Protein Kinase C- δ ( PKCδ) Regulates Proinflammatory Chemokine Expression through Cytosolic Interaction with the NF- κB Subunit p65 in Vascular Smooth Muscle Cells

Background: Proinflammatory chemokines released by vascular smooth muscle cells (VSMCs) play a critical role in vascular inflammation. Results: Promoting protein kinase C- (PKC) translocation or inhibition of NF-B pathway diminishes proinflammatory chemokine production. Conclusion: PKC regulates proinflammatory chemokine expression through cytosolic interaction with the NF-B subunit p65, thus modulating inflammation. Significance: Learning how PKC regulates proinflammatory chemokine expression is crucial for understanding vascular inflammation. Proinflammatory chemokines released by vascular smooth muscle cells (VSMCs) play a critical role in vascular inflammation. Protein kinase C- (PKC) has been shown to be up-regulated in VSMCs of injured arteries. PKC knock-out (Prkcd(-/-)) mice are resistant to inflammation as well as apoptosis in models of abdominal aortic aneurysm. However, the precise mechanism by which PKC modulates inflammation remains incompletely understood. In this study, we identified four inflammatory chemokines (Ccl2/Mcp-1, Ccl7, Cxcl16, and Cx3cl1) of over 45 PKC-regulated genes associated with inflammatory response by microarray analysis. Using CCL2 as a prototype, we demonstrated that PKC stimulated chemokine expression at the transcriptional level. Inhibition of the NF-B pathway or siRNA knockdown of subunit p65, but not p50, eliminated the effect of PKC on Ccl2 expression. Overexpressing PKC followed by incubation with phorbol 12-myristate 13-acetate resulted in an increase in p65 Ser-536 phosphorylation and enhanced DNA binding affinity without affecting IB degradation or p65 nuclear translocation. Prkcd gene deficiency impaired p65 Ser-536 phosphorylation and DNA binding affinity in response to TNF. Results from in situ proximity ligation analysis and co-immunoprecipitation performed on cultured VSMCs and aneurysmal aorta demonstrated physical interaction between PKC and p65 that took place largely outside the nucleus. Promoting nuclear translocation of PKC with peptide RACK diminished Ccl2 production, whereas inhibition of PKC translocation with peptide V1-1 enhanced Ccl2 expression. Together, these results suggest that PKC modulates inflammation at least in part through the NF-B-mediated chemokines. Mechanistically, PKC activates NF-B through an IB-independent cytosolic interaction, which subsequently leads to enhanced p65 phosphorylation and DNA binding affinity.