BARD1 mediates TGF-β signaling in pulmonary fibrosis
© André et al. 2015
Received: 1 April 2015
Accepted: 17 September 2015
Published: 29 September 2015
Idiopathic pulmonary fibrosis (IPF) is a rapid progressive fibro-proliferative disorder with poor prognosis similar to lung cancer. The pathogenesis of IPF is uncertain, but loss of epithelial cells and fibroblast proliferation are thought to be central processes. Previous reports have shown that BARD1 expression is upregulated in response to hypoxia and associated with TGF-β signaling, both recognized factors driving lung fibrosis. Differentially spliced BARD1 isoforms, in particular BARD1β, are oncogenic drivers of proliferation in cancers of various origins. We therefore hypothesized that BARD1 and/or its isoforms might play a role in lung fibrosis.
We investigated BARD1 expression as a function of TGF-β in cultured cells, in mice with experimentally induced lung fibrosis, and in lung biopsies from pulmonary fibrosis patients.
FL BARD1 and BARD1β were upregulated in response to TGF-β in epithelial cells and fibroblasts in vitro and in vivo. Protein and mRNA expression studies showed very low expression in healthy lung tissues, but upregulated expression of full length (FL) BARD1 and BARD1β in fibrotic tissues.
Our data suggest that FL BARD1 and BARD1β might be mediators of pleiotropic effects of TGF-β. In particular BARD1β might be a driver of proliferation and of pulmonary fibrosis pathogenesis and progression and represent a target for treatment.
Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive and fatal disease with limited treatment options [1, 2]. Of the main subtypes of IPF, usual interstitial pneumonia (UIP) is typically associated with extensive and persistent lung fibrosis, and non-specific interstitial pneumonia (NSIP) is associated with inflammation and fibrosis. The incidence of these diseases is increasing and is reported to be about 20 per 100,000 [3, 4].
The pathogenesis of IPF is poorly understood, but is thought to be caused by epithelial damage followed by a chronic progression of the healing response, resulting in excessive deposition of extracellular matrix proteins without resolution [5, 6]. Transforming Growth Factor-β (TGF-β) is generated via multiple molecular pathways associated with lung fibrosis [1, 7] and is one of a number of factors known to drive fibrosis. TGF-β signals through Smad3 to induce collagen production, a hallmark of lung fibrosis [8, 9]. Many inflammatory cells, central to fibrosis, store and secrete TGF-β. Coagulation cascade proteins, such as thrombin, are known to induce TGF-β activation , as does oxidative stress . TGF-β is thought to drive epithelial cell apoptosis, epithelial-mesenchymal transition (EMT), fibroblast proliferation, and collagen deposition, key processes to the initiation and progression of lung fibrosis.
Currently, there are no molecular biomarkers in widespread clinical use for IPF. Several susceptibility loci have been identified by genome wide association studies , but they have not been validated as biomarkers or drug targets as yet.
We hypothesized that BARD1 (BRCA1-associated RING domain 1), which is aberrantly expressed and correlated with poor prognosis in lung cancer , might be involved in lung fibrosis. BARD1 is a major binding protein of the breast cancer predisposition gene product BRCA1 and a tumor suppressor in its own right . Bound to BRCA1, BARD1 is essential for BRCA1’s E3 ubiquitin ligase and tumor suppressor activity in DNA repair and cell cycle control . Independently of BRCA1, BARD1 is an inducer of apoptosis by binding to and stabilizing p53 [16–18] and is upregulated in response to hormones, genotoxic stress, and hypoxia [16, 18–20]. However, BARD1 expression is also upregulated in proliferative tissues , where it has essential functions in mitosis [17, 22, 23]. Thus, there is strong evidence that BARD1 is involved in apoptosis and in the control of proliferation.
Alternatively spliced isoforms of BARD1 have been identified in various cancers correlated with disease progression and poor prognosis [13, 24–28]. These isoforms, abundantly expressed in tumors and cancer cell lines, lack the BRCA1-interacting N-terminal RING domain and do not retain tumor suppressor functions. In particular BARD1β has been shown to be essential for cell proliferation and for promoting fibroblast proliferation [23, 25]. BARD1β is considered a driver of tumorigenesis, as its expression correlates with tumor progression, poor prognosis, and decreased survival time of lung cancer patients .
