Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress the TNF-alpha gene expression by reducing phosphorylation of ERK1/2 and p38 MAPK in macrophages

We previously reported that excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress gene expression and production of tumour necrosis factor-alpha in murine macrophages stimulated with lipopolysaccharide. The present study investigated the suppressive mechanisms of tumour necrosis factor-alpha mRNA by excretory/secretory products in lipopolysaccharide-stimulated murine macrophages. Electrophoretic mobility shift assay and supershift assay revealed that neither nuclear translocation of nuclear factor-kappa B nor conformation of the p50/p65 nuclear factor-kappa B subunits was affected by the treatment of excretory/secretory products in lipopolysaccharide-stimulated macrophages. Inhibition of extracellular signal-regulated protein kinase 1/2 with PD98059 or p38 mitogen-activated protein kinase with SB203580 partially reduced tumour necrosis factor-alpha mRNA expression, and a combination of the two inhibitors additionally suppressed the level of tumour necrosis factor-alpha mRNA, revealing that both pathways are crucial for full induction of the gene. Northern blot analysis showed that excretory/secretory products additionally suppressed tumour necrosis factor-alpha mRNA expression in cells treated with PD98059 or SB208530 and, in turn, we found that excretory/secretory products reduced phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated macrophages by Western blot analysis. This is the first report demonstrating that excretory/secretory products from parasites suppress tumour necrosis factor-alpha mRNA expression by reducing phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase without any effect on nuclear factor-kappa B activity in macrophages stimulated with lipopolysaccharide. We hypothesise that excretory/secretory products may enable this parasite to survive within the host.     

Lipopolysaccharide stimulation of ERK1/2 increases TNF-alpha production via Egr-1

Lipopolysaccharide (LPS) is a potent activator of tumor necrosis factor-alpha (TNF-alpha) production by macrophages. LPS stimulates the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and increases TNF-alpha mRNA and protein accumulation in RAW 264.7 murine macrophages. However, the role of ERK1/2 activation in mediating LPS-stimulated TNF-alpha production is not well understood. Inhibition of ERK1/2 activation with PD-98059 or overexpression of dominant negative ERK1/2 decreased LPS-induced TNF-alpha mRNA quantity. LPS rapidly increased early growth response factor (Egr)-1 binding to the TNF-alpha promoter; this response was blunted in cells treated with PD-98059 or transfected with dominant-negative ERK1/2. Using a chloramphenicol acetyltransferase reporter gene linked to the Egr-1 promoter, we show that LPS increased Egr-1 promoter activity via an ERK1/2-dependent mechanism. These results delineate the role of ERK1/2 activation of Egr-1 activity in mediating LPS-induced increases in TNF-alpha mRNA expression in macrophages.     

Study of the insulin signaling pathways in the regulation of ACAT1 expression in cultured macrophages

Objective

To determine the signaling pathways and components involved in insulin-mediated regulation of Acyl-CoA: cholesterol acyltransferase1 (ACAT1).

Methods

THP-1 cells were cultured in RPMI 1640 medium and were induced into macrophages in the presence of 160 nM phorbol 12-myristate 13-acetate (PMA). Before insulin was added in, macrophages were preincubated with the inhibitors of the insulin signaling pathway, including wortmannin, phosphatidylinositol 3-kinase (PI3 K) inhibitor; PD98059, extracellular signal-regulated kinase (ERK) inhibitor; SB203580, p38 mitogen-activated protein kinase (p38MAPK) inhibitor; SP600125, c-Jun N-terminal kinase (JNK) inhibitor and U73122, phospholipase C-γ (PLC-γ) inhibitor. ACAT1 mRNA and protein expression level in macrophages were determined by real-time quantitative polymerase chain reaction and western blotting, respectively.

Results

Real-time quantitative polymerase chain reaction and western blotting demonstrated that PD98059, SB203580 or SP600125 down-regulated the expression of ACAT1 in a dose-dependent manner. However, no obvious alteration was found in wortmannin and U73122 groups.

Conclusion

These results suggest that the ERK, p38MAPK and JNK signaling pathways may be involved in insulin-mediated regulation of ACAT1, but no PI3K and PLC-γ signaling pathways were involved in the present study.     

