Induction of Antimicrobial Protein S100A15 Expression by Oral Microbial Pathogens Is Toll-like Receptors-Dependent Activation of c-Jun-N-Terminal Kinase (JNK), p38, and NF-κB Pathways.

The antimicrobial protein S100A15 belongs to the S100 family, which is differentially expressed in a variety of normal and pathological tissues. Although the function of S100A15 protein has been discussed in several studies, its induction and regulation in oral mucosa, so far, are largely unknown. In this study, we demonstrate that S100A15 is induced by the stimulation of oral mucosa with gram- or gram+ bacterial pathogens, as well as with the purified membrane components, namely lipopolysaccharides (LPS) and lipoteichoic acid (LTA). The stimulation of the human gingival fibroblast (GF) and the human mouth epidermal carcinoma (KB) cell lines with either gram- or gram+ bacterial pathogens or their purified membrane components (LPS and LTA) results in the activation of NF-κB, apoptosis-regulating kinase1 (ASK1), and MAP kinase signaling pathways including, c-Jun N-terminal kinase (JNK) and p38 together with their physiological substrates AP-1 and ATF-2, respectively. Inhibition of S100A15 by antibodies-mediated Toll-like receptor 4 (TLR4) or Toll-like receptor 2 (TLR2) neutralization reveals the induction of S100A15 protein by LPS/gram- bacterial pathogens to be TLR4- dependent mechanism, whereas induction by LTA/gram+ bacterial pathogens to be TLR2- dependent mechanism. Pre-treatment of GF and KB cells with JNK (SP600125), p38 (SB-203580), or NF-κB (Bay11-7082) specific inhibitors further demonstrates the importance of JNK, p38 and NF-κB pathways in the regulation of gram-/gram+ bacterial pathogen-induced S100A15 expression. Our data provide evidence that S100A15 is induced in cancer and non-cancer oral mucosa-derived cell lines by gram-/gram+ bacterial pathogens and provide insight into the molecular mechanisms by which gram- and gram+ bacterial pathogens induce S100A15 expression in the oral mucosa.

Performance of the Polydopamine-Graphene Oxide Composite Substrate in the Osteogenic Differentiation of Mouse Embryonic Stem Cells.

Graphene oxide (GO) is a biocompatible material considered a favorable stem cell culture substrate. In this study, GO was modified with polydopamine (PDA) to facilitate depositing GO onto a tissue culture polystyrene (PT) surface, and the osteogenic performance of the PDA/GO composite in pluripotent embryonic stem cells (ESCs) was investigated. The surface chemistry of the PDA/GO-coated PT surface was analyzed by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). A high cell viability of ESCs cultured on the PDA/GO composite-coated surface was initially ensured. Then, the osteogenic differentiation of the ESCs in response to the PDA/GO substrate was assessed by alkaline phosphatase (ALP) activity, intracellular calcium levels, matrix mineralization assay, and evaluation of the mRNA and protein levels of osteogenic factors. The culture of ESCs on the PDA/GO substrate presented higher osteogenic potency than that on the uncoated control surface. ESCs cultured on the PDA/GO substrate expressed significantly higher levels of integrin α5 and ß1, as well as bone morphogenetic protein receptor (BMPR) types I and II, compared with the control groups. The phosphorylation of extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun-N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) was observed in ESCs culture on the PDA/GO substrate. Moreover, BMP signal transduction by SMAD1/5/8 phosphorylation was increased more in cells on PDA/GO than in the control. The nuclear translocation of SMAD1/5/8 in cells was also processed in response to the PDA/GO substrate. Blocking activation of the integrin α5/ß1, MAPK, or SMAD signaling pathways downregulated the PDA/GO-induced osteogenic differentiation of ESCs. These results suggest that the PDA/GO composite stimulates the osteogenic differentiation of ESCs via the integrin α5/ß1, MAPK, and BMPR/SMAD signaling pathways.

Ca mobilization and signaling pathways induced by rRgpB in human gingival fibroblast.

