Effect of active vitamin D3 on VEGF-induced ADAM33 expression and proliferation in human airway smooth muscle cells: implications for asthma treatment
Kim et al. Respiratory Research
Effect of active vitamin D3 on VEGF- induced ADAM33 expression and proliferation in human airway smooth muscle cells: implications for asthma treatment
Sung-Ho Kim 0
Qing-Mei Pei 1
Ping Jiang 0
Min Yang 0
Xue-Jiao Qian 0
Jiang-Bo Liu 0
0 Department of Respiration, Tianjin First Central Hospital , Fukanglu-24, Nankaiqu, Tianjin 300192 , China
1 Department of Radiology, Tianjin Hospital of Integrated Traditional Chinese and Western Medicine , Tianjin , China
Background: Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodeling, which is associated with increased airway smooth muscle (ASM) mass. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodeling in asthma. Recent evidence indicates that a deficiency of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) may influence asthma pathogenesis. A disintegrin and metalloproteinase (ADAM)33 has been identified as playing a role in the pathophysiology of asthma. ADAM33, which is expressed in ASM cells, is suggested to play a role in the function of these cells. Recent studies show that 1,25-(OH)2D3 exerts direct inhibitory effects on passively sensitized human ASM cells in vitro, including inhibition of ADAM33 expression and cell proliferation; however, the mechanism has not been fully understood. Methods: In order to elucidate the precise mechanism underlying the effect of 1,25(OH)2D3 on VEGF-induced ADAM33 expression and ASM cell proliferation, we tested the effects of 1,25(OH)2D3 on cell cycle progression and evaluated the levels of phospho-VEGF receptor 2 (VEGFR2), phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), and phospho-Akt in VEGF-stimulated ASM cells. Results: We found that 1,25(OH)2D3 inhibited VEGF-induced ADAM33 expression and ASM cell proliferation, as well as cell cycle arrest. Additionally, VEGF-induced ADAM33 expression and ASM cell proliferation was suppressed via inhibition of ERK1/2 activity, but not that of Akt. Furthermore, 1,25(OH)2D3 treatment inhibited VEGF-induced activation of VEGFR2 as well as that of ERK and Akt in a concentration-dependent manner. 1,25(OH)2D3 also inhibited transforming growth factor (TGF)-β-induced VEGF secretion by ASM cells. Conclusions: Collectively, our findings suggest that 1,25(OH)2D3 inhibits VEGF-induced ASM cell proliferation by suppressing VEGFR2 and ERK1/2 activation and downregulating ADAM33. Further studies of these mechanisms are needed to facilitate the development of treatments for smooth muscle hyperplasia-associated diseases of the airway such as asthma.
Asthma; Vascular endothelial growth factor; ADAM33; 1; 25-dihydroxyvitamin D3
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 . 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 . A disintegrin and
metalloproteinase (ADAM)33, a recently discovered ADAM family
member, has been found to play a role in the
pathophysiology of asthma . 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 .
ADAM33 is preferentially expressed in smooth muscle
cells, fibroblasts, and myofibroblasts, but not in T cells,
epithelial cells, or inflammatory cells . 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 .
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).
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% CO2 as
previously described . 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’- CAGG
AATGCCAGCTATTATC −3’ and reverse, 5’-GTTTG
GTGTGGTTCAAGTTT-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.
were developed using an electrochemiluminescence (ECL)
solution (Pierce, Rockford, IL, USA) and exposed to
Kodak X-ray film.
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
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 , to establish ADAM33 overexpressing ASM
cells (ASM cells-ADAM33), and ASM cells infected with
retrovirus containing blank pMXs vector (ASM
cellsvector) 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
phosphatebuffered 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
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
cellsvector 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
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.
1,25-(OH)2D3 inhibits VEGF-induced ADAM33 expression
in ASM cells
In order to evaluate the direct effect of 1,25-(OH)2D3
on the VEGF-induced expression of ADAM33 in ASM
cells, we performed real-time PCR. ASM cells were
treated with various doses of 1,25-(OH)2D3, and at
various times after treatment or not with 50 ng/ml of VEGF
for 30 min; as a result, VEGF-induced ADAM33
expression was downregulated in a dose- and time-dependent
manner (Fig. 1a, b). In addition, we performed western
blot analysis to evaluate whether 1,25-(OH)2D3
additionally regulates VEGF-induced ADAM33 protein
expression in ASM cells. Interestingly, 1,25-(OH)2D3
also downregulated VEGF-induced ADAM33 protein
expression in ASM cells in a dose- and time-dependent
manner (Fig. 1c, d).We did not observe any VEGF- and/
or 1,25-(OH)2D3-induced cytotoxicity under the present
experimental conditions (data not shown).
