Expressed isolated integrin β1 subunit cytodomain induces endothelial cell death secondary to detachment

Summary

Expression of isolated β integrin cytoplasmic domains in cultured endothelial cells was reported to induce cell detachment and death. To test whether cell death was the cause or the consequence of cell detachment, we expressed isolated integrin β1 cytoplasmic and transmembrane domains (CH1) in cultured human umbilical vein endothelial cells (HUVEC), and monitored detachment, viability, caspase activation and signaling. CH1 expression induced dose-dependent cell detachment. At 24 h over 90% of CH1-expressing HUVEC were detached but largely viable (>85%). No evidence of pro-caspase-8, –3, and PARP cleavage or suppression of phosphorylation of ERK, PKB and Iκ-B was observed. The caspase inhibitor z-VAD did not prevent cell detachment. At 48 h, however, CH1-expressing cells were over 50% dead. As a comparison trypsin-mediated detachment resulted in a time-dependent cell death, paralleled by caspase-3 activation and suppression of ERK, PKB and Iκ-B phosphoyrylation at 24 h or later after detachment. HUVEC stimulation with agents that strengthen integrin-mediated adhesion (i.e.PMA, the Src inhibitor PP2 and COMP-Ang1) did not prevent CH1-induced detachment. Expression of CH1 in rat carotid artery endothelial cells in vivo caused endothelial cell detachment and increased nuclear DNA fragmentation among detached cells. A construct lacking the integrin cytoplasmic domain (CH2) had no effect on adhesion and cell viability in vitro and in vivo. These results demonstrate that isolated β1 cytoplasmic domain expression induces caspase-independent detachment of viable endothelial cells and that death is secondary to detachment (i.e. anoikis). They also reveal an essential role for integrins in the adhesion and survival of quiescent endothelial cells in vivo.

Keywords

Adhesion receptors / integrins - cell-matrix interactions - genetransfer - endothelial cells

^* These authors contributed equally to this work

^# Present addresses: Biocenter, University of Basel, Basel, Switzerland

^¶ F. Hoffmann-La Roche Ltd, Pharmaceutical Division, Clinical Science PDC-1, CH 4070 Basel, Switzerland

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