Idiopathic pulmonary fibrosis is a progressive lung disorder of unknown etiology. Previous studies have shown that aberrant activation of the Wnt/β-catenin signaling cascade occurs in lungs of patients with idiopathic pulmonary fibrosis. Given the important roles of the Wnt/β-catenin signaling pathway in the development of pulmonary fibrosis, we targeted this pathway for the intervention of pulmonary fibrosis with XAV939, a small molecule that specifically inhibits Tankyrase 1/2, eventually leading to the degradation of β-catenin and suppression of the Wnt/β-catenin signaling pathway. Our results demonstrated that XAV939 significantly inhibited the activation of Wnt/β-catenin signaling and attenuated bleomycin-induced lung fibrosis in mice, and thus improved the survival of mice with lung injury. Interestingly, previous investigations have confirmed that endogenous and exogenous mesenchymal stem cells could be recruited to the injured lung, although the exact effects of these cells are debatable. To determine the effect of Wnt/β-catenin signaling in the epithelial differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs), we established a coculture system that contains BM-MSCs and alveolar type II epithelial cells. The in vitro experiments demonstrated that XAV939 could promote the differentiation of BM-MSCs into an epithelium-like phenotype in the coculture system. We also found that XAV939 could inhibit the proliferation and myofibroblast differentiation of NIH/3T3 fibroblasts. This work supports that inhibition of the Wnt/β-catenin signaling pathway may be exploited for the treatment of idiopathic pulmonary fibrosis for which effective treatment strategies are still lacking.
- idiopathic pulmonary fibrosis
- Wnt/β-catenin signaling pathway
- bone marrow-derived mesenchymal stem cells
- alveolar type II epithelial cells
idiopathic pulmonary fibrosis (IPF) is a chronic lung disease, whose etiology remains a mystery (15). It is clinically characterized by a progressive loss of lung function leading to dyspnea and restricted respiration (3). The mean survival time is 2 to 3 years after diagnosis (9, 16). The pathogenesis of IPF is not fully understood and no effective drug treatment is available at present. Thus, it is important to unravel the mechanisms underlying the pathogenesis of pulmonary fibrosis to provide effective treatment targets.
Accumulating evidence suggests that the main pathological characteristics of IPF include aberrant proliferation of fibroblasts leading to formation of scar tissues, which may subsequently result in destruction of the lung architecture (16). Activation of the Wnt/β-catenin signaling pathway can increase the activation and proliferation of fibroblasts, promoting the secretion of collagen fiber (19). Being the classical Wnt signal pathway, the Wnt/β-catenin signaling pathway plays an important role in the proliferation, polarity determination, and growth direction of cells in embryonic development (20, 21). Furthermore, the aberrant activation of the Wnt/β-catenin signaling cascade has been observed in the lungs of patients with IPF (17). Taken together, abnormal activation of the Wnt/β-catenin signaling pathway is closely associated with the occurrence and development of pulmonary fibrosis. Hitherto no effective treatment for IPF has been reported. We therefore tested the effect of a Wnt/β-catenin-specific inhibitor in promoting the repair of the injured lung by suppressing the aberrant proliferation of fibroblasts and the development of pulmonary fibrosis. In addition, we also tried to unravel the possible mechanisms in this process.
