Novel phosphorylation-dependent ubiquitination of tristetraprolin by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1) and tumor necrosis factor receptor-associated factor 2 (TRAF2)

Acute versus chronic inflammation is controlled by the accurate activation and regulation of interdependent signaling cascades. TNF-receptor 1 engagement concomitantly activates NF-κB and JNK signaling. The correctly timed activation of these pathways is the key to account for the balance between NF-κB-mediated cell survival and cell death, the latter fostered by prolonged JNK activation. Tristetraprolin (TTP), initially described as an mRNA destabilizing protein, acts as negative feedback regulator of the inflammatory response: it destabilizes cytokine-mRNAs but also acts as an NF-κB inhibitor by interfering with the p65/RelA nuclear import pathway. Our biochemical studies provide evidence that TTP contributes to the NF-κB/JNK balance. We find that the MAP 3-kinase MEKK1 acts as a novel TTP kinase that, together with the TNF receptor-associated factor 2 (TRAF2), constitutes not only a main determinate of the NF-κB-JNK cross-talk but also facilitates "TTP hypermodification": MEKK1 triggers TTP phosphorylation as prerequisite for its Lys-63-linked, TRAF2-mediated ubiquitination. Consequently, TTP no longer affects NF-κB activity but promotes the activation of JNK. Based on our data, we suggest a model where upon TNFα induction, TTP transits a hypo- to hypermodified state, thereby contributing to the molecular regulation of NF-κB versus JNK signaling cascades.     

c-Jun N-terminal kinase attenuates TNFα signaling by reducing Nox1-dependent endosomal ROS production in vascular smooth muscle cells

Tumor necrosis factor-α (TNFα), a proinflammatory cytokine, causes vascular smooth muscle cell (VSMC) proliferation and migration and promotes inflammatory vascular lesions. Nuclear factor-kappa B (NF-κB) activation by TNFα requires endosomal superoxide production by Nox1. In endothelial cells, TNFα stimulates c-Jun N-terminal kinase (JNK), which inhibits NF-κB signaling. The mechanism by which JNK negatively regulates TNFα-induced NF-κB activation has not been defined. We hypothesized that JNK modulates NF-κB activation in VSMC, and does so via a Nox1-dependent mechanism. TNFα-induced NF-κB activation was TNFR1- and endocytosis-dependent. Inhibition of endocytosis with dominant-negative dynamin (DynK44A) potentiated TNFα-induced JNK activation, but decreased ERK activation, while p38 kinase phosphorylation was not altered. DynK44A attenuated intracellular, endosomal superoxide production in wild-type (WT) VSMC, but not in NADPH oxidase 1 (Nox1) knockout (KO) cells. siRNA targeting JNK1 or JNK2 potentiated, while a JNK activator (anisomycin) inhibited, TNFα-induced NF-κB activation in WT, but not in Nox1 KO cells. TNFα-stimulated superoxide generation was enhanced by JNK1 inhibition in WT, but not in Nox1 KO VSMC. These data suggest that JNK suppresses the inflammatory response to TNFα by reducing Nox1-dependent endosomal ROS production. JNK and endosomal superoxide may represent novel targets for pharmacologic modulation of TNFα signaling and vascular inflammation.     

In mouse embryonic fibroblasts, neither caspase-8 nor cellular FLICE-inhibitory protein (FLIP) is necessary for TNF to activate NF-κB, but caspase-8 is required for TNF to cause cell death, and induction of FLIP by NF-κB is required to prevent it

Binding of TNF to TNF receptor-1 can give a pro-survival signal through activation of p65/RelA NF-κB, but also signals cell death. To determine the roles of FLICE-inhibitory protein (FLIP) and caspase-8 in TNF-induced activation of NF-κB and apoptosis, we used mouse embryonic fibroblasts derived from FLIP and caspase-8 gene-deleted mice, and treated them with TNF and a smac-mimetic compound that causes degradation of cellular inhibitor of apoptosis proteins (cIAPs). In cells treated with smac mimetic, TNF and Fas Ligand caused wild-type and FLIP(-/-) MEFs to die, whereas caspase-8(-/-) MEFs survived, indicating that caspase-8 is necessary for death of MEFs triggered by these ligands when IAPs are degraded. By contrast, neither caspase-8 nor FLIP was required for TNF to activate p65/RelA NF-κB, because IκB was degraded, p65 translocated to the nucleus, and an NF-κB reporter gene activated normally in caspase-8(-/-) or FLIP(-/-) MEFs. Reconstitution of FLIP(-/-) MEFs with the FLIP isoforms FLIP-L, FLIP-R, or FLIP-p43 protected these cells from dying when treated with TNF or FasL, whether or not cIAPs were depleted. These results show that in MEFs, caspase-8 is necessary for TNF- and FasL-induced death, and FLIP is needed to prevent it, but neither caspase-8 nor FLIP is required for TNF to activate NF-κB.     

