Leukemia (2004) 18, 1176–1199 & 2004 Nature Publishing Group All rights reserved 0887-6924/04 $30.00 www.nature.com/leu

REVIEW

Apoptotic mechanisms in the control of erythropoiesis

U Testa1

1Department of Hematology and Oncology, Istituto Superiore di Sanita`, Rome, Italy

Erythropoiesis is a complex multistep process encompassing pluripotent stem cells, which have the unique property of both the differentiation of hemopoietic stem cells to mature erythro- self-renewal and differentiation through progressive commit- cytes. The steps involved in this complex differentiation process are numerous and involve first the differentiation to ment to multipotent progenitors, then to committed progenitors early erythoid progenitors (burst-forming units-erythroid, BFU- and finally to progressively maturing precursors. E), then to late erythroid progenitors (colony-forming units- In mouse (and also in humans) during embryonic/fetal erythroid) and finally to morphologically recognizable erythroid development, three distinct stages of erythropoiesis have precursors. A key event of late stages of erythropoiesis is been defined. The first stage corresponds to primitive embryonic nuclear condensation, followed by extrusion of the nucleus to erythropoiesis and occurs at the level of blood islands produce enucleated reticulocytes and finally mature erythro- cytes. During the differentiation process, the cells became that develop within the yolk sac around day 7 of murine progressively sensitive to erythropoietin that controls both the embryonic life; the distinctive feature of erythropoiesis at 1 survival and proliferation of erythroid cells. this stage corresponds to the Epo-independency. Primitive A normal homeostasis of the erythropoietic system requires erythroblasts differentiate within the bloodstream, remain an appropriate balance between the rate of erythroid cell predominantly nucleated at the end of their maturation and production and red blood cell destruction. Growing evidences have a very large size (accordingly, they are called ‘mega- outlined in the present review indicate that apoptotic mecha- nism play a relevant role in the control of erythropoiesis loblasts’). The second stage of erythropoietic development under physiologic and pathologic conditions. Withdrawal of corresponds to definitive erythropoiesis and occurs around day erythropoietin or stimulation of death receptors such as Fas 10 of gestation and consists of a rapid and marked proliferation or TRAIL-Rs leads to activation of a subset of caspases-3, -7 of erythroid elements in the fetal liver. The third stage of and -8, which then cleave the transcription factors GATA-1 and erythropoietic development is characterized by the migration TAL-1 and trigger apoptosis. In addition, there is evidence of hematopoietic stem cells at the level of the bone marrow, that a number of caspases are physiologically activated during erythroid differentiation and are functionally required where adult hemopoiesis and, particularly, adult erythropoiesis for erythroid maturation. Several caspase substrates are develops. cleaved in differentiating cells, including the protein acinus Erythropoiesis involves the progressive differentiation starting whose activation by cleavage is required for chromatin from hemopoietic stem cells to mature erythrocytes. The condensation. steps involved in this complex differentiation process are The studies on normal erythropoiesis have clearly indicated numerous and involve first the differentiation to multipotent that immature erythroid precursors are sensitive to apoptotic triggering mediated by activation of the intrinsic and extrinsic hemopoietic progenitors generating mixed colonies in vitro apoptotic pathways. These apoptotic mechanisms are fre- (CFU-Mix), and then to committed erythroid progenitors quently exacerbated in some pathologic conditions, associated subdivided in early erythroid progenitors (burst-forming unit- with the development of anemia (ie, thalassemias, multiple erythroid, BFU-E) generating in vitro large erythroid colonies myeloma, myelodysplasia, aplastic anemia). and late erythroid progenitors (colony-forming unit-erythroid, The considerable progress in our understanding of the CFU-E) generating small erythroid colonies and finally to apoptotic mechanisms underlying normal and pathologic erythropoiesis may offer the way to improve the treatment of morphologically recognizable erythroid precursors. During this several pathologic conditions associated with the development differentiation process, the cells became progressively sensitive of anemia. to erythropoietin due to the appearance of the erythropoietin Leukemia (2004) 18, 1176–1199. doi:10.1038/sj.leu.2403383 receptors on these cells. Keywords: erythropoiesis; apoptosis; anemia; myelodysplasia; The compartment of bone marrow erythroid precursors is well myeloma characterized and involves a proliferative–maturative compart- ment, involving maturation from proerythroblasts to polychro- matophilic erythroblasts through the basophilic stage, and a maturative nonproliferative compartment involving maturation Introduction from polychromatophilic erythroblasts to mature red blood cells Basic background through the reticulocyte stage. The end cells, erythrocytes, are uniform biconcave disks lacking a nucleus and organelles and composed in large part by a highly specialized protein, The hematopoietic system in vertebrates requires the presence hemoglobin. The bone marrow erythropoiesis is a highly of cells that ensure a continuous production of new cells needed efficient system that tunes the rate of erythropoietic production to replace mature blood elements endowed with only a limited to physiologic needs. In this system, the physiologic stimulus is lifespan. This continual blood cell production is ensured by hypoxia representing the signal inducing the synthesis of erythropoietin (Epo), the main cytokine involved in the control Correspondence: Dr U Testa, Department of Hematology and Oncology, Istituto Superiore di Sanita`, Viale Regina Elena 299, of erythroid production. In spite of the efficiency of the 00161 Rome, Italy; Fax: þ 390649387087; E-mail: [email protected] erythropoietic system, a small, but significant level of inefficient Received 15 December 2003; accepted 12 March 2004 erythropoiesis due to premature intramedullary death of Apoptotic mechanisms in the control of erythropoiesis U Testa 1177 hemoglobin concentration and an increase in chromatin density. This review will show the relevance of apoptotic mechanisms in the control of erythropoiesis under physiologic and patholo- gic conditions.

Role of the Epo EpoR in the control of erythropoiesis

Several decades of studies have shown that Epo represents the main cytokine involved in the control of erythropoiesis. Red blood cell production is strictly dependent on the interaction of Epo with its single transmembrane receptor (EpoR). Epo is secreted from the kidney and fetal liver according to a molecular mechanism triggered by hypoxia. The main targets of Epo are represented by bone marrow erythroid cells expressing EpoR: particularly, late erythroid progenitors (CFU-E) and proerythro- blasts, where the EpoR is maximally expressed (about 1000 receptors per cell). The crucial role of Epo and its receptor for red blood cell production is directly supported by knockout studies in mice of the Epo and EpoR: EpoÀ/À and EpoRÀ/À mice die at day 12.5 of embryonic life as a consequence of severe anemia. EpoÀ/À or EpoRÀ/À mice exhibited a normal number of erythroid progenitors (BFU-E and CFU-E) at the level of the fetal liver, thus showing that Epo and its receptor are not required for the commitment of hemopoietic stem cell to the erythroid lineage.1 The interaction of Epo with EpoR homodimer determines a conformational change of the EpoR subunits, with consequent activation of the signal transduction machinery. The signal transduction pathways induced by the activated EpoR are complex and have been elucidated only in part. As a class I cytokine receptor, EpoR initiates Epo-dependent signal transduction through the activation of the Janus kinase (JAK2), which binds the EpoR at the level of a conserved structure called Box1 motif (reviewed in Wojchowski et al2). Through JAK2 activation, EpoR induces multiple signalling pathways, which acting together orchestrate the complex cell Figure 1 Outline of the process of erythroid differentiation. In the machinery involved to prevent apoptosis and to support top panel, the main features of the erythroid cell morphology are terminal erythroid proliferation and differentiation. Among schematically represented. In the other three panels, the kinetics of them, the phosphatidylinositol 3 kinase (PI3K), AKT kinase and expression of several molecules during erythroid differentiation is Ras signalling pathways play a key role in the mediation of shown. These molecules are divided, according to their function, into many biological actions induced by Epo. three main groups represented by transcription factors, growth factor Another important pathway activated by EpoR is represented receptors and proteins important for erythroid cell structure and/or function. by Stat5, a member of the Stat family, acting downstream numerous cytokine receptors and regulating cell growth survival and differentiation via activation of target genes acting as a erythroid cells occurs. Recent studies indicate that this transcription factor. Given the potential role of Stat5 in the inefficient erythropoiesis is due to the apoptotic death of induction and modulation of antiapoptotic mechanisms elicited erythroblasts. by Epo, the role of this peculiar transcription factor in The main biochemical and molecular events occurring during erythropoiesis will be analyzed in detail below. the process of erythroid differentiation and maturation are In addition to Epo and its receptor, other cytokines play an briefly outlined in Figure 1. BFU-E progenitors take about 14 important role in the control of erythropoiesis. In this context, days to differentiate in vitro forming large colonies; CFU-E particularly relevant is the role of the stem cell factor (SCF), also progenitors take about 7 days to form colonies in vitro. CFU-E known as the kit ligand (KL) and its receptor (SCF-R or c-kit). c- progenitors undergo 3–5 mitotic divisions to differentiate to kit is expressed in the majority of CD34 þ hemopoietic terminal erythrois elements through different maturation steps: progenitors and its expression is maintained at high levels proerythroblasts, basophilic erythroblasts, polychromatophilic during the stages of differentiation from BFU-E to CFU-E; at later erythroblasts and orthochromatic erythroblasts. Finally, these stages of differentiation during the maturation of CFU-E, c-kit cells undergo terminal maturation by extruding their nuclei and expression progressively declines and disappears in polychro- progressively losing all organelles: as a consequence of these matophilic and orthochromatic erythroblasts.3 These findings processes, young erythrocytes, called reticulocytes, are formed. imply that SCF may exert effects on erythroid cells during early As erythroid precursors progress in their maturation, they and late stages of erythroid differentiation and may act also on undergo a progressive decrease in their size, an increase in immature erythroid precursors.3