Bone marrow-derived mesenchymal stem cells (BM-MSCs) are characterized by a number of features including easy isolation and culture, as well as multipotent differentiation potential along the osteogenic, chondrogenic, adipogenic, and many other lineages (24). Endogenous and exogenous mesenchymal stem cells can be recruited to the injured lung followed by differentiation into fibroblasts to facilitate the development of pulmonary fibrosis, or by differentiation into alveolar epithelial cells to repair the lung injury (30, 40). BM-MSCs have attracted attention for their antifibrotic effects. However, the role of BM-MSCs in pulmonary fibrosis is still inconclusive (11, 18, 27). BM-MSCs have the potential for multidirectional differentiation, including the capacity of differentiation into the epithelial cell lineage (24, 37), but the mechanism that accounts for the differentiation of BM-MSCs into alveolar epithelial cells remains unclear. Alveolar type II epithelial cells (ATII cells), which form the most important bioactive component of pulmonary epithelium, synthesize and secrete pulmonary surfactant (31). BM-MSCs may be induced by ATII cells to an epithelium-like phenotype. Rojas and colleagues (30) believed that the transplanted BM-MSCs are capable of promoting the repair of lung fibrosis by differentiating into alveolar epithelial cells. However, Hashimoto's research suggested that the transplanted BM-MSCs would differentiate into fibroblasts (11). Previous studies have demonstrated that the Wnt/β-catenin signaling pathway is also involved in the growth regulation of many types of stem cells to maintain these stem cells in a state of self-renewal (26). BM-MSCs express a certain level of the endogenous Wnt protein and regulate their own Wnt/β-catenin signaling to maintain proliferation and function through an autocrine or paracrine loop (8). Taking the effects of Wnt/β-catenin signaling in IPF into consideration, we speculated that a small-molecule compound that specifically inhibits the Wnt/β-catenin signaling may promote the differentiation of BM-MSCs into alveolar epithelial cells, and this process may play a role in the repair of bleomycin-induced pulmonary fibrosis.
XAV939 is a small-molecule compound that specifically targets Wnt/β-catenin signaling. XAV939 can maintain the integrity of the death complex by stabilizing the Axin2 structure via inhibiting the activity of the end anchorage polymerase PARP, eventually leading to the degradation of β-catenin and the suppression of Wnt/β-catenin signaling (13, 28). Unlike the constitutive expression of Axin1, the expression of Axin2 is induced by activated Wnt signaling, and Axin2 is involved in the negative feedback regulation (14). However, Axin2 has the same function as Axin1 (5). Therefore, XAV939 can restrain the abnormal activation of the Wnt/β-catenin signaling pathway without affecting the normal function of the cells. Compared with other Wnt signaling pathway inhibitors, one of the advantages of XAV939 lies in its strong specificity, as it does not inhibit other signaling pathways such as NF-κB and transforming growth factor-β (TGF-β) (13).
In the present study, by setting up a bleomycin-induced mouse pulmonary fibrosis model, we investigated the effects of XAV939 on the activation of Wnt/β-catenin signaling pathways and pulmonary fibrosis development in mice. Furthermore, we established a coculture system containing BM-MSCs and ATII cells to determine the effect of XAV939 on the epithelial differentiation of BM-MSCs. XAV939-mediated myofibroblastic differentiation of NIH/3T3 fibroblasts was also investigated.1
MATERIALS AND METHODS
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Experimentation Ethics Review Committee of Nanjing University (ethics approval no. A9089). All surgery was performed under pentobarbital sodium anesthesia, and all efforts were made to minimize suffering.
Mesenchymal stem cell culture and flow cytometric analysis.
C57BL/6 mice (6–7 wk old) were bought from the Animal Feeding Center of Yangzhou University (Yangzhou, China). Isolation and culture of mesenchymal stem cells and flow cytometric analysis were performed according to a previously described protocol (37). The cells were cultured in low-glucose Dulbecco's modified Eagle's medium (DMEM, Hyclone, Thermo Scientific, San Jose, CA) with 10% FBS (GIBCO, Paisley, UK; Invitrogen, Carlsbad, CA), 1% l-glutamine, and a 1% solution of penicillin and streptomycin. Cells were seeded at a density of 1 × 106 cells/ml onto uncoated flasks and cultured in a humidified incubator at 37°C in 5% CO2. The primary cells were split once they were confluent, and the immediate subculture was defined as passage 1. Passages 2, 3, 4, etc. were thus obtained accordingly following further passaging.
For flow cytometric analysis, 1 × 106 passage 5 BM-MSCs were incubated with fluorescence-conjugated primary antibodies at 37°C for 1 h in the dark followed by three washing steps with PBS. Cells were analyzed using a FACSCalibur flow cytometer (Becton-Dickson, San Jose, CA) and data were analyzed with BD CellQuest Pro software (Becton-Dickinson). The following antibodies were employed: phycoerythrin (PE)-conjugated CD29 from Bio-Legend (San Diego, CA), PE-conjugated CD44 from Antigenix America (Huntington Sta., NY), PE-conjugated CD34 from eBioscience (Santa Cruz, CA), FITC-conjugated SCA-1, FITC-conjugated CD106, FITC-conjugated CD11B, and FITC-conjugated CD45 from Millipore (Billerica, MA).