The role of BARD1 in the development and progression of proliferative lung diseases other than cancer remains unknown. Recent reports suggest that the pathogenesis of lung cancer and lung fibrosis share common features , and, for example, persistent epithelial cell damage and enhanced fibroblast proliferation are observed in both diseases, with IPF patients showing an 8–14 fold increase in lung cancer incidence .
Hallmarks of lung fibrosis are thought to be hypoxia, loss of epithelial cells due to apoptosis and/or EMT, and fibroblast proliferation [31, 32]. BARD1 was described as a mediator of stress signals, including hypoxia, towards apoptosis in vitro and in vivo . BARD1 might also be linked to TGF-β signaling, as upregulated BARD1 expression was shown along with TGF-β early response genes in breast cancer  and was associated with endoglin upregulation, a co-receptor for TGF-β .
Based on the key properties of BARD1 and its isoforms, and the observed features that characterize pulmonary fibrosis, we hypothesized that full length (FL) BARD1 and/or its isoforms might play a role in this disease. To test this hypothesis, we investigated the possible mechanisms of BARD1 actions by exploring the regulation of FL BARD1 and isoform expression by TGF-β and by their exogenous overexpression in vitro. We also analyzed BARD1 expression in lung tissues from patients with IPF and those of mice following experimentally induced lung fibrosis.
Materials and methods
Human lung biopsies of patients with Idiopathic Interstitial Pneumonias (IIP), IPF and NSIP, and human control lung tissue samples, were obtained in accordance to an approved protocol by the Institutional Ethics Committees of the University Hospitals Geneva (HUG) and the Hôpital Bichat, Paris. In total, 17 IIP samples and seven control lung tissue samples were analyzed. Control lung tissue samples were obtained from resected lung tissue collected during lung cancer surgery; samples were taken at sites distal to tumors. Five control samples were obtained from patients with other lung diseases, which included emphysema, tuberculosis, and carcinoid dysplasia. All patients were informed and approvals were obtained from the local ethics committees.
Paraffin-embedded sections from human tissue biopsies and air-inflated, perfusion-fixed lungs from mice were immune stained for BARD1 and α-smooth-muscle-actin (α-SMA). The primary antibodies used in the study were: BARD1-N19 (sc-7373; Santa Cruz; dilution 1:25) (BARD1 exon 1); BARD1-C20 (sc-7372; Santa Cruz; dilution 1:20) (BARD1 exon 11); BARD1-BL (A300-263A; Bethyl Laboratories; dilution 1:50) (middle exon 4); α-smooth muscle actin (ab5694; Abcam, dilution 1:100); BARD1-p25 (directed against sequence of alternative open reading frame of BARD1β) [23, 25] (dilution 1:50); p53 (sc-6243 Santa Cruz, dilution 1:20); Bax (sc-493; Santa Cruz; dilution 1:100). Secondary, horseradish-conjugated rabbit or goat antibodies were used at a 1:100 dilution. Then diaminobenzidine DAB staining was performed during 15 min at room temperature. Slides were counterstained with haematoxylin.
Mouse model of bleomycin-induced pulmonary fibrosis
C57BL/6J male mice aged 12–16 weeks were kept in specific pathogen-free conditions. All animal experiments were approved by the Institutional Ethics Committee of Animal Care in Geneva and the Cantonal Veterinary Office. Male mice, weighing 20–30 g, were anesthetized with ketamine/xylazine (i.p., 90/3.8 mg/kg). Bleomycin or saline was instilled intratracheally at a dose of 2U/kg to mice using an endotracheal catheter attached to a 50-ml Hamilton syringe (Hamilton Bonaduz) as previously described . Mice were killed at day 3 or 15 after bleomycin treatment and lungs were processed for subsequent analysis.
