Detrimental pro-senescence effects of vitamin D on lung fibrosis
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The multiple biological effects of vitamin D and its novel activities on inflammation and redox homeostasis have raised high expectations on its use as a therapeutic agent for multiple fibrogenic conditions. We have assessed the therapeutic effects of 1α,25-Dihydroxyvitamin D3, the biologically active form of vitamin D, in the context of lung fibrosis.
We have used representative cellular models for alveolar type II cells and human myofibroblasts. The extension of DNA damage and cellular senescence have been assessed by immunofluorescence, western-blot and senescence-associated β-galactosidase activity. We have also set up a murine model for lung fibrosis by intraperitoneal injections of bleomycin.
Vitamin D induces cellular senescence in bleomycin-treated alveolar epithelial type II cells and aggravates the lung pathology induced by bleomycin. These effects are probably due to an alteration of the cellular DNA double-strand breaks repair in bleomycin-treated cells.
The detrimental effects of vitamin D in the presence of a DNA damaging agent might preclude its use as an antifibrogenic agent for pulmonary fibrosis characterized by DNA damage occurrence and cellular senescence.
Actin, alpha 2, smooth muscle, aorta (α-smooth muscle actin, α-SMA)
Alveolar epithelial cell
Analysis of variance
Alveolar type I cell
Alveolar type II cell
Bovine serum albumin
Cyclin dependent kinase inhibitor 1A (p21)
Cyclin dependent kinase inhibitor 2A (p16)
Collagen type I alpha 1 chain
Collagen type III alpha 1 chain
Cytochrome P450 family 24 subfamily A member 1
DNA double-strand breaks
Fetal bovine serum
H2A histone family member X
Phosphate buffered saline
Focal adhesion kinase (FAK)
Reactive oxygen species
Senescence associated β-galactosidase activity
Sodium dodecyl sulfate
Standard error of the mean
Transforming growth factor beta 1
Tumor protein p53 binding protein 1
Tubulin alpha 1a
Vitamin D receptor
Vitamin D is a hydrophobic secosteroid involved in the homeostasis of calcium, magnesium, iron, phosphate and zinc. The biological form of vitamin D (1α,25-Dihydroxyvitamin D3, from now on vitamin D) exerts its activity through the binding to the vitamin D receptor (VDR), a member of the nuclear hormone receptor superfamily (Pike and Christakos 2017). Besides its role on mineral metabolism, vitamin D regulates some chronic conditions including autoimmune, cardiovascular and respiratory diseases (Finklea et al. 2011). Vitamin D and its analogs are also active in the regulation of fibrosis that characterizes multiple chronic diseases such as renal, cardiac, liver or pulmonary fibrosis (Ding et al. 2013; Ito et al. 2013; Meredith et al. 2015; Zhang et al. 2015). Regarding the latter condition a preventive treatment with vitamin D supplementation ameliorated the severity of lung fibrosis probably due to its anti-inflammatory effects. However, the “therapeutic” role of vitamin D supplementation has not so far been assessed in the context of lung fibrosis. Idiopathic pulmonary fibrosis (IPF) is a form of progressive interstitial pneumonia of unknown etiology (King Jr. et al. 2011). IPF is an example of aging with median diagnosis at 66 years and estimated survival of 3–4 years. In essence, IPF pathogenesis is the consequence of an excessive matrix deposition leading to tissue scarring and irreversible organ injury probably due to a persistent input of damage and tissue repair response. A cellular state called senescence is implicated in the tissue repair program and in IPF physiopathology. Essentially, cellular senescence is an irreversible state of proliferation induced by multiple stressors such as oxidative stress, DNA damage, protein instability and telomere attrition. Its primary role is to make the cell damage inflicted visible for the immune system in order to draw the elimination of damaged cells. Multiple senescence biomarkers accumulate in both fibroblasts and epithelial cells in IPF lungs including the expression of CDKN1A (p21), CDKN2A (p16), senescence-associated β-galactosidase activity (SA-βgal) and DNA damage (Kuwano et al. 1996; Schafer et al. 2017). The occurrence of cellular senescence in IPF, in contrast to other fibrogenic conditions, has a detrimental role in the progression of the disease (Hecker et al. 2014; Lv et al. 2013; Shivshankar et al. 2012).
