Arecoline‐induced myofibroblast transdifferentiation from human buccal mucosal fibroblasts is mediated by ZEB1

Abstract

Oral submucous fibrosis (OSF) is considered as a pre-cancerous condition of the oral mucosa and is highly associated with habitual areca quid chewing. Arecoline is the major alkaloid in areca quid and is thought to be involved in the pathogenesis of OSF. Our previous studies have demonstrated that arecoline could induce epithelial–mesenchymal transition (EMT)-related factors in primary human buccal mucosal fibroblasts (BMFs). Therefore, we investigated the expression of zinc finger E-box binding homeobox 1 (ZEB1), which is a well-known transcriptional factor in EMT, in OSF tissues and its role in arecoline-induced myofibroblast transdifferentiation from BMFs. The expression of ZEB1, as well as the myofibroblast marker α-smooth muscle actin (α-SMA), was significantly increased in OSF tissues, respectively. With immunofluorescence analysis, arecoline induced the formation of α-SMA-positive stress fibres in BMFs expressing nuclear ZEB1. Arecoline also induced collagen contraction of BMFs in vitro. By chromatin immunoprecipitation, the binding of ZEB1 to the α-SMA promoter in BMFs was increased by arecoline. The promoter activity of α-SMA in BMFs was also induced by arecoline, while knockdown of ZEB1 abolished arecoline-induced α-SMA promoter activity and collagen contraction of BMFs. Long-term exposure of BMFs to arecoline induced the expression of fibrogenic genes and ZEB1. Silencing of ZEB1 in fibrotic BMFs from an OSF patient also suppressed the expression of α-SMA and myofibroblast activity. Inhibition of insulin-like growth factor receptor-1 could suppress arecoline-induced ZEB1 activation in BMFs. Our data suggest that ZEB1 may participate in the pathogenesis of areca quid–associated OSF by activating the α-SMA promoter and inducing myofibroblast transdifferentiation from BMFs.

Elevation of Twist expression by arecoline contributes to the pathogenesis of oral submucous fibrosis

“…26 Our previous study has demonstrated that ZEB1 could bind to the E-box domain of a-SMA promoter to induce myofibroblast transdifferentiation activities in OSF. 11 However, the detailed molecular mechanisms involved in the regulatory links between Twist-mediated EMT and myofibroblast in OSF are still poorly understood. In this study, we attempted to investigate the role of Twist in the pathogenesis of areca quid chewing-associated OSF.…”

“…Previously, we have demonstrated that arecoline could induce myofibroblast activities in BMFs. 1,11 Targeting sh-Twist-1 abrogated arecoline-induced collagen gel contraction ( Figure 2C) and migration abilities ( Figure 2D) in BMFs.…”

The pharmacology, toxicology and potential applications of arecoline: a review

“…Therefore, Hu and Jian (2007) proposed that EMT was a new possible mechanism of OSF. Subsequently, a recent report clearly demonstrated that Zinc finger E-box binding homeobox 1 (ZEB1), a transcriptional repressor of E-cadherin, was increased in OSF tissues and was induced by arecoline in buccal mucosal fibroblasts followed by induction of myofibroblast activity (Chang et al 2014). Heme oxygenase-1 (HO-1) expression was significantly higher in OSF specimens, and it is expressed mainly by fibroblasts, endothelial cells and inflammatory cells ).…”

Aberrant SSEA-4 upregulation mediates myofibroblast activity to promote pre-cancerous oral submucous fibrosis

“…Our previous studies have demonstrated that arecoline could induce myofibroblast activities in BMFs1119. To further investigate whether SSEA-4 plays a role in maintaining arecoline-induced myofibroblastic differentiation activity, loss-of-function of ST3GAL2 was conducted in arecoline-stimulated BMFs.…”

“…Arecoline, the major areca nut alkaloid, could activate the transdifferentiation of oral keratinocytes into myofibroblasts8. Previously, we have also identified that the treatment of arecoline could induce myofibroblasts activities in human normal buccal mucosal fibroblasts (BMFs)91011, supporting the crucial roles of myofibroblasts transdifferentiation during the pathogenesis of OSF.…”