CXCL6‐EGFR‐induced Kupffer cells secrete TGF‐β1 promoting hepatic stellate cell activation via the SMAD2/BRD4/C‐MYC/EZH2 pathway in liver fibrosis

Abstract Liver fibrosis is the excessive accumulation of extracellular matrix proteins in response to the inflammatory response that accompanies tissue injury, which at an advanced stage can lead to cirrhosis and even liver failure. This study investigated the role of the CXC chemokine CXCL6 (GCP‐2) in liver fibrosis. The expression of CXCL6 was found to be elevated in the serum and liver tissue of high stage liver fibrosis patients. Furthermore, treatment with CXCL6 (100 ng/mL) stimulated the phosphorylation of EGFR and the expression of TGF‐β in cultured Kupffer cells (KCs). Although treatment with CXCL6 directly did not activate the hepatic stellate cell (HSC) line, HSC‐T6, HSCs cultured with media taken from KCs treated with CXCL6 or TGF‐β showed increased expression of α‐SMA, a marker of HSC activation. CXCL6 was shown to function via the SMAD2/BRD4/C‐MYC/EZH2 pathway by enhancing the SMAD3‐BRD4 interaction and promoting direct binding of BRD4 to the C‐MYC promoter and CMY‐C to the EZH2 promoter, thereby inducing profibrogenic gene expression in HSCs, leading to activation and transdifferentiation into fibrogenic myofibroblasts. These findings were confirmed in a mouse model of CCl4‐induced chronic liver injury and fibrosis in which the levels of CXCL6 and TGF‐β in serum and the expression of α‐SMA, SMAD3, BRD4, C‐MYC, and EZH2 in liver tissue were increased. Taken together, our results reveal that CXCL6 plays an important role in liver fibrosis through stimulating the release of TGF‐β by KCs and thereby activating HSCs.

pathogenesis of most types of chronic liver disease. This excessive deposition of ECM proteins at sites of tissue damage, resulting from, for example, viral infection or alcoholic liver disease, is a consequence of a dysregulated wound-healing response. Although reversible if the source of the injury is eliminated, unchecked liver fibrosis can develop into cirrhosis and even liver failure due to portal hypertension, with the only treatment option being transplantation. 1,2 Chronic liver disease is a major cause of mortality worldwide and correlates with an increased risk of hepatocellular carcinoma. 3 Hepatic stellate cells (HSCs), which are characterized by the storage of retinyl esters in cytoplasmic lipid droplets, constitute around 10% of resident liver cells and in healthy livers are phenotypically non-proliferative and quiescent. However, HSCs are the predominant precursor cells of liver myofibroblasts and upon activation, which is triggered by signalling pathways such as those involving plateletderived growth factor (PDGF) and transforming growth factor-beta (TGF-β), dramatic phenotypic changes are induced accompanied by transdifferentiation into proliferative, inflammatory, fibrogenic myofibroblasts. This process of HSC activation and transdifferentiation is the driver of fibrosis in liver injury. 4 However, the pathways and mediators involved in HSC activation are complex. Understanding the complex regulation of HSC activation is crucial for the development of novel therapeutic strategies to treat liver disease.
TGF-β, which is secreted by several cell types in the liver, is considered the predominant cytokine to trigger HSC activation and fibrogenic transdifferentiation. 5,6 Kupffer cells (or stellate macrophages) are specialized macrophages that reside in the liver and release proinflammatory and profibrogenic factors such as TGF-β in response to various stimuli (including CXC chemokines), that in turn modulate HSC behaviour and trigger HSC activation. 7 TGF-β-induced de novo synthesis of alpha smooth muscle cell actin (αSMA) fibres enhances the contractility of the cells and increases the expression of ECM proteins for ECM remodelling. 8 TGF-β also binds and phosphorylates the type I receptor, which in turn induces phosphorylation of downstream SMAD proteins, which translocate to the nucleus and activate various mitogen−activated protein kinase (MAPK) signalling pathways to trigger HSC activation and myofibroblast transformation, such as the extracellular signal−regulated kinase (ERK), p38 and c−jun N−terminal kinase (JNK) pathways. [9][10][11][12][13] Previous studies reported that SMAD proteins can bind to BRD4 in response to TGF-β stimulation. 14 BRD4 is a global regulator of enhancer−mediated profibrogenic gene expression in HSCs. 15 There is evidence from various cancer cell types that BRD4 can interact with the C-MYC promoter and directly regulate its transcriptional expression. [16][17][18] Furthermore, recent studies by one research group into the role of BRD4 in bladder cancer reported that BRD4 positively regulates enhancer of zeste homologue 2 (EZH2) transcription through the upregulation of the C-MYC promoter. 19,20 In this study, the role of CXCL6 (GCP-2) in liver fibrosis was investigated. The subfamily of CXC chemokines that possess an ELR motif are potent neutrophil chemoattractants and interact with the G protein-coupled receptors, CXCR1 and/or CXCR2. 21 Among this subfamily, CXCL6 has been shown to play a role in neutrophil recruitment leading to tissue damage and prolonged inflammatory responses. 22 CXCL6 has thereby been proposed to contribute to fibrosis and CXC chemokines have been proposed as prognostic biomarkers of liver fibrosis. 23 Our findings revealed a correlation between elevated CXCL6 levels in serum and liver tissues and high stage liver fibrotic disease in patients. By employing in vitro experiments and a carbon tetrachloride (CCl 4 )-induced fibrosis mouse model, 24 CXCL6 was shown to promote the release of TGF-β by Kupffer cells (KCs), leading to HSC activation. Our findings provide important insight into the complex mechanisms of HSC activation that contribute to liver fibrosis.

