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. 2011 Aug;25(8):1387-403.
doi: 10.1210/me.2011-0032. Epub 2011 Jun 9.

Gonadotropin-releasing hormone pulse sensitivity of follicle-stimulating hormone-beta gene is mediated by differential expression of positive regulatory activator protein 1 factors and corepressors SKIL and TGIF1

Devendra S Mistry  1 , Rie Tsutsumi, Marina Fernandez, Shweta Sharma, Steven A Cardenas, Mark A Lawson, Nicholas J G Webster
Affiliations

Gonadotropin-releasing hormone pulse sensitivity of follicle-stimulating hormone-beta gene is mediated by differential expression of positive regulatory activator protein 1 factors and corepressors SKIL and TGIF1

Devendra S Mistry et al. Mol Endocrinol. 2011 Aug.

Abstract

Gonadotropin synthesis and release is dependent on pulsatile stimulation by the hypothalamic neuropeptide GnRH. Generally, slow GnRH pulses promote FSH production, whereas rapid pulses favor LH, but the molecular mechanism underlying this pulse sensitivity is poorly understood. In this study, we developed and tested a model for FSHβ regulation in mouse LβT2 gonadotropes. By mining a previous microarray data set, we found that mRNA for positive regulators of Fshb expression, such as Fos and Jun, were up-regulated at slower pulse frequencies than a number of potential negative regulators, such as the corepressors Skil, Crem, and Tgif1. These latter corepressors reduced Fshb promoter activity whether driven by transfection of individual transcription factors or by treatment with GnRH and activin. Overexpression of binding or phosphorylation-defective ski-oncogene-like protein (SKIL) and TG interacting factor (TGIF1) mutants, however, failed to repress Fshb promoter activity. Knockdown of the endogenous repressors SKIL and TGIF1, but not cAMP response element-modulator, increased Fshb promoter activity driven by constant GnRH or activin. Chromatin immunoprecipitation analysis showed that FOS, SKIL, and TGIF1 occupy the FSHβ promoter in a cyclical manner after GnRH stimulation. Overexpression of corepressors SKIL or TGIF1 repressed induction of the Fshb promoter at the slow GnRH pulse frequency but had little effect at the fast pulse frequency. In contrast, knockdown of endogenous SKIL or TGIF1 selectively increased Fshb mRNA at the fast GnRH pulse frequency. Therefore, we propose a potential mechanism by which production of gonadotropin Fshb is modulated by positive transcription factors and negative corepressors with different pulse sensitivities.

