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1 Research Laboratory for Molecular Genetics, Yamagata University, Yamagata 990-9585, Japan
2 Center for Tsukuba Advanced Research Alliance
3 Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba 305-8577, Japan
4 JST-ERATO Environmental Response Project, University of Tsukuba, Tsukuba 305-8577, Japan
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Abstract |
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/ß globin chain imbalance. Despite the iron overload, transferrin receptors were highly expressed in the erythroid cells, suggesting they contribute to the formation of ring sideroblasts and siderocytes. These results indicate that a partially depleted heme supply provokes ring sideroblast formation. The experimental generation of ring sideroblasts in animals would contribute to our understanding of the iron metabolism and its disorder in erythroid cells. |
Introduction |
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There are ample lines of evidence obtained from in vitro and in vivo studies supporting the contention that ALAS-E plays critical roles in the regulation of heme biosynthesis in erythroid cells (Fujita et al. 1991; Meguro et al. 1995; Harigae et al. 1998; Nakajima et al. 1999). Expression of the Alas2 gene has been shown to be under the control of both GATA-1 (Surinya et al. 1997; Kramer et al. 2000) and the availability of cellular iron. The latter is mediated by the iron-responsive element (IRE) located in the 5'-UTR of ALAS-E mRNA (Cox et al. 1991; Dandekar et al. 1991).
While ALAS-E-deficient lines of zebrafish (Brownlie et al. 1998) and mouse have been generated (Nakajima et al. 1999), experimental generation of ring sideroblasts has not been observed in these genetically manipulated animals (reviewed in Yamamoto & Nakajima 2000). Since mouse embryos with an Alas2 gene disruption die by embryonic day 11.5 (E11.5), the consequence of an ALAS-E-deficiency in stages later than E11.5 cannot be examined, thus hampering our analysis of ring sideroblast formation in the ALAS-E-null mutant embryos. Indeed, in our previous study, we found a limited number of ring sideroblasts in the bone marrow of adult mouse chimeric for ALAS-E-null mutant cells and wild-type cells (Nakajima et al. 1999), suggesting that ring sideroblasts may be formed in hematopoietic tissues at more developed stages. An unexpected observation in the ALAS-E-null embryos was the presence of erythroid cells with diffuse iron accumulation in the cytosol, but not in the mitochondria, suggesting that primitive erythroid cells, which are predominant in the peripheral blood cells of embryos at E11.0, are capable of accumulating iron in the cytosol, but incapable of changing to ring sideroblasts (Nakajima et al. 1999).
To investigate the consequences of developmental stage-specific heme depletion and the mechanisms of ring sideroblast formation, we decided to adopt a transgenic complementation rescue approach. In this approach, the Alas2 knockout line of mice is rescued from embryonic lethality using transgenic mouse lines expressing ALAS-E. Especially, we surmised that partially rescued mice resulting from hypomorphic ALAS-E expression would give us important information. We exploited the Gata1 locus hematopoietic regulatory domain (G1-HRD; also referred to as IE3.9int), as IE3.9int has been characterized in vivo as an erythroid-specific gene regulatory domain. Indeed, transgenic expression of the erythropoietin receptor (Suzuki et al. 2002), GATA-1 (Takahashi et al. 2000) and small Maf proteins (Motohashi et al. 2000) under the transcriptional control of IE3.9int has been shown to rescue the knockout mice of each gene from embryonic lethality. Furthermore, this system has also been applied to the domain analysis of GATA-1 in vivo (Shimizu et al. 2001, 2004).
The IE3.9int vector activates transcription of the reporter genes in both primitive and definitive erythroid cells when tested by reporter gene transgenic mouse analyses. One of the useful features of the vector is that the regulatory domain has been characterized in detail and specific deletions that weaken its activity are already known (Nishimura et al. 2000; Ohneda et al. 2002). For instance, deletion of the first intron (resulting in the IE3.9 vector) affects the activity of IE3.9int; the IE3.9 vector activates transcription comparative to IE3.9int in primitive erythroid cells, but its activity in definitive erythroid cells is markedly attenuated (Onodera et al. 1997; Nishimura et al. 2000; Ohneda et al. 2002). Therefore, in order to fully and partially rescue the ALAS-E-null mutant mice, we generated transgenic lines of mice expressing human ALAS-E utilizing the IE3.9int and IE3.9 vectors, respectively. We selected low expressor lines from the IE3.9-AE (hereafter, the human ALAS-E transgene will be designated AE) transgenic mouse to achieve hypomorphic expression of ALAS-E in erythroid tissues.
We found in this study that the IE3.9int-AE transgene successfully restored ALAS-E-null mice to normal growth, demonstrating that the regulatory domain for Gata1 recapitulates the Alas2 gene expression profile sufficiently to sustain embryonic and adult erythropoiesis. To our surprise, in the ALAS-E-null mutant embryos partially rescued by a low level expression of the IE3.9-AE transgene, most of the primitive nucleated erythroid cells were transformed to ring sideroblasts. Whereas in these mice, the majority of definitive erythroid cells circulating in the stages of late gestation were transformed to siderocytes, which are enucleated erythrocytes harboring iron deposits. Definitive ring sideroblasts were also found among the nucleated circulating cells in the transgene-rescued embryos. Despite the iron overload, erythroid cells in the rescued mutant embryos expressed the transferrin receptor (TfR) at a high level, suggesting that the transferrin receptor participates in the formation of ring sideroblasts and siderocytes. These results thus suggest that the insufficient expression of ALAS-E or heme-deficiency in the mutant erythroid cells provokes a cellular iron overload and the formation of ring sideroblasts in vivo in the transgene-rescued embryos. In addition, closer examination revealed that the iron-overloaded erythroid cells suffered from an /ß globin chain imbalance.
