Regulation of Raf-1 kinase by TNF via its second messenger ceramide and cross-talk with mitogenic signalling.

Raf-1 kinase is a central regulator of mitogenic signal pathways, whereas its general role in signal transduction of tumour necrosis factor (TNF) is less well defined. We have investigated mechanisms of Raf-1 regulation by TNF and its messenger ceramide in cell-free assays, insect and mammalian cell lines. In vitro, ceramide specifically bound to the purified catalytic domain and enhanced association with activated Ras proteins, but did not affect the kinase activity of Raf-1. Cell-permeable ceramides induced a marked increase of Ras-Raf-1 complexes in cells co-expressing Raf-1 and activated Ras. Likewise, a fast elevation of the endogeneous ceramide level, induced by TNF treatment of human Kym-1 rhabdomyosarcoma cells, was followed by stimulation of Ras-Raf-1 association without significant Raf-1 kinase activation. Failure of TNF or ceramide to induce Raf-1 kinase was observed in several TNF-responsive cell lines. Both TNF and exogeneous C6-ceramide interfered with the mitogenic activation of Raf-1 and ERK by epidermal growth factor and down-regulated v-Src-induced Raf-1 kinase activity. TNF also induced the translocation of Raf-1 from the cytosolic to the particulate fraction, indicating that this negative regulatory cross-talk occurs at the cell membrane. Interference with mitogenic signals at the level of Raf-1 could be an important initial step in TNF's cytostatic action.     

RNAi‐mediated silencing of insulin receptor substrate‐4 enhances actinomycin D‐ and tumor necrosis factor‐α‐induced cell death in hepatocarcinoma cancer cell lines

Insulin receptor substrate‐4 (IRS‐4) transmits signals from the insulin‐like growth factor receptor (IGF‐IR) and the insulin receptor (IR) to the PI3K/AKT and the ERK1/2 pathways. IRS‐4 expression increases dramatically after partial hepatectomy and plays an important role in HepG2 hepatoblastoma cell line proliferation/differentiation. In human hepatocarcinoma, IRS‐4 overexpression has been associated with tumor development. Herein, we describe the mechanism whereby IRS‐4 depletion induced by RNA interference (siRNA) sensitizes HepG2 cells to treatment with actinomycin D (Act D) and combined treatment with Act D plus tumor necrosis factor‐α (TNF‐α). Similar results have been obtained in HuH 7 and Chang cell lines. Act D therapy drove the cells to a mitochondrial‐dependent apoptotic program involving cytochrome c release, caspase 3 activation, PARP fragmentation and DNA laddering. TNF‐α amplifies the effect of Act D on HepG2 cell apoptosis increasing c‐jun N‐terminal kinase (JNK) activity, IκB‐α proteolysis and glutathione depletion. IRS‐4 depleted cells that were treated with Act D showed an increase in cytochrome c release and procaspase 3 and PARP proteolysis with respect to control cells. The mechanism involved in IRS‐4 action is independent of Akt, IκB kinase and JNK. IRS‐4 down regulation, however, decreased γ‐glutamylcysteine synthetase content and cell glutathione level in the presence of Act D plus TNF‐α. These results suggest that IRS‐4 protects HepG2 cells from oxidative stress induced by drug treatment. J. Cell. Biochem. 108: 1292–1301, 2009. © 2009 Wiley‐Liss, Inc.     

