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.