scholarly journals Interleukin 1-induced phosphorylation of MAD3, the major inhibitor of nuclear factor κB of HeLa cells. Interference in signalling by the proteinase inhibitors 2,4-dichloroisocoumarin and tosylphenylalanyl chloromethylketone

1995 ◽  
Vol 307 (1) ◽  
pp. 287-295 ◽  
Author(s):  
F Guesdon ◽  
T Ikebe ◽  
E Stylianou ◽  
J Warwick-Davies ◽  
S Haskill ◽  
...  
Keyword(s):  
Hela Cells ◽  
Proteinase Inhibitors ◽  
Interleukin 1 ◽  
Serine Proteinase ◽  
Small Heat Shock Protein ◽  
Nuclear Factor Κb ◽  
Exchange Chromatography ◽  
Nuclear Factor ◽  
Phosphatase 2A

The regulation of the inhibitor of nuclear factor kappa B (I kappa B) by interleukin 1 (IL1) was investigated in HeLa cells. Two forms of I kappa B were resolved by ion-exchange chromatography. The major form (75%) was identified as MAD3 by specific antisera. IL1 generated rapidly (6 min) an electrophoretically retarded form of MAD3 that was stable in acid and was converted into the unmodified form by phosphatase 2A. It thus corresponded to a phosphorylation of the protein on serine or threonine. IL1 also caused the disappearance of MAD3 from the cells, which was complete 15 min after stimulation and coincided with a 46% reduction of cellular I kappa B activity. Newly-synthesized MAD3 accumulated to pre-stimulation levels between 60 and 90 min after stimulation and this coincided with the down-regulation of the phosphorylating activity. The serine proteinase inhibitors 3,4-dichloroisocoumarin (DCI) and tosylphenylalanyl chloromethylketone (TPCK) prevented phosphorylation and disappearance of MAD3. At the same concentrations (10-100 microM), they also increased basal phosphorylation of the small heat shock protein (hsp27) and prevented the IL1- and phorbol 12-myristate 13-acetate-induced increases of its phosphorylation. The inhibitors were thus interfering with protein kinases when blocking degradation of MAD3. Recombinant MAD3 phosphorylated in vitro by protein kinase C was not electrophoretically retarded, suggesting that MAD3 was phosphorylated by another kinase in IL1-stimulated cells. Our results suggest that the IL1-induced phosphorylation of MAD3 on serine or threonine leads to its degradation. DCI and TPCK blocked phosphorylation mechanisms and it could not be concluded that serine proteinases were involved in the breakdown of MAD3.

scholarly journals The IKKβ Subunit of IκB Kinase (IKK) is Essential for Nuclear Factor κB Activation and Prevention of Apoptosis

1999 ◽  
Vol 189 (11) ◽  
pp. 1839-1845 ◽  
Author(s):  
Zhi-Wei Li ◽  
Wenming Chu ◽  
Yinling Hu ◽  
Mireille Delhase ◽  
Tom Deerinck ◽  
...  
Keyword(s):  
Interleukin 1 ◽  
Nuclear Factor Κb ◽  
Regulatory Subunit ◽  
Nuclear Factor ◽  
Catalytic Subunits ◽  
Iκb Kinase ◽  
Ikk Complex ◽  
Factor Α ◽  
Necrosis Factor

The IκB kinase (IKK) complex is composed of three subunits, IKKα, IKKβ, and IKKγ (NEMO). While IKKα and IKKβ are highly similar catalytic subunits, both capable of IκB phosphorylation in vitro, IKKγ is a regulatory subunit. Previous biochemical and genetic analyses have indicated that despite their similar structures and in vitro kinase activities, IKKα and IKKβ have distinct functions. Surprisingly, disruption of the Ikkα locus did not abolish activation of IKK by proinflammatory stimuli and resulted in only a small decrease in nuclear factor (NF)-κB activation. Now we describe the pathophysiological consequence of disruption of the Ikkβ locus. IKKβ-deficient mice die at mid-gestation from uncontrolled liver apoptosis, a phenotype that is remarkably similar to that of mice deficient in both the RelA (p65) and NF-κB1 (p50/p105) subunits of NF-κB. Accordingly, IKKβ-deficient cells are defective in activation of IKK and NF-κB in response to either tumor necrosis factor α or interleukin 1. Thus IKKβ, but not IKKα, plays the major role in IKK activation and induction of NF-κB activity. In the absence of IKKβ, IKKα is unresponsive to IKK activators.

