NF-kappaB Signaling Pathway Research Articles

Pseudolaric acid B suppresses T lymphocyte activation through inhibition of NF‐κB signaling pathway and p38 phosphorylation

Pseudolaric acid B (PAB) is a major bioactive component of the medicinal plant Pseudolarix kaempferi. Traditional medicine practitioners in Asia have been using the roots of this plant to treat inflammatory and microbial skin diseases for centuries. In the current study, in vitro immunosuppressive effect of PAB and the underlying mechanisms have been investigated. The results showed that PAB dose-dependently suppressed human T lymphocyte proliferation, IL-2 production and CD25 expression induced by co-stimulation of PMA plus ionomycin or of anti-OKT-3 plus anti-CD28. Mechanistic studies showed that PAB significantly inhibited nuclear translocation of NF-kappaB p65 and phosphorylation and degradation of IkappaB-alpha evoked by co-stimulation of PMA plus ionomycin. PAB could also suppress the phosphorylation of p38 in the MAPKs pathway. Based on these evidences, we conclude that PAB suppressed T lymphocyte activation through inhibition of NF-kappaB and p38 signaling pathways; this would make PAB a strong candidate for further study as an anti-inflammatory agent.

Allylmethylsulfide Down-Regulates X-Ray Irradiation-Induced Nuclear Factor-kappaB Signaling in C57/BL6 Mouse Kidney.

Allylmethylsulfide (AMS), a volatile organosulfur derivative from garlic, has been shown to have radioprotective effects in radiation-challenged cell and animal models, but the mechanism of radioprotection is not well understood. To determine the mechanism of radioprotection in an in vivo model, we first verified the antioxidant capacity of AMS using 2,2'-azobis(2-amidinopropane) dihydrochloride-induced human embryonic kidney 293T cells by measuring reactive oxygen species generation, reduced glutathione, protein tyrosine kinase/protein tyrosine phosphatase balance, and nuclear factor-kappaB (NF-kappaB) protein levels. We then investigated the protective effects of AMS (55 and 275 micromol/kg, intraperitoneal treatment) on 15 Gy X-ray-irradiated mouse kidney. The results showed that AMS decreased the free radical-induced lipid peroxidation in mice exposed to X-rays. Moreover, the antioxidative AMS suppressed the activation of NF-kappaB and its dependent genes such as vascular cell adhesion molecule-1, inducible nitric oxide synthase, and cyclooxygenase-2 through inhibition of IkappaBalpha phosphorylation and activation of IkappaB kinase alpha/beta and mitogen-activated protein kinases (MAPKs). Based on these results, AMS may be a useful radioprotective agent by down-regulating the MAPKs and NF-kappaB signaling pathway that can be induced via X-ray irradiation.

Alpha-lipoic acid attenuates cisplatin-induced acute kidney injury in mice by suppressing renal inflammation

Cisplatin is a chemotherapeutic agent used in treatment of malignant tumours. However, cisplatin produces various side effects, such as nephrotoxicity, neurotoxicity, emetogenesis and ototoxicity. Inflammation is an important mechanism of cisplatin nephrotoxicity. Alpha-lipoic acid (alpha-LA) has anti-inflammatory effects that inhibit both adhesion molecule expression in human endothelial cells and monocyte adhesion by suppressing the nuclear factor-kappaB (NF-kappaB) signalling pathway. The goals of this study were to investigate the anti-inflammatory effects of alpha-LA during cisplatin-induced renal injury and to examine the mechanisms of protection. C57BL/6 mice were given cisplatin (20 mg/kg) with or without alpha-LA treatment (100 mg/kg for 3 days). Renal function, histological changes, adhesion molecule expression and inflammatory cell infiltration were examined. The effect of alpha-LA on NF-kappaB activity was evaluated by examining nuclear translocation and phosphorylation of NF-kappaB p65 subunits in kidney tissue. Cisplatin-induced decreases in renal function, measured by blood urea nitrogen, serum creatinine level and renal tubular injury scores, were attenuated by alpha-LA treatment. alpha-LA decreased the tissue levels of tumour necrosis factor-alpha, the expression of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1), and suppressed the infiltration of CD11b-positive macrophages. alpha-LA also attenuated the cisplatin-induced increases in the phosphorylation and nuclear translocation of NF- kappaB p65 subunits in kidney tissue. These results suggest that alpha-LA treatment ameliorates cisplatin-induced acute kidney injury by reducing inflammatory adhesion molecule expression and NF-kappaB activity.

