Functional expression of α7 nicotinic acetylcholine receptors in human periodontal ligament fibroblasts and rat periodontal tissues

Tobacco smoking is the main risk factor associated with chronic periodontitis, but the mechanisms that underlie this relationship are largely unknown. Recent reports proposed that nicotine plays an important role in tobacco-related morbidity by acting through the nicotinic acetylcholine receptors (nAChRs) expressed by non-neuronal cells. The aim of this study was to investigate whether α7 nAChR was expressed in periodontal tissues and whether it functions by regulating IL-1β in the process of periodontitis. In vitro, human periodontal ligament (PDL) cells were cultured with 10⁻¹² M of nicotine and/or 10⁻⁹ M of alpha-bungarotoxin (α-Btx), a α7 nAChR antagonist. The expression of α7 nAChR and IL-1β in PDL cells and the effects of nicotine/α-Btx administration on their expression were explored. In vivo, an experimental periodontitis rat model was established, and the effects of nicotine/α-Btx administration on expression of α7 nAChR and development of periodontitis were evaluated. We found that α7 nAChR was present in human PDL cells and rat periodontal tissues. The expressions of α7 nAChR and IL-1β were significantly increased by nicotine administration, whereas α-Btx treatment partially suppressed these effects. This study was the first to demonstrate the functional expression of α7 nAChR in human PDL cells and rat periodontal tissues. Our results may be pertinent to a better understanding of the relationships among smoking, nicotine, and periodontitis.     

IL-1β favors osteoclastogenesis via supporting human periodontal ligament fibroblasts

The balance between bone formation and bone resorption in inflammatory diseases is often disturbed. Periodontitis, a chronic inflammation of the tooth gums, leads to unwanted bone loss as a response to inflammatory compounds such as interleukin‐1β (IL‐1β). This excessive bone loss reflects an increased osteoclast formation and activity. Osteoclast formation is a multistep process driven by osteoclastogenesis supporting cells such as periodontal ligament fibroblasts. The inflammatory factors can induce osteoclastogenesis, probably also by affecting the periodontal ligament fibroblast. In this study we investigated how pre‐culture of periodontal ligament fibroblasts with IL‐1β affected osteoclastogenesis. Fibroblasts were pre‐cultured with IL‐1β and/or dexamethasone, a commonly used anti‐inflammatory compound, before being co‐cultured with peripheral blood mononuclear cells (PBMCs). Pre‐culture with IL‐1β (1–100 ng/ml) resulted in an increased number of adhered PBMCs as well as an increased mRNA expression of intercellular adhesion molecule‐1 (ICAM‐1), macrophage colony stimulating factor (M‐CSF) and IL‐1β. Pre‐culture with IL‐1β also caused retraction of fibroblasts and an augmented formation of TRACP⁺ multinucleated cells. Our data suggest that stimulation of fibroblasts with IL‐1β has a long‐lasting effect, leading to a significantly increased osteoclastogenesis. These results provide new insights for understanding excessive bone loss in periodontitis. J. Cell. Biochem. 112: 1890–1897, 2011. © 2011 Wiley‐Liss, Inc.     

Periostin inhibits hypoxia-induced apoptosis in human periodontal ligament cells via TGF-β signaling

Periostin (POSTN) is an extracellular matrix protein expressed predominantly in periodontal ligament (PDL) cells. The aim of this study was to investigate the effects of POSTN on human PDL cell apoptosis under hypoxic conditions. The percentage of apoptotic PDL cells under hypoxia was increased significantly when the endogenous POSTN gene was silenced using siRNA, but decreased when cells were treated with recombinant human POSTN (rhPOSTN), or when mouse Postn was overexpressed in vitro. Silencing POSTN during hypoxia decreased the expression of HIF prolyl-hydroxylase 2 (PHD2), but increased HIF-1α protein level. Conversely, treating hypoxic cells with rhPOSTN or overexpressing Postn increased PHD2 expression but decreased HIF-1α levels. The addition of rhPOSTN in the absence of a TGF-β receptor inhibitor (SB525334) significantly decreased hypoxia-induced apoptosis, while the effects of rhPOSTN were abolished when cells were co-treated with SB525334. Consistent with this, the phosphorylation of SMAD2 was increased in hypoxic PDL cells by the knockdown of POSTN, but decreased by treatment with rhPOSTN. Under normoxia, the PHD2 expression, HIF-1α level, and apoptosis were unaffected by POSTN siRNA, rhPOSTN, or Postn overexpression. These findings suggest that, under hypoxic conditions, POSTN regulates PHD2 expression and HIF-1α levels by modulating TGF-β1 signaling, leading to decreased apoptosis.     

