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Apoptosis is the prototype for a regulated form of cell death, but recent studies have revealed other types of regulated forms of cell death, including necroptosis and ferroptosis. The molecular mechanisms underlying the execution of these processes have been intensively investigated, yet the hallmarks of their morphology are not fully understood. Here, we report that electron lucent cytoplasm was a common feature of both necroptosis and ferroptosis, which was consistent with cytoplasmic vacuolization due to a defect in the cytoplasmic membrane integrity. Notably, the perinuclear space was dilated in necroptosis, but such dilation did not occur in ferroptosis. Cells undergoing ferroptosis, but not necroptosis, exhibited an electron lucent nucleus. We previously reported that one of the nuclear danger-associated molecular patterns (DAMPs), high mobility group box (HMGB)1, is rapidly released from the nucleus to the extracellular spaces of cells undergoing necroptosis through the ruptured nuclear and cytoplasmic membrane. Via time-lapse imaging of cells stably expressing HMGB1 fused to a fluorescence protein, we found that HMGB1 was also released from the nucleus to the cytosol, and then eventually released into the extracellular spaces in cells undergoing ferroptosis. Thus, nuclear membrane damage was induced prior to cytoplasmic membrane rupture in ferroptosis. Thus, dilation of the perinuclear space and an electron lucent nucleus may be the hallmarks of necroptosis and ferroptosis, respectively.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that plays a crucial role in protection of cells from electrophile-induced toxicity through up-regulating phase II detoxifying enzymes and phase III transporters. We previously reported that oxidative stress induces up-regulation of interleukin-11 (IL-11), a member of the IL-6 family that ameliorates acetaminophen-induced liver toxicity. However, a role for IL-11 in protection of cells from electrophile-induced toxicity remains unclear. Here we show that an environmental electrophile, 1,2-naphthoquinone (1,2-NQ), but not 15d-prostaglandin J2 (PGJ2) or tert-butylhydroxyquinone (tBHQ), induced IL-11 production. Consistent with a crucial role for prolonged ERK activation in H2O2-induced IL-11 production, 1,2-NQ, but not 15d-PGJ2 or tBHQ, elicited prolonged ERK activation. Conversely, inhibition of the ERK pathway by a MEK inhibitor completely blocked 1,2-NQ-induced IL-11 production at both protein and mRNA levels, further substantiating an intimate cross-talk between ERK activation and 1,2-NQ-induced IL-11 production. Promoter analysis of the Il11 gene revealed that two AP-1 sites were essential for 1,2-NQ-induced promoter activities. Among various members of the AP-1 family, Fra-1 was up-regulated by 1,2-NQ, and its up-regulation was blocked by a MEK inhibitor. Although NRF2 was not required for H2O2-induced IL11 up-regulation, NRF2 was essential for 1,2-NQ-induced IL11 up-regulation by increasing Fra-1 proteins possibly through promoting mRNA translation of FOSL1. Finally, intraperitoneal administration of 1,2-NQ induced body weight loss in wild-type mice, which was further exacerbated in Il11ra1−/− mice compared with Il11ra1+/− mice. Together, both Fra-1 and NRF2 play crucial roles in IL-11 production that protects cells from 1,2-NQ intestinal toxicity.

