Promotion of liver fibrosis by Y-box binding protein 1 via the attenuation of transforming growth factor-beta 3 transcription

Introduction

Liver fibrosis, a condition characterized by significant formation of scar tissue in the liver, is a critical stage in the progression of chronic liver injury to cirrhosis (1). It remains an important cause of death in developed countries and is increasing in incidence. Liver fibrosis may be caused by various liver injuries (2-4). It begins with hepatocyte necrosis and an accumulation of extracellular matrix (ECM), is followed by the degradation of the original hepatic lobular structure, which promotes scarring and pseudolobule formation, and finally the progression to cirrhosis. One study showed that terminating the fibrotic process can reverse progression and even cirrhosis (5). Therefore, it is of great significance to further explore and elucidate the cells and the molecular mechanisms involved in liver fibrosis.

Active hepatic stellate cells (HSCs) are the primary source of ECM during liver fibrosis (6). After their activation, HSCs are transformed from dormant vitamin A storage cells into myofibroblasts, a process characterized by the expression of markers such as alpha-smooth muscle actin (α-SMA) and ECM synthesis (7-9). The signals for HSC activation originate from a variety of cytokines, including transforming growth factor-beta 1 (TGF-β1) and platelet-derived growth factor (10,11). Additionally, HSC activation relies on changes in gene expression and epigenetic regulation (12).

Recent research has reported that all isoforms of the TGF family are increased in hepatitis C infection, which could inhibit viral propagation, but only TGF-β1 and TGF-β2, not TGF-β3, can induce liver fibrosis with a high expression of type I collagen alpha-1 (13). In pediatric liver transplantation patients, significantly elevated TGF-β3 and type I collagen levels were observed in children with fibrosis and no inflammation (14). In a rat model of liver fibrosis, TGF-β3 attenuated pathological damage and inhibited ECM production and deposition (15,16). Our previous chromatin immunoprecipitation (ChIP)-sequencing test results suggested that YB-1 was tightly bound to the TGF-β3 promoter (17).

YB-1 is a member of the cold-shock protein family and has been implicated in regulation of the cell cycle, cell differentiation, stress response, and the emergence of tumor drug resistance (18). YB-1 knockdown (KD) was shown to attenuate liver injury in a bile duct-ligated mouse model of liver fibrosis (19). In addition, novel small compounds targeting Smad3 can inhibit YB-1 nuclear transfer to antagonize TGF-β/SMAD3 pathway–mediated collagen gene transcription (19,20). A small azo phytotoxin molecule (SU056) has been found to effectively inhibit the growth and progression of tumors by inhibiting YB-1 (21). It has also been reported that targeting the YB-1 signaling axis can reverse tumor immune escape and multidrug resistance in hepatocellular carcinoma, which may provide a new therapeutic strategy for optimizing tumor-targeted therapy (22). There is abundant research concerning the role of YB-1 in the occurrence and development of liver diseases. It was reported that in a mouse model of hepatic fibrosis with bile duct ligation, liver lesions were observed after KD of YB-1 (19); surprisingly, the degree of liver injury was reduced, but the kidney injury caused by bile duct ligation was more severe than compared to their wild-type littermates (19). Another recent study has found that specific deletion of YB-1 in hepatic progenitor cells (HPC) inhibits the proliferation of HPC and that YB-1 negatively regulates the proliferation of HPC through p53 to alleviate liver fibrosis (23).

Xiong et al. confirmed the critical role of the TGF-β/SMAD signaling pathway in stimulating fibrosis gene transcription in fibroblasts and further noted that YB-1 and SMAD2 establish a positive feedback loop to promote liver fibrosis (24). However, the specific pathways through which YB-1 regulates collagen synthesis and HSC activation have not been fully elucidated.

