CpG ODN-induced matrix metalloproteinase-13 expression is mediated via activation of the ERK and NF-κB signalling pathways in odontoblast cells.

To investigate the effects of CpG ODN (CpG oligodeoxynucleotides) to model the action of bacterial challenge on pulpal matrix metalloproteinase-13 (MMP-13) expression and elucidate the associated intracellular signalling pathways. Real-time PCR was used to detect the effects of CpG ODN on MMP-13 mRNA expression levels in a murine odontoblast-lineage cell line (OLCs). The possible involvement of TLR9/MyD88, NF-κB or MAPK pathways involved in the CpG ODN-induced MMP-13 expression was examined by real-time PCR, transient transfection, luciferase activity assay and ELISA. Western blotting was performed to assay the phosphorylation of ERK at a range of time points. MMP-13 was constitutively expressed in OLCs, and their exposure to CpG ODN significantly increased MMP-13 expression. Pre-treatment of OLCs with the inhibitory peptide MyD88, or chloroquine, attenuated the CpG ODN-induced expression of MMP-13. Treatment of the OLCs with CpG ODN increased NF-κB-luciferase activity. This activity was decreased by the over-expression of a nondegrading mutant of IκBα (IκBαSR), although enhanced by the over-expression of NF-κB p65. MMP-13 expression induced by CpG ODN was markedly suppressed by NF-κB inhibitors (pyrrolidine dithiocarbamate, PDTC), IκBα phosphorylation inhibitors (Bay 117082) or IκB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone, TPCK). The inhibitor of ERK1/2, U0126, but not inhibitors of p38 MAPK and JNK, SB203580 and SP600125, decreased CpG ODN-mediated MMP-13 expression. The CpG ODN-induced MMP-13 expression in OLCs is mediated through TLR9, NF-κB and the ERK pathway indicating that potentially the recognition of CpG ODN by TLR9 on odontoblasts may regulate the remodelling of injured dental pulp and hard tissues by inducing MMP-13 expression.

Zhang J ，Zhu QL ，Huang P ，Yu Q ，Wang ZH ，Cooper PR ，Smith AJ ，He W ... -  《-》

Cytidine-phosphate-guanosine oligonucleotides induce interleukin-8 production through activation of TLR9, MyD88, NF-κB, and ERK pathways in odontoblast cells.

Odontoblasts are involved in innate immunity against invading microorganisms. However, the mechanisms of host inflammatory responses to bacterial DNA in odontoblasts are not fully understood. The purpose of this study was to investigate whether microbial cytidine-phosphate-guanosine (CpG) DNA influences interleukin-8 (IL-8) expression in odontoblasts and the signaling pathways involved. The effect of CpG oligonucleotide (CpG ODN) on IL-8 mRNA and protein expression levels in the mouse odontoblast-like cell line MDPC-23 was investigated by real-time polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA). Whether Toll-like receptor 9 (TLR9), myeloid differentiation marker 88 (MyD88), nuclear factor kappa B (NF-κB), or mitogen-activated protein kinase (MAPK) pathways were involved in the CpG ODN-induced IL-8 expression was determined by examined real-time PCR, ELISA, and luciferase activity assay. Extracellular signal-regulated kinase (ERK) activation and TLR9 protein expression were measured by Western blot analysis. Exposure to CpG ODN induced significant up-regulation of IL-8 mRNA and protein in MDPC-23 cells. CpG ODN-induced IL-8 up-regulation was attenuated by TLR9 inhibitor (chloroquine) and MyD88 inhibitory peptide. CpG ODN also increased the expression of TLR9 mRNA and protein in MDPC-23 cells. Treatment of MDPC-23 cells with NF-κB inhibitors (pyrrolidine dithiocarbamate), IκBα phosphorylation inhibitors (Bay 117082), or IκB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone) decreased CpG ODN-induced IL-8 expression. Furthermore, stimulation of cells with CpG ODN enhanced κB-luciferase activity, and the activity was diminished by the overexpression of dominant negative mutants of MyD88 and IκBα. In addition, CpG ODN-induced IL-8 expression was markedly suppressed by U0126, but not by SB203580 and SP600125. Moreover, CpG ODN activated ERK phosphorylation in a time-dependent manner. These data demonstrate that CpG ODN-induced IL-8 expression was mediated through TLR9, MyD88, NF-κB, and ERK pathways in MDPC-23 cells and suggest a possible role for the CpG DNA-mediated immune response in odontoblasts with involvement of TLR9, MyD88, and ERK pathways in this process.

He W ，Zhang Y ，Zhang J ，Yu Q ，Wang P ，Wang Z ，Smith AJ ... -  《-》

Lipopolysaccharide enhances decorin expression through the Toll-like receptor 4, myeloid differentiating factor 88, nuclear factor-kappa B, and mitogen-activated protein kinase pathways in odontoblast cells.

