MOLECULAR MECHANISMS OF ACCELERATED ERYTHROID APOPTOSIS DUE TO A GLYCOLYTIC ENZYME DEFECT

糖酵解酶缺陷加速红细胞凋亡的分子机制

• 批准号: 14570131
• 负责人: KANNO Hitoshi
• 金额: $ 2.18万
• 依托单位: TOKYO WOMEN'S MEDICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570131/
• 关键词: HEMOLYTIC ANEMIA; MUTATION; PYRUVATE KINASE; GLYCOLYSIS; ERYTHROCYTES; APOPTOSIS; フレンド細胞; RNAi; アイソザイム

Pyruvate kinase (PK) deficiency is one of the most prevalent causes of hereditary non-spherocytic hemolytic anemia due to glycolytic enzyme defects. We previously reported that hemolytic anemia of the Pk-1^<slc> mouse was due to a spontaneous mutant of the murine PKLR gene. Apoptotic erythroid cells were notably increased in spleen, and the transgenic rescue of the Pk-1^<slc> decreased apoptotic erythroid cells in the mutant spleen. SLC3 is a Friend erythroleukemic cell, which has been established from the Pk-1^<slc> mouse. The cell shows spontaneous apoptosis during routine passage and is more susceptible to apoptosis compared to control Friend cells, which is inducible with either glucose deprivation or glucose analogue, 2-deoxyglucose.In this study, possible adverse effects of PK deficiency on the maturation of erythroid progenitors were investigated. A four-year-old Japanese girl with severe PK deficiency underwent splenectomy to reduce her need for blood transfusions. The spleen w … More as examined a histologically and the hematopoietic progenitors in the spleen were assayed to evaluate the extramedullary hematopoiesis of the PK-deficient subject. The number of hematopoietic progenitors including CFU-GM, BFU-E and CFU-GEMM in the spleen of the PK-deficient patient was much higher than those found in control spleens, indicating enhanced extramedullary hematopoiesis. The TUNEL assay demonstrated apoptotic cells in the splenic red pulp of the PK-deficient patient. The expression of 7A6 antigen was detected in cells isolated from spleen and in cells cultured in vitro, but only in those cells that were positive for glycophorin A. These results provide evidence that the metabolic disturbances in PK deficiency affect not only the survival of red cells but also the maturation of erythroid progenitors, which results in premature cell death, i.e., apoptosis.To evaluate an involvement of R-PK in apoptosis of SLC3, we established two stable-transfectants with wild-type human R-PK cDNA, SLC3-hRPK.hi and lo, and examined gene expression profiles of these cells. DNA microarray analysis were performed by using Affymetrix GeneChip Mouse Expression Array 430A, and totally 22690 genes were analyzed. Approximately 7% of genes were significantly down-regulated both in SLC3-hRPK.hi and lo, including genes for ε-globin, erythropoietin receptor, β-spectrin, glycophorin A, or Rh-associated A glycoprotein. On the other hand, up-regulated genes was only about 0.2%, such as subtypes H1c and H2bc of histone 1, kcne3 potassium voltage-gated channel or adult α-globin.We identified that gene expression of several pro-apoptotic genes such as Bnip31, Pdcd6ip, Casp8ap2, Faf1 and Blk was significantly down-regulated by introducing RPK. These observations suggest that forced expression of RPK facilitated globin gene switching and terminal erythroid differentiation. Further studies will require elucidating mechanisms for apoptosis due to RPK deficiency. Less

