Nuclear Translocation Of P65 Subunit Research Articles

Signaling from the Substrate

Atherosclerotic plaques, which develop in response to a localized inflammatory response, occur at regions of disturbed blood flow. Fluid shear stress stimulates binding of endothelial cell integrins to the subendothelial extracellular matrix (ECM), leading to activation of the nuclear factor κB (NF-κB) signaling pathway and transcription of target genes such as ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1). Noting that endothelial cells express multiple integrins that preferentially bind to various matrix proteins and that inflammation promotes deposition of fibronectin and fibrinogen into the subendothelial ECM, Orr et al. investigated the role of the subendothelial ECM in mediating the response to shear stress. Changes in subendothelial matrix composition and activation of NF-κB target genes occurred at regions of disturbed flow in vivo before other atherosclerotic changes and were most pronounced in atherosclerosis-prone mice fed a high-fat diet. Fluid shear stress promoted phosphorylation of the NF-κB p65 subunit and NF-κB nuclear translocation in bovine aortic endothelial (BAE) cells cultured on fibrinogen or fibronectin. In contrast, shear stress--acting through integrin α2β1--promoted p38 mitogen-activated protein kinase activation in BAE cells grown on collagen, leading to reduced NF-κB activation. In cells grown on mixed matrices, collagen suppressed fibronectin-dependent NF-κB activation. However, growth on collagen failed to inhibit activation of NF-κB following exposure to tumor necrosis factor α; immunocytochemical analysis indicated that localized activation of p38 at integrin adhesion sites inhibited local--but not global--NF-κB activation. Intriguingly, NF-κB activation in cells grown on fibronectin was blocked by treatment with a fibronectin-derived peptide that alters matrix structure and stimulates p38 activation, which suggests that modification of the subendothelial matrix with external factors could provide a novel approach to treating atherosclerosis. A. W. Orr, J. M. Sanders, M. Bevard, E. Coleman, I. J. Sarembock, M. A. Schwartz, The subendothelial extracellular matrix modulates NF-κB activation by flow: A potential role in atherosclerosis. J. Cell Biol. 169 , 191-202 (2005). [Abstract] [Full Text]

A role for PKCζ in the LPS-induced translocation NF-κB p65 subunit in cultured myometrial cells

Human myometrial cells respond to the endotoxin lipopolysaccharide (LPS) by activation of protein kinase C (PKC) ζ and nuclear translocation of the p65 subunit of NF-kB. Our first objective was to determine the expression of TLR4 in cultured myometrial cells. Positive immunoreactivity observed for TLR4 suggests that myometrial cells have the potential to respond to LPS. To confirm that LPS signals via TLR4, the ability of an anti-TLR4 neutralizing antibody to block LPS-induced translocation of p65 was demonstrated. To determine whether LPS-induced nuclear translocation of p65 is mediated through the PKC pathway, myometrial cells were treated with various inhibitors of the PKC isoforms already characterized in human myometrium. Neither the selective conventional PKC inhibitor nor the inhibitor of PKCδ affected NF-kB activation. By contrast, we found that treatment of myometrial cells with an antisense against PKCζ affect LPS-induced nuclear translocation of the p65 subunit of NF-kB. Accordingly, our data support the notion that PKCζ is essential for LPS-induced NF-kB p65 subunit nuclear translocation in human myometrial cells.

Heme inhibits human neutrophil apoptosis: involvement of phosphoinositide 3-kinase, MAPK, and NF-kappaB.

High levels of free heme are found in pathological states of increased hemolysis, such as sickle cell disease, malaria, and ischemia reperfusion. The hemolytic events are often associated with an inflammatory response that usually turns into chronic inflammation. We recently reported that heme is a proinflammatory molecule, able to induce neutrophil migration, reactive oxygen species generation, and IL-8 expression. In this study, we show that heme (1-50 microM) delays human neutrophil spontaneous apoptosis in vitro. This effect requires heme oxygenase activity, and depends on reactive oxygen species production and on de novo protein synthesis. Inhibition of ERK and PI3K pathways abolished heme-protective effects upon human neutrophils, suggesting the involvement of the Ras/Raf/MAPK and PI3K pathway on this effect. Confirming the involvement of these pathways in the modulation of the antiapoptotic effect, heme induces Akt phosphorylation and ERK-2 nuclear translocation in neutrophils. Futhermore, inhibition of NF-kappa B translocation reversed heme antiapoptotic effect. NF-kappa B (p65 subunit) nuclear translocation and I kappa B degradation were also observed in heme-treated cells, indicating that free heme may regulate neutrophil life span modulating signaling pathways involved in cell survival. Our data suggest that free heme associated with hemolytic episodes might play an important role in the development of chronic inflammation by interfering with the longevity of neutrophils.

