Untersuchungen zu Wechselwirkungen von VE-Cadherin-Molekülen untereinander und mit TRPC4

Blutgefäße sind innen mit einem einschichtigen Verband aus Endothelzellen ausgekleidet, die die Barriere zwischen Blut und Interstitium bilden. Die parazelluläre Endothelbarriere besteht aus Occludens- und Adhärenskontakten. Occludenskontakte sorgen für eine Diffusionsbarriere, Adhärenskontakte sorg...

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Bibliographic Details
Main Author: Gutberlet, Judith Katharina
Contributors: Aumüller, G. (Prof. Dr.) , Drenckhahn, D. (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2006
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Endothelial cells form the inner surface of blood vessels and are responsible for maintenance of endothelial barrier function. These cells are connected by tight and adherens junctions. While tight junctions play a fundamental role in regulating transcellular permeability, adherens junctions contribute to the mechanical strength of intercellular adhesions. Strong adhesion in adherens junctions is mediated by the vascular endothelial-specific cadherin (VE-cadherin), which is a cytoskeleton-anchored membrane protein mediating Ca2+-dependent adhesion of endothelial cells by homophilic trans-interaction between cadherins of neighbouring cells. Therefore endothelial permeability is directly dependent on the regulation of VE-cadherin adhesion. Because cadherin trans-interaction is a low-affinity reaction (KD in the millimolar range), adhesive strength might be altered not only by Ca2+-dependent changes in cadherin-cytoskeletal linkage but also by dimerization or clustering of cadherins. In order to analyse the role of dimerization and oligomerization on cadherin-mediated cytoskeletal-independent adhesion, CHO cells were transfected with constructs encoding VE-cadherin fusion proteins, in which the cytoplasmic domain of VE-cadherin was substituted by a FK506 binding domain which allowed dimerization or oligomerization by a membrane-permeable FKBP crosslinker. The results of subsequent binding-assays indicate that the extracellular domain of VE-cadherin has - independent of its cytosolic domain - intrinsic binding activity, which appears to be dependent on cis-dimerization. This was supported by the observation that cis-dimerization was necessary for both, significant trans-interaction with soluble and microsphere-bound VE-cadherin-Fc-dimers. However, additional oligomerisation of VE-cadherin-molecules did not further increase homophilic binding activity. Tight binding occured only if cadherins were primarily mobile in the plane of the lipid bilayer thereby allowing them to accommodate to VE-cadherin molecules of the opposing side. In transfected cells with high VE-cadherin-FKBP expression cis-dimerization might also occur spontaneously to a certain degree and allow slightly enhanced binding of VE-cadherin-Fc-coated microspheres. In contrast, spontaneous cis-dimerization - if it occurs at all - was not sufficient for significant binding of soluble VE-cadherin dimers analysed by single molecule fluorescence. These data also suggest that VE-cadherin-fusion proteins become inserted into the plasma membrane as monomers followed by subsequent association into functional dimmers by addition of the membrane permeable dimerizer. Based on these findings it can be concluded that, VE-cadherin adhesive activity could reach from weak binding mediated by the intrinsic binding activity of its extracellular domain to strong binding caused by strong anchorage of the intracellular domain to the cytoskeleton. This further implies that, this adhesive activity could be regulated by controlling (i) the surface concentration, (ii) the formation of cis-dimers and (iii) the degree of cadherin-linkage to the cytoskeleton. In order to investigate binding forces between VE-cadherin monomers and directly compare them to forces between VE-cadherin dimers another VE-cadherin fusion protein was produced and purified by affinity chromatography. This protein was composed of the extracellular VE-cadherin domain fused to a dimerizing domain and a Histidin-tag. Gelfiltration analysis confirmed dimerization in the presence of dimerizer. Increased permeability is caused by opening of endothelial junctions during inflammation. This process requires an increase of the intracellular Ca2+-concentration [Ca2+]i due to influx of Ca2+ from the extracellular space. TRPC4, the predominate store-operated Ca2+-channel of endothelial cells, appears to be crucially involved in this process. As VE-cadherin-mediated adhesion is controlled by both [Ca2+]i and [Ca2+]e, we looked for a possible association of TRPC4 and VE-cadherin. Applying immunofluorescence using endothelial cells of different confluencies both proteins were found to co-localise continuously along cell borders. In addition, cotransfection of TRPC4-expressing HEK 293-cells with a VE-Cadherin-GFP-coding plasmid led to increased recruitment of diffusely distributed TRPC4 in control cells to VE-cadherin-containing cell-cell-contacts in transfected cells. These observations suggest a functional association between both proteins and opens up the interesting possibility that Ca2+-concentration regulated by TRPC4 in immediate vicinity of VE-cadherin might selectively control endothelial barrier properties without affecting the remaining cellular compartments.