scholarly journals Overexpression of an enzymically inactive interleukin-1-receptor-associated kinase activates nuclear factor-κB

1999 ◽  
Vol 339 (2) ◽  
pp. 227-231 ◽  
Author(s):  
Barbara MASCHERA ◽  
Keith RAY ◽  
Kimberly BURNS ◽  
Filippo VOLPE
Keyword(s):  
Kinase Activity ◽  
Interleukin 1 ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Wild Type ◽  
Defective Mutant ◽  
Asparagine Residue

Upon interleukin 1 (IL-1) stimulation, the IL-1-receptor (IL-1R)-associated kinase (IRAK) is rapidly recruited to the IL-1R complex and undergoes phosphorylation. Here we demonstrate that recombinant wild-type IRAK (IRAK-WT), but not a kinase-defective mutant with Asp340 replaced by an asparagine residue (IRAK-Asp340Asn), is highly phosphorylated and is capable of auto-phosphorylation in vitro. Overexpression of both IRAK-WT and IRAK-Asp340Asn caused activation of nuclear factor κB, suggesting that the kinase activity of IRAK is not required outside of the IL-1R complex.

scholarly journals 2-mercaptoethanol restores the ability of nuclear factor κB (NF κB) to bind DNA in nuclear extracts from interleukin 1-treated cells incubated with pyrollidine dithiocarbamate (PDTC). Evidence for oxidation of glutathione in the mechanism of inhibition of NFκB by PDTC

1996 ◽  
Vol 320 (3) ◽  
pp. 975-981 ◽  
Author(s):  
Paul BRENNAN ◽  
Luke A. J. O'NEILL
Keyword(s):  
Dna Binding ◽  
Interleukin 1 ◽  
Cell Types ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Oxygen Intermediates ◽  
Mechanism Of Inhibition ◽  
Nuclear Extracts ◽  
Anti Oxidant

The metal chelator and anti-oxidant pyrollidine dithiocarbamate (PDTC) has been used extensively in studies implicating reactive oxygen intermediates in the activation of nuclear factor κB (NFκB). In agreement with other studies, we have shown that PDTC inhibits NFκB activation in response to the pro-inflammatory cytokines interleukin 1 (IL1) and tumour necrosis factor (TNF). However, we have found that the inhibition was reversed by treatment of inhibited nuclear extracts with the reducing agent 2-mercaptoethanol. This was observed in extracts prepared from IL1-treated EL4.NOB-1 thymoma cells and TNF-treated Jurkat E6.1 lymphoma cells. These results suggested that the inhibition was caused by oxidation of NFκB on a sensitive thiol, possibly on the p50 subunit (which was detected in NFκB complexes in both cell types), and not by inhibition of the activation pathway. The possibility that PDTC was acting as a pro-oxidant was therefore investigated. PDTC caused an increase in oxidized glutathione, suggesting that it acts as an oxidizing agent in the cells tested rather than as an anti-oxidant. Similar results were obtained with diamide, a compound designed to oxidize glutathione. Finally, an increase in the ratio of oxidized to reduced glutathione was shown to inhibit NFκB–DNA binding in vitro. On the basis of these results we suggest that, while NFκB activation is unaffected by PDTC, DNA binding is inhibited through a mechanism involving a shift towards oxidizing conditions, and that this is the mechanism of action of both PDTC and diamide in the cells tested here.

