Repression Of NF-kappaB Activity Research Articles

Promyelocytic leukemia protein induces apoptosis due to caspase-8 activation via the repression of NFκB activation in glioblastoma

Promyelocytic leukemia (PML) protein plays an essential role in the induction of apoptosis; its expression is reduced in various cancers. As the functional roles of PML in glioblastoma multiforme (GBM) have not been clarified, we assessed the expression of PML protein in GBM tissues and explored the mechanisms of PML-regulated cell death in GBM cells. We examined the PML mRNA level and the expression of PML protein in surgical GBM specimens. PML-regulated apoptotic mechanisms in GBM cells transfected with plasmids expressing the PML gene were examined. The protein expression of PML was significantly lower in GBM than in non-neoplastic tissues; approximately 10% of GBM tissues were PML-null. The PML mRNA levels were similar in both tissue types. The overexpression of PML activated caspase-8 and induced apoptosis in GBM cells. In these cells, PML decreased the expression of transactivated forms of NFkappaB/p65, and c-FLIP gene expression was suppressed. Therefore, PML-induced apoptosis resulted from the suppression of the transcriptional activity of NFkappaB/p65. PML overexpression decreased phosphorylated IkappaBalpha and nuclear NFkappaB/p65 and increased the expression of the suppressor of cytokine signaling (SOCS-1). A proteasome inhibitor blocked the reduction of activated p65 by PML. The reduction of PML is associated with the pathogenesis of GBM. PML induces caspase-8-dependent apoptosis via the repression of NFkappaB activation by which PML facilitates the proteasomal degradation of activated p65 and the sequestration of p65 with IkappaBalpha in the cytoplasm. This novel mechanism of PML-regulated apoptosis may represent a therapeutic target for GBM.

Association of FOXJ1 polymorphisms with systemic lupus erythematosus and rheumatoid arthritis in Korean population

The forkhead-box J1 (FOXJ1) transcription factor could suppress a spontaneous activation of T cells and B cells through an induction of IkappaBbeta that results in repression of NF-kappaB activity. In Foxj1 deficiency mice, systemic autoimmune inflammation is quite common symptom. Therefore, deregulated Foxj1 is supposed to be associated with autoimmune diseases and/or other inflammatory diseases. Previously, we identified that polymorphisms of human FOXJ1 gene (g.??460C>T, g.1805G>T and g.3375G>C) are associated with allergic rhinitis in a Korean population. In present study, we compared the genotype and allele frequencies of these SNPs between healthy controls and systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA) patients. We also investigated the relationships between each genotype and the expression levels of anti- nuclear antibodies in SLE patients, and rheumatoid factor and anti-cyclic citrullinated peptide in RA patients. The frequencies of haplotypes constructed by these FOXJ1 SNPs were compared between controls and SLE (or RA) patients. The results of genotype and allele analysis showed that the prevalence of polymorphism g.3375G>C was associated with the susceptibility of SLE (P = 0.0072 and 0.0042, respectively). But no significant association was found with RA. In the haplotype analysis, however, the main CGG showed a weak association between controls and RA patients (P = 0.048).

Therapeutic Effects of Guggul and Its Constituent Guggulsterone: Cardiovascular Benefits

