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.
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-alpha reduced both EAAT promoter activity and cellular EAAT1 mRNA expression. These results enable studies of transcriptional regulation of EAAT1 gene at the promoter level.
Kim
Seon-Young
SY
Molecular Virology Division, St. Luke's-Roosevelt Hospital Center and Columbia University, New York, USA.
Choi
So-Young
SY
Chao
Wei
W
Volsky
David J
DJ
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003563:Cyclic CMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D004268:DNA-Binding Proteins; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004815:Epidermal Growth Factor / Q000494:pharmacology; D027341:Excitatory Amino Acid Transporter 1 / Q000235:genetics* / Q000378:metabolism; D005333:Fetus; D005786:Gene Expression Regulation; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives* / Q000494:pharmacology; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D024363:Transcription Initiation Site / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology*; D015533:Transcriptional Activation; D014162:Transfection; D016211:Transforming Growth Factor alpha / Q000494:pharmacology; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; D027341:Excitatory Amino Acid Transporter 1; D014157:Transcription Factors; D016211:Transforming Growth Factor alpha; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors; C497183:Usf1 protein, mouse; C497182:Usf1 protein, rat; D015124:8-Bromo Cyclic Adenosine Monophosphate; D003563:Cyclic CMP; D018698:Glutamic Acid; C020922:4-O-methyl-12-O-tetradecanoylphorbol 13-acetate; D004815:Epidermal Growth Factor; D012313:RNA; C053441:dibutyryl cyclic-3',5'-cytidine monophosphate; D008156:Luciferases; D013755:Tetradecanoylphorbol Acetate