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• W2114379972 abstract "To elucidate the function of Bcl10, recently cloned as an apoptosis-associated gene mutated in MALT lymphoma, we identified its binding partner TRAF2, which mediates signaling via tumor necrosis factor receptors. In mammalian cells, low levels of Bcl10 expression promoted the binding of TRAF2 and c-IAPs. Conversely, excessive expression inhibited complex formation. Overexpressed Bcl10 reduced c-Jun N-terminal kinase activation and induced nuclear factor κB activation downstream of TRAF2. To determine whether overexpression of Bcl10 could perturb the regulation of apoptosisin vivo, we generated Bcl10 transgenic mice. In these transgenic mice, atrophy of the thymus and spleen was observed at postnatal stages. The morphological changes in these tissues were caused by acceleration of apoptosis in T cells and B cells. The phenotype of Bcl10 transgenic mice was similar to that of TRAF2-deficient mice reported previously, indicating that excessive expression of Bcl10 might deplete the TRAF2 function. In contrast, in the other organs such as the brain, where Bcl10 was expressed at high levels, no apoptosis was detected. The altered sensitivities to overexpressed Bcl10 may have been due to differences in signal responses to Bcl10 among cell types. Thus, Bcl10 was suggested to play crucial roles in the modulation of apoptosis associated with TRAF2. To elucidate the function of Bcl10, recently cloned as an apoptosis-associated gene mutated in MALT lymphoma, we identified its binding partner TRAF2, which mediates signaling via tumor necrosis factor receptors. In mammalian cells, low levels of Bcl10 expression promoted the binding of TRAF2 and c-IAPs. Conversely, excessive expression inhibited complex formation. Overexpressed Bcl10 reduced c-Jun N-terminal kinase activation and induced nuclear factor κB activation downstream of TRAF2. To determine whether overexpression of Bcl10 could perturb the regulation of apoptosisin vivo, we generated Bcl10 transgenic mice. In these transgenic mice, atrophy of the thymus and spleen was observed at postnatal stages. The morphological changes in these tissues were caused by acceleration of apoptosis in T cells and B cells. The phenotype of Bcl10 transgenic mice was similar to that of TRAF2-deficient mice reported previously, indicating that excessive expression of Bcl10 might deplete the TRAF2 function. In contrast, in the other organs such as the brain, where Bcl10 was expressed at high levels, no apoptosis was detected. The altered sensitivities to overexpressed Bcl10 may have been due to differences in signal responses to Bcl10 among cell types. Thus, Bcl10 was suggested to play crucial roles in the modulation of apoptosis associated with TRAF2. caspase recruitment domain DNA Data Bank of Japan inhibitor of apoptosis c-Jun N-terminal kinase stress-activated protein kinase mucosa-associated lymphoid tissue nuclear factor transgenic mice tumor necrosis factor tumor necrosis factor receptor tumor necrosis factor receptor-associated factor TdT-mediated dUTP-biotin nick end labeling . hemagglutinin aminomethylcoumarin polymerase chain reaction secretory alkaline phosphatase Apoptosis is an indispensable phenomenon for development of most multicellular organisms (1.Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5085) Google Scholar, 2.Evan G. Littlewood T. Science. 1998; 281: 1317-1322Crossref PubMed Scopus (1359) Google Scholar). The components of the apoptosis pathways have been identified, and their functions have gradually been elucidated. Among these components, caspases are known to be major effector molecules of the execution of apoptosis. These molecules are cysteine proteases that possess a distinct fold and cleave specific aspartate-containing sites in many proteins involved in apoptosis (3.Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6112) Google Scholar). Caspases are regulated by adapter molecules mediated by a homophilic association of structurally related protein molecules. In these caspase-regulating proteins, three types of homology interaction domains have been identified including the death domain, the death effector domain, and the caspase recruitment domain (CARD)1 (4.Hofmann K. Bucher P. Tschopp J. Trends Biochem. Sci. 1997; 22: 155-156Abstract Full Text PDF PubMed Scopus (447) Google Scholar). All characterized proteins found to contain the CARD domain have been reported to act in apoptotic signaling and have been suggested to mediate the binding between adapter molecules and caspases (4.Hofmann K. Bucher P. Tschopp J. Trends Biochem. Sci. 1997; 22: 155-156Abstract Full Text PDF PubMed Scopus (447) Google Scholar). Apaf-1, a mitochondrial protein related to the Caenorhabditis elegans protein CED-4, contains a CARD domain in its N-terminal region and recruits CARD-containing caspase-9. The CARD-containing adapter molecule RAIDD recruits caspase-2 to the tumor necrosis factor receptor-1 (TNFR1) (5.Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2716) Google Scholar). Other CARD-containing molecules have recently been cloned, i.e. ARC, c-IAPs, and RICK/RIP2/CARDIAK (6.Koseki T. Inohara N. Chen S. Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5156-5160Crossref PubMed Scopus (305) Google Scholar, 7.Rothe M. Pan M.G. Henzel W.J. Ayres T.M. Goeddel D.V. Cell. 1995; 83: 1243-1252Abstract Full Text PDF PubMed Scopus (1045) Google Scholar, 8.Thome M. Hofmann K. Burns K. Martinon F. Bodmer J.L. Mattmann C. Tschopp J. Curr. Biol. 1998; 8: 885-888Abstract Full Text Full Text PDF PubMed Google Scholar). We attempted to clone the CARD-containing proteins that regulate apoptotic signaling. A human expressed sequence tag clone (GenBankTM accession no. AA455396) was found to have significant sequence homology to the CARD of RAIDD. The full-length cDNA was obtained using the expressed sequence tag cDNA fragment as a probe, and the gene products were identified with Bcl10, cloned previously as an apoptotic regulatory gene due to its direct involvement in the t(1;14)(p22;q32) of low grade B cell lymphomas of mucosa-associated lymphoid tissue (MALT lymphomas) (9.Willis T.G. Jadayel D.M. Du M.Q. Peng H. Perry A.R. Abdul R.M. Price H. Karran L. Majekodunmi O. Wlodarska I. Pan L. Crook T. Hamoudi R. Isaacson P.G. Dyer M.J. Cell. 1999; 96: 35-45Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). Overexpression of Bcl10 in cultured cells was reported to activate nuclear factor (NF)-κB transcriptional pathways (10.Koseki T. Inohara N. Chen S. Carrio R. Merino J. Hottiger M.O. Nabel G.J. Nunez G. J. Biol. Chem. 1999; 274: 9955-9961Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 11.Thome M. Martinon F. Hofmann K. Rubio V. Steiner V. Schneider P. Mattmann C. Tschopp J. J. Biol. Chem. 1999; 274: 9962-9968Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 12.Yan M. Lee J. Schilbach S. Goddard A. Dixit V. J. Biol. Chem. 1999; 274: 10287-10292Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and promote apoptosis. However, the mechanisms of regulation of apoptosis by Bcl10, and the pathophysiology of MALT lymphomas by Bcl10 mutation with the t (1;14)(p22;q32) are not known. Here, we report the identification of a protein associated with Bcl10 to examine the roles of Bcl10 in apoptotic signaling, using the yeast two-hybrid system. This protein is TRAF2, which is known to mediate signaling by the TNFR. We demonstrated that Bcl10 changes the interactions between TRAF2 and its associated proteins and modulates downstream signaling molecules. Furthermore, we generated transgenic (TG) mice expressing Bcl10 to investigate whether Bcl10 is also involved in the apoptotic pathway in vivo. Our results showed that Bcl10 plays significant roles in the modulation of apoptosis in vitro and in vivo. Animal studies were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Using the FASTA program, the partial nucleotide sequences of mouse cDNAs encoding peptides with homology of the CARD domain of RAIDD were found in the DDBJ expressed sequence tag data bases. We obtained the rat cDNA for the open reading frame by reverse transcription-PCR based on homology between mouse and rat. Reverse transcription-PCR was performed with a pair of degenerate primers, 5′-ATGACAGTGGATGCCCTCAN-3′ and 5′-CTGAATCAGGAAGCTCTGTN-3′. A cDNA library of rat forebrain at embryonic day 11, which was previously constructed in λZAP II (13.Yoneda T. Sato M. Maeda M. Takagi H. Brain Res. Mol. Brain Res. 1998; 62: 187-195Crossref PubMed Scopus (53) Google Scholar), was screened using the DNA fragment. The full-length cDNA was obtained, and we determined the entire nucleotide sequence by dideoxy sequencing (Perkin-Elmer). Although we submitted the sequence as the rat RCD (DDBJ accession no. AB016069), other groups reported mouse and human