Up-regulation of IL-23 p19 expression in human periodontal ligament fibroblasts by IL-1β via concurrent activation of the NF-κB and MAPKs/AP-1 pathways

Interleukin (IL)-23 is an essential cytokine involved in the expansion of a novel CD4(+) T helper subset known as Th17, which has been implicated in the pathogenesis of periodontitis recently. Our previous study first identified specialized human periodontal ligament fibroblasts (hPDLFs) as an important production source of IL-23. The present study was undertaken to investigate the effects of the pro-inflammatory and Th17-polarizing mediator IL-1β on hPDLFs-mediated IL-23 p19 production, and the molecular mechanism involved. IL-23 p19 expression was in situ detected in IL-1β-stimulated hPDLFs. IL-1β was capable of stimulating the expression of IL-23 p19 mRNA and protein in cultured hPDLFs, which was attenuated by IL-1 receptor antagonist (IL-1Ra) or myeloid differentiation primary response gene 88 (MyD88) inhibitor. Meanwhile, inhibitors of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1/2, c-Jun-N-terminal kinase (JNK), activator protein-1 (AP-1), or nuclear factor-kappaB (NF-κB) significantly suppressed IL-23 p19 production from IL-1β-stimulated hPDLFs. Moreover, IL-1β-initiated AP-1 activation was blocked by p38 MAPK, ERK 1/2, or JNK inhibition, whereas NF-κB activity remained unaltered by all the above pathway specific inhibitors. Thus, these results provide evidence that Th17-polarizing mediator IL-1β up-regulated the expression of IL-23 p19 in hPDLFs via NF-κB signaling and MAPKs-dependent AP-1 pathways. Taken together, our findings indicate that IL-1Ra may be used therapeutically to inhibit Th17-driven inflammatory diseases including periodontitis.     

Focal adhesion kinase mediates β-catenin signaling in periodontal ligament cells

Periodontal ligament (PDL) cells convert the orthodontic forces into biological responses by secreting signaling molecules to induce modeling of alveolar bone and tooth movement. Beta-catenin pathway is activated in response to mechanical loading in PDL cells. The upstream signaling pathways activated by mechanical loading resulting in the activation of β-catenin pathway through Wnt-independent mechanism remains to be characterized. We hypothesized that mechanical loading induces activation of β-catenin signaling by mechanisms that dependent on focal adhesion kinase (FAK) and nitric oxide (NO). We found that mechanical or pharmacological activation of β-catenin signaling in PDL cells upregulated the expression of β-catenin target genes. Pre-treatment of PDL cells with FAK inhibitor-14 prior to mechanical loading abolished the mechanical loading-induced phosphorylation of Akt and dephosphorylation of β-catenin. PDL cells pre-treated with NO donor or NO inhibitor and subjected to mechanical loading. Western blot analysis showed that the mechanical loading or pre-treatment with NO donor increased the levels of dephosphorylated β-catenin, pAkt, and pGSK-3β. Pre-treatment with NO inhibitor blocked the mechanical loading-induced phosphorylation of Akt and dephosphorylation of β-catenin. These data indicate that mechanical loading-induced β-catenin stabilization in PDL cells involves phosphorylation of Akt by two parallel pathways requiring FAK and NO.     