JNK3通过与RelA/p65相互作用抑制NF-κB信号通路减弱Bel-7402细胞的黏附能力

JNK信号通路在细胞的炎症、增殖与凋亡等生物学过程中发挥了重要的作用．我们采用酵母双杂交技术发现转录因子p65是JNK3的相互作用蛋白质．体内体外实验均证实JNK3与p65存在蛋白质相互作用．报告基因实验结果表明过表达JNK3抑制TNFα诱导NF-κB介导的转录激活．EMSA结果证明JNK3减弱NF-κB的DNA结合能力．实时定量PCR结果表明JNF3减少NF-κB靶基因的表达．综上所述,我们的研究结果表明JNK3做为一个调节分子在体内发挥了抑制p65转录活性的功能．     

Smac mimetic compounds potentiate interleukin-1beta-mediated cell death

Smac mimetic compounds (SMCs) potentiate TNFα-mediated cancer cell death by targeting the inhibitor of apoptosis (IAP) proteins. In addition to TNFα, the tumor microenvironment is exposed to a number of pro-inflammatory cytokines, including IL-1β. Here, we investigated the potential impact of IL-1β on SMC-mediated death of cancer cells. Synergy was seen in a subset of a diverse panel of 21 cancer cell lines to the combination of SMC and IL-1β treatment, which required IL-1β-induced activation of the NF-κB pathway. Elevated NF-κB activity resulted in the production of TNFα, which led to apoptosis dependent on caspase-8 and RIP1. In addition, concurrent silencing of cIAP1, cIAP2, and X-linked IAP by siRNA was most effective for triggering IL-1β-mediated cell death. Importantly, SMC-resistant cells that produced TNFα in response to IL-1β treatment were converted to an SMC-sensitive phenotype by c-FLIP knockdown. Reciprocally, ectopic expression of c-FLIP blocked cell death caused by combined SMC and IL-1β treatment in sensitive cancer cells. Together, our study indicates that a positive feed-forward loop by pro-inflammatory cytokines can be exploited by SMCs to induce apoptosis in cancer cells.     

TRAF2 phosphorylation promotes NF-κB-dependent gene expression and inhibits oxidative stress-induced cell death

Tumor necrosis factor α (TNF-α) receptor-associated factor 2 (TRAF2) regulates activation of the c-Jun N-terminal kinase (JNK)/c-Jun and the inhibitor of κB kinase (IKK)/nuclear factor κB (NF-κB) signaling cascades in response to TNF-α stimulation. Gene knockout studies have revealed that TRAF2 inhibits TNF-α-induced cell death but promotes oxidative stress-induced apoptosis. Here we report that TNF-α and oxidative stress both induce TRAF2 phosphorylation at serines 11 and 55 and that this dual phosphorylation promotes the prolonged phase of IKK activation while inhibiting the prolonged phase of JNK activation. Prolonged IKK activation trigged by TNF-α plays an essential role in efficient expression of a subset of NF-κB target genes but has no substantial role in TNF-α-induced cell death. On the other hand, TRAF2 phosphorylation in response to oxidative stress significantly promotes cell survival by inducing prolonged IKK activation and by inhibiting the prolonged phase of JNK activation. Notably, stable expression of phospho-null mutant TRAF2 in cancer cells leads to an increase in the basal and inducible JNK activation and B-cell lymphoma 2 (Bcl-2) phosphorylation. In addition, exposure of cells expressing phospho-null mutant TRAF2 to sublethal oxidative stress results in a rapid degradation of Bcl-2 and cellular inhibitor of apoptosis 1 as well as significantly increased cell death. These results suggest that TRAF2 phosphorylation is essential for cell survival under conditions of oxidative stress.     

Cellular FLICE-inhibitory protein (cFLIP) isoforms block CD95- and TRAIL death receptor-induced gene induction irrespective of processing of caspase-8 or cFLIP in the death-inducing signaling complex

