Evidence that genetic deletion of the TNF receptor p60 or p80 inhibits Fas mediated apoptosis in macrophages

Almost 19 members of the tumor necrosis factor (TNF) superfamily have been identified that interact with 29 different receptors. Whether these receptors communicate with each other is not understood. Recently, we have shown that receptor activator of NF-kappaB ligand signaling is modulated by genetic deletion of the TNF receptor. In the current report, we investigated the possibility of a cross-talk between Fas and TNF-alpha signaling pathway in macrophage cell lines derived from wild-type (WT) mice and from mice with genetic deletion of the type 1 TNF receptor (p60(-/-)), the type 2 TNF receptor (p80(-/-)), or both receptors (p60(-/-)p80(-/-)). We found that the macrophages expressing TNF receptors were highly sensitive to apoptosis induced by anti-Fas. The genetic deletion of TNF receptors, however, made the cells resistance to anti-Fas-induced apoptosis. Anti-Fas induced activation of caspase-3 and PARP cleavage in WT cells but not in TNF receptor-deleted cells. This difference was found to be independent of the expression of Fas, Fas-associated protein with death domain (FADD) or TNF receptor-associated death domain (TRADD). We found that anti-Fas induced recruitment of TNFR1 into Fas-complex. We also found that TRADD, which mediates TNF signaling, was constitutively bound to Fas receptor in TNF receptor-deleted cells but not in wild-type cells. Transient transfection of TNFR1 in TNFR1-deleted cells sensitized them to anti-Fas-induced apoptosis. Overall our results demonstrate that Fas signaling is modulated by the TNF receptors and thus provide the evidence of cross-talk between the receptors of two cytokines.     

Curcumin (diferuloylmethane) inhibits constitutive NF-kappaB activation, induces G1/S arrest, suppresses proliferation, and induces apoptosis in mantle cell lymphoma

Human mantle cell lymphoma (MCL), an aggressive B cell non-Hodgkin's lymphoma, is characterized by the overexpression of cyclin D1 which plays an essential role in the survival and proliferation of MCL. Because of MCL's resistance to current chemotherapy, novel approaches are needed. Since MCL cells are known to overexpress NF-kappaB regulated gene products (including cyclin D1), we used curcumin, a pharmacologically safe agent, to target NF-kappaB in a variety of MCL cell lines. All four MCL cell lines examined had overexpression of cyclin D1, constitutive active NF-kappaB and IkappaB kinase and phosphorylated forms of IkappaBalpha and p65. This correlated with expression of TNF, IkappaBalpha, Bcl-2, Bcl-xl, COX-2 and IL-6, all regulated by NF-kappaB. On treatment of cells with curcumin, however, downregulated constitutive active NF-kappaB and inhibited the consitutively active IkappaBalpha kinase (IKK), and phosphorylation of IkappaBalpha and p65. Curcumin also inhibited constitutive activation of Akt, needed for IKK activation. Consequently, the expression of all NF-kappaB-regulated gene products, were downregulated by the polyphenol leading to the suppression of proliferation, cell cycle arrest at the G1/S phase of the cell cycle and induction of apoptosis as indicated by caspase activation, PARP cleavage, and annexin V staining. That NF-kappaB activation is directly linked to the proliferation of cells, is also indicated by the observation that peptide derived from the IKK/NEMO-binding domain and p65 suppressed the constitutive active NF-kappaB complex and inhibited the proliferation of MCL cells. Constitutive NF-kappaB activation was found to be due to TNF, as anti-TNF antibodies inhibited both NF-kappaB activation and proliferation of cells. Overall, our results indicate that curcumin inhibits the constitutive NF-kappaB and IKK leading to suppression of expression of NF-kappaB-regulated gene products that results in the suppression of proliferation, cell cycle arrest, and induction of apoptosis in MCL.     

Reversal of chemoresistance and enhancement of apoptosis by statins through down-regulation of the NF-kappaB pathway

We recently found that simvastatin can modulate the nuclear factor-kappaB (NF-kappaB) activation pathway, but whether other statins have similar effects to those of simvastatin is unknown. Therefore, we evaluated the effect six different statins on TNF-induced NF-kappaB activation in human myeloid leukemia cells. We then determined whether the combination of statins and standard chemotherapeutic agents could overcome chemoresistance and augment apoptosis. Of the six statins evaluated, only the natural statins (simvastatin, mevastatin, lovastatin, and pravastatin), not the synthetic statins (fluvastatin and atorvastatin), inhibited TNF-induced NF-kappaB activation. Simvastatin suppressed the NF-kappaB activation and potentiated the apoptosis induced by doxorubicin, paclitaxel, and 5-fluorouracil. These results suggest that different statins behave differently from one another and that they may be useful in overcoming chemoresistance.     

Mechanisms of crosstalk between TNF-induced NF-kappaB and JNK activation in hepatocytes

Hepatocyte cell death is a universal feature of inflammatory liver diseases. The observation that mice deficient in the activation of nuclear factor-kappaB (NF-kappaB) are not viable because of excessive hepatocyte apoptosis induced by tumor necrosis factor (TNF) made it crystal-clear that NF-kappaB plays a central role in protecting hepatocytes against TNF-induced cell death. Also during TNF-mediated liver injury, NF-kappaB was shown to have an essential anti-apoptotic effect, underscoring the therapeutic importance of understanding its underlying molecular mechanisms. For a long time, the ability of NF-kappaB to induce the expression of a variety of anti-apoptotic proteins was thought to be solely responsible for its cytoprotective effects. However, during the past few years it has become clear that NF-kappaB-mediated inhibition of cell death also involves attenuating TNF-induced activation of c-Jun activating kinase (JNK). Whereas transient activation of JNK upon TNF treatment is associated with cellular survival, prolonged JNK activation contributes to cell death. Several studies have shown that NF-kappaB activation inhibits the sustained phase of TNF-induced JNK activation and thus protects cells against TNF cytotoxicity. In this review, we will discuss the various mechanisms by which NF-kappaB activation blunts TNF-induced JNK activation, including the induction of JNK inhibitory proteins and controlling the levels of reactive oxygen species (ROS). Moreover, because the cytoprotective effects of NF-kappaB activation are particularly important in liver physiology, we will put each of these JNK-inhibitory mechanisms into a 'hepatic perspective' by discussing their role in various mouse models of TNF-mediated liver injury.     

ABINs: A20 binding inhibitors of NF-κB and apoptosis signaling

ABINs have been described as three different proteins (ABIN-1, ABIN-2, ABIN-3) that bind the ubiquitin-editing nuclear factor-κB (NF-κB) inhibitor protein A20 and which show limited sequence homology. Overexpression of ABINs inhibits NF-κB activation by tumor necrosis factor (TNF) and several other stimuli. Similar to A20, ABIN-1 and ABIN-3 expression is NF-κB dependent, implicating a potential role for the A20/ABIN complex in the negative feedback regulation of NF-κB activation. Adenoviral gene transfer of ABIN-1 has been shown to reduce NF-κB activation in mouse liver and lungs. However, ABIN-1 as well as ABIN-2 deficient mice exhibit only slightly increased or normal NF-κB activation, respectively, possibly reflecting redundant NF-κB inhibitory activities of multiple ABINs. Other functions of ABINs might be non-redundant. For example, ABIN-1 shares with A20 the ability to inhibit TNF-induced apoptosis and as a result ABIN-1 deficient mice die during embryogenesis due to TNF-dependent fetal liver apoptosis. On the other hand, ABIN-2 is required for optimal TPL-2 dependent extracellularly regulated kinase activation in macrophages treated with TNF or Toll-like receptor ligands. ABINs have recently been shown to contain an ubiquitin-binding domain that is essential for their NF-κB inhibitory and anti-apoptotic activities. In this context, ABINs were proposed to function as adaptors between ubiquitinated proteins and other regulatory proteins. Alternatively, ABINs might disrupt signaling complexes by competing with other ubiquitin-binding proteins for the binding to specific ubiquitinated targets. Altogether, these findings implicate an important role for ABINs in the regulation of immunity and tissue homeostasis.     

Mitigation of tumor necrosis factor alpha cytotoxicity by aurintricarboxylic acid in human peripheral B lymphocytes

The aims of this study were to ascertain whether aurintricarboxylic acid (ATA), an endonuclease inhibitor, known to interfere, with the actions of cytokines such as interferons, is able to antagonize the toxic effects produced by tumor necrosis factor alpha (TNF-alpha) in human healthy peripheral B lymphocytes and try to elucidate the molecular machinery through which this possible antagonism takes place. Results evidenced that the balance of survival signals of human B lymphocytes in the presence of TNF-alpha was altered by the interaction of TNF-alpha with a salicylate compound, ATA. Apoptosis effected by TNF-alpha alone was suppressed in the presence of ATA, and this effect appeared essentially characterized by: (i) phosphorylation of phosphatidylinositol-3 kinase (PI-3K), influencing in turn protein kinase B/Akt (Akt) and Bad phosphorylation; (ii) nuclear translocation of the nuclear factor kappa B (NF-kappaB) and (iii) nuclear translocation of protein kinase C zed (PKCzeta). Reversal of TNF-alpha/ATA effects occurred in the presence of the PI-3K specific inhibitors wortmannin or LY294002 in the culture medium and was coincident with inhibition of the translocation of PKCzeta in the nucleus, while NF-kappaB was less affected. These results indicate, therefore, that PI-3K-mediated activation and nuclear transfer of PKCzeta might be essential steps of ATA antagonism against TNF-alpha, suggesting that possible ATA pharmacological applications might be taken into account for staving off systemic or local toxic effects produced by TNF-alpha.     