Redox modulation of NF-kappaB nuclear translocation and DNA binding in metastatic melanoma. The role of endogenous and gamma-glutamyl transferase-dependent oxidative stress.

The transcription factor NF-kappaB is implicated in the expression of genes involved in cell proliferation, apoptosis and metastasis. In melanoma, high constitutive levels of NF-kappaB activation are usually observed. NF-kappaB is regulated by oxidation/reduction (redox) processes, and the occurrence of constitutive oxidative stress in melanoma cells has been documented. Recent studies of our laboratories showed that the membrane-bound gamma-glutamyl transferase (GGT) enzyme activity--expressed by a number of malignancies, including melanoma--can act as a basal source of superoxide, hydrogen peroxide and other prooxidants. In the present study we utilized the 2/60 clone of Me665/2 human metastatic melanoma, which displays high levels of GGT activity, in order to verify if the presence of this enzyme--through the promotion of redox processes--may influence the activation status of NF-kappaB. The latter was evaluated by determining the nuclear translocation of the p65 subunit (by immunoblot), the DNA binding of NF-kappaB (by electrophoretic mobility shift assay) and its transcriptional activity (by gene transactivation studies). Me665/2/60 cells displayed a basal production of hydrogen peroxide. Stimulation of GGT activity by its substrates glutathione and glycyl-glycine caused additional production of hydrogen peroxide, up to levels approx. double the basal levels. Nuclear translocation of the NF-kappaB p65 subunit, DNA-binding and gene transactivation were thus investigated in Me665/2/60 cells whose GGT activity was modulated by means of substrates or inhibitors. Stimulation of GGT activity resulted in increased nuclear translocation of p65, while on the other hand NF-kappaB DNA binding and gene transactivation were paradoxically decreased. NF-kappaB DNA binding could be restored by treating cell lysates with the thiol-reducing agent dithiothreitol (DTT). Treatment of cells with exogenous hydrogen peroxide did not affect NF-kappaB activation status. Altogether, the data obtained indicate that GGT activity may impair the redox status of thiols that is critical for NF-kappaB DNA binding and gene transactivation, through the production of prooxidant species allegedly distinct from hydrogen peroxide. GGT activity therefore appears to be an additional factor in modulation of NF-kappaB transcriptional activity in melanoma, capable of hindering NF-kappaB DNA binding even in conditions where continuous oxidative stress would favor NF-kappaB nuclear translocation.

Inhibition of TNF-α, IL-1β, and IL-6 productions and NF-κB activation in lipopolysaccharide-activated RAW 264.7 macrophages by catalposide, an iridoid glycoside isolated from Catalpa ovata G. Don (Bignoniaceae)

Catalposide, the major iridoid glycoside isolated from the stem bark of Catalpa ovata G. Don (Bignoniaceae), was found to inhibit the productions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), and the activation of nuclear factor κB (NF-κB) in RAW 264.7 macrophages activated with lipopolysaccharide (LPS). Catalposide also inhibited the expressions of TNF-α, IL-1β, and IL-6 genes and the nuclear translocation of p65 subunit of NF-κB in LPS-activated RAW 264.7 cells. Flow cytometric analysis revealed that catalposide suppressed the binding of FITC-conjugated LPS to CD14 on the surface of cells, probably resulting in the inhibitory effects on TNF-α, IL-1β, and IL-6 productions and NF-κB activation. These findings suggest that catalposide could be an attractive candidate for adjunctive therapy in Gram-negative bacterial infections.

Inhibition of TNF-alpha and IL-6 production by Aucubin through blockade of NF-kappaB activation RBL-2H3 mast cells.

Antigen (Ag)-stimulated mast cells induce synthesis and production of cytokines including tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 with proinflammatory and immune regulatory properties. Expression of TNF-alpha and IL-6 was regulated by a transcription factor, nuclear factor (NF)-kappaB. The iridoid glycoside, aucubin, has been found as a natural constituent of many traditional oriental medicinal plants. We studied the effect of aucubin on the TNF-alpha and IL-6 expression in Ag-stimulated rat basophilic leukemia (RBL)-2H3 mast cells. We show that aucubin inhibited Ag-induced TNF-alpha and IL-6 production and expression in a dose-dependent manner with IC(50) of 0.101 and 0.19 microg/ml, respectively. Maximal inhibition of TNF-alpha and IL-6 production was 73 +/- 4.3% and 88.8 +/- 5%, respectively. Aucubin also inhibited Ag-induced nuclear translocation of p65 subunit of NF-kappaB and degradation of IkappaBalpha. Inhibition of NF-kappaB activation by aucubin might be specific since activator protein-1 binding activity was not affected. In conclusion, these results suggest that aucubin is a specific inhibitor of NF-kappaB activation in mast cells, which might explain its beneficial effect in the treatment of chronic allergic inflammatory diseases.

