NF-B Activation Research Articles

Nod2 in Normal and Abnormal Intestinal Immune Function

s a m h 3 s N I e h n m h f r a o i he search for genetic associations in Crohn’s disease has led to several fruitful discoveries that hold the otential to identify pathogenetic mechanisms and tarets for future therapy. The first gene to be identified for complex genetic disorder using genome wide scanning as the Nod2 gene. Simultaneous with the laborious ork of fine mapping chromosome 16 performed by ugot et al, the Nunez laboratory had identified the od2 gene in a search for genes containing caspase ctivation and recruitment domains (CARD). Both roups reported the now well-known association between olymorphisms in the Nod2 gene and susceptibility to rohn’s disease. These results were confirmed by many nvestigators in other ethnic groups with the notable xception of Asian populations. A subsequent wave of publications described the pheotype of Crohn’s disease patients carrying mutations in od2. Patients with Nod2 mutations are more likely to ave ileal disease with fibrostenotic complications. In dult populations, Nod2 mutations are not associated ith more frequent need for surgery or with rapid proression to complicated small bowel disease. By contrast, ugathasan et al have demonstrated that in a populaion-based, prospectively ascertained cohort of children ith Crohn’s disease that carriage of Nod2 mutations, in articular the 3020insC mutation resulted in an inreased need for surgery and earlier surgery. As a single genetic or stand-alone diagnostic test, utations in Nod2 are not likely to provide information hat will permit clinical decisions. More recently, the iminovitch group have found an interaction between utations in Nod2 and mutations in the Solute Carrier amily 22A4/22A5 (SLC22A/22A5) gene cluster. Mutaions in these organic cation transporter genes decrease xpression or function of carnitine transport in intestinal pithelial cells especially in response to heat shock or ellular stress. Other phenotypic associations such as the elationship of SLC22A/22A5 and perforating or fibrotenotic disease or need for surgery were not examined. hese data suggest interaction between Nod2 and the rganic cation transporter genes. One model to reconcile he relationship between SLC22A/22A5 and Nod2 is a ecrease in epithelial integrity caused by the former and n inability to mount an inflammatory response to inracellular bacteria caused by the latter (discussed later). e expect that a panel of genetic tests and biological ssays that address Nod2 function may soon provide rognostic and diagnostic information. The discovery of Nod2 immediately sparked tremenous enthusiasm given its homology to other immunoogically active molecules such as the pyrins (familial editerranean fever) and its role as a pattern recognition eceptor for pathogens. The true biologic role of Nod2 in ormal human physiology and in Crohn’s disease pathoenesis has proved elusive, however. Nod2 is normally xpressed in peripheral blood monocytes and Paneth ells. Cytokine stimulation of intestinal epithelial cells in ulture can also lead to expression of Nod2, and some ave described expression of Nod2 in the colon of paients with active Crohn’s disease. The ligand for Nod2 is muramyl dipeptide (MDP), a tructure contained within peptidoglycan and generated fter its lysosomal digestion. Using peripheral blood ononuclear cells (PBMC) from individuals who are omozygous for the truncation mutation of Nod2, 020insC (L1007fsinsC), the Nunez laboratory demontrated that homozygous individuals had decreased FB activation (by EMSA) and decreased expression of L-1 in response to MDP but not LPS. Importantly, ven unaffected individuals, ie, without Crohn’s disease, ad this biochemical defect suggesting that this alone is ot sufficient to cause Crohn’s disease. The more comon genotype of Crohn’s disease patients, however, is eterozygosity for Nod2 mutations. In this assay, PBMC rom heterozygotes behaved similarly to wild-type with espect to NFB activation and IL-1 expression. Using transient expression assay, Chamaillard et al have demnstrated that Nod2 variants can be categorized accordng to their basal ability to activate NFB and their

