Insights into glutamate transport regulation in human astrocytes: Cloning of the promoter for excitatory amino acid transporter 2 (EAAT2)

doi:10.1073/pnas.0136555100

Insights into glutamate transport regulation in human astrocytes: Cloning of the promoter for excitatory amino acid transporter 2 (EAAT2)

Departments of ^*Pathology, ^‡Neurosurgery, and ^§Urology, Herbert Irving Comprehensive Cancer Center, College of Physicians and Surgeons, Columbia University, New York, NY 10032; and ^†Molecular Virology Division, St. Luke's/Roosevelt Hospital Center, Columbia University, New York, NY 10582

Communicated by Allan H. Conney, Rutgers, The State University of New Jersey–New Brunswick, Piscataway, NJ (received for review May 21, 2002)

Abstract

Glutamate transport is central to neurotransmitter functions in the brain. Impaired glutamate transport induces neurotoxicity associated with numerous pathological processes, including stroke/ischemia, temporal lobe epilepsy, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, HIV-1-associated dementia, and growth of malignant gliomas. Excitatory amino acid transporter-2 (EAAT2) is a major glutamate transporter in the brain expressed primarily in astrocytes. We presently describe the cloning and characterization of the human EAAT2 promoter, demonstrating elevated expression in astrocytes. Regulators of EAAT2 transport, both positive and negative, alter EAAT2 transcription, promoter activity, mRNA, and protein. These findings imply that transcriptional processes can regulate EAAT2 expression. Moreover, they raise the intriguing possibility that the EAAT2 promoter may be useful for targeting gene expression in the brain and for identifying molecules capable of modulating glutamate transport that could potentially inhibit, ameliorate, or prevent various neurodegenerative diseases.

sequential progressive genomic scanning‖nuclear run-on assays‖promoter reporter assays‖mRNA and protein expression

Footnotes

↵ ¶ To whom correspondence should be addressed. E-mail: pbf1{at}columbia.edu.
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF510107).
Abbreviations:

EAAT,

excitatory amino acid transporter;

EAAT2-Prom,

EAAT2 promoter;

PHFA,

primary normal human fetal astrocytes;

PDTC,

pyrrolidinedithiocarbamate;

PKA,

protein kinase A;

EGF,

epidermal growth factor;

TGF-α,

transforming growth factor α;

TNF-α,

tumor necrosis factor α;

BAC,

bacterial artificial chromosome;

dBcAMP,

dibutyryl cAMP;

PI-3K,

phosphatidylinositol 3-kinase