Background
Asthma afflicts more than 300 million people worldwide and is one of the most common chronic disorders of childhood that affects an estimated 6.2 million children under the age of 18 [
1]. Asthma is a chronic inflammatory disease that results in the narrowing of the airways, tightening of the chest, shortness of breath, and coughing. The hallmarks of asthma include airway obstruction, chronic wheezing, airway hyperresponsiveness, airway remodeling, inflammation, and mucus hypersecretion. While current treatments include corticosteroids, leukotriene antagonists, and long-acting β2 agonists, these therapies are not effective in preventing or reversing airway remodeling in patients suffering from chronic allergic asthma [
2]. In addition, the beneficial anti-inflammatory effect of corticosteroids is not without many adverse effects. Therefore, further understanding of the mechanisms underlying airway remodeling is required to develop therapies that target the molecules involved in structural changes, including fibrosis and epithelial thickening.
Recently, vitamin D has received more attention as an effective immunomodulator in extra-musculoskeletal tissues. Vitamin D is a steroid hormone that is synthesized from cholesterol in the skin, or can be ingested through dietary sources. Vitamin D goes through sequential hydroxylation steps in the liver and kidney resulting in its final active form, 1,25(OH)
2D
3 or calcitriol. Calcitriol regulates bone, calcium, and phosphate metabolism through vitamin D receptor (VDR). The VDR forms a heterodimer with the retinoid X receptor and regulates gene expression in the nucleus. The 1,25(OH)
2D
3 can also bind to the VDR on the plasma membrane to exert rapid responses via production of second messengers [
3]. The increased incidence of asthma [
4] associated with increased vitamin D deficiency might suggest a link between the two in the pathogenesis of asthma [
5]-[
7]. Previous studies suggest that vitamin D status is a strong predictor of childhood asthma, with deficiency more frequent in children suffering from asthma compared to non-asthmatic controls [
8],[
9]. This association of vitamin D deficiency and asthma is not limited to children and includes prospective studies that suggest vitamin D insufficiency and deficiency are linked with severe and uncontrolled adult asthma [
10]. While this data suggests that vitamin D deficiency results in an increased risk for asthma and allergy, the amount of vitamin D that might be required to prevent or lessen the severity of an asthma attack is still unknown. A prospective study found that vitamin D supplementation in asthmatic children prevented asthma exacerbation triggered by acute respiratory infection [
11]. However, other reports do not suggest a role for vitamin D supplementation. In two recent studies vitamin D supplementation in asthma patients did not result in significant difference compared to the placebo group [
12],[
13].
Airway remodeling, a consequence of long-standing asthma, reduces lung function and is not regulated well with current therapies. There is currently limited information on the role of vitamin D as a potential inhibitor of airway remodeling in asthma. Previous work has demonstrated that vitamin D plays a role in airway smooth muscle, but the role of vitamin D in the epithelium is not well understood [
14],[
15]. One of the key cytokines involved in the airway remodeling process is TGF-β, which is released from degranulated eosinophils and mast cells to induce multiple localized effects resulting in airway remodeling [
16]-[
18].
Airway epithelial cells are located between the host and external environment, designating their key role in the protection of the host against microorganisms, dust, and other allergens. TGF-β has been suggested to initiate fibrosis in the airway epithelial cell through activation of epithelial mesenchymal transition (EMT) signals [
19],[
20]. One of the main transcription factors involved in this process is Snail. It has been demonstrated that Snail forms a transcriptional repressor with Smad3 and Smad4 to promote EMT [
21]. Activation of EMT signaling permits the cells to differentiate into myofibroblasts, enabling invasion and migration outside of the epithelium. Increased myofibroblasts in the submucosa secrete collagen and extracellular matrix, thereby, contributing to the subepithelial fibrosis in airway remodeling [
22].
While vitamin D has been shown to have immunomodulatory properties in immune cells [
10] and airway smooth muscle cells [
15], the properties of vitamin D in epithelial cells are currently lacking. Here, we investigated the effect of active form of vitamin D, calcitriol, and TGF-β on EMT markers in bronchial epithelial cells. While most studies examining EMT
in vitro utilize TGF-β1, there is little information on the effect of TGF-β2, which has also been identified as a contributor to the development of asthma [
23],[
24]. Our findings suggest that calcitriol can inhibit epithelial cell motility induced by both TGF-β isoforms, but calcitriol-induced inhibition of EMT characteristics in response to TGF-β1 and TGF-β2 could involve different molecular mechanisms.
