PERIOSTIN regulates MMP-2 expression via the αvβ3 integrin/ERK pathway in human periodontal ligament cells

ObjectiveDuring orthodontic tooth movement, activation of the vascular system in the compressed periodontal ligament (PDL), which becomes hypoxic, is essential for periodontal tissue remodelling. PERIOSTIN, an extracellular matrix protein, is expressed in PDL and its concentration is increased on the compressive side during orthodontic tooth movement. PERIOSTIN promotes angiogenesis through upregulation of matrix metalloproteinase (MMP)-2, which has been shown to be expressed via αvβ3 integrin/extracellular signal-related kinase (ERK) signalling pathway and vascular endothelial growth factor (VEGF). Therefore, we hypothesized that hypoxia-induced PERIOSTIN promotes MMP-2 expression via αvβ3 integrin/ERK signalling and VEGF in PDL cells.MethodsHuman PDL cells were cultured in condition medium containing desferrioxamine (DFO) to mimic hypoxia. The total RNA, cell lysates or supernatant were collected, and MMP2 and VEGF expression, PERIOSTIN expression and ERK phosphorylation, and MMP-2 activity were analysed by real-time RT-PCR, western blot analysis, and zymography, respectively. A recombinant human PERIOSTIN or PERIOSTIN siRNA was applied to the cells, then the total RNA was extracted to measure MMP-2 and VEGF expression. The cells were treated with αvβ3 integrin-blocking antibody or ERK inhibitor followed by PERIOSTIN stimulation. MMP-2 expression was measured by real-time RT-PCR.ResultsPERIOSTIN was upregulated in a time-dependent manner in human PDL cells treated with DFO, a chemical hypoxia mimic. MMP-2 and VEGF expression, and MMP-2 activity were increased by DFO or PERIOSTIN treatment, and decreased by PERIOSTIN silencing. PERIOSTIN treatment also induced ERK phosphorylation, and PERIOSTIN-induced MMP-2 was reduced by αvβ3 integrin-blocking antibody or ERK inhibitor.ConclusionThese data suggest that PERIOSTIN upregulates MMP-2 expression via the αvβ3 integrin/ERK signalling pathway and VEGF expression in human PDL cells.     

Intermittent PTH(1–34) signals through protein kinase A to regulate osteoprotegerin production in human periodontal ligament cells in vitro

Periodontal ligament (PDL) cells have been associated with the regulation of periodontal repair processes by the differential expression of osteoprotegerin and RANKL in response to intermittent parathyroid hormone (PTH) resulting in a modified activity of bone-resorbing osteoclasts. Here, we examined the intracellular signaling pathways that PDL cells use to mediate the PTH(1–34) effect on osteoprotegerin production and hypothesized that those would be dependent on the cellular maturation stage. Two stages of confluence served as a model for cellular maturation of 5th passage human PDL cells from six donors. Intermittent PTH(1–34) (10⁻¹² M) and PTH(1–31), the latter lacking the protein kinase C (PKC) activating domain, induced a significant decrease of osteoprotegerin production in confluent cultures, whereas the signal-specific fragments PTH(3–34) and PTH(7–34), which both are unable to activate protein kinase A (PKA), had no effect. The addition of the PKA inhibitor H8 antagonized the PTH(1–34) effect, whereas the PKC inhibitor RO-32-0432 did not. In pre-confluent, less mature cultures, intermittent PTH(1–34) resulted in a significant increase of osteoprotegerin. Similar results were obtained when PTH(1–31) substituted for PTH(1–34) as opposed to a lack of an effect of PTH(3–34) and PTH(7–34). Likewise, in confluent cultures, H8 inhibited the PTH(1–34) effect in pre-confluent cultures contrasted by RO-32-0432 which had no effect. These findings indicate that PTH(1–34) signaling targeting osteoprotegerin production in PDL cells involves a PKA-dependent pathway. The PTH(1–34) effect is dependent on cell status, whereas intracellular signal transduction is not. Clinical trials will have to prove whether those in vitro data are of physiological relevance for interference strategies.     

