TRAF1 Exacerbates Myocardial Ischemia Reperfusion Injury via ASK1-JNK/p38 Signaling.

Background After acute myocardial infarction, the recovery of ischemic myocardial blood flow may cause myocardial reperfusion injury, which reduces the efficacy of myocardial reperfusion. Ways to reduce and prevent myocardial ischemia/reperfusion (I/R) injury are of great clinical significance in the treatment of patients with acute myocardial infarction. TRAF1 (tumor necrosis factor receptor-associated factor 1) is an important adapter protein that is implicated in molecular events regulating immunity, inflammation, and cell death. Little is known about the role and impact of TRAF1 in myocardial I/R injury. Methods and Results TRAF1 expression is markedly induced in wild-type mice and cardiomyocytes after I/R or hypoxia/reoxygenation stimulation. I/R models were established in TRAF1 knockout mice and wild type mice (n=10 per group). We demonstrated that TRAF1 deficiency protects against myocardial I/R-induced loss of heat function, inflammation, and cardiomyocyte death. In addition, overexpression of TRAF1 in primary cardiomyocytes promotes hypoxia/reoxygenation-induced inflammation and apoptosis in vitro. Mechanistically, TRAF1 promotes myocardial I/R injury through regulating ASK1 (apoptosis signal-regulating kinase 1)-mediated JNK/p38 (c-Jun N-terminal kinase/p38) MAPK (mitogen-activated protein kinase) cascades. Conclusions Our results indicated that TRAF1 aggravates the development of myocardial I/R injury by enhancing the activation of ASK1-mediated JNK/p38 cascades. Targeting the TRAF1-ASK1-JNK/p38 pathway provide feasible therapies for cardiac I/R injury.

Targeting hepatic TRAF1-ASK1 signaling to improve inflammation, insulin resistance, and hepatic steatosis.

BACKGROUND & AIMS: Tumor necrosis factor receptor-associated factor 1 (TRAF1) is an important adapter protein that is largely implicated in molecular events regulating immunity/inflammation and cell death. Although inflammation is closely related to and forms a vicious circle with insulin dysfunction and hepatic lipid accumulation, the role of TRAF1 in hepatic steatosis and the related metabolic disorders remains unclear. METHODS: The participation of TRAF1 in the initiation and progression of hepatic steatosis was evaluated in high fat diet (HFD)-induced and genetic obesity. Mice with global TRAF1 knockout or liver-specific TRAF1 overexpression were employed to investigate the role of TRAF1 in insulin resistance, inflammation, and hepatic steatosis based on various phenotypic examinations. Molecular mechanisms underlying TRAF1-regulated hepatic steatosis were further explored in vivo and in vitro. RESULTS: TRAF1 expression was significantly upregulated in the livers of NAFLD patients and obese mice and in palmitate-treated hepatocytes. In response to HFD administration or in ob/ob mice, TRAF1 deficiency was hepatoprotective, whereas the overexpression of TRAF1 in hepatocytes contributed to the pathological development of insulin resistance, inflammatory response and hepatic steatosis. Mechanistically, hepatocyte TRAF1 promotes hepatic steatosis through enhancing the activation of ASK1-mediated P38/JNK cascades, as evidenced by the fact that ASK1 inhibition abolished the exacerbated effect of TRAF1 on insulin dysfunction, inflammation, and hepatic lipid accumulation. CONCLUSIONS: TRAF1 functions as a positive regulator of insulin resistance, inflammation, and hepatic steatosis dependent on the activation of ASK1-P38/JNK axis.

Structural asymmetry in the closed state of mitochondrial Hsp90 (TRAP1) supports a two-step ATP hydrolysis mechanism.

While structural symmetry is a prevailing feature of homo-oligomeric proteins, asymmetry provides unique mechanistic opportunities. We present the crystal structure of full-length TRAP1, the mitochondrial Hsp90 molecular chaperone, in a catalytically active closed state. The TRAP1 homodimer adopts a distinct, asymmetric conformation, where one protomer is reconfigured via a helix swap at the middle:C-terminal domain (MD:CTD) interface. This interface plays a critical role in client binding. Solution methods validate the asymmetry and show extension to Hsp90 homologs. Point mutations that disrupt unique contacts at each MD:CTD interface reduce catalytic activity and substrate binding and demonstrate that each protomer needs access to both conformations. Crystallographic data on a dimeric NTD:MD fragment suggests that asymmetry arises from strain induced by simultaneous NTD and CTD dimerization. The observed asymmetry provides the potential for an additional step in the ATPase cycle, allowing sequential ATP hydrolysis steps to drive both client remodeling and client release.

