Inactivation of the inhibitory kappaB protein kinase/nuclear factor kappaB pathway by Par-4 expression potentiates tumor necrosis factor alpha-induced apoptosis.

Par-4 is a novel protein identified in cells undergoing apoptosis. The ability of Par-4 to promote apoptotic cell death is dependent on the binding and inactivation of the atypical protein kinases C (PKCs). This subfamily of kinases has been reported to control nuclear factor kappaB (NF-kappaB) through the regulation of the IkappaB kinase activity. NF-kappaB activation by tumor necrosis factor alpha (TNFalpha) provides a survival signal that impairs the TNFalpha-induced apoptotic response. We show here that expression of Par-4 inhibits the TNFalpha-induced nuclear translocation of p65 as well as the kappaB-dependent promoter activity. Interestingly, Par-4 expression blocks inhibitory kappaB protein (IkappaB) kinase activity, which leads to the inhibition of IkappaB phosphorylation and degradation, in a manner that is dependent on its ability to inhibit lambda/iotaPKC. Of potential functional relevance, the expression of Par-4 allows TNFalpha to induce apoptosis in NIH-3T3 cells. In addition, the down-regulation of Par-4 levels by oncogenic Ras sensitizes cells to TNFalpha-induced NF-kappaB activation.     

Oxidative modification of IkappaB by monochloramine inhibits tumor necrosis factor alpha-induced NF-kappaB activation

We have previously reported that monochloramine (NH(2)Cl), a neutrophil-derived oxidant, inhibited tumor necrosis factor alpha (TNFalpha)-induced expression of cell adhesion molecules and nuclear factor-kappaB (NF-kappaB) activation (Free Radical Research 36 (2002) 845-852). Here, we studied the mechanism how NH(2)Cl inhibited TNFalpha-induced NF-kappaB activation, and compared the effects with taurine chloramine (Tau-NHCl). Pretreatment of Jurkat cells with NH(2)Cl at 70 microM resulted in suppression of TNFalpha-induced IkappaB phosphorylation and degradation, and inhibited NF-kappaB activation. In addition, a slow-moving IkappaB band appeared on SDS-PAGE. By contrast, Tau-NHCl for up to 200 microM had no effects. Interestingly, NH(2)Cl did not inhibit IkappaB kinase activation by TNFalpha. Protein phosphatase activity did not show apparent change. When recombinant IkappaB was oxidized by NH(2)Cl in vitro and phosphorylated by TNFalpha-stimulated Jurkat cell lysate, its phosphorylation occurred less effectively than non-oxidized IkappaB. In addition, when NF-kappaB-IkappaB complex was immunoprecipitated from NH(2)Cl-treated cells and phosphorylated in vitro by recombinant active IkappaB kinase, native IkappaB but not oxidized IkappaB was phosphorylated. Amino acid analysis of the in vitro oxidized IkappaB showed methionine oxidation to methionine sulfoxide. Although Tau-NHCl alone had little effects on TNFalpha-induced NF-kappaB activation, simultaneous presence of Tau-NHCl and ammonium ion significantly inhibited the NF-kappaB activation, probably through the conversion of Tau-NHCl to NH(2)Cl. These results indicated that NH(2)Cl inhibited TNFalpha-induced NF-kappaB activation through the oxidation of IkappaB, and that NH(2)Cl is physiologically more relevant than Tau-NHCl in modifying NF-kappaB-mediated cellular responses.     

