The S protein of hepatitis B virus promotes collagen type I expression in hepatic stellate cells by virtue of hepatocytes

Abstract

This study was conducted in order to investigate whether hepatitis B surface S protein (HBs) was able to directly or indirectly promote the proliferation and expression of collagen type I (Col I) and α-smooth muscle actin (α-SMA) in hepatic stellate cells (HSCs). The LX-2 human cell line and the HepG2 human hepatocellular carcinoma cell line were employed as HSCs and as hepatocytes, respectively. Recombinant HBs was added to the LX-2 cells for 48 h and the cell proliferation was assessed by the MTT assay. Col I and α-SMA were measured in the supernatant by ELISA, following treatment of the LX-2 and/or HepG2 cells with recombinant HBs. Transforming growth factor-β1 (TGF-β1) was also determined by ELISA in the HepG2 cell supernatants. The data demonstrated that high concentrations of recombinant HBs (10-50 ng/ml) inhibited the proliferation of LX-2 cells, whereas low concentrations (0.5-5 ng/ml) did not affect LX-2 cell proliferation. After treating LX-2 cells alone with recombinant HBs for 48 h, there was no significant increase in the Col I and α-SMA levels. However, Col I was increased ~1.7-fold in co-cultured (LX-2 and HepG2) cell supernatants following treatment with HBs for 24 h (HBs vs. control group: 48.51±3.51 vs. 28.23±2.55 ng/ml, respectively). Furthermore, TGF-β1 was significantly increased in the HepG2 cell supernatants following treatment with recombinant HBs. Therefore, we concluded that HBs directly affected the proliferation of HSCs, but promoted the Col I expression in HSCs possibly by virtue of hepatocytes secreting TGF-β1. This may provide a novel explanation of the fibrogenetic mechanism induced by hepatitis B virus-related proteins.

Keywords: LX-2 cell line; S protein; fibrosis; hepatic stellate cells; hepatitis B virus.

Figure 1

Effects of hepatitis B surface S protein (HBs) on LX-2 cell proliferation. The LX-2 cells were incubated with various concentrations of HBs. High concentrations of HBs (10–50 ng/ml) inhibited the proliferation of LX-2 cells, whereas low concentrations (0.5–5 ng/ml) did not affect LX-2 cell proliferation. No significant difference was observed in LX-2 cell survival, although the optical density (OD) value was increased, when HBs concentration reached a plateau of 1–5 ng/ml. ^*P<0.05. Error bars, ±2.00 SE.

Figure 2

Effect of hepatitis B surface S protein (HBs) on secretion of α-smooth muscle actin (α-SMA) and collagen type I (Col I) in LX-2 cells and the co-culture system of HepG2 and LX-2 cells. To detect the effect of HBs on the expression of Col I and α-SMA in LX-2 cells, the cells (2×10³ cells/well) were cultured for 24 h, then incubated with HBs (10 ng/ml) for 48 h. The change in Col I and α-SMA levels was no different between the HBs treatment and the control groups (data not shown): Col I, 28.61±3.25 vs. 26.30±3.69 ng/ml, t=0.47, P=0.648 and α-SMA, 25.08±5.33 vs. 24.48±2.62 ng/ml, t=0.101, P=0.962, respectively. HepG2 (2×10³ cells/well) and LX-2 cells (2×10³ cells/well) were co-cultured for 24 h, then incubated with HBs (10 ng/ml) for 24 h. Col I was found to be significantly increased following HBs treatment (48.51±3.51 vs. 28.23±2.55 ng/ml, t=4.674, P=0.001), whereas there was no obvious change in α-SMA (data not shown; 30.66±2.69 vs. 23.42±3.86 ng/ml, t=1.538, P=0.155). The HepG2 cells (2×10³ cells/well) were cultured alone with HBs for 24 h to eliminate the secretion of Col I and α-SMA. The results demonstrated that Col I was increased in HepG2 cell supernatants (37.63±3.43 vs. 18.49±3.58 ng/ml, t=3.856, P=0.003), although α-SMA was not (20.70±2.38 vs. 18.46±1.48 ng/ml, t=0.799, P=0.443). However, Col I was significantly lower in the supernatant of HepG2 cells stimulated by HBs than in the supernatant of the co-culture system (37.63±3.43 vs. 48.51±3.51 ng/ml, t=3.132, P=0.03), whereas the data did not reveal similar results for α-SMA. Error bars, ±2.00 SE.

Figure 3

Effect of hepatitis B surface S protein (HBs) on the secretion of transforming growth factor-β1 (TGF-β1) in HepG2 cells. To elucidate the mechanism underlying the effect of HepG2 on LX-2 cells, the HepG2 cells (2×10³ cells/well) were cultured for 24 h, then incubated with HBs (10 ng/ml) for 24 h. The results demonstrated that the TGF-β1 levels were higher in the HBs treatment group compared to those in the control group (9.80±1.89 vs. 6.49±1.41 ng/ml, t=3.635, P=0.005). ^*P<0.05. Error bars, ±2.00 SE.