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Results |
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To investigate how ALAS-E-deficiency affects the development of the hematopoietic system and iron metabolism, we performed transgenic complementation rescue experiments of ALAS-E-null mouse embryos. We generated transgenic lines of mice expressing human ALAS-E (hALAS-E) under the control of the mouse Gata1 gene hematopoietic regulatory domain (G1-HRD; also referred to as IE3.9int), as the IE3.9int has been shown to recapitulate erythroid and megakaryocytic lineage-specific expression of reporter genes in vivo (Onodera et al. 1997; Takahashi et al. 2000). In addition to full-size IE3.9int, we also utilized a truncated vector that lacks the first intron (called IE3.9; Fig. 1A, middle). We chose hALAS-E cDNA for the transgenic expression, because of the high level of similarity (> 90%) of the amino acid sequence of hALAS-E with that of mouse ALAS-E (mALAS-E), yet mouse and human ALAS-E are sufficiently divergent in their nucleotide sequences such that we can assess the transgenic vs. endogenous gene expression by RT-PCR. The transgenic constructs IE3.9int-AE and IE3.9-AE were injected into fertilized eggs and three and four transgenic mouse lines were established, respectively.
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Of the transgenic mouse lines generated, we selected one IE3.9int-AE line that showed a transgene expression level almost equal to the endogenous mALAS-E expression (lanes 1 and 2). We also selected two IE3.9-AE transgenic lines showing a transgene expression of approximately 50% (Line M) and 70% (Line L) less than the endogenous mALAS-E expression, respectively (see below for an explanation of this choice). The expression levels are shown in Fig. 1C.
Transgenic rescue of ALAS-E-null mutant mice from embryonic lethality
Since the mALAS-E (Alas2) gene is localized on the X-chromosome, hemizygous Alas2 knockout mice die by E11.5 due to severe anemia (Nakajima et al. 1999). Therefore, our scenario for the embryonic rescue experiments is that in ALAS-E-deficient erythroid cells bearing the transgene (Alas2–/Y::AE-Tg), the deficiency of endogenous mALAS-E should be compensated by transgenic hALAS-E expression directed by IE3.9int, and this should improve the profound anemia. To test this hypothesis, we first crossed male IE3.9int-AE transgenic mice (AE-Tg) with female Alas2 gene heterozygous knockout mice (Alas2–/Y). Upon genotyping the resulting embryos and pups ranging from E10.5 embryos to 20-day postgestation mice (P20), live embryos and pups of the Alas2–/Y::AE-Tg were observed and conformed to the Mendelian expectation (Table 1A). We refer to these rescued lines of mice as IE3.9int-AE-R (R stands for Rescued). This result demonstrates that hALAS-E expression directed by the G1-HRD (i.e. IE3.9int) recapitulated the expression of endogenous mALAS-E and complemented the major physiological function of endogenous mALAS-E.
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Remarkable fluctuation was observed in the efficiency of the rescue by the IE3.9-AE-M transgene. The anemic phenotype of IE3.9-AE-MR embryos at E14.5 (Fig. 2B,C) displayed substantial variation compared to wild-type embryos (Fig. 2A), as can be seen by the different densities in the red color of erythrocytes in the blood vessels and liver. Indeed, as for the two IE3.9-AE-MR embryos from the same litter, one was found to be dead at around E16.5 (Fig. 2E), while the other was anemic but alive at E17.5 (Fig. 2F). Since this fluctuation is in favor of investigating the effect of ALAS-E insufficiency at various developmental stages, we executed tissue analysis of partially rescued embryos throughout the gestation period beyond E11.5, focusing on IE3.9-AE-MR mice.
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Ring sideroblast formation in partially rescued embryos
Primitive erythroid cells in ALAS-E-null mutant mice suffer from iron overload starting from E9.5 and iron accumulated diffusely in the cytosol (Fig. 3A), but not in the mitochondria. The iron accumulation persisted until the knockout embryos died of anemia at E11.5; this embryonic lethality hampered further analyses of how iron accumulates in ALAS-E-deficient erythroid cells. Therefore, taking advantage of the hypomorphic transgene-rescued embryos, we examined iron metabolism in the mutant embryos by means of Prussian-Blue iron staining. To our surprise, typical ring sideroblasts with nuclei encircled by many iron deposits were identified in the IE3.9-AE-MR embryos at E12.5 (Fig. 3C, arrows), whereas such cells were not found in the wild-type embryos (Fig. 3B).
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Further analyses of IE3.9-AE-MR embryos revealed that the formation of ring sideroblasts became more extensive as the embryos grew and developed. Especially, in contrast to the wild-type embryos (Fig. 3G), most of the PBCs in the mutant embryos at E14.5 contained iron deposits (Fig. 3H). Upon Wright-Giemsa staining, we found that the majority of the PBCs were enucleated erythrocytes in wild-type embryos after E14.5 (Fig. 3I), but considerable numbers of nucleated primitive erythrocytes still remained in the circulating blood of IE3.9-AE-MR embryos (Fig. 3J). This observation suggests that the heme-deficiency might retard definitive erythropoiesis in the mutant embryos.