Aminosalicylic acid inhibits IkappaB kinase alpha phosphorylation of IkappaBalpha in mouse intestinal epithelial cells

Tumor necrosis factor alpha (TNFalpha)-stimulated nuclear factor (NF) kappaB activation plays a key role in the pathogenesis of inflammatory bowel disease (IBD). Phosphorylation of NFkappaB inhibitory protein (IkappaB) leading to its degradation and NFkappaB activation, is regulated by the multimeric IkappaB kinase complex, including IKKalpha and IKKbeta. We recently reported that 5-aminosalicylic acid (5-ASA) inhibits TNFalpha-regulated IkappaB degradation and NFkappaB activation. To determine the mechanism of 5-ASA inhibition of IkappaB degradation, we studied young adult mouse colon (YAMC) cells by immunodetection and in vitro kinase assays. We show 5-ASA inhibits TNFalpha-stimulated phosphorylation of IkappaBalpha in intact YAMC cells. Phosphorylation of a glutathione S-transferase-IkappaBalpha fusion protein by cellular extracts or immunoprecipitated IKKalpha isolated from cells treated with TNFalpha is inhibited by 5-ASA. Recombinant IKKalpha and IKKbeta autophosphorylation and their phosphorylation of glutathione S-transferase-IkappaBalpha are inhibited by 5-ASA. However, IKKalpha serine phosphorylation by its upstream kinase in either intact cells or cellular extracts is not blocked by 5-ASA. Surprisingly, immunodepletion of cellular extracts suggests IKKalpha is predominantly responsible for IkappaBalpha phosphorylation in intestinal epithelial cells. In summary, 5-ASA inhibits TNFalpha-stimulated IKKalpha kinase activity toward IkappaBalpha in intestinal epithelial cells. These findings suggest a novel role for 5-ASA in the management of IBD by disrupting TNFalpha activation of NFkappaB.     

Cytokine-induced activation of nuclear factor-kappa B is inhibited by hydrogen peroxide through oxidative inactivation of IkappaB kinase

Rapid activation of the IkappaB kinase (IKK) complex is considered an obligatory step in the activation of nuclear factor-kappaB (NF-kappaB) in response to diverse stimuli. Since oxidants have been implicated in the regulation of NF-kappaB, the focus of the present study was the activation of IKK by tumor necrosis factor alpha (TNFalpha) in the presence or absence of hydrogen peroxide (H(2)O(2)). Exposure of mouse alveolar epithelial cells to H(2)O(2) was not sufficient to activate IKK, degrade IkappaBalpha, or activate NF-kappaB. In contrast, TNFalpha induced IKK activity rapidly and transiently resulting in IkappaBalpha degradation and NF-kappaB activation. Importantly, in the presence of H(2)O(2), the ability of TNFalpha to induce IKK activity was markedly decreased and resulted in prevention of IkappaBalpha degradation and NF-kappaB activation. Neither tyrosine kinases nor phosphatidylinositol 3-kinases, known regulators of NF-kappaB by oxidants, were involved in IKK inhibition by H(2)O(2). Direct addition of H(2)O(2) to the immunoprecipitated IKK complex inhibited enzyme activity. Inhibition of IKK activity by H(2)O(2) was associated with direct oxidation of cysteine residues present in the IKK complex and occurred only in enzymatically active IKK. In contrast to previously published observations, our findings demonstrate that the oxidant H(2)O(2) reduces NF-kappaB activation by inhibiting activated IKK activity.     

Distinct role of spleen tyrosine kinase in the early phosphorylation of inhibitor of kappaB alpha via activation of the phosphoinositide-3-kinase and Akt pathways

Nuclear factor (NF)-kappaB activation is a critical step in the triggering of inflammatory responses by macrophages. Although numerous investigations have been reported, the precise regulatory mechanisms controlling inflammatory responses mediated by NF-kappaB remain unclear. In this study, we investigated the early signaling events responsible for modulating NF-kappaB activation using various parameters, such as the expression of pro-inflammatory genes and the phosphorylation levels of inhibitor of kappaB alpha (IkappaB alpha) and its upstream kinases. Lipopolysaccharide (LPS) treatment biphasically induced activation of IkappaB alpha phosphorylation at 5 and 30 min, which induced subsequent pro-inflammatory gene expression that was maximized at 45 and 90 min. Of the intracellular signals tested, a series of signaling cascades composed of spleen tyrosine kinase (Syk), phosphoinositide-3-kinase (PI3K), and Akt (protein kinase B) were involved in regulating early phosphorylation of IkappaB alpha, according to biochemical and pharmacological analyses. Therefore, our data suggests that Syk-mediated activation of intracellular signaling in response to LPS may play an important role in LPS-induced inflammatory signaling events. Thus, Syk may be a potential target for the development of potent anti-inflammatory drugs.     

