Essential role of nuclear factor (NF)-kappaB-inducing kinase and inhibitor of kappaB (IkappaB) kinase alpha in NF-kappaB activation through lymphotoxin beta receptor, but not through tumor necrosis factor receptor I

Both nuclear factor (NF)-kappaB-inducing kinase (NIK) and inhibitor of kappaB (IkappaB) kinase (IKK) have been implicated as essential components for NF-kappaB activation in response to many external stimuli. However, the exact roles of NIK and IKKalpha in cytokine signaling still remain controversial. With the use of in vivo mouse models, rather than with enforced gene-expression systems, we have investigated the role of NIK and IKKalpha in signaling through the type I tumor necrosis factor (TNF) receptor (TNFR-I) and the lymphotoxin beta receptor (LTbetaR), a receptor essential for lymphoid organogenesis. TNF stimulation induced similar levels of phosphorylation and degradation of IkappaBalpha in embryonic fibroblasts from either wild-type or NIK-mutant mice. In contrast, LTbetaR stimulation induced NF-kappaB activation in wild-type mice, but the response was impaired in embryonic fibroblasts from NIK-mutant and IKKalpha-deficient mice. Consistent with the essential role of IKKalpha in LTbetaR signaling, we found that development of Peyer's patches was defective in IKKalpha-deficient mice. These results demonstrate that both NIK and IKKalpha are essential for the induction of NF-kappaB through LTbetaR, whereas the NIK-IKKalpha pathway is dispensable in TNFR-I signaling.     

Soluble L-selectin attenuates tumor necrosis factor-alpha-mediated leukocyte adherence and vascular permeability: a protective role for elevated soluble L-selectin in sepsis

OBJECTIVE: We have previously demonstrated that leukocyte delivery to remote sites is decreased in sepsis and that increased concentrations of soluble L-selectin are, in part, responsible for this finding. Given that leukocytes have been implicated in the pathogenesis of vascular leakage, we hypothesized that the elevated soluble L-selectin concentrations in sepsis may translate into decreased inflammation-mediated leukocyte-endothelial cell interactions and vascular leakage at these sites. DESIGN: Prospective, controlled animal study. SETTING: Surgical research laboratory in a university hospital. SUBJECTS: Swiss white male mice weighing 25-35 g. INTERVENTIONS: Mice were randomized to one of three study groups: intracremaster tumor necrosis factor-alpha with subsequent intravenous bicarbonate buffered solution; intracremaster tumor necrosis factor-alpha with intravenous soluble L-selectin (10 microg/mL); and intracremaster bicarbonate buffered solution with intravenous bicarbonate buffered solution. The cremaster muscle was prepared for both light and fluorescence intravital microscopy 2 hrs after intracremaster injection, and fluorescein isothiocyanate-labeled albumin was injected intravenously. Leukocyte-endothelial interactions (rolling flux, rolling velocity, and adherence) were counted off-line. Postcapillary venule leakage was determined by the permeability index (perivenular/intravenular fluorescence) after intravenous injection of fluorescent albumin. MEASUREMENTS AND MAIN RESULTS: Soluble L-selectin significantly attenuated tumor necrosis factor-alpha-mediated increases in leukocyte adherence and vascular leakage. Leukocyte rolling velocity was restored to baseline with soluble L-selectin; however, rolling flux was not altered. Blood pressure, shear rate, and leukocyte counts did not differ between groups. CONCLUSIONS: Soluble L-selectin decreases local inflammation-mediated leukocyte adherence and vascular leakage in vivo. The increased concentrations of soluble L-selectin in sepsis may represent a protective mechanism by which the host attempts to diminish the deleterious systemic effects of activated leukocytes during sepsis.     