Oleogum resin (known as guggul) from the guggul tree, Commiphora mukul, found in India, Bangladesh, and Pakistan, has been used to treat various diseases including hyper-cholesterolemia, atherosclerosis, rheumatism, and obesity over several thousands of years. Guggulsterone isolated from guggul has been identified as the bioactive constituent responsible for guggul's therapeutic effects. Since the first study demonstrating the therapeutic effects of guggul in an animal model in 1966, numerous preclinical and clinical trails have been carried out. Although differences in study design, methodological quality, statistical analysis, sample size, and subject population result in certain inconsistencies in the response to therapy, the cumulative data from in vitro, preclinical, and clinical studies largely support the therapeutic claims for guggul described in the ancient Ayurvedic text. However, future clinical studies with much larger size and longer term are required to confirm these claims. The cardiovascular benefits of the therapy are derived from the multiple pharmacological activities associated with guggul or guggulsterone, notably its hypolipidemic, antioxidant, and antiinflammatory activities. It has been established that guggulsterone is an antagonist at farnesoid x receptor (FXR), a key transcriptional regulator for the maintenance of cholesterol and bile acid homeostasis. The FXR antagonism by guggulsterone has been proposed as a mechanism for its hypolipidemic effect. A recent study demonstrates that guggulsterone upregulates the bile salt export pump (BSEP), an efflux transporter responsible for removal of cholesterol metabolites, bile acids from the liver. Such upregulation of BSEP expression by guggulsterone favors cholesterol metabolism into bile acids, and thus represents another possible mechanism for its hypolipidemic activity. Guggulsterone has been found to potently inhibit the activation of nuclear factor-kappaB (NF-kappaB), a critical regulator of inflammatory responses. Such repression of NF-kappaB activation by guggulsterone has been proposed as a mechanism of the antiinflammatory effect of guggulsterone.

Identification of single nucleotide polymorphisms in FOXJ1 and their association with allergic rhinitis

Forkhead-box J1 (FOXJ1) is a presumed transcription factor that can suppress T cell activity, at least partially, through the repression of NFkappaB activity. Thus, dysregulation of FOXJ1 is thought to be associated with autoimmune diseases and/or other inflammatory diseases. To investigate the association between single nucleotide polymorphisms (SNPs) of human FOXJ1 and allergic rhinitis, we scanned the whole human FOXJ1 gene, including the promoter region, by direct sequencing of DNA from 32 individuals. We identified seven SNPs, three of which (g.-460C>T, g.1805G>T, and g.3375G>C) were chosen for large sample size genotyping (n=713), and to assess the genotype frequencies of these SNPs between controls and allergic rhinitis patients. We also investigated the relationships of each genotype with serum total IgE levels in allergic rhinitis patients, and compared the frequencies of haplotypes constructed by these SNPs between the two groups. Our results suggest that the SNPs g.-460C>T, g.1805G>T and g.3375G>C in the human FOXJ1 gene might be associated with susceptibility to allergic rhinitis (P=0.0184, 0.0076, and 0.0143, respectively). The main haplotype, CGG, also revealed a significant association with allergic rhinitis (P=0.000018). However, no significant association was found between serum total IgE levels and the genotypes of these polymorphisms.

Inhibition of the Nuclear Factor-κB Signaling Pathway by Leflunomide or Triptolide also Inhibits the Anthralin-Induced Inflammatory Response but Does Not Affect Keratinocyte Growth Inhibition

We performed this study to determine the relationship between activation of nuclear factor (NF)-kappaB and inhibition of keratinocyte growth by anthralin, which not only might be useful for a better understanding of the role of NF-kappaB in the pathogenesis of psoriasis, but also indicate whether the inflammatory reaction induced by anthralin is inseparable from its antipsoriatic activity. The involvement of NF-kappaB was assessed using the antipsoriatic drugs leflunomide and triptolide (T0) as effectors, since they can inhibit NF-kappaB activation induced by anthralin. The results showed that the inhibition of keratinocyte growth by anthralin was not related to the activation of NF-kappaB. Using sodium salicylate, a known NF-kappaB inhibitor, further confirmed this conclusion. Thus it might be possible to inhibit the inflammatory response induced by anthralin via repression of NF-kappaB activation. We found that leflunomide or T0 could significantly inhibit the mRNA overexpression of interleukin-8 and intercellular adhesion molecule-1 in keratinocytes induced by anthralin. Taken together, our data indicate that the growth inhibition of anthralin is related to the NF-kappaB-independent signaling pathway, and that leflunomide or T0 could control proinflammatory cytokine expression induced by anthralin via inhibiting the activation of NF-kappaB.

Repression of oxidant-induced nuclear factor-kappaB activity mediates placental cytokine responses in gestational diabetes.

