHa. Results are expressed as mean ± SEM from 4 independent experiments. Data were analyzed by One-way ANOVA followed by Dunnett’s test*: p<0.05, compared to no GnRHa.</p