| Objective: The study was to investigate the effect of transcriptional factor SP1 on the promoter activity of KiSS-1 gene in the hypothalamus GT1-7 neurons, as well as to explore the role of AMPK played in the translocation of SP1 in these cells.Methods: (1).We transfected GT1-7 cells with the 2-kb full-length KiSS-1 gene promoter (PGL3-KiSS-1 promoter) linked with a luciferase reporter, then treated them with SP1SiRNA or Mithramycin A, known as a pharmacological suppressor of SP1, and detected the luciferase activity by Dual-Luciferase Reporter Assay System. (2).We incubated GT1-7 cells with AICAR (an artificial activator of AMPK), Compound C (a suppressor of AMPK) or AMPKα1-siRNA, and determined the levels of SP1 protein from nuclear fraction, cytoplasmic fraction and total cell extracts by Western Blot. (3).We treated GT1-7 cells with AICAR or Compound C, and performed the immuno?uorescence analysis to directly visualize the effect of AMPK on the subcellular redistribution of SP1 protein.Results: (1).SP1SiRNA or Mithramycin A at different concentrations could decrease the promoter activity of KiSS-1 gene obviously in the hypothalamus GT1-7 cells. (2).AICAR decreased the nuclear fraction and increased the cytoplasm fraction of SP1. To the contrary, Compound C and AMPKα1-SiRNA played an inverse role compared with AICAR in the subcellular compartmentalization of SP1 in GT1-7 cells. AICAR and Compound C, as well as AMPKα1-SiRNA had no significant effect on the total extracts of SP1 in the GT1-7 cells. (3).The results from our immuno?uorescence analysis showed that AICAR inhibited the translocation of SP1, while Compound C enhanced the translocation of SP1 from cytoplasm to nucleus in the GT1-7 cells which confirmed the results of our Western Blot analysis. Conclusion: AMPK may inhibit the expression of KiSS-1 gene by decreasing the translocation of SP1 from cytoplasm to nucleus which followed by the weakened activation of KiSS-1 by SP1 in the hypothalamus GT1-7 neurons. |
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