Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-kappaB-dependent gene transcription
2000; Springer Nature; Volume: 19; Issue: 18 Linguagem: Inglês
10.1093/emboj/19.18.4976
ISSN1460-2075
Autores Tópico(s)Cell death mechanisms and regulation
ResumoArticle15 September 2000free access Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-κB-dependent gene transcription Madeleine Bonnard Madeleine Bonnard Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Christine Mirtsos Christine Mirtsos Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Shinobu Suzuki Shinobu Suzuki Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Kevin Graham Kevin Graham Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Jianing Huang Jianing Huang Tularik Incorporation, 2 Corporate Drive, South San Francisco, CA, 94080 USA Search for more papers by this author Michelle Ng Michelle Ng Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Annick Itié Annick Itié Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Andrew Wakeham Andrew Wakeham Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Arda Shahinian Arda Shahinian Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author William J. Henzel William J. Henzel Genentech Incorporation, 460 Point San Bruno Boulevard, South San Francisco, CA, 94080 USA Search for more papers by this author Andrew J. Elia Andrew J. Elia Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Wendy Shillinglaw Wendy Shillinglaw Genentech Incorporation, 460 Point San Bruno Boulevard, South San Francisco, CA, 94080 USA Search for more papers by this author Tak W. Mak Tak W. Mak Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Zhaodan Cao Zhaodan Cao Tularik Incorporation, 2 Corporate Drive, South San Francisco, CA, 94080 USA Search for more papers by this author Wen-Chen Yeh Corresponding Author Wen-Chen Yeh Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Madeleine Bonnard Madeleine Bonnard Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Christine Mirtsos Christine Mirtsos Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Shinobu Suzuki Shinobu Suzuki Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Kevin Graham Kevin Graham Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Jianing Huang Jianing Huang Tularik Incorporation, 2 Corporate Drive, South San Francisco, CA, 94080 USA Search for more papers by this author Michelle Ng Michelle Ng Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Annick Itié Annick Itié Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Andrew Wakeham Andrew Wakeham Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Arda Shahinian Arda Shahinian Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author William J. Henzel William J. Henzel Genentech Incorporation, 460 Point San Bruno Boulevard, South San Francisco, CA, 94080 USA Search for more papers by this author Andrew J. Elia Andrew J. Elia Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Wendy Shillinglaw Wendy Shillinglaw Genentech Incorporation, 460 Point San Bruno Boulevard, South San Francisco, CA, 94080 USA Search for more papers by this author Tak W. Mak Tak W. Mak Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Zhaodan Cao Zhaodan Cao Tularik Incorporation, 2 Corporate Drive, South San Francisco, CA, 94080 USA Search for more papers by this author Wen-Chen Yeh Corresponding Author Wen-Chen Yeh Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 Search for more papers by this author Author Information Madeleine Bonnard1, Christine Mirtsos1, Shinobu Suzuki1, Kevin Graham1, Jianing Huang2, Michelle Ng1, Annick Itié1, Andrew Wakeham1, Arda Shahinian1, William J. Henzel3, Andrew J. Elia1, Wendy Shillinglaw3, Tak W. Mak1, Zhaodan Cao2 and Wen-Chen Yeh 1 1Amgen Institute, Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, Canada, M5G 2C1 2Tularik Incorporation, 2 Corporate Drive, South San Francisco, CA, 94080 USA 3Genentech Incorporation, 460 Point San Bruno Boulevard, South San Francisco, CA, 94080 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:4976-4985https://doi.org/10.1093/emboj/19.18.4976 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Induction of NF-κB-dependent transcription requires phosphorylation and subsequent degradation of I-κB, an inhibitor of NF-κB, followed by nuclear translocation and DNA binding of NF-κB. Tumor necrosis factor receptor-associated factor 2 (TRAF2) plays a role