Inhibition of p38 kinase reveals a TNF-alpha-mediated, caspase-dependent, apoptotic death pathway in a human myelomonocyte cell line

TNF-alpha transduces signals of survival or death via its two receptors, R1/p55/p60 and RII/p80/p75. The role of caspases as effectors of cell death is universally accepted, although caspase inhibitors may potentiate TNF cytotoxicity in some instances. In conditions when macromolecular synthesis is blocked, caspases are part of the machinery that executes TNF-triggered apoptotic death in U937, a human myelomonocyte cell line, and in the Jurkat T cell line. However, inhibition of p38 mitogen-activated protein kinase (p38 MAPK) triggered TNF cytotoxicity in U937 cells and murine splenic macrophages, but not the Jurkat cell line. TNF induced expression of the antiapoptotic protein c-IAP2 (cytoplasmic inhibitor of apoptosis protein 2), and was blocked in the presence of a p38 MAPK inhibitor, which also induced caspase-dependent, TNF-mediated apoptosis in U937 cells. Thus, inhibition of p38 MAPK resulted in the activation of caspase 9 and cleavage of the adaptor molecule BH3 interacting domain death agonist, and blocked NF-kappaB-mediated transactivation, without affecting the nuclear translocation of NF-kappaB. Collectively, these data show that activation of p38 MAPK is critical to cell survival by TNF in U937 cells, and demonstrate lineage-specific regulation of TNF-triggered signals of activation or apoptosis.     

Effect of the polyamine analogue N1,N11-diethylnorspermine on cell survival and susceptibility to apoptosis of human chondrocytes

Chondrocyte survival is closely linked to cartilage integrity, and forms of chondrocyte apoptotic death can contribute to cartilage degeneration in articular diseases. Since growing evidence also implicates polyamines in the control of cell death, we have been investigating the role of polyamine metabolism in chondrocyte survival and apoptosis. Treatment of human C-28/I2 chondrocytes with N(1),N(11)-diethylnorspermine (DENSPM), a polyamine analogue with clinical relevance as an experimental anticancer agent, inhibited polyamine biosynthesis and induced polyamine catabolism, thus rapidly depleting all main polyamines. DENSPM did not increase significantly caspase activity, but provoked a late cell death associated to DNA fragmentation. A short treatment with DENSPM did not reduce cell viability when given alone, but enhanced caspase-3 and -9 activation in chondrocytes exposed to tumor necrosis factor-alpha (TNF) and cycloheximide (CHX). A longer treatment with DENSPM however reduced caspase response to TNF plus CHX. Depletion of all polyamines obtained by specific inhibitors of polyamine biosynthesis did not cause cell death and contrasted apoptosis by decreasing caspase activities. In conclusion, following DENSPM treatment, C-28/I2 chondrocytes are initially sensitized to caspase 9-dependent apoptosis in the presence of TNF and CHX and may eventually undergo a late and mainly caspase-independent cell death in the absence of other stimuli. Moreover, these results indicate that a reduction of polyamine levels not only leads to inhibition of cell proliferation, but also of caspase-mediated pathways of chondrocyte apoptosis.     

Fibroblast Growth Factor 2 Causes G2/M Cell Cycle Arrest in Ras-Driven Tumor Cells through a Src-Dependent Pathway

We recently reported that paracrine Fibroblast Growth Factor 2 (FGF2) triggers senescence in Ras-driven Y1 and 3T3^Ras mouse malignant cell lines. Here, we show that although FGF2 activates mitogenic pathways in these Ras-dependent malignant cells, it can block cell proliferation and cause a G2/M arrest. These cytostatic effects of FGF2 are inhibited by PD173074, an FGF receptor (FGFR) inhibitor. To determine which downstream pathways are induced by FGF2, we tested specific inhibitors targeting mitogen-activated protein kinase (MEK), phosphatidylinositol 3 kinase (PI3K) and protein kinase C (PKC). We show that these classical mitogenic pathways do not mediate the cytostatic activity of FGF2. On the other hand, the inhibition of Src family kinases rescued Ras-dependent malignant cells from the G2/M irreversible arrest induced by FGF2. Taken together, these data indicate a growth factor-sensitive point in G2/M that likely involves FGFR/Ras/Src pathway activation in a MEK, PI3K and PKC independent manner.     

