LPS promote the odontoblastic differentiation of human dental pulp stem cells via MAPK signaling pathway.

Human dental pulp stem cells (hDPSCs) show significant potential for exploitation in novel regeneration strategies, although lack of understanding of their responses to bacterial challenge constrains their application. The present study aimed to investigate whether lipopolysaccharide (LPS), the major pathogenic factor of Gram-negative bacteria, regulates the differentiation of hDPSCs and which intracellular signaling pathways may be involved. LPS treatment significantly promoted the differentiation of hDPSCs demonstrable by increased mineralized nodule formation and mRNA expression of several odontoblastic markers in a dose-dependent manner. While inhibition of TLR4, p38, and ERK signaling markedly antagonized LPS-mediated differentiation of hDPSCs. The inhibition of JNK and NF-κB signaling had no detectable effect on LPS activation of hDPSCs. LPS stimulation resulted in phosphorylation of NF-κB p65, IκB-α, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) in DPSCs in a time-dependent manner, which was markedly suppressed by their specific inhibitors, respectively. Data demonstrated that LPS promoted odontoblastic differentiation of hDPSCs via TLR4, ERK, and P38 MAPK signaling pathways, but not NF-κB signaling.

He W ，Wang Z ，Luo Z ，Yu Q ，Jiang Y ，Zhang Y ，Zhou Z ，Smith AJ ，Cooper PR ... -  《-》

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 ... -  《-》

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 ... -  《-》

Extracellular Signal-regulated Kinase Mitogen-activated Protein Kinase and Phosphatidylinositol 3-Kinase/Akt Signaling Are Required for Lipopolysaccharide-mediated Mineralization in Murine Odontoblast-like Cells.

Odontoblasts play an important role in post-developmental control of mineralization in response to external stimuli in the tooth. The present study investigated whether lipopolysaccharide (LPS), a major bacterial cell wall component, influenced mineralization in a murine odontoblast-like cell (OLC) line and the related intracellular signaling pathways involved. Alizarin red S staining was used to assess mineralized nodule formation in OLCs in response to LPS. The effects of LPS on gene expression of odontoblastic markers were investigated by using quantitative real-time reverse-transcriptase polymerase chain reaction. The potential involvement of toll-like receptor 4 (TLR4), nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), or phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways in the mineralized nodule formation, and mRNA expression of several odontoblastic markers of OLCs induced by LPS was assessed by using alizarin red S staining and quantitative real-time reverse-transcriptase polymerase chain reaction. Moreover, LPS stimulation resulted in phosphorylation of protein that was determined by Western blot analysis. OLCs showed reduced mineralized nodule formation and several odontoblastic markers expression in response to LPS exposure. Furthermore, inhibition of TLR4, extracellular signal-regulated kinase (ERK), and PI3K/Akt signaling noticeably antagonized LPS-mediated mineralization in OLCs. However, p38 MAPK, c-Jun N-terminal kinase, and NF-κB signaling inhibitors did not affect LPS-mediated mineralization in OLCs. Notably, LPS treatment resulted in a time-dependent phosphorylation of ERK and PI3K/Akt in OLCs, which was abrogated by their specific inhibitors. LPS decreased mineralization in OLCs via TLR4, ERK MAPK, and PI3K/Akt signaling pathways, but not p38, c-Jun N-terminal kinase, or NF-κB signaling.

Wang Z ，Ma F ，Wang J ，Zhou Z ，Liu B ，He X ，Fu L ，He W ，Cooper PR ... -  《-》