The normal human fibroblasts CCD-19Lu, bronchial epithelial cells 16HBE and NuLi-1, alveolar basal epithelial cells A549, and mouse fibroblasts L929 were cultured in RPMI 1640 Medium, GlutaMAX™ (Invitrogen), 10 % fetal calf serum (FCS), 200U/ml penicillin and 200U/ml streptomycin at 37 °C in a 5 % of CO2 incubator. For TGF-β1 treatment, cells were serum starved in 0.5 % FCS overnight prior to stimulation with 10 ng/ml recombinant TGF-β1 (Biolegend) for 24 or 48 h. In the Smad signaling inhibition assay, SB431542 (Abcam) was added to the media at a concentration of 10 μM 1 h prior TGF-β1. Following stimulation cell lysates were prepared for RNA extraction, using Qiagen RNAeasy kit (Qiagen), for protein extraction or cells were fixed for immunofluorescence assays. Details are available in the online supplement.
Results are expressed as mean-standard deviation as indicated and were analyzed with the two-tailed Student’s t test (GraphPad Prism). Significance level was set at p < 0.05.
FL BARD1 and BARD1β expression changes in response to TGF-β
After TGF-β treatment of CCD-19Lu lung fibroblasts, the expression of FL BARD1, a protein of 95 kD, as well as BARD1β, 82 kD, was upregulated after 24 and 48 h (Fig. 1a, b). We also used bronchial epithelial cells 16HBE and NuLi-1 to monitor BARD1 expression after TGF-β treatment. In these epithelial cells the increase in BARD1 expression in response to TGF-β was only moderate, and FL BARD1 expression increased, but less so BARD1β. To investigate whether BARD1 upregulation in response to TGF-β was observed in other cell lines we also tested L929 fibroblasts and A549 lung epithelial cells (Fig. 1c), as well as LL86 and HS888, normal lung fibroblasts and HeLa and HEK 293, epithelial cells (data not shown). FL BARD1 and BARD1β were upregulated in L929 cells with a similar pattern as observed for CCD-19Lu fibroblasts. The response in A549 cells was different from 16HBE and NuLI-1 epithelial cells, and a higher increase of BARD1β expression was observed (Fig. 1c). All other cell lines showed an increase at 24 h, but great variability after 48 h. To demonstrate that BARD1 expression was induced directly by TGF-β, we used a SMAD inhibitor and monitored BARD1 expression in response to TGF-β (Fig. 1d). In the presence of the SMAD inhibitor TGF-β-dependent FL BARD1 and BARD1β upregulation was reduced to levels without TGF-β stimulation.
As hypoxia is thought to also act as driver of lung fibrosis, we investigated whether BARD1 expression was modified by hypoxia in lung fibrosis. Both FL BARD1 and BARD1β were upregulated in lung epithelial cells and fibroblasts upon hypoxic treatment (Additional file 1: Figure S1). Therefore both factors hypoxia and TGF-β are activators of BARD1 expression.
We performed immunofluorescence microscopy to assess the subcellular distribution of BARD1 upon TGF-β treatment. We observed that BARD1 expression and localization changed in parallel to changes in expression of markers of EMT (Fig. 2c). In non-treated cells, although under starving conditions, E-cadherin was observed both at the cell-cell adhesion sites and close to the nuclear membrane. Fibronectin showed nuclear and cytoplasmic localization. BARD1 expression was observed mostly in the nucleus and less in the cytoplasm, and not at cell adhesion sites. In TGF-β treated cells, E-cadherin staining was lost at cell adhesion sites and appeared diffuse in the nucleus and cytoplasm and intense staining was observed in the nucleus in approximately 10 percent of cells. Fibronectin expression was increased with TGF-β treatment and localized to the nuclear membrane and to vesicles at the cell membrane, presumably cell adhesion points. As observed on Western blots, BARD1 staining was generally stronger in the TGF-β treated cells and localized to the nucleus and cytoplasm (Fig. 2c). Specifically elevated BARD1 expression was observed in dots at the cell membrane, as observed for fibronectin staining. Double labeling for fibronectin and BARD1 showed co-localization in the cytoplasm and in particular in the presumed cell adhesion points (Fig. 2d).
