The effects of transglutaminase 2 modulation on endothelial cell biology: focus on celiac disease pathogenesis

Objective: Transglutaminase 2 (TG2) is a multifunctional protein with an important role in vascular biology, where it is involved in cell–matrix interaction, cell attachment and cell population expansion. In efforts to elucidate the role of TG2 in endothelial cell biology, in this study, we measured several endothelial cell characteristics in cells where TG2 was specifically knocked down by RNAi. Materials and methods: The effect of small interfering RNA (siRNA)-TG2 on human umbilical vein endothelial cells was studied. Adhesion and cell viability were assessed by chemical reduction of MTT, and cell proliferation was analysed by flow cytometry. Apoptosis was evaluated by annexin V ⁄PI dual staining and protein expression level was assayed by western blotting. Results: We found that siRNA-TG2 reduced endothelial cell number, lead to cell adhesion deficiency, cell cycle arrest in G1 phase and induction of apoptosis. Our results show that exogenously added TG2 could reverse loss of adhesion but did not overcome the defect in cell proliferation, nor could it inhibit siRNA-TG2-induced apoptosis. Conclusion: We conclude that TG2 loss in endothelial cells causes reduction in cell number as a result of cell cycle arrest, flaws in adhesion and induction of apoptosis. Our results imply that reduction in cell number and increased apoptosis in response to TG2 silencing is independent of the cell adhesion process. Altogether, our findings underline the significance of TG2 in endothelial cell cycle progression and cell survival, in vitro. Introduction Transglutaminase 2 (TG2) is a member of the transglutaminase (TG) enzyme family (EC2.3.2.13), involved in a plethora of biological functions. TG2 is ubiquitously expressed in most mammalian tissues and is involved in a number of cell processes such as angiogenesis (1), apoptosis (2), cell differentiation (3) and wound healing (4,5). TG2 is predominantly an intracellular protein and functions inside the cell as a protein disulphide isomerase, an ATPase or a protein kinase (6–9), however, its best characterized feature is its G-protein function (10,11). In addition, TG2 can be found inside the nuclear compartment in association with histones, and histone phosphorylation activity has also been reported there (12). Moreover, TG2 can be secreted to the outside of cells, where it stabilizes the extracellular matrix, making it resistant to mechanical and proteolytic degradation (13,14). Furthermore, TG2 is present on the cell membrane, where it can associate with integrins and provide a binding site for fibronectin (FN) by simultaneously binding to b-integrins and FN. TG2-mediated adhesion also involves focal adhesion kinase (FAK), whose expression, maturation and activity TG2 is known to regulate (15). Altogether, TG2 plays a key role in a number of physiological processes and a cell survival function for TG2 is thus strongly implicated. As TG2 has an established role in angiogenesis (1,4), the molecular mechanism by which it affects proliferation, adhesion and apoptosis in endothelial cells has been examined here, using small interfering RNA (siRNA) knock-down. We have shown a central role for TG2 in cell survival in living cells, where it is needed for cell cycle progression from G1 to S phase. In addition to this, our data demonstrate that the roles of TG2 in Correspondence: K. Lindfors, Paediatric Research Centre, Medical School, FIN-33014 University of Tampere, Tampere, Finland. Tel. : +358 3 3551 8403; Fax: +358 3 3551 8402; E-mail: katri.lindfors@uta.fi 2010 Blackwell Publishing Ltd. 49 Cell Prolif. , 2011, 44, 49–58 doi: 10.1111/j.1365-2184.2010. 00716.x