Nanosecond pulsed electric fields (nsPEFs) activate intrinsic caspase-dependent and caspase-independent cell death in Jurkat cells

NsPEF ablation induces apoptosis markers, but specific cell death pathways have not been fully defined. To identify nsPEF-activated cell death pathways, wildtype human Jurkat cells and clones with deficiencies in extrinsic and intrinsic apoptosis pathways were investigated. NsPEFs activated caspase isozymes and induced identical electric field-dependent cell death in clones deficient in FADD or caspase-8, indicating that extrinsic apoptosis pathways were not activated. This was confirmed when cytochrome c release was shown to be unaffected by the pan caspase inhibitor, z-VAD-fmk. NsPEF-treated APAF-1-silenced cells did not exhibit caspase-3/7 and -9 activities and corresponding electric field-dependent cell death in this clone was attenuated compared to its vector control at low, but not at high electric fields. These data demonstrate that nsPEFs induce intrinsic apoptosis activate by cytochrome c release from mitochondria through an APAF-1- and caspase-dependent pathway as well as through caspase-independent mechanisms that remain to be defined. Furthermore, the results establish that nsPEFs can overcome natural and oncogenic mechanisms that promote cell survival through inhibition of apoptosis and other cell death mechanisms.     

The p42/p44 MAP kinase pathway prevents apoptosis induced by anchorage and serum removal

Anchorage removal like growth factor removal induces apoptosis. In the present study we have characterized signaling pathways that can prevent this cell death using a highly growth factor- and anchorage-dependent line of lung fibroblasts (CCL39). After anchorage removal from exponentially growing cells, annexin V-FITC labeling can be detected after 8 h. Apoptosis was confirmed by analysis of sub-G1 DNA content and Western blotting of the caspase substrate poly (ADP-ribose) polymerase. Growth factor withdrawal accelerates and potentiates suspension-induced cell death. Activation of Raf-1 kinase in suspension cultures of CCL39 or Madin-Darby canine kidney cells stably expressing an estrogen-inducible activated-Raf-1 construct (DeltaRaf-1:ER) suppresses apoptosis induced by growth factor and/or anchorage removal. This protective effect appears to be mediated by the Raf, mitogen- or extracellular signal-regulated kinase kinase (MEK), and mitogen-activated protein kinase module because it is sensitive to pharmacological inhibition of MEK-1 and it can be mimicked by expression of constitutively active MEK-1 in CCL39 cells. Finally, apoptosis induced by disruption of the actin cytoskeleton with the Rho-directed toxin B (Clostridium difficile) is prevented by activation of the DeltaRaf-1:ER chimeric construct. These findings highlight the ability of p42/p44 mitogen-activated protein kinase to generate survival signals that counteract cell death induced by loss of matrix contact, cytoskeletal integrity, and extracellular mitogenic factors.     

Inhibition of ceramide production reverses TNF-induced insulin resistance

Ceramide has been implicated as a mediator of insulin resistance induced by tumor necrosis factor-alpha (TNF) in adipocytes. Adipocytes contain numerous caveolae, sphingolipid and cholesterol-enriched lipid microdomains, that are also enriched in insulin receptor (IR). Since caveolae may be important sites for crosstalk between tyrosine kinase and sphingolipid signaling pathways, we examined the role of increased caveolar pools of ceramide in regulating tyrosine phosphorylation of the IR and its main substrate, insulin receptor substrate-1 (IRS-1). Neither exogenous short-chain ceramide analogs nor pharmacologic increases in endogenous caveolar pools of ceramide inhibited insulin-induced tyrosine phosphorylation of the IR and IRS-1. However, inhibition of TNF-induced caveolar ceramide production reversed the decrease in IR tyrosine phosphorylation in response to TNF. These results suggest that TNF-independent increases in caveolar pools of ceramide are not sufficient to inhibit insulin signaling but that in conjunction with other TNF-dependent signals, caveolar pools of ceramide are a critical component for insulin resistance by TNF.     