Leukemia Apoptotic mechanisms in the control of erythropoiesis U Testa 1178 These observations clearly showed that c-kit signalling is Erythroid differentiation is also influenced by other transcrip- indispensable for normal erythropoiesis. On the other hand, tion factors acting either at early or late stages of the numerous in vitro and in vivo experiments have shown that KL differentiation process. An important role in the control of early and Epo coadministration results in a stimulation of erythropoi- stages of erythropoiesis is played by Tal-1, a member of the esis and BFU-E and CFU-E colony stimulation: particularly, the basic helix-loop-helix (bHLH) family of transcription factors. in vitro administration of KL together with Epo resulted in a Tal-1 expression is essential for the early development of the marked enhancement of erythroid proliferation, associated with primitive and definitive hematopoietic systems. Tal-1 expression a delay in erythroid maturation. is strictly required for proper erythroid and megakaryocytic In addition to Epo and SCF, glucocorticoids also have a differentiation (ie, in the absence of tal-1, no erythroid and remarkable effect on erythropoiesis. These hormones act megakaryocytic differentiation is observed).13 Like other tissue- through nuclear receptors, and recent investigations indicate restricted bHLH transcription factors, Tal-1 binds DNA as a that mice deficient in glucocorticoid receptor expression exhibit heterodimer with the ubiquitously expressed E proteins, which a complete loss of stress erythropoiesis, that is, the capacity to recognizes the hexanucleotide sequence CANNTG found in a respond with an increased erythropoietic production to stress wide variety of eucaryotic transcriptional enhancers, particu- conditions, such as erythrolysis or hypoxia.4 larly at the level of erythroid and megakaryocytic genes. In normal adult tissues, Tal-1 expression is restricted to hemato- poietic and endothelial cells and, particularly in erythroid cells, Molecular mechanisms of control of erythropoiesis: role Tal-1 is initially expressed at low levels in quiescent erythroid of transcription factors progenitors, but becames highly expressed throughout the entire differentiative and maturative process and forms active hetero- The process of erythropoietic differentiation, as well as the dimers with E2A, participating in the activation of erythroid- whole process of hemopoietic differentiation, is orchestrated at specific genes.14 In fact, antisense oligomers to Tal-1 inhibit the molecular level by a complex network of transcription proliferation and self-renewal of erythroleukemia cells,15 and factors that act by regulating the expression of a set of target forced Tal-1 expression exerts a stimulatory effect on erythroid genes. development of normal hemopoietic progenitors.16 Among these different transcription factors, GATA-1 seems to It is of interest to note that several studies suggest that the play a key role in erythroid development. This zinc-finger capacity of Tal-1 to influence the hemopoietic differentiation transcription factor, expressed in erythroid and megakaryocytic depends more on the capacity to form transcriptional complexes cells, binds to GATA-binding motifs present in the promoters with other transcription factors than on its DNA-binding and/or enhancers of all erythroid-specific genes (reviewed in properties. This intriguing conclusion is directly supported by Cantor and Orkin5). GATA-1 is scarcely expressed in quiescent the observation that Tal-1 mutants that lack a DNA-binding erythroid progenitors, but is rapidly induced when these cells domain are able to rescue primitive erythropoiesis from in vitro are induced to erythroid differentiation by Epo and then differentiated Tal-1À/À embryonic stem cells.17 progressively accumulates during erythroid maturation, being abundantly expressed during all stages of erythroid matura- tion.6,7 The highest levels of GATA-1 are observed in CFU-Es Antiapoptotic mechanisms operating in erythroid cells and proerythroblasts.8 The hemopoietic phenotype of GATA-1 knockout mice showed severe anemia with the production of an Stat5 erythroid progeny, resulting in a maturational arrest at the level of proerythroblasts. Studies on GATA-1À/À embryonic stem cells The signal transducer and activator of transcription (Stat) induced in vitro to erythroid maturation showed that these cells proteins have a dual role as signal transducers and activators failed to mature beyond the proerythroblast stage and undergo of transcription. These proteins are latent in the cytoplasm and rapid apoptosis, thus suggesting an essential role for GATA-1 in are activated by extracellular signalling cytokines or growth erythroid survival, in addition to erythroid maturation.9 factors that bind to specific cell surface receptors.18 According to these observations, it seemed logical to assume Stat 5 is a transcription factor present in the cytoplasm in a that one or more GATA-1 target genes, which normally exert latent form and activated by the EpoR, as well as many other their physiological function at the proerythroblast/basophilic cytokine receptors. As a consequence of EpoR activation, Stat5 erythroblast stages, are strictly necessary for the survival and binds to phosphorylated tyrosines present on the cytoplasmic terminal maturation of erythroid precursors. However, surpris- tail of the EpoR, and itself becomes phosphorylated at the level ingly, an examination of the expression of several important of tyrosine residues. The activated Stat5 dimerizes and is potential target genes showed that they continued to be translocated from the cytoplasm to the nucleus, where it expressed at approximately normal levels in the absence of modulates the expression of several genes involved in the GATA-1.10 Among these potential target genes, one could be control of cell proliferation and differentiation. In addition to represented by Bcl-XL, an antiapoptotic protein whose expres- these genes, Stat5 activates the expression of several antiapop- 11 sion is strongly induced by GATA-1. totic genes in erythroid cells, most notably Bcl-XL, through a In addition to binding DNA, other transcription factors exert direct binding at the level of Stat5-binding consensus sequences 19–22 their activity through protein–protein interactions. A prototype present in the promoter of the Bcl-XL gene. These effects of of this category of factors is represented by FOG-1 (Friend of Stat5 on Bcl-XL activation may represent the molecular basis to GATA-1), a zinc-finger transcription factor that binds to the explain the antiapoptotic effects elicited by Stat5 in erythroid amino zinc-finger of GATA-1 (reviewed in Cantor and Orkin5). cell lines,19 while dominant-negative Stat5 induces a growth FOG-1, abundantly expressed in erythroid and megakaryocytic arrest and apoptosis of erythroid precursors.23 cells, is coexpressed with GATA-1 during erythroid develop- Studies on mice that lack the expression of both Stat5a and ment and plays a key role in the control of erythropoiesis: in Statb isoforms supported the protective role of Stat5 in erythroid fact, FOG-1 mice die of severe anemia caused by an erythroid cell survival. Stat5aÀ/À/Stat5bÀ/À mice embryos are severely maturation block similar to that observed in GATA-1À/À mice.12 anemic as a consequence of an impaired survival of liver

Leukemia Apoptotic mechanisms in the control of erythropoiesis U Testa 1179 differentiation from BFU-E to CFU-E and to immature erythroid precursors, followed by a rapid and marked decline at later stages of maturation (polychromatophilic and orthochromatic erythroblasts). (Figure 2). It is of interest to note that Stat5a5b deficiency also induces a premature death of myeloid precursors, which seems to be 25 related to a decrease of Bcl-2 and Bcl-XL.

Role of Bcl-XL as an antiapoptotic factor in erythropoiesis

Bcl-XL pertains to the family of Bcl-2-related proteins that act as important regulators of cell death, integrating competing apoptotic and antiapoptotic signals to modulate the activation of the cell death machinery. The Bcl-2 family members can be classified into three different groups on the basis of comparative analysis of their structure–function: (i) ‘multidomain’ death antagonists (Bcl-2, Bcl-XL, Bcl-w, Mcl-1 and A1); ‘multidomain’ death agonists (Bax, Box and Bok); and (iii) ‘BH3-only’ proapoptotic proteins (Bim, Bid, Bik and Bak). The first two groups of Bcl-2 members function to protect or disrupt the integrity of mitochondrial membranes, respectively, while BH3- only proteins trigger cell death through binding to the receptor domain of multidomain Bcl-2 members, thereby mediating the inactivation of the antiapoptotic members or the activation of 26,27 the proapoptotic members. Bcl-XL as well as Bcl-2 are supposed to be inserted into the outer mitochondrial membrane through a hydrophobic C-terminal motif. The main function of þ Figure 2 Kinetics of Stat5A and Sta5B expression in CD34 cells the antiapoptotic Bcl-2 family members seems to consist of selectively differentiating to erythroid (E), megakaryocytic (Mk) or promoting adaptation and maintaining the vitality of mitochon- monocytic (Mo) cells in unilineage cell cultures. The cells were 28 harvested at different days of culture, lysed and analyzed for Stat5A dria to various types of perturbations of cellular metabolism. and Stat5B content. For the E lineage, two lanes for each day of culture Particularly, the antiapoptotic mechanism of Bcl-XL, as well as are shown: the first corresponds to cells grown in the presence of Epo of other Bcl-2 family members, seems to be related to the alone, while the second corresponds to cells grown in the presence of inhibition of the activity of Bid and Bax, which cooperate in the Epo þ SCF. Samples have been normalized according to b-actin formation of pores in the membrane of mitochondria, allowing content. the release of components of these organelles in the cytoplasm, with a subsequent loss of their function.29 Recent studies, however, suggest a broader role of Bcl-2 in the control of erythroid progenitors. Particularly, fetal liver cells derived from apoptosis, which cannot be limited to the control of mitochon- Stat5aÀ/ÀStat5bÀ/À animals generated a low number of erythroid drial membrane integrity and also consists of a general control of colonies in vitro, showed a three-fold increase in the frequency caspase activation program independent of the cytochrome c/ of apoptotic cells and a pronounced increase in the percentage Apaf-1/caspase-9 apoptosome.30 Therefore, two models have of apoptotic cells when grown in vitro in the presence of Epo.19 been proposed to explain the antiapoptotic activity of Bcl-2/Bcl- À/À In spite of marked anemia during embryonic life, Stat5a XL. In the first model, BH3-only proteins can directly bind and Stat5bÀ/À mice exhibited a moderate condition of anemia at activate Bak/Bax, and this phenomenon may be inhibited by birth, which was progressively attenuated in animals of adult Bcl-2/Bcl-XL-mediated sequestration of BH3-only proteins. In age (at adult age only about 50% of Stat5aÀ/ÀStat5bÀ/À mice the second model, the binding of BH3-only proteins to their À/À À/À were anemic). However, the adult Stat5a Stat5b mice, in primary targets Bcl-2/Bcl-XL leads to a neutralization of these spite of their near-normal hematocrit levels, are deficient in antiapoptotic factors, resulting in the activation of Bax/Bak.31 generating high erythropoietic responses following a stress Gene targeting studies of antiapoptotic Bcl-2 family members stimulation (ie, the induction of a chemically induced haemo- provided clear evidence that they exhibit a unique physiologic lytic anemia).24 The analysis of erythropoiesis in Stat5aÀ/À5bÀ/À role, nonredundant with the activity of other cell death showed the existence of an increased pool of immature inhibitors, indicating that each Bcl-2 protein is essential for erythroblasts exhibiting reduced Bcl-XL levels and undergoing the survival of some cell types: thus, Bcl-2 is required for the apoptosis at a high rate with respect to the corresponding cells survival of kidney, melanocyte stem cells and mature lympho- 24 of wt animals. cytes; Bcl-XL for neuronal and erythroid cells; Bcl-w for sperm In conclusion, the analysis of Stat5 knockout mice strongly progenitors; A1 for neutrophils and Mcl1 for zygote implanta- suggests that the antiapoptotic effect of this transcription factor tion (reviewed in Cory and Adams31). Particularly interesting in erythroid cells is exerted at the level of immature erythroid were the phenotypic features observed in the Bcl-X gene À/À cells and is mediated mainly via modulation of Bcl-XL. In line knockout: in fact, Bcl-X mice die at E13, exhibiting massive with this observation, Stat5a and Stat5b and Bcl-XL exhibit a cell death at the level of neuronal elements of the central similar pattern of expression during normal erythroid matura- nervous system and of erythroid elements present in the fetal tion, with a progressive increase of expression during the liver.32

