NF-kappa B-dependent Transcription Research Articles

Akt‐mediated regulation of NFκB and the essentialness of NFκB for the oncogenicity of PI3K and Akt

The serine/threonine kinase Akt (cellular homolog of murine thymoma virus akt8 oncogene), also known as PKB (protein kinase B), is activated by lipid products of phosphatidylinositol 3-kinase (PI3K). Akt phosphorylates numerous protein targets that control cell survival, proliferation and motility. Previous studies suggest that Akt regulates transcriptional activity of the nuclear factor-kappaB (NFkappaB) by inducing phosphorylation and subsequent degradation of inhibitor of kappaB (IkappaB). We show here that NFkappaB-driven transcription increases in chicken embryonic fibroblasts (CEF) transformed by myristylated Akt (myrAkt). Accordingly, both a dominant negative mutant of Akt and Akt inhibitors repress NFkappaB-dependent transcription. The degradation of the IkappaB protein is strongly enhanced in Akt-transformed cells, and the loss of NFkappaB activity by introduction of a super-repressor of NFkappaB, IkappaBSR, interferes with PI3K- and Akt-induced oncogenic transformation of CEF. The phosphorylation of the p65 subunit of NFkappaB at serine 534 is also upregulated in Akt-transformed cells. Our data suggest that the stimulation of NFkappaB by Akt is dependent on the phosphorylation of p65 at S534, mediated by IKK (IkappaB kinase) alpha and beta. Akt phosphorylates IKKalpha on T23, and this phosphorylation event is a prerequisite for the phosphorylation of p65 at S534 by IKKalpha and beta. Our results demonstrate two separate functions of the IKK complex in NFkappaB activation in cells with constitutive Akt activity: the phosphorylation and consequent degradation of IkappaB and the phosphorylation of p65. The data further support the conclusion that NFkappaB activity is essential for PI3K- and Akt-induced oncogenic transformation.

HOX-Gene Expression and Inhibition of the Non-Canonical NF-KappaB Pathway in t(7;12) Leukemias

The chromosomal translocation t(7;12) was described recently in MLL negative acute myeloid leukemias of infancy associated with a very poor clinical outcome. The rearrangement involves the genes HLXB9 (7q36) and ETV6 (12p13) with a fusion transcript of exon1/HLXB9 and exon3/ETV6. An alternative in frame splicing variant of exon1/HLXB9 to exon2/ETV6 is also detectable. Leukemic bone marrow samples of 42 infants, diagnosed in Germany with AML, were screened for the fusion transcript HLXB9/ETV6 with RT-PCR. Inclusion criteria were diagnosis of AML, age<2 years, no MLL/AF9, MLL/AF4 or MLL/ENL rearrangement in RT-PCR, no chromosomal 11q23 rearrangement and no trisomy 21. Positive samples were cloned and sequenced. A fusion transcript was found in approx. ~17% of patients. Common features of HLXB9/ETV6 positive leukemias in this cohort are poorly differentiated FAB subtypes, trisomy 19 karyotype of the leukemic cells, coexpression of the T-cell markers CD4 and CD7 as well as high expression of the un-rearranged HLXB9 gene. Quantitative PCR showed no upregulation of HOXA9 and MEIS1 in HLXB9/ETV6 positive patients as reported for MLL+ leukemias. The mechanism of HLXB9/ETV6 induced leukemogenesis remains unknown. We show that in vitro retroviral transduction of murine hematopoietic precursor cells with either HLXB9/ETV6 or HLXB9 or ETV6/RUNX1 was not transforming. Furthermore altered activity of the transcription factor family NF-kappaB has been demonstrated for various types of cancer including acute myeloid leukemias. Therefore we analysed the impact of the HLXB9/ETV6 fusion on the NF-kappaB signaling pathway in a 293T cell-system. HLXB9/ETV6 and HLXB9 showed inhibition of the NF-kappaB dependent transcription signal in a reporter assay, whereas the typical ALL-fusion ETV6/RUNX1 showed no influence. Analysis of nuclear extracts by western blot confirmed, that the inhibition of the NF-kappaB signal is due to a reduced nuclear translocation of the non-canonical NF-kappaB components p52 and RelB, whereas the canonical pathway remains not influenced.Conclusion: The HLXB9/ETV6 fusion transcript can be found in 17% of infants with MLL-negative AML in a German cohort. HLXB9/ETV6 positive leukemias show no increased expression on HOXA9 and MEIS1, but coexpression of T-cell markers and inhibition of the non-canonical NF-kappaB pathway on protein level. HLXB9/ETV6 did not induce malignant transformation in murine hematopoietic precursor cells.