scholarly journals The role of TBK1 and IKKϵ in the expression and activation of Pellino 1

2011 ◽  
Vol 434 (3) ◽  
pp. 537-548 ◽  
Author(s):  
Hilary Smith ◽  
Xin-Yu Liu ◽  
Liang Dai ◽  
Eddy T. H. Goh ◽  
Aye-Thu Chan ◽  
...  
Keyword(s):  
Protein Expression ◽  
Interleukin 1 ◽  
E3 Ligase ◽  
Nuclear Factor Κb ◽  
Poly I:C ◽  
Tumour Necrosis Factor Receptor ◽  
Nuclear Factor ◽  
Gene Encoding ◽  
Gene And Protein Expression

Mammalian Pellino isoforms are phosphorylated by IRAK (interleukin receptor associated kinase) 1/IRAK4 in vitro, converting them into active E3 ubiquitin ligases. In the present paper we report a striking enhancement in both transcription of the gene encoding Pellino 1 and Pellino 1 protein expression when murine BMDMs (bone-marrow-derived macrophages) are stimulated with LPS (lipopolysaccharide) or poly(I:C). This induction occurs via a TRIF [TIR (Toll/interleukin-1 receptor)-domain-containing adaptor-inducing interferon-β]-dependent IRAK-independent pathway and is prevented by inhibition of the IKK [IκB (inhibitor of nuclear factor κB) kinase]-related protein kinases, TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1} and IKKϵ. Pellino 1 is not induced in IRF3 (interferon regulatory factor 3)−/− BMDMs, and its induction is only reduced slightly in type 1 interferon receptor−/− BMDMs, identifying Pellino 1 as a new IRF3-dependent gene. We also identify Pellino 1 in a two-hybrid screen using IKKϵ as bait, and show that IKKϵ/TBK1 activate Pellino 1 in vitro by phosphorylating Ser76, Thr288 and Ser293. Moreover, we show that the E3 ligase activity of endogenous Pellino 1 is activated in LPS- or poly(I:C)-stimulated macrophages. This occurs more rapidly than the increase in Pellino 1 mRNA and protein expression, is prevented by the inhibition of IKKϵ/TBK1 and is reversed by phosphatase treatment. Thus IKKϵ/TBK1 mediate the activation of Pellino 1's E3 ligase activity, as well as inducing the transcription of its gene and protein expression in response to TLR3 and TLR4 agonists.

A mechanism for the suppression of interleukin-1-induced nuclear factor κB activation by protein phosphatase 2Cη-2

2009 ◽  
Vol 423 (1) ◽  
pp. 71-78 ◽  
Author(s):  
Takeaki Henmi ◽  
Kazutaka Amano ◽  
Yuko Nagaura ◽  
Kunihiro Matsumoto ◽  
Seishi Echigo ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Interleukin 1 ◽  
Ectopic Expression ◽  
Transforming Growth Factor Β ◽  
Nuclear Factor Κb ◽  
Signalling Pathway ◽  
Nuclear Factor ◽  
Interfering Rna ◽  
Signalling Cascade