Although oxidative stress has been implicated in the pathogenesis of type 2 diabetes, limited data are available regarding its role in gestational diabetes mellitus (GDM), a disease of similar pathophysiology. The proinflammatory cytokines TNFalpha, IL-6, and IL-8 are released from the placenta at term and have been implicated in and/or associated with various metabolic events, including decreased insulin sensitivity. Previously we reported differences in the ex situ release of proinflammatory cytokines from placental and adipose tissues obtained from women with and without GDM. We proposed that these differences reflect preexposure and/or adaptation to oxidative stress by GDM tissues. In this study, we tested the hypothesis that placental tissue from women with GDM is less responsive to oxidative stress than tissue from normal women. Under basal conditions, release of TNFalpha, IL-6, and IL-8 was similar in both control and GDM groups. However, 8-isoprostane release was 2-fold greater in the GDM group (P < 0.01). In response to oxidative stress, TNFalpha and 8-isoprostane release and nuclear factor-kappaB (NF-kappaB) DNA-binding activity were significantly increased in normal tissues (20-fold, 2-fold, and 35%, respectively, P < 0.01). In contrast, the response of GDM tissues to oxidant stress was blunted, with no change in 8-isoprostane release, a 4-fold increase in TNFalpha release, and a 40% reduction in NF-kappaB DNA-binding activity. These data support the hypothesis that placentae from women with GDM display a reduced capacity, mediated by repression of NF-kappaB activity, to respond to oxidative stress.

Bcl-3 and NFκB p50-p50 Homodimers Act as Transcriptional Repressors in Tolerant CD4+ T Cells

The transcriptional events that control T cell tolerance are still poorly understood. To investigate why tolerant T cells fail to produce interleukin (IL)-2, we analyzed the regulation of NFkappaB-mediated transcription in CD4(+) T cells after tolerance induction in vivo. We demonstrate that a predominance of p50-p50 homodimers binding to the IL-2 promoter kappaB site in tolerant T cells correlated with repression of NFkappaB-driven transcription. Impaired translocation of the p65 subunit in tolerant T cells was a result from reduced activation of IkappaB kinase and poor phosphorylation and degradation of cytosolic IkappaBs. Moreover, tolerant T cells expressed high amounts of the p50 protein. However, the increased expression of p50 could not be explained by activation-induced de novo synthesis of the precursor p105, which was constitutively expressed in tolerant T cells. We also demonstrate the exclusive induction of the IkappaB protein B cell lymphoma 3 (Bcl-3) in tolerant T cells as well as its specific binding to the NFkappaB site. These results suggest that the cellular ratio of NFkappaB dimers, and thus the repression of NFkappaB activity and IL-2 production, are regulated at several levels in tolerant CD4(+) T cells in vivo.

TNF-alpha-induced sphingosine 1-phosphate inhibits apoptosis through a phosphatidylinositol 3-kinase/Akt pathway in human hepatocytes.

Human hepatocytes usually are resistant to TNF-alpha cytotoxicity. In mouse or rat hepatocytes, repression of NF-kappaB activation is sufficient to induce TNF-alpha-mediated apoptosis. However, in both Huh-7 human hepatoma cells and Hc human normal hepatocytes, when infected with an adenovirus expressing a mutated form of IkappaBalpha (Ad5IkappaB), which almost completely blocks NF-kappaB activation, >80% of the cells survived 24 h after TNF-alpha stimulation. Here, we report that TNF-alpha activates other antiapoptotic factors, such as sphingosine kinase (SphK), phosphatidylinositol 3-kinase (PI3K), and Akt kinase. Pretreatment of cells with N,N-dimethylsphingosine (DMS), an inhibitor of SphK, or LY 294002, an inhibitor of PI3K that acts upstream of Akt, increased the number of apoptotic cells induced by TNF-alpha in Ad5IkappaB-infected Huh-7 and Hc cells. TNF-alpha-induced activations of PI3K and Akt were inhibited by DMS. In contrast, exogenous sphingosine 1-phosphate, a product of SphK, was found to activate Akt and partially rescued the cells from TNF-alpha-induced apoptosis. Although Akt has been reported to activate NF-kappaB, DMS and LY 294002 failed to prevent TNF-alpha-induced NF-kappaB activation, suggesting that the antiapoptotic effects of SphK and Akt are independent of NF-kappaB. Furthermore, apoptosis mediated by Fas ligand (FasL) involving Akt activation also was potentiated by DMS pretreatment in Hc cells. Sphingosine 1-phosphate administration partially protected cells from FasL-mediated apoptosis. These results indicate that not only NF-kappaB but also SphK and PI3K/Akt are involved in the signaling pathway(s) for protection of human hepatocytes from the apoptotic action of TNF-alpha and probably FasL.