CHFR, a potential tumor suppressor, downregulates interleukin-8 through the inhibition of NF-κB

The mitotic checkpoint gene CHFR (checkpoint with forkhead and ring finger domains) is silenced in various human cancers by promoter hypermethylation, suggesting that CHFR is a tumor suppressor. Here, we show that CHFR functions as a negative regulator of the nuclear factor-kappaB (NF-kappaB) pathway. Expression of CHFR inhibited NF-kappaB reporter activity, whereas knockdown of CHFR activated reporter activity. These activities are independent of its RING finger domain. Furthermore, we found that CHFR physically interacts with p65 in cells. Electrophoretic mobility shift assays (EMSAs) and ELISA-based NF-kappaB-binding assays showed that CHFR negatively regulated transcriptional activity of p65. In addition, our data show that interleukin (IL)-8 is significantly downregulated by CHFR, and that the migration of human endothelial cells is suppressed in culture medium conditioned from CHFR-expressing cancer cells. Using a xenograft model, we show that neovascularization is suppressed by adenovirus-mediated transfer of CHFR. These results indicate that expression of CHFR markedly reduces the expression of IL-8 through the inhibition of NF-kappaB. As the NF-kappaB signaling pathway plays a critical role in the development and progression of cancer, our findings show the functional relationship between epigenetic alteration and inflammation/angiogenesis in human cancer cells, thereby showing several potential targets for therapeutic intervention.

Rosiglitazone Modulates the Innate Immune Response toPlasmodium falciparumInfection and Improves Outcome in Experimental Cerebral Malaria

For severe malarial syndromes such as cerebral malaria, adverse clinical outcomes are often mediated by the immune system rather than caused by the parasite directly. However, few therapeutic agents have been developed to modulate the host's immunopathological responses to infection. Here, we report that the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist rosiglitazone modulated the host response to malaria by enhancing phagocytic clearance of malaria-parasitized erythrocytes and by decreasing inflammatory responses to infection via inhibition of Plasmodium falciparum glycosylphosphatidylinositol-induced activation of the mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB) signaling pathways. We found that, in the Plasmodium berghei strain ANKA experimental model of cerebral malaria, rosiglitazone modified the inflammatory response to malarial infection and improved the survival rate even when treatment was initiated as late as day 5 after infection. Furthermore, rosiglitazone reduced the parasitemia in a CD36-dependent manner in the Plasmodium chabaudi chabaudi hyperparasitemia model. These data suggest that PPARgamma agonists represent a novel class of host immunomodulatory drugs that may be useful for treatment of severe malaria syndromes.

Multiple roles of the NF‐?B signaling pathway regulated by coupled negative feedback circuits

The NF-kappaB signaling pathway can perform multiple functional roles depending on specific cellular environments and cell types. Even in the same cell clones, the pathway can show different kinetic and phenotypic properties. It is believed that the complex networks controlling the NF-kappaB signaling pathway can generate these diverse and sometimes ambiguous phenomena. We noted, however, that the dynamics of NF-kappaB signaling pathway is highly stochastic and that the NF-kappaB signaling pathway contains multiple negative feedback circuits formed by IkappaB isoform proteins, IkappaBalpha and IkappaBepsilon in particular. Considering the topological similarity, their functional roles seem to be redundant, raising the question why different types of IkappaB isoforms need to exist. From extensive stochastic simulations of the NF-kappaB signaling pathway, we found that each IkappaB isoform actually conducts a different regulatory role through its own negative feedback. Specifically, our data suggest that IkappaBalpha controls the dynamic patterns of nuclear NF-kappaB, while IkappaBepsilon induces cellular heterogeneity of the NF-kappaB activities. These results may provide an answer to the question of how a single NF-kappaB signaling pathway can perform multiple biological functions even in the same clonal populations.

