The current studies were undertaken to test the approach of promoting erythropoiesis by suppressing endogenous erythroblast apoptosis. We used Nix null mice as the experimental model in these studies because Nix acts in opposition to EPO/Bcl-xl in erythroblast maturation, and elimination of Nix should de-repress erythroblast survival at baseline and in response to stress. The general principle was validated, as Nix null mice exhibit a high level of reticulocyte production that blunted the severity of experimental anemia and permitted more rapid hematocrit recovery, despite lower endogenous EPO levels.
Normal homeostatic erythropoiesis occurs continuously in order to replenish senescent erythrocytes that are removed from the circulation after a lifespan that, in the mouse, has a normal reported half-life of between 12 and 16.5 days 26,32
. Under these conditions, the number of earlier maturational stage erythroblasts, basophilic and chromatophilic erythroblasts, is held in check by intrinsic apoptosis pathways that are incompletely defined 33
. Thus, in the normal mouse spleen, which is more important to stress-mediated erythropoiesis than is bone marrow in this species 27,28
, examination of the erythroblast maturation sequence reveals a relative paucity of these early erythrocyte precursors, with an abundance of more mature orthochromatic erythroblasts that are readily available for terminal differentiation, enucleation, and release into the circulation during anemic or hypoxic stress. Although there may be multiple mechanisms for EPO-mediated erythropoiesis, EPO stimulation of Epo/Jak2/Stat5 34
, with or without involvement of GATA-1 35
, increases expression of the anti-apoptotic factor Bcl-xl, thereby enhancing survival of maturing erythroblasts. Nix is a developmentally regulated pro-apoptotic factor that is co-expressed in differentiating erythroblasts with, and functionally antagonized by, Bcl-xl 36,37
. Accordingly, ablation of the mouse Nix
gene alters the erythroblast maturational profile, causing enrichment of early- and mid-maturational sequence erythroblasts and increasing the overall proportion of erythroblasts to non-erythroblasts in spleen.
According to the above conceptual framework, Nix acts in opposition to EPO/Bcl-xl, and absence of Nix should therefore mimic EPO treatment. This was the case for in vitro CFU-E assays performed in cultured Nix
null splenocytes, which revealed EPO-independent CFU-E formation and increased sensitivity to exogenous EPO 19
. The current studies show that Nix
ablation closely mimics EPO in its effects on in vivo erythroblast maturation. In wild-type mice, EPO dramatically increased the numbers of basophilic erythroblasts in spleen, thus increasing the overall proportion of erythroblasts to non-erythroblasts. However, more mature orthochromatic erythroblasts continued to be present after EPO treatment. We observed that the erythroblast maturation profile of Nix
null mice was similar to that of wild-type mice recovering from acute anemia (endogenous EPO secretion), or who received exogenous EPO. Thus, Nix
ablation largely recapitulated the effects of exogenous EPO on erythroblast maturation.
Our results (current study and reference 19
), and recent observations implicating a role for Nix in mitochondrial clearance during erythrocyte maturation 29,30
, suggest that Nix has a dual role in red blood cell formation depending upon the developmental stage. During erythroblast development, Nix functions as a proapoptotic factor which serves as a mechanism to regulate erythrocyte ‘quantity’. Nix mediated apoptotic cell death in erythroblasts, in opposition to EPO induced survival signaling, may also be an important ‘triage’ mechanism exerting a ‘quality’ control in erythrocyte generation. Indeed, erythroblasts vary in their sensitivity to EPO signaling38
, which is likely a mechanism that acts in concert with Nix signaling to orchestrate removal of erythroblast subpopulations. The observed role for Nix in mediating mitochondrial clearance, which apparently can proceed unimpeded in a large proportion (~40-60%) of erythrocytes even in the absence of Nix 29,30
, is therefore an additional mechanism for quality control in a subpopulation of maturing erythroblasts. During anemic stress, when rapid mobilization of erythroblasts is required, stimulation of EPO production by the hypoxic stimulus in kidneys 12
, enhances survival signaling in all erythroblast populations, leading to formation of additional erythrocytes.
Our results show that targeting erythroblast apoptosis downstream of EPO signaling may be a viable strategy to prevent severe anemia in response to an acute anemic stress. Certain aspects of Nix-mediated erythroblast apoptosis are conducive to its therapeutic targeting to promote erythroblast survival: 1. It is an inducible pro-apoptotic protein, which is expressed in a highly regulated fashion during the erythroblast developmental sequence 37
. 2. Nix
ablation decreases apoptosis in basophilic and chromophilic erythroblasts 19
, which are the predominant population of cells upregulated in wild type mice in response to an acute anemic stress in the spleen (see and ). 3. Importantly targeting Nix does not impact the white blood cell compartment or hematopoietic stem cell population 19
, which suggests minimal potential for this approach to create undesirable hematopoietic side-effects.
Rapid and complete reconstitution of the erythrocyte compartment after PHZ induced anemia in Nix
null mice, suggests enhanced erythroblast survival as the primary mechanism uncovered by Nix
ablation. Indeed, in mice with enhanced erythroblast apoptosis, such as Lyn null mice 39
, with resultant impairment in STAT5/Bcl-xl mediated survival signaling, PHZ treatment did not result in accelerated recovery from anemia, despite stimulation of extramedullary hematopoiesis. Importantly, survival signaling through a functional EPO receptor (mediated via STAT5 activation) was necessary for recovery from PHZ induced anemic stress 40
. Based on our studies, we postulate that EPO induced Bcl-xl, antagonizes the proapoptotic action of Nix to promote erythroblast survival, both in the basal state 19
and during stress. PHZ induces hemolysis by causing oxidative damage to the erythrocyte membrane 41
. Therefore, if the major role for Nix was in mitochondrial clearance from maturing reticulocytes with increased oxidative stress, as postulated by Sandoval et al 30
, the response to PHZ treatment would be exactly opposite to what we observed, with much poorer recovery from anemia. Indeed, in p45NF-E2
null mice, with analogously elevated levels of reactive oxygen species in erythrocytes, PHZ treatment provoked a markedly increased severity of anemia 26
There are aspects of this work that suggest major challenges to implementing anti-apoptosis therapy for anemia. The normal hematocrit of unstressed Nix
null mice does not wholly reflect increased erythroblast production indicated by a reticulocyte count that is five-fold higher than normal. This is likely secondary to increased erythrocyte turn-over supported by the observation of morphologic changes in circulating erythrocytes (), increased osmotic fragility () and decreased life span of circulating red blood cells 30
. A similar phenomenon has been observed in polycythemic mice (induced by transgenically elevated circulating levels of EPO 42
); after EPO treatment, with anemia, and after Friend virus infection in mice 32,43
, as well as in human polycythemia vera 44
and a subset of patients having myelofibrosis with myeloid metaplasia, i.e. “spent polycythemia” 45-47
. In each of these conditions, an abbreviated erythrocyte life span is associated with increased activity of EPO or one of its downstream effectors, just as in Nix
null mice the stimulus for erythropoiesis is disinhibition of EPO-mediated cell survival signaling in erythroblasts.
The preponderance of evidence supports an important role for Nix as a key regulator of splenic erythroblast maturation. Since Nix gene ablation accelerated recovery from PHZ-induced anemia in mice, it is interesting to consider how erythroblast-specific apoptosis inhibition might prove useful to enhance rapid mobilization of erythroblast reserves, in the treatment of anemia.