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TNF-Induced Activation of NF-κB
Immunobiol., vol. 193, pp. 193-203 (1995)
©
1995 by Gustav Fischer Verlag, Stuttgart
Institut fur Medizinische Mikrobiologie und Hygiene, Technische Universitat Munchen, Munchen, Germany
TNF-Induced Activation of NF-xB STEFAN SCHUTZE':', KATJA WIEGMANN':., THOMAS MACHLEIDT\ and MARTIN KRONKE':'
Abstract Tumor Necrosis Factor (TNF) is one of the most potent physiological inducers of the nuclear transcription factor NF-xB. In light of the pivotal role of NF-xB in the development of immune responses and activation of HIV replication, the identification of TNF signal transduction pathways involved in NF-xB activation is of particular interest. Data from our laboratory demonstrate that the TNF signal transduction pathwaymediating NF-xB 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-xB inhibitor IxB as well as NF-xB activation. Besides endosomal acidic SMase, TNF also rapidly activates a plasmamembrane-associated neutral SMase (nSMase), that, however is not involved in TNF-induced NF-xB 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-xB. No apparent crosstalk was detected between nSMase and aSMase pathways, indicating that ceramide action depends on the topology of its production.
Introduction The cytokine Tumor Necrosis Factor (TNF) is a central polypeptide mediator of inflammation and cellular immune responses (1-3). The ability of TNF to affect the growth, differentiation and function of virtually every cell type investigated resides mainly in its potent gene regulatory properties of TNF (see ref. 4 for review). Evidently, the induction of some TNFresponsive genes of immunological relevance is mediated, at least in part, ". Present address: Institut fur Immunologie, Universitat Kiel, Brunswiker Str. 4, 24105
Kiel, Germany
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S. SCHOTZE,
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WIEGMANN ,
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MAC HLEIDT,
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M . KRONKE
through activation of the nuclear transcription factor system NF-xB (5-7). NF-xB is a ubiquitous transcription factor, present in the cytosol of most cell types as an inactive heterodimer composed of 50- and 65-kDa subunits, that is bound to an inhibitor molecule, IxB (8-12). Induction of NF-xB requires the release of IxB, followed by translocation of the NF-xB heterodimer to the nucleus, where it directly binds to its cognate DNA sequence (13). NF-xB activation can be achieved by treating cells with very different kinds of agents (reviewed in ref. 14), including the cytokines TNF and IL-1 (6, 7). In light of the pivotal role of NF-xB in the development of immune responses and activation of HIV replication (15), the identification of TNF signal transduction pathways involved in NF-xB activation is of particular interest. The signaling pathways mediating cellular responses to TNF are beginning to be elucidated. TNF signaling is initiated by TNF with two distinct cell surface receptor molecules with apparent molecular sizes of 55-60 kDa (TNF-R55) and 75-80 kDa (TNF-R75) (for reviews see ref. 16-18). While information about TNF-R75 is scarce, TNF-R55 has been shown to trigger specific signal cascades, including phospholipase A2 (PLA2), phosphatidylcholin-specific phospholipase C (PC-PLC), protein kinase C, mitogenactivated protein kinase (MAP-kinase) and two distinct types of sphingomyelinase (SMase), a plasmamembrane-bound neutral (nSMase) and a intracellular endo-/ lysosomal acidic (aSMase) (19-23). Several reports recently implicated the lipid second messenger ceramide, in various important pathways of TNF action (ref. 24-26 for recent reviews). Ceramide can be generated by sphingomyelin (SM) breakdown by two types of TNF-responsive SMases at various subcellular sites (27, 28). Neutral SMase (nSMase) hydrolyzes SM of the outer leaflet of the plasmamembrane (29), while an acid SMase (aSMase) produces ceramide in the endosomai/lysosomal compartments (27, 30). The range of immediate cerami de targets has not yet been fully elucidated but may include a membrane-associated protein kinase, a cytosolic protein phosphatase 2A and the zeta-isoform of protein kinase C (31-33). Among the further downstream signaling events triggered by ceramide are the MAP-kinase cascade, the down-regulation of c-Myc. In addition, ceramide was found to display a potent NF-xB inducing capacity (20, 34-36). Here we review recent findings from our laboratory on the characterization of the intracellular pathway resulting in TNF-induced NF-xB activation.
Exogenous PLe and synthetic 1,2-diacylglycerols induce NF-xB activation Within seconds after TNF binding to its receptor, the lipid second messenger 1,2-DAG is produced from phosphatidylcholine, catalyzed by a PCPLC (19). To ask whether PC-PLC mediates TNF-receptor induced NFxB activation, exogenous PLC and its reaction products, 1,2-diacylgly-
TNF-induced activation of NF-xB . 195
cerols, were tested for their NF-xB inducing activity (20). Exposure of Jurkat T cells either to PLC from Bac. cereus or to short-chain synthetic DAG like 1,2-dioctanoylglycerol (diC s), 1,2-dihexanoylglycerol (diC 6 ), and 1-0Ieoyl-2-acetylglycerol (1,2-0AG) resulted in NF-xB activation as estimated by electrophoretic mobility shift assays (EMSA) using a 29 basepair oligonucleotide encompassing the two xB sites present in the HIV enhancer. The NF-xB-inducing capacity of exogenous PLC and DAG, however, was less pronounced when compared to that of TNF, and moreover, could be observed only after long-term incubation (3-4 h). This delayed NF-xB induction may be due to the possibility that neither PLC nor DAG easily cross the membrane barrier, which may result in an inappropriate access to intracellular targets. The typical short-term NF-xB induction, characteristic of TNF-induced NF-xB activation could be observed when cells were permeabilized by streptolysin-O or using cell-free systems. Addition of increasing amounts of exogenous PLC from Bac. cereus to cell-free extracts resulted in dose-dependent NF-xB activation, suggesting that this enzyme is sufficient to initiate NF-xB activation. Similarly, Bac. cereus-derived PLC-induced NF-xB in Jurkat T cells permeabilized with streptolysin-O. Heat-treated PLC displayed drastically reduced NF-xB-inducing activity, which corresponded well to its residual heat-resistant enzymatic activity assayed in vitro. It is important to note that only 1,2-diacylglycerol but not 1,3-DAG like 1-palmitoyl-3-stearoylglycerol (1,3-PSG) and 1,3-dioctanoylglycerol (l-3-diC s) triggered NF-xB activation. These findings indicate, that the NF-xB complex is not dissociated by mere pH 5 treatment but rather specifically responds to 1,2-DAG physiologically generated by a TNF-responsive PC-PLC. Of note, NF-xB induction by 12,13-PMA appeared significantly reduced when compared to the NF-xB-inducing capacity of 1,2-DAG. It is important to stress that NF-xB induction was achieved at pH 5.0; at pH 7.4, activation of NF-xB by PLC or by diacylglycerols was less pronounced.