Regulation of Vinca alkaloid-induced apoptosis by NF-kappaB/IkappaB pathway in human tumor cells

Antimicrotubule Vinca alkaloids, such as vinblastine and vincristine, interfere with the dynamics of microtubules and have shown significant cell killing activity in a variety of tumor cells through induction of apoptosis. The mechanism by which Vinca alkaloids induce apoptosis is not entirely clear. In this study, we found that glucocorticoids inhibit Vinca alkaloid-induced apoptosis without affecting G(2)-M arrest in human breast cancer BCap37 cells and human epidermoid tumor KB cells, suggesting that Vinca alkaloid-induced apoptosis may occur via a pathway independent of cell cycle arrest. Further analyses indicated that Vinca alkaloids cause significant degradation of IkappaBalpha, which in turn results in nuclear factor-kappaB (NF-kappaB) activation. Transfection of antisense IkappaBalpha in BCap37 cells sensitizes Vinca alkaloid-induced apoptosis. Moreover, in vitro kinase assays show that the activity of IkappaB kinase (IKK) was activated by Vinca alkaloids and was not affected by glucocorticoids. Stable transfection of dominant-negative deletional mutant IkappaBalpha, which is insensitive to IKK-mediated phosphorylation and degradation, resulted in the inhibition of Vinca alkaloid-induced NF-kappaB activation and reduced sensitivity of tumor cells to Vinca alkaloid-induced apoptosis. These findings suggest that the NF-kappaB/IkappaB signaling pathway may contribute to the mediation of Vinca alkaloid-induced apoptosis in human tumor cells.     

17-Acetoxyjolkinolide B irreversibly inhibits IkappaB kinase and induces apoptosis of tumor cells

Nuclear factor-kappaB (NF-kappaB) is critically important for tumor cell survival, growth, angiogenesis, and metastasis. One of the key events in the NF-kappaB signaling is the activation of inhibitor of NF-kappaB kinase (IKK) in response to stimuli of various cytokines. We have identified 17-acetoxyjolkinolide B (17-AJB) from a traditional Chinese medicinal herb Euphorbia fischeriana Steud as a novel small-molecule inhibitor of IKK. 17-AJB effectively inhibited tumor necrosis factor-alpha-induced NF-kappaB activation and induced apoptosis of tumor cells. 17-AJB had no effect on binding of tumor necrosis factor-alpha to its receptor or on binding of NF-kappaB to DNA. It inhibited NF-kappaB nuclear translocation. Detailed analysis revealed that the direct target of 17-AJB was IKK. 17-AJB kept IKK in its phosphorylated form irreversibly. This irreversible modification of IKK inactivated its kinase activity, leading to its failure to activate NF-kappaB. The effect of 17-AJB on IKK was specific. It had no effect on other kinases such as p38, p44/42, and JNK. In addition, 17-AJB induced apoptosis in tumor cells. The effects of 17-AJB on apoptosis correlated with inhibition of expression of the NF-kappaB-regulated genes. Taken together, our data suggest that 17-AJB is a novel type NF-kappaB pathway inhibitor. Its unique interaction mechanism with IKK may render it a strong apoptosis inducer of tumor cells and a novel type anticancer drug candidate.     

Neurokinin-1 receptor antagonists protect mice from CD95- and tumor necrosis factor-alpha-mediated apoptotic liver damage

Previously, we have shown that primary afferent neurons are necessary for disease activity in immune-mediated liver injury in mice. These nerve fibers are detectable by substance P (SP) immunocytochemistry in the portal tract of rodent liver. Antagonists of the neurokinin-1 receptor (NK-1R), which is the prime receptor of SP, prevented liver damage by suppressing the synthesis of proinflammatory cytokines. Here, we investigated the influence of primary afferent nerve fibers, SP, and NK-1 receptor antagonists on hepatocyte apoptosis in vivo induced by administration of activating anti-CD95 monoclonal antibody (mAb) to mice. Depletion of primary afferent nerve fibers by neonatal capsaicin treatment prevented CD95-mediated activation of caspase-3, measured as enzymatic activity in liver homogenates or by demonstration of hepatocellular immunoreactivity for active caspase-3 in liver slices, and liver damage. This effect was reversed by administration of SP to anti-CD95 mAb-treated mice depleted from primary afferent neurons. The presence of the NK-1R on mouse hepatocytes was demonstrated by immunocytochemistry and flow cytometry. Intraperitoneal pretreatment with the NK-1 receptor antagonists (2S,3S)-cis-2-(diphenylmethyl)-N-([2-methoxyphenyl]-methyl)-1-azabicyclo(2.2.2.)-octan-3-amine (CP-96,345) or (2S,3S)3-([3,5-bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperadine (L-733,060) dose dependently protected mice from CD95-mediated liver injury. Similar results were obtained when apoptotic liver damage was induced by administration of tumor necrosis factor-alpha to d-galactosamine-sensitized mice. In conclusion, SP, probably by binding to its receptor on hepatocytes, might aggravate apoptotic signals in these cells. Because NK-1 receptor antagonists not only suppress the proinflammatory cytokine response in the liver but also prevent liver cell apoptosis in vivo, they might be suitable drugs for treatment of immune-mediated liver disease.     

