Up-regulation of Hedgehog pathway is associated with cellular permissiveness for hepatitis C virus replication - PubMed (original) (raw)
Up-regulation of Hedgehog pathway is associated with cellular permissiveness for hepatitis C virus replication
Steve S Choi et al. Hepatology. 2011 Nov.
Abstract
Studies of the hepatitis C virus (HCV) life-cycle rely heavily on 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 with Huh7 cells, have substantially decreased expression of epithelial markers, increased levels of mesenchymal markers, and markedly up-regulated Hh pathway activity: Shh, >100-fold, Gli1, >30-fold, Ptc, 2-fold. In Huh7.5 cells, we found that cyclopamine, an Hh pathway antagonist, reduced HCV RNA levels by 50% compared with 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.
Conclusion: We have identified a relationship between HCV and Hh signaling where up-regulated 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.
Copyright © 2011 American Association for the Study of Liver Diseases.
Figures
Figure 1. Huh7.5 are more permissive than Huh7 cells for HCV infection and RNA expression levels are markedly different for epithelial, mesenchymal and Hh pathway genes
A) Comparison of relative HCV RNA levels in Huh7.5 and Huh7 cells after infection with JFH1 HCV. RNA was isolated at 72 hours post-infection. Comparison of relative RNA levels of Huh7.5 cells compared to Huh7 cells for: B) epithelial markers E-cadherin and keratin 19 (Krt19); C) mesenchymal markers α–smooth muscle actin (αSMA), collagen, type I, alpha 1 (Col1α1) and S100 calcium binding protein A4 (S100A4); D) Hedgehog pathway and downstream targets sonic hedgehog (Shh), glioblastoma 1 (Gli1), Patched (Ptc). Results are expressed as relative fold expression with Huh7 expression indexed to 1. Key to statistical comparisons: *p<.05, ** p<.01, † p<.005.
Figure 2. Hh pathway blockade with cyclopamine results in reduced HCV RNA and protein levels
A) Huh7.5 cells grown on 12-well plates were infected with 1500 focus-forming units of JFH1 HCV virus alone, or virus plus vehicle control (DMSO), inactive analog (tomatidine 5μM) or Hh inhibitor (cyclopamine 5μM) and incubated for 72 hours. Relative RNA expression for HCV RNA, Shh and Gli1 were analyzed with virus-alone infected cells indexed at 1. Key to statistical comparisons: *p<.05, ** p<.01, † p<.005. B) Immunofluorescent analysis using α-HCV Core of Huh7.5 cells infected with virus plus DMSO, virus plus tomatidine or virus plus cyclopamine. C) Huh7.5 were grown as described above. Cells were infected with virus alone, virus plus antibody control (mouse IgG1, 10 μg/ml) or virus plus a Shh neutralizing antibody (5E1, 10 μg/ml) and incubated for 48 hours. Relative RNA expression for HCV RNA, Shh and Gli1 were analyzed with virus alone infected cells indexed at 1. Key to statistical comparisons: *p<.05.
Figure 2. Hh pathway blockade with cyclopamine results in reduced HCV RNA and protein levels
A) Huh7.5 cells grown on 12-well plates were infected with 1500 focus-forming units of JFH1 HCV virus alone, or virus plus vehicle control (DMSO), inactive analog (tomatidine 5μM) or Hh inhibitor (cyclopamine 5μM) and incubated for 72 hours. Relative RNA expression for HCV RNA, Shh and Gli1 were analyzed with virus-alone infected cells indexed at 1. Key to statistical comparisons: *p<.05, ** p<.01, † p<.005. B) Immunofluorescent analysis using α-HCV Core of Huh7.5 cells infected with virus plus DMSO, virus plus tomatidine or virus plus cyclopamine. C) Huh7.5 were grown as described above. Cells were infected with virus alone, virus plus antibody control (mouse IgG1, 10 μg/ml) or virus plus a Shh neutralizing antibody (5E1, 10 μg/ml) and incubated for 48 hours. Relative RNA expression for HCV RNA, Shh and Gli1 were analyzed with virus alone infected cells indexed at 1. Key to statistical comparisons: *p<.05.
Figure 3. Huh7 cells treated with a Hh pathway agonist demonstrate increased permissiveness for HCV replication
Using identical conditions to those described in figure 2A, Huh7 cells were mock infected (control), infected with JFH1 HCV plus vehicle (JFH+DMSO) and infected with JFH1 HCV and then treated with N-terminal fragment Shh ligand. After 72 hours, relative RNA expression was analyzed for HCV RNA, Shh and Gli1. Results are expressed as relative fold expression with mock-infected expression indexed to 1, except for HCV RNA sample, in which case JFH1+DMSO was indexed to 1. Key to statistical comparisons: *p<.05, ** p<.01, † p<.005.