Background A delicate balance between cell death and keratinocyte proliferation is crucial for normal skin development. Previous studies have reported that cellular FLICE (FADD-like ICE)-inhibitory protein plays a crucial role in prevention of keratinocytes from TNF-α–dependent apoptosis and blocking of dermatitis. However, a role for cellular FLICE-inhibitory protein in TNF-α–independent cell death remains unclear. Objective We investigated contribution of TNF-α–dependent and TNF-α–independent signals to the development of dermatitis in epidermis-specific Cflar -deficient (Cflar E-KO ) mice. Methods We examined the histology and expression of epidermal differentiation markers and inflammatory cytokines in the skin of Cflar E-KO ; Tnfrsf1a +/− and Cflar E-KO ; Tnfrsf1a −/− mice. Mice were treated with neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand to block TNF-α–independent cell death of Cflar E-KO ; Tnfrsf1a −/− mice. Results Cflar E-KO ; Tnfrsf1a −/− mice were born but experienced severe dermatitis and succumbed soon after birth. Cflar E-KO ;Tnfrsf1a +/− mice exhibited embryonic lethality caused by massive keratinocyte apoptosis. Although keratinocytes from Cflar E-KO ; Tnfrsf1a −/− mice still died of apoptosis, neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand substantially prolonged survival of Cflar E-KO ; Tnfrsf1a −/− mice. Expression of inflammatory cytokines, such as Il6 and Il17a was increased; conversely, expression of epidermal differentiation markers was severely downregulated in the skin of Cflar E-KO ; Tnfrsf1a −/− mice. Treatment of primary keratinocytes with IL-6 and, to a lesser extent, IL-17A suppressed expression of epidermal differentiation markers. Conclusion TNF receptor superfamily 1 (TNFR1)–dependent or TNFR1-independent apoptosis of keratinocytes promotes inflammatory cytokine production, which subsequently blocks epidermal differentiation. Thus blockade of both TNFR1-dependent and TNFR1-independent cell death might be an alternative strategy to treat skin diseases when treatment with anti–TNF-α antibody alone is not sufficient.

While apoptosis has been considered to be identical to programmed cell death, necroptosis, which is morphologically related to necrosis, has emerged as a novel type of programmed cell death. Necroptosis depends on two structurally related kinases, receptor-interacting serine-threonine kinase (RIPK)1 and RIPK3. RIPK1 is activated through oligomerization of upstream adaptor molecules such as Fas-associated protein with death domain (FADD) and TNF receptor-associated death domain (TRADD) that are triggered by TNFα or Fas ligand. Activated RIPK1 subsequently interacts with and activates RIPK3, resulting in necroptosis. However, contribution of oxidative stress to execution of necroptosis is still controversial. We found that a selective inhibitor for RIPK1, necrostatin-1 (Nec-1) significantly blocked TNFα-induced cell death and ROS accumulation in NF-κB activation-deficient cells. This suggests that these cells mostly died by necroptosis upon TNFα stimulation. Intriguingly, an antioxidant, butylated hydroxyanisole (BHA) blocked TNFα-induced necroptosis and ROS accumulation in NF-κB activation-deficient cells. However, Nec-1, but not BHA, inhibited TNFα-induced phosphorylation of RIPK1 in these cells, suggesting that ROS play a crucial role in execution of necroptosis downstream of RIPK1 activation. Structural and functional analyses using BHA related compounds revealed that both tert-butyl and hydroxy groups of BHA are crucial for its anti-necroptotic function. Together, these results suggest that TNFα-induced necroptosis is tightly associated with oxidative stress, and oxidative stress is induced downstream of RIPK1 activation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/death receptor 5 (DR5)-mediated cell death plays an important role in the elimination of tumor cells and transformed cells. Recently, recombinant TRAIL and agonistic anti-DR5 monoclonal antibodies have been developed and applied to cancer therapy. However, depending on the type of cancer, the sensitivity to TRAIL has been reportedly different, and some tumor cells are resistant to TRAIL-mediated apoptosis. Using confocal microscopy, we found that large amounts of DR5 were localized in the nucleus in HeLa and HepG2 cells. Moreover, these tumor cells were resistant to TRAIL, whereas DU145 cells, which do not have nuclear DR5, were highly sensitive to TRAIL. By means of immunoprecipitation and Western blot analysis, we found that DR5 and importin β1 were physically associated, suggesting that the nuclear DR5 was transported through the nuclear import pathway mediated by importin β1. Two functional nuclear localization signals were identified in DR5, the mutation of which abrogated the nuclear localization of DR5 in HeLa cells. Moreover, the nuclear transport of DR5 was also prevented by the knockdown of importin β1 using siRNA, resulting in the up-regulation of DR5 expression on the cell surface and an increased sensitivity of HeLa and HepG2 cells to TRAIL. Taken together, our findings suggest that the importin β1-mediated nuclear localization of DR5 limits the DR5/TRAIL-induced cell death of human tumor cells and thus can be a novel target to improve cancer therapy with recombinant TRAIL and anti-DR5 antibodies.

