Molecular mechanism of signaling by tumor necrosis factor

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1 Vol. 45 No. 2 SCIENCE IN CHINA (Series C) April 2002 Molecular mechanism of signaling by tumor necrosis factor ZHA Jikun ( ) 1 & SHU Hongbing ( ) 1,2 1. College of Life Sciences, Peking University, Beijing , China; 2. National Jewish Medical and Research Center, Denver, CO 80206, USA Correspondence should be addressed to Shu Hongbing ( shuh@njc.org) Received July 8, 2001 Abstract Tumor necrosis factor (TNF) is an important cytokine with multiple biological effects, including cell growth, differentiation, apoptosis, immune regulation and induction of inflammation. The effects of TNF are mediated by two receptors, TNF-R1 and TNF-R2. The major signal transduction pathways triggered by TNF include those that lead to apoptosis, activation of transcription factor NF-κ B and protein kinase JNK. This review will discuss the molecular mechanisms of these signaling pathways. Keywords: tumor necrosis factor, signal transduction, JNK kinase, NF-κB, apoptosis. In 1975, Carswell et al. discovered that bcg vaccinated mouse, after injection with lipopolysacricharide, produced a substance in its serum that could cause necrosis of tumors. This substance was named tumor necrosis factor (TNF) [1]. Shalaby et al. called the TNF produced by phagocytes TNF-α, and that produced by T cells TNF-β [2].TNF-α and TNF-β signal through the same cellular membrane receptors and function similarly. This review will focus on signaling by TNF-α, which we will refer as TNF. 1 Biological characteristics of TNF TNF is produced mainly by activated macrophages and monocytes, but also by many other cell types including T cells, B cells, NK cells and LAK cells, at lower levels. TNF is primarily expressed as a 26 ku type II transmembrane precursor. Its N-terminus extends to the extracellular space and C-terminus resides in the cytoplasm. The extracellular domain of the TNF precursor can be cleaved by metalloproteinases to form a 17 ku soluble cytokine [2]. Mature soluble TNF, probably its transmembrane precursor, exists as a trimer [3]. TNF signaling leads to multiple effects, including apoptosis, inflammation, and septic shock. 2 Biological characteristics of TNF receptors The wide range of TNF activities is mediated by TNF-R1 and TNF-R2, with molecular masses of 55 ku and 75 ku, Kd values of 500 pmol/l and 100 pmol/l respectively [4]. Both receptors are type I transmembrane proteins with their C-terminuses in the cytoplasm and N-terminuses extending to the extracellular space. TNF-R1 is expressed in a wide range of cell types, while TNF-R2 is mostly expressed in

2 114 SCIENCE IN CHINA (Series C) Vol. 45 immune cells [3,4]. The expression of the two receptors can be upregulated by TNF, IL-1, phorbol diesters, and camp. TNF-R1 and TNF-R2 share about 30% homology in their extracellular domains. However, their intracellular domains are unrelated. The extracellular domains of TNF-R1 and TNF-R2 contain four conserved cysteine-rich repeats which are involved in optimal TNF-binding. Fig. 1 is a schematic representation of the two TNF receptors. Fig. 1. Schematic representation of the two TNF receptors. 3 Signal transduction by TNF receptors The divergence of the intracellular domains of the two TNF receptors suggests that they have distinct signaling and functional properties. While both receptors can activate NF-κB and JNK, in most cases, only TNF-R1 can induce apoptosis. 3.1 TNF-R2 signaling complex Although the expression and effects of TNF-R2 are mainly limited to immune cells, the breakthrough of TNF signaling came from deciphering the TNF-R2 signaling complex. In 1994, Rothe et al. employed a biochemical method to purify TNF-R2 signaling complex and this effort identified four proteins, including TRAF1 (TNF-receptor-associated factor 1), TRAF2, ciap1 (cell inhibitor of apoptosis) and ciap2 [5, 6]. Wang et al. demonstrated that ciap1 and ciap2 are able to inhibit apoptosis induced by TNF by inhibiting Caspase-8 activation [7]. Rothe et al. discovered that TRAF2 is necessary for TNF-R2-mediated NF-κB activation. Overexpression of TRAF2 can activate NF-κB, while a deletion mutant of TRAF2 lacking N-terminal 86 amino acids can inhibit TNF-R2-mediated NF-κB activation [8]. It has also been suggested that TRAF2 is essential for TNF-R2-mediated JNK activation [9]. These studies suggest that TRAF2 is an important downstream molecule of TNF-R2.

