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