Sp1-binding Elements in the Promoter of RAGE Are Essential for Amphoterin-mediated Gene Expression in Cultured Neuroblastoma Cells

Abstract

Receptor for AGE (RAGE) and the polypeptide amphoterin are highly expressed and co-localized in neurons of the developing central nervous system of the rat. In vitro, the interaction of amphoterin with neuronal RAGE induces neurite outgrowth. We tested the hypothesis that interaction of amphoterin with neuronal cells enhances RAGE expression, thereby providing a mechanism by which amphoterin-mediated regulation of RAGE might contribute to promotion of neurite growth and spreading. Incubation of cultured neuroblastoma cells with amphoterin resulted in increased transcription and translation of RAGE, a process largely inhibited in the presence of anti-RAGE IgG but not by nonimmune IgG. To begin to delineate molecular mechanisms underlying these findings, we identified multiple putative binding elements within the 5-flanking region of the RAGE gene for Sp1, a transcription factor that has been critically linked to the process of normal development. DNase I footprinting and electrophoretic mobility shift assays demonstrated multiple functional Sp1-binding sites within the region ؊245 to ؊40 of the RAGE promoter. Transient transfection of cultured SK-N-SH neuroblastoma cells with chimeric 5-deletion constructs linked to luciferase reporter revealed that the region containing Sp1-binding elements did not contribute uniquely to basal expression of the RAGE gene. Simultaneous mutation of the multiple Sp1-binding elements in this region did not affect basal promoter function; however, promoter responsiveness to amphoterin was markedly attenuated. These results point to Sp1-dependent mechanisms underlying amphoterin-mediated increases in RAGE expression in neuroblastoma cells and further link amphoterin-RAGE interaction to development of the nervous system.The receptor for AGE (RAGE), 1 a member of the immunoglobulin superfamily of cell-surface molecules, interacts with a range of ligands (1-2). In pathophysiologic circumstances, advanced glycation end products (AGEs) result from nonenzymatic glycation and oxidation of proteins and lipids and accumulate in conditions such as diabetes, renal failure, and amyloidoses (3-4). AGEs interact with RAGE in a manner linked to perturbation of vascular and inflammatory cells (5-9). In addition, RAGE interacts with amyloid ␤-peptide, consequences of which include enhanced neuronal and microglial oxidant stress and dysfunction (10 -11). These processes may be important in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease. The expression of RAGE is strikingly enhanced in cells affected in settings associated with increased accumulation of AGEs and amyloid ␤-peptide (12-14). We recently identified that an important means by which the expression of RAGE might be regulated in inflammatorytype milieu such as diabetic vasculature and wounds, and Alzheimer brain, is via two functional NF-kB-binding elements located with the 5Ј-flanking region of the RAGE gene (15). Transfection of 5Ј-deletion luciferase reporter gene constructs containing two mutated NF-kB-binding s...

Receptor for Advanced Glycation End Products (RAGE)-mediated Neurite Outgrowth and Activation of NF-κB Require the Cytoplasmic Domain of the Receptor but Different Downstream Signaling Pathways

“…Interestingly, the analysis of the promoter region of RAGE gene revealed the presence of two functional NF-B-binding sites (46) suggesting a possible autoregulatory loop in the regulation of RAGE expression. However, when this manuscript was under preparation a finding was published (47) showing that amphoterin-induced increase in RAGE expression is mediated by binding of amphoterin to RAGE resulting in Sp1 activation rather than activation of NF-B. Soluble amphoterin was used in their study, whereas we have used matrix-bound amphoterin.…”

Central role of RAGE-dependent neointimal expansion in arterial restenosis

“…We speculate that enhanced MPO activity within the first hour after injury, induced at least in part by polymorphonuclear leukocytes attracted immediately to the injured arterial segment, triggers oxidant stress (39), thereby providing a definitive mechanism leading to formation of AGEs (38)(39)(40). We hypothesize that once formed, AGE ligands of the receptor likely stimulate enhanced expression of RAGE itself (55,56). In parallel, studies have suggested that acute injury, as well as proinflammatory stimuli, may enhance expression of S100/calgranulin ligands of RAGE (34-37), thus providing a mechanism for enhanced RAGE expression, as well.…”

HMGB1 and RAGE in Inflammation and Cancer

“…RAGE expression increases in circumstances when ligands, such as AGEs, and inflammatory mediators accumulate (106,116). Because the RAGE promoter contains multiple functional NF-κB and SP-1 transcription factor-binding sites (117,118), ligands and proinflammatory cytokines can promote the expression of RAGE, potentially triggering a receptor-dependent autoinflammatory loop.…”