Title: Chloride Intracellular Channels 1 and 4 function in distinct branches of S1P signaling to regulate endothelial cell behavior and vascular development
Description: Chloride intracellular channels (CLICs), 1 and 4 are expressed in endothelial cells where they promote cell proliferation, migration and vessel morphogenesis in vitro. Clic4-/- mice exhibit defects in retinal angiogenesis suggesting CLIC4 functions as an angiogenic regulator. S1P signaling, through S1P receptors S1P1 and S1P2, is essential for endothelial cell functions during vascular development. S1P treatment promotes CLIC4 localization to cell surface suggesting a link between CLICs and S1P pathways. Here we demonstrate that CLICs function in embryonic development, retinal angiogenesis and vascular permeability regulation. Clic1-/-;Clic4-/- embryos die in utero and exhibit severe growth restriction with vascular defects prior to death. Loss of Clic4 in murine endothelium (Clic4ECKO) caused aberrant retinal angiogenesis characterized by reduced vascular outgrowth and increased vessel sprouting. Clic4ECKO mice exhibited increased vessel leakiness as assessed by a lung permeability assay.

We establish that CLIC1 and CLIC4 function in distinct branches of the S1P pathway to promote angiogenesis. Knockdown of CLIC1 or CLIC4 in endothelial cells impeded S1P1-mediated induction of AKT and Rac1 and reduced endothelial cell migration and adherence junctions formation. CLIC1 knockdown alone inhibited RhoA activation and actin stress fibers downstream of S1P2. Using pharmacological perturbation of S1P signaling in Clic knockout mice we established that Clic4 is essential for S1P1-mediated regulation of retinal angiogenesis and vascular permeability. We conclude that CLIC1 and CLIC4 function as effectors in the S1P pathway, where they have overlapping functions in S1P1-PI3K signaling and CLIC1 uniquely acts as an effector in S1P2-RhoA signaling cascade. Through these findings, our work defines a molecular mechanism through which CLICs function in endothelium.