: To assess the () recombinant gingivalis gingipain R2 (rRgpB)-induced Ca mobilization in human gingival fibroblast (HGF) mediated by protease-activated receptor (PAR) and its downstream signal transduction pathways. : Flow cytometry was used to detect the expression of PAR in HGF. The proliferation of HGF was measured by CCK-8. The dynamic changes of intracellular Ca concentration in HGF induced by rRgpB and the blocking effect of PAR-1 antagonist were observed by laser confocal microscopy. Western blot was performed to determine the phosphorylation levels of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (p38 MAPK) and p65 in HGF. : PAR-1 and PAR-3 were expressed in HGF, and the rRgpB could promote the proliferation of HGF. rRgpB caused a transient increase in [Ca], which could be completely suppressed by vorapaxar, a PAR-1 antagonist. The phosphorylation levels of JNK, ERK1/2 and p65 were significantly up-regulated after the induction of rRgpB for and (all <0.05), which was completely inhibited by vorapaxar. However, the phosphorylation level of p38 MAPK had no significant change after rRgpB stimulation. : rRgpB causes an increase in [Ca] in HGF mediated by PAR-1. JNK, ERK1/2 and nuclear factor-κB may be involved in intracellular signal transduction after PAR-1 activation.

Photobiomodulation with 808-nm diode laser enhances gingival wound healing by promoting migration of human gingival mesenchymal stem cells via ROS/JNK/NF-κB/MMP-1 pathway.

Photobiomodulation (PBM) has been shown to improve wound healing by promoting mesenchymal stem cell migration and proliferation. However, it remains unknown whether an 808-nm diode laser can influence human gingival mesenchymal stem cells (HGMSCs), and which dose this works well. In the present study, it was found that PBM could promote the migration of HGMSCs but not the proliferation. Furthermore, PBM could activate mitochondrial ROS, which could elevate the phosphorylation levels of JNK and IKB in HGMSCs, and further activate NF-κB as the nuclear translocation of p65 is elevated. Taken together, these present results indicate that PBM might promote cell migration via the ROS/JNK/NF-κB pathway.

JNK Signaling as a Key Modulator of Soft Connective Tissue Physiology, Pathology, and Healing.

In healthy individuals, the healing of soft tissues such as skin after pathological insult or post injury follows a relatively predictable and defined series of cell and molecular processes to restore tissue architecture and function(s). Healing progresses through the phases of hemostasis, inflammation, proliferation, remodeling, and concomitant with re-epithelialization restores barrier function. Soft tissue healing is achieved through the spatiotemporal interplay of multiple different cell types including neutrophils, monocytes/macrophages, fibroblasts, endothelial cells/pericytes, and keratinocytes. Expressed in most cell types, c-Jun N-terminal kinases (JNK) are signaling molecules associated with the regulation of several cellular processes involved in soft tissue wound healing and in response to cellular stress. A member of the mitogen-activated protein kinase family (MAPK), JNKs have been implicated in the regulation of inflammatory cell phenotype, as well as fibroblast, stem/progenitor cell, and epithelial cell biology. In this review, we discuss our understanding of JNKs in the regulation of cell behaviors related to tissue injury, pathology, and wound healing of soft tissues. Using models as diverse as Drosophila, mice, rats, as well as human tissues, research is now defining important, but sometimes conflicting roles for JNKs in the regulation of multiple molecular processes in multiple different cell types central to wound healing processes. In this review, we focus specifically on the role of JNKs in the regulation of cell behavior in the healing of skin, cornea, tendon, gingiva, and dental pulp tissues. We conclude that while parallels can be drawn between some JNK activities and the control of cell behavior in healing, the roles of JNK can also be very specific modes of action depending on the tissue and the phase of healing.

Cell Cycle Arrest and Apoptosis Induced by Porphyromonas gingivalis Require Jun N-Terminal Protein Kinase- and p53-Mediated p38 Activation in Human Trophoblasts.

