Original Article

Yonago Acta Medica 2017;60:16–23

γ-Tocotrienol Inhibits TGF-β1-Induced Contractile Phenotype Expression of
Human Airway Smooth Muscle Cells
Takehito Fukushima,* Akira Yamasaki,* Tomoya Harada,* Hiroki Chikumi,* Masanari Watanabe,* Ryota
Okazaki,* Miki Takata,* Yasuyuki Hasegawa,* Jun Kurai,* Masaaki Yanai,* Akihiro Yamamoto,* Yuriko Sueda,*
Andrew J. Halayko† and Eiji Shimizu**Division of Medical Oncology and Molecular Respirology, Department of Multidisciplinary Internal Medicine, school of Medicine,
Tottori University Faculty of Medicine, Yonago 683-8504, Japan and †Departments of Physiology and Pathophysiology, and Internal
Medicine, University of Manitoba, Winnipeg, MB R3E 3P4 Canada

Airway wall remodeling is one of the characteristicfeatures of asthma. The structural changes in airway
remodeling include epithelial cell shedding, basement
membrane thickening, submucosal fibrosis and myofibroblast accumulation, airway smooth muscle (ASM)
cell hyperplasia and hypertrophy, and adventitial fibrosis.1 Several inflammatory mediators play importantroles in airway remodeling,2, 3 and among these, transforming growth factor b1 (TGF-b) plays a predominantrole through its capacity to induce extracellular matrix
protein synthesis and profibrotic differentiation of ASM
cells.4–6 TGF-β1 binds to its dimeric serine/threonine
kinase receptor which facilitates phosphorylation of
Smad2 and Smad3 to initiate a canonical signalingcascade.7 Sagara et al. have confirmed an association of
TGF-b1/Smad2 signaling with subsequent airway remodeling in asthma.8
Current asthma therapies, such as inhaled corticosteroid, b2 agonists, muscarinic receptor antagonists
and leukotriene receptor antagonists, have limited effects on airway remodeling.9 Bronchial thermoplasty isa nonpharmacological procedure that targets ASM bundles.10, 11 However, a recent study showed that the effectsof bronchial thermoplasty on bronchial wall thickening
may be transient.12
Vitamin E exhibits antioxidant activity and has
been evaluated for its effect in asthma since oxidative
stress is related to the pathogenesis of inflammatory airway disease.13 Nonetheless, the therapeutic efficacy ofvitamin E in asthma remains equivocal.14, 15
In nature, vitamin E exists in eight different isoforms; a, b, g, and d tocopherols and tocotrienols. The
major component of vitamin E is a-tocopherol, whereas
tocotrienols are less abundant. Beyond their antioxidant
capabilities, tocotrienols also have pharmacological effects on various cell types. Tocotrienol has been shownto inhibit proliferation of breast, prostate, and lungcancer cell lines.16 Yap et al. reported that g-tocotrienol
inhibits proliferation of a breast cancer cell line throughdownregulation of inhibitor of differentiation 1 (Id1),17
which has been reported to be crucial for differentiation

ABSTRACT

Tocotrienols, members of the vitamin E
family, exist in four different isoforms (a, b, g and d tocotrienol) that have can be protective against brain damage, as well as having anticancer effects in vivo and invitro. We have shown that g-tocotrienol inhibits human
airway smooth muscle cell proliferation and migrationinduced by platelet-derived growth factor (PDGF)-BB
by suppressing RhoA activation. In this study, we tested
whether g-tocotrienol modulates transforming growth
factor (TGF) -b-induced induction of human airway
smooth muscle (ASM) into a contractile phenotype andconcomitant synthesis of extracellular matrix proteins.
Methods ASM cells were stimulated with TGF-b1 (2ng/mL) for 48 hours and the effect of g-tocotrienol (50μM) on a-smooth muscle actin, fibronectin and collagen I expression was assessed using Western blotting.
The signaling pathways involved in TGF-b1 stimulation
were also investigated.
Results TGF-b1 increased a-smooth muscle actin, fibronectin and collagen Ⅰ abundance by 3- to 5-fold. This
response was inhibited significantly by g-tocotrienol.
Furthermore, g-tocotrienol suppressed RhoA activation,
but did not affect Smad2 or Smad3 phosphorylation.
Conclusion These results indicate that g-tocotrienol
has potential for benefit in modulating on airway remodeling in asthma, likely via a mechanism involving
the suppression of TGF-β activation of RhoA.
BackgroundKey words airway smooth muscle; asthma; g-Tocotrie-

