Cytochrome c oxidase deficiency accelerates mitochondrial apoptosis by activating ceramide synthase 6.

Although numerous pathogenic changes within the mitochondrial respiratory chain (RC) have been associated with an elevated occurrence of apoptosis within the affected tissues, the mechanistic insight into how mitochondrial dysfunction initiates apoptotic cell death is still unknown. In this study, we show that the specific alteration of the cytochrome c oxidase (COX), representing a common defect found in mitochondrial diseases, facilitates mitochondrial apoptosis in response to oxidative stress. Our data identified an increased ceramide synthase 6 (CerS6) activity as an important pro-apoptotic response to COX dysfunction induced either by chemical or genetic approaches. The elevated CerS6 activity resulted in accumulation of the pro-apoptotic C16 : 0 ceramide, which facilitates the mitochondrial apoptosis in response to oxidative stress. Accordingly, inhibition of CerS6 or its specific knockdown diminished the increased susceptibility of COX-deficient cells to oxidative stress. Our results provide new insights into how mitochondrial RC dysfunction mechanistically interferes with the apoptotic machinery. On the basis of its pivotal role in regulating cell death upon COX dysfunction, CerS6 might potentially represent a novel target for therapeutic intervention in mitochondrial diseases caused by COX dysfunction.

The power of bioenergy-related standards to protect biodiversity.

The sustainable production of bioenergy is vital to avoiding negative impacts on environmental goods such as climate, soil, water, and especially biodiversity. We propose three key issues that should be addressed in any biodiversity risk-mitigation strategy: conservation of areas of significant biodiversity value; mitigation of negative effects related to indirect land-use change; and promotion of agricultural practices with few negative impacts on biodiversity. Focusing on biodiversity concerns, we compared principles and criteria set to address biodiversity and other environmental and social issues in seven standards (defined here as commodity-based standards or roundtables, or relevant European legislation): five voluntary initiatives related to bioenergy feedstocks, the Renewable Transport Fuel Obligation (United Kingdom), and the European Renewable Energy Source Directive. Conservation of areas of significant biodiversity value was fairly well covered by these standards. Nevertheless, mitigation of negative impacts related to indirect land-use change was underrepresented. Although the EU directive, with its bonus system for the use of degraded land and a subquota system for noncrop biofuels, offered the most robust standards to mitigate potential negative effects, all of the standards fell short in promoting agricultural practices with low negative impacts on biodiversity. We strongly recommend that each standard be benchmarked against related standards, as we have done here, and that efforts should be made to strengthen the elements that are weak or missing. This would be a significant step toward achieving a bioenergy industry that safeguards Earth's living heritage.

TNF-alpha-converting enzyme (TACE/ADAM17)-dependent loss of CD30 induced by proteasome inhibition through reactive oxygen species.

Combinations with proteasome inhibitors are currently being investigated to improve the therapy of hematological malignancies. We previously found that proteasome inhibition by bortezomib failed to sensitize anti-CD30 antibody (Ab)-based lymphoma cell killing. In this study, we demonstrate in L540 Hodgkin's lymphoma cells that proteasome inhibition not only communicates apoptosis but also more rapidly causes a loss of CD30 antigen from cell membrane and a simultaneous release of soluble CD30, a targeting competitor. This shedding was catalyzed by the tumor necrosis factor (TNF)-alpha-converting enzyme (TACE, ADAM17) and blocked by the ADAM17-selective inhibitor, Ro32-7315. In parallel with CD30 shedding, bortezomib caused the generation of reactive oxygen species (ROS). As apoptosis and shedding were inhibited by the radical scavenger, N-acetyl-L-cysteine, ROS might have a pivotal function in both effects. In contrast, the pan-caspase inhibitor, zVAD-fmk, blocked bortezomib-induced apoptosis but not CD30 shedding, and Ro32-7315 blocked shedding but allowed apoptosis. This suggests independent terminal signaling pathways that are conflicting in Ab-based immunotherapy. Consequently, shedding inhibition substantially improved the synergistic antitumor efficacy of the human anti-CD30 Ab, MDX-060, and bortezomib. As proteasome inhibition also stimulated loss of TNF receptors, interleukin-6 receptor and syndecan-1 in different leukemia and lymphoma cell lines, we concluded that proteasome inhibition might impede targeted therapy against antigens susceptible to shedding.

Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain.

The magnesium-dependent, plasma membrane-associated neutral sphingomyelinase (N-SMase) catalyzes hydrolysis of membrane sphingomyelin to form ceramide, a lipid signaling molecule implied in intracellular signaling. We report here the biochemical purification to apparent homogeneity of N-SMase from bovine brain. Proteins from Nonidet P-40 extracts of brain membranes were subjected to four purification steps yielding a N-SMase preparation that exhibited a specific enzymatic activity 23,330-fold increased over the brain homogenate. When analyzed by two-dimensional gel electrophoresis, the purified enzyme presented as two major protein species of 46 and 97 kDa, respectively. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of tryptic peptides revealed at least partial identity of these two proteins. Amino acid sequencing of tryptic peptides showed no apparent homologies of bovine N-SMase to any known protein. Peptide-specific antibodies recognized a single 97-kDa protein in Western blot analysis of cell lysates. The purified enzyme displayed a K(m) of 40 microM for sphingomyelin with an optimal activity at pH 7-8. Bovine brain N-SMase was strictly dependent on Mg(2+), whereas Zn(2+) and Ca(2+) proved inhibitory. The highly purified bovine N-SMase was effectively blocked by glutathione and scyphostatin. Scyphostatin proved to be a potent inhibitor of N-SMase with 95% inhibition observed at 20 microM scyphostatin. The results of this study define a N-SMase that fulfills the biochemical and functional criteria characteristic of the tumor necrosis factor-responsive membrane-bound N-SMase.

Impaired neutral sphingomyelinase activation and cutaneous barrier repair in FAN-deficient mice.

The WD-40 repeat protein FAN binds to a distinct domain of the p55 receptor for tumor necrosis factor (TNF) and signals the activation of neutral sphingomyelinase (N-SMase). To analyze the physiological role of FAN in vivo, we generated FAN-deficient mice by targeted gene disruption. Mice lacking a functional FAN protein do not show any overt phenotypic abnormalities; in particular, the architecture and cellular composition of lymphoid organs appeared to be unaltered. An essential role of FAN in the TNF-induced activation of N-SMase was demonstrated using thymocytes from FAN knockout mice. Activation of extracellular signal-regulated kinases in response to TNF treatment, however, was not impaired by the absence of the FAN protein. FAN-deficient mice show delayed kinetics of recovery after cutaneous barrier disruption suggesting a physiological role of FAN in epidermal barrier repair. Although FAN exhibits striking structural homologies with the CHS/Beige proteins, FAN-deficient mice did not reproduce the phenotype of beige mice.

Inhibition of receptor internalization by monodansylcadaverine selectively blocks p55 tumor necrosis factor receptor death domain signaling.

The 55-kDa receptor for tumor necrosis factor (TR55) triggers multiple signaling cascades initiated by adapter proteins like TRADD and FAN. By use of the primary amine monodansylcadaverine (MDC), we addressed the functional role of tumor necrosis factor (TNF) receptor internalization for intracellular signal distribution. We show that MDC does not prevent the interaction of the p55 TNF receptor (TR55) with FAN and TRADD. Furthermore, the activation of plasmamembrane-associated neutral sphingomyelinase activation as well as the stimulation of proline-directed protein kinases were not affected in MDC-treated cells. In contrast, activation of signaling enzymes that are linked to the "death domain" of TR55, like acid sphingomyelinase and c-Jun-N-terminal protein kinase as well as TNF signaling of apoptosis in U937 and L929 cells, are blocked in the presence of MDC. The results of our study suggest a role of TR55 internalization for the activation of select TR55 death domain signaling pathways including those leading to apoptosis.

