Specific Inhibition of Nuclear Factor-κB–Dependent Inflammatory Responses by Cell Type-Specific Mechanisms upon A2A Adenosine Receptor Gene Transfer
- Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom
- Address correspondence to:
Dr. T. M. Palmer, 425 Davidson Bldg., University of Glasgow, Glasgow, G12 8QQ, UK. E-mail:t.palmer{at}bio.gla.ac.uk
Abstract
Adenosine is a potent inhibitor of inflammatory processes, and the A2A adenosine receptor (A2AAR) plays a key nonredundant role as a suppresser of inflammatory responses in vivo. In this study, we demonstrate that increasing A2AAR gene expression suppressed multiple inflammatory responses in both human umbilical vein endothelial cells (HUVECs) and rat C6 glioma cells in vitro. In particular, the induction of the adhesion molecule E-selectin by either tumor necrosis factor α (TNFα) or Escherichia coli lipopolysaccharide (LPS) was reduced by more than 70% in HUVECs, whereas inducible nitric-oxide synthase (iNOS) induction was abolished in C6 cells after exposure to interferon-γ in combination with LPS and TNFα, suggesting that the receptor inhibited a common step in the induction of each of these pro-inflammatory genes. Consistent with this hypothesis, A2AAR expression inhibited the activation of NF-κB, a key transcription factor whose proper function was essential for optimal iNOS and E-selectin induction. However, although NF-κB binding to target DNA was severely compromised in both cell types, the mechanisms by which this occurred were distinct. In C6 cells, A2AAR expression blocked IκBα degradation by inhibiting stimulus-induced phosphorylation, whereas in HUVECs, A2AAR expression inhibited NF-κB translocation to the nucleus independently of any effect on IκBα degradation. Together, these observations suggest that A2AAR-mediated inhibition NF-κB activation is a critical aspect of its anti-inflammatory signaling properties and that the molecular basis of this inhibition varies in a cell type-specific manner.
Footnotes
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This work was supported by project grants from the British Heart Foundation and National Heart Research Fund, equipment grants from Tenovus-Scotland and The Wellcome Trust (to T.M.P.) and studentships from the Medical Research Council (to A.F.M.) and British Heart Foundation (to E.W.S.).
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.104.001107.
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ABBREVIATIONS: AR, adenosine receptor; EC, endothelial cell; NECA, 5′-N-ethylcarboxamidoadenosine; CGS21680, 2-(p-carboxyethyl)phenylamino-5′-N-ethylcarboxamidoadenosine; CPA, N6-cyclopentyladenosine; HUVEC, human umbilical vein endothelial cell; TNFα, tumor necrosis factor α; iNOS, inducible nitric-oxide synthase; NF-κB, nuclear factor κB; LPS, lipopolysaccharide; IFN, interferon; AV, adenovirus; WT, wild type; GFP, green fluorescent protein; m.o.i., multiplicity of infection; DTT, dithiothreitol; pfu, plaque-forming units; PBS, phosphate-buffered saline; BSA, bovine serum albumin; JNK, c-Jun N-terminal kinase; PMSF, phenylmethylsulfonyl fluoride; GST, glutathione S-transferase; PBSGG, phosphate-buffered saline/goat serum/gelatin; ZM241385, 4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)phenol; PDTC, pyrrolidine dithiocarbamate; NAcCys, N-acetylcysteine; IKK, IκB kinase; PD, positive regulatory domain; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; TLR, Toll-like receptor; ELISA, enzyme-linked immunosorbent assay.
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- Received April 6, 2004.
- Accepted July 30, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