homologues of this gene as Bcl10/CIPER/CARMEN/mE10/hE10 (9.Willis T.G. Jadayel D.M. Du M.Q. Peng H. Perry A.R. Abdul R.M. Price H. Karran L. Majekodunmi O. Wlodarska I. Pan L. Crook T. Hamoudi R. Isaacson P.G. Dyer M.J. Cell. 1999; 96: 35-45Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, 10.Koseki T. Inohara N. Chen S. Carrio R. Merino J. Hottiger M.O. Nabel G.J. Nunez G. J. Biol. Chem. 1999; 274: 9955-9961Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 11.Thome M. Martinon F. Hofmann K. Rubio V. Steiner V. Schneider P. Mattmann C. Tschopp J. J. Biol. Chem. 1999; 274: 9962-9968Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 12.Yan M. Lee J. Schilbach S. Goddard A. Dixit V. J. Biol. Chem. 1999; 274: 10287-10292Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) while this manuscript was in preparation. To analyze the function of Bcl10 in the apoptotic pathway, we employed the yeast two-hybrid system to search for proteins that directly interact with Bcl10. Yeast cells, strain PJ69-2A, were transformed with an expression vector encoding the GAL4 DNA-binding domain combined with Bcl10. Then, the transformed yeast was mated with the yeast strain Y187, pretransformed with an expression vector containing the cDNA library fused to its DNA activation domain. We used two pretransformed MATCHMAKER libraries (mRNA sources: normal, whole brain pooled from 9 spontaneously aborted male/female Caucasian fetuses, aged 20–25 weeks (3.5 × 106 independent transformants) and normal, whole brain from a 37-year-old Caucasian man, cause of death; trauma (5 × 106 independent transformants)CLONTECH). Subsequent β-galactosidase assays were performed according to the manufacturer's protocol. As positive and negative controls, we used packaged transformants supplied with screening system. Aliquots of 5 × 106 293T cells were transfected with expression plasmids using LipofectAMINE transfection reagent (Life Technologies, Inc.) according to the manufacturer's protocol. The total amount of transfected plasmid DNA was adjusted to be the same within individual experiments. Cells were harvested at 24 h after transfection and lysed with 0.2% Nonidet P-40 in Dulbecco's phosphate-buffered saline. Subsequently, 1 mg of soluble protein was incubated with 1 μg/ml antibody for 2 h at 4 °C, and proteins captured with this antibody were coprecipitated with protein G-agarose (Life Technologies, Inc.). Immunoprecipitates or cell lysates were loaded onto 5–20% gradient SDS-polyacrylamide gels, electrophoresed, and immunoblotted with antibodies for detection. Materials used in the immunoprecipitation experiments were as follows: antibodies reacting specifically against FLAG-tag (Sigma), HA-tag (Babco), TRAF2, and c-IAP-1 and -2 (Santa Cruz). The peptide-column-purified rabbit polyclonal anti-Bcl10 antibody was raised against oligopeptides (amino acids 220–233) in our laboratory. Expression vectors were obtained from the following sources: pcDNA c-IAP-1 and -2 were kind gifts from Dr. Ryosuke Takahashi (14.Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1129) Google Scholar). pcDNA-Bcl10-HA, pcDNA-Bcl10-FLAG, and pcDNA-FLAG-TRAF2 were cloned in our laboratory. Plasmids carrying human or mouse Bcl10 were purchased from American Type Culture Collection. For in vivo metabolic labeling with32Pi, 293T cells in six-well plates were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum. One hour before labeling, medium was removed and the cells were washed twice with Dulbecco's phosphate-buffered saline (pH 7.4). The cells were resuspended in phosphate-free minimal essential medium (Sigma) supplemented with 10% dialyzed fetal calf serum (Life Technologies, Inc.) and incubated at 37 °C for 30 min. 32Pi (NEN Life Science Products) was added to the cultures at a final radioactivity of 150 μCi/ml. The cells were incubated at 37 °C for another 3 h. The 32Pi-labeled cells were washed three times with Dulbecco's phosphate-buffered saline and lysed with lysis buffer. Radiolabeled lysate from each sample was immunoprecipitated with anti-Bcl10 antibodies or anti-TRAF2. The precipitates were separated by 5–20% SDS-polyacrylamide gel electrophoresis and subsequently electrotransferred onto polyvinylidene difluoride filters (Millipore). The filters were exposed to x-ray film for detection of32P. In this procedure, to control for loading, the filters were stained with anti-Bcl10 antibody after