Cell apoptosis was measured by Annexin V-FITC and propidium iodide (PI) staining through flow cytometry. The cells were transplanted to six-well culture plates at a density of 1 × 106 cells/ml and were cultured for 12 h followed by treatment with 10−7 to 10−3 M XAV939 for 24 h. Cells were collected after centrifugation at 300 g for 5 min. Next, cells were washed twice with cold PBS and were resuspended in 500 μl binding buffer at a concentration of 1 × 106 cells/ml. Each cell sample was then stained with 5 μl Annexin V-FITC and 5 μl PI according to the manufacturer's instructions. The cells were then incubated in the dark at 25°C for 15 min. Samples were acquired on a FACSCalibur flow cytometer and analyzed with BD CellQuest Pro software (Becton-Dickinson).
ATII cell line culture.
The ATII cells from Yili Bio-technology (Shanghai, China) were cultured similarly as described for mesenchymal stem cells except that 5% FBS was used.
Indirect coculture experiments.
Indirect coculture was established by using cell culture inserts (0.4 μm pore, 4.5 cm2, Corning, NY) as described (37). Both BM-MSCs and ATII cells were plated at 105/ml at each plating time. We recorded the changes of cell morphology over a period of 14 days. Next, inserts were removed and BM-MSCs were harvested for RT-PCR, Western blotting, and immunofluorescent staining.
NIH/3T3 fibroblast culture.
NIH/3T3 fibroblasts (Chinese Academy of Sciences) were cultured in high DMEM (Hyclone) with 10% FBS (GIBCO), 1% l-glutamine, and a 1% solution of penicillin and streptomycin.
Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan), which measures cell viability, is based on the conversion of an orange-colored product from water-soluble tetrazolium salt (WST-8) by dehydrogenases in live cells. When NIH/3T3 fibroblasts or ATII cells reached 80% confluence, they were routinely passaged using 0.25% trypsin and were diluted 1:3 at each passage. NIH/3T3 fibroblasts or ATII cells were cultured in various concentrations of XAV939 (10−8 to 10−4 M) in 96-well plates followed by the CCK-8 assay at 12 h, 24 h, 36 h, or 48 h according to the instruction from the manufacturer.
FDA and PI staining for morphologic evaluation.
The integrity of the cell membrane was detected using fluorescein diacetate (FDA) (10)/PI staining. Cells were placed on a 24-well culture plate at 1 × 106 cells/ml in 0.5 ml culture medium and cultured for 12 h followed by treatment with 10−7-10−3 M XAV939 for 24 h. Next, the cells were stained with 5 μg/ml PI and 4 μg/ml FDA, followed by observation under a fluorescent microscope (Nikon ECLIPSE TE2000-S, Tokyo, Japan).
The bleomycin-induced mouse pulmonary fibrosis model.
Male C57BL/6 mice (6–7 wk old) were purchased from the Medical School of Yangzhou University. All mice were maintained under standard conditions with free access to water and laboratory rodent food. To set up the bleomycin-induced mouse pulmonary fibrosis model, mice were anesthetized with pentobarbital sodium (3 mg/kg). Next, mice received a single, slow intratracheal injection of 5 mg/kg bleomycin (Nippon Kayaku, Tokyo, Japan) dissolved in 50 μl of saline with MicroSprayer (Penn-Century, Wyndmoor, PA). Control mice received 50 μl of saline instead. Mice were killed on the indicated days. In some experiments, a more severe pulmonary fibrosis model was induced with 10 mg/kg bleomycin.
For the evaluation of the effect of XAV939 on bleomycin-induced pulmonary fibrosis, mice received both bleomycin and XAV939. Specifically, 10 days after administration of bleomycin, XAV939 was administered intraperitoneally at 5 mg/kg for 11 consecutive days. Mice were killed 21 days after bleomycin instillation.