Fish oil targets PTEN to regulate NFκB for downregulation of anti-apoptotic genes in breast tumor growth

The molecular mechanism for the beneficial effect of fish oil on breast tumor growth is largely undefined. Using the xenograft model in nude mice, we for the first time report that the fish oil diet significantly increased the level of PTEN protein in the breast tumors. In addition, the fish oil diet attenuated the PI 3 kinase and Akt kinase activity in the tumors leading to significant inhibition of NFkappaB activation. Fish oil diet also prevented the expression of anti-apoptotic proteins Bcl-2 and Bcl-XL in the breast tumors with concomitant increase in caspase 3 activity. To extend these findings we tested the functional effects of DHA and EPA, the two active omega-3 fatty acids of fish oil, on cultured MDA MB-231 cells. In agreement with our in vivo data, DHA and EPA treatment increased PTEN mRNA and protein expression and inhibited the phosphorylation of p65 subunit of NFkappaB in MDA MB-231 cells. Furthermore, DHA and EPA reduced expression of Bcl-2 and Bcl-XL. NFkappaB DNA binding activity and NFkappaB-dependent transcription of Bcl-2 and Bcl-XL genes were also prevented by DHA and EPA treatment. Finally, we showed that PTEN expression significantly inhibited NFkappaB-dependent transcription of Bcl-2 and Bcl-XL genes. Taken together, our data reveals a novel signaling pathway linking the fish oil diet to increased PTEN expression that attenuates the growth promoting signals and augments the apoptotic signals, resulting in breast tumor regression.

Human Immunodeficiency Virus Type 1 Tat Protein Inhibits the SIRT1 Deacetylase and Induces T Cell Hyperactivation

Symptoms of T cell hyperactivation shape the course and outcome of HIV-1 infection, but the mechanism(s) underlying this chronic immune activation are not well understood. We find that the viral transactivator Tat promotes hyperactivation of T cells by blocking the nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase SIRT1. Tat directly interacts with the deacetylase domain of SIRT1 and blocks the ability of SIRT1 to deacetylate lysine 310 in the p65 subunit of NF-kappaB. Because acetylated p65 is more active as a transcription factor, Tat hyperactivates the expression of NF-kappaB-responsive genes, a function lost in SIRT1-/- cells. These results support a model where the normal function of SIRT1 as a negative regulator of T cell activation is suppressed by Tat during HIV infection. These events likely contribute to the state of immune cell hyperactivation found in HIV-infected individuals.

NF-κB is a critical regulator of the survival of rodent and human hepatic myofibroblasts

Hepatic myofibroblast activation during injury causes deposition of extracellular matrix within the liver and promotes development of fibrosis. Hepatic myofibroblast apoptosis is associated with remodelling of fibrotic extracellular matrix and regression of fibrosis. Previous work showed that inhibition of constitutive NF-kappaB signaling promotes hepatic myofibroblast apoptosis and resolution of fibrosis in rodent models. However, to date agents used to target constitutive NF-kappaB transcriptional activity in hepatic myofibroblasts have been relatively non-specific with potential for off-target effects that may complicate data interpretation. Likewise, rat chronic liver disease models may not accurately recapitulate the activation of human hepatic myofibroblasts. We used a mutant recombinant IkappaBalpha super-repressor fused to the HIV-TAT domain to specifically target NF-kappaB signaling in hepatic myofibroblasts. Inhibition of NF-kappaB activity was measured using reporter assay. Apoptosis of hepatic myofibroblasts was assessed by morphological changes, cleavage of the PARP-1 protein and Caspase 3 activation. TAT-IkappaBalphaSR reduced NF-kappaB dependent transcription, Bcl-2 expression and promoted Jun-N-terminal kinase-dependent apoptosis in human and rat hepatic myofibroblasts. These data highlight the conserved role of NF-kappaB during fibrogenesis. Our data validate the use of rodent models for pre-clinical testing of NF-kappaB inhibitors as anti-fibrotics and stimulators of fibrotic extracellular matrix remodelling.

