An Anti-Apoptotic Molecule, Anamorsin, Is Upregulated in Anemia and Essential for Imperative Erytropoiesis
Authors: Tanimura Akira , Shibayama Hirohiko , Yuri Hamanaka , Fujita Natsuko , Yasuhiro Nagate , Ishibashi Tomohiko , Saitoh Norimitsu , Ichii Michiko , Yokota Takafumi , Ezoe Sachiko , Oritani Kenji , Kanakura Yuzuru 
Published: October 2019

Abstract
Introduction
Anamorsin (AM) is an anti-apoptotic molecule we originally isolated from hematopoietic cell line, Ba/F3. That is dependent on interleukin-3 (IL-3) stimulation. We showed that AM conferred resistance to apoptosis induced by the lack of IL-3 stimulation. AM has no structural homology to any known anti-apoptotic molecules such as Bcl-2 and IAP family members. AM-deficient mice are embryonic lethal at late gestation due to the defect of definitive hematopoiesis. Its embryos are anemic and very small in size. Thus AM is considered to be crucial in erythropoiesis but the mechanisms how AM works remain unclear. In this study, we tried to clarify the roles of AM in erythropoiesis.
Methods and results
At first, in order to examine the expression levels of AM in each differential stage of erythroid cells, we sorted those cells from eight-week-old mice bone marrow (n=4) using flow cytometry. The expression levels of AM were evaluated by Immunoblotting. The results showed that the proerythroblast population (CD71high Ter119low) expressed AM higher than any other erythroid cells. The expression of AM in CD71high Ter119 high cells and CD71low Ter119high cells, which correspond to basophilic erythroblasts and orthchromatophilic erythroblasts respectively, were markedly decreased. Next, we analyzed whether AM expression is induced by erythropoietin (EPO). Recombinant human EPO was injected intravenously (0.1mg/kg) with a single treatment (day0) to eight-week-old mice (n=3). The bone marrow and spleen cells were collected seven days after injection and the samples were subjected to immunoblotting. The results showed that EPO treatment led to increased expression of AM in both bone marrow and spleen. At 14 days after EPO injection, the expression of AM returned to the basal. Furthermore, we examined the AM expression in the acute anemia model. Blood of 0.2ml was exsanguinated from each mouse every day until hematocrit reached to the value less than 25 % (day 0).In this phlebotomized mice, the AM expression was strongly induced specially in proerythroblast population on day 1 and returned to the basal after the recovery from anemia. To explore the AM expression under the other stress, 5-FU (150 mg/kg), a chemotherapeutic drug, was administered to mice (n=3) with a single treatment (day 0). The increased expression of AM was also detected when the mice were most severely anemic (day7). To investigate the significance of AM expression induced by anemia, we transplanted 106 14.5 dpc wild-type (AM+/+) and AM-deficient (AM–/–) fetal liver cells into lethally irradiated congenic recipient mice. At 8 weeks after transplantation, we analyzed the response to anemia induced by phlebotomy. The results showed that donor-derived cells from AM+/+ fetal liver cells were detected the smooth recovery on day 3 after phlebotomy. On the other hand, donor-derived cells from AM–/– fetal liver cells showed an approximately 5-fold reduction compared to those from AM+/+fetal liver cells.
Conclusion and discussion
The proerythroblast population expressed AM at the highest level among various erythroid lineage populations. The AM expression level was increased by acute anemia induced by phlebotomy and AM-deficiency could show inefficient recovery of erythropoiesis from the induced acute anemia. Previously we reported that AM is essential for Fe/S cluster biogenesis and AM deficiency impaired the iron homeostasis (ASH abs. #610 (2012)). We also reported that the proerythroblast population in AM-deficient fetal liver cell showed more increased labile iron pool and significantly induced apoptosis. From our previous and present data, it is possible that AM may control erythropoiesis by preventing apoptosis when erythrocyte production is needed.

Disclosures
No relevant conflicts of interest to declare.