Indirubin enhances tumor necrosis factor-induced apoptosis through modulation of nuclear factor-kappa B signaling pathway

Although indirubin is known to exhibit anti-cancer and anti-inflammatory activities, very little is known about its mechanism of action. In this study, we investigated whether indirubin mediates its effects through interference with the NF-kappaB pathway. As examined by the DNA binding of NF-kappaB, we found that indirubin suppressed tumor necrosis factor (TNF)-induced NF-kappaB activation in a dose- and time-dependent manner. Indirubin also suppressed the NF-kappaB activation induced by various inflammatory agents and carcinogens. Further studies showed that indirubin blocked the phosphorylation and degradation of IkappaB alpha through the inhibition of activation of IkappaB alpha kinase and phosphorylation and nuclear translocation of p65. NF-kappaB reporter activity induced by TNFR1, TNF receptor-associated death domain, TRAF2, TAK1, NF-kappaB-inducing kinase, and IKKbeta was inhibited by indirubin but not that induced by p65 transfection. We also found that indirubin inhibited the expression of NF-kappaB-regulated gene products involved in antiapoptosis (IAP1, IAP2, Bcl-2, Bcl-xL, and TRAF1), proliferation (cyclin D1 and c-Myc), and invasion (COX-2 and MMP-9). This correlated with enhancement of the apoptosis induced by TNF and the chemotherapeutic agent taxol in human leukemic KBM-5 cells. Indirubin also suppressed cytokine-induced cellular invasion. Overall, our results indicate that anti-cancer and anti-inflammatory activities previously assigned to indirubin may be mediated in part through the suppression of the NF-kappaB activation pathway.     

IKKepsilon is part of a novel PMA-inducible IkappaB kinase complex

Here we report the identification of a novel PMA-inducible IkappaB kinase complex, distinct from the well-characterized high-molecular weight IkappaB kinase complex containing IKKalpha, IKKbeta, and IKKgamma. We have characterized one kinase from this complex, which we designate IKKepsilon. Although recombinant IKKepsilon directly phosphorylates only serine 36 of IKBalpha, the PMA-activated endogenous IKKepsilon complex phosphorylates both critical serine residues. Remarkably, this activity is due to the presence of a distinct kinase in this complex. A dominant-negative mutant of IKKepsilon blocks induction of NF-kappaB by both PMA and activation of the T cell receptor but has no effect on the activation of NF-KB by TNFalpha or IL-1. These observations indicate that the activation of NF-kappaB requires multiple distinct IkappaB kinase complexes, which respond to both overlapping and discrete signaling pathways.     

Detection of a novel parasite kinase activity at the Toxoplasma gondii parasitophorous vacuole membrane capable of phosphorylating host IkappaBalpha

Toxoplasma gondii activates the NF-kappaB pathway in the infected host cell resulting in upregulation of pro-survival genes and prevention of apoptosis. Manipulation of the NF-kappaB cascade by T. gondii correlates with the localization of phosphorylated IkappaB at the parasitophorous vacuole membrane (PVM). This suggests a parasite-mediated event, involving the recruitment and activation of the host IkappaB kinase (IKK) complex, as has been observed with the related protozoan Theileria parva. In contrast to Theileria, confocal microscopy studies showed no apparent hijacking of IKKalpha, IKKbeta, or their activated phosphorylated forms at the T. gondii PVM. Remarkably, phosphorylation of IkappaBalpha at Ser 32/36 was observed at the PVM of T. gondii-infected IKKalpha-/-, IKKbeta-/- and IKKalpha/beta double-knockout (IKKalpha/beta-/-) fibroblasts, suggesting the involvement of a parasite kinase activity independent of host IKK. The presence of a putative T. gondii IkappaB kinase was examined by in vitro kinase assays using GST-IkappaBalpha constructs and protein extracts from both extracellular parasites and PVM fractions. Interestingly, an activity capable of phosphorylating IkappaBalpha at the critical Ser 32/36 sites was identified in parasite extracts, a property restricted to the IKK signalosome. Taken together, our data support the role for a T. gondii kinase involved in phosphorylation of host cell IkappaBalpha and suggest an unusual mechanism utilized by an intracellular pathogen capable of manipulating the NF-kappaB pathway.     

