Raf-1 kinase mediates adenylyl cyclase sensitization by TNF- in human airway smooth muscle cells

Osawa Y, Yim PD, Xu D, Panettieri RA, Emala CW. Raf-1 kinase mediates adenylyl cyclase sensitization by TNFin human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 292: L1414–L1421, 2007. First published February 2, 2007; doi:10.1152/ajplung.00123.2006.—Tumor necrosis factor (TNF)is a potent inflammatory cytokine implicated in the exacerbation of asthma. Chronic exposure to TNFhas 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, TNFreceptors 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 TNFin the presence or absence of inhibitors of prostaglandin signaling, protein kinases, or Gi proteins. TNFcaused 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. TNFalso increased phosphorylation of Ser on raf-1 kinase, indicative of activation. IL-1 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: TNFtransactivation of the EGF receptor, with subsequent raf-1 kinase-mediated activation of adenylyl cyclase.