Publications by authors named "Rika Ouchida"

28 Publications

  • Page 1 of 1

Altered microbiota composition reflects enhanced communication in 15q11-13 CNV mice.

Neurosci Res 2020 Dec 18;161:59-67. Epub 2019 Dec 18.

RIKEN Brain Science Institute, Wako, Saitama, 351-0198, Japan; Graduate School of Biomedical and Health Sciences, Hiroshima University, Minami, Hiroshima, 734-8553, Japan; RIKEN Center for Science and Technology Hub, Medical Sciences Innovation Hub Program (MIH), Japan; Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Chuo, Kobe, 650-0017, Japan. Electronic address:

Autism spectrum disorder (ASD) is a complex and heterogeneous neurodevelopmental disorder. In addition to the core symptoms of ASD, many patients with ASD also show comorbid gut dysbiosis, which may lead to various gastrointestinal (GI) problems. Intriguingly, there is evidence that gut microbiota communicate with the central nervous system to modulate behavioral output through the gut-brain axis. To investigate how the microbiota composition is changed in ASD and to identify which microbes are involved in autistic behaviors, we performed a 16S rRNA gene-based metagenomics analysis in an ASD mouse model. Here, we focused on a model with human 15q11-13 duplication (15q dup), the most frequent chromosomal aberration or copy number variation found in ASD. Species diversity of the microbiome was significantly decreased in 15q dup mice. A combination of antibiotics treatment and behavioral analysis showed that neomycin improved social communication in 15q dup mice. Furthermore, comparison of the microbiota composition of mice treated with different antibiotics enabled us to identify beneficial operational taxonomic units (OTUs) for ultrasonic vocalization.
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http://dx.doi.org/10.1016/j.neures.2019.12.010DOI Listing
December 2020

Distinct roles of BCNP1 in B-cell development and activation.

Int Immunol 2020 01;32(1):17-26

Department of Immunology, School of Basic Medical Sciences.

B-cell novel protein 1 (BCNP1) has recently been identified as a new B-cell receptor (BCR) signaling molecule but its physiological function remains unknown. Here, we demonstrate that mice deficient in BCNP1 exhibit impaired B-cell maturation and a reduction of B-1a cells. BCNP1-deficient spleen B cells show enhanced survival, proliferation and Ca2+ influx in response to BCR cross-linking as compared with wild-type spleen B cells. Consistently, mutant B cells show elevated phosphorylation of SYK, B-cell linker protein (BLNK) and PLCγ2 upon BCR cross-linking. In vivo, BCNP1-deficient mice exhibit enhanced humoral immune responses to T-independent and T-dependent antigens. Moreover, aged mutant mice contain elevated levels of serum IgM and IgG3 antibodies and exhibit polyclonal and monoclonal B-cell expansion in lymphoid organs. These results reveal distinct roles for BCNP1 in B-cell development, activation and homeostasis.
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http://dx.doi.org/10.1093/intimm/dxz055DOI Listing
January 2020

The B cell novel protein 1 (BCNP1) regulates BCR signaling and B cell apoptosis.

Eur J Immunol 2019 06 28;49(6):911-917. Epub 2019 Mar 28.

Department of Immunology, School of Basic Medical Sciences, Fudan University, Shanghai, China.

The BCR plays a central role in B cell development, survival, activation, and differentiation. We have identified the B cell novel protein 1 (BCNP1) as a new regulator of BCR signaling. BCNP1 contains a pleckstrin homology domain, three proline-rich motifs, and a potential SH2 binding site, and is predominantly expressed by B cells. We found that BCNP1 overexpression in WEHI231 immature B cells potentiated α-IgM-induced apoptosis. Conversely, BCNP1-deficient WEHI231 cells, generated by CRISPR-Cas9-mediated genome editing, exhibited reduced apoptosis after BCR crosslinking. Biochemical analyses revealed that BCNP1 physically interacted with the B cell linker protein (BLNK), Grb2, and PLCγ2. Moreover, absence of BCNP1 resulted in accelerated dephosphorylation of BLNK, reduced phosphorylation of SYK and PLCγ2, and decreased Ca influx after BCR crosslinking. These results demonstrate that BCNP1 promotes BCR signaling by modulating the phosphorylation of BLNK, SYK, and PLCγ2.
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http://dx.doi.org/10.1002/eji.201847985DOI Listing
June 2019

FcμR interacts and cooperates with the B cell receptor To promote B cell survival.

J Immunol 2015 Apr 2;194(7):3096-101. Epub 2015 Mar 2.

Department of Immunology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China; Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan; Biotherapy Research Center, Fudan University, Shanghai 200032, China; and Immunobiology Institute, Fudan University, Shanghai 200032, China

The IgM FcR (FcμR) promotes B cell survival, but the molecular mechanism remains largely unknown. We show using FcμR(-/-) and wild-type mice that FcμR specifically enhanced B cell survival induced by BCR cross-linking with F(ab')2-anti-IgM Abs while having no effect on survival when the B cells were activated by CD40 ligation or LPS stimulation. FcμR expression was markedly upregulated by anti-IgM stimulation, which may promote enhanced FcμR signaling in these cells. Immunofluorescence and confocal microscopy analyses demonstrated that FcμR colocalized with the BCR on the plasma membrane of primary B cells. Coimmunoprecipitation analysis further revealed that FcμR physically interacted with the BCR complex. Because NF-κB plays a prominent role in B cell survival, we analyzed whether FcμR was involved in BCR-triggered NF-κB activation. FcμR did not affect BCR-triggered IκBα phosphorylation characteristic of the canonical NF-κB activation pathway but promoted the production of the noncanonical NF-κB pathway component p52. Consistent with the elevated p52 levels, FcμR enhanced BCR-triggered expression of the antiapoptotic protein BCL-xL. Importantly, FcμR stimulation alone in the absence of BCR signaling had no effect on either IκBα phosphorylation or the expression of p52 and BCL-xL. Therefore, FcμR relied on the BCR signal to activate the noncanonical NF-κB pathway and enhance B cell survival. These results reveal a cross-talk downstream of FcμR and BCR signaling and provide mechanistic insight into FcμR-mediated enhancement of B cell survival after BCR stimulation.
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http://dx.doi.org/10.4049/jimmunol.1402352DOI Listing
April 2015

LAPTM5 promotes lysosomal degradation of intracellular CD3ζ but not of cell surface CD3ζ.

