Reduced expression of tissue transglutaminase in a human endothelial cell line leads to changes in cell spreading, cell adhesion and reduced polymerisation of fibronectin

Abstract

Tissue transglutaminase (tTgase, type II) is a Ca2+-dependent GTP binding protein which crosslinks proteins via (epsilon)((gamma)-glutamyl)lysine bridges. Although essentially a cytosolic enzyme there is increasing evidence to suggest the enzyme is externalised where it may play a role in extracellular matrix organisation. To investigate the function of this enzyme in a human umbilical endothelial cell line ECV304 tTgase expression was reduced in these cells by up to 90% by stable transfection with a 1.1. kb antisense construct in the plasmid vector pSG5. Two clones showing a reduction in expression of tTgase activity of 70 and 90% have been isolated and characterised. These clones show a number of phenotypic differences when compared to the parent cell line and the transfected controls which include reduced cell spreading and a decreased adhesion of cells on different substrata as measured by their susceptibility to removal by trypsin. Reduced cell spreading in the antisense transfected clones was accompanied by a decrease in the crosslinking of fibronectin into polymeric multimers which could be correlated to the amount of tTgase externalised by cells. A novel assay was developed to measure externalised tTgase activity which is cell mediated, inhibited by preincubation of cells with anti-tTgase antibody and relies on the incorporation of biotinylated cadaverine into fibronectin. The results of these experiments suggest that externalised tTgase may play a key role in a number of cell behavioural patterns which might be related to the enzymes ability to bind and crosslink fibronectin.