Understanding the regulation of the PI(3,4,5)P3-Cdc42 signaling axis in macrophage podosomes

Abstract

Podosomes are actin-rich adhesion structures that play a crucial role in macrophage migration through various tissues. Phosphatidylinositol and GTPase signaling are essential factors in regulating the WASP-dependent F-actin polymerization that drives podosome formation. However, the signaling crosstalk and causal relationships between these pathways remain unclear. In this study, we hypothesized that phosphatidylinositol biogenesis serves as the upstream signal to regulate GTPase activation. Our findings confirm that inhibition of PI3K significantly reduces Cdc42-GTP levels, and a WASP-3D mutant defective in Cdc42-GTP binding fails to participate in podosome assembly. To identify the PI(3,4,5)P3-dependent Cdc42 GEFs, we performed quantitative imaging analysis to examine the spatial distribution and PI(3,4,5)P3-induced membrane association of various Cdc42 GEFs. The results reveal that the VAV1 GEFs are promptly recruited to the plasma membrane in a PI(3,4,5)P3-dependent manner and are a critical factor in triggering podosome assembly. Taken together, these findings demonstrate that PI3K activation serves as the upstream signal to recruit the Cdc42-GEF VAV1. The resulting elevated levels of Cdc42-GTP then trigger WASP-mediated F-actin polymerization, podosome assembly, and, ultimately, enhanced macrophage migration. This study provides important insights into the signaling mechanisms underlying the dynamics of podosome structures and macrophage mobility. By elucidating the PI3K-VAV1-Cdc42 axis regulating podosome formation, these results offer potential therapeutic targets for modulating macrophage function in various disease contexts.