Telomere-independent Rap1 induces cytokine production in pro-inflammatory macrophages through NFκB signaling

Abstract

Repressor activator protein 1 (Rap1), an established telomere-associated protein, is essential for maintaining telomere length and structural integrity, but it also exerts other non-telomeric functions. The present study tested the hypothesis that Rap1 is released into the cytoplasm and induces production of pro-inflammatory cytokines via nuclear factor kappa B (NFκB) signaling in macrophages, a cell type involved in the development and progression of atherosclerotic lesions. Western blotting analysis confirmed that Rap1 was present in the cytoplasm of differentiated human monocytic leukemia cells (THP-1, a macrophage-like cell line). Co-immunoprecipitation assay revealed a direct interaction between Rap1 and I kappa B kinase (IKK). Knockdown of Rap1 significantly suppressed lipopolysaccharide-mediated activation of NFκB (by 39.0%), and phosphorylation of inhibitor of kappa B α (IκBα) and p65 in THP-1 macrophages (by 52.7% and 32.0%, respectively). The reduction of NFκB activity was paralleled by a decreased production of NFκB-dependent pro-inflammatory cytokines [including interleukin (IL)-8, IL-1β and monocyte chemotactic protein-1] and an increased expression of IκBα (native NFκB inhibitor) in various macrophage models with pro-inflammatory phenotype, including THP-1, mouse peritoneal macrophages and bone marrow-derived M1 macrophages. These changes were observed selectively in pro-inflammatory macrophages but not in bone marrow-derived M2 macrophages (with an anti-inflammatory phenotype). Furthermore, in human atheroma, endothelial and smooth muscle cells elaborate cytokines and contribute to the overall inflammatory environment. However, knockdown or knockout of Rap1 does not influence the expression of NFκB-dependent targets in human umbilical vein endothelial cells, human aortic smooth muscle cells or mouse lung endothelial cells. Immunostaining revealed that Rap1 was localized mainly in macrophage-rich areas rather than in endothelial or smooth muscle cell-rich areas in human atherosclerotic plaques and that the presence of Rap1 was positively correlated with the advancement of the disease process. In conclusion, in pro-inflammatory macrophages, Rap1 promotes cytokine production via NFκB activation favoring a pro-inflammatory environment which may contribute to the development and progression of atherosclerosis.