Leukemia Apoptotic mechanisms in the control of erythropoiesis U Testa 1180 þ The level of action of Bcl-XL in the protection of erythroid Studies on human CD34 cells have shown that both Bcl-XL cells from apoptosis was investigated in detail using mouse and Bcl-2 are widely expressed in these cells; in contrast, in þ À embryonic stem cells in which both alleles of the Bcl-XL gene CD34 /38 cells (corresponding to early, immature hemopoie- 33 À/À were disrupted. The introduction of Bcl-XL embryonic stem tic progenitors), Bcl-XL is preferentially expressed as compared cells into mouse blastocysts clearly showed that these cells were to Bcl-2.43,44 unable to contribute to the generation of definitive erythroid The kinetics of Bcl-XL expression during erythroid differentia- 33 À/À cells. Bcl-XL embryonic stem cells generated in vitro a tion and maturation was explored using different cellular models number of erythroid colonies similar to those originating from wt of erythroid differentiation. Using human CD34 þ cells grown embryonic stem cells; however, a significant proportion of either in erythroid (SCF þ Epo) or in granulocytic (SCF þ G-CSF) erythroid precursors present in the erythroid colonies of cell culture medium, it was shown that at days 4 and 8 of À/À 33 Bcl-XL cells undergo apoptosis during their maturation. culture, Bcl-XL expression was markedly more pronounced in 45 These findings strongly suggested that Bcl-XL is a critical erythroid than in granulocytic precursors. In a second cellular antiapoptotic regulator of erythropoiesis. system, an immortalized murine cell line was used: these cells The role of Bcl-XL in adult erythropoiesis was explored correspond to immature murine erythroid precursors isolated through the study of transgenic mice conditionally deficient in from p53À/À mice that proliferate in medium containing SCF 34 Bcl-XL gene. At 3 months of age, these animals exhibited a and Dexamethasone, while they differentiate in medium condition of severe hemolytic anemia, associated with platelet containing Epo and Insulin.46 When shifted to a differentiating 34 deficiency. The analysis of the bone marrow of these animals medium, these cells undergo terminal maturation in 72 h. Bcl-XL showed a hyperplasia of both megakaryocytic and erythroid expression in these cells progressively increases, reaching precursors; the rate of apoptosis in erythroid precursors of Bcl- maximal expression at terminal erythroid maturation.46 In XL-deficient animals was only slightly increased as compared to another study, immature murine erythroblasts, mainly corre- that observed for the corresponding cells of normal animals. sponding to proerythroblasts, have been isolated from the spleen Since these animals suffered from a condition of severe anemia of mice infected 2 weeks earlier with an anemia-inducing strain and had increased numbers of reticulocytes, it was concluded of Friend virus and then induced in vitro to terminal erythroid 34 that Bcl-XL is essential for the survival of red blood cells. maturation in the presence of Epo; alternatively, cells corre- Studies on an immortal line of phenotypically normal mouse sponding to CFU-E have been isolated after 1 week of culture in erythroblasts provided further details about the antiapoptotic vitro of BFU-E in semisolid medium and then grown in vitro in 35 role of Bcl-XL in erythroid maturation. These cells were the presence of Epo up to terminal maturation. In both these cell maintained in an undifferentiated state by agents promoting self- culture systems, Bcl-XL was expressed at very low levels in renewal, such as SCF and glucocorticoids, while they were immature erythroblasts and its expression progressively and induced to differentiate by Epo. Overexpression of the Bcl-XL markedly increased during maturation up to terminal erythro- gene allowed these cells to undergo terminal differentiation to blasts.47 35 mature erythrocytes in the absence of Epo. We have reinvestigated Bcl-XL expression in erythroid The molecular basis responsible for the elevated expression of cells using a unique unilineage cell differentiation system Bcl-XL in erythroid cells is unknown at the moment. The involving the selective differentiation of purified peripheral þ promoter of the Bcl-XL gene used for the start of the transcription blood CD34 cells to erythroid cells in liquid suspension in in erythroid cells is localized at 50 in close proximity to the start serum-free medium in the presence of very low concentra- initiation codon ATG.36 This promoter region contains several tions of interleukin-3 (0.01 U/ml) and GM-CSF (0.001 ng/ml) and putative regulatory regions recognized by some transcription in the presence of saturating amounts of Epo (3 U/ml). This factors, including GATA-1, NF-E2 and Ets-1 and other transcrip- culture system allows exploration of the differentiation of tion factors pertaining to the Ets family36 This promoter region BFU-E to CFU-E (from day 0 to days 5–6 of culture) and the contains a Stat-binding element, able to bind Stat-5.37 GATA-1 maturation of CFU-E to mature erythroblasts and to reticulocytes plays an important positive role in the regulation of Bcl-X gene (from day 7 to days 14–15 of culture). In parallel, using expression.38 Particularly, it was shown through the analysis on different unilineage cell culture systems allowing the selective conditional knockout models that the expression of GATA-1 in differentiation of CD34 þ cells to granulocytic or monocytic embryonic stem cells strongly induces BCL-XL, but not Bcl-2, or megakaryocytic cells, we have compared the pattern of expression in erythroid cells and this may represent one of the Bcl-XL expression observed in other hematopoietic cell lineages. þ mechanisms responsible for the antiapoptotic effects exerted by In quiescent CD34 cells, we observed a low Bcl-XL expression 38 GATA-1 in erythroid cells. (Figure 3). A marked increase in Bcl-XL expression was Finally, more recent studies have led to the identification of observed during early stages of the differentiation of CD34 þ the major start site of Bcl-XL gene transcription in erythroid cells. to the erythroid lineage (day 4 and day 7, when the cells This site corresponds in the human Bcl-X gene at À654, relative correspond to a stage intermediate from a BFU-E to a CFU-E and to the ATG initiation codon; furthermore, an enhancer element to CFU-Es/proerythroblasts, respectively); Bcl-XL levels contin- was identified at position À1804 through À1734.39 ued to increase during the early and intermediate stages of Epo deprivation induces the activation of caspase-3, leading erythroid maturation, reaching a peak level of expression when to apoptosis of erythroblasts.40 Since activated caspase-3 was the majority of erythroid cells had reached the stage of 41 reported to cleave Bcl-XL, it is conceivable that Epo protects polychromatophilic erythroblasts; during terminal stages of erythroid cells from apoptosis in part via blockage of caspase-3- erythroid maturation (days 14–16 of culture), a marked decline dependent cleavage of the Bcl-XL protein. Furthermore, it was in Bcl-XL expression was observed (Figure 3). The administration shown that the activation of ERK1 and ERK2 by Epo upregulates of SCF to the erythroid cultures moderately potentiated Bcl-XL Bcl-XL expression via inhibition of caspase activities, thus expression, associated with terminal erythroid maturation resulting in the protection of erythroid cells from apoptosis.42 (Figure 3). In contrast, Bcl-2 expression was limited only to Few studies have explored the expression of Bcl-XL, as well as the early stages of erythroid differentiation and completely of Bcl-2, in hemopoietic progenitors (ie, in total CD34 þ cells or declined to very low or undetectable levels during the in fractions of CD34 þ cells, such as 34 þ /38À or 34 þ /38 þ ). maturation of erythroid cells.

Leukemia Apoptotic mechanisms in the control of erythropoiesis U Testa 1181 marked inhibition of hemoglobin synthesis after DMSO induc- tion, as well as an increased rate of apoptosis.48 Importantly, this inhibitory effect of Bcl-XL deficiency on hemoglobin synthesis was also observed in cells overexpressing Bcl-2.48 The inhibitory effect on hemoglobin synthesis caused by Bcl-XL deficiency was due to an inhibition of heme synthesis and not to a reduction of globin mRNA expression.48 Furthermore, studies of enforced expression i.v. of the FDCP-Mix multipotent progenitor cell line have indicated a novel role for Bcl-XL in cell fate decision beyond cell survival.49 In fact, the erythroid expression of Bcl-XL in these cells was associated with an induction of erythroid differentiation and prohibited the generation of myeloid cells.49 Abnormalities in Bcl-XL expression and/or function have been involved in the pathogenesis of some disorders of the erythroid lineage. In this context, the observations made in polycythemia vera are particularly relevant. Polycythemia vera (PV) is an acquired myeloproliferative disease character- ized by the accumulation of morphologically normal red cells, white cells and platelets.50 Since the accumulation of red cells dominates the clinical picture, the disease was called polycythemia. In spite of several decades of bio- chemical studies, the mechanisms responsible for the accumu- lation of erythrocytes in this disease remain unknown. However, some physiopathological mechanisms relevant for the develop- ment of the disease have been identified and they indicate that PV erythroid cells are resistant to some apoptotic stimuli.51 In line with this finding, it was shown that erythroid cells obtained from the bone marrow of patients with PV exhibit increased levels of Bcl-XL as compared to those observed in normal 52 erythroblasts. Bcl-XL overexpression may explain the acceler- ated differentiation of PV erythroid progenitors in vitro in the absence of Epo, in contrast to normal erythroblasts, as well as their survival advantage in vivo, where Epo production is very low. It is of interest to note that recent studies have indicated that Bcl-XL may represent a major target of some antitumor chemotherapeutic drugs. Therefore, Bcl-XL levels may represent a major determinant in the protection of the erythroblasts from apoptotic cell death induced by DNA-damaging chemother- þ þ Figure 3 Kinetics of Bcl-XL expression in CD34 (34 ) cells apeutic drugs. differentiating selectively either to erythroid (E) or megakaryocytic In spite of their structural heterogeneity and their different (Mk) or granulocytic (G) or monocytic (Mo) cells. The cells have been molecular targets, many chemotherapeutic agents kill tumor harvested at different days of culture, lysed and analyzed for Bcl-XL and Bcl-2 content by Western blotting. For the E lineage, two lanes are cells by inducing apoptosis. The majority of these drugs mediate shown for each day of culture: the first corresponds to cultures grown cell death through activation of the intrinsic apoptosis pathway in the presence of Epo alone, while the second corresponds to cultures (Figure 4); this pathway is activated by different types of grown in the presence of Epo and SCF. intracellular stresses, such as genetic damage or growth factor deprivation, and implicates the Apaf-1-dependent activation of caspase-9, following the release of cytochrome c and Smac/ 53 The study, in parallel, of Bcl-XL expression in other Diablo from mitochondria. This pathway is regulated by the hemopoietic lineages showed that this antiapoptotic protein proapoptotic multidomain and BH3-only Bcl-2 proteins and exhibits in the Mk cells, a pattern of expression similar to that their antiapoptotic counterparts, which also includes Bcl-XL.In observed in erythroid cells; in contrast, in granulocytic cells, addition to representing one of the main molecules involved in Bcl-XL was expressed at low levels only during initial stages of the cell protection in the intrinsic apoptotic pathway, Bcl-XL differentiation and thereafter declined to virtually undetectable may represent also a direct target of some chemotherapeutic levels (Figure 3). In contrast, Bcl-2 was detectable at low levels drugs. In fact, it was shown that Bcl-XL can be modified by only during the initial stages of Mk differentiation and then deamidation of asparagines residues, at the level of an declined to low or undetectable levels; in contrast, in unstructured loop separating the a1helix-BH4 domain and the 54 granulocytic cells, Bcl-2 was expressed at sustained levels a2helix-BH3 domain. Bcl-XL deamidation was observed in during all stages of differentiation and maturation (Figure 3). tumor cells or in mouse embryonic fibroblasts p53À/À treated Recent studies have suggested that in erythroid cells, Bcl-XL with different types of genotoxic agents, including cisplatin, 54 could exert other biologic functions in addition to its role as an etoposide, taxol and g-irradiation. The deamidation of Bcl-XL antiapoptotic factor. In fact, studies performed on the Friend has as a consequence led to its inactivation, with a consequent erythroleukemia cell line showed that the inhibition of Bcl-XL loss of Bcl-XL binding to Bim and subsequent induction of expression (by antisense transcripts) in these cells elicited a apoptosis. The same authors also showed that the hypopho-

Leukemia Apoptotic mechanisms in the control of erythropoiesis U Testa 1182

Figure 4 Signal transduction through the intrinsic apoptotic pathway. The intrinsic apoptotic pathway is initiated by different stimuli mainly represented by growth factor deprivation or genotoxic agents such as antitumor chemiotherapics or g-irradiation. The initial events are represented by the activation of ‘BH3-only’ proteins such as Bid and Bim; these activated proteins are subsequently translocated to the mitochondrial membrane, where they associate with and inactivate the antiapoptotic proteins Bcl-2 and Bcl-XL. The proapoptotic proteins, Bax and Bad, are activated and generate in the mitochondrial membrane a protein-permeable conduit, with consequent release of cytochrome c and other mitochondrial proteins. The release of cytochrome c induces the formation of apoptosome in association with Apaf1 and caspase-9. The Apoptosis-inducing factor (AIF) released by mitochondria is capable of inducing apoptosis independent of caspases. Smac/DIABLO released from the mitochondria interacts with and inactivates the ‘Inhibitor of apoptosis’ (IAP) with the consequent activation of caspases.

sphorylated active form of retinoblastoma protein (Rb) inhibits Hydroxyurea, another myelosuppressive agent, also elicited deamidation of Bcl-XL, suppressing through this mechanism the apoptosis of erythroid cells through a mechanism involving a apoptosis induced by DNA-damaging agents. strong upmodulation of the TRAIL receptor 2.56 Anemia is a common complication of cancer, often resulting in a decrease in the quality of life and influencing the outcomes of patient care. The myelosuppressive effects of chemotherapy Role of cell death receptors in the control of are a major cause of anemia in cancer patients. Chemotherapy- erythropoiesis induced anemia is a result of two different mechanisms acting at the level of: (a) the bone marrow progenitor/stem cell compart- Ligands and cell death receptors pertain to the tumor necrosis ment and (b) the immature erythoid precursors (proerythroblasts factor (TNF) and TNF receptor (TNF-R) superfamilies, respec- and basophilic erythroblasts).55 The latter mechanism was tively. Members of the TNF family of membrane-bound and recently clarified, showing that immature normal erythroblasts secreted ligands pair off with one or more specific cell surface are extremely sensitive to the cytotoxic effect of chemother- receptors that form a corresponding family of cognate receptors apeutic agents, such as cisplatin, etoposide or campothecin, (TNF-Rs). Each ligand–receptor pair is considered a system, and while mature erythroblasts (acidophilic erythroblasts) are almost actually more than 40 distinct ligand–receptor systems are completely resistant.55 Importantly, Epo also at high doses was currently recognized.57 Members of both the TNF ligand and unable to protect immature erythroblasts from chemotherapy- TNF-R superfamilies exhibit several remarkable structural induced apoptosis. According to these observations, it was similarities.58 The TNF ligands are type II transmembrane concluded that the primary target of chemotherapy-induced proteins, characterized by an extracellular C terminus domain, apoptosis is represented by proerythroblasts and basophilic named the ‘TNF homology domain’: this domain contains a erythroblasts.55 Interestingly, preincubation of erythroid cells conserved framework of aromatic and hydrophobic residues, with SCF resulted in a marked protection of immature erythroid responsible for the association of single molecules to form a cells from chemotherapy-induced apoptosis.55 The mechanism trimer.59 Most of the TNF ligands are synthesized as membrane- of this protection seems to be related to an SCF-mediated bound proteins, but soluble forms may be generated by a upregulation of Bcl-2 and Bcl-XL expression, associated with a process of limited proteolysis. The solubilization of some ligands consequent inhibition of caspase activation.55 At the moment, it is associated with a considerable loss of biological activity: this is unknown whether the inhibition of chemotherapic-induced is the case of the Fas ligand (Fas L) and of the TNF-related apoptosis could be related to an inhibition of Bcl-XL deamida- apoptosis-inducing ligand (TRAIL). The main molecules pertain- tion, possibly mediated by upregulation of hypophosphorylated ing to the TNF ligand superfamily are represented by TNF-a, Rb expression. FasL, TRAIL, TWEAK, RANKL, APRIL and Lymphotoxin-a (LTa).

