NF-kappaB activation represses tumor necrosis factor-alpha-induced autophagy

Activation of NF-kappaB and autophagy are two processes involved in the regulation of cell death, but the possible cross-talk between these two signaling pathways is largely unknown. Here, we show that NF-kappaB activation mediates repression of autophagy in tumor necrosis factor-alpha (TNFalpha)-treated Ewing sarcoma cells. This repression is associated with an NF-kappaB-dependent activation of the autophagy inhibitor mTOR. In contrast, in cells lacking NF-kappaB activation, TNFalpha treatment up-regulates the expression of the autophagy-promoting protein Beclin 1 and subsequently induces the accumulation of autophagic vacuoles. Both of these responses are dependent on reactive oxygen species (ROS) production and can be mimicked in NF-kappaB-competent cells by the addition of H2O2. Small interfering RNA-mediated knockdown of beclin 1 and atg7 expression, two autophagy-related genes, reduced TNFalpha- and reactive oxygen species-induced apoptosis in cells lacking NF-kappaB activation and in NF-kappaB-competent cells, respectively. These findings demonstrate that autophagy may amplify apoptosis when associated with a death signaling pathway. They are also evidence that inhibition of autophagy is a novel mechanism of the antiapoptotic function of NF-kappaB activation. We suggest that stimulation of autophagy may be a potential way bypassing the resistance of cancer cells to anti-cancer agents that activate NF-kappaB.     

The WTX tumor suppressor enhances p53 acetylation by CBP/p300

WTX encodes a tumor suppressor, frequently inactivated in Wilms tumor, with both plasma membrane and nuclear localization. WTX has been implicated in β-catenin turnover, but its effect on nuclear proteins is unknown. We report an interaction between WTX and p53, derived from the unexpected observation of WTX, p53, and E1B 55K colocalization within the characteristic cytoplasmic body of adenovirus-transformed kidney cells. In other cells without adenovirus expression, the C-terminal domain of WTX binds to the DNA-binding domain of p53, enhances its binding to CBP, and increases CBP/p300-mediated acetylation of p53 at Lys 373/382. WTX knockdown accelerates CBP/p300 protein turnover and attenuates this modification of p53. In p53-reconstitution experiments, cell-cycle arrest, apoptosis, and p53 target-gene expression are suppressed by depletion of WTX. Together, these results suggest that WTX modulates p53 function, in part through regulation of its activator CBP/p300.     

Cirp protects against tumor necrosis factor-alpha-induced apoptosis via activation of extracellular signal-regulated kinase

Mild hypothermia shows protective effects on patients with brain damage and cardiac arrest. To elucidate the molecular mechanisms underlying these effects, we analyzed the effects of low culture temperature (32 degrees C) and cold-inducible RNA-binding protein (Cirp) expression on apoptosis in vitro. In BALB/3T3 cells treated with tumor necrosis factor (TNF)-alpha and cycloheximide, the down-shift in temperature from 37 degrees C to 32 degrees C increased the expression of Cirp and suppressed the apoptosis. Activation of caspase-8 was suppressed, and the level of phosphorylated extracellular signal-regulated kinase (ERK) was increased. Transduction of Cirp into the Cirp-deficient mouse fibroblasts increased the level of phosphorylated ERK and suppressed the TNF-alpha-induced apoptosis both at 37 degrees C and 32 degrees C. The ERK-specific inhibitor PD98059 decreased the cytoprotective effect of Cirp as well as that of low culture temperature. These data suggest that mild hypothermia protects cells from TNF-alpha-induced apoptosis, at least partly, via induction of Cirp, and that Cirp protects cells by activating the ERK pathway.     

Protein phosphatase 4 is involved in tumor necrosis factor-alpha-induced activation of c-Jun N-terminal kinase.