Human cytomegalovirus-stimulated peripheral blood mononuclear cells induce HIV-1 replication via a tumor necrosis factor-alpha-mediated mechanism.

Human cytomegalovirus (HCMV) is a potential cofactor in HIV-1 infection. To investigate the mechanism whereby HCMV promotes HIV-1 replication, a PBMC coculture assay which measures HIV-1 p24 antigen release was used as an index of viral replication. HCMV-stimulated PBMC were capable of inducing HIV-1 replication in cocultures with acutely infected PBMC; however, this occurred only when the PBMC were from HCMV-seropositive donors (598 +/- 207 versus 27 +/- 10 pg/ml p24 antigen with PBMC from HCMV-seronegative donors on day 6 of coculture). Upon stimulation with HCMV, PBMC obtained exclusively from HCMV-seropositive donors released tumor necrosis factor (TNF)-alpha (270 +/- 79 pg/ml at 18 h of culture). Monoclonal antibodies to TNF-alpha blocked the activity of HCMV-stimulated PBMC in cocultures both with acutely HIV-1-infected PBMC and with the chronically infected promonocytic line U1. Also, treatment of HCMV-stimulated PBMC with pentoxifylline, an inhibitor of TNF-alpha mRNA, markedly reduced HIV-1 replication in cocultures both with acutely and chronically infected cells. These results indicate that TNF-alpha is a key mediator of HIV-1 replication induced by HCMV-stimulated PBMC and support the concept that this cytokine plays an important role in the pathogenesis of HIV-1 infection.     

Constitutive activation of NF-kappaB in human hepatocellular carcinoma: evidence of a cytoprotective role

Activation of nuclear factor-kappaB (NF-kappaB) can promote or inhibit apoptosis. Oxidative stress is an important mechanism by which certain anticancer drugs kill cancer cells, and is also one of the mechanisms that activate NF-kappaB. We therefore examined hepatic expression of the NF-kappaB monomer p65 in human hepatocellular carcinoma (HCC) tissue samples from eight patients and compared it with their respective samples of surrounding liver tissues. We also studied the effect of NF-kappaB inhibition in human HCC cells exposed to oxidative stress, by infecting HuH7 cells with a recombinant adenovirus carrying mutant IkappaBalpha (mIkappaBalpha). Cultured HuH7 cells were infected with mIkappaBalpha or beta-galactosidase (beta-Gal) for 24 hr followed by treatment with increasing concentrations of H2O2. Cytotoxicity, NF-kappaB translocation, NF-kappaB DNA binding, cell proliferation, and apoptosis were determined. The monomer p65 was overexpressed in six of eight human HCC tissues. In HuH7 cells, introduction of mIkappaBalpha potently inhibited the translocation, activation, and DNA binding of NF- kappaB. In control (beta-Gal-infected) HuH7 cells, exposure to H2O2 produced a dose-dependent increase in apoptosis, regardless of NF-kappaB status. mIkappaBalpha-mediated inhibition of NF-kappaB activation sensitized HuH7 cells to H2O2-induced inhibition of cell growth, and further promoted cell death. Addition of H2O2 (200-500 microM) to control or mIkappaBalpha-infected HuH7 cells enhanced caspase-3 activity and cleavage. Adenovirus-mediated transfer of mIkappaBalpha potently inhibits NF-kappaB activity in HuH7 cells, and this enhances oxidative stress-induced cell killing.     

