Elevation of S100A4 Expression in Buccal Mucosal Fibroblasts by Arecoline: Involvement in the Pathogenesis of Oral Submucous Fibrosis

BACKGROUND: S100A4, a member of the calcium-binding proteins, is dramatically elevated in a variety of fibrotic diseases. Areca quid chewing is the most important etiological factor in the pathogenesis of oral submucous fibrosis (OSF). OSF has been considered as a pre-cancerous condition of oral mucosa. The aim of this study was to determine the critical role of S100A4 expression in the pathogenesis of OSF both in vitro and in vivo. METHODOLOGY/PRINCIPAL FINDING: Thirty OSF tissues from areca quid chewers and ten normal buccal mucosa samples without areca quid chewing were analyzed by using immunohistochemistry for S100A4 expression in vivo. Collagen gel contraction capability and expression of tissue inhibitor of metalloproteinases 1 (TIMP1)/MMP9 in arecoline-stimulated BMFs with S100A4 knockdown was presented in vitro. Initially, S100A4 expression was higher in areca quid chewing-associated OSF specimens than normal buccal mucosa specimens (p = 0.001). Arecoline, a major areca nut alkaloid, led to dose- and time-dependent elevation of S100A4 expression in normal buccal mucosa fibroblasts BMFs (p<0.05). The additions of pharmacological agents rapamycin (mTOR inhibitor), PD98059 (ERK inhibitor), and Bay117082 (NF-κB inhibitor) were found to inhibit arecoline-induced S100A4 expression (p<0.05) in BMFs. Down-regulation of S100A4 by lentiviral infection significantly reversed arecoline-induced collagen gel contraction and TIMP1/MMP9 expression. CONCLUSION/SIGNIFICANCE: These results suggest that S100A4 expression is significantly up-regulated in OSF specimens. Arecoline-induced S100A4 expression was down-regulated by rapamycin, PD98059, and Bay117082. Targeting S100A4 might be a potential therapeutic target for OSF through TIMP1/MMP9 down-regulation.