Kinasing and Clipping Down the NF-κB Trail

Activation of T cells is a key requirement for a successful innate or adaptive immune response to pathogenic insult. T cell stimulation results in activation of the master transcription factor NF-B by a complicated signaling pathway that is only partially understood. In their Perspective, [van Oers and Chen][1] discuss new findings that implicate the kinase PDK1 ([ Lee et al .][2]) and a caspase (Su et al .) as crucial players in the T cell signaling pathway that activates NF-B. [1]: http://www.sciencemag.org/cgi/content/full/308/5718/65 [2]: http://www.sciencemag.org/cgi/content/short/308/5718/114

Carvedilol Inhibits Expressions of Vascular Cell Adhesion Molecule-1, Intercellular Adhesion Molecule-1, Monocyte Chemoattractant-1, and Interleukin-8 via NF-kappaB Inhibition in Human Endothelial Cells

Background and Objectives：Carvedilol is an anti-oxidative, the cardioprotective effects of which are mediated by the inhibition of NF-κB activation. The present study was designed to examine the effects of carvedilol, an α1- and β-blocker, on tumor necrosis factor (TNF)-α stimulated human umbilical vein endothelial cells (HUVEC). Materials and Methods：HUVEC were treated with TNF-α (10 ng/mL) in either the absence or presence of carvedilol. The levels of intracellular reactive oxygen species (ROS) were examined using a fluorescent dye DCFH-DA, with the adhesion of U-937 monocyte to the HUVEC. Nuclear factor kappa B (NF-κB) activation was determined by NF-κB p65 translocation to the nucleus using Western blotting and immunocytochemistry. The expressions of NF-κB dependent pro-inflammatory molecules, i.e., vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8, were measured by RT-PCR and ELISA. Bcl-2 and phosphorylation of c-Jun N-terminal protein kinase (JNK) were measured using Western blotting. Results：TNF-α treatment increased the activation of NFκB, suppressed Bcl-2, and increased the phosphorylation of JNK, the ROS level and the adhesion of U-937. The levels of mRNA and protein expressions of VCAM-1, ICAM-1, MCP-1 and IL-8 were up-regulated by TNFα. Carvedilol inhibited the phosphorylation of JNK, ROS formation and the adhesion of U-937 monocyte. In addition, carvedilol reduced the production of VCAM-1, ICAM-1, MCP-1 and IL-8 at the mRNA and protein levels, via the suppression of NF-B activation. Conclusion：These results suggested that the anti-inflammatory effects of carvedilol on TNF-α stimulated endothelial cells could be explained by its ROS-scavenging and NF-κ B inactivation properties. (Korean Circulation J 2005;35:576-582)

TLR5-mediated activation of p38 MAPK regulates epithelial IL-8 expression via a post-transcriptional mechanism

Yu, Yimin, Hui Zeng, Sean Lyons, Adam Carlson, Didier Merlin, Andrew S. Neish, and Andrew T. Gewirtz. TLR5-mediated activation of p38 MAPK regulates epithelial IL-8 expression via posttranscriptional mechanism. Am J Physiol Gastrointest Liver Physiol 285: G282–G290, 2003. First published April 17, 2003; 10.1152/ ajpgi.00503.2002.—Toll-like receptors (TLRs) activate antimicrobial gene expression in response to detection of specific bacterial products. Relatively little is known about TLR5, the only TLR thought to be preferentially expressed by epithelial cells, beyond that it confers activation of the transcription factor NFB in a MyD-88 dependent manner in response to flagellin. Because TLRs, in general, are also thought to signal through members of the MAPK family, we examined flagellin-induced MAPK activation (via examining its phosphorylation status) and its subsequent role in expression of the chemokine IL-8 in polarized intestinal epithelia. Flagellin, like other proinflammatory stimuli (TNF, Salmonella typhimurium), activated p38 MAPK in a TLR5-dependent manner, whereas aflagellate bacteria or EGF did not activate this kinase. Although ERK1 and -2 were also observed to be activated in response to flagellin, their activation was not restricted to proinflammatory stimuli because they were also potently activated by aflagellate bacteria (S. typhimurium or Escherichia coli) and EGF (neither of which activate NFB in these cells). Pharmacological inhibition of p38 MAPK (by SB-203580) potently (IC50 10 nM) reduced expression of IL-8 protein (maximal inhibition, 75%) but had no effect on NFB activation, only slightly attenuated upregulation of IL-8 mRNA levels in response to flagellin, and did not effect IL-8 mRNA stability. Together, these results indicate that epithelial TLR5 mediates p38 activation and subsequently regulates flagellin-induced IL-8 expression independently of NFB, probably by influencing IL-8 mRNA translation.