In ring sideroblasts, the nuclei are usually surrounded by dozens of iron deposits (see Fig. 3C,F). In contrast, the primitive erythrocytes found in the rescued IE3.9-AE-MR embryos contained dozens of cytoplasmic bodies that were stained by Wright-Giemsa (Fig. 3J). Since the distribution pattern of these cytoplasmic bodies appears to be different from that of the iron-overloaded mitochondria, it is unclear at present whether these cytoplasmic bodies originate from mitochondria or not. Cytoplasmic bodies did not exist in normal primitive erythroid cells (Fig. 3I), implying that they must be formed owing to the insufficient ALAS-E expression.
Ring sideroblasts originate from primitive erythroid lineage cells
To clarify the origin and nature of the ring sideroblasts, we made a comparative examination of the extent of iron accumulation in the PBCs of IE3.9-AE-MR and IE3.9int-AE-R embryos at E13.0. In the case of IE3.9-AE-MR, we found that most cells possessed a nucleus encircled by iron deposits (Fig. 3K), showing very good agreement with the results of E14.5 embryos (Fig. 3H). On the contrary, in IE3.9int-AE-R embryos, we could not find such typical ring sideroblasts (Fig. 3L), as was the case for wild-type embryos (Fig. 3G). These results strongly support the contention that the iron overload in the erythroid cells of rescued embryos was provoked by the insufficient expression of ALAS-E from the transgene or heme-deficiency in the mutant erythroid cells.
Diffuse iron accumulation in the cytosol, which was observed in E10.5 ALAS-E-null embryos (Fig. 3A), was also observed in the IE3.9-AE-MR embryos at E11.5 (Fig. 3N), suggesting that the iron-overloaded cells are most likely to be transformed into ring sideroblasts after E11.5. Cellular iron transport into mitochondria might be enhanced after E11.5 by certain mechanisms in the primitive erythroid cells.
We also carried out globin analysis of PBCs in E13.0 IE3.9-AE-MR embryos by immunocytochemistry using anti-
y-globin and anti-ß-major globin antibodies. Most nucleated cells in the embryonic circulation, which were ring sideroblasts (Fig. 3K), were stained with anti-y-globin antibody (Fig. 3O). On the contrary, enucleated erythrocytes were stained intensely with the anti-ß-major globin antibody (Fig. 3P). Based on these observations, we conclude that the ring sideroblasts appear after E11.5 through transformation from the primitive erythroid cells that harbor diffuse cytoplasmic iron-accumulation.
Ring sideroblasts are also formed in the definitive erythroid population
To examine whether ring sideroblasts are also formed in the definitive erythroid population, we then focused on fetal liver. As compared to the livers of E14.5 wild-type embryos (Fig. 4A), the livers of IE3.9int-AE-R embryos displayed a slight increase in the cells stained positively for iron (Fig. 4B; blue cells). In contrast, a high level of iron accumulation was observed in the livers of IE3.9-AE-MR embryos at E14.5 (Fig. 4C). As for the hemoglobin production, wild-type liver was strongly benzidine-positive (Fig. 4D), whereas benzidine-positive cells were markedly decreased in the livers of IE3.9int-AE-R (Fig. 4E) and IE3.9-AE-MR (Fig. 4F) embryos. Thus, IE3.9int-AE-R embryos showed a phenotype more similar to IE3.9-AE-MR embryos than to wild-type embryos in the case of hemoglobin production in the fetal liver. This suggests that definitive erythropoiesis at E14.5 was affected to a certain extent by the ALAS-E-deficiency even in the IE3.9int-AE-R embryos.
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We also stained PBCs from the E17.5 embryos with Prussian blue. While PBCs in the wild-type embryo at E17.5 showed no iron deposition (Fig. 4J), blood cells from the IE3.9-AE-MR embryos contained many iron deposits and some of them showed ring sideroblast morphology (Fig. 4K, arrows). It is generally accepted that primitive erythroid cells disappear at E17.5, so this result, along with the presence of the ß-major globin-positive nucleated cells at E17.5, argues that ring sideroblasts are also formed from definitive erythroid cells in the IE3.9-AE-MR embryos.
In addition to the identification of ring sideroblasts, we found that enucleated erythrocytes in the IE3.9-AE-MR embryos at E16.5 contained a number of iron deposits (Fig. 4M). Such aberrant erythrocytes are referred to as siderocytes, which usually increase in the PBCs of patients suffering from XLSA, hemolytic anemia, lead poisoning and similar conditions (Kappas et al. 1995). In smear samples of PBCs from the IE3.9-AE-MR embryos, the enucleated erythrocytes appeared to be basophilic (Fig. 4O) and there was prominent anisocytosis and poikilocytosis compared to those from the wild-type embryos (Fig. 4N).
Hematological analysis of circulating blood cells from IE3.9-AE-MR embryos
FACS analysis of embryonic PBCs by forward scatter, which reflects cell size, showed that the primitive and definitive erythroid cells in IE3.9-AE-MR embryos at E12.5 to E17.5 were microcytic. The PBCs in IE3.9-AE-MR embryos at E12.5, which were mostly primitive ring sideroblasts, were microcytic as compared to wild-type PBCs (Fig. 5A, left panel). The average size of the PBCs in the E16.5 IE3.9-AE-MR embryos, consisting mostly of siderocytes, was also smaller than the average size of the PBCs in wild-type embryos (right panel). Consistent with the results from cytological examination in the previous section, it was suggested that the transition from primitive to definitive erythropoiesis was delayed in the IE3.9-AE-MR embryos. This is because the embryos contained considerable numbers of large sized cells at E15.0 (center panel; arrow), which disappeared at E16.5 (right panel).