IL-1 (interleukin-1) is a pro-inflammatory cytokine that has a variety of effects during the process of inflammation. Stimulating cells with IL-1 initiates a signalling cascade that includes the activation of NF-κB (nuclear factor κB), and subsequently induces a variety of inflammatory genes. Although the molecular mechanism for the IL-1-induced activation of NF-κB has been well documented, much less is known about the mechanism by which protein phosphatases down-regulate this pathway. Here we show that mouse PP2Cη-2 (protein serine/threonine phosphatase 2Cη-2), a novel member of the protein serine/threonine phosphatase 2C family, inhibits the IL-1–NF-κB signalling pathway. Ectopic expression of PP2Cη-2 in human embryonic kidney HEK293IL-1RI cells inhibited the IL-1-induced activation of NF-κB. TAK1 (transforming-growth-factor-β-activated kinase 1) mediates the IL-1 signalling pathway to NF-κB, and we observed that the TAK1-induced activation of NF-κB was suppressed by PP2Cη-2 expression. Expression of IKKβ [IκB (inhibitory κB) kinase β], which lies downstream of TAK1, activates NF-κB, and this activation was also readily reversed by PP2Cη-2 co-expression. Additionally, PP2Cη-2 knockdown with small interfering RNA further stimulated the IL-1-enhanced phosphorylation of IKKβ and destabilization of IκBα in HeLa cells. PP2Cη-2 knockdown also increased the IL-1-induced expression of IL-6 mRNA. Furthermore, IKKβ was readily dephosphorylated by PP2Cη-2 in vitro. These results suggest that PP2Cη-2 inhibits the IL-1–NF-κB signalling pathway by selectively dephosphorylating IKKβ.

scholarly journals Periprosthetic Osteolysis: Mechanisms, Prevention and Treatment

2019 ◽  
Vol 8 (12) ◽  
pp. 2091 ◽  
Author(s):  
Stuart B. Goodman ◽  
Jiri Gallo
Keyword(s):  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Activator Protein 1 ◽  
Periprosthetic Osteolysis ◽  
Bone Implant ◽  
Joint Replacements ◽  
In Vivo Experiments ◽  
Mechanical Factors

Clinical studies, as well as in vitro and in vivo experiments have demonstrated that byproducts from joint replacements induce an inflammatory reaction that can result in periprosthetic osteolysis (PPOL) and aseptic loosening (AL). Particle-stimulated macrophages and other cells release cytokines, chemokines, and other pro-inflammatory substances that perpetuate chronic inflammation, induce osteoclastic bone resorption and suppress bone formation. Differentiation, maturation, activation, and survival of osteoclasts at the bone–implant interface are under the control of the receptor activator of nuclear factor kappa-Β ligand (RANKL)-dependent pathways, and the transcription factors like nuclear factor κB (NF-κB) and activator protein-1 (AP-1). Mechanical factors such as prosthetic micromotion and oscillations in fluid pressures also contribute to PPOL. The treatment for progressive PPOL is only surgical. In order to mitigate ongoing loss of host bone, a number of non-operative approaches have been proposed. However, except for the use of bisphosphonates in selected cases, none are evidence based. To date, the most successful and effective approach to preventing PPOL is usage of wear-resistant bearing couples in combination with advanced implant designs, reducing the load of metallic and polymer particles. These innovations have significantly decreased the revision rate due to AL and PPOL in the last decade.

scholarly journals A novel splice variant of mouse interleukin-1-receptor-associated kinase-1 (IRAK-1) activates nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK)

2003 ◽  
Vol 370 (1) ◽  
pp. 159-166 ◽  
Author(s):  
Ken YANAGISAWA ◽  
Kenji TAGO ◽  
Morisada HAYAKAWA ◽  
Motomichi OHKI ◽  
Hiroyuki IWAHANA ◽  
...  
Keyword(s):  
Splice Variant ◽  
Interleukin 1 ◽  
Nuclear Factor Κb ◽  
Tumour Necrosis Factor Receptor ◽  
Acceptor Site ◽  
Nuclear Factor ◽  
Death Domain ◽  
Signalling Molecule ◽  
Novel Variant ◽  
Nf Kappab