Porphyromonas gingivalis, a periodontal pathogen, has been implicated as a causative agent of preterm delivery of low-birth-weight infants. We previously reported that P. gingivalis activated cellular DNA damage signaling pathways and ERK1/2 that lead to G1 arrest and apoptosis in extravillous trophoblast cells (HTR-8 cells) derived from the human placenta. In the present study, we further examined alternative signaling pathways mediating cellular damage caused by P. gingivalis. P. gingivalis infection of HTR-8 cells induced phosphorylation of p38 and Jun N-terminal protein kinase (JNK), while their inhibitors diminished both G1 arrest and apoptosis. In addition, heat shock protein 27 (HSP27) was phosphorylated through both p38 and JNK, and knockdown of HSP27 with small interfering RNA (siRNA) prevented both G1 arrest and apoptosis. Furthermore, regulation of G1 arrest and apoptosis was associated with p21 expression. HTR-8 cells infected with P. gingivalis exhibited upregulation of p21, which was regulated by p53 and HSP27. These results suggest that P. gingivalis induces G1 arrest and apoptosis via novel molecular pathways that involve p38 and JNK with its downstream effectors in human trophoblasts.

Luminal Polyethylene Glycol Alleviates Intestinal Preservation Injury Irrespective of Molecular Size.

Intestinal preservation injury (IPI) and the resulting mucosa injury raise several serious challenges early after intestinal transplantation. The current clinical approach using only vascular perfusion allows the shortest preservation period among the abdominal organs. The experimental addition of luminal polyethylene glycol (PEG) solutions has been repeatedly suggested to alleviate preservation injury, improve graft quality, and prolong the preservation time. We investigated whether the molecular mass of PEG in solution influences the development of intestinal preservation injury. Small intestines of Sprague-Dawley rats were perfused with University of Wisconsin solution. Group 1 underwent vascular perfusion only (clinical control), group 2 received additional luminal PEG3350 Da, group 3 received luminal PEG10000 Da, and group 4 received luminal PEG20000 Da (n = 8/group). Tissue samples were obtained after 4, 8, and 14 hours. We studied the tissue damage (Chiu/Park score, Goblet cells, apoptosis, tight junctions), activation of c-Jun NH2-terminal kinase (JNK), and p38-mitogen-activated protein kinase (MAPK), and we performed Ussing chamber assessments. Mucosal morphologic and electrophysiologic parameters were significantly improved in the groups receiving luminal PEG. There was significantly less apoptotic activity in groups 2, 3, and 4. Both MAPKs revealed an activation peak after 4 hours with group 3 showing lesser p38-MAPK activation. PEG 20 kDa interfered with protein immunodetection. The results indicate that luminal solutions of PEG of medium and large molecular mass significantly delay the onset and development of IPI, providing further evidence that luminal interventions may allow for longer cold storage intervals of intestinal grafts.

Noscapine chemosensitization enhances docetaxel anticancer activity and nanocarrier uptake in triple negative breast cancer.

Chemosensitization and enhanced delivery to solid tumor are widely explored strategies to augment the anticancer efficacy of existing chemotherapeutics agents. The aim of current research was to investigate the role of low dose Noscapine (Nos) in potentiating docetaxel cytotoxicity and enhancing tumor penetration of nanocarriers. The objectives are; (1) To evaluate the chemo-sensitizing effect of Nos in combination with docetaxel (DTX), and to elucidate the possible mechanism (2) To investigate the effect of low dose Nos on tumor stroma and enhancing nanocarrier uptake in triple negative breast cancer (TNBC) bearing nude mice. Cytotoxicity and flow cytometry analysis of DTX in Nos (4µM) pre-treated MDA-MB-231 cells showed 3.0-fold increase in cell killing and 30% increase in number of late apoptotic cells, respectively. Stress transducer p38 phosphorylation was significantly upregulated with Nos exposure. DTX showed remarkable downregulation in expression of bcl-2, survivin and pAKT in Nos pre-treated MDA-MB-231 cells. Nos pre-sensitization significantly (p<0.02) enhanced the anti-migration effect of DTX. In vivo studies in orthotopic TNBC tumor bearing mice showed marked reduction in tumor collagen-I levels and significantly (p<0.03) higher intra-tumoral uptake of coumarin-6 loaded PEGylated liposomes (7-fold) in Nos treated group. Chemo-sensitization and anti-fibrotic effect of Nos could be a promising approach to increase anticancer efficacy of DTX which can be used for other nanomedicinal products.