Death receptors (DRs) induce apoptosis but also stimulate proinflammatory "non-apoptotic" signaling (e.g. NF-κB and mitogen-activated protein kinase (MAPK) activation) and inhibit distinct steps of DR-activated maturation of procaspase-8. To examine whether isoforms of cellular FLIP (cFLIP) or its cleavage products differentially regulate DR signaling, we established HaCaT cells expressing cFLIP(S), cFLIP(L), or mutants of cFLIP(L) (cFLIP(D376N) and cFLIP(p43)). cFLIP variants blocked TRAIL- and CD95L-induced apoptosis, but the cleavage pattern of caspase-8 in the death inducing signaling complex was different: cFLIP(L) induced processing of caspase-8 to the p43/41 fragments irrespective of cFLIP cleavage. cFLIP(S) or cFLIP(p43) blocked procaspase-8 cleavage. Analyzing non-apoptotic signaling pathways, we found that TRAIL and CD95L activate JNK and p38 within 15 min. cFLIP variants and different caspase inhibitors blocked late death ligand-induced JNK or p38 MAPK activation suggesting that these responses are secondary to cell death. cFLIP isoforms/mutants also blocked death ligand-mediated gene induction of CXCL-8 (IL-8). Knockdown of caspase-8 fully suppressed apoptotic and non-apoptotic signaling. Knockdown of cFLIP isoforms in primary human keratinocytes enhanced CD95L- and TRAIL-induced NF-κB activation, and JNK and p38 activation, underscoring the regulatory role of cFLIP for these DR-mediated signals. Whereas the presence of caspase-8 is critical for apoptotic and non-apoptotic signaling, cFLIP isoforms are potent inhibitors of TRAIL- and CD95L-induced apoptosis, NF-κB activation, and the late JNK and p38 MAPK activation. cFLIP-mediated inhibition of CD95 and TRAIL DR could be of crucial importance during keratinocyte skin carcinogenesis and for the activation of innate and/or adaptive immune responses triggered by DR activation in the skin.     

Nuclear-factor-kappaB (NF-kappaB) and radical oxygen species play contrary roles in transforming growth factor-beta1 (TGF-beta1)-induced apoptosis in hepatocellular carcinoma (HCC) cells

Nuclear-Factor-κB (NF-κΒ can counteract transforming growth factor-β1 (TGF-β1)-induced apoptosis in malignant hepatocytes through up-regulation of its downstream genes, such as X-linked inhibitor of apoptosis protein (XIAP). Reports have demonstrated that TGF-β1 can induce oxidative stress, and c-Jun N-terminal Kinase1 (JNK1) is indispensable for TGF-β1-induced apoptosis pathway, but the relationship between radical oxygen species (ROS) and the activation of JNKs is still unclear. In the present study, we found that ROS can induce JNK activation in TGF-β1 mediated apoptosis in hepatocytes. The inhibitors of hydrogen peroxide and superoxide, which were produced by mitochondria under stress, could inhibit the phosphorylation of c-Jun in XIAP knockdown cells. In conclusion, it is the first time to show that both NF-κB and antioxidants can counteract TGF-β1-induced apoptosis in hepatic cell death through JNK1 pathway.     

TWEAK induces apoptosis through a death-signaling complex comprising receptor-interacting protein 1 (RIP1), Fas-associated death domain (FADD), and caspase-8

The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TNFSF12, CD255) (TWEAK) can stimulate apoptosis in certain cancer cells. Previous studies suggest that TWEAK activates cell death indirectly, by inducing TNFα-mediated autocrine signals. However, the underlying death-signaling mechanism has not been directly defined. Consistent with earlier work, TWEAK assembled a proximal signaling complex containing its cognate receptor FN14, the adaptor TRAF2, and cellular inhibitor of apoptosis protein 1 (cIAP1). Neither the death domain adaptor Fas-associated death domain nor the apoptosis-initiating protease caspase-8 associated with this primary complex. Rather, TWEAK induced TNFα secretion and TNF receptor 1-dependent assembly of a death-signaling complex containing receptor-interacting protein 1 (RIP1), FADD, and caspase-8. Knockdown of RIP1 by siRNA prevented TWEAK-induced association of FADD with caspase-8 but not formation of the FN14-TRAF2-cIAP1 complex and inhibited apoptosis activation. Depletion of the RIP1 E3 ubiquitin ligase cIAP1 enhanced assembly of the RIP1-FADD-caspase-8 complex and augmented cell death. Conversely, knockdown of the RIP1 deubiquitinase CYLD inhibited these functions. Depletion of FADD, caspase-8, BID, or BAX and BAK but not RIP3 attenuated TWEAK-induced cell death. Pharmacologic inhibition of the NF-κB pathway or siRNA knockdown of RelA attenuated TWEAK induction of TNFα and association of RIP1 with FADD and caspase-8. These results suggest that TWEAK triggers apoptosis by promoting assembly of a RIP1-FADD-caspse-8 complex via autocrine TNFα-TNFR1 signaling. The proapoptotic activity of TWEAK is modulated by cIAP1 and CYLD and engages both the extrinsic and intrinsic signaling pathways.     