Fig. 1 1,25(OH)2D3 inhibits VEGF-induced ADAM33 expression at both mRNA and protein level. ASM cells were incubated with various doses of
1,25(OH)2D3 for 9 h before treatment or not with 50 ng/ml of VEGF for 30 min, and then real-time PCR performed (a). ASM cells were incubated at
indicated times of 100 nM of 1,25(OH)2D3, and then real-time PCR performed (b). The values are normalized relative to the GAPDH standard. ASM cells
were incubated with various doses of 1,25(OH)2D3 for 24 h before treatment or not with 50 ng/ml of VEGF for 30 min, and then western blotting
analysis for ADAM33 was performed (c). ASM cells were incubated at indicated times of 100 nM of 1,25(OH)2D3 before treatment or not with
50 ng/ml of VEGF for 30 min, and then western blotting analysis for ADAM33 was performed (d). β-actin was used as a loading control. All data are
representative of three independent experiments. Values represent the means ± SEM. *P < 0.05 vs. control, # P < 0.05, ## P < 0.005 vs. VEGF alone
1,25-(OH)2D3 inhibits VEGF-induced ASM cell proliferation
by downregulating ADAM33 expression
It has been reported that VEGF-D-enhanced ADAM33
plays an important role in tumor cell proliferation in the
gastric cancer cell line SNU-601. We initially tested the
effect of 1,25-(OH)2D3 on the VEGF-induced
proliferation of ASM cells. When ASM cells were treated with
various doses of 1,25-(OH)2D3, and at different times
after treatment or not with 50 ng/ml of VEGF for
30 min, 1,25-(OH)2D3 inhibited VEGF-enhanced BrdU
incorporation in a dose- and time-dependent manner in
ASM cells (Fig. 2a, b).
Next, to elucidate the effect of ADAM33 on the
proliferation of ASM cells, we constructed an ADAM33
siRNA transfection reagent. As shown in Fig. 2c and d,
we confirmed ADAM33 gene silencing at the mRNA
and protein level. To further confirm the silencing effect of
ADAM33 siRNA in ASM cells, a rescue experiment was
performed with ADAM33 siRNA in ASM cells-ADAM33.
Herein, western blot analysis was also performed to assess
ADAM33 expression in ASM cells-ADAM33 treated with
ADAM33 siRNA (ASM-ADAM33 siRNA). The result of
western blot analysis indicated that the expression of
ADAM33 was significantly downregulated in
ASMADAM33 siRNA compared with ASM cells-ADAM33 and
ASM cells-ADAM33 treated with nontargeting control
siRNA (ASM-control siRNA) (Fig. 2e). These results
indicated that the ADAM33 siRNA was effective in our study.
The cell proliferation ability was further evaluated. As
expected, When ASM cells-ADAM33 cells were
transfected with ADAM33 siRNA or control siRNA for 48 h in
the presence of 50 ng/ml VEGF and 100 nM
1,25(OH)2D3, BrdU incorporation was decreased in ADAM33
siRNA-transfected cells compared with negative control
siRNA-transfected cells (Fig. 2f ). These data indicate that
1,25-(OH)2D3 inhibits VEGF-induced proliferation of
ASM cells by downregulating ADAM33 expression.
1,25-(OH)2D3 induces G1-phase cell-cycle arrest in
VEGF-induced ASM cell proliferation
Flow cytometry analysis was performed to assess
whether the anti-proliferative effect of 1,25-(OH)2D3
was due to cell-cycle arrest in a specific phase. As shown
in Fig. 3, VEGF treatment significantly increased the
Fig. 2 1,25(OH)2D3 inhibits cell proliferation by down-regulation of ADAM33 expression. ASM cells were incubated with various doses of 1,25(OH)2D3
for 48 h before treatment or not with 50 ng/ml of VEGF for 30 min, and then cell proliferation was determined by BrdU incorporation (a). ASM cells
were incubated at indicated times of 100 nM of 1,25(OH)2D3 before treatment or not with 50 ng/ml of VEGF for 30 min, and then cell proliferation
was determined by BrdU incorporation (b). ASM cells were transfected with negative siRNA or ADAM33 siRNA, and then real-time PCR performed. The
values are normalized relative to the GAPDH standard (c). ASM cells (d) and ASM cells-ADAM33 (e) were transfected with negative siRNA or ADAM33
siRNA, and then western blotting analysis for ADAM33 was performed. β-actin was used as a loading control. ASM cells-ADAM33 were transfected with
negative siRNA or ADAM33 siRNA in the presence of VEGF (50 ng/ml) and 1,25-(OH)2D3 (100 nM) for 48 h, and then cell proliferation was determined
by BrdU incorporation (f). All experiments were done at least three times. Values represent the means ± SEM. *P < 0.05 vs. control or ASMs-vector;
# P < 0.05 vs. VEGF alone or control siRNA or ASMs-control siRNA
proportion of ASM cells in the S and G2/M phases of
the cell cycle, with a concomitant decrease in the
proportion in G1 phase relative to control cells. However,
1,25-(OH)2D3 treatment in the presence or absence of
VEGF markedly reduced the percentage of cells in the S
and G2/M phases, resulting in a significant
accumulation of cells in G1 phase, relative to VEGF-stimulated
ASM cells. Cell proliferation was only slightly affected
by 1,25-(OH)2D3 alone compared with the group
without VEGF challenge.
