McKenzie, Jenny Alison Gill; (2004) TNF-[alpha] signalling to the cell-cell junctions and the cytoskeleton in endothelial cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Tumour necrosis factor-[alpha] (TNF-[alpha]) is known to induce changes in endothelial cell morphology and paracellular permeability, but the mechanisms have not been extensively characterised. The purpose of this study was to establish the effects of TNF-[alpha] on human umbilical vein endothelial cell (HUVECs) paracellular permeability and tight junctions and relate these responses to changes in the actin cytoskeleton. TNF-[alpha] caused progressive changes to HUVECs over 24 h. TNF-[alpha] induced RhoA activation, myosin light chain phosphorylation, cortical F-actin thickening and the formation of tiny inter-cellular gaps within 10 min, A small increase in permeability accompanied these changes. By 24 h, TNF-[alpha] caused stress fibre formation, cell elongation and extensive gap formation. Occludin and JAM-A were lost from the tight junctions, ZO-1 was partially redistributed and permeability was increased. RhoA and ROCK inhibition prevented TNF-[alpha]-induced changes in F-actin and cell morphology, but ROCK inhibition did not re-establish the junctional localisation of ZO-1, nor did it prevent increased permeability. Myosin light chain kinase inhibition had no impact on TNF-[alpha]- induced stress fibres, cell elongation or permeability at 24 h. These results indicate that the TNF-[alpha]-induced morphological and cytoskeletal changes are not solely responsible for increased permeability and that signalling to the tight junction proteins may be more important for TNF-[alpha] regulation of barrier function. To identify potential interacting partners of occludin, a yeast two-hybrid experiment was performed using the C-terminus of occludin. The transcriptional co-activator protein, Ski-interacting protein (SKIP) and the Ser/Thr protein kinases, casein kinase I[epsilon] (CKI[epsilon]) and UNC-51 like kinase-1 (ULK-1) were identified in this screen. These novel interactions may be important for occludin regulation and function. During the course of these studies, adenoviruses were used to introduce genes into HUVECs. An inhibitory effect of control adenoviruses on TNF- -induced cytoskeletal changes and permeability was observed, suggesting that adenovirus binding and/or entry could modulate endothelial cell behaviour and responses.

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