Leukocyte-specific protein 1 links TNF receptor-associated factor 1 to survival signaling downstream of 4-1BB in T cells.

4-1BB is a member of the TNFR superfamily, which contributes to the activation of signaling pathways required for the survival of activated and memory T cells. We have shown previously that TRAF1, an adaptor protein recruited to 4-1BB, is required for 4-1BB-mediated CD8 T cell survival in vivo. With the use of a proteomics approach in primary T cells, we have identified LSP1 as a novel protein recruited to the 4-1BB signaling complex in a TRAF1-dependent manner. Further characterization of the interaction between TRAF1 and LSP1 revealed that LSP1 requires the TRAF-N domain of TRAF1 for direct association. Similarly to TRAF1(-/-) T cells, LSP1(-/-) T cells exhibit impaired ERK activation following stimulation through 4-1BB and consequently, are unable to down-modulate expression of the proapoptotic Bcl-2 family member Bim. Moreover, we demonstrate that the absence of LSP1 expression leads to defective expansion and survival of T cells in response to 4-1BB stimulation. Thus, we have identified LSP1 as a new mediator involved in 4-1BB signaling and T cell survival. Collectively, our work shows that TRAF1 and LSP1 cooperate downstream of 4-1BB to activate ERK signaling and down-modulate the levels of Bim leading to enhanced T cell survival.

Tumor necrosis factor receptor cross-talk.

Extensive research has been performed to unravel the mechanistic signaling pathways mediated by tumor necrosis factor receptor 1 (TNFR1), by contrast there is limited knowledge on cellular signaling upon activation of TNFR2. Recently published data have revealed that these two receptors not only function independently, but also can influence each other via cross-talk between the different signaling pathways initiated by TNFR1 and TNFR2 stimulation. Furthermore, the complexity of this cross-talk is also dependent on the different signaling kinetics between TNFR1 and TNFR2, by which a delicate balance between cell survival and apoptosis can be maintained. Some known signaling factors and the kinetics that are involved in the receptor cross-talk between TNFR1 and TNFR2 are the topic of this review.

Mitochondrial chaperone tumour necrosis factor receptor-associated protein 1 protects cardiomyocytes from hypoxic injury by regulating mitochondrial permeability transition pore opening.

Tumour necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial chaperone that plays a role in maintaining mitochondrial function and regulating cell apoptosis. The opening of the mitochondrial permeability transition pore (MPTP) is a key step in cell death after hypoxia. However, it is still unclear whether TRAP1 protects cardiomyocytes from hypoxic damage by regulating the opening of the pore. In the present study, primary cultured cardiomyocytes from neonatal rats were used to investigate changes in TRAP1 expression after hypoxia treatment as well as the mechanism and effect of TRAP1 on hypoxic damage. The results obtained showed that TRAP1 expression increased after 1 h of hypoxia and continued to increase for up to 12 h of treatment. Hypoxia caused an increase in cell death and decreased cell viability and mitochondrial membrane potential; overexpressing TRAP1 prevented hypoxia-induced damage to cardiomyocytes. The silencing of TRAP1 induced an increase in cell death and decreased both cell viability and mitochondrial membrane potential in cardiomyocytes under normoxic and hypoxic conditions. Furthermore, cell damage induced by the silencing of TRAP1 was prevented by the mitochondrial permeability transition pore inhibitor, cyclosporin A. These data demonstrate that hypoxia induces an increase in TRAP1 expression in cardiomyocytes, and that TRAP1 plays a protective role by regulating the opening of the mitochondrial permeability transition pore.

Tumor necrosis factor receptor-associated factor-1 enhances proinflammatory TNF receptor-2 signaling and modifies TNFR1-TNFR2 cooperation.