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Expression of the transferrin receptor in ring sideroblasts and siderocytes
We previously found that the transferrin receptor-1 (TfR) was not expressed in erythroid cells with a diffuse cytoplasmic iron overload, suggesting that the TfR does not play critical roles in iron overloading (Nakajima et al. 1999). To ascertain whether this is also true in the case of ring sideroblast formation in IE3.9-AE-MR embryos or if the TfR makes an important contribution to ring sideroblast formation in the rescued embryos, we examined the expression of the TfR in TER119-positive PBCs in IE3.9-AE-MR embryos using a FACS double sorting strategy.
In E12.5 wild-type embryos, the TER119-positive PBCs were found to be a single group highly expressing the TfR (Fig. 6A, left panel). In contrast, those in the circulating PBCs of IE3.9-AE-MR embryos, most of which were ring sideroblasts, were of two groups: the major group highly expressed the TfR, while the minor group moderately expressed the TfR (Fig. 6A, right panel). This indicates that there is not much difference between wild-type and rescued embryos in the expression of the TfR in erythroid cells. These results rather suggest that TfR may play important roles in the formation of ring sideroblasts in the rescued embryos.
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We then analyzed the surface expression of TER119, TfR, and c-Kit on the mononucleated cells from E15.0 fetal livers by FACS (Fig. 6C). In wild-type and IE3.9-AE-MR embryonic livers, erythroid-lineage cells were composed of TfR–TER119H (R2; 0.6% and 0.8%, respectively), TfRLTER119H (R3; 2.3% and 19.5%, respectively), TfRHTER119+ (R4; 82% and 53%, respectively), and TfRHTER119– (R5; 6.5% and 7.9%, respectively) cells. Similarly, the cells were divided into c-Kit–TER119H (wild-type, 82%; IE3.9-AE-MR, 66%) and c-KitdullTER119H (wild-type, 1.8%; IE3.9-AE-MR, 13.6%) cells (Fig. 6D). It is noteworthy that the TfRLTER119H fraction (R3) was remarkably larger in the livers of rescued embryos (19.5%) than in the livers of wild-type embryos (2.3%). Corresponding to this fraction, the c-KitdullTER119H cell fraction had also expanded in the rescued embryos (13.6%) compared to in the wild-type embryos (1.8%). These results suggest that maturation arrest might occur during erythropoiesis in the rescued embryonic livers.
Expression of globin chains in circulating blood cells of the rescued embryos
Circulating blood smears from the IE3.9-AE-MR embryos at E16.5 showed that approximately 25% of the cells contained Heinz-bodies (Fig. 7A, right panel), which are -globin precipitates and one of the clinical features of ß-thalassemia. These cells were rarely found among the PBCs of wild-type embryos (left panel). Heme should be incorporated into globin proteins as the prosthetic group in erythroid cells and globin chain expression is controlled in part by the heme supply mediated by heme-regulated inhibitor (HRI; Han et al. 2001). Therefore, one plausible explanation for the Heinz body formation is that there may be an /ß globin chain imbalance in the IE3.9-AE-MR erythrocytes.
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At E14.0, a significant increase was observed in the ratio of primitive ß-globin proteins (i.e. ßh0/1-, Y- and Y2-globins) to total ß-globin proteins in the blood cells of IE3.9-AE-MR embryos (Alas2+/Y 0.48; IE3.9-AE-MR 0.82), indicating a delayed transition from primitive to definitive erythropoiesis. This is in agreement with the finding that the major PBCs were nucleated in the IE3.9-AE-MR mutant embryos at E14.5 (Fig. 3H).
In addition, we found a reduction in the ß to -chain ratio in the blood cells of IE3.9-AE-MR embryos compared with that of wild-type littermates (Fig. 7C). From the gross morphology of the IE3.9-AE-MR embryos, the severity of the anemia seems to correlate well with such an /ß globin chain imbalance (Fig. 7D). When we plotted the relationship between the ß/ ratio and the MCH, the blood cells of the IE3.9-AE-MR embryos showed a globin chain imbalance and hypochromicity (Fig. 7E). Furthermore, to clarify the influence of heme deficiency in erythroid cells on globin chain expression, we performed TAU on the blood cells of ALAS-E-null embryos at E11.0 (Fig. 7F). The results showed that, compared to wild-type embryos, there was a decrease in the amounts of each kind of globin chain in the blood cells of E11.0 ALAS-E-null embryos. In primitive erythroid cells of the ALAS-E-null embryos, there seemed to be a delay in 
to -globin switching, such that -globin may be decreased or not detectable. In summary, heme deficiency in erythroid cells causes a reduction in globin proteins according to the severity of the deficiency, with adult ß-globin chains being most sensitive.
