Nicotine modulates gelatinase B (MMP-9) and epilysin (MMP-28) expression in reconstituted human oral epithelium Filippo Reno` 1 , Vincenzo Rocchetti 2 , Mario Migliario 2 , Mario Cannas 1 1 Human Anatomy Laboratory and 2 Dental Clinic, Department of Experimental and Clinical Medicine, University of Eastern Piedmont ‘‘A. Avogadro’’, Novara, Italy Oral epithelial keratinocytes express nicotinic cholinergic receptors which activation modulates keratinocytes dif- ferentiation and migration through different metabolic pathways. Matrix metalloproteinases (MMPs) are Zn- dependent enzyme involved in cell migration. Among them, gelatinase B (MMP-9) and epilysin (MMP-28) are two MMPs expressed by human keratinocytes during both wound healing and proliferation. Their expression has been investigated in a reconstituted human oral epi- thelium (HOE) exposed to nicotine (Nic, 1–50 lM) for 72 h both in the absence and presence of the nicotinic antagonist mecamylamine (Mec), H7, a PKC inhibitor and PD98059, a MAPK inhibitor (PD). At the end of treatment, MMP-28 expression has been analyzed in epithelium sections using an anti-MMP-28 antibody, whereas MMP-9 presence and activity has been measured in cell-conditioned medium analyzed by gelatine zymog- raphy. The expression of MMP-9 was reduced by Nic in a dose-dependent fashion and this effect was antagonized by Mec, H7 and PD. On the other hand, Nic increased the expression of MMP-28, and this effect was blocked both by H7 and PD, whereas Mec even enforced it. Nic effects on MMP-9 and MMP-28 expression by oral keratinocytes were not previously reported and these data suggest MMPs expression mediated by PKC and MAPK as a possible target for Nic toxicity in oral epithelium. J Oral Pathol Med (2011) 40: 33–36 Keywords: keratinocyte; MMP-9; MMP-28; nicotine Introduction Human keratinocytes produce, degrade and respond to achetylcholine (Ach) by rapidly modifying their gene expression trough the activation of multiple signal transduction pathways coupled to muscarinic and nic- otinic cholinergic receptors (1). In particular, oral epithelial keratinocytes express nicotinic cholinergic receptors (nAChRs) that bind nicotine (2) which acti- vation modulates keratinocytes differentiation (3, 4) and migration (5). Nicotine (Nic) is a vegetal alkaloid present in the cigarette smoking and its measured concentration in saliva from smokers is around 10 lM (2). Nicotine alone or in combination with others substances present in the cigarette smoking (6) is now recognized as an agent for the modulation of key cellular processes involved in the pathobiological effects of tobacco (7). In particular, nicotine has been shown to inhibit keratinocyte proliferation (3) and lateral migra- tion, a key step in wound-healing process (5). Oral epithelium keratinocytes express different matrix metal- loproteinases (MMPs) such as MMP-1, MMP-2, MMP- 9, MMP-10 and MMP-28 during wound repair (8). The synchronized expression of MMPs seems to be a key regulatory pathway for a correct and effective wound healing (9). The potential effect of Nic on the expression of MMPs involved in wound healing in oral keratino- cytes has not been explored so far. To evaluate Nic- MMPs interaction in oral epithelium, the expression of both gelatinase B (MMP-9) (10) and epilysin (MMP-28) (11), two MMPs expressed by human keratinocytes during both wound healing and proliferation, has been investigated in a reconstituted human oral epithelium (HOE) exposed to Nic. Materials and methods Reconstituted human oral epithelium TR146 cells were cultivated onto polycarbonate mem- brane in 24 wells plates (12) for 5 days to obtain a reconstituted human oral epithelium (HOE) (SkinEthic, Nice, France). HOE were then stimulated for 72 h in DMEM added with 10% FBS by nicotine (Nic, 1–50 lM) both in the absence and in the presence of the nicotinic antagonist mecamylamine (Mec, 10 lM). To Correspondence: Prof. F. Reno` , Experimental and Clinical Medicine Department, University of Eastern Piedmont ‘‘A.Avogadro’’, Via Solaroli 17, 28100 Novara, Italy. Tel ⁄ Fax: +39-0321-660634, E-mail: filippo.reno@med.unipmn.it Accepted for publication July 7, 2010 doi: 10.1111/j.1600-0714.2010.00949.x J Oral Pathol Med (2011) 40: 33–36 ª 2010 John Wiley & Sons A/S Æ All rights reserved wileyonlinelibrary.com/journal/jop