Liver fibrosis can be caused by hepatitis B virus or hepatitis C virus infection, alcohol, non-alcoholic fatty liver disease/non-alcoholic steatohepatitis, and other relatively rare conditions, such as autoimmune hepatitis, Wilson’s disease, and primary/secondary biliary cholangitis. Sustained chronic liver injury leads to normal cellular functional disruption and imbalance of the degradation and synthesis of extracellular matrix (ECM), which eventually develops the liver fibrosis [1
]. Hepatic stellate cells (HSCs) exist in the space between parenchymal cells and liver sinusoidal endothelial cells of the hepatic lobule. Following chronic liver injury, HSCs activate into α-SMA+
contractile myofibroblast-like cells, which are characterized by the increased proliferation and migration, the unbalanced matrix degradation and the release of pro-inflammatory and pro-fibrogenic factors [2
HSCs also have immunological functions—they express functional chemokine receptors and chemokines, including CXCL1, CXCL8, CXCL9, CXCL10, CCL2, CCL3 and CCL5 [3
]. CXC chemokine-ligand-1 (CXCL1), also known as Gro-alpha, is a ligand for G-protein-coupled receptor CXCR2. CXCL1 is considered to be involved in the activation of HSCs [4
], fibrogenesis and angiogenesis [5
]. CXCL1 has also been recognized as a biomarker associated with hepatocellular carcinoma in a serum level [7
CD147 is a transmembrane glycoprotein and a member of immunoglobulin superfamily. CD147 is widely expressed on numerous cells in carcinoma leading to the invasion, proliferation and survival of cancer cells, which is an important marker of tumor and poor prognosis [8
]. Our previous studies showed that transforming growth factor-β1-CD147 positive feedback loop promoted the activation of HSCs, and the expression of CD147 in liver fibrosis and cirrhosis was increased which was positively correlated with the Child–Pugh grade [11
In this study, we show that CD147 activates HSCs and upregulates CXCL1 expression in HSCs. In line with this, HSCs-specific CD147-knockout mice have greatly reduced HSCs activation and CXCL1 expression, leading to attenuated liver fibrosis. CD147 regulates CXCL1 expression in HSCs via PI3K/AKT pathway.
Patients with fibrosis often result from different etiologies of chronic liver injury. Cirrhosis is the terminal stage of progressive liver fibrosis, eventually developing to the hepatocellular carcinoma. Liver fibrosis is shown to be a reversible process [17
], withdrawal of the chronic injury results in decrease of pro-inflammatory and fibrogenic cytokines, increased collagenase activity, decreased ECM production, and disappearance of hepatic-activated HSCs [20
]. Our previous results show that CD147 upregulates fibrosis-related marker α-SMA and α1(I) collagen
in HSCs, and the positive loop of transforming growth factor-β1-CD147 promoted the activation of HSCs [11
]. Here, we provided additional evidence that knockout CD147 in mouse HSCs inhibited collagen deposition, inflammatory infiltration and HSCs activation, alleviating CCl4
-induced liver fibrosis. In addition, we found that overexpression of CD147 in LX-2 cells upregulated expression of chemokine CXCL1, which was upregulated in activated HSCs. Moreover, CXCL1 exerted a positive activation loop on HSCs, enhancing HSCs activation phenotypes. Taken together, these findings support our conclusions that CD147 stimulates the release of chemokine CXCL1 and promotes the development and progression of liver fibrosis.
Accumulated evidence suggests that chemokines play a critical role in acute and chronic liver diseases, mediating the immune infiltration in injured liver. Additionally, chemokines can also directly affect the biology of liver resident cells, such as HSCs, during inflammatory and fibrogenic tissue responses [23
]. It is reported the increased hepatic expression of pro-inflammatory genes TNF
in liver injury [24
]. In our study, we found that CXCL1 in activated HSCs was upregulated in CCl4
-induced fibrosis. CXCL1 binds to the CXCR2, which expresses on HSCs and neutrophils, and several types of cancer including melanoma [25
], lung [26
] and pancreatic cancers [27
]. Stimulation of HSCs with recombinant CXCL1 results in increased collagen type I and α-SMA production [5
]. Similarly, in our study rCXCL1 also upregulated the activation markers, α-SMA and α1(I) collagen
in LX-2 cells and promoted the cell contraction and proliferation.