Reduced tyrosine kinase activity of the insulin receptor in obesity-diabetes. Central role of tumor necrosis factor-alpha.

Insulin resistance is an important metabolic abnormality often associated with infections, cancer, obesity, and especially non-insulin-dependent diabetes mellitus (NIDDM). We have previously demonstrated that tumor necrosis factor-alpha produced by adipose tissue is a key mediator of insulin resistance in animal models of obesity-diabetes. However, the mechanism by which TNF-alpha interferes with insulin action is not known. Since a defective insulin receptor (IR) tyrosine kinase activity has been observed in obesity and NIDDM, we measured the IR tyrosine kinase activity in the Zucker (fa/fa) rat model of obesity and insulin resistance after neutralizing TNF-alpha with a soluble TNF receptor (TNFR)-lgG fusion protein. This neutralization resulted in a marked increase in insulin-stimulated autophosphorylation of the IR, as well as phosphorylation of insulin receptor substrate 1 (IRS-1) in muscle and fat tissues of the fa/fa rats, restoring them to near control (lean) levels. In contrast, no significant changes were observed in insulin-stimulated tyrosine phosphorylations of IR and IRS-1 in liver. The physiological significance of the improvements in IR signaling was indicated by a concurrent reduction in plasma glucose, insulin, and free fatty acid levels. These results demonstrate that TNF-alpha participates in obesity-related systemic insulin resistance by inhibiting the IR tyrosine kinase in the two tissues mainly responsible for insulin-stimulated glucose uptake: muscle and fat.     

Protein kinase C epsilon regulates tumor necrosis factor-alpha-induced stannin gene expression

Stannin (Snn) is a highly conserved vertebrate protein that has been closely linked to trimethyltin (TMT) toxicity. We have previously demonstrated that Snn is required for TMT-induced cell death. Others have shown that TMT exposure results in tumor necrosis factor-alpha (TNFalpha) production and that TNFalpha treatment induces Snn gene expression in human umbilical vein endothelial cells (HUVECs). In this study, we investigated a signaling mechanism by which Snn gene expression is regulated by TMT and demonstrated that TNFalpha stimulates Snn gene expression in a protein kinase C epsilon-dependent manner in HUVECs in response to TMT exposure. Supporting this, we show that TMT-induced toxicity is significantly blocked by pretreatment with an anti-TNFalpha antibody in HUVECs. Using a quantitative real-time polymerase chain reaction assay, we also show that the level of Snn gene expression is significantly increased in HUVECs in response to either TMT or TNFalpha treatment. This TNFalpha-induced Snn gene expression is blocked when HUVECs were pretreated with bisindolylmaleimide I, an inhibitor of protein kinase C (PKC). In contrast, when HUVECs were treated with phorbol 12-myristate 13-acetate, a PKC activator, we observed a significant increase in Snn gene expression. Using isotype-specific siRNA against PKC, we further show that knockdown of PKC epsilon, but not PKC delta or PKC zeta, significantly blocked TNFalpha-induced Snn gene expression. Together, these results indicate that TNFalpha-induced, PKC epsilon-dependent Snn expression may be a critical factor in TMT-induced cytotoxicity.     

The role of p38 map kinase in tumor necrosis factor-induced redistribution of vascular endothelial cadherin and increased endothelial permeability