Fine-tuning of host cell responses to commensal bacteria plays a crucial role in maintaining homeostasis of the gut. Here, we show that tumor necrosis factor receptor-associated factor (Traf)2(-/-) mice spontaneously developed severe colitis and succumbed within 3 weeks after birth. Histological analysis revealed that apoptosis of colonic epithelial cells was enhanced, and B cells diffusely infiltrated into the submucosal layer of the colon of Traf2(-/-) mice. Expression of proinflammatory cytokines, including Tnfa, Il17a, and Ifng, was up-regulated, whereas expression of antimicrobial peptides was down-regulated in the colon of Traf2(-/-) mice. Moreover, a number of IL-17-producing helper T cells were increased in the colonic lamina propria of Traf2(-/-) mice. These cellular alterations resulted in drastic changes in the colonic microbiota of Traf2(-/-) mice compared with Traf2(+/+) mice. Treatment of Traf2(-/-) mice with antibiotics ameliorated colitis along with down-regulation of proinflammatory cytokines and prolonged survival, suggesting that the altered colonic microbiota might contribute to exacerbation of colitis. Finally, deletion of Tnfr1, but not Il17a, dramatically ameliorated colitis in Traf2(-/-) mice by preventing apoptosis of colonic epithelial cells, down-regulation of proinflammatory cytokines, and restoration of wild-type commensal bacteria. Together, TRAF2 plays a crucial role in controlling homeostasis of the colon.

OTT/RBM15-BSAC/MAL/MKL1/MRTF-A was identified as a fusion transcript generated by t(1;22)(p13;q13) in acute megakaryoblastic leukemia. Previous studies have shown that BSAC (basic, SAP, and coiled-coil domain) activates the promoters containing CArG boxes via interaction with serum response factor, and OTT (one twenty-two) negatively regulates the development of megakaryocytes and myeloid cells. However, the mechanism by which OTT-BSAC promotes leukemia is largely unknown. Here we show that OTT-BSAC, but not BSAC or OTT strongly activates several promoters containing a transcription factor Yin Yang 1-binding sequence. In addition, although BSAC predominantly localizes in the cytoplasm and its nuclear translocation is considered to be regulated by the Rho-actin signaling pathway, OTT-BSAC exclusively localizes in the nucleus. Moreover, OTT interacts with histone deacetylase 3, but this interaction is abolished in OTT-BSAC. Collectively, these functional and spatial changes of OTT and BSAC caused by the fusion might perturb their functions, culminating in the development of acute megakaryoblastic leukemia.

Apoptosis signal-regulating kinase 1 (ASK1) plays a pivotal role in oxidative stress-induced cell death. Reactive oxygen species disrupt the interaction of ASK1 with its cellular inhibitor thioredoxin and thereby activates ASK1. However, the precise mechanism by which ASK1 freed from thioredoxin undergoes oligomerization-dependent activation has not been fully elucidated. Here we show that endogenous ASK1 constitutively forms a high molecular mass complex including Trx ( approximately 1,500-2,000 kDa), which we designate ASK1 signalosome. Upon H(2)O(2) treatment, the ASK1 signalosome forms a higher molecular mass complex at least in part because of the recruitment of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF6. Consistent with our previous findings that TRAF2 and TRAF6 activate ASK1, H(2)O(2)-induced ASK1 activation and cell death were strongly reduced in the cells derived from Traf2-/- and Traf6-/- mice. A novel signaling complex including TRAF2, TRAF6, and ASK1 may thus be the key component in oxidative stress-induced cell death.

We recently showed that TNF induces accumulation of reactive oxygen species (ROS) that mediates necrosis in murine embryonic fibroblasts (MEFs) derived from TRAF2- and TRAF5-double deficient (DKO) mice. To elucidate the defects that subsequently cause accumulation of ROS in DKO MEFs, we compared gene expression profiles of wild-type and DKO MEFs before and after TNF stimulation using cDNA microarrays. Interestingly, many antioxidant enzymes are induced by TNF in wild-type MEFs, induction of these genes is impaired in DKO MEFs. Taken that TNF induces accumulation of ROS in DKO, but not wild-type MEFs, upregulation of antioxidant enzyme(s) might play a crucial role in elimination of ROS.