Exogenous sphingomyelinases and ceramide induce NF-xB activation At first glance, the induction of NF-xB by DAG pointed to a possible involvement of PKC, because DAG is a well known activator of this enzyme. However, in a previous report, we have shown that TNF-induced NF-xB activation can occur independent of PKC (37). We therefore assumed, that 1,2-diacylglycerols activate enzyme(s) other than PKC, which mediate NF-xB activation. One candidate was acidic sphingomyelinase (SMase), which was previously shown to be activated by DAG in GH3 pituitary cells (38-40). To test the possible involvement of this C-type phospholipase, permeabilized Jurkat cells were treated with either commercially available sphingomyelinase preparations, or with the SMase reaction product, ce-
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ramide (20). Exogenous sphingomyelinase of human (acidic SMase from placenta) as well as of bacterial origin (neutral SMase from Bac. cereus) were both able to induce NF-xB activation. Of note, in a cell-free lysate system, neutral SMase exhibited the same Mg2+ dependency as described for the neutral SMase from human brain (41). Most pertinently, the SMase-specific sphingomyelin cleavage product ceramide, in a dose-dependent manner activated NF-xB when added to permeabilized cells (20). These experiments clearly underscore the potential of sphingomyelinases to activate NF-xB by generation of the second messenger-like molecule ceramide.
Dissection of neutral and acidic SMase in TNF-activation of NF-xB Recent studies indicate, that TNF activates two distinct sphingomyelinases, a membrane-bound neutral (nSMase) and endo-/lysosomal acidic (aSMase) enzyme (22). The development of assay systems that specifically distinguish between nSMase and aSMase allowed us to show that n- and aSMase are triggered independently from each other and coupled to distinct signaling pathways. The finding that synthetic 1,2-DAG was able to stimulate SMase activity as well as NF-xB in cell-free lysates at pH 5.0 (20) implicates an acidic SMase as a likely candidate for mediating NF-xB-activation in a DAG-dependent manner. The site of aSMase activation appears to be the endosome. Endosomal vesicles are rapidly acidified, which conditions for aSMase activation by DAG. Lysosomotropic agents like monensin, chloroquin and ammonium chloride raise the pH in endolysosomal compartments and were expected to inhibit the sequential activation of endosomal aSMase and NF-xB selectively, leaving the membrane-associated nSMase unaffected. Indeed, these agents blocked the activation of aSMase and NF-xB (22). In contrast, neither nSMase nor PC-PLC were inhibited, indicating that aSMase, but not nSMase, is involved in NF-xB activation. Furthermore, DAG-production appears not to suffice for NF-xB activation. The idea that aSMase is both required and sufficient for NF-xB activation is supported by the observation that recombinant aSMase overexpressed in Jurkat cells dose-dependently activated transcription from a chloramphenicol acetyltransferase (CAT) reporter plasmid containing four immunoglobulin/HIV -xB sites (22).
Sphingomyelinase activates proteolytic IxB-a degradation in a cell-free system A key event in the activation of NF-xB is the rapid release of the inhibitory subunit IxB-a (9, 13). Various inhibitors of serine-like proteases have been shown to block TNF-mediated NF-xB activation as well as the disappearance of IxB immunoreactivity in primary murine T lymphocytes and in
TNF-induced activation of NF-xB . 197
various human leukemic cell lines (35). The protease inhibitors did not block TNF-induced activation of either PC-PLC nor acidic SMase, indicating that the putative protease operates rather downstream of the TNF signal transduction cascade. IxB-degradation could be directly induced by addition of SMase or synthetic ceramide to a cell-free system, indicating a stringent coupling of aSMase to the NF-xB activation pathway. Similar to the TNF-induced NF-xB activation and TNF-induced IxB-degradation,
Nucleus Figure 1. TNF signaling pathways.
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also the SMase-induced IxB degradation was suppressed by the protease inhibitor dichloroisocoumarin (DCIC), indicating that the protease involved in NF-xB activation operates downstream of the aSMase at the level of IxB degradation, mediated by a chymotrypsin-like proteasome complex (42,43).
Independent activation of neutral SMase and acidic SMase/NF-xB by distinct domains of the 55 kDa TNF receptor We have recently reported that recombinant human TNF-R55 stably expressed in murine 70Z/3 cells, mediates TNF induction of several signaling enzymes (21 ). To achieve a pertinent dissection of nSMase and an aSMase pathway, structure-function analysis of human TNF-R55 was performed in 70Z/3 cells, demonstrating that nSMase and aSMase are triggered by distinct cytoplasmic domains. Wild-type human TNF-R55 and C-terminal deletions of TNF-R55 were stably expressed in 70Z/3 cells and assayed for their capability of mediating the activation of neutral SMase, acidic SMase and NF-xB (22). TNF-treatment of a 70Z/3 clone expressing wild-type TNF-R55 simultaneously elicited nSMase, aSMase and NF-xB activation, TNF-R55 mutants truncated C-terminally by 32, 43, 52, or 81 amino acids, respectively, were defective in signaling aSMase and NF-xB activation, indicating that the domain responsible for nSMase activation is N-terminal to amino acid residue 345. These findings illustrate that nSMase and aSMase are activated independently by distinct cytoplasmic domains of TNF-R55. TNF-R55 deletion mutants displayed a loss-of function phenotype also with regard to activation of PC-PLC, yet retained their capacity to signal stimulation of proline-directed protein kinases (PDPkinases) and phospholipase A2 (PLA 2) (22). PDP-kinases include the ceramide-activated protein kinase (CAPK) and mitogen-activated protein kinase (MAP-kinase) (44, 45). MAP-kinase in turn phosphorylates and thereby activates PLA2 (46, 47). These data can be reconciled with our results in a model (see Fig. 1) where nSMase couples to PDP kinase and PLAb yet has no access to the NF-xB activation pathway. In contrast, aSMase is secondary to PC-PLC and represents a crucial component of the NF-xB activation pathway, as recently proposed (20).
Discussion PKC or aSMase, either DAG-responsive enzyme has been implicated to mediate TNF-induced activation of NF-xB. However, NF-xB activation by TNF can occur clearly independent of PKC (37). A possible involvement of PC-PLC in NF-xB activation was suggested based on the observation that exposure of Jurkat cells to either B. cereus PC-PLC or short-chain synthetic diacylglycerol resulted in the activation of the nuclear transcrip-
TNF-induced activation of NF-xB . 199
tion factor NF-xB (20). Similarly, DOMINGUEZ et al. (48) recently demonstrated induction of NF-xB after microinjection of B. cereus PC-PLC in Xenopus laevis oocytes. Moreover, PC-PLC microinjected in oocytes transactivated xB-containing enhancer elements of the HIV long terminal repeat. Along these lines, overexpression of bacterial PC-PLC in transfected cells resulted in NF-xB activation (49). Strikingly, addition of a PIspecific PLC from B. cereus to U937 cells resulted in DAG production, yet failed to activate xB enhancer-directed CAT expression. These studies strongly suggest a specific role for PC-derived DAG distinct from that of PIPz-derived DAG . In addition to PC-PLC and synthetic diacylglycerols, exogenous SMase as well as its reaction product, ceramide, were found to induce NF-xB in permeabilized cells or in cell-free Iysates. Inducing of NFxB by exogenous SMase from S. aureus or a cell-permeable ceramide analog, Cg-ceramide, was recently demonstrated with intact HL60 cells by YANG et al. (34) and in SW480/~gal cells by JOHNS et al. (36). DBAIBO et al. (50) suggested a possible feed-back role for SMase in the TNF activation pathway of NF-xB, based on their findings that treatment of Jurkat cells with Crceramide was found to enhance TNF-induced NF-xB activation. Parallel to the induction of NF-xB binding activity in the nucleus, degradation of the inhibitor IxB-a could be directly induced by addition of PC-PLC, sphingomyelinase or synthetic ceramide to a cell-free system, indicating a stringent coupling of PC-PLC and SMase to the NF-xB activation pathway. The SMase-induced IxB degradation could be blocked by the inhibitor of serine-like proteases, dichloroisocoumarin (DCIC), indicating that the aSMase is linked to IxB degradation via the proteasome complex. A prerequisite for proteolytic IxB-degradation is phosphorylation of the IxB molecule (51). Recent work from]. MOSCATS group has suggested a role for PKC-zeta in NF-xB activation and IxB-degradation (48, 52). Interestingl y, this PKC isotype responds to synthetic ceramide (33) making this enzyme a likely candidate for transmitting the TNFresponsive SMase mediated signal to the NF-xB/ lxB complex. TNF activates two distinct SMases with neutral and acidic pH optima, respectively (22). Endolysosomal agents (monensin, chloroquin and ammonium chloride) inhibited the sequential TNF activation of acidic SMase and NF-xB, leaving neutral SMase and PC-PLC activities unaffected. These observations point to aSMase as the key enzyme in NF-xB activation. Furthermore, they demonstrate that nSMase is not involved in TNFinduced NF-xB activation . Overexpression of recombinant acidic SMase also resulted in NF-xB-driven CAT activity in Jurkat cells, confirming the results on the role of aSMase in the NF-xB activation pathway. Recent work from several groups has highlightened ceramide generated by sphingomyelin breakdown as an important intracellular signaling molecule (see 24, 25 for review). As demonstrated with TNF-R55 deletion mutants, the 55 kDa TNF-receptor independently triggers the activation of neutral and acidic SMase (22). Although both nSMase and aSMase generate the same lipid messenger ceramide, a previously unrecognized dichotomy
200 . S. SCHOTZE, K. WIEGMA NN, T. M ACHLEIDT, and M . KRONKE
of ceramide function becomes apparent, indicating that ceramide action is determined by the subcellular site of its production. While ceramide produced by a membrane-associated neutral SMase controls selected signaling pathways such as proline-directed protein kinases that include CAPK and MAP kinase, ceramide produced by the endosomal acidic SMase represents a prominent cofactor for the activation of NF-xB. Acknowledgement The work of our laboratory was supported b y the Deutsche Forschungsgemeinschaft (DFG) and the W ilhelm Sander Stiftung.