In Vitro Cellular Uptake and Effects of Fe3O4 Magnetic Nanoparticles on HeLa Cells

Fe3O4 magnetic nanoparticles (Fe3O4 MNPs) have attracted increasing attention in recent years due to their extensive applications. However, their effects on cells and relevant molecular mechanisms remain not very clear. In this paper, the effects of Fe3O4 MNPs on the proliferation and apoptosis of human HeLa cells, the activation of transcription factor NF-kappaB and the expressions of its target genes in HeLa cells were investigated. The entrance of nanoparticles into cells was demonstrated by Prussian blue staining and transmission electron microscopy (TEM) and it revealed that the majority of nanoparticles existed in cytoplasm in a collective format. The amount of nanoparticles in cells was dependent on the dose and incubating time acting to cells. It was revealed that Fe3O4 MNPs exerted no significant effects on the proliferation of HeLa cells and did not induce apoptosis of HeLa cells. The activation of NF-kappaB and the expressions of its typical target genes, IL-8, COX-2 and MCP-1, were not significantly changed under the various concentrations of Fe3O4 MNPs. Therefore, it is concluded that the Fe3O4 MNPs have a good biocompatibility to HeLa cells cultured in vitro and do not affect NF-kappaB signaling pathway under the dose and acting time used in this study.

Use of the Pharmacological Inhibitor BX795 to Study the Regulation and Physiological Roles of TBK1 and IκB Kinase ϵ: A DISTINCT UPSTREAM KINASE MEDIATES SER-172 PHOSPHORYLATION AND ACTIVATION

TANK-binding kinase 1 (TBK1) and IkappaB kinase epsilon (IKKepsilon) regulate the production of Type 1 interferons during bacterial and viral infection, but the lack of useful pharmacological inhibitors has hampered progress in identifying additional physiological roles of these protein kinases and how they are regulated. Here we demonstrate that BX795, a potent and relatively specific inhibitor of TBK1 and IKKepsilon, blocked the phosphorylation, nuclear translocation, and transcriptional activity of interferon regulatory factor 3 and, hence, the production of interferon-beta in macrophages stimulated with poly(I:C) or lipopolysaccharide (LPS). In contrast, BX795 had no effect on the canonical NFkappaB signaling pathway. Although BX795 blocked the autophosphorylation of overexpressed TBK1 and IKKepsilon at Ser-172 and, hence, the autoactivation of these protein kinases, it did not inhibit the phosphorylation of endogenous TBK1 and IKKepsilon at Ser-172 in response to LPS, poly(I:C), interleukin-1alpha (IL-1alpha), or tumor necrosis factor alpha and actually enhanced the LPS, poly(I:C), and IL-1alpha-stimulated phosphorylation of this residue. These results demonstrate that the phosphorylation of Ser-172 and the activation of TBK1 and IKKepsilon are catalyzed by a distinct protein kinase(s) in vivo and that TBK1 and IKKepsilon control a feedback loop that limits their activation by LPS, poly(I:C) and IL-1alpha (but not tumor necrosis factor alpha) to prevent the hyperactivation of these enzymes.

Mechanistic study of saikosaponin‐d (Ssd) on suppression of murine T lymphocyte activation

Saikosaponin-d (Ssd) is a triterpene saponin derived from the medicinal plant, Bupleurum falcatum L. (Umbelliferae). Previous findings showed that Ssd exhibits a variety of pharmacological and immunomodulatory activities including anti-inflammatory, anti-bacterial, anti-viral and anti-cancer effects. In the current study we have investigated the effects of Ssd on activated mouse T lymphocytes through the NF-kappaB, NF-AT and AP-1 signaling pathways, cytokine secretion, and IL-2 receptor expression. The results demonstrated that Ssd not only suppressed OKT3/CD28-costimulated human T cell proliferation, it also inhibited PMA, PMA/Ionomycin and Con A-induced mouse T cell activation in vitro. The inhibitory effect of Ssd on PMA-induced T cell activation was associated with down-regulation of NF-kappaB signaling through suppression of IKK and Akt activities. In addition, Ssd suppressed both DNA binding activity and the nuclear translocation of NF-AT and activator protein 1 (AP-1) of the PMA/Ionomycin-stimulated T cells. The cell surface markers like IL-2 receptor (CD25) were also down-regulated together with decreased production of pro-inflammatory cytokines of IL-6, TNF-alpha and IFN-gamma. These results indicate that the NF-kappaB, NF-AT and AP-1 (c-Fos) signaling pathways are involved in the T cell inhibition evoked by Ssd, so it can be a potential candidate for further study in treating T cell-mediated autoimmune conditions.

