Oxidative modulation of NF-kappaB signaling by oxidized low-density lipoprotein

Oxidized low-density lipoprotein (oxLDL) modifies macrophage inflammatory responses in the pathogenesis of atherosclerosis. In the present study, we focused on gamma-glutamylcysteine synthetase (gamma-GCS), a rate limiting enzyme of glutathione synthesis, and examined whether inflammatory stimulation of gamma-GCS gene in macrophages by lipopolysaccharide (LPS) is modified when the cells were exposed to oxLDL. We found that the nuclear factor-kappaB (NF-kappaB)-mediated induction of gamma-GCS by LPS (100 ng/ml) was suppressed by a 48-h pre-treatment with oxLDL (50 micro/ml), and this was due to a decrease in the DNA-binding activity of NF-kappaB. Furthermore, pre-treatment with oxLDL caused a carbonylation of NF-kappaB subunit p65. With alpha-tocopherol, the oxLDL-induced carbonylation of proteins decreased with a restoration of DNA-binding activity of NF-kappaB. Together, these indicate that oxidative modification of NF-kappaB suppresses LPS-induced expression of gamma-GCS gene in ox-LDL-treated cells, suggesting an implication of oxLDL-induced modulation of NF-kappaB signaling with atherosclerosis.     

The coiled-coil domain of TRAF6 is essential for its auto-ubiquitination

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a crucial signaling transducer that regulates a diverse array of physiological processes, including adaptive immunity, innate immunity, and bone metabolism. Importantly, it is essential for activating NF-kappaB signaling pathway in response to interleukin-1 and Toll-like receptor ligands. Previously, we characterized TRAF6 to be a ubiquitin ligase. In combination with the ubiquitin conjugating enzyme complex Ubc13/Uev1A, TRAF6 could catalyze the formation on itself of unique Lys-63 linked polyubiquitin chain that positively regulated NF-kappaB signaling pathway. However, it remains unknown how this auto-ubiquitination process is regulated. In this study, we found that the coiled-coil domain of TRAF6 was essential for its auto-ubiquitination and activating NF-kappaB signaling pathway. This domain served not as the specific target where the polyubiquitin chain was linked, but as a specific bridge to recruit Ubc13/Uev1A.     

Blunted activation of NF-kappaB and NF-kappaB-dependent gene expression by geranylgeranylacetone: involvement of unfolded protein response

Geranylgeranylacetone (GGA), an anti-ulcer agent, has anti-inflammatory potential against experimental colitis and ischemia-induced renal inflammation. However, molecular mechanisms involved in its anti-inflammatory effects are largely unknown. We found that, in glomerular mesangial cells, GGA blocked activation of nuclear factor-κB and consequent induction of monocyte chemoattractant protein 1 (MCP-1) by inflammatory cytokines. It was inversely correlated with induction of unfolded protein response (UPR) evidenced by expression of 78 kDa glucose-regulated protein (GRP78) and suppression of endoplasmic reticulum stress-responsive alkaline phosphatase. Various inducers of UPR including tunicamycin, thapsigargin, A23187, 2-deoxyglucose, dithiothreitol, and AB₅ subtilase cytotoxin reproduced the suppressive effects of GGA. Furthermore, attenuation of UPR by stable transfection with GRP78 diminished the anti-inflammatory effects of GGA. These results disclosed a novel, UPR-dependent mechanism underlying the anti-inflammatory potential of GGA.     

New ELISA-based method for the detection of O-GlcNAc transferase activity in vitro

O-GlcNAcylation is a dynamic, reversible, post-translational modification that regulates many cellular processes. O-GlcNAc transferase (OGT) is the sole enzyme transferring N-acetylglucosamine from uridine diphosphate (UDP)-GlcNAc to selected serine/threonine residues of cytoplasm and nucleus proteins. Aberrant of OGT activity is associated with several diseases, suggesting OGT as a novel therapeutic target. In this study, we created a new enzyme linked immunosorbent assays (ELISA)-based method for detection of OGT activity. First, casein kinase II (CKII), a well-known OGT substrate, was coated onto ELISA plate. Second, the GlcNAc transferred by OGT from UDP-GlcNAc to CKII was detected using an antibody to O-GlcNAc and then the horseradish peroxidase (HRP)-labeled secondary antibody. At last, 3,3′,5,5′-tetramethylbenzidine (TMB), the substrate of HRP, was used to detect the O-GlcNAcylation level of CKII which reflected the activity of OGT. Based on a series of optimization experiments, the RL2 antibody was selected for O-GlcNAc detection and the concentrations of CKII, OGT, and UDP-GlcNAc were determined in this study. ST045849, a commercial OGT inhibitor, was used to verify the functionality of the system. Altogether, this study showed a method that could be applied to detect OGT activity and screen OGT inhibitors.     