Elevated FL BARD1 and BARD1β expression in parallel to fibronectin upregulation and E-cadherin repression, both features of EMT, suggested that BARD1 played a role in this pathway. To determine whether FL BARD1 or BARD1β play a role in TGF-β-mediated EMT induction, we exogenously expressed FL BARD1 or BARD1β in A549 cells and monitored their expression, and the effect thereof on fibronectin and E-cadherin expression and localization.
As previously reported for fibroblasts [23, 25], overexpression of BARD1β induced increased proliferation of A459 lung epithelial cells, which was not observed with FL BARD1 expressing cells or controls (Fig. 3c). Overexpression of FL BARD1 and BARD1β in the fibroblast cell line L929 showed an even more pronounced increase of proliferation for BARD1β, but proliferation arrest for FL BARD1 (Fig. 3d).
As FL BARD1 was known as a mediator of apoptosis [16–18], we tested whether FL BARD1 induced apoptosis in A549 and L929 cells, and to what extent. We stained control cells and FL BARD1 or BARD1β overexpressing cells for annexin v and analysed them by FACS (Fig. 3e-g). Only FL BARD1, but not BARD1β significantly induced apoptosis in both cell lines.
Together these results suggest that FL BARD1 and BARD1β can be upregulated by TGF-β, and increased BARD1β might promote cell proliferation and act in a pathway regulating stability and/or intracellular localization of fibronectin. FL BARD1 upregulation leads to increased apoptosis of epithelial cells and fibroblasts.
BARD1 expression is upregulated in mouse model of lung fibrosis
Despite its limitations, several key features of human lung fibrosis are reproduced in the mouse model of lung fibrosis following administration of bleomycin . These features include epithelial cell apoptosis and fibroblast proliferation and differentiation. We thus investigated whether BARD1 expression in lung tissues from mice with bleomycin induced lung fibrosis reflected the expression pattern observed in vitro and whether it paralleled the progression of the disease in this model. Importantly, this model permits to investigate BARD1 expression at early stages of the disease.
These data show that BARD1 expression is modulated in the bleomycin model of lung fibrosis, and specifically BARD1β is upregulated in cells within fibrotic regions at 15 days post treatment.
BARD1 expression is associated with apoptosis
As FL BARD1 mediates apoptosis by binding to and stabilizing p53, and BARD1-repressed cells are resistant to apoptosis , and apoptosis of epithelial cells is thought to be one of the mechanisms leading to epithelial cell loss in fibrosis, we investigated FL BARD1 expression and its association with apoptosis markers in fibrotic lung tissues of bleomycin treated mice. We observed that BARD1 staining in fibrotic regions coincided with the expression of proteins implicated in the BARD1-induced apoptosis pathway , namely p53 and Bax, (Fig. 5c). BARD1, p53, and the p53–induced pro-apoptotic protein Bax showed prominent staining in the epithelium adjacent to fibrotic areas that also stained for BARD1. These data are consistent with the previously described role of FL BARD1 in apoptosis [16–18].
FL BARD1 and isoform BARD1β are expressed in human lung fibrosis
To determine the specific expression of FL BARD1 and/or BARD1 isoforms on the mRNA level, we performed RT-PCR on RNA extracted from lung biopsies from patients with pulmonary fibrosis (n = 17) and controls (Fig. 6a). Amplification of the entire BARD1 coding region (exon 1 to exon 11) showed no or very low expression of previously identified BARD1 isoforms in healthy tissues, but expression of FL BARD1 and BARD1β, as well as low expression levels of smaller isoforms was detected in IPF tissue samples (Fig. 6a). Using a BARD1β specific primer, we confirmed that BARD1β mRNA was the most frequently expressed in IPF samples.
