Hydrogen peroxide increases extracellular matrix mRNA through TGF-β in human mesangial cells

Background. Reactive oxygen species (ROS) are excessively produced in pathologic states, including many renal diseases. Transforming growth factor-β (TGF-β) may mediate renal fibrotic injury, and ROS may act through the TGF-β pathway to exert a profibrotic effect. Methods. The expression of TGF-β1 and extracellular matrix (ECM) components were assessed in cultured human mesangial cells (HMCs) incubated with glucose oxidase (GO), an enzyme that continuously generates hydrogen peroxide from glucose. A neutralizing anti-TGF-β antibody was added to test the hypothesis that hydrogen peroxide acts through activation of the TGF-β pathway to stimulate ECM expression. Results. Northern blot analysis revealed significantly increased steady-state levels of TGF-β1 and ECM proteins (collagen types I, III, and IV, and fibronectin) by approximately twofold. While no significant effect on mRNA stability after treatment with GO was observed, other studies employing promoter-reporter assays, competitive-quantitative reverse transcription-polymerase chain reaction, mink lung epithelial cell proliferation assay, and TGF-β1 enzyme-linked immunosorbent assay all demonstrated significant stimulation by GO (>1.5-fold) of TGF-β1 promoter activity, mRNA level, bioactivity, and protein production, respectively. Catalase pretreatment prevented the GO-induced stimulation of TGF-β1 mRNA. When incubations were performed with a panselective neutralizing anti-TGF-β antibody, the GO-stimulated expression of ECM molecules was prevented. Conclusions. GO-induced hydrogen peroxide production induces TGF-β1 synthesis and thereby increases ECM gene expression in cultured HMCs. These cellular responses may underlie the development and progression of renal diseases characterized by oxidative stress.