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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 798-805
Deficient Heme and Globin Synthesis in Embryonic Stem Cells
Lacking the Erythroid-Specific  -Aminolevulinate Synthase Gene
By
Hideo Harigae,
Naruyoshi Suwabe,
Peter H. Weinstock,
Mayumi Nagai,
Hiroyoshi Fujita,
Masayuki Yamamoto, and
Shigeru Sassa
From The Rockefeller University, New York, NY; the Departments of
Biochemistry and Molecular Biology and Applied Physiology, Tohoku
University School of Medicine, Sendai, Japan; and the Center for
Tsukuba Advanced Research Alliance and Institute for Basic Medical
Sciences, University of Tsukuba, Tsukuba, Japan.
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ABSTRACT |
The erythroid-specific isoform of  -aminolevulinate synthase
(ALAS-E) catalyzes the first step of heme biosynthesis in erythroid cells, and ALAS-E gene mutations are known to be responsible for x-linked sideroblastic anemia. To study the role of ALAS-E in erythroid
development, we prepared mouse embryonic stem (ES) cells carrying a
disrupted ALAS-E gene and examined the effect of the lack of ALAS-E
gene expression on erythroid differentiation. We found that mRNAs for
erythroid transcription factors and TER119-positive cells were
increased similarly both in the wild-type and mutant cells. In
contrast, heme content, the number of benzidine-positive cells, adult
globin protein, and mRNA for  -major globin were significantly
decreased in the mutant cells. These results were confirmed using
another ES differentiation system in vitro and suggest that ALAS-E
expression, hence heme supply, is critical for the late stage of
erythroid cell differentiation, which involves hemoglobin synthesis.
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INTRODUCTION |
-AMINOLEVULINATE synthase (ALAS) is
the first and rate-limiting enzyme in heme biosynthesis.1
There are two tissue-specific isozymes of this enzyme, ie, the
nonspecific isoform (ALAS-N) expressed ubiquitously and the
erythroid-specific isoform (ALAS-E) expressed exclusively in erythroid
cells.2 The human ALAS-N gene, ALAS1, has been
assigned to chromosome 3p21,3 while the ALAS-E gene,
ALAS2, to a distal subregion of band Xp11.2.4 In
Friend virus-transformed murine erythroleukemia (MEL) cells, mRNA for
ALAS-E was found to increase markedly when MEL cells were induced to
undergo erythroid differentiation by treatment with chemicals such as
dimethylsulfoxide, while ALAS-N mRNA decreased rapidly, suggesting that
the upregulation of the ALAS-E gene is essential in erythroid
development.5 Several point mutations in the ALAS-E gene
have been described in human patients with x-linked sideroblastic
anemia, also indicating the critical involvement of ALAS-E in the
development of anemia in this disorder.6,7
It is not clear, however, what effects the lack of ALAS-E expression
may have on erythropoiesis of normal hematopoietic cells. This question
is particularly intriguing because globin mRNA translation is known to
be regulated by the phosphorylation of the  -subunit of eukaryotic
translation initiation factor (eIF-2) by the heme-regulated eIF-2
kinase (HRI).8 It is possible therefore that deficient heme
synthesis may activate HRI and thus inhibit protein synthesis in
ALAS-E-deficient erythroid cells. Alternatively, heme may be required
in an early stage of erythroid cell differentiation, which involves
expression of various erythroid specific genes.
To address this question, we disrupted genetically the expression of
ALAS-E in embryonic stem (ES) cells and analyzed the effect of the lack
of ALAS-E on erythroid cell differentiation by using two different in
vitro differentiation systems of ES cells. ES cells are derived from
the inner cell mass of blastocysts9 and maintained in the
totipotent state in culture with feeder cells, or in the presence of
leukemia inhibitory factor (LIF), and they can contribute to somatic
and germ line tissues when reintroduced into blastocysts.10
In the first system, ES cells were cultivated in the presence of LIF
and were then let to differentiate by withdrawal of LIF from the
culture medium, and to form a three-dimensional structure termed
embryoid bodies (EBs), which contain various differentiated cell types,
including hematopoietic cells.11 Because the process of
hematopoietic development in EBs resembles normal hematopoiesis, this
system has been used for the analysis of genetic regulation of
hematopoietic cell differentiation12 and studies on the
effects of specifically introduced mutations on development of
hematopoietic cells.13,14 An alternative ES cell
differentiation system has recently been developed in which ES cells
are cocultured on a layer of OP9 feeder
cells,15 which permits ES cells to preferentially
differentiate into hematopoietic lineage cells, without forming
embryoid bodies.