Hypoxia enhances the effect of lipopolysaccharide-stimulated IL-1β expression in human periodontal ligament cells

Oral infection is inflammatory disease caused by bacteria. A major component of gram negative bacteria membrane associated with inflammation is lipopolysaccharide (LPS). Currently, evidence presenting the combined effect of LPS and hypoxia to inflammatory response in human periodontal ligament cells (HPDLs) was yet lacking. Here, we studied whether the influence of oxygen on LPS-stimulated inflammatory cytokines in HPDLs. HPDLs were stimulated with LPS in normoxia and hypoxia for 24 h. The mRNA and protein expression of inflammatory cytokines were examined by polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The intracellular mechanisms of these effects were investigated by chemical inhibitors and small interfering RNA (siRNA). The results showed that LPS-stimulated IL-1β, IL-6, IL-8 in HPDLs in both hypoxia and normoxia. Hypoxia condition enhanced the effect of LPS-stimulated cytokines expression. Apigenin, the hypoxia-inducible factors (HIF)-1α inhibitor, totally prevented LPS-stimulated IL-1β expression in both normoxia and hypoxia. Similar to knockout HIF-1α gene expression by siRNA did \not prevent LPS-stimulated IL-1β expression. These data concluded that hypoxia increased virulence of LPS-stimulated IL-1β production in HPDLs.     

Maresin 1 regulates autophagy and inflammation in human periodontal ligament cells through glycogen synthase kinase–3β/β-catenin pathway under inflammatory conditions

ObjectiveAccumulating lines of evidence suggest that maresin 1 (MaR-1) exerts anti-inflammatory effects in many cell types and plays beneficial roles in inflammatory disease, such as peritonitis and colitis. Moreover, it has been demonstrated that MaR-1 play protective roles against localized aggressive periodontitis. However, the function and mechanism of MaR-1 in human periodontal ligament cells (PDL) cells from periodontitis are poorly understood. The present study aimed to clarify the effects and molecular mechanism of MaR-1 in PDL cell survival and inflammation.MethodsPDL cells were isolated from the middle third of the root surface of premolars from four healthy humans; MTT assay and cell death detection ELISA assay were used to detect cell survival and apoptosis; Inflammatory cytokines level was measured by ELISA assay; RT-PCR and western blot was used to measure the mRNA and protein expression in this study.ResultsHere we found that MaR-1 treatment markedly promotes survival and inhibits apoptosis in PDL cell treated by LPS. MaR-1 treatment strikingly suppressed the production of LPS-induced pro-inflammatory cytokines IL-6, IL-8, TNF-α and IL-1β. MaR-1 also promotes autophagy by increasing the ratio of LC3II/LC3I, the level of beclin-1 and reduced the expression of p62 in LPS treated PDL cells, which is beneficial to cell survival. Moreover, the results showed that MaR-1-mediated autophagy is dependent on the glycogen synthase kinase–3β(GSK-3β)/β-catenin signal pathway. The inhibitor of autophagy 3-MA and the inhibitor of the GSK-3β/β-catenin signal pathway LiCL both reverse the effects of MaR-1 on LPS-treated PDL cell survival and inflammation.ConclusionMaR-1 promotes cell survival and alleviates cell inflammation by activating GSK-3β/β-catenin-dependent autophagy. These results provide new insights into the mechanism of chronic periodontitis.     

Inhibition of the receptor for advanced glycation promotes proliferation and repair of human periodontal ligament fibroblasts in response to high glucose via the NF-κB signaling pathway

ObjectiveTo observe if inhibition of the receptor for advanced glycation endproducts (RAGE) promotes proliferation and repair of human periodontal ligament fibroblasts (hPDLFs) stimulated by high glucose. In addition, we also discuss the effects of the NF-κB signaling pathway in relation to this process.MethodsPrimary cultured hPDLFs were exposed to either low glucose (5.5 mmol/L) or high glucose (25 mmol/L), and RAGE expression was measured by Western blot analysis. Cells were cultured in high glucose with different concentrations of the RAGE inhibitor, FPS-ZM1. We measured cell proliferation using the Cell Counting Kit-8 and expression of collagen type 1 and fibronectin by real-time PCR and ELISA, respectively. The relative protein expression levels of NF-κB p65 and phosphorylated p65 were measured by Western blot analysis.ResultsHigh glucose enhanced RAGE expression and suppressed cell growth. While FPS-ZM1 increased proliferation and expression of repair-related factors in high glucose, there was a concurrent decline in the phosphorylation level of NF-κB p65.ConclusionFPS-ZM1 rescued the proliferative capacity and repair capability of hPDLFs via the RAGE-NF-κB signaling pathway in response to high glucose.     

