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Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus.

Plissonnier ML, Lahlali T, Michelet M, Lebossé F, Cottarel J, Beer M, Neveu G, Durantel D, Bartosch B, Accardi R, Clément S, Paradisi A, Devouassoux-Shisheboran M, Einav S, Mehlen P, Zoulim F, Parent R - PLoS Biol. (2016)

Bottom Line: Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues.Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression.Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells.

View Article: PubMed Central - PubMed

Affiliation: Pathogenesis of Hepatitis B and C - Equipe labellisée LabEx DEVweCAN, INSERM U1052, Centre de Recherche en Cancérologie de Lyon, F-69003 Lyon, France, Université de Lyon, F-69003 Lyon, Université Lyon 1, ISPB, Lyon, F-69622, France, CNRS UMR5286, F-69083 Lyon, France, Centre Léon Bérard, F-69008 Lyon, France.

ABSTRACT
Hepatitis C virus (HCV) is an oncogenic virus associated with the onset of hepatocellular carcinoma (HCC). The present study investigated the possible link between HCV infection and Netrin-1, a ligand for dependence receptors that sustains tumorigenesis, in particular in inflammation-associated tumors. We show that Netrin-1 expression is significantly elevated in HCV+ liver biopsies compared to hepatitis B virus (HBV+) and uninfected samples. Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues. Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression. In vitro, HCV increased the level and translation of Netrin-1 in a NS5A-La-related protein 1 (LARP1)-dependent fashion. Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells. Netrin-1 enhanced infectivity of HCV particles and promoted viral entry by increasing the activation and decreasing the recycling of the epidermal growth factor receptor (EGFR), a protein that is dysregulated in HCC. Netrin-1 and HCV are, therefore, reciprocal inducers in vitro and in patients, as seen from the increase in viral morphogenesis and viral entry, both phenomena converging toward an increase in the level of infectivity of HCV virions. This functional association involving a cancer-related virus and Netrin-1 argues for evaluating the implication of UNC5 receptor ligands in other oncogenic microbial species.

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HCV induces the expression of Netrin-1 in vitro.A, B, C, D. HCV induces Netrin-1 mRNA in primary human hepatocytes. Cells were infected at a MOI of 1 with HCV genotype 2 strain 4 d after seeding (n = 4 independent preparations from four different patients, Wilcoxon test, p < 0.05). E. HCV induces Netrin-1 mRNA in primary human hepatocytes endogenously infected with HCV genotype 3 strain. F. HCV induces Netrin-1 mRNA in Huh7.5 cells. Proliferative Huh7.5 cells were infected at a MOI of 0.1 with HCV genotype 2 strain the day after seeding and trypsinized on day five post-infection. G. HCV induces Netrin-1 mRNA in Huh7.5 cells. Proliferative Huh7.5 cells were electroporated with HCV genotype 1a strain (H77). H. Differentiated Huh7.5 cells (right) were incubated with 2% DMSO 4 d after seeding and infected at a MOI of 0.05 3 d after DMSO treatment. The levels of HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR. Data are represented as mean ± standard deviation (n = 3 for each cell phenotype, Wilcoxon test, p < 0.05). The underlying data for panels in this figure can be found in S1 Data.
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pbio.1002421.g002: HCV induces the expression of Netrin-1 in vitro.A, B, C, D. HCV induces Netrin-1 mRNA in primary human hepatocytes. Cells were infected at a MOI of 1 with HCV genotype 2 strain 4 d after seeding (n = 4 independent preparations from four different patients, Wilcoxon test, p < 0.05). E. HCV induces Netrin-1 mRNA in primary human hepatocytes endogenously infected with HCV genotype 3 strain. F. HCV induces Netrin-1 mRNA in Huh7.5 cells. Proliferative Huh7.5 cells were infected at a MOI of 0.1 with HCV genotype 2 strain the day after seeding and trypsinized on day five post-infection. G. HCV induces Netrin-1 mRNA in Huh7.5 cells. Proliferative Huh7.5 cells were electroporated with HCV genotype 1a strain (H77). H. Differentiated Huh7.5 cells (right) were incubated with 2% DMSO 4 d after seeding and infected at a MOI of 0.05 3 d after DMSO treatment. The levels of HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR. Data are represented as mean ± standard deviation (n = 3 for each cell phenotype, Wilcoxon test, p < 0.05). The underlying data for panels in this figure can be found in S1 Data.

Mentions: Next, we investigated whether induction of Netrin-1 by HCV could also be seen in vitro, in a tissue inflammation-free environment. Primary human hepatocytes (PHH) were infected with an HCV japanese fulminant hepatitis 1 (JFH1; genotype 2) selected for its higher rate of propagation in cell cultures [24]. Results revealed a peak in Netrin-1 upregulation (by 5-fold to 20-fold) in the HCV-infected cultures at day two or day three (Fig 2A–2D). This was also confirmed in endogenously infected PHH with wild-type genotype 3 virus (Fig 2E). Our in vitro experiments were then conducted using a known hepatocytic cell line that is also amenable for mechanistic studies, in order to further examine Netrin-1 induction. We infected proliferating and dimethyl sulfoxide (DMSO)-differentiated [25] Huh7.5 cells [26] with an HCV JFH1 isolate bearing three adaptive mutations [27], or with a non-adapted genotype 1 strain [28]. Over the five- to ten-day kinetic follow-up study, HCV induced an 8-fold increase in the levels of Netrin-1 mRNA at day eight post-infection in proliferating cultures infected with an HCV JFH1 isolate (Fig 2F) or with a genotype 1 strain (Fig 2G) and in differentiated cells (Fig 2H). These results indicate that HCV induces Netrin-1 expression in hepatocytes both in vivo and in vitro.


Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus.