A number of evidences support the hypothesis that epithelial injury and impaired regeneration are sufficient to activate fibroblasts in the context of IPF. It has been proposed that cellular senescence induced by persistent epithelial damage may be the origin of such defective regeneration and the promotion of fibrosis. Indeed, accelerated epithelial senescence seems to play a role in the development of IPF (Aoshiba et al. 2013; Aoshiba et al. 2003; Chilosi et al. 2013; Kuwano et al. 2016). By other hand, vitamin D treatment or vitamin D receptor (VDR) have been implicated in the attenuation of fibrosis of various organs (Ding et al. 2013; Ito et al. 2013; Meredith et al. 2015; Zhang et al. 2015). Less is known, however, about its role in lung fibrosis. Some previous reports indicated that supplementation of vitamin D ameliorated the fibrotic effects induced by bleomycin (Zhang et al. 2015; Tan et al. 2016; Zhang et al. 2013). The common fact to these works is the preventive administration of vitamin D that, very probably, mediated these effects through its well-known anti-inflammatory properties.
We have evaluated the effects of the biological active form of vitamin D, 1α,25-Dihydroxyvitamin D3 or calcitriol, in two in vitro models for human alveolar type II (ATII) cells and myofibroblasts in terms of DNA damage, cellular senescence and fibrogenic activation. In addition, we have set up an IPF model consisting of the systemic exposure of mice to the antibiotic bleomycin in order to assess the potential “therapeutic” effect of vitamin D. The results indicate that only in the presence of the DNA damaging agent (bleomycin) vitamin D induces an alteration in the correct repair of DNA double-strand breaks (DSBs) and the induction of cellular senescence in epithelial cells. In our model of myofibroblasts, however, the response to bleomycin is the fibrogenic activation rather than their commitment to a senescence state. Moreover, our in vivo results indicate that rather than inhibiting lung fibrosis the treatment with vitamin D aggravates the disease probably due to the excessive presence of DNA damage in the form of DSBs especially in epithelial cells. Altogether our results suggest a detrimental role of vitamin D supplementation in lung fibrosis thus precluding its use as an antifibrogenic agent for IPF.
Materials and methods
Alveolar epithelial cells type II (A549, ATCC) and human myofibroblasts (Magro-Lopez et al. 2017) (Abyntek Biopharma) were maintained in DMEM medium supplemented with 10% FBS (Sigma), 2 mM glutamine and 100 U/mL of penicillin and streptomycin (Lonza). We used the active form of vitamin D (1α,25-Dihydroxyvitamin D3 or calcitriol; Sigma-Aldrich (cat.#D1530). Vitamin D stock was 10 μM in ethanol. Treatments were performed in cells maintained in DMEM supplemented with 10% hormone-depleted serum. This serum was prepared by using the anion exchange resin AGR1-X8 from BIO-RAD (cat.#1401441) as previously described (Zambrano et al. 2014). Bleomycin sulfate (cat.#CAYM13877–50) was purchased to VWR.
Analysis of proteins by western-blot
Cell monolayers were washed with ice-cold PBS and lysed in triple-detergent lysis buffer [50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 100 μg/ml PMSF, 2 μg/ml pepstatin, 2 μg/ml aprotinin, 2 μg/ml leupeptin, and phosphatase inhibitors cocktail 2 or 3 (cat.#P5726, P0044, Sigma-Aldrich)]. SDS-PAGE and immunoblotting were performed under standard conditions. Basically, samples in Laemmli buffer (30 μg/lane) were separated through 8% or 12% gels and transferred to nitrocellulose membranes for 90 min at RT in the presence of 20% methanol and 0,1% SDS. Membranes were blocked with 3% BSA in PBS-Tween 0,05% (PBST-BSA) and incubated O/N at 4 °C with specific antibodies diluted in PBST-BSA. Antibodies used were: γH2AFX (cat.#05–636, Millipore), CDKN1A (cat.#sc-6246, Santa Cruz Biotech), CDKN2A (cat.#04–239, Millipore), Cdkn2a (Millipore, cat.#PA5–20379), ACTA2 (cat.#MA5–11547, Thermoscientific), VIMENTIN (cat.#MA1–10459, Thermoscientific), Vitamin D receptor (VDR) (cat.#12550S, Cell Signaling), Tata box-binding protein (TBP) (cat.#818, abcam), tubulin (TUBA1A) (cat.#T9026, Sigma-Aldrich) and actin β (ACTB) (cat.#8224, abcam).
