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Sp1 is required for glucose-induced transcriptional regulation of mouse vesicular glutamate transporter 2 gene.

ABSTRACT: 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.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.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.We cloned the mouse VGLUT2 promoter and showed a novel molecular mechanism of glucose-induced mVGLUT2 transcription.

SUBMITTER: Li T

PROVIDER: S-EPMC2747381 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Sp1 is required for glucose-induced transcriptional regulation of mouse vesicular glutamate transporter 2 gene.

Li Tao T Bai Liqun L Li Jing J Igarashi Suzu S Ghishan Fayez K FK

Gastroenterology 20080229 7

<h4>Background & aims</h4>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.<h4>Methods</h4>A promoter region of mVGLUT2 was cloned by genomi ...[more]

PMID: 18440316

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Project description:Members of the superfamily of solute carrier (SLC) transmembrane proteins transport diverse substrates across distinct cellular membranes. Three SLC protein families transport distinct neurotransmitters into synaptic vesicles to enable synaptic transmission in the nervous system. Among them is the SLC17A6/7/8 family of vesicular glutamate transporters, which endows specific neuronal cell types with the ability to use glutamate as a neurotransmitter. The genome of the nematode Caenorhabditis elegans encodes three SLC17A6/7/8 family members, one of which, eat-4 /VGLUT, has been shown to be involved in glutamatergic neurotransmission. Here, we describe our analysis of the two remaining, previously uncharacterized SLC17A6/7/8 family members, vglu-2 and vglu-3 These two genes directly neighbor one another and are the result of a recent gene duplication event in C. elegans, but not in other Caenorhabditis species. Compared to EAT-4, the VGLU-2 and VGLU-3 protein sequences display a more distant similarity to canonical, vertebrate VGLUT proteins. We tagged both genomic loci with gfp and detected no expression of vglu-3 at any stage of development in any cell type of both C. elegans sexes. In contrast, vglu-2::gfp is dynamically expressed in a restricted set of distinct cell types. Within the nervous system, vglu-2::gfp is exclusively expressed in a single interneuron class, AIA, where it localizes to vesicular structures in the soma, but not along the axon, suggesting that VGLU-2 may not be involved in synaptic transport of glutamate. Nevertheless, vglu-2 mutants are partly defective in the function of the AIA neuron in olfactory behavior. Outside the nervous system, VGLU-2 is expressed in collagen secreting skin cells where VGLU-2 most prominently localizes to early endosomes, and to a lesser degree to apical clathrin-coated pits, the trans-Golgi network, and late endosomes. On early endosomes, VGLU-2 colocalizes most strongly with the recycling promoting factor SNX-1, a retromer component. Loss of vglu-2 affects the permeability of the collagen-containing cuticle of the worm, and based on the function of a vertebrate VGLUT1 protein in osteoclasts, we speculate that vglu-2 may have a role in collagen trafficking in the skin. We conclude that C. elegans SLC17A6/7/8 family members have diverse functions within and outside the nervous system.

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Sp1 is required for glucose-induced transcriptional regulation of mouse vesicular glutamate transporter 2 gene.

Publications

Sp1 is required for glucose-induced transcriptional regulation of mouse vesicular glutamate transporter 2 gene.

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