Toll-like receptor 4 mediates synergism between alcohol and HCV in hepatic oncogenesis involving stem cell marker Nanog

Abstract

Alcohol synergistically enhances the progression of liver disease and the risk for liver cancer caused by hepatitis C virus (HCV). However, the molecular mechanism of this synergy remains unclear. Here, we provide the first evidence that Toll-like receptor 4 (TLR4) is induced by hepatocyte-specific transgenic (Tg) expression of the HCV nonstructural protein NS5A, and this induction mediates synergistic liver damage and tumor formation by alcohol-induced endotoxemia. We also identify Nanog, the stem/progenitor cell marker, as a novel downstream gene up-regulated by TLR4 activation and the presence of CD133/Nanog-positive cells in liver tumors of alcohol-fed NS5A Tg mice. Transplantation of p53-deficient hepatic progenitor cells transduced with TLR4 results in liver tumor development in mice following repetitive LPS injection, but concomitant transduction of Nanog short-hairpin RNA abrogates this outcome. Taken together, our study demonstrates a TLR4-dependent mechanism of synergistic liver disease by HCV and alcohol and an obligatory role for Nanog, a TLR4 downstream gene, in HCV-induced liver oncogenesis enhanced by alcohol.

Fig. 1.

Increased susceptibility of NS5A transgenic mice to endotoxin challenge. (A and B) TLR4 mRNA and protein expressions in the liver of NS5A Tg mice were increased, as demonstrated by RT-PCR (A) and Western blot (B) analyses. (C) H&E-stained sections of livers collected 24 hours after LPS challenge (25 mg/kg weight, intraperitoneally) showed coagulative necrosis with hemorrhage in NS5A Tg mice (Middle) but no histological abnormality in wild-type mice (WT, Top) or NS5A Tg mice with TLR4 deficiency (Tlr4^−/−NS5A, Bottom) (Magnification: 100×). (D) Serum AST levels at 24 hours after LPS challenge. TLR4 deficiency (Tlr4^−/−) attenuated LPS-induced elevation of AST compared with wild-type mice (WT; *, P < 0.05). NS5A Tg mice showed an augmented AST elevation compared with WT (*, P < 0.05), and this effect was abrogated by TLR4 deficiency (Tlr4^−/−NS5A) (*, P < 0.05). (E) Time course changes in serum TNF-α level after LPS injection. Increased TNF-α levels in WT were reduced in Tlr4^−/− (*, P < 0.05) but augmented in NS5A mice (**, P < 0.05). Heightened TNF-α levels in NS5A mice were abrogated in Tlr4^−/−NS5A (***, P < 0.05). (F) LPS-induced signaling, such as TAK1 interaction with TRAF6 or IRAK, was enhanced in NS5A but not Tlr4^−/−NS5A mice. (G) LPS-induced phosphorylation of JNK and IκBα in the liver was increased in NS5A but not Tlr4^−/−NS5A mice.

Fig. 2.

HCV NS5A induced liver tumors after 12-month alcohol feeding, as shown in this representative photograph (Left) of gross liver appearance and a microphotograph (Right) of an H&E-stained section of the tumor. (Magnification: 100×).

Fig. 3.

(A) Confocal immunofluorescent microscopy demonstrates colocalization of the stem cell marker Nanog with CD133 or CD49f in the tumor-bearing tissue from NS5A mice fed alcohol for 12 months. (Magnification: 100×.) (B) Nanog protein was induced in the livers of NS5A mice fed alcohol, as determined by immunoblot analysis. Immunoblotting of β-actin served as a loading control. (C) Nanog mRNA was induced in the livers of NS5A mice fed ethanol, as determined by real-time PCR (*, P < 0.03 compared with ethanol-fed WT mice). This induction was attenuated in ethanol-fed Tlr4^−/−NS5A mice (*, P < 0.03 compared with ethanol-fed NS5A). (D) LPS induced Nanog mRNA in Huh7 cells transduced with an NS5A expression vector (*, P < 0.002 compared with the cells transduced with the empty vector and control shRNA). This induction was suppressed by retroviral expression of shRNA for TLR4 (**, P < 0.05). Nanog mRNA also was induced by LPS treatment in the cells transduced with a retroviral vector expressing wild-type TLR4 (TLR4+, *, P < 0.05 compared with the control cells), but not in the cells transduced with the mutant TLR4 vector (TLR4ΔCyp). (E) Efficient knockdown of TLR4 with the retroviral shRNA vector is evident in this immunoblot analysis and densitometric analysis revealing an 82% reduction (*, P < 0.05). (F) LPS added to the culture activated the Nanog promoter in Huh7 cells transfected with the promoter-luciferase construct (*, P < 0.002 compared with the control cells). TLR4 knockdown with shRNA as above abrogated the promoter activation (**, P < 0.05). Expression of wild-type TLR4 (TLR4+, *, P < 0.05) but not mutant TLR4 (TLR4ΔCyp) conferred the cells LPS-induced Nanog promoter activation. Relative light unit values were normalized by the activity of the Renilla luciferase construct driven by SV40 promoter as a transfection control.

