Tissue transglutaminase (tTG) is a Ca2+-dependent enzyme which cross-links proteins via ε(γ-glutamyl)lysine bridges. There is increasing evidence that tTG is involved in wound repair and tissue stabilization, as well as in physiological mechanisms leading to cell death. To investigate the role of this enzyme in tissue wounding leading to loss of Ca2+ homoeostasis, we initially used a model involving electroporation to reproduce cell wounding under controlled conditions. Two cell models were used whereby tTG expression is regulated either by antisense silencing in ECV 304 cells or by using transfected Swiss 3T3 cells in which tTG expression is under the control of the tet regulatory system. Using these cells, loss of Ca2+ homoeostasis following electroporation led to a tTG-dependent formation of highly cross-linked proteinaceous shells from intracellular proteins. Formation of these structures is dependent on elevated intracellular Ca2+, but it is independent of intracellular proteases and is near maximal after only 20min post-wounding. Using labelled primary amines as an indicator of tTG activity within these ‘wounded cells’, we demonstrate that tTG modifies a wide range of proteins that are present in both the perinuclear and intranuclear spaces. The demonstration of entrapped DNA within these shell structures, which showed limited fragmentation, provides evidence that the high degree of transglutaminase cross-linking results in the prevention of DNA release, which may serve to dampen any subsequent inflammatory response. Comparable observations were shown when monolayers of cells were mechanically wounded by scratching. In this second model of cell wounding, redistribution of tTG activity to the extracellular matrix was also demonstrated, an effect which may serve to stabilize tissues post-trauma, and thus contribute to the maintenance of tissue integrity.
Abbreviations used: BTC, biotin cadaverine [N-(5-aminopentyl)biotinamide]; DAB, 3,3′-diaminobenzidine; DTT, dithiothreitol; ECM, extracellular matrix; TG, transglutaminase; tTG, tissue TG; TUNEL, terminal transferase deoxytidyl uridine end labelling.
Present address: Division of Agricultural Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, U.K.