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.
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).


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.


CONCLUSIONS
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.
Lin
Feng-Yen
FY
Graduate Institute of Medical Sciences, Tri-service General Hospital, National Defense Medical Center, Taipei, Taiwan.
Chen
Yung-Hsiang
YH
Lin
Yi-Wen
YW
Tsai
Jen-Sung
JS
Chen
Jaw-Wen
JW
Wang
Hsiao-Jung
HJ
Chen
Yuh-Lien
YL
Li
Chi-Yuan
CY
Lin
Shing-Jong
SJ
D000818:Animals; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism*; D001011:Aorta / Q000378:metabolism / Q000473:pathology; D050197:Atherosclerosis / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D004731:Endotoxins / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D017539:Tunica Intima / Q000378:metabolism / Q000473:pathology
D000954:Antigens, Surface; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D004731:Endotoxins; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D019255:NADPH Oxidases; D020929:Mitogen-Activated Protein Kinase Kinases