Abstract
A20 protects against pathologic vascular remodeling by inhibiting the inflammatory transcription factor NF-κB. A20's function has been attributed to ubiquitin editing of receptor-interacting protein 1 (RIP1) to influence activity/stability. The validity of this mechanism was tested using a murine model of transplant vasculopathy and human cells. Mouse C57BL/6 aortae transduced with adenoviruses containing A20 (or β-galactosidase as a control) were allografted into major histocompatibility complex-mismatched BALB/c mice. Primary endothelial cells, smooth muscle cells, or transformed epithelial cells (all human) were transfected with wild-type A20 or with catalytically inactive mutants as a control. NF-κB activity and intracellular localization of RIP1 was monitored by reporter gene assay, immunofluorescent staining, and Western blotting. Native and catalytically inactive versions of A20 had similar inhibitory effects on NF-κB activity (-70% vs. -76%; P > 0.05). A20 promoted localization of RIP1 to insoluble aggresomes in murine vascular allografts and in human cells (53% vs. 0%) without altering RIP1 expression, and this process was increased by the assembly of polyubiquitin chains (87% vs. 28%; P < 0.05). A20 captures polyubiquitinated signaling intermediaries in insoluble aggresomes, thus reducing their bioavailability for downstream NF-κB signaling. This novel mechanism contributes to protection from vasculopathy in transplanted organs treated with exogenous A20.