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2-Perdida de La AHSP y Su Efecto en La Eritropoyesis y Beta-talasemia JCI 2004

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Research article The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 10 November 2004 1457 Loss of α-hemoglobin–stabilizing protein impairs erythropoiesis and exacerbates β-thalassemia Yi Kong, 1 Suiping Zhou, 2 Anthony J. Kihm, 2 Anne M. Katein, 3 Xiang Yu, 1 David A. Gell, 4 Joel P. Mackay, 4 Kazuhiko Adachi, 2 Linda Foster-Brown, 3 Calvert S. Louden, 3 Andrew J. Gow, 2 and Mitchell J. Weiss 2 1 Cell and Molecular Biology Graduat
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  Research article  The Journal of Clinical Investigation http://www.jci.org    Volume 114   Number 10   November 2004 1457 Loss of α -hemoglobin–stabilizing protein impairs erythropoiesis and exacerbates β -thalassemia  Yi Kong, 1  Suiping Zhou, 2  Anthony J. Kihm, 2  Anne M. Katein, 3  Xiang Yu, 1  David A. Gell, 4  Joel P. Mackay, 4  Kazuhiko Adachi, 2  Linda Foster-Brown, 3  Calvert S. Louden, 3  Andrew J. Gow, 2  and Mitchell J. Weiss 2 1 Cell and Molecular Biology Graduate Program, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA. 2 The Children’s Hospital of Philadelphia and The University of Pennsylvania, Philadelphia, Pennsylvania, USA. 3 Safety Assessment, Astra-Zeneca Pharmaceuticals, L.P., Wilmington, Delaware, USA. 4 School of Molecular and Microbial Biosciences, University of Sydney, New South Wales, Australia. Hemoglobin (Hb) A production during red blood cell development is coordinated to minimize the delete-rious effects of free α - and β -Hb subunits, which are unstable and cytotoxic. The α -Hb–stabilizing protein (AHSP) is an erythroid protein that specifically binds α -Hb and prevents its precipitation in vitro, which suggests that it may function to limit free α -Hb toxicities in vivo. We investigated this possibility through gene ablation and biochemical studies.  AHSP  –/–  erythrocytes contained hemoglobin precipitates and were short-lived. In hematopoietic tissues, erythroid precursors were elevated in number but exhibited increased apoptosis. Consistent with unstable α -Hb,  AHSP  –/–  erythrocytes contained increased ROS and evidence of oxidative damage. Moreover, purified recombinant AHSP inhibited ROS production by α -Hb in solution. Finally, loss of AHSP worsened the phenotype of β -thalassemia, a common inherited anemia characterized by excess free α -Hb. Together, the data support a model in which AHSP binds α -Hb transiently to stabilize its conformation and render it biochemically inert prior to Hb A assembly. This function is essential for normal erythropoiesis and, to a greater extent, in β -thalassemia. Our findings raise the possibility that altered AHSP expression levels could modulate the severity of β -thalassemia in humans. Introduction Late-stage erythroid development is largely dedicated to the produc-tion of the oxygen carrier hemoglobin (Hb) A, a tetramer consisting of two pairs of α -globin and β -globin protein subunits with each mono-mer bound to a heme moiety. Hb A synthesis is exquisitely coordinat-ed to minimize the accumulation of free α - or β -Hb subunits, which are cytotoxic. Excessive α -Hb is particularly damaging, as evidenced by β -thalassemia, a common inherited anemia in which mutations in the β -globin  gene impair the production of β -Hb with consequent buildup of the unpaired α -subunit (1–5). Intact monomeric α -Hb generates ROS that damage cellular proteins, lipids, and nucleic acids (6). In addition, α -Hb is structurally unstable, with a tendency to denature upon oxidation, filling the cytoplasm and cell membrane with precipitated α -globin polypeptides, free heme, porphyrins, and iron, which further propagate ROS production (reviewed in ref. 7). Together, these effects reduce the lifespan of circulating erythrocytes and also impair the viability of erythroid precursors in hematopoietic tissues, causing ineffective erythropoiesis.Most cells contain compensatory mechanisms to cope with