Tobacco smoking is an important risk factor for the development of severe periodontitis.Recently,we showed that nicotine affected mineralized nodule formation,and that nicotine andlipopolysaccharide stimulated the formation of osteoclast-like cells by increasing production of macrophagecolony-stimulating factor (M-CSF) and prostaglandin E_2 (PGE_2) by human osteoblastic Saos-2 cells.In thepresent study,we examined the effects of nicotine on the expression of matrix metalloproteinases (MMPs),tissue inhibitors of matrix metalloproteinases (TIMPs),the plasminogen activation system including thecomponent of tissue-type plasminogen activator (tPA),urokinase-type PA (uPA),and PA inhibitor type 1(PAI-1),α7 nicotine receptor,and c-fos.We also examined the effect of the nicotine antagonist D-tubocurarineon nicotine-induced expression of MMP-1.Gene expression was examined using real-time polymerase chainreaction (PCR) to estimate mRNA levels.In addition,expression of the MMP,TIMP,uPA,tPA,and PAI-1proteins was determined by Western blotting analysis.Nicotine treatment caused expression of MMP-1,2,3,and 13,but not MMP-14,to increase significantly after 5 or 10 d of culture;MMP-14 expression did notchange through day 14.Enhancement of MMP-1 expression by nicotine treatment was eliminated bysimultaneous treatment with D-tubocurarine.In the presence of nicotine,expression of uPA,PAI-1,orTIMP-1,2,3,or 4 did not change over 14 d of culture,whereas expression of tPA increased significantly byday 7.Nicotine also increased expression of the α7 nicotine receptor and c-fos genes.These results suggestthat nicotine stimulates bone matrix turnover by increasing production of tPA and MMP-1,2,3,and 13,thereby tipping the balance between bone matrix formation and resorption toward the latter process. Tobacco smoking is an important risk factor for the development of severe periodontitis. Recently, we showed that nicotine affected mineralized nodule formation, and that nicotine and lipopolysaccharide stimulated the formation of osteoclast-like cells by increasing production of macrophagecolony-stimulating factor (M-CSF) and prostaglandin E_2 (PGE_2) by human osteoblastic Saos-2 cells. in the present study, we examined the effects of nicotine on the expression of matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs), the plasminogen activation system including thecomponent of tissue-type plasminogen activator (tPA), urokinase-type PA (uPA), and PA inhibitor type 1 (PAI- 1), α7 nicotine receptor, and c- fos.We also examined the effect of the nicotine antagonist D-tubocurarineon nicotine -induced expression of MMP-1.Gene expression was examined using real-time polymerase chain reaction (PCR) to estimate mRNA levels. addition, expression of the MMP, TIMP, uPA, tPA, and PA I-1 proteins were determined by Western blotting analysis. Nicotine treatment caused expression of MMP-1, 2, 3, and 13, but not MMP-14, to increase significantly after 5 or 10 d of culture; day 14. Enhancement of MMP-1 expression by nicotine treatment was eliminated by simultaneous treatment with D -tubocurarine. the presence of nicotine, expression of uPA, PAI-1, orTIMP-1,2,3, or 4 did not change over 14 d of culture, whereas expression of tPA increased significantly by day 7. Nicotine also increased expression of the α7 nicotine receptor and c-fos genes. These results suggest that nicotine stimulates bone matrix turnover by increasing production of tPA and MMP-1, 2, 3, and 13, thereby tipping the balance between bone matrix formation and resorption toward the latter process.