The results of the two independent ES cell differentiation systems
showed that, whereas erythroid transcription factors were expressed
normally and a similar number of TER119-positive cells were observed
both in the wild-type and ALAS-E(-) ES cells, there was no increase in
the formation of heme and adult hemoglobin in ALAS-E(-) ES cells.
Especially, adult -major globin level was significantly lower in
ALAS-E(-) EBs than in the wild-type EBs, suggesting that HRI may be
activated by heme deficiency in ALAS-E(-) ES cell-derived erythroid
cells. These findings indicate that the early stage of erythroid
differentiation can be triggered without the presence of ALAS-E,
however, ALAS-E expression, hence increased heme biosynthesis, is
essential for the late stage in the development of erythroid cells
including hemoglobinized erythroid cells.
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MATERIALS AND METHODS |
Construction of a targeting vector.
A replacement-type ALAS-E targeting vector was assembled in a modified
plasmid pSP72 (Promega, Madison, WI), which had an additional
Not I site and a Pac I site introduced between the SP6
promoter sequence and Xho I site. As a selection marker, a Neomycin-resistance cassette from pMCneo, which contained a stop codon,16 was subcloned into Sal I site. As a
5 -homology region, a 0.8-kb Xho I/HindIII
fragment, including a part of exon 7, was subcloned into the
Xho I/HindIII site. As a 3-homology region, a
10-kb BamHI/Sal I fragment, including exon 11, was
subcloned into the BamHI site after converting an Sal I
site to a BamHI site. A herpes simplex virus thymidine-kinase
cassette was subcloned into an Not I site (Fig 1A). The final
construct was linearized by Pac I digestion.
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| Fig 1.
Disruption of the ALAS-E gene in mouse ES cells. (A)
Strategy for ALAS-E gene targeting. A phage clone encoding a part of mouse ALAS-E gene was cloned independently in our laboratory and is
shown in the upper side of (A) along with the normal mouse ALAS-E gene.
A probe used for Southern blotting in (B) is also shown. Maps of the
targeting construct and the predicted structure of the targeted ALAS-E
allele are shown in the lower lines of (A). (B) Southern blot analysis
of ES clones. A linearized construct was electroporated into J1 and CCE
cells and 20 mg of genomic DNA was isolated from clones selected in the
presence of Neomycin and Gancyclovir. The DNA samples were digested
with Pst I or EcoRI and examined by Southern blot
analysis. 6.8-kb and 4.6-kb bands detected after digestion with
Pst I represent the wild-type and the disrupted allele,
respectively, while 2.8-kb and 2.1-kb bands detected after digestion
with EcoRI represent the wild-type and the disrupted allele,
respectively. DNA samples are from wild-type CCE cells (lane 1),
ALAS-E(-) J1 cells (lane 2), or ALAS-E(-) CCE cells (lane 3). (C) Heme
content in differentiating EBs. Heme content of EBs of day 6, 8, and 10 was determined fluorometrically33 using 1 × 105 cells, which were dissociated from EBs into single
cells by incubation in a collagenase solution. Data are the mean of
three separate experiments.
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Transfection and screening of ES cells.
CCE ES cells,17 kindly provided by Dr P. Pandolfi (Memorial
Sloan-Kettering Cancer Center, New York, NY), were maintained on
mitomycin-treated STO feeder cells. J1 ES
cells18 were maintained on irradiated primary cultures of
embryonic mouse fibroblasts. Cell culture and electroporation were
performed as described previously.19 Clones selected in
G418 (400 µg/mL) and gancyclovir (2 mmol/L) were subjected to
Southern blot analysis to confirm a specific recombination allele. For
Southern blot analysis, genomic DNA was isolated by digesting cells in
a lysis buffer containing 1% sodium dodecyl sulfate (SDS), 625 µg/mL
proteinase K, 100 mmol/L NaCl, 10 mmol/L Tris (pH7.5), and 1 mmol/L
EDTA, at 55°C overnight and spooling after ethanol precipitation.