NF-κ B激活在兔急性心肌梗死再灌注后无复流中的意义

目的：观察心肌核因子-κB（NF-κB）在急性心肌梗死（AMI）再灌注后无复流的活化情况,探讨NF-κB促进无复流发生发展的作用机制。方法：24只新西兰大白兔随机分为假手术组（冠状动脉只穿线不结扎）和缺血再灌注组（结扎冠状动脉2小时,再灌注1小时）,每组12只。采用凝胶阻滞迁移分析方法（EMSA）检测正常区、缺血区和无复流区心肌组织中NF-κB活性;ELISA法测定不同时点血浆中白细胞介素-6（IL-6）、超敏C反应蛋白（CRP）以及肿瘤坏死因子-α（TNF-α）的含量;光镜、电镜观察心肌组织病理变化。结果：（1）与正常区相比,缺血区和无复流区心肌组织中NF-κB活性异常升高（P〈0.01）。（2）与结扎前相比结扎后2h、再灌注后1h血浆IL-6、CRP、TNF-α水平呈进行性升高（P均〈0.05）。（3）NF-κB的活性与无复流面积、血浆IL-6、CRP以及TNF-α水平呈正相关（分别为r=0.844,P〈0.01;r=0.682,P〈0.05;r=0.687,P〈0.05;r=0.893,P〈0.01）。（4）无复流面积与血浆IL-6、CRP以及TNF-α水平呈正相关（分别为r=0.861,P〈0.01;r=0.806,P〈0.01;r=0.877,P〈0.01）。（5）光镜及电镜结果显示无复流区的心肌组织损伤较缺血区更为严重。结论：急性心肌梗死再灌注后无复流现象的发生可能与局部心肌组织中NF-κB的过度活化有关,活化的NF-κB通过促进IL-6、TNF-α等炎症因子的表达,参与无复流的发生发展过程。     

Fhit protein inhibits cell growth by attenuating the signaling mediated by nuclear factor-kappaB in colon cancer cell lines

Fragile histidine triad (FHIT) gene is involved in the deletions at the 3p14.2 region in various cancers. We investigated the role of Fhit protein in cell growth by examining the signaling pathway affected by Fhit. We used 3 human colon cancer cell lines, SW480, DLD-1 and COLO201, in the study. SW480 cells, in which the expression of Fhit is completely absent, were transfected with pIRES1neo vector (SW/IRES cells), wild-type FHIT vector (SW/FHIT cells) or mt-FHIT (codon 96, His changed to Asn) vector (SW/mt-FHIT cells). The growth of SW/FHIT or SW/mt-FHIT cells was suppressed in comparison with that of parent or SW/IRES cells. Especially, the growth of SW/FHIT cells was considerably suppressed. On the other hand, the silencing of FHIT by an siRNA for it in SW/FHIT or DLD-1 cells harboring Fhit demonstrated that the growth of FHIT siRNA-treated cells was significantly enhanced in comparison with that of the vector control or nonspecific siRNA control. Thus, we found that Fhit negatively contributed to cell growth in the colon cancer cell lines. Moreover, SW/FHIT cells exhibited a higher sensitivity to oxidative stress evoked by inhibitors of mitochondrial electron transport or proteasomes compared with any of the control transfectants. The base line amount of phospho-IkappaB-alpha (p-IkappaB-alpha) was reduced in SW/FHIT cells compared with that in the other transfectants. On the contrary, the FHIT siRNA-treated SW/FHIT and DLD-1 cells exhibited an elevated p-IkappaB-alpha level in an RNAi experiment on FHIT. Perturbation of nuclear factor (NF)-kappaB signaling was strongly suggested by the fact that the wild-type Fhit expressants of SW480 cells tended to be sensitive to sulfasarazine or parthenolide, which are inhibitors of NF-kappaB. The time course of the level of IkappaB kinase (IKK) complex (IKKalpha/beta, phospho-IKKalpha/beta and IKKgamma) after the treatment with TNF-alpha was similar between the transfectants. Although p-IkappaB-alpha and phospho-NF-kappaB p65 (p-NF-kappaB) in SW/FHIT cells responded to TNF-alpha as those in other transfectants, the increase in the levels of p-IkappaB-alpha and p-NF-kappaB after a 5-min treatment was less in SW/FHIT cells than in the other transfectants. These results altogether suggest that Fhit functions as an anti-oncoprotein by inhibiting the phosphorylation of IkappaB-alpha and thereby blocking NF-kappaB signaling.     