Leukemia Apoptotic mechanisms in the control of erythropoiesis U Testa 1183 More than 40 TNF-Rs have been identified. The majority of In fact, caspase-8 and -10 directly cleave the ‘executione’ them are type I transmembrane proteins, with an extracellular caspase-3. Activated caspase-8 and -10 are also able to activate domain N terminus and with an intracellular domain C by proteolytic cleavage Bid, which is responsible for the terminus; a notable exception is represented by the TRAIL-R3, activation of the proapoptotic arm of the Bcl-2 gene super- which is anchored to the cell membrane through a covalently family, with the consequent release of apoptogenic factors from linked C-terminal glycolipid.58 Many of these receptors possess mitochondria. Particularly, SMAC/DIABLO released from mito- a stretch of amino acids, the death domain (DD), as part of their chondria promotes apoptosis through its capacity to bind and sequence.59 The DDs are required for apoptotic signalling. For neutralize the inhibitor of apoptosis proteins (IAP), thus some of these receptors, soluble forms may be generated preventing the activity of these factors as attenuators of the through a process of limited proteolysis (TNF-R1 and TNF-R2) or caspase activation. of alternative splicing (Fas). Recently, evidence was accumulated showing that death Each member of the TNF ligand superfamily binds at least one receptors and their ligands may act as negative regulators of receptor of the TNF-R superfamily: FasL binds selectively to Fas; erythropoiesis, and that their action may play a physiologically TRAIL may bind to four different membrane receptors, called relevant role in the control of the rate of erythropoiesis. TRAIL-R1, TRAIL-R2, TRAIL-R3 and TRAIL-R4; and TNF-a binds Furthermore, abnormalities of these receptors may play an two membrane receptors, TNF-R1 and TNF-R2. The majority of important role in the physiopathology of some anemic condi- the TNF-R superfamily members function as membrane recep- tions, as will be discussed later. Evidences are now analyzed tors transducing a signal following interaction with their ligands. showing the effect elicited by the activation of the main death These signalling receptors may be subdivided into two groups receptors on normal erythropoiesis. on the basis of the structure and function of their cytoplasmic region: one of the two receptor groups possesses a DD that mediates the association between the receptor and a death Effect of TNF-a on normal erythropoiesis: The binding of adaptor protein; the other group does not possess this death TNF-a to the TNF-R1 leads to a cascade of events, outlined cytoplasmic domain. The TNF-Rs, TRAIL-R1 and TRAIL-R2, as above, that determines the induction of apoptosis, as well as NF- well as Fas, are typical examples of death receptors. Two kB activation and JNK activation. Given the signal transduction examples of DD adaptors are represented by FADD (also known pathways activated by this cytokine, it is not surprising that TNF- as MORT1) and TRADD: FADD possesses two interfaces: one a is able to induce a variety of biological effects. Mouse TNF and interacting with the DD of Fas and the other one interacting with TNF-R1 gene knockout studies have clearly shown that this intracellular effector enzymes (caspases that initiate the apopto- cytokine plays a key role in protection against infection by tic process).60 The other adaptor called TRADD (TNFR- bacterial, fungal and parasitic pathogens. associated death domain) can initiate apoptosis through FADD In initial studies carried out in vivo64,65 and in vitro,66 or may stimulate protein kinases involved in the control of evidence was provided that TNF-a exerted an inhibitory effect phosphorylation cascades to induce the transcription of some on erythropoiesis. In addition, in a phase I study of TNF-a in immune-system modulation genes. TRADD seems to be cancer patients, a clear decrease in hemoglobin levels after 1 involved in the apoptotic signalling of the TNF-R, but not of month of TNF-a treatment was observed. Subsequent in vitro Fas and TRAIL-Rs.61 studies, however, showed that the inhibitory effect of TNF-a on Some of the receptors of the TNF receptor superfamily are erythropoiesis at the CFU-E level was likely mediated by IFN-b decoy receptors able to compete with signalling receptors for released by macrophages in response to TNF-a and not due to a ligand binding, thereby, inhibiting their function. Examples of direct effect of TNF-a on erythroid progenitors.67,68 However, in decoy receptors are given by DcR1 and DcR2 (also known as another study, a direct effect of TNF-a on erythroid progenitors TRAIL-R3 and TRAIL-R4), competing with TRAIL-R1 and TRAIL- (BFU-E) was suggested, mediated by the TNF-R1.69 R2, respectively, for binding of TRAIL and DcR3 (sFas) A direct effect of TNF-a on erythroid cells was supported by competing with Fas for binding of FasL.60 The physiologic role subsequent studies. Using preparations of virtually pure of these receptors is still unclear, but it has been suggested that erythroid precursors derived from unilineage erythroid cultures, they could protect some normal cells from their cytotoxic it was shown that TNF-a exerts a moderate, but significant, ligands. inhibitory effect on the proliferation of immature erythroblasts The series of biochemical events elicited by the interaction of (proerythroblasts and basophilic erythroblasts), associated with a death ligand with its receptor has in part been elucidated and a slight induction of apoptosis, in the presence of erythropoietin. can be summarized briefly as follows (Figure 5). The engage- Furthermore, TNF-a, like other death receptor ligands, exerted ment of death receptors by their ligands activates the extrinsic an inhibitory effect on erythroid maturation.70 A direct effect of apoptotic pathway. This pathway leads, as the intrinsic TNF-a on erythroid cells was also supported by a recent study apoptotic pathway, to the activation of caspases independent showing that: (i) both murine and human CFU-E release TNF-a; of p53. The activation of a death receptor by its ligand leads to (ii) the addition of neutralizing anti TNF-a antibody to cultures of the recruitment at the level of the receptor of a death adaptor, human CD34 þ cells stimulated with Epo increased the which in turn determines the rapid assembly of a death-inducing generation of erythroid cells; (iii) the number of BFU-E colonies signalling complex (abbreviated as DISC), with consequent was higher in the bone marrow of TNF-aÀ/À mice than in wt activation of the apoptosis initiating caspase-8 and -10. mice; and (iv) the addition of TNF-a to TNF-aÀ/À mice elicited a Caspase-10 is an initiator caspase found in primates but significant inhibition of BFU-E colony formation, while TNF-a not in rodents. The role of this caspase in initiating the exerted only a slight inhibitory effect on wt mice BFU-Es.71 apoptotic process is supported by two observations: (a) An inhibitory effect of TNF-a on the generation of erythroid lymphocytes from ALPS II patients bearing caspase-10 mutation cells from purified human CD34 þ cells was also reported in are resistant to TRAIL-mediated killing and62 (b) endogenous another recent study.72 In this study, it was also shown that a caspase-10 is activated and recruited at the level of the DISC detectable expression of TNF-R1 was observed only during the and is capable of transmitting an apoptotic signal in the absence initial stages of erythroid differentiation, while TNF-R2 expres- of caspase-8.63 sion was observed during all stages of erythroid differentiation.72

Leukemia Apoptotic mechanisms in the control of erythropoiesis U Testa 1184

Figure 5 Signal transduction through the extrinsic apoptotic pathway. Engagement of the either TNF or TRAIL or FasL with their cognate receptors TNF-R1 or TRAIL-R1 or -R2 or Fas results in the formation of a receptor proximal complex containing the adaptor proteins TRADD and FADD. These adaptor proteins in turn recruit additional key pathway-specific enzymes, such as caspase-8, and they became activated and initiate downstream events leading to apoptosis.

However, it is unlikely that the inhibitory effects of TNF-a on required for the induction of the expression of erythroid-specific erythropoiesis could be mediated via TNF-R2 signalling, in that genes.73 this receptor lacks a death domain in its cytoplasmic tail. The inhibitory effect of TNF-a on erythroid maturation seems to involve NF-kB induction.72 In fact, NF-kB activity is Effects of the Fas–FasL pathway on normal adult high in early erythroid progenitors and then declines at later erythropoiesis: Without death by apoptosis, the life of cells stages of erythroid maturation. In fact, low NF-kB levels are of the immune system and their precise function would not be

Leukemia Apoptotic mechanisms in the control of erythropoiesis U Testa 1185 possible: lymphocyte homeostasis is mainly mediated by the of quiescent CD34 þ cells to Fas-mediated apoptosis seems to Fas/FasL pathway.74 be related to the expression in these cells of elevated levels of c- Growing evidence indicates that the Fas/FasL system could FLIP (FLICE-inhibitory protein), a dominant-negative inhibitor of also play a relevant role in the regulation of hematopoiesis and, caspase 8.82 particularly, of erythropoiesis. Other studies have directly evaluated Fas/FasL expression in Initial studies on mice with mutations of either the Fas or FasL erythroid cells at various steps of differentiation and maturation. system failed to show significant modifications of the hemato- CFU-E constitutively express low levels of Fas, whose expression poiesis. However, a re-evaluation of the hematopoiesis in Fas or was potentiated by IFN-g;83 interestingly, CFU-E-like cells FasL-deficient mice showed a striking extramedullary increase possess low, but significant amounts of FasL, whose levels are in hematopoietic progenitors, including both erythroid and non- potentiated by IFN-g treatment and by differentiation.83 The erythroid progenitors; furthermore, CFU-S and CFU-C on expression of Fas and FasL was explored in detail by De Maria peripheral blood and in spleen clearly increased in these mice et al84 using a selective system of unilineage culture of human after birth.75 These observations clearly indicate that the Fas/ erythroid cells starting from purified CD34 þ peripheral blood FasL system can affect hematopoiesis during both the fetal and cells. Fas is clearly upregulated during the initial steps of adult life. These conclusions were supported by the observation erythroid differentiation from BFU-E to CFU-E, reaching showing that mice with mutations of the Fas (lpr mice) or of the elevated levels of expression at the stage of immature FasL gene (gld mice) show a pronounced increase in the number erythroblasts (proerythroblasts and basophilic erythroblasts) of CFU-GM.76 and the remaining expressed at significant levels up to terminal The expression of Fas/FasL during erythroid differentiation has stages of erythroid maturation (orthochromatic erythroblasts).84 been explored in detail. Initially, it showed low Fas mRNA These findings were also confirmed through the analysis of expression in CD34 þ cells isolated from human bone mar- erythroid cells present in normal bone marrow.84 Fas cross- row.77,78 Immature primitive hematopoietic progenitors linking was effective in inducing an apoptotic response only in (CD34 þ /38À) from human fetal liver express significant immature erythroblasts, while mature erythroblasts are resistant amounts of Fas antigen, whereas the more mature progenitors to this triggering.84 In contrast, an opposite pattern of FasL (CD34 þ /38 þ ) showed low Fas antigen expression.79 Both TNF- expression was observed, this membrane-bound ligand being a and interferon-g (IFN-g) induced a marked increase in Fas expressed in late differentiating Fas-insensitive erythroblasts, expression on CD34 þ cells.77,79,80 Interestingly, the expression mostly at the orthochromatic stage. FasL expressed on the of Fas antigen on CD34 þ cells was greatly increased following membrane of mature erythroblasts was found to be functional in the induction of the cycling of these cells with cytokines, such as that it was able to kill Fas-sensitive lymphoblast targets in a Fas- SCF, IL-3 and GM-CSF.79 dependent manner.84 Importantly, FasL-bearing mature erythro- Other studies have explored the consequences of Fas blasts displayed a Fas-mediated cytotoxicity against immature activation in progenitor cells on the induction of apoptosis, Fas-positive erythroblasts, which was in part abrogated by high proliferation and differentiation. Basically, these studies have concentrations of Epo.84 According to these findings, it was provided evidence that immature progenitor cells are resistant to suggested that erythropoiesis is regulated according to a Fas-mediated apoptosis. LinÀ/Sca þ /cKit þ stem cells reveal little negative feedback mediated by mature and immature erythro- or no constitutive expression of Fas and are resistant to the blasts, whereby the former cells might exert a cytotoxic Fas- apoptotic triggering by an anti-Fas agonistic antibody.81 How- mediated effect on the latter cells within the erythroblastic ever, if these stem cells are induced to cycle by incubation with island.84 (Figure 6). early-acting growth factors and incubated in the presence of As mentioned above, Epo was able to partially protect TNF-a, they exhibit a marked upmodulation of Fas antigen immature erythroid cells from a strong triggering of the Fas- expression and became sensitive to Fas-mediated apoptotic mediated apoptosis, as it is induced by Fas agonistic antibodies. triggering.81 These findings are also confirmed by studies on There is, however, evidence that other growth factors, like SCF, human CD34 þ cells derived from either peripheral blood or may exert a more pronounced protective effect than Epo. In this cord blood: these cells are found to be resistant to the apoptotic context, Lee et al85 showed that anti-HLA-DR monoclonal triggering elicited by either soluble FasL or anti-Fas agonistic antibody elicited a marked upregulation of Fas on primary antibody.82 However, if CD34 þ cells are induced to proliferate human bone marrow cells, thereby increasing their suscept- in the presence of flt3 ligand, SCF and thrombopoietin, they ibility to Fas-mediated cell death. SCF partially antagonized Fas- became sensitive to anti-Fas-mediated apoptosis. The resistance mediated apoptosis of primary cells, which suggests that SCF