References 1. OLD, L. J. 1987. Tumor necrosis factor: polypeptide mediator network. Nature 326: 330-331. 2. LE, J., and J. VILCEK. 1987. Bio logy of disease. Tumor necrosis factor and Interleukin-l: cytokines with multiple overlapping biological activities. Lab. Invest. 56: 234-248. 3. BEUTLER, B., and A. C FR AM I. 1988. The history, properties, and biological effects of cachectin . Biochemistry 27: 7575- 7582. 4. KRONK I·:,M., S. SCHUTZF, P. SCHEURICH, and K. PFIZENMA IER. 1992. TNF signal transdu ction and TNF responsive genes. In: AGGARWAL, B. B., and J. VILCEK (eds.), Tumor necrosis factor, Structure, Function, and Mechanism of Action. Marcel Dekker, New York, pp. 189-21 6. 5. LowFNTHM, .J. W., D. W. BALLARD, E. BOEHNLEIN, and W . C. GREENE. 1989. Tumor necrosis factor alpha induces proteins that bind specifically to kappa B-like enha ncer elements and regulate interleukin-2 receptor alpha chai n gene expression in primary human T lymphocytes . Proc. Nat!. Acad. Sci. USA 86: 2331- 2335. 6. OSBORN, L. , S. K UNKEL, and G. J. NABEL. 1989. Tumor necrosis factor alpha and interl eukin 1 stimulate th e human immunodeficiency virus enhancer by activation of the nuclear factor kappa B. Proc. N at\. Acad . Sci. USA 86: 2336- 2340. 7. DUH, E. J., W. J. MAURY, T. M . FOLKS, A. S. F AUCI, and A. B. RABSON. 1989. Tumor necrosis factor alpha activates human immunodeficiency virus type 1 through induction of nuclear factor bindin g to the NF-kappa B sites in the long terminal repeat. Proc. Nat!. Acad . Sci. USA 86: 5974-5978. 8. BAEUFRLF, P. A., and D. BAI.TIMORE . 1988. Activation of DNA-binding activity in an apparently cytoplasmatic precursor of the NF-xB transcripti on factor. Cell 53: 211-217. 9. BAEU FRU:, P. A., and D. BALTIMOR F. 1988. l-xB: a specific inhibitor of the NF-xB transcription factor. Science 242: 540-546. 10. BAEUE RLI, P. A., and D . BALTIMORE. 1989. A 65-kDa subunit of active NF-xB is required for inhibitio n of N F-xB by I-xB. Genes Dev. 3: 1689-1698. 11. GOS H, S., and D . BALTI MOKE. 1990. Activation in vitro of N F-xB by phosphorylation of its inhibitor IxB. N ature 344: 678-682. 12. RUB EN, S. M., P. J. DIII.ON, R. SCHRECK, T. H ENKEL, C. H . CH EN, M. MAAHER, P. A. BAFU FRIF, and C. A. R OSEN. 1991. Isolation of a rei- related human eDNA that potentially encodes th e 65-kDa subunit of NF-xB. Science 251: 1490-1493. 13. LIOU, H.-C., and D. BALTIMORE. 1993. Regulation of the NF-xBlrel transcription factor and IxB inhibitor system . Curr. Opin. Cell. BioI. 5: 477-487.
