Comparative structural and functional analysis of the GLT-1/EAAT-2 promoter from man and rat.

In the vertebrate CNS, glutamate transport predominantly occurs through the glutamate transporter subtype, GLT-1/EAAT-2, which prevails in astrocytes. GLT-1/EAAT-2 expression is impaired in many acute and chronic brain diseases, leading to increases in extracellular glutamate and subsequent excitotoxic neuronal cell death. An obvious therapeutical approach to prevent glutamate-induced brain damage would be targeting GLT-1/EAAT-2 expression. Since so far, insights into the mechanisms modulating GLT-1/EAAT-2 expression mostly originated from work with rat astrocytes, we now sought to determine whether this modulatory network would also apply to humans. To this end, we have cloned the previously unknown rat GLT-1/EAAT-2 promoter and compared it to the human promoter sequence. In reporter assays, the cloned 2.7-kb region immediately flanking the 5'-end of the rat GLT-1/EAAT-2 gene allowed for similar increases in constitutive gene expression as the human promoter sequence. Sequence analysis demonstrated the presence of highly conserved regions on the rat and human GLT-1/EAAT-2 promoters, which turned out to be likewise essential for constitutive GLT-1/EAAT-2 expression, stimulation of gene transcription by EGF, TGFalpha, and PACAP as well as inhibition of gene transcription by TNFalpha. Intriguingly, endothelin-1 which inhibits endogenous GLT-1/EAAT-2 expression, promoted activity of both rat and human reporter constructs, indicating the existence of (an) inhibitory mechanism(s) not operational in the reporter gene assay. Our findings establish close similarities in the regulation of GLT-1/EAAT-2 expression in rat and man and, hence, validate rat astrocytes as an assay system for studying the molecular mechanisms affecting glutamate homeostasis in the healthy and diseased human brain. (c) 2009 Wiley-Liss, Inc.