Immunol Cell Biol 2014 Jul 18;92(6):527-34. Epub 2014 Mar 18.

1] Department of Immunology, Shanghai Medical College, Fudan University, Shanghai, China [2] Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.

The lysosomal protein LAPTM5 has been shown to negatively regulate cell surface T cell receptor (TCR) expression and T-cell activation by promoting CD3ζ degradation in lysosomes, but the mechanism remains largely unknown. Here we show that LAPTM5 promotes lysosomal translocation of intracellular CD3ζ but not of the cell surface CD3ζ associated with the mature TCR complex. Kinetic analysis of the subcellular localization of the newly synthesized CD3ζ suggests that LAPTM5 targets CD3ζ in the Golgi apparatus and promotes its lysosomal translocation. Consistently, a Golgi-localizing mutant CD3ζ can be transported to and degraded in the lysosome by LAPTM5. A CD3ζ YF mutant in which all six tyrosine residues in the immunoreceptor tyrosine-based activation motif are mutated to phenylalanines is degraded as efficiently as is wild type CD3ζ, further suggesting that TCR signaling-triggered tyrosine phosphorylation of CD3ζ is dispensable for LAPTM5-mediated degradation. Previously, Src-like adapter protein (SLAP) and E3 ubiquitin ligase c-Cbl have been shown to mediate the ubiquitination of CD3ζ in the internalized TCR complex and its subsequent lysosomal degradation. We show that LAPTM5 and SLAP/c-Cbl function in distinct genetic pathways to negatively regulate TCR expression. Collectively, these results suggest that CD3ζ can be degraded by two pathways: SLAP/c-Cbl, which targets internalized cell surface CD3ζ dependent on TCR signaling, and LAPTM5, which targets intracellular CD3ζ independent of TCR signaling.
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http://dx.doi.org/10.1038/icb.2014.18DOI Listing
July 2014

Complement-activating IgM enhances the humoral but not the T cell immune response in mice.

PLoS One 2013 8;8(11):e81299. Epub 2013 Nov 8.

Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.

IgM antibodies specific for a certain antigen can enhance antibody responses when administered together with this antigen, a process believed to require complement activation by IgM. However, recent data show that a knock-in mouse strain, Cμ13, which only produces IgM unable to activate complement, has normal antibody responses. Moreover, the recently discovered murine IgM Fc receptor (FcµR or TOSO/FAIM3) was shown to affect antibody responses. This prompted the re-investigation of whether complement activation by specific IgM is indeed required for enhancement of antibody responses and whether the mutation in Cµ13 IgM also caused impaired binding to FcµR. The results show that IgM from Cµ13 and wildtype mice bound equally well to the murine FcµR. In spite of this, specific Cμ13 IgM administered together with sheep red blood cells or keyhole limpet hemocyanine was a very poor enhancer of the antibody and germinal center responses as compared with wildtype IgM. Within seconds after immunization, wildtype IgM induced deposition of C3 on sheep red blood cells in the blood. IgM which efficiently enhanced the T-dependent humoral immune response had no effect on activation of specific CD4(+) T cells as measured by cell numbers, cell division, blast transformation, or expression of the activation markers LFA-1 and CD44 in vivo. These observations confirm the importance of complement for the ability of specific IgM to enhance antibody responses and suggest that there is a divergence between the regulation of T- and B-cell responses by IgM.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081299PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3826713PMC
March 2015

FcμR (Toso/Faim3) is not an inhibitor of Fas-mediated cell death in mouse T and B cells.

Blood 2013 Mar;121(12):2368-70

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http://dx.doi.org/10.1182/blood-2012-12-470906DOI Listing
March 2013

Critical role of the IgM Fc receptor in IgM homeostasis, B-cell survival, and humoral immune responses.

Proc Natl Acad Sci U S A 2012 Oct 17;109(40):E2699-706. Epub 2012 Sep 17.

Laboratories for Immune Diversity, Bioenvironmental Epigenetics, Epithelial Immunobiology, and Lymphocyte Differentiation, RIKEN Research Center for Allergy and Immunology, Tsurumi, Yokohama 230-0045, Japan.

IgM antibodies have been known for decades to enhance humoral immune responses in an antigen-specific fashion. This enhancement has been thought to be dependent on complement activation by IgM-antigen complexes; however, recent genetic studies render this mechanism unlikely. Here, we describe a likely alternative explanation; mice lacking the recently identified Fc receptor for IgM (FcμR) on B cells produced significantly less antibody to protein antigen during both primary and memory responses. This immune deficiency was accompanied by impaired germinal center formation and decreased plasma and memory B-cell generation. FcμR did not affect steady-state B-cell survival but specifically enhanced the survival and proliferation induced by B-cell receptor cross-linking. Moreover, FcμR-deficient mice produced far more autoantibodies than control mice as they aged, suggesting that FcμR is also required for maintaining tolerance to self-antigens. Our results thus define a unique pathway mediated by the FcμR for regulating immunity and tolerance and suggest that IgM antibodies promote humoral immune responses to foreign antigen yet suppress autoantibody production through at least two pathways: complement activation and FcμR.
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http://dx.doi.org/10.1073/pnas.1210706109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3479561PMC
October 2012

A novel mechanism for the autonomous termination of pre-B cell receptor expression via induction of lysosome-associated protein transmembrane 5.

