Abstract 319: Protein Kinase C-delta Regulates Pro-inflammatory Chemokine Expression Through the NF?B Pathway in Vascular Smooth Muscle Cells

Abstract

Objective- Inflammation is a key contributor to a plethora of vascular diseases including atherosclerosis and aneurysm. Pro-inflammatory chemokines released by vascular smooth muscle cells (SMCs) play a critical role in vascular inflammation. Previously, we showed protein kinase C-delta (PKCδ) was upregulated in SMCs of inflamed arteries. PKCδ-/- mice are resistant to inflammation as well as apoptosis in models of abdominal aortic aneurysm. We hypothesize that PKCδ modulates the inflammatory response in part through regulation of pro-inflammatory chemokine expression in SMCs. Method and Results- To identify PKCδ-regulated chemokines, we used microarray to compare transcription profiles of SMCs infected with AdPKCδ or AdNull. Results showed that 45 out of 203 differentially expressed genes have known inflammatory response roles. RT-PCR analyses confirmed the regulatory effect of PKCδ on selected inflammatory chemokines, including MCP1/CCL2, CCL7, CXCL16 and CX3CL1. Using a mouse aneurysm model, we showed all of these chemokines were elevated in aortae of wildtype, but not PKCδ-/- mice. We postulate that PKCδ regulates chemokine expression via the NFκB pathway. Using MCP-1 as a prototype, a detailed analysis showed PKCδ induced MCP1 mRNA expression (4.25±0.57 fold, n=5, p<0.05) and protein secretion (4.41±0.21 fold, n=3, p<0.05). Analysis of pre-mRNA and mRNA indicated that PKCδ stimulated MCP1 expression at the transcription level rather than by controlling its mRNA stability. Using a NFκB inhibitor (Andrographolide) or siRNA knockdown of NFκB subunit p65 eliminated PKCδ’s effect on MCP1 expression. Physical interaction between PKCδ and p65 was shown in cultured SMCs and aneurysmal aortae by the in situ proximity ligation assay. PKCδ/p65 complexes were detected largely outside the nucleus, indicating the two pathways intersect in the cytosol. This notion is supported by the lack of effect of PKCδ translocation peptides on MCP1. Furthermore, we demonstrated that PKCδ enhanced Ser536 phosphorylation and DNA-binding affinity of p65. Conclusion- Our results show PKCδ plays a key role in inflammation through the NFκB-mediated chemokine expression pathway in vascular SMCs and may serve as a suitable target for novel anti-inflammatory therapy.