Background
Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodeling. Airway remodeling and airway obstruction have several features in common such as airway smooth muscle (ASM) cell hyperplasia and hypertrophy, as well as increase in vascular permeability and angiogenesis [
1,
2], both of which have been the target for numerous therapeutic regimens.
Recently, several growth factors and cytokines secreted by inflammatory cells have been implicated in ASM cell growth and division. Among these, Vascular endothelial growth factor (VEGF)-A (hereafter called VEGF) has been implicated in asthma-related inflammation and remodeling [
3,
4].
Various cytokines, cellular elements, oxidative stress, and protease/antiprotease levels affect lung fibroproliferation, remodeling, and function, which may be influenced by vitamin D levels [
5]. Moreover, previous studies have suggested the active involvement of VEGF in the pathogenesis of asthma, which may be mediated by 1,25-dihydroxycholecalciferol [1,25(OH)2 D3], the active form of vitamin D. In addition, 1,25(OH)2D3 has also been shown to inhibit the proliferation of airway smooth muscle cells [
6]. A disintegrin and metalloproteinase (ADAM)33, a recently discovered ADAM family member, has been found to play a role in the pathophysiology of asthma [
7]. Similar to other protease-type ADAM members, the active site sequence of ADAM33 lies in the metalloprotease domain, implying that this protein promotes the processing of growth factors, various adhesion molecules, cytokines, and cytokine receptors [
8]. ADAM33 is preferentially expressed in smooth muscle cells, fibroblasts, and myofibroblasts, but not in T cells, epithelial cells, or inflammatory cells [
9]. ADAM33 has been linked to allergic airway inflammation; however, its role in the pathophysiology of asthma remains to be proven. Recent studies show that 1,25-(OH)2D3 exerts direct inhibitory effects on passively sensitized HASMCs in vitro, including inhibition of cell proliferation and expression of ADAM33 [
10,
11]. However, the mechanisms underlying 1,25-(OH)2D3-induced inhibitory effects on ASM cell proliferation and expression of ADAM33 remain poorly defined.
Therefore, in the present study, we aimed to investigate the mechanisms underlying the inhibitory effects of 1,25-(OH)2D3 on VEGF-induced ADAM33 expression and ASM cell proliferation. Evaluation of cell proliferation by bromodeoxyuridine (BrdU) labeling has been described in several cell types and species. The key principle of this method is that BrdU is incorporated as a thymidine analog into nuclear DNA, thus acting as a marker that can be tracked with antibodies [
12].
Methods
Antibodies and reagents
Antibodies against phospho-ERK1/2 (Thr202/Tyr204), phospho-Akt (Ser473), phospho-VEGFR2 (Tyr1175), ERK1/2, Akt, and VEGFR2 were purchased from cell signaling technology (Danvers, MA). Antibody against ADAM33 and β-actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The secondary antibodies were obtained from (Jackson Immunoresearch, West Grove, PA). 1,25-(OH)2D3, TGF-β1, and VEGF were purchased from Sigma-Aldrich (St. Louis, MO). SU1498 was from CalBiochem (La Jolla, CA). PI3-K inhibitor LY294002, and the MAPK/ERK1/2 inhibitor U0126 were obtained from CalBiochem (La Jolla, CA).
Cell culture
Human ASM cells were obtained from ScienCell Research laboratories. Cells were cultured in six-well plates in Smooth Muscle Cell Medium (SMCM) containing 10% FBS and were maintained at 37 °C and 5% CO
2 as previously described [
13]. Cells from passage 3–6 maintained their SMC phenotype and were used in all experiments. ASM cells were characterized by smooth muscle cell markers including smooth muscle α-actin and smooth muscle heavy chain using immunofluorescence. In inhibition experiments, inhibitors of signal transduction pathways were added for 2 h before the addition of VEGF. All inhibitors were dissolved in dimethyl sulfoxide (DMSO; final concentration of 0.1%, vol/vol) and added to the medium. Vehicle controls contained the same amount of DMSO.
Real-time reverse transcriptase–PCR
Total RNA was isolated from ASM cells using a TRIzol regent (Invitrogen) after exposure to VEGF or 1,25-(OH)2D3. Total RNA (2 μg) was reverse transcribed using the oligo (dT) primer and MMLV reverse transcriptase (Promega, Madison, WI) at 42 °C for 90 min. Real-time PCR was performed using an ABI Prism 7500 instrument according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). The following primer pairs were used: ADAM33, forward 5’- CAGGAATGCCAGCTATTATC −3’ and reverse, 5’-GTTTGGTGTGGTTCAAGTTT-3’; and GAPDH, forward 5’-GGCCAAAAGGGTCATCA TC −3’ and reverse, 5’-GTGATGGCATGGACTGTGG-3’. After an initial hot start for 10 min, amplification was performed for 40 cycles consisting of denaturation for 10 s at 94 °C, annealing for 30 s at 56 °C, and extension for 40 s at 72 °C. The amplification kinetics was recorded as sigmoid progress curves for which fluorescence was plotted against the number of amplification cycles. The threshold cycle number (CT) was used to define the initial amount of each template. The CT was the first cycle for which a detectable fluorescent signal was observed. The mRNA expression levels were determined and compared with the GAPDH standard.