| Human serum and liver samples
Serum samples were taken from 50 patients with clinically diagnosed liver fibrosis who had been classified according to fibrotic staging (S) (n = 10 samples for each of the stages: S0, S1, S2, S3 and S4). Liver tissues were taken from 10 patients with clinically diagnosed liver hepatitis who had been classified according to fibrotic staging (S) (n = 6 samples from each of the stages: S0, S1, S2 and S4). All patients were admitted to our hospital from 2013 to 2015. Ethical approval for the study was provided by the independent ethics committee of Shanghai General Hospital, affiliated with Shanghai Jiao Tong University School of Medicine. Informed and written consent were obtained from all patients or their advisors according to the ethics committee guidelines.

| Liver histological observations
Slices of human liver were fixed in 10% phosphate-buffered saline (PBS)-formalin for at least 24 hour and then embedded in paraffin for histological assessment of tissue damage. Samples were subsequently sectioned (5 μm), stained with haematoxylin and eosin (H&E) using standard protocols and then examined microscopically under a light microscope (Olympus Corporation, Tokyo, Japan) to evaluate structural changes indicating liver damage.

| Immunohistochemistry
Liver tissue sections were initially treated by deparaffinization and hydration. Then EDTA (pH 8.0) was added and antigen retrieval was

| Biochemical analysis
ALT, AST, and hydroxyproline levels were analysed using commercial kits according to the manufacturers' protocols (Nanjing Jiancheng Bioengineering Institute).

| Cell culture
Hepatic stellate cell-T6 cells (HSCs) were purchased from the Cell Bank at Chinese Academy of Sciences (Shanghai, China) and the isolation of primary KCs and HSCs from rats was performed as previously described. 25   were used to calculate expression levels after normalization to β-actin, which was used as an internal standard. The primer sequences used in this study are listed in Table 1.

| Enzyme-linked immunosorbent assay (ELISA)
The levels of CXCL6, α−smooth muscle actin (α-SMA) and TGF-β in sera or cell culture supernatants were determined using an ELISA kit (Nanjing Jiancheng Bioengineering Institute) according to standard protocols.

| Immunofluorescent staining
Hepatic stellate cells were fixed in 4% paraformaldehyde solution

| Protein isolation and western blot analysis
Hepatic stellate cells or KCs were washed three times with PBS, harvested, and lysed in co-immunoprecipitation (co-IP) buffer, as previously described. 27 The total cell lysate (5 mg protein) was subjected to immunoprecipitation with the appropriate antibodies, as indicated, overnight at 4°C with gentle agitation, followed by incubation with (1:2000) and β-actin (1:5000), were all purchased from Santa Cruz Biotechnology. The expression levels of proteins were determined using ImageJ software.

| Chromatin immunoprecipitation (ChIP) assay
A ChIP assay was performed using an EZ-ChIP kit (Upstate Biotechnology, Lake Placid, NY, USA), as previously described. 20

| Statistical analysis
Statistical analyses were performed using the SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Statistically significant differences were detected using either the Student's t test for comparison between the means or one-way analysis of variance. Data are presented as the mean ± SD and are considered to be statistically significant at *P < 0.05, **P < 0.01, ***P < 0.001, # P < 0.05, ## P < 0.01 and ### P < 0.001.

| Elevated expression of CXCL6 in the sera and liver tissue of liver fibrosis patients
The sera of liver fibrosis patients categorized as belonging to different fibrotic stages (S1-S4) were analysed biochemically using commercial kits to assess ALT/AST activity and hydroxyproline levels. The serum ALT/AST ratio is a commonly used biomarker of liver health, and hydroxyproline is a major constituent of collagen. Overall, a steady increase in ALT/AST activity and hydroxyproline levels accompanied the progression of liver fibrosis, with the highest levels evident in patients with fibrotic stage S4, as expected ( Figure 1A, P < 0.05 for ALT and AST; Figure 1B, P < 0.001 for hydroxyproline vs the S0 group). Next, serum levels of TGF-β and CXCL6 were determined for the various fibrotic stages. Again, an overall increase in TGF-β and CXCL6 levels was detected with increased fibrotic staging, with the highest levels evident in patients with S4 fibrosis ( Figure 1C, P < 0.01 for TGF-β; Figure 1D, P < 0.01 for CXCL6 vs the S0 group). To con-   Figure 2F). Taken together, these findings confirmed

| CXCL6 can activate HSCs via KC conditioned medium
The ability of CXCL6 to activate HSCs was tested by analysing the α-SMA and TGF-β concentrations in HSCs (HSC-T6) incubated with CXCL6 over a 24 hour time-course by ELISA ( Figure 3A and B) Figure 3C and D). ELISA revealed that the α-SMA and TGF-β concentrations were significantly upregulated, peaking at 8 hour for α-SMA (P < 0.001 vs 0 hour group) and 18 hour for TGF-β (P < 0.001 vs 0 hour group). Immunofluorescent staining of α-SMA in HSCs cultured with KC conditioned medium confirmed α-SMA upregulation, peaking at 8 hour ( Figure 3E). These findings confirmed that exposure of HSCs to KC medium, conditioned by previous exposure to CXCL6, can activate these cells.