Quantitative real-time RT-PCR (RT-qPCR)
COLA1A (Collagen type I)
COLA3A (Collagen type III)
ACTA2 (α-smooth muscle actin, α-SMA)
Reactive oxygen species were detected with the probes DA-DCFH (D6883, Sigma-Aldrich) and MitoSOX (M36008, Invitrogen) by flow-cytometry.
ROS detection by 2′-7′-dichlorofluorescein diacetate (DA-DCFH)
Cells growing in DMEM supplemented with 10% hormone-depleted serum were seeded at a density of 32,000 cells/cm2 in MW12 plates. At the following day the medium was replaced by fresh medium and the cells were pre-treated for 2 h with 5 nmol/L vitamin D (1α,25-Dihydroxyvitamin D3) and then treated for 24 h with bleomycin (12,5 μg/mL) and/or 5 nmol/L vitamin D. The cultures were then washed twice with Hank’s balanced salt solution (HBSS) and incubated for 1 h with HBSS. After that, the cells were incubated with DA-DCFH (20 μM) in HBSS for 1 h, washed and trypsinized. Trypsin was neutralized with HBSS-FBS 2% and the cells were collected and analyzed immediately by flow cytometry. The measurements were carried out using FACSCanto (BD Bioscience) cytometer. Cell debris as represented by distinct low forward and side scatter were gated out for analysis; 25,000 gated events for each condition were analyzed with the FACSCanto and Flow Jo software.
Detection of mitochondrial ROS by MitoSOX
The treatments were carried-out as for the DA-DCFH probe. Cells were incubated for 1 h with 5 μM MitoSOX in HBSS and then processed for flow cytometry.
The controls for ROS induction employed in these experiments consisted of treatments for 1 h with 50 μM terbutyl hydroperoxide (cat.#416,665, Sigma-aldrich).
Senescence-associated β-galactosidase assay (SA-βgal)
SA-βgal assays were performed as previously described by (Dimri et al. 1995). Micrographs were taken in a microscope TS100F (Nikon) equipped with a digital camera DS-L1 (Nikon).
Cells were seeded in 8-well chambers (cat.#154,534; Thermofisher Scientific) at a density of 20,000 cells/well. The following day the cells were treated as indicated in the corresponding experiments. Immunofluorescence was performed as previously described (Zambrano et al. 2014). Basically, cells were fixed in 2% PFA in PBS for 10 min at RT and permeabilised with 0.1% Triton X-100 and 0.1% sodium citrate (5 min/RT). Preparations were then washed with PBS and washing solution (PBS/0.25% BSA/0.1% Tween 20), blocked for 30 min with blocking solution (washing solution + 2.5% BSA), and incubated overnight with antibodies against TP53BP1 (NB-100-304, Novus Biologicals), PTK2 (focal adhesion kinase FAK; cat.#PA5–16676 Thermosicentific), ACTA2 (α-SMA; cat.#MA5–11547, Thermoscientific). Preparations were then washed with washing solution and incubated with secondary antibodies conjugated with alexa fluor dyes (488, 546) from Life Technologies (cat.#A-11029, cat.#A-11035) for 1 h at RT. Nuclei were counterstained with DAPI, and samples were mounted with ProLong Diamond (cat.#P36961; Life Technologies). Cell images were captured with a fluorescence microscopy (Zeiss Axio) equipped with a camera (Axiocam MRm) and AxioVision software. DNA damage foci were counted from > 150 cells for each experimental condition.
