RESUMEN
BACKGROUND AND OBJECTIVE: Although heme oxygenase-1 (HO-1) plays a key role in inflammation, its anti-inflammatory effects and mechanism of action in periodontitis are still unknown. This study aimed to identify the effects of HO-1 on the proinflammatory mediators activated by nicotine and lipopolysaccharide (LPS) stimulation in human periodontal ligament (PDL) cells. MATERIAL AND METHODS: The production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) was evaluated using Griess reagent and an enzyme immunoassay, respectively. The expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and HO-1 proteins was evaluated by Western blot analysis. RESULTS: Lipopolysaccharide and nicotine synergistically induced the production of NO and PGE(2) and increased the protein expression of iNOS, COX-2 and HO-1. Treatment with an HO-1 inhibitor and HO-1 small interfering RNAs blocked the LPS- and nicotine-stimulated NO and PGE(2) release as well as the expression of iNOS and COX-2. CONCLUSION: Our data suggest that the nicotine- and LPS-induced inflammatory effects on PDL cells may act through a novel mechanism involving the action of HO-1. Thus, HO-1 may provide a potential therapeutic target for the treatment of periodontal disease associated with smoking and dental plaque.
Asunto(s)
Ciclooxigenasa 2/efectos de los fármacos , Hemo-Oxigenasa 1/farmacología , Lipopolisacáridos/farmacología , Nicotina/farmacología , Óxido Nítrico Sintasa de Tipo II/efectos de los fármacos , Ligamento Periodontal/efectos de los fármacos , Androstadienos/farmacología , Antracenos/farmacología , Línea Celular , Inhibidores de la Ciclooxigenasa 2/farmacología , Dinoprostona/análisis , Dinoprostona/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Hemo-Oxigenasa 1/antagonistas & inhibidores , Hemo-Oxigenasa 1/genética , Humanos , Mediadores de Inflamación/análisis , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Metaloporfirinas/farmacología , FN-kappa B/efectos de los fármacos , Óxido Nítrico/análisis , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Ligamento Periodontal/citología , Ligamento Periodontal/enzimología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Protoporfirinas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Transfección , WortmaninaRESUMEN
Although substance P (SP), a potent proinflammatory peptide, is involved in inflammation and immune responses, the effect of SP on the expression of macrophage inflammatory protein 3alpha[MIP-3alpha, chemokine C-C ligand 20 (CCL20)] in periodontal ligament (PDL) cells is unknown. Equally enigmatic is the link between SP, the stress protein heme oxygenase-1 (HO-1), and CCL20 production. We investigated whether SP induces the release of chemokine CCL20 from immortalized PDL (IPDL) cells, and further clarify SP-mediated pathways. We also examined the relationship between HO-1 and CCL20 by treating PDL cells with SP. Incubating IPDL cells with SP increased expression of CCL20 mRNA and CCL20 protein in a dose-time-dependent manner. Highly selective p38 and extracellular-regulated kinase 1/2 (ERK1/2) inhibitors abrogated SP-induced expression of CCL20 in IPDL cells. SP is also responsible for initiating phosphorylation of IkappaB, degradation of IkappaB and activation of nuclear factor (NF)-kappaB. SP induced expression of HO-1 in both a concentration- and time-dependent manner, and CCL20 reflected similar patterns. The inductive effects of SP on HO-1 and CCL20 were enhanced by HO-1 inducer hemin and the membrane-permeable guanosine 3',5'-monophosphate (cGMP) analogue 8-bromo-cGMP. Conversely, this pathway was inhibited by the HO-1 inhibitor zinc protoporphyrin IX (ZnPP IX) and the selective inhibitor of guanylate cyclase, 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ). We report herein the pathway that connects SP along with other modulators of neuroimmunoregulation to the induction of HO-1 and the inflammatory mediator macrophage inflammatory protein (MIP)-3alpha/CCL20 in IPDL cells, which play an important role in the development of periodontitis or inflammation during orthodontic tooth movement.
Asunto(s)
Quimiocina CCL20/metabolismo , Hemo-Oxigenasa 1/fisiología , Proteínas Inflamatorias de Macrófagos/metabolismo , Ligamento Periodontal/efectos de los fármacos , Sustancia P/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Transformada , Transformación Celular Viral , Células Cultivadas , Quimiocina CCL20/genética , Citocinas/farmacología , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Mediadores de Inflamación/farmacología , Proteínas Inflamatorias de Macrófagos/genética , Inhibidor NF-kappaB alfa , FN-kappa B/fisiología , Ligamento Periodontal/citología , Ligamento Periodontal/metabolismo , Fosforilación/efectos de los fármacos , ARN Mensajero/genéticaRESUMEN
BACKGROUND AND OBJECTIVE: Periodontal ligament cells and gingival fibroblasts are important in the remodeling of periodontal tissue, but human papilloma virus (HPV)16-immortalized cell lines derived from human periodontal ligament cells and gingival fibroblasts has not been characterized. The purpose of this study was to establish and differentially characterize the immortalized cell lines from gingival fibroblasts and periodontal ligament by HPV16 transfection. MATERIAL AND METHODS: Cell growth, cell cycle analysis, western blot for cell cycle regulatory proteins and osteogenic differentiation markers, and reverse transcription-polymerase chain reaction for periodontal ligament-specific markers were performed. RESULTS: Both immortalized cell lines (immortalized gingival fibroblasts and immortalized periodontal ligament cells) grew faster than primary cultured gingival fibroblasts or periodontal ligament cells. Immortalized gingival fibroblasts and immortalized periodontal ligament cells overexpressed proteins p16 and p21, and exhibited degradation of proteins pRb and p53, which normally cause cell cycle arrest in G2/M-phase. Western blotting and reverse transcription-polymerase chain reaction for periodontal ligament-specific and osteogenic differentiation marker studies demonstrated that a cell line, designated IPDL, mimicked periodontal ligament gene expression for alkaline phosphatase, osteonectin, osteopontin, bone sialoprotein, bone morphogenic protein-2, periostin, S-100A4 and PDLs17. CONCLUSION: These results indicate that IPDL and immortalized gingival fibroblast cell lines consistently retain normal periodontal ligament and gingival fibroblast phenotypes, respectively, and periodontal ligament markers and osteogenic differentiation in IPDL are distinct from immortalized gingival fibroblast cells.