nol; remodeling; vitamin E

Corresponding author: Akira Yamasaki, MD, PhD
yamasaki@med.tottori-u.ac.jp
Received 2016 December 2
Accepted 2016 December 26
Abbreviations: α-SMA,α-smooth muscle actin; ASM, airway
smooth muscle; BSA, bovine serum albumin; DAPI, 4’, 6-diamidino-2-phenylindole; EDTA, Ethylenediaminetetraacetic acid; FBS,
fetal bovine serum; hTERT, human telomerase reverse transcriptase; Id1, inhibitor of differentiation 1; PBS, phosphate bufferedsaline; PDGF, platelet-derived growth factor; Rho, Ras homolog;
TBS, cyto-Tris buffered saline; TGF, transforming growth factor

16γ-Tocotrienol and ASM differentiationof fibroblasts.18, 19
RhoA signaling is an important regulator of airway
remodeling in asthma since activation of the RhoA/
Rho kinase pathway is related to airway smooth muscle contraction, proliferation and cell migration.20 We
have reported that g-tocotrienol inhibits proliferationand migration of ASM cells through inhibition of RhoA
activation.21 Schaafsma et al reported that the synthesis
and secretion of extracellular matrix protein is mediatedthrough RhoA activation in ASM cells.22 Therefore, we
considered that g-tocotrienol could inhibit ASM cellulardifferentiation and synthesis of extracellular matrix protein.
In this study, we investigated if and how g-tocotrienol inhibits TGF-b1 induction of a hypertrophic,
contractile phenotype in human ASM cells, as well as
assessing its impact on TGF-β1-induced biosynthesis ofextracellular matrix proteins.

to-Tris buffered saline (TBS) with 0.1% Tween 20, thecoverslips were incubated with Alexa Fluor 568 conjugated goat anti-mouse IgG antibody (1:50; Molecular
Probes, Eugene, OR) for 1 h at room temperature. The
nuclei were then stained with 300 nM 4’, 6-diamidino-2-phenylindole (DAPI) (Molecular Probes) for 2 min.
The coverslips were mounted onto slides with anti-fade(Molecular Probes) and evaluated using fluorescencemicroscopy (Olympus, Tokyo, Japan).
Preparation of cell lysates and Western blotting

Cells were washed with ice-cold phosphate bufferedsaline (PBS) and lysed in radioimmunoprecipitation assay buffer (RIPA) (50 mM Tris HCl, pH 7.4; 1% NP-40;
0.25% sodium deoxycholate; 150 mM NaCl; 1 mM Ethylenediaminetetraacetic acid (EDTA), 1 mM Na3VO4,
1 mM NaF, and protease inhibitors). Equal amounts ofprotein were separated by electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes(GE Healthcare Lifescience, Buckinghamshire, UK).
After blocking with 5% milk in TBS (20 mM Tris, 150
mM NaCl, pH 7.6) containing 0.1% Tween 20 (TBS-T)
for 1 h at room temperature, blots were incubated overnight at 4 °C with 0.1% TBS-T containing monoclonalanti-a-smooth muscle actin antibody (1:2000; Sigma),monoclonal anti-b-actin antibody (1:10000; Cell Signaling Technology, Danvers, MA), monoclonal anti-fibronectin antibody (1:1000; Santa Cruz Biotechnology,
Santa Cruz, CA), polyclonal anti-collagen1A2 antibody(1:1000; Cell Signaling Technology), monoclonal anti-Smad2 antibody (1:1000; Cell Signaling Technology),
polyclonal anti-phospho-Smad2, anti-phospho Smad3,
and anti-Smad3 antibody (1:1000; Cell Signaling Technology), monoclonal anti-Id1 antibody (1:200; Cell
Signaling Technology) were used. After incubation
with the primary antibodies, membranes were washedand incubated with horseradish peroxidase-conjugatedanti-rabbit IgG (1:5000; GE Healthcare Lifescience) or
anti-mouse IgG (1:3000; GE Healthcare Lifescience)for 1 h at room temperature. Immunoreactive species
were visualized on an ImageQuant LAS 4000 mini (GEHealthcare Lifescience) with ECL reagents (GE Healthcare Lifescience) and bands were quantified by densitometry using TotalLab Quant software (Newcastle,
UK).

