P295: TNF alpha induces MCP 1 expression in vascular smooth muscle cells through a mechanism involving PKC delta

Abstract

Introduction: Monocycle chemo attractant protein 1 (MCP-1), produced by many types of vascular cells including smooth muscle cells (SMCs), plays an early and critical role in the development of vascular inflammation through the recruitment of monocytes and macrophages to the arterial wall. Targeted gene deletion of MCP-1 inhibits the development of intimal hyperplasia or abdominal aortic aneurysms in animal models. In this study, we investigated the molecular mechanism underlying the regulation of MCP-1 expression in SMCs, in particular the role of signaling molecule protein kinase C delta (PKCδ), which is known to be activated by pro-inflammation cytokines. Methods: The pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) was used as a prototype to stimulate rat aortic SMC line A10 as well as primary SMCs isolated from PKCδ knock-out mice and their wild-type littermates. MCP-1 protein in cell culture media was determined by an ELISA assay and MCP-1 mRNA by the semi-quantitative RT-PCR. Results: TNF-α, dose-dependently, elicited a marked increase in the amount of MCP-1 protein produced by A10 SMCs. A time-couse analysis showed that the induction was evident at 2 h and persistent up to 48 h. Similarly, TNF-α rapidly upregulated the level of MCP-1 mRNA in the cells, suggesting the induction may take place at the gene expression level. To understand the role of PKCδ in regulation of MCP-1 expression, we pretreated A10 cells with an increasing amount of rottlerin (0, 0.1, 0.5, 1 and 2 μM), a selective inhibitor to PKCδ. Rottlerin dose-dependently blocked the induction of MCP-1 protein and mRNA. To confirm these results obtained with the chemical inhibitor, we employed PKCδ knock-out mice. Aortic SMCs isolated from PKCδ null mice or their wild-type littermates were treated with TNF-α as above. PKCδ gene deficiency led to a complete loss of ability of SMCs to express MCP-1. Finally, we tested whether molecular activation of PKCδ is sufficient to induce MCP-1 expression. To this end, we ectopically expressed PKCδ in A10 cells via an adenoviral vector that expresses the wild-type PKCδ. Compared to the viral vector, Ad-PKCδ elicited an about two-fold increase in MCP-1 expression. Conclusions: Taken together, our data show that the pro-inflammatory cytokine TNF-α is a potent inducer of MCP-1 expression in vascular SMCs. TNF-α stimulates MCP-1 expression through a signaling pathway that involves PKCδ. Further studies are mandated to test whether inhibition of PKCδ could prevent MCP-1 expression thus monocytes recruitment in animal models following arterial injury.