Abstract 5849: Protein Phosphatase 2A Inhibition Prevents NO-induced Caspase-3 Activation and Apoptosis
Nitric oxide (NO), depending on its concentrations, can be either beneficial or detrimental in cardiovascular injury. For example, physiological levels of NO generated by eNOS protect postischemic hearts; whereas excessive NO from iNOS damage cells in heart failure. High levels of NO have been shown to activate caspase-3 leading to apoptosis. But the underlying mechanism is not fully understood. Here we identify protein phosphatase 2A (PP2A) as a crucial regulator in NO-induced caspase-3 activation and apoptosis. To explore the mechanism of NO-induced apoptosis, the HEK 293 cells were treated with NO donor SNAP. The concentrations of NO released by SNAP in the medium were monitored by the electrochemical detector. NO (0.1–10 μM) dose-dependently induced caspase-3 activation as evidenced by the increases of caspase-3 cleavage. Okadaic acid (100 nM) prevented caspase-3 activation in NO-treated cells, suggesting that PP2A was involved. Indeed, PP2A knockdown with siRNA largely abolished NO-induced caspase-3 activation. In contrast, protein phosphatase 1 knockdown had no effect on caspase-3 activation in NO-treated cells. The effect of PP2A on NO-induced caspase-3 activation was specific because PP2A inhibition did not affect TNF-α-initiated caspase-3 activation. Further studies showed that NO treatment caused Bcl-2 Ser70 dephosphorylation, which led to the loss of Bcl-2 function and subsequent caspase-3 activation. PP2A inhibition protected Bcl-2 by preserving its Ser70 phosphorylation. To validate the role of PP2A in modulating endogenous NO-induced apoptosis, we induced iNOS expression in macrophages with LPS (1 αg/ml) and IFN-γ (100 units/ml). NO generated from iNOS activated caspase-3 in macrophages. PP2A inhibition blocked caspase-3 activation in iNOS-induced cells by 84±5.6% (P<0.01, versus that in control cells, n=5). Consequently, the apoptosis in iNOS-induced cells was largely prevented. Collectively, these findings demonstrate PP2A as a critical regulator in NO-induced caspase-3 activation. PP2A inhibition protects cells against NO-induced apoptosis via preserving Bcl-2 phosphorylation and function. Thus, intervening PP2A may be a novel approach to prevent NO-induced apoptosis in cardiovascular injury.