Blockade of TNFR1 signaling: A role of oscillatory fluid shear stress in osteoblasts

Fluid shear stress protects cells from TNF‐α‐induced apoptosis. Oscillatory fluid shear stress (OFSS) is generally perceived as physiologically relevant biophysical signal for bone cells. Here we identify several cellular mechanisms responsible for mediating the protective effects of OFSS against TNF‐α‐induced apoptosis in vitro. We found that exposure of MC3T3‐E1 osteoblast‐like cells to as little as 5 min of OFSS suppressed TNF‐α‐induced activation of caspase‐3, cleavage of PARP and phosphorylation of histone. In contrast, H₂O₂‐induced apoptosis was not inhibited by OFSS suggesting that OFSS might not be protecting cells from TNF‐α‐induced apoptosis via stimulation of global pro‐survival signaling pathways. In support of this speculation, OFSS inhibition of TNF‐α‐induced apoptosis was unaffected by inhibitors of several pro‐survival signaling pathways including pI3‐kinase (LY294002), MAPK/ERK kinase (PD98059 or U0126), intracellular Ca2+ release (U73122), NO production (L‐NAME), or protein synthesis (cycloheximide) that were applied to cells during exposure to OFSS and during TNF‐α treatment. However, TNF‐α‐induced phosphorylation and degradation of IκBα was blocked by pre‐exposure of cells to OFSS suggesting a more specific effect of OFSS on TNF‐α signaling. We therefore focused on the mechanism of OFSS regulation of TNF‐receptor 1 (TNFR1) signaling and found that OFSS (1) reduced the amount of receptor on the cell surface, (2) prevented the association of ubiquitinated RIP in TNFR1 complexes with TRADD and TRAF2, and (3) reduced TNF‐α‐induced IL‐8 promoter activity in the nucleus. We conclude that the anti‐apoptotic effect of OFSS is not mediated by activation of universal pro‐survival signaling pathways. Rather, OFSS inhibits TNF‐α‐induced pro‐apoptotic signaling which can be explained by the down‐regulation of TNFR1 on the cell surface and blockade of TNFR1 downstream signaling by OFSS. J. Cell. Physiol. 226: 1044–1051, 2011. © 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.     

MAP kinase pathway signalling is essential for extracellular matrix determined mammary epithelial cell survival

Mammary epithelial cells in primary cell culture require both growth factors and specific extracellular matrix (ECM)-attachment for survival. Here we demonstrate for the first time that inhibition of the ECM-induced Erk 1/Erk 2 (p42/44 MAPK) pathway, by PD 98059, leads to apoptosis in these cells. Associated with this cell death is a possible compensatory signalling through the p38 MAP kinase pathway the inhibition of which, by SB 203580, leads to a more rapid onset of apoptosis. This provides evidence for a hitherto undescribed Erk 1/Erk 2 to p38 MAP kinase pathway 'cross-talk' that is essential for the survival of these cells. The cell death associated with inhibition of these two MAP kinase pathways however, occurred in the presence of insulin that activates the classical PI-3 kinase-dependent Akt/PKB survival signals and Akt phosphorylation. Cell death induced by inhibition of the MAP kinase pathways did not affect Akt phosphorylation and may, thus, be independent of PI-3 kinase signalling.     

Insulin-like growth factor-I and Bcl-X(L) inhibit c-jun N-terminal kinase activation and rescue Schwann cells from apoptosis