TNF-induced activation of NF-xB . 201 14. BAEUERLE, P. A. 1991. The inducible transcription activator NF-kappa B: regulation by distinct protein subunits. Biochim. Biophys. Acta 1072: 63-80. 15. BAEUERLE, P. A., and T. HENKEL. 1994. Function and activation of NF-kappa B in the immune system. Annu. Rev. Immunol. 12: 141-179. 16. TARTAGLIA, L. A., and D. V. GOEDDE!.. 1992. Two TNF receptors. Immunol. Today 13: 151-153. 17. ROTHE]., G. GEHR, H. LOETSCHER, and W. LESSLAUER. 1992. Tumor necrosis factor receptors: structure and function. Immunol. Res. 11: 81-90. 18. FIERS, W. 1991. Tumor necrosis factor. Characterization at the molecular, cellular and in vivo level. FEBS Lett. 285: 199-212. 19. SCHUTZE, S., D. BERKCJVIC, O. TOMSING, C. UNGER, and M. KRONKI'. 1991. Tumor necrosis factor induces rapid production of 1,2-diacylglycerol by a phosphatidylcholine-specific phospholipase. C. J. Exp. Med. 174: 975-988. 20. SCHUTZI', S., K. POTITIOII, T. MACHLEIDT, D. BERKOVIC, K. WIEGMANN, and M. KRONKI'. 1992. TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced «acidic» sphingomyelin breakdown. Cell 71: 765-776. 21. WIEGMANN, K., S. SCIIlJT7E, E. KAMPEN, A. HIMMEER, T. MACHEEIDT, and M. KRONKE. 1992. Human 55-kDa receptor for tumor necrosis factor coupled to signal transduction cascades. J. BioI. Chern. 267: 17997-18001. 22. WIEGMANN, K., S. SCIIOT7E, T. MACHLEIDT, D. WrITE, and M. KRONKE. 1994. Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 78: 1005-1015. 23. HELLER, R. A., and M. KRONKF. 1994. Tumor necrosis factor receptor-mediated signaling pathways. J. Cell BioI. 126: 5-9. 24. KOEESNICK, R., and D. W. GOlDE. 1994. The sphingomyelin pathway in tumor necrosis factor and intcrleukin-l signaling. Cell 77: 325-328. 25. HANNUN, Y. A. 1994. The sphingomyelin cycle and the second messenger function of ceramide. J. BioI. Chern. 269: 3125-3128. 26. SCHUTZE, S., T. MACHLElllT, and M. KRONKI'.. 1994. The role of diacylglycerol and ceramide in tumor necrosis factor and interleukin-l signal transduction. J. Leukoc. Bio!. 56: 533-541. 27. SPENCE, M. W. 1993. Sphingomyelinases. Adv. Lipid Res. 26: 3-23. 28. CHATTERJEE, S. 1993. Neutral sphingomyelinase. Adv. Lipid Res. 26: 25-48. 29. DAS, D. V. M., H. W. COOK, and M. W. SPENCE. 1984. Evidence that neutral sphingomyelinase of cultured murine neuroblastoma cells is orientated externally on the plasma membrane. Biochim. Biophys. Acta 777: 339-342. 30. MERIEL, A. H., Jr., Y. A. HANNUN, and R. M. BELL. 1993. Sphingolipids and their metabolites in cell regulation. Adv. Lipid Res. 25: 1-24. 31. MATHIAS, S., K. A. DRESSLER, and R. N. KOLESNICK. 1991. Characterization of a ceramide-activated protein kinase: stimulation by tumor necrosis factor alpha. Proc. Natl. Acad. Sci. USA 88: 10009-10013. 32. DOIlROWSKY, R. T., C. KAMIBAYASHI, M. C. MUMIlY, and Y. A. HANNUN. 1993. Ceramide activates heterotrimeric protein phosphatase 2A. J. BioI. Chern. 268: 15523-15530. 33. L07ANO,]., E. BERRA, M. M. MUNICIO, M. T. DIAZMECO, I. DOMINGUEZ, L. SANZ, and J. MOSCAT. 1994. Protein kinase C zeta isoform is critical for kappa B-dependent promoter activation by sphingomyelinase. J. Bio!. Chern. 269: 19200-19202. 34. YANG, Z., M. COSTANZO, D. W. GOLDE, and R. N. KOLESNICK. 1993. Tumor necrosis factor activation of the sphingomyelin pathway signals nuclear factor kappa B translocation in intact HL-60 cells. J. Bio!. Chern. 268: 20520-20523. 35. MACHLEIDT, T., K. WIF(;MANN, T. HENKEL, S. SCHOTZE, P. BAEUERLE, and M. KRONKI'. 1994. Sphingomyelinase activates proteolytic I kappa B-alpha degradation in a cell-free system. J. Bio!. Chern. 269: 13760-13765.
202 . S. SCHUTZI', K. WIEGMANN, T. MACHLEIDT, and M. KRONKE 36. JOHNS, L. D., T. SARR, and G. E. RANGES. 1994. Inhibition of ceramide pathway does not affect ability of TNF-alpha to activate nuclear factor-kappa B. J. Immunol. 152: 5877-5882. 37. MEICHLE, A., S. SCHUTZE, G. HENSEL, D. BRUNSING, and M. KRONKE. 1990. Protein kinase C-independent activation of nuclear factor kappa B by tumor necrosis factor. J. BioI. Chern. 265: 8339-8343. 38. QUINTERN, L. E., G. WEITZ, H. NEHRKORN, J. M. TAGER, A. W. SCHRAM, and K. SANDHOFF. 1987. Acid sphingomyelinase from human urine: purification and characterization. Biochim. Biophys. Acta 922: 323-336. 39. KOLESNICK, R. N. 1987. 1,2-Diacylglycerols but not phorbol esters stimulate sphingomyelin hydrolysis in GH3 pituitary cells. J. BioI. Chern. 262: 16759-16762. 40. KOLESNICK, R. N., and S. CLEGG. 1988. 1,2-diacylglycerols, but not phorbol esters, activate a potential inhibitory pathway for protein kinase C in GH3 pituitary cells. J. BioI. Chern. 263: 6534-6537. 41. RAO, B. G., and M. W. SPENCE. 1976. Sphingomyelinase activity at pH 7.4 in human brain and a comparison to activity at pH 5.0. J. Lipid Res. 17: 506-515. 42. ORLOWSKI, M., and C. MICHAUD. 1989. Pituitary multicatalytic proteinase complex. Specificity of components and aspects of proteolytic activity. Biochemistry 28: 9270-9278. 43. PALOMBELLA, V. J., A. L. GOLDBERG, and T. MANIATIS. 1994. The Ubiquitinproteasome pathway is required for processing the NF-xB1 precursor protein and the activation of NF-xB. Cell 78: 773-785. 44. JOSEPH, C. K., H. S. BVUN, R. BITTMANN, and R. N. KOLESNICK. 1993. Substrate recognition by ceramide-activated protein kinase. Evidence that kinase activity is proline-directed. J. BioI. Chern. 268: 20002-20006. 45. RAINES, M. A., R. N. KOLESNICK, and D. W. GOLDE. 1993. Sphingomyelinase and ceramide activate mitogen-activated protein kinase in myeloid HL-60 cells. J. BioI. Chern. 268: 14572-14575. 46. LIN, L. L., M. W ARTMANN, A. J. LIN, J. L. KNOPf, A. SETH, and R. j. DAVIS. 1993. cPLA 2 is phosphorylated and activated by MAP kinase. Cell 72: 269-278. 47. NEMENOFF, R. A., S. WrNITz, N. X. QIAN, V. VAN PUTTEN, G. L. JOHNSON, and L. E. HEASLFY. 1993. Phosphorylation and activation of a high molecular weight form of phospholipase A2 by p42 microtubule-associated protein 2 kinase and protein kinase C. J. BioI. Chern. 268: 1960-1964. 48. DOMINGUEZ, 1., L. SANZ, F. ARENZANA-SEISDEDOS, M. T. DIAZ MECO, J. L. VIRELIZIER, and J. MOSCAT. 1993. Inhibition of protein kinase C zeta subspecies blocks the activation of an NF-kappa B-like activity in Xenopus laevis oocytes. Mol. Cell. BioI. 13: 1290-1295. 49. ARENzANA-Sr·:IsDESDOS, F., B. FERNANDEZ, I. DOMINGUEZ, J. M. JACQUE, D. THOMAS, M. T. DIAZ MECO, J. MOSCAT, and J. L. VIRELIZIER. 1993. Phosphatidylcholine hydrolysis activates NF-xB and increases human immunodeficiency virus replication in human monocytes and T lymphocytes. J. Virol. 67: 6596-6604. 50. DBAIBO, G. S., L. M. OBEID, and Y. A. HANNUN. 1993. Tumor necrosis factor-alpha (TNF-alpha) signal transduction through ceramide. Dissociation of growth inhibitory effects of TNF-alpha from activation of nuclear factor-kappa B. J. BioI. Chern. 268: 17762-17766. 51. BEe, A. A., T. S. FINCO, P. V. NANTERMET, and A. S. BALDWIN. 1993. Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation. Mol. Cell BioI. 13: 3301-3310.
TNF-induced activation of NF-xB . 203 52 . DIAZ MEeo, M. T., I. DOMINGUEZ, L. SANZ, P. DENT, J. LOZANO, M. M. MUNICIO, E. BERR A, R. T. HA Y, T. W. STURGILL, and J. MOSCAT. 1994. zeta PKC induces phosphorylation and inactivation of I kappa B-alpha in vitro. EMBO J. 13: 2842-2848.
