u-0126 and pyrrolidine-dithiocarbamic-acid

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.

Topics: Adolescent; Adult; Alkaline Phosphatase; Anthracenes; Benzylamines; Butadienes; Calcium Compounds; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Culture Techniques; Cell Differentiation; Dental Pulp; Extracellular Matrix Proteins; Humans; Imidazoles; Integrin-Binding Sialoprotein; MAP Kinase Signaling System; NF-kappa B; Nitriles; Odontoblasts; Osteocalcin; Phosphoproteins; Protein Kinase Inhibitors; Pulp Capping and Pulpectomy Agents; Pyridines; Pyrrolidines; Sialoglycoproteins; Signal Transduction; Silicates; Stem Cells; Sulfonamides; Thiocarbamates; Young Adult

The aim of the present study was to investigate the effects of lipopolysaccharide (LPS) on the migration and adhesion of human dental pulp stem cells (hDPSCs) and the associated intracellular signalling pathways.. hDPSCs obtained from impacted third molars were exposed to LPS and in vitro cell adhesion and migration were evaluated. The effects of LPS on gene expression of adhesion molecules and chemotactic factors were investigated using quantitative real-time reverse-transcriptase polymerase chain (qRT-PCR). The potential involvement of nuclear factor NF-kappa-B (NF-κB) or mitogen-activated protein kinase (MAPK) signalling pathways in the migration and adhesion of hDPSCs induced by LPS was assessed using a transwell cell migration assay and qRT-PCR.. LPS promoted the adhesion of hDPSCs at 1μg/mL and 10μg/mL concentrations, 1μg/mL LPS showing the greater effect. Transwell cell migration assay demonstrated that LPS increased migration of hDPSCs at 1μg/mL concentration while decreasing it significantly at 10μg/mL. The mRNA expressions of adhesion molecules and chemotactic factors were enhanced significantly after stimulation with 1μg/mL LPS. Specific inhibitors for NF-κB and extracellular signal regulated kinases (ERK), c-Jun N-terminal kinase (JNK), and P38, markedly antagonised LPS-induced adhesion and migration of hDPSCs and also significantly abrogated LPS-induced up-regulation of adhesion molecules and chemotactic factors. In addition, specific inhibitors of SDF-1/CXCR4, AMD3100 significantly diminished LPS-induced migration of hDPSCs.. LPS at specific concentrations can promote cell adhesion and migration in hDPSCs via the NF-κB and MAPK pathways by up-regulating the expression of adhesion molecules and chemotactic factors.. LPS may influence pulp healing through enhancing the adhesion and migration of human dental pulp stem cells when it enters into pulp during pulp exposure or deep caries.

Topics: Adolescent; Adult; Anthracenes; Benzylamines; Butadienes; Cell Adhesion; Cell Adhesion Molecules; Cell Movement; Cells, Cultured; Chemokine CXCL12; Chemotactic Factors; Cyclams; Dental Pulp; Escherichia coli; Extracellular Signal-Regulated MAP Kinases; Heterocyclic Compounds; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; MAP Kinase Signaling System; NF-kappa B; Nitriles; p38 Mitogen-Activated Protein Kinases; Pyridines; Pyrrolidines; Receptors, CXCR4; Signal Transduction; Stem Cells; Thiocarbamates; Young Adult

We investigated the unknown molecular mechanisms of Interleukin-1 (IL-1β)-induced cartilage aggrecan degeneration by aggrecanase (ADAMTS-A Disintegrin And Metalloproteinase with ThromboSpondin motifs) in human articular chondrocytes, a model mimicking human arthritis.. Chondrocytes were pretreated with various pharmacological inhibitors and then stimulated with IL-1β for 24 h. ADAMTS-4 expression or activity was studied by RT-PCR or ELISA and other proteins measured by Western blotting.. MAP kinase kinase-specific inhibitor, U0126 inhibited IL-1-induced phosphorylation of ERK1/2 and down-regulated ADAMTS-4 expression and activity. Protein 38 inhibitor, SB203580 down-regulated the phosphorylation of p38 and its target, activating transcription factor-2 (ATF-2), ADAMTS-4 mRNA and activity. C-Jun N-terminal kinase (JNK) inhibitor, SP600125 diminished IL-1-stimulated JNK phosphorylation, ADAMTS-4 mRNA expression and enzyme activity. A c-fos/lipoxygenase pathway inhibitor and antioxidant, nordihydroguaiaretic acid (NDGA) significantly suppressed ADAMTS-4 mRNA induction and activity. Activating protein (AP-1) and nuclear factor kappa B (NF-ĸB) transcription factor inhibitors, curcumin and pyrrolidine dithiocarbamate (PDTC) partially inhibited ADAMTS-4 induction and activity.. These results suggest partial involvement of ERK-, p38-and JNK-MAPKs as well as AP-1, ATF-2 and NF-ĸB transcription factors in IL-1-induced ADAMTS-4 in chondrocytes. Inhibition of these targets by the specific pharmacological agents could be useful for reducing aggrecanase-driven cartilage resorption in arthritis.

