Role of hepatitis C virus inducted osteopontin in epithelial to mesenchymal transition, migration, and invasion of hepatocytes

Osteopontin (OPN) is a secreted phosphoprotein which has been linked to tumor progression and metastasis in a variety of cancers including hepatocellular carcinoma (HCC). Previous studies have shown that OPN is upregulated during liver injury and inflammation. However, the role of OPN in hepatitis C virus (HCV)-induced liver disease pathogenesis is not known. In this study, we determined the induction of OPN, and then investigated the effect of secreted forms of OPN in epithelial to mesenchymal transition (EMT), migration and invasion of hepatocytes. We show the induction of OPN mRNA and protein expression by HCV-infection. Our results also demonstrate the processing of precursor OPN (75 kDa) into 55 kDa, 42 kDa and 36 kDa forms of OPN in HCV-infected cells. Furthermore, we show the binding of secreted OPN to integrin αVβ3 and CD44 at the cell surface, leading to the activation of downstream cellular kinases such as focal adhesion kinase (FAK), Src, and Akt. Importantly, our results show the reduced expression of epithelial marker (E-cadherin) and induction of mesenchymal marker (N-cadherin) in HCV-infected cells. We also show the migration and invasion of HCV-infected cells using wound healing assay and matrigel coated Boyden chamber. In addition, we demonstrate the activation of above EMT markers, and the critical players involved in OPN-mediated cell signaling cascade using primary human hepatocytes infected with Japanese fulminant hepatitis (JFH)-1 HCV. Taken together, these studies suggest a potential role of OPN in inducing chronic liver disease and HCC associated with chronic HCV infection

Interaction of OPN with integrin αVβ3 and CD44.

<p>HCV-infected Huh7.5 cells were transfected with siGFP and siOPN as described in Materials and Methods. At 72 h posttransfection, equal amounts of cellular lysates from mock, HCV-infected cells and HCV-infected cells transfected with above siRNA were immunoprecipitated using anti-OPN (1∶100) antibody and immunoblotted with anti-integrin β3 and anti-CD44 (lane 1–4). Similarly the cellular lysates from HCV-infected cells were immunoprecipitated with anti-OPN and isotype control goat IgG antibodies in two different sets followed by immunoblot analysis using anti-β3 and anti-CD44 (lane 5–8).</p

Role of Hepatitis C Virus Induced Osteopontin in Epithelial to Mesenchymal Transition, Migration and Invasion of Hepatocytes

<div><p>Osteopontin (OPN) is a secreted phosphoprotein which has been linked to tumor progression and metastasis in a variety of cancers including hepatocellular carcinoma (HCC). Previous studies have shown that OPN is upregulated during liver injury and inflammation. However, the role of OPN in hepatitis C virus (HCV)-induced liver disease pathogenesis is not known. In this study, we determined the induction of OPN, and then investigated the effect of secreted forms of OPN in epithelial to mesenchymal transition (EMT), migration and invasion of hepatocytes. We show the induction of OPN mRNA and protein expression by HCV-infection. Our results also demonstrate the processing of precursor OPN (75 kDa) into 55 kDa, 42 kDa and 36 kDa forms of OPN in HCV-infected cells. Furthermore, we show the binding of secreted OPN to integrin αVβ3 and CD44 at the cell surface, leading to the activation of downstream cellular kinases such as focal adhesion kinase (FAK), Src, and Akt. Importantly, our results show the reduced expression of epithelial marker (E-cadherin) and induction of mesenchymal marker (N-cadherin) in HCV-infected cells. We also show the migration and invasion of HCV-infected cells using wound healing assay and matrigel coated Boyden chamber. In addition, we demonstrate the activation of above EMT markers, and the critical players involved in OPN-mediated cell signaling cascade using primary human hepatocytes infected with Japanese fulminant hepatitis (JFH)-1 HCV. Taken together, these studies suggest a potential role of OPN in inducing chronic liver disease and HCC associated with chronic HCV infection.</p></div

HCV induces EMT via OPN.

<p>HCV-infected cells were transfected with siOPN and siGFP as described in Materials and Methods. At 72 h posttransfection, cells were harvested and equal amounts of cellular lysates were immunoblotted with anti-E-cadherin (A), anti-N-cadherin (B), and anti-OPN (A, B). Actin was used as protein loading control.</p

Role of HCV-induced OPN in cell signaling cascade.

<p>(A) HCV-infected cells were transfected with siGFP, siOPN, siCD44, and siβ3 as described in Materials and Methods. At 72 h posttransfection, cells were harvested and equal amount of cellular lysates were subjected to western blot analysis using anti-OPN, anti-β3 and anti-CD44. (B) The above cellular lysates were subjected to western blot analysis using anti-FAK, anti-p-Akt (Ser ⁴⁷³), anti-p-Src (Tyr ⁴¹⁶), and anti-N-cadherin antibodies. HCV NS3 was used as a representative of HCV-infection. (C) Mock (Huh7.5) cells were treated with recombinant human OPN (rhOPN) (50 nM) for 48 h. Cells were harvested and equal amounts of cellular lysates were immunoblotted using anti-FAK and anti-p-Akt (Ser⁴⁷³) (lane 1, 2), anti-E-cadherin, anti-N-cadherin, anti-pSrc ⁴¹⁶ (lane 3, 4). (D) Cellular lysates from HepG2 cells incubated with cell culture supernatants from mock, HCV-infected cells and those infected cells transfected with siOPN and siGFP were immunoblotted using anti-E-cadherin and anti-N-cadherin (lane 1–4). Similarly cellular lysates from HepG2 cells incubated with cell culture supernatants from mock and HCV-infected cells with/without immunodepletion by anti-OPN, were immunoblotted using anti-E-cadherin and anti-N-cadherin (lane 5–7). Immunodepletion by isotype control goat IgG antibody was used as control (lane 8). Actin was used as protein loading control. The results shown are the representative of three independent experiments.</p

HCV promotes invasion of hepatoma cells.

