TAK1 mediates an activation signal from toll-like receptor(s) to nuclear factor-κB in lipopolysaccharide-stimulated macrophages

AbstractStimulation of monocytes/macrophages with lipopolysaccharide (LPS) results in activation of nuclear factor-κB (NF-κB), which plays crucial roles in regulating expression of many genes involved in the subsequent inflammatory responses. Here, we investigated roles of transforming growth factor-β activated kinase 1 (TGF-TAK1), a mitogen-activated protein kinase kinase kinase (MAPKKK), in the LPS-induced signaling cascade. A kinase-negative mutant of TAK1 inhibited the LPS-induced NF-κB activation both in a macrophage-like cell line, RAW 264.7, and in human embryonic kidney 293 cells expressing toll-like receptor 2 or 4. Furthermore, we demonstrated that endogenous TAK1 is phosphorylated upon simulation of RAW 264.7 cells with LPS. These results indicate that TAK1 functions as a critical mediator in the LPS-induced signaling pathway

Caffeic Acid Inhibited Receptor Activator of Nuclear Factor kappaB Ligand (RANKL)-Tumor Necrosis Factor (TNF) alpha-TNF Receptor Associated Factor (TRAF) 6 induced Osteoclastogenesis Pathway

BACKGROUND: Caffeic acid was reported in our previous study to have potential in inhibiting osteoclastogenesis through inhibition of nuclear factor κB (NFκB). Here in our current study, we would like to investigate further the caffeic acid-affected signaling pathway leading to NFκB inhibition. Since tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) plays important role in osteoclastogenesis, we applied TRAF6- transfected RAW264 cells D-Clone (RAW-D) cells as model in this study. METHODS: Caffeic acid in various concentrations was added to in vitro osteoclastogenesis of receptor activator nuclear factor κB ligand (RANKL)-TNFα-induced TRAF6-transfected RAW-D cells. Cells were collected, lysed and immunoblotted to detect TRAF6 expression. To detect tartrate resistant acid phosphatase (TRAP)+ polynucleated cells (PNCs), TRAP staining was performed. Meanwhile, to measure NFκB Activity, cells were transfected with pNFκB-TA-Luc and subjected to Dual Luciferase Reporter Assay System. RESULTS: Caffeic acid did not influence TRAF6 expression of RANKL-TNFα-induced TRAF6-transfected RAW-D cells. Caffeic acid diminished NFκB activity of RANKL-TNFα-induced TRAF6-transfected RAW-D cells in a concentration dependent manner. Significant NFκB activity inhibitions were seen under treatment of 1 and 10 μg/ml caffeic acid. By adding 10 μg/ml caffeic acid in RANKL-TNFα-induced TRAF6-transfected RAW-D cells, TRAP+ PNCs number was significantly suppressed. CONCLUSIONS: Caffeic acid inhibited RANKL-TNFα-TRAF6-induced osteoclastogenesis pathway. Since caffeic acid did not influence TRAF6 expression, TRAF6-RANK interactions and/or TRAF6 downstream signaling pathway should be further pursued to disclose inhibition mechanism of caffeic acid. KEYWORDS: caffeic acid, osteoclastogenesis, TRAF6, RANKL, TNFα, NFκB, RAW-D

Induction of cyclooxygenase-2 overexpression in human gastric epithelial cells by Helicobacter pylori involves TLR2/TLR9 and c-Srcdependent nuclear factor-

ABSTRACT Gastric epithelial cells were incubated with a panel of clinical isolates of Helicobacter pylori, including nonulcer dyspepsia with gastritis (HS, n ϭ 20), gastric ulcer (HU, n ϭ 20), duodenal ulcer (HD, n ϭ 21), and gastric cancer (HC, n ϭ 20). HC strains induced a higher cyclooxygenase-2 (COX-2) expression than those from HS, HD, and HU. The bacterial virulence factors and the host cellular pathways were investigated. Virulence genes of iceA, vacA, babA2, cagA 3Ј repeat region, and hrgA failed to show any association with the disease status and COX-2 expression. Methylation-specific polymerase chain reaction revealed HC strains not affecting the methylation status of COX-2 promoter. Nuclear factor (NF)-B, NF-interleukin 6, and cAMP response element were found to be involved in COX-2 induction. We explored a novel NF-B activation pathway. The mutants of TLR2 and TLR9, but not TLR4, inhibited H. pyloriinduced COX-2 promoter activity, and neutralizing antibodies for TLR2 and TLR9 abolished H. pylori-induced COX-2 expression

Thioredoxin-interacting protein gene expression via MondoA is rapidly and transiently suppressed during inflammatory responses.