Dr. MARTIN KRONKE, Institut fur Immunologie, Universitat Kid, Brunswiker Str. 4, 24105 Kid, Germany
©
1995 by Gustav Fischer Verlag, Stuttgart
Institut fur Medizinische Mikrobiologie und Hygiene, Technische Universitat Munchen, Munchen, Germany
TNF-Induced Activation of NF-xB STEFAN SCHUTZE':', KATJA WIEGMANN':., THOMAS MACHLEIDT\ and MARTIN KRONKE':'
Abstract Tumor Necrosis Factor (TNF) is one of the most potent physiological inducers of the nuclear transcription factor NF-xB. In light of the pivotal role of NF-xB in the development of immune responses and activation of HIV replication, the identification of TNF signal transduction pathways involved in NF-xB activation is of particular interest. Data from our laboratory demonstrate that the TNF signal transduction pathwaymediating NF-xB 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-xB inhibitor IxB as well as NF-xB activation. Besides endosomal acidic SMase, TNF also rapidly activates a plasmamembrane-associated neutral SMase (nSMase), that, however is not involved in TNF-induced NF-xB 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-xB. No apparent crosstalk was detected between nSMase and aSMase pathways, indicating that ceramide action depends on the topology of its production.
Introduction The cytokine Tumor Necrosis Factor (TNF) is a central polypeptide mediator of inflammation and cellular immune responses (1-3). The ability of TNF to affect the growth, differentiation and function of virtually every cell type investigated resides mainly in its potent gene regulatory properties of TNF (see ref. 4 for review). Evidently, the induction of some TNFresponsive genes of immunological relevance is mediated, at least in part, ". Present address: Institut fur Immunologie, Universitat Kiel, Brunswiker Str. 4, 24105
Kiel, Germany
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through activation of the nuclear transcription factor system NF-xB (5-7). NF-xB is a ubiquitous transcription factor, present in the cytosol of most cell types as an inactive heterodimer composed of 50- and 65-kDa subunits, that is bound to an inhibitor molecule, IxB (8-12). Induction of NF-xB requires the release of IxB, followed by translocation of the NF-xB heterodimer to the nucleus, where it directly binds to its cognate DNA sequence (13). NF-xB activation can be achieved by treating cells with very different kinds of agents (reviewed in ref. 14), including the cytokines TNF and IL-1 (6, 7). In light of the pivotal role of NF-xB in the development of immune responses and activation of HIV replication (15), the identification of TNF signal transduction pathways involved in NF-xB activation is of particular interest. The signaling pathways mediating cellular responses to TNF are beginning to be elucidated. TNF signaling is initiated by TNF with two distinct cell surface receptor molecules with apparent molecular sizes of 55-60 kDa (TNF-R55) and 75-80 kDa (TNF-R75) (for reviews see ref. 16-18). While information about TNF-R75 is scarce, TNF-R55 has been shown to trigger specific signal cascades, including phospholipase A2 (PLA2), phosphatidylcholin-specific phospholipase C (PC-PLC), protein kinase C, mitogenactivated protein kinase (MAP-kinase) and two distinct types of sphingomyelinase (SMase), a plasmamembrane-bound neutral (nSMase) and a intracellular endo-/ lysosomal acidic (aSMase) (19-23). Several reports recently implicated the lipid second messenger ceramide, in various important pathways of TNF action (ref. 24-26 for recent reviews). Ceramide can be generated by sphingomyelin (SM) breakdown by two types of TNF-responsive SMases at various subcellular sites (27, 28). Neutral SMase (nSMase) hydrolyzes SM of the outer leaflet of the plasmamembrane (29), while an acid SMase (aSMase) produces ceramide in the endosomai/lysosomal compartments (27, 30). The range of immediate cerami de targets has not yet been fully elucidated but may include a membrane-associated protein kinase, a cytosolic protein phosphatase 2A and the zeta-isoform of protein kinase C (31-33). Among the further downstream signaling events triggered by ceramide are the MAP-kinase cascade, the down-regulation of c-Myc. In addition, ceramide was found to display a potent NF-xB inducing capacity (20, 34-36). Here we review recent findings from our laboratory on the characterization of the intracellular pathway resulting in TNF-induced NF-xB activation.
Exogenous PLe and synthetic 1,2-diacylglycerols induce NF-xB activation Within seconds after TNF binding to its receptor, the lipid second messenger 1,2-DAG is produced from phosphatidylcholine, catalyzed by a PCPLC (19). To ask whether PC-PLC mediates TNF-receptor induced NFxB activation, exogenous PLC and its reaction products, 1,2-diacylgly-
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cerols, were tested for their NF-xB inducing activity (20). Exposure of Jurkat T cells either to PLC from Bac. cereus or to short-chain synthetic DAG like 1,2-dioctanoylglycerol (diC s), 1,2-dihexanoylglycerol (diC 6 ), and 1-0Ieoyl-2-acetylglycerol (1,2-0AG) resulted in NF-xB activation as estimated by electrophoretic mobility shift assays (EMSA) using a 29 basepair oligonucleotide encompassing the two xB sites present in the HIV enhancer. The NF-xB-inducing capacity of exogenous PLC and DAG, however, was less pronounced when compared to that of TNF, and moreover, could be observed only after long-term incubation (3-4 h). This delayed NF-xB induction may be due to the possibility that neither PLC nor DAG easily cross the membrane barrier, which may result in an inappropriate access to intracellular targets. The typical short-term NF-xB induction, characteristic of TNF-induced NF-xB activation could be observed when cells were permeabilized by streptolysin-O or using cell-free systems. Addition of increasing amounts of exogenous PLC from Bac. cereus to cell-free extracts resulted in dose-dependent NF-xB activation, suggesting that this enzyme is sufficient to initiate NF-xB activation. Similarly, Bac. cereus-derived PLC-induced NF-xB in Jurkat T cells permeabilized with streptolysin-O. Heat-treated PLC displayed drastically reduced NF-xB-inducing activity, which corresponded well to its residual heat-resistant enzymatic activity assayed in vitro. It is important to note that only 1,2-diacylglycerol but not 1,3-DAG like 1-palmitoyl-3-stearoylglycerol (1,3-PSG) and 1,3-dioctanoylglycerol (l-3-diC s) triggered NF-xB activation. These findings indicate, that the NF-xB complex is not dissociated by mere pH 5 treatment but rather specifically responds to 1,2-DAG physiologically generated by a TNF-responsive PC-PLC. Of note, NF-xB induction by 12,13-PMA appeared significantly reduced when compared to the NF-xB-inducing capacity of 1,2-DAG. It is important to stress that NF-xB induction was achieved at pH 5.0; at pH 7.4, activation of NF-xB by PLC or by diacylglycerols was less pronounced.
Exogenous sphingomyelinases and ceramide induce NF-xB activation At first glance, the induction of NF-xB by DAG pointed to a possible involvement of PKC, because DAG is a well known activator of this enzyme. However, in a previous report, we have shown that TNF-induced NF-xB activation can occur independent of PKC (37). We therefore assumed, that 1,2-diacylglycerols activate enzyme(s) other than PKC, which mediate NF-xB activation. One candidate was acidic sphingomyelinase (SMase), which was previously shown to be activated by DAG in GH3 pituitary cells (38-40). To test the possible involvement of this C-type phospholipase, permeabilized Jurkat cells were treated with either commercially available sphingomyelinase preparations, or with the SMase reaction product, ce-
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ramide (20). Exogenous sphingomyelinase of human (acidic SMase from placenta) as well as of bacterial origin (neutral SMase from Bac. cereus) were both able to induce NF-xB activation. Of note, in a cell-free lysate system, neutral SMase exhibited the same Mg2+ dependency as described for the neutral SMase from human brain (41). Most pertinently, the SMase-specific sphingomyelin cleavage product ceramide, in a dose-dependent manner activated NF-xB when added to permeabilized cells (20). These experiments clearly underscore the potential of sphingomyelinases to activate NF-xB by generation of the second messenger-like molecule ceramide.
