Calcium Signaling Regulates Leukocyte Transendothelial Migration through the Action of Endothelial Cell IQGAP1, Calmodulin, and CaMKIIδ
Authors: Sullivan David P. , Dalal Prarthana J. , Sacks David B. , Muller William A. 
Published: April 2020

When properly contained, inflammation leads to the recruitment and activation of circulating leukocytes to restore tissue and organism homeostasis. Although this process is critical for successful wound healing and the elimination of pathogens and infections, misdirected inflammation can exacerbate pathology and cause substantial morbidity and mortality. Inflammation a fundamental process that underlies the pathology of virtually all diseases including arthritis, atherosclerosis, and multiple sclerosis. As such, the regulation of inflammation represents a key target for potential therapeutic intervention.The transendothelial migration (TEM) of leukocytes out of the bloodstream and into tissue is the rate‐limiting and putative committed step of the inflammatory process. We recently identified the scaffolding protein IQ‐motif containing GTPase Activating Protein 1 (IQGAP1) in a proteomic screen for factors with a potential role in TEM. Knockdown of endothelial IQGAP1 substantially reduced leukocyte TEM. Using IQGAP1 domain truncation constructs we found that the IQ domain is required for its function in TEM. In other systems, the IQ domain binds calmodulin (CaM), a known calcium binding protein. Our group has also shown that a transient calcium influx, mediated by TRPC6, is required to support TEM. We hypothesized that IQGAP1 integrates the calcium signaling from TRPC6 through calmodulin to mediate TEM.We first confirmed that calcium signaling occurs during TEM using live animal intravital microscopy and a GCaMP3 reporter mouse. In this mouse, the fluorescence of a modified GFP construct increases with calcium concentration. Indeed, during leukocyte TEM we observed a local increase in endothelial calcium signal surrounding the transmigrating leukocyte. In vitro we then confirmed that IQGAP1 interacts with CaM via its IQ motif domain. We suspected that CaM propagated the calcium signal through calmodulin kinase II (CaMKII), an important CaM‐regulated kinase. Here we show that CaM interacts with CaMKIIδ in a calcium‐dependent manner. Both the inhibition of CaM with trifluoperazine and disruption of the CaM‐CaMKII interaction with the inhibitor KN‐93 caused a marked reduction in TEM. Similarly, the expression of both a dominant negative CaMKIIδ and CaMKIIN, a peptide inhibitor of CaMKII, substantially reduced leukocyte TEM in vitro.To further validate these findings, we developed two separate mouse models: one with an endothelial‐specific deletion of CaMKIIδ, the other with an endothelial‐specific expression of the CaMKIIN inhibitory peptide used in vitro. Both animal models showed significantly reduced TEM in the croton oil dermatitis model of inflammation confirming that endothelial CaMKIIδ is required for efficient leukocyte TEM in vivo. Overall, these findings uncover a novel connection between IQGAP1, CaM, and CaMKIIδ in coordinating the calcium signal that facilitates leukocyte TEM.Support or Funding InformationThis work was supported by NIH R01 HL046849 and R37 HL064774 to W.A.M.; F30HL134202, T32GM8152, and an Alpha Omega Alpha Carolyn L. Kuckein Student Research Fellowship to P.J.D.; and the Intramural Research Program of the NIH to D.B.S.