BrdU incorporation assay
ASM cells were seeded in 96-well plates and treated with various drugs as indicated in each experiment for indicated times. At the end of treatment, BrdU incorporation was assayed by incubating the cells with BrdU for 0.5–1 h using a BrdU Cell Proliferation Assay Kit (Calbiochem, San Diego, CA) according to the manufacturer’s instructions.
Establishment of ADAM33 overexpressing cell lines
To generate ADAM33 overexpressing vectors, the ADAM33-coding sequences were obtained by reverse transcription PCR and cloned into pMXs-based retroviral plasmid (Addgene). Human ASM cells were infected as described [
14], to establish ADAM33 overexpressing ASM cells (ASM cells-ADAM33), and ASM cells infected with retrovirus containing blank pMXs vector (ASM cells-vector) were used as the control group.
Cell cycle analysis
ASM cells were cultured in the complete medium with 1,25-(OH)2D3 for 2 h before treated or not with 50 ng/ml of VEGF for 48 h. All the cells were collected, and 1 × 106 cells were centrifuged, resuspended in ice-cold 70% ethanol and stored at −20 °C until further analysis. Washed cells were stained by 0.1% Triton X-100 in 0.01 M phosphate-buffered saline (pH 7.2) with 50 μg/ml propidium iodide (Sigma-Aldrich) and 1 mg/ml RNase A (Invitrogen), and incubated at 37 °C for 30 min in the dark. Samples of the cells were then analyzed for their DNA content using FACScan flow cytometry (Beckman, Miami, FL), and cell cycle phase distributions were analyzed by the Cell Quest acquisition software (BD Biosciences, Franklin Lanes, NJ). All experiments were performed in duplicate and repeated twice.
Western blot analysis
The cell extracts were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The membranes were blocked in blocking solution [5% non-fat dried milk in phosphate buffered saline (PBS)] for 2 h at room temperature and then probed with anti-ADAM33, anti-VEGFR2, anti-P-VEGFR2, anti-P-ERK1/2, anti-ERK1/2, anti-P-Akt, anti-Akt, and anti- β-actin for 1 h at room temperature. After washing three times in phosphate-buffered saline (PBS) containing 0 · 1% Tween-20 (PBS-T), the membranes were incubated with secondary antibodies for 1 h at room temperature. After washing an additional three times in PBS-T, the membranes were developed using an electrochemiluminescence (ECL) solution (Pierce, Rockford, IL, USA) and exposed to Kodak X-ray film.
Transfection of small interfering RNA (siRNA)
ADAM33 siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ADAM33 was transfected into ASM cells, ASM cells-ADAM33, and ASM cells-vector according to a siRNA transfection protocol provided by Santa Cruz Biotechnology. Briefly, after culturing ASM cells in antibiotic-free SMCM at 37 °C in a humidified atmosphere of 5% CO2 for 24 h, the siRNA duplex solution, which was diluted in siRNA transfection medium (Santa Cruz Biotechnology), was added to the ASM cells. After transfection for 24 h, the medium was replaced with normal SMCM, and ASM cells were treated with VEGF. Scrambled siRNA, purchased from Santa Cruz Biotechnology, was transfected to ASM cells as a negative standard.
Measurement of VEGF secretion
ASM cells were incubated with indicated doses of 1,25-(OH)2D3 for 48 h after stimulation with TGF-β1 for 30 min, and then the VEGF concentration in each supernatant was quantified using an ELISA kit for human VEGF (Invitrogen, Camarillo, CA) according to the manufacturer’s instructions.
Statistical analysis
All results are expressed as the mean ± SEM. The statistical evaluation of the results was performed by an independent t-test and an ANOVA with a Tukey post-hoc test. The results were significant with a value of p < 0.05.
Discussion
Elucidating the regulatory mechanisms of human ASM cell proliferation has potential clinical value, and may provide further insights into new strategies for the treatment of airway diseases, such as asthma, associated with smooth muscle hyperplasia.
VEGF is a potent stimulator of angiogenesis in asthma. Studies have found that epithelial cell-secreted VEGF promotes airway remodeling in asthma [
15]. In addition, VEGF levels are elevated in lung tissues and sputum of patients with asthma, and positively correlate with asthma disease severity. Furthermore, inhibition of VEGF leads to a significant reduction in goblet cell hyperplasia and basement membrane thickness [
16,
17]. A previous study found that ASM cells under strain promote angiogenesis
via secretion of VEGF, and that blocking VEGF attenuated these angiogenic changes [
18]. In our previous study, we found that basal VEGF release from human ASM cells was comparable to that from BEAS-2B cells, a human lung epithelial cell line. VEGF secreted by ASM cells is thought to play a role in extracellular matrix modulation and fibronectin secretion, as well as in smooth muscle hypertrophy, and therefore in remodeling [
19,
20]. Therefore, in the present study, we investigated the effect of 1,25(OH)2D3 on VEGF-induced cell proliferation and relevant signal transduction pathways, as well as on TGF-β1-induced VEGF secretion in human ASM cells.