Signalling to translational activation of tumour necrosis factor-alpha expression in human THP-1 cells

Monocytic THP-1 cells expressed tumour necrosis factor-alpha (TNF-alpha) mRNA, but hardly any detectable TNF-alpha protein and a partially activated MAP kinase ERK-2 in the unstimulated state. Stimulation with phorbol ester led to expression of TNF-alpha protein without significant changes in mRNA, a response that was sensitive to the MEK-1/2 inhibitors PD98059 and U0126. A calcium signal also led to expression of TNF-alpha protein, but now accompanied by a rapid increase in mRNA. A synergistic effect between phorbol ester and calcium ionophore was evident at the level of TNF-alpha protein, but not its mRNA. Stimulation with anisomycin led to a TNF-alpha expression that was sensitive to the p38 inhibitor SB203580. Actinomycin D lowered TNF-alpha mRNA in a similar way as PD98059 but was less inhibitory on PMA- or anisomycin-induced formation of TNF-alpha, thus confirming that these agents acted by causing translational derepression. Thus, in THP-1 cells MAP kinase pathways involving MEK-1/2 and possibly ERK-2 as well as the human p38 analogue were essential for basal TNF-alpha mRNA expression and translational activation.     

Mycobacteria-induced TNF-alpha and IL-10 formation by human macrophages is differentially regulated at the level of mitogen-activated protein kinase activity.

The clinical course of mycobacterial infections is linked to the capacity of pathogenic strains to modulate the initial antimycobacterial response of the macrophage. To elucidate some of the mechanisms involved, we studied early signal transduction events leading to cytokine formation by human monocyte-derived macrophages (MDM) in response to clinical isolates of Mycobacterium avium. TNF-alpha production induced by M. avium was inhibited by anti-CD14 mAbs, but not by Abs against the macrophage mannose receptor. Analysis of mitogen-activated protein (MAP) kinase activation (extracellular signal-regulated kinase 1/2, p38, and c-Jun NH(2)-terminal kinase) showed a rapid phosphorylation of all three subfamilies in response to M. avium, which was inhibited by anti-CD14 Abs. Using highly specific inhibitors of p38 (SB203580) and MAP kinase kinase-1 (PD98059), we found that activation of the extracellular signal-regulated kinase pathway, but not of p38, was essential for the M. avium-induced TNF-alpha formation. In contrast, IL-10 production was abrogated by the p38 inhibitor, but not by the MAP kinase kinase-1 inhibitor. In conclusion, M. avium-induced secretion of TNF-alpha and IL-10 by human macrophages is differentially regulated at the level of MAP kinase activity.     

Antagonistic effects of TNF-alpha on TGF-beta signaling through down-regulation of TGF-beta receptor type II in human dermal fibroblasts

Transforming growth factor-beta stimulates the production of the extracellular matrix, whereas TNF-alpha has antifibrotic activity. Understanding the molecular mechanism underlying the antagonistic activities of TNF-alpha against TGF-beta is critical in the context of tissue repair and maintenance of tissue homeostasis. In the present study, we demonstrated a novel mechanism by which TNF-alpha blocks TGF-beta-induced gene and signaling pathways in human dermal fibroblasts. We showed that TNF-alpha prevents TGF-beta-induced gene trans activation, such as alpha2(I) collagen or tissue inhibitor of metalloproteinases 1, and TGF-beta signaling pathways, such as Smad3, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases, without inducing levels of inhibitory Smad7 in human dermal fibroblasts. TNF-alpha down-regulates the expression of type II TGF-beta receptor (TbetaRII) proteins, but not type I TGF-beta receptor (TbetaRI), in human dermal fibroblasts. However, neither TbetaRII mRNA nor TbetaRII promoter activity was decreased by TNF-alpha. TNF-alpha-mediated decrease of TbetaRII protein expression was not inhibited by the treatment of fibroblasts with either a selective inhibitor of I-kappaB-alpha phosphorylation, BAY 11-7082, or a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, PD98059. Calpain inhibitor I (ALLN), a protease inhibitor, inhibits TNF-alpha-mediated down-regulation of TbetaRII. We found that TNF-alpha triggered down-regulation of TbetaRII, leading to desensitization of human dermal fibroblasts toward TGF-beta. Furthermore, these events seemed to cause a dramatic down-regulation of alpha2(I) collagen and tissue inhibitor of metalloproteinases 1 in systemic sclerosis fibroblasts. These results indicated that TNF-alpha impaired the response of the cells to TGF-beta by regulating the turnover of TbetaRII.     

Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress interleukin-1beta gene expression in murine macrophages

The present study shows that ES products from plerocercoids of Spirometra erinaceieuropaei suppressed interleukin-1beta mRNA expression in lipopolysaccharide-stimulated RAW 264.7 macrophages in the absence or presence of a cyclic AMP analogue, dibutyryl cyclic AMP. Investigation using the inhibitors of mitogen-activated protein kinase (MAPK) pathways revealed that extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase pathways are crucial for full induction of interleukin-1beta mRNA expression. ES products additionally suppressed interleukin-1beta mRNA expression in the cells treated with p38 mitogen-activated protein kinase inhibitor (SB203580) or extracellular signal-regulated protein kinase 1/2 inhibitor (PD98059). Western blot analysis showed that dibutyryl cyclic AMP enhanced lipopolysaccharide-induced phosphorylation of extracellular signal-regulated protein kinase 1/2, p38 mitogen-activated protein kinase and cyclic AMP responsive element binding protein (CREB) and, in turn, we demonstrated that ES products reduced the lipopolysaccharide and dibutyryl cyclic AMP-induced phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase, but not cyclic AMP responsive element binding protein. These data demonstrate that ES products from the plerocercoids of S. erinaceieuropaei may evade induction of interleukin-1beta mRNA by inhibiting extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase pathways in lipopolysaccharide and/or dibutyryl cyclic AMP-stimulated macrophages.     

Melatonin receptor activation regulates GnRH gene expression and secretion in GT1-7 GnRH neurons. Signal transduction mechanisms.

Melatonin plays a significant role in the control of the hypothalamic-pituitary-gonadal axis. Using the GT1-7 cell line, an in vitro model of GnRH-secreting neurons of the hypothalamus, we examined the potential signal transduction pathways activated by melatonin directly at the level of the GT1-7 neuron. We found that melatonin inhibits forskolin-stimulated adenosine 3'-, 5'-cyclic monophosphate accumulation in GT1-7 cells through an inhibitory G protein. Melatonin induced protein kinase C activity by 1.65-fold over basal levels, increased the phosphorylation of extracellular signal-regulated kinase 1 and 2 proteins, and activated c-fos and junB mRNA expression in GT1-7 cells. Using the protein kinase A inhibitor H-89, the protein kinase C inhibitor bisindolylmaleimide, and the mitogen-activated protein kinase kinase inhibitor PD98059, we found that the melatonin-mediated cyclical regulation of GnRH mRNA expression may involve the protein kinase C and the extracellular signal-regulated kinase 1 and 2 pathways, but not the protein kinase A pathway. We found that melatonin suppresses GnRH secretion by approximately 45% in the GT1-7 neurons. However, in the presence of the inhibitors H-89, bisindolylmaleimide, and PD98059 melatonin was unable to suppress GnRH secretion. These results provide insights into the potential signal transduction mechanisms involved in the control of GnRH gene expression and secretion by melatonin.     

Galectin-3 expression in macrophages is signaled by Ras/MAP kinase pathway and up-regulated by modified lipoproteins

To study the signaling pathway involved in the regulation of galectin-3 expression we used phorbol ester to stimulate macrophage differentiation of THP-1 cells. Treatment with phorbol 12-myristate 13-acetate (PMA) increased significantly the level of expression of galectin-3 in THP-1 cells. PMA-induced galectin-3 overexpression was blocked by: protein kinase C inhibitors staurosporine, calphostin C, and apigenin; tyrosine-specific protein kinase inhibitors genistein and tyrphostin A25; PD 98059, a selective inhibitor of mitogen-activated protein kinase (MAPK) kinase 1 (MEK1 or MKK1); and SB 203580, a specific inhibitor of p38 MAPK. Galectin-3 up-regulation was not affected by exposure to two inhibitors of cAMP-dependent protein kinase (PKA), H-89 and KT5720. Co-transfection of pPG3.5, a plasmid vector containing the rabbit galectin-3 promoter and the constructs pMCL-MKK1 N3 or pRC-RSV-MKK3Glu that constitutively express MKK1 and MKK3, raised the activity of galectin-3 promoter by 185% and 110%, respectively. Co-transfection with a Ha-Ras expression vector stimulated galectin-3 promoter activity approximately 10-fold. Expression of c-Jun or v-Jun raised the level of galectin-3 promoter activity more the three- and fourfold, respectively. Co-transfection of c-Jun and pPG3.5 5'-upstream deletion mutants resulted in a reduction of the galectin-3 promoter activity by 50% to 80%. Transfection of c-Jun, v-Jun or Ha-Ras increased significantly galectin-3 protein in THP-1 cells. These findings indicated that Ras/MEKK1/MKK1-dependent/AP-1 signal transduction pathway plays an important role in the expression of galectin-3 in PMA-stimulated macrophages. We further investigated the effect of modified lipoproteins on galectin-3 expression in macrophages. Murine resident peritoneal macrophages loaded with acetylated low-density lipoprotein (AcLDL) or oxidized LDL (OxLDL) showed increased galectin-3 protein and mRNA. These results showed that treatment of macrophages with PMA or modified lipoproteins results in galectin-3 overexpression. These findings may explain the enhanced expression of galectin-3 in atherosclerotic foam cells and suggest that Ras/MAPK signal transduction pathway is involved in controlling this gene.     