Improved Vascular Function upon Pioglitazone Treatment in Type 2 Diabetes is not Associated with Changes in Mononuclear NF-κB Binding Activity

Thiazolidinediones such as pioglitazone have been shown to exert anti-inflammatory effects independent of their insulin sensitizing effects by reducing activation of the proinflammatory transcription factor NF-kappaB in animal models of experimental diabetes. Furthermore, short-term pioglitazone treatment ameliorates endothelial dysfunction in conduit arteries of patients with type 2 diabetes. Since inflammation is supposed to impair flow-mediated vasodilatation, we studied the effects of an 8-week pioglitazone intervention on endothelial function and mononuclear NF-kappaB activation in patients with type 2 diabetes. Twenty patients were included in a randomized, double-blind, placebo-controlled study receiving 30 mg pioglitazone or placebo, respectively. Flow-mediated endothelium dependent vasodilatation (FMD) of the brachial artery, NF-kappaB binding activity in peripheral blood mononuclear cells [pBMC, determined by electrophoretic mobility shift assay (EMSA)] and interleukin-6 (IL-6)-transcription rates (determined by real-time PCR) were measured at study entry and after eight weeks of intervention. Pioglitazone treatment resulted in a significant improvement of FMD (4.3%+/-3.3; p=0.003), while no effect was seen under placebo medication (2.0%+/-2.7; p=0.71). The correction of FMD was neither paralleled by a pioglitazone-dependent reduction in mononuclear NF-kappaB binding activity (DeltaNF-kappaB activity: pioglitazone: 9.2%+/-6.7, p=0.24; placebo: 5.7%+/-19.6; p=0.82) nor in NF-kappaB dependent gene transcription as determined for IL-6 (DeltaIL-6 pioglitazone: +1.8%+/-12.0, p=0.93; placebo: -0.2%+/-9.7; p=0.92). These data demonstrate for the first time that pioglitazone treatment improves endothelial dysfunction in patients with type 2 diabetes without affecting NF-kappaB binding activity and NF-kappaB dependent proinflammatory gene expression in pBMC.

Neddylation of a breast cancer-associated protein recruits a class III histone deacetylase that represses NFκB-dependent transcription

Neddylation has an important role in ubiquitin-mediated protein degradation through modification of cullins, which are the main substrates for NEDD8 modification. Here, we show that breast cancer-associated protein 3 (BCA3) is a NEDD8 substrate. BCA3 suppressed NFkappaB-dependent transcription through its ability to bind to p65 and the cyclin D1 promoter in a neddylation-dependent manner. Transcriptional suppression mediated by BCA3 may be attributed to the ability of neddylated BCA3 to recruit SIRT1, a class III histone deacetylase. Silencing of endogenous BCA3 in DU145 and MCF7 cells enhanced NFkappaB transcription and inhibited tumour necrosis factor (TNF)alpha-induced apoptosis. Conversely, BCA3 silencing could be reversed by over-expression of wild-type BCA3 and SENP8, a NEDD8-specific protease, but not by neddylation-deficient BCA3 or a SENP8 mutant. These results provide a crucial link between neddylation and transcriptional regulation by SIRT1, a NAD-dependent histone deacetylase that prolongs life span in yeast and worms.

Identification of Novel and Cell Type Enriched Cofactors of the Transcription Activation Domain of RelA (p65 NF-κB)

RelA (NF-kappaB) is a transcription factor inducible by distinct stimuli in many different cell types. To find new cell type specific cofactors of NF-kappaB dependent transcription, we isolated RelA transcription activation domain binding proteins from the nuclear extracts of three different cell types. Analysis by electrophoresis and liquid chromatography tandem mass spectrometry identified several novel putative molecular partners. Some were strongly enriched in the complex formed from the nuclear extracts of specific cell types.