autoradiography. The activation of JNK/SAPK in 293T cells transfected with mock vector or with pcDNA-Bcl10 was examined at 30 min after treatment with 0, 5 or 20 ng/ml human TNF-α. These experiments were performed using a SAPK/JNK assay kit (New England Biolabs), and immunoprecipitation was performed with anti-JNK1 and -JNK2 antibodies (Santa Cruz) followed by Western blotting with phosphospecific anti-JNK antibody (Promega). Transfection, immunoprecipitation, and Western blotting were performed essentially as described above. To monitor the activation of NFκB, 293T cells were cotransfected with 1 μg of pNFκB-SEAP transcription reporter vector (CLONTECH) plus 2 μg of mock or Bcl10 expression plasmid. At 6 h after medium exchange with 0, 5, or 20 ng/ml human TNF-α, 20 μl of conditioned medium was collected, and activities of NFκB were determined according to the manufacturer's protocol. Total protein from 293T cells transfected with pcDNA-mock or pcDNA-Bcl10 vectors was obtained by homogenizing cells in a lysis buffer that contained 50 mm Tris-HCl (pH 7.4), 1 mm EDTA, 10 mm EGTA, 10 μm digitonin, 10 mmdithiothreitol. The amount of total protein in each sample was determined by the BCA protein assay (Pierce), and the samples were stored at −80 °C. Caspase-3/caspase-7-like activity was assessed by measuring cleavage of the fluorogenic substrateN-acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (Ac-DEVD-AMC) using a luminescence spectrometer. Cleavage of Ac-DEVD-AMC was measured for each sample of 40 μg of total protein (accumulation of fluorescence was linear for at least 2 h). The rate of fluorescence accumulation was calculated as the activity of a given enzyme. Experiments were repeated three times with each sample. Bcl10 TG mice were generated using the mouse prion promoter (a kind gift from Dr. D. R. Borchelt). The purified transgene fragments were injected into BCF1×BCF1 fertilized eggs, and incorporation of the transgene was examined by PCR of DNA extracted from the tail. Expression of Bcl10 was confirmed by immunoblotting analysis (data not shown). One-month-old Bcl10 TG mice and control littermates were sacrificed with chloroform. Organs were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned (5 μm thick). Sectioned tissues were stained with hematoxylin and eosin, and subjected to immunochemical and apoptotic assays. Histochemical analyses were performed with specific antibodies as described previously (15.Maeda M. Sugiyama T. Akai F. Jikihara I. Hayashi Y. Takagi H. Neurosci. Lett. 1998; 240: 69-72Crossref PubMed Scopus (45) Google Scholar). We used polyclonal antibodies to mouse CD3ε (PharMingen) and mouse CD45R (PharMingen) as T cell and B cell markers, respectively, and phosphospecific anti-JNK (Promega) for detection of activated JNK in vivo (data not shown). Three different methods were used to detect cell death in the thymus. First, sections were stained by the TUNEL method using an Apoptag in situapoptosis detection kit (Oncor) according to the manufacturer's instructions. Second, we used a monoclonal anti-ssDNA antibody supplied with an Apoptosis detection kit (Kamiya Biomedical Co.). Finally, morphological analyses of the ultrastructure of the outer thymic cortex were performed. Immunohistochemistry with anti-ssDNA antibody and electron microscopy were performed as described previously. Counterstaining was performed with methyl green. All characterized proteins found to contain the CARD domain have been reported to act in apoptotic signaling (1.Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5085) Google Scholar). Previous experiments have demonstrated that Bcl10 also plays an important role in apoptosis. We confirmed that Bcl10 promoted apoptosis in 293T cells (data not shown) and induced caspase-3/caspase-7-like protease activities (Fig. 3 C). To analyze the function of Bcl10 in the apoptotic pathway, we employed the yeast two-hybrid system to search for proteins that directly interact with Bcl10. Three positive clones encoding an identical protein, human TRAP3, were obtained from two cDNA sources (two clones from a human fetus brain cDNA library and one from a human adult brain cDNA library). Human TRAP3 is a human homologue of mouse TRAF2 (84.3% amino acid identity). Interactions between Bcl10 and TRAF2 were verified in yeast (Fig. 1).Figure 1Screening for proteins that interact with Bcl10 with the yeast two-hybrid system. Interactions