A TRAF2 binding independent region of TNFR2 is responsibl for TRAF2 depletion and enhancement of cytotoxicity driven b TNFR1

Tumor Necrosis Factor (TNF) interacts with two receptors known as TNFR1 and TNFR2. TNFR1 activation may result in either cell proliferation or cell death. TNFR2 activates Nuclear Factor-kappaB (NF-kB) and c-Jun N-terminal kinase (JNK) which lead to transcriptional activation of genes related to cell proliferation and survival. This depends on the binding of TNF Receptor Associated Factor 2 (TRAF2) to the receptor. TNFR2 also induces TRAF2 degradation. In this work we have investigated the structural features of TNFR2 responsible for inducing TRAF2 degradation and have studied the biological consequences of this activity. We show that when TNFR1 and TNFR2 are co-expressed, TRAF2 depletion leads to an enhanced TNFR1 cytotoxicity which correlates with the inhibition of NF-kB. NF-kB activation and TRAF2 degradation depend of different regions of the receptor since TNFR2 mutants at amino acids 343-349 fail to induce TRAF2 degradation and have lost their ability to enhance TNFR1-mediated cell death but are still able to activate NF-kB. Moreover, whereas NF-kB activation requires TRAF2 binding to the receptor, TRAF2 degradation appears independent of TRAF2 binding. Thus, TNFR2 mutants unable to bind TRAF2 are still able to induce its degradation and to enhance TNFR1-mediated cytotoxicity. To test further this receptor crosstalk we have developed a system stably expressing in cells carrying only endogenous TNFR1 the chimeric receptor RANK-TNFR2, formed by the extracellular region of RANK (Receptor activator of NF-kB) and the intracellular region of TNFR2.This has made possible to study independently the signals triggered by TNFR1 and TNFR2. In these cells TNFR1 is selectively activated by soluble TNF (sTNF) while RANK-TNFR2 is selectively activated by RANKL. Treatment of these cells with sTNF and RANKL leads to an enhanced cytotoxicity

Soluble tumor necrosis factor receptor 1 and 2 predict outcomes in advanced chronic kidney disease : a prospective cohort study

Background : Soluble tumor necrosis factor receptors 1 (sTNFR1) and 2 (sTNFR2) have been associated to progression of renal failure, end stage renal disease and mortality in early stages of chronic kidney disease (CKD), mostly in the context of diabetic nephropathy. The predictive value of these markers in advanced stages of CKD irrespective of the specific causes of kidney disease has not yet been defined. In this study, the relationship between sTNFR1 and sTNFR2 and the risk for adverse cardiovascular events (CVE) and all-cause mortality was investigated in a population with CKD stage 4-5, not yet on dialysis, to minimize the confounding by renal function. Patients and methods : In 131 patients, CKD stage 4-5, sTNFR1, sTNFR2 were analysed for their association to a composite endpoint of all-cause mortality or first non-fatal CVE by univariate and multivariate Cox proportional hazards models. In the multivariate models, age, gender, CRP, eGFR and significant comorbidities were included as covariates. Results : During a median follow-up of 33 months, 40 events (30.5%) occurred of which 29 deaths (22.1%) and 11 (8.4%) first non-fatal CVE. In univariate analysis, the hazard ratios (HR) of sTNFR1 and sTNFR2 for negative outcome were 1.49 (95% confidence interval (CI): 1.28-1.75) and 1.13 (95% CI: 1.06-1.20) respectively. After adjustment for clinical covariables (age, CRP, diabetes and a history of cardiovascular disease) both sTNFRs remained independently associated to outcomes (HR: sTNFR1: 1.51, 95% CI: 1.30-1.77; sTNFR2: 1.13, 95% CI: 1.06-1.20). A subanalysis of the non-diabetic patients in the study population confirmed these findings, especially for sTNFR1. Conclusion : sTNFR1 and sTNFR2 are independently associated to all-cause mortality or an increased risk for cardiovascular events in advanced CKD irrespective of the cause of kidney disease

Differential changes in gene expression in human neutrophils following TNF-α stimulation: Up-regulation of anti-apoptotic proteins and down-regulation of proteins involved in death receptor signaling.