Chromatin immunoprecipitation (ChIP) assays
Cells growing in DMEM supplemented with 10% hormone-depleted serum, were seeded in 150-mm dishes (6 × 106 cells/plate) and the next day pretreated with 5 nmol/L of vitamin D (1α,25-Dihydroxyvitamin D3) for 2 h. Cells were then treated with bleomycin (12,5 μg/mL) for 6 h, washed and incubated in the presence or absence of 5 nmol/L vitamin D. After 48 h, cells were washed, and lysed following specifications of the magnetic-bead based ChIP Assay Kit (17–10,085, EMD Millipore), and sonicated in a Bioruptor (UCD-200TM; Diagenode). Immunoprecipitated DNA was purified with the QIAquick PCR purification kit (cat.#28,106, Qiagen). In each immunoprecipitation, 2–3× 106 cells and 1 μg of the following antibodies: normal IgGs (cat.#sc-2027, sc-2025, Santa Cruz Biotech.), Histone H3 (cat.#ab1791; abcam), Acetylated Histone H3 (cat.#06–599; Upstate) and H3K27-3me (cat.#ab 6002; abcam) were used. DNA was amplified with primers P16(− 277): 5’-GTCCCTGCCCCTTTGCTATT and P16(+ 95): 5’-ACGGGTCGGGTGAGAGTC, that amplify a region encompassing the proximal P16 promoter (P16INK4a promoter) at the human CDKN2A locus. Real-time PCR was performed by using the powerUp SYBR Green mix and the Quantstudio-3 system from Applied Biosystems. The analysis was performed by the percentage input method (Haring et al. 2007).
Mice, bleomycin-based model of lung fibrosis and immunohistochemistry
Lung fibrosis was induced in pathogen-free 8- to 10-wk-old (weight 18–22 g) female C57BL/6 J mice by two intraperitoneal (i.p.) injections of bleomycin, diluted in 0,9% saline (0,1 mg/Kg b.w.), at days 0 and 2. At day 11, after the acute inflammatory response, the treatments with vitamin D (1α, 25-Dihydroxyvitamin D3) or vehicle were initiated (5 ng/ g b.w.). The doses employed and the protocol consisting of various i.p. injections have previously been described (Tan et al. 2016; Lee et al. 2015; Wang et al. 2016; Schapochnik et al. 2018). 1α,25-Dihydroxyvitamin D3 or the equivalent volume of vehicle was dissolved in saline immediately before the injections. Mice received an injection on days 11, 14, 17, 21 and 24 and were killed at day 28. The mice were divided randomly into the following experimental groups (five mice per group): a) saline only, b) bleomycin, c) bleomycin + vehicle and d) bleomycin + vitamin D (1α, 25-Dihydroxyvitamin D3). These experiments were repeated three times and were approved by the Committee of Bioethics and Animal Welfare of the Instituto de Salud Carlos III and Community of Madrid (file reference: PROEX-312). Protocols used followed the guidelines for animal protection reported by the Spanish national law RD 53/2013.
Lungs and liver were removed from each animal, dissected and fixed in 4% buffered formalin and embedded in paraffin wax or flash frozen with OCT compound. Four-micrometer sections from paraffin embedded tissue were stained with hematoxylin and eosin for assessment of tissue morphology or with trichrome to identify collagen.