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Figures

Fig. 1.
Fig. 1.
Induction of AP-1 factors and corepressor proteins by GnRH and the repression of Fshb promoter activity by SKIL, SKI, TGIF1, and CREM in LβT2 cells. A, Induction of protein expression by continuous 100 nm GnRH (left panel), after a single pulse of 100 nm GnRH (middle panel), or after multiple 5-min pulses of 1 or 10 nm GnRH every 30 or 120 min for 6 h in static culture (right panel). V, Vehicle control. Cell lysates were blotted for FOS, JUN, SKIL, TGIF1, CREM, or β-tubulin as a loading control. Each experiment was repeated at least three times. Quantification of protein induction is given in Supplemental Fig. 2. B–F, Cells were cotransfected with the −1.5-kb Fshb-luc reporter gene and the indicated expression vectors. B, Cotransfection of Fos, Jun, Skil, Ski, and Tgif1 or pCMVSPORT6. C, Cotransfection of Smad3, Smad4, Smad3/4, Skil, Ski, and Tgif1 or pCMVSPORT6. D, Cotransfection of Creb, DCreb, Crem, or pCMVSPORT6. E, Cotransfection of Skil, Ski, and Tgif1 or pCMVSPORT6 in presence of activin A and GnRH. Transfected cells were treated for 16 h with 25 ng/ml activin A, and then 100 nm GnRH was added for a further 6 h. F, Cotransfection of MCreb, KCreb, Crem, or pCMVSPORT6. Transfected cells were treated for 16 h with 25 ng/ml activin A, and then 100 nm GnRH was added for a further 6 h. Data represent the mean and se of at least three independent experiments. *, Significant difference (P < 0.05) compared with vehicle-treated control group; #, significant difference (P < 0.05) between designated groups.
Fig. 2.
Fig. 2.
Stimulation of Fshb promoter activity by corepressor knockdown or expression of mutant corepressors. In all panels, cells were cotransfected with the −1.5-kb Fshb-luc reporter gene and the indicated siRNA and/or expression vectors. A, Cotransfection of Fos/Jun and siRNA against Tgif1 or control siRNA. B, Cotransfection of Smad3 and siRNA against Skil or control siRNA. C, Cotransfection of DCreb and siRNA against Crem or control siRNA. D, Cotransfection of siRNA against the corepressors Tgif1, Skil, Crem, or control. Transfected cells were treated for 16 h with 25 ng/ml activin A, and then 100 nm GnRH was added for a further 6 h and harvested 48 h after siRNA transfection. E, Similar experiment to D but cells harvested 72 h after transfection. F, Cotransfection of wild-type human TGIF1 or mutant human TGIF1-2TV, which contains valine substitutions at the ERK phosphorylation site threonines 235 and 239. G, Cotransfection of human wild-type Skil or mutant Skil-S3,4W, which contains alanine substitutions at amino acids 85–88 to eliminate SMAD2 and SMAD3 binding and tryptophan 318 to eliminate SMAD4 binding. Data represent the mean and se of at least three independent experiments. *, Significant difference (P < 0.05) compared with vehicle-treated control group; #, significant difference (P < 0.05) between designated groups.
Fig. 3.
Fig. 3.
Corepressors SKIL and TGIF1 reduce Fshb promoter activity in response to pulsatile GnRH. A, Measurement of Fshb mRNA in LβT2 cells treated with 5-min pulses of 1 and 10 nm GnRH at intervals of 30 and 120 min over 6 h. Data represent the means and se of three independent experiments. *, Significant difference (P < 0.05) compared with vehicle-treated group; #, significant difference (P < 0.05) compared with respective 30-min GnRH pulse frequency-treated group. B–D, LβT2 cells were cotransfected with −1.5-kb Fshb-luc, tklacZ, and expression vectors for Tgif1, Skil, or control pCMVSPORT6 vector as indicated. Cells were treated with 5-min pulses of 10 nm GnRH in a pulse perifusion system at intervals of either 30 or 120 min for 6 h. Two nanograms per milliliter activin A were added to all of the groups at an interval of 120 min to maintain basal promoter activity. B, Effect of different pulse frequencies in the control-transfected cells. *, Significant (P < 0.05) increase relative to vehicle control; **, significant (P < 0.01) increase relative to vehicle control. C, Effect of TGIF1 or SKIL overexpression on Fshb induction by GnRH at 30-min pulse intervals. D, Effect of TGIF1 or SKIL overexpression on Fshb induction by GnRH at 120-pulse intervals. *, Significant decrease (P < 0.05) relative to pCMVSPORT6-transfected cells. E, Verification of siRNA knockdown in LβT2 cells electroporated with the indicated siRNA. Forty-eight hours later, cells were treated with 100 nm GnRH for 4 h to induce TGIF1 and SKIL. Protein expression was measured by immunoblotting using the indicated antibodies. Data were quantified using densitometry (Supplemental Fig. 2F). F–H, LβT2 cells were electroporated with the siRNA against Tgif1 or Skil for 48 h before being treated with 5-min pulses of 10 nm GnRH at intervals of either 30 or 120 min for 6 h in static culture. F, Induction of Fshb mRNA by GnRH pulses in the presence of the control siRNA. *, Significant difference from vehicle-treated control (P < 0.05); #, significant difference from 30 min pulse frequency treatment (P < 0.05). G, Effect of Tgif1 or Skil knockdown on Fshb induction by GnRH at 30-min pulse intervals. *, Significant difference from control siRNA-transfected group (P < 0.05). H, Effect of Tgif1 or Skil knockdown on Fshb induction by GnRH at 120-min pulse intervals.
