Arecoline induced Egr-1 synthesis via TGF-β1 signaling pathway in human buccal fibroblasts and epithelial cells: inhibition by EGCG
Date Issued
2016
Date
2016
Author(s)
Hsieh, Yu-Ping
Abstract
Areca nut (AN) chewing is the most important risk factor of oral submucous fibrosis (OSF). Early growth response-1 (Egr-1) protein has been shown to play a central role in the pathogenesis of many human fibrotic diseases. We immunohistochemically examined the expression of Egr-1 protein in OSF specimens. Positive Egr-1 staining was detected in subepithelial fibroblasts and inflammatory cells. We further observed Arecoline, a main alkaloid found in AN, increased Egr-1 synthesis in a dose- and time- dependent manner in buccal mucosal fibroblasts. Constitutive overexpression of Egr-1 during AN chewing may play a role in the pathogenesis of OSF. Pretreatment with antioxidant NAC, JNK inhibitor and ERK inhibitor significantly reduced Arecoline induced Egr-1 synthesis. Furthermore, epigallocatechin-3-gallate (EGCG) completely inhibited Arecoline-induced Egr-1 synthesis and the inhibition is dose-dependent. Transforming growth factor β (TGF-β) is a key regulator in the pathogenesis of OSF. Egr-1 is an important mediator of TGF-β-induced responses and is considered to play a central role in orchestrating fibrotic responses. Egr-1 may contribute to the pathogenesis of OSF as it is overexpressed in OSF specimens and in Arecoline-stimulated normal human buccal mucosal fibroblasts (BMFs). Previous study has shown that the stimulation of Egr-1 by TGF- in dermal fibroblasts occurs independent of the activin receptor-like kinase 5 (ALK5)/Smad3 signaling axis. Since Egr-1 is essential for the fibrotic response to TGF- and that TGF- signaling is cell type-and context-dependent, we aimed to examine the possible signal transduction pathways involved in TGF-β1-induced Egr-1 expression in BMFs. In the present study, we have shown that TGF-β1 induces Egr-1 expression and that Egr-1 mediates the TGF-β1-induced mRNAs expression of the α1- and α2-chains of type I collagen (COL1A1 and COL1A2) and the acid-soluble collagen production in BMFs. We have also shown that ALK5 inhibitor, ERK inhibitor, JNK inhibitor, and Smad3 inhibitor significantly abrogate the TGF-β1-induced levels of Egr-1 protein, indicating that ALK5/Smad3, ERK, and JNK are involved in the TGF-β1-induced Egr-1 in BMFs. We have further demonstrated that knocking down Smad3 using Smad2/3 siRNA completely abolishes the TGF1-induced Egr-1. Moreover, EGCG fully inhibits TGF-β1-induced Egr-1 expression and abrogates the TGF-1-stimulated production of acid-soluble collagens. We found TGF-β1 induced NADPH oxidase 4 (NOX4) expression in BMFs. Pretreatment with antioxidant NAC, NOX family inhibitor, and NOX4 inhibitor significantly reduced TGF-β1-induced Egr-1 expression and ROS production in BMFs. We found antioxidant NAC significantly inhibited TGF-β1-induced Src phosphorylation in BMFs. In addition, NOX4 inhibitor, Src inhibitor, and NOX4 siRNA significantly inhibited TGF-β1-induced Src, ERK, JNK, and Smad3 phosphorylation in BMFs. These results showed that TGF-β1-induced Src phosphorylation is regulated by NOX4/ROS, and Src is an upstream signaling transducer of ERK, JNK, and Smad3 in BMFs. We found Arecoline increased activated TGF-β1 level in BMFs. Pretreatment with antioxidant NAC and GSH significantly reduced Arecoline-induced activated TGF-β1 level in BMFs. We further found antioxidant NAC and Trolox, PEG-catalase, mitochondria-targeted O2- inhibitor, and MnTBAP significantly inhibited Arecoline-induced activated TGF-β1 level in BMFs. We found H2O2 increased activated TGF-β1 level in BMFs. Mitochondria-targeted O2- inhibitor significantly reduced Arecoline-induced mitochondrial superoxide in BMFs. These results showed that Arecoline increased activated TGF-β1 level via mitochondrial superoxide and H2O2 in BMFs. We further found EGCG significantly inhibited Arecoline-induced mitochondrial superoxide and activated TGF-β1 level in BMFs. Epithelial-mesenchymal transition (EMT) is a big issue in fibrosis disease. We investigate whether Egr-1 play a role in regulating EMT in TW2.6 cells. We found TGF-β1 induced Egr-1 and Vimentin expression and reduced E-cadherin expression in TW2.6 cells. Genetic targeting of Egr-1 by Egr-1 siRNA significantly inhibited TGF-β1-induced Egr-1 and Snail expression and reduced TGF-β1-induced EMT in TW2.6 cells. We found Src inhibitor, JNK inhibitor, antioxidant NAC, NOX family inhibitor, NOX4 inhibitor, ALK5 inhibitor, and Smad3 inhibitor significantly inhibited TGF-β1-induced Egr-1 and Snail expression in TW2.6 cells. We further found EGCG significantly inhibited TGF-β1-induced Egr-1 and Snail expression and reduced TGF-β1-induced EMT in TW2.6 cells. EGCG potentially qualifies as a useful reagent for the prevention and therapy of OSF.
Subjects
Oral submucous fibrosis（OSF）
Arecoline
Early growth response-1（Egr-1）
EGCG
Reactive oxygen species（ROS）
mitochondrial superoxide
latent Transforming growth factor-β1（latent TGF-β1）
myofibroblast differentiation
Epithelial-mesenchymal transition（EMT）
SDGs
Type
thesis
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