unstable proteins (8). These include molecular chaperones that stabilize proteins and in some cases facilitate their folding into native structures. In addition, there are degradation pathways that recognize and eliminate improperly folded polypeptides. Accord-ingly, tissues typically tolerate some protein instability, with disease ensuing only when the compensatory mechanisms become over-whelmed. Several findings indicate that mechanisms to neutralize free α -Hb exist in erythroid cells. First, erythroid precursors contain a small pool of excess free α -Hb with no apparent ill effects (9, 10). In addition, erythropoiesis is typically relatively normal in humans lacking one functional β -globin  gene ( β -thalassemia trait). Finally, there is frequent unexplained phenotypic diversity among individ-uals with the same β -thalassemia genotype (reviewed in ref. 11). The last observation could be explained by genetic variations in pro-cesses that stabilize or eliminate free α -Hb. Mechanisms to degrade excess free α -Hb in thalassemic erythroid cells were first recognized by Bank and O’Donnell in 1969 (12) and were later shown to be mediated through ubiquitin-dependent proteolytic pathways by Shaeffer and colleagues (13–15). More recently, we identified α -Hb– stabilizing protein (AHSP), also known as erythroid-associated fac-tor (ERAF), a candidate molecular chaperone for α -Hb (16, 17). AHSP was identified as an erythroid-specific protein whose gene was induced by the essential transcription factor GATA-1 (16, 17).  AHSP heterodimerizes with α -Hb (  K  d , approximately 100 nM), but does not bind β -Hb or Hb A. Moreover, α -Hb bound to AHSP is more resistant to oxidant-induced precipitation than α -Hb alone. Based on these findings, we hypothesized that AHSP might pro-tect erythroid cells from α -Hb toxicity by maintaining α -Hb in a stable state prior to its incorporation into Hb A. To test this, we generated  AHSP  –/–  mice by gene targeting. Preliminary analysis of these animals revealed abnormal erythrocyte morphology with Nonstandard abbreviations used:  AHSP, α -Hb–stabilizing protein; BFUe, burst-forming unit erythroid; CFUe, CFU erythroid; DCFH, 2 ′ ,7 ′ -dichlorofluorescin; DNPH, 2,4-dinitrophenylhydrazine; Epo, erythropoietin; Hb, hemoglobin; HDW, Hb distribution width; MCH, mean corpuscular Hb; MCV, mean corpuscular volume; NHS,  N  -hydroxysuccinimide; RDW, rbc distribution width; SBTI, soybean trypsin inhibitor; SCF, stem cell factor; TAU, Triton–acetic acid–urea; TMPD, tetramethyl-  p -phenylenediamine. Conflict of interest:  The authors have declared that no conflict of interest exists. Citation for this article:    J. Clin. Invest.   114 :1457–1466 (2004). doi:10.1172/JCI200421982.  research article 1458  The Journal of Clinical Investigation http://www.jci.org    Volume 114   Number 10   November 2004 hemoglobin precipitates (Heinz bodies) (17). Here we have exam-ined these mutant mice in greater detail to gain further insights into the molecular actions of AHSP in vivo. We found that loss of AHSP reduced the lifespan of circulating red blood cells and also caused increased apoptosis of erythroid precursors. These effects were mediated, at least in part, by increased production of ROS with consequent damage to Hb A and other cellular compo-nents. Moreover, AHSP blocked ROS production by α -Hb directly. Finally, through interbreeding of mutant mice, we show that loss of AHSP worsened the severity of β -thalassemia. Together, these findings indicate that AHSP acts as protein-specific molecular chaperone that detoxifies free α -Hb during normal erythropoiesis and in pathological states of α -Hb excess. Results  Loss of AHSP causes hemolytic anemia with globin chain precipitation . To study the hematopoietic consequences of AHSP loss, we disrupted the gene in mice as described previously (17). Our targeting strat-egy replaced the entire protein-encoding region with a  phosphoglyc-erate kinase promoter–neomycin-resistance  gene (  PGK-Neo  R  ) cassette flanked by lox P recombination sites. The hematopoietic abnor-malities in  AHSP  –/–  animals reported below were identical when the  PGK-Neo  R   expression cassette was either present or removed by Cre-mediated recombination (not shown).  