Mouse lungs were obtained for histopathology, collagen assay, RT-PCR, and Western blotting. Type I collagen is the most abundant fibrillar collagen and it is preferentially synthesized in pulmonary fibrosis (22, 33). Therefore an examination of the expression of collagen I can reflect the degree of fibrosis. The effect of XAV939 on the mortality rate in mice that received bleomycin (10 mg/kg) was also studied. Specifically, XAV939 was administered intraperitoneally at 5 mg/kg every day for 7 days beginning on day 10 after bleomycin dosing (10 mg/kg). Mouse survival rate was recorded.
Lower left lungs were fixed in 4% (wt/vol) neutral phosphate-buffered paraformaldehyde for 24 h, dehydrated, transparentized, and embedded in paraffin. Lung tissues were cut into 5-μm sections that were stained with hematoxylin-eosin (H&E) for structured observation, or with Masson's trichrome stain for detection of collagen deposits. Determination of hydroxyproline content was carried out using a kit from Nanjing JianCheng Bioengineering Institute (Nanjing, China) according to the instruction by the manufacturer.
RNA extraction and reverse transcriptase polymerase chain reaction.
cDNA was generated from RNA extracts derived from cultured BM-MSCs and lung tissues using a reverse transcription kit (Transgen, Beijing, China). GAPDH was used as an internal control. PCR was performed using the primers as shown in Table 1.
For detecting the effect of XAV939 on the differentiation of BM-MSCs or NIH/3T3, immunofluorescence analysis of BM-MSCs or NIH/3T3 was performed as described previously (35). The following primary antibodies were employed: Rabbit anti-collagen I, rabbit anti-occludin, and rabbit anti-SP-C (all antibodies were purchased from Abcam, Cambridge, MA). Alexa Fluor 488- or 594-conjugated goat anti-rabbit (Invitrogen) was used as the secondary antibody. The images were captured using a confocal fluorescence microscope (Olympus, Tokyo, Japan).
Proteins were purified from either BM-MSCs or lungs. Western blot analysis of cellular lysates was performed as previously described (39). Proteins were separated using 12% SDS-polyacrylamide gel electrophoresis and were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes using standard procedures. The following primary antibodies were employed: rabbit anti-β-catenin, rabbit anti-vimentin, rabbit anti-α-smooth muscle actin (α-SMA), rabbit anti-collagen I, rabbit anti-cytokeratin 18 (CK18), rabbit anti-cytokeratin 19 (CK19), rabbit anti-SP-C, rabbit anti-occludin, and mouse anti-β-actin (Abcam). Horseradish peroxidase-conjugated goat anti-rabbit/mouse IgG (Boster, Wuhan, China) was used as the secondary antibody. Immunoreactive protein bands were detected using an Odyssey Scanning System (LI-COR).
Experimental results are expressed as means ± SD. Differences were analyzed for significance (P < 0.05) by either one-way ANOVA or Fisher's exact test using SPSS for Windows (version 11.0; SPSS, Chicago, IL).
Development of pulmonary fibrosis in bleomycin-treated mice.
The development of pulmonary fibrosis in mice treated with 5 mg/kg bleomycin was assessed by H&E (Fig. 1A), Masson's trichrome staining (Fig. 1B), and the hydroxyproline content assay for collagen (Fig. 1C). We found that the alveolar structure was destroyed with hyperplasia of fibroblasts and collagen deposition from day 7 to day 21. A number of genes were significantly overexpressed after bleomycin administration. These include well-documented Wnt/β-catenin target genes as well as genes associated with epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) remodeling and fibrosis [i.e., S100A4, cyclin D1, fibronectin, lamin A, connective tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1), vimentin, α-SMA, and collagen I] (Fig. 1, D and E). The upregulation of β-catenin was also confirmed by Western blotting (Fig. 1F).
Effects of XAV939 on pulmonary fibrosis induced by bleomycin.