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Discussion |
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Ring sideroblasts appear in patients with myelodysplastic syndrome (MDS), especially in the refractory anemia with ring sideroblasts (RARS) subtype. The presence of ring sideroblasts in bone marrow cells is not confined to RARS, but it has been suggested that pathophysiological conditions aspiring to the formation of ring sideroblasts may also contribute to the development of other MDS (Greenberg et al. 2002). Thus, the molecular basis of ring sideroblast formation may also be important for understanding the transformation/development of leukemia.
It should be noted that in the transgene-rescued embryos, typical ring sideroblasts first appeared at E12.5 from primitive erythroid cells. Since simple ALAS-E-deficient mice died by E11.5 and ring sideroblasts were absent in the embryos, we surmise that a certain level of further maturation is necessary for the heme-deficient primitive erythroid cells to become ring sideroblasts. One plausible molecular process corresponding to this maturation seems to be enhanced transport of cytosolic iron into the mitochondria (TCIM in Fig. 8). Such a molecular process might be activated after the development of mutant embryos beyond E11.5. In this regard, we previously noted that, in ALAS-E-deficient embryos, the heme-deficiency resulted in the maturation arrest of primitive erythroid cells. Therefore, the development of erythroid cells in the ALAS-E-null mutant embryos might be delayed and this may also be the reason, albeit in part, why we could not see the ring sideroblasts in ALAS-E-null embryos at E11.5.
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By the same token, molecules related to ring sideroblast formation seem to be related to the utilization of iron from the transferrin and/or transfer of iron from the cytosol to the mitochondria (TCIM). However, while knowledge about cellular iron metabolism is rapidly expanding (Hentze et al. 2004), the molecular mechanisms governing mitochondrial iron metabolism remain largely unclear at present. For instance, mitochondrial ferritin, a newly identified ferritin with a mitochondrial targeting sequence (Levi et al. 2001) resides in the mitochondria of ring sideroblasts, but not in the mitochondria of normal erythroblasts (Cazzola et al. 2003). We preliminarily tried to detect the expression of mitochondria ferritin by RT-PCR, but we could not detect the expression of mitochondrial ferritin in PBCs of IE3.9-AE-MR embryos as well as those of wild-type embryos (unpublished observation). Frataxin, associated with the neurodegenerative disorder Friereich's ataxia, has also been suggested to be involved in mitochondrial iron import and/or export (Becker et al. 2002). However, direct evidence for the participation of mitochondrial ferritin and Frataxin in ring sideroblast formation awaits further analyses.
In spite of the mitochondrial iron overload, ring sideroblasts and siderocytes in the IE3.9-AE-MR embryos express TfR. This is in clear contrast to the PBCs in E10.5 ALAS-E-null embryos, which accumulate iron diffusely in the cytosol, but only marginally express TfR (Nakajima et al. 1999). All the iron required for hemoglobin synthesis is supposed to come from transferrin through TfR (Ponka 1997). It has been reported that heme-biosynthesis inhibitors, such as succinylacetone, inhibit the expression of TfR mRNA in DMSO-induced hemoglobin-synthesizing MEL cells, but that the inhibition does not occur in uninduced MEL cells (Chan et al. 1994), suggesting that heme-deficiency affects TfR expression distinctly depending on the stage of cellular differentiation.
TfR expression in erythroid cells is regulated by various signals, including hypoxia (Lok & Ponka 1999; Tacchini et al. 1999; Ang et al. 2002) and intracellular heme concentration (Hofer et al. 2003). An in vitro differentiation study with ALAS-E-null ES cells showed that ALAS-E-deficiency results in a profound iron accumulation in erythroblasts (Harigae et al. 2003). In IE3.9-AE-MR embryos, TfRdullTER119+ cells appeared in the PBCs at E12.5. Therefore, one attractive hypothesis is that TfR expression is sensitive to heme-deficiency in these cells. In contrast, the heme in sheep reticulocytes stimulates the loss of TfR associated with maturation (Ahn & Johnstone 1989). This observation rather suggests that the heme-deficiency in siderocytes might act to sustain the high-level of TfR expression. In addition, there is a possibility that in the IE3.9-AE-MR embryos, iron-dependent oxidation may convert IRP2 (Iwai et al. 1998) to repress the TfR expression post-transcriptionally. We surmise that elucidation of the molecular mechanisms of ring sideroblast formation should be useful for comprehending iron metabolism.
Globin biosynthesis in anemic patients with ring sideroblasts has been studied in special reference to the /ß globin chain ratios (White et al. 1971; Peters et al. 1983). However, as for the influence of heme-deficiency on the globin chain ratio or imbalance, a consistent conclusion has not been drawn from the studies. We found that a number of PBCs in the IE3.9-AE-MR embryos contain Heinz bodies and suffer from /ß globin chain imbalance similar to the case of ß-thalassemia. In addition, a severe reduction in globin proteins was observed in the primitive erythroid cells of ALAS-E-null embryos. Heme is known to stabilize hemoglobin and myoglobin compared to the respective apoglobins (Crumpton & Polson 1965; Kawahara et al. 1965), as heme binding confers a stable three-dimensional structure on the globin molecule. The -globin chain has a greater affinity for heme and binds to heme more rapidly than the ß-globin chain (Winterhalter & Deranleau 1967; Kawamura-Konishi & Suzuki 1985). This suggests that in heme-deficient erythroid cells most of the heme binds to the -globin chain and stabilizes the globin, whereas ß-globin becomes heme-free and unstable. In fact, ALAS-E-null mutant embryos showed a more severe abnormality in globin degradation than the rescued embryos. Thus, under the severe heme deficiency, a globin chain imbalance may occur due to a difference of affinity for heme between - and ß-globin chains. Alternatively, the imbalance of globin chains might be caused by an ineffective erythropoiesis. Whereas severe heme deficiency also causes delayed transition from primitive to definitive erythropoiesis in rescued embryos, we think that the globin chain production is relatively preserved and the ineffective erythropoiesis may be an independent phenomenon from the globin chain imbalance.
