Rb Plays an Essential Role in Erythroid Differentiation through Inhibition of Apoptosis Mediated by NFKB.

Inactivation of the retinoblastoma (Rb) gene results in embryonic lethality due to severe anemia and increased nucleated erythrocytes by day14. However, molecular mechanisms of the function of Rb in erythroid differentiation have been unclear. Recent studies have suggested that Rb has both intrinsic and extrinsic roles on erythroid differentiation. Using Rb-deficient (Rb^−/−) embryos(E12), we showed that Rb regulates terminal erythroid differentiation through inhibition of apoptosis mediated by NFKB. Enucleation of erythroblasts was impaired in semisolid culture of Rb^−/− hematopoietic progenitors in fetal liver. The lethally-irradiated recipient mice transplanted with Rb^−/− hematopoietic stem cells (HSCs) showed severe anemia with splenomegaly, whereas the number of leukocytes and platelets were normal. In Rb^−/− recipient mice, the nucleated erythrocytes and reticulocytes were significantly increased in the peripheral blood. We analyzed cell surface markers for erythroid lineage (TER119 and CD71) in the enlarged spleen. A block of erythroid differentiation at the early erythroblast stage (TER119^high CD71^high), accompanied with increased apoptosis, was observed in the recipient mice with Rb^−/− HSCs. We speculated that the defect in the erythroid differentiation of Rb^−/− HSCs might be caused by inappropriate cell death. Thus, we examined expression of apoptosis-related genes in early erythroblasts (CD71^high Ter119^high) and observed decrease of Bcl-XL expression. To clarify the function of Bcl-XL, we introduced exogenous cDNA of mouse Bcl-XL with GFP (Bcl-XL ires GFP) or GFP alone as control into HSCs and then transplanted them to lethally irradiated mice. From the point of CD71 and Ter119 expression pattern in GFP positive cells, Rb^−/− erythoblasts still showed the block in differentiation. In contrast, overexpression of Bcl-XL in Rb^−/− erythoblasts inhibited inappropriated apoptosis and restore the differentiation capacity. Further, we found that inactivation of NFKB, but not STAT5 in Rb^−/− erythroblasts. Treatment of NFKB inhibitor suppressed erythroid differentiation, accompanied by enucleation, and also inhibited upregulation of Bcl-XL. These data demonstrates that Rb is essential for erythroid differentiation through inhibition of apoptosis mediated by NFKB.