A novel signaling pathway in TNFα-induced neutrophil apoptosis

Comment on: Geering B, et al. Blood 2011; 117:5953-62.     

Shear stress regulates expression of death-associated protein kinase in suppressing TNFα-induced endothelial apoptosis

Death associated protein kinase (DAPK) is a positive regulator in tumor necrosis factor α (TNFα)‐induced apoptotic pathway, and DAPK expression is lost in cancer cells. In the vasculature, misdirected apoptosis in endothelial cells leads to pathological conditions such as inflammation and physiological shear stress is protective against apoptosis. Using bovine aortic endothelial cells, we found that DAPK expression increased, while the auto‐inhibitory phosphorylation of serine 308 decreased with shear stress at 12 dynes/cm² for 6 h. Quantitative RT‐PCR revealed a corresponding increase in DAPK mRNA [P < 0.01]. We found that after 18‐h TNFα induction, shearing cells for another 6 h significantly reduced apoptosis based on TUNEL staining [P < 0.05], although cell necrosis was not affected. Under the same conditions, we observed significantly decreased overall DAPK, as well as phospho‐serine 308 DAPK [P < 0.05] compared to TNFα treatment alone. Caspase‐3 and ‐7 activities downstream of DAPK were also attenuated. Shearing cells alone resulted in enhanced apoptosis, likely due to increased DAPK activity. Our findings were further supported by DAPK siRNA, which yielded contrary results. We present conclusive evidence for the first time that shear stress of up to 6 h up‐regulates DAPK expression and activation. However, in the presence of apoptotic stimuli such as TNFα, shear stress caused decrease in DAPK activity. In fact, long‐term shear stress of 24 h significantly reduced overall DAPK expression. Our findings strongly support a novel role for DAPK in mediating effects of shear stress in suppressing cytokine‐activated apoptosis. J. Cell. Physiol. 227: 2398–2411, 2012. © 2011 Wiley Periodicals, Inc.     

Pivotal role of the RanBP9-cofilin pathway in Aβ-induced apoptosis and neurodegeneration

Neurodegeneration associated with amyloid β (Aβ) peptide accumulation, synaptic loss, neuroinflammation, tauopathy, and memory impairments encompass the pathophysiological features of Alzheimer's disease (AD). We previously reported that the scaffolding protein RanBP9, which is overall increased in brains of AD patients, simultaneously promotes Aβ generation and focal adhesion disruption by accelerating the endocytosis of amyloid precursor protein (APP) and β1-integrin, respectively. Here, we show that RanBP9 protein levels are increased by fourfold in FAD mutant APP transgenic mice. Accordingly, RanBP9 transgenic mice demonstrate significantly increased synapse loss, neurodegeneration, gliosis, and spatial memory deficits. RanBP9 overexpression promotes apoptosis and potentiates Aβ-induced neurotoxicity independent of its capacity to promote Aβ generation. Conversely, RanBP9 reduction by siRNA or gene dosage mitigates Aβ-induced neurotoxicity. Importantly, RanBP9 activates/dephosphorylates cofilin, a key regulator of actin dynamics and mitochondria-mediated apoptosis, and siRNA knockdown of cofilin abolishes both Aβ and RanBP9-induced apoptosis. These findings implicate the RanBP9-cofilin pathway as critical therapeutic targets not only for stemming Aβ generation but also antagonizing Aβ-induced neurotoxicity.     

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.     

Oxidatively truncated phospholipids are required agents of tumor necrosis factor α (TNFα)-induced apoptosis