in NF-κB activation in response to cytokines such as tumor necrosis factor α (TNFα). In this study, we purified and characterized a novel kinase (T2K, also known as TBK1 or NAK), which associates with TRAF2 and exhibits kinase activity towards I-κBα in vitro. The physiological function of T2K was investigated using T2K-deficient mice. Heterozygotes appear normal, but t2k−/− animals die at ∼E14.5 of massive liver degeneration and apoptosis. Never theless, hematopoietic progenitors from T2K-deficient fetal liver support normal lymphocyte development. Furthermore, t2k−/− embryonic fibroblasts and thymocytes do not display increased sensitivity to TNFα-induced apoptosis. In response to either TNFα or IL-1 induction, t2k−/− embryonic fibroblasts exhibit normal degradation of I-κB and κB-binding activity. However, NF-κB-directed transcription is dramatically reduced. These results demonstrate that, like I-κB kinase β and the RelA subunit of NF-κB, T2K is critical in protecting embryonic liver from apoptosis. However, T2K has a unique role in the activation of NF-κB-directed transcription, apparently independent of I-κB degradation and NF-κB DNA binding. Introduction Rel-family NF-κB transcription factors, including the prototypical RelA–p50 heterodimer, are critical regulators of genes involved in immune and inflammatory responses, as well as protecting against apoptosis (Ghosh et al., 1998; Foo and Nolan, 1999). NF-κB is normally sequestered in the cytoplasm of resting cells, where it is bound to I-κB inhibitory proteins (I-κBα, I-κBβ and I-κBϵ). This interaction masks the nuclear localization signal of NF-κB, preventing its nuclear translocation (Ghosh et al., 1998). Various extracellular stimuli, including pro-inflammatory cytokines such as tumor necrosis factor α (TNFα) and interleukin-1 (IL-1), virus infection, lipopolysaccharide (LPS) and lymphocyte activation, result in the phosphorylation of I-κB proteins on two conserved serine residues (Ser32 and -36 on I-κBα, and Ser19 and -23 on I-κBβ) (Ghosh et al., 1998). This phosphorylation targets I-κB for ubiquitylation and subsequent proteosome- mediated degradation, resulting in the release of NF-κB. NF-κB then translocates to the nucleus where it binds to specific κB sites in promoter sequences and activates transcription (Ghosh et al., 1998). Phosphorylation of I-κB in response to extracellular stimuli is carried out by the I-κB kinase (IKK) complex, which is comprised of (at least) two catalytic subunits, IKKα and IKKβ (DiDonato et al., 1997; Mercurio et al., 1997; Regnier et al., 1997; Woronicz et al., 1997; Zandi et al., 1997), as well as the modulator NEMO (NF-κB essential modulator) (Rothwarf et al., 1998; Yamaoka et al., 1998). Although IKKα and IKKβ appear to have analogous activities in vitro, IKKα- and IKKβ-deficient mice have dramatically different phenotypes. IKKα−/− mice exhibit severe limb and skin abnormalities. Surprisingly, the induction of NF-κB in ikkα−/− murine embryonic fibroblasts (EFs) by either TNFα or IL-1 is normal (Hu et al., 1999; Takeda et al., 1999). In contrast, the phenotype of IKKβ-deficient mice resembles that of relA−/− mice (Beg et al., 1995) but is more severe. IKKβ−/− animals display massive liver degeneration due to apoptosis and die between E12.5 and E14.5 (Q.Li et al., 1999b; Z.W.Li et al., 1999; Tanaka et al., 1999). IKKβ−/− EFs show enhanced sensitivity to TNFα-induced apoptosis, and NF-κB activation in response to TNFα and IL-1 is significantly reduced compared with the wild type (Q.Li et al., 1999b; Z.W.Li et al., 1999; Tanaka et al., 1999). These results indicate that it is IKKβ that mediates responses to TNFα and IL-1 (Q.Li et al., 1999b; Z.W.Li et al., 1999; Tanaka et al., 1999), while IKKα may respond to an as yet uncharacterized morphogenetic signal (Hu et al., 1999; Q.Li et al., 1999a; Takeda et al., 1999). The phenotype of NEMO-deficient mice is similar to that of ikkβ−/− animals, although even more severe. Mutant mice display massive liver degeneration due to apoptosis and die between E12.5 and E13 (Makris et al., 2000; Rudolph et al., 2000). EFs from nemo−/− embryos show enhanced sensitivity to TNFα-induced apoptosis, and NF-κB activation in response to TNFα and IL-1 is dramatically reduced compared with wild-type EFs, demonstrating that NEMO is an essential component of the IKK complex (Makris et al., 2000; Rudolph et al., 