Validation of IKKβ as therapeutic target in airway inflammatory disease by adenoviral‐mediated delivery of dominant‐negative IKKβ to pulmonary epithelial cells

Asthma is an inflammatory disease of the lungs and the transcription factor NF-kappa B regulates the production of numerous inflammatory mediators that may have a role in the pathogenesis of asthma. Hence, the signalling pathways leading to NF-kappa B activation are considered prime targets for novel anti-inflammatory therapies. The prevention of NF-kappa B activity in mice, through the knockout of IKK beta or p65, causes fatal liver degeneration in utero making it difficult to determine the full implications of inhibiting NF-kappaB activity in tissues physiologically relevant to human diseases. This study used adenovirus delivery of a dominant inhibitor of NF-kappaB (I kappa B alpha delta N) and dominant-negative IKK alpha (IKK alpha(KM)) and IKK beta (IKK beta(KA)) to investigate the role of the individual IKKs in NF-kappa B activation and inflammatory gene transcription by human pulmonary A549 cells. Overexpression of IKK beta(KA) or I kappa B alpha delta N prevented NF-kappa B-dependent transcription and DNA binding. IKK beta(KA) also prevented I kappa B alpha kinase activity. Similarly, IKK beta(KA) and I kappa B alpha delta N overexpression also inhibited IL-1beta- and TNF alpha-dependent increases in ICAM-1, IL-8 and GM-CSF in addition to IL-1beta-mediated increases in cyclooxygenase-2 expression, whereas IKK alpha(KM) overexpression had little effect on these outputs. IKK beta(KA) also reduced cell viability and induced caspase-3 and PARP cleavage regardless of the stimuli, indicating the induction of apoptosis. This effect seemed to be directly related to IKK beta kinase activity since I kappa B alpha delta N only induced PARP cleavage in TNF alpha-treated cells. These results demonstrate that inhibition of IKK beta and NF-kappa B suppresses inflammatory mediator production and reduces A549 cell viability. Thus, novel therapies that target IKK beta could have potent anti-inflammatory effects and may be beneficial in the treatment of certain cancers.

Intracellular LPS inhibits the activity of potassium channels and fails to activate NFkappaB in human macrophages.

Although much has been learned about signal transduction mechanisms and binding proteins involved in lipopolysaccharides (LPS)-induced activation of monocytes/macrophages, little is known about the ability of internalized LPS to activate cells. To approach this question we either exposed macrophages to LPS or microinjected the cells with LPS before studying early cellular events associated with LPS-mediated macrophage activation. We measured membrane currents and translocation of NFkappaB to the nucleus. Using the whole-cell patch clamp technique ion channels were analyzed and characterized as K+ sensitive inward and outward currents. Exogenous LPS was shown to increase the voltage-dependent outward current whereas the voltage-dependent inward current was unaffected. However when cells were microinjected with LPS both inward and outward current were completely abolished. The presence of LPS within the cells did not prevent them to perform phagocytosis or to respond to fMLP with an appropriate increase in [Ca2+]i. The immunocytological detection of NFkappaB p65 translocation revealed that exogenous LPS led to the nuclear localization of the p65 subunit of NFkappaB, whereas only the cytoplasmic localization of p65 was observed following microinjection of LPS. These data show that one major process in macrophage activation, the NFkappaB dependent transcription of a number of genes encoding for many inflammatory mediators cannot be induced by intracellular LPS but requires the interaction of LPS with external membrane components. However intracellular LPS causes a drastic decrease in potassium currents which by keeping the cell membrane at a depolarized potential may result in changed biological answers of these cells.

