Abstract 18711: The Adaptor Protein Skap2 is Required for Integrin-Mediated Signaling in Macrophages
Background: Integrins mediate macrophage migration and phagocytosis, processes that are central to atherogenesis. The adaptor protein Skap2 is phosphorylated in response to integrin engagement in myeloid cells, but the manner in which Skap2 facilitates integrin signaling remains unclear. We hypothesized that Skap2 is required to propagate integrin-mediated signals in macrophages, leading to cytoskeletal reorganization and cell migration.
Methods and Results: Skap2-deficient bone marrow derived macrophages exhibit impaired actin polymerization upon plating on the integrin substrate fibrinogen (normalized F-actin content as measured by phalloidin staining, 1.6 ± 0.1 for Skap2+/− vs. 1.0 ± 0.1 for Skap2−/−, p<0.01). Furthermore, macrophages depend on Skap2 for migration in scrape assays and in transwell motility responses to chemoattractants (normalized migration index in response to CSF1, 2.7 ± 0.3 for Skap2+/− vs. 1.0 ± 0.1 for Skap2−/−; in response to CCL2, 2.6 ± 0.1 for Skap2+/− vs. 1.0 ± 0.1 for Skap2−/−; p<0.01). In a new assay of local actin polymerization upon ligating αV integrins with antibody-coated microbeads, the central role of Skap2 in integrin-mediated actin rearrangement is confirmed (actin ruffling index, 3.3 ± 0.6 for Skap2+/− vs. 1.0 ± 0.5 for Skap2−/−, with no response to control antibodies, n=10, p<0.01). Immunoprecipitation and immunoblotting experiments show that integrin-induced activation of Src-family kinases (SFK) is defective in the absence of Skap2, leading to impaired tyrosine phosphorylation of Syk, Vav1 and paxillin. Phosphorylation of the Skap2-associated molecules Adap and Sirpα is also diminished in Skap2−/− macrophages, although integrin-stimulated activation of Pyk2 and MAP kinases is normal, indicating that Skap2 participates selectively in signaling pathways downstream of integrins. Given the importance of macrophage-based integrin signaling in atherogenesis, future work will focus on preliminary data suggesting that Skap2 modulates this process in apoE−/− mice.
Conclusions: These data suggest that Skap2 controls cytoskeletal rearrangement and migration of macrophages in response to integrin engagement, most likely through SFK activation, with potential implications for atherogenesis.
- © 2010 by American Heart Association, Inc.