between Bcl10 and TRAF2 in yeast with filter assays for β-galactosidase activity. A transformant with murine p53 and SV40 large T antigen, which are known to interact in the yeast two-hybrid assay, was used as a positive control. Positive signals at the same level as the positive control were obtained with the Bcl10 and TRAF2 transformants. (Clone E was from the human fetus brain cDNA library and clone A from the human adult brain cDNA library.) No positive reaction and scarce growth were observed with the Bcl10 and SV40 large T antigen transformant as a negative control. Quantitative liquid β-galactosidase assay was performed on individual colonies.View Large Image Figure ViewerDownload (PPT) To confirm the interaction in mammalian cells, expression vectors of wild-type or truncated forms of Bcl10 tagged with the HA epitope at their C termini and TRAF2 were co-transfected into 293T cells, and we carried out immunoprecipitation analyses followed by Western blotting. Immunoprecipitation with anti-TRAF2 antibody revealed that the 32-kDa full-length Bcl10 (Bcl10-F-HA) and the deletion mutant (amino acids 1–110) containing the CARD domain (Bcl10-N-HA) were co-immunoprecipitated with TRAF2, but the other deletion mutant (amino acids 120–233) containing the C-terminal region of Bcl10 was not (Fig.2 A). Co-immunoprecipitation was also performed in the opposite direction (Fig. 2 B). These results indicated that TRAF2 interacts with Bcl10 and that the binding is CARD-dependent. TRAF2 is an intracellular signal-transducing protein recruited to TNFR1 and TNFR2 following TNF stimulation. TNF affects the activities of the transcription factors NFκB and the stress-activated protein kinase JNK/SAPK, leading to induction of proinflammatory and immunomodulatory genes (16.Costanzo A. Guiet C. Vito P. J. Biol. Chem. 1999; 274: 20127-20132Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). To determine whether the activation of these signaling pathways is changed in the presence of excessive levels of Bcl10, we examined the activities of JNK/SAPK and activation of NFκB in 293T cells transfected with Bcl10 constructs. After treatment with TNF for 30 min, JNK/SAPK was activated in 293T cells transfected with mock vector. Expression of Bcl10 inhibited the activation of JNK/SAPK by TNF stimulation (Fig. 3 A). Consistent with this result, immunoprecipitation with anti-JNK1 antibody followed by Western blotting with phosphospecific anti-JNK antibody showed that the level of phosphorylated-JNK1 was reduced in 293T cells expressing Bcl10 (Fig. 3 A). We next examined the effects of Bcl10 expression on the NFκB signaling pathway. Expression of Bcl10 in 293T cells increased alkaline phosphatase reporter activities in a dose-dependent manner, which was consistent with previous findings (9.Willis T.G. Jadayel D.M. Du M.Q. Peng H. Perry A.R. Abdul R.M. Price H. Karran L. Majekodunmi O. Wlodarska I. Pan L. Crook T. Hamoudi R. Isaacson P.G. Dyer M.J. Cell. 1999; 96: 35-45Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, 10.Koseki T. Inohara N. Chen S. Carrio R. Merino J. Hottiger M.O. Nabel G.J. Nunez G. J. Biol. Chem. 1999; 274: 9955-9961Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 11.Thome M. Martinon F. Hofmann K. Rubio V. Steiner V. Schneider P. Mattmann C. Tschopp J. J. Biol. Chem. 1999; 274: 9962-9968Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Treatment of various cells with TNF has been shown to induce NFκB activity (16.Costanzo A. Guiet C. Vito P. J. Biol. Chem. 1999; 274: 20127-20132Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), and this was confirmed in the present study. Transient transfection of 293T cells with Bcl10 cDNA enhanced the activation of NFκB by TNF stimulation, suggesting that Bcl10 could regulate the TNF-signaling pathway directly or indirectly (Fig. 3 B). TRAF2 is known to be involved in the activation of JNK and NFκB pathways and to interact with c-IAPs directly, complexes of which affect the regulation of apoptosis (1.Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5085) Google Scholar, 7.Rothe M. Pan M.G. Henzel W.J. Ayres T.M. Goeddel D.V. Cell. 1995; 83: 1243-1252Abstract Full Text PDF PubMed Scopus (1045) Google Scholar). We considered that Bcl10 may alter the complex formation of TRAF2 and c-IAPs, and the proportion of binding to TRAF2 by these molecules may determine the efficiency of TNF signaling because Bcl10 potentially interacts with TRAF2 and the expression of Bcl10 affects the signaling pathways of JNK and NFκB. Therefore, we examined whether Bcl10 expression regulates complex formation between TRAF2 and c-IAP1 and c-IAP2 (Fig.4 A). Intracellular binding between c-IAP1 and TRAF2 is enhanced in the presence of low levels of Bcl10. Surprisingly, the migration of c-IAP1 molecules that interacted with TRAF2 was slower, suggesting that post-translational modification of c-IAP1 is caused by expression of Bcl10. In contrast, the presence of excessive amounts of Bcl10 reduced the interaction between c-IAP1 and TRAF2. Under these conditions, Bcl10 showed direct binding or indirect binding through TRAF2 to c-IAP1 (Fig. 4 A). Similar results were also obtained with c-IAP2 substituted for c-IAP1 (data not shown). Western blotting analysis for Bcl10 showed a 32–35-kDa double band (Fig. 4 A). This shifted band was demonstrated to be phosphorylated Bcl10 by metabolic labeling experiments using32Pi (Fig. 4 B). Our results also showed that phosphorylated Bcl10 was co-immunoprecipitated with TRAF2. Having demonstrated that Bcl10 binds to TRAF2 directly, and alters the levels of complex formation of TRAF2-c-IAPs, regulating the signaling pathways of NFκB and JNK, it was of interest to determine whether overexpression of Bcl10 in vivo could perturb the regulation of apoptosis in various tissues. We generated TG mice expressing Bcl10 under the control of the prion promoter. Twelve independently derived mouse lines behaved similarly in all subsequent analyses, thereby confirming that the observed effects were due to transgene expression and not chromosomal integration site. No developmental or morphological abnormalities were observed in TG mice during embryonic or neonatal stages. In contrast, all lines of Bcl10-TG mice showed normal development of their four extremities, but their body trunks were devoid of fat deposits and they showed reduced body weight gain compared with wild-type littermates (Fig.5 A). The TG animals became smaller with time, and 6 TG mice died by 4 weeks of age with only 3 surviving longer than 2 months. At the time of death, the body weights of TG animals ranged from 40% to 70% of those of wild-type littermates. Histological studies revealed that most organs (brain, heart, lung, stomach, small intestine, colon, liver, pancreas, kidney, adrenal grand) of Bcl10 TG mice, while proportionally smaller than those of control littermates, showed no gross developmental abnormalities. However, TG mice exhibited distinct changes in the architecture of the thymus and spleen with accelerated involution. The thymus of TG mice was extremely atrophic, its cortex was thinner, the number of lymphocytes was markedly decreased, and the lobules consisted almost entirely of medullary structures with cystic changes of Hassall's corpuscles (Fig. 5 C). The spleen of TG mice was also atrophic, and on histological analyses lymphoid nodules were seen to be reduced in both number and size (data not shown). One case showed complete disappearance of lymphoid nodules. The remaining pulp cells showed disseminated massive nuclear condensation or fragmentation of lymphocytes. Immunohistochemical analyses showed that CD45R, a B cell marker, immunoreactivity was markedly decreased in the TG spleen (Fig.5 B) and was depleted in the TG thymus (data not shown). Cells immunoreactive for the T cell marker CD3ε were also significantly decreased in number in the TG thymus (data not shown). To elucidate whether lymphoid depletion in the thymus cortex and spleen of Bcl10 TG animals was due to acceleration of abnormal cell death, we carried out ultrastructual analysis. Degeneration of thymocytes in the cortex was characterized by atrophy of cell bodies and nuclear condensation or fragmentation (Fig.6 C). Analysis by the in situ TUNEL method and immunohistochemistry with anti-ssDNA antibody confirmed that the cell death was due to apoptosis (Fig. 6,A and B). We identified TRAF2 as a binding partner of Bcl10 using the yeast two-hybrid system. Bcl10 directly interacted with TRAF2 in a CARD-dependent manner. Some other groups reported that Bcl10 failed to interact with TRAF2 using immunoprecipitation analyses (10.Koseki T. Inohara N. Chen S. Carrio R. Merino J. Hottiger M.O. Nabel G.J. Nunez G. J. Biol. Chem. 1999; 274: 9955-9961Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 11.Thome M. Martinon F. Hofmann K. Rubio V. Steiner V. Schneider P. Mattmann C. Tschopp J. J. Biol. Chem. 1999; 274: 9962-9968Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 20.Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (968) Google Scholar). We cannot explain these different results. One possibility is the differences of antibodies used in the immunoprecipitated experiments. We detected binding between Bcl10 and TRAF2 only with the anti-N-terminal TRAF2 antibody. Anti-C-terminal TRAF2 antibody may block the binding site of TRAF2. Several proteins have been reported to bind with Bcl10 such as TRAF1, TRAF5, and TRADD (11.Thome M. Martinon F. Hofmann K. Rubio V. Steiner V. Schneider P. Mattmann C. Tschopp J. J. Biol. Chem. 1999; 274: 9962-9968Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 20.Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (968) Google Scholar). Interactions of Bcl10 with other members of the TRAF family may be due to their structural similarity with TRAF2. TRADD may be co-immunoprecipitated with Bcl10 through TRAF2 indirectly, or may bind with both TRAF2 and Bcl10. Further studies are needed to clarify the mechanisms of complex formation between Bcl10 and other binding partners. The members of the TRAF family are cytoplasmic adapter proteins known to mediate signaling events specifically for members of the TNFR family. The TNF stimulation induces NFκB and JNK/SAPK mediated by TRAF2 and its associated proteins such as c-IAPs. In this study, we analyzed the binding of TRAF2 to Bcl10 and c-IAP1. Binding activities of TRAF2 and c-IAP1 were increased at low levels of Bcl10 expression. Conversely, at high levels, Bcl10 inhibited complex formation. Although the mechanism by which complex formation was regulated by the Bcl10 expression level remains to be elucidated, Bcl10 may activate its cellular targets, such as an undefined kinase, which promotes complex formation of TRAF2 and c-IAPs. Indeed, a low level of Bcl10 expression altered the molecular weights of c-IAPs, which are likely to be phosphorylated forms. In contrast, high levels of Bcl10 decreased the amounts of TRAF2 and c-IAPs complexes, and conversely promoted the binding of Bcl10 both to TRAF2 and to c-IAPs. Thus, binding of TRAF2 and c-IAP1 was likely to be competitively inhibited by excessive amounts of Bcl10. Further analyses are needed to determine the mechanisms responsible for regulation of complex formation by these molecules. However, it is possible that Bcl10 regulates the signaling pathways of NFκB and JNK by altering the patterns of complex formation. Bcl10 TG mice revealed lymphoid depletion in the thymus and spleen, caused by acceleration of apoptosis in both B and T lymphocytes. The mouse prion promoter is known to be capable of driving low levels of transgene expression in a variety of tissues, with the highest expression occurring in the brain and heart (18.Borchelt D.R. Davis J. Fischer M. Lee M.K. Slunt H.H. Ratovitsky T. Regard J. Copeland N.G. Jenkins N.A. Sisodia S.S. Price D.L. Genet. Anal. 1996; 13: 159-163Crossref PubMed Scopus (297) Google Scholar). We detected no morphological abnormalities in any organs other than lymphoid tissues. The altered sensitivities to exogenous expression of Bcl10 may have been due to the differences of signal responses to Bcl10 among cell types. Similar findings of the thymus and spleen in Bcl10 TG mice were previously reported in TRAF2-deficient mice (19.Yeh W.C. Shahinian A. Speiser D. Kraunus J. Billia F. Wakeham A. de la Pompa J. Ferrick D. Hum B. Iscove N. Ohashi P. Rothe M. Goeddel D.V. Mak T.W. Immunity. 1997; 7: 715-725Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). In addition, similarly to Bcl10 TG mice, TRAF2-deficient mice appeared normal at birth and became smaller and devoid of fat deposits during postnatal development. TRAF2−/− mutants also died prematurely. The TRAF2-deficient cells showed severe reduction of TNF-mediated JNK/SAPK activation. Overexpression of Bcl10 in cultured cells attenuated the JNK signaling pathway activated by TNF stimulation, and immunoreactivity of phosphorylated JNK was reduced in the thymus of the Bcl10 TG mice (data not shown). The phenotypic similarities in both animal models and cultured cells imply that Bcl10 depletes the function of TRAF2, which is known to act as an anti-apoptotic factor in nature (20.Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (968) Google Scholar). The results of binding analysis of TRAF2, c-IAPs, and Bcl10 suggested that excessive expression of Bcl10 in lymphatic tissues of the TG mice inhibits TRAF2 and c-IAPs complex formation (Fig. 4 A). Consequent down-regulation of the TNFR-TRAF2 signaling pathway, which has an anti-apoptotic effect, could be attenuated and cause the reduction of JNK/SAPK activities. Thymocytes from TRAF2-deficient mice were reduced in total cell number and were devoid of CD4+/CD8+ cells at terminal stages. However, those of younger knock-out mice showed a profile of surface markers similar to the wild-type (19.Yeh W.C. Shahinian A. Speiser D. Kraunus J. Billia F. Wakeham A. de la Pompa J. Ferrick D. Hum B. Iscove N. Ohashi P. Rothe M. Goeddel D.V. Mak T.W. Immunity. 1997; 7: 715-725Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). Bcl10-TG mice also showed normal development of the thymus at birth, but subsequently exhibited progressive atrophy of the thymic cortex, in which apoptotic cell numbers were increased. Taken together with the findings in TRAF2-deficient mice, thymocytes appeared to develop normally even when Bcl10 was expressed at excessive levels or TRAF2 was absent in the thymus, indicating no requirement of the TNFR-TRAF2 pathway regulated by Bcl10 and c-IAPs. After the maturation of T cells, deregulation of the TNFR signaling pathway is considered to promote apoptosis in thymocytes through reduction of phosphorylated JNK levels. In the case of B cells, levels of immunoreactivities of the B cell marker CD45R in secondary follicles of the Bcl10-TG spleen were markedly decreased. In addition, TUNEL- and ssDNA-positive cells were frequently observed in the same regions, suggesting that overexpression of Bcl10 also causes apoptosis of B cells as well as T cells. Our results suggested that Bcl10 plays important roles in lymphocyte apoptosis, but we have not elucidated the precise roles of Bcl10 during the lymphocyte differentiation in detail. Studies to clarify the detailed roles of Bcl10 are currently in progress in our laboratory. Bcl10 was cloned due to its direct involvement in t(1;14)(p22;q32) of low grade MALT lymphoma (9.Willis T.G. Jadayel D.M. Du M.Q. Peng H. Perry A.R. Abdul R.M. Price H. Karran L. Majekodunmi O. Wlodarska I. Pan L. Crook T. Hamoudi R. Isaacson P.G. Dyer M.J. Cell. 1999; 96: 35-45Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, 21.Zhang Q. Siebert R. Yan M. Hinzmann B. Cui X. Xue L. Rakestraw K.M. Naeve C.W. Beckmann G. Weisenburger D.D. Sanger W.G. Nowotny H. Vesely M. Callet B.E. Salles G. Dixit V.M. Rosenthal A. Schlegelberger B. Morris S.W. Nat. Genet. 1999; 22: 63-68Crossref PubMed Scopus (335) Google Scholar). Bcl10 expressed in a MALT lymphoma is known to exhibit a frameshift mutation resulting in truncation distal to the CARD. Similar mutations were detected in other tumor types including follicular lymphoma and Sezary syndrome. Although overexpression of Bcl10 induces apoptosis, truncation mutants of Bcl10 lost their proapoptotic functions, suggesting that these mutants have lost their apoptotic activity and concomitant gain of proproliferative function in human cancers, i.e. loss of Bcl10 function, might cause tumor development. Conversely, as shown in the present study, the excessive expression of Bcl10 in vivo was confirmed to induce apoptosis in specific tissues. These results indicated that a small change in the Bcl10 expression level could perturb the precise regulation of apoptosis and proliferation in specific cells through the modification of NFκB and JNK pathways. In conclusion, the present study suggested that Bcl10 affects the signaling pathway associated with TRAF2 and plays significant roles in the regulation of apoptosis in vitro and in vivo. Our results also seem to be important for understanding the pathogenesis of a wide range of cancers. Furthermore, regulation of Bcl10 functions or the TNFR-TRAF2 pathway could allow for the development of therapeutic strategies for human cancers in hematopoietic and lymphatic tissues. We thank our colleagues who participated in this work (in alphabetical order): Sachiyo Funai, Ikuyo Jikihara, Kaoru Nakano, and Eri Yasuoka." @default.
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• W2114379972 title "Regulatory Mechanisms of TRAF2-mediated Signal Transduction by Bcl10, a MALT Lymphoma-associated Protein" @default.
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