Phosphate is critical in multiple biological processes, including phosphorylation reactions, ATP production, and DNA structure and synthesis, and is likely an important determinant of cell growth. It remains unclear how individual cells initially sense changes in extracellular phosphate concentration and the cellular consequences of these changes. PiT1 is a constitutively expressed, high affinity sodium-dependent phosphate import protein and studies in nonhematopoietic cells suggest it plays a role in governing cellular proliferation. Recently, we reported that conditional deletion of Pit1 in neonatal mice causes a profound macrocytic anemia, characterized by a block in terminal erythroid differentiation, dyserythropoiesis, and increased apoptosis. The animals also have marked thrombocytosis and mild neutropenia. Importantly, the phenotype is intrinsic to the hematopoietic system (ASH Annual Meeting Abstracts, November 2011;118:681). Further characterization of their hematopoietic phenotype reveals equivalent numbers of marrow-derived hematopoietic progenitor cells (common myeloid, megakaryocyte-erythroid, and granulocyte-macrophage progenitors) compared to controls. Deleted mice demonstrate a relative expansion in Lin−c-KithighSca-1−CD16/CD32highCD34− cells, which is absent in control animals; morphology and additional flow cytometric characterization (high endoglin and CD150 expression) of this population suggest it includes early erythroid precursors (Pre CFU-E) with aberrant expression of the myeloid antigen CD16. Thus, erythroid differentiation is impacted by a lack of PiT1 from the early CFU-E through basophilic erythroblast stages. Additionally, we discovered that the animals have a marked B cell lymphocytopenia (0.4 K/uL ± 0.1 vs. 2.6 ± 0.5, p<1.0E−4, deleted n=11, control n=4, mean±SEM, Student's t-test) due to a defect in B cell development prior to the pre-pro B cell stage and an additional defect in B cell development unique to late, early pro-B cell development. We confirmed that the defect in B cell development in Pit1-deleted mice is intrinsic to the hematopoietic system by demonstrating B cell lymphocytopenia in lethally irradiated mice transplanted with Pit-1flox/flox;Mx-cre marrow and then treated with poly(I)poly(C) to delete Pit-1 specifically in engrafted cells (1.1 K/uL ± 0.3 vs. 4.9 ± 0.4, p<1.0E−3, deleted n=4, control n=3, mean±SEM, Student's t-test). Cell cycle profiles and BrdU studies show that erythroid cells and B cells lacking PiT1 have impaired cell cycle progression akin to that seen in siRNA knockdown studies of PiT1 in nonhematopoietic cells. Total and sodium-dependent phosphate uptake in flow-cytometrically sorted basophilic erythroblasts/proerythroblasts (CD71highCD44highFSChighB220−Gr1−Mac1−) and whole bone marrow cells are equivalent in deleted and control populations, proving that the phenotype is independent of phosphate uptake (p>0.5). We hypothesize that the profound anemia in mice lacking PiT1 reflects a unique vulnerability of proerythroblasts/basophilic erythroblasts to defects in cell cycle progression due to their high proliferative requirements and the unique coordinate control of cell cycle exit with terminal erythroid differentiation. Late, early pro-B cells may also be particularly vulnerable to perturbations in the cell cycle since they undergo a proliferative burst, likely dependent on the assembly and signaling of the pre-B cell receptor. Ongoing genomic and proteomic studies of flow-cytometrically sorted proerythroblasts/basophilic erythroblasts from Pit1-deleted and control mice are aimed at defining activated signaling networks to account for the anemia in mice lacking PiT1. Our work may offer further insight into how erythroid differentiation is intimately coupled with cellular proliferation, one possible mechanism of ineffective erythropoiesis in low grade acquired myelodysplastic syndromes, and the proliferative stresses shared between terminal erythroid differentiation and early B cell development.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.