The prevalence of TNFα-induced necrosis over apoptosis is determined by TAK1-RIP1 interplay

Death receptor-induced programmed necrosis is regarded as a secondary death mechanism dominating only in cells that cannot properly induce caspase-dependent apoptosis. Here, we show that in cells lacking TGFβ-activated Kinase-1 (TAK1) expression, catalytically active Receptor Interacting Protein 1 (RIP1)-dependent programmed necrosis overrides apoptotic processes following Tumor Necrosis Factor-α (TNFα) stimulation and results in rapid cell death. Importantly, the activation of the caspase cascade and caspase-8-mediated RIP1 cleavage in TNFα-stimulated TAK1 deficient cells is not sufficient to prevent RIP1-dependent necrosome formation and subsequent programmed necrosis. Our results demonstrate that TAK1 acts independently of its kinase activity to prevent the premature dissociation of ubiquitinated-RIP1 from TNFα-stimulated TNF-receptor I and also to inhibit the formation of TNFα-induced necrosome complex consisting of RIP1, RIP3, FADD, caspase-8 and cFLIP(L). The surprising prevalence of catalytically active RIP1-dependent programmed necrosis over apoptosis despite ongoing caspase activity implicates a complex regulatory mechanism governing the decision between both cell death pathways following death receptor stimulation.     

Quantitative phosphoproteomic analysis of RIP3‐dependent protein phosphorylation in the course of TNF‐induced necroptosis

Tumor necrosis factor (TNF) induced cell death in murine fibrosarcoma L929 cells is a model system in studying programed necrosis (also known as necroptosis). Receptor interacting protein 3 (RIP3), a serine–threonine kinase, is known to play an essential role in TNF‐induced necroptosis; however, the phosphorylation events initiated by RIP3 activation in necroptotic process is still largely unknown. Here, we performed a quantitative MS based analysis to compare TNF‐induced changes in the global phosphoproteome of wild‐type (RIP3^+/+) and RIP3‐knockdown L929 cells at different time points after TNF treatment. A total of 8058 phosphopeptides spanning 6892 phosphorylation sites in 2762 proteins were identified in the three experiments, in which cells were treated with TNF for 0.5, 2, and 4 h. By comparing the phosphorylation sites in wild‐type and RIP3‐knockdown L929 cells, 174, 167, and 177 distinct phosphorylation sites were revealed to be dependent on RIP3 at the 0.5, 2, and 4 h time points after TNF treatment, respectively. Notably, most of them were not detected in a previous phosphoproteomic analysis of RIP3‐dependent phosphorylation in lipopolysaccharide‐stimulated peritoneal macrophages and TNF‐treated murine embryonic fibroblasts (MEFs), suggesting that the data presented in this report are highly relevant to the study of TNF‐induced necroptosis of L929 cells.     

Regulator of G-Protein Signaling 19 (RGS19) and Its Partner Gα-Inhibiting Activity Polypeptide 3 (GNAI3) Are Required for zVAD-Induced Autophagy and Cell Death in L929 Cells

Autophagy has diverse biological functions and is involved in many biological processes. The L929 cell death induced by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone (zVAD) was shown to be an autophagy-mediated death for which RIP1 and RIP3 were both required. It was also reported that zVAD can induce a small amount of TNF production, which was shown to be required for zVAD-induced L929 cell death, arguing for the contribution of autophagy in the zVAD-induced L929 cell death. In an effort to study RIP3 mediated cell death, we identified regulator of G-protein signaling 19 (RGS19) as a RIP3 interacting protein. We showed that RGS19 and its partner Gα-inhibiting activity polypeptide 3 (GNAI3) are involved in zVAD-, but not TNF-, induced cell death. The role of RGS19 and GNAI3 in zVAD-induced cell death is that they are involved in zVAD-induced autophagy. By the use of small hairpin RNAs and chemical inhibitors, we further demonstrated that zVAD-induced autophagy requires not only RIP1, RIP3, PI3KC3 and Beclin-1, but also RGS19 and GNAI3, and this autophagy is required for zVAD-induced TNF production. Collectively, our data suggest that zVAD-induced L929 cell death is a synergistic result of autophagy, caspase inhibition and autocrine effect of TNF.     