Flavones Mitigate Tumor Necrosis Factor-α-Induced Adhesion Molecule Upregulation in Cultured Human Endothelial Cells: Role of Nuclear Factor-κB

Flavones have been classified as anti-atherogenic agents that inhibit monocyte adhesion to stimulated endothelium, possibly by blocking induction of cell adhesion molecules (CAM). This anti-atherogenic feature of these flavonoids appears to be related to their chemical structures. Flavones may interfere with key signaling events involved in endothelial cell activation by inflammatory mediators. This study examined the effects of flavones on the induction of CAM and the translocation and DNA binding of nuclear factor-kappa B (NF-kappa B) in TNF-alpha-activated human umbilical vein endothelial cells (HUVEC). The effects of flavones, luteolin and apigenin, on adhesion of THP-1 monocytes to the TNF-alpha-activated HUVEC, protein expression and mRNA levels of vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin, and nuclear appearance and DNA binding activity of NF-kappa B were determined. Flavanols, flavonols, and flavanones were used for comparison. TNF-alpha significantly induced HUVEC protein expression of VCAM-1, ICAM-1, and E-selectin with increasing mRNA levels. Luteolin and apigenin inhibited the TNF-alpha-induced upregulation of THP-1 adhesion and VCAM-1 expression; these inhibitory effects were dose-dependent. The flavones at doses of > or =25 micromol/L almost completely abolished the increased CAM protein and mRNA regardless of their anti-oxidative activity. With the exception of the flavonol quercetin, flavonoids had no such effect; quercetin substantially attenuated the CAM induction. The flavones inhibited nuclear translocation and DNA binding activity of the NF-kappa B-containing binding site in the promoter region of the CAM genes in TNF-alpha-activated HUVEC. The inhibition of endothelial CAM induction by flavones is mediated by their interference with the NF-kappa B-dependent transcription pathway. Thus, the flavones may hamper initial atherosclerotic events involving endothelial CAM induction.

Ineffectiveness of Histone Deacetylase Inhibitors to Induce Apoptosis Involves the Transcriptional Activation of NF-κB through the Akt Pathway

Histone deacetylase (HDAC) inhibitors are emerging as a new class of anticancer agents for the treatment of solid and hematological malignancies. Although HDAC inhibitors induce cell death through an apoptotic process, little is known about the molecular events that control their effectiveness. In this study, we demonstrate that HDAC inhibitors are limited in their ability to induce apoptosis in non-small cell lung cancer (NSCLC) cell lines despite their ability to effectively inhibit deacetylase activity. Because the anti-apoptotic transcription factor NF-kappa B has been shown to be under the control of HDAC-mediated repression, we analyzed whether HDAC inhibitors activated NF-kappa B in NSCLC cells. HDAC inhibitors effectively stimulated endogenous NF-kappa B-dependent gene expression by up-regulating IL-8, Bcl-XL, and MMP-9 transcripts. The ability of HDAC inhibitors to increase NF-kappa B transcriptional activity was not associated with signaling events that stimulated nuclear translocation, but rather modulated the transactivation potential of the RelA/p65 subunit of NF-kappa B. The inhibition of HDAC activity was associated with the recruitment of the p300 transcriptional co-activator to chromatin in an Akt-dependent manner. Moreover, Akt directly phosphorylated p300 in vitro and was required for stimulating the transactivation potential of the co-activator following the addition of HDAC inhibitors. Selective inhibition of either the phosphoinositide 3-kinase/Akt pathway, or NF-kappa B itself blocked the ability of HDAC inhibitors to activate NF-kappa B and dramatically sensitized NSCLC cells to apoptosis following of the addition of HDAC inhibitors. Our study indicates that the ineffectiveness of HDAC inhibitors to induce apoptosis in NSCLC cancer cells is associated with the ability of these molecules to stimulate NF-kappa B-dependent transcription and cell survival.

Endothelial activation by angiotensin II through NFkappaB and p38 pathways: Involvement of NFkappaB-inducible kinase (NIK), free oxygen radicals, and selective inhibition by aspirin.