Whereas accumulating evidence indicates that a number of inflammatory genes are induced by activation of nuclear factor-κB and other transcription factors, less is known about genes that are suppressed by proinflammatory stimuli. Here we show that expression of thioredoxin-interacting protein (Txnip) is dramatically suppressed both in mRNA and protein levels upon stimulation with lipopolysaccharide in mouse and human macrophages. In addition to lipopolysaccharide, a Toll-like receptor 4 ligand, stimulation with other Toll-like receptor ligands such as CpG DNA also suppressed Txnip expression. Not only the Toll-like receptor ligands, but also other proinflammatory stimulators, such as interleukin-1β and tumor necrosis factor-α elicited the similar response in fibroblasts. Suppression of Txnip by lipopolysaccharide is accompanied by a decrease of the glucose sensing transcription factor MondoA in the nuclei and dissociation of the MondoA:Mlx complex that bound to the carbohydrate-response elements in the Txnip promoter in unstimulated cells. Lipopolysaccharide-mediated decrease of nuclear MondoA was inhibited in the presence of 2-deoxyglucose. Furthermore, blockage of glyceraldehyde-3-phosphate dehydrogenase by iodoacetate alleviated the suppression of Txnip mRNA by lipopolysaccharide, suggesting the involvement of glucose-metabolites in the regulation. Since Txnip is implicated in the regulation of glucose metabolism, this observation links between inflammatory responses and metabolic regulation

Caffeic Acid Inhibited Receptor Activator of Nuclear Factor kappaB Ligand (RANKL)-Tumor Necrosis Factor (TNF) alpha-TNF Receptor Associated Factor (TRAF) 6 induced Osteoclastogenesis Pathway

BACKGROUND: Caffeic acid was reported in our previous study to have potential in inhibiting osteoclastogenesis through inhibition of nuclear factor κB (NFκB). Here in our current study, we would like to investigate further the caffeic acid-affected signaling pathway leading to NFκB inhibition. Since tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) plays important role in osteoclastogenesis, we applied TRAF6- transfected RAW264 cells D-Clone (RAW-D) cells as model in this study.METHODS: Caffeic acid in various concentrations was added to in vitro osteoclastogenesis of receptor activator nuclear factor κB ligand (RANKL)-TNFα-induced TRAF6-transfected RAW-D cells. Cells were collected, lysed and immunoblotted to detect TRAF6 expression. To detect tartrate resistant acid phosphatase (TRAP)+ polynucleated cells (PNCs), TRAP staining was performed. Meanwhile, to measure NFκB Activity, cells were transfected with pNFκB-TA-Luc and subjected to Dual Luciferase Reporter Assay System.RESULTS: Caffeic acid did not influence TRAF6 expression of RANKL-TNFα-induced TRAF6-transfected RAW-D cells. Caffeic acid diminished NFκB activity of RANKL-TNFα-induced TRAF6-transfected RAW-D cells in a concentration dependent manner. Significant NFκB activity inhibitions were seen under treatment of 1 and 10 μg/ml caffeic acid. By adding 10 μg/ml caffeic acid in RANKL-TNFα-induced TRAF6-transfected RAW-D cells, TRAP+ PNCs number was significantly suppressed.CONCLUSION: Caffeic acid inhibited RANKL-TNFα-TRAF6-induced osteoclastogenesis pathway. Since caffeic acid did not influence TRAF6 expression, TRAF6-RANK interactions and/or TRAF6 downstream signaling pathway should be further pursued to disclose inhibition mechanism of caffeic acid.KEYWORDS: caffeic acid, osteoclastogenesis, TRAF6, RANKL, TNFα, NFκB, RAW-