Role of the Ether-a-gò-gò-Related Gene 1B Isoform in Hematopoiesis

Abstract 1222

Normal hematopoiesis is characterized by the tuned regulation of self renewal, proliferation, differentiation and migration of hematopoietic stem cells and HSC-derived multipotent and lineage-committed hemopoietic progenitor cells. This regulation is a complex process, which requires several levels of control provided by the activity of numerous membrane receptors and soluble proteins, which mediate the communication among hematopoietic cells, and between the cells and the microenvironment. In this contest, ion channels must be mentioned. Indeed, besides their canonical role in cell excitability, they can also modulate different cellular functions, such as proliferation, apoptosis and differentiation, in both excitable and non excitable cells. This role is also relevant in hematopoietic cells, where ion channels have a clear role in different functions of fully differentiated cells (Int Rev Cell Mol Biol. 2010;279:135–190).

On these bases, we analyzed the role of ether a gò-gò-related gene 1 (ERG1) channels in normal hematopoiesis. In particular, we performed lack of function studies using a murine ERG1 knock out (KO) model (in SV129 strain). Since mice with a general and complete KO of the whole ERG1 gene die during early development, we analyzed mice with a selective deletion of the ERG1B isoform (ERG1B^−/− mice, Mol Cell Biol. 1003;23(6):1856–1862), which is the ERG1 isoform mostly expressed in leukemic blasts (Blood. 2007;110(4):1238–1250). Such mice are viable and do not show any life threatening physical or behavioral abnormalities. First, we verified ERG1 transcripts expression in wild type SV129 mice (WT): both transcripts were expressed in spleen and thymus with higher values for the ERG1A isoform; ERG1B isoform presented a good expression level in bone marrow (BM) especially in the Sca-1⁺ population. Consequently we performed experiments to evaluate the role of ERG1B in normal hematopoiesis. Young (0–3 months old) KO mice presented a reduced number of CFUs (colony forming units) in the BM. CFUs levels were restored in adult mice. BM of KO mice showed hypocellularity and an increased number of megakaryocytes intriguingly associated with a reduction of erythrocytes (Ter119⁺). As evidenced by the histological analysis, splenomegaly of KO mice could be traced back to a great amount of mature red blood cells, filling the interfollicular space of the red pulp and subcapsulary space. Such splenic congestion in ERG1B^−/− mice is accompanied by a relative decrease in the number of megakaryocytes, as well as by a reduced capacity to develop CFUs. On the whole, these data are suggestive of a failure of spleen hemopoiesis, with a concomitant red cell engulfment that lead to a putative erythropoiesis that occurs locally in the spleen maybe due to reprogrammation of hematopoietic cells of different lineage. To better characterize hematopoiesis in ERG1B^−/− mouse model we performed two different stress tests: myelotoxicity and acute hemolytic anemia induction. Myelotoxicity was induced by single dose injection of cyclophosphamide (450 mg/Kg) in both WT and KO mice. From this induction we expected a decreased myelopoiesis, mostly affecting granulocytes and monocytes, followed by a rebound due to the capacity of the mice to undertake myelopoiesis. In KO model this compensation was absent suggesting a functional defect into the myeloid lineage correlated with ERG1B deletion. Finally, we induced acute anemia in mice testing their response to phenylhydrazine (PHZ, 60 mg/Kg). As expected, in WT mice, RBCs value rapidly declined followed by a compensatory erythropoiesis. In ERG1B^−/− mice, we observed a reduced capacity to recover physiological RBCs values. Such results suggest that a functional defect occurred also into the erythroid lineage.

On the whole, the present study provides evidence that the ERG1B isoform exerts a relevant role in hematopoiesis, driving the commitment and maturation of different hematopoietic cell populations.