Dichotomy between RIP1- and RIP3-mediated necroptosis in tumor necrosis factor-α-induced shock

Tumor necrosis factor receptor (TNFR) signaling may result in survival, apoptosis or programmed necrosis. The latter is called necroptosis if the receptor-interacting protein 1 (RIP1) inhibitor necrostatin-1 (Nec-1) or genetic knockout of RIP3 prevents it. In the lethal mouse model of TNFα-mediated shock, addition of the pan-caspase inhibitor zVAD-fmk (zVAD) accelerates time to death. Here, we demonstrate that RIP3-deficient mice are protected markedly from TNFα-mediated shock in the presence and absence of caspase inhibition. We further show that the fusion protein TAT-crmA, previously demonstrated to inhibit apoptosis, also prevents necroptosis in L929, HT29 and FADD-deficient Jurkat cells. In contrast to RIP3-deficient mice, blocking necroptosis by Nec-1 or TAT-crmA did not protect from TNFα/zVAD-mediated shock, but further accelerated time to death. Even in the absence of caspase inhibition, Nec-1 application led to similar kinetics. Depletion of macrophages, natural killer (NK) cells, granulocytes or genetic deficiency for T lymphocytes did not influence this model. Because RIP3-deficient mice are known to be protected from cerulein-induced pancreatitis (CIP), we applied Nec-1 and TAT-crmA in this model and demonstrated the deterioration of pancreatic damage upon addition of these substances. These data highlight the importance of separating genetic RIP3 deficiency from RIP1 inhibition by Nec-1 application in vivo and challenge the current definition of necroptosis.     

Activation and caspase-mediated inhibition of PARP: a molecular switch between fibroblast necrosis and apoptosis in death receptor signaling

Death ligands not only induce apoptosis but can also trigger necrosis with distinct biochemical and morphological features. We recently showed that in L929 cells CD95 ligation induces apoptosis, whereas TNF elicits necrosis. Treatment with anti-CD95 resulted in typical apoptosis characterized by caspase activation and DNA fragmentation. These events were barely induced by TNF, although TNF triggered cell death to a similar extent as CD95. Surprisingly, whereas the caspase inhibitor zVAD prevented CD95-mediated apoptosis, it potentiated TNF-induced necrosis. Cotreatment with TNF and zVAD was characterized by ATP depletion and accelerated necrosis. To investigate the mechanisms underlying TNF-induced cell death and its potentiation by zVAD, we examined the role of poly(ADP-ribose)polymerase-1 (PARP-1). TNF but not CD95 mediated PARP activation, whereas a PARP inhibitor suppressed TNF-induced necrosis and the sensitizing effect of zVAD. In addition, fibroblasts expressing a noncleavable PARP-1 mutant were more sensitive to TNF than wild-type cells. Our results indicate that TNF induces PARP activation leading to ATP depletion and subsequent necrosis. In contrast, in CD95-mediated apoptosis caspases cause PARP-1 cleavage and thereby maintain ATP levels. Because ATP is required for apoptosis, we suggest that PARP-1 cleavage functions as a molecular switch between apoptotic and necrotic modes of death receptor-induced cell death.     

RIP1-dependent and independent effects of necrostatin-1 in necrosis and T cell activation

Background

Programmed necrosis/necroptosis is an emerging form of cell death that plays important roles in mammalian development and the immune system. The pro-necrotic kinases in the receptor interacting protein (RIP) family are crucial mediators of programmed necrosis. Recent advances in necrosis research have been greatly aided by the identification of chemical inhibitors that block programmed necrosis. Necrostatin-1 (Nec-1) and its derivatives were previously shown to target the pro-necrotic kinase RIP1/RIPK1. The protective effect conferred by Nec-1 and its derivatives in many experimental model systems was often attributed to the inhibition of RIP1 function.

Methodology/Principal Findings

We compared the effect of Nec-1 and siRNA-mediated silencing of RIP1 in the murine fibrosarcoma cell line L929. Treatment of L929 cells with the pan-caspase inhibitor zVAD-fmk or exogenous TNF induces necrosis. Strikingly, we found that siRNA-mediated silencing of RIP1 inhibited zVAD-fmk induced necrosis, but not TNF-induced necrosis. TNF-induced cell death in RIP1 knocked down L929 cells was inhibited by Nec-1, but not the caspase inhibitor zVAD-fmk. We found that PKA-C§ expression, but not Jnk or Erk activation, was moderately inhibited by Nec-1. Moreover, we found that Nec-1 inhibits proximal T cell receptor signaling independent of RIP1, leading to inhibition of T cell proliferation.

