rSjp40 inhibits activated hepatic stellate cells by promoting nuclear translocation of YB1 and inducing BMP-7/Smad1/5/8 pathway

Background Activation of hepatic stellate cells is the dominant pathogenic event during the process of liver fibrosis. Bone morphogenic protein (BMP)-7 has recently been identified as an anti-fibrotic factor and leads to phosphorylation of Smad1/5/8 in activated hepatic stellate cells. Its expression can be upregulated by the transcriptional activator, Y-Box protein-1 (YB1). Previous studies have found that the recombinant Schistosoma japonicum protein p40 (rSjp40) can inhibit the activation of hepatic stellate cells, and based on this evidence we attempted to investigate whether or not BMP-7 is involved in rSjp40’s inhibition. Methods A human hepatic stellate cell line, the LX-2 cell line, was cultured and treated with rSjp40. The role of BMP-7 was analyzed by Western blot. Results Our findings testified that knockdown of BMP-7 impaired rSjp40-induced downregulation of α-SMA and phosphorylation of Smad1/5/8 in LX-2 cells. Furthermore, rSjp40 upregulated expression of BMP-7 at both mRNA and protein levels depending on YB1. Interestingly, YB1 was translocated from the cytoplasm to the nucleus upon treatment of rSjp40. Conclusions These results suggest that rSjp40 inhibits the activation of hepatic stellate cells by promoting nuclear translocation of YB1 and inducing BMP-7/Smad1/5/8 pathway, which provide a new clue to guide ongoing research into the anti-fibrosis of rSjp40.


Background
Liver fibrosis is a pathological process due to most chronic liver injuries like viral hepatitis, alcohol abuse, metabolic disease, and parasite infection. Both quantitative and qualitative changes in the composition of liver extracellular matrix (ECM) occur during the development of liver fibrosis. The total ECM increases 3-5-fold, accompanied by the shift in the type of ECM from the normal low density basement membrane-like matrix to interstitial type matrix containing types I and III collagens [1]. Accordingly, activated hepatic stellate cells (HSCs), characterized by the appearance of α-smooth muscle actin (α-SMA), are the main ECM-producing cells [2]. Of note, HSC activation is particularly promoted by profibrogenic cytokines, with transforming growth factor-β1 (TGF-β1) being the most potent [3]. The signaling of TGF-β1 leads to the phosphorylation of receptor-specific Smad (R-Smad) proteins, Smad2 and Smad3. Then R-Smads together with a common mediator Smad4 translocate to the nucleus, where they enhance transcription of collagen genes, inhibit synthesis of ECM degradation enzymes and upregulate expression of tissue inhibitors of metalloproteinases to promote liver fibrosis [4].

