Nrf2 signaling, a mechanism for cellular stress resistance in long-lived mice

Abstract

Transcriptional regulation of the antioxidant response element (ARE) by Nrf2 is important for the cellular adaptive response to toxic insults. New data show that primary skin-derived fibroblasts from the long-lived Snell dwarf mutant mouse, previously shown to be resistant to many toxic stresses, have elevated levels of Nrf2 and of multiple Nrf2-sensitive ARE genes. Dwarf-derived fibroblasts exhibit many of the traits associated with enhanced activity of Nrf2/ARE, including higher levels of glutathione and resistance to plasma membrane lipid peroxidation. Treatment of control cells with arsenite, an inducer of Nrf2 activity, increases their resistance to paraquat, hydrogen peroxide, cadmium, and UV light, rendering these cells as stress resistant as untreated cells from dwarf mice. Furthermore, mRNA levels for some Nrf2-sensitive genes are elevated in at least some tissues of Snell dwarf mice, suggesting that the phenotypes observed in culture may be mirrored in vivo. Augmented activity of Nrf2 and ARE-responsive genes may coordinate many of the stress resistance traits seen in cells from these long-lived mutant mice.

FIG. 1.

Measurement of the cellular redox status of nicotinamide derived molecules. The graphs show the levels of NAD and related molecules, measured by HPLC, using the culture conditions that are typically used for assessment of PMRS function. (A) Amount of NADH measured after alkaline extraction, of dwarf and control cells that had been cultured in control (complete) medium, low-glucose medium (0.25 mg/ml glucose and 20% dialyzed serum), and rotenone-containing medium (high glucose and 10 μΜ rotenone). (B) Under the conditions described for panel A, NAD was measured in a parallel acid extraction. (C and D) Data for NADPH and NADP. (E and F) Ratios between the redox molecules, showing clear effects of culture conditions but no differences between dwarf and control cells. Error bars indicate the standard error of the mean (SEM; n = 12). *, P < 0.05, by a paired t test.

FIG. 2.

PMRS function and lipid peroxidation. (A) Catalytic function of PMRS enzymes measured in purified membranes from dwarf and control fibroblasts (n = 12). Cyto, cytochrome; Red, reductase. *, P < 0.05, by a paired t test. (B and C) Relative levels of lipid peroxidation with (B) or without (C) serum under conditions including control (complete medium and DMEM), hydrogen peroxide (50 μM)-treated (H₂O₂), low-glucose (0.01 mM), rotenone (Rot; 10 μM), t-butyl hydroperoxide (t-BHPX; 50 μM), and cadmium chloride (CdC_l2; 10 μM), as measured by the fluorescent dye DPPP (n = 12; *, P < 0.05, by paired t test). t-BHPX can oxidize DPPP even in the absence of cells; such background values were measured and subtracted from each sample. (D) The LD₅₀ for H₂O₂ of normal cells either untreated or exposed to low-glucose medium (0.2 mg/ml) or rotenone (10 μM). Error bars indicate SEM for all figures. (E and F) Relative fluorescence of two dyes that measure cellular production of superoxide, DHE and Mitosox. The results (n = 4) show dwarf and control cellular production of superoxide in complete medium and in serum-free medium and after exposure to rotenone (40 μM for 4 h). Rotenone treatment increased the production of superoxide in both cell lines, but the effect did not differ between genotypes.

FIG. 3.

Effects of Nrf2 activators on WST-1 reduction. The effects of 24 h of Nrf2 activation by arsenite (3 μM) or tBHQ (5 μM) treatment on dwarf and control cells are shown; these agents induce 20 to 60% increase in WST-1 reduction over untreated controls. Each symbol represents a different cell line tested with and without activation and is connected by a line to the same cell line after activation. Neither treatment showed differences in the responses by dwarfs and controls (data not shown).

FIG. 4.

