Sp1 is required for glucose-induced transcriptional regulation of mouse vesicular glutamate transporter 2 gene.
OBJECTIVE
Vesicular glutamate transporter (VGLUT) has been reported to be involved in glucose-induced insulin secretion. It has been shown that glucose stimulates the expression of VGLUT isoform 2 (VGLUT2) in beta cells via transcriptional mechanism. In this study, we identified the mouse VGLUT2 (mVGLUT2) promoter and characterized the transcriptional mechanism of glucose-stimulated mVGLUT2 expression in beta-cells.


METHODS
A promoter region of mVGLUT2 was cloned by genomic polymerase chain reaction. The mechanism of Sp1 in glucose-induced transactivation of mVGLUT2 was investigated by luciferase assay, electrophoretic mobility shift assay, chromatin immunoprecipitation assay, and Western blot analysis.


RESULTS
A promoter containing 2133 base pairs of upstream sequence of the 5'-flanking region of mVGLUT2 complementary DNA was cloned. Transient transfection of various 5'-end deletion constructs of the mVGLUT2 promoter/luciferase reporter indicated that the region between -96 to +68 base pair contains the basal promoter for mVGLUT2. Mutational analysis and electromobility shift assay showed an important role for the transcription factor Sp1 in both basal and glucose-induced mVGLUT2 transcription. The interaction between Sp1 and mVGLUT2 was confirmed by chromatin immunoprecipitation assays. Glucose stimulates the phosphorylation of Sp1 via mitogen-activated protein kinase P38 and P44/42. This leads to increased binding activity of Sp1 to the mVGLUT2 promoter and results in activation of the gene.


CONCLUSIONS
We cloned the mouse VGLUT2 promoter and showed a novel molecular mechanism of glucose-induced mVGLUT2 transcription.
Li
Tao
T
Department of Pediatrics, Steele Children's Research Center, University of Arizona Health Science Center, Tucson, Arizona 85724, USA.
Bai
Liqun
L
Li
Jing
J
Igarashi
Suzu
S
Ghishan
Fayez K
FK
D024506:5' Flanking Region; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D003001:Cloning, Molecular; D024202:Electrophoretic Mobility Shift Assay; D017930:Genes, Reporter; D005947:Glucose / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000201:enzymology / Q000378:metabolism*; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D050599:Vesicular Glutamate Transport Protein 2 / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
C493477:Slc17a6 protein, mouse; D016329:Sp1 Transcription Factor; D050599:Vesicular Glutamate Transport Protein 2; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D005947:Glucose