2000). Members of the TNF receptor-associated factor (TRAF) family of adaptor proteins are involved in coupling stimulation of receptors for pro-inflammatory cytokines, such as the IL-1 receptor (IL-1R) and the TNF receptors (TNFRs) to NF-κB activation, and other downstream events (Arch et al., 1998). TRAF2 plays a critical role in signal transduction mediated by both TNFR1 and TNFR2, and has been implicated in TNFα-induced activation of NF-κB and the stress-activated protein kinase (SAPK/JNK) (Rothe et al., 1995; Liu et al., 1996; Lee et al., 1997; Natoli et al., 1997; Yeh et al., 1997). Similarly, TRAF6 is important for the transduction of IL-1-induced signals, including those resulting in NF-κB activation (Cao et al., 1996b; Lomaga et al., 1999). However, the signaling pathway linking the TRAF proteins to NF-κB activation remains to be defined. A MAP-3K homolog termed NF-κB-inducing kinase (NIK) has been shown to bind both TRAF2 and TRAF6, linking these adaptors to downstream signaling (Song et al., 1997). However, recent studies highlighting the role of NIK in lymph node organogenesis and B-lymphocyte activation indicate that the function of NIK may be restricted to certain cell types, implying that additional molecules must also be involved in regulating NF-κB activation downstream of TRAF proteins (Shinkura et al., 1999; Garceau et al., 2000). In this report, we describe the characterization of a novel TRAF2-associated kinase, T2K, and study its physiological function by gene targeting. T2K-deficient mice die of liver apoptosis, but t2k−/− EFs show normal sensitivity to TNFα-induced apoptosis, and normal I-κB degradation and NF-κB DNA binding in response to TNFα or IL-1 stimulation. Nevertheless, NF-κB-directed transcription in response to these pro-inflammatory cytokines is reduced in t2k−/− EF cells. These results indicate that T2K is critical in protecting embryonic liver from massive apoptosis, and that T2K may regulate NF-κB activation in a way that is distinct from the process involving I-κB degradation. Results Isolation of T2K Since TRAF2 plays a role in the activation of NF-κB, it seemed reasonable that TRAF2 might interact, directly or indirectly, with a mediator involved in the signaling cascade leading to activation of the transcription factor. We investigated this possibility using the 293 cell line stably expressing a FLAG-tagged TRAF2 molecule. TRAF2 and associated proteins were immunoprecipitated and assayed for kinase activity by determining their ability to phosphorylate I-κBα in vitro. A kinase activity was indeed identified in TRAF2 complexes that could phosphorylate I-κBα in vitro. After further purification by gel filtration and monoQ column chromatography, the amino acid sequence of the putative kinase was determined and used to search EST sequences held in the National Center of Biological Information. A matching EST sequence (R19830) was used to clone a full-length cDNA encoding a putative protein kinase, which was named T2K. Both recombinant T2K and immunoprecipitated TRAF2 complexes could phosphorylate a wild-type I-κBα peptide (25–41) and a mutant peptide in which Ser32 was mutated to Ala, but not an altered pepide in which Ser36 was mutated to Ala (Figure 1A). These results indicate that the kinase activity is specific for Ser36 of I-κBα. Furthermore, since both recombinant T2K and the immunoprecipitated TRAF2 complexes had the same substrate specificity, T2K is likely to account for the kinase activity detected in the TRAF2 complexes. The identity of T2K was further confirmed by western blot analysis in which polyclonal rabbit antiserum raised against recombinant T2K recognized a band of ∼80 kDa only in anti-TRAF2 immunoprecipitates of untransfected 293 cells, not in anti-IKKβ or anti-TRAF6 immunoprecipitates (Figure 1B). While this manuscript was in preparation, two groups reported the isolation of molecules identical to T2K. Tojima et al. (2000) reported cloning of a cDNA encoding a kinase, called NAK, capable of phosphorylating IKKβ and I-κBα Ser36 in vitro. In addition, Pomerantz and Baltimore (1999) reported the cloning of a cDNA encoding TANK-binding kinase 1 (TBK1). TANK (I-TRAF) is a TRAF-binding protein with both stimulatory and inhibitory properties that has been implicated in NF-κB activation (Cheng and Baltimore, 1996; Rothe et al., 1996). These researchers also observed