Requirement of FADD for tumor necrosis factor-induced activation of acid sphingomyelinase.

The generation of mice strains deficient for select members of the signaling complex of the 55-kDa tumor necrosis factor receptor (TNF-R55) has allowed the assignment of specific cellular responses to distinct TNF-R55-associated proteins. In particular, the TNF-R55-associated protein FADD seems to be responsible for recruitment and subsequent activation of caspase 8. In this report we demonstrate the requirement of FADD for TNF-induced activation of endosomal acid sphingomyelinase (A-SMase). In primary embryonic fibroblasts from FADD-deficient mice the activation of A-SMase by TNF-R55 ligation was almost completely impaired. This effect is specific in that other TNF responses like activation of NF-kappaB or neutral (N-)SMase remained unaffected. In addition, interleukin-1-induced activation of A-SMase in FADD-deficient cells was unaltered. In FADD-/- embryonic fibroblasts reconstituted by transfection with a FADD cDNA expression construct, the TNF responsiveness of A-SMase was restored. The results of this study suggest that FADD, in addition to its role in triggering a proapoptotic caspase cascade, is required for TNF-induced activation of A-SMase.

Immunoaugmenting effect of FK 506 on experimental allergic encephalomyelitis in Lewis rats.

The effect of the immunosuppressive drug FK 506 on encephalomyelitis (EAE) in Lewis rats was studied. Treatment that began during EAE induction delayed EAE onset, but when the disease started it was chronic/progressive and of unusual severity and duration, leading to death in many animals. Treatment started after onset of EAE shortened the disease. Forty seven days after immunization, extensive demyelination and inflammation were observed in the spinal cords of rats treated with FK 506 from the day of EAE induction. Rats treated after EAE onset had only minimal pathological abnormalities.

Induction of stress-activated protein kinases/c-Jun N-terminal kinases by the p55 tumour necrosis factor receptor does not require sphingomyelinases.

Ceramide has been implicated in the activation of stress-activated protein kinases/c-Jun N-terminal kinases (SAPK/JNK). Binding of tumour necrosis factor (TNF) to its 55 kDa receptor (TR55) leads to the generation of ceramide through activation of either acid or neutral sphingomyelinase (A/N-SMase) as well as to potent activation of SAPK/JNK. We have examined a putative role of both N- and A-SMase in the TR55-dependent activation of SAPK/JNK. The analysis of TR55 deletion mutants expressed in 70Z/3 pre-B cells revealed that activation of SAPK/JNK occurs independently of N-SMase. Although both SAPK/JNK and A-SMase are activated by the death domain of TR55, pharmacological prevention of the TR55-dependent activation of A-SMase, or proteolytic degradation of A-SMase in 70Z/3 cells, did not impair SAPK/JNK activation, indicating that SAPK/JNK are not secondary to A-SMase. In addition, proteolytic degradation of A-SMase also did not affect SAPK/JNK activation by ultraviolet (UV-C) irradiation, arguing against a general role of A-SMase in stress-mediated responses. Furthermore, fibroblasts from Niemann-Pick A patients deficient in A-SMase did not show altered activation of SAPK/JNK in response to either TNF or UV-C. These results suggest that TR55 can activate SAPK/JNK without direct participation of sphingomyelinases or ceramide.

TNF receptor death domain-associated proteins TRADD and FADD signal activation of acid sphingomyelinase.