Role of mitogen-activated protein kinase pathways in migration of gingival epithelial cells in response to stimulation by cigarette smoke condensate and infection by Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Previous studies have shown that cigarette smoke (CS) and periodontal pathogens could alter wound healing responses of gingival epithelial cells. To elucidate molecular mechanisms leading to these epithelial changes, we studied the signaling pathway involved in the modulation of cell migration by CS condensate (CSC) and the infection by a prominent periodontal pathogen, Porphyromonas gingivalis. MATERIAL AND METHODS: Human gingival epithelial cells (Ca9-22) were treated with CSC or vehicle control for 24 h. Activation of mitogen-activated protein kinases (MAPK) in cells with or without infection by P. gingivalis was assessed by polymerase chain reaction array and immunoblotting using phospho-specific antibodies. Cell migration was assessed using in vitro wound closure model, and specific pharmacologic inhibitors of MAPK pathways were used to characterize further the extent of involvement of the MAPK pathways. RESULTS: Polymerase chain reaction array showed that gene expression of several members of the MAPK, particularly p38 and JNK, was upregulated more than twofold in Ca9-22 cells stimulated with 10 µg/mL CSC. Coincubation with P. gingivalis induced a different pattern of gene expression for MAPK pathways, but it did not suppress the MAPK-related genes upregulated by CSC. A significant phosphorylation of ERK1/2 and p38 was observed in cells stimulated with 10 µg/mL CSC (p < 0.05), whereas coincubation with a higher concentration of CSC (250 µg/mL) evoked no such activation. P. gingivalis infection resulted in a tendency to reduce the phosphorylation of ERK1/2 and p38, which had been enhanced by stimulation with 10 µg/mL CSC. Incubation with ERK1/2 and p38 inhibitors significantly reduced the wound closure of CSC-stimulated cells, by approximately 43% and 46%, respectively (p < 0.05). CONCLUSION: CSC exerts effects on the migration of human gingival epithelial cells through the activation of the MAPK ERK1/2 and p38 signaling pathways. P. gingivalis infection attenuates the CSC-induced migration at least partly by suppressing the phosphorylation of ERK1/2 and p38, but other pathways are likely to be involved in this modulatory process.

Inhibition of AXUD1 attenuates compression-dependent apoptosis of cementoblasts.

OBJECTIVES: Root resorptions are common undesirable side effects of orthodontic treatment. In most patients, these defects are repaired by cementoblasts. However, in 1-5 % of patients, the repair fails. The repair mechanism is not well understood. Apoptosis of cementoblasts might contribute to an impaired repair of root resorptions induced by orthodontic forces. MATERIALS AND METHODS: To gain insight into putative molecular pathways leading to compression-induced apoptosis of human primary cementoblasts (HPCBs), three independent cell populations were subjected to compressive loading at 5, 20, and 30 g/cm2 for 1, 6, and 10 h. The mRNA expression of AXUD1, a novel pro-apoptotic gene, was monitored by quantitative reverse transcription PCR (qRT-PCR). To identify a possible function in compression-dependent apoptosis, AXUD1 was silenced in cementoblasts using an siRNA approach. Apoptosis of cementoblasts was measured by annexin V staining and flow cytometry. The phosphorylation of c-Jun-N-terminal kinases (JNKs) was investigated by Western blotting. RESULTS: AXUD1 was significantly induced in a time- and force-dependent manner. The rate of apoptotic HPCBs increased by 20-40 % after 10 h of compression (30 g/cm2). Phosphorylation of JNKs was detected after 10 h at 30 g/cm2. SiRNA-mediated knockdown of AXUD1 led to decreased phosphorylation of JNKs and reduced apoptosis rates in compressed HPCBs. CONCLUSIONS: Compression-induced apoptosis of HPCBs is mediated by AXUD1 via a JNK-dependent pathway. CLINICAL RELEVANCE: AXUD1-dependent apoptosis of human cementoblasts might contribute to an impaired repair of root resorptions during orthodontic tooth movement. Further studies are needed to develop treatment strategies aiming to minimize root resorption during orthodontic tooth movement.