We used the transgenic mouse GFAP-Cre;Bsgfl/fl
to further verify the CD147 function in HSCs in our study. Liver injury is usually accompanied with inflammatory response. A high-fat diet combined with acute ethanol consumption synergistically induces acute liver inflammation and injury via upregulation of hepatic CXCL1 expression and promotion of hepatic neutrophil infiltration [29
]. In CCl4
-induced mouse fibrosis, GFAP-Cre;Bsgfl/fl
mice showed less hepatic inflammatory infiltration and serum CXCL1 expression. We had proven that overexpression of CD147 promoted α1(I) collagen
synthesis, while GFAP-Cre;Bsgfl/fl
mice showed less amount of collagen deposition. Liver fibrosis is due to the unbalanced degradation and synthesis of ECM. Activated HSCs are a major source of collagen and ECM and play a key role in the formation of hepatic fibrosis [30
]; quantitative assessment of HSCs activity is one of the means to detect the development of fibrosis. The GFAP-Cre;Bsgfl/fl
mice showed a reduction of α-SMA and desmin expression, which indicates that inhibiting CD147 contributes to the HSCs deactivation and, thus, the reversal of HSC activation contributes to termination of fibrogenesis [32
The activated HSCs have activation signatures (MAPK and PI3K-AKT phosphorylation) and upregulated genes (COL1A1
, and CXCL1
) expression [33
]. As integrin is important for HSCs transdifferentiation [34
], in integrin-mediated signaling transduction process, FAK is a key molecule and acts like an integrator that accepts and amplifies signals in the cell and activates downstream signal pathway [35
]. CD147 can bind to integrin and activates the downstream FAK/PI3K signaling pathway [36
]. CD147 overexpression induced the AKT phosphorylation; however, treating with FAK/PI3K inhibitor LY294002, CD147-induced AKT phosphorylation and CXCL1 expression were significantly inhibited. Taken together, CD147 mediated CXCL1 expression probably by directly binding to integrin in HSCs through the PI3K/AKT cell signal activation.
Liver fibrosis shows complex interplay between the epithelial cells, inflammatory cells, myofibroblasts and ECM components of the wound-healing response [2
]. In summary, we demonstrate that CD147 promotes the CXCL1 expression and modulates the liver fibrosis. Therapeutically, our results strongly suggest a potential for inhibition of CD147 as a treatment strategy in liver fibrosis.
4. Materials and Methods
4.1. Cells Culture and Reagents
Human HSCs cell line, LX-2 was cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Hycolon, Logan, UT, USA) supplemented with antibiotics (100 U/mL of penicillin and 100 mg/mL of streptomycin) and 10% fetal bovine serum in a humidified atmosphere containing 5% CO2
at 37 °C. Mouse primary HSCs were isolated from the mice livers mainly by in situ pronase/collagenase perfusion of mouse liver, followed by density gradient-based separation, as described [37
]. The cells were cultured in DMEM (Hycolon) supplemented with antibiotics (100 U/mL of penicillin and 100 mg/mL of streptomycin) and 15% fetal bovine serum. Human recombinant CXCL1 was from Peprotech (Princeton, NJ, USA). PI3K/AKT inhibitor, LY294002 was from Cell Signaling Technology (Danvers, MA, USA).
All experimental protocols were approved by the Laboratory Animal Ethics Committee of the Fourth Military Medical University (No. 20121003, 16 October 2012) and performed in strict accordance with the People’s Republic of China Legislation Regarding the Use and Care of Laboratory Animals. HSCs-specific Cre recombinase transgenic mice (glial fibrillary acidic protein (GFAP
) purchased from Shanghai Biomodel Organism Science & Technology Development Co., Ltd. (Shanghai, China) and conditional CD147 targeting mice (Bsgfl/fl
) were constructed by Dr. Yao Hui [13
] in our laboratory. All gene identification primers were synthesized by the Beijing Genomics Institute (BGI, Beijing, China) and listed in Table 1
. To induce chronic liver injury, mice were treated with 10% carbon tetrachloride (CCl4
) intraperitoneally at a dose of 5 μL/g, three times per week.
4.3. siRNA Transfection
Silencing was performed by the transient transfection of siRNA oligos using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. We used two silencing oligos from GenePharma (Shanghai, China): si-CD147-1: 5′-GTA CAA GAT CAC TGA CTC T-3′; si-CD147-2: 5′-GTT CTT CGT GAG TTC CTC-3′.
