CLIC1 and CLIC4 mediate endothelial S1P receptor signaling to facilitate Rac1 and RhoA activity and function

Chloride intracellular channels 1 (CLIC1) and 4 (CLIC4) are expressed in endothelial cells and regulate angiogenic behaviors in vitro, and the expression of Clic4 is important for vascular development and function in mice. Here, we found that CLIC1 and CLIC4 in endothelial cells regulate critical G protein-coupled receptor (GPCR) pathways associated with vascular development and disease. In cultured endothelial cells, we found that CLIC1 and CLIC4 transiently translocated to the plasma membrane in response to sphingosine 1-phosphate (S1P). Both CLIC1 and CLIC4 were essential for mediating S1P-induced activation of the small guanosine triphosphatase (GTPase) Rac1 downstream of S1P receptor 1 (S1PR1). In contrast, only CLIC1 was essential for S1P-induced activation of the small GTPase RhoA downstream of S1PR2 and S1PR3. Neither were required for other S1P-S1PR signaling outputs. Rescue experiments revealed that CLIC1 and CLIC4 were not functionally interchangeable, suggesting distinct and specific functions for CLICs in transducing GPCR signaling. These CLIC-mediated mechanisms were critical for S1P-induced stimulation of the barrier function in endothelial cell monolayers. Our results define CLICs as previously unknown players in the pathways linking GPCRs to small GTPases and vascular endothelial function.