Hyperoxia induces Egr-1 expression through activation of extracellular signal-regulated kinase 1/2 pathway

Early growth response gene (Egr-1) is a stress response gene activated by various forms of stress and growth factor signaling. We report that supraphysiologic concentrations of O(2) (hyperoxia) induced Egr-1 mRNA and protein expression in cultured alveolar epithelial cells, as well as in mouse lung in vivo. The contribution of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK), p38 MAPK and PI3-kinase pathways to the activation of Egr-1 in response to hyperoxia was examined. Exposure to hyperoxia resulted in a rapid phosphorylation of ERK 1/2 kinases in mouse alveolar epithelial cells LA4. MEK inhibitor PD98059, but not inhibitors of p38 MAPK or PI3-kinase pathway, prevented Egr-1 induction by hyperoxia. The signaling cascade preceding Egr-1 activation was traced to epidermal growth factor receptor (EGFR) signaling. Hyperoxia is used as supplemental therapy in some diseases and typically results in elevated levels of reactive oxygen intermediates (ROI) in many lung cell types, the organ that receives highest O(2) exposure. Our results support a pathway for the hyperoxia response that involves EGF receptor, MEK/ERK pathway, and other unknown signaling components leading to Egr-1 induction. This forms a foundation for analysis of detailed mechanisms underlying Egr-1 activation during hyperoxia and understanding its consequences for regulating cell response to oxygen toxicity.     

Differential regulation of CD44 expression by lipopolysaccharide (LPS) and TNF-alpha in human monocytic cells: distinct involvement of c-Jun N-terminal kinase in LPS-induced CD44 expression

Alterations in the regulation of CD44 expression play a critical role in modulating cell adhesion, migration, and inflammation. LPS, a bacterial cell wall component, regulates CD44 expression and may modulate CD44-mediated biological effects in monocytic cells during inflammation and immune responses. In this study, we show that in normal human monocytes, LPS and LPS-induced cytokines IL-10 and TNF-alpha enhance CD44 expression. To delineate the mechanism underlying LPS-induced CD44 expression, we investigated the role of the mitogen-activated protein kinases (MAPKs), p38, p42/44 extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) by using their specific inhibitors. We demonstrate the involvement, at least in part, of p38 MAPK in TNF-alpha-induced CD44 expression in both monocytes and promonocytic THP-1 cells. However, neither p38 nor p42/44 MAPKs were involved in IL-10-induced CD44 expression in monocytes. To further dissect the TNF-alpha and LPS-induced signaling pathways regulating CD44 expression independent of IL-10-mediated effects, we used IL-10 refractory THP-1 cells as a model system. Herein, we show that CD44 expression induced by the LPS-mediated pathway predominantly involved JNK activation. This conclusion was based on results derived by transfection of THP-1 cells with a dominant-negative mutant of stress-activated protein/extracellular signal-regulated kinase kinase 1, and by exposure of cells to JNK inhibitors dexamethasone and SP600125. All these treatments prevented CD44 induction in LPS-stimulated, but not in TNF-alpha-stimulated, THP-1 cells. Furthermore, we show that CD44 induction may involve JNK-dependent early growth response gene activation in LPS-stimulated monocytic cells. Taken together, these results suggest a predominant role of JNK in LPS-induced CD44 expression in monocytic cells.     

Globular adiponectin decreases leptin-induced tumor necrosis factor-alpha expression by murine macrophages: involvement of cAMP-PKA and MAPK pathways

Several lines of evidence have supported a link between obesity and inflammation. The present study investigated the capacity of leptin and globular adiponectin to affect tumor necrosis factor alpha (TNF-alpha) production in murine peritoneal macrophages. Leptin stimulated TNF-alpha production at mRNA as well as protein levels in a dose- and time-dependent manner. Intracellular cAMP concentration was increased and protein kinase A (PKA) was activated with the treatment of leptin, subsequently downstream MAPK signal proteins, ERK1/2 and p38, were phosphorylated. Specific inhibitors for the signal proteins, Rp cAMPS, H89, PD98059, and U0126, or SB203580, suppressed the signaling pathway and TNF-alpha expression. Although gAd partially increased cAMP concentration and PKA activity, it directly reduced leptin-induced ERK1/2 and p38 MAPK phosphorylation thus inhibiting TNF-alpha production. In conclusion, leptin promotes inflammation by stimulating TNF-alpha production, which is mediated by cAMP-PKA-ERK1/2 and p38 MAPK pathways. gAd inhibited leptin-induced TNF-alpha production through suppressing phosphorylation of ERK1/2 and p38 pathways.     

