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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
Author(s) -
Kim SeonYoung,
Choi SoYoung,
Chao Wei,
Volsky David J.
Publication year - 2003
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2003.02128.x
Subject(s) - biology , microbiology and biotechnology , promoter , transcription factor , tata box , caat box , gene expression , gene , biochemistry
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.