Conclusions/Significance

Our results reveal that besides RIP1, Nec-1 also targets other factors crucial for necrosis induction in L929 cells. In addition, high doses of Nec-1 inhibit other signal transduction pathways such as that for T cell receptor activation. These results highlight the importance to independently validate results obtained using Nec-1 with other approaches such as siRNA-mediated gene silencing. We propose that some of the previous published results obtained using Nec-1 should be re-evaluated in light of our findings.     

KiSS1 suppresses TNFα‐induced breast cancer cell invasion via an inhibition of RhoA‐Mediated NF‐κB activation

Tumor necrosis factor‐alpha (TNFα) induces cancer development and metastasis, which is prominently achieved by nuclear factor‐kappa B (NF‐κB) activation. TNFα‐induced NF‐κB activation enhances cellular mechanisms including proliferation, migration, and invasion. KiSS1, a key regulator of puberty, was initially discovered as a tumor metastasis suppressor. The expression of KiSS1 was lost or down‐regulated in different metastatic tumors. However, it is unclear whether KiSS1 regulates TNFα‐induced NF‐κB activation and further tumor cell migration. In this study, we demonstrate that KiSS1 suppresses the migration of breast cancer cells by inhibiting TNFα‐induced NF‐κB pathway and RhoA activation. Both KiSS1 overexpression and KP10 (kisspeptin‐10) stimulation inhibited TNFα‐induced NF‐κB activity, suppressed TNFα‐induced cell migration and cell attachment to fibronectin in breast cancer cells while KP10 has little effect on cancer cell proliferation. Furthermore, KP10 inhibited TNFα‐induced cell migration and RhoA GTPase activation. Therefore, our data demonstrate that KiSS1 inhibits TNFα‐induced NF‐κB activation via downregulation of RhoA activation and suppression of breast cancer cell migration and invasion. J. Cell. Biochem. 107: 1139–1149, 2009. © 2009 Wiley‐Liss, Inc.     

MicroRNA‐155 prevents necrotic cell death in human cardiomyocyte progenitor cells via targeting RIP1

To improve regeneration of the injured myocardium, cardiomyocyte progenitor cells (CMPCs) have been put forward as a potential cell source for transplantation therapy. Although cell transplantation therapy displayed promising results, many issues need to be addressed before fully appreciating their impact. One of the hurdles is poor graft‐cell survival upon injection, thereby limiting potential beneficial effects. Here, we attempt to improve CMPCs survival by increasing microRNA‐155 (miR‐155) levels, potentially to improve engraftment upon transplantation. Using quantitative PCR, we observed a 4‐fold increase of miR‐155 when CMPCs were exposed to hydrogen‐peroxide stimulation. Flow cytometric analysis of cell viability, apoptosis and necrosis showed that necrosis is the main cause of cell death. Overexpressing miR‐155 in CMPCs revealed that miR‐155 attenuated necrotic cell death by 40 ± 2.3%via targeting receptor interacting protein 1 (RIP1). In addition, inhibiting RIP1, either by pre‐incubating the cells with a RIP1 specific inhibitor, Necrostatin‐1 or siRNA mediated knockdown, reduced necrosis by 38 ± 2.5% and 33 ± 1.9%, respectively. Interestingly, analysing gene expression using a PCR‐array showed that increased miR‐155 levels did not change cell survival and apoptotic related gene expression. By targeting RIP1, miR‐155 repressed necrotic cell death of CMPCs, independent of activation of Akt pro‐survival pathway. MiR‐155 provides the opportunity to block necrosis, a conventionally thought non‐regulated process, and might be a potential novel approach to improve cell engraftment for cell therapy.     