Extracellular signal-regulated kinases 1 and 2 participate in interleukin-17 plus tumor necrosis factor-alpha-induced stabilization of interleukin-6 mRNA in human pancreatic myofibroblasts

In human pancreatic myofibroblasts, interleukin (IL)-17 markedly enhances tumor necrosis factor (TNF)-alpha-induced IL-6 secretion through the induction of IL-6 mRNA stabilization. Induced stability of IL-6 mRNA was markedly decreased by the inhibitors of extracellular signal-regulated kinase (ERKs), PD98059 and U0216. This indicates that activation of the ERK pathway is involved in the induction of IL-6 mRNA stabilization by IL-17 plus TNF-alpha.     

Inducible expression of the megakaryocyte-specific gene glycoprotein IX is mediated through an Ets binding site and involves upstream activation of extracellular signal-regulated kinase.

Glycoprotein IX is a megakaryocyte-specific gene crucial for adequate and functional expression of the Glycoprotein Ib-IX complex. This study used phorbol 12-myristate 13-acetate (PMA) and thrombopoietin (TPO)-induced differentiation of Dami and UT-7 cells, respectively, to investigate the regulation of inducible Glycoprotein IX expression during megakaryocyte differentiation. PMA and TPO were able to modulate GPIX expression at mRNA and protein levels. Transient transfection studies using nested 5'-deletions and mutations of the GPIX promoter demonstrated the absolute requirement of an inverted Ets site 5'-ACTTCCT-3' for inducible reporter gene expression. The upstream signaling events associated with PMA and TPO-inducible expression of GPIX were also investigated. The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase inhibitor PD98059 inhibited both PMA and TPO-inducible reporter activity in a dose-dependent manner, whereas inhibition of p38/MAPK had no significant effect. The protein kinase C inhibitor GF109203X failed to inhibit TPO-activation of the GPIX promoter in UT-7 cells. This study demonstrates that inducible expression in response to either PMA or TPO is mediated through the Ets site in the proximal promoter of GPIX and is dependent upon the upstream activation of MAPK/extracellular signal-regulated kinase.     

c-Jun N-terminal kinase negatively regulates lipopolysaccharide-induced IL-12 production in human macrophages: role of mitogen-activated protein kinase in glutathione redox regulation of IL-12 production

Although c-Jun N-terminal kinase (JNK) plays an important role in cytokine expression, its function in IL-12 production is obscure. The present study uses human macrophages to examine whether the JNK pathway is required for LPS-induced IL-12 production and defines how JNK is involved in the regulation of IL-12 production by glutathione redox, which is the balance between intracellular reduced (GSH) and oxidized glutathione (GSSG). We found that LPS induced IL-12 p40 protein and mRNA in a time- and concentration-dependent manner in PMA-treated THP-1 macrophages, and that LPS activated JNK and p38 mitogen-activated protein (MAP) kinase, but not extracellular signal-regulated kinase, in PMA-treated THP-1 cells. Inhibition of p38 MAP kinase activation using SB203580 dose dependently repressed LPS-induced IL-12 p40 production, as described. Conversely, inhibition of JNK activation using SP600125 dose dependently enhanced both LPS-induced IL-12 p40 production from THP-1 cells and p70 production from human monocytes. Furthermore, JNK antisense oligonucleotides attenuated cellular levels of JNK protein and LPS-induced JNK activation, but augmented IL-12 p40 protein production and mRNA expression. Finally, the increase in the ratio of GSH/GSSG induced by glutathione reduced form ethyl ester (GSH-OEt) dose dependently enhanced LPS-induced IL-12 p40 production in PMA-treated THP-1 cells. GSH-OEt augmented p38 MAP kinase activation, but suppressed the JNK activation induced by LPS. Our findings indicate that JNK negatively affects LPS-induced IL-12 production from human macrophages, and that glutathione redox regulates LPS-induced IL-12 production through the opposite control of JNK and p38 MAP kinase activation.