We therefore designed this study to evaluate the correlation between YB-1 and TGF-β3 expression levels during liver fibrosis. We hypothesized that overexpression (OE) of YB-1 would result in HSC activation and increased liver fibrosis. Our results showed that the activation signals generated by YB-1 could be transmitted to adjacent HSCs, thereby amplifying the signal effect, and that TGF-β3 may be involved in YB-1-mediated activation of HSCs. Taken together, our results showed that YB-1 OE in HSCs attenuated TGF-β3 transcription and promoted liver fibrosis. We present the following article in accordance with the ARRIVE reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-23-835/rc).

Methods

HSC activation model of TGF-β1–induced the LX-2 cell line

The human HSC line LX-2 (Shanghai Cell Bank of the Chinese Academy of Sciences, Shanghai, China) was cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum at 37 °C under 5% CO₂. The culture medium was replenished every 2–3 days, and cells were passaged using 0.25% trypsin when they reached 80–90% confluence. For the generation of the in vitro liver fibrosis model, 2×10⁵ LX-2 cells/mL were seeded into culture plates and incubated for 24 h before 10 ng/mL TGF-β1 was added to the cells for 24 h to stimulate fibrosis.

Mouse model of carbon tetrachloride–induced liver fibrosis

Male wild-type C57BL/6 mice aged 8 weeks old (20–25 g) were obtained from Bikai Company (Shanghai, China) and maintained in a specific-pathogen-free room on a 12 h day–night cycle, with ad libitum access to food and water. Experiments were approved by the Committee on the Ethics of Animal Experiments of Shanghai General Hospital, Nanjing Medical University (No. 2018KY071, Shanghai, China), in compliance with the institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration. The mice were randomly divided into 2 groups (5 mice in both groups): an experimental and control group. Carbon tetrachloride (CCl₄) in corn oil (corn oil:CCl₄ = 9:1, v/v) was administered to the mice in the experimental group at a dose of 0.01 mL/g body weight, twice weekly for 8 weeks. The mice in the control group were injected with an equal volume of corn oil, twice weekly for the same period. All mice were humanely killed 48 h after the final injection.

To clarify the effect of YB-1 OE on CCl₄-induced liver fibrosis, we constructed a mouse-derived YB-1 OE plasmid and screened its expression efficacy (25). The lentiviral vector pCDH-CMV-MCS-EF1-Puro from Asia-Vector Biotechnology (Shanghai, China) was used to express the targeting sequence of YB-1 (m-Ybx1-F:5'-CTCGGATCCGCCACCATGAGCAGCGAGGCCGAGAC-3' and m-Ybx1-R:5'-CCCTCTAGACTCGAGCTCAGCCCCGCCCTGCTCAG-3'). Briefly, pCDH-CMV-m-Ybx1 plasmids were transfected into HEK 293T cells with packaging vectors. According to the screening results, we packaged the lentivirus, which was then purified to a titer power of >10⁹. The corresponding lentivirus was injected via the tail vein in the second week of the CCl₄-induced liver fibrosis model. All mice were randomly divided into 3 groups (5 mice in each group): (I) a normal control group, (II) a CCl₄ + control virus group, and (III) a CCl₄ + YB-1 OE group. At the end of the experiment, liver tissues of a total of 15 mice from the 3 different groups were harvested.

Plasmid transfection

Lipofectamine (Lipo2000) was used for all the plasmid transfection experiments according to the manufacturer’s instructions. Each 500 µL of transfection solution consisted of 0.8 µg of DNA dissolved in 100 µL of a serum-free minimal essential medium (Opti-MEM) and 2 µL of Lipo2000 that had been mixed for 20 min. The cells in each well of 24-well plates were transfected with 500 µL of the transfection mixture for 4–6 h, after which the medium was replaced with serum-free Opti-MEM (at 400 µL per well).