Open Access
Parasites & Vectors *Correspondence: yinongduan@aliyun.com † Liuting Chen, Qi Zhou and Ertao Liu contributed equally to this work 1 Department of Pathogen Biology, School of Medicine, Nantong University, Nantong 226001, Jiangsu, People's Republic of China Full list of author information is available at the end of the article Bone morphogenic protein (BMP)-7, a member of the TGF-β superfamily, has been reported to counteract some of the profibrogenic actions of TGF-β1 [5]. It initiates the signal transduction cascade by allowing phosphorylation of Smad1/5/8. Smad1/5/8 forms a complex via binding to Smad4, thus, being translocated to the nucleus to suppress the accumulation of Smad3 by TGF-β1 stimulation [6,7]. In liver fibrosis, the involvement of BMP-7 has only recently been suggested. Oral administration of recombinant adeno-associated virus carrying BMP-7 in mice led to an increased circulating BMP-7 concentration and resulted in amelioration of CCl 4 -induced liver fibrosis and HSC activation [8]. Injection of adenoviral BMP-7 promoted the reversion of liver fibrosis/cirrhosis in the CCl 4 -induced cirrhotic hamster model [9]. And in other animal models like thioacetamide-or repeated intraperitoneal injection of porcine serum-induced liver fibrosis in rats, ectopic expression of BMP-7 can also inhibit the fibrogenic progress [10,11]. Furthermore, in the Schistosoma japonicum-induced liver fibrosis mouse model, administration of recombinant BMP-7 intraperitoneally significantly decreased the degree of collagen deposition and the expression of α-SMA in the liver, measured by Masson's staining and α-SMA staining [12].
Y-Box protein-1 (YB1) is a DNA-and RNA-binding protein and identified as a transcriptional activator of BMP-7, which binds to the proximal region of BMP-7 promoter (−192 to +3 bp). It is reported that knockdown or knockout of YB1 can reduce BMP-7 expression. In the high glucose condition, YB1 was translocated to the nucleus to increase the BMP-7 promoter activity [13,14].
Schistosomiasis is an infectious disease that affected 207.7 million people worldwide in 2016, according to the latest Global Health Observatory (GHO) data. Most evidence suggests that schistosome eggs induce the morbidity caused by schistosome infection [15]. However, studies in recent years have found that schistosome eggs can restrain the activation of HSCs and induce the downregulation of fibrogenesis [16,17]. In our laboratory, we also found that soluble egg antigens (SEA) from Schistosoma japonicum can induce the suppression of activated human stellate cell line, LX-2 cells and primary mouse HSCs through the TGF-β1 and PPARγ signaling pathways [18]. SEA is a complex mixture which is isolated from schistosome eggs. And Sjp40 is a major component of SEA from Schistosoma japonicum [19]. In previous research, we have expressed and purified the recombinant Sjp40 protein (rSjp40), and the preceding studies have demonstrated that rSjp40 potently inhibits the activation of HSCs to exert its anti-fibrotic effect [20][21][22].
In this study, we identified that BMP-7 is involved in rSjp40's inhibition on HSC activation. Knockdown of BMP-7 inhibited rSjp40-induced phosphorylation of Smad1/5/8 and downregulation of α-SMA. Mechanistically, YB1 was translocated to the nucleus and promoted BMP-7 expression after rSjp40 treatment. Our findings reveal a previously unidentified target and provide insight into the mechanisms of rSjp40's inhibition on HSC activation.
After blocked in 5% nonfat milk, the membranes were incubated with the indicated primary and secondary antibodies. Protein bands were visualized with ECL system (Biorad, Berkeley, CA, USA).

Isolation of cytoplasmic and nuclear fraction
LX-2 cells left untreated or treated with rSjp40 (5 μg/ml) for 48 h were harvested and separated using the Nuclear and Cytoplasmic Protein Extraction kit (ApplyGen, Beijing, China) according to the manufacturer's instructions.

Immunofluorescence analysis
LX-2 cells were seeded on 24-well plates and left untreated or treated with rSjp40 (5 μg/ml) on the following day. Forty-eight hours after treatment, cells were fixed by 4% paraformaldehyde and permeabilized by 0.1% Triton X-100. Then cells were incubated with the primary Ab against YB1 at 4 °C overnight, followed by Alex-aFluor 594 secondary Ab (Molecular Probes, Waltham, MA, USA) for 2 h at room temperature. The cells were also stained with Hoechst 33342 (Sigma-Aldrich, Saint Louis, MO, USA) for 15 min and observed by fluorescent microscopy.

Statistical analysis
All experiments were analyzed by the Student's t-test. A P-value < 0.05 was considered significant.

rSjp40 promotes the nuclear translocation of YB1 in LX-2 cells
We next investigated the mechanism of rSjp40 in the regulation of YB1. Since rSjp40 had little effect on total level of YB1 expression (Fig. 3b), we wondered whether rSjp40 is involved in YB1 nuclear translocation. It has been reported that YB1 regulated gene transcription in the nucleus only [14], therefore, nuclear translocation of YB1 is essential for it to exert its function. Cell fractionation experiments indicated that a major fraction of YB1 was located in the cytoplasm and only trace amount of YB1 was detected in the nucleus in untreated cells (Fig. 4a). Interestingly, upon rSjp40 treatment, the nuclear level of YB1 was increased (t-test: t (4) = 4.553, P = 0.0052), whereas cytoplasmic level of YB1 was decreased (t-test: t (4) = 6.503, P = 0.0014), with total level of YB1 unchanged (Fig. 4a). Fluorescent microscopy further confirmed that YB1 was translocated from the cytoplasm to the nucleus after rSjp40 treatment (Fig. 4b). These results suggest that rSjp40 promotes the nuclear translocation of YB1 in LX-2 cells.