Essential role for IkappaB kinase beta in remodeling Carma1-Bcl10-Malt1 complexes upon T cell activation

T cell receptor (TCR) signaling to IkappaB kinase (IKK)/NF-kappaB is controlled by PKCtheta-dependent activation of the Carma1, Bcl10, and Malt1 (CBM) complex. Antigen-induced phosphorylation of Bcl10 has been reported, but its physiological function is unknown. Here we show that the putative downstream kinase IKKbeta is required for initial CBM complex formation. Further, upon engagement of IKKbeta/Malt1/Bcl10 with Carma1, IKKbeta phosphorylates Bcl10 in the C terminus and thereby interferes with Bcl10/Malt1 association and Bcl10-mediated IKKgamma ubiquitination. Mutation of the IKKbeta phosphorylation sites on Bcl10 enhances expression of NF-kappaB target genes IL-2 and TNFalpha after activation of primary T cells. Thus, our data provide evidence that IKKbeta serves a dual role upstream of its classical substrates, the IkappaB proteins. While being essential for triggering initial CBM complex formation, IKKbeta-dependent phosphorylation of Bcl10 exhibits a negative regulatory role in T cell activation.     

Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO

The receptor interacting protein kinase 1 (RIP1) is essential for the activation of nuclear factor kappaB (NF-kappaB) by tumor necrosis factor alpha (TNFalpha). Here, we present evidence that TNFalpha induces the polyubiquitination of RIP1 at Lys-377 and that this polyubiquitination is required for the activation of IkappaB kinase (IKK) and NF-kappaB. A point mutation of RIP1 at Lys-377 (K377R) abolishes its polyubiquitination as well as its ability to restore IKK activation in a RIP1-deficient cell line. The K377R mutation of RIP1 also prevents the recruitment of TAK1 and IKK complexes to TNF receptor. Interestingly, polyubiquitinated RIP1 recruits IKK through the binding between the polyubiquitin chains and NEMO, a regulatory subunit of the IKK complex. Mutations of NEMO that disrupt its polyubiquitin binding also abolish IKK activation. These results reveal the biochemical mechanism underlying the essential signaling function of NEMO and provide direct evidence that signal-induced site-specific ubiquitination of RIP1 is required for IKK activation.     

Sequestration of NF-kappaB signaling complexes in lipid rafts contributes to repression of NF-kappaB in T lymphocytes under hyperthermia stress

Sepsis causes extensive apoptosis of lymphocytes, a pathological condition that is frequently associated with hyperthermia. Heat stress has been implicated to repress the activation of an inflammatory mediator, nuclear factor of kappaB (NF-kappaB), which sensitizes cells to apoptosis mediated by inflammatory cytokine, tumor necrosis factor alpha. However, the molecular mechanism of hyperthermia-associated loss of T cells remains unclear. We show that hyperthermia causes rapid translocation of IkappaB kinase (IKK) and NF-kappaB complexes into the plasma membrane-associated lipid rafts in T cells. Heat stress induces aggregation of Carma1 in lipid rafts, which in turn recruits protein kinase C theta (PKC theta) and Bcl10 to the microdomains, causing subsequent membrane translocation of the IKK and NF-kappaB signalosomes. Depletion of Carma1 and inhibition of PKC theta impair accumulation of NF-kappaB complexes in lipid rafts. Heat stress prohibits IkappaB kinase activity by sequestrating the IKK and NF-kappaB complexes in lipid rafts and by segregating the chaperone protein Hsp90, an essential cofactor for IKK, from the IKK complex. This process ultimately results in functional deficiency of NF-kappaB and renders T cells resistant to tumor necrosis factor alpha-induced activation of IKK, thereby contributing to the apoptotic loss of T lymphocytes in sepsis-associated hyperthermia.     