Polyacrylic acid-coated and non-coated iron oxide nanoparticles induce cytokine activation in human blood cells through TAK1, p38 MAPK and JNK pro-inflammatory pathways.

Iron oxide nanoparticles (ION) can have a wide scope of applications in biomedicine, namely in magnetic resonance imaging, tissue repair, drug delivery, hyperthermia, transfection, tissue soldering, and as antimicrobial agents. The safety of these nanoparticles, however, is not completely established, namely concerning their effect on immune system and inflammatory pathways. The aim of this study was to evaluate the in vitro effect of polyacrylic acid (PAA)-coated ION and non-coated ION on the production of six cytokines [interleukin 1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), interleukin 8 (IL-8), interferon gamma (IFN-γ) and interleukin 10 (IL-10)] by human peripheral blood cells, and to determine the inflammatory pathways involved in this production. The obtained results showed that PAA-coated and non-coated ION were able to induce all the tested cytokines and that activation of transforming growth factor beta (TGF-ß)-activated kinase (TAK1), p38 mitogen-activated protein kinases (p38 MAPK) and c-Jun N-terminal kinases (JNK) were involved in this effect.

Effects of Intravenous Injection of Porphyromonas gingivalis on Rabbit Inflammatory Immune Response and Atherosclerosis.

The effects of intravenous injection of Porphyromonas gingivalis (Pg) on rabbit inflammatory immune response and atherosclerosis were evaluated by establishing a microamount Pg bacteremia model combined with high-fat diet. Twenty-four New Zealand rabbits were randomly divided into Groups A-D (n = 6). After 14 weeks, levels of inflammatory factors (C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1)) in peripheral blood were detected by ELISA. The aorta was subjected to HE staining. Local aortic expressions of toll-like receptor-2 (TLR-2), TLR-4, TNF-α, CRP, IL-6, matrix metallopeptidase-9, and MCP-1 were detected by real-time PCR, and those of nuclear factor-κB (NF-κB) p65, phospho-p38 mitogen-activated protein kinase (MAPK), and phospho-c-Jun N-terminal kinase (JNK) proteins were detected by Western blot. Intravenous injection of Pg to the bloodstream alone induced atherosclerotic changes and significantly increased systemic and local aortic expressions of inflammatory factors, NF-κB p65, phospho-p38-MAPK, and JNK, especially in Group D. Injection of microamount Pg induced inflammatory immune response and accelerated atherosclerosis, in which the NF-κB p65, p38-MAPK, and JNK signaling pathways played important roles. Intravenous injection of Pg is not the same as Pg from human periodontitis entering the blood stream. Therefore, our results cannot be extrapolated to human periodontitis.

Nitric oxide induces apoptosis in human gingival fibroblast through mitochondria-dependent pathway and JNK activation.

AIM: To investigate the molecular mechanisms of nitric oxide (NO)-induced cytotoxic effect in human gingival fibroblast (HGF) cells. METHODOLOGY: After sodium nitroprusside (SNP), as NO donor, was treated to HGF, viability was measured by MTT assay and apoptosis was determined by TUNEL and DNA fragmentation assay. Mitochondrial membrane potential was detected using confocal microscopy, and caspase activity assay was measured by spectrophotometer. Mitogen-activated protein kinases (MAPK) activation, Bax/Bcl-2 ratio and cytochrome c release were analysed by Western blot analyses. Cells were exposed to MAPK inhibitors (U0126, SB203580 and SP600125) before SNP treatment to investigate the effects of MAPK kinases on the NO-induced apoptosis in HGF. Statistical analysis was performed using one-way analysis of variance with the Student-Newman-Keuls post hoc test for multiple group comparison. RESULTS: Apoptosis was significantly increased (P = 0.011 and 0.0004, respectively) in the presence of SNP (1 and 3 mmol L(-1) ) after 12 h in HGF. However, 1H-[1,2,4] oxadiatolo [4, 3-a] cluinoxaline-1-one (ODQ), a soluble guanylate cyclase inhibitor, did not block the decrement of cell viability by NO. SNP treatment induced the loss of mitochondrial membrane potential, release of cytochrome c, increased Bax/Bcl-2 ratio and activation of caspases in HGF. Also, SNP treatment increased phosphorylation of MAPKinases and c-Jun N-terminal kinase (JNK) inhibitor (5 and 10 µmol L(-1) ) rescued cell viability decreased by SNP in HGF (P = 0.024 and 0.0149, respectively). CONCLUSION: Nitric oxide induced apoptosis in human gingival fibroblast through the mitochondria-mediated pathway by regulation of Bcl-2 family and JNK activation.