Angiotensin-II (AII), the dominant effector of the renin-angiotensin system, is involved in the pathogenesis of cardiovascular diseases, such as atherosclerosis. Upregulation of the adhesion molecules VCAM-1, ICAM-1, and E-selectin in endothelial cells by inflammatory cytokines through nuclear factor kappa B (NFkappaB) activation is implicated in formation and progression of atherosclerotic plaque. Here we show that AII induces NFkappaB-dependent transcription in primary endothelial cell lines, leading to the upregulation of ICAM-1 and VCAM-1 expression. NFkappaB activation by AII is mediated by the NFkappaB-inducing kinase (NIK), a common mediator of NFkappaB activation by inflammatory cytokines, such as TNF-alpha. However, NFkappaB stimulation by AII differs from that of TNF-alpha since a TNF-receptor associated factor 2 (TRAF-2) dominant negative mutant does not prevent AII-mediated NFkappaB activation. In analogy with TNF-alpha-dependent activation of NFkappaB, treatment with either the anti-oxidant N-acetyl cysteine (NAC) or the cyclooxygenase (COX) inhibitor acetyl salicylic acid (aspirin), but not indometacin, prevents the induction of NFkappaB-dependent transcription by AII. Thus, production of reactive oxygen species, aspirin (asp)-sensitive enzymes of the arachidonate metabolism, and NIK are common transducers of AII- and TNF-dependent pathways to NFkappaB. AII also activates the inflammatory p38 kinase in endothelial cells, an effect inhibited by exposure to either NAC or asp. Pharmacological interference of the p38 pathway, with the inhibitor SB 202190, prevented AII-mediated activation of the NFkappaB target V-CAM, without affecting degradation of IkappaBalpha. These results support a pro-inflammatory effect of the vasoactive peptide AII in endothelial cells, through at least two pathways-NFkappaB and p38-both of which are sensitive to asp and antioxidants.

Tranilast inhibits cytokine-induced nuclear factor kappaB activation in vascular endothelial cells.

Tranilast [N-(3,4-dimethoxycinnamoyl)anthranilic acid] inhibits vascular inflammation. However, the relevant anti-inflammatory mechanisms are not completely understood. We studied the effects of tranilast on nuclear factor-kappaB (NF-kappaB)-dependent endothelial cell adhesion molecule expression and transcriptional regulation. Cultured human umbilical vein endothelial cells were preincubated with 12.5 to 100 microg/ml tranilast. Tumor necrosis factor-alpha (TNF-alpha)-induced endothelial VCAM-1, ICAM-1, and E-selectin surface expression was inhibited dose dependently. Maximal inhibition achieved with 100 microg/ml tranilast was 38 +/- 6.9, 31.8 +/- 1.5, and 31.9 +/- 1.9%, respectively (mean +/- S.E.M., p < 0.001, n = 5). Secretion of interleukin 6, which is also NF-kappaB-sensitive, was significantly inhibited by tranilast. Endothelial MHC-I expression, which is independent of NF-kappaB, was not inhibited. Although cytokine-induced degradation of NF-kappaB inhibitor proteins (IkappaB-alpha, -beta, and -epsilon), nuclear translocation of NF-kappaB, and binding of NF-kappaB to kappaB cis-acting elements in the adhesion molecule promoters were not affected by tranilast, ICAM-1-kappaB and E-selectin-kappaB reporter gene activity was inhibited by 53% (n = 5, p < 0.01) and 51% (n = 5, p < 0.001), respectively. In contrast, using SP-1 and C/EBP constructs, reporter gene activity was not altered. Expression of the transcriptional coactivator cAMP response element binding protein binding protein (CBP) was inhibited by tranilast, resulting in a loss of interaction between NF-kappaB and CBP. Therefore, in therapeutically relevant concentrations (50 microg/ml), tranilast inhibits NF-kappaB-dependent transcriptional activation by interfering with the NF-kappaB/CBP association. We propose that inhibition of NF-kappaB dependent gene transcription contributes to the anti-inflammatory effects of tranilast.

Activation of the transcription factor NF-kappaB by Campylobacter jejuni.