It has been shown that tumor necrosis factor receptor-2 (TNFR2) stimulation leads to degradation of TNF receptor associated factor-2 (TRAF2) and inhibition of TNFR1-induced activation of NFkappaB and JNK. Here, we show that TRAF1 inhibits TNFR2-induced proteasomal degradation of TRAF2 and relieves TNFR1-induced activation of NFkappaB from the inhibitory effect of TNFR2. TRAF1 co-recruited with TRAF2 to both TNF receptors. Despite lacking an amino-terminal RING/zinc-finger domain, TRAF1 did not interfere with TNFR1-induced activation of JNK and NFkappaB. It is noted that physiological expression levels of TRAF1 enhanced NFkappaB activation and interleukin-8 (IL8) production induced by TNFR2. Thus, TRAF1 shifts the quality of integrated TNFR1-TNFR2 signaling from apoptosis induction to proinflammatory NFkappaB signaling.

Biological risks for neurological abnormalities associated with hyperbilirubinemia.

Unconjugated bilirubin (UCB) injury to glial cells leads to the secretion of glutamate and elicits a typical inflammatory response. Release of pro-inflammatory cytokines may influence gliogenesis and neurogenesis, and lead to deficits in learning and memory. Glutamate metabolism dysregulation and overexpression of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta are consistent with schizophrenia neuropathology. Recently, an increased prevalence of schizophrenia was reported in individuals with Gilbert's syndrome and among those who have had elevated levels of UCB in the neonatal life. In this review, we explore the reactivity of astrocytes, neurons and microglia to UCB, the cascade of events implicated in the immunostimulant effects of UCB, as well as the role of each nerve cell type and maturation state in the neuropathology of UCB. Identification of the signaling events promoted by UCB will be relevant for developing novel therapies that might reduce the risk of brain injury and disabilities.

ERK-dependent Bim modulation downstream of the 4-1BB-TRAF1 signaling axis is a critical mediator of CD8 T cell survival in vivo.

During an acute immune response, CD8 T cells undergo rapid expansion followed by a contraction phase during which the majority of activated T cells die, leaving a few survivors to persist as memory cells. The regulation of T cell survival is critical at each stage of this response. 4-1BB, a TNFR family member, has been implicated in prolonging the survival of activated and memory CD8 T cells; however, the precise mechanisms by which 4-1BB sustains T cell survival are incompletely understood. Upon aggregation on T cells, 4-1BB associates with two TNFR-associated factors (TRAF), TRAF1 and TRAF2. TRAF2 is essential for downstream signaling from 4-1BB; however, the role of TRAF1 in 4-1BB signaling has not been elucidated and there have been conflicting data as to whether TRAF1 provides a positive or a negative signal in T cells. In this study, we report that TRAF1 plays a critical role in survival signaling downstream of 4-1BB during CD8 T cell expansion in response to viral infection in vivo. Further analysis reveals that TRAF1-deficient cells are impaired in their ability to up-regulate the prosurvival Bcl-2 family member Bcl-x(L) and show increased levels of the proapoptotic Bcl-2 family member Bim following 4-1BB signaling. TRAF1-deficient CD8 T cells fail to activate ERK in response to 4-1BB ligation and inhibition of ERK signaling downstream of 4-1BB in wild-type cells leads to increased Bim levels. Thus, TRAF1 has a prosurvival effect in CD8 T cells via the 4-1BB-mediated up-regulation of Bcl-x(L) and ERK-dependent Bim down-modulation.

Blockade of tumor necrosis factor (TNF) receptor type 1-mediated TNF-alpha signaling protected Wistar rats from diet-induced obesity and insulin resistance.

