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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 macrophage colony-stimulating factor (M-CSF) and prostaglandin E2 (PGE2) 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 the component of tissue-type plasminogen activator (tPA), urokinase-type PA (uPA), and PA inhibitor type 1 (PAI-1), alpha7 nicotine receptor, and c-fos. We also examined the effect of the nicotine antagonist D-tubocurarine on nicotine-induced expression of MMP-1. Gene expression was examined using real-time polymerase chain reaction (PCR) to estimate mRNA levels. In addition, expression of the MMP, TIMP, uPA, tPA, and PAI-1 proteins 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 not change through day 14. Enhancement of MMP-1 expression by nicotine treatment was eliminated by simultaneous treatment with D-tubocurarine. In the presence of nicotine, expression of uPA, PAI-1, or TIMP-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 alpha7 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.

Previous studies have indicated that lipopolysaccharide (LPS) from Gram-negative bacteria in plaque induces the release of prostaglandin E(2) (PGE(2)), which promotes alveolar bone resorption in periodontitis, and that tobacco smoking might be an important risk factor for the development and severity of periodontitis. We determined the effect of nicotine and LPS on alkaline phosphatase (ALPase) activity, PGE(2) production, and the expression of cyclooxygenase (COX-1, COX-2), PGE(2) receptors Ep1>4, and macrophage colony stimulating factor (M-CSF) in human osteoblastic Saos-2 cells. The cells were cultured with 10(-3) M nicotine in the presence of 0, 1, or 10 mug/ml LPS, or with LPS alone. ALPase activity decreased in cells cultured with nicotine or LPS alone, and decreased further in those cultured with both nicotine and LPS, whereas PGE(2) production significantly increased in the former and increased further in the latter. By itself, nicotine did not affect expression of COX-1, COX-2, any of the PGE(2) receptors, or M-CSF, but when both nicotine and LPS were present, expression of COX-2, Ep3, Ep4, and M-CSF increased significantly. Simultaneous addition of 10(-4) M indomethacin eliminated the effects of nicotine and LPS on ALPase activity, PGE(2) production, and M-CSF expression. Phosphorylation of protein kinase A was high in cells cultured with nicotine and LPS. These results suggest that LPS enhances the production of nicotine-induced PGE(2) by an increase in COX-2 expression in osteoblasts, that nicotine-LPS-induced PGE2 interacts with the osteoblast Ep4 receptor primarily in autocrine or paracrine mode, and that the nicotine-LPS-induced PGE(2) then decreases ALPase activity and increases M-CSF expression.