Methods
Cell culture
Bronchial epithelial cells, BEAS-2B cells (ATCC, Manassas, VA) were seeded on 6-well culture dishes at 1.5 × 105 cells/ml and cultured in BepiCM media (ScienCell, Carlsbad, CA) supplemented with 10% FBS. When cells reached 50% confluence, the media was changed and cells received serum-free media for 24 hours. Cells were then subjected to stimulation with either 0.1% 95% ethanol (vehicle) or 100 nM calcitriol (Sigma-Aldrich, St. Louis, MO) for 24 hours. TGF-β1 or TGF-β2 (PeproTech, Rocky Hill, NJ) were then added to the cells for an additional 48 hours.
RNA isolation and qPCR
Total RNA was isolated using the RNeasy Plus Mini kit (Qiagen, Valencia, CA) according to manufacturer's instructions. RNA concentration was quantified using a Nanodrop (Thermo-Scientific, Rockford, IL). First-strand cDNA synthesis was performed using 1 μg total RNA with oligo dT, 5X reaction buffer, MgCl2, dNTP mix, RNAse inhibitor and Improm II reverse transcriptase as per manufacturer's instructions in the Improm II reverse transcription kit (Promega, Madison, WI). Following the first strand synthesis, real time PCR was performed using 3.2 ng of cDNA, 10 μl SYBR Green PCR Master Mix (BioRad Laboratories, Hercules, CA) and forward and reverse primers (10 pmol/μl) (Integrated DNA Technologies, Coralville, IA) using a real time PCR system (CFX96, BioRad Laboratories, Hercules, CA). Relative mRNA transcripts levels were normalized against an internal housekeeping gene and sample differences determined using
ΔΔCt relative method. A complete list of the primers and their sequences is provided in Table
1.
Table 1
Primer sequences used for qPCR experiments
E-cadherin | Forward | AAG AAG CTG GCT GAC ATG TAC GGA |
| Reverse | CCA CCA GCA ACG TGA TTT CTG CAT |
Snail | Forward | TTT CTG GTT CTG TGT CCT CTG CCT |
| Reverse | TGA GTC TGT CAG CCT TTG TCC TGT |
MMP2 | Forward | AGA AGG ATG GCA AGT ACG GCT TCT |
| Reverse | AGT GGT GCA GCT GTC ATA GGA TGT |
MMP9 | Forward | ATT TCT GCC AGG ACC GCT TCT ACT |
| Reverse | CAG TTT GTA TCC GGC AAA CTG GCT |
18S | Forward | TCA ACT TTC GAT GGT AGT CGC CGT |
| Reverse | TCC TTG GAT GTG GTA GCC GTT TCT |
GAPDH | Forward | TCG ACA GTC AGC CGC ATC TTC TTT |
| Reverse | ACC AAA TCG GTT GAC TCC GAC CTT |
N-cadherin | Forward | TGT GGG AAT CCG ACG AAT GGA TGA |
| Reverse | TGG AGC CAC TGC CTT CAT AGT CAA |
Vimentin | Forward | AGA ACC TGC AGG AGG CAG AAG AAT |
| Reverse | TTC CAT TTC ACG CAT CTG GCG TT |
Western blot
BEAS-2B cells were washed with ice-cold PBS and incubated with 0.15 ml of modified RIPA lysis buffer with protease and phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich, St. Louis, MO). Protein concentration was determined by the BCA protein assay kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions. Each sample of protein containing 15 μg of protein was mixed with equal volume of Laemmli buffer containing 10% 2-mercaptoethanol. Proteins were resolved on 10-20% polyacrylamide gel (BioRad, Hercules, CA). Proteins were transferred onto a nitrocellulose membrane (BioRad, Hercules, CA), which were subsequently blocked with 5% non-fat dry milk for one hour. The membrane was then incubated overnight at 4°C with E-cadherin (ab15148, Abcam, Cambridge, MA), Snail (NBP1-19529, Novus, Littleton, CO), and Vimentin (sc-6260, Santa Cruz, Dallas, Texas). Following, a horseradish peroxidase-conjugated secondary antibody (Novus, Littleton, CO) was added to the membrane. The protein expression was detected by ECL chemiluminescence detection reagents (Bio-Rad, Hercules, CA). The immunoreactivity was captured by the ChemiDoc™ MP System (Bio-Rad Laboratories, Hercules, CA). Membranes were stripped and reprobed for N-cadherin and GAPDH. Results were normalized against housekeeping gene GAPDH.
