Identification of an oxygen responsive enhancer element in the glyceraldehyde-3-phosphate dehydrogenase gene

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is i nduced by hypoxia in endothelial cells (EC). Upregulation occurs primarily at the level of transcription and occurs to a much greater extent in EC tha n in other cell types. To characterize EC specific hypoxia response element s within the GAPDH gene, we performed transient transfection studies in EC, fibroblasts and smooth muscle cells using portions of the GAPDH promoter l inked to a CAT reporter gene. These initial studies identified an EC specif ic hypoxia responsive region that was further characterized (using SV40-pro moter-CAT reporter constructs) as a 19-nucleotide sequence (-130 to -112) c ontaining both an hypoxia inducible factor-1 (HIF-1)-binding site and a nov el flanking sequence. Electrophoretic mobility shift assays confirmed induc ible EC protein binding to this fragment. Mutation of either the HIF-1-bind ing site or the flanking sequence resulted in complete loss of function and loss of inducible protein binding. Thus, a single HIF-1-binding site is ne cessary, but not sufficient, for hypoxic regulation of GAPDH in EC, Further more, the novel HIF-1 flanking sequence required for GAPDH upregulation and the protein(s) that bind to it may be EC specific. (C) 1999 Elsevier Scien ce B.V. All rights reserved.