We then performed IHC with antibody C20 recognizing a C-terminal epitope of BARD1 on tissue samples of NSIP as well as UIP patients. C20 showed BARD1 expression in NSIP and UIP, while only weak staining was observed in control tissues (Fig. 6b). BARD1 expression specifically localized to the fibrotic regions and was observed in all patients tested. The number of positive cells appeared to increase with progression of the fibrotic lesions, while minimal staining was observed in unaffected tissue. These data thus demonstrate that FL BARD1 and the spliced isoform BARD1β are expressed in fibrotic lung tissues and are associated with the disease.
We report here for the first time that the tumor suppressor BARD1 and its pro-proliferative isoform BARD1β are associated with pulmonary fibrosis and presumably involved in its pathogenesis.
BARD1 a novel biomarker and possible treatment target for lung fibrosis
Several proteins, including NADPH oxidase 4, Toll-like receptor 3, CCL18, matrix metalloproteinase-7, and interleukin-8, besides TGF-β, are widely accepted as key players in pulmonary fibrosis . In recent years several genes were identified that are critically involved in the pathogenesis of lung fibrosis [12, 40]. Some of the newly identified genes are involved in hitherto ignored pathways, namely cell-cell adhesion, host defense, and DNA repair .
The BARD1 gene was not discovered as one associated with lung fibrosis in previously reported genetic screens. This could be due to the switching of the splicing pattern of the BARD1 gene that is associated with lung fibrosis, which would need other approaches for detection than screening for mutations or overall expression changes. Indeed there is evidence that SNPs in BARD1 that are associated with a subgroup of high-risk, aggressive, neuroblastoma, promote alternative splicing and expression of BARD1β [25, 41]. Exon and intron sequencing of the BARD1 gene has identified mutations in exons and introns that affect splicing in breast and ovarian cancer . It is therefore possible that SNPs in BARD1 might be found associated with specific subclasses of lung fibrosis.
We hypothesized that the tumor suppressor BARD1, with functions in DNA repair pathways, plays a role in the pathogenesis of lung fibrosis, as previous reports suggested a role of BARD1 in the regulation of proliferation: i) BARD1 expression is upregulated in proliferating cells, ii) BARD1 isoforms are highly upregulated in cancer cells, and associated with uncontrolled or deregulated growth. As uncontrolled growth is also the feature of lung fibrosis, BARD1 expression was expected to be associated with lung fibrosis.
Distinct expression patterns of BARD1 and its isoforms in lung fibrosis and cancer
The pro-proliferative functions of isoform BARD1β were first described in in vitro cultures of epithelial cells  and confirmed in non-transformed fibroblasts . Consistent with its pro-proliferative action BARD1β was found expressed in most epithelial cell-derived cancers, but in combination with other isoforms and absence of FL BARD1 [13, 24, 26, 28]. In fibrotic lung tissues, the most significant expression was found for FL BARD1 and BARD1β. This difference is striking and might reflect the less complex nature of the cells forming the fibrotic tissue. Cancer cells have the capacity to evade apoptosis, grow independently of growth signals, have activated telomerase and unlimited growth, escape immune surveillance, and become invasive and metastatic . Compared to these changes the proliferating fibroblasts have acquired only little cancer cell-like capacities. The activation of telomerase might be associated with lung fibrosis . Fibrotic fibroblasts and epithelial cells express FL BARD1 and BARD1β. Loss of FL BARD1 leads to genetic instability, a hallmark of cancer cells, but not so much of lung fibrosis.
A role of TGF-β in up-regulating FL BARD1 and its isoforms
The data presented here confirm that TGF-β induces BARD1 expression and demonstrate for the first time that this occurs in lung fibrosis. Specifically, we observe upregulation of BARD1β on the mRNA and protein level in epithelial cells and fibroblasts. The upregulation of BARD1β in fibroblasts is consistent with the view that BARD1β acts as a pro-proliferative factor in a TGF-β pathway in lung fibrosis. Importantly, upregulation is cell type specific with marked differences between epithelial cells and fibroblasts. One explanation of these differences could be the differential expression of TGF-β receptors on epithelial cells and fibroblasts. It is also possible that other regulatory pathways are involved, such as microRNAs (miRNAs). Several miRNAs are implicated in TGF-β signaling, as recently reported for lung cancer . The BARD1 3’UTR is target of a large number of miRNAs, including those induced by TGF-β signaling, that regulate the pattern of BARD1 isoform expression . Further studies are required to unravel miRNA-mediated regulation of the TGF-β-dependent BARD1 expression.