that TBK1/T2K is a kinase capable of forming a complex with TANK and TRAF2 (Pomerantz and Baltimore, 1999), consistent with our findings. Figure 1.Identification of a TRAF2-associated kinase (T2K). (A) TRAF2 complexes or recombinant T2K were used in in vitro kinase assays to assess the phosphorylation of peptide substrates encompassing amino acids 25–41 of either wild-type or mutated I-κBα (mutated amino acids are underlined). (B) Co-immunoprecipitation of endogenous T2K. Extracts of 293 cells either left untreated or stimulated with 100 ng/ml TNFα for 10 min were incubated with pre-immune rabbit serum or rabbit anti-serum specific for either IKKβ, TRAF2 or TRAF6 monoclonal antibody followed by western blotting using antiserum specific for recombinant T2K. Download figure Download PowerPoint Generation of T2K-deficient mice To characterize the function of T2K in vivo, we disrupted the murine t2k gene in embryonic stem (ES) cells, using a targeting vector designed to replace exons 1 and 2 of the endogenous locus with a PGK-neo cassette (Figure 2A). Correctly targeted ES cell clones were identified by Southern blotting and injected into C57BL/6 blastocysts to generate chimeric mice. Male chimeras with germline transmission were utilized to generate t2k+/− animals, which were interbred to generate t2k−/− mice. Genotypes were confirmed by Southern blotting (Figure 2B) and PCR analysis (Figure 2C). Although t2k+/− mice appeared normal and were fertile, no viable homozygous mutant mice were observed in the first 111 pups derived from t2k+/− intercrosses, suggesting that T2K deficiency results in embryonic lethality. EFs were subsequently derived from early mutant embryos and total RNA was prepared for northern blot analysis. The results confirmed the absence of detectable T2K mRNA in t2k−/− cells (Figure 2D). Figure 2.Targeted disruption of the murine t2k gene. (A) Top, a portion of the endogenous t2k locus containing three exons (shown as solid boxes). Middle, the targeting construct. Bottom, the mutant locus resulting from homologous recombination. The SacI (S) restriction site and the flanking probe used to detect homologous recombination are indicated. (B) Genomic Southern blot of SacI-digested DNA from E13.5 embryos of the genotypes indicated, showing the 6.6 kb wild-type band and the 14.6 kb mutant band detected by the flanking probe in (A). (C) Genotyping by PCR of DNA from E13.5 embryos using the primers described in Materials and methods. (D) Northern blot analysis of T2K RNA expression in E13.5 EFs of the genotypes indicated. The cells were either left untreated or stimulated with 10 ng/ml TNFα for the time points indicated. Download figure Download PowerPoint Liver degeneration in T2K-deficient embryos Embryos from timed matings of t2k+/− animals were analyzed to determine the time of lethality (Table I). Offspring of all three expected genotypes were present at the normal Mendelian ratio until E14.5 when t2k−/− embryos appeared pale and exhibited dramatic liver degeneration (Figure 3A–D). Development of all other organs appeared normal. Histological analysis of embryo sections revealed liver hemorrhage and severe tissue loss (Figure 3C–F). To determine whether the liver degeneration was attributable to apoptosis, TUNEL assays were performed on sections of E12.5, E13.5 and E14.5 wild-type and mutant embryos. Sections of wild-type livers at any of these stages showed very few TUNEL-positive cells (Figure 3G and data not shown). In contrast, while sections of E12.5 t2k−/− embryos appeared normal, livers of E13.5 t2k−/− embryos displayed mild, focal liver degeneration and apoptosis (data not shown). By E14.5, extensive apoptosis was detected throughout liver sections of t2k−/− embryos (Figure 3H), indicating that apoptosis is a major cause of liver degeneration in these mutants, and consequently, of embryonic death. The liver apoptosis occurring in the absence of T2K was strikingly similar to that observed in nemo−/−, ikkβ−/− and relA−/− animals (Beg et al., 1995; Q.Li et al., 1999b; Z.W.Li et al., 1999; Tanaka et al., 1999; Rudolph et al., 2000), prompting us to speculate that T2K might be involved in the activation of NF-κB. Figure 3.Massive liver apoptosis in the absence of T2K. (A and B) Low power view of E14.5 wild-type (A) and t2k−/− (B) embryos. (C–F) Histological analysis of E14.5 liver sections. Views of H&E staining