title: Protein kinase C-δ (PKCδ) regulates proinflammatory chemokine expression through cytosolic interaction with the NF-κB subunit p65 in vascular smooth muscle cells.
abstract: Proinflammatory chemokines released by vascular smooth muscle cells (VSMCs) play a critical role in vascular inflammation. Protein kinase C-δ (PKCδ) has been shown to be up-regulated in VSMCs of injured arteries. PKCδ knock-out (Prkcd(-/-)) mice are resistant to inflammation as well as apoptosis in models of abdominal aortic aneurysm. However, the precise mechanism by which PKCδ modulates inflammation remains incompletely understood. In this study, we identified four inflammatory chemokines (Ccl2/Mcp-1, Ccl7, Cxcl16, and Cx3cl1) of over 45 PKCδ-regulated genes associated with inflammatory response by microarray analysis. Using CCL2 as a prototype, we demonstrated that PKCδ stimulated chemokine expression at the transcriptional level. Inhibition of the NF-κB pathway or siRNA knockdown of subunit p65, but not p50, eliminated the effect of PKCδ on Ccl2 expression. Overexpressing PKCδ 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 IκB degradation or p65 nuclear translocation. Prkcd gene deficiency impaired p65 Ser-536 phosphorylation and DNA binding affinity in response to TNFα. Results from in situ proximity ligation analysis and co-immunoprecipitation performed on cultured VSMCs and aneurysmal aorta demonstrated physical interaction between PKCδ and p65 that took place largely outside the nucleus. Promoting nuclear translocation of PKCδ with peptide ψδRACK diminished Ccl2 production, whereas inhibition of PKCδ translocation with peptide δV1-1 enhanced Ccl2 expression. Together, these results suggest that PKCδ modulates inflammation at least in part through the NF-κB-mediated chemokines. Mechanistically, PKCδ activates NF-κB through an IκB-independent cytosolic interaction, which subsequently leads to enhanced p65 phosphorylation and DNA binding affinity.
authors: From the Division of Vascular Surgery, Department of Surgery, University of Wisconsin, Madison, Wisconsin 53705 and.
authors: Jun
authors: J
authors: Ren
authors: Qiwei
authors: Q
authors: Wang
authors: Stephanie
authors: S
authors: Morgan
authors: Yi
authors: Y
authors: Si
authors: Aarthi
authors: A
authors: Ravichander
authors: Changlin
authors: C
authors: Dou
authors: K Craig
authors: KC
authors: Kent
authors: Bo
authors: B
authors: Liu
mesh_terms: D000818:Animals; D017544:Aortic Aneurysm, Abdominal / Q000473:pathology; D018925:Chemokines / Q000235:genetics*; D003600:Cytosol / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D007249:Inflammation / Q000378:metabolism; D008297:Male; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000473:pathology; D010766:Phosphorylation; D011485:Protein Binding; D051745:Protein Kinase C-delta / Q000378:metabolism*; D051381:Rats; D051996:Transcription Factor RelA / Q000378:metabolism*; D014158:Transcription, Genetic; D015854:Up-Regulation
chemical_list: D018925:Chemokines; D051996:Transcription Factor RelA; D004247:DNA; D051745:Protein Kinase C-delta
keywords: Chemokines; Gene Expression; Inflammation; Nuclear Factor κ-Light Chain Enhancer of Activated B Cells (NF-κB); Protein Kinase C-δ (PKCδ); Protein-Protein Interactions; Vascular Smooth Muscle Cells