We previously reported that Schwann cells undergo apoptosis after serum withdrawal. Insulin-like growth factor-I, via phosphatidylinositol-3 kinase, inhibits caspase activation and rescues Schwann cells from serum withdrawal-induced apoptosis. In this study, we examined the role of c-jun N-terminal protein kinase (JNK) in Schwann cell apoptosis induced by serum withdrawal. Activation of both JNK1 and JNK2 was detected 1 h after serum withdrawal with the maximal level detected at 2 h. A dominant negative JNK mutant, JNK (APF), blocked JNK activation induced by serum withdrawal and Schwann cell apoptosis, suggesting JNK activation participates in Schwann cell apoptosis. Serum withdrawal-induced JNK activity was caspase dependent and inhibited by a caspase 3 inhibitor, Ac-DEVD-CHO. Because insulin-like growth factor-I and Bcl-X(L) are both Schwann cell survival factors, we tested their effects on JNK activation during apoptosis. Insulin-like growth factor-I treatment decreased both JNK1 and JNK2 activity induced by serum withdrawal. LY294002, a phosphatidylinositol-3 kinase inhibitor, blocked insulin-like growth factor-I inhibition on JNK activation, suggesting that phosphatidylinositol-3 kinase mediates the effects of insulin-like growth factor-I. Overexpression of Bcl-X(L) also resulted in less Schwann cell death and inhibition of JNK activation after serum withdrawal. Collectively, these results suggest JNK activation is involved in Schwann cell apoptosis induced by serum withdrawal. Insulin-like growth factor-I and Bcl family proteins rescue Schwann cells, at least in part, by inhibition of JNK activity.     

Regulation of the Akt/Glycogen synthase kinase-3 axis by insulin-like growth factor-II via activation of the human insulin receptor isoform-A

Insulin-like growth factor II (IGF-II) plays a key role in mitogenesis during development and tumorigenesis and is believed to exert its mitogenic functions mainly through the IGF-I receptor. Recently, we identified the insulin receptor isoform A (IR(A)) as an additional high affinity receptor for IGF-II in both fetal and cancer cells. Here we investigated the mitogenic signaling of IGF-II via the Akt/Glycogen synthase kinase 3 (Gsk3) axis employing R-IR(A) cells that are IGF-I receptor null mouse embryonic fibroblasts expressing the human IR(A). IGF-II induced activation of the proto-oncogenic serine kinase Akt, reaching maximal at 5-10 min. IGF-II also caused the rapid and sustained deactivation of glycogen synthase kinase 3-beta (Gsk3beta), reaching maximal at 1-3 min, shortly preceding, therefore, maximal activation of Akt. Under our conditions, IGF-II and insulin induced 70-80% inhibition of Gsk3betaactivity. In these cells IGF-II also deactivated Gsk3alpha although less effectively than Gsk3beta. In parallel experiments, we found that IGF-II induced transient activation of extracellular-signal-regulated kinases (Erk) reaching maximal at 5-10 min and decreasing thereafter. Time courses and potencies of regulation of both mitogenic pathways (Akt/Gsk3beta and Erk) by IGF-II via IR(A) were similar to those of insulin. Furthermore, IGF-II like insulin effectively stimulated cell cycle progression from the G0/G1 to the S and G2/M phases. Interestingly, AP-1-mediated gene expression, that was reported to be negatively regulated by Gsk3beta was only weakly increased after IGF-II stimulation. Our present data suggest that the coordinated activation or deactivation of Akt, Gsk3beta, and Erk may account for IGF-II mitogenic effects and support an active role for IR(A) in IGF-II action.     

Caspase activation by BCR cross-linking in immature B cells: differential effects on growth arrest and apoptosis.