Caspase blockade induces RIP3-mediated programmed necrosis in Toll-like receptor-activated microglia

Microglia are the resident immune cells in the central nervous system and key players against pathogens and injury. However, persistent microglial activation often exacerbates pathological damage and has been implicated in many neurological diseases. Despite their pivotal physiological and pathophysiological roles, how the survival and death of activated microglia is regulated remains poorly understood. We report here that microglia activated through Toll-like receptors (TLRs) undergo RIP1/RIP3-dependent programmed necrosis (necroptosis) when exposed to the pan caspase inhibitor zVAD-fmk. Although zVAD-fmk and the caspase-8 inhibitor IETD-fmk had no effect on unstimulated primary microglia, they markedly sensitized microglia to TLR1/2,3,4,7/8 ligands or TNF treatment, triggering programmed necrosis that was completely blocked by R1P1 kinase inhibitor necrostatin-1. Interestingly, necroptosis induced by TLR ligands and zVAD was restricted to microglial cells and was not observed in astrocytes, neurons or oligodendrocytes even though they are known to express certain TLRs. Deletion of genes encoding TNF or TNFR1 failed to prevent lipopolysaccharide- and poly(I:C)-induced microglial necroptosis, unveiling a TNF-independent programmed necrosis pathway in TLR3- and TLR4-activated microglia. Microglia from mice lacking functional TRIF were fully protected against TLR3/4 activation and zVAD-fmk-induced necrosis, and genetic deletion of rip3 also prevented microglia necroptosis. Activation of c-jun N-terminal kinase and generation of specific reactive oxygen species were downstream signaling events required for microglial cell death execution. Taken together, this study reveals a robust RIP3-dependent necroptosis signaling pathway in TLR-activated microglia upon caspase blockade and suggests that TLR signaling and programmed cell death pathways are closely linked in microglia, which could contribute to neuropathology and neuroinflammation when dysregulated.     

RIP1 mediates the protection of geldanamycin on neuronal injury induced by oxygen-glucose deprivation combined with zVAD in primary cortical neurons

Caspase-dependent apoptosis is considered one of the most important cell death pathways. When the apoptotic process is blocked, a form of programmed necrosis called necroptosis occurs. Apoptosis and necroptosis may share some regulatory mechanisms. Recent studies indicated that receptor interacting protein 1 (RIP1), an Hsp90-associated kinase, is an important regulatory switch between apoptosis and necroptosis. In this study, we showed that oxygen-glucose deprivation (OGD) combined with a caspase inhibitor zVAD (OGD/zVAD)-induced RIP1 protein expression in a time-dependent manner. We found that geldanamycin (GA), a benzoquinone ansamycin, protected against neuronal injury induced by OGD/zVAD treatment in cultured primary neurons. More importantly, GA decreased RIP1 protein level in a time- and concentration-dependent manner. In this study, we found that GA also decreased the Hsp90 protein level, which caused instability of RIP1 protein, resulting in decreased RIP1 protein level but not RIP1 mRNA level after GA treatment. We concluded that the GA-mediated protection against OGD/zVAD-induced neuronal injury was associated with enhanced RIP1 protein instability by decreasing Hsp90 protein level. GA and its derivatives may be promising for the prevention of neuronal injury during ischemic injury.     

TNFAIP8: A new effector for Galpha(i) coupling to reduce cell death and induce cell transformation

Galpha(i)‐coupled receptors comprise a diverse family of receptors that induce transformation by largely unknown mechanisms. We previously found that the Galpha(i)‐coupled dopamine‐D2short (D2S) receptor transforms Balb‐D2S cells via Gαi3. To identify new Gαi effectors, a yeast two‐hybrid screen was done using constitutively active Gαi3‐Q204L as bait, and tumor necrosis factor‐alpha (TNFα)‐induced protein 8 (TNFAIP8, SCC‐S2/NDED/GG2‐1) was identified. In contrast, TNFAIP8‐related TIPE1 and TIPE2 showed a very weak interaction with Gαi3. In yeast mating, in vitro pull‐down, co‐immunoprecipitation and bioluminescence resonance energy transfer (BRET) assays, TNFAIP8 preferentially interacted with activated Gαi proteins, consistent with direct Gαi‐TNFAIP8 coupling. Over‐expression or depletion of TNFAIP8 using antisense constructs in Balb‐D2S cells did not affect D2S‐induced signaling to Gαi‐dependent inhibition of cAMP. In contrast, antisense depletion of TNFAIP8 completely inhibited spontaneous and D2S‐induced foci formation, consistent with a role for TNFAIP8 in Gαi‐dependent transformation. To address possible mechanisms, the effect of D2S signaling via TNFAIP8 on TNFα action was examined. D2S receptor activation inhibited TNFα‐induced cell death in Balb‐D2S cells, but not in cells depleted of TNFAIP8. However, depletion of TNFAIP8 did not prevent D2S‐induced inhibition of TNFα‐mediated caspase activation, suggesting that D2S/TNFAIP8‐induced protection from TNFα‐induced cell death is caspase‐independent. The data suggest that Gαi‐TNFAIP8‐mediated rescue of pre‐oncogenic cells enhances progression to oncogenic transformation, providing a selective target to inhibit cellular transformation. J. Cell. Physiol. 225: 865–874, 2010. © 2010 Wiley‐Liss, Inc.     

