rSJYB1 inhibits collagen type I protein expression in hepatic stellate cells via down-regulating activity of collagen α1 (I) promoter

Abstract

YB1 is a negative regulator in liver fibrosis. We wondered whether SJYB1, a homologous protein of YB1 from Schistosoma japonicum, has an effect on liver fibrosis in vitro. Recombinant SJYB1 (rSJYB1) protein was expressed in a bacterial system and purified by Ni-NTA His·Bind Resin. A human hepatic stellate cell line, the LX-2 cell line, was cultured and treated with rSJYB1. The role of rSJYB1 on LX-2 cells was then analysed by Western blot and luciferase assay. We succeeded in expressing and purifying SJYB1 in a bacterial system and the purified rSJYB1 could be recognized by S japonicum-infected rabbit sera. Western bolt analysis showed that rSJYB1 inhibited the expression of collagen type I, but had little effect on α-smooth muscle actin (α-SMA). Further analysis revealed that rSJYB1 inhibited the activity of collagen α1 (I) (COL1A1) promoter and functioned at -1592/-1176 region of COL1A1 promoter. Our data demonstrate that rSJYB1-mediated anti-fibrotic activity involves inhibiting the activity of COL1A1 promoter and subsequently suppressing the expression of collagen type I in hepatic stellate cells.

Keywords: SJYB1; collagen type I; hepatic stellate cell; liver fibrosis.

Figure 1

Detection of anti‐SJYB1 antibody in Schistosoma japonicum‐infected rabbit serum. A, Western blot analysis of purified recombinant SJYB1 (rSJYB1) using an anti‐His antibody. Lane 1: molecular weight marker; Lane 2: purified rSJYB1; Lane 3: control protein from empty pET28a (+) vector. B, Western blot of rSJYB1 probed with S japonicum‐infected rabbit serum. Lane 1: molecular weight marker; Lane 2: control protein from empty pET28a (+) vector; Lane 3: purified rSJYB1. C, Western blot of rSJYB1 probed with normal rabbit serum. Lane 1: molecular weight marker; Lane 2: control protein from empty pET28a (+) vector; Lane 3: purified rSJYB1. The sera were used at 1:1000 dilution

Figure 2

Recombinant SJYB1 (rSJYB1) inhibits collagen type I expression in LX‐2 cells. A, LX‐2 cells (1 × 10⁶) were left unstimulated or stimulated with 2.5 μg/mL rSJYB1 for the indicated times, and then, immunoblot analysis was performed with the indicated Abs. B, LX‐2 cells (1 × 10⁶) were left unstimulated or stimulated with several doses of rSJYB1, and then, immunoblot analysis was performed with the indicated Abs. The histograms show the relative intensities of the bands, which were quantitated by densitometry using Image Lab and normalized to GAPDH levels. Graphs show mean ± SD, n = 3. **P < 0.01

Figure 3

Recombinant SJYB1 (rSJYB1) inhibits collagen α1 (I) (COL1A1) promoter activity in LX‐2 cells. LX‐2 cells (1 × 10⁵) were transfected with the empty pGL3‐basic vector or pGL3‐COL1A1 reporter plasmid (1 μg) and pRL‐TK reporter plasmid (0.02 μg). Twenty‐four hours after transfection, cells were left unstimulated or stimulated with rSJYB1 (2.5 μg/mL) for 72 h before luciferase assays were performed. The Graph shows mean ± SD, n = 3. *P < 0.05, ***P < 0.001

Figure 4

Recombinant SJYB1 (rSJYB1) functions at −1592/−1176 region of collagen α1 (I) (COL1A1) promoter to inhibit its activity. A, Diagram of the construction of COL1A1 promoter truncated fragments. B, LX‐2 cells (1 × 10⁵) were transfected with the empty pGL3‐basic vector, pGL3‐COL1A1, pGL3‐COL1A1a, pGL3‐COL1A1b, pGL3‐COL1A1c, pGL3‐COL1A1d or pGL3‐COL1A1e reporter plasmid (1 μg) and pRL‐TK reporter plasmid (0.02 μg). Twenty‐four hours after transfection, luciferase assays were performed. C, LX‐2 cells (1 × 10⁵) were transfected with the empty pGL3‐basic vector, pGL3‐COL1A1, pGL3‐COL1A1a, pGL3‐COL1A1b, pGL3‐COL1A1c, pGL3‐COL1A1d or pGL3‐COL1A1e reporter plasmid (1 μg) and pRL‐TK reporter plasmid (0.02 μg). Twenty‐four hours after transfection, cells were left unstimulated or stimulated with rSJYB1 (2.5 μg/mL) for 72 h before luciferase assays were performed. The graph shows mean ± SD, n = 3. **P < 0.01