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.     

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.     

Effects of TGF-β1 on the proliferation and differentiation of human periodontal ligament cells and a human periodontal ligament stem/progenitor cell line

Periodontal ligament (PDL) is a specialized connective tissue that influences the lifespan of the tooth. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine, but little is known about the effects of TGF-β1 on PDL cells. Our aim has been to demonstrate the expression of TGF-β1 in rat PDL tissues and to evaluate its effects on the proliferation and gene expression in human PDL cells (HPLCs) and a human PDL stem/progenitor cell line, line 1-11, that we have recently developed. The expression of TGF-β1 in the entire PDL tissue was confirmed immunohistochemically, and both HPLCs and cell line 1-11 expressed mRNA from the TGF-β1, TGF-β type I receptor, and TGF-β type II receptor genes. Although exogenous TGF-β1 stimulated the proliferation of HPLCs, it did not upregulate the expression of alpha-smooth muscle actin (α-SMA), type I collagen (Col I), or fibrillin-1 (FBN1) mRNA or of α-SMA protein in HPLCs, whereas expression for these genes was attenuated by an anti-TGF-β1 neutralizing antibody. In contrast, exogenous TGF-β1 reduced the proliferation of cell line 1-11, although it upregulated the expression of α-SMA, Col I, and FBN1 mRNA and of α-SMA protein in this cell line. In addition, interleukin-1 beta stimulation significantly reduced the expression of TGF-β1 mRNA and protein in HPLCs. Thus, TGF-β1 seems to play an important role in inducing fibroblastic differentiation of PDL stem/progenitor cells and in maintaining the PDL apparatus under physiological conditions.     

Human periodontal ligament fibroblasts stimulate osteoclastogenesis in response to compression force through TNF-α-mediated activation of CD4+ T cells

Periodontal ligament fibroblasts (PLF) sense and respond to mechanical stimuli and participate in alveolar bone resorption during orthodontic treatments. This study examined how PLF influence osteoclastogenesis from bone marrow-derived macrophages (BMM) after application of tension or compression force. We also investigated whether lymphocytes could be a primary stimulator of osteoclastic activation during alveolar bone remodeling. We found that mechanical forces inhibited osteoclastic differentiation from BMM in co-cultures with PLF, with PLF producing predominantly osteoprotegerin (OPG) rather than receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL). In particular, PLF increased the expression of tumor necrosis factor (TNF)-α in response to compression. Additional experiments showed the presence of CD4- and B220-positive cells with a subsequent increase in tartrate-resistant acid phosphatase (TRAP)-positive cells and RANKL expression only at the compression side of the force-subjected periodontal tissues. Exogenous TNF-α increased the number of TRAP-positive cells and pit formation in the co-cultures of BMM with Jurkat, but not with BJAB cells and this effect was almost completely inhibited by antibodies to TNF-α or TNF receptor. Collectively, the current findings suggest that PLF secrete relatively higher levels of TNF-α at the compression side than at the tension side and this imbalance leads to RANKL expression by activating CD4+ T cells, thereby facilitating bone resorption during orthodontic tooth movement.     

Mechanical loading prevents the stimulating effect of IL-1β on osteocyte-modulated osteoclastogenesis

Ever since the technique of coaxing ordinary skin cells into becoming pluripotent stem cells (iPSCs) has been developed, which have the potential to become any cell or tissue in the body, efforts were made to improve the approach because some major challenges. Increasing evidence suggests that several microRNAs (miRNAs) are involved in early embryonic development and embryonic stem cell formation, known as embryonic stem cell (ESC)-specific miRNAs, particularly the miR-302 family. We summarized here a novel approach to generate iPSCs by using miR-302 and its related miRNAs such as miR-367. The development of this miR-302/367-mediated iPSC (termed mirPSC) may provide tools to deal with the obstacles facing some current iPSC reprogramming methods. The mechanism by which miR-302/367 induce iPSC reprogramming is proposed.     

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.     

Exosomes from IL-1β stimulated synovial fibroblasts induce osteoarthritic changes in articular chondrocytes

Introduction

Osteoarthritis (OA) is a whole joint disease, and characterized by progressive degradation of articular cartilage, synovial hyperplasia, bone remodeling and angiogenesis in various joint tissues. Exosomes are a type of microvesicles (MVs) that may play a role in tissue-tissue and cell-cell communication in homeostasis and diseases. We hypothesized that exosomes function in a novel regulatory network that contributes to OA pathogenesis and examined the function of exosomes in communication among joint tissue cells.