MAPKs, activator protein-1 and nuclear factor-κB mediate production of interleukin-1β-stimulated cytokines, prostaglandin E₂ and MMP-1 in human periodontal ligament cells

Murayama R, Kobayashi M, Takeshita A, Yasui T, Yamamoto M. MAPKs, activator protein‐1 and nuclear factor‐κB mediate production of interleukin‐1β‐stimulated cytokines, prostaglandin E ₂ and MMP‐1 in human periodontal ligament cells. J Periodont Res 2011; 46: 568–575. © 2011 John Wiley & Sons A/S Background and Objective: Determination of the interleukin‐1 (IL‐1) signaling cascades that lead to the production of various inflammatory mediators and catabolic factors may clarify attractive targets for therapeutic intervention for periodontitis. We comprehensively assessed the involvement of MAPKs, activator protein‐1 (AP‐1) and nuclear factor‐κB (NF‐κB) in IL‐1β‐induced production of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), prostaglandin E₂ (PGE₂) and MMP‐1 in human periodontal ligament cells. Material and Methods: Human periodontal ligament cells were pretreated with an inhibitor for each of the MAPKs or NF‐κB and subsequently treated with IL‐1β. Following treatment, phosphorylation of three types of MAPK (ERK, p38 MAPK and c‐Jun N‐terminal kinase), IκB kinase (IKK) α/β/γ and IκB‐α, as well as the DNA binding activity of AP‐1 and NF‐κB and the production of IL‐6, IL‐8, PGE₂ and MMP‐1, were determined by western blotting, a gel mobility shift assay and ELISA, respectively. Results: The three MAPKs, simultaneously activated by IL‐1β, mediated the subsequent DNA binding of AP‐1 at various magnitudes, while IKKα/β/γ, IκB‐α and NF‐κB were also involved in the IL‐1 signaling cascade. Furthermore, IL‐1β stimulated the production of IL‐6, IL‐8, PGE₂ and MMP‐1 via activation of the three MAPKs and NF‐κB, because inhibitors of these significantly suppressed the IL‐1β‐stimulated production of these factors. Conclusion: Our results strongly suggest that MAPK, AP‐1 and NF‐κB mediate the IL‐1β‐stimulated synthesis of IL‐6, IL‐8, PGE₂ and MMP‐1 in human periodontal ligament cells. Therefore, inhibition of activation of MAPK, AP‐1 and/or NF‐κB may lead to therapeutic effects on progression of periodontitis.     

Effect of hydrogen sulphide on the expression of osteoprotegerin and receptor activator of NF-κB ligand in human periodontal ligament cells induced by tension-force stimulation

ObjectiveHydrogen sulphide (H₂S) is an endogenous gaseous signalling molecule, the generation rate of which is affected by mechanical force in cells. Recently, it was reported that mechanical force plays an important role in some pathways such as osteoprotegerin (OPG) and the receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) in human periodontal ligament cells (hPDLCs). Here, we investigated whether H₂S played a regulatory role within the periodontal remodelling process by addressing the expression level of OPG/RANKL in hPDLCs.DesignhPDLCs were first applied with tension force for 0–120 min to select the optimal time for force application. These cells were treated with H₂S for 24 h followed by stimulation with tension-force application. Cell proliferation and apoptosis were assessed by the methyl thiazolyl tetrazolium (MTT) assay and flow cytometry analysis. For OPG, RANKL and cystathionine-γ-lyase (CSE), real-time polymerase chain reaction (PCR) was used to analyse the messenger RNA (mRNA) expression; enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of OPG and soluble RANKL (sRANKL).ResultsTension force promoted the mRNA expression of CSE and the optimal application time was 60 min. The expression of OPG was increased in a concentration-dependent manner by H₂S treatment. Importantly, the relative OPG/RANKL expression ratio was significantly increased upon induction by H₂S, an effect that was enhanced by tension-force application.ConclusionsH₂S could promote osteogenic differentiation by regulating the relative OPG/RANKL expression ratio of hPDLCs, which is enhanced by tension force. These findings may be valuable for understanding the mechanism of H₂S in the periodontal remodelling, especially in the process of tooth movement.     

Comparison of the properties of human CD146+ and CD146− periodontal ligament cells in response to stimulation with tumour necrosis factor α

