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J Biol Chem, Vol. 273, Issue 47, 30870-30878, November 20, 1998
From the Division of Surgical Science, Department of Surgery, and
Departments of Physiology and Medicine, Columbia University College of
Physicians and Surgeons, New York, New York 10032
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.
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