Nrf1 is critical for redox balance and survival of liver cells during development - PubMed (original) (raw)

Nrf1 is critical for redox balance and survival of liver cells during development

Linyun Chen et al. Mol Cell Biol. 2003 Jul.

Abstract

The Nrf1 transcription factor belongs to the CNC subfamily of basic leucine zipper proteins. Knockout of Nrf1 is lethal in mouse embryos, but nothing is known about the cell types that absolutely require its function during development. We show by chimera analysis that Nrf1 is essential for the hepatocyte lineage. Mouse embryonic stem cells lacking Nrf1 developed normally and contributed to most tissues in adult chimeras where Nrf1 is normally expressed. Nrf1-deficient cells contributed to fetal, but not adult, liver cells. Loss of Nrf1 function resulted in liver cell apoptosis in late-gestation chimeric fetuses. Fetal livers from mutant embryos exhibited increased oxidative stress and impaired expression of antioxidant genes, and primary cultures of nrf1(-/-) fetal hepatocytes were sensitive to tert-butyl hydroperoxide-induced cell death, suggesting that impaired antioxidant defense may be responsible for the apoptosis observed in the livers of chimeric mice. In addition, cells deficient in Nrf1 were sensitized to the cytotoxic effects of tumor necrosis factor (TNF). Our results provide in vivo evidence demonstrating an essential role of Nrf1 in the survival of hepatocytes during development. Our results also suggest that Nrf1 may promote cell survival by maintaining redox balance and protecting embryonic hepatocytes from TNF-mediated apoptosis during development.

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Figures

FIG. 1.

FIG. 1.

Nrf1-deficient ES cells do not contribute to the livers of adult chimeric mice. The contributions of ES cells to tissues of nrf1−/− and nrf1+/− adult chimeric mice were estimated by GPI isoenzyme assay. The percentages of the GPI-A isoform (C57BL/6 host blastocyst-specific isoform) in tissues of chimeric mice are shown. Each bar is the value for one mouse. Seven nrf1−/− adult chimeric mice (a) and three nrf1+/− adult chimeric mice (b) were analyzed.

FIG. 2.

FIG. 2.

Targeting strategy to generate the nrf1lacZ allele. (a) A partial genomic structure of the nrf1 gene and restriction map is shown at the top of the figure. The targeting vector and the targeted allele are shown at the bottom. Exons of the nrf1 gene (shaded boxes) and the DNA-binding and leucine zipper domain that was replaced with the IRES-βGeo cassette (black box) are indicated. A probe external to the targeting vector was used for Southern blotting, and the sizes of _Nsi_I (N) and _Eco_RI (R) fragments of the targeted and wild-type (Wt) loci are indicated. (b) Southern blot analysis of genomic DNAs from ES cell clones. The nrf1 genotypes of ES cell clones are indicated above the lanes. The positions of the 16-kb mutant (Mut) and 14-kb wild-type (Wt) bands are indicated by arrows.

FIG. 3.

FIG. 3.

X-Gal staining shows the absence of nrf1lacZ/lacZ ES cells in the hepatic parenchyma of adult chimeric mice. (a and b) Whole-mount X-Gal staining of livers from two representative adult mice, both of which have extensive chimerism determined from coat color and X-Gal staining of other tissues. Note the absence of blue staining in the hepatic parenchyma. (c) Gall bladder showing intense blue staining.

FIG.4.

FIG.4.

Chimeric embryos with livers with large contributions of ES cells are anemic. (a to d) Four E14.5 nrf1lacZ/lacZ chimeric littermates whole mount stained with X-Gal. (e) Cross section of an E14.5 chimeric fetal liver stained with X-Gal showing intense blue staining of hepatocytes. (f) Hematocrits of the chimeric mice shown in panels a to d. Note that chimeras 3 and 4 (c and d), which showed blue staining in their livers (arrows), have low hematocrits. (g and h) E16.5 nrf1lacZ/lacZ chimeric littermates whole mount stained with X-Gal. (i and j) Blood smears of E16.5 nrf1lacZ/lacZ chimeric littermates. (i) Note that the blood of chimera 5 (g), which showed intense blue staining in the liver, contains large number of yolk sac-derived nucleated red cells (arrows) and few nonnucleated red cells of fetal liver origin. (j) Blood of chimera 6 (h), which showed intense X-Gal staining in the embryo (inset) but not the liver, contains both nucleated erythrocytes and nonnucleated red cells.

FIG.5.

FIG.5.

Late-gestation fetal livers of nrf1lacZ/lacZ chimeric mice show increased apoptosis and necrosis. (a and b) Livers of E18.5 chimeric fetuses showing patches of necrotic tissue. (c) Liver shown in panel b that was whole mount stained with X-Gal. (d) Cross section of E18.5 liver showing X-Gal staining in hepatocytes. (e) Cross section of E18.5 liver stained with hematoxylin and eosin. Note the presence of pkynotic and fragmented nuclei (red arrows in panels d and e). (f) TUNEL showing apoptotic cells. (g) Section of an E18.5 chimeric liver showing the border between healthy (upper left) and necrotic (lower right) tissues. (h) Higher-magnification photomicrograph of an E18.5 fetal liver section showing clusters of nucleated erythroid cells (red arrows) scattered between necrotic tissue.

FIG.6.

FIG.6.

Nrf1-deficient fetal livers exhibit increased oxidative stress. (a) ROS levels of E13.5 liver samples from wild-type (white bars), nrf1−/− (black bars), and nrf2−/− (hatched bars) mice as determined by flow cytometric analysis of intracellular DCF fluorescence. Results from three experiments are shown. Levels are expressed relative to the signal for the wild-type sample in each experiment, which was arbitrarily assigned a value of one. In experiment 3, primary hepatocytes were used; fetal liver cells were cultured overnight on collagen-coated plates to remove nonadhering hematopoietic cells. (b) GSH levels in the livers of wild-type and nrf1+/− (control) (n = 8), nrf1−/− (n = 4), and nrf2−/− (n = 4) mice. (c) GSH levels of primary hepatocytes from E13.5 control (n = 5) and nrf1−/− (n = 3) fetuses. (d) GSSG levels of wild-type and nrf1−/− livers. Mean values ± standard deviations (error bars) are shown. Values that were statistically significantly different (P < 0.05 by Student's t test) are indicated by an asterisk.

FIG. 7.

FIG. 7.

Nrf1-deficient primary hepatocytes are sensitive to tBHP-induced cell death. (a) Percent survival of control (white bars) and nrf1−/− (shaded bars) hepatocytes that were treated with 0.25 mM tBHP (+ TBHP) or not treated with tBHP (− TBHP). (b and c) Detection of apoptosis (arrows) by DAPI staining of hepatocytes treated with tBHP.

FIG. 8.

FIG. 8.

Nrf1-deficient cells are sensitized to TNF-induced cell death. (a) Percent survival of wild-type and nrf1−/− fibroblasts treated with various concentrations of TNF. (b) Percent survival of wild-type and nrf1−/− fibroblasts treated with cycloheximide and various concentrations of TNF. (c) Caspase 3-like enzyme activity of wild-type and nrf1−/− fibroblasts after treatment with cycloheximide and TNF. Changes in caspase 3-like activity are expressed relative to the activity in untreated cells. Wild-type cells (black bars), nrf1−/− cells (gray bars), and NAC-pretreated nrf1−/− cells (hatched, light gray bars in panel b) were studied. Mean values ± standard deviations (error bars) are shown.

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