Fig. 4.

TLR4-dependent development of liver tumors by hepatoblast transplantation. (A) A schematic diagram depicting the generation of liver tumors following retroviral transduction of TLR4 in purified E-cadherin⁺ hepatoblasts from p53^−/− mice, transplantation and engraftment of the hepatoblasts in a recipient C57BL/6 mouse, and repetitive LPS injection. (B) Gross photographs of livers following transplantation of p53^−/− hepatoblasts transduced with c-Myc, Tlr4, Nanog, shRNA for Nanog, or scrambled shRNA and 25 weeks of LPS treatment (2 mg/kg, intraperitoneally, every other day, the mice with c-Myc-transduced cells did not receive LPS). (C) Tumor incidence of recipient mice after transplantation of p53^−/− hepatoblasts transduced with the indicated gene and/or shRNA. Mice that were received a transplant of p53^−/−, c-Myc-transduced cells mostly developed liver tumors without LPS treatment. The mice receiving a transplant of TLR4-transduced p53^−/− cells also developed liver tumors at the incidence rate of 40% (*P < 0.03). This tumor incidence was suppressed by cotransduction of Nanog shRNA but not control scrambled shRNA (**, P < 0.05 compared with the cells transduced with control shRNA). Transduction of Nanog without LPS injection also produced liver tumors, but with much less frequency (***, P < 0.05 compared with empty vector-transduced cells without LPS). (D) GFP imaging of the tumor-bearing livers of LPS-treated mice transplanted with p53^−/− hepatoblasts transduced with TLR4 plus Nanog shRNA or control shRNA. (E) Survival curves of mice after transplantation of the cells transduced with the indicated gene and/or shRNA. (F) Tumor volume measurement was performed by 3-dimensional GFP imaging of nude mice at various times following s.c. transplantation of p53^−/− hepatoblasts transduced with TLR4, a deletion mutant of cytoplasmic domain of TLR4 (ΔCyt), or c-Myc. Note a progressive tumor growth with the c-Myc-transduced cells even without LPS treatment serving as a positive control. Liver tumors arose and grew in mice receiving a transplant of TLR4-transduced cells in response to repetitive LPS injection but not without LPS injection. The cells transduced with the mutant TLR4 failed to form a tumor mass. (*, P < 0.05 compared with empty vector-transduced cells with LPS.) (G) Hepatoblasts (p53^−/−) transduced with a Nanog or control retrovirus were subcutaneously transplanted into nude mice, and the tumor mass growth was monitored as above. Immunoblot analysis was performed on the transduced cells after 10 days following infection and just before transplantation. This analysis confirms the expression of Nanog (Inset). Nanog-transduced progenitor cells led to a small but significantly increased tumor mass compared with the cells transduced with the control vector (*, P < 0.05). (H) Hepatic progenitor cells expressing TLR4 gave rise to growing tumors in nude mice repetitively injected with LPS, and this growth was significantly attenuated with Nanog shRNA (*, P < 0.04). Immunoblotting of lysates from the hepatoblasts collected 10 days after the transplantation and LPS injection confirms induction of Nanog in TLR4-transduced cells and effective knockdown of Nanog by cotransduction of the specific shRNA (Inset).