TNF-alpha plays an important role in the pathogenesis of obesity and insulin resistance in which the effect of TNF-alpha signaling via TNF receptor type 1 (TNFR1) largely remains controversial. To delineate the role of TNFR1-mediated TNF-alpha signaling in the pathogenesis of this disorder, a TNFR1 blocking peptide-Fc fusion protein (TNFR1BP-Fc) was used for the present study. Wistar rats were fed a high-fat/high-sucrose (HFS) diet for 16 wk until obesity and insulin resistance developed. In comparison with increased body weight and fat weight, enlarged adipocytes, and hypertriglyceridemia in the obese state, the subsequent 4-wk treatment with TNFR1BP-Fc resulted in significant weight loss characterized by decreased fat pad weight and adipocyte size and reduced plasma triglycerides. Furthermore, obesity-induced insulin resistance, including hyperinsulinemia, elevated C-peptide, higher degree of hyperglycemia after glucose challenge, and less hypoglycemic response to insulin, was markedly improved, and the compensatory hyperplasia and hypertrophy of pancreatic islets were reduced. Interestingly, treatment with TNFR1BP-Fc markedly suppressed systemic TNF-alpha release and its local expression in pancreatic islets and muscle and adipose tissues. In addition, blockage of TNFR1-mediated TNF-alpha signaling in obese rats significantly enhanced tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in the muscle and fat tissues. Our results strongly suggest a pivotal role for TNFR1-mediated TNF-alpha signaling in the pathogenesis of obesity and insulin resistance. Thus, TNFR1BP-Fc may be a good candidate for the treatment of this disease.

TNF receptor-associated factor 1 expressed in resident lung cells is required for the development of allergic lung inflammation.

TNF is a major therapeutic target in a range of chronic inflammatory disorders, including asthma. TNFR-associated factor (TRAF)1 is an intracellular adaptor molecule important for signaling by TNFR. In this study, we investigated the role of TRAF1 in an adoptive transfer model of allergic lung inflammation. Mice deficient in TRAF1 (TRAF1(-/-)) and wild-type (WT) control animals were adoptively transferred with WT OVA-immune CD4(+) T cells, exposed to an aerosol of LPS-free OVA, and analyzed for the development of allergic lung inflammation. In contrast to WT mice, TRAF1(-/-) recipients failed to display goblet cell hyperplasia, eosinophilic inflammation, and airway hyperresponsiveness in this model of asthma. Neither T cell recruitment nor expression of the proinflammatory cytokines IL-4, IL-5, IL-13, or TNF occurred in the lungs of TRAF1(-/-) mice. Although purified myeloid TRAF1(-/-) dendritic cells (DCs) exhibited normal Ag-presenting function and transmigratory capacity in vitro and were able to induce OVA-specific immune responses in the lung draining lymph nodes (LNs) following adoptive transfer in vivo, CD11c(+)CD11b(+) DCs from airways of TRAF1(-/-) recipients were not activated, and purified draining LN cells did not proliferate in vitro. Moreover, transfer of WT or TRAF1(-/-) DCs failed to restore T cell recruitment and DC activation in the airways of TRAF1(-/-) mice, suggesting that the expression of TRAF1 in resident lung cells is required for the development of asthma. Finally, we demonstrate that T cell-transfused TRAF1(-/-) recipient mice demonstrated impaired up-regulation of ICAM-1 expression on lung cells in response to OVA exposure.

TRAF1 and its biological functions.

Tumor necrosis factor (TNF) receptor-associated factor (TRAF)1 was originally identified based on its ability to interact with the cytosolic domain ofTNF receptor type 2 (TNFR2). TRAF1 is unique among TRAF proteins in that it lacks RING domain found in the N-terminal regions of other TRAFs. TRAF1 can associate with multiple TNFR family members and can also bind several protein kinases and adaptor proteins suggesting that this protein likely possesses multiple functions in cytokine signaling networks. Although our understanding ofTRAF 1 functions and the underlying mechanisms at molecular and cellular levels has been advanced in recent years, much still needs to be learned before we have a full grasp of TRAF1 biology.

Mechanisms involved in Korean mistletoe lectin-induced apoptosis of cancer cells.

