Figure 6. Epiregulin is an LPS-inducible promoter of hepatocarcinogenesis.

A. Ereg and Hgf mRNA were determined in Tlr4^mut mice (n=14), Tlr4^mut (n=15) and antibiotics-treated Tlr4^WT (n=9) after DEN and 2×CCl₄ injections. Fold induction vs. untreated livers. B. Ereg and Hgf mRNA were determined in TLR4-chimeric mice with Tlr4^WT resident cells and Tlr4^WT BM (n=6) or Tlr4^mut resident cells and Tlr4^WT BM (n=6) after treatment with DEN and 2 CCl₄ injections. Fold induction vs. untreated livers. C. Western blot of HGF from Tlr4^WT mice, Tlr4^mut mice and antibiotics-treated mice after DEN and 2×CCl₄ injections. D. Hepatocytes (Hep), Kupffer cells (KC) and hepatic stellate cells (HSC) were isolated from liver treated with DEN and 2 injections of CCl₄ followed by analysis of Ereg mRNA expression by qPCR (left panel). Shown is a fold-induction vs. whole liver. Ereg mRNA was isolated in liver extracts from CCl₄-treated mice injected with LPS (10 mg/kg i.v.) or PBS 4 days after the last CCl₄ injection (second panel from left). Activated hepatic stellate cells were isolated from CCl₄-treated and bile duct-ligated livers. Epiregulin was determined by ELISA 24hr after plating. E. Hepatic stellate cells from DEN plus 2×CCl₄ treated mice were stimulated with LPS (100 ng/ml) in the presence of either an adenoviral IκB superrepressor (AdIκBsr) or empty shuttle virus (AdSh) by qPCR (left panel) or ELISA (right panel) F. EREG expression was determined in normal liver (“NL”, n=4) and livers of patients with alcoholic hepatitis (“AH”, n=10, left panel) and in activated human hepatic stellate cells stimulated with LPS (100 ng/ml, right panel). G. WT (n=11) and Ereg^KO mice (n=14) were treated with DEN plus 22 injections of CCl₄. Mice were sacrificed 6.5 months after DEN. Shown are representative images of tumors as well as tumor number, size of the largest tumor, and liver body weight ratio. H. Ereg expression was analyzed in non-tumorous regions of DEN plus CCl₄-treated Tlr4^WT (n=12), Tlr4^mut mice (n=9) and antibiotics-treated Tlr4^WT mice (n=6) 10.5 months after DEN. Fold induction vs. untreated liver. Data are represented as means ± SD. * p<0.05, ** p<0.01. nd, non-detectable