Betel-derived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis

Oral submucous fibrosis (OSF) is a premalignant, fibrosing disorder of the mouth, pharynx, and oesophagus, with a malignant transformation rate of 7-13%. OSF is strongly associated with areca (betel) nut chewing and worldwide, over 5 million people are affected. As alpha v beta 6 integrin is capable of promoting both tissue fibrosis and carcinoma invasion, we examined its expression in fibroepithelial hyperplasia and OSF. alpha v beta 6 was markedly up-regulated in OSF, with high expression detected in 22 of 41 cases (p < 0.001). We investigated the functional role of alpha v beta 6 using oral keratinocyte-derived cells genetically modified to express high alpha v beta 6 (VB6), and also NTERT-immortalized oral keratinocytes, which express low alpha v beta 6 (OKF6/TERT-1). VB6 cells showed significant alpha v beta 6-dependent activation of TGF-beta 1, which induced transdifferentiation of oral fibroblasts into myofibroblasts and resulted in up-regulation of genes associated with tissue fibrosis. These experimental in vitro findings were confirmed using human clinical samples, where we showed that the stroma of OSF contained myofibroblasts and that TGF-beta 1-dependent Smad signalling was detectable both in keratinocytes and in myofibroblasts. We also found that arecoline, the major alkaloid of areca nuts, up-regulated keratinocyte alpha v beta 6 expression. This was modulated through the M-4 muscarinic acetylcholine receptor and was suppressed by the M-4 antagonist, tropicamide. Arecoline-dependent alpha v beta 6 up-regulation promoted keratinocyte migration and induced invasion, raising the possibility that this mechanism may support malignant transformation. Over 80% of OSF-related oral cancers examined had moderate/high alpha v beta 6 expression. These data suggest that the pathogenesis of OSF may be epithelial-driven and involve arecoline-dependent up-regulation of alpha v beta 6 integrin. Copyright (C) 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.