Characterization and functional analysis of the promoter of RAGE, the receptor for advanced glycation end products.
The receptor for advanced glycation end products, RAGE, is a member of the immunoglobulin superfamily of cell surface molecules differentially expressed on a range of cell types. Ligation of RAGE perturbs homeostatic mechanisms and, potentially, provides a basis for cellular dysfunction in pathologic situations in which its ligands accumulate. To understand factors underlying RAGE expression, we cloned the 5'-flanking region of the RAGE gene and characterized putative regulatory motifs. Analysis of the putative promoter region revealed the presence of three potential NF-kappaB-like and two SP1 binding sites. Transient transfection of vascular endothelial and smooth muscle cells using chimeric 5'-deletion constructs linked to luciferase reporter revealed that the region -1543/-587 contributed importantly to both basal and stimulated expression of the RAGE gene. This region of the RAGE gene contained three putative NF-kappaB-like binding sites and was responsible for increased luciferase activity observed when endothelial or smooth muscle cells were stimulated with lipopolysaccharide. DNase I footprinting assays and electrophoretic mobility shift assay revealed that two of the three NF-kappaB-like binding sites (1 and 2) were likely functional and responsive to stimuli. Upon simultaneous mutation of NF-kappaB-like sites 1 and 2, both basal promoter expression and response to stimulation with LPS, as measured by relative luciferase activity, were significantly diminished. These results point to NF-kappaB-dependent mechanisms regulating cellular expression of RAGE and suggest a means of linking RAGE to the inflammatory response.
Li
J
J
Departments of Physiology, Medicine and Surgery, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
Schmidt
A M
AM
D001483:Base Sequence; D001665:Binding Sites; D008070:Lipopolysaccharides / Q000494:pharmacology; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000235:genetics*; D016385:TATA Box
D008070:Lipopolysaccharides; D016328:NF-kappa B; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic