Overexpression of catalase in cytosolic or mitochondrial compartment protects HepG2 cells against oxidative injury.

HepG2 cells were transfected with vectors containing human catalase cDNA and catalase cDNA with a mitochondrial leader sequence to allow comparison of the effectiveness of catalase overexpressed in the cytosolic or mitochondrial compartments to protect against oxidant-induced injury. Overexpression of catalase in cytosol and in mitochondria was confirmed by Western blot, and activity measurement and stable cell lines were established. The intracellular level of H(2)O(2) induced by exogenously added H(2)O(2) or antimycin A was lower in C33 cell lines overexpressing catalase in the cytosol and mC5 cell lines overexpressing catalase in the mitochondria as compared with Hp cell lines transfected with empty vector. Cell death caused by H(2)O(2), antimycin A, and menadione was considerably suppressed in both the mC5 and C33 cell lines. C33 and mC5 cells were also more resistant to apoptosis induced by H(2)O(2) and to the loss of mitochondrial membrane potential induced by H(2)O(2) and antimycin A. In view of the comparable protection by catalase overexpressed in the cytosol versus the mitochondria, catalase produced in both cellular compartments might act as a sink to decompose H(2)O(2) and move diffusable H(2)O(2) down its concentration gradient. The present study suggests that catalase in cytosol and catalase in mitochondria are capable of protecting HepG2 cells against cytotoxicity or apoptosis induced by oxidative stress.     

TNF-alpha/cycloheximide-induced apoptosis in intestinal epithelial cells requires Rac1-regulated reactive oxygen species

Previously we have shown that both Rac1 and c-Jun NH(2)-terminal kinase (JNK1/2) are key proapoptotic molecules in tumor necrosis factor (TNF)-alpha/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells, whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The present studies tested the hypothesis that Rac1-mediated ROS production is involved in TNF-alpha-induced apoptosis. In this study, we showed that TNF-alpha/CHX-induced ROS production and hydrogen peroxide (H(2)O(2))-induced oxidative stress increased apoptosis. Inhibition of Rac1 by a specific inhibitor NSC23766 prevented TNF-alpha-induced ROS production. The antioxidant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrial electron transport chain inhibitor, attenuated mitochondrial ROS production and apoptosis. Rot also prevented JNK1/2 activation during apoptosis. Inhibition of Rac1 by expression of dominant negative Rac1 decreased TNF-alpha-induced mitochondrial ROS production. Moreover, TNF-alpha-induced cytosolic ROS production was inhibited by Rac1 inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and NAC. In addition, DPI inhibited TNF-alpha-induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Rac1 or cytosolic ROS dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus these results indicate that TNF-alpha-induced apoptosis requires Rac1-dependent ROS production in intestinal epithelial cells.     

Polyamines are required for activation of c-Jun NH2-terminal kinase and apoptosis in response to TNF-alpha in IEC-6 cells

Intracellular polyamine homeostasis is important for the regulation of cell proliferation and apoptosis and is necessary for the balanced growth of cells and tissues. Polyamines have been shown to play a role in the regulation of apoptosis in many cell types, including IEC-6 cells, but the mechanism is not clear. In this study, we analyzed the mechanism by which polyamines regulate the process of apoptosis in response to tumor necrosis factor-alpha (TNF-alpha). TNF-alpha or cycloheximide (CHX) alone did not induce apoptosis in IEC-6 cells. Significant apoptosis was observed when CHX was given along with TNF-alpha, as indicated by a significant increase in the detachment of cells, caspase-3 activity, and DNA fragmentation. Polyamine depletion by treatment with alpha-difluoromethylornithine significantly reduced the level of apoptosis, as judged by DNA fragmentation and the caspase-3 activity of attached cells. Apoptosis in IEC-6 cells was accompanied by the activation of upstream caspases-6, -8, and -9 and NH2-terminal c-Jun kinase (JNK). Inhibition of JNK activation prevented caspase-9 activation. Polyamine depletion prevented the activation of JNK and of caspases-6, -8, -9, and -3. SP-600125, a specific inhibitor of JNK activation, prevented cytochrome c release from mitochondria, JNK activation, DNA fragmentation, and caspase-9 activation in response to TNF-alpha/CHX. In conclusion, we have shown that polyamine depletion delays and decreases TNF-alpha-induced apoptosis in IEC-6 cells and that apoptosis is accompanied by the release of cytochrome c, the activation of JNK, and of upstream caspases as well as caspase-3. Polyamine depletion prevented JNK activation, which may confer protection against apoptosis by modulation of upstream caspase-9 activation.     