Molecular basis for the unique deubiquitinating activity of the NF-kappaB inhibitor A20

Nuclear factor κB (NF-κB) activation in tumor necrosis factor, interleukin-1, and Toll-like receptor pathways requires Lys63-linked nondegradative polyubiquitination. A20 is a specific feedback inhibitor of NF-κB activation in these pathways that possesses dual ubiquitin-editing functions. While the N-terminal domain of A20 is a deubiquitinating enzyme (DUB) for Lys63-linked polyubiquitinated signaling mediators such as TRAF6 and RIP, its C-terminal domain is a ubiquitin ligase (E3) for Lys48-linked degradative polyubiquitination of the same substrates. To elucidate the molecular basis for the DUB activity of A20, we determined its crystal structure and performed a series of biochemical and cell biological studies. The structure reveals the potential catalytic mechanism of A20, which may be significantly different from papain-like cysteine proteases. Ubiquitin can be docked onto a conserved A20 surface; this interaction exhibits charge complementarity and no steric clash. Surprisingly, A20 does not have specificity for Lys63-linked polyubiquitin chains. Instead, it effectively removes Lys63-linked polyubiquitin chains from TRAF6 without dissembling the chains themselves. Our studies suggest that A20 does not act as a general DUB but has the specificity for particular polyubiquitinated substrates to assure its fidelity in regulating NF-κB activation in the tumor necrosis factor, interleukin-1, and Toll-like receptor pathways.     

A self-assembled quantum dot probe for detecting beta-lactamase activity

This communication describes a quantum dot probe that can be activated by a reporter enzyme, beta-lactamase. Our design is based on the principle of fluorescence resonance energy transfer (FRET). A biotinylated beta-lactamase substrate was labeled with a carbocyanine dye, Cy5, and immobilized on the surface of quantum dots through the binding of biotin to streptavidin pre-coated on the quantum dots. In assembling this nanoprobe, we have found that both the distance between substrates and the quantum dot surface, and the density of substrates are important for its function. The fluorescence emission from quantum dots can be efficiently quenched (up to 95%) by Cy5 due to FRET. Our final quantum dot probe, assembled with QD605 and 1:1 mixture of biotin and a Cy5-labeled lactam, can be activated by 32microg/mL of beta-lactamase with 4-fold increase in the fluorescence emission.     

Expression of APOBEC2 is transcriptionally regulated by NF-kappaB in human hepatocytes

Apolipoprotein B mRNA-editing enzyme catalytic subunit 2 (APOBEC2) is a member of the nucleic-acid-editing enzymes. However, the physiological function of APOBEC2 remains unclear. We demonstrate that APOBEC2 expression is strongly enhanced in response to both tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta. Inhibition of NF-kappaB activation invariably blocks TNF-alpha-induced APOBEC2 expression. The promoter region of APOBEC2 contains functional NF-kappaB response elements in the 5' untranslated region of the gene at -625/-616. These results show that APOBEC2 expression is regulated by pro-inflammatory cytokines via NF-kappaB activation and suggest a possible role of APOBEC2 in the pathophysiology of hepatic inflammation.     

