Chloride Intracellular Channel (CLIC) Proteins Regulate Endothelial G-Protein Coupled Receptor Signaling

Chloride intracellular channels (CLICs) are a family of six proteins proposed, based on structure, to function as ion channels or glutathione-S-transferases. CLIC1 and CLIC4 are expressed in endothelial cells and are vital for angiogenesis. CLICs respond to G protein-coupled receptor (GPCR) agonists such as sphingosine-1-phosphate (S1P) by undergoing relocalization to the cell surface membrane. We hypothesized that CLICs function in endothelial GPCR signaling and function. Using loss of function studies, we found that reduced expression of either CLIC1 or CLIC4 abrogated S1P receptor 1 (S1PR1) -mediated endothelial barrier enhancement and Rac1 activation in human umbilical vein endothelial cells (HUVECs). CLIC1, but not CLIC4, was required for S1PR2/S1PR3 -mediated actin stress fiber formation and RhoA activation. We expanded our studies to investigate CLIC function downstream of protease-activated receptors (PARs), a class of GPCRs that respond to thrombin. Activation of PAR1 in HUVEC by thrombin promoted the cell membrane localization of CLIC4, but not CLIC1. CLIC4 was required for thrombin-mediated endothelial barrier disruption and RhoA activation. While CLIC1 did not function in the initial thrombin response, it was necessary for normal recovery of endothelial cells following thrombin-mediated changes. Thus, this work demonstrates that endothelial CLICs function in S1PR and PAR signaling, acting to facilitate the regulation of small GTPases Rac1 and RhoA. We evaluated CLIC function in endothelial barrier regulation by non-GPCR mechanisms and found that CLIC1 and CLIC4 are not required for vascular endothelial growth factor (VEGF) -mediated barrier disruption. This survey of three signaling pathways (S1PR, PAR, VEGFR) suggests that CLICs are specific to GPCR signaling. To evaluate the role of CLICs in murine vasculatures, we developed an endothelial specific knockout mouse model of Clic4 (Clic4ECKO). In mice, PAR1 activation by a specific PAR1-activating peptide induces lung edema by endothelial barrier disruption. Our preliminary data demonstrates that loss of endothelial Clic4 reduces PAR1-induced lung edema. We conclude that CLIC1 and CLIC4 function as mediators of endothelial GPCR signaling, functioning upstream of Rac1 and RhoA. This work uncovers a novel family of GPCR effector proteins, CLICs, whose membrane localization in response to GPCR activation facilitates the activation of small GTPases.