Genetic deletion of the Rho GEF Net1 impairs mouse macrophage motility and actin cytoskeletal organization

Abstract

Macrophages are innate immune cells that constantly patrol an organism to fulfill protective and homeostatic roles. Previous studies have shown that Rho GTPase activity is required for macrophage mobility, yet the roles of upstream regulatory proteins controlling Rho GTPase function in these cells are not well defined. Previously we have shown that the RhoA GEF Net1 is required for human breast cancer cell motility and extracellular matrix invasion. To assess the role of Net1 in macrophage motility, we isolated bone marrow macrophage (BMM) precursors from wild type and Net1 knockout mice. Loss of Net1 did not affect the ability of BMM precursors to differentiate into mature macrophages in vitro, as measured by CD68 and F4/80 staining. However, Net1 deletion significantly reduced RhoA activation, F-actin accumulation, adhesion, and motility in these cells. Nevertheless, similar to RhoA/RhoB double knockout macrophages, Net1 deletion did not impair macrophage recruitment to the peritoneum in a mouse model of sterile inflammation. These data demonstrate that Net1 is an important regulator of RhoA signaling and motility in mouse macrophages in vitro, but that its function may be dispensable for macrophage recruitment to inflammatory sites in vivo.

Keywords: Net1; RhoA; actin; adhesion; macrophage; motility.

Figure 1.

Net1 deletion impairs RhoA activation in mouse bone marrow macrophages. (A) Net1 mRNA expression in M-CSF differentiated mouse bone marrow macrophages (BMMs). (B, C) Representative examples of F4/80 and CD68 expression in wild type (B) and Net1 KO (C) BMMs. (D) Quantification of the percent of cells with CD68 and F4/80 expression in differentiated BMMs. (E) Representative example of RhoA activity in BMMs, as measured by GST-RBD pulldown. (F) Quantification of RhoA activity in BMMs. For all plots, bars = median values. n.s. = not significant. ** = p < 0.01. *** = p < 0.001.

Figure 2.

Net1 deletion reduces F-actin content in BMMs. (A) Representative images of pMLC2 staining of BMMs from wild type and Net1 KO mice. Scale bar is 10 µm. (B) Quantification of pMLC2 staining in multiple fields from three different BMM isolates for each genotype. (C) Western blot for pS19-MLC2 and total MLC2 in three different isolates of wild type and Net1 knockout BMMs. (D) Quantification of pS19-MLC2/MLC2 Western blots. (E) Representative images of F-actin staining in BMMs from wild type and Net1 KO mice. Scale bar is 10 µm. (F) Quantification of F-actin staining in multiple fields from three different BMM isolates for each genotype. (G) Western blot for pS3 cofilin and total cofilin in three different isolates of wild type and Net1 knockout BMMs. (H) Quantification of pS3-cofilin/cofilin Western blots. Bars are median values for all plots. n.s. = not significant. * = p < 0.05. *** = p < 0.001.

Figure 3.

Net1 deletion impairs BMM adhesion and motility, but does not affect Matrigel invasion or peritoneal recruitment. (A) Adhesion of wild type and Net1 KO BMMs to fibronectin 30 minutes after plating. (B) Migration of wild type and Net1 KO BMMs in transwell inserts with MCP-1 in the bottom well. (C) Invasion of wild type and Net1 KO BMMs through Matrigel coated transwell inserts with MCP-1 in the bottom well. For all experiments, three different BMM isolates were assayed in triplicate for each genotype. (D) Representative examples of F4/80 and CD68 expressing cells in peritoneal isolates from wild type and Net1 KO mice three days after thioglycollate injection. (E, F, G) Quantification of F4/80⁺CD68⁺ (E), CD45⁺ (F), and CD45⁺CD11b⁺ cells in peritoneal isolates from thioglycollate injected wild type and Net1 KO mice. For all plots, bars = median values. n.s. = not significant; ** = p < 0.01; *** = p < 0.001.