Shikonin, a Chinese plant-derived naphthoquinone, induces apoptosis in hepatocellular carcinoma cells through reactive oxygen species: A potential new treatment for hepatocellular carcinoma

Although shikonin, a naphthoquinone derivative, has showed anti-cancer activity, its precise molecular anti-tumor mechanism remains to be elucidated. In this study, we investigated the effects of shikonin on human hepatocellular carcinoma (HCC) in vitro and in vivo. Our results showed that shikonin induced apoptosis of Huh7 and BEL7402 but not nontumorigenic cells. ROS generation was detected, and ROS scavengers completely inhibited shikonin-induced apoptosis, indicating that ROS play an essential role. Although the JNK activity was significantly elevated after shikonin treatment, JNK was not linked to apoptosis. However, downregulation of Akt and RIP1/NF-κB activity was found to be involved in shikonin-induced apoptosis. Ectopic expression of Akt or RIP1 partly abrogated the effects of shikonin, and Akt inhibitor and RIP1 inhibitor synergistically induced apoptosis in conjunction with shikonin treatment. ROS scavengers blocked shikonin-induced inactivation of Akt and RIP1/NF-κB, but Akt or RIP1/NF-κB did not regulate ROS generation, suggesting that Akt and RIP1/NF-κB signals are downstream of ROS generation. In addition, the results of xenograft experiments in mice were consistent with in vitro studies. Taken together, our data show that shikonin, which may be a promising agent in the treatment of liver cancer, induced apoptosis in HCC cells through the ROS/Akt and RIP1/NF-κB pathways.     

c-Rel deficiency increases caspase-4 expression and leads to ER stress and necrosis in EBV-transformed cells

LMP1-mediated activation of nuclear factor of kappaB (NF-κB) is critical for the ligand independent proliferation and cell survival of in vitro EBV-transformed lymphoblastoid cell lines (LCLs). Previous experiments revealed that a majority of LMP1-dependent responses are regulated by NF-κB. However, the extent that individual NF-κB family members are required for these responses, in particular, c-Rel, whose expression is restricted to mature hematopoietic cells, remains unclear. Here we report that low c-Rel expression in LCLs derived from a patient with hyper-IgM syndrome (Pt1), resulted in defects in proliferation and cell survival. In contrast to studies that associated loss of NF-κB with increased apoptosis, Pt1 LCLs failed to initiate apoptosis and alternatively underwent autophagy and necrotic cell death. Whereas the proliferation defect appeared linked to a c-Rel-associated decrease in c-myc expression, identified pro-survival and pro-apoptotic targets were expressed at or near control levels consistent with the absence of apoptosis. Ultrastructural examination of Pt1 LCLs revealed a high level of cellular and ER stress that was further supported by gene expression profiling showing the upregulation of several genes involved in stress and inflammation. Apoptosis-independent cell death was accompanied by increased expression of the inflammatory marker, caspase-4. Using gene overexpression and siRNA knockdown we demonstrated that levels of c-Rel directly modulated expression of caspase-4 as well as other ER stress genes. Overall, these findings reveal the importance of c-Rel in maintaining LCL viability and that decreased expression results in ER stress and a default response leading to necrotic cell death.     

Anti-inflammatory, pro-apoptotic, and anti-proliferative effects of a methanolic neem (Azadirachta indica) leaf extract are mediated via modulation of the nuclear factor-κB pathway

Azadirachta indica (neem tree) is used in traditional Indian medicine for its pharmacological properties including cancer prevention and treatment. Here, we studied a neem extract's anti-inflammatory potential via the nuclear factor-κB (NF-κB) signaling pathway, linked to cancer, inflammation, and apoptosis. Cultured human leukemia cells were treated with a methanolic neem leaf extract with or without tumor necrosis factor (TNF)-α stimulation. Inhibition of NF-κB activity was demonstrated by luciferase assay and electrophoretic mobility shift assay (EMSA). Inhibition of viability by neem extracts was assessed by luminescent assays. Western blot analysis allowed assessing the inhibitory effect of the neem extract on TNF-α-induced degradation of inhibitor of κB (IκB) and nuclear translocation of the NF-κB p50/p65 heterodimer. Inhibition of IκB kinase (IKK) activity was shown as well as the effect of neem extract on the induction of apoptotic cell death mechanisms by nuclear fragmentation analysis and flow cytometry analysis. In conclusion, our data provide evidence for a strong effect of the neem extract on pro-inflammatory cell signaling and apoptotic cell death mechanisms, contributing to a better understanding of the mechanisms triggered by Azadirachta indica.     

活性氧对NF-κB活性及JNK信号通路的调节

活性氧（ROS）是生物体有氧代谢过程中产生的一类活性含氧化合物的总称,机体细胞可通过多种途径维持ROS产生与降解的动态平衡。研究表明,活性氧可作为第二信使调节与细胞增殖、分化、凋亡相关的信号转导通路。c-JunN端激酶（JNK）通路可以介导氧化应激、细胞因子、紫外照射等引起的细胞凋亡。另外,κ基因结合核因子（NF-κB）是氧化应激调节的靶因子之一,同样也能诱导促进细胞内的氧化应激反应,还可通过活性氧蓄积抑制JNK的激活。简要综述活性氧对NF-κB和JNK信号通路的调节。