Campylobacter jejuni is a food-borne pathogen responsible for infectious enterocolitis. The early-response transcription factor NF-kappa B triggers the expression of genes associated with cellular immune and inflammatory responses. Co-incubation of HeLa cells with viable C. jejuni leads to the activation of the transcription factor NF-kappa B as determined by specific induction of a cellular luciferase-based reporter. Boiled cell-free extracts of C. jejuni are also potent dose-dependent stimulators of NF-kappa B-dependent transcription, the levels of which can reach up to 1000-fold as compared with independent controls. Using both cultured HeLa cells and human colonic epithelial (HCA-7) cells, the activation of NF-kappa B by C. jejuni boiled extract has been monitored through the degradation of IKB alpha and DNA binding of the nuclear translocated p50/p65 heterodimer of NF-kappa B. These events are co-ordinated with elaboration of the pro-inflammatory cytokine interleukin-8. Fractionation of the boiled C. jejuni extract suggests that the majority of the bioactive component has a molecular mass of 3 kDa or less, which is insensitive to proteinase K treatment.

Tissue-specific glucocorticoid resistance-hypersensitivity syndromes: Multifactorial states of clinical importance

Tissue-specific glucocorticoid resistance-hypersensitivity syndromes: Multifactorial states of clinical importance

PYK2 Links Gqα and G13α Signaling to NF-κB Activation

Signaling via a variety of G-protein-coupled receptors (GPCRs) leads to activation of nuclear factor (NF)-kappa B. Evidence exists for a signaling pathway initiated by the B2 type bradykinin receptor via G(q) activation, which leads to the sequential stimulation of phosphoinositide 3-kinase (PI3K), the serine/threonine kinase Akt, I kappa B kinases, and finally nuclear factor NF-kappa B-dependent transcription. GPCR-mediated G(q)alpha or G(13)alpha activation also potently stimulates the tyrosine kinase PYK2. In this study we tested whether G(q)alpha- and/or G(13)alpha-induced PYK2 activation contributes to GPCR-mediated NF-kappa B activation. Among the GTPase-deficient forms of G alpha tested, G(13)alpha and G(q)alpha most potently stimulated an NF-kappa B-dependent reporter gene. PYK2 activated the same reporter gene and synergized with either G(q)alpha Q209L (QL) or G(13)alpha Q226L (QL). Placing PYK2 upstream of both PI3K and Akt activation, PYK2 activated Akt through a PI3K-dependent pathway, and either a dominant negative form of Akt or the PI3K inhibitor LY294002 blocked PYK2-stimulated NF-kappa B-dependent transcription. Placing PYK2 downstream of G-protein activation, a kinase-dead form of PYK2, PYK2 (KD), blocked NF-kappa B-dependent transcription triggered by signaling through the muscarinic receptor type 1 and either G(q)alpha QL or G(13)alpha QL. PYK2 (KD) also blocked Akt activation by the same stimuli. These results indicate that PYK2 can link G-protein activation through PI3K, Akt, and I kappa B kinase to NF-kappa B activation.

Human labour is associated with nuclear factor-kappaB activity which mediates cyclo-oxygenase-2 expression and is involved with the 'functional progesterone withdrawal'.

Human labour is associated with the up-regulation of prostaglandins within the uterus, synthesized via the type-2 cyclo-oxygenase enzyme (COX-2). These lead to remodelling of the fetal membranes and cervix and to stimulation of myometrial contractions. In the human, the principal source of prostaglandins is the amnion. Progesterone acts to promote myometrial quiescence, and in many species the onset of labour is preceded by withdrawal of progesterone. Humans show no systemic progesterone withdrawal, although biochemical changes within the uterus are similar to those in other species. A mutual negative interaction between the transcription factor nuclear factor (NF)-kappaB and the progesterone receptor (PR) has been reported. Using transient transfections and assays for transcriptional activation and promoter binding, we have shown that there is constitutive activity of NF-kappaB in amnion cells at the time of labour, and that COX-2 expression depends upon NF-kappaB. In cells obtained before labour, in which NF-kappaB activity is low, increasing the concentration of PR represses NF-kappaB dependent transcription, while stimulation with IL-1beta both increases NF-kappaB activity and represses PR activity. Our data suggest that human labour is associated with constitutive NF-kappaB activity within the amnion, which functions to increase the expression of COX-2 and appears to contribute to the 'functional progesterone withdrawal'.