Mechanisms Involved in Alleviation of Intestinal Inflammation by Bifidobacterium Breve Soluble Factors

ObjectivesSoluble factors released by Bifidobacterium breve C50 (Bb) alleviate the secretion of pro-inflammatory cytokines by immune cells, but their effect on intestinal epithelium remains elusive. To decipher the mechanisms accounting for the cross-talk between bacteria/soluble factors and intestinal epithelium, we measured the capacity of the bacteria, its conditioned medium (Bb-CM) and other Gram(+) commensal bacteria to dampen inflammatory chemokine secretion.MethodsTNFα-induced chemokine (CXCL8) secretion and alteration of NF-κB and AP-1 signalling pathways by Bb were studied by EMSA, confocal microscopy and western blotting. Anti-inflammatory capacity was also tested in vivo in a model of TNBS-induced colitis in mice.Results Bb and Bb-CM, but not other commensal bacteria, induced a time and dose-dependent inhibition of CXCL8 secretion by epithelial cells driven by both AP-1 and NF-κB transcription pathways and implying decreased phosphorylation of p38-MAPK and IκB-α molecules. In TNBS-induced colitis in mice, Bb-CM decreased the colitis score and inflammatory cytokine expression, an effect reproduced by dendritic cell conditioning with Bb-CM.Conclusions Bb and secreted soluble factors contribute positively to intestinal homeostasis by attenuating chemokine production. The results indicate that Bb down regulate inflammation at the epithelial level by inhibiting phosphorylations involved in inflammatory processes and by protective conditioning of dendritic cells.

Biomechanical Thresholds Regulate Inflammation through the NF-κB Pathway: Experiments and Modeling

BackgroundDuring normal physical activities cartilage experiences dynamic compressive forces that are essential to maintain cartilage integrity. However, at non-physiologic levels these signals can induce inflammation and initiate cartilage destruction. Here, by examining the pro-inflammatory signaling networks, we developed a mathematical model to show the magnitude-dependent regulation of chondrocytic responses by compressive forces.Methodology/Principal FindingsChondrocytic cells grown in 3-D scaffolds were subjected to various magnitudes of dynamic compressive strain (DCS), and the regulation of pro-inflammatory gene expression via activation of nuclear factor-kappa B (NF-κB) signaling cascade examined. Experimental evidences provide the existence of a threshold in the magnitude of DCS that regulates the mRNA expression of nitric oxide synthase (NOS2), an inducible pro-inflammatory enzyme. Interestingly, below this threshold, DCS inhibits the interleukin-1β (IL-1β)-induced pro-inflammatory gene expression, with the degree of suppression depending on the magnitude of DCS. This suppression of NOS2 by DCS correlates with the attenuation of the NF-κB signaling pathway as measured by IL-1β-induced phosphorylation of the inhibitor of kappa B (IκB)-α, degradation of IκB-α and IκB-β, and subsequent nuclear translocation of NF-κB p65. A mathematical model developed to understand the complex dynamics of the system predicts two thresholds in the magnitudes of DCS, one for the inhibition of IL-1β-induced expression of NOS2 by DCS at low magnitudes, and second for the DCS-induced expression of NOS2 at higher magnitudes.Conclusions/SignificanceExperimental and computational results indicate that biomechanical signals suppress and induce inflammation at critical thresholds through activation/suppression of the NF-κB signaling pathway. These thresholds arise due to the bistable behavior of the networks originating from the positive feedback loop between NF-κB and its target genes. These findings lay initial groundwork for the identification of the thresholds in physical activities that can differentiate its favorable actions from its unfavorable consequences on joints.

The effects of PDTC plus leflunomide and cyclosporine on the NF-κB signaling pathway in mouse-to-rat cardiac xenografts