1,25-(OH)2D3 inhibits VEGF-induced ADAM33 expression
and ASM cell proliferation through inactivation of VEGFR2
Several studies have reported that VEGF-induced cell
proliferation is mediated by the interaction of VEGF with
VEGFR2 (also known as KDR or FLK1). In order to
determine whether blocking the VEGF-VEGFR2 interaction
prevents VEGF-mediated ADAM33 upregulation and
ASM cell proliferation, we used SU1498, an inhibitor of
the tyrosine kinase activity of VEGFR2. SU1498 blocks the
interaction of VEGF with VEGFR2 but not with VEGFR1
(FLT1). As shown in Fig. 4a, VEGF-increased ADAM33
expression was inhibited by SU1498 in a dose-dependent
manner. In addition, SU1498 blocked VEGF-induced
BrdU incorporation in ASM cells (Fig. 4b). Finally, we
performed western blot analysis to evaluate whether
1,25-(OH)2D3 also regulates VEGF-induced VEGFR2
activation in ASM cells. Interestingly, 1,25-(OH)2D3
also downregulated VEGF-induced VEGFR2 activation
in ASM cells in a dose-dependent manner. These data
indicate that 1,25-(OH)2D3 inhibits VEGF-induced
ADAM33 expression and cell proliferation by
suppression of the VEGF/VEGFR2 interaction.
1,25-(OH)2D3-mediated inhibition of VEGF-induced
ADAM33 expression and cellular proliferation involves
the MAPK/ERK1/2 pathway
The majority of in vitro reports suggest that PI3-K and
ERK1/2 activation represents the major signal transduction
Fig. 3 1,25(OH)2D3 inhibits cell cycle in ASM cells. ASM cells were incubated with 100 nM of 1,25(OH)2D3 in the presence or absence of VEGF
(50 ng/ml) for 48 h, and then Flow cytometric analysis for cell cycle was performed. All experiments were done at least three times. Values
represent the means ± SEM
pathway for cytokine-stimulated,
G-protein-coupledreceptor (GPCR)-mediated, or receptor tyrosine kinase
(RTK)-mediated proliferation of ASM cells. It is
known that the interaction of VEGF with VEGFR2
activates the MAPK/ERK1/2-dependent and
PI3-k/Aktdependent signaling transduction pathways. In order
to evaluate the effects of VEGF and 1,25-(OH)2D3 on
the activation of ERK1/2 and Akt in ASM cells, the
levels of phosphorylated ERK1/2 and Akt expression
were investigated by western blotting. When cells
were incubated in the absence or presence of 50 ng/
ml VEGF for the indicated duration, an increase in
the phosphorylation of ERK1/2 and Akt was observed,
with no effect on the total levels of these proteins
(Fig. 5a and b). In addition, treatment with
1,25(OH)2D3 significantly attenuated VEGF-stimulated
ERK1/2 and Akt phosphorylation in a dose-dependent
manner (Fig. 4c and d). The levels of phospho-ERK1/2
and phospho-Akt were not affected in
1,25-(OH)2D3treated cells compared with the group without VEGF
In order to determine the signaling pathways that play
a role in VEGF-induced ADAM33 expression and ASM
cell proliferation, we used specific inhibitors of ERK1/2
and Akt signaling pathways. The inhibitors tested included
U0126 (a MAPK/ERK1/2 inhibitor) and LY294002 (a
PI3K inhibitor). Pretreatment of ASM cells with U0126
was found to significantly decrease ADAM33 expression
in VEGF-treated ASM cells relative to control (Fig. 5e).
However, VEGF-induced ADAM33 expression was not
affected by the addition of LY294002 (Fig. 5e). These results
demonstrate that VEGF increases ADAM33 expression
through the activation of ERK1/2. We additionally
investigated whether U0126 and LY294002 inhibit the
VEGF-induced proliferation of ASM cells; when cells
were incubated with U0126 or LY294002in the
presence of VEGF, U0126 blocked VEGF-induced BrdU
incorporation (Fig. 5f ). However, LY294002 had no
Fig. 4 1,25(OH)2D3 inhibits VEGF-induced ADAM33 expression and cell proliferation by inactivation of VEGFR2. ASM cells were incubated with
indicated doses of SU1498 for 2 h before treatment with VEGF (50 ng/ml) for 24 h, and then western blotting analysis for ADAM33 was performed.
