Abstract
Oral submucous fibrosis (OSF) is a chronic inflammatory disease characterized by the accumulation of excess collagen, and areca nut chewing has been proposed as an important etiological factor for disease manifestation. Activation of transforming growth factor-β signaling has been postulated as the main causative event for increased collagen production in OSF. Oral epithelium plays important roles in OSF, and arecoline has been shown to induce TGF-β in epithelial cells. In an attempt to understand the role of areca nut constituents in the manifestation of OSF, we studied the global gene expression profile in epithelial cells (HaCaT) following treatment with areca nut water extract or TGF-β. Interestingly, 64% of the differentially regulated genes by areca nut water extract matches with the TGF-β induced gene expression profile. Out of these, expression of 57% of genes was compromised in the presence of ALK5 (TβRI) inhibitor and 7% were independently induced by areca nut, highlighting the importance of TGF-β in areca nut actions. Areca nut water extract treatment induced p-SMAD2 and TGF-β downstream targets in HaCaT cells but not in human gingival fibroblast cells (hGF), suggesting epithelial cells could be the source of TGF-β in promoting OSF. Water extract of areca nut consists of polyphenols and alkaloids. Both polyphenol and alkaloid fractions of areca nut were able to induce TGF-β signaling and its downstream targets. Also, SMAD-2 was phosphorylated following treatment of HaCaT cells by Catechin, Tannin and alkaloids namely Arecoline, Arecaidine and Guvacine. Moreover, both polyphenols and alkaloids induced TGF-β2 and THBS1 (activator of latent TGF-β) in HaCaT cells suggesting areca nut mediated activation of p-SMAD2 involves up-regulation and activation of TGF-β. These data suggest a major causative role for TGF-β that is induced by areca nut in OSF progression.
Highlights
Oral submucous fibrosis (OSF) is a chronic inflammatory disease characterized by epithelial atrophy and fibrosis in submucosa of the oral tissues that can cause difficulty in chewing, swallowing, speaking, and mouth opening [1]
Areca nut induces transforming growth factor-b (TGF-b) signaling in epithelial cells In order to study the areca nut effects on epithelial cells, gene expression profiling was performed using Agilent whole human genome 4644 K arrays with the RNAs extracted from HaCaT cells treated with areca nut water extract (5 mg/ml; 5H). 1331 genes were found to be differentially expressed following treatment with areca nut water extract (P#0.05, fold change $1.5)
HaCaT cells were treated with areca nut water extract in the presence of ALK5 inhibitor (TbRI inhibitor, SB431542) and gene expression profiling was compared with areca nut extract regulated genes
Summary
Oral submucous fibrosis (OSF) is a chronic inflammatory disease characterized by epithelial atrophy and fibrosis in submucosa of the oral tissues that can cause difficulty in chewing, swallowing, speaking, and mouth opening [1]. TGF-b was shown to be up-regulated in OSF tissues [8] and its activation has been shown by the nuclear localization of p-SMAD2 in OSF tissues compared to normals [9,10]. Lysyl oxidase catalyzes formation of aldehydes from lysine residues in collagen and elastin precursors while Transglutaminase-2 (TGM-2) catalyzes transamidating acyltransferase reaction leading to matrix stabilization. These crosslinking enzymes are known to be affected by the pro-fibrotic cytokines like TGF-b, highlighting the probable role of pro-fibrogenic cytokines in OSF [13,14]. TGF-b activation by arecoline in oral keratinocytes was shown to
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