Parathyroid hormone(1–34) mediates proliferative and apoptotic signaling in human periodontal ligament cells in vitro via protein kinase C-dependent and protein kinase A-dependent pathways

Periodontal ligament (PDL) cells exhibit several osteoblastic traits and are parathyroid hormone (PTH)-responsive providing evidence for a role of these cells in dental hard-tissue repair. To examine the hypothesis that PDL cells respond to PTH stimulation with changes in proliferation and apoptotic signaling through independent but convergent signaling pathways, PDL cells were cultured from human bicuspids obtained from six patients. PDL cells at different states of maturation were challenged with PTH(1–34) intermittently for 0, 1, or 24 h/cycle or exposed continuously. Specific inhibitors to protein kinases A and C (PKA, PKC) and the mitogen-activated protein kinase cascade (MAPK) were employed. At harvest, the cell number, BrdU incorporation, and DNA fragmentation were determined by means of cell counting and immunoassays. Intermittent PTH(1–34) caused a significant increase in cell number in confluent cells as opposed to a reduction in pre-confluent cells. In confluent cells, the effect resulted from a significant increase in proliferation, whereas DNA fragmentation was reduced when PTH(1–34) was administered for 1 h/cycle but increased after PTH(1–34) for 24 h/cycle. Inhibition of PKC inhibited PTH(1–34)-induced proliferation but enhanced apoptosis. Inhibition of PKA enhanced proliferation and DNA fragmentation. Similar results were obtained in less mature cells, although, in the presence of the PKA inhibitor, the PTH(1–34)-induced changes were more pronounced than in confluent cells. In the presence of the MAPK inhibitor, all of the parameters examined were reduced significantly in both maturation states. Thus, PTH(1–34) mediates proliferative and apoptotic signaling in human PDL cells in a maturation-state-dependent manner via PKC-dependent and PKA-dependent pathways.This research was supported by research grants from the BONFOR program (O-135.0006) of the University of Bonn, Bonn, Germany and the Deutsche Forschungsgemeinschaft (DFG; LO-1181/1-1).     

Exposure to transforming growth factor-β1 after basic fibroblast growth factor promotes the fibroblastic differentiation of human periodontal ligament stem/progenitor cell lines

Basic fibroblast growth factor (bFGF) is a cytokine that promotes the regeneration of the periodontium, the specialized tissues supporting the teeth. bFGF, does not, however, induce the synthesis of smooth muscle actin alpha 2 (ACTA2), type I collagen (COL1), or COL3, which are principal molecules in periodontal ligament (PDL) tissue, a component of the periodontium. We have suggested the feasibility of using transforming growth factor-β1 (TGFβ1) to induce fibroblastic differentiation of PDL stem/progenitor cells (PDLSCs). Here, we investigated the effect of the subsequent application of TGFβ1 after bFGF (bFGF/TGFβ1) on the differentiation of PDLSCs into fibroblastic cells. We first confirmed the expression of bFGF and TGFβ1 in rat PDL tissue and primary human PDL cells. Receptors for both bFGF and TGFβ1 were expressed in the human PDLSC lines 1-11 and 1-17. Exposure to bFGF for 2 days promoted vascular endothelial growth factor gene and protein expression in both cell lines and down-regulated the expression of ACTA2, COL1, and COL3 mRNA in both cell lines and the gene fibrillin 1 (FBN1) in cell line 1-11 alone. Furthermore, bFGF stimulated cell proliferation of these cell lines and significantly increased the number of cells in phase G2/M in the cell lines. Exposure to TGFβ1 for 2 days induced gene expression of ACTA2 and COL1 in both cell lines and FBN1 in cell line 1-11 alone. BFGF/TGFβ1 treatment significantly up-regulated ACTA2, COL1, and FBN1 expression as compared with the group treated with bFGF alone or the untreated control. This method might thus be useful for accelerating the generation and regeneration of functional periodontium.     

