Elevated Nrf-2 responses are insufficient to mitigate protein carbonylation in hepatospecific PTEN deletion mice
Dennis R Petersen1, Laura M Saba1, Volkan I Sayin2,3
1Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America.
Objective:
In the liver, a contributing factor in the pathogenesis of non-alcoholic fatty liver disease (NASH) is oxidative stress, which leads to the accumulation of highly reactive electrophilic α/β unsaturated aldehydes. The objective of this study was to determine the impact of NASH on protein carbonylation and antioxidant responses in a murine model.
Methods:
Liver-specific phosphatase and tensin homolog (PTEN)-deletion mice (PTENLKO) or control littermates were fed a standard chow diet for 45-55 weeks followed by analysis for liver injury, oxidative stress and inflammation.
Results:
Histology and Picrosirius red-staining of collagen deposition within the extracellular matrix revealed extensive steatosis and fibrosis in the PTENLKO mice but no steatosis or fibrosis in controls. Increased steatosis and fibrosis corresponded with significant increases in inflammation. PTEN-deficient livers showed significantly increased cell-specific oxidative damage, as detected by 4-hydroxy-2-nonenal (4-HNE) and acrolein staining. Elevated staining correlated with an increase in nuclear DNA repair foci (γH2A.X) and cellular proliferation index (Ki67) within zones 1 and 3, indicating oxidative damage was zonally restricted and was associated with increased DNA damage and cell proliferation. Immunoblots showed that total levels of antioxidant response proteins induced by nuclear factor erythroid-2-like-2 (Nrf2), including GSTμ, GSTπ and CBR1/3, but not HO-1, were elevated in PTENLKO as compared to controls, and IHC showed this response also occurred only in zones 1 and 3. Furthermore, an analysis of autophagy markers revealed significant elevation of p62 and LC3II expression. Mass spectrometric (MS) analysis identified significantly more carbonylated proteins in whole cell extracts prepared from PTENLKO mice (966) as compared to controls (809). Pathway analyses of identified proteins did not uncover specific pathways that were preferentially carbonylated in PTENLKO livers but, did reveal specific strongly increased carbonylation of thioredoxin reductase and of glutathione-S-transferases (GST) M6, O1, and O2.
Conclusions:
Results show that disruption of PTEN resulted in steatohepatitis, fibrosis and caused hepatic induction of the Nrf2-dependent antioxidant system at least in part due to elevation of p62. This response was both cell-type and zone specific. However, these responses were insufficient to mitigate the accumulation of products of lipid peroxidation.
Insights
PTEN deletion in mice caused non-alcoholic steatohepatitis (NASH) with liver fibrosis and oxidative stress. The Nrf2 antioxidant system was induced but insufficient to prevent lipid peroxidation product accumulation.
Area of Science:
- Hepatology
- Oxidative Stress Research
- Molecular Pathology
Background:
- Non-alcoholic fatty liver disease (NASH) pathogenesis involves oxidative stress and reactive aldehydes.
- Protein carbonylation is a marker of oxidative damage in liver disease.
Purpose of the Study:
- To investigate the impact of PTEN deletion on protein carbonylation and antioxidant responses in a murine model of NASH.
Main Methods:
- Utilized liver-specific PTEN-deletion (PTENLKO) mice and control littermates.
- Assessed liver injury, steatosis, fibrosis, inflammation, oxidative damage (4-HNE, acrolein), DNA damage (γH2A.X), proliferation (Ki67), antioxidant proteins (Nrf2 pathway), autophagy markers (p62, LC3II), and protein carbonylation via mass spectrometry.
Main Results:
- PTENLKO mice developed extensive steatosis, fibrosis, and inflammation.
- Significant increases in oxidative damage, DNA damage, and cell proliferation were observed, restricted to specific liver zones.
- Elevated Nrf2-dependent antioxidant proteins (GSTs, CBR1/3) and autophagy markers (p62, LC3II) were detected.
- Mass spectrometry identified increased carbonylated proteins, notably thioredoxin reductase and specific glutathione-S-transferases.
Conclusions:
- PTEN disruption induces steatohepatitis and fibrosis, activating a zone-specific Nrf2-dependent antioxidant response partly mediated by p62.
- Despite antioxidant system induction, these responses were inadequate to prevent lipid peroxidation product accumulation in PTEN-deficient livers.
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