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. 2007 May 30;270(1-2):64-72.
doi: 10.1016/j.mce.2007.02.007. Epub 2007 Feb 24.

Egr-1 binds the GnRH promoter to mediate the increase in gene expression by insulin

Sara A DiVall  1 , Sally Radovick, Andrew Wolfe
Affiliations

Egr-1 binds the GnRH promoter to mediate the increase in gene expression by insulin

Sara A DiVall et al. Mol Cell Endocrinol. .

Abstract

Insulin increases gonadotropin-releasing hormone (GnRH) gene expression in in vitro models of GnRH neurons. Early growth response-1 (Egr-1) is a transcription factor that mediates the effect of insulin on target genes. In the GN11 cell line--an immortalized GnRH-secreting neuronal cell line--insulin maximally increases Egr-1 mRNA after 30min of treatment and Egr-1 protein and GnRH mRNA after 60min of treatment. Egr-1 small interfering RNA blocks the insulin-induced increase in GnRH promoter activity, measured as luciferase expression. Chromatin immunoprecipitation using Egr-1 antibody precipitates DNA in a proximal region of the GnRH promoter but not DNA in a distal region. Mutagenesis of a putative Egr-1 binding site within the proximal region blocks the insulin-induced increase in GnRH promoter activity. Thus, Egr-1 binds the GnRH promoter at a site between -67 and -76bp from the transcriptional start site to mediate the insulin-induced increase in GnRH gene transcription.

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Figures

Fig. 1
Fig. 1. Dose response and time course of insulin induction of Egr-1 gene and protein expression
GN11 cells were serum deprived overnight then treated with insulin A) for 30 minutes at the specified doses or B) with 30 nM at the specified times. Total RNA was extracted, RT-PCR performed and the resultant cDNA amplified using quantitative PCR with primers specific for mouse Egr-1. Relative mRNA levels were calculated using the ΔΔCt method and graphed as fold increase in mRNA levels in insulin treated cells relative to untreated cells. Each graph represents three (3) separate experiments performed in duplicate. In comparison of insulin treated versus untreated controls, statistical significance of p<0.05 is indicated by the asterisk. C) Nuclear extract was made from GN11 cells after treatment with 30 nM insulin and subjected to western blot analysis using an antibody specific for Egr-1.
Fig. 2
Fig. 2. Dependency of the insulin-induced increase in Egr-1 expression on the MAP kinase pathway
GN11 cells were serum deprived overnight, pretreated with 10 μM of the MAP kinase pathway specific inhibitor PD98059, 10μM of the PI3 kinase inhibitor Wortmannin or vehicle for 30 minutes then treated with 30 nM of insulin for 30 minutes. Relative mRNA levels were calculated using the ΔΔCt method and graphed as fold increase in mRNA levels of insulin treated cells relative to untreated cells. In comparison of treated versus untreated controls, statistical significance of p<0.05 is indicated by the asterisk. Graph represents three (3) separate experiments performed in duplicate.
Fig. 3
Fig. 3. Dose response and time course of insulin induction of GnRH gene expression in GN11 cells
GN11 cells were serum deprived overnight then treated with insulin at the indicated doses for one hour (A) or with 30 nM of insulin for the specified times (B). Relative mRNA levels were calculated using the ΔΔCt method and graphed as fold increase in mRNA levels in insulin treated cells relative to untreated cells. In comparison of insulin treated versus untreated controls, statistical significance of p<0.05 is indicated by the asterisks. Each graph represents four (4) separate experiments performed in duplicate.
Fig. 4
Fig. 4. Egr-1 siRNA interferes with the insulin induced increase in Egr-1 protein and GnRH promoter activity
200 ng of scrambled siRNA or Egr-1 siRNA was transfected into GN11 cells. After 24 hours, media was replaced by serum free media and the next morning, cells were stimulated with 30 nM insulin or vehicle for 60 minutes. A) Nuclear extract was made from GN11 cells and subjected to western blot analysis using an antibody specific for Egr-1. B) Cells were co-transfected with a 1250LUC construct then luciferase activity (RLU1) and renilla luciferase activity (RLU2) were measured. Luciferase activity was corrected for transfection efficiency using the equation RLU1/RLU2. The RLU1/RLU2 for insulin treated conditions relative to the RLU1/RLU2 for untreated conditions is graphed. Upon comparison of the 1250LUC activity alone with 1250LUC co-transfected with siRNA activity, statistical significance of p<0.05 was assigned and is indicated by the asterisk. Graph represents five (5) separate experiments performed in triplicate.
Fig. 5
Fig. 5. Egr-1 interacts with the GnRH promoter in response to insulin
A) The proximal region of the GnRH gene promoter contains a GC rich region between −67 and −75 bp that has high homology to the Egr-1 consensus sequence (underlined). The distal region of the GnRH gene promoter between −829 and −838 bp also contains a GC rich region. B) GN11 cells were treated with 30 nM of insulin after serum starvation for 16 hours. Cells were then cross linked using formaldehyde, sonicated, then subjected to immunoprecipitation with no antibody or 10 ug of Egr-1 antibody. DNA was extracted and subjected to quantitative PCR. Data are expressed as fold differences relative to control conditions (in which no antibody is used during the immunoprecipitation). DNA was subjected to PCR using primers that amplified a region of the GnRH promoter between −176 bp and +28 bp (“proximal primers”; left panel) or primers that amplified a region between −936 bp and −775 bp (“distal primers”; right panel). In comparison between conditions, statistical significance of p<0.05 is assigned and indicated by an asterisk. Each ChiP assay was performed on three different occasions; for each assay, samples were subjected to PCR in triplicate.
Fig. 6
Fig. 6. Mutation of the putative Egr-1 binding site in the proximal region of the GN11RH gene promoter blocks the insulin induced increase in GnRH promoter activity
The putative binding sites of the −1250LUC construct of the GnRH gene promoter were subjected to site directed mutagenesis. A) Top panel: sequence of the putative Egr-1 binding site in the proximal mouse GnRH gene promoter. Bottom panel: sequence generated by site directed mutagenesis within the −1250LUC construct and used in subsequent studies (−67 mutant). B) Top panel: sequence of the putative Egr-1 binding site in the distal proximal mouse GnRH gene promoter. Bottom panel: sequence generated by site directed mutagenesis within the −1250LUC construct and used in subsequent studies (−819 mutant). C) GN11 cells were transfected with 200 ng of wild type −1250LUC construct, −67 mutant, or −829 mutant. After 24 hours, media was changed to serum free media and 16 hours later, cells were stimulated with 30 nM insulin for 60 minutes. Luciferase activity (RLU1) and renilla luciferase activity (RLU2) were measured. Luciferase activity was corrected for transfection efficiency using the equation RLU1/RLU2. The corrected luciferase activity for the insulin treated samples were normalized to the corresponding untreated sample and this normalized value graphed. The graph represents three (3) separate experiments with triplicates of each condition. In comparison of wildtype and mutant constructs, statistical significance of p<0.05 is assigned and indicated by an asterisk.
Fig. 7
Fig. 7. Model of the Role of Egr-1 in GnRH Neurons
Insulin binds to its receptor on G11 cells and activates the MAP kinase pathway to increase Egr-1 transcription, mRNA, and protein levels. Egr-1, in turn, binds to the GnRH promoter to increase GnRH transcription and mRNA levels.
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