Age-dependent regulation of antioxidant genes by p38α MAPK in the liver

Abstract

p38α is a redox sensitive MAPK activated by pro-inflammatory cytokines and environmental, genotoxic and endoplasmic reticulum stresses. The aim of this work was to assess whether p38α controls the antioxidant defense in the liver, and if so, to elucidate the mechanism(s) involved and the age-related changes. For this purpose, we used liver-specific p38α-deficient mice at two different ages: young-mice (4 months-old) and old-mice (24 months-old). The liver of young p38α knock-out mice exhibited a decrease in GSH levels and an increase in GSSG/GSH ratio and malondialdehyde levels. However, old mice deficient in p38α had higher hepatic GSH levels and lower GSSG/GSH ratio than young p38α knock-out mice. Liver-specific p38α deficiency triggered a dramatic down-regulation of the mRNAs of the key antioxidant enzymes glutamate cysteine ligase, superoxide dismutase 1, superoxide dismutase 2, and catalase in young mice, which seems mediated by the lack of p65 recruitment to their promoters. Nrf-2 nuclear levels did not change significantly in the liver of young mice upon p38α deficiency, but nuclear levels of phospho-p65 and PGC-1α decreased in these mice. p38α-dependent activation of NF-κB seems to occur through classical IκB Kinase and via ribosomal S6 kinase1 and AKT in young mice. However, unexpectedly the long-term deficiency in p38α triggers a compensatory up-regulation of antioxidant enzymes via NF-κB activation and recruitment of p65 to their promoters. In conclusion, p38α MAPK maintains the expression of antioxidant genes in liver of young animals via NF-κΒ under basal conditions, whereas its long-term deficiency triggers compensatory up-regulation of antioxidant enzymes through NF-κΒ.

Keywords: And catalase; Glutamate cysteine ligase; Glutathione; Nuclear factor ƙB; Superoxide dismutase 1; Superoxide dismutase 2.

Graphical abstract

Fig. 1

Levels of GSH (A) and GSSG (B), GSSG/GSH ratio (C) and malondialdehyde levels (D) in the liver of young WT and p38α KO mice and in old WT and p38α KO mice. The number of samples per group was 4–6. Statistical significance is indicated as * p < 0.05 WT vs. KO; #p < 0.05 and ##p < 0.01 old vs. young.

Fig. 2

mRNA relative expression of Gclc (A), Sod1 (B), Sod2 (C) and catalase (D) vs Tbp in the liver of young WT and KO mice and in the liver of old WT and p38α KO mice. The number of samples per group was 4–6. Statistical significance is indicated as *p < 0.05 and * *p < 0.01 WT vs. KO; #p < 0.05 and ##p < 0.01 old vs. young.

Fig. 3

Representative image of western blotting of nuclear levels of Nrf-2, PGC-1α, p-p65 (Ser536), p65, and TBP (TATA-binding protein) as loading control in the liver of young WT and p38α KO mice (A) and in the liver of old WT and p38α KO mice (B). The number of samples per group was 4. Representative images of immunohistochemistry of p65 in liver histological sections (C). Scale bar = 30 µm; selected squares with magnification 200%.

Fig. 4

Representative image of western blotting of p-p38α (Thr180/Tyr182), p38α, p-IKKα/β (Ser173/180), IKKα and IKKβ in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice. α-tubulin was used as a loading control (A). Representative image of western blotting of p-Rsk1 (Ser380) and Rsk-1 in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice. α-tubulin was used as a loading control (B). Representative image of western blotting of p-Akt (Ser473) and Akt in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice (C). The number of samples per group was 4.

Fig. 5

Histograms showing the recruitment of p65 in the promoter regions of Gclc, Sod1, Sod2 and catalase in the liver of young WT and p38α KO mice (A) and in the liver of old WT and p38α KO mice (B), measured by chromatin immunoprecipitation (ChIP) assay. The number of samples per group was 4. Statistical significance is indicated as * *p < 0.01 WT vs. KO.

Fig. 6

Histograms showing chromatin immunoprecipitation (ChIP) assay of the histone modification H3K4me3 in the promoter regions of Gclc, Sod1, Sod2 and catalase in the liver of young WT and p38α KO (A) and in the liver of old WT and p38α KO mice (B). The number of mice per group was 4. Statistical significance is indicated as **p < 0.01 WT vs. KO.

Fig. 7

mRNA relative expression of Tnf-α and Cxcl-1 vs Rplp0 in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice (A). Histograms showing the recruitment of p65 in the promoter regions of Tnf-α and Cxcl-1 in the liver of young WT and p38α KO mice (B) and in the liver of old WT and p38α KO mice (C), measured by chromatin immunoprecipitation (ChIP) assay. The number of samples per group was 4–6 for RT-PCR and 4 for ChIP assay. Statistical significance is indicated as * p < 0.05 WT vs. KO; #p < 0.05 old vs. young.