PU.1, a hematopoietic cell-specific Ets family transcription factor, is involved in the generation of murine erythroleukemia (MEL). To identify the target gene(s) of PU.1 in MEL cells, we carried out differential display (DD) analysis and isolated a novel gene whose expression was up-regulated after overexpression of PU.1 in MEL cells. Because the gene exhibited about 90% homology with the human calcium–calmodulin-dependent kinase I-like kinase (CKLiK) gene, it was identified as a mouse homologue of human CKLiK. The mCKLiK gene was mapped to the mouse chromosome 2A1-A3 region and shown to be expressed predominantly in T cells lymphoma and embryonal carcinoma cell lines and primary thymus and brain. Two types of transcripts were present showing a difference in the 3′ portion of the coding region and CREB-activating ability. Overexpression of each isoform of mCKLiK in MEL cells revealed that one of them induces, while the other inhibits apoptosis under low serum condition. Differentiation inhibition and lineage switch to myelomonocytes, which were previously observed in MEL cells overexpressing PU.1, were not provoked in the cells overexpressing mCKLiK. These results suggest that mCKLiK is up-regulated by PU.1 in MEL cells and involved in apoptosis of the cells.

LIGHT (homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes) is a member of the tumor necrosis factor superfamily that can interact with lymphotoxin-beta receptor (LTbetaR), herpes virus entry mediator, and decoy receptor (DcR3). In our previous study, we showed that LIGHT is able to induce cell death via the non-death domain containing receptor LTbetaR to activate both caspase-dependent and caspase-independent pathway. In this study, a LIGHT mutein, LIGHT-R228E, was shown to exhibit similar binding specificity as wild type LIGHT to LTbetaR, but lose the ability to interact with herpes virus entry mediator. By using both LIGHT-R228E and agonistic anti-LTbetaR monoclonal antibody, we found that signaling triggered by LTbetaR alone is sufficient to activate both caspase-dependent and caspase-independent pathways. Cross-linking of LTbetaR is able to recruit TRAF3 and TRAF5 to activate ASK1, whereas its activity is inhibited by free radical scavenger carboxyfullerenes. The activation of ASK1 is independent of caspase-3 activation, and kinase-inactive ASK1-KE mutant can inhibit LTbetaR-mediated cell death. This suggests that ASK1 is one of the factors involved in the caspase-independent pathway of LTbetaR-induced cell death.

Phosphorylation of several serine residues especially in the transactivation (TA) domain of p65 NF-kappaB subunit has been suggested to be important for its transcriptional activity. However, the responsible phosphorylation site of p65 remains controversial. To investigate the biological significance of phosphorylation and to determine the critical phosphorylation sites of p65, we reconstituted murine embryonic fibroblasts (MEFs) from p65(-/-) mice with various serine to alanine (SA)-substituted mutants of p65. Unexpectedly, mutants in the TA domain, including S529A, S536A, and S529A/S536A, completely rescued the defect of p65(-/-) MEFs as assessed by tumor necrosis factor (TNF)- or interleukin-1 (IL-1)-induced IL-6 production and protection from TNF-induced cell death. On the other hand, S276A mutant had an impaired ability to rescue these responses. Moreover, TNF-induced phosphorylation of p65 was severely impaired in S276A mutant, indicating that S276 is the major phosphorylation site of p65 and its phosphorylation is essential for p65-dependent cellular responses.

