Requirement for a Complex of Wiskott-Aldrich Syndrome Protein (WASP) with WASP Interacting Protein in Podosome Formation in Macrophages

Abstract

Chemotactic migration of macrophages is critical for the recruitment of leukocytes to inflamed tissues. Macrophages use a specialized adhesive structure called a podosome to migrate. Podosome formation requires the Wiskott-Aldrich syndrome protein (WASP), which is a product of the gene defective in an X-linked inherited immunodeficiency disorder, the Wiskott-Aldrich syndrome. Macrophages from WASP-deficient Wiskott-Aldrich syndrome patients lack podosomes, resulting in defective chemotactic migration. However, the molecular basis for podosome formation is not fully understood. I have shown that the WASP interacting protein (WIP), a binding partner of WASP, plays an important role in podosome formation in macrophages. I showed that WASP bound WIP to form a complex at podosomes and that the knockdown of WIP impairs podosome formation. When WASP binding to WIP was blocked, podosome formation was also impaired. When WASP expression was reduced by small interfering RNA transfection, the amount of the complex of WASP with WIP decreased, resulting in reduced podosome formation. Podosomes were restored by reconstitution of the WASP-WIP complex in WASP knockdown cells. These results indicate that the WASP-WIP complex is required for podosome formation in macrophages. When podosome formation was reduced by blocking WASP binding to WIP, transendothelial migration of macrophages, the most crucial process in macrophage trafficking, was impaired. These results suggest that a complex of WASP with WIP plays a critical role in podosome formation, thereby mediating efficient transendothelial migration of macrophages.

Wiskott-Aldrich Syndrome Protein Is a Key Regulator of the Phagocytic Cup Formation in Macrophages

“…WIP also binds directly to F-actin and inhibits Ccd42-mediated actin polymerization; it synergizes with N-WASP to induce filopodia when overexpressed in fibroblasts (39). A general role in filopodia formation is consistent with the requirement of WIP in pathogenic settings, notably the intracellular motility of vaccinia virus and Shigella, which involves the formation of an actin comet-tail mediated by the N-WASP⅐WIP complex (40), and the transendothelial migration of macrophages, which involves the formation of the podosomes mediated by the WASP⅐WIP complex (35,41).…”

“…The mouse cDNA contains 4 mismatches with the 19 bases of the human WASP siRNA sequence. When human WASP expression was silenced by the human WASP siRNA, mouse WASP was successfully expressed, and the mouse WASP expression reversed reduced formation of podosomes, the actin-based structures for macrophage migration, in the human WASP knockdown cells (35). FLAG-tagged mouse WASP was expressed in the WASP knockdown THP-1 cells (Fig.…”

“…Human primary monocytes were differentiated into macrophages by incubating with macrophage-colony stimulating factor-1 (35,43). In both PMA-differentiated THP-1 cells and human primary macrophages, typical phagocytic cups were observed in the presence of 3-MA, when cells were incubated with IgG-opsonized latex beads (Fig.…”

“…To do this, PMA-differentiated THP-1 cells were co-transfected with human WASP siRNA and mouse WASP cDNA and then examined for the phagocytic cup formation in the transfected cells. This experiment was designed based on our previous study (35). A mouse WASP cDNA and the human WASP siRNA were used for co-transfection experiments.…”

“…WIP is a widely expressed protein and is the best characterized of the mammalian verprolins. WIP plays a key role in regulating WASP, with which it appears to form a constitutive complex that functions as a unit in many cellular processes (26,27,35,36). For example, WIP is required for targeting WASP to the immune synapse after T-cell receptor ligation (6,17).…”