Nicotinic acetylcholine receptor α7 and β4 subunits contribute nicotine-induced apoptosis in periodontal ligament stem cells

Nicotine, a major component of cigarette smoking, is the important risk factor for the development of periodontal disease. However, the mechanisms that underlie the cytotoxicity of nicotine in human periodontal ligament stem cells (PDLSCs) are largely unknown. Thus, the purpose of this study was to determine the cytotoxic effect of nicotine by means of nicotinic acetylcholine receptor (nAChR) activation in PDLSCs. We first detected α7 and β4 nAChRs in PDLSCs. The gene expressions of α7 and β4 nAChR were increased by nicotine administration. Nicotine significantly decreased cell viability at a concentration higher than 10⁻⁵ M. DNA fragmentation was also detected at high doses of nicotine treatment. Moreover, the detection of sub G1 phase and TUNEL assay demonstrated that nicotine significantly induced apoptotic cell death at 10⁻² M concentration. Western blot analysis confirmed that p53 proteins were phosphorylated by nicotine. Under various doses of nicotine, a decrease in the anti-apoptotic protein Bcl-2, but an increase in p53 and cleaved caspase-3 protein levels, was detected in a dose-dependent manner. However, the apoptotic effect of nicotine was inhibited by the pretreatment of α-bungarotoxin, a selective α7 nAChR antagonist or mecamylamine, a non-selective nAChR antagonist. Finally, increases in the subG1 phase and DNA fragmentation by nicotine was attenuated by each nAChR antagonist. Collectively, the presence of α7 and β4 nAChRs in PDLSCs supports a key role of nAChRs in the modulation of nicotine-induced apoptosis.     

JNK and AKT/GSK3β signaling pathways converge to regulate periodontal ligament cell survival involving XIAP

Periodontal ligament cells (PDLCs) were incubated with H₂O₂ and the levels of XIAP protein, protein kinase B (AKT), phosphorylated forms of AKT (pAKT), c-Jun N-terminal kinase (JNK), and glycogen synthase kinase-3β (GSK3β) were determined by western immunoblotting or immunocytochemistry. After overexpression and knockdown of XIAP, the AKT, pAKT, JNK and GSK3β levels were determined in PDLCs exposed to H₂O₂.     

Double-edged-sword effect of IL-1β on the osteogenesis of periodontal ligament stem cells via crosstalk between the NF-κB,MAPK and BMP/Smad signaling pathways