Responses of human neutrophils to TNF-α are complex and multifactorial. Exposure of human neutrophils to TNF-α in vitro primes the respiratory burst, delays apoptosis and induces the expression of several genes including chemokines, and TNF-α itself. This study aimed to determine the impact of TNF-α exposure on the expression of neutrophil genes and proteins that regulate apoptosis. Quantitative PCR and RNA-Seq, identified changes in expression of several apoptosis regulating genes in response to TNF-α exposure. Up-regulated genes included TNF-α itself, and several anti-apoptotic genes, including BCL2A1, CFLAR (cFLIP) and TNFAIP3, whose mRNA levels increased above control values by between 4-20 fold (n = 3, P < 0.05). In contrast, the expression of pro-apoptotic genes, including CASP8, FADD and TNFRSF1A and TNFRSF1B, were significantly down-regulated following TNF-α treatment. These changes in mRNA levels were paralleled by decreases in protein levels of caspases 8 and 10, TRADD, FADD, TNFRSF1A and TNFRSF1B, and increased cFLIP protein levels, as detected by western blotting. These data indicate that when neutrophils are triggered by TNF-α exposure, they undergo molecular changes in transcriptional expression to up-regulate expression of specific anti-apoptotic proteins and concomitantly decrease expression of specific proteins involved in death receptor signaling which will alter their function in TNF-α rich environments

Trisomy of a Down Syndrome Critical Region Globally Amplifies Transcription via HMGN1 Overexpression

Down syndrome (DS, trisomy 21) is associated with developmental abnormalities and increased leukemia risk. To reconcile chromatin alterations with transcriptome changes, we performed paired exogenous spike-in normalized RNA and chromatin immunoprecipitation sequencing in DS models. Absolute normalization unmasks global amplification of gene expression associated with trisomy 21. Overexpression of the nucleosome binding protein HMGN1 (encoded on chr21q22) recapitulates transcriptional changes seen with triplication of a Down syndrome critical region on distal chromosome 21, and HMGN1 is necessary for B cell phenotypes in DS models. Absolute exogenous-normalized chromatin immunoprecipitation sequencing (ChIP-Rx) also reveals a global increase in histone H3K27 acetylation caused by HMGN1. Transcriptional amplification downstream of HMGN1 is enriched for stage-specific programs of B cells and B cell acute lymphoblastic leukemia, dependent on the developmental cellular context. These data offer a mechanistic explanation for DS transcriptional patterns and suggest that further study of HMGN1 and RNA amplification in diverse DS phenotypes is warranted. How trisomy 21 contributes to Down syndrome phenotypes, including increased leukemia risk, is not well understood. Mowery et al. use per-cell normalization approaches to reveal global transcriptional amplification in Down syndrome models. HMGN1 overexpression is sufficient to induce these alterations and promotes lineage-associated transcriptional programs, signaling, and B cell progenitor phenotypes

Microtubule interfering agents and KSP inhibitors induce the phosphorylation of the nuclear protein p54(nrb), an event linked to G2/M arrest

Microtubule interfering agents (MIAs) are anti-tumor drugs that inhibit microtubule dynamics, while kinesin spindle protein (KSP) inhibitors are substances that block the formation of the bipolar spindle during mitosis. All these compounds cause G2/M arrest and cell death. Using 2D-PAGE followed by Nano-LC-ESI-Q-ToF analysis, we found that MIAs such as vincristine (Oncovin) or paclitaxel (Taxol) and KSP inhibitors such as S-tritil-l-cysteine induce the phosphorylation of the nuclear protein p54(nrb) in HeLa cells. Furthermore, we demonstrate that cisplatin (Platinol), an anti-tumor drug that does not cause M arrest, does not induce this modification. We show that the G2/M arrest induced by MIAs is required for p54(nrb) phosphorylation. Finally, we demonstrate that CDK activity is required for MIA-induced phosphorylation of p54(nrb)

Neutrophil cell surface receptors and their intracellular signal transduction pathways

AbstractNeutrophils play a critical role in the host defense against bacterial and fungal infections, but their inappropriate activation also contributes to tissue damage during autoimmune and inflammatory diseases. Neutrophils express a large number of cell surface receptors for the recognition of pathogen invasion and the inflammatory environment. Those include G-protein-coupled chemokine and chemoattractant receptors, Fc-receptors, adhesion receptors such as selectins/selectin ligands and integrins, various cytokine receptors, as well as innate immune receptors such as Toll-like receptors and C-type lectins. The various cell surface receptors trigger very diverse signal transduction pathways including activation of heterotrimeric and monomeric G-proteins, receptor-induced and store-operated Ca2+ signals, protein and lipid kinases, adapter proteins and cytoskeletal rearrangement. Here we provide an overview of the receptors involved in neutrophil activation and the intracellular signal transduction processes they trigger. This knowledge is crucial for understanding how neutrophils participate in antimicrobial host defense and inflammatory tissue damage and may also point to possible future targets of the pharmacological therapy of neutrophil-mediated autoimmune or inflammatory diseases