AHSP  –/–  and  AHSP  +/–  mice were born at the expected mendelian ratios and displayed no gross abnormalities compared with wild-type (  AHSP  +/+ ) littermates.  AHSP  –/–  mice exhibited normal lifespans up to at least 18 months of age. As expected, there was no  AHSP   RNA or AHSP protein detected in hematopoietic tissues of  AHSP  –/–  animals (17). Hematological analysis of the gene-targeted mice per-formed using an automated analyzer (Bayer ADVIA 120) revealed several new findings not appreciated previously (Table 1). No abnor-malities in platelets or white blood cells were detected, but there were several obvious erythroid defects.  AHSP  –/–  animals exhibited a mild but significant anemia associated with small red blood cells (low mean corpuscular volume [MCV]) containing decreased Hb (low mean corpuscular Hb [MCH]). There was significant variation in the size and hemoglobin content of the mutant erythrocytes, as evidenced by increased rbc distribution width (RDW) and Hb distri-bution width (HDW), respectively. The reticulocyte count was ele- vated in a subset of  AHSP  –/–  mice, indicating increased erythrocyte production to compensate for accelerated loss or destruction.The blood smears of  AHSP  –/–  mice showed numerous morphologic abnormalities, including irregular size and shape, target cells, and spiculated cells (Figure 1A, upper right panel). There were increased polychromatophilic cells representing newly synthesized erythro-cytes; many of these contained eosinophilic inclusions. Inclusion bodies were also apparent in  AHSP  –/–  erythrocytes after staining with Table 1 Erythrocyte indices of AHSP  –/–   mice AHSP   genotype +/+ ( n   = 8) –/– ( n   = 9) P   value Hb 16.9 ± 0.6 14.5 ± 1.0 < 0.001Hct 55.8 ± 2.1 48.7 ± 3.2 < 0.001Reticulocytes (%) 2.2 ± 1.0 4.4 ± 1.8 0.19MCV 53.5 ± 1.6 45.6 ± 1.2 < 0.001MCH 16.2 ± 0.5 13.6 ± 0.3 < 0.001RDW 13.1 ± 2.2 19.1 ± 2.0 < 0.001HDW 1.5 ± 0.1 2.3 ± 0.1 < 0.001 Hb, g/dl; hematocrit (Hct), %; MCV, fl; MCH, pg; RDW, %; HDW, g/dl. Values shown are mean ± standard deviation; n   =  number of mice ana-lyzed. Hematological indices from  AHSP  heterozygotes did not differ significantly from those of wild-type mice. Figure 1  AHSP  –/–   erythrocytes exhibit abnormal morphology, hemoglobin precip-itates (Heinz bodies), and reduced lifespan. ( A ) Wright-Giemsa stain-ing (upper panels) shows eosinophilic inclusion bodies (*) in  AHSP  –/–   erythrocytes. Heinz body staining, which detects denatured globin chains (lower panels), is weakly positive in some  AHSP +/–   erythro-cytes (indicated by carets) and is strongly positive in  AHSP  –/–   cells. The boxed area in the top right panel shows an enlargement of an inclusion body. Original magnification, × 1,000. ( B ) Erythrocyte survival kinetics determined by biotin labeling. Circulating erythrocytes in 5 animals of each genotype were biotinylated at days –2 and –1. Beginning at day 0, approximately 5 μ l of blood was removed from the tail vein at the indi-cated time points, and the fraction of biotin-labeled erythrocytes was quantified by flow cytometry. The half-life of wild-type red blood cells was 22 days, whereas that of the  AHSP  –/–   red blood cells was 12 days.  AHSP +/–   erythrocytes exhibited normal survival kinetics (not shown). ( C ) Prussian blue staining for cellular iron in spleen. Increased iron in the  AHSP  –/–   spleen reflects accelerated clearance of erythroid cells by the reticuloendothelial system. Original magnification, × 200.  