Topics: Anthracenes; Butadienes; Cartilage, Articular; Cells, Cultured; Chondrocytes; Curcumin; Endopeptidases; Gene Expression Regulation; Humans; Imidazoles; Interleukin-1beta; JNK Mitogen-Activated Protein Kinases; Lipoxygenases; Masoprocol; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; NF-kappa B; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Pyrrolidines; RNA, Messenger; Thiocarbamates; Transcription Factor AP-1

Black soybean (Glycine max L. merr.) is an edible Chinese medicine for nourishment spleen. In the present study, effects of characterized polysaccharides from black soybean (PGM) on granulocyte colony-stimulated factor (G-CSF) production in human peripheral blood mononuclear cells (PBMC) were determined and their action mechanisms were examined. The results indicated that PGM concentration-dependently enhanced G-CSF production in PBMC through modulation of mRNA expression. Data from Western blotting showed that PGM significantly induced the extracellular signal-regulated protein kinase (ERK) activation in PBMC. The nuclear factor (NF)-κB activation in PBMC was increased with PGM by modulation of IκB degradation and PKC θ activation. The levels of G-CSF mRNA in PGM-treated PBMC could be reduced by ERK inhibitor U0126 and NF-κB inhibitor pyrrolidine dithiocarbamate, respectively. Furthermore, the data showed that PGM stimulated phosphoinositide 3-kinase (PI3K)-regulated Akt phosphorylation. The PI3K inhibitor, Ly294002, blocked ERK, NF-κB, and PKC θ activation and G-CSF mRNA expression in PBMC induced by PGM. Thus, we first proved that the enhancement mechanisms of PGM on G-CSF production, appeared to be mediated, at least in part, through activation of PI3K, ERK, PKC θ, and NF-κB signaling pathways in PBMC. We suggest that PGM from black soybean is a potential G-CSF stimulator.

Topics: Adult; Blotting, Western; Butadienes; Chromones; Extracellular Signal-Regulated MAP Kinases; Gene Expression; Glycine max; Granulocyte Colony-Stimulating Factor; Humans; Leukocytes, Mononuclear; Male; Morpholines; NF-kappa B; NFATC Transcription Factors; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Polysaccharides; Protein Kinase C; Pyrrolidines; RNA, Messenger; Thiocarbamates

In the present study, we confirmed the ability of M. hyopneumoniae to induce the secretion of large amount of proinflammatory cytokine and nitric oxide (NO) in murine macrophage RAW 264.7 cells. Moreover, M. hyopneumoniae-induced activation of the MAPK and NF-кB pathways by phosphorylation of ERK1/2, p38 and JNK/SAPK and by dissociation of IκB from NF-κB. Translocation of transcription factor NF-κB and its binding was confirmed through western blot and electromobility shift assay. From these results, we further hypothesized that these signal proteins were involved in M. hyopneumoniae-induced proinflammatory cytokines and NO productions in macrophages. Hence, we utilized specific blockers of MAPK and NF-κB to investigate the signaling pathway involvement in cytokine and NO production through pharmacological approaches. The results demonstrated significant inhibition of TNF-α, IL-1β, IL-6 and NO by MAPK inhibitors. NF-κB inhibitor PDTC significantly inhibited IL-1β and NO production. These findings contribute to the understanding of the mechanisms of immune reactivity and may ultimately prove useful in the development of new therapeutic strategies. In summary, we found critical evidence for the involvement of NF-κB and MAPK signaling pathways in the upregulation of proinflammatory cytokine and NO induced by M. hyopneumoniae.

Topics: Animals; Blotting, Western; Butadienes; Cell Line; Cytokines; Electrophoretic Mobility Shift Assay; Enzyme Activation; Imidazoles; Macrophages; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinases; Mycoplasma hyopneumoniae; NF-kappa B; Nitric Oxide; Nitriles; Phosphorylation; Pneumonia of Swine, Mycoplasmal; Protein Kinase Inhibitors; Pyridines; Pyrrolidines; Swine; Thiocarbamates

Tumor necrosis factor-alpha (TNF-alpha) is a skeletal catabolic agent that stimulates osteoclastogenesis and inhibits osteoblast function. Although TNF-alpha inhibits the mineralization of osteoblasts, the effect of TNF-alpha on mesenchymal stem cells (MSC) is not clear. In this study, we determined the effect of TNF-alpha on osteogenic differentiation of stromal cells derived from human adipose tissue (hADSC) and the role of NF-kappaB activation on TNF-alpha activity. TNF-alpha treatment dose-dependently increased osteogenic differentiation over the first 3 days of treatment. TNF-alpha activated ERK and increased NF-kappaB promoter activity. PDTC, an NF-kappaB inhibitor, blocked the osteogenic differentiation induced by TNF-alpha and TLR-ligands, but U102, an ERK inhibitor, did not. Overexpression of miR-146a induced the inhibition of IRAK1 expression and inhibited basal and TNF-alpha- and TLR ligand-induced osteogenic differentiation. TNF-alpha and TLR ligands increased the expression of transcriptional coactivator with PDZ-binding motif (TAZ), which was inhibited by the addition of PDTC. A ChIP assay showed that p65 was bound to the TAZ promoter. TNF-alpha also increased osteogenic differentiation of human gastroepiploic artery smooth muscle cells. Our data indicate that TNF-alpha enhances osteogenic differentiation of hADSC via the activation of NF-kappaB and a subsequent increase of TAZ expression.