We explored the inhibitory effects of XAV939 on bleomycin-induced pulmonary fibrosis following intratracheal spray of bleomycin (5 mg/kg). XAV939 significantly reduced the severity of pulmonary fibrosis in the lungs compared with saline controls as assessed by H&E (Fig. 2A), Masson's trichrome staining (Fig. 2B), and hydroxyproline content assay for collagen (Fig. 2C). To confirm the inhibitory mechanisms of Wnt/β-catenin signaling in bleomycin-induced pulmonary fibrosis, the expression of a number of well-documented Wnt/β-catenin target genes was measured. The pulmonary expression of these genes, as well as genes associated with EMT and ECM remodeling and fibrosis, was significantly increased after bleomycin administration, However, the increased expression of these genes was suppressed in bleomycin-treated mice that received XAV939 (Fig. 2, D and E). Compared with saline controls, expression of vimentin, α-SMA, and β-catenin after bleomycin administration was markedly increased, which was significantly inhibited by XAV939. Epithelial markers such as CK18, CK19, and occludin were significantly downregulated in lungs following bleomycin-induced injury. However, treatment with XAV939 resulted in marked increases of the expression of these proteins in lung tissues, compared with the levels seen in animals that received bleomycin alone (Fig. 2, F and G).
Effect of XAV939 on survival rate of mice injured by a high dose of bleomycin.
We chose a high dose of bleomycin (10 mg/kg) to investigate the effect of XAV939 on the survival rate after bleomycin-induced injury in mice. Only 20% of mice survived on day 17 after bleomycin treatment. However, mice demonstrated a significantly higher survival rate (65%) following intraperitoneal injection of XAV939 at a daily dose of 5 mg/kg (Fig. 2H).
Effect of XAV939 on proliferation and myofibroblast differentiation of NIH/3T3 fibroblasts.
Idiopathic pulmonary fibrosis is characterized by aberrant proliferation and recruitment of fibroblasts and finally by formation of scar tissues leading to the subsequent destruction of the lung architecture. To explore the effect of XAV939 on fibroblast proliferation, NIH/3T3 fibroblasts were cultured with various concentrations of XAV939 and cell proliferation was measured. Our results demonstrated that XAV939, at a concentration higher than 1 × 10−6 M, could remarkably inhibit the proliferation of NIH/3T3 fibroblasts (Fig. 3A). To determine the effect of XAV939 on myofibroblast differentiation of NIH/3T3 fibroblasts and suppression of the Wnt signaling pathway in the cells, levels of α-SMA, collagen I, and β-catenin were measured. The results indicated that the expression of α-SMA, collagen I, and β-catenin in NIH/3T3 fibroblasts was restrained by XAV939 (Fig. 3, B and C), suggesting compromised myofibroblast differentiation. We also demonstrated that XAV939, at a concentration of 1 × 10−3 M, could significantly increase death of NIH/3T3 fibroblasts (7.28 ± 0.08% vs. control 5.82 ± 0.05%, P < 0.05). No significant differences were observed between control and exposure at concentrations ranging from 10−7 M to 10−4 M (Fig. 3, D and E).
Impact of XAV939 on apoptosis and differentiation of ATII cells.
At a concentration higher than 1 × 10−5 M, XAV939 could remarkably inhibit the proliferation of ATII cells (Fig. 4A). XAV939 even at 10 μM had no effect on the levels of CK18, occludin, and SP-C (Fig. 4B), although the expression of β-catenin was suppressed significantly (Fig. 4C). After exposure to XAV939 at 10−4 M for 24 h, no significantly different levels of cell death were observed. However, significantly elevated cell death could be observed when concentrations of XAV939 were raised to 10−3 M (1.93% ± 0.03% vs. control, 1.28 ± 0.03%, P < 0.05; Fig. 4, D and E).
Identification of BM-MSCs.
Following culture for 5 days, the cells isolated from mouse bone marrow demonstrated the morphology of a long spindle, spiral, and radial arrangement (Fig. 5A). These cells expressed CD29, CD44, CD106, and SCA-1, but not CD11b, CD45, CD34 (Fig. 5B). Taken together, these results suggest that the cells possessed the main features of mesenchymal stem cells.
Impact of XAV939 on differentiation of BM-MSCs cocultured with ATII cells.
