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(Chest. 2001;120:S32-S33.)
© 2001 American College of Chest Physicians

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

Victor J. Thannickal, MD; Jose M. Larios, BS and Barry L. Fanburg, MD

* 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

Myofibroblasts play an important role in the pathobiology of pulmonary fibrosis. Transforming growth factor (TGF)-ß1 is a strong inducer of myofibroblast differentiation both in vivo and in vitro. We have previously demonstrated the ability of TGF-ß1 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-ß1–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 {alpha}-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, {alpha}-actinin was found to localize to actin stress fibers in response to TGF-ß1, while protein expression was unaltered. Concurrently, TGF-ß1 markedly upregulated the protein expression of nonmuscle myosin heavy chain type A and {alpha}-smooth muscle actin in a time-dependent manner. All of these phenotypic changes suggestive of myofibroblast differentiation as well as the TGF-ß1-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-ß1 were found to be cell adhesion-dependent. Taken together, these results suggest that myofibroblast differentiation of lung fibroblasts by TGF-ß1 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.

Footnotes

Abbreviation: TGF = transforming growth factor

This work was supported by National Institutes of Health grants K08-03552 and HL-42376.

References

  1. Thannickal, VJ, Fanburg, BL (1995) Activation of an H2O2-generating NADH oxidase in human lung fibroblasts by transforming growth factor ß1. J Biol Chem 270,30334-30338
  2. Thannickal, VJ, Aldweib, KD, Fanburg, BL (1998) Tyrosine phosphorylation regulates H2O2 production in lung fibroblasts stimulated by transforming growth factor ß1. J Biol Chem 273,23611-23615




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