Dissection of neutral and acidic SMase in TNF-activation of NF-xB Recent studies indicate, that TNF activates two distinct sphingomyelinases, a membrane-bound neutral (nSMase) and endo-/lysosomal acidic (aSMase) enzyme (22). The development of assay systems that specifically distinguish between nSMase and aSMase allowed us to show that n- and aSMase are triggered independently from each other and coupled to distinct signaling pathways. The finding that synthetic 1,2-DAG was able to stimulate SMase activity as well as NF-xB in cell-free lysates at pH 5.0 (20) implicates an acidic SMase as a likely candidate for mediating NF-xB-activation in a DAG-dependent manner. The site of aSMase activation appears to be the endosome. Endosomal vesicles are rapidly acidified, which conditions for aSMase activation by DAG. Lysosomotropic agents like monensin, chloroquin and ammonium chloride raise the pH in endolysosomal compartments and were expected to inhibit the sequential activation of endosomal aSMase and NF-xB selectively, leaving the membrane-associated nSMase unaffected. Indeed, these agents blocked the activation of aSMase and NF-xB (22). In contrast, neither nSMase nor PC-PLC were inhibited, indicating that aSMase, but not nSMase, is involved in NF-xB activation. Furthermore, DAG-production appears not to suffice for NF-xB activation. The idea that aSMase is both required and sufficient for NF-xB activation is supported by the observation that recombinant aSMase overexpressed in Jurkat cells dose-dependently activated transcription from a chloramphenicol acetyltransferase (CAT) reporter plasmid containing four immunoglobulin/HIV -xB sites (22).
Sphingomyelinase activates proteolytic IxB-a degradation in a cell-free system A key event in the activation of NF-xB is the rapid release of the inhibitory subunit IxB-a (9, 13). Various inhibitors of serine-like proteases have been shown to block TNF-mediated NF-xB activation as well as the disappearance of IxB immunoreactivity in primary murine T lymphocytes and in
TNF-induced activation of NF-xB . 197
various human leukemic cell lines (35). The protease inhibitors did not block TNF-induced activation of either PC-PLC nor acidic SMase, indicating that the putative protease operates rather downstream of the TNF signal transduction cascade. IxB-degradation could be directly induced by addition of SMase or synthetic ceramide to a cell-free system, indicating a stringent coupling of aSMase to the NF-xB activation pathway. Similar to the TNF-induced NF-xB activation and TNF-induced IxB-degradation,
Nucleus Figure 1. TNF signaling pathways.
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also the SMase-induced IxB degradation was suppressed by the protease inhibitor dichloroisocoumarin (DCIC), indicating that the protease involved in NF-xB activation operates downstream of the aSMase at the level of IxB degradation, mediated by a chymotrypsin-like proteasome complex (42,43).
Independent activation of neutral SMase and acidic SMase/NF-xB by distinct domains of the 55 kDa TNF receptor We have recently reported that recombinant human TNF-R55 stably expressed in murine 70Z/3 cells, mediates TNF induction of several signaling enzymes (21 ). To achieve a pertinent dissection of nSMase and an aSMase pathway, structure-function analysis of human TNF-R55 was performed in 70Z/3 cells, demonstrating that nSMase and aSMase are triggered by distinct cytoplasmic domains. Wild-type human TNF-R55 and C-terminal deletions of TNF-R55 were stably expressed in 70Z/3 cells and assayed for their capability of mediating the activation of neutral SMase, acidic SMase and NF-xB (22). TNF-treatment of a 70Z/3 clone expressing wild-type TNF-R55 simultaneously elicited nSMase, aSMase and NF-xB activation, TNF-R55 mutants truncated C-terminally by 32, 43, 52, or 81 amino acids, respectively, were defective in signaling aSMase and NF-xB activation, indicating that the domain responsible for nSMase activation is N-terminal to amino acid residue 345. These findings illustrate that nSMase and aSMase are activated independently by distinct cytoplasmic domains of TNF-R55. TNF-R55 deletion mutants displayed a loss-of function phenotype also with regard to activation of PC-PLC, yet retained their capacity to signal stimulation of proline-directed protein kinases (PDPkinases) and phospholipase A2 (PLA 2) (22). PDP-kinases include the ceramide-activated protein kinase (CAPK) and mitogen-activated protein kinase (MAP-kinase) (44, 45). MAP-kinase in turn phosphorylates and thereby activates PLA2 (46, 47). These data can be reconciled with our results in a model (see Fig. 1) where nSMase couples to PDP kinase and PLAb yet has no access to the NF-xB activation pathway. In contrast, aSMase is secondary to PC-PLC and represents a crucial component of the NF-xB activation pathway, as recently proposed (20).
Discussion PKC or aSMase, either DAG-responsive enzyme has been implicated to mediate TNF-induced activation of NF-xB. However, NF-xB activation by TNF can occur clearly independent of PKC (37). A possible involvement of PC-PLC in NF-xB activation was suggested based on the observation that exposure of Jurkat cells to either B. cereus PC-PLC or short-chain synthetic diacylglycerol resulted in the activation of the nuclear transcrip-
TNF-induced activation of NF-xB . 199
tion factor NF-xB (20). Similarly, DOMINGUEZ et al. (48) recently demonstrated induction of NF-xB after microinjection of B. cereus PC-PLC in Xenopus laevis oocytes. Moreover, PC-PLC microinjected in oocytes transactivated xB-containing enhancer elements of the HIV long terminal repeat. Along these lines, overexpression of bacterial PC-PLC in transfected cells resulted in NF-xB activation (49). Strikingly, addition of a PIspecific PLC from B. cereus to U937 cells resulted in DAG production, yet failed to activate xB enhancer-directed CAT expression. These studies strongly suggest a specific role for PC-derived DAG distinct from that of PIPz-derived DAG . In addition to PC-PLC and synthetic diacylglycerols, exogenous SMase as well as its reaction product, ceramide, were found to induce NF-xB in permeabilized cells or in cell-free Iysates. Inducing of NFxB by exogenous SMase from S. aureus or a cell-permeable ceramide analog, Cg-ceramide, was recently demonstrated with intact HL60 cells by YANG et al. (34) and in SW480/~gal cells by JOHNS et al. (36). DBAIBO et al. (50) suggested a possible feed-back role for SMase in the TNF activation pathway of NF-xB, based on their findings that treatment of Jurkat cells with Crceramide was found to enhance TNF-induced NF-xB activation. Parallel to the induction of NF-xB binding activity in the nucleus, degradation of the inhibitor IxB-a could be directly induced by addition of PC-PLC, sphingomyelinase or synthetic ceramide to a cell-free system, indicating a stringent coupling of PC-PLC and SMase to the NF-xB activation pathway. The SMase-induced IxB degradation could be blocked by the inhibitor of serine-like proteases, dichloroisocoumarin (DCIC), indicating that the aSMase is linked to IxB degradation via the proteasome complex. A prerequisite for proteolytic IxB-degradation is phosphorylation of the IxB molecule (51). Recent work from]. MOSCATS group has suggested a role for PKC-zeta in NF-xB activation and IxB-degradation (48, 52). Interestingl y, this PKC isotype responds to synthetic ceramide (33) making this enzyme a likely candidate for transmitting the TNFresponsive SMase mediated signal to the NF-xB/ lxB complex. TNF activates two distinct SMases with neutral and acidic pH optima, respectively (22). Endolysosomal agents (monensin, chloroquin and ammonium chloride) inhibited the sequential TNF activation of acidic SMase and NF-xB, leaving neutral SMase and PC-PLC activities unaffected. These observations point to aSMase as the key enzyme in NF-xB activation. Furthermore, they demonstrate that nSMase is not involved in TNFinduced NF-xB activation . Overexpression of recombinant acidic SMase also resulted in NF-xB-driven CAT activity in Jurkat cells, confirming the results on the role of aSMase in the NF-xB activation pathway. Recent work from several groups has highlightened ceramide generated by sphingomyelin breakdown as an important intracellular signaling molecule (see 24, 25 for review). As demonstrated with TNF-R55 deletion mutants, the 55 kDa TNF-receptor independently triggers the activation of neutral and acidic SMase (22). Although both nSMase and aSMase generate the same lipid messenger ceramide, a previously unrecognized dichotomy
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of ceramide function becomes apparent, indicating that ceramide action is determined by the subcellular site of its production. While ceramide produced by a membrane-associated neutral SMase controls selected signaling pathways such as proline-directed protein kinases that include CAPK and MAP kinase, ceramide produced by the endosomal acidic SMase represents a prominent cofactor for the activation of NF-xB. Acknowledgement The work of our laboratory was supported b y the Deutsche Forschungsgemeinschaft (DFG) and the W ilhelm Sander Stiftung.
