貪食による要除去細胞排除の分子機構

金沢大学医薬保健研究域医学系アポトーシス細胞表面には貪食目印分子が出現し、これが食細胞の受容体に認識されることによりアポトーシス細胞選択的な貪食が規定される。平成14年度は、前年度の成果をふまえて精巣セルトリ細胞の貪食誘導機構を解析した。また、新規課題としてマクロファージ遊走の分子機構を検討した。1)SR-BIとPSを介した貪食反応の情報伝達機構と生理学的意義セルトリ細胞は貪食受容体SR-BIを使ってアポトーシス精子形成細胞表層のホスファチジルセリン(PS)を認識し、両者の直接結合により貪食誘導する。本研究により、膜貫通タンパク質であるSR-BIは既知貪食誘導アダプター分子CED-6とセルトリ細胞内で結合することがわかった。しかし、SR-BIの細胞内領域にはCED-6結合領域は存在せず、両者は間接的に結合して情報伝達すると考えられた。一方、PS結合タンパク質アネキシンVまたはSR-BIのPS結合領域を精巣精細管に注入すると、組織中のアポトーシス精子形成細胞数の増加と産生される精子数の減少とが認められた。したがって、アポトーシス細胞貪食反応は精子形成の進行に必要であると考えられた。2)マクロファージ遊走因子産生と貪食機構哺乳類卵巣では退縮時黄体に黄体細胞のアポトーシスとマクロファージ浸潤とが観察され、マクロファージ遊走因子macrophage chemoattractant protein-1(MCP-1)の発現誘導も認められる。今回の解析により、発情期ラットで非アポトーシス黄体細胞がMCP-1を発現するとわかった。また、アポトーシスとMCP-1発現とはいずれも酸化ストレスが原因となって誘導されることが判明した。これより、マクロファージは性周期に伴って黄体内で産生されるMCP-1濃度変化を感知して、アポトーシス細胞周辺に移動すると考えられ、これによりアポトーシス黄体細胞が貪食除去されると予想された。研究課題/領域番号:13780500, 研究期間(年度):2001-2002出典：「貪食による要除去細胞排除の分子機構」研究成果報告書　課題番号13780500（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-13780500/)を加工して作

セルトリ細胞による細胞膜リン脂質を介した精子形成細胞の貧食機構

取得学位：博士(学術)，学位授与番号：博甲第239号，学位授与年月日：平成10年3月25日,学位授与年：199

食細胞による死細胞貪食の細胞内情報経路の研究

金沢大学医薬保健研究域医学系生体内で生じる変性自己細胞や侵入した微生物は,近隣の食細胞に貪食され分解されることで,生体内の恒常性が保たれている。取り込みには,食細胞内の低分子量G蛋白質を介する細胞骨格再編成が必要だが,その詳細は不明であり,本研究はG蛋白質を介する貪食経路解明が目的である。SR-BIを介する低分子量G蛋白質活性化機構SR-BI依存貪食を行うマクロファージ株に,低分子量G蛋白質Rac1,Cdc42,Rhoそれぞれのドミナントネガティブ体発現ベクターを導入し,貪食効率を測定した。Rac1,Cdc42が本貪食反応に必要であることが判明した。次に,これらのG蛋白質経路へのMAPキナーゼの必要性を特異阻害剤を用いて調べると,ERK,p38,JNKのいずれも貪食時のRac1,Cdc42活性化に必要ないとわかった。MAPキナーゼはSR-BI依存貪食時に活性化することより,G蛋白質経路とMAPキナーゼ経路は,SR-BI下流で独立に働く可能性がある。G蛋白質共役型受容体(GPCR)による微生物貪食排除の調節GPCRに属するカンナビノイド受容体(CB1,CB2)のマクロファージ貪食能への関わりを検証した。エンドカンナビノイドの2-アラキドノイルグリセロールは,CB2を介して貪食受容体dectin-1の機能を促進し,真菌の貪食排除を亢進することがわかった。G蛋白質により感染免疫が調節される例が提唱された。研究課題/領域番号:18057009, 研究期間(年度):2006 – 2007出典：「食細胞による死細胞貪食の細胞内情報経路の研究」研究成果報告書　課題番号18057009（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-18057009/)を加工して作

Mechanisms and consequences of phagocytosis on influenza virus-infected cells

金沢大学医薬保健研究域薬学系Influenza virus-infected cells are induced to undergo apoptosis and become susceptible to phagocytosis. Data from our in vitro and in vivo experiments have suggested that 1) alveolar macrophages and neutrophils phagocytose influenza virus-infected cells in an apoptosis-dependent manner; 2) the membrane phospholipid phosphatidylserine and viral neuraminidase-processed carbohydrates at the surface of target cells and phagocytes, respectively, are involved in the association of the two types of cells; and 3) phagocytic elimination of virus-infected cells leads to a reduction in the pathogenesis of influenza. These findings could lead to the development of a novel antiviral agent against influenza. © 2008 Bentham Science Publishers Ltd.全文公開20090

A presumed human nuclear autoantigen that translocates to plasma membrane blebs during apoptosis