MATERIALS AND METHODS
Human ASM cells and culture conditions

ASM cells immortalized by stable expression of humantelomerase reverse transcriptase (hTERT) were a kind
gift from Dr. Andrew J. Halayko at the University of
Manitoba. Cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) containing 10% fetal bovine
serum (FBS), 100 mg/mL penicillin, and 100 mg/mL
streptomycin at 37 °C in a humidified 5% CO2 atmosphere. All experiments were performed in minimum of
three different cell lines.
Stimulation of ASM cells with TGF-β1

ASM cells were seeded in a 60 mm dish and allowed to
grow to confluence. The cells were then serum starved
for 24 h and incubated with 10 to 50 mM g-tocotrienolfor 1 h, followed by stimulation with 2 ng/mL TGF-b1
for the intended duration.
Immunocytochemical analysis

ASM cells were seeded on coverslips and allowed to
grow to confluence. The cells were then serum starved
for 24 h and stimulated with TGF-b1 in the presence orabsence of 50 mM g-tocotrienol for 48 h. The cells were
washed with CB buffer [10 mM 2-(4-Morpholino) ethanesulfonic acid, 150 mM NaCl, 5 mM glucose, and 5
mM MgCl2] and fixed with 3% paraformaldehyde and3% triton-X for 5 min. After washing with CB buffer,the coverslips were incubated with 5% bovine serum
albumin (BSA) for 1 h, and then mouse anti-a-smoothmuscle actin antibody (1:100; Sigma, St Louis, Mo)
was added to the coverslips and incubated overnight at
4 °C in a humidified chamber. After washing with cy-

RhoA activation assay

RhoA activity was measured using an Active Rho detection kit (Cell Signaling Technology, Danvers, MA)
according to the manufacturer’s protocol. Samples wereseparated by electrophoresis and transferred to PVDF
membranes (GE Healthcare Lifescience). After blocking
17

T. Fukushima et al.

with 5% milk in TBS containing 0.05% Tween 20 for 1
h at room temperature, blots were incubated with TBS-T
containing the primary mouse monoclonal antibody for
RhoA (1:1000; Santa Cruz Biotechnology) overnight at
4 °C. The membranes were washed and incubated withhorseradish peroxidase-conjugated anti-mouse IgG(1:10000) for 1 h at room temperature. Bands were visualized on an ImageQuant LAS 4000 mini with ECL
reagents.

Statistical Analyses

Reagents

Western blotting and densitometry analysis showedthat incubation of the ASM cells with TGF-b1 led tosignificant accumulation of a-SMA (Figs. 1A and 1B).
Notably, co-treatment of cells with g-tocotrienol was
sufficient to inhibit TGF-b1-induced a-SMA expres-

Data from individual assays are expressed as the mean
± SD. Statistically significant differences were assessedby ANOVA (Bonferroni multiple comparisons test) orStudent’s t-test with P-values < 0.05 considered significant.
RESULTS

γ-Tocotrienol inhibits TGF-β1-induced expressionof α-smooth muscle actin

g-Tocotrienol was purchased from Cayman Chemical(Ann Arbor, MI). Y-27632 was purchased from Wako
(Osaka, Japan). Simvastatin was purchased from Calbiochem (San Diego, CA).

(A)A

α-SMA
β-actin(B)B

α−SMA / β−actin(Fold-change relative to control)

Control TGF-β1(2 ng/mL)

10

25

10

50

γ-Tocotorienol (µM)

25

50

TGF-β1 + γ-tocotorienol

4
3

*

2
1

Fig.	
  01	
  	
  
Fig.	
  1	
  	
  
(C)

Control TGF-β1(2 ng/mL)

10

25

50

γ­Tocotorienol (µM)

10

25

50

TGF-β1 + γ-tocotorienol

Fig. 1. g-Tocotrienol inhibits the TGF-b1-induced expression of a-smooth muscle actin.
hTERT-ASM cells were pretreated withg-tocotrienol for 1 h and then stimulated with
TGF-b1 (2 ng/mL) for 48 h. Western blotting
analysis showed that relative abundance of
a-SMA was increased by TGF-b1 and g-tocotrienol inhibited the TGF-b1-induced expression of a-SMA in a dose-dependent manner.
Panel A shows a representative blot and panel
B shows the results of densitometry analysis
from seven cell lines. Data are expressed asmean value ± SD (n = 7). *P < 0.05 versus
TGF-b1 (A and B). TGF-b1 enhanced a-SMA
expression compared with control. g-Tocotrienol inhibited the induction of a-SMA expression by TGF-b1. g-Tocotrienol alone did notaffect a-SMA expression (C). Bar = 50 mM.
α-SMA, α-smooth muscle actin; TGF, transforming growth factor.