AIM: To investigate the anti-cancer mechanisms of Korean mistletoe lectin (Viscum album coloratum agglutinin, VCA) using a human colon cancer cell line (COLO). METHODS: Cytotoxic effects of VCA on COLO cells were determined by 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) assay in vitro and tumor-killing effects in vivo. To study the mechanisms involved, the expression of various pro-caspases, anti-apoptotic proteins, and death receptors was determined by western blot. To determine which death receptor is involved in VCA-induced apoptosis of COLO cells, cytotoxicity was examined by MTT assay after treatment with agonists or antagonists of death receptors. RESULTS: VCA killed COLO cells in a time- and dose-dependent manner and induced complete regression of tumors in nude mice transplanted with COLO cells. Treatment of COLO cells with VCA activated caspase-2, -3, -8, and -9 and decreased expression of anti-apoptotic molecules including receptor interacting protein, nuclear factor-kappaB, X-linked inhibitor of apoptosis protein, and Akt/protein kinase B. We then examined the involvement of death receptors in VCA-induced apoptosis. Only tumor necrosis factor receptor 1, among the death receptors examined, was involved in apoptosis of COLO cells, evidenced by inhibition of VCA-induced apoptosis and decreased activation of caspases, particularly caspase-8, by tumor necrosis factor receptor 1 antagonizing antibody. CONCLUSION: VCA-induced apoptotic COLO cell death is due to the activation of caspases and inhibition of anti-apoptotic proteins, in part through the tumor necrosis factor receptor 1 signaling pathway.

TNF family ligands define niches for T cell memory.

Immunological memory is a critical feature of the adaptive immune system and the underlying principal behind vaccination. The mechanisms that maintain memory T cell survival between the initial and subsequent encounter with antigen remain incompletely defined. Although the cytokines IL-15 and IL-7 are important in memory T cell homeostasis, additional signals by way of TNFR family members are required for maximal maintenance of T cell memory. Here we propose a unifying model in which subsets of TNF family ligands distinguish the competitive niches for maintenance of CD4 versus CD8 T cell memory. Understanding the unique 'memory niches' defined by TNF family ligand expression will provide new insights into the mechanisms of memory T cell maintenance.

Differential expression of TRAF1 aids in the distinction of cutaneous CD30-positive lymphoproliferations.

Lymphomatoid papulosis (LyP), primary cutaneous anaplastic large T-cell lymphoma (cALCL), and cutaneous infiltrates of systemic anaplastic large cell lymphoma (sALCL) are CD30-positive lymphoproliferative disorders of the skin that overlap clinically, histopathologically, immunophenotypically, and genetically but differ considerably in their prognosis. In particular, lesions of LyP regress spontaneously, whereas those of cALCL and sALCL persist and may progress and spread to extracutaneous sites. In contrast to patients with cALCL, LyP patients do not benefit from an aggressive radio- and/or chemotherapeutic approach. We generated a novel tumor necrosis factor receptor (TNFR)-associated factor 1 (TRAF1) antibody that recognizes a formalin-resistant epitope (Ber-TRAF1A) and investigated the expression of TRAF1, an intracellular component of TNFR signaling, in LyP and ALCL. We could show a strong TRAF1 expression in the tumor cells of most LyP cases (42/49, 84%). In contrast, tumor cells of primary and secondary cALCL revealed TRAF1 expression in only a few cases (3/41, 7%) as shown for sALCL without skin manifestation. The data indicate that TRAF1 expression reliably distinguishes LyP from primary or secondary cALCL. This might be of crucial diagnostic importance and has a strong impact on the treatment decision for patients with cALCL and LyP.

A critical role for TNF receptor-associated factor 1 and Bim down-regulation in CD8 memory T cell survival.

The mechanisms that allow the maintenance of immunological memory remain incompletely defined. Here we report that tumor necrosis factor receptor (TNFR)-associated factor (TRAF) 1, a protein recruited in response to several costimulatory TNFR family members, is required for maximal CD8 T cell responses to influenza virus in mice. Decreased recovery of CD8 T cells in vivo occurred under conditions where cell division was unimpaired. In vitro, TRAF1-deficient, antigen-activated T cells accumulated higher levels of the proapoptotic BH3-only family member Bim, particularly the most toxic isoform, Bim(S). In the presence of excess IL-15, memory phenotype T cells with similar surface phenotype and comparable levels of Bcl-2 family members could be generated from WT or TRAF1-deficient T cell receptor transgenic OT-I T cells. However, when the memory CD8 T cells were allowed to compete for survival signals in the absence of antigen in vivo, the TRAF1-deficient T cells showed decreased recovery compared with TRAF1-sufficient T cells. This defect in T cell recovery in vivo was alleviated by introduction of siRNA to down-modulate Bim in TRAF1-deficient memory T cells. These studies identify the TRAF1 signaling axis and Bim down-regulation as critical for CD8 memory T cell survival in vivo.