After digestion with EcoRI or Pst I, the Southern blots
were probed with a 0.6-kb fragment corresponding to an external
5-homology region of the targeting vector, or the Neo cassette
(Fig 1A).
In vitro differentiation of ES cells.
Differentiation of ES cells in semisolid culture was induced according
to the method described previously.20 To remove feeder layer cells, cells were plated onto a gelatin-coated plastic dish and
incubated at 37°C for 1 hour. After incubation, ES cells in suspension were seeded to bacterial-grade dishes at 1 × 105 cells/mL in Iscove's Modified Dulbecco's medium
supplemented with 15% fetal bovine serum (FBS) (GIBCO-BRL,
Gaithersburg, MD), 450 µmol/L monothio-glycerol (MTG) (Sigma, St
Louis, MO) and 2 U/mL rh erythropoietin (EPO, Kirin Brewery Co, Tokyo,
Japan) for 24 hours to allow formation of aggregates. The suspension
was then diluted to achieve 50 to 100 aggregates per 35-mm dish, and the cells were directly added to a 0.9% methylcellulose medium containing 15% FBS, 450 µmol/L MTG, and 2 U/mL EPO.
For cell differentiation on a layer of stromal cells, we first
performed a one-step differentiation method. Collected ES cells were
seeded onto a confluent layer of OP9 cells in a Falcon 6-flat well
tissue culture plate (Becton Dickinson Co, Lincoln Park, NJ) at 1 × 104 cells/well and incubated for up to 3 weeks as described previously.15 The two-step method
described by Nakano et al15 was used to induce more
efficient in vitro differentiation of hematopoietic cells from ES cells
on OP9 cells. Approximately 2 weeks after seeding ES cells on OP9
cells, floating cells from the stromal cell layer were collected with
media. These cells were analyzed by fluorescence-activated cell sorting
(FACS) and dianisidine staining. FACS analysis was performed with
fluorescein isothiocyanate (FITC)-labeled TER119
antibody,21 kindly provided by Dr Tatsuo Kina (Kyoto
University, Kyoto, Japan), and FITC-conjugated Mac1 antibody
(Pharmingen, San Diego, CA) as markers of erythroid and myeloid
lineages, respectively. Dianisidine-stained cells were fixed to slide
glass by using Cytospin (Shandon Southern Products, Cheshire, UK), and
the nuclei of these cells were counter-stained with methylgreen.
Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.
EBs were harvested at various times in culture and RNA was extracted
according to the method described previously.22 A total of
2 µg of total RNA were reverse-transcribed in 30 µL of reaction mixture containing the RT buffer (GIBCO-BRL), 10 mmol/L dithiothreitol (DTT), 1 µg of oligo (dT), 1 mmol/L each of
deoxynucleotide triphosphates (dNTP), 14 U of RNase inhibitor
(GIBCO-BRL) and 300 U of Moloney murine leukemia virus
(M-MLV) reverse transcriptase. PCR reactions were
performed in a final volume of 70 µL of the PCR reaction buffer
(Perkin-Elmer-Cetus, Norwalk, CT) containing 0.1 mmol/L each of dNTP,
50 pmol each of the primer, 3 µCi
[-32P]deoxycytidine triphosphate (dCTP)
(3,000 Ci/mmol), and 2.5 U of Taq polymerase (Promega). The amount of
cDNAs added to the reaction mixture was normalized by the intensity of
the -actin amplicon. Aliquots were electrophoresed in 7%
polyacrylamide gels. Gels were dried, autoradiographed, and subjected
to densitometric analysis. An optimal cycle number for each primer was
determined by preliminary PCR by removing aliquots at various numbers
of cycles and examining intensity of amplicons at each time. Linear amplification was verified at the determined cycle numbers for each
primer. Primers used in this study were as follows:
-actin23 sense, 5-GTGACGAGGCCCAGAGCAAG;
antisense, 5-AGGGGCCGGACTCATCGTAC; ALAS-E24 sense,
5-GTGGTGCAGCCAAGTTTGTC; antisense,
5-AGCATAGGTGGTAACATATT; GATA-125 sense,
5-ACTCGTCATACCACTAAGGT; antisense,
5-AGTGTCTGTAGGCCTCAGCT; EKLF26 sense,
5-GATCGCCGGAGACGCAGGCT; antisense,
5-TCCCCAGTCCTTGTGCAGGA; p4527 sense,
5-TCAGCAGAACAGGAACAGGT; antisense,
5-GCTTTGACACTGGTATAGCT; h1 globin28
sense, 5-CTCAAGGAGACCTTTGCTCA; antisense,
5-GCCTAATTCAGTCCCCATGG; -major globin29
sense, 5-CACAACCCCAGAAACAGACA; antisense, 5-CTGACAGATGCTCTCTTGGG.
