Flow antagonizes TNF-α signaling in endothelial cells by inhibiting caspase-dependent PKCζ processing

Unidirectional laminar flow is atheroprotective, in part by inhibiting cytokine-mediated endothelial cell (EC) inflammation and apoptosis. Previously, we showed that flow inhibited TNF-α signaling by preventing activation of JNK. Recently, PKCζ was identified as the PKC isoform most strongly regulated by flow pattern, with increased PKCζ activity in regions of disturbed flow versus unidirectional flow. Interestingly, PKCζ is cleaved by caspases after TNF-α stimulation to generate a 50-kDa truncated form (CATζ, catalytic domain of PKCζ) with a higher kinase activity than the full-length protein. We hypothesized that flow would inhibit TNF-α-mediated PKCζ cleavage and thereby CATζ formation. We found that PKCζ activity was required for TNF-α-mediated JNK and caspase-3 activation in ECs. PKCζ was rapidly cleaved to generate CATζ in cultured bovine and human aortic ECs and in intact rabbit vessels stimulated with TNF-α. This truncated form of PKCζ enhanced JNK and caspase-3 activation. Interestingly, PKCζ cleavage was prevented by inhibitors of PKCζ, JNK, and caspase activities, suggesting that these enzymes, via regulating CATζ formation, modulate caspase-3 activity in ECs. Finally, we found that flow reduced caspase-dependent processing of PKCζ and caspase-3 activation. These results define a novel role for PKCζ as a shared signaling mediator for flow and TNF-α, and important for flow-mediated inhibition of proinflammatory and apoptotic events in ECs.