title: Spatial compartmentalization of tumor necrosis factor (TNF) receptor 1-dependent signaling pathways in human airway smooth muscle cells. Lipid rafts are essential for TNF-alpha-mediated activation of RhoA but dispensable for the activation of the NF-kappaB and MAPK pathways.
abstract: Tumor necrosis factor (TNF)-alpha-induced activation of RhoA, mediated by TNF receptor 1 (TNFR1), is a prerequisite step in a pathway that leads to increased 20-kDa light chain of myosin (MLC20) phosphorylation and airway smooth muscle contraction. In this study, we have investigated the proximal events in TNF-alpha-induced RhoA activation. TNFR1 is localized to both lipid raft and nonraft regions of the plasma membrane in primary human airway smooth muscle cells. TNF-alpha engagement of TNFR1 recruited the adaptor proteins TRADD, TRAF-2, and RIP into lipid rafts and activated RhoA, NF-kappaB, and MAPK pathways. Depletion of cholesterol from rafts with methyl-beta-cyclodextrin caused a redistribution of TNFR1 to nonraft plasma membrane and prevented ligand-induced RhoA activation. By contrast, TNF-alpha-induced activation of NF-kappaB and MAPKs was unaffected by methyl-beta-cyclodextrin indicating that, in airway smooth muscle cells, activation of these pathways occurred independently of lipid rafts. Targeted knockdown of caveolin-1 completely abrogated TNF-alpha-induced RhoA activation, identifying this raft-resident protein as a positive regulator of the activation process. The signaling adaptors TRADD and RIP were also found to be necessary for ligand-induced RhoA activation. Taken together, our results suggest that in airway smooth muscle cells, spatial compartmentalization of TNFR1 provides a mechanism for generating distinct signaling outcomes in response to ligand engagement and define a mechanistic role for lipid rafts and caveolin-1 in TNF-alpha-induced activation of RhoA.
authors.affiliation: School of Medical Sciences, University of Aberdeen, IMS Building, Foresterhill, Aberdeen AB25 2ZD, United Kingdom. i.hunter@abdn.ac.uk
authors.forename: Irene
authors.initials: I
authors.lastname: Hunter
authors.forename: Graeme F
authors.initials: GF
authors.lastname: Nixon
mesh_terms: D001980:Bronchi / Q000378:metabolism*; D051242:Caveolin 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D015151:Immunoblotting; D047468:Immunoprecipitation; D021962:Membrane Microdomains / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000494:pharmacology; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism*; D012293:Rifampin / Q000031:analogs & derivatives / Q000378:metabolism; D015398:Signal Transduction; D053199:TNF Receptor-Associated Death Domain Protein / Q000378:metabolism; D047992:TNF Receptor-Associated Factor 2 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: C084369:3-formyl-23-(O-(beta-glucopyranosyl))rifamycin SV; C495540:CAV1 protein, human; D051242:Caveolin 1; D016328:NF-kappa B; D034741:RNA, Small Interfering; D047888:Receptors, Tumor Necrosis Factor, Type I; D053199:TNF Receptor-Associated Death Domain Protein; D047992:TNF Receptor-Associated Factor 2; D014409:Tumor Necrosis Factor-alpha; D051550:I-kappa B Kinase; D020928:Mitogen-Activated Protein Kinases; D020742:rhoA GTP-Binding Protein; D012293:Rifampin