In this study, the effects of pirrolidine dithiocarbamate (PDTC) plus leflunomide (Lef) and cyclosporine (CsA) on the NF-kappaB signaling pathway in mouse-to-rat cardiac xeno-transplantation models were investigated. NIH mice and Wistar rats served as donors and recipients respectively. Mouse-to-rat cardiac xenotransplantation was performed. The recipients were divided into 5 groups: group A (the control group), group B (PDTC group), group C (PDTC plus CsA group), group D (PDTC plus Lef group) and group E (PDTC plus Lef and CsA group). The expressions of IKKalpha/beta, NF-kappaB-P65, IkappaBalpha, ICAM-1 and NF-kappaB DNA binding activity in xenograft tissues were determined by immunohistochemistry and Western blot as well as electrophoretic mobility shift assay (EMSA). The median survival time of cardiac xenografts in the control group, PDTC group, PDTC plus CsA group, PDTC plus Lef group and PDTC plus Lef and CsA group was (2.17+/-0.41), (2.33+/-0.52), (4.67+/-1.21), (7.00+/-1.79) and (9.00+/-1.41) days respectively. The survival time of xenografts in the PDTC plus Lef and CsA group was significantly longer than that in other four groups (P<0.05 for all), that in the PDTC plus Lef group longer than that in the control group, PDTC group and PDTC plus CsA group (P<0.05 for all), that in PDTC plus CsA group longer than the control group and PDTC group (P<0.05 for all). The expressions of IKKalpha/beta, NF-kappaB-P65, IkappaBalpha and ICAM-1 and NF-kappaB DNA binding activity were notably increased in mouse-to-rat cardiac xenografts. The expressions were decreased in the control group, PDTC group, PDTC plus CsA group, PDTC plus Lef and PDTC plus Lef and CsA group in turn. It was concluded that PDTC plus Lef and CsA can significantly suppress the expressions of IKKalpha/beta, NF-kappaB-P65, IkappaBalpha, ICAM-1 and NF-kappaB DNA binding activity, thereby prolonging the survival of the cardiac xenografts.

The type III transforming growth factor- receptor negatively regulates nuclear factor kappa B signaling through its interaction with -arrestin2

Transforming growth factor-beta (TGF-beta) increases or decreases nuclear factor kappa B (NFkappaB) signaling in a context-dependent manner through mechanisms that remain to be defined. The type III transforming growth factor-beta receptor (TbetaRIII) is a TGF-beta superfamily co-receptor with emerging roles in both mediating and regulating TGF-beta superfamily signaling. We have previously reported a novel interaction of TbetaRIII with the scaffolding protein, beta-arrestin2, which results in TbetaRIII internalization and downregulation of TGF-beta signaling. beta-arrestin2 also scaffolds interacting receptors with the mitogen-activated protein kinase and NFkappaB-signaling pathways. Here, we demonstrate that TbetaRIII, through its interaction with beta-arrestin2, negatively regulates NFkappaB signaling in MCF10A breast epithelial and MDA-MB-231 breast cancer cells. Increasing TbetaRIII expression reduced NFkappaB-mediated transcriptional activation and IkappaBalpha degradation, whereas a TbetaRIII mutant unable to interact with beta-arrestin2, TbetaRIII-T841A, had no effect. In a reciprocal manner, short hairpin RNA-mediated silencing of either TbetaRIII expression or beta-arrestin2 expression increased NFkappaB-mediated transcriptional activation and IkappaBalpha degradation. Functionally, TbetaRIII-mediated repression of NFkappaB signaling is important for TbetaRIII-mediated inhibition of breast cancer cell migration. These studies define a mechanism through which TbetaRIII regulates NFkappaB signaling and expand the roles of this TGF-beta superfamily co-receptor in regulating epithelial cell homeostasis.

Ultrasound stimulates NF‐κB activation and iNOS expression via the Ras/Raf/MEK/ERK signaling pathway in cultured preosteoblasts

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and non-operatively clinical uses. Nitric oxide (NO) is a crucial early mediator in mechanically induced bone formation. Here we found that US-mediated inducible nitric oxide synthase (iNOS) expression was attenuated by Ras inhibitor (manumycin A), Raf-1 inhibitor (GW5074), MEK inhibitor (PD98059), NF-kappaB inhibitor (PDTC), and IkappaB protease inhibitor (TPCK). US-induced Ras activation was inhibited by manumycin A. Raf-1 phosphorylation at Ser(338) by US was inhibited by manumycin A and GW5074. US-induced MEK and ERK activation was inhibited by manumycin A, GW5074, and PD98059. Stimulation of preosteoblasts with US activated IkappaB kinase alpha/beta (IKK alpha/beta), IkappaBalpha phosphorylation, p65 phosphorylation at Ser(276), p65, and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. US-mediated an increase of IKK alpha/beta, IkappaBalpha, and p65 phosphorylation, kappaB-luciferase activity and p65 and p50 binding to the NF-kappaB element was inhibited by manumycin A, GW5074, and PD98059. Our results suggest that US increased iNOS expression in preosteoblasts via the Ras/Raf-1/MEK/ERK/IKKalphabeta and NF-kappaB signaling pathways.