When BM-MSCs and ATII cells were cocultured for 14 days, BM-MSCs transformed to a paving stone shape resembling epithelial cells and the transformation was further promoted by XAV939 (Fig. 6A). To confirm the differentiation, we measured the expression of some epithelial cell markers of BM-MSCs on day 14 following coculture by RT-PCR and Western blotting. Expression levels of occludin and SP-C were further confirmed by immunofluorescence assay. Increased expression of CK18, CK19, occludin, and SP-C on BM-MSCs following coculture with ATII cells was observed, and the expression was further increased by the addition of XAV939. In contrast, BM-MSCs expressed no epithelial cell markers without coculture (Fig. 6, B–F).
Effect of XAV939 on suppression of β-catenin expression in BM-MSCs.
The β-catenin content in BM-MSCs declined following coculture with ATII cells. The addition of XAV939 could further reduce the β-catenin levels in BM-MSCs either in the presence or absence of XAV939. These results suggest that under the condition of coculture with epithelial cells, the Wnt/β-catenin signaling pathway of BM-MSCs was restrained, and XAV939 could further promote such inhibition (Fig. 6, E and F).
Pulmonary fibrosis is a systemic disorder that involves many pathogenic factors. Currently, it is particularly desirable to develop new therapeutic approaches to inhibit fibrosis. The lung injury model induced by bleomycin is appropriate for investigating pulmonary fibrosis and the development of effective treatments. Although the model has certain limitations, such as the incomplete simulation of IPF, some of the activated signaling pathways in IPF patients can also be identified in the model (7). The Wnt/β-catenin signaling pathway is associated with fibroblast activation, tissue repair, and collagen synthesis (4, 32). It is reported that the Wnt signaling pathway is activated and the levels of Wnt isoforms are increased in the lungs of IPF and patients with other forms of pulmonary fibrosis (6). Along this line, recent studies have identified ICG-001, a small molecule that selectively inhibits T-cell factor (TCF)/β-catenin transcription in a Creb-binding protein (CBP)-dependent fashion by repressing the β-catenin-driven gene transcription in a TOPflash luciferase reporter assay and attenuates bleomycin-induced lung fibrosis in mice. Administration of ICG-001 can not only inhibit the occurrence and development of pulmonary fibrosis, but also reverse established fibrosis (12). In this study, we investigated the effect of XAV939 on bleomycin-induced lung fibrosis and tried to reveal detailed pathogenic mechanisms. XAV939 has also been previously used to block the canonical Wnt pathway induced by miRNA 154, which participates in lung fibrosis and seems to upregulate the canonical Wnt/β-catenin signaling (23).
In the present study, we demonstrated that a number of well-documented Wnt/β-catenin target genes were significantly increased after bleomycin administration in the lung, so were genes associated with EMT and ECM remodeling, and fibrosis. These results were consistent with the findings in IPF. Mesenchymal stem cells did not abrogate the histopathologic changes of HCl-induced acute lung injury and pulmonary fibrosis. We have previously shown that inhibition of Wnt/β-catenin signaling by DKK-1, a natural inhibitor of the Wnt/β-catenin signaling pathway, can promote the differentiation of exogenous BM-MSCs into epithelial cells in vivo and thus regulate lung injury and repair, although only a fraction of the exogenous cells are recruited (34). In addition, recruitment of endogenous mesenchymal stem cells to the injured lung is also observed (40). Therefore, we speculated that inhibition of the Wnt/β-catenin signaling may directly promote the repair of lung injury. As the targeting property and efficiency of DKK-1 are relatively poor due to its relatively large size, perhaps a small-molecule compound that promotes the differentiation of BM-MSCs into alveolar epithelial cells by specifically inhibiting the Wnt/β-catenin signaling may have a more efficient therapeutic potential for the repair of pulmonary fibrosis.
Moeller divided the pathological processes involved in bleomycin-induced rodent pulmonary fibrosis into two stages, namely the early inflammatory reaction stage (7 days) and the subsequent fibrosis stage (apparent on day 14, peak on day 21) (25). To avoid the influence of XAV939 on the role of inflammation, we injected XAV939 intraperitoneally starting from day 10 for 11 consecutive days after the administration of bleomycin by the intratracheal route. Our results showed that XAV939 could inhibit the activation of the Wnt/β-catenin signaling in the lung tissue. Furthermore, XAV939 could to a great extent restrain the development of pulmonary fibrosis induced by bleomycin. XAV939 could significantly increase the survival rate of mice following lung injury induced by high-concentration bleomycin. These results suggest that pulmonary fibrosis can be improved by inhibition of the Wnt/β-catenin signaling.