In vivo, the fibrotic lung tissues from humans and mice showed an upregulation of FL BARD1 and BARD1β. In vitro experiments confirmed the induction of FL BARD1 and BARD1β on the transcriptional and protein level in response to TGF-β to some extent. In vivo, however, their expression might also be driven by hypoxia. Hypoxia is also a driver of miRNA expression in cancers and could play an additive role on the regulation of BARD1 expression, as BARD1 upregulation was observed in response to hypoxia in vivo [16, 20]. This could be reproduced in lung epithelial cells and fibroblast in culture (Additional file 1: Figure S1).
Mechanism of action of FL BARD1 and its isoforms in epithelial cells and fibroblasts
The intracellular functions of FL BARD1 and its isoforms are not well understood. FL BARD1 is mostly bound to BRCA1, and the BRCA1-BARD1 heterodimer has E3 ubiquitin ligase activity [14, 15]. Several proteins have been discovered that are ubiquitinated and degraded by the BRCA1-BARD1 ubiquitin ligase [14, 47], including proteins that act in a signaling pathway from genotoxic stress to repair . Proteins that play a role in cell cycle control, such as the centrosomal protein gamma-tubulin, and Aurora kinases, have also been identified as targets of the BRCA1-BARD1 E3 ligase [22, 23]. The isoforms lacking the BRCA1 interaction domain still retain the domain required for interaction with the ubiquitin ligase target protein. This is the case for BARD1β, which lacks the RING domain, but contains the Aurora kinase binding domain [23, 25]. BARD1β, by binding to Aurora A and B, antagonizes their degradation. Aurora kinases are required for progressing through cell cycle checkpoints and for completion of cytokinesis . In non-transformed cells, Aurora kinases are synthesized during S-phase and gradually degraded during mitosis, in cancer cells their upregulation leads to uncontrolled progression through mitosis. This is also observed by FL BARD1 repression and/or BARD1β upregulation [23, 25]. Thus, BARD1β antagonizes the function of FL BARD1 and BRCA1 and acts pro-proliferative by overriding mitotic checkpoints, in epithelial cells and fibroblasts. The expression of BARD1β in fibrotic lung tissues is consistent with the view that BARD1β might drive fibroblast proliferation by its binding to Aurora kinases. It is possible that BARD1β also binds to other hitherto not identified target proteins of the FL BARD1-BRCA1 ubiquitin ligase.
Fibrotic fibroblasts produce excessive collagen, and FL BARD1 and/or other isoforms could be involved in lung fibrosis due to their ubiquitin ligase function which could contribute to the accumulation of collagen via (de)regulation of collagen or other extra cellular matrix protein production and/or turnover, which remains to be investigated.
Hypothesis and future directions
TGF-β has long been proposed as a key molecule central to the pathogenesis of lung fibrosis . However, it has thus far proved to be an intractable target largely due to the pleiotropic effects of this molecule, as well as its potential tumorigenic effects, and indirect approaches to modulate TGF-β signaling are considered [51, 52]. We suggest that BARD1 might act as downstream activator of epithelial cell apoptosis and fibroblast proliferation and therefore might present a strong candidate target molecule for the treatment of lung fibrosis and possibly other diseases in which fibro-proliferation is a feature.
We are grateful to Dr. YongQiang Zhang for help with immunohistochemistry experiments, to Dr. Eva Dizin, Dr. David CJ Howell and Dr. Maxim Pilyugin for discussions, technical advice, and for providing invaluable samples. This work was supported by grant from the Swiss National Research Foundation to IIF (SNF no 31003A-110038), and a Wellcome Trust Programme grant (no GR071124MA) to GJL and RCC.
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