Tumor necrosis factor-alpha-induced caspase-1 gene expression. Role of p73

Tumour necrosis factor-alpha (TNF-alpha) is a cytokine that is involved in many functions, including the inflammatory response, immunity and apoptosis. Some of the responses of TNF-alpha are mediated by caspase-1, which is involved in the production of the pro-inflammatory cytokines interleukin-1beta, interleukin-18 and interleukin-33. The molecular mechanisms involved in TNF-alpha-induced caspase-1 gene expression remain poorly defined, despite the fact that signaling by TNF-alpha has been well studied. The present study was undertaken to investigate the mechanisms involved in the induction of caspase-1 gene expression by TNF-alpha. Treatment of A549 cells with TNF-alpha resulted in an increase in caspase-1 mRNA and protein expression, which was preceded by an increase in interferon regulatory factor-1 and p73 protein levels. Caspase-1 promoter reporter was activated by the treatment of cells with TNF-alpha. Mutation of the interferon regulatory factor-1 binding site resulted in the almost complete loss of basal as well as of TNF-alpha-induced caspase-1 promoter activity. Mutation of the p53/p73 responsive site resulted in reduced TNF-alpha-induced promoter activity. Blocking of p73 function by a dominant negative mutant or by a p73-directed small hairpin RNA reduced basal as well as TNF-alpha-induced caspase-1 promoter activity. TNF-alpha-induced caspase-1 mRNA and protein levels were reduced when p73 mRNA was down-regulated by small hairpin RNA. Caspase-5 gene expression was induced by TNF-alpha, which was inhibited by the small hairpin RNA-mediated down-regulation of p73. Our results show that TNF-alpha induces p73 gene expression, which, together with interferon regulatory factor-1, plays an important role in mediating caspase-1 promoter activation by TNF-alpha.     

Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3

In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway.     

Evidence for serpinB2-independent protection from TNF-alpha-induced apoptosis

Clade B serine proteinase inhibitors (serpins) are intracellular proteins, whereas most of their identified targets are extracellular. A proposed intracellular role for these inhibitors is protection from apoptosis. We investigated the contribution of serpinB2 (plasminogen activator inhibitor-2, PAI-2) activity in TNF-alpha-induced apoptosis. PAI-2 is expressed in many normal and transformed cell types, particularly after stimulation with inflammatory cytokines. PAI-2 has been linked to protection from TNF-alpha-induced apoptosis, and a stabilizing interaction with the retinoblastoma protein (Rb1) has been proposed. We examined the activity of PAI-2 in TNF-alpha-induced apoptosis using HeLa, Isreco-1 and HT1080 cell lines. Stimulation with TNF-alpha protected each cell type from apoptosis induced by TNF-alpha and cycloheximide. Protection correlated with an increase in PAI-2 expression in IS-1 and HT1080 cells but not in HeLa cells where PAI-2 mRNA and protein were undetectable. PAI-2 was overexpressed in each cell type but gave no protection from TNF-alpha-induced apoptosis measured by cell viability, annexinV binding and caspase-3/7 activity. We detected wild-type Rb1, unchanged TNF receptor levels and induction of other apoptosis-protective factors in all cell types. In conclusion, elevated PAI-2 levels do not protect cells from TNF-alpha-induced apoptosis, and the protective effect of prior stimulation with TNF-alpha does not require PAI-2.     

Synergistic induction of apoptosis in murine hepatoma Hepa1-6 cells by IFN-gamma and TNF-alpha

In this study, we examined the susceptibility of murine hepatoma Hepa1-6 cells to undergo IFN-gamma- and/or TNF-alpha-induced apoptosis. IFN-gamma or TNF-alpha alone had no demonstrable cytotoxic effects, whereas IFN-gamma and TNF-alpha in combination induced apoptosis drastically in Hepa1-6 cells. During this apoptosis, an increase in caspase-3- and -8-like protease activities and activation of caspase-3, identified by the appearance of its p17 fragment, were observed. Moreover, the cytotoxic induction and caspase-3 activation were effectively inhibited by Z-Asp-CH(2)-DCB (Z-Asp), a caspase inhibitor. Further, an elevation of cytochrome c in the cytosol, in a parallel to activation of caspase-3, was observed in a time-dependent manner. Concurrently, up-regulation of caspase-11 gene expression and processing of procaspase-11 were detected during this apoptosis. These results suggest that the caspase-3 activation, the release of cytochrome c from mitochondria, and increased caspase-11 gene expression involve in synergistic induction of apoptosis in Hepa1-6 cells by IFN-gamma and TNF-alpha.     