Mol Cell Biol 2012 Nov 4;32(21):4462-71. Epub 2012 Sep 4.

Department of Immune Regulation, Tokyo Medical and Dental University Graduate School, Tokyo, Japan.

The expression of the pre-B cell receptor (BCR) is confined to the early stage of B cell development, and its dysregulation is associated with anomalies of B-lineage cells, including leukemogenesis. Previous studies suggested that the pre-BCR signal might trigger the autonomous termination of pre-BCR expression even before the silencing of pre-BCR gene expression to prevent sustained pre-BCR expression. However, the underlying mechanism remains ill defined. Here we demonstrate that the pre-BCR signal induces the expression of lysosome-associated protein transmembrane 5 (LAPTM5), which leads to the prompt downmodulation of the pre-BCR. While LAPTM5 induction had no significant impact on the internalization of cell surface pre-BCR, it elicited the translocation of a large pool of intracellular pre-BCR from the endoplasmic reticulum to the lysosomal compartment concomitantly with a drastic reduction of the level of intracellular pre-BCR proteins. This reduction was inhibited by lysosomal inhibitors, indicating the lysosomal degradation of the pre-BCR. Notably, the LAPTM5 deficiency in pre-B cells led to the augmented expression level of surface pre-BCR. Collectively, the pre-BCR induces the prompt downmodulation of its own expression through the induction of LAPTM5, which promotes the lysosomal transport and degradation of the intracellular pre-BCR pool and, hence, limits the supply of pre-BCR to the cell surface.
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http://dx.doi.org/10.1128/MCB.00531-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486144PMC
November 2012

LAPTM5 protein is a positive regulator of proinflammatory signaling pathways in macrophages.

J Biol Chem 2012 Aug 25;287(33):27691-702. Epub 2012 Jun 25.

Program in Cell Biology, the Hospital for Sick Children, and Biochemistry Department, University of Toronto, Toronto, Ontario M5G 1L7, Canada.

LAPTM5 (lysosomal-associated protein transmembrane 5) is a protein that is preferentially expressed in immune cells, and it interacts with the Nedd4 family of ubiquitin ligases. Recent studies in T and B cells identified LAPTM5 as a negative regulator of T and B cell receptor levels at the plasma membrane. Here we investigated the function of LAPTM5 in macrophages. We demonstrate that expression of LAPTM5 is required for the secretion of proinflammatory cytokines in response to Toll-like receptor ligands. We also show that RAW264.7 cells knocked down for LAPTM5 or macrophages from LAPTM5(-/-) mice exhibit reduced activation of NF-κB and MAPK signaling pathways mediated by the TNF receptor, as well as multiple pattern recognition receptors in various cellular compartments. TNF stimulation of LAPTM5-deficient macrophages leads to reduced ubiquitination of RIP1 (receptor-interacting protein 1), suggesting a role for LAPTM5 at the receptor-proximate level. Interestingly, we find that macrophages from LAPTM5(-/-) mice display up-regulated levels of A20, a ubiquitin-editing enzyme responsible for deubiquitination of RIP1 and subsequent termination of NF-κB activation. Our studies thus indicate that, in contrast to its negative role in T and B cell activation, LAPTM5 acts as a positive modulator of inflammatory signaling pathways and hence cytokine secretion in macrophages. They also highlight a role for the endosomal/lysosomal system in regulating signaling via cytokine and pattern recognition receptors.
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http://dx.doi.org/10.1074/jbc.M112.355917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431655PMC
August 2012

Rapid cell division contributes to efficient induction of A/T mutations during Ig gene hypermutation.

Mol Immunol 2011 Sep 2;48(15-16):1993-9. Epub 2011 Jul 2.

Laboratory for Immune Diversity, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama, Kanagawa 230-0045, Japan.

Ig gene hypermutation is initiated by the activation-induced cytidine deaminase (AID), which converts cytosine to uracil and generates a U:G lesion. One of the unsolved mysteries is how AID-triggered U:G lesions result in efficient induction of mutations at non-damaged A/T bases in the V(H) genes of germinal center (GC) B cells. Genetic and biochemical evidence suggests that components of the mismatch repair pathway and the low fidelity DNA polymerase η are required for the induction of A/T mutations. However, mismatch repair proficient NIH3T3 cells are unable to generate a high frequency of A/T mutations, even after DNA polymerase η overexpression, suggesting that additional mechanisms are involved. Since GC B cells undergo enormous expansion while undergoing hypermutation, we hypothesized that rapid cell division might play a role in the induction of A/T mutations. To test this hypothesis, we utilized an efficient in vitro mutagenesis system, which closely mirrors physiological Ig gene hypermutation, in the human GC-like B cell line Ramos. Ramos cells transduced with AID-IRES-GFP retrovirus were cultured for 10 days in medium supplemented with 20% or 2% fetal bovine serum (FBS) to allow rapid and slow proliferation, respectively. Analysis of the V(H) gene mutations revealed that A/T mutations were significantly reduced in 2% FBS compared with 20% FBS, with transitions more affected than transversions. These results demonstrate that rapid cell division contributes to efficient induction of A/T mutations and suggest that the rate of DNA replication has a profound effect on the processing of AID-triggered U:G lesions.
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http://dx.doi.org/10.1016/j.molimm.2011.06.218DOI Listing
September 2011

A role for lysosomal-associated protein transmembrane 5 in the negative regulation of surface B cell receptor levels and B cell activation.