The 15-deoxy-delta12,14-prostaglandin J2 inhibits the inflammatory response in primary rat astrocytes via down-regulating multiple steps in phosphatidylinositol 3-kinase-Akt-NF-kappaB-p300 pathway independent of peroxisome proliferator-activated receptor gamma

Ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), such as 15-deoxy-12,14-PGJ2 (15d-PGJ2), have been proposed as a new class of anti-inflammatory compounds because 15d-PGJ2 was able to inhibit the induction of inflammatory response genes such as inducible NO synthase (iNOS) and TNF (TNF-alpha) in a PPAR-dependent manner in various cell types. In primary astrocytes, the anti-inflammatory effects (inhibition of TNF-alpha, IL-1beta, IL-6, and iNOS gene expression) of 15d-PGJ2 are observed to be independent of PPARgamma. Overexpression (wild-type and dominant-negative forms) of PPARgamma and its antagonist (GW9662) did not alter the 15d-PGJ2-induced inhibition of LPS/IFN-gamma-mediated iNOS and NF-kappaB activation. The 15d-PGJ2 inhibited the inflammatory response by inhibiting IkappaB kinase activity, which leads to the inhibition of degradation of IkappaB and nuclear translocation of p65, thereby regulating the NF-kappaB pathway. Moreover, 15d-PGJ2 also inhibited the LPS/IFN-gamma-induced PI3K-Akt pathway. The 15d-PGJ2 inhibited the recruitment of p300 by NF-kappaB (p65) and down-regulated the p300-mediated induction of iNOS and NF-kappaB luciferase reporter activity. Coexpression of constitutive active Akt and PI3K (p110) reversed the 15d-PGJ2-mediated inhibition of p300-induced iNOS and NF-kappaB luciferase activity. This study demonstrates that 15d-PGJ2 suppresses inflammatory response by inhibiting NF-kappaB signaling at multiple steps as well as by inhibiting the PI3K/Akt pathway independent of PPARgamma in primary astrocytes.     

Novel synergistic mechanism for sst2 somatostatin and TNFalpha receptors to induce apoptosis: crosstalk between NF-kappaB and JNK pathways

Somatostatin is a multifunctional hormone that modulates cell proliferation, differentiation and apoptosis. Mechanisms for somatostatin-induced apoptosis are at present mostly unsolved. Therefore, we investigated whether somatostatin receptor subtype 2 (sst2) induces apoptosis in the nontransformed murine fibroblastic NIH3T3 cells. Somatostatin receptor subtype 2 expression induced an executioner caspase-mediated apoptosis through a tyrosine phosphatase SHP-1 (Src homology domain phosphatase-1)-dependent stimulation of nuclear factor kappa B (NF-kappaB) activity and subsequent inhibition of the mitogen-activated protein kinase JNK. Tumor necrosis factor alpha (TNFalpha) stimulated both NF-kappaB and c-Jun NH2-terminal kinase (JNK) activities, which had opposite action on cell survival. Importantly, sst2 sensitized NIH3T3 cells to TNFalpha-induced apoptosis by (1) upregulating TNFalpha receptor protein expression, and sensitizing to TNFalpha-induced caspase-8 activation; (2) enhancing TNFalpha-mediated activation of NF-kappaB, resulting in JNK inhibition and subsequent executioner caspase activation and cell death. We have here unraveled a novel signaling mechanism for a G protein-coupled receptor, which directly triggers apoptosis and crosstalks with a death receptor to enhance death ligand-induced apoptosis.