Methods

Human synovial fibroblasts (SFB) and articular chondrocytes were obtained from normal knee joints. Exosomes isolated from conditioned medium of SFB were analyzed for size, numbers, markers and function. Normal articular chondrocytes were treated with exosomes from SFB, and Interleukin-1β (IL-1β) stimulated SFB. OA-related genes expression was quantified using real-time PCR. To analyze exosome effects on cartilage tissue, we performed glycosaminoglycan release assay. Angiogenic activity of these exosomes was tested in migration and tube formation assays. Cytokines and miRNAs in exosomes were analyzed by Bio-Plex multiplex assay and NanoString analysis.

Results

Exosomes from IL-1β stimulated SFB significantly up-regulated MMP-13 and ADAMTS-5 expression in articular chondrocytes, and down-regulated COL2A1 and ACAN compared with SFB derived exosomes. Migration and tube formation activity were significantly higher in human umbilical vein endothelial cells (HUVECs) treated with the exosomes from IL-1β stimulated SFB, which also induced significantly more proteoglycan release from cartilage explants. Inflammatory cytokines, IL-6, MMP-3 and VEGF in exosomes were only detectable at low level. IL-1β, TNFα MMP-9 and MMP-13 were not detectable in exosomes. NanoString analysis showed that levels of 50 miRNAs were differentially expressed in exosomes from IL-1β stimulated SFB compared to non-stimulated SFB.

Conclusions

Exosomes from IL-1β stimulated SFB induce OA-like changes both in vitro and in ex vivo models. Exosomes represent a novel mechanism by which pathogenic signals are communicated among different cell types in OA-affected joints.     

Low extracellular pH stimulates the production of IL-1β by human monocytes

The development of acidic environments is a hallmark of inflammatory processes of different etiology. We have previously shown that transient exposure to acidic conditions, similar to those encountered in vivo, induces the activation of neutrophils and the phenotypic maturation of dendritic cells. We here report that extracellular acidosis (pH 6.5) selectively stimulates the production and the secretion of IL-1β by human monocytes without affecting the production of TNF-α, IL-6 and the expression of CD40, CD80, CD86, and HLA-DR. Stimulation of IL-1β production by pH 6.5-treated monocytes was shown to be dependent on caspase-1 activity, and it was also observed using peripheral blood mononuclear cells instead of isolated monocytes. Contrasting with the results in monocytes, we found that pH 6.5 did not stimulate any production of IL-1β by macrophages. Changes in intracellular pH seem to be involved in the stimulation of IL-1β production. In fact, monocytes cultured at pH 6.5 undergo a fall in the values of intracellular pH while the inhibitor of the Na+/H+ exchanger, 5-(N-ethyl-N-isopropyl)amiloride induced both, a decrease in the values of intracellular pH and the stimulation of IL-1β production. Real time quantitative PCR assays indicated that monocytes cultured either at pH 6.5 or in the presence of 5-(N-ethyl-N-isopropyl)amiloride expressed higher levels of pro-IL-1β mRNA suggesting that low values of intracellular pH enhance the production of IL-1β, at least in part, by stimulating the synthesis of its precursor.     

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).     

Macrophage-induced expression and release of matrix metalloproteinase 1 and 3 by human preadipocytes is mediated by IL-1β via activation of MAPK signaling

Obesity is associated with a chronic low‐grade inflammation and increased macrophage infiltration in adipose tissue. Matrix metalloproteinases (MMPs) are involved in adipose tissue remodeling and inflammatory responses in obesity. This study investigated whether macrophage‐derived factors modulate expression and secretion of MMP1 and MMP3 in human preadipocytes. The potential mediators and signaling pathways were also explored. MMP1 and MMP3 were primarily expressed and secreted by preadipocytes and dramatically reduced post‐differentiation. Preadipocytes were incubated with RPMI 1640 medium (control) or THP‐1 macrophage‐conditioned (MC) medium (25% and 100%) for 24 h. MC medium markedly increased mRNA levels of MMP1 (up to 122‐fold) and MMP3 (up to 59‐fold), as well as protein release of MMP1 (up to 378‐fold) and MMP3 (up to 10‐fold) in a dose‐dependent manner. Treatment with IL‐1β or TNFα, the major products of macrophages, also induced MMP1 and MMP3 secretion by preadipocytes. Neutralizing IL‐1β abolished the induction of MMP1 and MMP3 in preadipocytes by MC medium while the effects of TNFα neutralization were modest. Furthermore, MC medium or IL‐1β led to the phosphorylation of p38, ERK and JNK MAPKs. Inhibition of p38, ERK and JNK reversed the stimulatory effects of MC or IL‐1β on MMP1 and MMP3 production. MC medium and IL‐1β also activated NF‐κB p65 whereas reduced IκBα protein expression in preadipocytes. These results suggest that macrophage accumulation in adipose tissue has a central role in stimulating MMP1 and MMP3 production by preadipocytes, and this is partially mediated by IL‐1β via activation of the MAPK and NF‐κB signaling pathways. J. Cell. Physiol. 226: 2869–2880, 2011. © 2011 Wiley‐Liss, Inc.     

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.