Upregulation of Tissue Transglutaminase in Transforming Growth Factor-b Induced Epithelial Mesenchymal TG2Transdifferentiation In Rat Tubular Epithelial Cells

Abstract
Background: Epithelial-Mesenchymal Transdifferentiation (EMT) is believed to be a key step in the course of glomerular and tubulointerstitial fibrosis. Many of the regulators of this process have been identified and Transforming Growth Factor-b  (TGF-b) plays a central role. Tissue transglutaminase (TG2) has a crucial role in extracellular matrix formation and stabi-lization.
Aim of Study: This study was designed to explore whether TG2 is essential in EMT induced by TGF-b  in renal tubular epithelial cells.
Material and Methods: EMT was achieved by treating NRK52-E renal tubular epithelial cells with high concentration of rhTGF-b  (10ng/ml) for 24, 48 and 72h. Development of mesenchymal cell markers Fibroblast Specific Protein (FSP-1) and a  Smooth Muscle Actin (a-SMA) and decrease in tubular epithelial cell surface marker E-Cadherin were used to confirm EMT. The NTU283; TG2 inhibitor was used for the inhibition assays. TG2 activity was quantified using a 14C putrescine incorporation assay. Protein expression and localisation of different markers were assessed by western blot and immunofluorescent dual immunofluorescent staining. Phase analysis was used to quantify the amount of staining for each of the markers used.
Results: A significant increase in TG2 activity and protein expression were both observed with TGF-b  induced transdif-ferentiation at 48h. This observation was associated with loss of epithelial cell marker E-Cadherin and appearance of intra-cellular a-SMA and FSP-1, characteristic of myofibroblasts. Dual staining showed high TG2 levels in cells (0.254±0.07), compared to the control (0.02±0.008). This also showed loss of epithelial phenotype, decrease of E-cadherin and acquisition of myofibroblast characteristics a-SMA (0.671±0.13) com-pared to the control (0.076±0.016) and FSP-1 (0.492±0.017) compared to control (0.03±0.017). Cells also demonstrated high degree of co-localization of Collagen I (ColI) and a-SMA with TG2. The NTU283; TG2 inhibitor ameliorated TGF-b  induced EMT. This was also associated with changed pattern of collagen I and a-SMA co-localization.
Conclusion: This data demonstrated upregulation of TG2 during the process of TGF-b  induced EMT. TG2 inhibitor showed non-significant reduction of both TG2 and mesenchy-mal markers but demonstrated changes in the molecular configuration of mesenchymal markers a-SMA and Col I, raising the possibility of a role of TG2 in EMT.