| CXCL6 activates HSCs by stimulating TGF-β and its downstream Smad3-BRD4
To determine the pathway by which CXCL6 induces activation of HSCs, KCs were treated with CXCL6 with or without the addi-  Figure 5A and B).
The expression of phosphorylated SMAD3 was downregulated following treatment with all three of the inhibitors ( Figure 5B).
Next, KCs were treated with CXCL6 for 18 hour, then the KC conditioned medium was transferred onto 3-day HSCs and cultured for 8 hour. Whole cell extracts were immunoprecipitated with SMAD3 antibody or an equal amount of rabbit IgG and blotted with an BRD4 antibody ( Figure 5C). Exposure to CXCL6 led to the coimmunoprecipitation of the SMAD3 and BRD4 antibodies indicating that CXCL6 stimulates the interaction between Smad3 and BRD4.
Finally, a ChIP assay was performed on the same set of cells using the SMAD3 and BRD4 antibodies ( Figure 5D). The results indicated that CXCL6 stimulates the direct binding of BRD4 to the C-MYC promoter and C-MYC to the EZH2 promoter, but not BRD4 to the EZH2 promoter.

| DISCUSSION
The targeting of signalling pathways that perpetuate myofibroblast activation is a promising therapeutic strategy to combat chronic fibrosis. Fibrosis contributes to the pathogenesis of a number of chronic disorders affecting the liver, kidney, lungs and heart, which collectively account for a substantial proportion of disease-related mortalities in developed countries. 28   KCs are specialized macrophages that reside in the liver and secrete a variety of proinflammatory and profibrogenic factors such as cytokines, chemokines, prostaglandins, leukotrienes and complement factors in response to various stimuli. 31 Chemokines reported to be released by KCs include CXCL1, CXCL2 and CXCL8, which attract neutrophils, 32 CCL1, CCL2, CCL25 and CX3CL1, which promote the infiltration of bone marrow−derived monocytes, [33][34][35][36] and CXCL16, which attract natural killer T cells. 37 We therefore speculate that activated KCs may be the source of CXCL6 in fibrotic livers and that the CXCL6 secreted by these macrophages, in turn, promotes the release of TGF-β by KCs via the CXCR1/2-EGFR pathway. Kupffer cells reside alongside HSCs in the liver, therefore the secreted TGF-β has profibrogenic effects on neighbouring HSCs.
As stated above, our findings revealed that CXCL6 activated the CXCR1/2-EGFR pathway in KCs. However, interestingly, CXCL6 did not activate HSCs via this same pathway, despite the fact that CXCR1/2-EGFR is also expressed in HSCs. In HSCs, CXCL6 must induce activation via a distinct pathway. This may be investigated further in subsequent studies.
One of the limitations of our study was that we only investigated the secretion of TGF-β in KCs in response to CXCL6. TGF-β is considered the predominant cytokine to trigger HSC activation and fibrogenic transdifferentiation; however, it is possible that CXCL6 may alter the secretion of other fibrosis-related cytokines in KCs.
This should be addressed in future studies. Another limitation was that we did not genetically knock down the expression of liver CXCL6 to confirm its role in promoting the secretion of TGF-β and F I G U R E 6 CXCL6 is Highly Expressed in a Long-Term CCl 4 -Induced Liver Fibrosis Mouse Model. A, Serum ALT/AST activity was detected after mice had been treated with CCl 4 (200 or 300 mg/kg) for 0, 2, 4, 6 or 8 wk. B-D, Serum hydroxyproline, TGF-β and CXCL6 levels were determined. E, Representative images of H&E stained liver sections (×400 magnification). F-H, Relative mRNA and protein levels in the mouse liver tissues were detected by RT-PCR and western blotting (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001 vs 0 wk group activating HSCs in vivo. Future studies analysing the effects of knockdown mutants in the animal model would verify our findings.
In conclusion, our findings reveal the role of CXCL6 in promoting the release of TGF-β by KCs. TGF-β is a potent inducer of HSC activation via a signalling cascade that involves the SMAD2 protein and the BRD4/C-MYC/EZH2 axis. HSC activation and the resulting transdifferentiation into fibrogenic myofibroblasts play a pivotal role in the pathogenesis of liver fibrosis. Our findings, therefore, provide important insight into the complex mechanisms of HSC activation that contribute to liver fibrosis.

ACKNOWLEDGEMENTS
This work was supported by The National Natural Science Foundation of China (Grant no. 81400629)

CONF LICTS OF INTEREST
The authors declare that they have no conflict of interests to disclose.