References 1. OLD, L. J. 1987. Tumor necrosis factor: polypeptide mediator network. Nature 326: 330-331. 2. LE, J., and J. VILCEK. 1987. Bio logy of disease. Tumor necrosis factor and Interleukin-l: cytokines with multiple overlapping biological activities. Lab. Invest. 56: 234-248. 3. BEUTLER, B., and A. C FR AM I. 1988. The history, properties, and biological effects of cachectin . Biochemistry 27: 7575- 7582. 4. KRONK I·:,M., S. SCHUTZF, P. SCHEURICH, and K. PFIZENMA IER. 1992. TNF signal transdu ction and TNF responsive genes. In: AGGARWAL, B. B., and J. VILCEK (eds.), Tumor necrosis factor, Structure, Function, and Mechanism of Action. Marcel Dekker, New York, pp. 189-21 6. 5. LowFNTHM, .J. W., D. W. BALLARD, E. BOEHNLEIN, and W . C. GREENE. 1989. Tumor necrosis factor alpha induces proteins that bind specifically to kappa B-like enha ncer elements and regulate interleukin-2 receptor alpha chai n gene expression in primary human T lymphocytes . Proc. Nat!. Acad. Sci. USA 86: 2331- 2335. 6. OSBORN, L. , S. K UNKEL, and G. J. NABEL. 1989. Tumor necrosis factor alpha and interl eukin 1 stimulate th e human immunodeficiency virus enhancer by activation of the nuclear factor kappa B. Proc. N at\. Acad . Sci. USA 86: 2336- 2340. 7. DUH, E. J., W. J. MAURY, T. M . FOLKS, A. S. F AUCI, and A. B. RABSON. 1989. Tumor necrosis factor alpha activates human immunodeficiency virus type 1 through induction of nuclear factor bindin g to the NF-kappa B sites in the long terminal repeat. Proc. Nat!. Acad . Sci. USA 86: 5974-5978. 8. BAEUFRLF, P. A., and D. BAI.TIMORE . 1988. Activation of DNA-binding activity in an apparently cytoplasmatic precursor of the NF-xB transcripti on factor. Cell 53: 211-217. 9. BAEU FRU:, P. A., and D. BALTIMOR F. 1988. l-xB: a specific inhibitor of the NF-xB transcription factor. Science 242: 540-546. 10. BAEUE RLI, P. A., and D . BALTIMORE. 1989. A 65-kDa subunit of active NF-xB is required for inhibitio n of N F-xB by I-xB. Genes Dev. 3: 1689-1698. 11. GOS H, S., and D . BALTI MOKE. 1990. Activation in vitro of N F-xB by phosphorylation of its inhibitor IxB. N ature 344: 678-682. 12. RUB EN, S. M., P. J. DIII.ON, R. SCHRECK, T. H ENKEL, C. H . CH EN, M. MAAHER, P. A. BAFU FRIF, and C. A. R OSEN. 1991. Isolation of a rei- related human eDNA that potentially encodes th e 65-kDa subunit of NF-xB. Science 251: 1490-1493. 13. LIOU, H.-C., and D. BALTIMORE. 1993. Regulation of the NF-xBlrel transcription factor and IxB inhibitor system . Curr. Opin. Cell. BioI. 5: 477-487.
TNF-induced activation of NF-xB . 201 14. BAEUERLE, P. A. 1991. The inducible transcription activator NF-kappa B: regulation by distinct protein subunits. Biochim. Biophys. Acta 1072: 63-80. 15. BAEUERLE, P. A., and T. HENKEL. 1994. Function and activation of NF-kappa B in the immune system. Annu. Rev. Immunol. 12: 141-179. 16. TARTAGLIA, L. A., and D. V. GOEDDE!.. 1992. Two TNF receptors. Immunol. Today 13: 151-153. 17. ROTHE]., G. GEHR, H. LOETSCHER, and W. LESSLAUER. 1992. Tumor necrosis factor receptors: structure and function. Immunol. Res. 11: 81-90. 18. FIERS, W. 1991. Tumor necrosis factor. Characterization at the molecular, cellular and in vivo level. FEBS Lett. 285: 199-212. 19. SCHUTZE, S., D. BERKCJVIC, O. TOMSING, C. UNGER, and M. KRONKI'. 1991. Tumor necrosis factor induces rapid production of 1,2-diacylglycerol by a phosphatidylcholine-specific phospholipase. C. J. Exp. Med. 174: 975-988. 20. SCHUTZI', S., K. POTITIOII, T. MACHLEIDT, D. BERKOVIC, K. WIEGMANN, and M. KRONKI'. 1992. TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced «acidic» sphingomyelin breakdown. Cell 71: 765-776. 21. WIEGMANN, K., S. SCIIlJT7E, E. KAMPEN, A. HIMMEER, T. MACHEEIDT, and M. KRONKE. 1992. Human 55-kDa receptor for tumor necrosis factor coupled to signal transduction cascades. J. BioI. Chern. 267: 17997-18001. 22. WIEGMANN, K., S. SCIIOT7E, T. MACHLEIDT, D. WrITE, and M. KRONKE. 1994. Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 78: 1005-1015. 23. HELLER, R. A., and M. KRONKF. 1994. Tumor necrosis factor receptor-mediated signaling pathways. J. Cell BioI. 126: 5-9. 24. KOEESNICK, R., and D. W. GOlDE. 1994. The sphingomyelin pathway in tumor necrosis factor and intcrleukin-l signaling. Cell 77: 325-328. 25. HANNUN, Y. A. 1994. The sphingomyelin cycle and the second messenger function of ceramide. J. BioI. Chern. 269: 3125-3128. 26. SCHUTZE, S., T. MACHLElllT, and M. KRONKI'.. 1994. The role of diacylglycerol and ceramide in tumor necrosis factor and interleukin-l signal transduction. J. Leukoc. Bio!. 56: 533-541. 27. SPENCE, M. W. 1993. Sphingomyelinases. Adv. Lipid Res. 26: 3-23. 28. CHATTERJEE, S. 1993. Neutral sphingomyelinase. Adv. Lipid Res. 26: 25-48. 29. DAS, D. V. M., H. W. COOK, and M. W. SPENCE. 1984. Evidence that neutral sphingomyelinase of cultured murine neuroblastoma cells is orientated externally on the plasma membrane. Biochim. Biophys. Acta 777: 339-342. 