The role of phosphatidylinositol 3-kinase and the mitogen-activated protein kinases in insulin-like growth factor-I-mediated effects in vascular endothelial cells.

Despite an improved understanding of the molecular mechanisms of insulin-like growth factor-I (IGF-I) signaling and the recognition that IGF-I mediates many effects in endothelial cells, some of which may be important for atherosclerosis, little is known about the signal transduction pathways that mediate the effects of IGF-I in endothelial cells. To that end, we examined the signaling pathways activated by IGF-I in endothelial cells and their contribution to IGF-I-stimulated endothelial cell migration and nuclear factor (NF)-kappaB-dependent transcription. Treatment of bovine pulmonary artery endothelial cells (PAEC) with IGF-I activated the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)1/2 and ERK5. In contrast, IGF-I had no effect on either c-Jun amino-terminal kinase or p38 kinase activity. IGF-I also activated phosphatidylinositol (PI) 3-kinase, as reflected by increased phosphorylation of AKT: There was no evidence of cross-talk between the ERK and PI 3-kinase pathways in PAEC. In PAEC transiently transfected with pTK81-NFkappaB-Luc, which contained four copies of the NF-kappaB DNA binding site 5' to a minimal promoter and the luciferase gene, treatment with 50 ng/ml IGF-I increased luciferase activity 1.8-fold. Inhibition of ERK activity using PD98059 and PI 3-kinase activity with LY 294002 abrogated the induction of NF-kappaB-dependent transcription by IGF-I, suggesting that both pathways contribute to the effect of IGF-I on NF-kappaBdependent transcription. In contrast to the effect of tumor necrosis factor-alpha on NF-kappaB activation, Western blot analyses demonstrated that IGF-I had no effect on IkappaB phosphorylation and degradation or nuclear translocation and DNA binding of NF-kappaB. These data suggest a direct of effect of IGF-I on nuclear NF-kappaB. IGF-I also increased endothelial cell migration approximately 2-fold, as demonstrated using a Boyden chamber apparatus. IGF-I-induced endothelial cell migration was inhibited, in part, by LY 294002 but not PD98059. Together, these studies demonstrate that IGF-I activates multiple signaling pathways in endothelial cells with little evidence for cross-talk between the pathways. Moreover, these pathways appear to mediate both overlapping and distinct effects in that activation of both PI 3-kinase and the ERKs contributed to the stimulation of NF-kappaB-dependent transcription by IGF-I, whereas only PI 3-kinase mediated IGF-I-stimulated endothelial cell migration.

A novel transformation suppressor, Pdcd4, inhibits AP-1 transactivation but not NF-kappaB or ODC transactivation.

Pdcd4 is a novel transformation suppressor that is highly expressed in promotion-resistant (P-) mouse epidermal JB6 cells but not in susceptible (P+) cells. Overexpression of pdcd4 cDNA in stably transfected P+ cells rendered cells resistant to tumor promoter-induced transformation, indicating that elevated expression of Pdcd4 protein is sufficient to suppress neoplastic transformation. To determine whether Pdcd4 suppresses neoplastic transformation through inhibiting known transformation required events, we examined the possibility that pdcd4 inhibited the activation of AP-1 or NF-kappaB dependent transcription or of ornithine decarboxylase (ODC) activity. Activation of AP-1-dependent transcriptional activity was inhibited by pdcd4 expression in a concentration dependent manner. In contrast, Pdcd4 slightly increased NF-kappaB-dependent transcription and did not alter ODC enzymatic activity. Previous studies suggested that activation of AP-1 was required for P+ cell transformation as well as for tumor promotion in vivo. These results indicate that Pdcd4 functions as a transformation suppressor, possibly through inhibiting AP-1 activation in combination with other factors such as enhancing NF-kappaB activation. Pdcd4 may thus constitute a useful molecular target for cancer prevention.