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

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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