TNFα generates reactive oxygen species (ROS) at the cell surface that induce cell death, but how ROS communicate to mitochondria and their specific apoptotic action(s) are both undefined. ROS oxidize phospholipids to hydroperoxides that are friable and fragment adjacent to the (hydro)peroxide function, forming truncated phospholipids, such as azelaoyl phosphatidylcholine (Az-PC). Az-PC is relatively soluble, and exogenous Az-PC rapidly enters cells to damage mitochondrial integrity and initiate intrinsic apoptosis. We determined whether this toxic phospholipid is formed within cells during TNFα stimulation in sufficient quantities to induce apoptosis and if they are essential in TNFα-induced cytotoxicity. We found that TNFα induced ROS formation and phospholipid peroxidation in Jurkat cells, and either chemical interference with NADPH oxidase activity or siRNA suppression of the NADPH oxidase-4 subunit blocked ROS accumulation and phospholipid peroxidation. Mass spectrometry showed that phospholipid peroxides and then Az-PC increased after TNFα exposure, whereas ROS inhibition abolished Az-PC accumulation and TNFα-induced cell death. Glutathione peroxidase-4 (GPx4), which specifically metabolizes lipid hydroperoxides, fell in TNFα-stimulated cells prior to death. Ectopic GPx4 overcame this, reduced peroxidized phospholipid accumulation, blocked Az-PC accumulation, and prevented death. Conversely, GPx4 siRNA knockdown enhanced phospholipid peroxidation, increasing TNFα-stimulated Az-PC formation and apoptosis. Truncated phospholipids were essential elements of TNFα-induced apoptosis because overexpression of PAFAH2 (a phospholipase A(2) that selectively hydrolyzes truncated phospholipids) blocked TNFα-induced Az-PC accumulation without affecting phospholipid peroxidation. PAFAH2 also abolished apoptosis. Thus, phospholipid oxidation and truncation to apoptotic phospholipids comprise an essential element connecting TNFα receptor signaling to mitochondrial damage and apoptotic death.     

BimL displacing Bcl-xL promotes Bax translocation during TNFα-induced apoptosis

Bcl-2 family proteins are implicated as essential regulators in tumor necrosis factor-α (TNFα)-induced apoptosis. Bim_L, a BH3-only member of Bcl-2 family, can directly or indirectly activate the proapoptotic Bax and the subsequent mitochondrial apoptotic pathway. However, the molecular mechanism of Bim_L activating Bax activation during TNFα-induced apoptosis is not fully understood. In this study, the role of Bim_L in Bax activation during TNFα-induced apoptosis was investigated in differentiated PC12 and MCF7 cells, with real-time single-cell analysis. The experimental results show that Bax translocated to mitochondria and cytochrome c (Cyt c) released from mitochondria after TNFα treatment. Furthermore, SP600125 (specific inhibitor of JNK) could inhibit the Cyt c release from mitochondria. Co-immunoprecipitation results show that, the interaction between Bcl-x_L and Bax decreased after TNFα treatment, while that between Bcl-x_L and Bim_L increased. Bax did not co-immunoprecipitate with Bim_L before or after the TNFα treatment. In addition, the increased interaction between Bim_L and Bcl-x_L was dynamically monitored by using fluorescence resonance energy transfer (FRET) technique. Most importantly, there was no evidence of Bim_L redistribution to mitochondria until cell apoptosis. By comprehensively analyzing these data, it is concluded that Bim_L displaces Bcl-x_L in the mitochondria and promotes Bax translocation during TNFα-induced apoptosis.     

A novel PKC-ι inhibitor abrogates cell proliferation and induces apoptosis in neuroblastoma

Protein Kinase C-iota (PKC-ι), an atypical protein kinase C isoform manifests its potential as an oncogene by targeting various aspects of cancer cells such as growth, invasion and survival. PKC-ι confers resistance to drug-induced apoptosis in cancer cells. The acquisition of drug resistance is a major obstacle to good prognosis in neuroblastoma. The focus of this research was to identify the efficacy of [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) as a novel PKC-ι inhibitor in neuroblastoma cell proliferation and apoptosis. ICA-1 specifically inhibits the activity of PKC-ι but not that of PKC-zeta (PKC-ζ), the closely related atypical PKC family member. The IC(50) for the kinase activity assay was approximately 0.1μM which is 1000 times less than that of aurothiomalate, a known PKC-ι inhibitor. Cyclin dependent kinase 7 (Cdk7) phosphorylates cyclin dependent kinases (cdks) and promotes cell proliferation. Our data shows that PKC-ι is an in vitro Cdk7 kinase and the phosphorylation of Cdk7 by PKC-ι was potently inhibited by ICA-1. Furthermore, our data shows that neuroblastoma cells proliferate via a PKC-ι/Cdk7/cdk2 cell signaling pathway and ICA-1 mediates its antiproliferative effects by inhibiting this pathway. ICA-1 (0.1μM) inhibited the in vitro proliferation of BE(2)-C neuroblastoma cells by 58% (P=0.01). Additionally, ICA-1 also induced apoptosis in neuroblastoma cells. Interestingly, ICA-1 did not affect the proliferation of normal neuronal cells suggesting its potential as chemotherapeutic with low toxicity. Hence, our results emphasize the potential of ICA-1 as a novel PKC-ι inhibitor and chemotherapeutic agent for neuroblastoma.     