Immunohistochemistry was performed on 4 μ-sections of paraffin-fixed embedded tissue. Antigen retrieval was performed with citrate buffer (pH 6) using a Microwave Tender Cooker (Nordic Ware) and a 700 W microwave (15 min 700 W/15 min 350 W). Endogenous peroxidase activity was inhibited with 0.3% H2O2 in methanol (25 min) and after that the sections were permeabilized and blocked simultaneously with PBS + 1%BSA + 0,1% TX-100, during 30 min. The slides were then washed once with PBS for 5 min. Endogenous biotin, biotin receptors and avidin binding sites were blocked by using the avidin/biotin blocking kit from Vector Laboratories (cat.#SP-2001). The slides were washed with PBS and incubated ON, in a humidity chamber, with the primary antibody diluted in blocking solution (PBS + 1%BSA). Incubations with the secondary biotinylated antibodies and development were performed with the kits MP-7402 (ImmPRESS) and SK-4605 (ImmPACT VIP HRP substrate) from Vector Laboratories, following their instructions. Methyl green (cat.#M8884-5G; Sigma-Aldrich) was used as counterstaining and xylene substitute mountant (cat.#1,900,231; Thermofisher) as mounting medium. Primary antibody used was γH2AFX (1:1000, cat.#05–636 Millipore). Damaged cells were counted from representative immunohistochemical fields for each experimental condition.
Statistical significance of data was determined by applying a two-tailed Student’s t test or the analysis of variance followed by the Newman–Keuls or Bonferroni post-tests for the experiments with more than two experimental groups. P < 0.05 is considered significant. Significance of the analysis of variance post-test or the Student’s t test is indicated in the figures as *, P < 0.05; **, P < 0.01; and ***, P < 0.001. Statistics were calculated with the Prism 7 software (GraphPad Software). Data were subjected to the Shapiro-Wilk test and D’Agostino and Pearson omnibus test to verify their normality. The results presented in the figures are means ±SEM. Experiments were repeated at least two times.
Effects of vitamin D and bleomycin in A549 cells and human lung fibroblasts
Effects of vitamin D in A549 cells [alveolar type II cells (ATII)]
Effects of vitamin D in human lung myofibroblasts
The increase in the levels of DNA damage induced by vitamin D in the presence of bleomycin is not mediated by ROS
The increase in the levels of CDKN2A induced by vitamin D in the presence of bleomycin is due to a posttranscriptional mechanism
Vitamin D treatment aggravates bleomycin-induced lung fibrosis by increasing DNA damage
We have analyzed the effects of the biological active form of vitamin D (calcitriol or 1α, 25-Dihydroxyvitamin D3) on representative cellular models for ATII cells and myofibroblasts, in terms of DNA damage, cellular senescence and fibrogenic activation. We have also assessed its potential antifibrotic therapeutic role in a mouse model of IPF based on intraperitoneal injections of bleomycin.
Our results indicate that low concentrations of bleomycin induce DSBs and cellular senescence more efficiently in A549 cells. In a previous report, Aoshiba et al. also described that immortalized A549 cells rapidly become senescent upon bleomycin treatment (Aoshiba et al. 2003). In the presence of a DNA damaging agent, the decrease in DNA damage expression as a consequence of DNA repair was lower in vitamin D treated cells than in the control ones, suggesting an alteration in the cellular DSBs repair capabilities of A549 cells. This alteration would only take place in cells treated with bleomycin as cells treated with vitamin D in the absence of bleomycin do not exhibit more DNA damage than the controls and cells treated with vitamin D and subjected to 3Gy of γ radiation showed no alterations in the cellular repair capacities of DSBs, at least, at the times assessed.
Vitamin D receptor is expressed in both cell types and fully functional as determined by the ligand induction of CYP24A1 expression. Neither the replication rates nor the ROS levels were increased in cells exposed to 5 nmol/L vitamin D. However, under the presence of a DNA damaging agent, vitamin D induced a posttranscriptional induction of CDKN2A in A549 cells that together with the expression of DNA damage and the SA-βgal activity contributed to the establishment of a canonical DNA damage-induced senescence program or premature senescence.
Bleomycin causes fibrotic lesions in C57BL/6 mice within a short period of time. It has been reported that the main histological hallmarks of IPF are present in bleomycin treated animals but it largely depends on the route of bleomycin administration (Chua et al. 2005). For instance, intratracheal instillation, the standard route of administration, produces a bronchiocentric fibrosis, whereas intravenous or intraperitoneal administration induces subpleural scarring similar to IPF (Chua et al. 2005). Bleomycin rapidly induces an acute inflammatory response lasting up to 8 days followed by the pure fibrotic response consisting of matrix deposition and lung architecture distortion out to 28 or 35 days (Moeller et al. 2008). Assuming this sequence of events, treatments during the first 7 days can be considered as preventive while treatments during the later stages after day 7–10 can be considered as therapeutic (Moeller et al. 2008). Some reports have indicated that supplementation of vitamin D ameliorated the fibrotic effects induced by bleomycin (Zhang et al. 2015; Tan et al. 2016; Zhang et al. 2013) but these effects were probably due to a preventive, anti-inflammatory treatment of vitamin D.