c‐FLIP is involved in erythropoietin‐mediated protection of erythroid‐differentiated cells from TNF‐α‐induced apoptosis

The TNF‐α (tumour necrosis factor) affects a wide range of biological activities, such as cell proliferation and apoptosis. Cell life or death responses to this cytokine might depend on cell conditions. This study focused on the modulation of factors that would affect the sensitivity of erythroid‐differentiated cells to TNF‐α. Hemin‐differentiated K562 cells showed higher sensitivity to TNF‐induced apoptosis than undifferentiated cells. At the same time, hemin‐induced erythroid differentiation reduced c‐FLIP (cellular FLICE‐inhibitory protein) expression. However, this negative effect was prevented by prior treatment with Epo (erythropoietin), which allowed the cell line to maintain c‐FLIP levels. On the other hand, erythroid‐differentiated UT‐7 cells – dependent on Epo for survival – showed resistance to TNF‐α pro‐apoptotic action. Only after the inhibition of PI3K (phosphatidylinositol‐3 kinase)‐mediated pathways, which was accompanied by negative c‐FLIP modulation and increased erythroid differentiation, were UT‐7 cells sensitive to TNF‐α‐triggered apoptosis. In summary, erythroid differentiation might deregulate the balance between growth promotion and death signals induced by TNF‐α, depending on cell type and environmental conditions. The role of c‐FLIP seemed to be critical in the protection of erythroid‐differentiated cells from apoptosis or in the determination of their sensitivity to TNF‐mediated programmed cell death. Epo, which for the first time was found to be involved in the prevention of c‐FLIP down‐regulation, proved to have an anti‐apoptotic effect against the pro‐inflammatory factor. The identification of signals related to cell life/death switching would have significant implications in the control of proliferative diseases and would contribute to the understanding of mechanisms underlying the anaemia associated with inflammatory processes.     

Tumour necrosis factor‐α promotes liver ischaemia‐reperfusion injury through the PGC‐1α/Mfn2 pathway

Tumour necrosis factor (TNF)‐α has been considered to induce ischaemia‐reperfusion injury (IRI) of liver which is characterized by energy dysmetabolism. Peroxisome proliferator–activated receptor‐γ co‐activator (PGC)‐1α and mitofusion2 (Mfn2) are reported to be involved in the regulation of mitochondrial function. However, whether PGC‐1α and Mfn2 form a pathway that mediates liver IRI, and if so, what the underlying involvement is in that pathway remain unclear. In this study, L02 cells administered recombinant human TNF‐α had increased TNF‐α levels and resulted in down‐regulation of PGC‐1α and Mfn2 in a rat liver IRI model. This was associated with hepatic mitochondrial swelling, decreased adenosine triphosphate (ATP) production, and increased levels of reactive oxygen species (ROS) and alanine aminotransferase (ALT) activity as well as cell apoptosis. Inhibition of TNF‐α by neutralizing antibody reversed PGC‐1α and Mfn2 expression, and decreased hepatic injury and cell apoptosis both in cell culture and in animals. Treatment by rosiglitazone sustained PGC‐1α and Mfn2 expression both in IR livers, and L02 cells treated with TNF‐α as indicated by increased hepatic mitochondrial integrity and ATP production, reduced ROS and ALT activity as well as decreased cell apoptosis. Overexpression of Mfn2 by lentiviral‐Mfn2 transfection decreased hepatic injury in IR livers and L02 cells treated with TNF‐α. However, there was no up‐regulation of PGC‐1α. These findings suggest that PGC‐1α and Mfn2 constitute a regulatory pathway, and play a critical role in TNF‐α‐induced hepatic IRI. Inhibition of the TNF‐α or PGC‐1α/Mfn2 pathways may represent novel therapeutic interventions for hepatic IRI.     