Interleukin-1 (IL-1)-receptor-associated kinase (IRAK) is an indispensable signalling molecule for host-defence responses initiated by a variety of ligands that bind to members of the Toll/IL-1 receptor family. Here we report a novel splice variant of mouse IRAK-1, IRAK-1-S, which is generated by utilizing a new splicing acceptor site within exon 12. IRAK-1-S cDNA is shorter than the originally reported IRAK-1 (IRAK-1-W) cDNA by 271 nucleotides, and the subsequent frameshift causes a premature termination of translation after 23 amino acids, which are unique to the IRAK-1-S protein. To elucidate the physiological function of IRAK-1-S, we overexpressed it in 293T cells and studied the effects on the IL-1 signalling cascade. As it lacks the C-terminal region of IRAK-1-W that has been reported to contain the TRAF6 (tumour necrosis factor receptor-associated factor 6) binding domain, IRAK-1-S was unable to bind TRAF6 protein, which is a proposed downstream signalling molecule. However, IRAK-1-S overexpressed in 293T cells induced constitutive activation of nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) independent of stimulation by IL-1, as did IRAK-1-W. To clarify the mechanism of NF-κB activation by IRAK-1-S in the absence of binding to TRAF6, we demonstrated that IRAK-1-S binds to IRAK-1-W through its death domain; the findings suggested that overexpressed IRAK-1-S may bind endogenous IRAK-1-W and activate TRAF6 through IRAK-1-W. These results also indicate that this novel variant may play roles in the activation of NF-κB and JNK by IL-1 and other ligands whose signal transduction is dependent on IRAK-1 under physiological conditions.

scholarly journals The nuclear factor κB inhibitor (E)-2-fluoro-4′-methoxystilbene inhibits firefly luciferase

2012 ◽  
Vol 32 (6) ◽  
pp. 531-537 ◽  
Author(s):  
Albert Braeuning ◽  
Silvia Vetter
Keyword(s):  
Photon Emission ◽  
Gene Promoter ◽  
Firefly Luciferase ◽  
Nuclear Factor Κb ◽  
Signalling Pathway ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Reporter System ◽  
Reporter Assays

Photinus pyralis (firefly) luciferase is widely used as a reporter system to monitor alterations in gene promoter and/or signalling pathway activities in vitro. The enzyme catalyses the formation of oxyluciferin from D-luciferin in an ATP-consuming reaction involving photon emission. The purpose of the present study was to characterize the luciferase-inhibiting potential of (E)-2-fluoro-4′-methoxystilbene, which is known as a potent inhibitor of the NF-κB (nuclear factor κB) signalling pathway that is used to modulate the NF-κB signalling pathway in vitro. Results show that (E)-2-fluoro-4′-methoxystilbene effectively inhibits firefly luciferase activity in cell lysates and living cells in a non-competitive manner with respect to the luciferase substrates D-luciferin and ATP. By contrast, the compound has no effect on Renilla and Gaussia luciferases. The mechanism of firefly luciferase inhibition by (E)-2-fluoro-4′-methoxystilbene, as well as its potency is comparable to its structure analogue resveratrol. The in vitro use of trans-stilbenes such as (E)-2-fluoro-4′-methoxystilbene or resveratrol compromises firefly luciferase reporter assays as well as ATP/luciferase-based cell viability assays.

Is the Effect of Interleukin-1 on Glutathione Oxidation in Cultured Human Fibroblasts Involved in Nuclear Factor-κB Activation?

2001 ◽  
Vol 3 (2) ◽  
pp. 329-340 ◽  
Author(s):  
Patricia Renard ◽  
Edouard Delaive ◽  
Martine Van Steenbrugge ◽  
José Remacle ◽  
Martine Raes
Keyword(s):  
Interleukin 1 ◽  
Human Fibroblasts ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Cultured Human Fibroblasts ◽  
Glutathione Oxidation

In Vitro and in Vivo Nuclear Factor-κB Inhibitory Effects of the Cell-Penetrating Penetratin Peptide

2006 ◽  
Vol 69 (6) ◽  
pp. 2027-2036 ◽  
Author(s):  
Tamás Letoha ◽  
Erzsébet Kusz ◽  
Gábor Pápai ◽  
Annamária Szabolcs ◽  
József Kaszaki ◽  
...  
Keyword(s):  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Inhibitory Effects ◽  
Cell Penetrating
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