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.

rSjP40 Inhibited the Activity of Collagen Type I Promoter via Ets-1 in HSCs

“…However, rSjP40 could not affect the promoter activities in cells transfected with these truncated plasmids (Figure 2B). Compared the sequences of pGL3-COL1A1 and pGL3-COL1A1a (Chen et al, 2019a), we confirmed that rSjP40 down-regulated the promoter activity of COL1A1 at the core region of -1,722/-1,592. Ets-1 May Bind to COL1A1 Promoter at the Region of -1,679/-1,673…”

“…We used bioinformatics to analyze COL1A1 promoter sequences and predict possible binding sites for common transcription factors. Truncated mutation of transcription factor binding site was carried out and five luciferase reporter gene plasmids were constructed for experimental study ( Chen et al, 2019a ): pGL3-COL1A1a (-1,592/+21), pGL3-COL1A1b (-1,167/+21), pGL3-COL1A1c (-443/+21), pGL3-COL1A1d (-239/+21), pGL3-COL1A1e (-215/+21) ( Figure 2A ). Then the plasmids containing the truncated sequences of COL1A1 promoter ( Chen et al, 2019a ) were transfected into LX-2 cells, respectively.…”

“…Truncated mutation of transcription factor binding site was carried out and five luciferase reporter gene plasmids were constructed for experimental study ( Chen et al, 2019a ): pGL3-COL1A1a (-1,592/+21), pGL3-COL1A1b (-1,167/+21), pGL3-COL1A1c (-443/+21), pGL3-COL1A1d (-239/+21), pGL3-COL1A1e (-215/+21) ( Figure 2A ). Then the plasmids containing the truncated sequences of COL1A1 promoter ( Chen et al, 2019a ) were transfected into LX-2 cells, respectively. We found that rSjP40 could only inhibit the promoter activity in cells transfected with pGL3-COL1A1 ( Figure 2B ).…”

“…Mouse mAbs against COL1A1 (Abcam, United Kingdom), rabbit mAbs against GAPDH (Goodhere, China) or Ets-1 (Proteintech, United Kingdom), horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Santa Cruz, United States) and HRP-conjugated anti-rabbit IgG (Biosharp, China) were purchased from the indicated companies. Plasmids containing COL1A1 promoter sequences constructed as previously described ( Chen et al, 2019a ) were preserved in our lab.…”

Crosstalk network among multiple inflammatory mediators in liver fibrosis

“…Activated HSCs produce endothelin-1 to promote fibrogenesis[3]. A homologous protein of YB1 (a negative mediator for liver fibrosis) mediated anti-fibrotic activity by suppressing the expression of collagen type I in HSCs[4]. Moreover, Wnt signaling can also enhance HSC activation and promote liver fibrosis[5].…”