Abstract 356: Sam68 Promotes NF-kB Signaling and Inflammation in Macrophages and Impedes the Recovery of Arterial Injury

Abstract

Background: The role of Src-associated-in-mitosis-of-68-kDa (Sam68) protein in vascular biology has not been studied. A recent report suggests that Sam68 suppresses TNF-a-mediated NF-kB activation in embryonic fibroblasts. Since NF-kB plays a critical role in vascular inflammation and injury, we sought to dissect the molecular mechanism by which Sam68 regulates NF-kB signaling and its functional significance in vascular injury. Methods & Results: The endothelial denudation injury was induced in the carotid arteries of Sam68 -/- mice and WT littermates. Sam68 -/- mice displayed a significantly accelerated re-endothelialization and attenuated neointimal hyperplasia, which was associated with reduced macrophage infiltration and lowered expression of pro-inflammatory cytokines (i.e., TNF-a, MCP-1, IL-1, and IL-6) in the injured vessels. The improved carotid recovery in Sam68 -/- mice was recapitulated in WT mice that had received Sam68 -/- bone-marrow (BM) transplantation, suggesting that Sam68 impedes vascular recovery primarily by its function in BM cells, likely pro-inflammatory cells. In cultured Raw264.7 macrophages, knockdown of Sam68 resulted in a significant reduction in the TNF-a-induced expression of pro-inflammatory cytokines and in the level of P-IKKαβ, P-IkBa (in cytosol) and P-P65 (in nucleus), indicating attenuated NF-kB activation. These results were confirmed in peritoneal and BM-derived macrophages from Sam68 -/- and WT mice. Furthermore, Sam68 co-immunoprecipitation and mass-spectrometric analyses identified cytoskeleton protein Filamin A (FLNA) as a novel Sam68-interacting protein in response to TNF-a stimulation. Reverse co-immunoprecipitation and truncational mutagenesis confirmed that Sam68-FLNA interactions require the N-terminus of Sam68. Finally, analyses in Raw264.7 cells with FLNA knockdown revealed that the effects of Sam68 on TNF-a[[Unsupported Character - Codename ­]]-induced NF-kB signaling and pro-inflammatory cytokine expression were dependent on FLNA. Conclusions: Our data suggest that Sam68 promotes pro-inflammatory response in injured arteries and impedes recovery, and this effect may be attributable, at least partially, to the exaggerated NF-kB activity in macrophages.