192 VISFATIN/NAMPT: A POTENTIAL TARGET FOR NGF-TRIGGERED PAIN IN OSTEOARTHRITIS

Abstract

3. TGase 2 assay. The expression and enzyme activity of TGase 2 was examined with western blot and immunocytochemistry. Total cellular proteins were isolated and western blotted as described previously. Enzyme activity was evaluated by determining the incorporated biotinylated pentylamine using horseradish peroxidase-conjugated streptavidin and measuring the absorbance at 492 nm using a microplate spectrophotometer. Results: 1. RA up-regulated TGase 2 expression and enzyme activity in human chondrocytes. Human chondrocytes treated with RA resulted in up-regulation of TGase 2 protein as shown by the western blot and immunocytochemistry. RA-induced TGase 2 expression increased time dependently. The enzyme activity of TGase 2 was also increased in RA treated cells compared with normal human chondrocyte cells. 2. TGase 2 induced by RA results in decreased apoptosis in human chondrocytes. Apoptosis in RA-treated chondrocytes was decreased, measured by Annexin-V FACS analysis, when compared with H2O2-treated cells chondrocytes. Similar patterns were observed in 3 independent experiments using chondrocytes obtained from different patients. Conclusions: We have previously reported that endogenous TGase 2 expression was increased in human chondrocytes undergoing apoptosis. Inhibition of TGase 2 by MDC and TGase 2 siRNA was also shown to increase apoptosis and suggest a possible protective role of TGase 2 in chondrocyte apoptosis. The protective role of TGase 2 was further validated in this study as the RA-induced overexpression of TGase 2 decreased apoptosis of human chondrocytes. These results implicate a protective role of TGase 2 against apoptosis in human chondrocytes and the possibility of TGase 2 as a potential modulator of osteoarthritis.