Intersectin Regulates Endothelial Cell Junction Integrity

Abstract

Our recent work indicated that the intersectin-2L (ITSN-2L), via its DH-PH domain with Cdc42-specific guanine nucleotide exchange factor activity, promotes actin polymerization and decrease in caveolae internalization. Expression of the DH-PH domain of ITSN-2L in cultured endothelial cells revealed a selective activation of Cdc42. Biochemical analyses of endothelial cell lysates and double fluorescent labeling of cultured endothelial cells indicate that ITSN interacts and co-localizes with members of the Cdc42-N-WASp-Arp2/3 actin polymerization pathway. Moreover, phalloidin staining of ECs expressing the DH-PH domain of ITSN-2L showed actin cytoskeletal changes such as increases in filopodia and cortical actin, while a biochemical assay for actin polymerization indicated a shift from G- to F-actin. We now show that these actin cytoskeletal rearrangements caused by ITSN-2L also have an effect on the integrity of interendothelial junctions. Morphological analysis of ECs transfected with the DH-PH domain of ITSN-2L demonstrated the appearance of gaps between cells. Moreover, functional studies in mouse lungs, upon activation of Cdc42, indicated increased vascular permeability via the interendothelial junctions. When coupled to our previous data indicating a decrease in caveolae internalization with ITSN overexpression, these current data show the involvement of ITSN in the regulation of both transcellular and paracellular transport pathways in the endothelium. In this sense, ITSN may serve to regulate endothelial permeability by controlling both pathways. Thus, the regulation of Cdc42 activity and actin polymerization by ITSN may be crucial for the loss of endothelial junctional integrity. This research is supported in part by NIH T32 HL07289.