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Updated: Apr 29, 2026
Cell Type-specific Gene Expression Profiling in the Mouse Liver
Published on: September 17, 2019
Identification and quantification of the basal and inducible Nrf2-dependent proteomes in mouse liver: biochemical,
Joanne Walsh1, Rosalind E Jenkins1, Michael Wong1
1MRC Centre for Drug Safety Science, School of Biomedical Sciences, University of Liverpool, Sherrington Buildings, Liverpool, Merseyside L69 3GE, United Kingdom.
Abstract:
The transcription factor Nrf2 is a master regulator of cellular defence: Nrf2 null mice (Nrf2((-/-))) are highly susceptible to chemically induced toxicities. We report a comparative iTRAQ-based study in Nrf2((-/-)) mice treated with a potent inducer, methyl-2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate (CDDO-me; bardoxolone -methyl), to define both the Nrf2-dependent basal and inducible hepatoproteomes. One thousand five hundred twenty-one proteins were fully quantified (FDR <1%). One hundred sixty-one were significantly different (P<0.05) between WT and Nrf2((-/-)) mice, confirming extensive constitutive regulation by Nrf2. Treatment with CDDO-me (3mg/kg; i.p.) resulted in significantly altered expression of 43 proteins at 24h in WT animals. Six proteins were regulated at both basal and inducible levels exhibiting the largest dynamic range of Nrf2 regulation: cytochrome P4502A5 (CYP2A5; 17.2-fold), glutathione-S-transferase-Mu 3 (GSTM3; 6.4-fold), glutathione-S-transferase Mu 1 (GSTM1; 5.9-fold), ectonucleoside-triphosphate diphosphohydrolase (ENTPD5; 4.6-fold), UDP-glucose-6-dehydrogenase (UDPGDH; 4.1-fold) and epoxide hydrolase (EPHX1; 3.0-fold). These proteins, or their products, thus provide a potential source of biomarkers for Nrf2 activity. ENTPD5 is of interest due to its emerging role in AKT signalling and, to our knowledge, this protein has not been previously shown to be Nrf2-dependent. Only two proteins altered by CDDO-me in WT animals were similarly affected in Nrf2((-/-)) mice, demonstrating the high degree of selectivity of CDDO-me for the Nrf2:Keap1 signalling pathway.
Biological Significance:
The Nrf2:Keap1 signalling pathway is attracting considerable interest as a therapeutic target for different disease conditions. For example, CDDO-me (bardoxolone methyl) was investigated in clinical trials for the treatment of acute kidney disease, and dimethyl fumarate, recently approved for reducing relapse rate in multiple sclerosis, is a potent Nrf2 inducer. Such compounds have been suggested to act through multiple mechanisms; therefore, it is important to define the selectivity of Nrf2 inducers to assess the potential for off-target effects that may lead to adverse drug reactions, and to provide biomarkers with which to assess therapeutic efficacy. Whilst there is considerable information on the global action of such inducers at the mRNA level, this is the first study to catalogue the hepatic protein expression profile following acute exposure to CDDO-me in mice. At a dose shown to evoke maximal Nrf2 induction in the liver, CDDO-me appeared highly selective for known Nrf2-regulated proteins. Using the transgenic Nrf2((-/-)) mouse model, it could be shown that 97% of proteins induced in wild type mice were associated with a functioning Nrf2 signalling pathway. This analysis allowed us to identify a panel of proteins that were regulated both basally and following Nrf2 induction. Identification of these proteins, which display a large magnitude of variation in their expression, provides a rich source of potential biomarkers for Nrf2 activity for use in experimental animals, and which may be translatable to man to define individual susceptibility to chemical stress, including that associated with drugs, and also to monitor the pharmacological response to Nrf2 inducers.
Insights
This study defines the proteins regulated by the Nrf2 (Nuclear factor erythroid 2-related factor 2) pathway in the liver. We identified key proteins, like CYP2A5 and GSTs, that can serve as biomarkers for Nrf2 activity and drug response.
Area of Science:
- Proteomics
- Cellular Biology
- Toxicology
Background:
- Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of cellular defense mechanisms.
- Nrf2-deficient mice exhibit heightened susceptibility to chemical toxicities.
- The Nrf2:Keap1 signaling pathway is a significant therapeutic target for various diseases.
Purpose of the Study:
- To comprehensively define the Nrf2-dependent basal and inducible hepatoproteome using a comparative proteomic approach.
- To identify potential protein biomarkers for Nrf2 activity and therapeutic efficacy of Nrf2 inducers.
- To assess the selectivity of the Nrf2 inducer methyl-2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate (CDDO-me).
Main Methods:
- iTRAQ-based quantitative proteomics was employed in wild-type (WT) and Nrf2 null (Nrf2((-/-)))) mice.
- Mice were treated with CDDO-me (bardoxolone methyl), a potent Nrf2 inducer.
- Statistical analysis was performed to identify differentially expressed proteins (FDR <1%, P<0.05).
Main Results:
- 1,521 proteins were quantified, with 161 showing significant constitutive regulation by Nrf2.
- CDDO-me treatment altered 43 proteins in WT mice; 97% were Nrf2-dependent.
- Six proteins, including CYP2A5, GSTM3, GSTM1, ENTPD5, UDPGDH, and EPHX1, were regulated both basally and inducibly, exhibiting the largest dynamic range.
Conclusions:
- CDDO-me demonstrates high selectivity for the Nrf2:Keap1 pathway, with minimal off-target effects.
- The identified proteins, particularly those with large expression changes, serve as robust biomarkers for Nrf2 activity.
- These findings support the therapeutic potential of Nrf2 modulators and provide tools for monitoring drug response and individual susceptibility to chemical stress.