金沢大学医薬保健研究域薬学系The structure and subcellular localization of a number of molecules change during apoptosis. These molecules are recognized by the immune system, leading to the development of autoimmunity when apoptotic cells fail to be effectively cleared by phagocytosis. We searched for such molecules by analyzing sera from 12 individuals who suffered from autoimmune diseases and from 3 patients with amyotrophic lateral sclerosis. One serum sample, designated 681, detected an antigen that fulfilled the above criteria. In Western blotting of lysates of human Jurkat T cells, the 681 antigen appeared as a distinct signal with a molecular mass of 60 kDa in normal cells, and 2 additional signals with faster mobilities were detected in apoptotic cells. The results of subcellular fractionation and immunofluorescence experiments revealed this antigen to be strictly localized in the nucleus of normal cells, but to be translocated to a region near the plasma membrane, to membrane blebs in particular, after the induction of apoptosis. Under conditions in which membrane blebbing was inhibited in apoptotic cells, the antigen still moved away from the nucleus, but its accumulation at the periplasmic region was completely abolished. The apparent partial cleavage and intracellular redistribution of the 681 antigen in apoptotic cells mimics changes previously reported for the nuclear autoantigen La, but the 681 antigen was clearly distinct from La. These results suggest that cleavage-dependent exit from the nucleus during apoptosis is a phenomenon common to nuclear autoantigens

Structural change of ribosomes during apoptosis: Degradation and externalization of ribosomal proteins in doxorubicin-treated Jurkat cells

金沢大学医薬保健研究域薬学系Changes in the amount and localization of human ribosomal proteins during apoptosis were determined. When total lysates of Jurkat cells undergoing apoptosis induced by doxorubicin were analyzed by Western blotting, degradation of three ribosomal proteins, S18, L5, and L14, was detected at 48 h after the induction of apoptosis. Decreases in the amounts of these three ribosomal proteins were also observed in ribosome-enriched fractions. These changes were partly abolished by the addition of the pan-caspase inhibitor z-VAD-fmk. Moreover, formation of the 80S ribosome complex appeared to be inhibited at 48 h after apoptosis induction. On the other hand, the rate of protein synthesis, assessed by measuring the incorporation of [35S]Met into bulk proteins, decreased as early as 12 h after the addition of doxorubicin. These results indicate that changes in the amount of ribosomal proteins and the overall structure of ribosomes in apoptosing cells occur after protein synthesis declines. Finally, analyses by flow cytometry, immunofluorescence, and Western blotting showed that six ribosomal proteins, S15, PO, L5, L6, L36a, and L41, were relocalized and expressed at the cell surface during apoptosis. The above results collectively indicate that ribosomes are structurally altered in apoptotic cells following inactivation of protein synthesis

Isolation of a Drosophila gene coding for a protein containing a novel phosphatidylserine-binding motif

金沢大学医薬保健研究域薬学系To elucidate the molecular basis of the binding of proteins to the membrane phospholipid phosphatidylserine (PS), we characterized PS-binding peptides isolated from a phage display library. Amino acid sequences deduced from the nucleotide sequences of over 60 phage clones isolated revealed that there was no common primary structure among these peptides, but all peptides were rich in basic amino acid residues. In particular, 15 clones encoded peptides that contained contiguous arginine residues. Characterization of two such peptides in more detail showed that they bound to PS, and to a much lower extent to other phospholipids, including phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine. Unlike other Ca2+-dependent PS-binding proteins, these peptides did not require Ca2+ for binding to PS, and the addition of Ca2+ did not alter the phospholipid specificity. Substitution of one of the two RR sequences in one peptide by alanine had no effect, but that of both sequences completely abolished the activity. Furthermore, we identified a Drosophila gene coding for a presumed nuclear protein that shares an amino acid sequence, including a RR residue, with one of the two PS-binding peptides. This protein bound to PS partly depending on the presence of the RR residue. These results allowed us to conclude that an amino acid sequence including contiguous arginine residues is a novel motif that defines Ca2+-independent PS-binding activity. © 2005 The Japanese Biochemical Society

Signalling pathway involving GULP, MAPK and Rac1 for SR-BI-induced phagocytosis of apoptotic cells.

金沢大学医薬保健研究域薬学系Class B scavenger receptor type I (SR-BI) is a phosphatidylserine (PS)-recognizing receptor of testicular Sertoli cells responsible for the phagocytosis of spermatogenic cells undergoing apoptosis. Here, we determined signal mediators that compose a signalling pathway for SR-BI-induced phagocytosis. Results of a yeast two-hybrid analysis and a cell-free binding assay indicated that SR-BI binds to engulfment adapter protein (GULP) using the C-terminal intracellular domain. A co-immunoprecipitation analysis showed the existence of a complex of GULP and SR-BI in cells prior to the activation of SR-BI by PS. A reduction of GULP expression in phagocytes decreased the SR-BI-mediated phagocytosis of apoptotic cells. Administration to phagocytes of PS-containing liposomes increased the levels of the GTP-bound form of Rac1 and the phosphorylated forms of mitogen-activated protein kinases (MAPK) p38 and extracellular signal-related kinase 1 and 2. Finally, lowering the expression of GULP abrogated MAPK phosphorylation, and the presence of MAPK inhibitors reduced the level of GTP-bound Rac1 in PS-activated phagocytes. These results collectively suggested the following signalling pathway for the SR-BI-induced phagocytosis: (i) PS-recognizing SR-BI activates associated GULP; (ii) activated GULP induces MAPK phosphorylation; (iii) activated MAPK increases GTP-bound Rac1; and (iv) activated Rac1 induces a rearrangement of the actin cytoskeleton