Sphingomyelinase (SMase) activation and ceramide generation have emerged as an important signaling pathway transducing diverse biological effects of cytokine receptors like p55 tumor necrosis factor (TNF) receptor or Fas. Here we describe the TNF-dependent activation of acid SMase (A-SMase) through the p55 TNF receptor-associated proteins TRADD and FADD. Overexpression of TRADD and FADD in 293 cells did not change basal activity of A-SMase but enhanced TNF-induced stimulation of A-SMase. Other TNF R55-associated proteins like TRAF2 and RIP, which were reported to mediate TNF R55-mediated activation of nuclear factor kappaB, did not affect activation of A-SMase. Caspase inhibitors markedly reduced A-SMase activity, suggesting the involvement of an ICE-like protease in TRADD/FADD-mediated activation of A-SMase. Overexpression of caspase-8/a (FLICE/MACH) or caspase-10/b (FLICE2) did not change A-SMase activity, suggesting that TRADD/FADD-mediated activation of A-SMase involves a yet to be defined caspase-like protease distinct from caspase-8/a or -10/b.

Activation of acid sphingomyelinase by interleukin-1 (IL-1) requires the IL-1 receptor accessory protein.

The cytokine interleukin-1 (IL-1) plays an important role in inflammation and regulation of immune responses, but the mechanisms of its signal transduction and cell activation processes are incompletely understood. Ceramide generated by sphingomyelinases (SMases) is known to function as an important second messenger molecule in the signaling pathway of IL-1 and tumor necrosis factor. To investigate the activation of SMases by IL-1, we used an IL-1 receptor type I (IL-1RI)-positive EL4 thymoma cell line, which is defective in IL-1R accessory protein (IL-1RAcP) expression. In this cell line (EL4D6/76), tumor necrosis factor induced ligand/receptor internalization, NFkappaB nuclear translocation, IL-2 production, and the activation of neutral (N)-SMase and acid (A)-SMase. In contrast, stimulation with IL-1 resulted only in the activation of N-SMase whereas ligand/receptor internalization, NFkappaB translocation, IL-2 production, and activation of A-SMase were not detected. Transfection of this functionally defective EL4D6/76 with IL-1RAcP cDNA restored these functions. These data suggest that A-SMase activity is strongly linked with the internalization of IL-1RI mediated by IL-1RAcP and that A-SMase and N-SMase are activated by different pathways.

FAN, a novel WD-repeat protein, couples the p55 TNF-receptor to neutral sphingomyelinase.

The initiation of intracellular signaling events through the 55 kDa tumor necrosis factor-receptor (TNF-R55) appears to depend on protein intermediates that interact with specific cytoplasmic domains of TNF-R55. By combined use of the yeast interaction trap system and a peptide scanning library, the novel WD-repeat protein FAN has been identified, which specifically binds to a cytoplasmic nine amino acid binding motif of TNF-R55. This region has been previously recognized as a distinct functional domain that is both required and sufficient for the activation of neutral sphingomyelinase (N-SMase). Overexpression of full-length FAN enhanced N-SMase activity in TNF-treated cells, while truncated mutants of FAN produced dominant negative effects. The data suggest that FAN regulates ceramide production by N-SMase, which is a crucial step in TNF signaling.

Function of the p55 tumor necrosis factor receptor "death domain" mediated by phosphatidylcholine-specific phospholipase C.

Tumor necrosis factor (TNF) is a pleiotropic mediator of inflammation that has been implicated in the pathogenesis of devastating clinical syndromes including septic shock. We have investigated the role of a TNF-responsive phosphatidylcholine-specific phospholipase C (PC-PLC) for the cytotoxic and proinflammatory activity of TNF. We show here that the cytotoxicity signaled for by the so-called "death domain" of the p55 TNF receptor is associated with the activation of PC-PLC. The xanthogenate tricyclodecan-9-yl (D609), a specific and selective inhibitor of PC-PLC, blocked the cytotoxic action of TNF on L929 and Wehi164 cells. In vivo, D609 prevented both adhesion molecule expression in the pulmonary vasculature and the accompanying leukocyte infiltration in TNF-treated mice. More strikingly, D609 protects BALB/c mice from lethal shock induced either by TNF, lipopolysaccharide, or staphylococcal enterotoxin B. Together these findings imply PC-PLC as an important mediator of the pathogenic action of TNF, suggesting that PC-PLC may serve as a novel target for anti-inflammatory TNF antagonists.