BAFF et al.: novel members of the TNF ligand and receptor families as therapeutic targets

Tumour necrosis factor (TNF) family ligands and their corresponding receptors play important roles in the immune system, where they regulate lymphoid organ development and influence immune cell proliferation, differentiation, activation and death. Several TNF family members, including TNF-α and CD154, have already provided valuable therapeutic targets for the treatment of immune-mediated diseases. Over the past three years, a new subfamily of TNF-related ligands/receptors, which also plays a role in immunity, has been discovered by sequence-homology database searches. This subfamily, which appears to be particularly important in B cell immunity, includes two ligands termed B cell activating factor of the TNF family (BAFF) and a proliferation-inducing ligand (APRIL) and at least three receptors: B cell maturation protein (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), which bind both ligands, and BAFF receptor (BAFF-R), which binds only BAFF. Besides promoting the survival of B cells and regulating their proliferation and differentiation in collaboration with signals from the B cell antigen receptor, BAFF and APRIL may modulate T cell activation and APRIL may also act as an autocrine growth factor for certain tumour cells. There is evidence that overproduction of BAFF in mice results in a B cell-mediated autoimmune syndrome resembling systemic lupus erythematosus (SLE), and that elevated levels of this cytokine are associated with SLE and rheumatoid arthritis in humans. The discovery of this novel ligand/receptor network has provided novel insights into the mechanisms of immunoregulation. Most importantly, it offers the opportunity for developing novel treatment strategies for autoimmune diseases, immunodeficiencies, lymphoma and cancer. The prospect of exploiting this new information for therapeutic purposes has generated a flurry of recent patent applications that are discussed here.     

Inhibition of lipopolysaccharide-induced expression of inducible nitric oxide synthase and tumor necrosis factor-alpha by 2'-hydroxychalcone derivatives in RAW 264.7 cells

In cultures of the murine macrophage cell line RAW 264.7, effects of four 2'-hydroxychalcone derivatives, 2'-hydroxy-4'-methoxychalcone (compound 1), 2',4-dihydroxy-4'-methoxychalcone (compound 2), 2',4-dihydroxy-6'-methoxychalcone (compound 3) and 2'-hydroxy-4,4'-dimethoxychalcone (compound 4), on lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and tumor necrosis factor (TNF)-alpha were examined. Compounds 1, 2 and 3 at 3-30microM inhibited the production with almost the same potency. Compound 4 showed no inhibitory activity. Compounds 1, 2 and 3 at 3-30microM inhibited the LPS-induced expression of inducible nitric oxide synthase (iNOS) and TNF-alpha mRNA. To clarify the mechanism involved, effects of compounds 1, 2 and 3 on the activation of nuclear factor (NF)-kappaB and activator protein-1 (AP-1) were examined. Both the LPS-induced activation of NF-kappaB and AP-1 were blocked by compounds 1, 2 and 3 at 3-30microM. Moreover, the three compounds at such concentrations inhibited the LPS-induced IkappaB degradation and the phosphorylation of c-jun N-terminal kinase (JNK) and c-jun. These findings suggest that the inhibition of the LPS-induced production of NO and TNF-alpha by the 2'-hydroxychalcone derivatives is due to the inhibition of NF-kappaB and AP-1 activations.     

Novel TNF receptor-like molecules and their biological uses

Tumour necrosis factor (TNF) ligands and receptors play a central role in the regulation of immune responses. Aberrant expression or activation of specific TNF ligand or receptor molecules may lead to autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosis. Amgen describes new members of the TNF receptor family, the MK61 series of molecules. Preclinical data demonstrating a role for the MK61 molecules in regulating B cell response to antigens are presented. The clinical utility of either agonists or antagonists of the members of the MK61 family of molecules remains to be proven.     