Microenvironmental conditions can interfere with the functional role and differentiation of mesenchymal stem cells (MSCs). Recent studies suggest that an inflammatory microenvironment can significantly impact the osteogenic potential of periodontal ligament stem cells (PDLSCs), but the precise effects and mechanisms involved remain unclear. Here, we show for the first time that interleukin-1β (IL-1β) has dual roles in the osteogenesis of PDLSCs at concentrations ranging from physiologically healthy levels to those found in chronic periodontitis. Low doses of IL-1β activate the BMP/Smad signaling pathway to promote the osteogenesis of PDLSCs, but higher doses of IL-1β inhibit BMP/Smad signaling through the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling, inhibiting osteogenesis. These results demonstrate that crosstalk between NF-κB, MAPK and BMP/Smad signaling mediates this dual effect of IL-1β on PDLSCs. We also show that the impaired osteogenesis of PDLSCs results in more inflammatory cytokines and chemokines being released, inducing the chemotaxis of macrophages, which further clarifies the role of PDLSCs in the pathogenesis of periodontitis.Approximately 90% of the population suffers from periodontitis,^{1, 2} which is characterized by chronic bacterial infections in the supporting structures of the teeth and a homeostatic imbalance between two coupled process in the periodontal system – bone resorption by osteoclasts and bone formation by osteoblasts. This disease involves interactions with bacterial products, numerous cell populations and different inflammatory mediators, and it can lead to tooth loss in adults.^{1, 2}Periodontal ligament stem cells (PDLSCs), a newly recognized sub-population of mesenchymal stem cells (MSCs), have attracted increasing attention in relation to their multipotency. As PDLSCs can easily be obtained from periodontal tissue, they are considered important for prospective cell-based therapies. Recently, PDLSCs have been shown to migrate to the site of periodontal lesions and to mediate periodontal regeneration.^{3, 4, 5} However, recent studies have found that the osteogenic capacity of stem cells is impaired in inflammatory microenvironments^6,7 and that there are complex interactions between stem cells and the microenvironment under pathological conditions. Our previous studies found that disrupted and disease-associated microenvironments could influence the characteristics and functions of MSCs.8-10 Additionally, some studies have indicated that MSCs act in an immunomodulatory manner to regulate the function and chemotaxis of immune cells and that environmental factors may determine which immunomodulatory pathways are operational in MSCs.¹¹ Thus, we assume that the mutual interactions between stem cells and inflammatory microenvironments are crucial to harnessing the regenerative potential of PDLSCs for therapeutic use.Interleukin-1 (IL-1) is a pleiotropic cytokine and a central mediator of innate immunity and inflammation.¹² In clinical studies, IL-1β has been found in increased concentrations in gingival crevicular fluid (GCF) and at sites of periodontal damage,^{13, 14} and levels of IL-1β have been reported to decrease after periodontal treatment.^{15, 16} Compared with levels at healthy sites, local IL-1β and tumor necrosis factor-α (TNF-α) levels in the microenvironments of chronic periodontitis have been found to be significantly elevated and to be associated with periodontal tissue destruction.17–19 IL-1 stimulates bone resorption by promoting osteoclast activation17^,20^,21 and mediates the osteoclastogenic effects of TNF-α by enhancing the expression of RANKL.15 In inflammatory microenvironments, IL-1 and TNF have a prominent role in the pathogenesis of periodontitis.19 Although TNF-α has activity similar to that of IL-1β, IL-1β is present at higher levels in inflamed gingival tissues, and its expression is limited to the connective tissue layer.22 Multiple studies have investigated the effect of IL-1β on osteoblast differentiation,^{23, 24} but conflicting data has been presented and the underlying mechanism of its effects remains unclear.25 A previous study has shown that the concentration of IL-1β in GCF is 145±167 pg/ml in healthy subjects and 6452±2289 pg/ml in patients with chronic periodontitis.26 In this study, we mimicked an inflammatory microenvironment using IL-1β at different concentrations that ranged from healthy physiological levels to those observed in the GCF in cases of chronic periodontitis26 and tried to establish an in vitro osteogenesis model to investigate the effects of different doses of IL-1β on PDLSCs.Previously, it has been reported that the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways have crucial roles in the regulation of inflammation and bone metabolism.²⁷²⁸ In addition, the BMP/Smad signaling pathways have important roles in the regulation of osteoblast differentiation.29 However, the roles these signaling pathways have in the osteogenesis of MSCs in inflammatory microenvironments remain unclear. In the present study, we investigated the interactions of BMP/Smad, MAPK and NF-κB signaling pathways in mediating the IL-1β-regulated osteogenic differentiation of PDLSCs. Because the resident periodontal cells can produce various inflammatory mediators that induce inflammatory cells to invade the tissue and affect bone resorption,30 we further examined the role of PDLSCs in the pathogenesis of periodontitis by determining the production of inflammatory cytokines and chemokines by PDLSCs in which osteogenesis was inhibited by IL-1β.     

Nuclear factor-κB modulates osteogenesis of periodontal ligament stem cells through competition with β-catenin signaling in inflammatory microenvironments

Inflammation can influence multipotency and self-renewal of mesenchymal stem cells (MSCs), resulting in their awakened bone-regeneration ability. Human periodontal ligament tissue-derived MSCs (PDLSCs) have been isolated, and their differentiation potential was found to be defective due to β-catenin signaling indirectly regulated by inflammatory microenvironments. Nuclear factor-κB (NF-κB) is well studied in inflammation by many different groups. The role of NF-κB needs to be studied in PDLSCs, although genetic evidences have recently shown that NF-κB inhibits osteoblastic bone formation in mice. However, the mechanism as to how inflammation leads to the modulation of β-catenin and NF-κB signaling remains unclear. In this study, we investigated β-catenin and NF-κB signaling through regulation of glycogen synthase kinase 3β activity (GSK-3β, which modulates β-catenin and NF-κB signaling) using a specific inhibitor LiCl and a phosphatidylinositol 3-kinase (PI3K) inhibitor LY 294002. We identified that NF-κB signaling might be more important for the regulation of osteogenesis in PDLSCs from periodontitis compared with β-catenin. BAY 11-7082 (an inhibitor of NF-κB) could inhibit phosphorylation of p65 and partly rescue the differentiation potential of PDLSCs in inflammation. Our data indicate that NF-κB has a central role in regulating osteogenic differentiation of PDLSCs in inflammatory microenvironments. Given the molecular mechanisms of NF-κB in osteogenic differentiation governed by inflammation, it can be said that NF-κB helps in improving stem cell-mediated inflammatory bone disease therapy.     

