Hydrogen peroxide increases extracellular matrix mRNA through TGF-beta in human mesangial cells
M C Iglesias-De La Cruz1, P Ruiz-Torres, J Alcamí
1Departments of Physiology and Medicine, Alcalá University, Madrid, Spain.
Background:
Reactive oxygen species (ROS) are excessively produced in pathologic states, including many renal diseases. Transforming growth factor-beta (TGF-beta) may mediate renal fibrotic injury, and ROS may act through the TGF-beta pathway to exert a profibrotic effect.
Methods:
The expression of TGF-beta1 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-beta antibody was added to test the hypothesis that hydrogen peroxide acts through activation of the TGF-beta pathway to stimulate ECM expression.
Results:
Northern blot analysis revealed significantly increased steady-state levels of TGF-beta1 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-beta1 enzyme-linked immunosorbent assay all demonstrated significant stimulation by GO (>1.5-fold) of TGF-beta1 promoter activity, mRNA level, bioactivity, and protein production, respectively. Catalase pretreatment prevented the GO-induced stimulation of TGF-beta1 mRNA. When incubations were performed with a panselective neutralizing anti-TGF-beta antibody, the GO-stimulated expression of ECM molecules was prevented.
Conclusions:
GO-induced hydrogen peroxide production induces TGF-beta1 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.
Insights
Oxidative stress from hydrogen peroxide (H2O2) increases transforming growth factor-beta 1 (TGF-β1) and extracellular matrix (ECM) gene expression in kidney cells. This pathway may drive fibrotic kidney disease progression.
Area of Science:
- Nephrology
- Cell Biology
- Biochemistry
Background:
- Reactive oxygen species (ROS) are implicated in renal diseases.
- Transforming growth factor-beta (TGF-β) signaling may mediate fibrotic injury.
- ROS might exacerbate renal fibrosis via the TGF-β pathway.
Purpose of the Study:
- To investigate if hydrogen peroxide (H2O2) stimulates extracellular matrix (ECM) expression through the TGF-β pathway in human mesangial cells (HMCs).
Main Methods:
- Human mesangial cells (HMCs) were treated with glucose oxidase (GO) to generate H2O2.
- TGF-β1 and ECM component expression was assessed.
- Neutralizing anti-TGF-β antibody was used to block TGF-β signaling.
- Various assays measured TGF-β1 promoter activity, mRNA levels, bioactivity, and protein production.
Main Results:
- Glucose oxidase (GO) significantly increased TGF-β1 and ECM protein levels (collagen types I, III, IV, fibronectin) by approximately twofold.
- GO stimulated TGF-β1 promoter activity, mRNA levels, bioactivity, and protein production.
- Catalase prevented GO-induced TGF-β1 mRNA increase.
- Anti-TGF-β antibody blocked GO-stimulated ECM expression.
Conclusions:
- H2O2-induced TGF-β1 synthesis increases ECM gene expression in HMCs.
- These findings suggest a mechanism linking oxidative stress to fibrotic kidney disease progression.
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