The role of human antigen R, an RNA-binding protein, in mediating the stabilization of toll-like receptor 4 mRNA induced by endotoxin: A novel mechanism involved in vascular inflammation

Feng Yen Lin, Yung Hsiang Chen, Yi Wen Lin, Jen Sung Tsai, Jaw Wen Chen, Hsiao Jung Wang, Yuh Lien Chen, Chi Yuan Li, Shing Jong Lin

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

OBJECTIVE - 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. TLR4 mRNAs often contain AU-rich elements (AREs) in their 3′ untranslated regions (3′UTR) which have a high affinity for RNA-binding proteins. It is not know whether the RNA-binding protein, human antigen R (HuR), regulates TLR4 expression in human aortic smooth muscle cells (HASMCs). METHODS AND RESULTS - Stimulation of HASMCs with LPS significantly increased the cytosolic HuR 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. Knock-down 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, knock-down 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. CONCLUSION - Activation of NADPH oxidase and the MAPK-signaling pathway contribute to HuR-mediated stabilization of TLR4 mRNA induced by LPS in HASMCs. In the balloon injured rabbit aorta model, systemic inflammation induced by LPS caused intimal hyperplasia and increased TLR4 and HuR expression.

Original languageEnglish
Pages (from-to)2622-2629
Number of pages8
JournalArteriosclerosis, Thrombosis, and Vascular Biology
Volume26
Issue number12
DOIs
Publication statusPublished - Dec 2006
Externally publishedYes

Fingerprint

Toll-Like Receptor 4
RNA-Binding Proteins
Endotoxins
Blood Vessels
Inflammation
Antigens
Messenger RNA
Lipopolysaccharides
Smooth Muscle Myocytes
3' Untranslated Regions
Luciferases
Gene Expression
NADPH Oxidase
RNA Stability
Reporter Genes
Vascular Smooth Muscle
AU Rich Elements
Toll-Like Receptor 3
Tunica Intima
p38 Mitogen-Activated Protein Kinases

Keywords

  • Human antigen R
  • Inflammation
  • LPS
  • Toll-like receptor
  • Vascular smooth muscle cell (VSMC)

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine

Cite this

The role of human antigen R, an RNA-binding protein, in mediating the stabilization of toll-like receptor 4 mRNA induced by endotoxin : A novel mechanism involved in vascular inflammation. / Lin, Feng Yen; Chen, Yung Hsiang; Lin, Yi Wen; Tsai, Jen Sung; Chen, Jaw Wen; Wang, Hsiao Jung; Chen, Yuh Lien; Li, Chi Yuan; Lin, Shing Jong.

In: Arteriosclerosis, Thrombosis, and Vascular Biology, Vol. 26, No. 12, 12.2006, p. 2622-2629.

Research output: Contribution to journalArticle

Lin, Feng Yen ; Chen, Yung Hsiang ; Lin, Yi Wen ; Tsai, Jen Sung ; Chen, Jaw Wen ; Wang, Hsiao Jung ; Chen, Yuh Lien ; Li, Chi Yuan ; Lin, Shing Jong. / The role of human antigen R, an RNA-binding protein, in mediating the stabilization of toll-like receptor 4 mRNA induced by endotoxin : A novel mechanism involved in vascular inflammation. In: Arteriosclerosis, Thrombosis, and Vascular Biology. 2006 ; Vol. 26, No. 12. pp. 2622-2629.
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abstract = "OBJECTIVE - 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. TLR4 mRNAs often contain AU-rich elements (AREs) in their 3′ untranslated regions (3′UTR) which have a high affinity for RNA-binding proteins. It is not know whether the RNA-binding protein, human antigen R (HuR), regulates TLR4 expression in human aortic smooth muscle cells (HASMCs). METHODS AND RESULTS - Stimulation of HASMCs with LPS significantly increased the cytosolic HuR 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. Knock-down 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, knock-down 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. CONCLUSION - Activation of NADPH oxidase and the MAPK-signaling pathway contribute to HuR-mediated stabilization of TLR4 mRNA induced by LPS in HASMCs. In the balloon injured rabbit aorta model, systemic inflammation induced by LPS caused intimal hyperplasia and increased TLR4 and HuR expression.",
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T2 - A novel mechanism involved in vascular inflammation

AU - Lin, Feng Yen

AU - Chen, Yung Hsiang

AU - Lin, Yi Wen

AU - Tsai, Jen Sung

AU - Chen, Jaw Wen

AU - Wang, Hsiao Jung

AU - Chen, Yuh Lien

AU - Li, Chi Yuan

AU - Lin, Shing Jong

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AB - OBJECTIVE - 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. TLR4 mRNAs often contain AU-rich elements (AREs) in their 3′ untranslated regions (3′UTR) which have a high affinity for RNA-binding proteins. It is not know whether the RNA-binding protein, human antigen R (HuR), regulates TLR4 expression in human aortic smooth muscle cells (HASMCs). METHODS AND RESULTS - Stimulation of HASMCs with LPS significantly increased the cytosolic HuR 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. Knock-down 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, knock-down 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. CONCLUSION - Activation of NADPH oxidase and the MAPK-signaling pathway contribute to HuR-mediated stabilization of TLR4 mRNA induced by LPS in HASMCs. In the balloon injured rabbit aorta model, systemic inflammation induced by LPS caused intimal hyperplasia and increased TLR4 and HuR expression.

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KW - Toll-like receptor

KW - Vascular smooth muscle cell (VSMC)

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