Oxidative protein cross-linking reactions involving L-tyrosine in transforming growth factor-beta1-stimulated fibroblasts.
The mechanisms by which ligand-stimulated generation of reactive oxygen species in nonphagocytic cells mediate biologic effects are largely unknown. The profibrotic cytokine, transforming growth factor-beta1 (TGF-beta1), generates extracellular hydrogen peroxide (H2O2) in contrast to intracellular reactive oxygen species production by certain mitogenic growth factors in human lung fibroblasts. To determine whether tyrosine residues in fibroblast-derived extracellular matrix (ECM) proteins may be targets of H2O2-mediated dityrosine-dependent cross-linking reactions in response to TGF-beta1, we utilized fluorophore-labeled tyramide, a structurally related phenolic compound that forms dimers with tyrosine, as a probe to detect such reactions under dynamic cell culture conditions. With this approach, a distinct pattern of fluorescent labeling that seems to target ECM proteins preferentially was observed in TGF-beta1-treated cells but not in control cells. This reaction required the presence of a heme peroxidase and was inhibited by catalase or diphenyliodonium (a flavoenzyme inhibitor), similar to the effect on TGF-beta1-induced dityrosine formation. Exogenous addition of H2O2 to control cells that do not release extracellular H2O2 produced a similar fluorescent labeling reaction. These results support the concept that, in the presence of heme peroxidases in vivo, TGF-beta1-induced H2O2 production by fibroblasts may mediate oxidative dityrosine-dependent cross-linking of ECM protein(s). This effect may be important in the pathogenesis of human fibrotic diseases characterized by overexpression/activation of TGF-beta1.
Pulmonary and Critical Care Division, Department of Medicine, New England Medical Center/Tupper Research Institute, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
J M
JM
Larios
R
R
Budhiraja
B L
BL
Fanburg
V J
VJ
Thannickal
D001713:Biphenyl Compounds / Q000494:pharmacology; D002374:Catalase / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D019281:Dimerization; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D006735:Horseradish Peroxidase / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000494:pharmacology; D008168:Lung; D009861:Onium Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D001713:Biphenyl Compounds; D016326:Extracellular Matrix Proteins; D005456:Fluorescent Dyes; D009861:Onium Compounds; D016212:Transforming Growth Factor beta; C031291:diphenyliodonium; D014443:Tyrosine; D006861:Hydrogen Peroxide; C007543:dityrosine; D006735:Horseradish Peroxidase; D002374:Catalase