Porphyromonas gingivalis-induced reactive oxygen species activate JAK2 and regulate production of inflammatory cytokines through c-Jun.

Pathogen-induced reactive oxygen species (ROS) play a crucial role in host innate immune responses through regulating the quality and quantity of inflammatory mediators. However, the underlying molecular mechanisms of this effect have yet to be clarified. In this study, we examined the mechanism of action of ROS stimulated by Porphyromonas gingivalis in gingival epithelial cells. P. gingivalis induced the rapid production of ROS, which lead to the phosphorylation of JAK2 and increased levels of secreted proinflammatory cytokines interleukin-6 (IL-6) and IL-1ß. Neutralization of ROS by N-acetyl-l-cysteine (NAC) abrogated the phosphorylation of JAK2 and suppressed the production of IL-6 and IL-1ß. ROS-mediated phosphorylation of JAK2 induced the phosphoactivation of c-Jun amino-terminal protein kinase (JNK) and the downstream transcriptional regulator c-Jun. Inhibition of JAK2, either pharmacologically or by small interfering RNA (siRNA), reduced both the phosphorylation of these molecules and the production of proinflammatory cytokines in response to P. gingivalis. Furthermore, pharmacological inhibition or siRNA-mediated gene silencing of JNK or c-Jun mimicked the effect of JAK2 inhibition to suppress P. gingivalis-induced IL-6 and IL-1ß levels. The results show that ROS-mediated activation of JAK2 is required for P. gingivalis-induced inflammatory cytokine production and that the JNK/c-Jun signaling axis is involved in the ROS-dependent regulation of IL-1ß and IL-6 production.

Streptococcus mutans wall-associated protein A promotes TLR4-induced dendritic cell maturation.

Dendritic cells orchestrate innate and adaptive immune responses, which are central to establishing efficient responses to vaccination. Wall-associated protein A (WapA) of Streptococcus mutans was previously used as a vaccine in animal studies for immunization against dental caries. However, as a cell surface protein, whether WapA activates innate immune responses and the effects of WapA on DCs remain unclear. In this study, WapA was cloned into the GST fusion vector pEBG, which can be expressed efficiently in mammalian cells. We found that when added before stimulation with LPS, purified WapA-GST protein increased TLR4-induced NF-κB and MAPK signalling pathway activation. Pretreatment with WapA-GST also increased LPS-induced proinflammatory cytokine production by DCs, including IL-12, IL-6 and TNF-α. Furthermore, expression of the DC maturation markers CD80/86, CD40 and MHC II was also increased by WapA pretreatment. These data indicate that WapA is recognized by DCs and promotes DC maturation.

JNK MAPK is involved in BMP-2-induced odontoblastic differentiation of human dental pulp cells.

