Oxidative protein cross-linking reactions involving L-tyrosine in transforming growth factor-beta 1-stimulated fibroblasts

The mechanisms by which ligand-stimulated generation of reactive oxygen spe cies in nonphagocytic cells mediate biologic effects are largely unknown. T he profibrotic cytokine, transforming growth factor-beta1 (TGF-beta1), gene rates extracellular hydrogen peroxide (H2O2) in contrast to intracellular r eactive oxygen species production by certain mitogenic growth factors in hu man 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 comp ound that forms dimers with tyrosine, as a probe to detect such reactions u nder dynamic cell culture conditions. With this approach, a distinct patter n of fluorescent labeling that seems to target ECM proteins preferentially was observed in TGF-beta1-treated cells but not in control cells. This reac tion required the presence of a heme peroxidase and was inhibited by catala se or diphenyliodonium (a flavoenzyme inhibitor), similar to the effect on TGF-beta1-induced dityrosine formation. Exogenous addition of H2O2 to contr ol cells that do not release extracellular H2O2 produced a similar fluoresc ent labeling reaction. These results support the concept that, in the prese nce of heme peroxidases in vivo, TGF-beta1-induced H2O2 production by fibro blasts may mediate oxidative dityrosine-dependent cross-linking of ECM prot ein(s), This effect may be important in the pathogenesis of human fibrotic diseases characterized by overexpression/activation of TGF-beta1.