of wild-type (C and E) and mutant (D and F) livers at either low (C and D) or high (E and F) magnification are shown. (G and H) Enhanced apoptosis in livers of E14.5 T2K-deficient embryos. TUNEL assays were performed on sections of wild-type (G) and mutant (H) E14.5 embryos. A high power view of the liver is shown in both cases. Download figure Download PowerPoint Table 1. Genotypes of embryos derived from t2k+/− intercrosses Stage +/+ +/− −/− Total Normal Abnormal E12.5 8 26 6 0 40 E13.5 36 82 19 21 158 E14.5 17 28 4 12 61 E15.5 10 20 0 4a 34 a In resorption. Normal sensitivity of thymocytes and EFs to TNFα-induced apoptosis Stimulation of TNFR1 results in signal transduction that contributes both to apoptosis and cell survival (Hsu et al., 1996b; Yeh et al., 1999). TRAF2 and NF-κB play important roles in the survival pathway, as shown by the finding that relA−/−, ikkβ−/−, nemo−/− and traf2−/− cells display enhanced sensitivity to TNFα-induced apoptosis. Wild-type EFs are resistant to treatment with TNFα alone, but relA−/−, nemo−/− and ikkβ−/− EFs are exquisitely sensitive to TNFα-induced cell death (Beg et al., 1995; Q.Li et al., 1999b; Z.W.Li et al., 1999; Tanaka et al., 1999; Rudolph et al., 2000). TRAF2-deficient EFs are mildly sensitive to apoptosis induced by TNFα alone, but exhibit a dramatic increase in sensitivity in the presence of TNFα combined with low concentrations of the protein synthesis inhibitor cycloheximide (Yeh et al., 1997). At higher concentrations, cycloheximide can also render wild-type EFs sensitive to TNFα treatment. In this context, we assessed the response of t2k−/− EFs to TNFα alone or in combination with various concentrations of cycloheximide. Sensitivity to staurosporine was used as a positive control. T2K-deficient EFs behaved like wild-type EFs rather than relA−/−, ikkβ−/−, nemo−/− or traf2−/− EFs, being resistant to apoptosis induced by TNFα at all but the highest concentrations of cycloheximide (Figure 4A). Figure 4.Normal sensitivity of thymocytes and EFs to TNFα-induced cell death in the absence of T2K. (A) TNFα-induced apoptosis of EFs. Wild-type and T2K-deficient EFs were either left untreated or stimulated with staurosporine (2 μM) or TNFα (10 ng/ml) in combination with the indicated concentrations of cycloheximide for 18 h. Cells were harvested and stained for viability as described in Materials and methods. Values shown are the percentage of viable cells after treatment relative to untreated controls. Individual treatments were measured in triplicate. (B) TNFα-induced apoptosis of CD4+CD8+ (DP) thymocytes. Thymocytes from the reconstituted mice described in the text were cultured at 37°C for 20 h in the presence of either 1 μM dexamethasone or the indicated concentrations of TNFα. Cell viability was determined by 7-amino actinomycin D (7-AAD) staining. Results are expressed as the percentage of total viable DP cells remaining after treatment relative to the untreated control of the same genotype. Individual treatments were measured in quadruplicate. Download figure Download PowerPoint The importance of the role of TRAF2 in protection against TNFα-induced apoptosis was highlighted further by the demonstration that traf2−/− thymocytes undergo enhanced apoptosis in response to TNFα (Yeh et al., 1997). In order to obtain t2k−/− thymocytes to assess their sensitivity to apoptotic stimuli, we used fetal liver cells from either wild-type or t2k−/− E12.5 to reconstitute the lymphoid compartment of irradiated rag1−/− mice. Bone marrow, thymus, lymph nodes, spleens and peripheral blood of all reconstituted animals had normal proportions of immature, and mature, T- and B-cell populations (data not shown). T2K-deficient double positive (DP) thymocytes were as sensitive as wild-type DP thymocytes to dexamethasone-induced apoptosis. In contrast to traf2−/− DP thymocytes, t2k−/− DP thymocytes were as resistant to TNFα treatment as were wild-type DP thymocytes (Figure 4B). Importantly, the embryonic lethality and liver apoptosis of animals lacking either RelA or IKKβ can be attributed to TNFR1 signaling. Thus, animals lacking both RelA and TNFR1 (Beg and Baltimore, 1996), like those lacking both IKKβ and TNFR1 (Q.Li et al., 1999b), develop to term. Results from Table II show that six of the first 94 pups born from intercrossing t2k+/− tnfr1+/− mice were t2k−/− tnfr1−/− mice (expected Mendelian