WOX1 is essential for tumor necrosis factor-, UV light-, staurosporine-, and p53-mediated cell death, and its tyrosine 33-phosphorylated form binds and stabilizes serine 46-phosphorylated p53

WW domain-containing oxidoreductase WOX1, also named WWOX or FOR, undergoes Tyr33 phosphorylation at its first N-terminal WW domain and subsequent nuclear translocation in response to sex steroid hormones and stress stimuli. The activated WOX1 binds tumor suppressor p53, and both proteins may induce apoptosis synergistically. Functional suppression of WOX1 by antisense mRNA or a dominant negative abolishes p53-mediated apoptosis. Here, we determined that UV light, anisomycin, etoposide, and hypoxic stress rapidly induced phosphorylation of p53 at Ser46 and WOX1 at Tyr33 (phospho-WOX1) and their binding interactions in several tested cancer cells. Mapping by yeast two-hybrid analysis and co-immunoprecipitation showed that phospho-WOX1 physically interacted with Ser46-phosphorylated p53. Knockdown of WOX1 protein expression by small interfering RNA resulted in L929 fibroblast resistance to apoptosis by tumor necrosis factor, staurosporine, UV light, and ectopic p53, indicating an essential role of WOX1 in stress stimuli-induced apoptosis. Notably, UV light could not induce p53 protein expression in these WOX1 knockdown cells, although p53 mRNA levels were not reduced. Suppression of WOX1 by dominant negative WOX1 (to block Tyr33 phosphorylation) also abolished UV light-induced p53 protein expression. Time course analysis showed that the stability of ectopic wild type p53, tagged with DsRed, was decreased in WOX1 knockdown cells. Inhibition of MDM2 by nutlin-3 increased the binding of p53 and WOX1 and stability of p53. Together, our data show that WOX1 plays a critical role in conferring cellular sensitivity to apoptotic stress and that Tyr33 phosphorylation in WOX1 is essential for binding and stabilizing Ser46-phosphorylated p53.     

JNK1 physically interacts with WW domain-containing oxidoreductase (WOX1) and inhibits WOX1-mediated apoptosis

Transient activation of c-Jun N-terminal kinase (JNK) promotes cell survival, whereas persistent JNK activation induces apoptosis. Bovine testicular hyaluronidase PH-20 activates JNK1 and protects L929 fibroblasts from staurosporine-mediated cell death. PH-20 also induces the expression of a p53-interacting WW domain-containing oxidoreductase (WOX1, also known as WWOX or FOR) in these cells. WOX1 enhances the cytotoxic function of tumor necrosis factor and mediates apoptosis synergistically with p53. Thus, the activated JNK1 is likely to counteract WOX1 in mediating apoptosis. Here it is demonstrated that ectopic JNK1 inhibited WOX1-mediated apoptosis of L929 fibroblasts, monocytic U937 cells, and other cell types. Also, JNK1 blocked WOX1 prevention of cell cycle progression. By stimulating cells with anisomycin or UV light, JNK1 became activated, and WOX1 was phosphorylated at Tyr(33). The activated JNK1 physically interacted with the phosphorylated WOX1, as determined by co-immunoprecipitation. Alteration of Tyr(33) to Arg(33) in WOX1 abrogated its binding interaction with JNK1 and its activity in mediating cell death, indicating that Tyr(33) phosphorylation is needed to activate WOX1. A dominant negative WOX1 was developed and shown to block p53-mediated apoptosis and anisomycin-mediated WOX1 phosphorylation but could not inhibit JNK1 activation. This mutant protein bound p53 but could not interact with JNK1, as determined in yeast two-hybrid analysis. Taken together, phosphorylation of JNK1 and WOX1 is necessary for their physical interaction and functional antagonism.     

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.     

TIAF1 and p53 functionally interact in mediating apoptosis and silencing of TIAF1 abolishes nuclear translocation of serine 15-phosphorylated p53

TIAF1 is a TGF-beta 1-induced factor that protects L929 fibroblasts from TNF-mediated apoptosis. In contrast, overexpressed TIAF1 induces growth inhibition and apoptosis of monocytic U937 and various nonfibroblast cells. TIAF1-mediated apoptosis of U937 cells involves upregulation of p53, p21, and Smad2/4, but downregulation of ERK phosphorylation. To determine whether p53 and TIAF1 functionally interact in regulating cell death, ectopic TIAF1 and p53 were shown to induce apoptosis of U937 cells in both synergistic and antagonistic manners. At optimal levels both TIAF1 and p53 mediated apoptosis cooperatively. Also, both proteins suppressed adherence-independent growth of L929 cells. In contrast, initiation of apoptosis by overexpressed TIAF1 was blocked by low doses of p53, and vice versa. Furthermore, ectopic p53 blocked an ongoing apoptosis in U937 cells stably expressing TIAF1. Yeast two-hybrid analyses failed to demonstrate the binding of p53 with TIAF1, suggesting an unidentified protein that links the p53/TIFA1 interaction. Suppression of TIAF1 expression by siRNA could not inhibit Ser15 phosphorylation in p53 in response to UV and etoposide. However, nuclear translocation of these Ser15-phosphorylated p53 was significantly reduced in TIAF1-silenced cells. Taken together, TIAF1 and p53 functionally interact in regulating apoptosis, and TIAF1 is likely to participate in the nuclear translocation of activated p53.     

Transforming growth factor-β1 blocks the enhancement of tumor necrosis factor cytotoxicity by hyaluronidase Hyal-2 in L929 fibroblasts

Background  

Functional antagonism between transforming growth factor beta (TGF-β) and hyaluronidase has been demonstrated. For example, testicular hyaluronidase PH-20 counteracts TGF-β1-mediated growth inhibition of epithelial cells. PH-20 sensitizes various cancer cells to tumor necrosis factor (TNF) cytotoxicity by upregulating proapoptotic p53 and WW domain-containing oxidoreductase (WOX1). TGF-β1 blocks PH-20-increased TNF cytotoxicity. In the present study, the functional antagonism between TGF-β1 and lysosomal hyaluronidases Hyal-1 and Hyal-2 was examined.     

Modulation of Sonic hedgehog signaling and WW domain containing oxidoreductase WOX1 expression enhances radiosensitivity of human glioblastoma cells

WW domain containing oxidoreductase, designated WWOX, FOR or WOX1, is a known pro-apoptotic factor when ectopically expressed in various types of cancer cells, including glioblastoma multiforme (GBM). The activation of sonic hedgehog (Shh) signaling, especially paracrine Shh secretion in response to radiation, is associated with impairing the effective irradiation of cancer cells. Here, we examined the role of Shh signaling and WOX1 overexpression in the radiosensitivity of human GBM cells. Our results showed that ionizing irradiation (IR) increased the cytoplasmic Shh and nuclear Gli-1 content in GBM U373MG and U87MG cells. GBM cells with exogenous Shh treatment exhibited similar results. Pretreatment with Shh peptides protected U373MG and U87MG cells against IR in a dose-dependent manner. Cyclopamine, a Hedgehog/Smoothened (SMO) inhibitor, reversed the protective effect of Shh in U87MG cells. Cyclopamine increased Shh plus IR-induced H2AX, a marker of DNA double-strand breaks, in these cells. To verify the role of Shh signaling in the radiosensitivity of GBM cells, we tested the effect of the Gli family zinc finger 1 (Gli-1) inhibitor zerumbone and found that it could sensitize GBM cells to IR. We next examined the role of WOX1 in radiosensitivity. Overexpression of WOX1 enhanced the radiosensitivity of U87MG (possessing wild type p53 or WTp53) but not U373MG (harboring mutant p53 or MTp53) cells. Pretreatment with Shh peptides protected both WOX1-overexpressed U373MG and U87MG cells against IR and increased the cytoplasmic Shh and nuclear Gli-1 content. Zerumbone enhanced the radiosensitivity of WOX1-overexpressed U373MG and U87MG cells. In conclusion, overexpression of WOX1 preferentially sensitized human GBM cells possessing wild type p53 to radiation therapy. Blocking of Shh signaling may enhance radiosensitivity independently of the expression of p53 and WOX1. The crosstalk between Shh signaling and WOX1 expression in human glioblastoma warrants further investigation.     

Inhibition of p38 mitogen-activated protein kinase reduces TNF-induced activation of NF-kappaB, elicits caspase activity, and enhances cytotoxicity

Among other cellular responses, tumor necrosis factor (TNF) induces different forms of cell death and the activation of the p38 mitogen-activated protein kinase (MAPK). The influence of p38 MAPK activation on TNF-induced apoptosis or necrosis is controversially discussed. Here, we demonstrate that pharmacological inhibition of p38 MAPK enhances TNF-induced cell death in murine fibroblast cell lines L929 and NIH3T3. Furthermore, overexpression of dominant-negative versions of p38 MAPK or its upstream kinase MKK6 led to increased cell death in L929 cells. While overexpression of the p38 isoforms alpha and beta did not protect L929 cells from TNF-induced toxicity, overexpression of constitutively active MKK6 decreased TNF-induced cell death. Although the used inhibitors of p38 MAPK decreased the phosphorylation of the survival kinase PKB/Akt, this effect could be ruled out as cause of the observed sensitization to TNF-induced cytotoxicity. Finally, we demonstrate that the nuclear factor kappaB (NF-kappaB)-dependent gene expression, shown as an example for the anti-apoptotic gene cellular inhibitor of apoptosis (c-IAP2), was reduced by p38 MAPK inhibition. In consequence, we found that inhibition of p38 MAPK led to the activation of the executioner caspase-3.     

TIAF1 self-aggregation in peritumor capsule formation, spontaneous activation of SMAD-responsive promoter in p53-deficient environment, and cell death

Self-aggregation of transforming growth factor β (TGF-β)1-induced antiapoptotic factor (TIAF1) is known in the nondemented human hippocampus, and the aggregating process may lead to generation of amyloid β (Aβ) for causing neurodegeneration. Here, we determined that overexpressed TIAF1 exhibits as aggregates together with Smad4 and Aβ in the cancer stroma and peritumor capsules of solid tumors. Also, TIAF1/Aβ aggregates are shown on the interface between brain neural cells and the metastatic cancer cell mass. TIAF1 is upregulated in developing tumors, but may disappear in established metastatic cancer cells. Growing neuroblastoma cells on the extracellular matrices from other cancer cell types induced production of aggregated TIAF1 and Aβ. In vitro induction of TIAF1 self-association upregulated the expression of tumor suppressors Smad4 and WW domain-containing oxidoreductase (WOX1 or WWOX), and WOX1 in turn increased the TIAF1 expression. TIAF1/Smad4 interaction further enhanced Aβ formation. TIAF1 is known to suppress SMAD-regulated promoter activation. Intriguingly, without p53, self-aggregating TIAF1 spontaneously activated the SMAD-regulated promoter. TIAF1 was essential for p53-, WOX1- and dominant-negative JNK1-induced cell death. TIAF1, p53 and WOX1 acted synergistically in suppressing anchorage-independent growth, blocking cell migration and causing apoptosis. Together, TIAF1 shows an aggregation-dependent control of tumor progression and metastasis, and regulation of cell death.     

Structure/Function analysis of p55 tumor necrosis factor receptor and fas-associated death domain. Effect on necrosis in L929sA cells

Tumor necrosis factor (TNF) induces a typical apoptotic cell death program in various cell lines by interacting with the p55 tumor necrosis factor receptor (TNF-R55). In contrast, triggering of the fibrosarcoma cell line L929sA gives rise to characteristic cellular changes resulting in necrosis. The intracellular domain of TNF-R55 can be subdivided into two parts: a membrane-proximal domain (amino acids 202-325) and a C-terminal death domain (DD) (amino acids 326-413), which has been shown to be necessary and sufficient for apoptosis. Structure/function analysis of TNF-R55-mediated necrosis in L929sA cells demonstrated that initiation of necrotic cell death, as defined by swelling of the cells, rapid membrane permeabilization, absence of nuclear condensation, absence of DNA hypoploidy, and generation of mitochondrial reactive oxygen intermediates, is also confined to the DD. The striking synergistic effect of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone on TNF-induced necrosis was also observed with receptors solely containing the DD. TNF-R55-mediated necrosis is not affected by the dominant negative deletion mutant of the Fas-associated death domain (FADD-(80-205)) that lacks the N-terminal death effector domain. Moreover, overexpression of FADD-(80-205) in L929sA is cytotoxic and insensitive to CrmA, while the cytotoxicity due to overexpression of the deletion mutant FADD-(1-111) lacking the DD is prevented by CrmA. These results demonstrate that the death domain of FADD can elicit an active necrotic cell death pathway.     

Ras signaling in tumor necrosis factor-induced apoptosis.

Tumor necrosis factor (TNF) exerts cytotoxicity on many types of tumor cells but not on normal cells. The molecular events leading to cell death triggered by TNF are still poorly understood. Our previous studies have shown that enforced expression of an activated H-ras oncogene converted non-tumorigenic, TNF-resistant C3H 10T1/2 fibroblasts into tumorigenic cells that also became very sensitive to TNF-induced apoptosis. This finding suggested that Ras activation may play a role in TNF-induced apoptosis. In this study we investigated whether Ras activation is an obligatory step in TNF-induced apoptosis. Introduction of two different molecular antagonists of Ras, the rap1A tumor suppressor gene or the dominant-negative rasN17 gene, into H-ras-transformed 10TEJ cells inhibited TNF-induced apoptosis. Similar results were obtained with L929 cells, a fibroblast cell line sensitive to TNF-induced apoptosis, which does not have a ras mutation. While Ras is constitutively activated in TNF-sensitive 10TEJ cells, TNF treatment increased Ras-bound GTP in TNF-sensitive L929 cells but not in TNF-resistant 10T1/2 cells. Moreover, RasN17 expression blocked TNF-induced Ras-GTP formation in L929 cells. These results demonstrate that Ras activation is required for TNF-induced apoptosis in mouse fibroblasts.     

Human papillomavirus type 16 E6-enhanced susceptibility of L929 cells to tumor necrosis factor alpha correlates with increased accumulation of reactive oxygen species.

Human papillomavirus type 16 (HPV-16) E6 has been shown to prevent or enhance apoptosis depending on the stimulus and cell type. Here we present evidence that HPV-16 E6 sensitized murine fibrosarcoma L929 cells to tumor necrosis factor alpha (TNF)-induced cytolysis. The E6-enhanced cytolysis correlated with a precedent increase in reactive oxygen species (ROS) level and antioxidant treatment could completely block the E6-dependent sensitization. These findings represent the first demonstration of a link between a viral oncogene-sensitized cytolysis and ROS. Previous studies have shown conflicting results regarding whether TNF-induced cytolysis of L929 cells is through necrosis or apoptosis. Here we report that, although L929 cells underwent DNA fragmentation after exposure to TNF, they retained the morphology of intact nuclei while gaining permeability to propidium iodide, features characteristic of necrosis rather than apoptosis. We confirmed that the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone markedly increased the susceptibility of L929 cells to TNF, and further demonstrated that E6 enhanced this susceptibility, which again correlated with increased ROS accumulation. We showed that the expression of E6 in L929 cells did not alter the stability of p53, and the cells retained a p53 response to actinomycin D. Furthermore, two E6 mutants defective for p53 degradation in other systems exhibited differential effects on TNF sensitization. These results suggest that the enhancement of TNF-induced L929 cytolysis by E6 is independent of p53 degradation. We also found that TNF-induced activation of NF-kappaB did not account for the enhanced TNF susceptibility by E6.     

IkappaBalpha is essential for maintaining basal c-Jun N-terminal kinase (JNK) activation and regulating JNK-mediated resistance to tumor necrosis factor cytotoxicity in L929 cells.

Early activation of c-Jun N-terminal kinase (JNK) is believed to block apoptosis in response to death signals such as tumor necrosis factor (TNF). Brief exposure of murine L929 fibroblasts to anisomycin for 1 hr to activate JNK resulted in resistance to TNF killing. TNF rapidly induced cytoplasmic shrinkage in control cells, but not in the anisomycin-pretreated L929 cells. However, the induced TNF resistance was suppressed in the L929 cells which were engineered to stably inhibit IkappaBalpha protein expression by antisense mRNA ( approximately 80% reduction in protein expression). No constitutive NF-kappaB nuclear translocation and increased TNF resistance were found in these IkappaBalpha antisense cells. Notably, these cells had a significantly reduced basal level of JNK activation (50-70%), compared to vector control cells. Furthermore, brief exposure of L929 cells to wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), resulted in resistance to TNF killing, probably due to preconsumption of caspases by wortmannin. Nonetheless, wortmannin-induced TNF resistance was suppressed in the IkappaBalpha antisense cells. Thus, these observations indicate that IkappaBalpha is essential for maintaining the basal level of JNK activation and regulating the JNK-induced TNF resistance.     

The lysosome-associated apoptosis-inducing protein containing the pleckstrin homology (PH) and FYVE domains (LAPF), representative of a novel family of PH and FYVE domain-containing proteins, induces caspase-independent apoptosis via the lysosomal-mitochondrial pathway

Lysosomes have recently been identified as important apoptotic signal integrators in response to various stimuli. Here we report the functional characterization of LAPF, a novel lysosome-associated apoptosis-inducing protein containing PH and FYVE domains. LAPF is a representative of a new protein family, the Phafins (protein containing both PH and FYVE domains), which consists of 14 unidentified proteins from various species. Overexpression of LAPF in L929 cells induces apoptosis and also increases cell sensitivity to TNFalpha-induced apoptosis, concomitant with its translocation to lysosomes. Two mutants of LAPF, either lacking the PH or FYVE domain, failed to induce cell death and translocate to lysosomes, suggesting that both domains are required for its apoptosis-inducing activity and relocation. We demonstrate that LAPF may induce apoptosis via the following steps: LAPF translocation to lysosomes, lysosomal membrane permeabilization (LMP), release of cathepsin (cath) D and L, mitochondrial membrane permeabilization (MMP), release of apoptosis-inducing factor (AIF), and caspase-independent apoptosis. The cath D-specific inhibitor attenuates LAPF-induced apoptosis, indicating a pivotal role of lysosomes in LAPF-initiated apoptosis. We also demonstrate that the lysosomal pathway was employed in the typical apoptotic model in which high dose TNFalpha was used to stimulate L929 cells. Silencing of LAPF expression by small RNA interference protected L929 cells from hTNFalpha-induced apoptosis by impairing hTNFalpha-triggered LMP and MMP. Therefore, LAPF may launch caspase-independent apoptosis through the lysosomal-mitochondrial pathway.     

Zfra is an inhibitor of Bcl-2 expression and cytochrome c release from the mitochondria

Zfra is a small size 31-amino-acid C2H2 zinc finger-like protein, which is known to interact with c-Jun N-terminal kinase 1 (JNK1), WW domain-containing oxidoreductase (WWOX, FOR or WOX1), TNF receptor-associated death domain protein (TRADD) and nuclear factor kappaB (NF-kappaB) during stress response. Here, we show that Zfra became phosphorylated at Ser8 (as determined by specific antibody) and translocated to the mitochondria in response to inducers of mitochondrial permeability transition (MPT) (e.g. staurosporine and betulinic acid). Overexpressed Zfra induced cell death. This event is associated, in part, with increased dissipation of mitochondrial membrane potential (MMP) and increased chromosomal DNA fragmentation. Intriguingly, Zfra significantly downregulated Bcl-2 and yet blocked cytochrome c release from the mitochondria. Overexpression of an S8G-Zfra mutant (Ser8 to Gly8 alteration) could not induce cell death, probably due to its failure of translocating to the mitochondria and causing MMP dissipation. Over-expressed proapoptotic WOX1 induced cytochrome c release from the mitochondria. Zfra bound and blocked the effect of WOX1. Taken together, Ser8 is essential for overexpressed Zfra to exert cell death via the mitochondrial pathway. Zfra downregulates Bcl-2 and induces MMP dissipation but causes no cytochrome c release, indicating a novel death pathway from the mitochondria.     

Apoptin induces apoptosis by changing the equilibrium between the stability of TAp73 and ΔNp73 isoforms through ubiquitin ligase PIR2

Apoptin, a protein derived from the chicken anaemia virus, induces cell death in various cancer cells but shows little or no cytotoxicity in normal cells. The mechanism of apoptin-induced cell death is currently unknown but it appears to induce apoptosis independent of p53 status. Here we show that p73, a p53 family member, is important in apoptin-induced apoptosis. In p53 deficient and/or mutated cells, apoptin induced the expression of TAp73 leading to the induction of apoptosis. Knockdown of p73 using siRNA resulted in a significant reduction in apoptin-induced cytotoxicity. The p53 and p73 pro-apoptotic target PUMA plays an important role in apoptin-induced cell death as knockdown of PUMA significantly reduced cell sensitivity to apoptin. Importantly, apoptin expression resulted in a marked increase in TAp73 protein stability. Investigation into the mechanisms of TAp73 stability showed that apoptin induced the expression of the ring finger domain ubiquitin ligase PIR2 which is involved in the degradation of the anti-apoptotic ?Np73 isoform. Collectively, our results suggest a novel mechanism of apoptin-induced apoptosis through increased TAp73 stability and induction of PIR2 resulting in the degradation of ?Np73 and activation of pro-apoptotic targets such as PUMA causing cancer cell death.     