Discussion
In this study, we found that rSjp40 increased the phosphorylation level of Smad1/5/8 and decreased the expression of α-SMA in LX-2 cells. Interestingly, knockdown of BMP-7 inhibited rSjP40-triggered phosphorylation of Smad1/5/8 and downregulation of α-SMA. Based on the evidence that the anti-fibrotic actions of BMP-7 are due to its ability to counteract TGF-β1 signaling through increasing the phosphorylation level of Smad1/5/8 [7,10,25,26], we therefore concluded that rSjp40 inhibits activation of HSCs partially through BMP-7/Smad1/5/8 signaling pathway. However, our results showed that the phosphorylation level of Smad1/5/8 in LX-2 cells transfected with BMP-7-siRNA and treated with rSjP40 was even lower than the LX-2 cells transfected with BMP-7-siRNA only (t-test: t (4) = 3.276, P = 0.0153) (Fig. 2). It is possible that rSjp40 could directly inhibit the phosphorylation level of Smad1/5/8 via an unknown mechanism. YB1 can be shuttled between the cytoplasm and the nucleus. Many compounds such as oxymatrine and Hsc025 can induce the nuclear translocation of YB1 [27,28]. In contrast, TGF-β1 reduces the nuclear translocation of YB1 from the cytoplasm in Madin-Darby Canine Kidney (MDCK)-cells [14]. In this study, we found that the nuclear translocation of YB1 could also be induced by rSjP40 treatment. It is reported that p53 was required for the nuclear translocation of YB1, and the frequency of nuclear YB1 was dependent on the amount of p53 but not formation of p53/YB1 complex [29]. Our previous study showed that rSjp40 upregulated p53 expression Fig. 2 rSjp40 inhibits activation of HSCs via BMP-7/Smad1/5/8-dependent mechanism. LX-2 cells (2-3 × 10 5 ) were transfected with Con-siRNA or BMP-7-siRNA (300 pmol each). Forty-eight hours after transfection, cells were left untreated or treated with rSjp40 (5 μg/ml) for 48 hours before immunoblot analysis was performed with the indicated Abs. The bar graphs show the relative intensities of the bands, which were quantitated by densitometry using Image Lab, normalizing to GAPDH levels. Data are represented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 in LX-2 cells [23]. Therefore, it is possible that rSjp40induced nuclear translocation of YB1 is also dependent on p53.
According to previous reports, YB1 is also a negative regulator in liver fibrosis. One direct evidence is that overexpression of YB1 improved liver fibrosis induced by CCl 4 in mice [30]. Adding to its mechanism, some studies showed that YB1 inhibited TGF-β1 signaling via acting on different targets [31,32], while others reported that YB1 had the repressive effect on collagen type I gene transcription [33][34][35]. In our study, we showed that YB1 exerts anti-fibrotic action by increasing the level of BMP-7 expression in LX-2 cells.

Conclusions
In conclusion, these present findings demonstrated that BMP-7 is required for rSjp40's inhibition on HSC activation. Upon rSjp40 treatment, YB1 was translocated to the nucleus to promote BMP-7 expression, leading to phosphorylation of Smad1/5/8. Our studies provide a new Fig. 3 rSjp40-induced BMP-7 expression is dependent on YB1 in LX-2 cells. a LX-2 cells (2-3 × 10 5 ) were left untreated or treated with rSjp40 (5 μg/ml) for 48 hours, and then total RNA was extracted for qPCR analysis. Data are represented as the mean ± SD (n = 3). ****P < 0.0001. b LX-2 cells (2-3 × 10 5 ) were transfected with Con-siRNA or YB1-siRNA (300 pmol each). Forty-eight hours after transfection, cells were left untreated or treated with rSjp40 (5 μg/ml) for 48 hours before immunoblot analysis was performed with the indicated Abs. The bar graphs show the relative intensities of the bands, which were quantitated by densitometry using Image Lab, normalizing to GAPDH levels. Data are represented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01 clue to guide ongoing research into the anti-fibrosis of rSjp40.

Availability of data and materials
The data supporting the conclusions of this article are included within the article. Fig. 4 rSjp40 promotes the nuclear translocation of YB1 in LX-2 cells. a LX-2 cells (1-2 × 10 6 ) were left untreated or treated with rSjp40 (5 μg/ ml) for 48 hours. Then the cells fractionated, and the subcellular fractions, including cytoplasm and nucleus were equilibrated to equal volumes and analyzed by immunoblots with the indicated Abs. The bar graphs show the relative intensities of the bands, which were quantitated by densitometry using Image Lab, normalizing to GAPDH or LaminB1 levels. Data are represented as the mean ± SD (n = 3). **P < 0.01. b LX-2 cells (5-6 × 10 4 ) were left untreated or treated with rSjp40 (5 μg/ml) for 48 hours. Then cells were fixed with 4% paraformaldehyde, and immunostaining was performed before observed by fluorescent microscopy. Scale-bars: b, 20 μm