J Immunol 2010 Jul 2;185(1):294-301. Epub 2010 Jun 2.

Laboratory for Immune Diversity, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama, Japan.

Mechanisms by which cell surface levels of the BCR are regulated remain largely unknown. We found that B cells lacking the lysosomal-associated protein transmembrane 5 (LAPTM5) expressed higher levels of cell surface BCR than did wild-type (WT) B cells after Ag stimulation in vitro and in vivo. In addition, LAPTM5-deficient mice contained an increased frequency of Ag-specific B cells and produced greater amounts of Abs than did WT mice after immunization with a T-dependent Ag. Adoptive transfer of LAPTM5-deficient B cells with WT T cells into RAG1-deficient mice revealed that the increased surface BCR levels and the enhanced B cell activation and Ab production were due to a B cell intrinsic defect. As they aged, the LAPTM5-deficient mice had increased titers of serum IgM and autoantibodies and immune complex deposition in the kidney. Immunofluorescent and biochemical analysis revealed that LAPTM5 physically interacted with the BCR complex and promoted its degradation in the lysosomal compartment in mouse B cells. These results demonstrate a role for LAPTM5 in the negative regulation of cell surface BCR levels and B cell activation.
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http://dx.doi.org/10.4049/jimmunol.1000371DOI Listing
July 2010

Deficiency of the oxidative damage-specific DNA glycosylase NEIL1 leads to reduced germinal center B cell expansion.

DNA Repair (Amst) 2009 Nov 24;8(11):1328-32. Epub 2009 Sep 24.

Laboratory for Immune Diversity, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.

Mammalian cells possess multiple DNA glycosylases, including OGG1, NTH1, NEIL1, NEIL2 and NEIL3, for the repair of oxidative DNA damage. Among these, NEIL1 and NEIL2 are able to excise oxidized bases on single stranded or bubble-structured DNA and has been implicated in repair of oxidative damage associated with DNA replication or transcription. We found that Neil1 was highly constitutively expressed in the germinal center (GC) B cells, a rapidly dividing cell population that is undergoing immunoglobulin (Ig) gene hypermutation and isotype switching. While Neil1(-/-) mice exhibited normal B and T cell development and maturation, these mice contained a significantly lower frequency of GC B cells than did WT mice after immunization with a T-dependent antigen. Consistent with the reduced expansion of GC B cells, Neil1(-/-) mice had a decreased frequency of Ig gene hypermutation and produced less antibody against a T-dependent antigen during both primary and secondary immune responses. These results suggest that repair of endogenous oxidative DNA damage by NEIL1 is important for the rapid expansion of GC B cells and efficient induction of humoral immune responses.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915481PMC
http://dx.doi.org/10.1016/j.dnarep.2009.08.007DOI Listing
November 2009

A critical role for REV1 in regulating the induction of C:G transitions and A:T mutations during Ig gene hypermutation.

J Immunol 2009 Aug 8;183(3):1846-50. Epub 2009 Jul 8.

Laboratory for Immune Diversity, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama, Japan.

REV1 is a deoxycytidyl transferase that catalyzes the incorporation of deoxycytidines opposite deoxyguanines and abasic sites. To explore the role of its catalytic activity in Ig gene hypermutation in mammalian cells, we have generated mice expressing a catalytically inactive REV1 (REV1AA). REV1AA mice developed normally and were fertile on a pure C57BL/6 genetic background. B and T cell development and maturation were not affected, and REV1AA B cells underwent normal activation and class switch recombination. Analysis of Ig gene hypermutation in REV1AA mice revealed a great decrease of C to G and G to C transversions, consistent with the disruption of its deoxycytidyl transferase activity. Intriguingly, REV1AA mice also exhibited a significant reduction of C to T and G to A transitions. Moreover, each type of nucleotide substitutions at A:T base pairs was uniformly reduced in REV1AA mice, a phenotype similar to that observed in mice haploinsufficient for Polh. These results reveal an unexpected role for REV1 in the generation of C:G transitions and A:T mutations and suggest that REV1 is involved in multiple mutagenic pathways through functional interaction with other polymerases during the hypermutation process.
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http://dx.doi.org/10.4049/jimmunol.0901240DOI Listing
August 2009

Induction of a:T mutations is dependent on cellular environment but independent of mutation frequency and target gene location.

J Immunol 2008 Dec;181(11):7835-42

Laboratory for Immune Diversity, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Tsurumi, Yokohama, Japan.

Based on its substrate specificity, activation-induced cytidine deaminase can directly induce C:G mutations in Ig genes. However the origin of A:T mutations, which occur in a similar proportion in germinal center (GC) B cells, is unclear. Genetic evidence suggests that the induction of A:T mutations requires the components of the mismatch repair system and DNA polymerase eta (POLH). We found that fibroblasts and GC B cells expressed similar levels of the mismatch repair components, but nonetheless the fibroblasts failed to generate a significant proportion of A:T mutations in a GFP reporter gene even after POLH overexpression. To investigate whether the ability to generate A:T mutations is dependent on the cellular environment (i.e., GC B cell or fibroblast) or the target gene (i.e., Ig or GFP), we developed a mutation detection system in a human GC-like cell line. We introduced a GFP gene with a premature stop codon into Ramos cells and compared the activation-induced cytidine deaminase-induced mutations in the endogenous V(H) and the transgenic GFP genes. Remarkably, a high proportion of A:T mutations was induced in both genes. Ectopic expression of POLH did not further increase the proportion of A:T mutations but diminished the strand bias of these mutations that is normally observed in V(H) genes. Intriguingly, the total mutation frequency in the GFP gene was consistently one-fifth of that in the V(H) gene. These results demonstrate that the ability to generate A:T mutations is dependent on the GC B cell environment but independent of the mutation frequency and target gene location.
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http://dx.doi.org/10.4049/jimmunol.181.11.7835DOI Listing
December 2008

DNA polymerase eta is a limiting factor for A:T mutations in Ig genes and contributes to antibody affinity maturation.