3 No. 2 MOLECULAR MECHANISM OF SIGNALING BY TUMOR NECROSIS FACTOR TNF-R1 signaling complex The molecular mechanisms of TNF-R1 signaling have been mostly understood in recent years. Binding of TNF to TNF-R1 induces trimmerization of TNF-R1, and this triggers the recruitment of downstream molecules, including TRADD, FADD, TRAF2 and RIP, to the TNF-R1 signaling complex. It has been suggested that a cytoplasmic protein, SODD, is associated with the intracellular domain of TNF-R1 and prevents TNF-R1 auto-aggregation. Upon TNF stimulation, SODD is dissociated with TNF-R1 [10] and this enables the recruitment of the adaptor protein TRADD to TNF-R1 [11]. It has been demonstrated that the interaction between TRADD and TNF-R1 is TNF dependent [12, 13]. TRADD functions as an adapter to recruit FADD through their death domains. FADD in turn recruits caspase-8 (also called FLICE/MACH) which subsequently activates an apoptotic cascade and leads to apoptosis [14ü17]. TRADD can also recruit TRAF2 and RIP, and these interactions lead to NF-κB and JNK activation. 3.3 Downstream signaling pathways JNK activation pathways. The mitogen-activated protein kinase (MAPK) cascades, which are conserved in the eukerocytes, play important roles in cell migration, division, differentiation, apoptosis and survival. TNF employs these MAPK activation cascades to activate JNK [18]. The MAPK cascades consist of three types of kinases. The first type is MAPKKK, which contains five kinase families: MEKK (MEKK1, 2, 3, 4), MLK (MLK1, 2, 3, DLK, LZK), ASK (ASK1, 2), TAK1 and TPL2 [19]. The second type is MAPKK, which contains several members, among them, MKK4/SEK1 [19] and MKK7 can activate JNK. The third type is MAPK, which contains P38, ERK, and JNK. Under physiological condition, TRAF2 is associated with two proteins I-TRAF and c-iap1. Upon TNF stimulation, TRAF2-cIAP1 is recruited to TNF-R1 complex through TRADD-TRAF2 interaction, while I-TRAF is dissociated with TRAF2 [13,20]. Yeh et al. performed knock-out study of TRAF2 in mice and found that TNF-triggered JNK activation is completely lost in traf2 -/- mice. This study demonstrated that TRAF2 is indispensable in JNK activation [21], however, TNF-triggered NF-κB activation was only slightly reduced in traf2 -/- cells. Recruitment of TRAF2 to the TNF-R1 complex causes activation of JNK through a MEKK1-MKK4 dependent pathway [22ü24]. It has also been suggested that GCKR (germinal center kinase related) [25] and ASK1 (apoptosis signal-regulating kinase 1) [26] may be also involved in TRAF2-mediated JNK activation NF-κB activation pathway. NF-κB (nuclear factor kapper B) was first discovered as a nuclear factor binding to the enhancer sequence GGGACTTTCC of the immunoglobulin light chain in lymph cells [27]. It was then found to be expressed in all mammalian cells. NF-κB exists as a heterodimer, mostly P50/RelA. NF-κB regulates a variety of target genes involved in inflamma-

4 116 SCIENCE IN CHINA (Series C) Vol. 45 tion, immune response and protection of cells from apoptosis [28ü30]. In most cell types, NF-κB is sequestered in cytoplasm through interaction with one of its inhibitory proteins called IκBs. In response to TNF and other stimulation, IκBs undergo rapid phosphorylation at two conserved N-terminal serine residues, which targets them for polyubiquitination followed by degradation through the 26s proteasome. This results in release of NF-κB, which then translocates to the nucleus to activate transcription of its downstream genes [28]. The kinase that phosphorylates IκB isiκb Kinase (IKK), which consists of three subunits: IκBα (IKK1, CHUK, 85kD) [31,32],IκBβ (IKK2, 87kD) [33,34] and IκBγ (NEMO, 48kD) [35,36].IKKα and IKKβ are catalytic subunits, while IKKγ is a regulatory subunit. IKKα and IKKβ, once activated, can directly phosphorylate Ser32 