The protease DDI2 regulates NRF1 activation in response to cadmium toxicity

Abstract

DNA-damage inducible 1 homolog 2 (DDI2) is a protease that activates the transcription factor NRF1. Cellular models have shown that this pathway contributes to cell-stress adaptation, for example, on proteasome inhibition. However, DDI2 physiological function is unknown. Ddi2 Knock-out (KO) mice were embryonic lethal. Therefore, we generated liver-specific Ddi2-KO animals and used comprehensive genetic analysis to identify the molecular pathways regulated by DDI2. Here, we demonstrate that DDI2 contributes to metallothionein (MT) expression in mouse and human hepatocytes at basal and upon cadmium (Cd) exposure. This transcriptional program is dependent on DDI2-mediated NRF1 proteolytic maturation. In contrast, NRF1 homolog NRF2 does not contribute to MT production. Mechanistically, we observed that Cd exposure inhibits proteasome activity, resulting in DDI2-mediated NRF1 proteolytic maturation. In line with these findings, DDI2 deficiency sensitizes cells to Cd toxicity. This study identifies a function for DDI2 that links proteasome homeostasis to heavy metal mediated toxicity.

Keywords: Biological sciences; Molecular biology; Molecular mechanism of gene regulation.

Graphical abstract

Figure 1

DDI2 modulates metallothionein gene expression in vivo (A) Schematic of mouse models: Full DDI2-knockout (KO); wild-type (WT) and liver specific DDI2-KO (up) and representation of DDI2 locus highlighting the LoxP sites used to generate the DDI2^flox/flox mice (down). (B) Volcano plot from RNA-sequencing data from liver samples of DDI2-KO compared to WT littermate control mice. The fold change (FC) is plotted on the xaxis, and the false discovery rate (FDR) (adjusted pvalue) is plotted on the yaxis. The represented genes have an FDR lower than 0.05 (horizontal dash line). Some genes are highlighted. The full raw data can be found on the supplementary material. Data points generated from two biological replicates.

Figure 2

DDI2 activates metallothionein in response to cadmium exposure (A and B) Relative mRNA levels (A) and protein levels (B) from liver samples of DDI2-KO (KO) and wild-type (WT) mice after 16 h of intraperitoneal injection of PBS or 8 mg/kg of CdCl₂ (Cd). (A) mRNA levels of metallothioneins, Mt1 and Mt2, normalized with Actin mRNA levels, each dot represents a single animal. n = 11, 14 for WT and n = 13, 17 for DDI2-KO, treated with PBS or Cd, respectively. (B) Representative immunoblotting (n = 3) showing the relative protein levels of DDI2, NRF1, NRF2, MT and tubulin as loading control, of liver samples as in (A). Each line represents one animal and the protein molecular weights in KDa are indicated. ◀ indicates the full-length protein, ◁ indicates the cleaved protein, se: short image exposure, le: long image exposure. (C) Representative micrographs (n = 3) of immunohistochemistry staining of MT proteins from liver tissues as in (A). (D) mRNA levels of Mt2, normalized with Actin, from liver samples from WT, DDI2-KO, NRF2-KO, and DDI2- and NRF2- double-KO (d-KO) from animals treated as in (A) (n = 5). p values were calculated using two-tailed unpaired Mann-Whitney t-tests and error bars denote SD. ∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001; B, blood vessel; arrow, epithelial cells.

Figure 3

Cadmium induces DDI2-mediated cleavage and nucleus translocation of NRF1 (A) Activation of MT1 promoter-driven luciferase activity in HepG2 cells after 6 h exposure to CdCl₂ (5 μM), ZnSO₄ (100 μM), tBHQ (50 μM), K₂Cr₂O₇ (50 μM), NaAsO₂ (12.5 μM), H₂O₂ (1 mM), bortezomib (BTZ, 10 nM), carfilzomib (CFZ, 10 nM) or DMSO as a control (Ctr). (B) Representative immunoblotting (n = 3) showing the relative protein levels of DDI2, NRF1, NRF2 and tubulin as loading control, of CRISPR KO (Cr)-DDI2 or Cr-luciferase (luci) HepG2 cells treated with Cd (5 μM) during the indicated time in hours (h). (C) Representative immunoblotting (n = 3) showing subcellular fractionation of endogenous DDI2, NRF1, NRF2, Ubiquitin and Lamin A/C in total cell lysates, nucleus and cytoplasm from HepG2 cells treated with or without Cd (5 μM) during 6 h. (D) Activation of MT1 promoter-driven luciferase activity in Cr-DDI2, Cr-NRF1 or HepG2 parental cells (WT) after 6 h exposure to Cd (5 μM). Each bar represents the mean, and error bars denote SD of at least three independent experiments. p values were calculated using one-way ANOVA followed by Bonferroni’s multiple comparison test. ∗p<0.05; ∗∗p<0.01. ◀ indicates the full-length protein, ◁ indicates the cleaved protein.

Figure 4

Cadmium indirectly inhibits the proteasome (A) Relative proteasome activity of HepG2 cells treated with CdCl₂ (Cd; + 1 μM or ++ 5 μM) or carfilzomib (CFZ, 10 nM) during 6 h. Each bar represents the mean, and error bars denote SD of at least three independent experiments. (B) Relative proteasome activity of purified 26S proteasome complexes incubated with the substrate Suc-LLVY-AMC and treated with or without Cd (1 μM) or CFZ (10 nM) during the indicated time in minutes (n = 3).

Figure 5

DDI2 promotes cell survival during metal toxicity (A) Relative mRNA levels of MT2A, MT1G and NQO1 in CRISPR KO (Cr)-DDI2 or Cr-luciferase (luci) control HepG2 cells treated with CdCl₂ (Cd, as indicated concentrations) for 6 h. Target genes were normalized with GAPDH mRNA levels. (n = 3). (B) Left, Viability of Cr-DDI2 and Cr-luci control HepG2 cells after 48 h treatment with indicated doses of Cd and correspondent IC50 values presented in the bar graph, right (n = 5). Relative viability assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTS)/1-methoxyphenazine methosulfate (PMS) assay. (C) Viability of Cr-DDI2, Cr-NRF1 and Cr-luci control HepG2 cells after 48 h treatment with 500 μM of cisplatin, normalized to non-treated cells (n = 3). p values were calculated using two-tailed unpaired Mann-Whitney t-tests and error bars denote SD. ∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001.