Western blot analysis

Total proteins were extracted from the HSCs or liver tissues using a radioimmunoprecipitation assay (RIPA) protein buffer and then quantified. Equal amounts of each protein extract were then denatured and separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The separated protein bands were then transferred from the gel to a polyvinylidene difluoride membrane using the wet transfer method. The membrane was blocked in a bovine serum albumin (BSA) solution for 1 h before incubation with primary antibodies against TGF-β1, TGF-β3, α-SMA, collagen III, collagen I, TGF-β3 receptor (TGFBR), p-SMAD2, SMAD2, p-SMAD3, SMAD3, chitinase-3-like protein 1 (YKL-40), YB-1, metalloproteinase inhibitor 1 (Timp1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was applied. All the primary antibodies were purchased from Abcam (Cambridge, UK). The membranes were rinsed and incubated with horseradish peroxidase–conjugated secondary antibodies before being visualized with an enhanced chemiluminescence developer solution. The expression level of each target protein was measured using ImageJ software (US National Institutes of Health, Bethesda, MD, USA).

Quantitative polymerase chain reaction (PCR) analysis

Total RNA was extracted from the HSCs using the chloroform–isopropanol method and finally completely dissolved in diethylpyrocarbonate-treated water. After the determination of its concentration, 600 ng was used for reverse transcription, which was performed according to the instructions of the PrimeScript RT Reagent Kit (Takara Bio, Kusatsu, Japan). A 10 L SYBR Green system (Fermentas, Ontario, Canada) was used for the quantitative PCR, and the 2^−ΔΔCT method was used to calculate the relative expression level of the target genes normalized to that of GAPDH.

ChIP assay

For the ChIP assay, we first added 210 µL of 37% formaldehyde to the adherent cells with 8 mL culture medium before gently shaking the mixture at room temperature for 10 min. After 400 µL of 2.5-M glycine was used to stop the reaction, the mixture continued to be shaken gently until it turned yellow. The culture medium was discarded, and 1 mL of phosphate-buffered saline (PBS) was added. The cells were harvested by centrifugation (8,000 g, 30 s, room temperature) and resuspended in 400 µL of lysis buffer (containing 1% SDS). After incubation on ice for 10 min, the resuspension was sonicated 5 times on ice (10 s, 22% power). The chromatin was harvested after centrifugation (12,000 g, 10 min, 4 °C), a small proportion was used for electrophoretic identification, and the remaining solution was stored at –80 °C.

Next, we added 600 µL of dilution buffer (containing phenylmethylsulfonyl fluoride [PMSF]) to the chromatin. After washing with agaroseA or agaroseG using Tris-EDTA (TE) buffer (10 mM of Tris-HCl and 1 mM of EDTA dissolved in non-RNA enzyme water), we added 300 µL of chromatin and 1.2 mL of dilution buffer to each tube (three 1.5-mL tubes for each group) and then the washed beads and prewashed the chromatin for 2 h. The beads for immunoprecipitation (IP) were incubated in 1 mL of TE and 10 µL of BSA (50 mg/mL) at 4 °C for 1 h. The beads were harvested with centrifugation (3,000 g, 2 min), and the resuspension was used as the input. The chromatin was divided into 2 parts and incubated overnight with the target antibody (2 ug) and normal immunoglobin G (IgG), respectively. The beads for IP were divided into each tube (three 1.5-mL tubes for each group), and incubated for 1 h, before 1 low-salt wash, 2 high-salt washes, 1 LiCl wash, and 2 TE washes were applied in turn to wash the beads for IP. After each wash, the beads were centrifuged at 3,000 g for 2 min, and the supernatant was discarded. After the last wash, the supernatant was completely discarded. Finally, 200 µL of freshly prepared elute buffer (0.1 M NaHCO₃, 1% SDS) was added per tube, and the supernatant was harvested by centrifugation 2 times after 15 min of shaking. Then, 16 µL of 5-M NaCl was added to each input, IgG, and target antibody groups for reverse cross-linking at 65 °C for 5–16 h. After purifying the DNA with a PCR purification kit, we completed detection using quantitative PCR analysis.

Enzyme-linked immunosorbent assay (ELISA)

Antibodies were diluted in buffer solution and then added to ELISA plates for overnight incubation at 4 °C. On the following day, the plates were blocked and washed, and the diluted test sample was added. Enzyme-labeled antibodies were added and incubated at 37 °C for 1 h. A solution of the chromogenic substrate 3, 3', 5, 5'-tetramethylbenzidine was added, and the reaction was terminated with 2 M of sulfuric acid. The absorbance (optical density value) in each well was measured at 450 nm on a microplate reader.