and Ser36 of IκBα or Ser19 or Ser 23 of IκBβ [37].IKKα and IKKβ have distinct effects: IKKα has a higher efficiency to phosphorylate Ser23 than Ser19 of IκBα, while IKKβ has an equal efficiency [34]. Gene knock-out experiments suggest that IKKβ is essential for proper activation of NF-κB in response to proinflammatory stimuli and prevention of TNF-induced apoptosis, while IKKα is dispensable for this process [38,39]. How TRAF2 and RIP, when recruited to TNF-R1, activate IKK is controversial. One possibility is that an MAPKKK kinase, NIK, may link TRAF2/RIP to downstream IKK [40]. However, gene knockout experiments indicated that NIK is not required for TNF-R1-mediated NF-κB activation [41]. The second possibility is that RIP can activate two atypical forms of PKC ξpkc or λ/ιpkc, which then activate IKKβ [42]. The third possibility is a direct interaction between RIP and IKKγ [43,44]. Finally, it has recently been proposed that MEKK3 is essential for RIP-mediated IKK activation [45] Apoptosis signaling pathway. Upon TNF stimulation, TRADD can also recruit FADD to the TNF-R1 signaling complex through interaction of their respective death domains [15]. FADD, in turn, recruits caspase-8 (originally called FLICE/MACH) [16,17]. Once caspase-8 is recruited to TNF-R1 signaling complex, it is activated by induced proximity. The activated caspase-8 then activates a downstream caspase cascade leading to apoptosis [46]. 3.4 Feedback regulatory mechanisms in TNF signaling Down-regulation of membrane-bound TNF-receptors. In most cell types, binding of TNF to TNF-R1 not only activates downstream signaling cascades but also causes the TNF-TNF-R1 complex to be pinocytosised in a clathrin dependent manner. The pinocytosomes are then fused with lysosomes and the TNF-TNF-R1 complex is hydrolyzed. This process causes the decrease of signaling time of activated TNF-R1 [47]. Binding of TNF to TNF-R2 leads to shedding of the extracellular domain of TNF-R2, which is released to blood to neutralize TNF [47, 48] Regeneration of IκB. After IκB is polyubiquitinized and degraded, NF-κB is released into nucleus to promote target genes transcription. Interestingly, IκB is one of the first genes tran-

5 No. 2 MOLECULAR MECHANISM OF SIGNALING BY TUMOR NECROSIS FACTOR 117 scriptionally induced by NF-κB, which provides a negative feedback control for NF -κb activation [28] Inhibition of NF-κB activation by A20. A20 is zinc finger protein transcriptionally induced by TNF and NF-κB activation [49]. A20 can interact with multiple signaling proteins in NF-κB activation pathways and inhibit NF-κB activation mediated by these signaling proteins [50]. 4 Conclusion In the last decade, considerable progress has been made in understanding the mechanisms of TNF signaling. Fig. 2 is a schematic model of TNF-R1 signaling pathways. Fig. 2. A schematic model of TNF signal pathway. Although the model of TNF-R1 signaling has become a paradigm on how all other TNF receptor family members signal, some details on TNF-R1 signaling still need to be elucidated. For example, TNF-R1 can simultaneously trigger apoptotic and anti-apoptotic NF-κB activation pathways. However, we do not know how the fate of TNF-stimulated cells (live or die) is determined. It is also controversial on how IKK is activated by the TRAF2 and RIP. Experiments with untransfected cells, especially gene knock-out studies, will certainly help to resolve the unknown details of TNF signaling. Acknowledgements This work was supported by the National Outstanding Youth Foundation of China (Grant No ), National Basic Science Researches Program (Grant No. G ) and Peking University. References 1. Carswell, E. A., Old, L. J., Kassel, R. L. et al., Endotoxin-induced serum factor that causes necrosis of tumors, Proc. Natl. Acad. Sci. USA, 1975, 72 (9):