research article  The Journal of Clinical Investigation http://www.jci.org    Volume 114   Number 10   November 2004 1459 crystal violet, which detects denatured globin chains (Heinz bodies, Figure 1A, lower panels). Of note,  AHSP  +/–  erythrocytes also contained occasional Heinz bodies, suggesting haploinsufficiency effects (see below). Unstable denatured Hbs can accelerate erythrocyte destruc-tion by causing intravascular hemolysis and/or sequestration by the reticuloendothelial system. In support of this, the presence of large inclusions only in polychromatophilic cells suggests that these are rapidly cleared from the circulation. This preferential loss of nascent erythroid cells could result in a reticulocyte count that is lower than expected for the degree of erythrocyte destruction.To determine the effects of AHSP loss on erythrocyte lifespan more directly, we injected animals with  N  -hydroxysuccinimide–biotin (NHS-biotin), which labeled nearly all circulating red blood cells with biotin (Figure 1B). The survival of tagged erythrocytes was then monitored over a 50-day period by removal of small sam-ples (approximately 5 μ l) of blood from the tail vein, staining with FITC-streptavidin, and quantification of the fraction of labeled cells using flow cytometry. Erythrocytes from normal littermates exhibited a half-life of about 22 days, in accordance with previ-ous studies (18). In contrast, the half-life of  AHSP  –/–  erythrocytes was significantly shortened to about 12 days. Hence, loss of AHSP causes significant premature destruction of circulating red blood cells. Histological examination of spleens from  AHSP  –/–  mice showed engulfment of erythroid cells by macrophage (erythropha-gocytosis; not shown) and increased macrophage iron, as detected by Prussian blue staining (Figure 1C). These findings are consistent with accelerated removal of mutant erythrocytes and/or erythroid precursors by the reticuloendothelial system (also see below).  Hyperplasia and excessive apoptosis of AHSP  –/–  erythroid precursors . In most disorders of erythrocyte destruction, there is a compensatory increase in production of erythroid precursors in hematopoietic tissues. The spleen, a major site of erythropoiesis in mice, was sig-nificantly enlarged in  AHSP  –/–  mice (Figure 2A). Flow cytometry showed an increased proportion of erythroid precursors in  AHSP  –/–  spleens, as measured by expression of the late-stage erythroid-spe-cific cell surface marker Ter119 (Figure 2B). To further examine Figure 2 Erythroid hyperplasia in  AHSP  –/–   mice. ( A ) Increased spleen weight in  AHSP  –/–   mice. ( B ) Elevated proportion of Ter119 +  erythroid cells in  AHSP  –/–   mice, as determined by flow cytometry. ( C ) Methylcellulose progenitor assays.* P  < 0.005. n   =  6 for each genotype. Figure 3 Ineffective erythropoiesis in  AHSP  –/–   mice. ( A ) Analysis of splenic erythroid precursors according to levels of Ter119 and CD71 expres-sion. Regions a through d (boxed areas) represent increasingly mature stages of erythroid development (19). APC, allophycocyanin. PE, phycoerythrin. ( B ) Ratio of mature to immature erythroid precursors in spleen calculated according to levels of Ter119 and CD71 expression defined by flow cytometry gates in panel A : Percent mature cells = number of cells in d / (a + b + c + d); n   =  6 animals for each genotype; ** P  < 0.05. ( C – E ) Detection of apoptosis in erythroid precursors. Spleen sections were stained for the erythroid surface marker Ter119 (brown) and for nuclear endonucleolytic cleavage using the TUNEL assay (red). Examples of normal viable erythroid precursors (Ter119 + TUNEL – ) are indicated by arrows. Examples of apoptotic erythroid precursors (Ter119 + TUNEL + ) are indicated by arrowheads. AHSP genotypes are indicated. E  is an enlargement of the boxed region in D . Original mag-nification in C – E , × 400. ( F ) Percent apoptotic erythroid precursors in  AHSP  –/–   mice and wild-type littermates ( n   =  4 mice of each genotype).  