Histone h3 modifications in rat hepatic stellate cells by ethanol.

AIMS: Hepatic stellate cells (HSCs) play critical roles in the development of hepatic fibrosis caused by various agents including alcohol. Ethanol causes post-translational modification in histone. The goal of this study is to investigate whether ethanol affected acetylation and methylation of histone H3 in rat HSCs.

METHODS

We isolated and separated HSCs using collagenase perfusion of liver followed by Nycodenz density gradient centrifugation. HSCs were divided and treated with different concentrations of ethanol for various times. Histone was isolated using acid extraction method. Acetylation and methylation of histone H3 at Lys9 was analysed by both western blot and fluorescein isothiocyanate (FITC) immunochemical stain. Acetylation of histone H3 at Lys9 (Ac-H3-lys9), Lys14 (Ac-H3-Lys14), Lys18 (Ac-H3-lys18), or Lys23 (Ac-H3-lys23) was checked by western blotting.

RESULTS

At lysine 9, ethanol caused dose-dependent increase of Ac-H3 up to 200 mM. Ac-H3-lys9 increased with a maximum of 86-fold at 72 h and 200 mM ethanol treatment, and decreased thereafter. This increase was confirmed by both western blotting and FITC stain. At high dose, ethanol increased acetylation of histone H3 at Lys23 (Ac-H3-lys23), but it had no effect on Ac-H3-lys14 or Ac-H3-lys18. The intensity of the FITC-labelled dimethyl-histone H3 at Lys9 (Me-H3-lys9) antibody appeared to decrease slightly with increasing dose of ethanol. But this did not appear to change when monitored by western blotting.

CONCLUSIONS

Ethanol caused dose and time-dependent increase in acetylation of histone H3 at Lys9, but not at Lys14 or Lys18. Compared with hepatocytes the Ac-H3-lys9 in HSCs required longer ethanol exposure. Levels of Me-H3-lys9 seemed to remain unaltered. Thus increase in Ac-H3-lys9 represents a nuclear-chromatin modification event in HSCs exposed to ethanol.