PP125(FAK) IS REQUIRED FOR STRETCH DEPENDENT MORPHOLOGICAL RESPONSE OF ENDOTHELIAL-CELLS

In this study, critical signaling pathway required for the stretch ind uced morphological changes of human umbilical endothelial cells (HUVEC s) was investigated. Uniaxial cyclic stretch (1 Hz, 20% in length) of the cells cultured on an elastic silicon membrane induced a gradual mo rphological change in the cells from a polygonal shape to an elongated spindle-like shape whose long axis was aligned perpendicular to the s tretch axis, We found that protein tyrosine phosphorylation of cellula r proteins increased and peaked at 20 min in response to cyclic stretc h. Either treatment of cells with gadolinium (Gd3+), a potent blocker for stretch-activated channels, or removal of extracellular Ca2+ block ed the tyrosine phosphorylation of the proteins, suggesting that stret ch-activated (SA) ion channels regulated stretch specific tyrosine pho sphorylation, The major phosphorylated proteins had molecular masses o f approximately 120-135 kDa, and 70 kDa, Immunoprecipitation experimen ts revealed that paxillin, focal adhesion kinase (pp125(FAK)) and pp13 0(CAS) were included in the 70 kDa and 120-135 kDa bands, respectively , The morphological change was inhibited by herbimycin A and genistein , inhibitors of tyrosine kinases, suggesting that tyrosine phosphoryla tion was required for the morphological change, In addition, the kinas e activation of pp125(FAK) was observed in response to cyclic stretch, Moreover, suppression of pp125(FAK) expression by the antisense phosp horothioate oligodeoxynucleotides (S-ODN) in HUVECs resulted in inhibi tion of tyrosine phosphorylation of paxillin and the stretch-dependent morphological changes. These results suggest that an activation of ty rosine kinase(s) by an increase in intracellular Ca2+ and pp125(FAK) p lay a critical role in the unique morphological change specifically ob served in endothelial cells subjected to uni-axial cyclic stretch.