Protein phosphatase 4 (PP4, previously named protein phosphatase X (PPX)), a PP2A-related serine/threonine phosphatase, has been shown to be involved in essential cellular processes, such as microtubule growth and nuclear factor kappa B activation. We provide evidence that PP4 is involved in tumor necrosis factor (TNF)-alpha signaling in human embryonic kidney 293T (HEK293T) cells. Treatment of HEK293T cells with TNF-alpha resulted in time-dependent activation of endogenous PP4, peaking at 10 min, as well as increased serine and threonine phosphorylation of PP4. We also found that PP4 is involved in relaying the TNF-alpha signal to c-Jun N-terminal kinase (JNK) as indicated by the ability of PP4-RL, a dominant-negative PP4 mutant, to block TNF-alpha-induced JNK activation. Moreover, the response of JNK to TNF-alpha was inhibited in HEK293 cells stably expressing PP4-RL in comparison to parental HEK293 cells. The involvement of PP4 in JNK signaling was further demonstrated by the specific activation of JNK, but not p38 and ERK2, by PP4 in transient transfection assays. However, no direct PP4-JNK interaction was detected, suggesting that PP4 exerts its positive regulatory effect on JNK in an indirect manner. Taken together, these data indicate that PP4 is a signaling component of the JNK cascade and involved in relaying the TNF-alpha signal to the JNK pathway.     

Tumor necrosis factor-alpha-induced cyclooxygenase-2 expression in human tracheal smooth muscle cells: involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-kappaB

This study was to determine the mechanism of tumor necrosis factor-alpha (TNF-alpha)-enhanced cyclooxygenase (COX)-2 expression associated with prostaglandin E2 (PGE2) synthesis in human tracheal smooth muscle cells (HTSMCs). TNF-alpha markedly increased COX-2 expression and PGE2 synthesis in a time- and concentration-dependent manner, whereas COX-1 remained unaltered. Tyrosine kinase inhibitor (genistein), phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D-609) and PKC inhibitor (GF109203X) attenuated TNF-alpha-induced COX-2 expression and PGE2 synthesis in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis were also inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 and SB202190 (inhibitors of p38 MAPK), respectively, suggesting the involvement of p42/p44 and p38 MAPKs in these responses. This hypothesis was further supported by that TNF-alpha induced a transient activation of p42/p44 and p38 MAPKs in a time-and concentration-dependent manner. Furthermore, TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) reversely correlated with the degradation of IkappaB-alpha in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis was also inhibited by NF-kappaB inhibitor pyrrolidinedithiocarbamate (PDTC). These findings suggest that the increased expression of COX-2 correlates with the release of PGE2 from TNF-alpha-challenged HTSMCs, at least in part, mediated through p42/p44 and p38 MAPKs as well as NF-kappaB signaling pathways in HTSMCs.     

Ebselen inhibits tumor necrosis factor-alpha-induced c-Jun N-terminal kinase activation and adhesion molecule expression in endothelial cells

Tumor necrosis factor-alpha (TNF-alpha) stimulates expression of endothelial cell (EC) genes that may promote atherosclerosis in part by an activation of mitogen-activated protein (MAP) kinases. Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one), a selenoorganic compound, is effective for acute ischemic stroke; however, its effect on EC has not yet been elucidated. We examined the effect of ebselen on TNF-alpha-induced MAP kinase activation and adhesion molecule expression in cultured human umbilical vein endothelial cells (HUVEC). Extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 were rapidly and significantly activated by TNF-alpha in HUVEC. TNF-alpha-induced JNK activation was inhibited by ebselen, whereas ERK1/2 and p38 were not affected. Apoptosis signal-regulated kinase 1 (ASK1) was suggested to be involved in TNF-alpha-induced JNK activation because transfection of kinase-inactive ASK1 inhibited TNF-alpha-induced JNK activation. Ebselen inhibited TNF-alpha-induced TNF receptor-associated factor 2 (TRAF2)-ASK1 complex formation and phosphorylation of stress-activated protein kinase ERK kinase 1 (SEK1), which is an upstream signaling molecule of JNK. Finally, TNF-alpha-induced activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) activation and resultant intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions were inhibited by ebselen. Specific inhibitors for JNK and NF-kappaB also inhibited TNF-alpha-induced ICAM-1 and VCAM-1 expressions in HUVEC. These findings suggest that ebselen prevents TNF-alpha-induced EC activation through the inhibition of TRAF2-ASK1-SEK1 signaling pathway, which leads to JNK activation. Inhibition of JNK by ebselen may imply its usefulness for the prevention of atherosclerosis relevant to EC activation.     

Insulin-like growth factor binding protein-3 mediates tumor necrosis factor-alpha-induced apoptosis: role of Bcl-2 phosphorylation.