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Experimental procedures |
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Human ALAS-E (hALAS-E) cDNA (Furuyama et al. 1997), which contains an intact IRE in 5'-UTR, was ligated to the 3' end of the IE3.9int or IE3.9 genomic fragment (Onodera et al. 1997). The transgenes isolated from the plasmid vector were used for the generation of transgenic mice. Fertilized eggs from BDF1 parents were employed. Transgenic lines of mice were identified using primer sets to detect hALAS-E cDNA in the transgenes: sense, 5'-GAAGGTCACACACCTGATTC-3'; anti-sense 5'-ACAGCATGGACCTCATCCAG-3'. Female mice heterozygous for the Alas2 knockout (Nakajima et al. 1999) were crossed with hALAS-E transgenic heterozygous mice to generate hALAS-E-expressing embryos harboring the ALAS-E-null mutant allele (compound mutation designated IE3.9int-AE-R). The Alas2 knockout mutant allele was detected using the primer set sense 5'-AATGGGCTGACCGCTTCCTCGTGCT-3' and anti-sense 5'-TGTGCATAATTCCATCACGC-3', which amplifies a genomic fragment composed of exon 8 and the Neo gene. We determined the sex of the embryos by PCR amplification of the Y chromosome-specific Zfy-1 gene (Nakajima et al. 1999).
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from the livers of E16.5 embryos or from single cell suspensions prepared from E10.0 yolk sac cells and cDNAs were synthesized using Superscript II (Invitrogen). The primer sets used were designed so that they could detect both mouse (endogenous) and human (transgene-derived) ALAS-E cDNAs (sense 5'-CCATGCTGTAGGACTGTATG-3' and anti-sense 5'-GGTAGTTGATGGCCTGCAC-3'). To compare the amount of mRNAs derived from the transgene with the amount of endogenous mouse ALAS-E mRNA, PCR products were digested with HindIII and subjected to electrophoresis.
Histological and cytological analyses of embryos
Paraffin-embedded sections of 6 µm were stained with benzidine or Prussian blue to examine the presence of hemoglobin and ferric iron, respectively, and immunostained with anti-mouse y-globin or ß-major globin antibody as previously described (Nakajima et al. 1999). Blood smears were stained with Wright-Giemsa or with crystal violet to detect Heinz bodies. To examine the presence of ferric iron, blood smears were stained with Prussian blue as previously described (Nakajima et al. 1999). The hematological indices of embryonic peripheral blood cells (PBCs) were analyzed with an automatic blood counter (Celltac alpha, NIHON KOHDEN). As for Hb and RBC, the ratios to those of wild-type littermates are presented. Electron microscopic analysis was carried out by standard procedures.
FACS analysis
Single cell suspensions of fetal liver cells or PBCs were prepared with a 27G needle and passed through a 40-µm nylon mesh. The expressions of TfR and TER119 in each sample were analyzed by two-color FACS analysis with FITC-conjugated anti-CD71 antibody (HANA5 (Kina et al. 2000) or anti-mouse TfR antibody) and Phycoerythrin (PE)-conjugated anti-TER119 antibody, respectively.
Globin electrophoresis and MALDI–TOF–MS analysis
Triton–acid–urea (TAU) gel electrophoresis was performed for separation of the globin chains as described (Lopez et al. 2002). Protein bands were visualized by Coomassie blue staining and the gel was dried in cellophane. Specific bands in the gels were excised as 1-mm blocks. Proteins in the gels were digested in situ with trypsin and extracted following standard procedures. An aliquot (1 µL) of digested supernatant was desalted with Ziptip (C18 reverse-phase column, Millipore) and spotted on to a MALDI–TOF–MS sample plate with 1 µL matrix (saturated alpha-Cyano-4-hydroxycinnamic acid (BRUKER DALTONICS) in 0.1% TFA-AcCN (2 : 1)). An Autoflex (BRUKER DALTONICS) was used to generate peptide mass fingerprints. These were searched against the MASCOT database supplied from Matrix Science. A mass fingerprint was considered a significant match if the score was equal to or greater than 75.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: masi{at}tara.tsukuba.ac.jp
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Ang, S.O., Chen, H., Hirota, K., et al. (2002) Disruption of oxygen homeostasis underlies congenital Chuvash polycythemia. Nature Genet. 32, 614–621.[CrossRef][Medline]
Becker, E.M., Greer, J.M., Ponka, P. & Richardson, D.R. (2002) Erythroid differentiation and protoporphyrin IX down-regulate frataxin expression in Friend cells: characterization of frataxin expression compared to molecules involved in iron metabolism and hemoglobinization. Blood
99, 3813–3822.
Bishop, D.F., Henderson, A.S. & Astrin, K.H. (1990) Human delta-aminolevulinate synthase: assignment of the housekeeping gene to 3p21 and the erythroid-specific gene to the X chromosome. Genomics 7, 207–214.[CrossRef][Medline]
Brittenham, G. (2000) Disorders of iron metabolism: iron deficiency and overload. In: Hematology: Basic Principles and Practice (eds R. Hoffman, E. J. Benz Jr, S. Shattil, et al.), pp. 397–428. Philadelphia: Churchill Livingstone.