Figure 6 Negative regulation of erythropoiesis mediated by the interaction of FasL expressed on the membrane of mature erythroblasts with its receptor Fas present on the surface of immature erythroblasts. Following this interaction that takes place within the erythroblastic islands, the cells undergo either a maturation block/delay if the Epo concentration is high or an apoptotic process if the Epo concentration is low.

Leukemia Apoptotic mechanisms in the control of erythropoiesis U Testa 1186

Figure 7 Negative regulation of erythropoiesis triggered by death receptor activation or by erythropoietin deprivation: both these induce the apoptotic cascade, with the consequent activation of caspases that cleave the transcription factors GATA-1 and Tal-1. The cleavage of these transcription factors is responsible for either the maturation arrest or apoptosis of erythroid cells.

protects hemopoietic precursor cells from Fas-mediated apop- tion elicited by Epo deprivation or by death receptor triggering tosis.85 SCF may protect CFU-E from Fas-induced apoptosis: the involve as a necessary step the caspase-mediated cleavage of protective effect of SCF was significantly more effective than the two transcription factors, Tal-1 and GATA-1, whose integrity effect elicited by Epo.86 This protective effect of SCF involves the and activity are essential for erythroid maturation and survival activation of Src-kinases.86 The simultaneous addition of IFN-g (Figure 7). and FasL to CFU-E elicited a marked activation of caspase-8 and -3 with a consequent apoptotic response; this phenomenon was greatly inhibited by SCF addition, via a mechanism involving Effect of TRAIL on normal erythropoiesis: Initial studies the upregulation of c-FLIP expression.87 performed by De Maria et al70 have shown that erythroid cells, In another set of studies, the mechanisms through which Fas- in addition to Fas and TNF-R1, also express other death mediated apoptosis exert a negative control on erythropoiesis receptors, such as TRAIL-R1 and TRAIL-R2. The expression of were explored in detail. These experiments take advantage of these two membrane receptors was higher in immature than in the observation that Fas triggering in the presence of high Epo mature erythroblasts.70 TRAIL-R3 and TRAIL-R4 are not concentrations elicited a low apoptotic response in immature expressed at any stage of erythroid maturation.89 Furthermore, erythroblasts, but induced a blockade of erythroid maturation.70 it was shown that the membrane-bound form of the TRAIL was This last mechanism requires caspase activation.70 One of the also expressed in normal erythroblasts, with a preferential major effects of caspase activation consisted in the degradation expression at the level of mature erythroblasts.70 Therefore, the of the transcription factor GATA-1; the induction of GATA-1 pattern of TRAIL/TRAIL-R system in erythroid cells was highly degradation seems responsible for the Fas-mediated blockade of comparable to that observed for the Fas/FasL system. erythroid cell maturation since the transduction in erythroid Immature erythroid cells are sensitive to the antidifferentiative progenitors of a caspase-resistant GATA-1 mutant resulted in a (in the presence of Epo) or cytotoxic (at low Epo concentrations) complete protection against Fas-mediated blockade of erythroid effects induced following the interaction of TRAIL with the maturation.70 Interestingly, the erythropoiesis blockade elicited TRAIL-Rs expressed on these cells.70 These findings were by Epo deprivation was in part inhibited by the transduction of confirmed in subsequent studies, showing that hemopoietic caspase-resistant GATA-1 in erythroid progenitors.70 In a second progenitors as well as mature erythroblasts are resistant to the study, it was shown that another transcription factor, Tal-1, is an apoptotic effects induced by TRAIL.89,90 The blockade of additional target protein cleaved by activated caspases in erythroid maturation induced by TRAIL-R triggering involves erythroid cells. The transduction in hemopoietic progenitors of ERK1/2 activation, as suggested by the inhibition by pharmaco- a Tal-1 mutant resistant to caspase cleavage protected erythroid logical inhibitors of the ERK pathway.89 cells from Fas-mediated inhibitory effect on erythroid matura- The mechanism of TRAIL-induced apoptosis of erythroid cells tion and cleavage of GATA-1.88 Interestingly, the expression of was explored in experimental models of erythroid differentia- the Tal-1 caspase-resistant mutant in erythroid cells completely tion. Studies in erythroleukemia K562 cell line showed a protects these cells from the apoptosis elicited by erythropoietin sensitivity of these cells to TRAIL only after induction with deprivation.88 hemin.91 The induction of TRAIL sensitivity was not related to The ensembling of these observations indicates that the the stimulation of expression of TRAIL receptors, but to a apoptotic effects and the inhibitory actions on cell differentia- downmodulation of c-FLIP.91

Leukemia Apoptotic mechanisms in the control of erythropoiesis U Testa 1187 Death receptors also influence the survival of other loss of the nucleus occurring at terminal stages of matura- hemopoietic lineages: The activation of death receptors tion.97,98 was also shown to affect the survival of other types of Recent studies have provided evidence that the ‘spontaneous’ hemopoietic cells. In neutrophils, FasL, TRAIL and TNF interact activation of caspases was observed during later steps of cell with their respective receptors present on the membrane of maturation in various cell lineages, which include monocytic, neutrophils and per se do not induce apoptosis, but block the megakaryocytic and erythroid lineages. During the process of survival of these cells induced by growth factors, such as GM- normal maturation of monocytes to macrophages, an activation CSF and G-CSF.92 This inhibitory effect on growth factor- of caspase-3 and -9 was observed, associated with the release of dependent survival seems to be mediated via the capacity of cytochrome c from the mitochondria and cleavage of the protein activated death receptors to bind in a caspase-independent acinus, but without induction of cell death.99 The blockade of manner tyrosine phosphatase SHP-1, which dephosphorylates the caspase activity using synthetic cell-permeable inhibitors JAK-2.92 As a consequence of the binding of SHP-1, an inhibited the cell maturation process.99 Similar observations inhibition of the growth factor receptor antiapoptotic signalling have been made in megakaryocytic cells. Caspase activation was observed; also, this last event was caspase independent.92 occurred at two stages of megakaryocytic maturation: (i) a first This type of mechanism of action of activated death receptors step of caspase activation (caspase-3 and -9 became sponta- was not explored in erythroid cells. neously activated) occurred before proplatelet activation and was limited only to some cellular compartments and (ii) a Animal studies indicating a role of death receptors in the second step of diffuse caspase activation occurred at the end of 100 control of erythropoiesis: Mice harboring targeted null the maturation process after platelet release. The first process mutations in Fas and TNFR-1 exhibit no significant defects in the of caspase activation did not lead to cell death, while the second 100 erythroid lineage. However, studies performed in zebrafish have wave of caspase activation was associated with cell death. shown a very interesting observation. In fact, a hemopoietic- Importantly, the addition of caspase inhibitors to megakaryocy- specific death receptor was identified in this animal species.93 tic cultures inhibited platelet release. In line with these Very interestingly, the generation of transgenic mice expressing observations, transgenic mice overexpressing the antiapoptotic 101 a dominant-negative inhibitor showed a specific phenotypic gene Bcl-XL exhibited impaired platelet fragmentation. abnormality: the production of a surplus of erythroid cells.93 At Several lines of evidence indicate that caspase activation also the moment, there is no evidence in mammals about a death occurs during the process of erythroid maturation and could receptor with a pattern of expression limited to the hemopoietic play an important role in this process. In this context, an initial lineage.93 study showed that caspase-1, -2, -3, -5, -6, -7, -8 and -9 are expressed in erythroid cells.102 The levels of procaspase-2, -3 g g and -8 were markedly higher in immature erythroblasts than in Induction of erythroid apoptosis by IFN- : IFN- is an 102 example of a cytokine that does not induce an apoptotic mature erythroblasts. As expected, Epo deprivation elicited a signalling per se, but may induce apoptosis of erythroid marked increase in caspase activation. A transient caspase-3 progenitors through an indirect mechanism involving upmodu- and -7 activation was observed during the stages of erythroid lation of both Fas and FasL, as mentioned above. In addition, maturation corresponding to proerythroblasts and basophilic g erythroblasts; this phenomenon was transient in that the caspase IFN- upmodulates the level of expression and activates several 103 caspases, including caspase-8 and -3, in erythroid progenitors.94 activation regressed at later stages of erythroid maturation. Paradoxically, IFN-g may exert a protective effect on apoptosis These caspases, transiently activated through the mitochondrial on erythroid cells at later stages of differentiation.95 pathway, cleave proteins involved in nucleus integrity (lamin B) and chromatin condensation (acinus) without inducing cell Ceramide, an intracellular second messenger produced by 103 sphingommyelin hydrolysis, has been involved as a mediator of death. Inhibitors of caspases, such as z-VAD, added to apoptosis induced by a number of cytokines, including IFN-g erythroid cultures just before the moment of caspase activation a elicited a block of erythroid maturation at the basophilic and TNF- . Ceramide induces an inhibition of CFU-E colony 103 96 stage. These findings have also been confirmed in murine formation. Interestingly, the ceramide antagonist sphingosine- 104 1-phosphate significantly reversed the CFU-E colony inhibition erythroblasts. Furthermore, it was shown that the over- induced by IFN-g.96 These observations suggest that ceramide is expression of Raf-1, which prevents caspase activation, impairs erythroid maturation by reducing differentiation-associated one of the key mediators of the inhibition of CFU-E colony 104 formation by IFN-g.96 caspase activation. An opposite phenomenon was observed in Raf-1À/À mice.104 A possible involvement of caspase in the control of erythroid Role of caspases in erythroid maturation cell production is also supported by the analysis of the phenotype of mice with the targeting of the genes encoding There is evidence that these caspases may also display, in some of these caspases. In this context, the most interesting addition to a role in the apoptotic process, a function in the findings originated from the analysis of caspase-8À/À mice.105 process of cell differentiation/maturation. Furthermore, some Caspase-8 deficiency resulted in embryonic lethality, associated caspases are required for the process of normal erythroid with two salient features: impaired heart muscle development differentiation. and congested accumulation of erythrocytes.105 These features The first evidences of a role for caspases in cell differentiation resemble the phenotype reported also in mice with targeted were derived from studies carried out in lens epithelial cells and disruption of FADD gene.106,107 in keratinocytes: caspase-3 is activated during terminal stages of The analysis of the defects in the erythroid lineage observed in lens epithelial cell differentiation; furthermore, caspase inhibi- these animals was, however, limited to the analysis of tors block the denucleation of these cells occurring at the erythrocytes and it is, therefore, impossible to know whether terminal stages of lens epithelial cell maturation.97 Similarly, it mice with caspase-8 and FADD deficiency also exhibit was shown that caspases are activated during normal keratino- abnormalities of erythroid cell maturation, in addition to an cyte differentiation and this activation is required for the normal increased expansion of the number of mature erythrocytes. It is