Diesel exhaust particles enhance lung injury related to bacterial endotoxin through expression of proinflammatory cytokines, chemokines, and intercellular adhesion molecule-1.

Epidemiologic studies demonstrate acute and serious adverse effects of particulate air pollution on respiratory health, especially in people who are susceptible to bacterial infection. However, the underlying mechanism remains to be elucidated. To provide experimental evidence for the epidemiologic data, we determined the effects of diesel exhaust particles (DEP), major participants in particulate pollutants, on lung injury related to bacterial endotoxin in mice. Intratracheal instillation of DEPs synergistically enhanced lung injury related to endotoxin from gram-negative bacteria, which was characterized by neutrophil sequestration, interstitial edema, and alveolar hemorrhage. In the presence of endotoxin, DEPs further activated the nuclear translocation of p65 subunit of nuclear factor-kappaB (NF-kappaB) in the lung and increased the lung expression of intercellular adhesion molecule-1, interleukin-1beta, macrophage chemoattractant protein-1, keratinocyte chemoattractant (KC), macrophage inflammatory protein-1alpha, and Toll-like receptors. DEPs given alone increased the lung expression of Toll-like receptor 4 and the nuclear localization of p50 subunit of NF-kappaB. The combined exposure to DEPs and endotoxin decreased nuclear localization of CCAAT/enhancer binding protein beta. These results provide the first experimental evidence that DEPs enhance neutrophilic lung inflammation related to bacterial endotoxin. The enhancement is mediated by the induction of proinflammatory molecules, likely through the expression of Toll-like receptors and the activation of p65-containing dimer(s) of NF-kappaB, such as p65/p50.

VEGI, a new member of the TNF family activates nuclear factor-kappa B and c-Jun N-terminal kinase and modulates cell growth.

Recently a new member of the human tumor necrosis factor (TNF) family named as VEGI was reported. However, very little is known about the biological activities displayed by this cytokine. In this report, we show that in myeloid cells VEGI activated the transcription factor kappa B (NF-kappa B) as determined by the electrophoretic mobility shift assay, induced degradation of I kappa B alpha, and nuclear translocation of p65 subunit of NF-kappa B. VEGI also activated NF-kappa B-dependent reporter gene expression. In addition, VEGI activated c-Jun N-terminal kinase. When examined for growth modulatory effects, VEGI inhibited the proliferation of breast carcinoma (MCF-7), epithelial (HeLa), and myeloid (U-937 and ML-1a) tumor cells; and activated caspase-3 leading to PARP cleavage. VEGI-induced cytotoxicity was potentiated by inhibitors of protein synthesis. VEGI also induced proliferation of normal human foreskin fibroblast cells. The activity of VEGI could neither be neutralized by antibodies against TNF, nor could it compete with TNF binding, indicating that the activity of VEGI is not due to TNF and it binds to a distinct receptor. These results suggest that VEGI, a new member of the TNF family, has a signaling pathway similar to TNF and is most likely a multifunctional cytokine.

IL-13 Suppresses TNF-Induced Activation of Nuclear Factor-κB, Activation Protein-1, and Apoptosis

AbstractIL-13 is known to suppress the production of inflammatory cytokines such as TNF. Whether IL-13 also modulates the biologic effects of TNF is not known. In the present report we examined the effect of IL-13 on TNF-induced activation of nuclear transcription factors NF-κB and activation protein-1 (AP-1) and apoptosis. Pretreatment of cells with IL-13 blocked TNF-induced NF-κB activation, nuclear translocation of p65 subunit, and degradation of IκBα. IL-13 also inhibited NF-κB activation by LPS, okadaic acid, H2O2, and ceramide. TNF-induced NF-κB-dependent gene transcription was also blocked by IL-13. TNF-induced activation of another nuclear transcription factor, AP-1, was suppressed by IL-13. The activation of N-terminal c-Jun kinase and mitogen-activated protein kinase kinase, implicated in the regulation of AP-1 and NF-κB, was also down-regulated by IL-13. TNF-mediated cytotoxicity and activation of caspase-3 were abolished by IL-13. The inhibitory effects of IL-13 on TNF were sensitive to H-7, neomycin, and wortmannin, suggesting that the pathway consisting of protein kinase C, phosphatidylinositol 3-kinase, and phospholipase C must be involved in IL-13 signaling. Thus, overall, these results demonstrate that IL-13 is a potent inhibitor of TNF-mediated activation of NF-κB, AP-1, and apoptosis, which may contribute to its previously described immunosuppressive and anti-inflammatory effects.