Up-regulation of Hedgehog pathway is associated with cellular permissiveness for hepatitis C virus replication

Abstract

Studies of the Hepatitis C virus (HCV) life-cycle rely heavily upon Huh7.5 cells, but the reasons why these cells are exceptionally permissive for HCV replication are not clear. Based on recent clinical observations, we hypothesized that the Hedgehog (Hh) pathway, which has not been previously associated with HCV replication, may be involved in the Huh7.5 phenotype of increased permissiveness. We tested this hypothesis by comparing levels of a variety of Hh related cellular markers in Huh7.5 cells with the parental Huh7 cells, which are far less permissive. Here, we demonstrate that Huh7.5 cells, when compared to Huh7 cells, have substantially decreased expression of epithelial markers, increased levels of mesenchymal markers and markedly upregulated Hh pathway activity: Shh, >100 fold, Gli1, >30 fold, Ptc, 2 fold. In Huh7.5 cells, we found that cyclopamine, a Hh pathway antagonist, reduced HCV RNA levels by 50% compared to vehicle and inactive isomer controls. Moreover, in Huh7 cells, treatment with recombinant Shh ligand and SAG, both Hh pathway agonists, stimulated HCV replication by 2 fold and 4 fold, respectively. These effects were observed with both viral infections and a subgenomic replicon. Finally, we demonstrated that GDC-0449 decreased HCV RNA levels in a dose response manner. Conclusions We have identified a relationship between HCV and Hh signaling where upregulated pathway activity during infection promotes an environment conducive to replication. Given that Hh activity is very low in most hepatocytes, these findings may serve to further shift the model of HCV liver infection from modest widespread replication in hepatocytes to one where a subset of cells support high level replication. These findings also introduce Hh pathway inhibitors as potential anti-HCV therapeutics.

Osteopontin is up-regulated in chronic hepatitis C and is associated with cellular permissiveness for hepatitis C virus replication

“…We had previously reported that permissiveness of HCV replication was associated with increased Hedgehog (Hh) activity (15), and that inhibition of Hh signaling led to a reduction in cell-associated HCV RNA levels. Consistently, we find that basal expression of OPN, a Hh-regulated target gene and a proximal effector of the Hh pathway (8), is 30-fold higher in Huh7.5 than Huh7 cells (Fig 1B–C).…”

“…Treatment with SAG (associated with increased HCV RNA (15)) led to a 2-fold increase in OPN mRNA, while inhibiting the Hh pathway (associated with reduced HCV RNA) repressed OPN mRNA to basal (control) levels. The aggregate data supports our hypothesis that OPN supports HCV replication in vitro.…”

“…In separate experiments, Huh7.5 cells were treated with the Hh agonist SAG (0.3uM) or the Hh antagonist GDC-0449 (5uM) at the time of infection with JFH1 virus or mock infection (control), and RNA harvested as previously described (15). …”

“…Recently, we showed that activation of the Hh pathway also occurs during chronic HCV (15, 16). Intriguingly, Hh signaling could enhance viral permissivity and replication in HCV-infected liver cells.…”

Sonic hedgehog pathway inhibitor mitigates mouse hepatocellular carcinoma

“…41 GDC-0449 has anti-hepatitis C virus (HCV) activity in a dose-dependent manner in human HCC cell line Huh 7.5. 42 Vismodegib reduced TNFrelated apoptosis-inducing ligand-mediated liver injury in mouse model. 43 Furthermore, GDC-0449 alters the homeostasis of intracellular Ca 21 , inhibits cell growth, and reduces side population in lung cancer.…”