Nitric oxide modulates osteoblastic differentiation with heme oxygenase-1 via the mitogen activated protein kinase and nuclear factor-kappaB pathways in human periodontal ligament cells.
ABSTRACT Nitric oxide (NO) and heme oxygenase-1 (HO-1) play important roles in the regulation of stem cell proliferation and differentiation. However, it has not been examined whether human periodontal ligament (PDL) cells can differentiate into osteoblast-like cells by NO activity mediated via HO-1. The objective of this study was to determine the effect of NO on proliferation and differentiation in human PDL cells, and to identify the underlying mechanism of its actions. Primary human PDL cells were cultured with NO donor sodium nitroprusside (SNP); cell proliferation and differentiation were measured. NO production, cell viability and cell proliferation were evaluated using the Griess reagent, MTT assay and BrdU incorporation, respectively. To analyze differentiation, we measured alkaline phosphatase (ALP) activity, osteocalcin (OC), osteonectin (ON) expression, and bone sialoprotein (BSP) by Western blotting. SNP-induced NO production is associated with inducible nitric oxide synthase induction in a time and dose-dependent manner. SNP resulted in decreased cell proliferation and increased expression of osteogenic differentiation markers such as ALP, OC, ON and BSP. Maximal HO-1 was reached with 0.05 mM SNP and gradually decreased with 1.0 mM. Treatment with an HO-1 inhibitor and selective inhibitors of extracellular regulated kinase 1/2 and nuclear factor-kappaB blocked the SNP-induced growth inhibition, as well as osteoblastic differentiation. These data suggest that NO-induced osteogenic differentiation through HO-1 may be an important mediator of periodontal regeneration or bone tissue engineering.
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ABSTRACT: 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.Clinical & Experimental Immunology 01/2008; 150(3):567-75. · 3.41 Impact Factor
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ABSTRACT: This study examined the effects of exogenous nitric oxide (NO) on human pulp cells and the involvement of cyclic 3',5'-monophosphate (cGMP) in pulpal protection induced by heme oxygenase-1 (HO-1) against NO-induced cytotoxicity. This study investigated cytotoxicity and HO-1 induction in pulp cells induced by the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP), by using Western blotting and a cell viability assay. It also investigated whether HO-1 contributes to the cytoprotective effect against the cytotoxicity caused by NO and the relationship between HO-1 and cGMP in the signaling pathway. S-nitroso-N-acetyl-D,L-penicillamine decreased cell viability, but increased HO-1 expression in a concentration- and time-dependent manner in human pulp cells. NO-induced cytotoxicity was inhibited in the presence of hemin (inducer of HO-1), whereas it was enhanced in the presence of zinc protoporphyrin IX (ZnPP IX, HO-1 inhibitor); therefore, the NO-induced cytotoxicity was correlated with HO-1 expression. Pretreatment with a membrane-permeable cGMP analog, 8-bromo-cGMP, restored cell death and enhanced the HO-1 protein expression induced by SNAP. By contrast, 1 mM SNAP inhibited guanylate cyclase in pulp cells pretreated with 1H-[1,2,4]oxadiazole[4,3-alpha]quinoxalin-1-one (ODQ), resulting in marked cytotoxicity. These findings of a link between HO-1, regulated via the cGMP system and NO-induced cytotoxicity in human pulp cells, suggest a protective role for HO-1 in pulpal inflammation.Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics 01/2007; 102(6):803-8. · 1.50 Impact Factor
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ABSTRACT: This study was conducted to evaluate the pulpal response to direct capping with either mineral trioxide aggregate (MTA) or calcium hydroxide (CH) cement in humans, with a focus on dentin bridge formation and dentin sialoprotein (DSP) and heme oxygenase-1 (HO-1) expression. Direct pulp capping was performed in 20 cases of caries-free human third molars. The pulps were exposed and capped with either MTA or hard-setting CH. After 2 months, the teeth were extracted, and the specimens were prepared for histologic and immunohistochemical evaluations. Histologically, 100% of the MTA group and 60% of the CH group developed dentin bridges. The mean thickness of the dentin bridges observed in the MTA group was statistically greater than that of CH group. In addition, DSP and HO-1 were expressed in the odontoblast-like cells and pulp fibroblasts beneath the dentin bridge; furthermore, significantly greater immunostaining was observed in the MTA group than in the CH group. Collectively, these results indicate that MTA is superior to CH in terms of inducing the dentinogenic process in human pulp capping.Journal of endodontics 07/2008; 34(6):666-70. · 2.95 Impact Factor