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- Title
Transcriptional regulation of human excitatory amino acid transporter 1 (EAAT1): cloning of the EAAT1 promoter and characterization of its basal and inducible activity in human astrocytes.
- Authors
Kim, Seon-young; Choi, So-young; Chao, Wei; Voisky, David J.
- Abstract
Excitatory amino acid transporter 1 (EAAT1) is one of the two glial glutamate transporters that clear the extracellular glutamate generated during neuronal signal transmission. Here, we cloned and characterized a 2.1-kb promoter region of human EAAT1 and investigated its function in the transcriptional regulation of the EAAT1 gene in human primary astrocytes. The full-length promoter region lacked TATA and CCAAT boxes and an initiator element, it contained several potential transcription factor-binding sites and it exhibited promoter activity in primary astrocytes and in several types of transformed cells. Consecutive 5′-deletion analysis of the EAAT1 promoter indicated the presence of negative and positive regulatory regions and a putative core promoter between −57 bp and +20 bp relative to the transcription start site (TSS). The core promoter contained a single GC-box in position −52/−39 and one E-box near the TSS and the GC-box site that was responsible for 90% of the basal promoter activity as determined by mutational analysis. Electrophoretic mobility shift, supershift and competition assays demonstrated binding of stimulating proteins (Sp) 1 and 3 to the GC-box and upstream stimulating factor (USF) 1 to the E-box. Treatment of primary human astrocytes with cellular modulators 8-bromo cyclic AMP and epidermal growth factor increased EAAT1 promoter activity in transient transfection assays and increased cellular EAAT1 mRNA expression and glutamate uptake by astrocytes. Conversely, tumor necrosis factor-α reduced both EAAT promoter activity and cellular EAAT1 mRNA expression. These results enable studies of transcriptional regulation of EAAT1 gene at the promoter level.
- Subjects
AMINO acids; ASTROCYTES; MOLECULAR genetics; NEURAL transmission; GENETIC transcription; MOLECULAR cloning
- Publication
Journal of Neurochemistry, 2003, Vol 87, Issue 6, p1485
- ISSN
0022-3042
- Publication type
Article
- DOI
10.1046/j.1471-4159.2003.02128.x