The role of nuclear receptor co-activator 4 in erythropoiesis (Reply to Nai et al.)

Abstract

We thank Nai and colleagues for their interest in our recently published paper, Santana-Codina et al., 1 and their comment, also published in Haematologica. 2 Their comment, containing new unpublished experimental data, questions the importance of nuclear receptor coactivator 4 (NCOA4) in acute erythropoietic expansion. Here, we respond to clarify inconsistencies in the interpretation of their new data with respect to our experiments and highlight the multiple lines of evidence that support our overall conclusions regarding the cell autonomous and non-autonomous roles of NCOA4 in supporting murine erythropoiesis. With respect to their concerns over the use of tamoxifen in our inducible model of acute whole body Ncoa4 genetic ablation: we stand by the conclusions of our experiments as they were tightly controlled with all appropriate genetic backgrounds and treatment controls utilized. While we agree that tamoxifen may have off-target effects on red blood cells (RBC), our experiments as designed and carried out were well-controlled. Specifically, mice in each group had no baseline alterations in systemic iron parameters or erythropoiesis prior to tamoxifen administration making the alterations observed most likely to be a result of acute Ncoa4 ablation leading to an acute loss of flux of ferritin through the autophagic degradation pathway thereby altering systemic iron homeostasis and basal erythropoiesis. The key difference in the new data presented by Nai and colleagues in their comment 2 is the presence of long-standing Ncoa4 knockout in their mouse model prior to administration of tamoxifen, which we argue makes their conclusions uninterpretable with respect to our data in an inducible knockout mouse model. Specifically, they use a mouse model with constitutive (from birth) total body knockout of Ncoa4, which is a distinct situation compared to acute ablation of Ncoa4 expression in an adult mice. The critical point here is that with a constitutive total body ablation of Ncoa4 from birth, erythropoiesis is altered in such a way that RBC at the time of tamoxifen administration already have accumulated long-standing significant changes that alter the baseline response to any agent that has potential deleterious effects on the RBC, such as tamoxifen. For that matter, Nai et al. do not present hemoglobin levels in wild-type mice that were subjected to gavage solely with the vehicle (corn oil) that was used to dissolve tamoxifen. Given that repeated gavage is expected to induce a profound stress response, it is unclear if the reduction in hemoglobin levels observed in wild type mice (and which form the basis of their arguments) can be attributed specifically to tamoxifen. While we cannot fully evaluate their claim regarding the lack of alterations in systemic iron parameters in the Sv129/J background they use given their reference to unpublished data, we do note that these Ncoa4 knockout animals appear to have altered baseline hematologic parameters with microcytosis and a trend towards decreased hemogolobin and he...

Role of Nuclear Receptors in Controlling Erythropoiesis

“…Interestingly, the NCOA4 knock-out results in transient anemia during embryonic development but this phenotype must take into account the role of NCOA4 in regulating iron metabolism that could, in turn, contribute to regulate red cells production [ 42 , 43 ]. The discrepancy of the results obtained in constitutive versus tamoxifen-induced NCOA4 knock-out mice add a further layer of complexity in the interpretation of the role of NCOA4 in erythropoiesis [ 44 , 45 ].…”