6 118 SCIENCE IN CHINA (Series C) Vol Shalaby, M. R., Pennica, D., Palladino, M. A., An overview of the history and biological properties of tumor necrosis factors, Springe. Semin. Immun., 1986, 9 (1): Bazzoni, F., Beutler, B., How do tumor necrosis factor receptors work? J. Immfla., 1995, 45: Vandenabeele, P., Declercq, W., Bayaert, R. et al., Two tumor necrosis factor receptors: structure and function, Trends in Cell Biology, 1995, 5: Rothe, M., Wang, S. C., Henzel, W. J. et al., A novel family of putative signal transducers associated with the cytoplasmic domain of the 75kDa tumor necrosis factor receptor, Cell, 1994, 78: Rothe, M., Pan, M. G., Henzel, W. J. et al., The TNFR2-TRAF signaling complex contains two novel proteins related to Baculovirus inhibitor of apoptosis proteins, Cell, 1995, Wang, C. Y., Mayo, M. Y., Korneluk, R. G. et al., NF-κB antiapoptosis: Induction of TRAF1 and TRAF2 and c-iap1 and c-iap2 to suppress Caspase-8 activation, Science, 1998, 281: Rothe, M., Sarma, V., Dixit, V. M. et al., TRAF2-mediated activation of NF-κB by TNF-receptor 2 and CD40, Science, 1995, 269: Reinhard, C., Shamoon, B., Shyamala, V. et al., Tumor necrosis factor α-induced activation of c-jun N-terminal kinase is mediated by TRAF2, EMBO J., 1997, 16(5): Jiang, Y. P., Woronicz, J. D., Liu, W. et al., Prevention of constitutive TNF receptor 1 signaling by silencer of death domains, Science, 1999, 283: Hsu, H., Xiong, J., Goeddel, D. V., The TNF receptor 1 associated protein TRADD signals cell death and NF-κB activation, Cell, 1995, 83: Hsu, H., Shu, H. B., Pan, M. G. et al., TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways, Cell, 1996, 84: Shu, H. B., Takeuchi, M., Goeddel, D. V., The tumor necrosis factor receptor 2 signal tranducers TRAF2 and c-iap1 are components of the tumor necrosis factor receptor 1 signaling complex, Proc. Natl. Acad. Sci. USA, 1996, 93: Chinnaiyan, A. M., Rourke, K. O., Tewari, M. et al., FADD, a novel death domain containing protein, interacts with the death domain of Fas and initiates apoptosis, Cell, 1995, 81: Boldin, M. P., Varfolomeev, E. E., Pancer, Z. et al., A novel protein interacts with the death domain of Fas/Apo1 contains a sequence motif related to the death domain, J. Biol. Chem., 1995, 270: Muzio, M., Chinnaiyan, J. A., Frank, C. et al., FLICE, a novel FADD- homologous ICE/CED-3 like protease is recruited to the CD95( Fas/Apo-1) death- inducing signaling complex, Cell, 1996, 85: Boldin, M. P., Goneharou, T. M., Goltlsev, Y. V. et al., Involvement of MACH, a novel MORT1/FADD- interacting protease, in Fas/Apo1 and TNF receptor induced cell death, Cell, 1996, 85: Davis, R. J., Signal translation by the JNK group of MAP kinase, Cell, 2000, 103: Derijard, B., Raingeaud, J., Barrett, T. et al., Independent human MAP kinase signal transduction pathways defined by MEK and MKK isoforms, Science, 1995, 267: Rothe, M., Xiong, J., Shu, H. B. et al., I-TRAF is a novel TRAF-interaction protein that regulates TRAF mediates signal transduction, Proc. Natl. Acad. Sci. USA, 93: Yeh, W. C., Arda, S., Daniel, S. et al., Early lethality, functional NF-κB and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice, Immunity, 1997, 7: Baud, V., Liu, Z. G., Bennett, B. et al., Signaling by proinflammatory cyokines: Oligomerization of TRAF2 and TRAF6 is sufficient for JNK and IKK activation and target for gene induction via an amino-terminal effector domain, Genes & Deve., 1999, 13: Yan, M., Dan, T., Deak, J. C. et al., Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEK1, Nature, 1994, 372: Lee, F. S., Reters, R. T., Dang, L. C. et al., MEKK1 activates both IκB kinaseα and IκB kinaseβ, Proc. Natl. Acad. Sci. USA, 1998, 95: Shi, C. S., Kehrl, J. H., Activation of stress-activated protein kinase /c-jun N-terminal kinase, but not NF-κB bythetumor necrosis factor 2 and germinal center kinase related-dependent pathway, J. Biol. Chem., 1997, 272 (51):

