Bile acids promote HCV replication through the EGFR/ERK pathway in replicon-harboring cells

Abstract

Objectives: Bile acids promoted the replication of hepatitis C virus (HCV) and compromised the anti-HCV effects of interferon-α (IFN-α) in replicon-harboring cells. To explore a potential mechanism for the observation, we studied the effects of bile acids on the epidermal growth factor receptor (EGFR) and the extracellular signal-regulated kinase (ERK) pathway in association with HCV replication in genotype 1a or 1b replicon-harboring cells.

Methods: Replicon-harboring cells were treated with various bile acids, IFN-α and small molecule inhibitors either individually or combined together. The effects of these treatments were measured using cell cycle analysis, qRT-PCR, and Western blot analysis.

Results: Bile acids induced the activation of EGFR/ERK pathway and extended S-phase of cells, which was correlated with the increased levels of viral replication. The inhibitors of EGFR (AG1478) or ERK (U0126) significantly mitigated the bile acid-mediated promotion of HCV replication. When AG1478 or U0126 were added to the treatment of bile acids and IFN-α, they were able to restore the anti-HCV effects of IFN-α.

Conclusion: Our data suggest that the addition of an EGFR or ERK inhibitor to the current IFN-α-based regimen may improve overall treatment efficacy by blocking the bile acid-mediated promotion of HCV replication.

Fig. 1

Enhancement of HCV replication after bile acid treatment in 1A7 cells. Semiconfluent cells were treated with mock medium, CDCA, GCDCA, or UDCA for 24 or 48 h. HCV RNA (a) or NS5b (b) was measured by real-time qRT-PCR or Western blot analysis, respectively. a qRT-PCR levels after treatment with mock medium, CDCA 100 μM (CDCA100), CDCA 20 μM (CDCA20), GCDCA 200 μM (GCDCA 200), GCDCA 100 μM (GCDCA100) and UDCA 200 μM (UDCA200) for 24 or 48 h. Asterisk (∗) indicates that the RNA levels by the treatment were significantly increased compared to those by control (mock medium) treatment (p < 0.05). b Western blot analysis detecting NS5b after the treatment for 24 h. Upper panel, lane 1: mock medium; lane 2: CDCA (100 μM); lane 3: CDCA (20 μM); lane 4: GCDCA (200 μM); lane 5: GCDCA (100 μM). Lower panel, as a loading control, Western blot analysis of β-actin was performed with the same samples. c Flow cytometry analysis of NS5b levels in 1A7 cells with the treatment of mock medium or CDCA (100 μM) for 24 h. Staining control was prepared using the same procedure without the incubation with the NS5b antibody.

Fig. 2

Enhancement of luciferase activity under AP-1 or SRE promoter control after bile acid treatment in GS4.1 cells. The luciferase-based plasmid under AP-1 or SRE promoter control was transfected into semiconfluent GS4.1 cells. The cells were treated with CDCA 100 μM (CDCA-100), DCA 100 μM (DCA100), GCDCA 200 μM (GCDCA200), UDCA 200 μM (UDCA200), or CDCA100 + U0126 20 μM (U20) 4 h after transfection and incubated for an additional 24 h. Asterisk (∗) indicates that the luciferase activities by the treatment were significantly increased compared to those by the control treatment (p < 0.05). a Treatment of various bile acids in the luciferase activity under the control of signaling pathways, AP-1 or SRE. b The effects of U0126 on CDCA-mediated induction of luciferase expression under AP-1 or SRE. Asterisk (∗) indicates that the luciferase activities by co-treatment with CDCA and U0126 were significantly reduced compared to those by the treatment with CDCA alone (p < 0.05).

Fig. 3

The activation of EFGR by the treatment with bile acids in GS4.1 cells. Confluent GS4.1 cells were treated with mock medium, CDCA, GCDCA, or UDCA for 30 min, and then cell lysates were prepared and Western blot analysis detecting phosphor-EGFR or EGFR. Lane 1: parental Huh-7 cells with mock treatment; lane 2: mock treatment; lane 3: CDCA 100 μM; lane 4: GCDCA 200 μM; lane 5: UDCA 200 μM.

Fig. 4

Inhibitors of EGFR or ERK in bile acid-mediated promotion of HCV replication. Semiconfluent GS4.1 or 1A7 cells were incubated with mock medium, CDCA, AG1478 or CDCA + AG1478 for 24 h, and HCV replication was measured by detecting HCV RNA (a, b) or protein (c). a Real-time qRT-PCR analysis of HCV RNA levels after the treatment with CDCA 100 μM (CDCA100), AG1478 30 μM (AG30), CDCA100 + AG30, or CDCA100 + AG1478 15 μM (AG15). b Real-time qRTPCR analysis of HCV RNA levels after the treatment with CDCA100, U0126 20 μM (U20), CDCA100 + U20, or CDCA100 + U0126 10 μM (U10). Asterisk (∗) indicates that the RNA levels by double treatment with CDCA and AG1478 or U0126 were significantly reduced compared to those by the treatment with CDCA alone (p < 0.05). c Western blot analysis of HCV protein NS5B in GS4.1 cells after treatment with CDCA, AG1478 or CDCA + AG1478. Lane 1: Mock; lane 2: CDCA 100 μM; lane 3: AG1478 30 μM + CDCA 100 μM; lane 4: AG1478 5 μM + CDCA 100 μM, and lane 5: AG1478 30 μM. As a loading control, Western blot analysis of β-actin was performed with the same samples.

Fig. 5

Inhibitors of EGFR or ERK in anti-HCV action of IFN in the presence of CDCA. HCV RNA levels were measured after 24 h treatments with IFN 5 U/ml (IFN5), CDCA 100 μM (CDCA100) + IFN5, or CDCA100 + IFN5 + (AG1478 30 μM [AG30] or U0126 20 μM [U20]). Asterisk (∗) indicates that the RNA levels by IFN 5 U/ml treatment were significantly reduced compared to those by mock treatment (p < 0.05). Double asterisk (∗∗) indicates that the RNA levels by triple treatment were significantly reduced compared to those by double treatments of IFN5 + CDCA100 (p < 0.05).