Brownlie, A., Donovan, A., Pratt, S.J., et al. (1998) Positional cloning of the zebrafish sauternes gene: a model for congenital sideroblastic anaemia. Nature Genet. 20, 244–250.[CrossRef][Medline]
Cazzola, M., Invernizzi, R., Bergamaschi, G., et al. (2003) Mitochondrial ferritin expression in erythroid cells from patients with sideroblastic anemia. Blood
101, 1996–2000.
Chan, R.Y., Seiser, C., Schulman, H.M., Kuhn, L.C. & Ponka, P. (1994) Regulation of transferrin receptor mRNA expression. Distinct regulatory features in erythroid cells. Eur. J. Biochem. 220, 683–692.[Medline]
Cotter, P.D., Baumann, M. & Bishop, D.F. (1992) Enzymatic defect in X-linked sideroblastic anemia: molecular evidence for erythroid 
-aminolevulinate synthase deficiency. Proc. Natl. Acad. Sci. USA
89, 4028–4032.
Cox, T.C., Bawden, M.J., Martin, A. & May, B.K. (1991) Human erythroid 5-aminolevulinate synthase: promoter analysis and identification of an iron-responsive element in the mRNA. EMBO J. 10, 1891–1902.[Medline]
Cox, T.C., Bottomley, S.S., Wiley, J.S., Bawden, M.J., Matthews, C.S. & May, B.K. (1994) X-linked pyridoxine-responsive sideroblastic anemia due to a THR388-to-SER substitution in erythroid 5-aminolevulinate synthase. New Engl. J. Med.
330, 675–679.
Crumpton, M.J. & Polson, A. (1965) A comparison of the conformation of whale sperm metmyoglobin with that of apomyoglobin. J. Mol. Biol. 11, 722–729.[Medline]
Dandekar, T., Stripecke, R., Gray, N.K., et al. (1991) Identification of a novel iron-responsive element in murine and human erythroid delta-aminolevulinic acid synthase mRNA. EMBO J. 10, 1903–1909.[Medline]
Fujita, H., Yamamoto, M., Yamagami, T., Hayashi, N. & Sassa, S. (1991) Erythroleukemia differentiation. Distinctive responses of the erythroid-specific and the nonspecific delta-aminolevulinate synthase mRNA. J. Biol. Chem.
266, 17494–17502.
Furuyama, K., Fujita, H., Nagai, T., et al. (1997) Pyridoxine refractory X-linked sideroblastic anemia caused by a point mutation in the erythroid 5-aminolevulinate synthase gene. Blood
90, 822–830.
Greenberg, P.L., Young, N.S. & Gattermann, N. (2002) Myelodysplastic syndromes. Hematology (Am. Soc. Hematol. Educ. Program) 136–161.
Han, A.P., Yu, C., Lu, L., et al. (2001) Heme-regulated eIF2alpha kinase (HRI) is required for translational regulation and survival of erythroid precursors in iron deficiency. EMBO J. 20, 6909–6918.[CrossRef][Medline]
Harigae, H., Nakajima, O., Suwabe, N., et al. (2003) Aberrant iron accumulation and oxidized status of erythroid-specific delta-aminolevulinate synthase (ALAS2)-deficient definitive erythroblasts. Blood
101, 1188–1193.
Harigae, H., Suwabe, N., Weinstock, P.H., et al. (1998) Deficient heme and globin synthesis in embryonic stem cells lacking the erythroid-specific delta-aminolevulinate synthase gene. Blood
91, 798–805.
Hentze, M.W., Muckenthaler, M.U. & Andrews, N.C. (2004) Balancing acts: molecular control of mammalian iron metabolism. Cell 117, 285–297.[CrossRef][Medline]
Hofer, T., Wenger, R.H., Kramer, M.F., Ferreira, G.C. & Gassmann, M. (2003) Hypoxic up-regulation of erythroid 5-aminolevulinate synthase. Blood
101, 348–350.
Iwai, K., Drake, S.K., Wehr, N.B., et al. (1998) Iron-dependent oxidation, ubiquitination, and degradation of iron regulatory protein 2: implications for degradation of oxidized proteins. Proc. Natl. Acad. Sci. USA
95, 4924–4928.
Kappas, A., Sassa, S., Galbraith, R.A. & Nordmann, Y. (1995) The Porphyrias. In: The Metabolic and Molecular Basis of Inherited Disease (eds C.R. Scriver, A.L. Beaudet, W.S. Sly, et al.), pp. 2103–2159. New York, NY: McGraw-Hill.
Kawahara, K., Kirshner, A.G. & Tanford, C. (1965) Dissociation of human CO-hemoglobin by urea, guanidine hydrochloride, and other reagents. Biochemistry 4, 1203–1213.[CrossRef][Medline]
Kawamura-Konishi, Y. & Suzuki, H. (1985) Binding reaction of hemin to globin. J. Biochem.
98, 1181–1190.