The insulin-like growth factor (IGF)-independent effects of insulin-like growth factor binding protein-3 (IGFBP-3) to effect cellular apoptosis have now been described in various cellular systems. IGFBP-3 mediates transforming growth factor-beta-induced apoptosis. Other growth-inhibitory and apoptosis-inducing agents such as tumor necrosis factor-alpha (TNF-alpha) and the tumor suppressor gene p53 also induce IGFBP-3. In this report, we demonstrate the role of IGFBP-3 as a mediator of apoptosis induced by TNF-alpha and elucidate the process involved in its signaling mechanism. Treatment of PC-3 cells with TNF-alpha resulted in the induction of IGFBP-3 expression in a dose- and time-dependent fashion and also induced apoptosis. TNF-alpha-induced apoptosis was prevented by cotreatment with IGFBP-3 neutralizing antibodies or IGFBP-3-specific antisense thiolated oligonucleotides. Both IGFBP-3 and TNF-alpha treatment increased the levels of the inactive, serine phosphorylated form of the survival protein Bcl-2. The effect of TNF-alpha on Bcl-2 serine phosphorylation was blocked by IGFBP-3 antisense oligomers. These findings confirm that IGFBP-3 is essential for TNF-alpha-induced apoptosis in PC-3 cells and that this IGFBP-3 effect includes the inactivation of Bcl-2 through serine phosphorylation.     

Vagotomy attenuates tumor necrosis factor-alpha-induced sleep and EEG delta-activity in rats

Much evidence suggests that tumor necrosis factor-alpha (TNF-alpha) is involved in the regulation of physiological sleep. However, it remains unclear whether peripheral administration of TNF-alpha induces sleep in rats. Furthermore, the role of the vagus nerve in the somnogenic actions of TNF-alpha had not heretofore been studied. Four doses of TNF-alpha were administered intraperitoneally just before the onset of the dark period. The three higher doses of TNF-alpha (50, 100, and 200 microg/kg) dose dependently increased nonrapid eye movement sleep (NREMS), accompanied by increases in electroencephalogram (EEG) slow-wave activity. TNF-alpha increased EEG delta-power and decreased EEG alpha- and beta-power during the initial 3 h after injection. In vagotomized rats, the NREMS responses to 50 or 100 microg/kg of TNF-alpha were attenuated, while significant TNF-alpha-induced increases in NREMS were observed in a sham-operated group. Moreover, the vagotomized rats failed to exhibit the increase in EEG delta-power induced by TNF-alpha intraperitoneally. These results suggest that peripheral TNF-alpha can induce NREMS and vagal afferents play an important role in the effects of peripheral TNF-alpha and EEG synchronization on sleep. Intraperitoneal TNF-alpha failed to affect brain temperature at the doses tested, thereby demonstrating that TNF-alpha-induced sleep effects are, in part, independent from its effects on brain temperature. Results are consistent with the hypothesis that a cytokine network is involved in sleep regulation.     

RelB/p50 dimers are differentially regulated by tumor necrosis factor-alpha and lymphotoxin-beta receptor activation: critical roles for p100

Tumor necrosis factor-alpha (TNF-alpha) and lymphotoxin-beta receptor (LTbetaR) signaling both play important roles in inflammatory and immune responses through activation of NF-kappaB. Using various deficient mouse embryonic fibroblast cells, we have compared the signaling pathways leading to NF-kappaB induction in response to TNF-alpha and LTbetaR activation. We demonstrate that LTbetaR ligation induces not only RelA/p50 dimers but also RelB/p50 dimers, whereas TNF-alpha induces only RelA/p50 dimers. LTbetaR-induced binding of RelB/p50 requires processing of p100 that is mediated by IKKalpha but is independent of IKKbeta, NEMO/IKKgamma, and RelA. Moreover, we show that RelB, p50, and p100 can associate in the same complex and that TNF-alpha but not LTbeta signaling increases the association of p100 with RelB/p50 dimers in the nucleus, leading to the specific inhibition of RelB DNA binding. These results suggest that the alternative NF-kappaB pathway based on p100 processing may account not only for the activation of RelB/p52 dimers but also for that of RelB/p50 dimers and that p100 regulates the binding activity of RelB/p50 dimers via at least two distinct mechanisms depending on the signaling pathway involved.