ADAM33 was investigated as a potentially important molecule in ASM cell proliferation for a number of reasons. ADAM33 has been shown to be expressed in ASM cells and airway fibroblasts, suggesting a role for this gene in modifying cellular functions such as proliferation, migration, and differentiation [
7,
9].
Indeed, ADAM33 expression was significantly higher in the airways of human subjects with asthma compared to those of controls. Further, increased expression correlated with asthma severity progression, from mild to severe lung function [
21,
22]. As ADAM33 is predominantly expressed in ASM cells, Lin et al. investigated whether ADAM33 protein expression correlates with ASM cell mechanics in an ovalbumin- (OVA-) sensitized rat model; ADAM33 expression was found to be elevated in ASM cells from OVA-sensitized rats relative to non-sensitized rats. Importantly, ADAM33 expression positively correlated with cell traction force, stiffness, and expression of F-actin and vinculin, suggesting that ADAM33 is a mediator of ASM cell dysfunction in asthma [
23]. Although the mechanisms for ADAM33-mediated remodeling are not clear, it has been reported that a soluble form of ADAM33 causes rapid induction of neovascularization both ex vivo and in vivo, as well as endothelial cell differentiation in vitro, suggesting that ADAM33 promotes angiogenesis and elicits airway remodeling [
24]. Ito et al. investigated ADAM33 expression in ASM cells and found that ADAM33 mRNA and protein levels are significantly higher in these cells from subjects with asthma than in ASM cells from normal control subjects [
9]. In this study, we demonstrated that 1,25(OH)2D3 inhibits VEGF-induced ADAM33 expression at both the mRNA and protein level, suggesting that expression may be primarily regulated at the mRNA level; however, it is necessary to study the ADAM33 promoter region as well as the transcription factors that putatively interact with this region in order to elucidate the pathways involved in the regulation of ADAM33 expression.
1,25(OH)2D3 has been shown to inhibit the proliferation of airway smooth muscle cells [
6]. Moreover, in utero vitamin D deficiency in mice leads to increased airway smooth muscle mass and airway resistance [
25]. Furthermore, in children with severe asthma, lower levels of vitamin D have been shown to be associated with increased airway smooth muscle mass [
26,
27]. Recently, 1,25(OH)2D3 has been reported to exert a negative regulatory effect on VEGF secretion [
5,
28]. The mechanism by which 1,25(OH)2D3 regulates VEGF secretion is currently unclear. However, several possible underlying mechanisms have been suggested such as the rapid induction of non-transcriptional responses, which may occur
via activation of transmembrane signal transduction pathways,
e.g. protein kinase C, phosphatidylinositol 3-kinase/Akt, and p42/p44 MAP kinase, all of which are closely associated with VEGF expression [
5,
29]. In addition, it has been shown that 1,25(OH)2D3 induces rapid and sustained activation of phosphatidylinositol 3-kinase/Akt in a nongenomic manner [
5]. Swain et al. reported that 1,25(OH)2D3 may regulate phopholipase C production by cells, which, in turn, may modulate signal transduction by receptors with tyrosine kinase activity, including VEGF [
5,
30]. Second, 1,25(OH)2D3 may modulate the expression of growth factor receptors [
5,
31]. Finally, growth factors may modulate the expression of the nuclear vitamin D receptor [
32]. Further studies are needed to elucidate the mechanisms by which 1,25(OH)2D3 decreases VEGF secretion; this information should facilitate the development of new therapeutic strategies for the treatment of asthma.
It has been found that ERK1/2 activation is involved in cell growth, morphogenesis, and migration of endothelial cells stimulated by angiogenic factors [
33,
34]. Moreover, the activation of PI3K/Akt is considered to play a role in a variety of biological functions such as cell growth, vascular remodeling, angiogenesis, and survival [
34,
35]. VEGF promotes endothelial cell growth and survival
via the ERK1/2 and PI3K/Akt pathways, respectively [
36‐
38]. Walker et al. [
39] compared the extent to which ERK1/2 or PI3K cascades contributed to α-thrombin-stimulated or PDGF-stimulated proliferation of bovine tracheal smooth muscle, and found that, although the PI3K pathway was essential, the ERK1/2 pathway was required for a full mitogenic response. Such findings suggest that although active PI3K is sufficient for the stimulation of ASM DNA synthesis, either by GPCR-coupled or RTK-coupled pathways, parallel ERK1/2-dependent signaling events are required for maximal proliferation. Recent studies have shown that ERK1/2 activation, but not Akt activation, is required for HASM cell proliferation [
40‐
42]. In the present study, we observed that ERK inhibition, but not PI3K inhibition, suppressed ADAM33 expression induced by VEGF. Further research is needed to elucidate the mechanism by which the ERK1/2 pathway enhances the transcription of ADAM33.