u-0126 and sphingosine-1-phosphate

To investigate the cell status of dental pulp cells (DPCs) in a sphingosine-1-phosphate (S1P)-induced microinflammation environment and the possible mechanisms of cell homoeostasis maintenance by S1P.. Sphingosine-1-phosphate receptor (S1PR) expression was examined in DPCs within a local S1P-induced microinflammation model established using 1 μmol L(-1) S1P. U0126 [extracellular signal-regulated kinase (ERK) inhibitor], LY294002 (AKT inhibitor) and Y27632 (ROCK inhibitor) were used to inhibit corresponding signalling pathways of DPCs. CCK8 and cell cycle analysis tested cell proliferation. Immunofluorescence staining JC-1 detected changes of mitochondrial membrane potential (ΔΨm). Tests for apoptosis and the apoptosis-related proteins Bax and Bcl-2 were assessed by flow cytometry and western blot analysis, respectively. Expressions of ERK and AKT were evaluated by western blot analysis. The results were analysed using the Student's t-test and the significance level set at P < 0.05.. Expressions of S1PR1, S1PR2 and S1PR3 in DPCs differed amongst individuals. DPCs maintained self-homoeostasis in response to S1P-induced microinflammation via S1PRs. During this repair process, ERK, AKT and ROCK had a short-term complementary interaction at 60 min, but then AKT and ERK gradually played decisive roles after 24 h in proliferation enhancement and apoptosis inhibition, respectively (P > 0.05).. The AKT-ERK balance may determine whether DPC homoeostasis in S1P-induced microinflammation is maintained by synergistic regulation of cell growth and apoptosis.

Topics: Adolescent; Adult; Amides; Apoptosis; Blotting, Western; Butadienes; Cell Culture Techniques; Cell Proliferation; Chromones; Dental Pulp; Homeostasis; Humans; In Vitro Techniques; Lysophospholipids; Membrane Potential, Mitochondrial; Molar, Third; Morpholines; Nitriles; Pyridines; Signal Transduction; Sphingosine

Sphingosine 1-phosphate (S1P) has been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. S1P increases the expression of several proteins including COX-2 in vascular smooth muscle cells (VSMCs) and contributes to arteriosclerosis. However, the mechanisms regulating COX-2 expression by S1P in VSMCs remain unclear. Western blotting and RT-PCR analyses showed that S1P induced the expression of COX-2 mRNA and protein in a time- and concentration-dependent manner, which was attenuated by inhibitors of MEK1/2 (U0126) and PI3K (wortmannin), and transfection with dominant negative mutants of p42/p44 mitogen-activated protein kinases (ERK2) or Akt. These results suggested that both p42/p44 MAPK and PI3K/Akt pathways participated in COX-2 expression induced by S1P in VSMCs. In accordance with these findings, S1P stimulated phosphorylation of p42/p44 MAPK and Akt, which was attenuated by U0126, LY294002, or wortmannin, respectively. Furthermore, this up-regulation of COX-2 mRNA and protein was blocked by a selective NF-kappaB inhibitor helenalin. Consistently, S1P-stimulated translocation of NF-kappaB into the nucleus was revealed by immnofluorescence staining. Moreover, S1P-stimulated activation of NF-kappaB promoter activity was blocked by phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and helenalin, but not by U0126, suggesting that involvement of PI3K/Akt in the activation of NF-kappaB. COX-2 promoter assay showed that S1P induced COX-2 promoter activity mediated through p42/p44 MAPK, PI3K/Akt, and NF-kappaB. These results suggested that in VSMCs, activation of p42/p44 MAPK, Akt and NF-kappaB pathways was essential for S1P-induced COX-2 gene expression. Understanding the mechanisms involved in S1P-induced COX-2 expression on VSMCs may provide potential therapeutic targets in the treatment of arteriosclerosis.

Topics: Animals; Butadienes; Cells, Cultured; Cyclooxygenase 2; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Enzymologic; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NF-kappa B; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Promoter Regions, Genetic; Protein Kinase Inhibitors; Protein Transport; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction; Sphingosine; Transcription, Genetic