30. MERIEL, A. H., Jr., Y. A. HANNUN, and R. M. BELL. 1993. Sphingolipids and their metabolites in cell regulation. Adv. Lipid Res. 25: 1-24. 31. MATHIAS, S., K. A. DRESSLER, and R. N. KOLESNICK. 1991. Characterization of a ceramide-activated protein kinase: stimulation by tumor necrosis factor alpha. Proc. Natl. Acad. Sci. USA 88: 10009-10013. 32. DOIlROWSKY, R. T., C. KAMIBAYASHI, M. C. MUMIlY, and Y. A. HANNUN. 1993. Ceramide activates heterotrimeric protein phosphatase 2A. J. BioI. Chern. 268: 15523-15530. 33. L07ANO,]., E. BERRA, M. M. MUNICIO, M. T. DIAZMECO, I. DOMINGUEZ, L. SANZ, and J. MOSCAT. 1994. Protein kinase C zeta isoform is critical for kappa B-dependent promoter activation by sphingomyelinase. J. Bio!. Chern. 269: 19200-19202. 34. YANG, Z., M. COSTANZO, D. W. GOLDE, and R. N. KOLESNICK. 1993. Tumor necrosis factor activation of the sphingomyelin pathway signals nuclear factor kappa B translocation in intact HL-60 cells. J. Bio!. Chern. 268: 20520-20523. 35. MACHLEIDT, T., K. WIF(;MANN, T. HENKEL, S. SCHOTZE, P. BAEUERLE, and M. KRONKI'. 1994. Sphingomyelinase activates proteolytic I kappa B-alpha degradation in a cell-free system. J. Bio!. Chern. 269: 13760-13765.
202 . S. SCHUTZI', K. WIEGMANN, T. MACHLEIDT, and M. KRONKE 36. JOHNS, L. D., T. SARR, and G. E. RANGES. 1994. Inhibition of ceramide pathway does not affect ability of TNF-alpha to activate nuclear factor-kappa B. J. Immunol. 152: 5877-5882. 37. MEICHLE, A., S. SCHUTZE, G. HENSEL, D. BRUNSING, and M. KRONKE. 1990. Protein kinase C-independent activation of nuclear factor kappa B by tumor necrosis factor. J. BioI. Chern. 265: 8339-8343. 38. QUINTERN, L. E., G. WEITZ, H. NEHRKORN, J. M. TAGER, A. W. SCHRAM, and K. SANDHOFF. 1987. Acid sphingomyelinase from human urine: purification and characterization. Biochim. Biophys. Acta 922: 323-336. 39. KOLESNICK, R. N. 1987. 1,2-Diacylglycerols but not phorbol esters stimulate sphingomyelin hydrolysis in GH3 pituitary cells. J. BioI. Chern. 262: 16759-16762. 40. KOLESNICK, R. N., and S. CLEGG. 1988. 1,2-diacylglycerols, but not phorbol esters, activate a potential inhibitory pathway for protein kinase C in GH3 pituitary cells. J. BioI. Chern. 263: 6534-6537. 41. RAO, B. G., and M. W. SPENCE. 1976. Sphingomyelinase activity at pH 7.4 in human brain and a comparison to activity at pH 5.0. J. Lipid Res. 17: 506-515. 42. ORLOWSKI, M., and C. MICHAUD. 1989. Pituitary multicatalytic proteinase complex. Specificity of components and aspects of proteolytic activity. Biochemistry 28: 9270-9278. 43. PALOMBELLA, V. J., A. L. GOLDBERG, and T. MANIATIS. 1994. The Ubiquitinproteasome pathway is required for processing the NF-xB1 precursor protein and the activation of NF-xB. Cell 78: 773-785. 44. JOSEPH, C. K., H. S. BVUN, R. BITTMANN, and R. N. KOLESNICK. 1993. Substrate recognition by ceramide-activated protein kinase. Evidence that kinase activity is proline-directed. J. BioI. Chern. 268: 20002-20006. 45. RAINES, M. A., R. N. KOLESNICK, and D. W. GOLDE. 1993. Sphingomyelinase and ceramide activate mitogen-activated protein kinase in myeloid HL-60 cells. J. BioI. Chern. 268: 14572-14575. 46. LIN, L. L., M. W ARTMANN, A. J. LIN, J. L. KNOPf, A. SETH, and R. j. DAVIS. 1993. cPLA 2 is phosphorylated and activated by MAP kinase. Cell 72: 269-278. 47. NEMENOFF, R. A., S. WrNITz, N. X. QIAN, V. VAN PUTTEN, G. L. JOHNSON, and L. E. HEASLFY. 1993. Phosphorylation and activation of a high molecular weight form of phospholipase A2 by p42 microtubule-associated protein 2 kinase and protein kinase C. J. BioI. Chern. 268: 1960-1964. 48. DOMINGUEZ, 1., L. SANZ, F. ARENZANA-SEISDEDOS, M. T. DIAZ MECO, J. L. VIRELIZIER, and J. MOSCAT. 1993. Inhibition of protein kinase C zeta subspecies blocks the activation of an NF-kappa B-like activity in Xenopus laevis oocytes. Mol. Cell. BioI. 13: 1290-1295. 49. ARENzANA-Sr·:IsDESDOS, F., B. FERNANDEZ, I. DOMINGUEZ, J. M. JACQUE, D. THOMAS, M. T. DIAZ MECO, J. MOSCAT, and J. L. VIRELIZIER. 1993. Phosphatidylcholine hydrolysis activates NF-xB and increases human immunodeficiency virus replication in human monocytes and T lymphocytes. J. Virol. 67: 6596-6604. 50. DBAIBO, G. S., L. M. OBEID, and Y. A. HANNUN. 1993. Tumor necrosis factor-alpha (TNF-alpha) signal transduction through ceramide. Dissociation of growth inhibitory effects of TNF-alpha from activation of nuclear factor-kappa B. J. BioI. Chern. 268: 17762-17766. 51. BEe, A. A., T. S. FINCO, P. V. NANTERMET, and A. S. BALDWIN. 1993. Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation. Mol. Cell BioI. 13: 3301-3310.
TNF-induced activation of NF-xB . 203 52 . DIAZ MEeo, M. T., I. DOMINGUEZ, L. SANZ, P. DENT, J. LOZANO, M. M. MUNICIO, E. BERR A, R. T. HA Y, T. W. STURGILL, and J. MOSCAT. 1994. zeta PKC induces phosphorylation and inactivation of I kappa B-alpha in vitro. EMBO J. 13: 2842-2848.
Dr. MARTIN KRONKE, Institut fur Immunologie, Universitat Kid, Brunswiker Str. 4, 24105 Kid, Germany