Overexpression of truncated IκBα potentiates TNF-α-induced apoptosis in mesangial cells

Background. Dysregulation of apoptosis is one of the likely underlying mechanisms of mesangial proliferative glomerulonephritis (GN), a disease in which proinflammatory cytokines exhibit a wide range of biological activities. Among them, tumor necrosis factor-alpha (TNF-alpha) induces two conflicting pathways, one leading to activation of the nuclear factor-kappa B (NF-kappa B), and the other leading to caspase-mediated apoptosis. We investigated whether or not specific inhibition of NF-kappa B affects TNF-alpha-induced apoptosis in rat mesangial cells (MCs). Methods. To specifically inhibit NF-kappa B activation, we constructed a recombinant adenovirus vector expressing a truncated form of I kappa B alpha (AdexI kappa B Delta N) that lacks the phosphorylation sites essential for the activation of NF-kappa B. Electrophoretic mobility shift assay was performed to evaluate NF-kappa B activity. Nuclear morphology was observed by staining with Hoechst-33258. DNA fragmentation was detected using an ELISA kit with an antihistone antibody. To investigate the regulation of apoptosis, we measured caspase-3 and caspase-8 activity by ELISA. and examined the Bcl-2 and Bar protein level by Western blot. Results. TNF-alpha-induced NF-kappa B activation was blocked by overexpression of I kappa B Delta N. Overexpression of I kappa B Delta N potentiated TNF-alpha-induced apoptosis compared to mock transfection, and the potentiation was abolished by treatment with a caspase-3 inhibitor, Z-DEVD-FMK. Overexpression of I kappa B Delta N augmented TNF-alpha-induced caspase-3 and caspase-8 activity, but did not affect Bcl-2 or Bar protein expression. Conclusion. Overexpression of I kappa B Delta N potentiates TNF-alpha-induced apoptosis and augments caspase-8 and caspase-3 activity in rat MCs without changing Bcl-2 or Bar protein expression. These results suggest the potential usefulness of AdexI kappa B Delta N to induce apoptosis in MCs under inflammatory conditions.