Laminar shear stress elicit distinct endothelial cell e‐selectin expression pattern via TNFα and IL‐1β activation

The ability to discriminate cell adhesion molecule expression between healthy and inflamed endothelium is critical for therapeutic intervention in many diseases. This study explores the effect of laminar flow on TNFα‐induced E‐selectin surface expression levels in human umbilical vein endothelial cells (HUVECs) relative to IL‐1β‐induced expression via flow chamber assays. HUVECs grown in static culture were either directly (naïve) activated with cytokine in the presence of laminar shear or pre‐exposed to 12 h of laminar shear (shear‐conditioned) prior to simultaneous shear and cytokine activation. Naïve cells activated with cytokine in static served as control. Depending on the cell shear history, fluid shear is found to differently affect TNFα‐induced relative to IL‐1β‐induced HUVEC expression of E‐selectin. Specifically, E‐selectin surface expression by naïve HUVECs is enhanced in the 8–12 h activation time range with simultaneous exposure to shear and TNFα (shear‐TNFα) relative to TNFα static control whereas enhanced E‐selectin expression is observed in the 4–24 h range for shear‐IL‐1β treatment relative to IL‐1β static control. While exposure of HUVECs to shear preconditioning mutes shear‐TNFα‐induced E‐selectin expression, it enhances or down‐regulates shear‐IL‐1β‐induced expression dependent on the activation period. Under dual‐cytokine‐shear conditions, IL‐1β signaling dominates. Overall, a better understanding of E‐selectin expression pattern by human ECs relative to the combined interaction of cytokines, shear profile and history can help elucidate many disease pathologies. Biotechnol. Bioeng. 2013; 110: 999–1003. © 2012 Wiley Periodicals, Inc.     

HSP70 interacts with TRAF2 and differentially regulates TNFα signalling in human colon cancer cells

Members of tumour necrosis factor (TNF) family usually trigger both survival and apoptotic signals in various cell types. Heat shock proteins (HSPs) are conserved proteins implicated in protection of cells from stress stimuli. However, the mechanisms of HSPs in TNFα‐induced signalling pathway have not been fully elucidated. We report here that HSP70 over‐expression in human colon cancer cells can inhibit TNFα‐induced NFκB activation but promote TNFα‐induced activation of c‐Jun N‐terminal kinase (JNK) through interaction with TNF receptor (TNFR)‐associated factor 2 (TRAF2). We provide evidence that HSP70 over‐expression can sequester TRAF2 in detergent‐soluble fractions possibly through interacting with TRAF2, leading to reduced recruitment of receptor‐interacting protein (RIP1) and IκBα kinase (IKK) signalosome to the TNFR1–TRADD complex and inhibited NFκB activation after TNFα stimuli. In addition, we found that HSP70–TRAF2 interaction can promote TNFα‐induced JNK activation. Therefore, our study suggests that HSP70 may differentially regulate TNFα‐induced activation of NFκB and JNK through interaction with TRAF2, contributing to the pro‐apoptotic roles of HSP70 in TNFα‐induced apoptosis of human colon cancer cells.     

Stromal cell‐derived factor‐1/CXCR4 enhanced motility of human osteosarcoma cells involves MEK1/2, ERK and NF‐κB‐dependent pathways

