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.2013 Aug;280(15):3609-20.
doi: 10.1111/febs.12350. Epub 2013 Jun 18.

Nuclear factor-erythroid 2-related factor 1 regulates expression of proteasome genes in hepatocytes and protects against endoplasmic reticulum stress and steatosis in mice

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Nuclear factor-erythroid 2-related factor 1 regulates expression of proteasome genes in hepatocytes and protects against endoplasmic reticulum stress and steatosis in mice

Candy S Lee et al. FEBS J.2013 Aug.

Abstract

The ubiquitin-proteasome system is important in maintaining protein homeostasis. NFE2-related factor 1 (Nrf1), a transcription factor in the cap 'n' collar basic-leucine zipper family, regulates expression of cytoprotective genes. It was previously shown that liver-specific knockout of Nrf1 (Nrf1LKO) leads to hepatic cell death, steatohepatitis and cancer. However, the mechanisms underlying these pathologies are not clear. Here, we report that Nrf1 is critical for proteasome gene expression in the liver. Liver-specific knockout of Nrf1 results in impaired basal and induced expression of proteasome genes, and diminished proteasome activity in hepatocytes. In addition, our findings demonstrated that endoplasmic reticulum stress signaling pathway was also activated in Nrf1LKO livers. Inhibition of proteasome activity leads to endoplasmic reticulum stress in Nrf1-deficient hepatocytes, prompting the development of steatosis in the liver. Our results indicate that Nrf1 plays an integral role in the maintenance of proteasome function in hepatocytes and in the prevention of liver steatosis development. Moreover, these results highlight an association between proteasome dysfunction, endoplasmic reticulum stress and steatosis.

Keywords: ER stress; Nrf1; proteasome; steatohepatitis; transcriptional regulation.

© 2013. This article is a U.S. Government work and is in the public domain in the USA.

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Figures

Figure 1
Figure 1. Nrf1 but not Nrf2 is required for both basal and induced proteasome gene expression in hepatocytes
(A) Comparison of mRNA encoding proteasomal genes in wild type and Nrf1LKO livers by real time RTPCR analysis. Mean values ± SEM of 3-5 mice per group, and significance was assessed by Student’s t test (*P< 0.05). (B) Comparison of mRNA encoding proteasome genes in vehicle-treated or bortezomib-treated wild type and Nrf1LKO livers by real time RTPCR analysis. Mice were intraperitoneally injected with DMSO or bortezomib (2.5 mg/kg), and livers were harvested 24 hr after for RNA extractions. Mean values ± SEM of 3-5 mice per group, and significance was assessed by Student’s t test within genotype between treatments (*P< 0.05). (C) Levels of proteasome alpha-subunits analyzed by Western blotting. (D) Comparison of mRNA encoding proteasome genes in vehicle-treated or bortezomib-treated wild type and Nrf2KO liver by real time RTPCR analysis. Mice were intraperitoneally injected with DMSO or bortezomib (2.5 mg/kg), and livers were harvested 24 hr after for RNA extractions. (E) Comparison of mRNA encoding proteasome genes and oxidative stress genes in vehicle-treated or Sulforaphane-treated wild type and Nrf1LKO livers by real time RTPCR analysis. Mice were intraperitoneally injected with DMSO or sulfurophane (5 mg/kg), and livers were harvested 24 hr after for RNA extractions. Mean values ± SEM of 3 mice per group, and significance was assessed by Student’s t test within genotype between treatments. *P< 0.05 compared to vehicle-treated wild type livers; #P< 0.05 compared to Nrf1LKO vehicle-treated livers.
Figure 2
Figure 2. Nrf1LKO livers show accumulation of ubiquitin and decreased proteasome activity
(A) Measurement of chymotrypsin-like activity by in-gel assay of 1-month old wild type and Nrf1LKO liver homogenates. Fluorescence from free AMC was visualized on a UV transilluminator. Two representative samples from each genotype are shown. The upper band represents the 26S particle and the lower band represents the 20S core. Densitometric quantitations for 26S and 20S levels are shown. (B) Chymotrypsin-like activities in wild type and Nrf1LKO liver homogenates. Mean values ± SEM (n=6 per genotype). *P< 0.05. (C) Western blot analysis of wild type and Nrf1LKO liver lysates with ubiquitin antibody. Note presence of high molecular weight ubiquinated proteins running as a smear in Nrf1LKO samples. (D) Liver sections of 1-month old wild type and Nrf1LKO mice immunostained for ubiquitin.
Figure 3
Figure 3. ER stress pathway is activated in Nrf1LKO livers
(A) Liver lysates from wild type and Nrf1LKO mice were analyzed for PERK and eIF2α phosphorylation, and total levels of PERK, eIF2α, ATF4, CHOP by immunoblotting. Beta-actin levels were used as loading control. (B) Expression of CHOP, BiP, GADD45b and ATF4 mRNA in control and Nrf1LKO livers. Data represents the mean ± SEM of 3 mice per group, and significance was assessed by Student’s t test (*P< 0.05).
Figure 4
Figure 4. Livers of Nrf1 heterozygous mice exhibit enhanced ER stress and steatosis in response to proteasome inhibition
(A) H&E and Oil Red O staining of livers of vehicle-treated or bortezomib-treated (2.5 μg/kg) wild type and Nrf1+/− mice. (B) Liver triglyceride (TG) content of wild type and Nrf1+/− mice treated with vehicle or bortezomib (2.5 mg/kg) or bortezomib and PBA (120 mg/kg). Bars represent the mean ± SEM of 3 independent samples in each group, and significance was assessed within genotype by Student’s t test. *P< 0.05 compared to bortezomib-treated wild type livers; **P< 0.05 compared to bortezomib-treated wild type livers; and # P< 0.05 compared to bortezomib-treated Nrf1+/− livers. (C) Expression of ER stress genes in livers of wild type and Nrf1+/− mice treated with bortezomib or bortezomib and PBA. Bars represent the mean ± SEM of 3 different samples in each group, and significance was assessed within genotype by Student’s t test. *P< 0.05 compared bortezomib-treated wild type livers; **P< 0.05 compared to bortezomib-treated wild type livers; and # P< 0.05 compared to bortezomib-treated Nrf1+/− livers. [32]
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