Abstract 5528: Novel Role of PKCζ-ERK5 Complex in TNFα-mediated eNOS Protein De-stabilization
Background: Laminar steady (s−) flow-induced activation of ERK5 leads to KLF2 activation and subsequent eNOS expression, thus maintaining normal endothelial cell (EC) function. In contrast, PKCζ has emerged as a pathological mediator of EC dysfunction based on
a positive correlation between PKCζ activity and disturbed (d−) flow; and
the essential role of PKCζ activation in TNFα stimulation of JNK, caspase-3 and EC apoptosis.
Emerging evidence demonstrates the reduction of eNOS expression in d-flow area, but the molecular mechanisms remain unknown.
Methods and Results: First, both TNFα and d-flow activated PKCζ, and TNFα inhibited s-flow-induced eNOS protein expression in EC. Interestingly, the reduction of eNOS expression by TNFα was abolished by adenoviral infection of a dominant negative PKCζ (Ad.DN-PKCζ). However, Ad.DN-PKCζ could not reverse the TNFα-mediated reduction of KLF2/eNOS mRNA expression. These data portend a post-translational downregulation of eNOS expression via PKCζ. Pretreatment with MG132 (proteosome inhibitor) inhibited TNFα-mediated reduction of eNOS protein, suggesting involvement of the ubiquitin-proteosome system. In vitro run-off assays showed that TNFα de-stabilized eNOS, and transduction of Ad.DN-PKCζ stabilized eNOS, which suggested a key role of PKCζ activation on eNOS stability. Using a mammalian two-hybrid assay we found a direct association between PKCζ and ERK5. Further, we found that the catalytic domain of PKCζ (aa 405–591) and the C-terminal region of ERK5 (aa 571– 806) appeared to be the domains required for this direct interaction. In vitro kinase assays revealed that Ser 486 of ERK5 is a novel phosphorylation site of PKCζ. Then, we investigated the contribution of both PKCζ-ERK5 binding and phosphorylation on eNOS stability. First, overexpression of the ERK5 fragment (aa 571– 806) inhibited PKCζ-ERK5 binding and PKCζ-mediated reduction of eNOS. Second, we found that an ERK5S486A mutant (Ad.ERK5 S486A) significantly inhibited eNOS de-stabilization compared with ERK5 wild type (Ad.ERK5-WT).
Conclusion: These data suggest a key role of PKCζ- ERK5 complex in TNFα-mediated eNOS protein de-stabilization and subsequent EC dysfunction.