The Effects of Lipopolysaccharide on the Toll-like Receptor 4 Expression in Vascular Smooth Muscle Cells

• Main Authors: Feng-Yen Lin, 林豐彥
• Other Authors: Shing-Jong Lin
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/48598089637446169500

博士 === 國防醫學院 === 醫學科學研究所 === 95 === Lipopolysaccharide (LPS) interacts with toll-like receptor 4 (TLR4) and induces proliferation of vascular smooth muscle cells (VSMCs) which plays a causal role in atherogenesis. The role of TLR4 expression and regulation in LPS-stimulated VSMCs remains unclear. This study was conducted to investigate the mechanisms involved in lipopolysaccharide (LPS)-induced TLR4 expression in VSMCs. Stimulation of human aortic smooth muscle cells (HASMCs) with LPS significantly increased TLR4 expression. The increase was regulated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (including the activation of subunits p47phox and Rac1), which mediates the production of reactive oxygen species and the activation of intracellular mitogen-activated protein kinase signaling pathways. Treatment with polyethylene-glycol-conjugated superoxide dismutase, N-acetylcysteine (NAC), diphenylene iodonium (DPI), or apocynin significantly decreased LPS-induced TLR4 expression. An actinomycin D chase experiment showed that LPS increased the half-life of TLR4 mRNA. Inhibition of NADPH oxidase activity by DPI, apocynin, or NAC significantly decreased TLR4 mRNA stability, as did the knock down of RAC1 gene expression by RNA interference. TLR4 mRNAs often contain AU-rich elements (AREs) in their 3’ untranslated regions (3’UTR) which have a high affinity for RNA-binding proteins. The current study demonstrated that stimulation of HASMCs with LPS significantly increased the cytosolic HuR, a kind of RNA-binding proteins, level in vitro. Immunoprecipitation and RT-PCR demonstrated that LPS markedly increased the interaction of HuR and 3’UTR of TLR4 mRNA. The reporter plasmid, which contains the 3’UTR of TLR4 mRNA, significantly increased luciferase reporter gene expression in LPS-induced HASMCs. These data suggest that the 3’UTR of TLR4 mRNA confers LPS responsiveness and that HuR modulates 3’UTR-mediated gene expression. Knockdown of HuR inhibited LPS-induced TLR4 mRNA stability in HASMCs and luciferase reporter gene expression in CMV-Luciferase-TLR4 3’UTR-transfected HASMCs. In addition, inhibition of NADPH oxidase activity by diphenylene iodonium, knockdown of Rac1 gene expression by siRNA, and decrease of p38 MAPK activity by SB203580 significantly decreased the cytosolic HuR level, which mediates TLR4 mRNA stability. We also demonstrated in an animal model that LPS administration led to a significant increase of balloon-injury-induced neointimal hyperplasia, and of TLR4 expression, in rabbit aorta. These findings suggest that NADPH oxidase activation, mRNA stabilization, and MAPK signaling pathways play critical roles in LPS-enhanced TLR4 expression in HASMCs, which contributes to vascular inflammation and cardiovascular disorders.