Bone morphogenetic protein-2 (BMP-2) is a multi-functional growth factor belonging to the transforming growth factor ß superfamily that has a broad range of activities that affect many different cell types. BMP-2 induces odontoblastic differentiation of human dental pulp cells (DPCs), but the underlying mechanism remains unclear. In this study, we investigated the potential role of the JNK mitogen-activated protein kinases (MAPK) pathway in BMP-2-induced odontoblastic differentiation of DPCs. The levels of phosphorylated and unphosphorylated JNK MAPK were quantified by Western blot analysis following treatment with BMP-2 and the JNK inhibitor SP600125. The role of JNK MAPK in the BMP-2-induced odontoblastic differentiation of DPCs was determined by measuring alkaline phosphatase (ALP) activity and by examining the expression of odontoblastic markers using quantitative real-time polymerase chain reaction analysis. The effect of JNK MAPK silencing on odontoblastic differentiation was also investigated. BMP-2 upregulated the phosphorylation of JNK in DPCs in a dose- and time-dependent manner. Early markers of odontoblastic differentiation, including ALP activity, osteopontin and dentin matrix protein-1, were not inhibited by the JNK inhibitor. However, the JNK inhibitor, SP600125, significantly inhibited late-stage differentiation of odontoblasts, including the gene expression of osteocalcin, dentin sialophosphoprotein and bone sialoprotein, and also reduced the formation of mineralized nodules in BMP-2-treated DPCs. Consistent with this observation, silencing of JNK MAPK also decreased late-stage odontoblastic differentiation. Taken together, these findings suggest that JNK activity is required for late-stage odontoblastic differentiation induced by BMP-2.

Neural epidermal growth factor-like 1 protein (Nell-1) alleviates periodontal tissue destruction in periodontitis by regulating the ratio of M2/M1 macrophage phenotypes.

BACKGROUND: Periodontitis is a common oral disease with high prevalence worldwide. Neural epidermal growth factor-like 1 protein (Nell-1) has recently been reported to have anti-inflammation effects and may be a drug candidate for osteoarthritis. However, its immunotherapeutic effects in periodontitis remain unknown. Therefore, this study aimed to investigate the effects of Nell-1 on periodontitis in terms of macrophage polarization and analyze its possible underlying mechanism. METHODS: A rat ligation-induced experimental periodontitis model was established and locally injected with Nell-1 (n = 6/group). Periodontal tissue destruction and macrophage polarization in vivo were analyzed using micro-CT, histology analysis, and western blot. Enzyme-linked immunosorbent assay was used to evaluate serum inflammatory cytokines. Then, the RAW 264.7 macrophage cells were treated with lipopolysaccharide (LPS), Nell-1, and the c-Jun N-terminal kinases (JNK) inhibitor (SP600125). RT-PCR, western blot, and flow cytometry were performed to further analyze the effect of Nell-1 on macrophage polarization and the underlying mechanism in vitro. RESULTS: Local treatment with Nell-1 significantly alleviated the destruction of alveolar bone and fibers in periodontitis, and upregulated the ratio of M2/M1 macrophages in periodontal tissues (P < 0.05). In vitro, Nell-1 at the concentrations of 200 and 500 ng/mL could significantly inhibit the expression of M1-related inflammatory factors in LPS-stimulated macrophages, and increase the expression of M2-related markers, regulating the macrophage phenotype switch into M2 (P < 0.05). The mRNA of JNK and relative protein level of phospho-JNK/JNK were also upregulated by Nell-1 (P < 0.05). Additionally, the JNK inhibitor (SP600125) could reverse the effect of Nell-1 on macrophage polarization (P < 0.05). CONCLUSIONS: Nell-1 could modulate the ratio of M2/M1 macrophages possibly through the JNK/MAPK signaling pathway, subsequently attenuating the inflammation and destruction of periodontal tissues caused by periodontitis.

EGF positively regulates the proliferation and migration, and negatively regulates the myofibroblast differentiation of periodontal ligament-derived endothelial progenitor cells through MEK/ERK- and JNK-dependent signals.

