In this study, critical signaling pathway required for the stretch induced morphological changes of human umbilical endothelial cells (HUVECs) was investigated. Uni-axial cyclic stretch (1 Hz, 20% in length) of the cells cultured on an elastic silicon membrane induced a gradual morphological change in the cells from a polygonal shape to an elongated spindle-like shape whose long axis was aligned perpendicular to the stretch axis. We found that protein tyrosine phosphorylation of cellular proteins increased and peaked at 20 min in response to cyclic stretch. Either treatment of cells with gadolinium (Gd3+), a potent blocker for stretch-activated channels, or removal of extracellular Ca2+ blocked the tyrosine phosphorylation of the proteins, suggesting that stretch-activated (SA) ion channels regulated stretch specific tyrosine phosphorylation. The major phosphorylated proteins had molecular masses of approximately 120-135 kDa, and 70 kDa. Immunoprecipitation experiments revealed that paxillin, focal adhesion kinase (pp125(FAK)) and pp130(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 phosphorylation was required for the morphological change. In addition, the kinase activation of pp125(FAK) was observed in response to cyclic stretch. Moreover, suppression of pp125(FAK) expression by the antisense phosphorothioate oligodeoxynucleotides (S-ODN) in HUVECs resulted in inhibition of tyrosine phosphorylation of paxillin and the stretch-dependent morphological changes. These results suggest that an activation of tyrosine kinase(s) by an increase in intracellular Ca2+ and pp125(FAK) play a critical role in the unique morphological change specifically observed in endothelial cells subjected to uni-axial cyclic stretch.
- Antisense phosphorothioate oligodeoxynucleotides
- Stretch-activated ion channel
- Uni-axial cyclic stretch
ASJC Scopus subject areas
- Molecular Biology
- Cancer Research