Nrf2 protein levels are increased in cells from dwarf mice. (A) Representative immunoblot for Nrf2 in fibroblasts from normal and dwarf mice under complete and arsenite (4 h) conditions. (B) Quantification of results from 12 blots from control cells and those preincubated in the presence of arsenite (10 μM for 4 h). *, P < 0.05 by a paired t test. (C and D) Immunoblot assays for Keap1 (n = 4) and Nrf1 (n = 5). Error bars indicate SEM. (E) Control blot for Nrf2 protein. A whole control membrane probing for Nrf2 protein with molecular weight (left lane) and Nrf2 positive control (sc-127242; Santa Cruz Biotechnology; right lane) markers is shown. All lysates were from control cells removed from serum (left two lanes), treated with arsenite (middle three lanes), or untreated (right three lanes). Lanes were loaded with 75 μg of protein for cell lysates and 5 μg of protein for Nrf2 controls.

FIG. 5.

Fibroblasts from dwarf mice have high levels of several ARE-dependent mRNAs. Each panel shows transcriptional levels of RNA derived from dwarf and control cells in complete medium or after arsenite treatment (10 μM for 24 h). Six pairs of cell cultures were evaluated for each transcript. *, P < 0.05 by a paired t test; #, P < 0.1 for GSTA1. Error bars indicate SEM.

FIG. 6.

Dwarf cells have increased glutathione levels and resist arsenite toxicity. (A) Levels of total reduced thiols in arbitrary units in control cells and in cells exposed to arsenite (10 μM for 24 h) or low glucose (0.25 mg/ml for 2 h). *, P < 0.02, by a paired t test (n = 8 pairs). (B) Total glutathione content under each of the indicated conditions. (C) Cell survival at various arsenite concentrations; the figure shows results for two individual control and two dwarf mice. (D) Compilation of LD₅₀ results from experiments like those shown in panel C for six pairs of mice; each symbol represents a different donor mouse. Error bars indicate SEM.

FIG. 7.

Nrf2 activation augments cellular stress resistance and does so to a greater extent in control cells. Data represent the stress resistance of cells from normal and dwarf mice to four agents: cadmium chloride (CdC_l2), hydrogen peroxide (H₂O₂), paraquat, and UV light before (white bars) and after (black bars) arsenite (5 μM for 24 h)-mediated activation of Nrf2. Significance is indicated as follows for each panel: ∧, a significant increase in resistance after arsenite treatment (P < 0.05); ∧∧, a nearly significant increase (P < 0.10); *, a significant difference between dwarf and control cells (without arsenite exposure). For each panel, n = 5, except for H₂O₂, where there were five normal cell lines and only three dwarf cell lines. Error bars indicate SEM.

FIG. 8.

Multiple ARE genes are upregulated in dwarf tissues. Data represent the RNA levels of four Nrf2-regulated ARE genes, metallothionein 1 (MT1), heme oxygenase-1 (HMOX), glutamate cysteine ligase, modifier subunit (GCLM), and thioredoxin reductase (TXNRD), in dwarf and control tissues derived from heart, liver, or brain tissue. Each bar represents the mean fold change normalized to control levels of mRNA; error bars indicate the SEM (n = 6 pairs for each RNA transcript). *, P < 0.05; **, P < 0.10 (by a paired t test).

FIG. 9.

Model of dwarf cellular resistance to toxic and metabolic stress. A speculative model of differences between dwarf and normal cells in various aspects of cellular function; upward-pointing arrows indicate augmentation in cells from dwarf mice compared to control cells. The model postulates that hormonal differences in the dwarf mice initiate and maintain stable epigenetic changes, perhaps through PI3K, Akt, PKC, or MAPK, all of which are known to affect Nrf2 levels or function. These changes act to modify Nrf2, releasing it from Keap1 and promoting activation of ARE genes. Downstream of Nrf2 activation, proteins important for glutathione synthesis and reduction, peroxide detoxification, and the PMRS combine to increase cellular resistance to oxidative stresses, such as peroxide, cadmium, arsenite, paraquat, and to the nonoxidative stress of UV irradiation. Furthermore, the increase in PMRS activity and in glutathione electron donation allows these cells to resist the nontoxic effects of metabolic stresses such as glucose deprivation and rotenone exposure.