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H2O2 Production by Myofibroblasts Is Dependent on Src Kinase(s) and Actin Cytoskeletal Regulation*

Victor J. Thannickal, MD; Jose M. Larios, BS; Barry L. Fanburg, MD
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*From the Pulmonary and Critical Care Division, New England Medical Center and Tufts University School of Medicine, Boston, MA.

Correspondence to: Victor J. Thannickal, MD, Pulmonary and Critical Care Division, New England Medical Center, 750 Washington St, NEMC #257, Boston, MA 02111



Chest. 2001;120(1_suppl):S32-S33. doi:10.1378/chest.120.1_suppl.S32
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Myofibroblasts play an important role in the pathobiology of pulmonary fibrosis. Transforming growth factor (TGF)-β1 is a strong inducer of myofibroblast differentiation both in vivo and in vitro. We have previously demonstrated the ability of TGF-β1 to activate a novel cell surface-associated H2O2-generating nicotanimide adenine dinucleotide oxidase in cultured human lung fibroblast,1 along with the tyrosine phosphorylation of an approximately 115-kd protein.2 To identify this protein and potentially others that may associate with it, we immunoprecipitated TGF-β1–treated cell lysates with an antiphosphotyrosine antibody (PY-20) under nondenaturing conditions and then separated the immunoprecipitated complexes by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Two proteins, human α-actinin and nonmuscle myosin heavy chain type A, were identified by band excision and sequence analysis by microcapillary reverse-phase high-performance liquid chromatography nanoelectrospray tandem mass spectrometry (μLC/MS/MS; Harvard Microchemistry Facility; Cambridge MA). Using immunofluorescence techniques, α-actinin was found to localize to actin stress fibers in response to TGF-β1, while protein expression was unaltered. Concurrently, TGF-β1 markedly upregulated the protein expression of nonmuscle myosin heavy chain type A and α-smooth muscle actin in a time-dependent manner. All of these phenotypic changes suggestive of myofibroblast differentiation as well as the TGF-β1-induced tyrosine phosphorylation and nicotanimide adenine dinucleotide oxidase activation/H2O2 production were inhibited in the presence of the Src kinase inhibitor, PP2 (10 μM). Moreover, both tyrosine phosphorylation and oxidase activation induced by TGF-β1 were found to be cell adhesion-dependent. Taken together, these results suggest that myofibroblast differentiation of lung fibroblasts by TGF-β1 is associated with Src kinase(s)-dependent actin cytoskeletal regulation that is required for the assembly and/or activation of oxidase components at the cell surface, possibly involving focal contacts.

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