P281	Vascular hyperpermeability inducer thrombin causes beta-catenin translocation in human umbilical vein endothelial cells.
C.M.L.Beckers, M.A.Engelse, R.J.P.Musters, V.W.M.van Hinsbergh, G.P.van Nieuw Amerongen
VU University Medical Center, ICaR-VU, Laboratory of Physiology, Amsterdam, NL.

Disruption of cell-cell contacts, which is followed by formation of gaps in the endothelial barrier, is an important step in the development of vascular hyperpermeability. Beta-catenin is an important protein of the adherens junction and plays a major role in the maintenance of the cell-cell contacts. Endothelial activation by hyperpermeability-inducers might result in dissociation of the adherens junctional complex. Using live cell fluorescence imaging in HUVECs, we have now monitored gap formation upon stimulation with the potent hyperpermeability-inducer thrombin. Thrombin-activated cells contract in a RhoA-dependent manner, resulting in formation of gaps between the cells. At places were gaps are formed, the membranes of two adjacent cells remain connected by finger-like structures. In resting HUVECs, β-catenin is localized as a thin lining at the cell periphery. High power 3D-deconvolution fluorescence microscopy reveals that between the peripheral fine web of F-actin filaments β-catenin forms a honey-comb-like network, resembling the structure that has been shown for VE-cadherin. In thrombin-activated HUVECs, at those places where gaps are formed, β-catenin has disappeared from the membrane. Interestingly, upon thrombin stimulation we also observed alterations in RhoA activity in the junctional areas as evidenced by fluorescence resonance energy transfer (FRET) microscopy. Western blot analysis demonstrated a thrombin-initiated redistribution of β-catenin from the membrane to the cytosolic fraction. In addition, upon thrombin stimulation part of the β-catenin co-localizes with vinculin, suggesting translocation of β-catenin to focal contact sites. In conclusion, we have shown that in confluent endothelial cells β-catenin is localized in a honey-comb-like structure around the F-actin filaments in junctional areas between the cells and that upon thrombin stimulation the adherens junctions are disrupted so that gaps, and remaining finger-like bridges, are formed. Associated with altered RhoA activity, β-catenin is redistributed to focal contact sites and the cytosol. Future studies have to reveal whether b-catenin is degraded or is stabilized in the cytosol due to the Wnt signalling pathway and can translocate to the nucleus to induce gene-expression.

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