Live Animal Imaging of Calcium Signaling During Transendothelial Migration Demonstrates Tight Temporospatial Regulation Coordinated by Endothelial Cell TRPC6, IQGAP1, and CaMKIIδ

Abstract

Proper regulation of inflammatory cell recruitment is critical to fighting infection and maintaining organism homeostasis. Misdirected inflammation can exacerbate pathology and underlies the pathology of virtually all diseases including arthritis, atherosclerosis, and multiple sclerosis. 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. As such, the regulation of TEM represents a key target for potential therapeutic intervention. Our lab has previously reported that the divalent cation channel TRPC6 was responsible for the increase in cytosolic free calcium (↑[Ca2+]i) in endothelial cells that is required for TEM. TRPC6 is activated downstream of PECAM engagement and is concentrated at the site of leukocyte-endothelial contact during TEM. However, the temporal and spatial regulation of ↑[Ca2+]i during TEM was not known. To visualize calcium signaling during TEM in live animals we developed an intravital microscopy system using the GCaMP3 reporter mouse. In this model, the fluorescence of a modified GFP construct increases with calcium concentration. Indeed, during leukocyte TEM we observed a highly localized increase in endothelial calcium signal surrounding the transmigrating leukocyte that corresponded temporally to the transmigration event. This is the first demonstration of endothelial cell calcium signaling during TEM in vivo. Our lab has also previously reported that the scaffolding protein IQ-motif containing GTPase Activating Protein 1 (IQGAP1) in endothelial cells is required for TEM and functions downstream of PECAM engagement. Here we show that the signals propagated by TRPC6 and IQGAP1 converge into one pathway that results in the activation of calmodulin kinase II δ (CaMKIIδ) that is required for TEM. More specifically, we show that the IQ domain of IQGAP1 binds calmodulin (CaM), a known calcium binding protein. This interaction is required for the downstream activation of CaMKIIδ, which binds to CaM in a calcium-dependent manner. Disruption of either the IQGAP1-CaM or the CaM-CaMKIIδ interaction substantially reduces TEM in vitro. To further validate these findings, we developed two separate genetically-altered mouse lines: one with an inducible endothelial-specific deletion of CaMKIIδ, the other with an inducible endothelial-specific expression an intracellular CaMKIIN inhibitory peptide. Both mice showed significantly reduced TEM in the croton oil dermatitis model of inflammation compared to uninduced controls, confirming that endothelial CaMKIIδ is required for efficient leukocyte TEM in vivo. Overall, these findings uncover a new role for CaMKIIδ and novel connections between it and TRPC6, IQGAP1, and CaM in coordinating the calcium signal that facilitates leukocyte TEM. These results also identify CAMKIIδ as a potential target for anti-inflammatory therapeutics.