Deletion of integrin linked kinase in endothelial cells results in defective RTK signaling caused by caveolin 1 mislocalization

Abstract

Integrin linked kinase (ILK) connects the ILK-Pinch-Parvin complex with integrin adhesion sites. Because of the functional relevance of integrin-linked signaling for endothelial cell (EC) biology, we have explored this pathway in Ilk–/– embryonic stem (ES) cells differentiated into ECs and vessel-like structures. We have focused in particular on the mechanistic relevance of ILK-Pinch-Parvin complex-related signaling for EC development and tube formation. Our analysis revealed that the formation of vessel-like structures was strongly reduced in Ilk–/– ES cells and that this phenotype could be rescued by re-expression of ILK in ES cells. ECs were MACS sorted from wild-type (WT) and Ilk–/– ES cells and functional analysis using intracellular calcium imaging as the read-out yielded a complete lack of vascular endothelial growth factor- and epidermal growth factor-dependent responses. The possibility of a caveolin 1-related defect was investigated by transfecting WT and Ilk–/– ECs with a caveolin 1-EGFP fusion protein. Time-lapse microscopy showed that the prominent phenotype is due to altered dynamics of caveolin 1 and to a lack of positioning of caveolin 1 in the vicinity of the plasma membrane and that it is rescued by re-expressing ILK in the Ilk–/– ES cells. We also found that the defect is caused by the perturbed organization of microtubules and cortical actin filaments. Thus, ILK is required as a scaffold to allow actin-microtubule interactions and correct positioning of caveolin 1 close to the plasma membrane. This is crucial for signaling compartmentalization in ECs and explains the key role of ILK for EC development and function.