The mechanism by which tumor necrosis factor alpha (TNFalpha) increases endothelial permeability is unclear. Vascular endothelial (VE) cadherin (cadherin 5) is an important contributor to endothelial monolayer integrity. The purpose of our study was to determine the effect of TNFalpha on VE-cadherin cell-surface expression and to identify the signaling pathways involved in TNF-induced changes in cadherin expression. Human umbilical vein endothelial cell monolayer permeability was measured by enzyme-linked immunosorbent assay for biotin-labeled albumin. Immunofluorescence, laser confocal microscopy, and Western immunobloting were used to assess VE cadherin distribution. Mitogen-activated protein kinase (MAPK) activity was determined using functional kinase assays and was inhibited with the compounds SB202190 and PD98059. TNFalpha significantly increased permeability and induced p38 and ERK MAPK activation compared with controls (P < 0.05). These changes were associated with a loss of membrane-associated VE cadherin. Inhibition of p38 but not ERK MAPK significantly reduced the effect of TNFalpha on endothelial permeability and cell-surface VE cadherin expression. p38 MAP kinase activation appears to be an important upstream signaling event associated with increased endothelial permeability and vascular endothelial cadherin redistribution.     

Regulation of peritoneal and systemic neutrophil-derived tumor necrosis factor-alpha release in patients with severe peritonitis: role of tumor necrosis factor-alpha converting enzyme cleavage

OBJECTIVE: Polymorphonuclear neutrophil (PMN) influx and peritoneal tumor necrosis factor (TNF)-alpha production are key host defense mechanisms during peritonitis. The aim of this study was to explore the potential interactions between TNF-alpha production and TNF-alpha converting enzyme (TACE) expression by PMN in the blood and peritoneum of patients with severe peritonitis. DESIGN: A prospective study. SETTING: A surgical adult intensive care unit in a university hospital. PATIENTS: A total of 29 consecutive immunocompetent patients with severe sepsis within 48 hrs of onset were enrolled and underwent laparotomy for a diffuse secondary peritonitis. Thirteen volunteers served as controls. MEASUREMENTS: Blood and peritoneal fluid recovered during laparotomy were analyzed and compared for 1) soluble TNF-alpha, soluble L-selectin, and type I and II TNF-alpha receptor levels; 2) PMN membrane TNF-alpha, membrane L-selectin, and TACE expression (flow cytometry); and 3) TNF-alpha production by cultured PMN. Correlations between these forms of PMN-derived TNF-alpha and the severity of the peritonitis and patient's outcome were investigated. MAIN RESULTS: Elevated soluble TNF-alpha levels in both plasma and peritoneal fluid from the patients were found, together with decreased expression of membrane TNF-alpha and TACE up-regulation at the PMN surface. Soluble L-selectin and type I and II TNF receptors were highly released, suggesting also the role of TACE. In contrast, the capacity of both blood and peritoneal PMN to synthesize TNF-alpha in vitro, in optimal conditions of stimulation (lipopolysaccharide + interferon-gamma), was impaired as compared with controls' blood PMN. Regulation of PMN-derived TNF-alpha was similar in the two compartments, but responses were more pronounced in the peritoneum. TACE up-regulation at the surface of blood-derived PMN correlated with the Sequential Organ Failure Assessment score and vital outcome. CONCLUSION: These human data demonstrate that mTACE is up-regulated at the PMN surface during severe peritonitis. This finding could be related to a paracrine regulatory loop involving some TACE substrates such as TNF-alpha, L-selectin, and TNF receptors.     

Maturational differences in lung NF-kappaB activation and their role in tolerance to hyperoxia

Neonatal rodents are more tolerant to hyperoxia than adults. We determined whether maturational differences in lung NF-kappaB activation could account for the differences. After hyperoxic exposure (O2 > 95%), neonatal (<12 hours old) lung NF-kappaB binding was increased and reached a maximum between 8 and 16 hours, whereas in adults no changes were observed. Additionally, neonatal NF-kappaB/luciferase transgenic mice (incorporating 2 NF-kappaB consensus sequences driving luciferase gene expression) demonstrated enhanced in vivo NF-kappaB activation after hyperoxia in real time. In the lungs of neonates, there was a propensity toward NF-kappaB activation as evidenced by increased lung I-kappaB kinase protein levels, I-kappaBalpha phosphorylation, beta-transducin repeat-containing protein levels, and total I-kappaBalpha degradation. Increased lung p-JNK immunoreactive protein was observed only in the adult lung. Inhibition of pI-kappaBalpha by BAY 11-7085 resulted in decreased Bcl-2 protein levels in neonatal lung homogenates and decreased cell viability in lung primary cultures after hyperoxic exposure. Furthermore, neonatal p50-null mutant (p50(-/-)) mice showed increased lung DNA degradation and decreased survival in hyperoxia compared with WT mice. These data demonstrate that there are maturational differences in lung NF-kappaB activation and that enhanced NF-kappaB may serve to protect the neonatal lung from acute hyperoxic injury via inhibition of apoptosis.     

