Gα13-Mediated β2 Integrin Outside-in Signaling in Neutrophil Migration
Authors: Chang Claire W , Cheng Ni , Skidgel Randal , Bai Yanyan , Du Xiaoping 
Published: November 2021

Abstract
Transendothelial migration of neutrophils requires chemoattractant signals and also integrin family of adhesion receptors, particularly the β 2 family of integrins, including Mac-1 and LFA-1. Signals transmitted by G protein-coupled receptors (GPCR) for chemoattractants and cytokines induces inside-out signaling activating the ligand binding function of integrins. Conversely ligand binding to integrins stimulates outside-in signaling, leading to cell spreading, retraction and migration. The heterotrimeric G protein subunit, Gα13, is important for GPCR signaling leading to RhoA activation but also binds to integrins, including β2 integrins to stimulate outside-in signaling. To study the roles of Gα13 in neutrophil migration, we tested the effect of Gα13 knockout on transendothelial migration of neutrophils stimulated by chemoattractant fMIVIL. We demonstrate that transendothelial migration of Gα13 knockout neutrophils was significantly but partially reduced as compared with wild type mice. Transendothelial migration of Gα13 knockout neutrophils is similar to wild type neutrophil migration neutralized with an anti-Mac1 (anti-αm) antibody, and was not further inhibited by the anti-Mac1 antibody, suggesting that transendothelial migration mediated by integrin αmβ2 was predominantly Gα13-dependent. Interestingly, either anti-β2 antibody or anti-LFA1 (anti-αL) antibody appeared to inhibit transendothelial migration of not only wild type neutrophils, but also to a degree, Gα13-knockout neutrophils, suggesting a minor LFA1-dependent but Gα13-independent component of transendothelial migration in addition to the Gα13-dependent transendothlial migration. Furthermore, even though the fibrinogen and ICAM-1 are both β2 ligands, we show that more neutrophils migrated through ICAM-1-coated transwells than fibrinogen-coated transwells, and only ICAM-1-mediated neutrophil migration is Gα13 dependent, suggesting that Gα13-dependent neutrophil migration is selective for certain β2 integrin ligand (ICAM-1). Importantly, Gα13 knockout selectively inhibited the velocity of neutrophil migration on integrin ligand ICAM-1, but had no effect on the directionality of neutrophil migration which requires GPCR-dependent chemoattactant signaling. To understand whether and how Gα13 regulate integrin signaling, we show that Gα13 knockout did not affect the static adhesion of neutrophils to ICAM1, but significantly inhibited neutrophil spreading on ICAM-1. Furthermore, Gα13 bound to β2 integrins in neutrophils adherent on ICAM-1, and this binding was inhibited by the ExE motif peptide MB2mP6 derived from the Gα13 binding site of β2 integrin cytoplasmic domain. MB2mP6 also inhibited transendothelial cell migration similarly as Gα13 knockout. These data suggest that Gα13 plays an important role in promoting β2-integrin dependent neutrophil transendothelial migration mainly by mediating integrin outside-in signaling. Consistent with previous findings of the role of Gα13-dependent outside-in signaling in negative regulation of RhoA in other integrin subtypes, both Gα13 knockout and MB2mP6 abolished the transient inhibition in RhoA during adhesion of neutrophils on ICAM-1. These data suggest that Gα13 mediates outside-in signaling and transient inhibition of RhoA, and thus promotes neutrophil spreading and migration on integrin ligands. To test the role of Gα13 in neutrophil migration in vivo, we showed that neutrophil infiltration in vivo was reduced in leukocyte-selective Gα13 knockout mice using both thioglycolate-induced peritoneal neutrophil infiltration model and LPS-induced neutrophil lung infiltration model in vivo. Furthermore, MB2mP6 inhibited neutrophil infiltration in cardiac tissues in the cardiac ischemia-reperfusion injury model in mice. These data suggest that Gα13-integrin interaction plays an essential role in the integrin-dependent transendothelial migration and is likely to be important in neutrophils' immune and inflammatory functions.

Disclosures
No relevant conflicts of interest to declare.