Tumor necrosis factor (TNF) is a multifunctional cytokine, which induces proliferation or death in a cell type-dependent manner. We previously showed that murine embryonic fibroblasts (MEFs) from TNF receptor-associated factor 2 (Traf2) and Traf5 double-deficient (double knockout (DKO)) mice were highly susceptible to TNF-induced cell death. By functional cloning to rescue DKO MEFs from TNF-induced cell death, we have identified a novel gene, Bsac. BSAC is composed of N-terminal basic, SAP (SAF-A/B, Acinus, PIAS), and coiled-coil domains. BSAC is a nuclear protein, and overexpression of BSAC potently activates promoters containing A + T-rich sequences named CArG boxes. Domain mapping analysis revealed that both N-terminal basic and C-terminal proline-rich sequence are required for the transcriptional activity. Overexpression of BSAC in DKO MEFs partially inhibited TNF-induced cell death by suppressing activation of caspases. Interestingly, inhibition of TNF-induced cell death was not observed in DKO MEFs transfected with either N-terminal or C-terminal deletion mutant of BSAC, revealing an intimate correlation between transcriptional activity and antiapoptotic function. Recently, a human homologue of BSAC named MAL/MKL1 (megakaryocytic acute leukemia/megakaryoblastic leukemia-1) was identified as a fusion transcript generated by t(1,22) translocation in acute megakaryoblastic leukemia. Collectively, BSAC is a novel transcriptional activator with antiapoptotic function, which may be involved in the leukemogenesis.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) were identified as signal transducers for the TNF receptor superfamily. However, the exact roles of TRAF2 and TRAF5 in TNF-induced NF-kappaB activation still remain controversial. To address this issue, we generated TRAF2 and TRAF5 double knockout (DKO) mice. TNF- but not interleukin-1-induced nuclear translocation of NF-kappaB was severely impaired in murine embryonic fibroblasts (MEFs) derived from DKO mice. Moreover, DKO MEFs were more susceptible to TNF-induced cytotoxicity than TRAF2 knockout MEFs. Collectively, these results indicate that both TRAF2 and TRAF5 are involved in TNF-induced NF-kappaB activation and protection from cell death.

Antigen recognition through T cell receptor (TCR)-CD3 complex transduces signals into T cells, which regulate activation, function, and differentiation of T cells. The TCR-CD3 complex is composed of two signaling modules represented by CD3zeta and CD3epsilon. Signaling through CD3zeta has been extensively analyzed, but that via CD3epsilon, which is also crucial in immature thymocyte development, is still not clearly understood. We isolated cDNA encoding a novel CD3epsilon-binding protein CAST. CAST specifically interacts in vivo and in vitro with CD3epsilon but not with CD3zeta or FcRgamma via a unique membrane-proximal region of CD3epsilon. CAST is composed of 512 amino acids including a single tyrosine and undergoes tyrosine phosphorylation upon TCR stimulation. Overexpression of two dominant-negative types of CAST, a minimum CD3epsilon-binding domain and a tyrosine-mutant, strongly suppressed NFAT activation and interleukin-2 production. These results demonstrate that CAST serves as a component of preformed TCR complex and transduces activation signals upon TCR stimulation and represents a new signaling pathway via the CD3epsilon-containing TCR signaling module.

Mechanisms by which the human T-cell leukemia virus type I Tax oncoprotein activates NF-kappaB remain incompletely understood. Although others have described an interaction between Tax and a holo-IkappaB kinase (IKK) complex, the exact details of protein-protein contact are not fully defined. Here we show that Tax binds to neither IKK-alpha nor IKK-beta but instead complexes directly with IKK-gamma, a newly characterized component of the IKK complex. This direct interaction with IKK-gamma correlates with Tax-induced IkappaB-alpha phosphorylation and NF-kappaB activation. Thus, our findings establish IKK-gamma as a key molecule for adapting an oncoprotein-specific signaling to IKK-alpha and IKK-beta.

