title: The synthetic cannabinoid R(+)WIN 55,212-2 inhibits the interleukin-1 signaling pathway in human astrocytes in a cannabinoid receptor-independent manner.
abstract: R(+)WIN 55,212-2 is a synthetic cannabinoid that controls disease progression in models of multiple sclerosis. This is associated with its ability to reduce migration of leukocytes into the central nervous system. Because leukocyte migration is dependent on induction of adhesion molecules and chemokines by pro-inflammatory cytokines, we examined the effects of R(+)WIN 55,212-2 on their expression. Using 1321N1 astrocytoma and A-172 glioblastoma as cell models we show that R(+)WIN 55,212-2, but not its inactive chiral form S(-)WIN 55,212-2, strongly inhibits the interleukin-1 (IL-1) induction of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and the chemokine IL-8. This inhibition is not mediated via the CB1 or CB2 cannabinoid receptors, because their selective antagonists and pertussis toxin failed to affect the inhibitory effects of R(+)WIN 55,212-2. Furthermore reverse transcription-PCR analysis did not detect the expression of either receptor in 1321N1 cells. R(+)WIN 55,212-2 was shown to inhibit adhesion molecule and chemokine expression at the level of transcription, because it strongly inhibited the IL-1 induction of ICAM-1, VCAM-1, and IL-8 mRNAs and blocked the IL-1 activation of their promoters. The NFkappaB pathway was then assessed as a lead target for R(+)WIN 55,212-2. NFkappaB was measured by expression of a transfected NFkappaB-regulated reporter gene. Using this assay, R(+)WIN 55,212-2 strongly inhibited IL-1 activation of NFkappaB. Furthermore R(+)WIN 55,212-2 inhibited the ability of overexpressed Myd88, Tak-1, and IKK-2 to induce the reporter gene suggesting that R(+)WIN 55,212-2 acts at or downstream of IKK-2 in the IL-1 pathway. However R(+)WIN 55,212-2 failed to inhibit IL-1-induced degradation of IkappaBalpha, excluding IKK-2 as a direct target. In addition electrophoretic mobility shift and chromatin immunoprecipitation assays showed that R(+)WIN 55,212-2 does not regulate the IL-1-induced nuclear translocation of NFkappaB or the ability of the latter to bind to promoters regulating expression of ICAM-1 and IL-8. These data suggest that R(+)WIN 55,212-2 blocks IL-1 signaling by inhibiting the transactivation potential of NFkappaB.
authors.affiliation: UCD School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Niamh M
authors.initials: NM
authors.lastname: Curran
authors.forename: Bryan D
authors.initials: BD
authors.lastname: Griffin
authors.forename: Daniel
authors.initials: D
authors.lastname: O'Toole
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Brady
authors.forename: Stephen N
authors.initials: SN
authors.lastname: Fitzgerald
authors.forename: Paul N
authors.initials: PN
authors.lastname: Moynagh
mesh_terms: D021581:Active Transport, Cell Nucleus; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001254:Astrocytoma / Q000378:metabolism / Q000473:pathology; D048588:Benzoxazines; D015153:Blotting, Western; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002186:Cannabinoids / Q000378:metabolism*; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002465:Cell Movement; D047369:Chromatin Immunoprecipitation; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D017930:Genes, Reporter; D005909:Glioblastoma / Q000473:pathology; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D047468:Immunoprecipitation; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D007375:Interleukin-1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D007962:Leukocytes / Q000166:cytology; D009025:Morpholines / Q000494:pharmacology*; D009103:Multiple Sclerosis / Q000473:pathology; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology*; D010766:Phosphorylation; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
chemical_list: D048588:Benzoxazines; D002121:Calcium Channel Blockers; D002186:Cannabinoids; D020672:I-kappa B Proteins; D007375:Interleukin-1; D016209:Interleukin-8; D009025:Morpholines; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D009281:Naphthalenes; D012333:RNA, Messenger; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1; D000072000:NF-KappaB Inhibitor alpha; D012694:Serine; C070417:(3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone; D012313:RNA; D004247:DNA