Immunofluorescence assay

After the cell medium in the 24-well plate was discarded, cells were fixed with 4% formaldehyde for 30 min. The formaldehyde was washed off, which was followed by blocking for 30 min and incubation with a α-SMA antibody for 2 h and a secondary antibody (1:1,000) for 1 h in the dark at room temperature. After a 5-min DAPI staining (1:10,000) in the dark, fluorescence microscopy was performed.

Luciferase reporter assay

Primers were designed to clone the required fragment of the TGF-β3 promoter region 2 from genomic DNAs using PCR, and the fragment was then inserted into a luciferase reporter plasmid (pGL3-basic). Screening of the positive clones and sequencing of the plasmids were then performed to identify the correct clone, after which the plasmids were purified for further use. HEK293T human embryonic kidney cells were cultured and inoculated in 24-well plates prior to their cotransfection with either the YB-1 OE or KD plasmid and the luciferase reporters. At the end of the transfection period, the protein was extracted for detection, and the luciferase activity was measured after the addition of substrate. The relative fluorescence intensity was calculated and compared with that of the control group.

Masson and Sirius red staining

Mouse liver tissues were fixed with 4% formaldehyde and used to prepare 5-µm thick paraffin sections that were routinely dewaxed to water before being stained (xylene I and II for 20 min each, absolute ethanol I and II and 75% alcohol for 5 min each, and a wash with water).

We used a Masson staining kit (G1006; Servicebio, Wuhan, China). The sections were immersed in Masson A solution overnight and washed on the second day. After being stained with a mixture of Masson B and C solution for 1 min, the sections were differentiated with 1% hydrochloric acid alcohol and stained with Masson D solution for 6 min, Masson E solution for 1 min, and Masson F solution for 2–30 s, successively. Following this, 1% glacial acetic acid was used for rinsing and final differentiation. Sirius red staining solution (G1018; Servicebio) was used to stain the sections for 8 min.

All sections were dehydrated 2–3 times with absolute ethanol and then treated with xylene for 5 min. After mounting with neutral resin was completed, images were taken using a microscope.

Statistical analysis

All data are presented as the mean ± standard error of the mean. The unpaired Student t test was used to compare the data from each group, and 1-way analysis of variance was used to test the significance of differences between the mean values of 3 or more groups. Differences with a P value <0.05 were considered statistically significant.

Results

Upregulation of YB-1 and downregulation of TGF-β3 in liver fibrosis

After 8 weeks of CCl₄ injections, the mouse livers were confirmed to be fibrotic through histological assessment. Masson and Sirius red staining showed that compared with the untreated control mice, the CCl₄-treated mice had necrotic and disordered hepatocytes and increased collagen deposition in the portal area, which are typical of the pathological changes associated with liver fibrosis (Figure 1A,1B). Western blot analysis revealed that the expression of YB-1 was upregulated but that of TGF-β3 was downregulated in the fibrotic liver tissue (Figure 1C,1D).

Figure 1 YB-1 was upregulated but TGF-β3 was downregulated in liver fibrosis. (A) Masson staining and Sirius red staining of liver sections from the control and CCl₄ groups. (B) Quantitative statistics of Masson and Sirius red staining. ***, P<0.001 control vs. CCl₄. (C) Western blots of YB-1 and TGF-β3 in the control and CCl₄ groups. (D) Quantitative statistics of Western blotting. **, P<0.01 Ctrl vs. CCl₄. Ctrl, control group.

