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 MicroRNA 132 Regulates Nutritional Stress-Induced Chemokine Production through Repression of SirT1

2009 ◽  
Vol 23 (11) ◽  
pp. 1876-1884 ◽  
Author(s):  
Jay C. Strum ◽  
Jennifer H. Johnson ◽  
James Ward ◽  
Hongbo Xie ◽  
John Feild ◽  
...  
Keyword(s):  
Human Adipose Tissue ◽  
Cell Types ◽  
Nuclear Factor Κb ◽  
Serum Deprivation ◽  
Nuclear Factor ◽  
Chemokine Production ◽  
Monocyte Chemoattractant Protein 1 ◽  
Regulatory Pathways ◽  
Proinflammatory Mediators

Abstract Human adipose tissue secretes a number of proinflammatory mediators that may contribute to the pathophysiology of obesity-related disorders. Understanding the regulatory pathways that control their production is paramount to developing effective therapeutics to treat these diseases. Using primary human adipose-derived stem cells as a source of preadipocytes and in vitro differentiated adipocytes, we found IL-8 and monocyte chemoattractant protein-1 (MCP-1) are constitutively secreted by both cell types and induced in response to serum deprivation. MicroRNA profiling revealed the rapid induction of microRNA 132 (miR-132) in these cells when switched to serum-free medium. Furthermore, miR-132 overexpression was sufficient to induce nuclear factor-κB translocation, acetylation of p65, and production of IL-8 and MCP-1. Inhibitors of miR-132 decreased acetylated p65 and partially inhibited the production of IL-8 and MCP-1 induced by serum deprivation. MiR-132 was shown to inhibit silent information regulator 1 (SirT1) expression through a miR-132 binding site in the 3′-untranslated region of SirT1. Thus, in response to nutritional availability, induction of miR-132 decreases SirT1-mediated deacetylation of p65 leading to activation of nuclear factor-κB and transcription of IL-8 and MCP-1 in primary human preadipocytes and in vitro differentiated adipocytes.

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 Interleukin-2 transcription is regulated in vivo at the level of coordinated binding of both constitutive and regulated factors.

1994 ◽  
Vol 14 (3) ◽  
pp. 2159-2169 ◽  
Author(s):  
P A Garrity ◽  
D Chen ◽  
E V Rothenberg ◽  
B J Wold
Keyword(s):  
T Cells ◽  
Dna Binding ◽  
Cyclosporin A ◽  
Interleukin 2 ◽  
Cell Types ◽  
Regulatory Elements ◽  
In Vitro Assays ◽  
Nuclear Extracts

Interleukin-2 (IL-2) transcription is developmentally restricted to T cells and physiologically dependent on specific stimuli such as antigen recognition. Prior studies have shown that this stringent two-tiered regulation is mediated through a transcriptional promoter/enhancer DNA segment which is composed of diverse recognition elements. Factors binding to some of these elements are present constitutively in many cell types, while others are signal dependent, T cell specific, or both. This raises several questions about the molecular mechanism by which IL-2 expression is regulated. Is the developmental commitment of T cells reflected molecularly by stable interaction between available factors and the IL-2 enhancer prior to signal-dependent induction? At which level, factor binding to DNA or factor activity once bound, are individual regulatory elements within the native enhancer regulated? By what mechanism is developmental and physiological specificity enforced, given the participation of many relatively nonspecific elements? To answer these questions, we have used in vivo footprinting to determine and compare patterns of protein-DNA interactions at the native IL-2 locus in cell environments, including EL4 T-lymphoma cells and 32D clone 5 premast cells, which express differing subsets of IL-2 DNA-binding factors. We also used the immunosuppressant cyclosporin A as a pharmacological agent to further dissect the roles played by cyclosporin A-sensitive factors in the assembly and maintenance of protein-DNA complexes. Occupancy of all site types was observed exclusively in T cells and then only upon excitation of signal transduction pathways. This was true even though partially overlapping subsets of IL-2-binding activities were shown to be present in 32D clone 5 premast cells. This observation was especially striking in 32D cells because, upon signal stimulation, they mobilized a substantial set of IL-2 DNA-binding activities, as measured by in vitro assays using nuclear extracts. We conclude that binding activities of all classes fail to stably occupy their cognate sites in IL-2, except following activation of T cells, and that specificity of IL-2 transcription is enforced at the level of chromosomal occupancy, which appears to be an all-or-nothing phenomenon.