E1A sensitizes cells to tumor necrosis factor alpha by downregulating c-FLIP S

Tumor necrosis factor alpha (TNF-alpha) activates both apoptosis and NF-kappaB-dependent survival pathways, the former of which requires inhibition of gene expression to be manifested. c-FLIP is a TNF-alpha-induced gene that inhibits caspase-8 activation during TNF-alpha signaling. Adenovirus infection and E1A expression sensitize cells to TNF-alpha by allowing apoptosis in the absence of inhibitors of gene expression, suggesting that it may be disabling a survival signaling pathway. E1A promoted TNF-alpha-mediated activation of caspase-8, suggesting that sensitivity was occurring at the level of the death-inducing signaling complex. Furthermore, E1A expression downregulated c-FLIP(S) expression and prevented its induction by TNF-alpha. c-FLIP(S) and viral FLIP expression rescued E1A-mediated sensitization to TNF-alpha by restoring the resistance of caspase-8 to activation, thereby preventing cell death. E1A inhibited TNF-alpha-dependent induction of c-FLIP(S) mRNA and stimulated ubiquitination- and proteasome-dependent degradation of c-FLIP(S) protein. Since elevated c-FLIP levels confer resistance to apoptosis and promote tumorigenicity, interference with its induction by NF-kappaB and stimulation of its destruction in the proteasome may provide novel therapeutic approaches for facilitating the elimination of apoptosis-refractory tumor cells.     

The effect of specific caspase inhibitors on TNF-alpha and butyrate-induced apoptosis of intestinal epithelial cells

Tumour necrosis factor-alpha (TNF-alpha)-induced intestinal epithelial cell apoptosis may contribute to mucosal injury in inflammatory bowel disease. Inhibition of TNF-alpha-induced apoptosis, using specific caspase inhibitors could, therefore, be of benefit in the treatment of disease. In vitro, CaCo-2 colonic epithelial cells are refractory to apoptosis induced by TNF-alpha alone; however, TNF-alpha can act synergistically with the short-chain fatty acid (SCFA) and colonic fermentation product, butyrate, to promote apoptosis. TNF-alpha/butyrate-induced apoptosis was characterised by nuclear condensation and fragmentation and caspase-3 activation. Inhibitors of caspase-8 (z-IETD.fmk) and caspase-10 (z-AEVD.fmk) significantly reduced TNF-alpha/butyrate-induced apoptosis, based on nuclear morphology and terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labelling (TUNEL), although caspase inhibition was associated with a significant increase in cells demonstrating atypical nuclear condensation. Inclusion of atypical cells in calculations of total cell death, still demonstrated that z-IETD.fmk and z-AEVD.fmk (in combination) significantly reduced cell death. Reduction in cell death was associated with maintenance of viable cell number. Transmembrane resistance was also used a measure of the ability of caspase inhibitors to prevent TNF-alpha/butyrate-mediated damage to epithelial monolayers. TNF-alpha/butyrate resulted in a significant fall in transmembrane resistance, which was prevented by pre-treatment with z-IETD.fmk, but not z-AEVD.fmk. In conclusion, synthetic caspase inhibitors can reduce the apoptotic response of CaCo-2 colonic epithelial cells to TNF-alpha/butyrate, improve the maintenance of viable cell numbers and block loss of transmembrane resistance. We hypothesise that caspase inhibition could be a useful therapeutic goal in the treatment of inflammatory bowel conditions, such as ulcerative colitis.     