Fig. 4.
Fig. 4.
Fshb promoter occupancy by FOS, JUN, SKIL, and TGIF1 in response to GnRH. A, ChIP assay was performed with LβT2 cells treated with continuous 100 nm GnRH for the indicated times. Chromatin was immunoprecipitated using antibodies to FOS, JUN, TGIF1, and SKIL. Precipitated chromatin was amplified using three independent sets of primers to the proximal Fshb promoter. Representative PCR are shown. Amplification of input DNA is shown as a control for chromatin recovery. B, Quantification of the ChIP assay. Results are the mean of at least three experiments with three independent primer pairs. Data are normalized to respective inputs and then to their respective zero time points. *, Significant difference from respective 0 h (P < 0.05). C, ChIP assay was performed with LβT2 cells treated with a single 5-min pulse of 100 nm GnRH for the indicated times using antibodies to FOS, JUN, TGIF1, and SKIL as before. The panels show representative PCR. D, Quantification of ChIP after a single pulse of GnRH. Data were normalized and analyzed as before. *, Significant difference from respective 0 h (P < 0.05). Data represent the mean and se of at least two independent experiments.
Fig. 5.
Fig. 5.
Kinetics of transcription factor expression and Fshb promoter occupancy in cells exposed to fast and slow GnRH pulses. LβT2 cells were serum starved for 24 h and then treated with 5-min pulses of 1 or 10 nm GnRH every 30 or 120 min for 6 h. After 6 h, cells were treated with one final synchronizing pulse of GnRH and then harvested immediately or 30, 60, or 120 min after the last pulse. A, Time course of induction of FOS, JUN, TGIF1, and SKIL protein expression. Representative blots are shown. Blots were reblotted for β-tubulin for loading control and were quantified by densitometry (Supplemental Fig. 5). B, Time course of promoter occupancy by ChIP after the last GnRH pulse. Chromatin was immunoprecipitated with antibodies to FOS, JUN, TGIF1, and SKIL. The panels show representative PCR. C, Quantification of the ChIP data after a multiple pulse of GnRH. Data were normalized to respective inputs and then to vehicle-treated cells as before. *, Significant difference from respective 0 time point (P < 0.05). Data represent the mean and se of at least two independent experiments.
Fig. 6.
Fig. 6.
Ratio of transcription factor to corepressor occupancy after the final pulse and kinetics of Fshb mRNA induction in cells exposed to fast and slow GnRH pulses. A, Quantification of the ChIP data from Fig. 5 plotted as FOS/TGIF1, FOS/SKIL, JUN/TGIF1, or JUN/SKIL promoter occupancy ratios. B, Fshb mRNA induction after final pulse using the experimental paradigm from Fig. 5. #, Significant difference compared with the respective 30-min pulse interval group (P < 0.05); *, significant difference compared with the vehicle-treated, zero time point (P < 0.05). The promoter occupancy and mRNA induction data were fit using linear regression; ‡, the linear regression line has a significantly nonzero slope (P < 0.01). Refer to Supplemental Table 2 for specific r2 and P values. The time-course data represent the mean and se of three independent experiments.
Fig. 7.
Fig. 7.
Model for pulse regulation by the Fshb gene. The transcription factors FOS and JUN are induced at slow GnRH pulse frequencies, but the corepressors SKIL and TGIF1 do not change. This leads to an increased ratio of promoter occupancy by stimulatory transcription factors to inhibitory corepressors causing increased Fshb mRNA induction. Because the GnRH pulse frequency increases, there is additional induction of the corepressors SKIL and TGIF1 so the ratio of promoter occupancy by stimulatory transcription factors to inhibitory corepressors does not change. The presence of the corepressors on the Fshb promoter dampens induction of the Fshb mRNA at the faster GnRH pulse frequency, preventing further induction.
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