A reappraisal of the role of Zn2+ in TGF-β1-induced apoptosis in primary hepatocytes

There is now a wealth of information regarding the apoptotic mode of cell death and its importance in toxicological studies in many mammalian organs including the liver. In this study, we investigated the modulatory effects of the heavy metal Zn²⁺ on transforming growth factor-β₁ (TGF-β₁)-induced apoptosis in primary rat hepatocytes. Apoptosis induced by TGF-β₁ (1 ng/ml) in hepatocytes was accompanied by nuclear condensation as assessed morphologically by staining with Hoechst 33258 and DNA cleavage as detected biochemically by in situ end-labeling, field inversion and conventional gel electrophoresis. Pretreatment with 100 μmol/L Zn²⁺ abrogated the nuclear condensation, in situ end-labeling, and DNA laddering in TGF-β₁-treated hepatocytes. Surprisingly, Zn²⁺ did not inhibit the formation of high-molecular-weight DNA fragments (30–50 kbp to 250–300 kbp). These data provide evidence that Zn²⁺ exerts its effects on the endonucleases that act downstream in the execution phase of TGF-β₁-induced apoptosis in hepatocytes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Involvement of both tumor necrosis factor-α-induced necrosis and p53-mediated caspase-dependent apoptosis in nephrotoxicity of cisplatin

We previously reported that necrosis occurs predominantly in porcine renal tubular LLC-PK₁ cells, when the cells were exposed transiently to a high concentration of cisplatin. Moreover, we demonstrated that generation of reactive oxygen species and subsequent production of tumor necrosis factor-α (TNF-α) through phosphorylation of p38 MAPK are implicated in the pathogenesis of cisplatin-induced renal cell injury. However, some TUNEL-positive cells appeared in renal proximal tubules of rats after systemic injection of cisplatin, suggesting an involvement of apoptosis. In the present study, we found in LLC-PK₁ cells that both apoptosis and necrosis were elicited when the cells were exposed to 200 μM cisplatin for 1 h followed by incubation for 24 h in the presence of 20 μM cisplatin. The cisplatin-induced necrosis was largely attenuated by the antioxidant N-acetylcysteine, while apoptosis was prevented by the specific inhibitors for caspases-2, -8, and -3 and a p53 inhibitor pifithrin-α but not by the p38 MAPK inhibitor SB203580. On the other hand, SB203580 attenuated the cisplatin-induced increase in TNF-α production. These findings suggest that p53-mediated activations of caspases-2, -8 and -3 play a key role in cisplatin-induced renal cell apoptosis, while oxidative stress-induced TNF-α synthesis via p38 MAPK phosphorylation contributed to the necrosis.     

A selective estrogen receptor modulator inhibits tumor necrosis factor-α-induced apoptosis through the ERK1/2 signaling pathway in human chondrocytes