Leukemia Apoptotic mechanisms in the control of erythropoiesis U Testa 1188 of interest that in a recently described family exhibiting a aging could participate in the degradation of crucial erythrocyte complete deficiency of caspase-8, no hemopoietic abnormal- membrane proteins involved in the maintenance of shape and ities have been described.108 The discrepancy between the function. In line with these findings, it was observed that a loss phenotype of caspase-8 deficiency in humans and mice could of band 3 is associated with premature erythroid cell death be related to the function in humans of caspase-10, the closest (dyserythropoiesis).116 paralogue of caspase-8 in humans (caspase-10 has no known Since an increase in Ca2 þ concentration was associated with orthologue in mice). the aging of erythrocytes and was considered as one of the In addition to caspases, recent studies also suggest a possible molecular mechanisms responsible for the senescence of these role of p53 during late stages of erythroblast maturation. cells, additional studies have explored whether the induction of Particularly, a high expression of the p53 protein was observed an increase of Ca2 þ concentration in erythrocytes could trigger in late orthochromatic erythroblasts: this p53 activation may be an apoptotic or apoptotic-like response. Following the induction related to the nuclear degradation occurring in these cells, of an increase of cytosolic Ca2 þ concentration, erythrocytes without a fully executed apoptotic process.109 undergo a rapid self-destruction process exhibiting several features of an apoptotic process, such as plasma membrane microvesciculation, phosphatidylserine externalization; this Apoptotic mechanisms in mature red blood cells process culminates either in erythrocyte destruction or, in the presence of macrophages, in macrophage phagocytosis with The occurrence of apoptotic processes and particularly of a subsequent destruction.114 This process is inhibited by cysteine possible activation of caspases was also explored in mature protease inhibitors, but not by caspase inhibitors.114 Impor- erythrocytes. These cells lack a nucleus and after a lifespan of tantly, cysteine protease inhibitors allowed erythrocyte survival about 120 days undergo a process of senescence and are then both in vitro and in vivo.114 According to these findings, it was removed from the circulation. During this process, senescent proposed that an increase in intracellular Ca2 þ concentration in erythrocytes undergo typical morphological changes, such as erythrocytes activates cysteine protease calpain that mediates shrinkage as a consequence of the progressive release of spectrin cleavage and other morphological changes leading to microvescicles from the cell membrane and shape transforma- cell shrinkage (Figure 8). tion from a discocyte to a spherocyte form, enabling first their recognition and then their phagocytosis by macrophages. In addition to these changes, there are a series of modifications at Role of deoxyribonuclease IIa in erythroid maturation the level of the cell membrane, such as a progressive loss of the structural membrane protein spectrin and the loss of membrane During the terminal stages of erythroid maturation, erythroblasts asymmetry associated with the externalization of phosphatidyl- exhibit several features typical of an apoptotic process, such as a serine residues, resembling those observed in the process of reduction of cell size, withdrawal from the cell cycle, nuclear apoptotic cell death in nucleated cells. These observations condensation and nuclear expulsion. The progressive transition prompted studies aimed at evaluating whether mature RBCs from the rapid proliferating compartment of immature erythro- possess an apoptotic machinery similar to that observed in the blasts to the terminal slowly proliferating compartment of majority of nucleated cells. Human erythrocytes do not undergo mature erythroblasts is regulated by growth-inhibitory genes.117 programmed cell death when either treated with staurosporine Many endonucleases have been implicated in the apoptotic and cycloheximide or when cultured in the absence of serum, process and particularly in DNA digestion occurring during conditions that induce apoptosis in all types of nucleated apoptosis, such as caspase-activated DNase (CAD), deoxyribo- cells.110 According to these observations, it was concluded that nuclease I (DNaseI) and deoxyribonuclease II (DNaseII). Two mature red blood cells do not have an apoptotic machinery and types of DNase II exist in mammalians that have been therefore do not undergo an apoptotic program. This issue was designated as DNase IIa and DNase IIb. DNase IIa is re-explored by the same authors on chicken erythrocytes, ubiquitously expressed, while DNase IIb is expressed only in showing that serum deprivation or treatment with staurosporine the salivary gland. and cycloheximide induces the death of these cells; however, Recent studies aimed at elucidating the physiological role of although these erythrocyte deaths displayed many features that DNase IIa have shown a role of this enzyme in erythroid are typical of apoptotic cells, they are not blocked or inhibited maturation and, particularly, in the process of enucleation by different types of caspase inhibitors.111 According to these occurring at very late stages of maturation. Mice deficient in observations, it was concluded that chicken erythrocytes die DNase IIa expression were generated. These animals die of without apparently activating caspases. severe anemia and show a marked decrease of circulating red More recently, the problem was carefully re-evaluated in blood cells (ie, they have only 1/10 of the physiological number human erythrocytes. In this context, it was observed that mature of circulating erythrocytes), associated with the presence in the erythrocytes contain considerable amounts of caspase-3 and circulation of definitive nucleated erythroblasts, a cell type caspase-8, while other main components of the apoptotic normally present only in the bone marrow and not in peripheral machinery such as caspase-9, Apaf-1 and cytochrome c are blood.118 The reduced presence of anucleated red blood cells in absent.112 Although present at relatively high levels, caspase-3 the peripheral blood of DNase IIaÀ/À mice cannot be related to a and caspase-8 were not activable by various types of proapop- defect in erythroid commitment in that these animals exhibit a totic stimuli.112 normal number of both BFU-E and CFU-E in their fetal liver.118 Recent studies suggest a possible role for caspase-3 in However, mutant fetal liver erythroblasts showed a normal erythrocyte aging.113 First, it was observed that activated maturation after transfer into irradiated normal mice, thus caspase-3 can be detected in old, but not in young red blood suggesting that the defect of DNase IIÀ/À erythroblasts is not cells;114 second, this activated caspase-3 is able to cleave cell intrinsic to these cells. In fact, histological experiments membrane band 3, disrupting its interaction with the peripheral suggested that the source of DNase II responsible for the rescue membrane protein 4.2.115 According to these observations, it of the maturation of DNase IIÀ/À erythroblasts is represented by was suggested that some caspases activated during red cell macrophages. In fact, it was suggested that central macrophages

Leukemia Apoptotic mechanisms in the control of erythropoiesis U Testa 1189

Figure 8 Outline of the main apoptotic processes occurring during the process of erythroid differentiation. Mature erythroid cells exert a negative control on immature erythroblasts via FasL and other death receptor ligands. During the late stages of erythroid maturation, the activity of deoxyribonuclease II via macrophages is required for enucleation of orthochromatic erythroblasts with their consequent maturation to anucleated reticulocytes. During the senescence of red blood cells, an apoptotic-like process is observed, related to an increase in cellular Ca2 þ concentration that leads to the activation of the proteolytic enzyme calpain with consequent plasma membrane micriveciculation, phosphatidylserine externalization and subsequent recognition, engulfment and destruction by macrophages. present in erythroblastic islands may represent the source of Role of AKT pathway in the apoptotic control of the erythroid DNase IIa.118 lineage This conclusion was directly supported by the analysis of a second knockout of DNase IIa. In fact, the generated DNase IIÀ/À In many cell types, the phosphoinoinositide-3-kinase (PI3K)/AKT mice die at birth, exhibiting at the level of the liver macrophages (also known as protein kinase B) pathway plays a key role in the large DNA bodies resulting from the engulfment of undigested survival signalling in response to a variety of growth factors and nuclei extruded from erythroblasts.119 According to these this seems also to be the case in erythroid cells. The AKT/PKB findings, it was concluded that macrophages play an essential pathway involves a cascade of activation/signalling steps that role in erythroid maturation through a biochemical mechanism can be summarized as follows and that have been carefully involving DNase IIa. reviewed in several recent papers:121–124 The ensemble of these observations indicates that a regulated process of DNA fragmentation is strictly required for normal tissue homeostasis.120 Furthermore, it is evident that the (a) the first step involves the activation and modulation of PI3K; enucleation event occurring during late erythroid maturation is PI3K may be activated through different mechanisms, a regulated process requiring extensive signalling between involving either the binding of the complex p85/p110 erythroblasts and macrophages. (p85 is the regulatory subunit, while p110 is the catalytic

Leukemia Apoptotic mechanisms in the control of erythropoiesis U Testa 1190 subunit) to phosphorylated tyrosine residues present at the sively during erythroid maturation.134 In immature erythroid level of the cytoplasm domain of activated receptor tyrosine precursors, FKHR-L1 was rapidly phosphorylated by Epo kinases or the interaction with members of the small GTPase through a PI3K-dependent pathway.134 family, such as Ras; An additional target of PKB/AKT is represented by the (b) the second step consists in the generation of 30-phosphory- glycogen synthetase kinase-3 (GSK 3), a serine/threonine kinase lated inositol lipids (PIP3) that function as classical second involved in metabolic processes (glycogen metabolism), andut messengers by binding proteins that harbor PIP3-binding also implicated in apoptosis regulation in primary human domains; erythroid progenitors and its activity is suppressed by Epo, as 135 (c) PIP3 binds the binding domain of the serine/threonine PKB/ well as by SCT. Importantly, the inhibition of GSK3 using AKT, with its consequent activation. specific chemical inhibitors prevents apoptosis of erythroid (d) The activated PKB/AKT phosphorylates several components progenitors by Epo deprivation.135 of the apoptotic machinery and through this mechanism The ensemble of these observations implicates at least two induces antiapoptotic effects. PKB/AKT targets, FKHR-L1 and GSK3, in the regulation of the survival of erythroid cells. Particularly, PKB/AKT promotes cell survival by multiple mechanisms: (i) phosphorylation and inactivation of the proapoptotic protein Bad; (ii) maintenance of mitochondrial Other signalling pathways involved in the apoptotic integrity; (iii) decrease of the expression of death genes, such as regulation of erythroid cells FasL and Bim, via the phosphorylation of forkhead transcription factor FKHR-L1: phosphorylated FKHR-L1 is exported from the In addition to the PKB/AKT, other signalling pathways play a nucleus to the cytoplasm where it is sequestered by 14-3-3 role in the survival of erythroid cells. proteins; and (iv) increase of the transcription of survival genes The transcription activator protein 1 (AP1) could play a role in through the activation of NF-kB and CREB transcription the regulation of apoptosis mediated by Epo in erythroid cells. factors.121–123 Furthermore, recent studies indicate that the This is a transcriptional complex comprising members of the Jun PKB/AKT pathway is involved in the upregulation of survivin and of the Fos families of transcription factors. Evidence was expression observed during cell cycle entry.125 Survivin is a provided both in primary human hemopoietic progenitors and in member of the IAP family that inhibits apoptosis by inactivating Epo-dependent cell lines that both Epo addition or Epo several caspases.126 deprivation induces AP-1 activity, and this AP-1 induction is Initial studies on erythropoiesis and PKB/AKT signalling were required both for induction of erythroid proliferation and 136 based on the use of chemical inhibitors of PI3K. The PI3K apoptosis. The analysis of the composition of AP-1 transcrip- inhibitor wortmannin blocked the PI3K activation elicited by tional complex in these two conditions showed an interesting Epo and considerably decreased the proliferation of erythroid finding: JunB is present in erythroid cells, which triggered precursors expanded in vitro.127 Using a more specific and apoptosis by Epo deprivation, while c-Jun was present in 136 potent PI3K inhibitor (LY294002), an inhibitory effect on erythroid cells induced to proliferate. erythroid cell proliferation was confirmed, but it was also Finally, one of the two negative regulators of EpoR signalling, shown that PKB/AKT inhibition elicited the apoptotic death of a the small cytokine-inducible SH2-domain containing protein 137 significant proportion of erythroid precursors.128 In a subsequent (CIS) plays a role in the control of erythroid cell apoptosis. study, the effects of the PI3K inhibitor LY294002 at earlier stages CIS belongs to the family of suppressor of cytokine signaling of erythroid differentiation have been explored, showing a proteins and acts by reducing the Stat-5 activation elicited by marked inhibitory effect on the differentiation of CD34 þ to Epo. The enforced expression of this protein in erythroid erythroid precursors: particularly, the addition of the inhibitor to progenitors not only reduced their proliferation but also induced 137 CD34 þ cultures grown in the presence of Epo and SCF resulted a significant level of apoptosis. in a marked inhibition of the generation of glycophorin-A þ 129 cells. Apoptotic mechanisms underlying pathologic conditions of The essential role of the PI3K in the mechanism of induction erythropoiesis of erythroid proliferation and survival was also confirmed in a 130 recent study. It was also shown that three different mechan- The improvement of our understanding of the apoptotic isms equally active in erythroid cells are responsible for PI3K mechanisms and their role in the control of erythropoiesis has activation: direct association of the PI3K to the EpoR; prompted a series of studies focused in determining the phosphorylation of Gab via either Tyr 343 or Tyr 401 of the abnormalities of these mechanisms occurring under some 130 EpoR; and phosphorylation of the IRS2 adaptor protein. pathologic conditions involving the erythroid lineage. Usually, The activation of PKB/AKT following Epo stimulation requires under pathologic conditions, an exacerbation of the apoptotic a normal protein kinase C (PKC) activity, as suggested by the mechanisms is observed, leading to an increased destruction of 131 experiments carried out with PKC inhibitors. erythroid precursors. These conditions are briefly analyzed here. As mentioned above, the FOXO family of Forkhead transcrip- tion factors is an important target of the activated PKB/AKT: activated PKB/AKT directly phosphorylates FKHR-L1 and other Apoptotic mechanisms underlying anemia occurring in forkhead transcription factors resulting in cytoplasmic retention rheumatoid arthritis and inactivation, thus inhibiting the activity of these transcrip- tion factors required for the control of the expression of several About 50% of patients with rheumatoid arthritis develop anemia genes involved in cell cycle control, cell death and oxidative of moderate entity, exhibiting all the laboratory features of an stress.132 There is evidence that the activation of FKHR-L1 alone anemia associated with a chronic disease. can recapitulate all known elements of the apoptotic program Recent studies suggest that the increased release of TNF-a normally induced by cytokine withdrawal.133 FKHR-L1 is could also be responsible for the development of anemia expressed in erythroid cells, its expression decreasing progres- through the induction of an apoptotic mechanism. Studies