Zymography
Supernatant from the stimulated BEAS-2B cells were collected, centrifuged to remove debris, and subjected to gelatin zymography. An equal volume of conditioned media from each sample was then run on an 8% SDS-PAGE containing gelatin (1.0 mg/ml). Following electrophoresis, the gels were washed in Triton X-100 and incubated for 18 hours in 50 mM Tris-HCl buffer containing 0.2 mol/L NaCl and 10 mMol/L CaCl2. Gels were stained with Brilliant Blue R250 and destained. MMP activity was assessed by observing white bands against a dark background.
Invasion assay
Following stimulation with calcitriol and/or TGF-β1/TGF-β2, BEAS-2B cells were isolated and reseeded onto 250 μg/ml of growth factor reduced Matrigel™ coated transwell inserts at 1.5 × 105 cells/ml. Supernatant from these cells was used as the chemoattractant. After 48 hours, cells on the top of the inserts were removed and the remaining cells on the bottom were fixed and stained with the Diff-Quick stain kit (Fisher Scientific, Waltham, MA). The density of the invasive cells on the bottom of the insert was examined by counting the cells in five fields per insert locations under a light microscope at 20x magnification.
Scratch wound healing assay
BEAS-2B cells were seeded, allowed to reach 75% confluency, and serum starved for 24 hours and subsequently stimulated with 100 nM calcitriol for 24 hr. A wound line was generated by using a sterile 10 μl pipette tip followed by the addition of 10 ng/ml TGF-β1 or TGF-β2 for an additional 48 hours. Images were obtained at 0 and 48 hours using a light microscope at 20x magnification with a digital camera under bright field illumination using an Olympus CKX41 microscope.
The distance between the edges of the wound was measured at six different areas from the wound edge-to-edge using ImageJ software. The area between the wound edges was measured at each time point using ImageJ software (as described previously by Dr. Kees Straatman, Advanced Imaging Facilities, University of Leicester, Leicester, UK). The measurements were then converted into a percentage using the formula: % of wound closure = (measurement at 48 h/measurement at time 0 h) * 100; then to obtain the % of wound closure: 100% - % of wound remaining [
25].
Data analysis
Data is expressed as mean × SEM from three or four independent experiments. Multiple group comparison was performed using one-way analysis of variance with a Dunnett or Tukey post-hoc test. GraphPad Prism v5.0 was used to analyse data with a p value of <0.05 considered significant.
Discussion
In normal epithelium, injury to the epithelium leads to the release of soluble factors including cytokines, chemokines, prostaglandins, transforming growth factor (TGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), matrix metalloproteinase (MMP), which can all promote cell adhesion junction remodeling and migration during wound repair [
27]. Activation of fibroblasts and myofibroblasts is important for wound closure and healing, but may become aberrant in the presence of increased inflammation. Myofibroblasts can originate from different precursor cells including fibroblasts, mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, smooth muscle cells, and epithelial cells which are derived through induction of a process termed epithelial-mesenchymal transition (EMT). Following injury to the epithelium, an epithelial cell will go through architectural rearrangement to enable spreading, migration and secretion of extracellular matrix (ECM) [
27],[
28].
Chronic asthma is characterized by structural changes in the lung termed airway remodeling resulting in a decline in lung function regardless of anti-inflammatory treatment [
2]. The airway epithelial cells are at the frontlines of protection in the airway. Following damage from pollutants or allergens, the epithelium can repair itself through initiation of the EMT process. In general, epithelial stress will initiate the generation of mesenchymal cells for tissue generation and tissue closure, with attenuation of EMT following the completion of repair. However, it is thought in the context of inflammation in the asthmatic airway, the release of EMT signaling is not discontinued, but amplified [
29]. During the repair process, cell-cell and cell-adhesion contacts are remodeled with loss of the epithelial marker such as E-cadherin and increased expression of mesenchymal markers of vimentin, N-cadherin, and α-smooth muscle actin (α-SMA). Additionally, epithelial cells will also secrete MMPs in response TGF-β1, indicating a possible role in migration of epithelial cells through the basement membrane [
30]. These changes in the polarized epithelial cell result in functional changes, allowing the epithelial cells to become motile and degrade the underlying ECM and move below the epithelium. There is evidence to suggest that airway remodeling and hyperresponsiveness in asthma may be driven by repeated exposures to allergens or environmental toxins, leading to the inappropriate repair of airway epithelial cells [
31],[
32].