NF‐κB promotes iNOS and VEGF expression in salivary gland adenoid cystic carcinoma cells and enhances endothelial cell motility in vitro

Tumour cell-derived angiogenic-related factors and endothelial cell mobility are essential for neovascularization and haematogenous metastasis of adenoid cystic carcinoma (ACC) of the human salivary glands. Our previous study demonstrated that vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and nuclear factor kappaB (NF-kappaB) staining correlated with ACC microvessel density. However, there still remains a lack of direct evidence to clarify the function of liposaccharide-induced NF-kappaB activity in ACC angiogenesis. This study aimed to demonstrate the function of liposaccharide-induced NF-kappaB signalling pathway (which would be relevant to angiogenesis) of ACC cell lines. A PCMV-IkappaBalphaM vector transfection assay was performed to inhibit NF-kappaB activity. Constitutive and liposaccharide-induced NF-kappaB activity, along with VEGF and iNOS expression, was detected by electrophoretic mobility shift assays, reverse transcription-polymerase chain reaction and Western blot assay. Tumour cell-derived NO(2) (-) was evaluated by a nitrite determination assay, and endothelial cell mobility was investigated by endothelial cell proliferation, migration and tube formation assays. We demonstrated that regulation of VEGF and iNOS expression utilized the NF-kappaB signalling pathway in ACC cell lines. In the constitutive and liposaccharide-induced condition, NF-kappaB activity was blocked by phosphorylation-defective IkappaBalpha. Accordingly, mRNA and protein expression of VEGF and iNOS were significantly decreased, with a great drop in endogenous NO(2) (-). Furthermore, supernatant from ACC-2/IkBaM and ACC-M/IkBaM cells resulted in much lower endothelial cell mobility than that from ACC-2 and ACC-M cells. Our results suggest that NF-kappaB promotes iNOS and VEGF expression in salivary gland ACC cells and enhances endothelial cell motility in vitro.

Phenanthrene-based tylophorine-1 (PBT-1) inhibits lung cancer cell growth through the Akt and NF-kappaB pathways.

Tylophorine and related natural compounds exhibit potent antitumor activities. We previously showed that PBT-1, a synthetic C9-substituted phenanthrene-based tylophorine (PBT) derivative, significantly inhibits growth of various cancer cells. In this study, we further explored the mechanisms and potential of PBT-1 as an anticancer agent. PBT-1 dose-dependently suppressed colony formation and induced cell cycle G2/M arrest and apoptosis. DNA microarray and pathway analysis showed that PBT-1 activated the apoptosis pathway and mitogen-activated protein kinase signaling. In contrast, PBT-1 suppressed the nuclear factor kappaB (NF-kappaB) pathway and focal adhesion. We further confirmed that PBT-1 suppressed Akt activation accelerated RelA degradation via IkappaB kinase-alpha and down-regulated NF-kappaB target gene expression. The reciprocal recruitment of RelA and RelB on COX-2 promoter region led to down-regulation of transcriptional activity. We conclude that PBT-1 induces cell cycle G2/M arrest and apoptosis by inactivating Akt and by inhibiting the NF-kappaB signaling pathway. PBT-1 may be a good drug candidate for anticancer chemotherapy.

GFP‐p65 knock‐in mice as a tool to study NF‐κB dynamics in vivo

The nuclear factor kappaB (NF-kappaB) signaling pathway regulates immune and inflammatory responses and is implicated in the pathogenesis of multiple diseases. The principal mechanism controlling NF-kappaB activation depends on the association of NF-kappaB transcription factor dimers with IkappaB molecules, which prevents the accumulation of NF-kappaB in the nucleus and the activation of target gene transcription. Monitoring the nucleocytoplalsmic shuttling of NF-kappaB factors is a reliable method to study the dynamic regulation of NF-kappaB activity. Here, we generated knock-in mice expressing a fusion protein between the green fluorescent protein (GFP) and the p65/RelA NF-kappaB subunit (GFP-p65) from the endogenous p65 genomic locus. Homozygous GFP-p65 mice developed normally and showed normal NF-kappaB activation, demonstrating that the GFP-p65 fusion protein functionally substitutes for wild-type p65. Live imaging of primary cells from GFP-p65 mice allowed real-time monitoring of p65 nucleocytoplasmic shuttling upon NF-kappaB activation. Therefore, the GFP-p65 knock-in mice constitute an invaluable tool for studying the dynamic regulation of NF-kappaB.