7 No. 2 MOLECULAR MECHANISM OF SIGNALING BY TUMOR NECROSIS FACTOR Gotoh, Y., Cooper, J. A., Reaction oxygen species and dimerization-induced activation of apoptosis signal regulation by kinase 1 in tumor necrosis factor-α signal transduction, J. Biol. Chem., 1998, 273(8): Sen, R., Baltimore, D., Inducibility of the immunoglobin enhancer-binding protein in NF-κB by a post translational mechanism, Cell, 1986, 47: Baldwin, A. S., The NF-κBandI-κB Proteins: New discoveries and insights, Annu. Rev. Immunol., 1996, 14: Stark, G. R., Kerr, I. M., Williams, B. R. et al., How cells respond to interferons, Annu. Rev. Biochem., 1998, 67: Baichwal, V. R., Baeuerle, P. A., Activation of NF-κB or die? Curr. Biol., 1997, 7: Didonato, J. A., Hayakawa, M., Rathwarf, D. M., A cytokine responsive I-κB kinase that activates the transcription factor NF-κB, Nature, 1997, 388: Reginier, C. H., Song, H. Y., Gao, X. et al., Identification and characterization of an IκB kinase, Cell, 1997, 90: Mercurio, F., Zhu, H., Murray, B. W. et al., IKK-1 and IKK-2: cytokine-activation IκB kinase essential for NF-κB action, Science, 1997, 278: Woronicz, J. D., Gao, X., Cao, Z. D. et al., IκB kinase-β: NF-κB activation and complex formation with IκB kinase-α and NIK, Science, 1997, 278: Yamaoka, S., Courtois, G., Bessia, C. et al., Complementation cloning of NEMO, a component of the IκB kinase complex: Essential for NF-κ B activation, Cell, 1998, 93: Rothwarf, D. M., Zandi, E., Natoli, G. et al., IKK-γ is an essential regulatory subunits of the IκB Kinase complex, Nature, 1998, 395: Didonato, J., Mercurco, F., Rosette, C. et al., Mapping of the inducible IκB phosphorylation sites that signal its ubiquitination and regulation, Mol. Cell. Biol., 1996, 16: Li, Q., Antwerp, D. V., Mercurio, F. et al., Severe liver degeneration in mice lacking the IκB kinase 2 gene, Science, 1999, 284: Hu, Y., Baud, V., Delhase, M. et al., Abnormal morphogenesis but intact IKK activation in mice lacking the IKKα subunit of IκB kinase, Science, 1999, 284: Ling, L., Cao, Z., Goeddel, D. V., NF-κB inducing kinase activates IKK-α by phosphorylation of Ser-176, Proc. Natl. Acad. Sci. USA, 1998, 95: Li, Y., Lin, W., Wesche, H. et al., Defective lymphotoxin-β receptor-induced NF-κB transcriptional activity in NIK-deficient mice, Science, 2001, 291: Lallena, M. J., Diazmeco, M. T., Bren, G. etal., ActivationofIκBkinaseβ by protein kinase C isoform, Mole. Cell Biol., 1999, 19(3): Poyer, J. L., Srinivasula, S. M., Lin, J. H. et al., Activation of the IκB kinasebyripviaikkγ/nemo mediated oligomerization, J. Biol. Chem., 2000, 275(48): Zhang, S. Q., Kroalenk, A., Cantaarella, G. et al., Recruitment of the IKK signalsome to the p55 to the TNF receptor: RIP and A20 bind to NEMO (IKKγ) upon receptor stimulations, Immunity, 2000, 12: Yang, J., Lin, Y., Guo, Z. J. et al., The essential role of MEKK3 in TNF-induced NF-κB activation, Nature Immunology, 2000, 2(7): S. 46. Salvesen, C. S., Dixit, V. M., Caspase activation: the induced-proximity model, Proc. Natl. Acad. Sci. USA,1999, 96: Porteu, F., Hieblot, C., Tumor necrosis factor induces a selective shedding of its p75 receptor from human neutrophils, J. Biol. Chem., 1994, 269(4): Kohno, T., Brewe, M. T., Baker, S. L. et al., A second tumor necrosis factor receptor gene product can shed a naturally occurring tumor necrosis factor inhibitor, Proc. Natl. Acad. Sci. USA, 1990, 87: Song, H. Y., Rothe, M. Godden, D. V., The tumor necrosis factor inducible zinc finger protein A20 interacts with TRAF1/ TRAF2 and inhibits NF-κB activation, Proc. Natl. Acad. Sci. USA, 1996, 93: Lee, E. G., Boon, D. L. E., Ma, A. et al., Fail to regulate TNF-induced NF-κB and cell death responses in A20-deficient mice, Science, 2000, 289: