EphA2/EphA4変異マウスを用いた腎嚢胞性疾患発症の分子メカニズムの解明

金沢大学学際科学実験センターEphA受容体型チロシンキナーゼはephrinAをリガンドとし,複数の受容体が複数のリガンドを共有しているごとが知られている。私の作成したEphA2/EphA4ダブル変異マウスにおいては胎生11.5目頃より尿管芽の拡張が見られ,腎嚢胞性疾患を発症したので,その分子メカニズムについて調べた。In situ hybridizationやRT-PCRにより,尿管芽形成の場においてはEphA2やEphA4が尿管芽に特異的に発現し,ephrinA1-5の全てが尿管芽と周辺の間絨織細胞に発現していることがわかった。EphAのシグナルカスケードの下流にはMAPKシグナルカスケードが存在することがこれまでに知られている。しかし,Tsaoらとの共同研究により,EphA2シグナルの下流ではなく上流にMAPKシグナルカスケードが存在し,EphA2の発現を誘導する可能性もあることがわかった(Zhang, et. al. 2008)。尿管芽形成に必須であるRet-GDNFシグナルカスケードの下流にはMAPKカスケードが存在することが知られており、Sprouty1変異マウスなどの腎嚢胞性疾患発症にはMAPKのカスケードの亢進が見られることがわかっている。よって,EphAシグナルとMAPKシグナルが複雑に関係して異所的な尿管芽細胞の増殖を抑制している可能性が出てきた。また,慶応大学の松尾先生との共同研究により,破骨細胞におけるEphA2およびEphA4の機能解析を進めた。その結果,EphA2/EphA4ダブル変異マウスの大腿骨骨量および骨密度が増加することがわかった。現在,分子メカニズムについてさらに解析中である。研究課題/領域番号:18700395, 研究期間(年度):2006 – 2007出典：「EphA2/EphA4変異マウスを用いた腎嚢胞性疾患発症の分子メカニズムの解明」研究成果報告書　課題番号18700395（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-18700395/)を加工して作

HP-1γ Controls High-Affinity Antibody Response to T-Dependent Antigens

In vitro observations suggest a role for the mouse heterochromatin protein 1γ (HP-1γ) in the immune system. However, it has not been shown if and how HP-1γ contributes to immunity in vivo. Here we show that in mice, HP-1γ positively regulates the germinal center reaction and high-affinity antibody response to thymus (T)-dependent antigens by limiting the size of CD8+ regulatory T-cell (Treg) compartment without affecting progenitor B- or T-cell-development. Moreover, HP-1γ does not control cell proliferation or class switch recombination. Haploinsufficiency of cbx-3 (gene encoding HP-1γ) is sufficient to expand the CD8+ Treg population and impair the immune response in mice despite the presence of wild-type HP-1α and HP-1β. This is the first in vivo evidence demonstrating the non-redundant role of HP-1γ in immunity

Beta-1,4-galactosyltransferase-3 deficiency suppresses the growth of immunogenic tumors in mice

BackgroundBeta-1,4-galactosyltransferase-3 (B4GALT3) belongs to the family of beta-1,4-galactosyltransferases (B4GALTs) and is responsible for the transfer of UDP-galactose to terminal N-acetylglucosamine. B4GALT3 is differentially expressed in tumors and adjacent normal tissues, and is correlated with clinical prognosis in several cancers, including neuroblastoma, cervical cancer, and bladder cancer. However, the exact role of B4GALT3 in the tumor immune microenvironment (TIME) remains unclear. Here, we aimed to elucidate the function of B4GALT3 in the TIME.MethodsTo study the functions of B4GALT3 in cancer immunity, either weakly or strongly immunogenic tumor cells were subcutaneously transplanted into wild-type (WT) and B4galt3 knockout (KO) mice. Bone marrow transplantation and CD8+ T cell depletion experiments were conducted to elucidate the role of immune cells in suppressing tumor growth in B4galt3 KO mice. The cell types and gene expression in the tumor region and infiltrating CD8+ T cells were analyzed using flow cytometry and RNA sequencing. N-glycosylated proteins from WT and B4galt3 KO mice were compared using the liquid chromatography tandem mass spectrometry (LC-MS/MS)-based glycoproteomic approach.ResultsB4galt3 KO mice exhibited suppressed growth of strongly immunogenic tumors with a notable increase in CD8+ T cell infiltration within tumors. Notably, B4galt3 deficiency led to changes in N-glycan modification of several proteins, including integrin alpha L (ITGAL), involved in T cell activity and proliferation. In vitro experiments suggested that B4galt3 KO CD8+ T cells were more susceptible to activation and displayed increased downstream phosphorylation of FAK linked to ITGAL.ConclusionOur study demonstrates that B4galt3 deficiency can potentially boost anti-tumor immune responses, largely through enhancing the influx of CD8+ T cells. B4GALT3 might be suppressing cancer immunity by synthesizing the glycan structure of molecules on the CD8+ T cell surface, as evidenced by the changes in the glycan structure of ITGAL in immune cells. Importantly, B4galt3 KO mice showed no adverse effects on growth, development, or reproduction, underscoring the potential of B4GALT3 as a promising and safe therapeutic target for cancer treatment

Digenic inheritance of mutations in EPHA2 and SLC26A4 in Pendred syndrome

Enlarged vestibular aqueduct (EVA) is one of the most commonly identified inner ear malformations in hearing loss patients including Pendred syndrome. While biallelic mutations of the SLC26A4 gene, encoding pendrin, causes non-syndromic hearing loss with EVA or Pendred syndrome, a considerable number of patients appear to carry mono-allelic mutation. This suggests faulty pendrin regulatory machinery results in hearing loss. Here we identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4. EphA2 forms a protein complex with pendrin controlling pendrin localization, which is disrupted in some pathogenic forms of pendrin. Moreover, point mutations leading to amino acid substitution in the EPHA2 gene are identified from patients bearing mono-allelic mutation of SLC26A4. Ephrin-B2 binds to EphA2 triggering internalization with pendrin inducing EphA2 autophosphorylation weakly. The identified EphA2 mutants attenuate ephrin-B2- but not ephrin-A1-induced EphA2 internalization with pendrin. Our results uncover an unexpected role of the Eph/ephrin system in epithelial function

Protection of Macaques with Diverse MHC Genotypes against a Heterologous SIV by Vaccination with a Deglycosylated Live-Attenuated SIV

HIV vaccine development has been hampered by issues such as undefined correlates of protection and extensive diversity of HIV. We addressed these issues using a previously established SIV-macaque model in which SIV mutants with deletions of multiple gp120 N-glycans function as potent live attenuated vaccines to induce near-sterile immunity against the parental pathogenic SIVmac239. In this study, we investigated the protective efficacy of these mutants against a highly pathogenic heterologous SIVsmE543-3 delivered intravenously to rhesus macaques with diverse MHC genotypes. All 11 vaccinated macaques contained the acute-phase infection with blood viral loads below the level of detection between 4 and 10 weeks postchallenge (pc), following a transient but marginal peak of viral replication at 2 weeks in only half of the challenged animals. In the chronic phase, seven vaccinees contained viral replication for over 80 weeks pc, while four did not. Neutralizing antibodies against challenge virus were not detected. Although overall levels of SIV specific T cell responses did not correlate with containment of acute and chronic viral replication, a critical role of cellular responses in the containment of viral replication was suggested. Emergence of viruses with altered fitness due to recombination between the vaccine and challenge viruses and increased gp120 glycosylation was linked to the failure to control SIV. These results demonstrate the induction of effective protective immune responses in a significant number of animals against heterologous virus by infection with deglycosylated attenuated SIV mutants in macaques with highly diverse MHC background. These findings suggest that broad HIV cross clade protection is possible, even in hosts with diverse genetic backgrounds. In summary, results of this study indicate that deglycosylated live-attenuated vaccines may provide a platform for the elucidation of correlates of protection needed for a successful HIV vaccine against diverse isolates

ヒストン修飾によるインプリンティング遺伝子制御の解析と周産期致死疾患モデルの開発

京都大学 / 金沢大学学際科学実験センターHP1γは神経幹細胞の条件的ヘテロクロマチン領域においてヒストンH3K9me3やH3K9me2だけでなく，H3K27me3の維持に関わることが示唆された。また，Jmjd3欠損マウス，Jmjd3酵素活性特異的変異マウスおよびUtx欠損マウスを作製し，UtxではなくJmjd3がHox遺伝子の制御に関わること，Jmjd3はHox遺伝子の発現開始制御に関わること，Jmjd3は脱メチル化酵素活性を介してHox遺伝子の制御に関わることを明らかにした。さらに，胎盤におけるインプリンティング遺伝子の発現異常がポリコーム因子のリクルートができないことによる可能性が示唆された。HP1gamma mutant neurospheres had tendency to differentiate into neurons and astrocytes, and not only H3K9 but H3K27 methylation decreased in HP1gamma mutant neurospheres. Jmjd3 and Utx are H3K27 demethylases and thought to be involved in the many human diseases, however, the functional differences between Jmjd3 and Utx in mammals are still unclear. We examined both Jmjd3 and Utx deficient embryos. The results suggest that Jmjd3, but not Utx is involved in the axial patterning through Hox regulation in mice in contrast to previous reports that not Jmjd3 but Utx determines the axis formation via Hox regulation in zebrafish and nematode. Furthermore, Jmjd3 mouse mutants lacking only demethylase activity were examined since Jmjd3 functions in two ways: demethylase-dependent and independent. They showed the same phenotypes as Jmjd3 deficient embryos, suggesting that demethylase activity of Jmjd3 is crucial for the axial patterning in mice.研究課題/領域番号:25430085, 研究期間(年度):2013-04-01 – 2016-03-3

ヘテロクロマチンプロテイン1γ変異マウスが不妊症を呈する分子メカニズムの解析

金沢大学学際科学実験センター我々が作製したHP1γの変異マウスは雌雄ともに不妊であったので、その原因について研究を行った。その結果、減数分裂の開始時期にセントロメア近傍領域の集積が認められないこと、及び、野生型精原細胞において集積のみられるヒストンメチル化酵素がHP1γ変異マウスの精原細胞では集積できないことがわかり、これらのことから、HP1γはセントロメア近傍におけるヒストン修飾因子のリクルートに必須であり、減数分裂の正常な進行にはセントロメアのヒストン修飾が必要であることが明らかになった。We obtained HP1 gamma mutant mice and found that all homozygous mice were infertile as a result of defects in meiosis. Some kinds of histone methylation at pericentromeric heterochromatin were reduced in the mutant spermatocytes and spermatogonia. Moreover, a methyltransferase of H3K9 in spermatocytes could not accumulate in the mutant spermatocytes. These findings suggest that HP1 gamma is essential for recruitment of histone modification factors at pericentromeric heterochromatin and histone modifications are required for progression of meiosis.研究課題/領域番号:20700363, 研究期間(年度):2008 – 200

ヒストン修飾因子変異マウスが不妊症を呈するメカニズムの解析

金沢大学学際科学実験センター我々が作製したHP1γ変異マウスを解析した結果、減数分裂の進行時にHP1γはセントロメア近傍におけるヒストン修飾因子のリクルートに必須であることが明らかになった。また、減数分裂以前の始原生殖細胞(PGC)の増殖にもHP1γが必須であることを明らかにした。さらに、ヒストン脱メチル化酵素の変異マウスは骨格の発生に異常を起こして出生直後致死となること、骨格の発生異常はHox遺伝子の発現制御の異常によることを明らかにした。We generated HP1 gamma mutant mice and found that histone methylation at pericentromeric heterochromatin was reduced in the mutant germ cells at meiosis. Next, we found that HP1 gamma is essential for cell cycle progression of primordial germ cells(PGC). Moreover, we found that histone demethylase regulates expression of Hox genes, and the demethylase-deficient mice died perinatally.研究課題/領域番号:22700451, 研究期間(年度):2010-201

A degron system targeting endogenous PD-1 inhibits the growth of tumor cells in mice

必要な時だけ標的タンパク質を壊すがん治療 --薬剤投与によるマウス内在性PD-1の分解--. 京都大学プレスリリース. 2022-06-20.A smashing solution for cancer therapy: KyotoU develops protein degrading system to repress cancer in mice. 京都大学プレスリリース. 2022-08-12.Recently, targeted protein degradation systems have been developed using the ubiquitin-proteasome system. Here, we established Programmed cell death-1 (PD-1) knockdown mice as a model system for subjecting endogenous mouse proteins to the small molecule-assisted shutoff (SMASh) degron system. SMASh degron-tagged PD-1-mCherry in Jurkat cells and CD3+ splenocytes were degraded by the NS3/4A protease inhibitors, asunaprevir (ASV) or grazoprevir (GRV). Growth of MC-38 colon adenocarcinoma cells injected in Pdcd1-mCherry-SMASh homozygous knock-in (KI) mice was repressed by ASV or GRV. Moreover, growth of MC-38 cells was suppressed in wild-type mice transplanted with KI bone marrow cells after GRV treatment. This is the first study to use a degron tag targeting an endogenous mouse protein in vivo. Our experimental system using the SMASh degron may be employed for treating diseases and characterizing the cellular functions of essential proteins