research article 1460  The Journal of Clinical Investigation http://www.jci.org    Volume 114   Number 10   November 2004 erythroid precursors, we disaggregated spleens into single-cell suspensions and seeded them into semisolid medium. This assay assesses the developmental potential of individual cells and iden-tifies 2 types of committed erythroid precursors: burst-form-ing unit erythroid (BFUe) cells represent early-stage precursors that give rise to large hemoglobinized colonies in the presence of erythropoietin (Epo) and stem cell factor (SCF); CFU erythroid (CFUe) precursors represent a later stage of development and give rise to smaller colonies in the presence of Epo alone. As shown in Figure 2C, the proportions of both BFUe and CFUe precur-sors were elevated in  AHSP  –/–  mice. In contrast, myeloid progeni-tors, which give rise to granulocyte- and macrophage-containing colonies in appropriate cytokines, were unchanged. Finally, his-tological analysis showed notable expansion of the splenic red pulp in the knockout animals (not shown). Therefore, splenic enlargement observed in  AHSP  –/–  mice is due to expansion of the erythroid compartment. We also detected significant erythroid hyperplasia in the bone marrow of  AHSP  –/–  mice, although to a lesser extent than observed in the spleen (not shown). These results reflect increased erythropoietic drive to compensate for accelerated destruction of mature  AHSP  –/–  erythrocytes.In β -thalassemia, accumulation of excess free α -Hb not only dam-ages mature erythrocytes but also induces apoptosis of erythroid precursors in hematopoietic tissues in a process called ineffective erythropoiesis (1). To investigate whether loss of AHSP causes inef-fective erythropoiesis, we examined splenocytes for coexpression of Ter119 and the transferrin receptor CD71, which allows subdi- vision of erythroid precursors according to their maturation stage (Figure 3A) (19).  AHSP  –/–  mice exhibited an elevated proportion of immature erythroid precursors (Ter119 high, CD71 high) com-pared with that of mature ones (Ter119 high, CD71 low), suggest-ing that the transition from immature to mature precursor was partially blocked (Figure 3, A and B). This effect could reflect inef-fective erythropoiesis, which is characterized by premature death of erythroid precursors in hematopoietic tissues. We assessed this by direct immunohistochemical examination (Figure 3, C–F). Erythroid cells were identified by expression of Ter119 (brown staining in Fig-ure 3, C and D). Apoptotic cells were identified by TUNEL staining, which detects endonucleolytic cleavage of nuclear DNA (red staining in Figure 3, C–E).  AHSP  –/–  spleens contained an increased propor-tion of Ter119-staining cells, consistent with the flow cytometry data in Figure 2B, above. In addition, apoptosis of erythroid precursors Figure 4 Unstable hemoglobins in  AHSP  –/–   erythrocytes. ( A ) TAU gel analysis of membrane-associated globin chains with the  AHSP  genotypes indicated at the top. Each lane represents membrane skeletons pre-pared from the same number of erythrocytes. Right margin: α , α -globin; β , β -globin. ( B ) Isopropanol hemoglobin stability test. Fresh hemoly-sates were incubated with isopropanol (17% volume/volume) at 37°C, and hemoglobin precipitation was quantified at the indicated times.  AHSP  genotypes are indicated. The hemoglobin stabilities were signifi-cantly different at all time points for the two genotypes ( n   =  5 animals of each genotype; P  < 0.01). Figure 5 Oxidative stress in  AHSP  –/–   erythrocytes. ( A ) Relative ROS levels in erythrocytes at baseline and with added H 2 O 2 . ROS were measured by incubation of cells with DCFH, which is converted by ROS to the fluorescent product 2 ′ ,7 ′ -dichlorofluorescein (DCF). ( B ) Protein oxi-dation in erythrocyte lysates. Twenty micrograms of hemolysate was treated with DNPH for derivatization of carbonyl groups (by products of protein oxidation). Protein-associated DNP was detected by Western blotting. An identical blot probed with anti-actin (bottom) indicates equal protein loading in each lane. ( C ) Susceptibility to phenylhydrazine-induced hemolytic anemia. Drug was administered on days –1 and 0. Blood was first sampled on day 0, then hematocrit (top panel) and reticulocyte counts (bottom panel) were assessed daily until recovery. No abnormalities were detected in  AHSP +/–   mice in the experiments described in panels A – C  (not shown).
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