A novel cytoplasmic domain of the p55 tumor necrosis factor receptor initiates the neutral sphingomyelinase pathway.

The human p55 tumor necrosis factor (TNF) receptor (TR55) initiates at least two independent signaling cascades. The acidic sphingomyelinase (A-SMase) pathway involves a phosphatidylcholine-specific phospholipase C, an endosomal A-SMase, and controls expression of multiple TNF-responsive genes through induction of transcription factors such as NF-kappaB. The neutral sphingomyelinase (N-SMase) pathway comprises a membrane-bound N-SMase, proline-directed protein kinases, as well as phospholipase A2 and appears critical for the inflammatory responses induced by TNF. While the domain of TR55 that induces A-SMase is probably identical to the death domain, the exact location and extent of a putative N-SMase activation domain are still unknown. Structure-function analysis of TR55 deletion mutants revealed a novel region of 11 amino acids at position 309-319 that is both necessary and sufficient for activation of N-SMase. The N-SMase activation domain is distinct from the death domain and incapable of induction of A-SMase, NF-kappaB, and cytotoxicity. Taken together, our results suggest that a functionally independent region of TR55 is responsible for selectively initiating the N-SMase pathway that couples to an important inflammatory signaling cascade.

Experimental allergic encephalomyelitis, beta-adrenergic receptors and interferon gamma-secreting cells in beta-adrenergic agonist-treated rats.

Treatment with the beta 2-adrenergic agonist terbutaline or the beta-adrenergic agonist isoproterenol suppresses experimental allergic encephalomyelitis, decreases the number of IFN gamma-producing splenic cells, and decreases the number of beta-adrenergic receptors on splenic lymphocytes in Lewis rats. The effects of terbutaline are greater when the drug is given from the day of immunization through the acute phase of the illness or from day 15 postimmunization until recovery, than when given for the first 12 days after immunization.

TNF-induced activation of NF-kappa B.

Tumor Necrosis Factor (TNF) is one of the most potent physiological inducers of the nuclear transcription factor NF-kappa B. In light of the pivotal role of NF-kappa B in the development of immune responses and activation of HIV replication, the identification of TNF signal transduction pathways involved in NF-kappa B activation is of particular interest. Data from our laboratory demonstrate that the TNF signal transduction pathway-mediating NF-kappa B activation involves two phospholipases, a phosphatidylcholine-specific phospholipase C (PC-PLC) and an endosomal acidic sphingomyelinase (aSMase). The aSMase activation by TNF is secondary to the generation of 1,2-diacylglycerol (DAG) produced by a TNF-responsive PC-PLC. SMase and its product ceramide induce degradation of the NF-kappa B inhibitor I kappa B as well as NF-kappa B activation. Besides endosomal acidic SMase, TNF also rapidly activates a plasmamembrane-associated neural SMase (nSMase), that, however is not involved in TNF-induced NF-kappa B activation. NSMase and aSMase are activated by different cytoplasmic domains of the 55 kDa TNF-receptor and are coupled to select pathways of TNF signaling. Ceramide generated by nSMase directs the activation of proline-directed serin/threonine protein kinases and phospholipase A2 and ceramide produced by aSMase triggers the activation of NF-kappa B. No apparent crosstalk was detected between nSMase and aSMase pathways, indicating that ceramide action depends on the topology of its production.

CD28 signals through acidic sphingomyelinase.