Eur J Immunol 2008 Oct;38(10):2796-805

Laboratory for Immune Diversity, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama, Japan.

DNA polymerase eta (POLH) is required for the generation of A:T mutations during the somatic hypermutation of Ig genes in germinal center B cells. It remains unclear, however, whether POLH is a limiting factor for A:T mutations and how the absence of POLH might affect antibody affinity maturation. We found that the heterozygous Polh+/- mice exhibited a significant reduction in the frequency of A:T mutations in Ig genes, with each type of base substitutions at a level intermediate between the Polh+/+ and Polh(-/-) mice. These observations suggest that Polh is haplo-insufficient for the induction of A:T mutations in Ig genes. Intriguingly, there was also a reduction of C to T and G to A transitions in Polh+/- mice as compared with WT mice. Polh(-/-) mice produced decreased serum titers of high-affinity antibodies against a T-dependent antigen, which was associated with a significant reduction in the number of plasma cells secreting high-affinity antibodies. Analysis of the V region revealed that aa substitutions caused by A:T mutations were greatly reduced in Polh(-/-) mice. These results demonstrate that POLH is a limiting factor for A:T mutations and contributes to the efficient diversification of Ig genes and affinity maturation of antibodies.
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http://dx.doi.org/10.1002/eji.200838502DOI Listing
October 2008

A lysosomal protein negatively regulates surface T cell antigen receptor expression by promoting CD3zeta-chain degradation.

Immunity 2008 Jul;29(1):33-43

Laboratory for Immune Diversity, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama 230-0045, Japan.

Modulation of surface T cell antigen receptor (TCR) expression is an important mechanism for the regulation of immune responses and the prevention of T cell hyperactivation and autoimmunity. The TCR is rapidly internalized after antigen stimulation and then degraded in lysosomes. However, few of the molecules involved in this process have been identified. We demonstrate that the lysosomal protein LAPTM5 negatively regulated surface TCR expression by specifically interacting with the invariant signal-transducing CD3zeta chain and promoting its degradation without affecting other CD3 proteins, CD3epsilon, CD3delta, or CD3gamma. TCR downmodulation required the polyproline-tyrosine motifs and the ubiquitin-interacting motif of LAPTM5. LAPTM5 deficiency resulted in elevated TCR expression on both CD4(+)CD8(+) thymocytes and spleen T cells after CD3 stimulation, as well as enhanced T cell responses in vitro and in vivo. These results identify a lysosomal protein important for CD3zeta degradation and illustrate a unique mechanism for the control of surface TCR expression and T cell activation.
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http://dx.doi.org/10.1016/j.immuni.2008.04.024DOI Listing
July 2008

Genetic analysis reveals an intrinsic property of the germinal center B cells to generate A:T mutations.

DNA Repair (Amst) 2008 Aug 17;7(8):1392-8. Epub 2008 Jun 17.

Laboratory for Immune Diversity, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Tsurumi, Yokohama, Japan.

The immunoglobulin genes undergo a high frequency of point mutations at both C:G and A:T pairs in the germinal center (GC) B cells. This hypermutation process is initiated by the activation-induced cytidine deaminase (AID), which converts cytosine to uracil and generates a U:G lesion. Replication of this lesion, or its repair intermediate the abasic site, could introduce C:G mutations but the mechanisms leading to mutations at non-damaged A:T pairs remain elusive. Using a lacZ-transgenic system in which endogenous genome mutations can be detected with high sensitivity, we found that GC B cells exhibited a much higher ratio of A:T mutations as compared to naïve B, non-GC B, and cells of other tissues. This property does not require AID or active transcription of the target gene, and is dependent on DNA polymerase eta. These in vivo results demonstrate that GC B cells are unique in having an intrinsic propensity to generate A:T mutations during repair of endogenous DNA damage. These findings have important implications in understanding how AID, which can only target C:G base pairs, is able to induce the entire spectrum of mutations observed in immunoglobulin variable region genes in GC B cells.
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http://dx.doi.org/10.1016/j.dnarep.2008.04.014DOI Listing
August 2008

DNA polymerases eta and theta function in the same genetic pathway to generate mutations at A/T during somatic hypermutation of Ig genes.

J Biol Chem 2007 Jun 20;282(24):17387-94. Epub 2007 Apr 20.

Laboratory for Immune Diversity, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama 230-0045, Japan.

Somatic hypermutation of the Ig genes requires the activity of multiple DNA polymerases to ultimately introduce mutations at both A/T and C/G base pairs. Mice deficient for DNA polymerase eta (POLH) exhibited an approximately 80% reduction of the mutations at A/T, whereas absence of polymerase (POLQ) resulted in approximately 20% reduction of both A/T and C/G mutations. To investigate whether the residual A/T mutations observed in the absence of POLH are generated by POLQ and how these two polymerases might cooperate or compete with each other to generate A/T mutations, here we have established mice deficient for both POLH and POLQ. Polq(-/-)Polh(-/-) mice, however, did not show a further decrease of A/T mutations as compared with Polh(-/-) mice, suggesting that POLH and POLQ function in the same genetic pathway in the generation of these mutations. Frequent misincorporation of nucleotides, in particular opposite template T, is a known feature of POLH, but the efficiency of extension beyond the misincorporation differs significantly depending on the nature of the mispairing. Remarkably, we found that POLQ catalyzed extension more efficiently than POLH from all types of mispaired termini opposite A or T. Moreover, POLQ was able to extend mispaired termini generated by POLH albeit at a relatively low efficiency. These results reveal genetic and biochemical interactions between POLH and POLQ and suggest that POLQ might cooperate with POLH to generate some of the A/T mutations during the somatic hypermutation of Ig genes.
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http://dx.doi.org/10.1074/jbc.M611849200DOI Listing
June 2007

Absence of DNA polymerase theta results in decreased somatic hypermutation frequency and altered mutation patterns in Ig genes.

DNA Repair (Amst) 2006 Nov 4;5(11):1384-91. Epub 2006 Aug 4.

Laboratory for Antigen Receptor Diversity, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama 230-0045, Japan.

Multiple DNA polymerases participate in somatic hypermutation of immunoglobulin (Ig) genes. Mutations at A/T are largely dependent on DNA polymerase eta (POLH) whereas mutations at C/G appear to be generated by several DNA polymerases. We have previously shown that mice expressing a catalytically inactive POLQ (Polq-inactive) have a reduction in C/G mutations. Here we have generated mice that completely lack Polq expression (Polq-null). Polq-null mice have no obvious abnormality in B or T cell differentiation, and their splenic B cells responded normally to various activation signals and underwent normal Ig gene class switching. The mutant mice mounted relatively normal immune responses against a T-dependent antigen although there was a slight decrease in antigen specific antibodies. Polq-null mice exhibited a mild reduction in the overall mutation frequency, however, in contrast to Polq-inactive mice where the reduction mostly affected mutations at C/G, Polq-null mice showed a reduction of both C/G and A/T mutations and there was a significant increase of G to C transversions. These results confirm a role for POLQ in somatic hypermutation and suggest that in the complete absence of POLQ other polymerases may functionally substitute, resulting in a mutation pattern different from that found in Polq-inactive mice.
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http://dx.doi.org/10.1016/j.dnarep.2006.06.006DOI Listing
November 2006

Role of DNA polymerase theta in tolerance of endogenous and exogenous DNA damage in mouse B cells.

Genes Cells 2006 Feb;11(2):111-21

Laboratory for Antigen Receptor Diversity, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama 230-0045, Japan.

DNA polymerase theta (Poltheta) is a family A polymerase that contains an intrinsic helicase domain. To investigate the function of Poltheta in mammalian cells, we have inactivated its polymerase activity in CH12 mouse B lymphoma cells by targeted deletion of the polymerase core domain that contains the catalytic aspartic acid residue. Compared to parental CH12 cells, mutant cells devoid of Poltheta polymerase activity exhibited a slightly reduced growth rate, accompanied by increased spontaneous cell death. In addition, mutant cells showed elevated sensitivity to mitomycin C, cisplatin, etoposide, gamma-irradiation and ultraviolet (UV) radiation. Interestingly, mutant cells were more sensitive to the alkylating agent methyl methanesulfonate (MMS) than parental cells. This elevated MMS sensitivity relative to WT cells persisted in the presence of methoxyamine, an inhibitor of the major base excision repair (BER) pathway, suggesting that Poltheta is involved in tolerance of MMS through a mechanism that appears to be different from BER. These results reveal an important role for Poltheta in preventing spontaneous cell death and in tolerance of not only DNA interstrand cross-links and double strand breaks but also UV adducts and alkylation damage in mammalian lymphocytes.
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http://dx.doi.org/10.1111/j.1365-2443.2006.00922.xDOI Listing
February 2006

DNA polymerase theta contributes to the generation of C/G mutations during somatic hypermutation of Ig genes.

Proc Natl Acad Sci U S A 2005 Sep 19;102(39):13986-91. Epub 2005 Sep 19.

Laboratories for Antigen Receptor Diversity, Cell Signaling, and Developmental Genetics, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama, Kanagawa 230-0045, Japan.

Somatic hypermutation of Ig variable region genes is initiated by activation-induced cytidine deaminase; however, the activity of multiple DNA polymerases is required to ultimately introduce mutations. DNA polymerase eta (Poleta) has been implicated in mutations at A/T, but polymerases involved in C/G mutations have not been identified. We have generated mutant mice expressing DNA polymerase (Pol) specifically devoid of polymerase activity. Compared with WT mice, Polq-inactive (Polq, the gene encoding Pol) mice exhibited a reduced level of serum IgM and IgG1. The mutant mice mounted relatively normal primary and secondary immune responses to a T-dependent antigen, but the production of high-affinity specific antibodies was partially impaired. Analysis of the J(H)4 intronic sequences revealed a slight reduction in the overall mutation frequency in Polq-inactive mice. Remarkably, although mutations at A/T were unaffected, mutations at C/G were significantly decreased, indicating an important, albeit not exclusive, role for Pol activity. The reduction of C/G mutations was particularly focused on the intrinsic somatic hypermutation hotspots and both transitions and transversions were similarly reduced. These findings, together with the recent observation that Pol efficiently catalyzes the bypass of abasic sites, lead us to propose that Pol introduces mutations at C/G by replicating over abasic sites generated via uracil-DNA glycosylase.
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http://dx.doi.org/10.1073/pnas.0505636102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1236561PMC
September 2005

HEXIM1 forms a transcriptionally abortive complex with glucocorticoid receptor without involving 7SK RNA and positive transcription elongation factor b.

Proc Natl Acad Sci U S A 2005 Jun 7;102(24):8555-60. Epub 2005 Jun 7.

Division of Clinical Immunology and Department of Rheumatology and Allergy, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

The HEXIM1 protein has been shown to form a protein-RNA complex composed of 7SK small nuclear RNA and positive transcription elongation factor b (P-TEFb), which is composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1, and to inhibit the kinase activity of CDK9, thereby suppressing RNA polymerase II-dependent transcriptional elongation. Here, we biochemically demonstrate that HEXIM1 forms a distinct complex with glucocorticoid receptor (GR) without RNA, CDK9, or cyclin T1. HEXIM1, through its arginine-rich nuclear localization signal, directly associates with the ligand-binding domain of GR. Introduction of HEXIM1 short interfering RNA and adenovirus-mediated exogenous expression of HEXIM1 positively and negatively modulated glucocorticoid-responsive gene activation, respectively. In the nucleus, HEXIM1 was shown to localize in a distinct compartment from that of the p160 coactivator transcriptional intermediary factor 2. Overexpression of HEXIM1 decreased ligand-dependent association between GR and transcriptional intermediary factor 2. Antisense-mediated disruption of 7SK blunted the negative effect of HEXIM1 on arylhydrocarbon receptor-dependent transcription but not on GR-mediated one, indicating that a class of transcription factors are direct targets of HEXIM1. These results indicate that HEXIM1 has dual roles in transcriptional regulation: inhibition of transcriptional elongation dependent on 7SK RNA and positive transcription elongation factor b and interference with the sequence-specific transcription factor GR via a direct protein-protein interaction. Moreover, the fact that the central nuclear localization signal of HEXIM1 is essential for both of these actions may argue the crosstalk of these functions.
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http://dx.doi.org/10.1073/pnas.0409863102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1150813PMC
June 2005

Bcl-2 prevents hypoxia/reoxygenation-induced cell death through suppressed generation of reactive oxygen species and upregulation of Bcl-2 proteins.

J Cell Biochem 2003 Dec;90(5):914-24

Division of Cell Biochemistry, Hiroshima Prefectural University School of BioSciences, Shobara, Hiroshima 727-0023, Japan.

The function of bcl-2 in preventing cell death is well known, but the mechanisms whereby bcl-2 functions are not well characterized. One mechanism whereby bcl-2 is thought to function is by alleviating the effects of oxidative stress upon the cell. To examine whether Bcl-2 can protect cells against oxidative injury resulting from post-hypoxic reoxygenation (H/R), we subjected rat fibroblasts Rat-1 and their bcl-2 transfectants b5 to hypoxia (5% CO2, 95% N2) followed by reoxygenation (5% CO2, 95% air). The bcl-2 transfectants exhibited the cell viability superior to that of their parent non-transfectants upon treatment with reoxygenation after 24-, 48-, or 72-h hypoxia, but not upon normoxic serum-deprivation or upon serum-supplied hypoxic treatment alone. Thus bcl-2 transfection can prevent cell death of some types, which occurred during H/R but yet not appreciably until termination of hypoxia. The time-sequential events of H/R-induced cell death were shown to be executed via (1) reactive oxygen species (ROS) production at 1-12 h after H/R, (2) activation of caspases-1 and -3, at 1-3 h and 3-6 h after H/R, respectively, and (3) loss of mitochondrial membrane potential (DeltaPsi) at 3-12 h after H/R. These cell death-associated events were prevented entirely except caspase-1 activation by bcl-2 transfection, and were preceded by Bcl-2 upregulation which was executed as early as at 0-1 h after H/R for the bcl-2 transfectants but not their non-transfected counterpart cells. Thus upregulation of Bcl-2 proteins may play a role in prevention of H/R-induced diminishment of cell viability, but may be executed not yet during hypoxia itself and be actually operated as promptly as ready to go immediately after beginning of H/R, resulting in cytoprotection through blockage of either ROS generation, caspase-3 activation, or DeltaPsi decline.
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http://dx.doi.org/10.1002/jcb.10723DOI Listing
December 2003

Role of the glucocorticoid receptor for regulation of hypoxia-dependent gene expression.

J Biol Chem 2003 Aug 16;278(35):33384-91. Epub 2003 Jun 16.

Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 08-8639, Japan.

Glucocorticoids are secreted from the adrenal glands and act as a peripheral effector of the hypothalamic-pituitary-adrenal axis, playing an essential role in stress response and homeostatic regulation. In target cells, however, it remains unknown how glucocorticoids fine-tune the cellular pathways mediating tissue and systemic adaptation. Recently, considerable evidence indicates that adaptation to hypoxic environments is influenced by glucocorticoids and there is cross-talk between hypoxia-dependent signals and glucocorticoid-mediated regulation of gene expression. We therefore investigated the interaction between these important stress-responsive pathways, focusing on the glucocorticoid receptor (GR) and hypoxia-inducible transcription factor HIF-1. Here we show that, under hypoxic conditions, HIF-1-dependent gene expression is further up-regulated by glucocorticoids via the GR. This up-regulation cannot be substituted by the other steroid receptors and is suggested to result from the interaction between the GR and the transactivation domain of HIF-1 alpha. Moreover, our results also indicate that the ligand binding domain of the GR is essential for this interaction, and the critical requirement for GR agonists suggests the importance of the ligand-mediated conformational change of the GR. Because these proteins are shown to colocalize in the distinct compartments of the nucleus, we suggest that these stress-responsive transcription factors have intimate communication in close proximity to each other, thereby enabling the fine-tuning of cellular responses for adaptation.
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http://dx.doi.org/10.1074/jbc.M302581200DOI Listing
August 2003

Anti-apoptotic defense of bcl-2 gene against hydroperoxide-induced cytotoxicity together with suppressed lipid peroxidation, enhanced ascorbate uptake, and upregulated Bcl-2 protein.

