Disruption of canonical AHR-mediated induction of hepatocyte PKM2 expression compromises antioxidant defenses and increases TCDD-induced hepatotoxicity

Abstract

Metabolic reprogramming by the pyruvate kinase M2 isoform is associated with cell proliferation and reactive oxygen species (ROS) defenses. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an environmental contaminant that induces ROS and hepatotoxicity, dose-dependently induces pyruvate kinase muscle isoform M2 (PKM2) in the liver. To further investigate its role in combating TCDD hepatotoxicity, a Pkm^ΔDRE mouse was constructed lacking the dioxin response element mediating aryl hydrocarbon receptor (AHR) induction. TCDD failed to induce hepatic PKM2 in Pkm^ΔDRE mice and in primary hepatocytes isolated from an AHR knockout model (AHR^V375Afl/flAlb-Cre^ERT2), demonstrating induction is AHR dependent. Both wild-type (WT) and Pkm^ΔDRE mice exhibited dose-dependent increases in liver weight after treatment with TCDD every 4 days for 28 days. Glutathione (GSH) levels increased in WT mice while oxidized glutathione (GSSG) levels increased in both models with a 24-fold decrease in the GSH/GSSG ratio in Pkm^ΔDRE mice suggesting lower antioxidant and recycling capacity. Moreover, TCDD-induced fibrosis was more severe in Pkm^ΔDRE mice while Pkm^ΔDRE hepatocytes exhibited greater cytotoxicity following co-treatment with TCDD and hydrogen peroxide. TCDD also induced PKM2 in human HepaRG™ cells with AHR enrichment at a conserved DRE core within the locus. These results suggest AHR-mediated PKM2 induction is a novel antioxidant response to TCDD.

Fig. 1

Pkm^ΔDRE mice development and validation. A) CRISPR-Cas9 was used to specifically delete the 5bp DRE core (5’ – GCGTG - 3′) in the pDRE located in the Pkm locus (mm10:chr9:59,668,103–59,668,107). Protospacer of gRNA is annotated, PAM – underlined sequence, a single G > A mutation in the PAM was caused to prevent re-cutting of the targeted allele. Sanger sequencing of the edited allele is aligned with the reference sequence to demonstrate the DRE deletion. B) Hepatic AHR enrichment in mice at 2 h following oral gavage with either sesame oil vehicle (control) or 30 μg/kg TCDD measured by ChIP-PCR. C) The in vivo effect of TCDD on Pkm2 mRNA expression assessed in livers from wild type (WT, blue) and Pkm^ΔDRE (red) mice. D) The in vitro effect of TCDD on Pkm2 mRNA expression in isolated WT and Pkm^ΔDRE primary mouse hepatocytes (n = 5) treated for 24 h. E) The dose dependent in vitro effect of TCDD on PKM2 protein levels in isolated WT and Pkm^ΔDRE primary mouse hepatocytes (n = 5) treated for 24 h. F) The dose dependent effect of TCDD on Pkm2 mRNA expression after 24 h on primary hepatocytes isolated from AHR^V372Afl/flAlb-Cre^ERT2 mice following treatment with vehicle (control) or tamoxifen (AHR null). G) The effect of TCDD on pyruvate kinase (PK) activity assessed in liver extracts from wild type (WT, blue) and Pkm^ΔDRE (red) mice orally gavaged with sesame oil (vehicle) or 30 μg/kg TCDD, respectively, every 4 days for 28 days. H) The effect of 100 nM TCDD on PKM2 mRNA expression in differentiated human HepaRG cells (n = 2) after 24 h. I) AHR enrichment in human HepaRG cells after 45 min of vehicle or 100 nM TCDD treatment assessed by ChIP-PCR. A motif was identified within the enriched region (hg38:chr15:72,211,159–72,211,173) and assessed as a functional DRE. Gene expression was assessed by qRT-PCR. Protein levels were examined using an automated capillary-based immunoassay system. Bars represent mean ± standard deviation (SD). Asterisk indicates significance (∗ - p ≤ 0.05; ∗∗ - p ≤ 0.01; ∗∗∗ - p ≤ 0.001; ∗∗∗∗ - p ≤ 0.0001) determined by two-way ANOVA and Tukey's post-hoc test for panels A–G and I, and t-test for panel H.

Fig. 2

Histological evaluation of liver sections from wild type (WT) and Pkm^ΔDRE mice gavaged with sesame oil vehicle or 30 μg/kg TCDD every 4 days for 28 days. (A) Representative micrographs of hematoxylin and eosin staining (H&E) to assess hepatic lesions, F4/80 to determine macrophage infiltration, and PicroSirius Red (PSR) to detect collagen deposition. Scale bar represents 100 μm. (B) Quantitation of F4/80 and PSR staining used QuPath and the Quantitative Histological Analysis Tool (n = 8–10) (Nault et al., 2015), respectively. C) Effect of TCDD on the expression of fibrosis-related genes in male WT and Pkm^ΔDRE mice (n = 5) orally gavaged every 4 days for 28 days with TCDD. Gene expression was evaluated by qRT-PCR. Bars represent mean ± SD. Asterisk indicates significance (∗ - p ≤ 0.05; ∗∗ - p ≤ 0.01; ∗∗∗∗ - p ≤ 0.0001) determined by two-way ANOVA and Tukey's post-hoc test.

Fig. 3

Protective effect of AHR-induced PKM2. (A) Reduced (GSH) and (B) oxidized (GSSG) glutathione levels in wild type (WT) and Pkm^ΔDRE mice (n = 6) following oral gavage with sesame oil vehicle (control) or 0.3–30 μg/kg TCDD. (C) GSH:GSSG ratio. Bars represent mean ± SEM. (D) Viability of WT and Pkm^ΔDRE primary hepatocytes after treatment with DMSO or 10 nM TCDD for 24 h followed by cotreatment with H₂O₂ for 48 h. Bars represent mean ± SD. Asterisk denotes significance (∗p < 0.05, ∗∗p < 0.01) determined by two-way ANOVA and Tukey's post-hoc test.

Fig. 4

Induction of cytokine levels in serum from wild type (WT) and Pkm^ΔDRE mice following oral gavage with TCDD (n = 6). Bars represent mean ± SD. Asterisk indicates significance (∗ - p ≤ 0.05) determined by two-way ANOVA and Tukey's post-hoc test.