title: Raf-1 kinase mediates adenylyl cyclase sensitization by TNF-alpha in human airway smooth muscle cells.
abstract: Tumor necrosis factor (TNF)-alpha is a potent inflammatory cytokine implicated in the exacerbation of asthma. Chronic exposure to TNF-alpha has been reported to induce G protein-coupled receptor desensitization, but adenylyl cyclase sensitization, in airway smooth muscle cells by an unknown mechanism. Cyclic AMP, which is synthesized by adenylyl cyclases in response to G protein-coupled receptor signals, is an important second messenger involved in the regulation of the airway muscle proliferation, migration, and tone. In other cell types, TNF-alpha receptors transactivate the EGF receptor, which activates raf-1 kinase. Further studies in transfected cells show that raf-1 kinase can phosphorylate and activate some isoforms of adenylyl cyclase. Cultured human airway smooth muscle cells were treated with TNF-alpha in the presence or absence of inhibitors of prostaglandin signaling, protein kinases, or G(i) proteins. TNF-alpha caused a significant dose- (1-10 ng/ml) and time-dependent (24 and 48 h) increase in forskolin-stimulated adenylyl cyclase activity, which was abrogated by pretreatment with GW5074 (a raf-1 kinase inhibitor), was partially inhibited by an EGF receptor inhibitor, but was unaffected by pertussis toxin. TNF-alpha also increased phosphorylation of Ser(338) on raf-1 kinase, indicative of activation. IL-1beta and EGF sensitization of adenylyl cyclase activity was also sensitive to raf-1 kinase inhibition by GW5074. Taken together, these studies link two signaling pathways not previously characterized in human airway smooth muscle cells: TNF-alpha transactivation of the EGF receptor, with subsequent raf-1 kinase-mediated activation of adenylyl cyclase.
authors.affiliation: Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Osawa
authors.forename: Peter D
authors.initials: PD
authors.lastname: Yim
authors.forename: Dingbang
authors.initials: D
authors.lastname: Xu
authors.forename: Reynold A
authors.initials: RA
authors.lastname: Panettieri
authors.forename: Charles W
authors.initials: CW
authors.lastname: Emala
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D002478:Cells, Cultured; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D008168:Lung / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D010766:Phosphorylation / Q000187:drug effects; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D007527:Isoenzymes; D014409:Tumor Necrosis Factor-alpha; C512478:EGFR protein, human; D066246:ErbB Receptors; D019908:Proto-Oncogene Proteins c-raf; D000262:Adenylyl Cyclases; D007213:Indomethacin