Epigenetics of human melanoma： promises and challenges

Melanoma is the deadliest form of skin cancer with rising incidence and mortality rates. Although early-stage melanoma is highly curable, advanced-stage melanoma is refractory to treatment. This underscores the importance of prevention and early detection as well as the need to improve treatment and prognostication of human melanoma. Elucidating the underlying mechanisms of the initi- ation and progression of human melanoma can help identify potential targets of intervention for prevention, diagnosis, therapy, and prognosis of this disease. Aberrant DNA methylation and histone modifications are the best-established epigenetic mechanisms of carcinogenesis. The occurrence of epigenetic changes prior to clinical diagnosis of cancer and their reversibility through pharmaco-logic/genetic approaches offer a promising avenue for basic and translational research on human melanoma. Candidate gene（s） or genome-wide aberrant DNA methylation and histone modifications have been observed in human melanoma tumor tissues and cell lines, and correlated to cellular and functional characteristics and/or clinicopathologicai features of this malignancy. The present review summarizes the published researches on aberrant DNA methylation and histone modifications in connection with human melanoma. Representative studies are highlighted to set forth the current state of knowledge, gaps in the knowledgebase, and future directions in these epigenetic fields of research. Examples of epigenetic therapy applied for human melanoma in vitro, and the challenges of its in vivo application for clinical treatment of solid tumors are discussed.     

Influence of the human blood serum on contractility and β-adrenoreactivity of the isolated human myocardium

Influence of adrenaline (10⁻⁹ to 10⁻⁴ g/ml) on the contraction amplitude caused by electrostimuli (1Hz, 5 ms, 25–30 V) and inotropic and adrenomodulation activities of blood serum of nonpregnant women (at dilutions of 1 : 10 000, 1 : 1000, 1 : 500, 1 : 100, 1 : 50, 1 : 10, and 1 : 5) have been studied. The study has been carried out on isolated myocardium strips of the right atrial auricle that were taken from 43 patients with ischemic illness of the heart and 9 patients with valvular heart diseases of various etiologies upon venous cannula insertion during an aortocoronary bypass. Direct dependence of the contraction amplitude on the cardiac output according to Teicholz has been found. This meant that strips of the right atrial auricle reflected the contractility of the left ventricle myocardium. Adrenaline has been shown to dose-dependently increase the amplitude of evoked contractions in the concentration interval from 10⁻⁷ to 10⁻⁶ g/ml and had no influence from 10⁻⁹ to 10⁻⁸ g/ml (dissociation constant, 2 × 10⁻⁷ g/ml), which proved a decrease in the β-adrenoreceptor’s (β-AR) activation. Blood serum in a dilution range from 1 : 10 000 to 1 : 50 had no effect on the contraction amplitude, but an enhanced effect has been found in a dilution range from 1 : 10 and 1 : 5. The presence of the endogenous activator of myocytes contractility (EAMC) has explained this enhanced effect. The β-adrenomodulation activity of blood serum has been explained by the presence of the endogenous sensitizer of β-AR (ESBAR) and the endogenous blocker of β-AR (EBBAR). The ESBAR activity of blood serum (dilutions: 1 : 1000, 1 : 500, 1 : 100, and 1 : 50) has been found in experiments with a subthreshold adrenaline concentration (10⁻⁸ g/ml). ESBAR (dilutions: 1 : 50 and 1 : 10) and EBBAR (dilution 1 : 500) activities of blood serum have been found in experiments with the maximum effective concentration of adrenaline (10⁻⁶ g/ml). Therefore, blood serum endogenous modulators of β-adrenergic reactivity, ESBAR and EBBAR, can modulate the activation of β-AR of human cardiomyocytes. These prove the prospects of the ESBAR analogue application in cardiology.