ObjectivesPeriodontal ligament stem cells (PDLSCs) can be used in periodontal regeneration. Tumour necrosis factor-alpha (TNF-α) participates in the regulation of cell proliferation, apoptosis, differentiation, and migration. However, whether TNF-α can affect the biological features of PDLSCs is still unclear. The objective of this study was to illustrate the biological effects (proliferation, apoptosis, osteogenesis and migration) of TNF-α on human CD146 positive periodontal ligament cells (CD146+PLDCs) and CD146 negative periodontal ligament cells (CD146?PDLCs).MethodsCD146 ± PDLCs were isolated from human PDLCs and analyzed using a fluorescence-activated cell sorter. The biological effects of TNF-α on CD146 ± PDLCs were evaluated by CCK-8 assay (proliferation), DAPI staining (apoptosis), alizarin red staining and alkaline phosphatase activities assay (osteogenesis), and wounding assay and transwell assay (migration).ResultsCD146+PDLCs, which expressed MSC surface markers CD105, CD90, CD73, CD44, and Stro-1, showed higher proliferative and osteogenic potential than CD146?PDLCs. TNF-α at a dose of 2.5 ng/ml was found to enhance both proliferation and osteogenesis in CD146+PDLCs. At 5 ng/ml, TNF-α promoted proliferation, osteogenesis, and apoptosis in CD146+PDLCs and enhanced osteogenesis in CD146?PDLCs. At 10 ng/ml, TNF-α only aggravated apoptosis in CD146+PDLCs. The migratory ability of both CD146+PDLCs and CD146?PDLCs was not altered by TNF-α.ConclusionsCD146+PDLCs were subpopulation of MSC. It showed greater proliferative and osteogenic potential than CD146?PDLCs. At low concentration, TNF-α was beneficial to CD146+PDLCs on proliferation and osteogenesis, and at high concentration it was detrimental. CD146?PDLCs were found to be less sensitive to TNF-α.     

Nicotine and lipopolysaccharide stimulate the production of MMPs and prostaglandin E(2) by hypoxia-inducible factor-1α up-regulation in human periodontal ligament cells

Kim Y‐S, Shin S‐I, Kang K‐L, Herr Y, Bae W‐J, Kim E‐C. Nicotine and lipopolysaccharide stimulate the production of MMPs and prostaglandin E₂ by hypoxia‐inducible factor‐1α up‐regulation in human periodontal ligament cells. J Periodont Res 2012; 47: 719–728. © 2012 John Wiley & Sons A/S Background and Objective: Although hypoxia‐inducible factor 1α (HIF‐1α) is up‐regulated in the periodontal pockets of periodontitis patients, the expression and precise molecular mechanisms of HIF‐1α remain unknown in human periodontal ligament cells (PDLCs). The aim of this study was to explore the effects, as well as the signaling pathway, of nicotine and lipopolysaccharide (LPS) on the expression of HIF‐1α and on the production of its target genes, including cyclooxygenase‐2 (COX‐2)‐derived prostaglandin E₂ (PGE₂), MMP‐2 and MMP‐9 in PDLCs. Material and Methods: The expression of COX‐2 and HIF‐1α proteins was evaluated using western blotting. The production of PGE₂ and MMPs was evaluated using enzyme immunoassays and zymography, respectively. Results: LPS and nicotine synergistically induced the production of PGE₂, MMP‐2 and MMP‐9, and increased the expression of MMP‐2, MMP‐9, COX‐2 and HIF‐1α proteins. Inhibition of HIF‐1α activity by chetomin or knockdown of HIF1α gene expression by small interfering RNA markedly attenuated the production of LPS‐ and nicotine‐stimulated PGE₂ and MMPs, as well as the expression of COX‐2 and HIF‐1α. Furthermore, pretreatment with inhibitors of COX‐2, p38, extracellular signal‐regulated kinase, Jun N‐terminal kinase, protein kinase C, phosphatidylinositol 3‐kinase and nuclear factor‐kappaB decreased the expression of nicotine‐ and LPS‐induced HIF‐1α and COX‐2, as well as the activity of PGE₂ and MMPs. Conclusion: These data demonstrate novel mechanisms by which nicotine and LPS promote periodontal tissue destruction, and provide further evidence that HIF‐1α is a potential target in periodontal disease associated with smoking and dental plaque.     

Age-related production of osteoclasts and the changes of serum levels of vascular endothelial growth factor (VEGF) and receptor activator for nuclear factor (NF)-κB ligand (RANKL) in osteopetrotic (op/op) mice

ObjectiveExpression of osteoclasts in osteopetrotic (op/op) mice is substantially reduced by the absence of functional macrophage colony-stimulating factor (M-CSF). However, it has been reported that osteoclasts do gradually appear in the bones of op/op mice and spontaneously correct the osteopetrosis.DesignAge-related production of osteoclasts and the changes of serum levels of vascular endothelial growth factor (VEGF) and receptor activator for nuclear factor (NF)-κB ligand (RANKL) in op/op mice were examined.ResultsThe number of femoral osteoclasts, and the serum levels of VEGF, both gradually increased in op/op mice after birth and reached a peak in 120- and 60-day-old mice, respectively. However, the serum levels of RANKL showed an inverse relationship to osteoclast number.ConclusionsThese findings suggest that the appearance of osteoclasts may be influenced by the serum levels of VEGF and that the serum levels of RANKL may be influenced by the appearance of osteoclasts.