The B cell lymphoma WEHI-231 has been used as a model to study immature B cell tolerance, based on its capacity to undergo growth arrest and programmed cell death on B cell receptor (BCR) cross-linking. Using this model to identify the molecular mechanisms underlying these processes, we found that BCR cross-linking results in the selective activation of caspase 7/Mch3, but not of the other two members of the CPP32 family, caspase 2/Nedd2 and caspase 3/CPP32. This was evidenced by the induction of proteolytic activity against the substrate for the CPP32 subfamily of caspases (z-DVED-AMC) in vitro, as well as PARP proteolysis in vivo and by the processing of the 35 kDa Mch3 into a 32 kDa species, which was later further proteolyzed. The general caspase inhibitor z-VAD-fmk, but not the CPP32 family inhibitor Ac-DEVD-CHO, blocked anti- micro-induced apoptosis, indicating that a caspase not belonging to the CPP32-like family is also implicated in anti- micro-triggered apoptosis. In contrast, z-VAD-fmk was not able to counteract growth arrest induced by anti- micro treatment, suggesting that caspase activation is not necessary for induction of growth arrest. Neither of the inhibitors prevented Mch3 processing; however, z-VAD-fmk prevented proteolysis of the p32 subunit, suggesting that further processing of this subunit is associated with apoptosis. Bcl-2 overexpression prevented anti- micro induction of CPP32-like activity and apoptosis, and blocked further processing of the Mch3 p32 subunit. In contrast, CD40 stimulation completely blocked the appearance of the p32 subunit in addition to blocking CPP32-like activity and apoptosis induced by BCR cross-linking. Moreover, only CD40 stimulation was able to prevent anti- micro-induced growth arrest, which was correlated with inhibition of retinoblastoma and of cyclin A down-regulation. In splenic B cells, Mch3 is also specifically proteolyzed ex vivo after induction of apoptosis by BCR cross-linking, demonstrating the specific involvement of caspase-7/Mch3 in apoptosis induced in B cell tolerance.     

Cleavage of focal adhesion proteins and PKCdelta during lovastatin-induced apoptosis in spontaneously immortalized rat brain neuroblasts

We have previously shown that lovastatin induces apoptosis in spontaneously immortalized rat brain neuroblasts. Focal adhesion proteins and protein kinase Cdelta (PKCdelta) have been implicated in the regulation of apoptosis. We found that lovastatin exposure induced focal adhesion kinase, Crk-associated substrate (p130(Cas)), PKCdelta cleavage and caspase-3 activation in a concentration-dependent manner. Lovastatin effects were fully prevented by mevalonate. The cleavage of p130(Cas) was almost completely inhibited by z-DEVD-fmk, a specific caspase-3 inhibitor, and z-VAD-fmk, a broad spectrum caspase inhibitor, indicating that cleavage is mediated by caspase-3. In contrast, the lovastatin-induced cleavage of PKCdelta was only blocked by z-VAD-fmk suggesting that PKCdelta cleavage is caspase-dependent but caspase-3-independent. Additionally, z-VAD-fmk partially prevented lovastatin-induced neuroblast apoptosis. The present data show that lovastatin may induce neuroblast apoptosis by both caspase-dependent and independent pathways. These findings may suggest that the caspase-dependent component leading to the neuroblast cell death is likely to involve the cleavage of focal adhesion proteins and PKCdelta, which may be partially responsible for some biochemical features of neuroblast apoptosis induced by lovastatin.     

Mechanisms of resistance of human glioma cells to Apo2 ligand/TNF-related apoptosis-inducing ligand.

BACKGROUND: Many tumor cells are resistant to Apo2L/TRAIL-induced apoptosis in the absence of inhibitors of protein synthesis. Apo2L/TRAIL, in addition to induction of apoptosis, may therefore also activate survival pathways. METHODS: Here we investigated whether such survival pathways mediate resistance to Apo2L.0-induced apoptosis in human glioma cells. RESULTS: Apo2L.0 induced the phosphorylation of ERK1/2, but not of Akt. This effect was unaffected by caspase inhibition. Inhibitors of protein synthesis, PI3 kinase, ERK kinase, NF-kappaB or casein kinase 2 sensitized for Apo2L.0-induced apoptosis to a different extent in a panel of human malignant glioma cell lines. However, none of the sensitizers overcame resistance mediated by ectopic expression of the viral caspase 8 inhibitor, crm-A. Primary glioma cultures were almost completely resistant to Apo2L.0-induced cell death even in the presence of the inhibitors. Caspase-8 was expressed in these cells whereas only weak expression of DR5 was detected. Transient expression of DR5 conferred sensitivity to Apo2L.0. CONCLUSION: These data challenge the view that specific cell lines harbour specific mechanisms of resistance to Apo2L/TRAIL. Weak expression of DR5 in primary glioma might limit the therapeutic application of Apo2L/TRAIL in human glioblastoma patients.