4.4. Western Blot
Western blot was performed as previously described [11
]. The primary antibodies used were mouse anti-human CD147 antibody (1:1000) prepared by our laboratory [38
], anti-AKT (1:2000; ARE6004), anti-phospho-AKT (1:500; ARE6002) were purchased from Antibody revelation (San Diego, CA, USA), rat anti-mouse CD147 (1:500; ab34016) were purchased from Abcam (Cambridge, UK), anti-β-actin (1:1000; M1210-2) were purchased from Hubio (Hangzhou, China), and anti-α-tubulin (1:1000; 66031-1-Ig) were purchased from Proteintech (Wuhan, China). A Western-Light chemiluminescent detection system (Image Station 4000 MM Pro, XLS180, Kodak, Rochester, NY, USA) was used to visualize the signals.
4.5. Real-Time PCR
First, total RNA was extracted by a Total RNA Kit II (Omega, Riverside, CA, USA), then using a PrimeScript™ RT reagent kit (TaKaRaBio, Otsu, Japan) reverse transcript to cDNA. All the steps were based on the product protocol. Single-stranded cDNA was amplified by quantitative RT-PCR using a SYBR Premix ExTaq™ kit (TaKaRaBio) on a Stratagene Mx3005P™ Real-Time PCR System (Agilent Technologies, Waldbronn, Germany). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used to normalize RNA inputs. RT reactions were performed as previously described [11
]. All primers were synthesized by the Beijing Genomics Institute (BGI, Beijing, China) and listed in Table 1
4.6. Enzyme-Linked Immunosorbent Assay (ELISA)
First, we used serum separator tube and allowed mice blood samples to clot for one hour at room temperature, then centrifuged for 15 min at approximately 3000× g, at 4 °C. Serum CXCL1 detection was performed using a CXCL1 enzyme-linked immunosorbent assay kit (Mlbio, Shanghai, China) according to the manufacturer’s instructions. The concentration in each sample well was determined by interpolation from a standard curve. Each sample was tested in triplicate.
4.7. Cell Contraction Assay
We used a standard kit assay (Cell Biolabs, San Diego, CA, USA), operated according to the manufacturer’s instructions. The gel area was quantified by ImageJ software (ImageJ 1.48, National Institutes of Health). The gel contraction area was calculated by the initial gel area (outer white circle) minus the terminal gel area (inner white circle). The normalized contraction of corresponding control was a ratio of control contraction area to rCXCL1 contraction area.
4.8. Collagen Staining
Collagen accumulation was detected with sirius red, which was performed as previously described [37
]. Liver tissues were fixed with 4% formalin and embedded in paraffin. Following deparaffinization and hydration, the sections were stained with sirius red (Sigma, Darmstadt, Germany).
Liver tissues were fixed with 4% formalin and embedded in paraffin. Sections were deparaffinized and incubated with primary antibodies including anti-CXCL1 (1:100; ab86436), anti-α-SMA (1:100; ab7817), and anti-desmin (1:100; ab86592) (Abcam, Cambridge, UK). Staining was performed as described previously [39
Immunofluorescence was carried out as described previously [39
]. Primary antibodies included anti-α-SMA (1:100; ab7817), anti-CXCL1 (1:100; ab86436) and anti-CD147 antibodies (1:100; ab34016) (Abcam, Cambridge, UK).
4.11. Fluorescence Activated Cell Sorting (FACS)
FACS analysis was carried out as described previously [40
]. Primary antibodies included anti-α-SMA (1:100; ab7817) (Abcam, Cambridge, UK) and anti-CXCL1 (1:100; MAB4542) (RD, Minneapolis, MN, USA). The cells were analyzed by flow cytometry (BD FACSCalibur™ Flow Cytometer, BD Bioscience, San Jose, CA, USA) using FlowJo software (FlowJo 7.6, BD Bioscience).
LX-2 cells (5 × 104 cells/well) were seeded in 96-well plates and incubated with 100 ng/mL rCXCL1 for 24 h. Ten µL of the CCK8 (Engreen, Beijing, China) was added to each well using the pipettor. The cells were incubated for 2 h. The absorbance of each sample was measured using a microplate reader at a wavelength of 450 nm.
4.13. Statistical Analysis
Each experiment was repeated at least three times. Student’s t-test was used to compare the two mean values. A one-way analysis of variance was performed to compare the multiple mean values. Data were presented as the mean ± SD from three independent experiments unless otherwise indicated. The Graphpad Prism software and SPSS 17.0 software were used for statistical analysis. A p value < 0.05 was considered statistically significant.