Abstract 353: Interaction of the RAGE Cytoplasmic Domain & Diaphanous-1: a Novel Receptor Signaling Mechanism Linked to Cardiovascular Disease
The Receptor for Advanced Glycation Endproducts (RAGE) has been linked to the pathogenesis of cardiovascular disease. How RAGE activates signal cascades leading to vascular dysfunction has yet to be fully elucidated. Here, we have identified the interacting signaling partner of the RAGE cytoplasmic domain. Using a yeast two-hybrid cDNA library, we identified the RAGE cytoplasmic tail bound Diaphanous-1 (Dia-1), a molecule that mediates intracellular signaling and cellular motility. Four key studies confirmed this interaction; first, in vivo, co-immunoprecipitation (IP) of RAGE/Dia-1 from RAGE expressing cells was demonstrated, but not from DN-RAGE (RAGE tail deleted) expressing cells. Second, interaction between epitope tagged RAGE tail and Dia-1 was confirmed in vivo by co-IP. Third, in vitro, RAGE tail pull-down of Dia-1 indicated the interaction between RAGE and Dia-1 was direct. Fourth, by confocal microscopy RAGE and Dia-1 was co-localized, with significantly less seen with DN-RAGE and Dia-1 expressing cells. The key test of the relevance of this interaction was confirmed by functional studies using Dia-1 siRNA knockdown or scramble siRNA as a control (confirmed by real-time PCR and by western blot). Critically, compared to transfected siRNA scramble control, Dia-1 siRNA transfection blocked RAGE ligand stimulated cellular migration and invasion (p<0.0001). Consistent with key roles for RAGE/Dia-1 interaction in the vasculature, we identified expression of Dia-1 and co-localization with RAGE in ECs, aortic SMC and monocyte-derived cells. In murine femoral artery denudation, striking upregulation and co-localization of RAGE/Dia-1 was noted. Western blot of arterial lysates revealed a time-dependent increase in Dia-1, with confocal microscopy confirming increased expression and co-localization of RAGE and Dia-1 in the injured femoral artery. Further, in apoE null mice, RAGE/Dia-1 was found co-localize and be upregulated in atherosclerotic plaques. In conclusion, these data identify a novel mechanism linking RAGE to Diaphanous-1, which may bridge RAGE-induced signal transduction and cellular motility. We conclude that blocking RAGE/Dia-1 interaction is a novel target for therapy in vascular disease.