Kina, T., Ikuta, K., Takayama, E., et al. (2000) The monoclonal antibody TER-119 recognizes a molecule associated with glycophorin A and specifically marks the late stages of murine erythroid lineage. Br. J. Haematol. 109, 280–287.[CrossRef][Medline]
Kramer, M.F., Gunaratne, P. & Ferreira, G.C. (2000) Transcriptional regulation of the murine erythroid-specific 5-aminolevulinate synthase gene. Gene 247, 153–166.[CrossRef][Medline]
Levi, S., Corsi, B., Bosisio, M., et al. (2001) A human mitochondrial ferritin encoded by an intronless gene. J. Biol. Chem.
276, 24437–24440.
Lok, C.N. & Ponka, P. (1999) Identification of a hypoxia response element in the transferrin receptor gene. J. Biol. Chem.
274, 24147–24152.
Lopez, R.A., Schoetz, S., DeAngelis, K., O’Neill, D. & Bank, A. (2002) Multiple hematopoietic defects and delayed globin switching in Ikaros null mice. Proc. Natl. Acad. Sci. USA
99, 602–607.
Meguro, K., Igarashi, K., Yamamoto, M., Fujita, H. & Sassa, S. (1995) The role of the erythroid-specific delta-aminolevulinate synthase gene expression in erythroid heme synthesis. Blood
86, 940–948.
Motohashi, H., Katsuoka, F., Shavit, J.A., Engel, J.D. & Yamamoto, M. (2000) Positive or negative MARE-dependent transcriptional regulation is determined by the abundance of small Maf proteins. Cell 103, 865–875.[CrossRef][Medline]
Nakajima, O., Takahashi, S., Harigae, H., et al. (1999) Heme deficiency in erythroid lineage causes differentiation arrest and cytoplasmic iron overload. EMBO J. 18, 6282–6289.[CrossRef][Medline]
Nishimura, S., Takahashi, S., Kuroha, T., et al. (2000) A GATA box in the GATA-1 gene hematopoietic enhancer is a critical element in the network of GATA factors and sites that regulate this gene. Mol. Cell. Biol.
20, 713–723.
Ohneda, K., Shimizu, R., Nishimura, S., et al. (2002) A minigene containing four discrete cis elements recapitulates GATA-1 gene expression in vivo. Genes Cells 7, 1243–1254.[Abstract]
Onodera, K., Takahashi, S., Nishimura, S., et al. (1997) GATA-1 transcription is controlled by distinct regulatory mechanisms during primitive and definitive erythropoiesis. Proc. Natl. Acad. Sci. USA
94, 4487–4492.
Peters, R.E., May, A. & Jacobs, A. (1983) Globin chain synthesis ratios in sideroblastic anaemia. Br. J. Haematol. 53, 201–209.[Medline]
Ponka, P. (1997) Tissue-specific regulation of iron metabolism and heme synthesis: distinct control mechanisms in erythroid cells. Blood
89, 1–25.
Riddle, R.D., Yamamoto, M. & Engel, J.D. (1989) Expression of -aminolevulinate synthase in avian cells: separate genes encode erythroid-specific and nonspecific isozymes. Proc. Natl. Acad. Sci. USA
86, 792–796.
Shimizu, R., Ohneda, K., Engel, J.D., Trainor, C.D. & Yamamoto, M. (2004) Transgenic rescue of GATA-1-deficient mice with GATA-1 lacking a FOG-1 association site phenocopies patients with X-linked thrombocytopenia. Blood
103, 2560–2567.
Shimizu, R., Takahashi, S., Ohneda, K., Engel, J.D. & Yamamoto, M. (2001) In vivo requirements for GATA-1 functional domains during primitive and definitive erythropoiesis. EMBO J. 20, 5250–5260.[CrossRef][Medline]
Surinya, K.H., Cox, T.C. & May, B.K. (1997) Transcriptional regulation of the human erythroid 5-aminolevulinate synthase gene. Identification of promoter elements and role of regulatory proteins. J. Biol. Chem.
272, 26585–26594.
Suzuki, N., Ohneda, O., Takahashi, S., et al. (2002) Erythroid-specific expression of the erythropoietin receptor rescued its null mutant mice from lethality. Blood
100, 2279–2288.
Tacchini, L., Bianchi, L., Bernelli-Zazzera, A. & Cairo, G. (1999) Transferrin receptor induction by hypoxia. HIF-1-mediated transcriptional activation and cell-specific post-transcriptional regulation. J. Biol. Chem.
274, 24142–24146.
Takahashi, S., Shimizu, R., Suwabe, N., et al. (2000) GATA factor transgenes under GATA-1 locus control rescue germ line GATA-1 mutant deficiencies. Blood
96, 910–916.
White, J.M., Brain, M.C. & Ali, M.A. (1971) Globin synthesis in sideroblastic anaemia. I. Alpha and beta peptide chain synthesis. Br. J. Haematol. 20, 263–275.[Medline]
Winterhalter, K.H. & Deranleau, D.A. (1967) The structure of a hemoglobin carrying only two hemes. Biochemistry 6, 3136–3143.[Medline]
Yamamoto, M. & Nakajima, O. (2000) Animal models for X-linked sideroblastic anemia. Int. J. Hematol. 72, 157–164.[Medline]
Yamamoto, M., Yew, N.S., Federspiel, M., Dodgson, J.B., Hayashi, N. & Engel, J.D. (1985) Isolation of recombinant cDNAs encoding chicken erythroid delta-aminolevulinate synthase. Proc. Natl. Acad. Sci. USA
82, 3702–3706.
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