丙酮酸激酶（PK）缺乏症是由于糖酵解酶缺陷引起的遗传性非细胞溶血性贫血的最普遍原因之一。我们先前报道说，PK-1^<slc>小鼠的溶血性贫血是由于鼠PKLR基因的赞助突变体引起的。脾脏中凋亡的红细胞细胞显着增加，而PK-1^<slc>的转基因营救改善了突变体臂中的凋亡性红细胞细胞。 SLC3是一个朋友红血球血症细胞，已从PK-1^<slc>鼠标建立。该细胞在常规通道过程中显示出自发的凋亡，与对照友好细胞相比，与对照友好细胞相比更容易受到凋亡的影响，与对照的友谊细胞相比，这是葡萄糖剥夺或葡萄糖模拟的诱导性，2-脱氧葡萄糖。在这项研究中，PK缺乏对erythroid祖先的成熟的不利影响。一个四岁的日本女孩严重缺乏症，进行了脾切除术，以减少她对输血的需求。分配了脾脏的脾脏……脾脏和脾脏中的造血祖细胞被分配，以评估PK缺陷受试者的外卵外造血。 PK缺陷患者的脾脏中包括CFU-GM，BFU-E和CFU-GEMM在内的造血祖细胞的数量远高于对照脾脏中的患者，表明增强了硫外造血。 TUNEL分析表明，PK缺陷患者的脾脏红色果肉中的凋亡细胞。在从脾脏分离的细胞和体外培养的细胞中检测到7A6抗原的表达，但仅在那些对糖蛋白A呈阳性的细胞中。这些结果证明，PK缺乏症中的代谢灾害不仅会影响红细胞的存活，而且还会影响伴有促成幼虫的成熟量，即在ape eftery ape中的成熟，即在eapeption cyl ape and ape and ape consection appop ins ap ape con。 SLC3的凋亡，我们建立了两种稳定的转移剂，它具有野生型人R-PK cDNA，SLC3-HRPK.HI和LO，并检查了这些细胞的基因表达谱。通过使用Affymetrix Genechip小鼠表达阵列430a进行DNA微阵列分析，并分析了22690个基因。在Slc3-hrpk.hi和LO中，大约7％的基因都显着下调，包括ε-珠蛋白，红细胞生成素受体，β-谱蛋白，糖蛋白A，糖蛋白A或RH相关的基因。另一方面，上升的基因仅约0.2％，例如组蛋白1的亚型H1C和H2BC，KCNE3钾电压门控通道或成人α-珠蛋白。我们确定了几种基因表达了几种促凋亡基因的表达，例如BNIP31，PDCD6IP，casp8ap，faffk1和Blk，BNERP的强大。这些观察结果表明，RPK的强迫表达制备的球蛋白基因开关和末端红细胞分化。进一步的研究将需要阐明由于RPK缺乏而导致凋亡的机制。较少的

项目成果

Morimoto A, Ueda I, Hirashima Y, Sawai Y, Usuku T, Kano G, Kuriyama K, Todo S, Sugimoto T, Kanno H, Fujii H, Imashuku S: "A novel missense mutation (1060G→C) in the phosphoglycerate kinase gene in a Japanese boy with chronic hemolytic anemia, developmenta

Morimoto A、Ueda I、Hirashima Y、Sawai Y、Usuku T、Kano G、Kuriyama K、Todo S、Sugimoto T、Kanno H、Fujii H、Imashuku S：“磷酸甘油酸激酶中的一种新型错义突变 (1060G→C)患有慢性溶血性贫血的日本男孩的基因，正在发育

Sakimoto, T. et al.: "A novel nonsense mutation with a compound heterozygous mutation in TGFBI gene in lattice corneal dystrophy type I"Jpn J Ophthalmol. 47. 13-17 (2003)

Sakimoto, T. 等人：“I 型格子角膜营养不良中 TGFBI 基因中具有复合杂合突变的新型无义突变”Jpn J Ophamol。

Murakami K, Kanno H, Tancabelic J, Fujii H: "Gene expression and biological significance of hexokinase in erythroid cells."Acta Haematol. 108. 204-209 (2002)

Murakami K、Kanno H、Tancabelic J、Fujii H：“红系细胞中己糖激酶的基因表达和生物学意义。”Acta Haematol。

Kanno, H. et al.: "Homozygous intragenic deletion of type-I hexokinase gene causes lethal hemolytic anemia of the affected fetus"BLOOD. 100. 1930 (2002)

Kanno, H. 等人：“I 型己糖​​激酶基因的纯合基因内缺失会导致受影响胎儿发生致命的溶血性​​贫血”BLOOD。

Kanno H et al.: "Phrsiological significance and molecular genetics of red cell enzymes involved in the ribonucleotide metabolism"Proc Jpn Acad. 78. 287-292 (2002)

Kanno H 等人：“参与核糖核苷酸代谢的红细胞酶的生理学意义和分子遗传学”Proc Jpn Acad。