Stimulation of monocytes/macrophages with lipopolysaccharide (LPS) results in activation of nuclear factor-kappaB (NF-kappaB), which plays crucial roles in regulating expression of many genes involved in the subsequent inflammatory responses. Here, we investigated roles of transforming growth factor-beta 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-kappaB 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.
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"Expression vectors for overexpression of FLAG-tagged versions of MyD88 were constructed with the pcDNA3 vector (Life Technologies , Madison, WI) as described previously . A reporter plasmid for the 165-nt mouse IkB-z 3 0 -UTR was cloned into the pGL4.12 "
[Show abstract][Hide abstract] ABSTRACT: Posttranscriptional regulatory elements in the 3'-untranslated regions (UTRs) of mRNAs play an important role in mRNA stabilization. Induction of IκB-ζ, a critical transcriptional regulator in the innate immune response, is mediated via specific mRNA stabilization by lipopolysaccharide (LPS) and interleukin (IL)-1β. It is known that the 3'-UTR of IκB-ζ, especially 165 nucleotides after the stop codon, plays a crucial role in mRNA stability. Herein, we show that AU-rich elements and miRNA targets in these 165 3'-UTR nucleotides are dispensable for stability of IκB-ζ mRNA. Additionally, NF-κB activation is important for IκB-ζ transcription, but dispensable for IκB-ζ mRNA stability. Interestingly, high-throughput screening results show that MyD88, a signal molecule responsive to LPS/IL-1β stimulation, is key for stabilizing IκB-ζ mRNA expression. Moreover, MyD88-deficient macrophages exhibited a decreased half-life of IκB-ζ mRNA expression. These results indicate that the LPS/IL-1β-MyD88 axis plays a crucial role for stabilization of IκB-ζ mRNA.
Full-text · Article · Dec 2015 · Biochemical and Biophysical Research Communications
"Drosophila TAK1 is essential for antibacterial innate immunity27. When TAK1 is kinase-negatively mutated, the LPS induced NF-κB activation is inhibited28. Thus, TAK1 is a critical mediator in the LPS-induced signaling pathway. "
[Show abstract][Hide abstract] ABSTRACT: Aim:
Recent studies have shown that constitutive activation of the nuclear factor κB (NF-κB) plays a key role in chronic inflammation and cancers. The aim of this study was to characterize lobolide, a cembrane diterpene, as a drug candidate targeting the NF-κB signaling pathway.
A HEK 293/NF-κB-Luc stable cell line was constructed to evaluate the effect of lobolide on NF-κB activation. THP-1 human monocytes and peripheral blood mononuclear cells (PBMCs) from healthy volunteers were tested. Lipopolysaccharide (LPS)-induced TNFα and IL-1β production and activation of the TAK1-IKK-NF-κB pathway were studied using ELISA and Western blot analysis.
In HEK 293/NF-κB-Luc stable cells, lobolide (0.19–50 μmol/L) inhibited NF-κB activation in a concentration-dependent manner with an IC50 value of 4.2±0.3 μmol/L. Treatment with lobolide (2.5–10 μmol/L) significantly suppressed LPS-induced production of TNFα and IL-1β in both THP-1 cells and PBMCs. In THP-1 cells, the suppression was partially caused by blockade of the translocation of NF-κB from the cytoplasm to the nucleus via affecting the TAK1-IKK-NF-κB pathway and p38 and ERK MAPK activity.
Lobolide is a potential inhibitor of the NF-κB pathway, which blocks the translocation of NF-κB from the cytoplasm to the nucleus. Lobolide inhibits LPS-stimulated TNFα and IL-1β release, suggesting that the compound might be an anti-inflammatory compound.
Full-text · Article · Aug 2012 · Acta Pharmacologica Sinica
"TAK1 regulates NF-κB-inducing kinase activity that activates IKKα/β downstream of MyD88 and TRAF6. TAK1 is also a MAP kinase kinase kinase for p38 that is critical for the production of pro-inflammatory cytokines (Irie et al., 2000). Stimulation of TLRs results in the downstream activation of the cytoplasmic Toll/IL-1 receptor (TIR) domain portion of the TLR, which then recruits MyD88/IRAK/TRAF6 and activates the MAPK superfamily cascade (Dalpke and Heeg, 2002; O'Neill, 2002; Akira, 2003) and the transcription factors, NF-κB and AP- 1 that leads to the expression of genes that participate in the innate immune response including pro-inflammatory cytokines. "
[Show abstract][Hide abstract] ABSTRACT: Previously conducted studies using two chicken lines (A and B) show that line A birds have increased resistance to a number of bacterial and protozoan challenges and that heterophils isolated from line A birds are functionally more responsive. Furthermore, when stimulated with toll-like receptor (TLR) agonists, heterophils from line A expressed a totally different cytokine and chemokine mRNA expression pattern than heterophils from line B. A large-scale gene expression profile using an Agilent 44K microarray on heterophils isolated from line A and line B also revealed significantly differential expression in many immune-related genes following Salmonella enteritidis (SE) stimulation, which included genes involved in the TLR pathway. Therefore, we hypothesize the differences between the lines result from distinctive TLR pathway signaling cascades that mediate heterophil function and, thus, innate immune responsiveness to SE. Using quantitative RT-PCR on mRNA from heterophils isolated from control and SE-stimulated heterophils of each line, we profiled the expression of all chicken homologous genes identified in a reference TLR pathway. Several differentially expressed genes found were involved in the TLR-induced My88-dependent pathway, showing higher gene expression in line A than line B heterophils following SE stimulation. These genes included the toll-like receptor genes TLR4, TLR15, TLR21, MD2, the adaptor proteins toll-interleukin 1 receptor domain containing adaptor protein (TIRAP), Tumor necrosis factor-receptor associated factor 3 (TRAF3), the IκB kinases TGF-β-activating kinase 1 (TAK1), IKKε and IKKα, the transcription factors NFkB2 and interferon regulatory factor 7 (IRF7), phosphoinositol-3 kinase (PI-3K), and the mitogen-activated protein kinase (MAPK) p38. These results indicate that higher expression of TLR signaling activation of both MyD88-dependent and TRIF-dependent pathways are more beneficial to avian heterophil-mediated innate immunity and
Full-text · Article · Jul 2012 · Frontiers in Genetics