Exposure of renal mesangial cells to sphingosine 1-phosphate (S1P) leads to a rapid and transient activation of the mitogen- and stress-activated protein kinases but also the protein kinase B. Here, we show that S1P also induces phosphorylation of Smad proteins, which are members of the transforming growth factor-beta (TGF-beta) signaling device. However, Smad phosphorylation occurred more slowly with a maximal effect after 20-30 min of S1P stimulation when compared with the rapid activation of the MAPKs. Interestingly, Smad phosphorylation is increased by pertussis toxin, which is in contrast to the complete inhibition of S1P-induced MAPK phosphorylation by pertussis toxin. TGF-beta is a potent anti-inflammatory cytokine, which in mesangial cells attenuates the expression of (i) inducible nitricoxide synthase (iNOS) caused by interleukin (IL)-1beta, (ii) secreted phospholipase A(2) (sPLA(2)), and (iii) matrix metalloproteinase-9 (MMP-9). These gene products are also down-regulated by S1P in a concentration-dependent manner. Furthermore, the expression of connective tissue growth factor is enhanced by both TGF-beta(2) and S1P. These effects of S1P are not mediated by the MAPK cascade as neither pertussis toxin nor the MAPK cascade inhibitor U0126 are able to reverse this inhibition. Overexpression of the inhibitory Smad-7 or down-regulation of co-Smad-4 lead to a reversal of the blocking effect of S1P on IL-1beta-induced NO release. Moreover, down-regulating the TGF-beta receptor type II by the siRNA technique or antagonizing the S1P(3) receptor subtype with suramin abrogates S1P-stimulated Smad phosphorylation. In summary, our data show that S1P trans-activates the TGF-beta receptor and triggers activation of Smads followed by activation of connective tissue growth factor gene transcription and inhibition of IL-1beta-induced expression of iNOS, sPLA(2), and MMP-9.

Topics: Animals; Butadienes; Cells, Cultured; DNA-Binding Proteins; Enzyme Inhibitors; Glomerular Mesangium; Interleukin-1; Lysophospholipids; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Nitriles; Phosphorylation; Rats; Receptors, Transforming Growth Factor beta; Signal Transduction; Sphingosine; Transcriptional Activation; Transforming Growth Factor beta

Sphingosine 1-phosphate (S1P) is a lipid agonist that regulates smooth muscle cell (SMC) and endothelial cell functions by activating several members of the S1P subfamily of G-protein-coupled Edg receptors. We have shown previously that SMC differentiation is regulated by RhoA-dependent activation of serum response factor (SRF). Because S1P is a strong activator of RhoA, we hypothesized that S1P would stimulate SMC differentiation. Treatment of primary rat aortic SMC cells with S1P activated RhoA as measured by precipitation with a glutathione S-transferase-rhotekin fusion protein. In SMC and 10T1/2 cells, S1P treatment up-regulated the activities of several transiently transfected SMC-specific promoters, and these effects were inhibited by the Rho-kinase inhibitor, Y-27632. S1P also increased smooth muscle alpha-actin protein levels in SMC but had no effect on SRF binding to the smooth muscle alpha-actin CArG B element. Quantitative reverse transcriptase-PCR showed that S1P treatment of SMC or 10T1/2 cells did not increase the mRNA level of either of the recently identified SRF co-factors, myocardin or myocardin-related transcription factor-A (MRTF-A). MRTF-A protein was expressed highly in SMC and 10T1/2 cultures, and importantly the effects of S1P were inhibited by a dominant negative form of MRTF-A indicating that S1P may regulate the transcriptional activity of MRTF-A. Indeed, S1P treatment increased the nuclear localization of FLAG-MRTF-A, and the effect of MRTF-A overexpression on smooth muscle alpha-actin promoter activity was inhibited by dominant negative RhoA. S1P also stimulated SMC growth by activating the early growth response gene, c-fos. This effect was not attenuated by Y-27632 but could be inhibited by the MEK inhibitor, UO126. S1P enhanced SMC growth through ERK-mediated phosphorylation of the SRF co-factor, Elk-1, as measured by gel shift and Elk-1 activation assays. Taken together these results demonstrate that S1P activates multiple signaling pathways in SMC and regulates proliferation by ERK-dependent activation of Elk-1 and differentiation by RhoA-dependent activation of MRTF-A.

Topics: Amides; Animals; Aorta; Apoptosis Regulatory Proteins; Blotting, Western; Butadienes; Cell Differentiation; Cell Division; Cells, Cultured; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Endothelial Cells; Enzyme Inhibitors; ets-Domain Protein Elk-1; Fibroblasts; Genes, Dominant; Genes, Reporter; Glutathione Transferase; GTP-Binding Proteins; Intracellular Signaling Peptides and Proteins; Lysophospholipids; Mice; Mitogen-Activated Protein Kinases; Muscle, Smooth; Myocytes, Smooth Muscle; NIH 3T3 Cells; Nitriles; Nuclear Proteins; Phosphorylation; Polymerase Chain Reaction; Proto-Oncogene Proteins; Pyridines; Rats; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; rhoA GTP-Binding Protein; RNA, Messenger; Serum Response Factor; Signal Transduction; Sphingosine; Time Factors; Trans-Activators; Transcription Factors; Transfection; Up-Regulation