ratio 6.4%, 1:16). These double mutant animals developed to term rather than dying at ∼E14.5. The apparent rescue of embryonic lethality by inactivation of the TNFR1 gene supports the hypothesis that embryonic lethality of t2k−/− animals is due to TNFα-induced liver apoptosis, and that T2K is an important mediator in TNFR1 signaling. Together, these results indicate that although T2K plays an important role in protection against TNFα-induced apoptosis, it is dispensable for resistance of certain cell types, such as thymocytes and EFs, to TNFα-induced apoptosis. Table 2. Genotypes of embryos derived from t2k+/− tnfr1+/− intercrosses t2k+/+ tnfr1+/+ t2k+/+ tnfr1+/− t2k+/+ tnfr1−/− t2k+/− tnfr1+/+ t2k+/− tnfr1+/− t2k+/− tnfr1−/− t2k−/− tnfr1+/+ t2k−/− tnfr1+/− t2k−/− tnfr1−/− Total 6 18 12 16 18 18 0 0 6 94 6.4% 19.1% 12.8% 17% 19.1% 19.1% 6.4% 100% Normal I-κBα phosphorylation/degradation and κB-binding activity in t2k−/− cells Given the ability of T2K to phosphorylate I-κBα in vitro, and the similarities in liver phenotype observed in t2k−/−, relA−/−, nemo−/− and ikkβ−/− mice, we investigated various steps leading to activation of NF-κB-dependent transcription in t2k−/− EFs. Cytoplasmic and nuclear protein extracts were prepared from wild-type and T2K-deficient EFs stimulated with either TNFα or IL-1 for 1, 3, 9, 27 or 90 min. Phosphorylation of Ser32 of I-κBα and degradation of I-κBα, I-κBβ and I-κBϵ were assessed by probing western blots of cytoplasmic extracts with either a phospho-I-κBα-specific antibody, or I-κBα-, I-κBβ- and I-κBϵ-specific antibodies, respectively. Rapid phosphorylation of I-κBα Ser32, comparable to that in the wild type, was observed in t2k−/− EFs in response to either TNFα or IL-1 (Figure 5A). Degradation of I-κBα (Figure 5A), I-κBβ and I-κBϵ (data not shown) in response to either stimulus was also normal in the mutant cells. Figure 5.Normal I-κBα phosphorylation, degradation and NF-κB DNA binding activity in t2k−/− EFs. (A) I-κBα phosphorylation/degradation. Wild-type and T2K-deficient EFs were treated with either TNFα (10 ng/ml) or IL-1β (10 ng/ml) for the times indicated. Cytoplasmic extracts were analyzed by western blotting using monoclonal antibodies specific for either phospho-I-κBα (top), I-κBα (middle) or the loading control actin (bottom). (B) κB binding activity. Wild-type and t2k−/− EFs were incubated with either mouse recombinant TNFα (10 ng/ml) (top) or IL-1β (10 ng/ml) (bottom) for the times indicated. NF-κB activation in 10 μg of nuclear extract was determined by EMSA as described in Materials and methods. For both (A) and (B), one result representative of three independent experiments is shown. (C) RelA binding activity. Wild-type and t2k−/− EFs were incubated with 10 ng/ml of either mouse recombinant TNFα or IL-1β for 27 min. RelA binding activity was assessed by incubation of 10 μg of nuclear extract with RelA-specific antibody followed by electrophoretic mobility supershift assay, as described in Materials and methods. Download figure Download PowerPoint NF-κB DNA binding activity in nuclear extracts of wild-type and t2k−/− EFs was then assessed by electrophoretic mobility shift assay (EMSA) using a labeled oligonucleotide containing κB binding sites. Stimulation with either TNFα or IL-1, as above, resulted in a rapid induction of κB binding activity in nuclear extracts from cells of both genotypes (Figure 5B). Together these data show that, at least in EFs, T2K is not essential for either phosphorylation-induced degradation of I-κB or NF-κB DNA binding in response to TNFα or IL-1. To assess the contribution of RelA to the κB DNA binding activity observed in Figure 5B, nuclear extracts from either wild-type or t2k−/− EFs, stimulated with either TNFα or IL-1, were pre-incubated with RelA-specific Ab prior to EMSA analysis. The κB DNA binding activity induced by either cytokine was reactive to the RelA-specific antibody, causing a supershift (Figure 5C). Furthermore, RelA-reactive κB DNA binding activity could account for the majority of the activity detected in extracts from both wild-type and t2k−/− EFs (Figure 5C), indicating that T2K is not essential for RelA DNA binding in response to TNFα or IL-1. Impaired NF-κB-dependent gene expression in t2k−/− cells NF-κB-dependent gene expression was assessed both by a reporter assay
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