(C) C

Non-­‐s)mulated

γ-­‐tocotrienol	
  (50	  μM)

Non-stimulatedNon-­‐s)mulated(50mM)
γ-Tocotrienol
γ-­‐tocotrienol	
  (50	  μM)

	  TGF-β1	  	  	
  TGF-­‐β1	
  (2ng/mL)(2ng/mL)	  	
  TGF-β	  	
  TGF-­‐β1	  +	
  γ-­‐tocotrienol	  + γ-tocotrienol

	
  	
  	
  	
  TGF-­‐β1	
  (2ng/mL)

18

	
  	
  	
  	
  TGF-­‐β1	
  +	
  γ-­‐tocotrienol	  γ-Tocotrienol and ASM differentiation

sion in a dose-dependent manner (P < 0.05) (Fig. 1B).
In addition, immunocytochemical staining of the ASM
showed that TGF-b1 markedly enhanced the densityof a-smooth muscle actin (a-SMA) stress fibers, andthough this accumulation was inhibited by g-tocotrienol,g-tocotrienol had no obvious impact on a-SMA fibers in
the absence of TGF-β1 (Fig. 1C).

fibronectin and collagen I protein (Figs. 2A and B).
Induction of these extracellular matrix proteins was
significantly inhibited by g-tocotrienol (Figs. 2C and D),
whereas g-tocotrienol alone had no significant effect on
the expression of fibronectin or collagen I.

γ-Tocotrienol inhibits TGF-β1-induced expression

As expected, TGF-b1 induced rapid and sustained phosphorylation of Smad2 and Smad3, which peaked 60120 min after treatment. However, in contrast to effects
on TGF-β1-induced α-SMA, fibronectin and collagen I,
pretreatment with g-tocotrienol did not prevent
TGF-b1-induced phosphorylation of Smad2 andSmad3 (Fig. 3A and 3B).

Effects of γ-tocotrienol on TGF-β1-induced cellsignaling

Fig.	
  2	
   of ﬁbronectin and collagen Ⅰ

TGF-b1 treatment greatly increased the abundance of(A)A
Fibronec)nβ-­‐ac)n	
  TGF-­‐β1	  Control	
10(2	
  ng/mL)	

50

10

25

50

TGF-­‐β1	
  +	
  γ-­‐tocotorienol

Collagen	
  Iβ-­‐ac)n	  Control	

TGF-­‐β1	
  10(2	
  ng/mL)	

Fig.	
  2	
  

C
(C)

(D)D

Collagen	
  I/β-­‐ac)n	
  Fibronec)n/β-­‐ac)n	
  (Fold-­‐change	
  rela)ve	
  to	
  control) (Fold-­‐change	
  rela)ve	
  to	
  control)

(B)

B

25

γ-­‐Tocotorienol	
  (µM)

25

50

γ-­‐Tocotorienol	
  (µM)

1025

50

TGF-­‐β1	
  +	
  γ-­‐tocotorienol

5
4
3

*

2
1
0
Control	

TGF-­‐β1	
  10(2	
  ng/mL)	

25

50

γ-­‐Tocotorienol	
  (µM)

1025

50

TGF-­‐β1	
  +	
  γ-­‐tocotorienol

2

*

1

0
Control	

TGF-­‐β1	
  10(2	
  ng/mL)	

25

50

γ-­‐Tocotorienol	
  (µM)

1025

50

TGF-­‐β1	
  +	
  γ-­‐tocotorienol

Fig. 2. g-Tocotrienol inhibits the TGF-b1-induced expression of fibronectin and collagen I.
hTERT-ASM cells were pre-treated with g-tocotrienol for 1 h and thenstimulated with TGF-b1 for 48 h. TGF-b1 increased the abundance offibronectin and collagen I (A and B). g-Tocotrienol significantly attenuatedthe TGF-b1-induced expression of fibronectin and collagen I (C and D).
g-Tocotrienol alone did not affect the expression of fibronectin and collagen I (C and D). Data are expressed as mean ± SD (n = 8 for fibronectin,n = 7 for collagen I). *P < 0.05 versus TGF-b1. TGF, transforming growth
factor.

