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 ... -  《-》

CpG oligonucleotides induce an immune response of odontoblasts through the TLR9, MyD88 and NF-kappaB pathways.

Odontoblasts are the first-line defense cells against invading microorganisms. Toll-like receptors (TLRs) play a crucial role in innate immunity, and TLR9 is involved in the recognition of microbial DNA. This study aimed to investigate whether odontoblasts can respond to CpG DNA and to determine the intracellular signaling pathways triggered by CpG DNA. We found that the mouse odontoblast-like cell line MDPC-23 constitutively expressed TLR9. Exposure to CpG ODN induced a potent proinflammatory response based on an increase of IL-6 and TNF-alpha expression. Pretreatment with an inhibitory MyD88 peptide or a specific inhibitor for TLR9, NF-kappaB or IkappaBalpha markedly inhibited CpG ODN-induced IL-6 and TNF-alpha expression. Moreover, the CpG ODN-mediated increase of kappaB-luciferase activity in MDPC-23 cells was suppressed by the overexpression of dominant negative mutants of TLR9, MyD88 and IkappaBalpha, but not by the dominant negative mutant of TLR4. This result suggests a possible role for the CpG DNA-mediated immune response in odontoblasts and indicates that TLR9, MyD88 and NF-kappaB are involved in this process.

He W ，Yu Q ，Zhou Z ，Wang P ... -  《-》

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 ... -  《-》

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 ... -  《-》

LPS induces IL-8 expression through TLR4, MyD88, NF-kappaB and MAPK pathways in human dental pulp stem cells.

To evaluate the effects of lipopolysaccharide (LPS) on interleukin-8 (IL-8) and related intracellular signalling pathways in human dental pulp stem cells (hDPSCs). Human pulp tissues were isolated from human impacted third molars, and the hDPSCs were cultured and characterized. The effects of LPS on IL-8 and Toll-like receptor 4 (TLR4) gene expression in hDPSCs were investigated using real-time quantitative RT-PCR and ELISA. Whether TLR4/MyD88/NF-кB was involved in the LPS-induced up-regulation of IL-8 in hDPSCs was determined using transient transfection, luciferase assay and ELISA. The involvement of MAPKs in the LPS-induced up-regulation of IL-8 in hDPSCs was investigated via transient transfection, luciferase assay, ELISA and western blot. The data were statistically analysed using Student's t-test or one-way anova followed by the Student-Neumann-Keuls test. Cells exposed to LPS not only displayed an enhanced expression of TLR4 but also showed an elevated IL-8 gene expression; exposure to LPS also resulted in the induction of IL-8 gene transcription via promoter activation. The LPS-induced IL-8 promoter activation was inhibited through dominant-negative mutations in TLR4 and MyD88, but not in TLR2. The LPS-induced IL-8 protein release was attenuated through the administration of TLR4-neutralizing antibody or MyD88 inhibitory peptide and a dominant-negative mutation in IκBα. In contrast, IL-8 protein release was enhanced through the expression of NF-κB p65. Treatment with PDTC, TPCK or Bay117082 effectively antagonized LPS-induced IL-8 protein release. Moreover, both the promoter activity and the LPS-induced release of IL-8 were diminished upon the administration of U0126 and SB203580, but not SP600125. Moreover, the exposure to LPS activated ERK1/2 and p38 MAPK phosphorylation in cells. This study reports the LPS-mediated transcriptional and post-translational up-regulation of IL-8, which is a process that also involves TLR4, MyD88, NF-κB and MAPK.

He W ，Qu T ，Yu Q ，Wang Z ，Lv H ，Zhang J ，Zhao X ，Wang P ... -  《-》