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Updated: Feb 7, 2026
Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models
Published on: October 22, 2012
NADPH oxidase 5 promotes proliferation and fibrosis in human hepatic stellate cells
Aitor Andueza1, Naiara Garde1, Antonia García-Garzón1
1Department of Biochemistry and Genetics, University of Navarra, Pamplona, Spain.
Abstract:
NADPH oxidase (Nox) variants Nox1, Nox2 and Nox4 are implicated in the progression of liver fibrosis. However, the role of Nox5 is not yet known, mainly due to the lack of this enzyme in rat and mouse genomes. Here we describe the expression and functional relevance of Nox5 in the human cell line of hepatic stellate cells (HSC) LX-2. Under basal conditions, three long (Nox5-L: Nox5α, -β, and -δ) and a short (Nox5-S or Nox5ε) splice variants were detected, which were silenced with specific siRNAs for Nox5. The most abundant isoform was Nox5-S, accounting for more than 90% of Nox5 protein. Overexpression of Nox5β generated reactive oxygen species (ROS) in the presence of calcium, as judged by the production of hydrogen peroxide, L-012 luminescence and cytochrome c reduction. Nox5ε did not generated ROS under these conditions, and a reduced ROS production was observed when co-expressed with Nox5β. In contrast, dihydroethidium oxidation was increased by Nox5β or Nox5ε, suggesting that Nox5ε induced intracellular oxidative stress by an unknown mechanism. Functional studies showed that both Nox5β and Nox5ε stimulated the proliferation of LX-2 cells and the collagen type I levels, while Nox5 siRNAs inhibited these effects. Interestingly, TGF-β and angiotensin II upregulated Nox5 expression, which was reduced in cells pre-incubated with catalase. Further studies silencing Nox5 in TGF-β-treated cells resulted in a reduction of collagen levels via p38 MAPK. Collectively, these results show for the first time that Nox5 can play a relevant role in the proliferation and fibrosis on human HSC.
Insights
This study reveals that NADPH oxidase 5 (Nox5) promotes human hepatic stellate cell proliferation and collagen production, key factors in liver fibrosis. Silencing Nox5 reduces these pro-fibrotic effects, highlighting Nox5 as a potential therapeutic target.
Area of Science:
- Biochemistry
- Cell Biology
- Hepatology
Background:
- NADPH oxidase (Nox) enzymes are implicated in liver fibrosis progression.
- The specific role of Nox5 in liver fibrosis remains largely unknown due to its absence in rodent models.
Purpose of the Study:
- To investigate the expression and functional significance of Nox5 in human hepatic stellate cells (HSCs).
- To determine Nox5's role in HSC proliferation and collagen production.
Main Methods:
- Detection and characterization of Nox5 splice variants (Nox5-L, Nox5-S) in LX-2 cells.
- Assessment of reactive oxygen species (ROS) generation using various assays (hydrogen peroxide, luminescence, cytochrome c reduction, dihydroethidium oxidation).
- Functional studies involving Nox5 overexpression and siRNA-mediated silencing, assessing cell proliferation and collagen type I levels.
- Investigation of regulatory pathways involving TGF-β, angiotensin II, and p38 MAPK.
Main Results:
- Multiple Nox5 splice variants, predominantly Nox5-S, were detected in human HSCs.
- Nox5β overexpression generated ROS in a calcium-dependent manner, while Nox5ε increased intracellular oxidative stress via an uncharacterized mechanism.
- Both Nox5β and Nox5ε stimulated HSC proliferation and collagen type I production.
- TGF-β and angiotensin II upregulated Nox5 expression.
- Nox5 silencing inhibited proliferation and collagen levels, particularly in TGF-β-treated cells, involving the p38 MAPK pathway.
Conclusions:
- Nox5 is expressed in human HSCs and its variants play a significant role in regulating cell proliferation and collagen synthesis.
- Nox5 contributes to pro-fibrotic processes in human HSCs, suggesting it as a potential therapeutic target for liver fibrosis.
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