19

γ-Tocotrienol inhibits TGF-β1-induced RhoA
activation

We next examined the effects of pre-treatment of
ASM cells with g-tocotrienol on RhoA activationin response to acute exposure to TGF-b1. Notably, TGF-b1-induced activation of RhoA wasinhibited significantly by g-tocotrienol (Fig. 4).
γ-Tocotrienol does not change the TGF-β1induced increase of Id1 expression

We also examined the effects of TGF-b1 andg-tocotrienol on expression of the basic HLHtranscription factor repressor Id1. After TGF-b1
stimulation Id1 abundance increased markedly,
peaking from 3 to 6 h later, then returning to nearbase-line levels after 12 hours. In the presenceof g-tocotrienol, although TGF-β1 induced an increase in Id1 from 1-3 hours, this was lower thanseen with TGF-β1-alone, and returned to baseline
within 6 hours of TGF-β1 exposure However,these changes were not statistically significant(Fig. 5).
Effect of the ROCK inhibitor on Id1 expression induced by TGF-β1

Finally, we examined the relationship between
RhoA activation and Id1 expression. ASM cells
were pretreated with Y27632 (10 mM) or simvastatin (10 mM) for 1 h and then stimulated with
TGF-b1 (2 ng/mL) for 3 h. While TGF-b1 significantly increased Id1 expression, this increase was
not inhibited by Y27632 or simvastatin (Fig. 6).
DISCUSSION
In this study, we showed that g-tocotrienol inhibited the TGF-b1-induced differentiation of human
ASM cells into a contractile phenotype and the

T. Fukushima et al.

concomitant synthesis of collagen I and fibronectin. Our
results also revealed the signaling pathways that are affected by g-tocotrienol in TGF-β1–exposed ASM cells,with a suppression of RhoA activation, but with no effects on Id1 expression or Smad phosphorylation. Thesefindings are important because ASM cell differentiationand synthesis of extracellular matrix protein contributeto the airway remodeling observed in asthma. Our data
indicate that g-tocotrienol may prevent airway remodeling in asthma through inactivation of the RhoA/Rhokinase signaling pathway.
Fig.	
  3	
  	
  
In nature, four different isoforms of tocotrienol exist (a, b, g and d). From these, we selected g-tocotrienol

pSmad3	
  /	
  Smad3	  (Fold	  increase	
  over	  basal)pSmad2	
  /	
  Smad2	
  (Fold	  increase	
  over	  basal)since g-tocotrienol has shown to inhibit breast cancer
cells proliferation and ASM cells proliferation and migration.21, 23 The effects of other isoforms on human
ASM cells functions has not been investigated yet, other isoforms might have similar effects on human ASM
cells and have beneficial effects on airway remodelingof asthma. Further study is needed to evaluate the effectof a, b and d tocotrienol.
In this study, we used 10 to 50 mM of g-tocotrienol
as in our previous study.21 We also confirmed that the
maximum concentration of g-tocotrienol (50 mM) had
no cytotoxic effects on human ASM cells (data notshown). However, this concentration of g-tocotrienol exhibits cytotoxic effect on other cell types.24, 25 Moreover,clinical application of g-tocotrienol by oral(A)administration at this concentration mightA
be difficult since serum level of tocotrienol
pSmad2	  was 0.54 ± 0.45 mM when hypercholesterolemia subjects received 250 mg/day of g-toSmad2	
  cotrienol supplementation by oral administration.26 Inhalation of g-tocotrienol might
pSmad3	  be one of the best delivery methods to reacheffective concentration in the human lung.
Smad3	
  Id1 is a member of the helix-loop-helix proteins that act as negative regulatorsTime	
  (min)
0
15
30
60
120
15
30
60
120
of DNA-binding of basic helix-loop-helix
Fig.	
  3	
  
