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.


| INTRODUC TI ON
inhibiting transforming growth factor-β (TGF-β) signalling, a major pathway to initiate HSC activation. 2 Overexpression of YB1 can partially rescue tissue in a CCl 4 -induced liver damage model 3 and this beneficial effect is linked with the repressive effect of YB1 on collagen type I gene transcription. 4 Furthermore, it is reported that a novel small compound Hsc025 ameliorates CCl 4 -induced liver fibrosis in mice by promoting nuclear translocation of YB1 and subsequently inhibiting TGF-β-stimulated collagen gene expression. 5 In this study, we expressed and purified a YB1 homologue, the recombinant SJYB1 protein (rSJYB1) from Schistosoma japonicum (S japonicum) in a bacterial system. Results from this study demonstrated that rSJYB1 inhibits collagen type I protein expression in HSCs via down-regulating activity of collagen α1 (I) (COL1A1) promoter.

| Amplification of SJYB1
Based on the nucleotide sequence of SJYB1 (NCBI Accssion No. AF367371), two primers were designed to amplify SJYB1 from S japonicum egg cDNA (Table 1). PCR parameters were as follows: denaturation at 98°C for 10 seconds, primer annealing at 58°C for 5 seconds, primer extension at 72°C for 1 minute, cycled for 30 cycles, with a final extension at 72°C for 10 minutes before storing the sample at 4°C. The PCR product was sequenced and the sequence was characterized using nucleotide basic local alignment search tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and named SJYB1.

| Construction of SJYB1 prokaryotic expression vector
PCR products of SJYB1 were digested with BamHI and HindIII enzymes and ligated to BamHI-and HindIII-digested expression vector pET28a (+). Insert DNA was sequenced to ensure authenticity of the clone nucleotide sequence.

| Expression and purification of rSJYB1
The empty pET28a (+) vector or the recombinant construct of SJYB1

Primer
Sequence (5′→3′) Purpose TA B L E 1 Primers used in this study

| Western blot analysis
Cells were lysed in radio-immunoprecipitation assay buffer with 1%

| Construction of plasmids containing COL1A1 promoter sequence
Genomic DNA was extracted from LX-2 cells according to instruc- promoter-associated truncated plasmids, the indicated fragments were amplified by PCR from pGL3-COL1A1 and PCR primers were designed as shown in Table 1. The PCR products were also digested with SacI and XhoI and then subcloned into pGL3-basic vector.

| Transfection and dual-luciferase reporter assay
LX-2 cells were cotransfected with the indicated plasmids of sults showed that rSJYB1 was recognized strongly by S japonicuminfected rabbit sera ( Figure 1B). In contrast, sera from uninfected rabbits could not recognize rSJYB1 ( Figure 1C) and neither S japonicum-infected rabbit sera nor uninfected rabbit sera could recognize protein sample from empty pET28a (+) vector ( Figure 1B,C). These results indicated that native SJYB1 induces circulating antibodies in rabbits during S japonicum infection.

| rSJYB1 inhibits collagen type I expression in LX-2 cells
To determine the effect of rSJYB1 on liver fibrosis in vitro, we used

| rSJYB1 inhibits COL1A1 promoter activity in LX-2 cells
Next, to examine whether the effect of rSJYB1 on collagen type I expression takes place at the transcriptional level, we constructed a luciferase reporter plasmid of COL1A1 promoter, pGL3-COL1A1.
In our reporter assays, pGL3-COL1A1 had markedly higher activity compared to pGL3-basic in LX-2 cells (Figure 3). However, its activity was decreased after treatment with rSJYB1 for 72 hours (Figure 3).
These results showed that rSJYB1 has a crucial role in the regulation of COL1A1 promoter activity.

| rSJYB1 functions at −1592/−1176 region of COL1A1 promoter to inhibit its activity
In order to narrow down the activity region, we established five luciferase reporter plasmids of truncated fragments of COL1A1 promoter, named pGL3-COL1A1a, pGL3-COL1A1b, pGL3-COL1A1c, pGL3-COL1A1d and pGL3-COL1A1e ( Figure 4A). The activities of these truncated fragments of COL1A1 promoter were shown in Figure 4B. Next, these truncated fragments of COL1A1 promoter as well as pGL3-COL1A1 were transfected into LX-2 cells, respectively, and then, transfected cells were treated with rSJYB1 for 72 hours. Results from reporter assays indicated that COL1A1 promoter activity was apparently decreased in rSJYB1-  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

| D ISCUSS I ON
The YB1 proteins were first recognized as transcriptional factors binding to the Y-box element. However, it has since become apparent that the YB1 proteins are multifunctional and participate in translational regulation, DNA repair and pre-mRNA splicing 6 Extensive studies have shown that the YB1 proteins are related to proliferation and transformation of eukaryotic cells, 7,8 being also correlated with tumour aggressiveness and as a promising molecular target in the therapy of cancer and inflammation. 9   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 SJYB1 inhibited the expression of collagen type I, but not α-SMA in LX-2 cells, suggesting that rSJYB1 specifically regulates synthesis of collagen type I rather than HSC activation. Unexpectedly, another study on mice infected with S mansoni has revealed that infected mice immunized with rSMYB1 exhibited reductions in the number of adult worm and eggs/granuloma retained in the liver, but no significant decrease was detected in the area of fibrosis. 13 It is possible that the roles of YB1 from schistosomes in vitro are not entirely consistent with that in vivo. In addition, whether the minor differences in protein sequences lead to the function difference between SJYB1 and SMYB1 has not been determined. Furthermore, it is demonstrated that SMYB1 could not bind to promoters of the S mansoni genes in vivo indicating that SMYB1 may not act directly as a transcription factor. 11 Therefore, it is possible that rSJYB1 regulates COL1A1 promoter activity indirectly, which may be via relevant transcription factors or signalling pathways, just as P40 regulates p27 promoter activity via transcription factor E2F1. 24 Further studies will be performed to elucidate the molecular mechanism.
In conclusion, our data suggest that rSJYB1-mediated anti-fibrotic activity involves inhibiting the activity of COL1A1 promoter and subsequently suppressing the expression of collagen type I in HSCs.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict interests.