Furthermore, we investigated the possible mechanisms of XAV939 on the suppression of pulmonary fibrosis. Substantial experimental evidence now suggests that fibroblasts can be recruited to the injured region and participate in the repair, followed by the formation of fiber and the secretion of extracellular matrix (1). Upon activation, fibroblasts differentiate into myofibroblasts, which have a substantially increased potential for the synthesis of ECM, resulting in aggravated fibrosis. Wei and colleagues (38) found that Wnt-3a-induced β-catenin activation could stimulate the proliferation and migration of fibroblasts, collagen gel contraction and myofibroblast differentiation, and enhance profibrogenic gene expression. In the present study, we demonstrated that XAV939 could inhibit myofibroblast differentiation and ECM accumulation in vitro and it can also suppress the proliferation of NIH/3T3 fibroblasts. However, no significantly different levels of apoptosis were observed upon administration of XAV939 within a relevant concentration range. Taken together, these results strongly suggest a direct effect of XAV939 on fibroblasts and suggest that cytotoxicity may not be the major effect mediating the inhibition of myofibroblast differentiation.
Recent studies have shown that bone marrow-derived circulating progenitor cells could accumulate in the lung and contribute to pulmonary fibrosis after lung injury (2). In experimental pulmonary fibrosis in mice, bone marrow progenitor recruitment accounts for ∼20% of the fibroblast pool (36). BM-MSCs can be recruited to the damaged tissues and differentiate into specific types of cells involved in tissue repair, but the differentiation direction is uncertain. In this study, we screened the BM-MSCs that were isolated from bone marrow and the results showed that cell surface marker expression was as expected (29). In our in vitro experiment, BM-MSCs differentiated into an epithelium-like phenotype following coculture with alveolar epithelial cells, evidenced by the expression of specific molecular markers of alveolar epithelial cells. XAV939 could significantly suppress Wnt/β-catenin signaling and promote the epithelial differentiation but had little impact on ATII cells. Previous reports suggested that when the Wnt/β-catenin signaling is activated, BM-MSCs can differentiate into fibroblasts or myofibroblasts, and this process can be inhibited by DKK-1 (34). These findings prompted us to propose that inhibition of Wnt/β-catenin signaling could promote the alveolar epithelial cell-mediated differentiation of autologous BM-MSCs into epithelial cells in the damaged lung, and hence the improvement in lung function.
In conclusion, our data demonstrate that XAV939 could significantly improve the pulmonary fibrosis induced by bleomycin through inhibition of the Wnt/β-catenin signaling pathway, and increase mouse survival after bleomycin-induced injury. XAV939 is capable of promoting the differentiation of BM-MSCs to an epithelium-like phenotype in an in vitro coculture system. These results confirm that the Wnt/β-catenin signaling pathway plays an important role in the pathological processes involving pulmonary fibrosis, and that the suppression of Wnt/β-catenin signaling by specific inhibitors could restrain the development of pulmonary fibrosis. We believe that our work has certain clinical significance for developing therapeutic strategies for pulmonary fibrosis.
This work was supported by National Natural Science Foundation of China (81170054), Natural Science Foundation of Jiangsu Province of China (BK2011570), the National Basic Research Program of China (973 program 2010CB945103), and Open Research Fund of State Key Laboratory of Bioelectronics, Southeast University.
No conflicts of interest, financial or otherwise, are declared by the author(s).
C.W., H.Z., and X.H. conception and design of research; C.W., H.Z., Z.S., Y.G., C.N., and Z.L. performed experiments; C.W., H.Z., and W.Q. analyzed data; C.W. and W.Q. interpreted results of experiments; C.W. prepared figures; C.W. drafted manuscript; Z.X. edited and revised manuscript; X.H. approved final version of manuscript.
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