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. 2017 Mar 9;60(1):16-23.
eCollection 2017 Mar.

γ-Tocotrienol Inhibits TGF-β1-Induced Contractile Phenotype Expression of Human Airway Smooth Muscle Cells

Takehito Fukushima  1 , Akira Yamasaki  1 , Tomoya Harada  1 , Hiroki Chikumi  1 , Masanari Watanabe  1 , Ryota Okazaki  1 , Miki Takata  1 , Yasuyuki Hasegawa  1 , Jun Kurai  1 , Masaaki Yanai  1 , Akihiro Yamamoto  1 , Yuriko Sueda  1 , Andrew J Halayko  2 , Eiji Shimizu  1
Affiliations

γ-Tocotrienol Inhibits TGF-β1-Induced Contractile Phenotype Expression of Human Airway Smooth Muscle Cells

Takehito Fukushima et al. Yonago Acta Med. .

Abstract

Background: Tocotrienols, members of the vitamin E family, exist in four different isoforms (α, β, γ and δ tocotrienol) that have can be protective against brain damage, as well as having anticancer effects in vivo and in vitro. We have shown that γ-tocotrienol inhibits human airway smooth muscle cell proliferation and migration induced by platelet-derived growth factor (PDGF)-BB by suppressing RhoA activation. In this study, we tested whether γ-tocotrienol modulates transforming growth factor (TGF) -β-induced induction of human airway smooth muscle (ASM) into a contractile phenotype and concomitant synthesis of extracellular matrix proteins.

Methods: ASM cells were stimulated with TGF-β1 (2 ng/mL) for 48 hours and the effect of γ-tocotrienol (50 μM) on α-smooth muscle actin, fibronectin and collagen I expression was assessed using Western blotting. The signaling pathways involved in TGF-β1 stimulation were also investigated.

Results: TGF-β1 increased α-smooth muscle actin, fibronectin and collagen Ⅰ abundance by 3- to 5-fold. This response was inhibited significantly by γ-tocotrienol. Furthermore, γ-tocotrienol suppressed RhoA activation, but did not affect Smad2 or Smad3 phosphorylation.

Conclusion: These results indicate that γ-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.

Keywords: airway smooth muscle; asthma; remodeling; vitamin E; γ-Tocotrienol.

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Figures

Fig. 1.
Fig. 1.
γ-Tocotrienol inhibits the TGF-β1-induced expression of α-smooth muscle actin. hTERT-ASM cells were pretreated with γ-tocotrienol for 1 h and then stimulated with TGF-β1 (2 ng/mL) for 48 h. Western blotting analysis showed that relative abundance of α-SMA was increased by TGF-β1 and γ-tocotrienol inhibited the TGF-β1-induced expression of α-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 as mean value ± SD (n = 7). *P < 0.05 versus TGF-β1 (A and B). TGF-β1 enhanced α-SMA expression compared with control. γ-Tocotrienol inhibited the induction of α-SMA expression by TGF-β1. γ-Tocotrienol alone did not affect α-SMA expression (C). Bar = 50 μM. α-SMA, α-smooth muscle actin; TGF, transforming growth factor.
Fig. 2.
Fig. 2.
γ-Tocotrienol inhibits the TGF-β1-induced expression of fibronectin and collagen I. hTERT-ASM cells were pre-treated with γ-tocotrienol for 1 h and then stimulated with TGF-β1 for 48 h. TGF-β1 increased the abundance of fibronectin and collagen I (A and B). γ-Tocotrienol significantly attenuated the TGF-β1-induced expression of fibronectin and collagen I (C and D). γ-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-β1. TGF, transforming growth factor.
Fig. 3.
Fig. 3.
γ-Tocotrienol does not inhibit TGF-β1-induced phosphorylation of Smad2 and Smad3. A: Phosphorylation of Smad2 and Smad3 peaked 60 to 120 min after stimulation with TGF-β1 (2 ng/mL). γ-Tocotrienol (50 μM) did not inhibit the TGF-β1-induced phosphorylation of Smad2 and Smad3. B: The phosphorylated Smad2 and Smad3 bands were quantified by densitometry and expressed as the fold change, relative to control. Neither phosphorylation of Smad2 nor Smad3 were inhibited by γ-tocotrienol. Data are expressed as mean ± SD (n = 5). TGF, transforming growth factor.
Fig. 4.
Fig. 4.
γ-Tocotrienol inhibits TGF-β-induced activation of RhoA. A: Effect of γ-tocotrienol on the activation of RhoA induced by TGF-β1 was studied. Rho activation peaked 5 min after TGF-β1 stimulation; this effect was inhibited by γ-tocotrienol (10 to 50 μM). B: The activated RhoA bands were quantified by densitometry and expressed as a fold change relative to control unstimulated. The TGF-β1-induced activation of RhoA was inhibited significantly by γ-tocotrienol. Data are expressed as mean ± SD (n = 5). *P < 0.05 versus TGF-β1. Rho, Ras homolog; TGF, transforming growth factor.
Fig. 5.
Fig. 5.
Effect of γ-tocotrienol on Id1 expression induced by TGF-β1. hTERT-ASM cells were pretreated with γ-tocotrienol for 1 h and then stimulated with TGF-β1 (2 ng/mL). TGF-β1 increased the expression of Id1 from 3 to 6 h. γ-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 cell lines. Id1, inhibitor of differentiation; TGF, transforming growth factor.
Fig. 6.
Fig. 6.
Simvastatin and the Rho-kinase inhibitor Y-27632 do not inhibit the TGF-β1-induced expression of Id1. hTERT-ASM cells were pretreated with Y27632 (10 μM) or simvastatin (10 μM) for 1 h and then stimulated with TGF-β1 (2 ng/mL) for 3 h. TGF-β1 increased the expression of Id1, but this was not affected by either Y27632 or simvastatin. Data are expressed as mean ± SD (n = 4). Id, inhibitor of differentiation; TGF, transforming growth factor.
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