Critical role of the carboxyl terminus of proline-rich tyrosine kinase (Pyk2) in the activation of human neutrophils by tumor necrosis factor: separation of signals for the respiratory burst and degranulation

Transduction of Tat-tagged fusion proteins confirmed a hypothesis based on pharmacologic inhibitors (Fuortes, M., M. Melchior, H. Han, G.J. Lyon, and C. Nathan. 1999. J. Clin. Invest. 104:327-335) that proline-rich tyrosine kinase (Pyk2) plays a critical role in the activation of adherent human neutrophils, and allowed an analysis of individual Pyk2 domains not possible with chemical inhibitors. Acting as a dominant negative, the COOH terminus of Pyk2 fused to a Tat peptide (Tat-CT), but not other regions of Pyk2, specifically inhibited the respiratory burst of cells responding to tumor necrosis factor (TNF), Salmonella, or Listeria, while sparing responses induced by phorbol ester. Tat-CT suppressed TNF-triggered cell spreading and the phosphorylation of endogenous Pyk2 and the associated tyrosine kinase Syk without blocking the ability of neutrophils to degranulate and kill bacteria. Thus, separate signals control the respiratory burst and degranulation, and a normal rate of killing of some bacteria can be sustained by granule products in conjunction with a minimal residual respiratory burst. Inhibition of select inflammatory functions without impairment of antibacterial activity may commend the Pyk2 pathway as a potential target for antiinflammatory therapy.     

Bruton's tyrosine kinase is required for lipopolysaccharide-induced tumor necrosis factor alpha production

Lipopolysaccharide (LPS), a product of Gram-negative bacteria, is potent mediator of tumor necrosis factor (TNF)alpha production by myeloid/macrophage cells. Inhibitors capable of blocking the signaling events that result in TNF alpha production could provide useful therapeutics for treating septic shock and other inflammatory diseases. Broad spectrum tyrosine inhibitors are known to inhibit TNF alpha production, however, no particular family of tyrosine kinases has been shown to be essential for this process. Here we show that the Bruton's tyrosine kinase (Btk)-deficient mononuclear cells from X-linked agammaglobulinemia patients have impaired LPS-induced TNF alpha production and that LPS rapidly induces Btk kinase activity in normal monocytes. In addition, adenoviral overexpression of Btk in normal human monocytes enhanced TNF alpha production. We examined the role of Btk in TNF alpha production using luciferase reporter adenoviral constructs and have established that overexpression of Btk results in the stabilization of TNF alpha mRNA via the 3' untranslated region. Stimulation with LPS also induced the activation of related tyrosine kinase, Tec, suggesting that the Tec family kinases are important components for LPS-induced TNF alpha production. This study provides the first clear evidence that tyrosine kinases of the Tec family, in particular Btk, are key elements of LPS-induced TNF alpha production and consequently may provide valuable therapeutic targets for intervention in inflammatory conditions.     

Protein kinase regulates tumor necrosis factor mRNA stability in virus- stimulated astrocytes

Infection of astrocytes with Newcastle disease virus stimulated the production of 1,2-diacylglycerol, and resulted in the kinase-dependent expression of mRNAs encoding tumor necrosis factor (TNF), interferon alpha and beta, and interleukin 6. The half-life of TNF mRNA was significantly decreased in the presence of protein kinase inhibitors H-7 and staurosporine, but not in the presence of HA1004. In contrast to the decay of TNF mRNA, the half-lives of other cytokine mRNAs were only minimally affected by the kinase inhibitors. These data indicated that the stability of TNF mRNA was regulated through a novel, kinase-dependent pathway.