β-actin was used as a loading control (a). ASM cells were incubated with indicated doses of SU1498 for 2 h before treatment with VEGF (50 ng/ml)
for 48 h, and then cell proliferation was determined by BrdU incorporation (b). ASM cells were incubated with various doses of 1,25(OH)2D3 for 24 h
before treatment or not with 50 ng/ml of VEGF for 30 min, and then western blotting analysis for VEGFR2 was performed (c). All experiments were
done at least three times. Values represent the means ± SEM. *P < 0.05 vs. control; # P < 0.05, ## P < 0.005 vs. VEGF alone
effect on VEGF-induced BrdU incorporation (Fig. 5f ).
These data show that 1,25-(OH)2D3 inhibits
VEGFinduced ADAM33 expression and proliferation of
ASM cells through the suppression of the MAPK/
Effect of 1,25-(OH)2D3 on VEGF secretion in ASM cells
VEGF released from airway epithelial cells aggravates
airway inflammation and remodeling. TGF-β1
expression is elevated in asthmatic airways as well as in the
bronchoalveolar lavage (BAL) fluid of patients with
asthma. Therefore, we studied the effects of
1,25(OH)2D3 on TGF-β1-induced VEGF expression in ASM
cells. The VEGF levels in culture supernatants from
TGF-β1-stimulated ASM cells were significantly higher
than those in unstimulated cells (Fig. 6). However,
1,25(OH)2D3 inhibited the release of VEGF from ASM cells
stimulated with TGF-β1 in a dose-dependent manner
(Fig. 6). TGF-β1 contributes to airway inflammation by
enhancing VEGF release via the PI3-K pathway in
human ASM cells. Therefore, these data suggest that
1,25(OH)2D3 inhibits TGF-β1-induced VEGF release, likely
by attenuating Akt phosphorylation.
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 . 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
. 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
Fig. 5 1,25(OH)2D3 inhibits VEGF-induced ERK 1/2 phosphorylation in ASM cells. ASM cells were incubated at indicated times of VEGF (50 ng/ml),
and then western blotting analysis for phospho-ERK 1/2 (a) and phospho-Akt (b) was performed. ASM cells were incubated with various doses of
1,25(OH)2D3 for 24 h before treatment or not with 50 ng/ml of VEGF for 30 min, and then western blotting analysis for phospho-ERK 1/2 (c) and
phospho-Akt (d) was performed. The total ERK1/2 and Akt was used as a loading control. ASM cells were incubated with 20 μM U0126 or 20 μM
LY294002 for 2 h before treatment with VEGF (50 ng/ml) for 24 h, and then western blotting analysis for ADAM33 was performed. β-actin was
used as a loading control (e). ASM cells were incubated with 20 μM U0126 or 20 μM LY294002 for 2 h before treatment with VEGF (50 ng/ml) for
48 h, and then cell proliferation was determined by BrdU incorporation (f). All experiments were done at least three times. Values represent the
means ± SEM. *P < 0.05 vs. control; # P < 0.05 vs. VEGF alone
Fig. 6 1,25(OH)2D3 inhibits TGF-β1-stimulated VEGF secretion by
ASM cells. ASM cells were incubated with indicated doses of
1,25(OH)2D3 for 48 h after stimulation with 10 ng/ml of TGF-β1 for
30 min, and then the VEGF concentration in each supernatant was
quantified using a human ELISA kit. Experiments were done at least
three times. Values represent the means ± SEM. *P < 0.05 vs.
control; # P < 0.05 vs. TGF-β1 alone
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 . 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 . 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 . 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 . Moreover, in
utero vitamin D deficiency in mice leads to increased
airway smooth muscle mass and airway resistance .
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 . 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 . 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.  compared the extent to
which ERK1/2 or PI3K cascades contributed to
αthrombin-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.
In conclusion, our results provide important insights
into the mechanisms by which 1,25(OH)2D3 regulates
VEGF-induced ADAM33 expression and ASM cell
proliferation, as the effects of this compound on various
underlying cellular signaling pathways such as the
suppression of VEGFR2 and ERK1/2 phosphorylation. The
present findings expand our knowledge of the role of
1,25(OH)2D3 in airway remodeling, and are expected to
enable the development of effective therapies for airway
diseases such as asthma.
1,25(OH)2D3: 1,25-dihydroxyvitamin D3; ADAM-33: A disintegrin and
metalloproteinase33; ASM: Airway smooth muscle; ERK1/2:
Phosphoextracellular signal-regulated kinase 1/2; GPCR: G-protein-coupled-receptor;
RTK: Receptor tyrosine kinase; TGF-β: Transforming growth factor-β;
VEGF: Vascular endothelial growth factor; VEGFR2: Vascular endothelial
growth factor receptor 2
Availability of data and material
Source data and material will be made available upon reasonable request.
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