Leukemia Apoptotic mechanisms in the control of erythropoiesis U Testa 1191 performed in the bone marrow of anemic patients with chronic of patients with aggressive myeloma and high FasL levels, there arthritis showed a significant decrease in the number of is an increase of immature erythroblasts in the bone marrow, erythroid progenitors.138,139 The analysis of the erythroid associated with a decrease of mature erythroid cells.142 compartment in these patients showed interesting findings: (i) These observations argue in favor of the induction of a decreased number of mature erythroblasts; and (ii) an apoptosis of erythroid cells mediated by the induction of FasL increased rate of apoptosis within the compartment of immature expressed on the surface of myeloma cells with Fas expressed on erythroblasts.138 Interestingly, patients undergoing therapy with the membrane of immature erythroblasts. This hypothesis was a blocking anti-TNF-a monoclonal antibody exhibit an improve- supported by coculture experiments showing that myeloma cells ment in anemia and in the proportion of apoptotic erythro- are able to induce the cell death of erythroblasts, this blasts.139 phenomenon being inhibited by agents that block either Fas or These observations may have potential implications in our FasL.142 At the level of erythroid cells, the cell population more understanding of the mechanisms underlying anemia not only in sensitive to apoptosis, mediated by myeloma plasmocytes, is rheumatoid arthritis but also in other chronic inflammatory represented by immature erythroblasts (ie, proerythroblasts and diseases. basophilic erythroblasts). Furthermore, immature erythroblasts from myeloma patients are more sensitive to FasL-mediated apoptosis than their normal counterparts.142 Finally, apoptotic Apoptotic mechanisms in the genesis of anemia in erythroblasts were observed in vivo in the bone marrow of multiple myeloma myeloma patients; these cells showed a considerable expression of caspases-3 and -8 in both their proenzymatic forms and in Anemia is a complication frequently observed in multiple cleaved, active subunits.143 myeloma, in that up to 70–80% of these patients exhibit a Erythroblasts isolated from the bone marrow of patients with moderate to severe form of anemia (about 10% of these patients aggressive myeloma displayed reduced levels of GATA-1, a exhibit a severe form of anemia). A series of recent studies transcription factor essential for erythroid cell survival. indicate that the anemia of multiple myeloma could be According to these findings, it was proposed that the anemia mediated through an apoptotic mechanism. Recent studies have observed in multiple myeloma may be related to two mechan- showed FasL overexpression in multiple myeloma:140 there is isms: one dependent upon an elevated expression of FasL on the evidence that FasL expressed on tumor cells may mediate a membrane of myeloma plasmocytes and the other dependent on tumor escape mechanism through elimination of the Fas þ an increased sensitivity of Fas þ immature erythroblasts to the effector lymphoid cells committed to tumor surveillance. The apoptotic triggering elicited through the interaction of FasL þ level of FasL expressed on myeloma cells usually correlates with plasmocytes with these cells; as a consequence of this the grade of malignancy: high-grade myeloma patients display interaction, there is the activation of the apoptotic machinery, higher levels of FasL on their membrane, compared with activation of caspases and cleavage of GATA-1 with consequent patients with a low-grade disease.141 Similar observations have arrest of cell maturation or induction of apoptotic cell death been made for TRAIL, whose expression was observed (Figure 9). particularly in the tumor cells of patients exhibiting an aggressive disease.141 Studies performed on myeloma patients at different stages of the disease have shown that in the bone Ineffective erythropoiesis and apoptosis in thalassemias marrow, there is an inverse relationship between the level of FasL expressed on myeloma plasmocytes and the hemoglobin The unbalanced globin chain synthesis occurring in thalasse- level.142 Furthermore, it was observed that in the bone marrow mias leads to the formation of either free a-chains (b-

Figure 9 Apoptotic mechanisms underlying the development of anemia in multiple myeloma patients. FasL and TRAIL highly expressed on the surface of malignant plasmocytes interact with their respective receptors present on the surface of immature erythroid cells with the consequent induction of the apoptosis of these cells.

Leukemia Apoptotic mechanisms in the control of erythropoiesis U Testa 1192 thalassemias) or b-chains (a-thalassemias), which precipitate which does not correspond to any known membrane antigen of into the cytoplasm of erythroid precursors leading to the macrophages.153 premature death of these cells and to hemolysis of mature red blood cells.144 Under normal conditions, hemoglobin A (HbA) synthesis is coordinated to ensure a virtually equal Apoptotic mechanisms in megaloblastic anemias synthesis of a- and b-globin chains and to minimize the accumulation of free subunits that form cytotoxic precipitates, Megaloblastic anemia is caused by the deficiency of folate or resulting in premature cell death. Erythroid cells possess cobalamin (Vitamin B12). This type of anemia is characterized mechanisms to block a moderate imbalance in globin chain by pancytopenia resulting from the death of differentiating synthesis through the action of the alfa hemoglobin stabilizing hemopoietic cells, and particularly of erythroid precursors, protein (AHSP), an abundant erythroid-specific protein that is before they reach the terminal maturation. induced by GATA-1 and forms a stable complex with free The demonstration that apoptosis plays an important role in a-globin chains.145 This mechanism of protection of erythroid the genesis of anemia observed in megaloblastic anemias was cells, however, is not sufficient to protect these cells when the obtained in studies in animal models. An in vitro model of globin chain synthesis is greatly unbalanced, as it occurs in megaloblastic anemia was based on the culture of early thalassemias. erythroid precursors isolated from the spleens of mice that were Erythrokinetics studies performed in b-thalassemia patients fed with a folate-free diet during the acute erythroblastosis phase have provided clear evidence that the bone marrow is the main of disease induced by the Friend virus.154 When these site of erythroid cell death: in fact, in these patients as many as erythroblasts were grown in folate-rich medium they survived, 80% of the erythroid precursors die in the marrow.146 The cause proliferated and underwent terminal maturation.154 Interest- of the premature death of b-thalassemic erythroid precursors is ingly, the addition of thymidine to the folate-free medium due to apoptosis; in fact, the analysis of DNA laddering provided inhibited the apoptosis, thus suggesting that folate deficiency evidence that in b-thalassemia major, erythroid precursors, but leads to a defect in thymidine synthesis with consequent uracil not myeloid or lymphoid precursors, underwent an increased misincorporation into DNA. This misincorporation of uracil into level of apoptosis.147 Using flow cytometric techniques, it was DNA may induce double-stranded breakage of DNA with shown that b-thalassemic erythroid precursors undergo apopto- consequent induction of an apoptotic process. This hypothesis sis at a rate three to four times that of normal controls.148 If the was directly confirmed showing that erythroblasts undergoing evaluation is carried out in absolute terms, it corresponds to an apoptosis due to folate deficiency have increased uracil absolute increase in erythroid precursor apoptosis of about 15 misincorporation into DNA and increased p53 levels.155 More times over the normal controls.148 Analysis of the rate of recent studies based on the same cellular model showed that apoptosis of erythroid precursors in different types of thalasse- megaloblastic erythroblasts first accumulate in the S-phase and mic patients, characterized by different levels of ineffective then undergo apoptosis.156 Finally, experiments in p53À/À mice erythropoiesis, provided clear evidence that there is a direct provided clear evidence that the induction of apoptosis in folate- correlation in these patients between the level of ineffective deficient erythroblasts does not require p53.156 erythropoiesis and the percentage of apoptotic erythroblasts.149 Hemopoietic progenitors have been purified from the bone marrow of b-thalassemic patients and induced in vitro to Apoptotic mechanisms underlying the defective erythroid differentiation/maturation. Using this approach, it erythropoiesis in myelodysplasias was clearly shown that erythroid cultures that originated from b-thalassemic patients exhibit a limited expansion, compared to Myelodysplasias are an example of a disease in which the control cultures, due to the death of erythroid cells when they development of pathologic apoptotic mechanisms is responsible reach the maturation stage corresponding to polychromatophi- for several features of the hematologic manifestations of this lic/orthochromatic erythroblasts.150 Particularly, in this study, a disease. Myelodysplastic syndromes comprise a heterogeneous marked increase in the proportion of apoptotic polychromato- group of disorders of hemopoietic stem cells characterized by a philic and orthochromatic erythroblasts was observed in b- hypercellular bone marrow with several morphologic features of thalassemic cultures.150 The occurrence of apoptosis at the dysplasis, associated with peripheral blood cytopenias affecting polychromatophilic stage coincides with the stage where red blood cells, granulocytes and platelets, and a variable intensive hemoglobinization occurs and could be explained incidence of transformation into acute myeloid leukemia (AML). by the increase in a-globin production and precipitation. The presence of anemia dominates the clinical picture. In spite of the markedly increased rate of apoptosis of b- According to the probability of progressing to AML, myelodys- thalassemic erythroid precursors in the bone marrow, the plasias are divided into low and high risk: the low-risk inspection of bone marrow aspirates of these patients does not myelodysplastic syndromes include refractory anemia and show an increased number of dying erythroblats. This apparent refractory anemia with ringed sideroblasts and have a low paradox was explained by the recent demonstration that dying probability of progression and the high-risk patients exhibit b-thalassemic erythroblasts are rapidly phagocytosed by macro- severe cytopenia, increased percentage of blasts or cytogenetic phages, whose number and activation are enhanced.151 The abnormalities. mechanisms involved in the recognition of apoptotic erythro- A hallmark of the disease consists in the presence of a blasts by macrophages are not fully understood, but they seem hypercellular bone marrow, associated with peripheral blood to involve at least the CD36 present on the membrane of cytopenia. It has been suggested that a high level of apoptosis is macrophages and phosphatidylserine residues exposed on the responsible for the ineffective erythropoiesis observed in membrane of apoptotic erythroblasts.151 The mechanism of myelodysplastic syndromes (MDS).157 This increased rate of recognition and engulfment of apoptotic cells by macrophages apoptosis is typical of the early and intermediate stages of is a complex phenomenon, which seems to involve several development of the disease. An increased sensitivity to receptors on the membrane of activated macrophages, such as apoptosis is replaced by an increased resistance to apoptosis CD14 and CD36152 and a receptor of recent identification, during progression of the disease to leukemia.158