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 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.

scholarly journals Nuclear factor κB (NF-κB) activation primes cells to a pro-inflammatory polarized response to a Toll-like receptor 7 (TLR7) agonist

2009 ◽  
Vol 421 (2) ◽  
pp. 301-310 ◽  
Author(s):  
Jongdae Lee ◽  
Masaaki Hayashi ◽  
Jeng-Fan Lo ◽  
Colleen Fearns ◽  
Wen-Ming Chu ◽  
...  
Keyword(s):  
Immune Cell ◽  
Interleukin 1 ◽  
Cell Types ◽  
Nuclear Factor Κb ◽  
Proximal Region ◽  
Binding Motif ◽  
Nuclear Factor ◽  
Viral Immunity ◽  
Toll Like Receptor ◽  
Tlr7 Agonist

TLR7 (Toll-like receptor 7) mediates anti-viral immunity by recognizing ssRNA (single-stranded RNA) viruses. Small-molecular-mass TLR7 agonists have been approved, or are being evaluated, for treatment of cancers or infectious diseases. Although TLR7 is predominantly expressed in a restricted set of immune cell types, including pDCs (plasmacytoid dendritic cells), it is also expressed in non-native expressing cells (e.g. hepatocytes) under certain circumstances. To elucidate the molecular basis of TLR7 induction by pro-inflammatory stimulation and the subsequent cellular responses in these non-native TLR7-expressing cell types, we first cloned and characterized the 5′-promoter region of TLR7. The proximal region of this promoter drives the transcription of the TLR7 gene. Pro-inflammatory stimuli activated TLR 7 transcription via a NF-κB (nuclear factor κB)-binding motif in this region, and this activation could be blocked by mutation of the NF-κB binding site or addition of NF-κB inhibitors. Further studies showed that pretreatment of the Hep3B hepatocytes with TNF-α (tumour necrosis factor-α) or IL-1 (interleukin-1) rendered them responsive to TLR7 activation by a TLR7 agonist. However, distinct from TLR7 activation in pDCs, which respond to stimulation with Th1 polarized cytokine production, TLR7 induction by pro-inflammatory signals in hepatocytes reconstitutes the NF-κB-dependent cascade but not the IRF7 (interferon regulatory factor 7)-dependent cascade, resulting in a pro-inflammatory polarized response rather than a Th1 polarized response. These results indicate that inflammatory stimulation is capable of priming cells to respond to TLR7 agonist with an immune response that differs from that in native TLR7-expressing cells.

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 Interleukin-2 transcription is regulated in vivo at the level of coordinated binding of both constitutive and regulated factors

1994 ◽  
Vol 14 (3) ◽  
pp. 2159-2169
Author(s):  
P A Garrity ◽  
D Chen ◽  
E V Rothenberg ◽  
B J Wold
Keyword(s):  
T Cells ◽  
Dna Binding ◽  
Cyclosporin A ◽  
Interleukin 2 ◽  
Cell Types ◽  
Regulatory Elements ◽  
In Vitro Assays ◽  
Nuclear Extracts

Interleukin-2 (IL-2) transcription is developmentally restricted to T cells and physiologically dependent on specific stimuli such as antigen recognition. Prior studies have shown that this stringent two-tiered regulation is mediated through a transcriptional promoter/enhancer DNA segment which is composed of diverse recognition elements. Factors binding to some of these elements are present constitutively in many cell types, while others are signal dependent, T cell specific, or both. This raises several questions about the molecular mechanism by which IL-2 expression is regulated. Is the developmental commitment of T cells reflected molecularly by stable interaction between available factors and the IL-2 enhancer prior to signal-dependent induction? At which level, factor binding to DNA or factor activity once bound, are individual regulatory elements within the native enhancer regulated? By what mechanism is developmental and physiological specificity enforced, given the participation of many relatively nonspecific elements? To answer these questions, we have used in vivo footprinting to determine and compare patterns of protein-DNA interactions at the native IL-2 locus in cell environments, including EL4 T-lymphoma cells and 32D clone 5 premast cells, which express differing subsets of IL-2 DNA-binding factors. We also used the immunosuppressant cyclosporin A as a pharmacological agent to further dissect the roles played by cyclosporin A-sensitive factors in the assembly and maintenance of protein-DNA complexes. Occupancy of all site types was observed exclusively in T cells and then only upon excitation of signal transduction pathways. This was true even though partially overlapping subsets of IL-2-binding activities were shown to be present in 32D clone 5 premast cells. This observation was especially striking in 32D cells because, upon signal stimulation, they mobilized a substantial set of IL-2 DNA-binding activities, as measured by in vitro assays using nuclear extracts. We conclude that binding activities of all classes fail to stably occupy their cognate sites in IL-2, except following activation of T cells, and that specificity of IL-2 transcription is enforced at the level of chromosomal occupancy, which appears to be an all-or-nothing phenomenon.

Novel Mechanism of Inhibition of Nuclear Factor-κB DNA-Binding Activity by Diterpenoids Isolated from Isodon rubescens

2005 ◽  
Vol 68 (2) ◽  
pp. 286-297 ◽  
Author(s):  
Chung-Hang Leung ◽  
Susan P. Grill ◽  
Wing Lam ◽  
Quan-Bin Han ◽  
Han-Dong Sun ◽  
...  
Keyword(s):  
Dna Binding ◽  
Nuclear Factor Κb ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Dna Binding Activity ◽  
Mechanism Of Inhibition
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