J Cell Biochem 2003 May;89(2):321-34

Division of Cell Biochemistry, Hiroshima Prefectural University School of BioSciences, Nanatsuka 562, Shobara, Hiroshima 727-0023, Japan.

Although it is well known that Bcl-2 can prevent apoptosis, the Bcl-2's anti-apoptotic mechanism is not fully understood. Here, we investigate the mechanism of oxidant-induced cell death and to investigate the role of Bcl-2 in the tert-butyl hydroperoxide (t-BuOOH)-induced oxidant injury in Rat-1 fibroblasts and their bcl-2 transfected counterparts, b5 cells. Treatment with t-BuOOH causes mitochondrial disfunction and induced morphological features consistent with apoptosis more markedly in Rat-1 cells than in b5 cells. The hydroperoxide t-BuOOH at concentrations less than 100 nM for as long as 48 h or with higher concentrations (up to 100 microM) for only 3 h induces death in Rat-1 cells, whereas their bcl-2 transfectants were significantly resistant to cytotoxicity by both time and all concentration other than 100 microM. The similar results were obtained also for DNA strand cleavages as detected by TUNEL stain. The bcl-2 transfectants significantly suppressed t-BuOOH-induced increases in both lipid peroxidation and caspase-3 activation 3 and 1 h after t-BuOOH exposure, respectively, but failed to suppress either caspase-1 activation or an enhanced production of the intracellular reactive oxygen species (ROS). Intracellular uptake of [1-(14)C] ascorbic acid (Asc) into the bcl-2 transfectants was superior to that into the non-transfectants always under examined conditions regardless of serum addition to culture medium and cell density. Upregulation of Bcl-2 proteins was rapidly induced after t-BuOOH exposure in the transfectants, but not in non-transfectants, and restored till 24 h to the normal Bcl-2 level. Thus suppressions of both lipid peroxidation and the subsequent cell death events such as caspase-3 activation and DNA cleavage were concerned with the inhibitory effects of Bcl-2 on the t-BuOOH-induced cytotoxicity. And some of these events may correlate with Bcl-2 expression-induced partial enhanced anti-oxidant cellular ability including enrichment of intracellular Asc and oxidative stress-induced upregulation of Bcl-2 protein. On the other hand, ROS production and caspase-1 activation were not related to cytoprotection by Bcl-2.
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http://dx.doi.org/10.1002/jcb.10506DOI Listing
May 2003

Suppression of NF-kappaB-dependent gene expression by a hexamethylene bisacetamide-inducible protein HEXIM1 in human vascular smooth muscle cells.

Genes Cells 2003 Feb;8(2):95-107

Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Background: Neointima formation is a characteristic feature of atherosclerosis and post-angioplasty restenosis, in which various soluble factors and mechanical injury stimulate signalling pathways in vascular smooth muscle cells (VSMC), promoting their migration and proliferation, and the eventual formation of the neointima. The transcription factor NF-kappaB has been shown to play a pivotal role in this process. Hexamethylene bisacetamide, an inhibitor of VSMC proliferation, induces the mRNA expression of HEXIM1 (hexamethylene bisacetamide-inducible protein 1). However, the protein expression and function of HEXIM1 remain unknown.

Results: In the present study, we demonstrated that HEXIM1 localizes in the cytoplasm and nucleus, and its nuclear expression is restricted to discrete speckled areas. Treatment of VSMC with hexamethylene bisacetamide up-regulated HEXIM1 expression, not only in mRNA but also protein levels. Moreover, HEXIM1 is shown to suppress the transcriptional activity of NF-kappaB via its C-terminal leucine-rich domain. A glutathione-S-transferase pull down assay indicated that HEXIM1 interacts with the p65 subunit of NF-kappaB. In VSMC, treatment with hexamethylene bisacetamide resulted in a down-modulation of the transcription of NF-kappaB target genes.

Conclusion: We may therefore conclude that HEXIM1 plays an inhibitory role in NF-kappaB-dependent gene expression in VSMC and is the candidate of a novel therapeutic target for inhibition of VSMC proliferation.
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http://dx.doi.org/10.1046/j.1365-2443.2003.00618.xDOI Listing
February 2003

Cytokine receptor common beta subunit-mediated STAT5 activation confers NF-kappa B activation in murine proB cell line Ba/F3 cells.

J Biol Chem 2002 Feb 7;277(8):6254-65. Epub 2001 Dec 7.

Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

The cytokine receptor common beta subunit (beta(c)) transmits intracellular signals upon binding ligand such as granulocyte-macrophage colony-stimulating factor or interleukin-3 (IL-3); however, transcriptional regulation under the control of signaling events downstream of the beta(c) is not fully understood. Using murine Ba/F3 cells, here we demonstrate that the beta(c)-mediated signals stimulate NF-kappa B-driven gene expression of not only the reporter construct but also endogenous target genes such as IL-6. Analyzing the effects of several inhibitors or mutant receptors revealed that this NF-kappa B activation is mediated neither by MEK/ERK/MAPK nor by the phosphatidylinositol 3-kinase pathway but by STAT5. Overexpression experiments of the wild-type or constitutive active form of STAT5 further confirmed this notion. In addition, STAT5-dependent NF-kappa B activation is mediated not through an inducible nuclear translocation but via up-regulation of both DNA binding activity and transactivation potential of NF-kappa B. Furthermore, we also show that as yet undefined humoral factor(s) may be involved in this NF-kappa B activation process. Taken together, we may propose that cytokine receptor-mediated STAT5 activation and expression of its target genes culminates in a unique mode of NF-kappa B activation and gene expression.
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http://dx.doi.org/10.1074/jbc.M109878200DOI Listing
February 2002