Topics: Acyltransferases; Adipose Tissue; Binding Sites; Butadienes; Calcinosis; Cell Differentiation; Cells, Cultured; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Interleukin-1 Receptor-Associated Kinases; Male; Mesenchymal Stem Cells; MicroRNAs; Middle Aged; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NF-kappa B; Nitriles; Osteogenesis; Promoter Regions, Genetic; Protein Kinase Inhibitors; Pyrrolidines; Recombinant Proteins; RNA Interference; Thiocarbamates; Time Factors; Toll-Like Receptors; Transcription Factor RelA; Transcription Factors; Transfection; Tumor Necrosis Factor-alpha; Up-Regulation

Angiotensin II (Ang II) has been recognized as an apoptosis inducer in podocytes, but the mechanism of apoptosis induced by Ang II is unclear. Transient receptor potential cation channel 6 (TRPC6) is a calcium channel located in podocyte membrane. The present study evaluated the alteration of TRPC6 expression and the Ca(2+) influx involved in Ang II-induced podocyte apoptosis. The possible pathways related to TRPC6 in Ang II-induced podocyte apoptosis were also investigated. The apoptosis of mouse podocytes (MPC5) was induced by Ang II. The protein level of TRPC6 was increased markedly in response to Ang II stimulation, and the intracellular Ca(2+) concentration was elevated. By transfection with TRPC6 siRNA, Ang II-induced podocyte apoptosis and the transient Ca(2+) influx were inhibited. Treated with extracellular signal-regulated kinase (ERK) pathway specific inhibitor U0126 or nuclear factor-kappaB (NF-kappaB) pathway specific inhibitor ammonium pyrrolidinedithiocarbamate (PDTC) and Ang II, respectively in podocytes, not only was the TRPC6 up-regulation reduced, but the podocyte apoptosis was also decreased. Moreover, the translocation of NF-kappaB in nucleus resulted from Ang II was reduced by treatment with U0126. In conclusion, the enhancement expression of TRPC6 as well as the increased Ca(2+) influx mediated by TRPC6 channels contributed to the podocyte apoptosis. The activation of ERK pathway and subsequent translocation of NF-kappaB was possibly necessary for the up-regulation TRPC6 induced by Ang II.

Topics: Angiotensin II; Animals; Apoptosis; Butadienes; Calcium; Cell Line; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Mice; NF-kappa B p50 Subunit; Nitriles; Podocytes; Pyrrolidines; Thiocarbamates; TRPC Cation Channels; TRPC6 Cation Channel; Up-Regulation

Asthma is an inflammatory disease, in which eotaxin, MCP-1 and MCP-3 play a crucial role. These chemokines have been shown to be expressed and produced by IL-1beta-stimulated human airway smooth muscle cells (HASMC) in culture. In the present study we were interested to unravel the IL-1beta-induced signal transduction leading to chemokine production. Using Western blot, we observed an activation of p38 MAPK, JNK kinase and p42/p44 ERK when HASMC were stimulated with IL-1beta. We also observed a significant decrease in the expression and the release of eotaxin, MCP-1 and MCP-3 in the presence of SB203580, an inhibitor of p38 MAPK (71 +/- 6%, P < 0.05, n = 8 and 39 +/- 10% P < 0.01, n = 10 respectively), curcumin, an inhibitor of JNK kinase (83 +/- 4.9% and 88 +/- 3.4% respectively, P < 0.01, n = 4). U0126, an inhibitor of p42/p44 ERK, also produced a significant decrease in chemokine production (46.3 +/- 9%, P < 0.01 n = 10 and 67.8 +/- 12%, P < 0.01, n = 12). Pyrrolydine dithiocarbamate, an inhibitor of NF-kappaB was also able to reduce the eotaxin, MCP-1 and MCP-3 expression and production (50 +/- 13%, P < 0.05, n = 10 and 23 +/- 7%, P < 0.05, n = 12). We conclude that p38 MAPK, JNK kinase, ERK and NF-kappaB are involved in the IL-1beta-induced eotaxin, MCP-1, and MCP-3 expression and release in HASMC.

Topics: Asthma; Blotting, Northern; Butadienes; Cells, Cultured; Chemokine CCL11; Chemokine CCL2; Chemokine CCL7; Chemokines; Chemokines, CC; Curcumin; Cytokines; Enzyme Activation; Enzyme Inhibitors; Humans; Imidazoles; Immunoblotting; Interleukin-1; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Monocyte Chemoattractant Proteins; Muscle, Smooth; NF-kappa B; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Pyrrolidines; Stimulation, Chemical; Thiocarbamates