Regulation of glutathione S-transferase P1-1 gene expression by NF-kappaB in tumor necrosis factor alpha-treated K562 leukemia cells

Glutathione S-transferases (GSTs) play an important role in the protection of cells against xenobiotics and lipid hydroperoxides generated by oxidative stress. In human, the GSTP1-1 expression is commonly increased in many tumors and involved in the development of antineoplastic drug resistance. Reactive oxygen species are released at inflammation sites and oxidative stress conditions enhance the expression of genes encoding antioxidant enzymes such as GSTs. Here we investigated the regulation of the GSTP1-1 gene expression in the K562 cell line by nuclear factor kappaB (NF-kappaB) and the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha). By studying GSTP1-1 mRNA expression and NF-kappaB/GSTP1-1 promoter interactions, we showed the implication of NF-kappaB in the GSTP1-1 gene expression and we described a new specific TNFalpha-inducible NF-kappaB binding site upstream of the minimal promoter. Moreover, TNFalpha treatment as well as cotransfection of NF-kappaB signaling pathway intermediates induced an activation of the GSTP1-1 gene promoter in K562 cells. Site-directed mutagenesis of the NF-kappaB site strongly inhibited TNFalpha- and NF-kappaBp65-induced promoter activation. Altogether, we showed that a sequence located at -323/-314 within the GSTP1-1 promoter bound NF-kappaB p50/65 and p65/p65 dimers and that this kappaB site was involved in the regulation of the gene by TNFalpha.     

Pentoxifylline induces apoptosis in vitro in cutaneous T cell lymphoma (HuT-78) and enhances FasL mediated killing by upregulating Fas expression

Constitutive nuclear factor-kappaB (NF-kappaB) is known to play an important role in the survival of HuT-78 cells, a cutaneous T cell lymphoma (CTCL) cell line. Here, we have demonstrated that pentoxifylline (PTX), a phosphodiesterase inhibitor, can trigger a series of events leading to apoptosis in HuT-78 cells without affecting NF-kappaB. Apoptosis was ascertained by sub-G1 peak analysis and TUNEL assay. Apoptosis induced by PTX in HuT-78 cells involved mitochondrial hyperpolarization, cytochrome c release, caspase-3 activation and PARP cleavage. Further, it was found that PTX treatment downregulated Bcl-xl and c-FLIP expression without affecting constitutive NF-kappaB but upregulated activator protein-1 (AP-1). Low concentration of PTX upregulated Fas and TRAIL expression in HuT-78 cells. In addition, PTX can act as a scavenger of reactive oxygen intermediate and it could enhance FasL mediated killing in HuT-78 cells. Our results taken together indicated that PTX may be a potential agent for killing CTCL cells.     

Evidence that activation of nuclear factor-kappaB is essential for the cytotoxic effects of doxorubicin and its analogues

Several reports within the last 5 years have suggested that nuclear factor (NF)-kappaB activation suppresses apoptosis through expression of anti-apoptotic genes. In the present report, we provide evidence from four independent lines that NF-kappaB activation is required for the cytotoxic effects of doxorubicin. We used doxorubicin and its structural analogues WP631 and WP744, to demonstrate that anthracyclines activate NF-kappaB, and this activation is essential for apoptosis in myeloid (KBM-5) and lymphoid (Jurkat) cells. All three anthracyclines had cytotoxic effects against KBM-5 cells; analogue WP744, was most potent, with an IC(50) of 0.5 microM, and doxorubicin was least active, with an IC(50) of 2 microM. We observed maximum NF-kappaB activation at 1 microM with WP744 and at 50 microM with doxorubicin and WP631, and this activation correlated with the IkappaBalpha degradation. Because the anthracycline analogue (WP744), most active as a cytotoxic agent, was also most active in inducing NF-kappaB activation and the latter preceded the cytotoxic effects, suggests that NF-kappaB activation may mediate cytotoxicity. Second, receptor-interacting protein-deficient cells, which did not respond to doxorubicin-induced NF-kappaB activation, were also protected from the cytotoxic effects of all the three anthracyclines. Third, suppression of NF-kappaB activation by pyrrolidine dithiocarbamate, also suppressed the cytotoxic effects of anthracyclines. Fourth, suppression of NF-kappaB activation by NEMO-binding domain peptide, also suppressed the cytotoxic effects of the drug. Overall our results clearly demonstrate that NF-kappaB activation and IkappaBalpha degradation are early events activated by doxorubicin and its analogues and that they play a critical pro-apoptotic role.     

NF-kappaB and inflammation in genetic disease

By responding to pro-inflammatory cytokines, such as IL-1beta and TNF-alpha, and controlling itself the expression of numerous mediators of inflammation, NF-kappaB plays a pivotal role in controlling the proper sequence of events characterizing the inflammation process. Although excessive NF-kappaB activation is often associated with inflammatory signs in many different tissues, impaired NF-kappaB activation can also generate inflammation. This is the case in humans suffering from the genetic disease incontinentia pigmenti that exhibit severe skin inflammation. Identifying the molecular basis of this pathology, mutations affecting the gene coding for NEMO, has allowed production of mouse models for investigating the disease. Their characterization supports the view that a very tight positive and negative regulation of the NF-kappaB signaling pathway is required in vivo to ensure not only a fine-tuned response to injury or infection but also to maintain tissue homeostasis.