Figure 4. Huh7 cells treated with SAG, a Hh pathway agonist with a different target, also demonstrate increased permissiveness for HCV replication
A) Huh7 cells were mock infected (control), infected with JFH1 HCV alone, JFH1 HCV plus vehicle (JFH+DMSO) and JFH1 HCV plus SAG 0.3μM. After 72 hours, relative RNA expression was analyzed for HCV RNA, Shh and Gli1. Results are expressed as relative fold expression with mock-infected expression indexed to 1, except for HCV RNA sample, in which case JFH1 HCV alone was indexed to 1. Key to statistical comparisons: *p<.05, ** p<.01, † p<.005. B) Protein lysates were created from the above described experiment. Antibodies to HCV Core, Shh and α-tubulin were used for analysis.
Figure 5. SAG treatment of Huh7.5 cells further increases their permissivity for HCV replication
Huh7.5 cells were mock infected (control), infected with JFH1 HCV alone, JFH1 HCV plus vehicle (JFH+DMSO) and JFH1 HCV plus SAG 0.3μM. After 72 hours, relative RNA expression was analyzed for HCV RNA, Shh and Gli1. Results are expressed as relative fold expression with mock infected expression indexed to 1, except for HCV RNA sample, in which case JFH1 HCV alone was indexed to 1. Key to statistical comparisons: *p<.05, ** p<.01, † p<.005. B) Protein lysates were created from the above described experiment. Antibodies to HCV Core, Shh and α-tubulin were used for analysis.
Figure 6. Blockade of Hh pathway with Cyclopamine in Huh7.5 cells reduces replication of HCV subgenomic replicon
Huh7.5 cells harboring HCV Con1 subgenomic replicon RNA were treated with vehicle control (DMSO), inactive analog (tomatidine 5μM) or Hh pathway antagonist (cyclopamine 5μM). After 48 hours, relative RNA expression was analyzed for HCV RNA, Shh and Gli1. Results are expressed as relative fold expression with vehicle control sample (DMSO) indexed to 1. Key to statistical comparisons: *p<.05, ** p<.01, † p<.005.
Figure 7. GDC-0449, a pre-clinical Hh pathway inhibitor, inhibits replication of JFH1 HCV in a dose response manner
A) Huh7.5 cells were mock infected (control), infected with JFH1 HCV alone, JFH1 HCV plus vehicle (JFH+DMSO) and JFH1 HCV plus GDC-0449 5μM concentration. After 72 hours, relative RNA expression was analyzed for HCV RNA, Shh and Gli1. Results are expressed as relative fold expression with mock infected expression indexed to 1, except for HCV RNA sample, in which case JFH1 HCV alone was indexed to 1. B) Protein lysates were created from the above described experiment. Antibodies to HCV Core, Shh and α-tubulin were used for analysis. C) The above experiment was replicated with varying concentrations of GDC-0449 to assess dose response of anti-HCV activity. Concentrations used were: 0 μM, 0.05 μM, 0.5 μM, 5 μM and 25 μM. After 72 hours, relative RNA expression was analyzed for HCV RNA, Shh and Gli1. Results are expressed as relative fold expression with mock infected expression indexed to 1, except for HCV RNA sample, in which case JFH1 HCV alone was indexed to 1. Key to statistical comparisons: *p<.05, ** p<.01, † p<.005.
Figure 7. GDC-0449, a pre-clinical Hh pathway inhibitor, inhibits replication of JFH1 HCV in a dose response manner
A) Huh7.5 cells were mock infected (control), infected with JFH1 HCV alone, JFH1 HCV plus vehicle (JFH+DMSO) and JFH1 HCV plus GDC-0449 5μM concentration. After 72 hours, relative RNA expression was analyzed for HCV RNA, Shh and Gli1. Results are expressed as relative fold expression with mock infected expression indexed to 1, except for HCV RNA sample, in which case JFH1 HCV alone was indexed to 1. B) Protein lysates were created from the above described experiment. Antibodies to HCV Core, Shh and α-tubulin were used for analysis. C) The above experiment was replicated with varying concentrations of GDC-0449 to assess dose response of anti-HCV activity. Concentrations used were: 0 μM, 0.05 μM, 0.5 μM, 5 μM and 25 μM. After 72 hours, relative RNA expression was analyzed for HCV RNA, Shh and Gli1. Results are expressed as relative fold expression with mock infected expression indexed to 1, except for HCV RNA sample, in which case JFH1 HCV alone was indexed to 1. Key to statistical comparisons: *p<.05, ** p<.01, † p<.005.
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