JNK: a killer on a transcriptional leash.

Whether to live or die is arguably the most important decision a cell has to make, and the NF-B/Rel group of transcription factors is a key element in this decision. Since the discovery of the Anti-Apoptotic Function of NF-B in 1996, the number of publications dealing with the control of apoptosis by NF-B has increased at an astonishing pace – over 600 papers were published in the last 18 months alone! This plethora of publications underscores the crucial role that this particular biological activity of NF-B plays in widespread human diseases, including cancer; and indeed, blockers of NF-B have already been used successfully to treat otherwise recalcitrant malignancies. As with other functions of NF-B, the suppression of apoptosis involves activation of a program of gene expression. While progress in understanding this program has undoubtedly been made, it remains unclear as to which genes are most critical to this activity of NF-B, and ultimately, by which mechanisms their products inhibit the apoptotic signaling cascade. Recently, an important piece of this puzzle – pertaining to the control of tumor necrosis factor (TNF)-induced apoptosis – has been found, with the demonstration that activation of NF-B shuts down the c-Jun-N-terminal kinase (JNK) cascade1,2,3,4 (Figure 1).

Overexpression of redox factor-1 protects against postischemic liver injury by reducing oxidative stress and NF-κB activity

REPERFUSION of ischemic tissue results in the generation of reactive oxygen species (ROS). Parenchymal cells in the ischemic tissue contribute to oxidant stress through intracellular ROS generation early after reperfusion. In addition to direct tissue damage, ROS generated in postischemic tissue also functions as signaling intermediates that cause up-regulation of a variety of pro-inflammatory and pro-apoptotic molecules. Many of these substances are produced following activation of the redoxsensitive transcription factor NFB, an inflammatory reaction that compounds and perpetuates early reperfusion injury. For example, redox factor-1 (Ref-1), a bi-functional protein that is up-regulated in response to a host of stimuli that result in intracellular ROS generation, serves as an endonuclease in the base excision repair pathway, and also displays a reducing capacity that facilitates the DNAbinding activities of many redox-sensitive transcription factors including NFB. Both of these properties of Ref-1 may explain its role in cytoprotection against hypoxic and hyperoxic cell stresses in vitro. The present study examines the cytoprotective effects of Ref-1 in hepatic inflammation, apoptosis, and injury responses induced by ischemia-reperfusion in vivo.

The expression of membrane-bound CD14 renders mouse B-1 cells susceptible to LPS

The surface expression of CD14 on mouse B-1 cells and its role on their response to lipopolysaccharide (LPS) were studied by using the murine TH2.52 B-1 cell line and peritoneal B-1 cells. TH2.52 cells with the B-1 phenotype were found to express membrane-bound CD14. Furthermore, CD14 was expressed on physiological peritoneal CD5+ B-1 cells. The stimulation of CD14-expressing TH2.52 cells with a low concentration of LPS resulted in the activation of nuclear factor (NF)-B and a mitogen-activated protein kinase (MAPK). The LPS-induced NF-B and MAPK activation was markedly inhibited by anti-CD14 antibody. These results suggest that B-1 cells may respond to LPS via membrane-bound CD14.

Reply to 'LMP2 expression and proteasome activity in NOD mice'

In our paper, we described developmentally expressed defects in splenocytes and macrophages in NF-B activation and LMP2 expression in the NOD mouse4. These defects parallel disease expression, which occurs in 30% of males and 70% of females by 30 weeks of age13, and we emphasized that such abnormalities are never seen early in life. In contrast, we and others have reported normal or augmented proteasome and NF-B activity in target tissue in autoimmune mice4, 14. Thus, phenotypic derangements in proteasome and NF-B depend on gender, age, tissue and disease course in individual NOD mice.