Role of COX-2 in epithelial-stromal cell interactions and progression of ductal carcinoma in situ of the breast.

Epithelial-stromal cell interactions have an important role in breast tumor progression, but the molecular mechanisms underlying these effects are just beginning to be understood. We previously described that fibroblasts promote, whereas normal myoepithelial cells inhibit, the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinomas by using a xenograft model of human DCIS. Here, we report that the tumor growth and progression-promoting effects of fibroblasts are at least in part due to increased COX-2 expression in tumor epithelial cells provoked by their interaction with fibroblasts. Up-regulation of COX-2 in DCIS xenografts resulted in increased VEGF and MMP14 expression, which may contribute to the larger weight and invasive histology of COX-2-expressing tumors. Administration of celecoxib, a selective COX-2 inhibitor, to tumor-bearing mice decreased xenograft tumor weight and inhibited progression to invasion. Coculture of fibroblasts with DCIS epithelial cells enhanced their motility and invasion, and this change was associated with increased MMP14 expression and MMP9 protease activity. We identified the NF-kappaB pathway as one of the mediators of stromal fibroblast-derived signals regulating COX-2 expression in tumor epithelial cells. Inhibition of NF-kappaB and COX-2 activity and down-regulation of MMP9 expression attenuated the invasion-promoting effects of fibroblasts. These findings support a role for COX-2 in promoting the progression of DCIS to invasive breast carcinomas, and suggest that therapeutic targeting of the NF-kappaB and prostaglandin signaling pathways might be used for the treatment and prevention of breast cancer.

CARMA1‐mediated NF‐κB and JNK activation in lymphocytes

Activation of transcription factor nuclear factor-kappaB (NF-kappaB) and Jun N-terminal kinase (JNK) play the pivotal roles in regulation of lymphocyte activation and proliferation. Deregulation of these signaling pathways leads to inappropriate immune response and contributes to the development of leukemia/lymphoma. The scaffold protein CARMA1 [caspase-recruitment domain (CARD) membrane-associated guanylate kinase (MAGUK) protein 1] has a central role in regulation of NF-kappaB and the JNK2/c-Jun complex in both B and T lymphocytes. During last several years, tremendous work has been done to reveal the mechanism by which CARMA1 and its signaling partners, B cell CLL-lymphoma 10 and mucosa-associated lymphoid tissue 1, are activated and mediate NF-kappaB and JNK activation. In this review, we summarize our findings in revealing the roles of CARMA1 in the NF-kappaB and JNK signaling pathways in the context of recent advances in this field.

Chemosensitization and Immunosensitization of Resistant Cancer Cells to Apoptosis and Inhibition of Metastasis by the Specific NF-&amp;#954;B Inhibitor DHMEQ

Tumors develop resistance to cytotoxic apoptotic stimuli induced by various chemotherapeutic drugs and immunotherapies. Therefore, there is a need to overcome chemo- and immuno-resistance of tumors through the development of small molecules, as sensitizing agents, aimed at targeting gene products that regulate the apoptotic pathways and allow therapeutics to be effective. The constitutively activated NF-kappaB (nuclear factor kappa B) signaling pathway is involved in cell survival, inflammation and metastasis and is invariably constitutively activated in most cancers. Consequently, NF-kappaB is intimately involved in the regulation of resistance to cytotoxic drugs. A novel NF-kappaB inhibitor, DHMEQ (dehydroxymethylepoxyquinomicin), inhibits the translocation of NF-kappaB into the nucleus as well as inhibits DNA binding of NF-kappaB components and was shown to be a potent chemo- and immuno-sensitizing agent and in combination with cytotoxic therapeutics resulted in significant reversal of resistance and tumor cell death. This review will present various lines of evidence supporting the therapeutic efficacy of DHMEQ when used in combination with conventional/new cytotoxic drugs in the treatment of resistant tumor cells as well as in the prevention of metastasis.