Promotion of liver fibrosis by YB-1 in the in vivo mouse model of carbon tetrachloride–induced liver fibrosis

Based on the results from the in vivo model of CCl₄-induced liver fibrosis, the corresponding lentivirus was used to affect YB-1 expression. The results of Masson and Sirius red staining showed that YB-1 OE enhanced collagen deposition in liver tissue (Figure 2A). α-SMA expression detected with immunofluorescence showed a further increase in the CCl₄ + YB-1 OE group (Figure 2B,2C). Western blot analysis confirmed that YB-1 was successfully overexpressed by the lentivirus (Figure 2D). Collagen I expression was significantly increased by YB-1 OE. Furthermore, key factors of TGF-β signaling were detected using Western blot analysis: the expression of TGF-β3 was reduced in the CCl₄ group, while YB-1 OE further suppressed TGF-β3 expression (Figure 2D,2E). In addition, the expression of other TGF pathway–related proteins, including TGFBR, p-SMAD2/SMAD2, and p-SMAD3/SMAD3, showed opposite results to those of TGF-β3 (Figure 2D,2E).

Figure 2 YB-1 promoted liver fibrosis in an in vivo mouse model of CCl₄-induced liver fibrosis. (A) Masson and Sirius red staining of liver sections from the control, CCl₄*, and CCl₄ + YB-1-OE groups. (B) Immunofluorescence of α-SMA in the control, CCl₄* and CCl₄ + YB-1-OE groups. (C) Quantitative statistics of immunofluorescence. ***, P<0.01 control vs. CCl₄; ^##, P<0.01 CCl₄* vs. CCl₄ + YB-1 OE. (D) Western blots of YB-1, collagen I, TGF-β3, TGFBR, p-SMAD2/SAMD2, and p-SMAD3/SAMD3 in the control, CCl₄*, and CCl₄ + YB-1-OE groups. (E) Quantitative statistics of Western blotting. *, P<0.05 control vs. CCl₄; **, P<0.01 Ctrl vs. CCl₄*; ^#, P<0.05 CCl₄vs. CCl₄ + YB-1-OE; ^##, P<0.01 CCl₄* vs. CCl₄ + YB-1 OE; ^###, P<0.001 CCl₄* vs. CCl₄ + YB-1 OE. Ctrl, control group; OE, overexpression.

Promotion of liver fibrosis by YB-1 in the TGF-β1–induced HSC activation model

TGF-β1 is an important activator protein closely linked to HSC activation and liver fibrosis. We established a model of TGF-β1–induced HSC activation using the LX-2 cell line to evaluate how the regulation of YB-1 expression affects this liver condition. Western blotting results showed that α-SMA expression in the HSCs was significantly increased after TGF-β1 stimulation (Figure 3A,3B). Additionally, the expression levels of YKL-40, Timp1, and ECM proteins (i.e., collagen I and collagen III) were all upregulated, suggesting increased ECM synthesis and pathological remodeling in response to TGF-β1 stimulation (Figure 3A,3B). We also examined the effects of YB-1 KD and OE on liver fibrosis in vitro using the LX-2 cell–based model. YB-1 KD significantly decreased α-SMA and collagen I expressions (Figure 3A,3B). Similarly, the expression of YKL-40 and Timp1 was decreased when YB-1 was knocked down in the presence of TGF-β1 (Figure 3A,3B). Conversely, YB-1 OE by cells transfected with the YB-1-carrying plasmid increased the expression of α-SMA, ECM proteins, and YKL-40, promoting liver fibrosis in response to TGF-β1 and increasing fibrotic collagen production (Figure 3A,3B). These results were verified using an immunofluorescence assay, which showed that α-SMA expression increased in response to TGF-β1 stimulation, and this effect was further increased in response to YB-1 OE and decreased in response to YB-1 KD (Figure 3C,3D). These findings suggested that the OE of YB-1 induced LX-2 cell activation in response to TGF-β1 signaling, whereas the inhibition of YB-1 expression prevented liver fibrosis in LX-2 cells.

