Our recent studies showed that transglutaminase-1 (TGase-1) is uniquely expressed in

Our recent studies showed that transglutaminase-1 (TGase-1) is uniquely expressed in mouse renal proximal tubular cells (RPTC) and mediates cell proliferation. Pretreatment of cells with MDC or TGase-1 siRNA inhibited phosphorylation of all these molecules. Inhibition of either the AKT or STAT3 pathway potentiated H2O2-induced cell death and increased GSK-3β activity by dephosphorylation at serine 9. Furthermore treatment with GSK-3β inhibitors reduced H2O2-induced apoptosis and abolished the death-promoting effect of AKT and STAT3 inhibition. Therefore we have identified TGase-1 as a novel survival factor in renal epithelial cells and it contributes to cell survival through activation of the AKT and STAT3 signaling pathways following oxidant injury. < 0.05 was considered statistically significant. RESULTS Activation of TGase-1 is required for RPTC survival following oxidant injury. Intracellular ROS has Tariquidar (XR9576) been reported to be involved in the activation of TGases (6 19 However the role of TGases in RPTC death following oxidant injury is not clear. To address this issue RPTC were exposed to 1 mM H2O2 in the presence or absence of MDC a pseudosubstrate inhibitor of TGases that is widely used for inhibition of TGase activity (4 49 and cell viability was examined using the MTT assay. Cell viability was decreased to 60% in RPTC treated with H2O2 alone for 4 h and further reduced to 38 and 25% in the presence of 50 and 100 μM MDC respectively (Fig. 1and and and and and and and and and and and release and apoptotic cell death in a variety of cell types in response to oxidant injury (9 24 AKT can induce its inactivation by direct phosphorylation at serine 9 (24). Since the above data revealed that TGase-1 mediated AKT activation following oxidant injury it is possible that TGase-1 would also regulate GSK-3β activity. To test this hypothesis we examined the effect of TGase-1 inhibition on phosphorylation of GSK-3β at serine 9. GSK-3β is constitutively activated and its phosphorylation at serine 9 is inactive. As shown in Fig. 8 and and and and and F). Cell … Tariquidar (XR9576) Tariquidar (XR9576) The above data (Figs. 5-8) show that blockade of either the PI3K/AKT or STAT3 pathway potentiates cell death and inactivates GSK-3β by phosphorylation at serine 9 suggesting that activation of the PI3K/AKT and STAT3 pathways may contribute to cell survival through inactivation of GSK-3β. If this is indeed the case inactivation of GSK-3β should block the death-promoting effect of AKT and STAT3 inhibition. To test this hypothesis RPTC were treated with the PI3K/Akt pathway inhibitor (LY294002) or STAT3 inhibitor (S3I201) in the absence or presence of TDZD-8 before H2O2 exposure. As shown in Fig. 10 TDZD-8 treatment abolished the inhibitory effect of LY294002 and S3I201 on cell survival under oxidant stress. Similar results were obtained when RPTC overexpressing TGase-1 were treated with those inhibitors (data not shown). These data together with the inhibitory effect of MDC and TGase-1 siRNA on GSK-3β phosphorylation (Fig. 8) suggest that TGase-1 induces cell survival through the AKT/STAT3/GSK-3β pathway in RPTC after oxidant injury. Fig. 10. TDZD-8 treatment abolished the Tariquidar (XR9576) death-promoting effect of LY294002 and S3I201 in RPTC following oxidant Rabbit Polyclonal to KIF4A. injury. RPTC were treated with 1 mM H2O2 for 4 h in the absence or presence of LY29400 (20 μM) or S3I201 (50 μM) with/without TDZD-8 … DISCUSSION ROS including H2O2 are generated following I/R and toxicant exposure and are critically involved in the pathogenesis of AKI (5 7 22 In this study we demonstrated that the exposure of RPTC to H2O2 increased TGase activation and induced apoptosis. Inhibition of TGase activity by a pharmacological inhibitor (MDC) and reduction of TGase-1 expression with siRNA potentiated H2O2-induced apoptotic cell death. Conversely overexpression of TGase-1 inhibited the apoptosis and increased the cell viability. Therefore we have identified the novel function of TGase-1 as a survival factor in renal epithelial cells and its activation protects RPTC from apoptosis following oxidant injury. The ability of cells to survive a variety of stresses including oxidant stress often depends on the activation of survival signaling pathways. PI3K/AKT and JAK2-STAT3 pathways have been shown to be activated by H2O2 (15 32 37 41 and mediate cell.