N-acetylcysteine attenuates TNF-alpha induced changes in secretion of interleukin-6, plasminogen activator inhibitor-1 and adiponectin from 3T3-L1 adipocytes

TNF-alpha is a key molecule in obesity-related metabolic disturbances. This study was designed to determine whether N-acetylcysteine (NAC), an antioxidant, prevents the activation of nuclear factor-kappaB (NF-kappaB) by exogenously administered TNF-alpha in adipocytes, and whether such change affects the production of adipocytokines. The treatment of well-differentiated 3T3-L1 cells with 20 mM of NAC significantly increased the reduced glutathione concentration up to 150% of control. The treatment with 10 ng/ml of TNF-alpha decreased antioxidant enzyme levels such as CuZn-superoxide dismutase (SOD), MnSOD and catalase, and activated NF-kappaB in 3T3-L1 adipocytes. The activation of NF-kappaB was significantly prevented by the pretreatment with 20 mM of NAC. TNF-alpha (1-10 ng/ml) dose-dependently increased interleukin (IL)-6 and plasminogen activator inhibitor-1 (PAI-1) secretion from 3T3-L1 adipocytes, while decreased adiponectin secretion. NAC (5-20 mM) attenuated the TNF-alpha-induced changes in these adipocytokine secretions in a dose-dependent manner. The effect of TNF-alpha and NAC on the adipocytokine productions was exerted at the m-RNA level, judging from results of the real time RT-PCR analysis. The present study revealed that NAC inhibited the TNF-alpha-mediated activation of NF-kappaB and improved the adverse changes in the levels of IL-6, PAI-1 and adiponectin in 3T3-L1 adipocytes. NAC may have the potential to improve the obesity-related abnormal adipocytokine metabolism by attenuating the TNF-alpha-induced oxidant-antioxidant imbalance in adipocytes.     

TAK1 is critical for IkappaB kinase-mediated activation of the NF-kappaB pathway

Cytokine treatment stimulates the IkappaB kinases, IKKalpha and IKKbeta, which phosphorylate the IkappaB proteins, leading to their degradation and activation of NF-kappaB regulated genes. A clear definition of the specific roles of IKKalpha and IKKbeta in activating the NF-kappaB pathway and the upstream kinases that regulate IKK activity remain to be elucidated. Here, we utilized small interfering RNAs (siRNAs) directed against IKKalpha, IKKbeta and the upstream regulatory kinase TAK1 in order to better define their roles in cytokine-induced activation of the NF-kappaB pathway. In contrast to previous results with mouse embryo fibroblasts lacking either IKKalpha or IKKbeta, which indicated that only IKKbeta is involved in cytokine-induced NF-kappaB activation, we found that both IKKalpha and IKKbeta were important in activating the NF-kappaB pathway. Furthermore, we found that the MAP3K TAK1, which has been implicated in IL-1-induced activation of the NF-kappaB pathway, was also critical for TNFalpha-induced activation of the NF-kappaB pathway. TNFalpha activation of the NF-kappaB pathway is associated with the inducible binding of TAK1 to TRAF2 and both IKKalpha and IKKbeta. This analysis further defines the distinct in vivo roles of IKKalpha, IKKbeta and TAK1 in cytokine-induced activation of the NF-kappaB pathway.     

Evaluation of an rRNA-targeted oligonucleotide probe for the detection of mosquitocidal strains of Bacillus sphaericus in soils: characterization of novel strains lacking toxin genes

Abstract: Colonies of Bacillus sphaericus on primary isolation have been identified using an oligonucleotide probe targeted to a specific region of the 16S rRNA. Of 3440 colonies from soil samples from Brazil, 57 hybridized to the probe in colony blots but when purified DNA was used in slot-blots the probe was more specific and only 27 isolates hybridized. Of these, 20 strains were confirmed as members of DNA homology group IIA (potential mosquitocidal strains) by ribotyping and isoenzyme analysis. However, none of these strains was toxic to Anopheles or Culex larvae, nor did they contain recognized toxin genes. This is the first demonstration of such non-pathogenic strains of B. sphaericus DNA homology group IIA and their common occurrence suggests that pathogenicity is not an important contribution to the success of these bacteria in the environment. Similar screening of strains from Scottish soils indicated that B. sphaericus DNA homology group IIA strains were less common in this habitat and none were recovered on this occasion.