Rb Regulates Erythroid Differentiation through Bcl-XL-Dependent Anti-Apoptotic Effect.

Inactivation of the retinoblastoma (Rb) gene results in embryonic lethality due to severe anemia and increased nucleated erythrocytes by day E14.5. 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. Here we showed that Rb regulates terminal erythroid differentiation through inhibition of apoptosis. 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 (Hgb 8.2±1.47 g/dl versus WT :12.0±1.22 g/dl) 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, 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 profiling for apoptosis-related genes in early erythroblasts (CD71^highTer119^high) by RT-PCR. Interestingly, the dramatic decrease of Bcl-XL expression was detected in the erythroblasts, whereas other Bcl-2 family members including Bcl-2 and Bax were unchanged. 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, a ratio of late erythroblasts in Bcl-XL expressing cells was higher than that in GFP control cells in Rb^−/− recipient mice and a number of annexin V positive cells were much reduced in the mice transplanted with Bcl-XL expressing cells, indicating that overexpression of Bcl-XL inhibited inappropriated apoptosis and restored the differentiation. These data demonstrates that Rb regulates erythroid differentiation through Bcl-XL-dependent anti-apoptotic effect.