Caracterización del procesamiento del receptor de muerte DR6

DR6 es un receptor de muerte de la superfamilia TNFR, caracterizada por poseer dominios ricos en cisteína en su región extracelular. DR6 posee cuatro CRDs y una región espaciadora característica de este receptor situada entre los CRDs y la región transmembrana. Al igual que otros DRs, está descrito que DR6 es capaz de inducir apoptosis, aunque esto depende de la línea celular, así como de activar a JNK y a NF-kappaB. No obstante, se desconocen las vías de señalización que conducen a estas tres respuestas, así como su regulación. Recientemente se ha descrito que DR6 es procesado por la metaloproteasa MT1-MMP en células tumorales, y en nuestro laboratorio hemos determinado que el receptor expresado por células HEK293 transfectadas transitoriamente también es procesado proteolíticamente. En este estudio mostramos que el procesamiento de DR6 exógeno en las células HEK293 es parcialmente inhibido por GM6001 (un inhibidor general de metaloproteasas) a concentraciones hasta 10 veces más altas de las normalmente utilizadas. Además, si bloqueamos la activación de la metaloproteasa MT1-MMP mediante el inhibidor de furina II, no se detecta inhibición del procesamiento de DR6. Mediante RT-PCR demostramos que las células HEK293 no expresan MT1-MMP, confi rmando que esta metaloproteasa no está implicada en el procesamiento de DR6 en estas células. La deleción de la región espaciadora (aa 239-345) genera un receptor que no sufre procesamiento y que es funcional, ya que se comporta de forma similar al receptor completo en cuanto a la activación de JNK y NF-kappaB. También mostramos que DR6 endógeno es procesado en las células de carcinoma de pulmón A549 mediante un proceso que es dependiente de TNF, sin que se observe un aumento en la expresión ni del receptor ni de la MT1-MMP

Fosforilación del EF1B-GAMMA de eucariotas durante la parada en mitosis producida por los mias y los inhibidoresde KSP

Los MIAs o agentes que interfi eren con los microtúbulos incluyen compuestos como el nocodazol y fármacos antitumorales como paclitaxel y docetaxel. Estos agentes y los inhibidores de KSP (STLC, etc.) son compuestos capaces de inhibir la dinámica de los microtúbulos, parar el ciclo celular en mitosis e inducir muerte celular. En nuestro laboratorio se ha descrito que el paclitaxel induce la fosforilación del eEF1B-gamma (Prado, M.A. y cols., Proteomics, 2007, Vol.7, 3299-3304), siendo esta proteína parte fundamental del complejo eEF1B. Este complejo, de gran importancia en el proceso de elongación proteica, se encarga de activar el eEF1A (transportador del aminoaciltRNA al ribosoma) gracias a la actividad intercambiadora de nucleótidos de guanina que posee. Utilizando 2D-PAGE del proteoma de células HeLa demostramos que el cisplatino, un fármaco antitumoral independiente de fase, no induce la fosforilación del eEF1B-gamma, mientras que otros compuestos antimitóticos, como docetaxel, nocodazol y STLC, sí inducen la fosforilación de esta proteína. El posterior análisis del proteoma de células HeLa tratadas con paclitaxel, pero paradas previamente al comienzo de fase S por acción de la afi dicolina, revela que la parada en mitosis es necesaria para la fosforilación del eEF1B-gamma. Una 2D-PAGE de extractos tratados con roscovitina, un inhibidor de CDK1, mostró que esta vía de fosforilación del eEF1B-gamma es dependiente de esta quinasa. Finalmente, un análisis por espectrometría de masas MALDI-ToF del eEF1B-gamma digerido con la endoproteinasa GluC, mostró que el péptido comprendido entre los residuos 224 y 234 se fosforila tras el tratamiento con paclitaxel, aunque no es posible determinar si se produce en el residuo T224 o T230