TGF-­‐β1
TGF-­‐β1+	  γ-­‐tocotrienol	
  transcriptional factors.27 Id1 binds caveolin-1 and induces epithelial-mesenchymal(B)B
6cell transition and migration of prostatic
5cancer cell lines.28 Furthermore, caveolin-1
4
is required for expression of the contractile
3
phenotype.29 Id1 is a critical mediator of2
TGF-b1-induced transdifferentiation in a
1
rat hepatic stellate cell line.30 Furthermore,
0
0
15
30
60
120
15
30
60
120
Je et al. reported that Id1 inhibits TGF-b1induced collagen expression in a dermal
TGF-­‐β1
TGF-­‐β1+	  γ-­‐tocotrienol	
  fibroblast cell line.31 Since Id1 is decreased
5by g-tocotrienol in breast cancer cell lines,174
we considered that downregulation of Id1
3
was involved in the mechanism underlying
2
the g-tocotrienol inhibition of the fibrotic1
phenotype change of ASM cells. However,0
g-tocotrienol did not change the expression
0
15
30
60
120
15
30
60
120
of Id1 in this study. Therefore, Id1 is not
TGF-­‐β1
TGF-­‐β1+	  γ-­‐tocotrienol	  involved in this inhibitory effect of g-tocotrienol on the profibrotic changes induced by
Fig. 3. g-Tocotrienol does not inhibit TGF-b1-induced phosphorylation of Smad2
and Smad3.
TGF-b1 in ASM cells.
A: Phosphorylation of Smad2 and Smad3 peaked 60 to 120 min after stimulaWe demonstrated that the TGF-b1tion with TGF-b1 (2 ng/mL). g-Tocotrienol (50 mM) did not inhibit the TGF-b1induced activation of RhoA was prevented
induced phosphorylation of Smad2 and Smad3. B: The phosphorylated Smad2
by g-tocotrienol. RhoA and its downstream
and Smad3 bands were quantified by densitometry and expressed as the foldRho kinase play a key role in ASM cellschange, relative to control. Neither phosphorylation of Smad2 nor Smad3 were
inhibited by g-tocotrienol. Data are expressed as mean ± SD (n = 5). TGF, transcontraction and airway hyperresponsiveforming growth factor.

20γ-Tocotrienol and ASM differentiation

Fig.	
  4	
  
(A)

A

Ac)vate	  RhoA
Total	
  RhoA

TGF-­‐β1	
  +	
  γ-­‐tocotrienolγ-­‐Tocotrienol

RhoA	
  	
  ac)vity	
  (Fold	  of	
  control)

3
2

*

1
0

A

ol

en

i
otr

c

o
γ-T

Id1	
  
β-­‐ac)n	
  
Time	
  
(h)

B

0

1

3

6

12

1

TGF-­‐β1	  (2	  ng/mL)

3

6

12

TGF-­‐β1+	  γ-­‐tocotrienol	  	
  (50	  µM)

Id1	
  /β-­‐ac)n	
  (Fold	  increase	
  over	  basal)

15
10
5
0
0

1

3

6

12

TGF-­‐β1	  (2	  ng/mL)

1

3

6

12

TGF-­‐β1+	  γ-­‐tocotrienol	  	
  (50	  µM)

Fig. 5. Effect of g-tocotrienol on Id1 expression induced
by TGF-b1.
hTERT-ASM cells were pretreated with g-tocotrienol for
1 h and then stimulated with TGF-b1 (2 ng/mL). TGF-b1
increased the expression of Id1 from 3 to 6 h. g-Tocotrienol did not affect Id1 expression at any of the time
points. Panel A shows a representative blot and panel B
shows the results of densitometry analysis from six celllines. Id1, inhibitor of differentiation; TGF, transforming
growth factor.

Id1β-­‐ac)n	  Control

Id1/β-­‐ac)n	
  (Fold	  increase	
  over	  basal)(B)

B

Fig. 4. g-Tocotrienol inhibits TGF-b-induced activation of RhoA.
A: Effect of g-tocotrienol on the activation of RhoA
induced by TGF-b1 was studied. Rho activation
peaked 5 min after TGF-b1 stimulation; this effect was inhibited by g-tocotrienol (10 to 50 mM).
B: The activated RhoA bands were quantified bydensitometry and expressed as a fold change relative to control unstimulated. The TGF-b1-induced
activation of RhoA was inhibited significantly byg-tocotrienol. Data are expressed as mean ± SD (n= 5). *P < 0.05 versus TGF-b1. Rho, Ras homolog;
TGF, transforming growth factor.