BACKGROUND/AIMS: Remodeling of fibrous and vascular tissues in the periodontal ligament (PDL) around the tooth root was observed during tooth movement by orthodontic force application. We previously demonstrated that a single cell-derived culture (SCDC) of primarily cultured PDL fibroblasts, called SCDC2, has an endothelial progenitor cell (EPC)-like character and can form endothelial cell (EC) marker-positive blood vessel-like structures. However, the types of molecular mechanisms that control the in vivo kinetic properties and the differentiation of the PDL-derived EPC-like cells into myofibroblasts (MFs), which are known to expand fibrous tissues, require clarification. METHODS: Using specific mitogen activated protein kinase (MAPK) inhibitors, we examined how epidermal growth factor (EGF)-mediated MAPK signals affected the proliferation, migration, and MF differentiation of these cells. RESULTS: EGF induced SCDC2 cell proliferation in MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)- and c-Jun N-terminal kinase (JNK)-dependent manners. In addition, EGF suppressed the expression of MF differentiation markers in these cells in a MEK/ERK-dependent manner, and, moreover, stimulated the cell migration in a MEK/ERK-dependent manner. CONCLUSION: EGF regulates fibrous tissue remodeling in PDLs through MEK/ERK- and JNK-mediated signals by affecting the proliferation, migration, and MF differentiation of the PDL-derived EPC-like cells.

(-)-Epigallocatechin-3-gallate inhibits CC chemokine ligand 11 production in human gingival fibroblasts.

BACKGROUND: CC chemokine ligand 11 (CCL11) is related to Th2 cells migration via CC chemokine receptor 3 (CCR3). Th2 cells are involved in the etiology of periodontal disease. However, how the infiltration of Th2 cells is controlled in periodontally diseased tissues is unknown. (-)-Epigallocatechin gallate (EGCG), the major catechin in green tea, has multiple beneficial effects, but the effects of EGCG on CCL11 production are uncertain. In this study, we investigated whether cytokines could induce CCL11 production in human gingival fibroblasts (HGFs). Moreover, we examined the effects of EGCG on CCL11 production in HGFs. METHODS AND RESULTS: ELISA analysis disclosed that interleukin (IL)-4 synergistically enhanced CCL11 production in IL-1ß or tumor necrosis factor (TNF)-α-stimulated HGFs. EGCG prevented IL-1ß/ IL-4 or TNF-α/IL-4-mediated CCL11 production in a concentration dependent manner. CCL11 production in HGFs was positively regulated by p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-Jun N terminal kinase (JNK). Western blot analysis revealed that EGCG treatment prevented IL-1ß/IL-4 or TNF-α/IL-4-induced ERK and JNK activation in HGFs. CONCLUSIONS: These data provide that CCL11 production in HGFs could be associated with Th2 cells infiltration in periodontal lesions. Moreover, EGCG is useful for periodontitis treatment to inhibit CCL11 production.

Adenosine receptor stimulation by polynucleotides (PDRN) reduces inflammation in experimental periodontitis.

AIM: Adenosine receptors modulate inflammation in periodontal tissues. No data are available regarding the effects of adenosine A(2A) receptor stimulation in experimental periodontitis (EPD). The aim of this study was to investigate the effects of polynucleotides (also known as polydeoxyribonucleotide, PDRN), a ligand of A(2A) receptor, in EPD in rats. MATERIALS AND METHODS: EPD was induced ligating the cervix of the lower left first molar. Sham-EPD had no ligature. After 7 days, EPD animals were randomized to a daily treatment with vehicle gel or 0.75% PDRN gel or PDRN gel with a specific A(2A) antagonist (DMPX). Treatments lasted 7 days. Animals were then euthanized and the periodontium and surrounding gingival tissue were excised for histological evaluation and bio-molecular analysis of inflammatory (p-JNK, p-ERK, TNF-α, IL-6, HMGB-1) and apoptotic proteins (BAX and Bcl-2). RESULTS: Vehicle-treated EPD rats showed severe inflammatory infiltrate in both gingival and periodontal ligament, as well as an enhanced expression of p-JNK, p-ERK, TNF-α, IL-6, HMGB-1 and BAX and a reduction in Bcl-2. PDRN gel restored the histological features, blunted inflammatory and apoptotic proteins expression and preserved Bcl-2 expression. DMPX abrogated PDRN positive effects. CONCLUSION: Our data suggest that adenosine receptor stimulation by PDRN might represent a new therapeutic strategy for periodontitis.