ERKs/p53 signal transduction pathway is involved in doxorubicin-induced apoptosis in H9c2 cells and cardiomyocytes

The cardiotoxic effects of doxorubicin, a potent chemotherapeutic agent, have been linked to DNA damage, oxidative mitochondrial damage, and nuclear translocation of p53, but the exact molecular mechanisms causing p53 transactivation and doxorubicin-induced cardiomyopathy are not clear. The present study was carried out to determine whether extracellular signal-regulated kinases (ERKs), which are known to be activated by DNA damaging agents, are responsible for doxorubicin-induced p53 activation and oxidative mitochondrial damage in H9c2 cells. Cell death was measured by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling, annexin V-fluorescein isothiocyanate, activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase (PARP). We found that doxorubicin produced cell death in H9c2 cells in a time-dependent manner, beginning at 6 h, and these changes are associated decreased expression of Bcl-2, increases in Bax and p53 upregulated modulator of apoptosis-alpha expression, and collapse of mitochondria membrane potential. The changes in cell death and Bcl-2 family proteins, however, were preceded by earlier activation and nuclear translocation of ERKs, followed by increased phosphorylation at Ser15 and nuclear translocation of the phosphorylated p53. The functional importance of ERK1/2 and p53 in doxorubicin-induced toxicity was further demonstrated by the specific ERK inhibitor U-0126 and p53 inhibitor pifithrin (PFT)-alpha, which abrogated the changes in Bcl-2 family proteins and cell death produced by doxorubicin. U-0126 blocked the phosphorylation and nuclear translocation of both ERK1/2 and p53, whereas PFT-alpha blocked only the changes in p53. Doxorubicin and ERK inhibitors produced similar changes in ERK1/2-p53, PARP, and caspase-3 in neonatal rat cultured cardiomyocytes. Thus we conclude that ERK1/2 are functionally linked to p53 and that the ERK1/2-p53 cascade is the upstream signaling pathway responsible for doxorubicin-induced cardiac cell apoptosis. ERKs and p53 may be considered as novel therapeutic targets for the treatment of doxorubicin-induced cardiotoxicity.     

TGF-beta-induced matrix proteins inhibit p42/44 MAPK and JNK activation and suppress TNF-mediated IkappaBalpha degradation and NF-kappaB nuclear translocation in L929 fibroblasts

The role of transforming growth factor beta1 (TGF-beta1)-induced extracellular matrix proteins in the modulation of cellular response to the cytotoxic effect of tumor necrosis factor (TNF) or Fas ligand was investigated. Murine L929 fibroblasts were prestimulated with or without TGF-beta1 for 1-24 h and the resulting extracellular protein matrices were prepared. Unstimulated control L929 cells were then cultured on these matrices. Compared to control matrix-stimulated L929 cells, the TGF-beta1 matrix-stimulated cells resisted TNF killing in the presence of actinomycin D (ActD), but became more susceptible to killing by anti-Fas antibodies/ActD. The induced TNF resistance is independent of the NF-kappaB antiapoptotic effect. For example, exposure of TGF-beta1 matrix-stimulated L929 cells to TNF failed to result in IkappaBalpha degradation and NF-kappaB nuclear translocation or activation. Also, control matrix stimulated the activation of p42/44 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) in L929 cells, whereas TGF-beta1 matrix suppressed the activation. Nonetheless, in response to TNF, JNK activation was restored in the TGF-beta1 matrix-stimulated cells. By metabolic labeling, ammonium sulfate precipitation and N-terminal amino acid microsequencing, TGF-beta1 was shown to induce a novel matrix protein of 46 kDa (p46) from L929 cells. Adsorption of p46 by peptide antibodies against its N-terminus removed the TGF-beta1 matrix protein-mediated protection against TNF/ActD cytotoxicity and its enhancement of anti-Fas/ActD killing, indicating that p46 is responsible for these effects. Immunostaining of L929 cells revealed that the antibodies were bound to a membrane protein of 100 kDa (p100). Thus, the matrix p46 is likely derived from the released membrane p100.