CD27 is a member of the tumor necrosis factor (TNF) receptor superfamily and is expressed on T, B, and NK cells. The signal via CD27 plays pivotal roles in T-T and T-B cell interactions. Here we demonstrate that overexpression of CD27 activates NF-kappaB and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). Deletion analysis of the cytoplasmic domain of CD27 revealed that the C-terminal PIQEDYR motif was indispensable for both NF-kappaB and SAPK/JNK activation and was also required for the interaction with TNF receptor-associated factor (TRAF) 2 and TRAF5, both of which have been implicated in NF-kappaB activation by members of the TNF-R superfamily. Co-transfection of a dominant negative TRAF2 or TRAF5 blocked NF-kappaB and SAPK/JNK activation induced by CD27. Recently, a TRAF2-interacting kinase has been identified, termed NF-kappaB-inducing kinase (NIK). A kinase-inactive mutant NIK blocked CD27-, TRAF2-, and TRAF5-mediated NF-kappaB and SAPK/JNK activation. These results indicate that TRAF2 and TRAF5 are involved in NF-kappaB and SAPK/JNK activation by CD27, and NIK is a common downstream kinase of TRAF2 and TRAF5 for NF-kappaB and SAPK/JNK activation.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are signal transducers for members of the TNF receptor superfamily. We previously identified murine TRAF5 (mTRAF5) and showed that it specifically interacts with the lymphotoxin-beta receptor (LT-beta R) and activates the transcription factor NF-kappa B. Here we have cloned the human TRAF5 homologue (hTRAF5) by cross hybridization with mTRAF5 cDNA. hTRAF5 cDNA is composed of 2894 nucleotides with a 557-amino-acid open reading frame that exhibits 77.5 and 80% identity to mTRAF5 at the nucleotide and amino acid levels, respectively. Northern blot analysis revealed that hTRAF5 mRNA is expressed in all visceral organs. Western blotting revealed that hTRAF5 protein was abundantly expressed in the human follicular dentritic cell line, FDC-1, and to a much lesser degree in several tumor cell lines. Interspecific backcross mapping revealed that Traf5 is located in the distal region of mouse chromosome 1, which shares a region of homology with human chromosome 1q. Fluorescence in situ hybridization confirmed regional localization to human chromosome 1q32.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are signal transducers for several members of the TNF receptor superfamily. We have identified a novel member of the TRAF family by degenerate oligonucleotide polymerase chain reaction amplification that contains a zinc RING finger and zinc finger motifs, a coiled-coil region, and a C-terminal "TRAF" homology domain. In vitro translated TRAF5 binds to the cytoplasmic region of the lymphotoxin-beta receptor (LT-betaR) but not to several other related receptors including CD40, both TNF receptors, Fas, and nerve growth factor receptor. TRAF5 and LT-betaR coimmunoprecipitate when overexpressed in COS7 cells. TRAF5 mRNA expression is found in all visceral organs and overlaps with LT-betaR. These features distinguish TRAF5 from the other members of the TRAF family. The transcription factor NF-kappaB is activated in HEK293 cells by overexpression of full-length TRAF5 but not a truncated form lacking the zinc binding region. Furthermore, overexpression of LT-betaR in HEK293 cells also results in activation of NF-kappaB, which is partially inhibited by the truncated TRAF5 mutant. These results show TRAF5 is functionally similar to TRAF2 in that both mediate activation NF-kappaB and implicate TRAF5 as a signal transducer for LT-betaR.

T cell antigen receptor (TCR)-CD3 complex is composed of six different subunits: TCR alpha and TCR beta and CD3 gamma, CD3 delta, CD3 epsilon, and CD3 eta. Antigen recognition signals are transduced from TCR to the cytoplasm through the cytoplasmic domain of the CD3 chains. To understand the downstream signal transduction pathways, we cloned genes encoding proteins capable of binding to CD3 epsilon with a probe of glutathione S-transferase fused to the cytoplasmic region of CD3 epsilon. One of these clones was found to encode topoisomerase II beta (topoII beta). The binding region of CD3 epsilon is located within the N-terminal 12 amino acids containing the motif of a basic amino acid cluster. A similar motif was found in the gamma chain of Fc receptors (FcR gamma) but not in the CD33 eta chain, and indeed, FcR gamma but not CD3 eta bound to topoII beta. The binding region of topoII beta was determined to be the C terminus. Since this region appears to be the regulatory region of the enzymatic activity, the binding of CD3 epsilon might affect the function of topoII beta. Although topoII beta is localized mainly in the nucleus and CD3E is a membrane protein, we demonstrated the presence of CD3 epsilon in the nuclear fraction of thymocytes, which increased upon T cell activation. The specific interaction in cells was evidenced by co-immunoprecipitation of topoII beta and CD3E from the nuclear fraction of T cells. The possible function of this interaction is discussed.