Figure 3 YB-1 promoted liver fibrosis in a model of TGF-β1–induced HSC. (A) Western blot of α-SMA, collagen I, collagen III, YKL-40, and Timp1 in YB-1 KD or YB-1 OE HSCs following TGF-β1 stimulation. (B) Quantitative statistics of Western blotting. ***, P<0.001 NC* vs. NC; ^#, P<0.05 TGF-β1 + YB-1 KD/OE vs. NC*; ^##, P<0.01 TGF-β1 + YB-1 KD/OE vs. NC*; ^###, P<0.001 TGF-β1 + YB-1 KD/OE vs. NC*. (C) Immunofluorescence images of α-SMA in YB-1 KD or YB-1 OE HSCs following TGF-β1 stimulation (blue: nucleus; green: α-SMA). (D) Quantitative statistics of immunohistochemical assays. **, P<0.01 NC* vs. NC; ^##, P<0.01 TGF-β1 + YB-1 KD/OE vs. NC*. NC, normal control; NC*, NC + TGF-β1. HSC, hepatic stellate cell; KD, knockdown; OE, overexpression.

YB-1 promoted liver fibrosis in the model of TGF-β1–induced HSC activation via the attenuation of TGF-β3 transcription

Other studies have linked YB-1 expression and TGF-β signaling to fibrosis in the liver and kidneys (24,26). In this study, we evaluated the potential mechanism underlying this interaction. The promoter of TGF-β3 was divided into 5 regions, after which ChIP analysis was used to confirm the interaction between the promoter and YB-1 and to identify the exact region of interaction. In the YB-1-overexpressing HSCs, strong enrichment of TGF-β3 promoter region 2 in comparison with the control and YB-1-DNA complex was noted, which indicated that the YB-1 transcription factor–binding site of TGF-β3 lies in region 2 of its promoter (Figure 4A). This interaction was confirmed by the luciferase reporter assay, in which LX-2 cells were transfected with luciferase reporter constructs harboring the TGF-β3 promoter sites, and their luciferase activity was then monitored under various conditions. These assays revealed that luciferase activity was increased in the YB-1 KD groups, whereas there was no significant difference in enzymatic activity in the YB-1 OE groups (Figure 4B). ELISA results showed that relative to the control values, the concentration of TGF-β3 was increased in response to YB-1 KD and decreased in response to YB-1 OE (Figure 4C), suggesting that inhibition of YB-1 induces TGF-β3 expression. Taken together, these results indicated that TGF-β1 stimulation activated YB-1 expression in the LX-2 cell line, which in turn attenuated TGF-β3 transcription and facilitated liver fibrosis.

Figure 4 YB-1 promoted liver fibrosis in the LX-2 cell line by attenuating TGF-β3 transcription. (A) Enrichment of each region of the TGF-β3 promoter in the YB-1 DNA fragments from chromatin immunoprecipitation. ***, P<0.001 YB-1 OE vs. NC. (B) Luciferase reporter assay showing the interactions between YB-1 and the TGF-β3 promoter region. ***, P<0.001 YB-1 KD vs. NC. (C) ELISA of TGF-β3 from YB-1 OE or YB-1 KD hepatic stellate cells following TGF-β1 stimulation. ***, P<0.001 NC + TGF-β1 vs. NC, ^##, P<0.01 TGF-β1 + YB-1 KD/OE vs. NC + TGF-β1. KD, knockdown; OE, overexpression; NC, normal control; NC*, NC + TGF-β1; ELISA, enzyme-linked immunosorbent assay.

Activation of HSCs by YB-1 further activated surrounding cells via TGF-β3

As demonstrated, YB-1 induced liver fibrosis in the LX-2 cell line via TGF-β3 downregulation. Coculture experiments were carried out to verify the regulation of HSCs by YB-1, which may further affect surrounding cells via TGF-β3 secretion.

To confirm this, we designed an experiment using 2 groups of HSCs: control, YB-1 KD, and YB-1 OE. The YB-1 KD and YB-1 OE HSCs were treated with TGF-β1, whereas the control HSCs transfected with a blank plasmid were treated with PBS as blank controls. The culture supernatant from the respective groups was collected and treated with quiescent LX-2 cells. Western blot and immunofluorescence assays were then used to detect the expression of HSC activation markers and collagen synthesis proteins. Our results were consistent with those for the direct treatment assays. The culture supernatant of the YB-1 OE group induced the expression of both activation markers and collagen synthesis proteins (collagen I, collagen III, α-SMA, YKL-40, and Timp1), whereas that from the YB-1-inhibition group attenuated liver fibrosis in the LX-2 cell line (Figure 5). We found that YB-1 OE might indirectly amplify this effect on neighboring HSCs.