Cyclophosphamide enhances TNF-alpha-induced apoptotic cell death in murine vascular endothelial cell

Cyclophosphamide (CPA) is one of the therapeutic agents for systemic inflammatory disorders. In murine dermal endothelial cells (F-2), 4-hydroxycyclophosphamide (4-HC), which is active metabolite of CPA, enhanced TNF-alpha-induced DNA fragmentation. In addition, 4-HC was shown to elevate TNF-alpha-induced caspase-3 activation. Caspase-8 activation was identified by the treatment of TNF-alpha, whereas 4-HC was no effect. In contrast, only when treated with 4-HC, caspase-9 activation and the increase in the intracellular expression of Bax were detected. These results suggest that CPA may sensitize endothelial cells to TNF-alpha-induced apoptosis through a mitochondria-dependent pathway and clinically may contribute to the limitation of inflammatory process.     

Protein phosphatase 2A regulates apoptosis in intestinal epithelial cells

Polyamine depletion prevents apoptosis by increasing serine/threonine phosphorylation leading to either inactivation or activation of pro- and anti-apoptotic proteins, respectively. Despite evidence that protein kinases are regulators of apoptosis, a specific role for protein phosphatases in regulating cell survival has not been established. In this study, we show that polyamine depletion inhibits serine/threonine phosphatase 2A (PP2A). Inhibition of PP2A in cells depleted of polyamines correlated well with increased phosphorylation of Bad at Ser112. Bad Ser112 phosphorylation in response to tumor necrosis factor (TNF)-alpha treatment decreased with time in cells grown in control as well as those grown in the presence of alpha-difluoromethylornithine plus putrescine. However, a sustained increase in the levels of Bad Ser112 phosphorylation was maintained in response to TNF-alpha treatment in cells grown in the presence of alpha-difluoromethylornithine. Inhibition of PP2A by okadaic acid and fostriecin or PP2A small interfering RNA transfection significantly decreased TNF-alpha-induced apoptosis in control and polyamine-depleted cells. Inhibition of PP2A by okadaic acid: 1) increased Bad and Bcl-2 phosphorylation at Ser112 and Ser70, respectively; 2) increased ERK activity; 3) prevented JNK activation; 4) prevented cytochrome c release, and activation of caspases-9 and -3 in response to TNF-alpha. Inhibition of MEK1 by U0126 prevented phosphorylation of Bad at Ser112. These results indicate that polyamines regulate PP2A activity, and inhibition of PP2A in response to polyamine depletion increases steady state levels of Bad and Bcl-2 proteins and their phosphorylation and thereby prevents cytochrome c release, caspase-9, and caspase-3 activation.     

TNF-alpha-induced impairment of mitochondrial integrity and apoptosis mediated by caspase-8 in adult ventricular myocytes

Tumor necrosis factor (TNF)-alpha has been shown to induce apoptosis in a variety of cell types including cardiac myocytes. Sphingosine/ceramide and nitric oxide have been associated with apoptosis induced by TNF-alpha; however, signaling mechanisms of TNF-alpha-induced apoptosis in cardiac myocytes are not well defined. This study examined whether alterations in mitochondrial integrity are involved in TNF-alpha-induced apoptosis in adult ventricular myocytes (ARVM) and determined the roles of caspase-8 (an upstream mediator of TNF-alpha receptor-associated signaling) in this process. After incubation for 24-48 h in serum-free culture medium, ARVM underwent spontaneous apoptosis, which remained stable and was not affected by Z-IETD-FMK, a selective caspase-8 inhibitor. Meanwhile, exposure to TNF-alpha resulted in an increase in apoptosis that was detectable at 6 h and became significant after 12 h, when compared to time-controls. After 24-h exposure, TNF-alpha increased caspase-8 activities, mitochondrial cytochrome C (Cyt C) release to the cytosol, accompanied by loss of mitochondrial transmembrane potential (delta psi(m)). Inhibition of caspase-8 activation in the presence of Z-IETD-FMK abolished the TNF-alpha-induced increases in mitochondrial Cyt C release, loss of delta psi(m) and apoptosis. Therefore, these results suggest that TNF-alpha-induced increase in apoptosis in ARVM results from caspase-8-dependent impairment of mitochondrial integrity.     

TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation

Crohn's disease (CD) patients have an abnormal increase in intestinal epithelial permeability. The defect in intestinal tight junction (TJ) barrier has been proposed as an important etiologic factor of CD. TNF-alpha increases intestinal TJ permeability. Because TNF-alpha levels are markedly increased in CD, TNF-alpha increase in intestinal TJ permeability could be a contributing factor of intestinal permeability defect in CD. Our purpose was to determine some of the intracellular mechanisms involved in TNF-alpha modulation of intestinal epithelial TJ permeability by using an in vitro intestinal epithelial system consisting of filter-grown Caco-2 monolayers. TNF-alpha produced a concentration- and time-dependent increase in Caco-2 TJ permeability. TNF-alpha-induced increase in Caco-2 TJ permeability correlated with Caco-2 NF-kappa B activation. Inhibition of TNF-alpha-induced NF-kappa B activation by selected NF-kappa B inhibitors, curcumin and triptolide, prevented the increase in Caco-2 TJ permeability, indicating that NF-kappa B activation was required for the TNF-alpha-induced increase in Caco-2 TJ permeability. This increase in Caco-2 TJ permeability was accompanied by down-regulation of zonula occludens (ZO)-1 proteins and alteration in junctional localization of ZO-1 proteins. TNF-alpha modulation of ZO-1 protein expression and junctional localization were also prevented by NF-kappa B inhibitors. TNF-alpha did not induce apoptosis in Caco-2 cells, suggesting that apoptosis was not the mechanism involved in TNF-alpha-induced increase in Caco-2 TJ permeability. These results demonstrate for the first time that TNF-alpha-induced increase in Caco-2 TJ permeability was mediated by NF-kappa B activation. The increase in permeability was associated with NF-kappa B-dependent downregulation of ZO-1 protein expression and alteration in junctional localization.     

Molecular mechanisms of TNF-alpha-induced ceramide formation in human glioma cells: P53-mediated oxidant stress-dependent and -independent pathways

The present study was designed to examine the roles of p53, reactive oxygen species (ROS), and ceramide, and to determine their mutual relationships during tumor necrosis factor (TNF)-alpha-induced apoptosis of human glioma cells. In cells possessing wild-type p53, TNF-alpha stimulated ceramide formation via the activation of both neutral and acid sphingomyelinases (SMases), accompanied by superoxide anion (O2-*) production, and induced mitochondrial depolarization and cytochrome c release, whereas p53-deficient cells were partially resistant to TNF-alpha and lacked O2-* generation and neutral SMase activation. Restoration of functional p53 sensitized glioma cells expressing mutant p53 to TNF-alpha by accumulation of O2-*. z-IETD-fmk (benzyloxycarbonyl-Ile-Glu-Thr-Asp fluoromethyl ketone), but not z-DEVD-fmk (benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone), blocked TNF-alpha-induced ceramide formation through both SMases as well as O2-* generation. Caspase-8 was processed by TNF-alpha regardless of p53 status of cells or the presence of antioxidants. Two separate signaling cascades, p53-mediated ROS-dependent and -independent pathways, both of which are initiated by caspase-8 activation, thus contribute to ceramide formation in TNF-alpha-induced apoptosis of human glioma cells.     

Mechanisms of the induction of apoptosis in human hepatoma cells by tumour necrosis factor-alpha.

Tumour necrosis factor alpha (TNF-alpha) at 20 ng/ml induced apoptosis in human hepatoma cells in vitro. The effect of TNF-alpha-induced apoptosis was exacerbated by the hypoxanthine-xanthine oxidase (HX/XO) system and cycloheximide (CHX), but alleviated by superoxide dismutase (SOD), suggesting that TNF-alpha-induced apoptosis may be due to oxidative stress, and independent of protein synthesis. TNF-alpha elevated free Ca(2+)concentration, triggered lipid peroxidation and decreased the expression of bcl-2 protein. The findings suggest that TNF-alpha-induced apoptosis may be involved in stimulating Ca(2+)-dependent endonuclease activity and increasing membrane lipid peroxidation. Bcl-2 may play a pivotal role in serving as a Ca(2+)regulator or antioxidant, preventing lipid peroxidation in the process.     