Lipopolysaccharide (LPS) has been shown to regulate the function of odontoblasts. However, the molecular mechanisms of the effect of LPS on odontoblasts are poorly understood. Decorin (DCN), one of the major matrix proteoglycans, is known to affect the mineralization of teeth. In this study, we investigated whether LPS can regulate the expression of DCN in odontoblasts and determined the intracellular signaling pathways triggered by LPS. The DCN messenger RNA and protein expression changes in mouse odontoblast-lineage cells (OLCs) were detected by real-time polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA). Whether TLR4, myeloid differentiating factor 88 (MyD88), nuclear factor-kappa B (NF-κB), or mitogen-activated protein kinase (MAPK) pathways were involved in the LPS-induced DCN expression was determined by examined real-time PCR, ELISA, and luciferase activity assay. The activation of extracellular signal-regulated kinase (ERK), p38, and JNK in OLCs was measured by Western blot analysis. We found that the mouse OLCs expressed DCN. DCN messenger RNA was rapidly induced by LPS in a time- and dose-dependent manner. Pretreatment with a MyD88 inhibitory peptide, a TLR4 antibody, or a specific inhibitor for NF-κB or I Kappa B alpha (IκBα) significantly inhibited LPS-induced DCN expression. Moreover, the LPS-mediated increase in κB-luciferase activity in OLCs was suppressed by the overexpression of dominant negative mutants of TLR4, MyD88, and IκBα but not by a dominant negative mutant of TLR2. In addition, LPS stimulation activated the ERK, p38, and JNK MAPK pathways. The pretreatment of OLCs with specific inhibitors of the ERK, p38, and JNK MAPK pathways markedly offset the LPS-induced up-regulation of DCN expression. Our results show that LPS stimulation can up-regulate the gene expression of DCN via the TLR4, MyD88, NF-κB, and MAPK pathways in odontoblast cells.

He W ，Qu T ，Yu Q ，Wang Z ，Wang H ，Zhang J ，Smith AJ ... -  《-》

Biodentine induces human dental pulp stem cell differentiation through mitogen-activated protein kinase and calcium-/calmodulin-dependent protein kinase II pathways.

Biodentine (Septodont, Saint-Maur-des-Fossès, France), a new tricalcium silicate cement formulation, has been introduced as a bioactive dentine substitute to be used in direct contact with pulp tissue. The aim of this study was to investigate the response of human dental pulp stem cells (hDPSCs) to the material and whether mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), and calcium-/calmodulin-dependent protein kinase II (CaMKII) signal pathways played a regulatory role in Biodentine-induced odontoblast differentiation. hDPCs obtained from impacted third molars were incubated with Biodentine. Odontoblastic differentiation was evaluated by alkaline phosphatase activity, alizarin red staining, and quantitative real-time reverse-transcriptase polymerase chain reaction for the analysis of messenger RNA expression of the following differentiation gene markers: osteocalcin (OCN), dentin sialophosprotein (DSPP), dentin matrix protein 1 (DMP1), and bone sialoprotein (BSP). Cell cultures in the presence of Biodentine were exposed to specific inhibitors of MAPK (U0126, SB203580, and SP600125), NF-κB (pyrrolidine dithiocarbamate), and CaMKII (KN-93) pathways to evaluate the regulatory effect on the expression of these markers and mineralization assay. Biodentine significantly increased alkaline phosphatase activity and mineralized nodule formation and the expression of OCN, DSPP, DMP1, and BSP. The MAPK inhibitor for extracellular signal-regulated kinase 1/2 (U0126) and Jun N-terminal kinase (SP600125) significantly decreased the Biodentine-induced mineralized differentiation of hDPSCs and OCN, DSPP, DMP1, and BSP messenger RNA expression, whereas p38 MAPK inhibitors (SB203580) had no effect. The CaMKII inhibitor KN-93 significantly attenuated and the NF-κB inhibitor pyrrolidine dithiocarbamate further enhanced the up-regulation of Biodentine-induced gene expression and mineralization. Biodentine is a bioactive and biocompatible material capable of inducing odontoblast differentiation of hDPSCs. Our results indicate that this induction is regulated via MAPK and CaMKII pathways.

Luo Z ，Kohli MR ，Yu Q ，Kim S ，Qu T ，He WX ... -  《-》

Lipopolysaccharide enhances Wnt5a expression through toll-like receptor 4, myeloid differentiating factor 88, phosphatidylinositol 3-OH kinase/AKT and nuclear factor kappa B pathways in human dental pulp stem cells.

Lipopolysaccharide (LPS) has been implicated in mesenchymal stem cell differentiation processes. Wnt5a, one of the "non-canonical" Wnt family members, is important in signaling stem cell differentiation and in the inflammatory responses of immune cells. Here we studied whether LPS can regulate the expression of Wnt5a in human dental pulp stem cells (hDPSCs) and investigated the intracellular signaling pathways activated by LPS. Wnt5a mRNA and protein expression changes in hDPSCs were investigated by real-time polymerase chain reaction analysis and enzyme-linked immunosorbent assay. In addition, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and luciferase activity assays were used to determine whether toll-like receptor 4 (TLR4), myeloid differentiating factor 88 (MyD88), nuclear factor kappa B (NF-kB), or the phosphatidylinositol 3-OH kinase (PI3K)/AKT pathways are involved in LPS-induced Wnt5a expression. The activation of PI3K and AKT in hDPSCs was measured by Western blot analysis. Wnt5a mRNA and protein expression was rapidly increased in response to LPS in a time- and dose-dependent manner. LPS-induced Wnt5a expression was effectively attenuated by administration of a TLR4 neutralizing antibody, MyD88 inhibitory peptide, PI3-kinase inhibitors (LY294002 and wortmannin), an AKT inhibitor, or NF-κB inhibitor (pyrrolidine dithiocarbamate), IκBa phosphorylation inhibitor (Bay 117082), or IκB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone). Treatment of hDPSCs with LPS activated PI3-kinase (p85) and AKT signaling in a time-dependent manner. Moreover, LPS-mediated increases in κB-luciferase activity were diminished by the overexpression of dominant negative mutants of TLR4, MyD88, p85, AKT, and IκBa. These results demonstrated that LPS-induced Wnt5a expression was mediated through the TLR4/MyD88/PI3-kinase/AKT pathway, which then initiated NF-κB activation in hDPSCs.

He W ，Wang Z ，Zhou Z ，Zhang Y ，Zhu Q ，Wei K ，Lin Y ，Cooper PR ，Smith AJ ，Yu Q ... -  《-》