Leukemia Apoptotic mechanisms in the control of erythropoiesis U Testa 1193 Increased apoptosis of bone marrow precursors is a feature According to these observations, it was suggested that the typically observed in the majority of MDS patients.159 The increased susceptibility to apoptosis in MDS cells is caused by analysis of the mechanisms involved in the increased rate of events sensitizing these cells to death receptor triggering. apoptosis in MDS showed an involvement of both the Particularly, a current conceivable model to explain apoptosis extrinsic and intrinsic apoptotic pathways. A large number of in RA and RA with excess of ringed sideroblasts implies that studies provided evidence that the activation of the Fas/FasL increased sensitivity to triggering of death receptors is depen- system may represent an important pathogenetic mechanism in dent on constitutive mitochondrial signalling, with consequent MDS.160–162 These initial observations were carefully confirmed cytochrome c release and activation of caspase-9.168 and extended in a recent study163 where CD34 þ cells were It is important to note that in MDS, apoptosis is markedly isolated from the bone marrow of MDS patients and grown in a increased not only in the erythroid lineage but also in the two-step liquid suspension culture system suitable for selective myeloid, megakaryocytic, B-lymphoid and stromal cellular erythroid differentiation. Using this cell culture system, the elements of the bone marrow.171 authors showed that MDS CD34 þ cells grow normally during the initial proliferation/differentiation steps until they reach the stages of proerythroblasts, while their growth significantly Apoptotic mechanisms in aplastic anemia declines as compared to controls due to an increased rate of apoptosis.163 A large set of studies has suggested that the triggering of Interestingly, Fas antigen expression in MDS patients was apoptotic mechanisms could play a relevant role in the more elevated than in controls during all stages of erythroid pathogenesis of aplastic anemia. differentiation/maturation; furthermore, in mature erythroblasts, Two main types of aplastic anemia are observed in children: FasL expression was significantly higher in MDS than in the Diamond–Blackfan anemia and the Fanconi anemia. controls.163 According to these findings, it was concluded that Diamond–Blackfan anemia (BDA) is characterized by a chronic MDS erythroid cells exhibit an exacerbation of the physiologic aregenerative disease, starting during the neonatal period or mechanisms of Fas-mediated control of erythropoiesis and infancy. The hallmark of this condition consists in a bone exhibit a Fas-dependent apoptosis, even in the presence of high marrow exhibiting a selective marked decrease of erythroid Epo levels. Interestingly, Fas-triggered apoptosis in bone marrow precursors in the bone marrow. Initial studies have shown that cells of MDS patients with refractory anemia with ringed both BFU-E and CFU-E derived from the bone marrow of sideroblasts is significantly improved by G-CSF treatment; Diamond–Blackfan patients are more sensitive to Epo depriva- particularly, the addition of G-CSF to CD34 þ cells improves tion than their normal counterparts; particularly, apoptotic erythroid colony formation in these patients.164 changes, such as DNA fragmentation, were detected within As mentioned above, patients with more advanced stages of 3 h after Epo deprivation, while they appeared after 9 h in the MDS disease and with poor prognosis due to the presence of controls.172 In another study, a marked increase in serum cytogenetic abnormalities may exhibit a condition different from concentration of FasL was observed in the majority of DBA that reported in MDS patients with refractory anemia (RA) or RA patients.173 Although this observation suggests that activation of with ringed sideroblasts. In fact, some of these patients may cytotoxic lymphocytes occurs in DBA, the origin of increased exhibit an increased resistance to apoptosis instead of an serum FasL level is unclear. It may be suggested that certain increased sensitivity.165 This issue was recently reinvestigated, subsets of cytotoxic lymphocytes overproduce FasL and through providing clear evidence that the heterogeneity displayed by this this mechanism exert an inhibitory effect on erythroid progeni- group of MDS patients is due to the heterogeneity of the tors/precursors. cytogenetic abnormalities:166 in fact, these patients have three In 25% of cases, it was shown that DBA occurs due to the different types of cytogenetic abnormalities represented by mutation of the gene encoding the ribosomal protein S19.174 trisomy 8, monosomy 7 and 5qÀ deletion.166 There is clear evidence that the mutation of this gene is There is also evidence that the TRAIL/TRAILRs system may responsible for the development of erythroblastopenia in DBA play a role in the ineffective hemopoiesis observed in MDS. patients.175 However, it remains largely unclear why the loss of Both TRAIL and TRAILR1 and -R2 are expressed at increased this ribosomal protein, whose synthesis is observed particularly levels in the bone marrow of MDS patients compared to the in immature erythroblasts and decreasing progressively during respective levels observed in normal controls.166 Furthermore, erythroid maturation,176 induces the development of anemia hemopoietic procursors derived from the bone marrow of MDS and to the increased sensitivity to apoptosis observed in DBA patients are supranormally sensitive to TRAIL-mediated apopto- patients. sis.167 Fanconi anemia (FA) is a rare autosomal recessive disease There is evidence, however, that in MDS, in addition to the characterized by congenital abnormalities, progressive bone extrinsic apoptotic pathway the intrinsic apoptotic pathway is marrow failure and cancer susceptibility. The major clinical also activated. The hallmark of activation of the intrinsic problems of this condition depend on the development of apoptosis signalling pathway is the release of apoptogenic pancytopenia. One of the major features of the disease is related factors from mitochondria. In fact, markers of mitochondrial to the development of a progressive megaloblastic anemia. damage are commonly seen in MDS, including cytochrome c Recent studies have provided evidence that FA results from the release,168 accumulation of iron in the mitochondrial matrix of mutation of one of the eight genes (FANC A, B, C, D1, D2, E, F ringed sideroblasts169 and the occurrence of mitochondrial and G).177,178 Many of these proteins form a nuclear complex DNA mutations.170 Interestingly, in a recent study carried out on that seems to be involved in the repair of double-strand DNA refractory anemia with ringed sideroblasts, it was shown that a breaks.177–179 Furthermore, one of these proteins, FANC C, significant proportion of erythroid progenitors display a sponta- plays a role in the modulation of apoptotic response to an neous release of cytochrome c from mitochondria with oxidative stress.177,178 consequent activation of caspase-9.168 The addition of G-CSF Several studies have provided evidence that FA hemopoietic significantly inhibited cytochrome c release and caspase progenitors exhibit an increased sensitivity to cell death induced activation. by death receptor triggering. In this context, initial studies

Leukemia Apoptotic mechanisms in the control of erythropoiesis U Testa 1194 showed that bone marrow CD34 þ cells of FA patients exhibited The role of the renin–angiotensin system is particularly relevant. an increased expression of Fas that correlated with increased In fact, angiotensin II stimulates the proliferation of normal sensitivity to anti-Fas antibody-mediated inhibition of colony erythroid progenitors,200 while a lack of angiotensin-converting formation.180 These findings have been extended in other enzyme causes anemia.201 studies showing that both erythroid and granulo-monocytic The mechanism through which angiotensin II stimulates the progenitors are highly sensitive to the inhibitory effect exerted proliferation of erythroid progenitors is unknown, but it seems to by IFN-g: particularly, very low doses of this cytokine inhibited involve an enhanced survival of erythroid cells. This conclusion colony formation in FA patients, but not in normal controls.181 is strongly supported by studies carried out using inhibitors of In line with these findings, HPCs from mice nullizygous for the the angiotensin-convertory enzyme: these inhibitors induce FANC C locus are hypersensitive to the inhibitory effects apoptosis of erythroid progenitors/precursors,202 via induction induced by IFN-g.181 The increased sensitivity of FA HPCs to of death receptor pathways.203 It is of interest to note that these IFN-g was related to the capacity of this cytokine to upregulate inhibitors have been successfully used in vivo for the treatment Fas. of these forms of erythrocytosis. In addition to IFN-g hypersensitivity, FA bone marrow cells exhibited a high rate of spontaneous apoptosis and hypersensi- 182 tivity to FasL-mediated apoptosis. Interestingly, the over- Concluding remarks expression of the FA group C gene protects HPCs from death induced by Fas-mediated apoptosis.183 Experiments performed Growing evidences outlined in the present review indicate that on FAN C gene knockout mice provided further evidence that an apoptotic mechanisms play an important role in the control of increased sensitivity of erythroid progenitors to TNF-a and FasL- erythropoiesis under physiologic and pathologic conditions. mediated apoptosis could represent the main mechanism Here, it is important to underline some general conclusions. involved in the genesis of anemia in FA patients.184 As it is largely expected, the apoptotic responses in FA hemopoietic cells to IFN-g, TNF-a and FasL are mediated via the caspase-8- (1) Studies on the role of Fas/FasL system in erythropoiesis have dependent activation of caspase-3.185 provided evidences suggesting that mature erythroblasts The inhibitory effects of IFN-g and TNF-a in FA on may exert a negative control on the level and rate of erythropoiesis and more generally on hematopoiesis are related maturation of immature erythroid cells acting through a not only to a marked activation of the death receptor apoptotic ligand/receptor mechanism, with death ligands expressed signalling but also to induction of the release of nitric oxide, on mature erythroid cells and the death receptor being which exerts an inhibitory effect on hematopoiesis.186 present on the surface of immature erythroblasts. This In addition to pediatric aplastic anemia, there is evidence also mechanism may account for the low, but significant, level in acquired aplastic anemia about the involvement of apoptotic of ineffective erythropoiesis associated with premature mechanisms. The majority of acquired aplastic anemias are death of erythroid cells observed in normal subjects.204 related to physiopathological mechanisms dependent upon a T- Furthermore, this mechanism may represent an additional cell-mediated, organ-specific destruction of bone marrow tool of control of erythropoiesis, in addition to the negative hemopoietic cells.187 Thus, there is evidence that a significant feedback mechanism of control of erythropoiesis based on proportion of these patients exhibit an increased expression of modulation of erythropoietin production according to Fas on CD34 þ cells and abnormal levels of increased oxygen level.204 apoptosis.188 Increased FasL levels are observed in T lympho- (2) There is evidence not only for the erythroid lineage but also cytes of these patients189 and these cells seem to be responsible for the granulocytic system that death ligands/receptors may for the destruction of hemopoietic progenitors in aplastic exert an important physiologic homeostatic control. These anemia. findings are reminiscent of those observed in lymphocytes, Interestingly, the gene expression profile of CD34 þ cells of where there is clear evidence of about the key role of death aplastic anemia patients (mostly adult patients) demonstrated an receptors/ligands in the homeostatic control of their increased expression of a large number of genes implicated in number.205,206 The continuous death of a part of lympho- apoptosis and cell death, compared to CD34 þ cells isolated cytes is an essential process needed to maintain a constant from normal subjects.190 This important observation indicates lymphocyte population in the face of a continuous flux on that in aplastic anemia, CD34 þ cells represent the main target new lymphocytic elements.205,206 of an immunologically mediated damage. (3) Some apoptotic mechanisms are spontaneously activated and are required for the process of normal erythroid maturation. This is the case of a mild caspase activation Apoptotic mechanisms in the genesis of erythrocytosis occurring during late stages of erythroid maturation at the associated with various pathologic conditions polychromatophilic stage and required for a normal process of erythroid maturation. This is a general phenomenon Recent studies suggest an implication of apoptotic mechanisms observed also during megakaryocytic maturation and in the genesis of erythrocytosis associated with various types of monocyte to macrophage maturation. It is of interest to pathologic conditions. Particularly, these studies have shown a note that caspase activation is also required for the possible involvement of the renin angiotensin system in the differentiation of other cell types, such as keratinocytes genesis of erythrocytosis observed following a kidney trans- and corneal cells. plant191 or in association with congestive heart failure,192 (4) Immature erythroid cells are sensitive to the apoptotic chronic obstructive pulmonary disease,193,194 essential, malig- stimuli originating from death receptor signalling. It was nant, or renovascular hypertension195–197 or with chronic shown that the activation of death receptors in these cells in hemodialysis.198,199 The pathogenesis of erythrocytosis ob- the presence of exogenous Epo determines a clear inhibition served in these conditions is complex in that erythropoietin, of erythroid maturation, a phenomenon dependent on angiotensin II and insulin-like growth factor I are all involved. caspase activation. The target of activated caspases is

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