Hepatocytes Sensitized to Tumor Necrosis Factor-α Cytotoxicity Undergo Apoptosis through Caspase-dependent and Caspase-independent Pathways

Hepatocytes can be sensitized to tumor necrosis factor (TNF)-alpha toxicity by repression of NF-kappaB activation or inhibition of RNA synthesis. To determine whether both forms of sensitization lead to TNF-alpha cytotoxicity by similar mechanisms, TNF-alpha-induced cell death in RALA255-10G hepatocytes was examined following infection with an adenovirus, Ad5IkappaB, that blocks NF-kappaB activation or following cotreatment with actinomycin D (ActD). TNF-alpha treatment of Ad5IkappaB-infected cells resulted in 44% cell death within 6 h. ActD/TNF-alpha induced no death within 6 h but did lead to 37% cell death by 24 h. In both instances, cell death occurred by apoptosis and was associated with caspase activation, although caspase activation in ActD-sensitized cells was delayed. CrmA and chemical caspase inhibitors blocked Ad5IkappaB/TNF-alpha-induced cell death but did not inhibit ActD/TNF-alpha-induced apoptosis. A Fas-associated protein with death domain (FADD) dominant negative decreased Ad5IkappaB/TNF-alpha- and ActD/TNF-alpha-induced cell death by 81 and 47%, respectively. However, downstream events differed, since Ad5IkappaB/TNF-alpha but not ActD/TNF-alpha treatment caused mitochondrial cytochrome c release. These results suggest that NF-kappaB inactivation and inhibition of RNA synthesis sensitize RALA255-10G hepatocytes to TNF-alpha toxicity through distinct cell death pathways that diverge below the level of FADD. ActD-induced hepatocyte sensitization to TNF-alpha cytotoxicity occurs through a FADD-dependent, caspase-independent pathway of apoptosis.

Induction of TNFalpha in macrophages by vanadate is dependent on activation of transcription factor NF-kappaB and free radical reactions.

Vanadium-induced TNFalpha production is believed to play an important role in respiratory disease associated with air pollution and occupational exposure. While vanadium is able to induce TNFalpha in macrophages or airway epithelial cells, the underlying mechanism is not well defined. In the present study, mechanisms of vanadate-induced TNFalpha production were analyzed in the murine Raw264.7 cells. Vanadate induces a significant amount of TNFalpha at both the protein and mRNA levels, and the induction is vanadate dose-dependent. The mechanism analysis was focused on transcriptional regulation of TNFalpha gene by vanadate. Transient transfection studies show that the TNFalpha gene promoter was activated by vanadate and this activation was associated with an increase in DNA binding activity of the nuclear factor-kappaB (NF-kappaB). Mutation of the NF-kappaB binding site in the gene promoter led to a loss of the promoter responsiveness to vanadate, indicating requirement of NF-kappaB. This is supported by evidence that inhibition of NF-kappaB activation by SN50, a specific NF-kappaB inhibitor, resulted in a decrease in the TNFalpha production. A role of reactive oxygen species (ROS) was explored in vanadate activity. The result shows that vanadate-induced TNFalpha production is elevated by NADPH, which enhances vanadate-mediated generation of ROS, but is inhibited by an antioxidant, N-acetyl-L-cysteine (NAC). Modification of TNFalpha production is associated with an enhancement or a repression of NF-kappaB activity by NADPH or NAC, respectively. Taken together, these results indicate that: (a) activation of the TNFalpha gene promoter contributes to the vanadate-induced TNFalpha production; (b) NF-kappaB is required for the vanadate-induced promoter activity of TNFalpha gene; (c) free radical reactions are involved in the vanadate-induced TNFalpha production and NF-kappaB activation.