O fator de transcrição NF-kapaB nos mecanismos moleculares de ação de psicofármacos

Nos últimos anos muitos esforços têm sido empregados para elucidar os mecanismos envolvidos na regulação da transcrição gênica. Moléculas que participam desses processos regulatórios, como os fatores de transcrição, têm recebido atenção especial. A participação desses fatores em diversas funções neurais enfatiza sua importância para a compreensão de distúrbios relacionados ao sistema nervoso central (SNC) e para delinear novos caminhos de acesso terapêutico. O fator de transcrição NF-kapaB destaca-se pela sua vasta gama de ações e pelo fato de diversas proteínas estarem integradas na dinâmica de sua ativação. Evidências recentes apontam o envolvimento desse fator na plasticidade, desenvolvimento e neurodegeneração, com funções essenciais e específicas em neurônios e células da glia. Nesse sentido, o glutamato desponta como um ativador específico (tanto via receptores NMDA e AMPA/KA), juntamente com as neurotrofinas. Este artigo aborda a complexa regulação bioquímica do NF-kapaB e destaca as prováveis contribuições provenientes dos novos conhecimentos junto ao campo da psicofarmacologia. Portanto, alterações da atividade do NF-kapaB poderão trazer novas perspectivas no desenvolvimento de novos psicofármacos.

The Acid Sphingomyelinase Inhibitor SR33557 Counteracts TNF-α-Mediated Potentiation of IL-1β-Induced NF-κB Activation in the Insulin-Producing Cell Line RINm5F

Cytokines induce nitric oxide (NO) production and cell death in insulin-producing cells in vitro but the signaling pathways mediating the cytokine effects are not well characterized. The aim of this study was to determine whether sphingomyelinase (SMase) participates in cytokine signaling leading to NF- B activation, iNOS induction and cell death in insulin-producing cells. Acute exposure to IL-1β or TNF-α did not affect SMase activities in rat insulinoma (RINm5F) cells. TNF-α activated NF-B in gel shift experiments without inducing iNOS - as assessed by nitrite formation - whereas IL-1β stimulated both NF-B activation and iNOS induction. Natural ceramide did not activate NF-B or iNOS. However, both cera-mide and TNF-α potentiated IL-1β- induced activation of NF-B and iNOS. Moreover, the potentiating effects of TNF-α were counteracted by the acid SMase inhibitor SR33557. The combination of IL-1β and IFN-γ induced apoptosis in RINm5F cells, which was paralleled by a modest increase in acid SMase, whereas ceramide mainly induced necrosis. It is concluded that cytokine-induced p-cell signaling is associated with the induction of iNOS but not with enhanced SMase activities. However, TNF-α-mediated potentiation of the IL-1 β effect may involve an increased sensitivity to basal acid SMase activity. An increased acid SMase activity may participate in the execution of cytokine-induced P-cell apoptosis

Characterization of the NF-kappaB activation induced by TR8, an osteoclastogenic tumor necrosis factor receptor family member.

TR8 is a recently identified member of the tumor necrosis factor (TNF) receptor superfamily. TR8 seems to play important roles in bone metabolism as stimulation of this receptor with its ligand, TL8 or osteoclast differentiation factor (ODF), induced the differentiation and activation of osteoclasts. Despite its important biological functions, the biochemical events ensuing from TR8 activation have not been revealed in detail. Most of TNF receptor family proteins provoke the activation of the NF-kappaB transcription factor. In the present study, we examined the signaling potential of TR8 to induce NF-B activation. When overexpressed in a human embryonic kidney cell line by transient transfection, TR8 caused a strong activation of NF-kappaB, which was further increased upon stimulation with TL8. The TR8-induced NF-B activation was abrogated by the co-expression of the TRAF6 mutant lacking the Ring and zinc finger domains and that of the kinase-inactive mutant NIK. Taken together, our study suggests that the presence of intact TRAF6 and the kinase activity of NIK may be essential for TR8 to induce NF-kappaB activation.