T cell receptor recognition of antigen can lead either to T lymphocyte differentiation and proliferation or to a state of unresponsiveness, which is dependent on whether appropriate costimulatory signals are provided to the mature T cell. We have investigated a novel intracellular signaling pathway provided by the costimulatory molecule CD28. CD28 engagement triggers the activation of an acidic sphingomyelinase (A-SMase), which results in the generation of ceramide, an important lipid messenger intermediate. A-SMase activation by CD28 occurred in resting as well as in activated primary T cells or leukemic Jurkat cells. In contrast, ligation of either CD3 or CD2 did not result in A-SMase activation. Overexpression of recombinant A-SMase in Jurkat T cells substituted for CD28 with regard to nuclear factor-kB activation. These data suggest that CD28 provides an important costimulatory signal by activation of an acidic sphingomyelinase pathway.

Tumor necrosis factor (TNF)-alpha activates c-raf-1 kinase via the p55 TNF receptor engaging neutral sphingomyelinase.

TNF-alpha mediates proliferation, functional activation and apoptotic death of cells depending upon its concentration and target cell type. The signaling pathways used by TNF-alpha to mount these responses are, at present, not completely understood. We report here that TNF-alpha promotes dose- and time-dependent phosphorylation and activation of the c-raf-1 kinase engaging the type I p55 TNF receptor (TNF-R). c-raf-kinase activation was duplicated by an agonistic monoclonal antibody directed against the p55 TNF-R. Moreover, ectopic expression of the human p55 TNF-R in murine pre-B 70Z/3 cells was sufficient to confer c-raf-1-kinase activation by human TNF-alpha. By inhibiting intracellular activation of acidic sphingomyelinase (SMase) and by using deleted forms of the type I TNF-R it was shown that the neutral, but not the acidic SMase, participated in TNF-alpha-mediated phosphorylation and activation of the c-raf kinase. TNF-alpha-induced transcriptional activation of a heterologous promoter construct harboring the AP-1 binding site was also mediated by the type I p55 TNF-R. In this case the initiation of transcription required the same cytoplasmic domain as that responsible for activation of c-raf-1 kinase and was liberated in the presence of a dominant negative mutant of c-raf-1.

Regulation of the human TNF promoter by the transcription factor Ets.

Tumor necrosis factor (TNF) affects the growth, differentiation, and function of a multitude of cell types and is viewed as a potent mediator of inflammation and cellular immune responses. In order to delineate functional domains that control TNF gene transcription, we have analyzed a 5' flanking region of the human TNF promoter spanning base pairs -115 to -98. This region contains a PEA3/Ets-1 binding motif 5' GAGGA 3' in direct juxtaposition to an AP-1/ATF-like palindromic sequence motif 5' TGAGCTCA 3'. Specific binding of Ets and Jun to their respective elements is demonstrated by competition analysis as well as by supershift assays. As shown by promoter deletion analysis, these two binding sites were essential for both basal promoter activity and responsiveness to the phorbol ester phorbol 12-myristate 13-acetate. Co-transfection of c-ets or c-jun expression plasmids along with TNF promoter-CAT reporter constructs revealed the participation of both transcription factors in the regulation of TNF gene transcription. Correspondingly, site-specific mutation of either Ets or Jun sites led to a complete loss of responsiveness to the respective transcription factor. These data suggest an essential role of Ets for the activation of TNF gene transcription.

Beta-adrenergic agonists suppress chronic/relapsing experimental allergic encephalomyelitis (CREAE) in Lewis rats.

Chronic/relapsing experimental allergic encephalomyelitis (CREAE) serves as an animal model for relapsing/remitting multiple sclerosis. Treatment with the beta-adrenergic agonist isoproterenol or the beta 2-adrenergic agonist terbutaline significantly suppressed both the first acute attack and the number of relapses in CREAE Lewis rats. The number of relapses was decreased even when treatment with beta-adrenergic agonist was started after the onset of the first acute attack of CREAE. beta-adrenergic receptor number was increased significantly on splenocytes from CREAE rats as compared to healthy controls or CFA-injected rats. Terbutaline treatment of CREAE rats lowered the splenocyte receptor number to normal values.