Abstract 94: Scaffolding Protein EBP50 Regulates Vascular Smooth Muscle Nox1 Activity by Binding p47phox
The NADPH oxidase (Nox) enzyme family is implicated in the pathogenesis of cardiovascular diseases (CVDs) including hypertension. In large vessel smooth muscle cells (SMCs), a unique hybrid Nox1 oxidase exists which propagates SMC dysfunction and hypertrophy. We recently demonstrated that these actions involve activation of Nox1 by hydrogen peroxide (H2O2) leading to feed-forward production of reactive oxygen species and activation of apoptosis signal regulating kinase 1 (Ask1). Here we postulate that Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50), a PDZ-domain scaffolding protein, is required for H2O2-mediated activation of SMC Nox1. In SMCs, knockdown of EBP50 by shRNA abolished superoxide (O2•-) production in 50 μM H2O2-treated cells (37.7 ± 4.5, 79.9 ± 2.3 and 29.0 ± 3.5 x104 O2•--mediated RLU for GFP plasmid vehicle-treated, GFP plasmid H2O2-treated and EBP50 shRNA plasmid H2O2-treated groups, respectively). Furthermore, H2O2-induced O2•- production was absent in SMCs isolated from EBP50 knockout (KO) mice (29.0 ± 2.7 vs 79.1 ± 2.4 x104 RLU for EBP50 KO vs wildtype after H2O2 treatment). Intriguingly, p47phox, an essential cytosolic subunit of the hybrid SMC Nox1 system, upon phosphorylation co-immunoprecipitated with EBP50 in COS-7 cells transfected with both proteins (Fig 1). Taken together, our data suggest that EBP50 promotes Nox activity by binding active p47phox. These studies identify a novel partner in Nox1 activation and potentially provide new insights into the broader regulation of this enzyme family. Moreover, this added level of complexity could yield new therapeutic strategies to disrupt Nox in the treatment of hypertension and other CVDs.
- © 2012 by American Heart Association, Inc.