Eosinophils commonly secrete TGF-β. However other cells including macrophages, lymphocytes, fibroblasts, epithelial cells, and mast cells can also secrete TGF-β [
33]. The number of eosinophils found in the asthmatic lung has been found to be increased in bronchial biopsies of asthmatic patients [
34] and BAL fluid in mice [
35]. One of the major cytokines secreted by eosinophils is that of TGF-β [
36]. TGF-β has also been found to be increased in the BAL fluid and tissue of asthmatic patients. However, which isoform is expressed and the tissue location is still debatable. Overall, TGF-β1 [
37] and TGF-β2 [
24] were found to be in higher amounts in the BAL fluid of asthmatics compared to control patients. Both TGF-β1 and TGF-β2 were found to be expressed by eosinophils and contribute to increase number of eosinophils in the airway [
23],[
36],[
38]. The pathogenesis of airway remodeling has been thought to be an activation of the epithelial-mesenchymal trophic unit in which increased levels of TGF-β contribute to the activation of epithelial cells to transform into myofibroblasts [
39]. While TGF-β1 has been found to be involved in the EMT process, it has not been established whether TGF-β2 has the same effect. Our results here are consistent with previous contributions in that we observe BEAS-2B cells treated with TGF-β1 have increased markers of EMT with a loss of E-cadherin. Conversely, TGF-β2 treatment of BEAS-2B cells presented an expression pattern of EMT markers which was different from that induced by TGF-β1.
Although the contribution of EMT in asthma is still highly debated, there is evidence that epithelial cells contribute to the pool of myofibroblasts [
22]. This increase in myofibroblasts may be a part of the airway remodeling process as noted in chronic asthma. TGF-β1 has been demonstrated to be responsible for differentiation into myofibroblasts and this effect was found not be abrogated by corticosteroid treatment [
40],[
41]. Therefore, finding an effective alternative therapy to anti-inflammatory drugs is of great significance. Calcitriol was shown to prevent the effects of EMT in rat lung and mouse lung fibroblasts treated with TGF-β [
42]. Anti-inflammatory effect of vitamin D on a steroid resistant gene was also observed in airway smooth muscle cells treated with TNFα and IFNγ [
43]. We were, therefore, interested in observing the effects of calcitriol on TGF-β treated BEAS-2B cells. Markers of EMT, such as loss of E-cadherin, increased expression of N-cadherin, and vimentin, were observed at the mRNA and protein level following treatment with TGF-β1. This increase in TGF-β was impeded by pre-stimulation with calcitriol, as indicated by qPCR and Western blot. Our data suggest that both TGF-β1 and TGF-β2 enhance BEAS-2B cell invasion and MMP2 mRNA expression and that this process is abrogated by calcitriol.
A recent study included adult asthma patients on the inhaled ciclesonide and levalbuterol combined with an initial dose of 100,000 IU of vitamin D
3 followed by a daily dose of 4,000 IU. In this study, there were no differences in the indices of asthma control, asthma attacks, or improved quality of life in adult asthmatics compared to the placebo group. Sputum eosinophilia, lung function, or airway hyperreactivity were also not improved following treatment with Vitamin D
3 [
12]. However, in patients that had responded well to the vitamin D
3 treatment or had reached a 25-hydroxyvitmain D level of 30 ng/ml or greater, these patients had a lower rate of first exacerbation rates, and lower overall rates of exacerbation and treatment failures [
11]. Unfortunately, the authors did not determine if vitamin D treatment modified sputum or blood inflammatory biomarkers. None-the-less, these results support the therapeutic benefit of vitamin D in allergic asthma, perhaps as an adjunct therapy. Another recent study investigated the role of vitamin D supplementation in asthmatic children aged 6-18 years with mild asthma and insufficient vitamin D levels. Following 6 weeks of 2,000 IU of vitamin D daily, there was no difference in the effect of vitamin D compared to placebo on methacholine challenge test, IgE levels, airway cytokines, and eosinophilia although there was a significant increase in serum vitamin D levels. However, the relatively small sample size of 36 patients of this study prevents any discernable conclusions [
13].
