TGF-β1-driven reduction of cytoglobin leads to oxidative DNA damage in stellate cells during non-alcoholic steatohepatitis

Abstract

Background & aims: Cytoglobin (CYGB) is a respiratory protein that acts as a scavenger of reactive oxygen species. The molecular role of CYGB in human hepatic stellate cell (HSC) activation and human liver disease remains uncharacterised. The aim of this study was to reveal the mechanism by which the TGF-β1/SMAD2 pathway regulates the human CYGB promoter and the pathophysiological function of CYGB in human non-alcoholic steatohepatitis (NASH).

Methods: Immunohistochemical staining was performed using human NASH biopsy specimens. Molecular and biochemical analyses were performed by western blotting, quantitative PCR, and luciferase and immunoprecipitation assays. Hydroxyl radicals (^•OH) and oxidative DNA damage were measured using an ^•OH-detectable probe and 8-hydroxy-2'-deoxyguanosine (8-OHdG) ELISA.

Results: In culture, TGF-β1-pretreated human HSCs exhibited lower CYGB levels - together with increased NADPH oxidase 4 (NOX4) expression - and were primed for H₂O₂-triggered ^•OH production and 8-OHdG generation; overexpression of human CYGB in human HSCs reversed these effects. Electron spin resonance demonstrated the direct ^•OH scavenging activity of recombinant human CYGB. Mechanistically, pSMAD2 reduced CYGB transcription by recruiting the M1 repressor isoform of SP3 to the human CYGB promoter at nucleotide positions ⁺2-⁺13 from the transcription start site. The same repression did not occur on the mouse Cygb promoter. TGF-β1/SMAD3 mediated αSMA and collagen expression. Consistent with observations in cultured human HSCs, CYGB expression was negligible, but 8-OHdG was abundant, in activated αSMA⁺pSMAD2⁺- and αSMA⁺NOX4⁺-positive hepatic stellate cells from patients with NASH and advanced fibrosis.

Conclusions: Downregulation of CYGB by the TGF-β1/pSMAD2/SP3-M1 pathway brings about ^•OH-dependent oxidative DNA damage in activated hepatic stellate cells from patients with NASH.

Lay summary: Cytoglobin (CYGB) is a respiratory protein that acts as a scavenger of reactive oxygen species and protects cells from oxidative DNA damage. Herein, we show that the cytokine TGF-β1 downregulates human CYGB expression. This leads to oxidative DNA damage in activated hepatic stellate cells. Our findings provide new insights into the relationship between CYGB expression and the pathophysiology of fibrosis in patients with non-alcoholic steatohepatitis.

Keywords: Cytoglobin; Hepatic stellate cells; Liver fibrosis; NAFLD/NASH; Oxidative stress; TGF-β1.