Bleomycin exposure of myofibroblasts however resulted in a fibrogenic activation that could be inhibited by the treatment with vitamin D as previously described. This differential response to DNA damage might be due to an inherent resistance of myofibroblasts to bleomycin ROS-induced DNA damage. A growing body of evidence indicates that epithelial fibroblast interactions play an essential role in the development of IPF (Noble 2008; Selman and Pardo 2006; Yanagi et al. 2015). Two types of AECs line up the alveolar wall, ATI and ATII cells. As result of the persistent tissue damage there is a net loss of alveolar epithelium with the subsequent integrity loss of the basement membrane. ATII cells are more resistant than ATI cells to damage and become activated to stimulate fibroblasts to produce extra cellular matrix components. Our results support this notion. Severely damaged cells with a phenotype compatible with ATII cells are lining the alveoli and in areas of abnormal re-epithelization. In addition, epithelial cells from bronchioles show clear signs of DNA damage in contrast to myofibroblasts from fibrotic foci such as those embedded at subpleural foci. These observations suggest that epithelial cells assimilate the bulk of the damage inflicted and trigger the activation and proliferation of fibroblasts.
These findings indicate vitamin D exacerbates the occurrence of fibrosis probably by increasing DNA damage primarily in epithelial cells of lungs subjected to a bleomycin insult. The presence of DNA damage and probably the onset of cellular senescence in ATII cells would compromise its function in the alveolar homeostasis maintenance. This abnormal alveolar regeneration has been observed in lungs from patients with IPF (Noble 2008; Selman and Pardo 2006; Kawanami et al. 1982).
Although some epidemiology studies suggest a potential relationship between vitamin D deficiency and advanced lung disease (Forli et al. 2004; Mascitelli et al. 2010), a systematic review of studies related to health outcome and vitamin D intakes shows no relationship between vitamin D supplement and the reduction in overall mortality of any cause (Chung et al. 2009; Manson et al. 2018; Newberry et al. 2014). Our study suggests that vitamin D supplementation could have an adverse effect in patients with signs of lung fibrosis.
Vitamin D exhibited an unexpected detrimental activity in the presence of a DNA damaging agent. This activity is probably due to a suboptimal capability repair of DSBs associated to the establishment of a senescence phenotype of ATII cells and to the exacerbation of the lung pathology induced by bleomycin. The detrimental effects of vitamin D in the presence of a DNA damaging agent might preclude its use as an antifibrogenic agent in pulmonary fibrosis characterized by DNA damage occurrence and cellular senescence such as the idiopathic pulmonary fibrosis.
We thank the core facilities of the Functional Unit for Research into Chronic Diseases (UFIEC-ISCIII) for technical help.
This work was supported by Grants MPY-1038/14 and MPY-1146/16 from Institute of Health Carlos III (ISCIII) to Alberto Zambrano and SAF-2015-71140-R from Ministry of Economy and Competitiveness (MINECO) to Isabel Liste.
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TG and EM-L performed, designed experiments and analyzed data. JM and RG-M performed experiments and analyzed data. MJF-A designed experiments and analyzed data, IL performed, designed experiments and analyzed data. AZ performed, designed experiments, analyzed data, wrote paper and conceived the project. All authors read and approved the final manuscript.
The experiments involving animals were approved by the Committee of Bioethics and Animal Welfare of the Instituto de Salud Carlos III and Community of Madrid (file reference: PROEX-312). Protocols used followed the guidelines for animal protection reported by the Spanish national law RD 53/2013.
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