Tumor necrosis factor α (TNF-α) is a pleiotropic cytokine mediating inflammatory as well as cell death activities, and is thought to induce chondrocytic chondrolysis in inflammatory and degenerative joint diseases. Selective estrogen receptor modulators (SERMs), such as raloxifene, which are commonly used in clinical settings act as estrogen agonists or antagonists. It is assumed that estrogens have a potential role in cartilage protection; however, the precise molecular mechanism for the protective effects of estrogens is unclear. This study was designed to examine whether raloxifene inhibits TNF-α-induced apoptosis in human chondrocytes and to clarify the mechanisms involved. We also investigated the signaling pathways responsible for the anti-apoptotic effect of raloxifene. Apoptosis in chondrocytes was determined by DNA fragmentation assay and caspase-3 activation. Raloxifene significantly inhibited TNF-α-induced caspase-3 activation and cell DNA fragmentation levels in chondrocytes. The inhibitory effect of raloxifene was abolished by the estrogen receptor antagonist ICI 182,780. Extracellular signal-regulated kinase 1/2 (ERK1/2) regulates apoptosis, acting as an apoptotic or anti-apoptotic signal. TNF-α-induced apoptosis was significantly enhanced by the ERK1/2 pathway inhibitor PD98059. Raloxifene stimulated a further increase in ERK1/2 phosphorylation in TNF-α-treated chondrocytes. Furthermore, the anti-apoptotic effects of raloxifene were inhibited by PD98059. In addition, the anti-apoptotic effects of raloxifene were completely abolished in ERK1/2 siRNA-treated chondrocytes. These results suggest that raloxifene prevents caspase-3-dependent apoptosis induced by TNF-α in human chondrocytes by activating estrogen receptors and the ERK1/2 signaling pathway.     

TRIM32 protein sensitizes cells to tumor necrosis factor (TNFα)-induced apoptosis via its RING domain-dependent E3 ligase activity against X-linked inhibitor of apoptosis (XIAP)

TRIM32, which belongs to the tripartite motif (TRIM) protein family, has the RING finger, B-box, and coiled-coil domain structures common to this protein family, along with an additional NHL domain at the C terminus. TRIM32 reportedly functions as an E3 ligase for actin, a protein inhibitor of activated STAT y (PIASy), dysbindin, and c-Myc, and it has been associated with diseases such as muscular dystrophy and epithelial carcinogenesis. Here, we identify a new substrate of TRIM32 and propose a mechanism through which TRIM32 might regulate apoptosis. Our overexpression and knockdown experiments demonstrate that TRIM32 sensitizes cells to TNFα-induced apoptosis. The RING domain is necessary for this pro-apoptotic function of TRM32 as well as being responsible for its E3 ligase activity. TRIM32 colocalizes and directly interacts with X-linked inhibitor of apoptosis (XIAP), a well known cancer therapeutic target, through its coiled-coil and NHL domains. TRIM32 overexpression enhances XIAP ubiquitination and subsequent proteasome-mediated degradation, whereas TRIM32 knockdown has the opposite effect, indicating that XIAP is a substrate of TRIM32. In vitro reconstitution assay reveals that XIAP is directly ubiquitinated by TRIM32. Our novel results collectively suggest that TRIM32 sensitizes TNFα-induced apoptosis by antagonizing XIAP, an anti-apoptotic downstream effector of TNFα signaling. This function may be associated with TRIM32-mediated tumor suppressive mechanism.     

Death receptor 3 mediates TNFSF15- and TNFα-induced endothelial cell apoptosis

Tumor necrosis factor superfamily 15 (TNFSF15) suppresses angiogenesis by specifically inducing apoptosis in proliferating endothelial cells. Death receptor 3 (DR3), a member of the TNF receptor superfamily (TNFRSF25), has been identified as a receptor for TNFSF15 to activate T cells. It is unclear, however, whether DR3 mediates TNFSF15 activity on endothelial cells. Here we show that siRNA-mediated knockdown of DR3 in an in vivo Matrigel angiogenesis assay, or in adult bovine aortic endothelial (ABAE) cell cultures, leads to resistance of endothelial cells to TNFSF15-induced apoptosis. Interestingly, DR3-depleted cells also exhibited markedly diminished responsiveness to TNFα cytotoxicity, even though DR3 is not a receptor for TNFα. Treatment of the cells with either TNFSF15 siRNA or a TNFSF15-neutralizing antibody, 4-3H, also results in a significant inhibition of TNFα-induced apoptosis. Mechanistically, DR3 siRNA treatment gives rise to an increase of ERK1/2 MAPK activity, and up-regulation of the anti-apoptotic proteins c-FLIP and Bcl-2, thus strengthening apoptosis-resisting potential in the cells. These findings indicate that DR3 mediates TNFSF15-induced endothelial cell apoptosis, and that up-regulation of TNFSF15 expression stimulated by TNFα is partly but significantly responsible for TNFα-induced apoptosis in endothelial cells.