Figure 5 HSCs activated by YB-1 further activated surrounding cells through TGF-β3. (A) Western blots of α-SMA, collagen I, collagen III, YKL-40, and Timp1 expression levels in HSCs treated with the culture supernatant of YB-1 OE or YB-1 KD HSCs following TGF-β1 stimulation. (B) Quantitative statistics of Western blotting. *, P<0.05 NC + TGF-β1 vs. NC; **, P<0.01 NC + TGF-β1 vs. NC; ***, P<0.001 NC + TGF-β1 vs. NC; ^#, P<0.05 TGF-β1 + YB-1 KD/OE vs. NC + TGF-β1; ^##, P<0.01 TGF-β1 + YB-1 KD/OE vs. NC + TGF-β1; ^###, P<0.001 TGF-β1 + YB-1 KD/OE vs. NC + TGF-β1. (C) Immunofluorescence of α-SMA in HSCs exposed to the culture supernatant of YB-1 OE or YB-1 KD HSCs following TGF-β1 stimulation (blue: nucleus; green: α-SMA). (D) Quantitative statistics of immunohistochemical assays. ***, P<0.001 NC + TGF-β1 vs. NC; ^###, P<0.001 TGF-β1 + YB-1 KD/OE vs. NC + TGF-β1. HSC, hepatic stellate cell; KD, knockdown; OE, overexpressing; NC, normal control; NC*, NC + TGF-β1.

Finally, we evaluated the expression of key factors involved in TGF-β signaling in the mouse model of CCl₄-induced liver fibrosis. We found that the expression levels of TGF-β1 and other proteins in its downstream signaling pathways were significantly increased in the CCl₄ group (Figure 6). In particular, the levels of phosphorylated SMAD2 and SMAD3 were increased, indicating their activation during fibrosis (Figure 6). By contrast, TGF-β3 expression was significantly decreased in the fibrotic liver tissue, likely owing to YB-1 OE.

Figure 6 TGF-β signaling in liver fibrosis. (A) Western blots of TGFBR, p-SMAD2/SAMD2, and p-SMAD3/SAMD3 in the control and CCl₄ groups. (B) Quantitative statistics of Western blotting. *, P<0.05 vs. Ctrl; **, P<0.01 vs. Ctrl; ***, P<0.001 Ctrl vs. CCl₄. KD, knockdown; Ctrl, control group.

Discussion

Liver fibrosis is characterized by an accumulation of ECM and pathological disorders of the liver. As the major source of ECM, activated HSCs act as the effectors of liver fibrosis and are regarded as the most important cells during the progression of this condition. The TGF-β pathway is considered as the key fibrogenic pathway driving HSC activation and inducing ECM production, with TGF-β1 being the most critical factor. TGF-β3, contrary to TGF-β1, has the effect of inhibiting fibrosis (16), and as a regulator, YB-1 plays a role in a variety of liver fibrosis–related pathways, including the TGF-β/SMAD pathway (19,20,24).

YB-1 is a member of the cold-shock protein family, which includes both transcription and translation factors that are widely involved in regulating gene expression (19,27,28). YB-1 is involved in the fibrosis of solid organs, including the kidney and liver, and acts as a crucial orchestrator of the expression of several fibrosis-related genes (19,24,26,29,30). It has been reported that YB-1 affects the activity of the TGF-β/SMAD pathway via the regulation of collagen and/or the activation of HSCs. YB-1 improves the stability of SMAD2 by reducing its ubiquitin-mediated degradation (24). Meanwhile, YB-1 also attenuates TGF-β1–induced collagen type 1 alpha 2 chain transcription by blocking the interaction between SMAD3 and p300, which mediates the function of interferon gamma in liver fibrosis (24).

