Abstract 15382: p62 Binding to Protein Kinase C ζ Regulates Tumor Necrosis Factor α-Induced Inflammatory and Apoptotic Pathways in Endothelial Cells
Background: Protein kinase C (PKC)ζ is a key pathological mediator of tumor necrosis factor (TNF)α-induced vascular inflammation. It is the only PKC isoform that is specifically activated in disturbed flow regions of the vasculature. We have shown that TNFα-induced PKCζ signaling induces activation of c-Jun N-terminal kinase (JNK) and cleavage of caspase-3 in endothelial cells (EC). The specific mechanisms by which TNFα stimulates PKCζ activity in EC are unclear. p62 is a scaffold protein that regulates several cell signaling pathways by binding to target proteins. Because p62 is known to bind PKCζ, we hypothesized that interaction of PKCζ with p62 is required for TNFα-induced PKCζ signaling and vascular inflammation.
Methods and Results: By en face immunohistochemistry of aortas from ApoE knockout mice, we found increased p62 expression in disturbed flow areas, but not in steady flow. PKCζ activation correlated with the magnitude of p62 expression, suggesting a potential role in atheropromotion. To model vascular inflammation, we treated cultured EC with 10 ng/ml TNFα for 24 hr. Consistent with in vivo data, p62 was significantly increased by 2.91±0.34 -fold in response to TNFα. Importantly, pretreatment with 10 mM Tiron, a reactive oxygen species (ROS) scavenger, inhibited TNFα-induced p62 expression, suggesting a key role for ROS in chronic inflammation associated with disturbed flow. To study the effect of TNFα on the interaction of PKCζ and p62 we used the Duolink proximity ligation assay, which showed that the interaction was significantly increased by TNFα (10 ng/ml): 2.16±0.14 at 15 min and 2.73±0.36 fold at 30 min. Activation of PKCζ required interaction with p62 since transfection of the dominant-negative p62K7A mutant that cannot bind to PKCζ reduced PKCζ activation, JNK activation, and caspase-3 cleavage in response to TNFα. In addition, when we depleted p62 by siRNA, TNFα-induced PKCζ activation, JNK activation and caspase-3 cleavage were dramatically suppressed.
Conclusions: These data suggest that binding of PKCζ and p62 is critical for TNFα-induced PKCζ activation and PKCζ-dependent pathological signaling in EC exposed to atheroprone disturbed flow.
- © 2011 by American Heart Association, Inc.