The Institute of Medicine recommendations for adequate vitamin D intake to maintain 25(OH)D
3 serum value at 20 ng/ml or 50 nM. However, many clinical laboratories continue to routinely report a value of 20 ng/ml as inadequate and that value 21-29 ng/ml (52.5-72.5 nM) are insufficient [
44]. Vitamin D supplemented in the diet of OVA-sensitized mice resulted in a decreased severity of airway remodeling [
45]. These mice, supplemented with 10,000 IU/kg or 2,000 IU/kg of vitamin D ended up with serum 25(OH)D levels of 67.13 ng/ml and 31.00 ng/ml, respectively [
46]. Vitamin D supplementation reduced airway hyperresponsiveness, airway remodeling, and BALF cytokine levels [
45]. However, vitamin D supplementation did not fully reverse the effects of allergic airway inflammation. The physiological range of calcitriol has been observed to be around 0.05 to 0.16 nmol/L. The ability of bronchial epithelial cells to convert inactive vitamin D to its active form has recently been demonstrated, indicating that the local concentration of calcitriol is much higher at the local cellular level [
47]. Therefore, to address the difference between sufficient and supplementation values, higher calcitriol levels of 100 nM were used in this study. While the results of clinical trials utilizing vitamin D in asthma patients have inconsistent results, vitamin D supplementation may still prove to be beneficial to those suffering with asthma Additional careful and controlled studies are warranted to address the controversy on the role of vitamin D deficiency in the pathogenesis of asthma.
The differences in mRNA and protein levels following TGF-β1 or TGF-β2 stimulation suggest that the induction of N-cadherin, snail, and vimentin may occur through different receptors. TGF-β binds to heterodimeric type I and type II TGF-β receptors. There are seven type I receptors and five type II receptors, of which the heterodimeric association of these serine/threonine receptors determines the specificity of the ligand signaling [
48]. Co-receptors, such as betaglycan, can also modulate TGF-β1 signaling which is imperative for TGF-β2 signaling [
45]. The mechanisms mediating EMT may be dependent on Smad when stimulated with TGF-β1. This process may be increased in the presence of cytokines, including TNF-α [
30], IL-22 [
49], IL-4 and IL-17 [
50]. Johnson
et al. [
22] observed increased expression and nuclear translocation of Snail, a transcriptional repressor of E-cadherin and a potent inducer of EMT, in the airway epithelial cells of HDM-exposed mice and increased TGF-β upregulation following allergen challenge. Furthermore, the authors also found increased phosphorylated-Smad3 and Snail1 in TGF-β/EGF-induced EMT [
22]. Additionally, deficiency in the PI3K inhibitor phosphatase and tensin homolog (PTEN), has been associated with increased myofibroblast differentiation [
51]. Cytokines such as TNF-α could also induce EMT through Snail stabilization and increased migration and invasion of tumor cells. The cytokine IL-6 was recently shown to induce migration of bronchial epithelial cells in PI3K/Akt/GSK-3β/β-catenin dependent manner suggesting that this pathway may be contributing to the mesenchymal population of cells often found in asthma through induction of EMT signals [
52],[
53]. TGF-β1 has also been found to induce nuclear translocation of relA/p65 of NF-κB, inducing NF-kB gene activity and down-regulating PTEN promoter activity and protein expression via NF-kB [
54].
The findings in this study suggest that calcitriol prevents the migration and invasion of TGF-β-treated bronchial epithelial cells. The differences in mRNA and protein data (Figure
5) indicate that calcitriol mediates this process through different mechanisms. Calcitriol has been shown to inhibit NF-κB nuclear translocation into human bronchial smooth muscle cells by decreasing importin α3 expression via VDR [
15]. VDR expression was found to be decreased in OVA-sensitized and challenged mice fed with a vitamin D deficient diet [
55]. Therefore, importins represent a potential target for vitamin D in alleviating allergic immune responses [
56]. Also, deletion of VDR in mouse embryonic fibroblasts reduced the levels of NF-κB p65 protein and the activity of translation regulators eIF2α and protein kinase R [
57]. Therefore, the underlying mechanisms of vitamin D regulation and EMT and its involvement in airway remodeling in the airway need further elucidation and may be a point of therapeutic interest.
Competing interests
The authors declare that they have no competing interests.