ABSTRACT
BACKGROUND: The aim of the present study is to investigate the expression of phospholipase D (PLD) 1 and PLD2 in periodontal patients and in human periodontal ligament cells (HPDLCs) exposed to nicotine plus lipopolysaccharide (LPS) from Porphyromonas gingivalis (Toll-like receptor 2 ligand). Furthermore, the effects of PLD isoform inhibition on the inflammatory response and osteoclast differentiation and its mechanisms were determined. METHODS: Proinflammatory mediators were examined by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. To silence the gene expression of the PLD isoforms, cells were transfected with small interfering RNA (siRNA) targeting PLD1 or PLD2. Mouse bone marrow-derived macrophages (BMMs) were used as osteoclast precursor cells for in vitro osteoclastogenesis. Western blot analysis and immunofluorescence were used to assess signaling pathways. RESULTS: Chronic smokers with periodontitis exhibited significantly higher PLD1 and PLD2 messenger RNA (mRNA) expression than non-smokers with periodontitis and healthy controls. Nicotine and LPS upregulated PLD1 and PLD2 mRNA expression in a dose-dependent manner in HPDLCs. Pharmacologic and siRNA-mediated inhibition of PLD1 and PLD2 attenuated the nicotine- and LPS-induced upregulation of inducible nitric oxide (NO) synthase and cyclooxygenase-2, production of NO, and prostaglandin E2, and mRNA expression and secretion of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-8. The conditioned media from HPDLCs treated with PLD isoform inhibitors or siRNA against PLD inhibited receptor activator of nuclear factor-κB (NF-κB) ligand-mediated osteoclast differentiation, as well as protein expression of nuclear factor of activated T cells c1 and c-Fos, in BMMs. In addition, PLD isoform inhibitors and siRNA inhibited the nicotine- and LPS-induced activation of phosphoinositide 3-kinase, protein kinase C, p38, extracellular signal-regulated kinase, c-Jun N-terminal protein kinase, mitogen-activated protein kinase, and NF-κB. CONCLUSION: To the best of the authors' knowledge, this study is the first to demonstrate that PLD isoform inhibition has anti-inflammatory and antiosteoclastogenic effects and thus may be a therapeutic target for the treatment of periodontitis.