Pseudomonas aeruginosa delays Kupffer cell death via stabilization of the X-chromosome-linked inhibitor of apoptosis protein

Kupffer cells are important for bacterial clearance and cytokine production during infection. We have previously shown that severe infection with Pseudomonas aeruginosa ultimately results in loss of Kupffer cells and hepatic bacterial clearance. This was associated with prolonged hepatic inflammation. However, there is a period of time during which there is both preserved hepatic bacterial clearance and increased circulating TNF-alpha. We hypothesized that early during infection, Kupffer cells are protected against TNF-alpha-induced cell death via activation of survival pathways. KC13-2 cells (a clonal Kupffer cell line) were treated with P. aeruginosa (strain PA103), TNF-alpha, or both. At early time points, TNF-alpha induced caspase-mediated cell death, but PA103 did not. When we combined the two exposures, PA103 protected KC13-2 cells from TNF-alpha-induced cell death. PA103, in the setting of TNF exposure, stabilized the X-chromosome-linked inhibitor of apoptosis protein (XIAP). Stabilization of XIAP can occur via PI3K and Akt. We found that PA103 activated Akt and that pretreatment with the PI3K inhibitor, LY294002, prevented PA103-induced protection against TNF-alpha-induced cell death. The effects of LY294002 included decreased levels of XIAP and increased amounts of cleaved caspase-3. Overexpression of Akt mimicked the effects of PA103 by protecting cells from TNF-alpha-induced cell death and XIAP cleavage. Transfection with a stable, nondegradable XIAP mutant also protected cells against TNF-alpha-induced cell death. These studies demonstrate that P. aeruginosa delays TNF-alpha-induced Kupffer cell death via stabilization of XIAP.     

Rac1 mediates intestinal epithelial cell apoptosis via JNK

Apoptosis plays a key role in the maintenance of a constant cell number and a low incidence of cancer in the mucosa of the intestine. Although the small GTPase Rac1 has been established as an important regulator of migration of intestinal epithelial cells, whether Rac1 is also involved in apoptosis is unclear. The present study tested the hypothesis that Rac1 mediates TNF-alpha-induced apoptosis in IEC-6 cells. Rac1 is activated during TNF-alpha-induced apoptosis as judged by the level of GTP-Rac1, the level of microsomal membrane-associated Rac1, and lamellipodia formation. Although expression of constitutively active Rac1 does not increase apoptosis in the basal condition, inhibition of Rac1 either by NSC-23766 (Rac1 inhibitor) or expression of dominant negative Rac1 protects cells from TNF-alpha-induced apoptosis by inhibiting caspase-3, -8, and -9 activities. Inhibition of Rac1 before the administration of apoptotic stimuli significantly prevents TNF-alpha-induced activation of JNK1/2, the key proapoptotic regulator in IEC-6 cells. Inhibition of Rac1 does not modulate TNF-alpha-induced ERK1/2 and Akt activation. Inhibition of ERK1/2 and Akt activity by U-0126 and LY-294002, respectively, increased TNF-alpha-induced apoptosis. However, inhibition of Rac1 significantly decreased apoptosis in the presence of ERK1/2 and Akt inhibitors, similar to the effect observed with NSC-23766 alone in response to TNF-alpha. Thus, Rac1 inhibition protects cells independently of ERK1/2 and Akt activation during TNF-alpha-induced apoptosis. Although p38 MAPK is activated in response to TNF-alpha, inhibition of p38 MAPK did not decrease apoptosis. Rac1 inhibition did not alter p38 MAPK activity. Thus, these results indicate that Rac1 mediates apoptosis via JNK and plays a key role in proapoptotic pathways in intestinal epithelial cells.     

Contrasting effects of IG20 and its splice isoforms, MADD and DENN-SV, on tumor necrosis factor alpha-induced apoptosis and activation of caspase-8 and -3

We identified a novel cDNA (IG20) that is homologous to cDNAs encoding a protein differentially expressed in normal and neoplastic cells (DENN-SV) and human MADD (MAPK-activating death domain-containing protein). Furthermore, we show that the above variants most likely result from alternative splicing of a single gene. Functional analyses of these variants in permanently transfected HeLa cells revealed that IG20 and DENN-SV render them more susceptible or resistant to tumor necrosis factor alpha (TNF-alpha)-induced apoptosis, respectively. All variants tested could interact with TNF receptor 1 and activate ERK and nuclear factor kappaB. However, relative to control cells, only cells expressing IG20 showed enhanced TNF-alpha-induced activation of caspase-8 and -3, whereas cells expressing DENN-SV showed either reduced or no caspase activation. Transfection of these cells with a cDNA encoding CrmA maximally inhibited apoptosis in HeLa-IG20 cells. Our results show that IG20 can promote TNF-alpha-induced apoptosis and activation of caspase-8 and -3 and suggest that it may play a novel role in the regulation of the pleiotropic effects of TNF-alpha through alternative splicing.