The role of IQGAP1 in transendothelial migration: from in vitro identification to in vivo validation

Abstract

Although inflammation is the body's stereotyped response to tissue damage, the clinical outcomes of this complex process, whether beneficial or detrimental, are rooted in the recruitment and subsequent function of the leukocytes at site of insult. It therefore follows that the regulation of leukocyte recruitment represents an acutely tractable target for therapeutic intervention. In order to reach the target tissue, leukocytes must undergo a carefully choreographed series of sequential intercellular interactions with the endothelium which culminate in transendothelial migration (TEM). As the leukocyte prepares to traverse the endothelial cell layer requisite protein‐protein interactions between the two cells directs the recruitment of the endothelial cell sub‐junctional Lateral Border Recycling Compartment (LBRC). Several of the endothelial proteins crucial for this process reside in the LBRC and its function in TEM is required for efficient leukocyte efflux.To identify additional proteins that are involved in LBRC function in an unbiased approach, we adapted a biochemical purification method known as the diaminobenzidine‐induced density shift to specifically label and recover membrane fractions enriched the LBRC proteins. Through this process we identified IQ domain containing GTPase Activating Protein 1 (IQGAP1) as a previously unknown LBRC associated proteins. IQGAP1 is a cytoplasmic multidomain scaffolding protein that has been shown in other systems to integrate and propagate complex signaling pathways. Genetic ablation of IQGAP1 in endothelial cell cultures using shRNA transfection substantially reduced their ability to support efficient monocyte TEM. Re‐expression of full‐length IQGAP1 rescued this defect, confirming its essential function in this process. Interestingly, re‐expression of IQGAP1 constructs that lack the actin‐binding or IQ‐motif domains were not able to rescue the TEM defect shRNA mediated reduction of IQGAP1 expression did not affect the physical amount of LBRC membrane that could be isolated from endothelial cells suggesting instead that IQGAP1 plays a role in some signaling aspect or other function of the LBRC. To extend these studies in vivo we created bone marrow chimeras (i.e. wild type leukocytes in IQGAP1 knockout animals) and investigated the requirement of endothelial cell IQGAP1 in inflammation. We used two separate mouse models; croton oil dermatitis visualized with conventional confocal microscopy and IL‐1β induced inflammation in the cremaster muscle visualized by 4D intravital microscopy. In both models the ability of neutrophils to extravasate across the endothelium into inflamed tissues was significantly inhibited in animals that lacked IQGAP1. Together with our in vitro studies, these findings provide important insight into the critical role of endothelial cell IQGAP1 in leukocyte diapedesis.Support or Funding InformationThis research was supported by F32 AI084454 to DPS and R21 HL102519, R01 HL046849, and R37 HL064774 to WAM.