Plissonnier ML, Lahlali T, Michelet M, Lebossé F, Cottarel J, Beer M, Neveu G, Durantel D, Bartosch B, Accardi R, Clément S, Paradisi A, Devouassoux-Shisheboran M, Einav S, Mehlen P, Zoulim F, Parent R - PLoS Biol. (2016)

HCV induces the expression of Netrin-1 in vitro.A, B, C, D. HCV induces Netrin-1 mRNA in primary human hepatocytes. Cells were infected at a MOI of 1 with HCV genotype 2 strain 4 d after seeding (n = 4 independent preparations from four different patients, Wilcoxon test, p < 0.05). E. HCV induces Netrin-1 mRNA in primary human hepatocytes endogenously infected with HCV genotype 3 strain. F. HCV induces Netrin-1 mRNA in Huh7.5 cells. Proliferative Huh7.5 cells were infected at a MOI of 0.1 with HCV genotype 2 strain the day after seeding and trypsinized on day five post-infection. G. HCV induces Netrin-1 mRNA in Huh7.5 cells. Proliferative Huh7.5 cells were electroporated with HCV genotype 1a strain (H77). H. Differentiated Huh7.5 cells (right) were incubated with 2% DMSO 4 d after seeding and infected at a MOI of 0.05 3 d after DMSO treatment. The levels of HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR. Data are represented as mean ± standard deviation (n = 3 for each cell phenotype, Wilcoxon test, p < 0.05). The underlying data for panels in this figure can be found in S1 Data.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4816328&req=5

pbio.1002421.g002: HCV induces the expression of Netrin-1 in vitro.A, B, C, D. HCV induces Netrin-1 mRNA in primary human hepatocytes. Cells were infected at a MOI of 1 with HCV genotype 2 strain 4 d after seeding (n = 4 independent preparations from four different patients, Wilcoxon test, p < 0.05). E. HCV induces Netrin-1 mRNA in primary human hepatocytes endogenously infected with HCV genotype 3 strain. F. HCV induces Netrin-1 mRNA in Huh7.5 cells. Proliferative Huh7.5 cells were infected at a MOI of 0.1 with HCV genotype 2 strain the day after seeding and trypsinized on day five post-infection. G. HCV induces Netrin-1 mRNA in Huh7.5 cells. Proliferative Huh7.5 cells were electroporated with HCV genotype 1a strain (H77). H. Differentiated Huh7.5 cells (right) were incubated with 2% DMSO 4 d after seeding and infected at a MOI of 0.05 3 d after DMSO treatment. The levels of HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR. Data are represented as mean ± standard deviation (n = 3 for each cell phenotype, Wilcoxon test, p < 0.05). The underlying data for panels in this figure can be found in S1 Data.
Mentions: Next, we investigated whether induction of Netrin-1 by HCV could also be seen in vitro, in a tissue inflammation-free environment. Primary human hepatocytes (PHH) were infected with an HCV japanese fulminant hepatitis 1 (JFH1; genotype 2) selected for its higher rate of propagation in cell cultures [24]. Results revealed a peak in Netrin-1 upregulation (by 5-fold to 20-fold) in the HCV-infected cultures at day two or day three (Fig 2A–2D). This was also confirmed in endogenously infected PHH with wild-type genotype 3 virus (Fig 2E). Our in vitro experiments were then conducted using a known hepatocytic cell line that is also amenable for mechanistic studies, in order to further examine Netrin-1 induction. We infected proliferating and dimethyl sulfoxide (DMSO)-differentiated [25] Huh7.5 cells [26] with an HCV JFH1 isolate bearing three adaptive mutations [27], or with a non-adapted genotype 1 strain [28]. Over the five- to ten-day kinetic follow-up study, HCV induced an 8-fold increase in the levels of Netrin-1 mRNA at day eight post-infection in proliferating cultures infected with an HCV JFH1 isolate (Fig 2F) or with a genotype 1 strain (Fig 2G) and in differentiated cells (Fig 2H). These results indicate that HCV induces Netrin-1 expression in hepatocytes both in vivo and in vitro.

Bottom Line: Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues.Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression.Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells.

View Article: PubMed Central - PubMed

Affiliation: Pathogenesis of Hepatitis B and C - Equipe labellisée LabEx DEVweCAN, INSERM U1052, Centre de Recherche en Cancérologie de Lyon, F-69003 Lyon, France, Université de Lyon, F-69003 Lyon, Université Lyon 1, ISPB, Lyon, F-69622, France, CNRS UMR5286, F-69083 Lyon, France, Centre Léon Bérard, F-69008 Lyon, France.

ABSTRACT
Hepatitis C virus (HCV) is an oncogenic virus associated with the onset of hepatocellular carcinoma (HCC). The present study investigated the possible link between HCV infection and Netrin-1, a ligand for dependence receptors that sustains tumorigenesis, in particular in inflammation-associated tumors. We show that Netrin-1 expression is significantly elevated in HCV+ liver biopsies compared to hepatitis B virus (HBV+) and uninfected samples. Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues. Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression. In vitro, HCV increased the level and translation of Netrin-1 in a NS5A-La-related protein 1 (LARP1)-dependent fashion. Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells. Netrin-1 enhanced infectivity of HCV particles and promoted viral entry by increasing the activation and decreasing the recycling of the epidermal growth factor receptor (EGFR), a protein that is dysregulated in HCC. Netrin-1 and HCV are, therefore, reciprocal inducers in vitro and in patients, as seen from the increase in viral morphogenesis and viral entry, both phenomena converging toward an increase in the level of infectivity of HCV virions. This functional association involving a cancer-related virus and Netrin-1 argues for evaluating the implication of UNC5 receptor ligands in other oncogenic microbial species.

Show MeSH
Related in: MedlinePlus