<p>(A) HCV-infected cells (from the same pool of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087464#pone-0087464-g002" target="_blank">figure 2A</a>) were transfected with siGFP and siOPN. At 24 h posttransfection, approximately 3×10⁵ cells were seeded in transwell chamber for 48 h and images of invaded cells were recorded under microscope at least three individual fields per well at 10× magnification. (B) The invaded cells were counted in at least three individual fields per insert and represented by bar diagrams. The results shown are representative of two independent experiments performed in duplicate. (C) The above invaded cells were quantified in 96 wells plate at OD 560 nm using extraction buffer. Data represent means ± SD of two independent experiments performed in duplicate. *denotes p<0.05 compared to mock-infected Huh7.5 cells. **denotes p<0.05 compared to HCV-infected cells transfected with siGFP.</p

Model illustrating the OPN mediated signaling cascade in HCV-infected hepatocytes.

<p>HCV induces OPN expression, which is cleaved by unknown mechanism in response to HCV-infection and the active forms are secreted out from the cells. The secreted form of OPN binds to cell surface receptors on the same or other cells via integrin αVβ3 and CD44. This interaction leads to EMT, cell migration and invasion through the activation/phosphorylation of FAK, Akt and Src mediated signaling pathways.</p

Colocalization of OPN with integrin αVβ3 and CD44.

<p>(A, B) Mock and HCV-infected cells (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087464#pone-0087464-g002" target="_blank">figure 2A</a>) were transfected with siGFP and siOPN. At 72 h posttransfection, cells were permeabilized and incubated with anti-OPN, anti-αVβ3, anti-CD44 and anti-HCV NS5A antibodies for 1 h at RT, followed by incubation with secondary antibodies; for OPN (anti-goat Alexa Fluor 546), αVβ3 (anti-mouse Alexa Fluor 488), CD44 (anti-mouse Alexa Fluor 488) and HCV NS5A (anti-rabbit Alexa Fluor 633). DAPI was used as a nuclear stain. Arrows represent colocalization of OPN with αVβ3 and CD44 respectively. HCV NS5A represents HCV infection. Scale bar 10 µM. (C) Colocalization of OPN with pan-cadherin (plasma membrane marker). Mock and HCV-infected cells (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087464#pone-0087464-g002" target="_blank">figure 2A</a>) were permeabilized and incubated with anti-OPN, anti-pan-cadherin and anti-HCV NS5A antibodies for 1 h at RT, followed by incubation with secondary antibodies; for OPN (anti-goat Alexa Fluor 546), pan-cadherin (anti-rabbit Alexa Fluor 488) and HCV NS5A (anti-rabbit Alexa Fluor 633). (D) Similarly, non-permeabilized mock and HCV-infected cells were incubated with anti-OPN and anti-pan-cadherin antibodies for 1 h at RT and then cells were permeabilized and incubated with anti-HCV NS5A antibody for 1 h at RT followed by 1 h incubation with above secondary antibodies. DAPI was used as a nuclear stain. Arrows represent colocalization of OPN with pan-cadherin. (E) Colocalization of OPN with PDI (ER marker). As described in panel C and D, permeabilized cells were incubated with anti-OPN, anti-PDI and anti-HCV NS5A antibodies for 1 h at RT, followed by incubation with secondary antibodies; for OPN (anti-goat Alexa Fluor 546), PDI (anti-rabbit Alexa Fluor 488) and HCV NS5A (anti-rabbit Alexa Fluor 633). (F) Simultaneously, non-permeabilized cells were incubated with anti-OPN and anti-PDI antibodies for 1 h at RT and then cells were permeabilized and incubated with anti-HCV NS5A antibody for 1 h at RT followed by 1 h incubation with above secondary antibodies. DAPI was used as a nuclear stain. Arrows represent colocalization of OPN with ER marker. HCV NS5A represents HCV infection. Scale bar 10 µM.</p

HCV induces hepatoma cells migration.

<p>(A) HCV-infected cells (from the same pool of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087464#pone-0087464-g002" target="_blank">figure 2A</a>) were transfected with siGFP and siOPN and migration was examined by wound healing assay. Images were taken at 0 h and 48 h postwounding. Arrows indicate the wound of monolayer cells scratched using pipette tips. The results shown are representative of three independent experiments. (B) The percent migrated depth of above cells was measured in three independent experiments represented by bar diagram. *denotes p<0.05 compared to mock-infected Huh7.5 cells. **denotes p<0.05 compared to HCV-infected cells transfected with siGFP.</p

Effect of HCV-induced OPN on αVβ3 and CD44 expression.

<p>(A) Huh7.5 cells were incubated with HCV (m.o.i. of 1). At day 3 postinfection, cells were immunostained using anti-NS5A antibody as described in Materials and Methods. (B) Silencing of OPN mRNA expression. The above HCV-infected cells (panel A) were transfected with siGFP and siOPN using lipofectamine 2000 as per manufacturer’s instruction (Invitrogen). At 72 h posttransfection, cellular RNA was extracted and expression of OPN mRNA was quantified by QRT-PCR. OPN mRNA expression was normalized by 18S rRNA. Data represent means+standard deviations of two independent experiments performed in duplicate. *denotes p<0.05 compared to mock-infected Huh7.5 cells. **denotes p<0.05 compared to HCV-infected Huh7.5 cells transfected with siGFP. (C, D) Cellular lysates from the above siRNA transfected cells (panel B) were immunoblotted using anti-β3, anti-β6 and anti-CD44 antibodies. Actin was used as protein loading controls.</p