Abstract
Anandamide (arachidonoylethanolamine, AEA), an endogenous agonist for both the cannabinoid CB1 receptor and the vanilloid VR1 receptor, elicits neurobehavioral, anti-inflammatory, immunomodulatory, and proapoptotic effects. Because of the central role of nuclear factor-κB (NF-κB) in the inflammatory process and the immune response, we postulated that AEA might owe some of its effects to the suppression of NF-κB. This study shows that AEA inhibits tumor necrosis factor-α (TNFα)-induced NF-κB activation by direct inhibition of the IκB kinase (IKK)β and, to a lesser extent, the IKKα subunits of κB inhibitor (IκB) kinase complex, and that IKKs inhibition by AEA correlates with inhibition of IκBα degradation, NF-κB binding to DNA, and NF-κB-dependent transcription in TNFα-stimulated cells. AEA also prevents NF-κB-dependent reporter gene expression induced by mitogen-activated protein kinase kinase kinase and NF-κB-inducing kinase. The NF-κB inhibitory activity of AEA was independent of CB1 and CB2activation in TNFα-stimulated 5.1 and A549 cell lines, which do not express vanilloid receptor 1, and was not mediated by hydrolytic products formed through the activity of the enzyme fatty acid amide hydrolase. Chemical modification markedly affected AEA inhibitory activity on NF-κB, suggesting rather narrow structure-activity relationships and the specific interaction with a molecular target. Substitution of the alkyl moiety with less saturated fatty acids generally reduced or abolished activity. However, replacement of the ethanolamine “head” with a vanillyl group led to potent inhibition of TNFα-induced NF-κB-dependent transcription. These findings provide new mechanistic insights into the anti-inflammatory and proapoptotic activities of AEA, and should foster the synthesis of improved analogs amenable to pharmaceutical development as anti-inflammatory agents.
Footnotes
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This work was supported by MCyT grant BIO 2000-1091-C01 (to E.M.), by European Union grant QLK3-CT-2000-00463 (to E.M. and G.A.), and by INTAS grant 97/1297 (to V.D.M.).
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R. S. and M.A.C. contributed equally to this study.
- Abbreviations:
- AEA
- anandamide
- 2-AG
- 2-arachidonoylglicerol
- CB
- cannabinoid receptor
- FAAH
- fatty acid amide hydrolase
- VR1
- vanilloid receptor type 1
- NF-κB
- nuclear factor-κB
- IκB
- κB inhibitor
- IKK
- IκB kinase
- IKC
- IκB kinase complex
- NIK
- NF-κB inducing kinase
- TNFα
- tumor necrosis factor-α
- MEKK
- mitogen-activated protein kinase kinase kinase
- COX-2
- cyclooxygenase 2
- HIV-LTR
- HIV long terminal repeat
- mAb
- monoclonal antibody
- ATFMK
- arachidonoyl trifluoromethyl ketone
- N-AVAM
- N-acylvanillamide
- DTT
- dithiothreitol
- NP-40
- Nonidet-P40
- EMSA
- electrophoretic mobility shift assay
- RT-PCR
- reverse transcriptase-polymerase chain reaction
- bp
- base pair(s)
- ERK
- extracellular signal-regulated kinase
- cyPG
- cyclopentenone prostaglandin
- SR141716A
- N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride
- Received June 5, 2002.
- Accepted October 21, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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