The inhibitor of apoptosis protein fusion c-IAP2.MALT1 stimulates NF-kappaB activation independently of TRAF1 AND TRAF2.

The inhibitors of apoptosis (IAPs) are a family of cell death inhibitors found in viruses and metazoans. All members of the IAP family have at least one baculovirus IAP repeat (BIR) motif that is essential for their anti-apoptotic activity. The t(11, 18)(q21;q21) translocation fuses the BIR domains of c-IAP2 with the paracaspase/MALT1 (mucosa-associated lymphoid tissue) protein, a critical mediator of T cell receptor-stimulated activation of NF-kappaB. The c-IAP2.MALT1 fusion protein constitutively activates the NF-kappaB pathway, and this is considered critical to malignant B cell transformation and lymphoma progression. The BIR domains of c-IAP1 and c-IAP2 interact with tumor necrosis factor receptor-associated factors 1 and 2 (TRAF1 and TRAF2). Here we investigated the importance of TRAF1 and TRAF2 for c-IAP2.MALT1-stimulated NF-kappaB activation. We identified a novel epitope within the BIR1 domains of c-IAP1 and c-IAP2 that is crucial for their physical interaction with TRAF1 and TRAF2. The c-IAP2.MALT1 fusion protein associates with TRAF1 and TRAF2 using the same binding site. We explored the functional relevance of this interaction and established that binding to TRAF1 and TRAF2 is not required for c-IAP2.MALT1-stimulated NF-kappaB activation. Furthermore, gene ablation of TRAF2 or combined down-regulation of TRAF1 and TRAF2 did not affect c-IAP2.MALT1-stimulated signaling. However, TRAF1/2-binding mutants of c-IAP2.MALT1 still oligomerize and activate NF-kappaB, suggesting that oligomerization might be important for signaling of the fusion protein. Therefore, the t(11, 18)(q21;q21) translocation creating the c-IAP2.MALT1 fusion protein activates NF-kappaB and contributes to human malignancy in the absence of signaling adaptors that might otherwise regulate its activity.

Major histocompatibility complex (MHC2+) perivascular macrophages in the axotomized facial motor nucleus are regulated by receptors for interferon-gamma (IFNgamma) and tumor necrosis factor (TNF).

The major histocompatibility complex (MHC) glycoproteins, MHC1 and MHC2, play a key role in the presentation of antigen and the development of the immune response. In the current study we examined the regulation of the MHC2 in the mouse brain after facial axotomy. The normal facial motor nucleus showed very few slender and elongated MHC2+ cells. Transection of the facial nerve led to a gradual but strong upregulation in the number of MHC2+ cells, beginning at day 2 and reaching a maximum 14 days after axotomy, correlated with the induction of mRNA for tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta and interferon-gamma (IFNgamma) and a peak in neuronal cell death. In almost all cases, MHC2 immunoreactivity was restricted to perivascular macrophages that colocalized with vascular basement membrane laminin and macrophage IBA1-immunoreactivity, with no immunoreactivity on phagocytic microglia, astrocytes or invading T-cells. Heterologous transplantation and systemic injection of endotoxin or IFNgamma did not affect this perivascular MHC2 immunoreactivity, and transgenic deletion of the IL1 receptor type I, or TNF receptor type 1, also had no effect. However, the deletion of IFNgamma receptor subunit 1 caused a significant increase, and that of TNF receptor type 2 a strong reduction in the number of MHC2+ macrophages, pointing to a counter-regulatory role of IFNgamma and TNFalpha in the immune surveillance of the injured nervous system.

Modulation of life and death by the tumor necrosis factor receptor-associated factors (TRAFs).

The TNF receptor-associated factor (TRAF) family is a group of adapter proteins that link a wide variety of cell surface receptors. Including the TNF and IL-1 receptor superfamily to diverse signaling cascades, which lead to the activation of NF-kappaB and mitogen-activated protein kinases. In addition, TRAFs interact with a variety of proteins that regulate receptor-induced cell death or survival. Thus, TRAF-mediated signals may directly induce cell survival or interfere with the death receptor-induced apoptosis.