3
2
1
0

Y27632	
   Simvasta)n	
   TGF-­‐β1	
   Y27632	
   Simvasta)n	
  
+	
  
(10	
  µM) (10	
  µM)
(2	
  ng/mL) +	  	
  TGF-­‐β1
	  TGF-­‐β1

Fig. 6. Simvastatin and the Rho-kinase inhibitor
Y-27632 do not inhibit the TGF-b1-induced expressionof Id1.
hTERT-ASM cells were pretreated with Y27632 (10mM) or simvastatin (10 mM) for 1 h and then stimulatedwith TGF-b1 (2 ng/mL) for 3 h. TGF-b1 increased theexpression of Id1, but this was not affected by either
Control	
 Y27632	
   Simvasta)n	
   TGF-­‐β1	
   Y27632	
   Simvasta)n	
   Y27632 or simvastatin. Data are expressed as mean ±
+	
  
(10	
  µM) (10	
  µM)
+	
  
SD (n = 4). Id, inhibitor of differentiation; TGF, trans	
  TGF-­‐β1
	  TGF-­‐β1
forming growth factor.

21

T. Fukushima et al.

ness.32 In ASM cells, simvastatin suppresses the TGFb1-induced expression of collagen I and fibronectin.22
Schaafsma et al. reported that PDGF induced RhoA
activation and ASM cell contraction.33 We also reported that g-tocotrienol inhibits the PDGF-BB-induced
ASM cells proliferation and migration via inactivationof RhoA.21 These data suggest RhoA play a key rolein ASM cells functions and g-tocotrienol might inhibit
RhoA activation and as a result, inhibit airway remodeling.
Cheung et al. reported that Id1 induced epithelial
cell invasion, which was regulated by Rho GTPases.34 In
this study, we demonstrated that inhibition of the RhoA/
ROCK pathway by simvastatin or Y27632 did not affectthe induction of Id1 by TGF-b1. Our results indicate thatg-tocotrienol directly inhibits RhoA activation without upregulating or downregulating Id1. However, theprecise mechanism by which RhoA is inactivated byg-tocotrienol in the ASM remains unclear. Qureshi et al.
reported that tocotrienols lower serum cholesterol,35, 36
which is mediated by inhibition of HMG-CoA reductase
by stimulating the ubiquitination and degradation of reductase and by blocking sterol regulatory element-binding proteins.37 Future investigations will determine
whether these pathways are involved in ASM cells.
We haven’t studied the role of reactive oxygen
species (ROS) in this study. It has been reported thatvitamin E has antioxidant activity and g-tocotrienol decreased PDGF-BB-induced ROS levels in human airway
smooth muscle cells.21, 38 Since TGF-b1 generates ROS
and ROS is related to airway remodeling in asthma andfibrosis in several organs,13, 39 reduction of ROS might be
one of the inhibitory mechanisms of g-tocotrienol in thisstudy.
The effect of current asthma therapies on airway
remodeling in asthma has been investigated in several
studies (reviewed in 40). Since the feature of ASM remodeling correlates with asthma severity,41 therapies
targeting ASM are important. Bergeron et al. reported
that inhaled corticosteroid (ICS) decrease a-smoothmuscle area in peripheral airways. However, ICS did
not decrease collagen III or a-smooth muscle area in thecentral airways nor did it affect the number of TGF-b
positive cells in the submucosa.42 Chakir et al. also reported that oral corticosteroid did not change TGF-b,
collagen I and III levels in moderate to severe asthma.43
Therefore, new drugs targeting the TGF-b1-induced
profibrotic changes in airway mesenchymal cells are
an unmet need. g-Tocotrienol exhibits anti-proliferativeand inhibitory effects on migration of ASM cells. Furthermore, we have demonstrated the anti-fibrotic effectof g-tocotrienol in TGF-b1 stimulated ASM cells. Thus,

g-Tocotrienol might be possible therapeutic option targeting airway remodeling in asthma.
In conclusion, we demonstrated the effect of g-tocotrienol on the TGF-b1-induced differentiation ofhuman ASM cells and extracellular matrix deposition.
We also defined the key elements of the downstream
signaling cascade inhibited by g-tocotrienol in the ASM
cells activated by TGF-b1. These new findings suggestthat g-tocotrienol could become a therapeutic agent toregulate airway remodeling in asthma.
Acknowledgments: This work was supported by JPPS KAKENHI
24500976.
The authors declare no conflict of interest.

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