Role of paxillin in the early phase of orientation of the vascular endothelial cells exposed to cyclic stretching

Abstract

Paxillin, a structural and signaling scaffold molecule in focal adhesions (FAs), is considered to be important in intracellular signaling transduction and the cell shape changes in response to cyclic stretching. However, the detailed role of paxillin in stretch-induced morphological changes of endothelial cells (ECs) has not fully determined until date. In this study, in order to understand the role of paxillin in the orientation of ECs exposed to cyclic stretching, we examined the time course of changes in the shape and distribution of FA proteins of paxillin knockdown ECs. Non-treated ECs subjected to 20% cyclic stretching at 0.5Hz oriented perpendicularly to the direction of stretching after 10min of exposure. On the other hand, the orientation of paxillin knockdown ECs was abolished at 10min, but it was observed after 60min of cyclic stretching exposure. Immunofluorescent microscopy revealed that accumulation and redistribution of FA proteins, including focal adhesion kinase (FAK) and integrin β1, were observed at 10min of exposure to cyclic stretching in non-treated ECs. The accumulation of FAK and integrin β1 was not prominent in paxillin knockdown ECs under static conditions and after 10min of exposure to cyclic stretching. However, we found that accumulation of FA proteins in paxillin knockdown ECs at 30 and 60min was similar to that in non-transfected ECs. Because paxillin is an adaptor protein offering binding sites for FAK and integrin β1, which are critical molecules for the early signaling events of focal adhesion formation in ECs, these results suggest that paxillin is required for the early phase of EC orientation in response to cyclic stretching by scaffolding for accumulation of FA proteins.