Osteosarcoma is characterized by a high malignant and metastatic potential. The chemokine stromal‐derived factor‐1α (SDF‐1α) and its receptor, CXCR4, play a crucial role in adhesion and migration of human cancer cells. Integrins are the major adhesive molecules in mammalian cells, and has been associated with metastasis of cancer cells. Here, we found that human osteosarcoma cell lines had significant expression of SDF‐1 and CXCR4 (SDF‐1 receptor). Treatment of osteosarcoma cells with SDF‐1α increased the migration and cell surface expression of αvβ3 integrin. CXCR4‐neutralizing antibody, CXCR4 specific inhibitor (AMD3100) or small interfering RNA against CXCR4 inhibited the SDF‐1α‐induced increase the migration and integrin expression of osteosarcoma cells. Pretreated of osteosarcoma cells with MAPK kinase (MEK) inhibitor PD98059 inhibited the SDF‐1α‐mediated migration and integrin expression. Stimulation of cells with SDF‐1α increased the phosphorylation of MEK and extracellular signal‐regulating kinase (ERK). In addition, NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) also inhibited SDF‐1α‐mediated cell migration and integrin up‐regulation. Stimulation of cells with SDF‐1α induced IκB kinase (IKKα/β) phosphorylation, IκB phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. Furthermore, the SDF‐1α‐mediated increasing κB‐luciferase activity was inhibited by AMD3100, PD98059, PDTC and TPCK or MEK1, ERK2, IKKα and IKKβ mutants. Taken together, these results suggest that the SDF‐1α acts through CXCR4 to activate MEK and ERK, which in turn activates IKKα/β and NF‐κB, resulting in the activations of αvβ3 integrins and contributing the migration of human osteosarcoma cells. J. Cell. Physiol. 221: 204–212, 2009. © 2009 Wiley‐Liss, Inc     

Necroptosis is a key mediator of enterocytes loss in intestinal ischaemia/reperfusion injury

Cell death is an important biological process that is believed to have a central role in intestinal ischaemia/reperfusion (I/R) injury. While the apoptosis inhibition is pivotal in preventing intestinal I/R, how necrotic cell death is regulated remains unknown. Necroptosis represents a newly discovered form of programmed cell death that combines the features of both apoptosis and necrosis, and it has been implicated in the development of a range of inflammatory diseases. Here, we show that receptor‐interacting protein 1/3 (RIP1/3) kinase and mixed lineage kinase domain‐like protein recruitment mediates necroptosis in a rat model of ischaemic intestinal injury in vivo. Furthermore, necroptosis was specifically blocked by the RIP1 kinase inhibitor necrostatin‐1. In addition, the combined treatment of necrostatin‐1 and the pan‐caspase inhibitor Z‐VAD acted synergistically to protect against intestinal I/R injury, and these two pathways can be converted to one another when one is inhibited. In vitro, necrostatin‐1 pre‐treatment reduced the necroptotic death of oxygen‐glucose deprivation challenged intestinal epithelial cell‐6 cells, which in turn dampened the production of pro‐inflammatory cytokines (tumour necrosis factor‐α and interleukin‐1β), and suppressed high‐mobility group box‐1 (HMGB1) translocation from the nucleus to the cytoplasm and the subsequent release of HMGB1 into the supernatant, thus decreasing the activation of Toll‐like receptor 4 and the receptor for advanced glycation end products. Collectively, our study reveals a robust RIP1/RIP3‐dependent necroptosis pathway in intestinal I/R‐induced intestinal injury in vivo and in vitro and suggests that the HMGB1 signalling is highly involved in this process, making it a novel therapeutic target for acute ischaemic intestinal injury.     

Death of HT29 adenocarcinoma cells induced by TNF family receptor activation is caspase-independent and displays features of both apoptosis and necrosis

The HT29 adenocarcinoma is a common model of epithelial cell differentiation and colorectal cancer and its death is an oft-analyzed response to TNF family receptor signaling. The death event itself remains poorly characterized and here we have examined the involvement of caspases using pan-caspase inhibitors. zVAD-fmk did not block death of HT29 cells in response to activation of the Fas, TRAIL, TNF, TWEAK and LTbeta receptors. The secondary induction of TNF or the other known bona fide death inducing ligands did not account for death following LTbeta receptor activation indicating that TNF family receptors can trigger a caspase-independent death pathway regardless of the presence of canonical death domains in the receptor. To provide a frame of reference, the phenotype of HT29 death was compared to four other TNF family receptor triggered death events; Fas induced Jurkat cell apoptosis, TNF/zVAD induced L929 fibroblast necrosis, TNF induced death of WEHI 164 fibroblastoid cells and TNF/zVAD induced U937 death. The death of HT29 and U937 cells under these conditions is an intermediate form with both necrotic and apoptotic features. The efficient coupling of TNF receptors to a caspase-independent death event in an epithelial cell suggests an alternative approach to cancer therapy.