We first observed that YB-1 was upregulated in the fibrotic liver of mice and then used TGF-β1–stimulated LX-2 cells to evaluate the function of YB-1 during liver fibrosis (31). KD of YB-1 prevented the function of TGF-β1, resulting in reduced expression of the α-SMA and ECM synthesis genes both in vitro and in vivo. By contrast, YB-1 OE promoted liver fibrosis in TGF-β1–induced LX-2 activation and CCl₄-induced liver fibrosis.

In the liver fibrosis models and in the TGF-β1–induced HSC activation model, TGF-β3 was downregulated, especially under YB-1 OE. A previous study confirmed that the TGF-β–YB-1-Atg7 axis promotes HPC proliferation and liver fibrogenesis (32). Results from RNA sequencing in a previous study indicated that differentially expressed genes between YB-1 KD HPCs and the control were involved in the TGF-β signaling pathway (26). TGF-β3 is identified as one of the target genes of YB-1, whose binding site is located in the promoter region of TGF-β3, showing an enrichment state (17). YB-1 seems to be related to the antihepatic fibrosis feedback pathway of TGF-β3. We speculated that increased expression of YB-1 may inhibit TGF-β3 transcription, so we needed to further consider and verify whether there is a correlation between YB-1 and TGF-β3 in the occurrence and development of liver fibrosis.

Accordingly, we carried out TGF-β1–induced HSC activation as a liver fibrosis model, constructed YB-1 OE and interference plasmids, and transfected HSCs. ChIP analysis showed that YB-1 interacted with region 2 of the TGF-β3 promoter, and the luciferase assay revealed that this interaction reduced TGF-β3 expression, confirming the direct interaction between these proteins and our original study hypothesis. It showed that YB-1 expression promoted TGF-β1–induced liver fibrosis in the LX-2 cell line by attenuating the transcription of TGF-β3.

The fibrosis-initiating stimulus acts on HSCs and induces cytokine and inflammatory factor production, resulting in the primary stimulation response (33,34). The activated HSCs then amplify this fibrosis-initiating stimulus and further activate the surrounding cells; however, the underlying mechanism for this signaling is not well understood (35). Having shown that YB-1 is a vital activator of HSCs during pro-fibrogenic factor stimulation, we designed an experiment to determine whether the profibrotic signals mediated by YB-1 can be transmitted to the extracellular environment, resulting in the indirect activation of neighboring HSCs and thus amplifying the initial activation effect. For this purpose, we collected the supernatants of the YB-1 OE and YB-1 KD cell cultures, used them to treat HSCs respectively, and then monitored the cells for signs of activation. We found that the signal from the YB-1-overexpressing HSCs could be transmitted to the surrounding cells via extracellular materials. Additionally, we detected the TGF-β pathway in these activated surrounding cells, which indicated that YB-1 signaling plays an essential role in the fibrotic process. However, as we did not enrich or deplete TGF-β3 from the culture supernatant, which contained many other factors, so this conclusion needs further experimental confirmation.

There are some limitations to this study which should be acknowledged. First, the initial factors that promote the high expression of YB-1 in the process of liver fibrosis were not identified. Second, only a single cell line was examined in our experiments. Our understanding of the relationship between YB-1 and TGF-β3 will expand and deepen through the continuing focus of our subsequent research.

Conclusions

YB-1 is an inhibitory signal for TGF-β3 transcription, and its expression leads to liver fibrosis. Additionally, YB-1 expression appears to influence HSC activation in nearby tissues by regulating TGF-β3 secretion. Taken together, our results revealed that YB-1 promotes liver fibrosis by attenuating TGF-β3 transcription. These findings may form a theoretical basis for selecting more appropriate drugs in treating liver fibrosis.

Acknowledgments

The authors sincerely thank Dr. Fei Li (Department of Gastroenterology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China), for his contributions to the subject design and his assistance with the study.

Funding: None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-23-835/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-23-835/dss

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Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-23-835/coif). The authors have no conflicts of interest to declare.

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