Immunoblot analysis.
For immunoblot analysis, EBs were homogenized in 20 mmol/L Tris-Cl
(pH7.4) containing 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, and 1 mmol/L phenylmethylsulfonyl fluoride (PMSF). A total of 15 µg of
protein was loaded onto a 12% Laemmli gel30 and
electrophoretically separated. Immunoblotting and detection by an
enhanced chemiluminescence (ECL, Amersham International
plc, Buckinghamshire, UK) was performed as described
previously.31 Primary antibodies used in this study were a
rat antimouse GATA-1 antiserum (Santa Cruz Biotechnology, Santa Cruz,
CA) and a rabbit antimouse hemoglobin antiserum (CAPPEL/Organon Teknica, Durham, NC). A goat antirat IgG for GATA-1 and a goat antirabbit-IgG for hemoglobin, both of which had been coupled with
horseradish peroxidase, were used as the secondary antibody in the ECL
assay. The specificity of rat antimouse GATA-1 antibody (N6) has been
previously established by its exclusive binding to mouse GATA-1 protein
in immunoblot analysis.32 The antihemoglobin antibody used
was a polyclonal antibody raised in a rabbit immunized by mouse adult
hemoglobin(s).
Heme assay.
EBs were dissociated by incubation in phosphate-buffered saline
containing 0.25% collagenase and then incubated with 20% FBS for 1 hour.12 After incubation, a single cell suspension was prepared by passing cells through a syringe with a 20-gauge needle. Heme content was determined in triplicate using 1 × 105 cells per assay by fluorometry as described
previously.33
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RESULTS |
Preparation of a targeting vector for ALAS-E gene.
Before ALAS-E gene targeting, we examined the chromosomal location of
the mouse ALAS-E gene. Our finding indicates that the mouse ALAS-E gene
was located on the X-chromosome (data not shown), as is the case with
the human ALAS-E gene (ALAS2).4
To prepare a disrupted ALAS-E gene in mouse ES cells, we constructed a
replacement vector that contained a 0.8-kb 5-homologous region
and a 10-kb 3-homologous region of the ALAS-E gene flanking a
NEO cassette (Fig 1A). Because a 4.4-kb
genomic fragment, including exon 8 through exon 10, was replaced with
the NEO cassette by the homologous recombination, the mutant allele
lacked a domain including the binding site for pyridoxal
5-phosphate,34 an essential cofactor for ALAS
activity (Fig 1A). The linearized targeting vector was electroporated
into two ES cell lines, CCE and J1, and clones were screened for
resistance to G418 and gancyclovir. Southern blot analysis of the
selected clones showed that one each of the CCE and J1 clone contained
the targeted ALAS-E gene (Fig 1B).
Heme content in ALAS-E(-) EBs.
To evaluate the consequence of ALAS-E deficiency in erythroid cell
development, ALAS-E(-) ES cells were incubated in a semisolid medium to
allow EB formation and cell differentiation. EBs thus developed were
harvested at various time points and heme content was determined. The
heme content in the wild-type EBs increased from day 6, continued to
increase during cell differentiation, and reached a maximum by day 10 (Fig 1C). Development of hemoglobinized erythroid cells was also
observed in the wild-type EBs (see below), which was similar to the
rate of heme synthesis. In contrast to the wild-type EBs, heme content
in ALAS-E(-) EBs did not increase at all (Fig 1C).
Erythroid differentiation of ALAS-E(-) ES cells.
To examine whether cells in ALAS-E(-) EBs differentiate along with the
erythroid lineage, both the wild-type and ALAS-E(-) EBs were harvested
on day 8 and were dissociated using a collagenase solution and stained
for hemoglobin with benzidine. Significant numbers of
benzidine-positive cells were detected in the wild-type EBs
(Fig 2A), whereas no benzidine-positive
cells were observed in ALAS-E(-) EBs (Fig 2B). This finding indicates
that cells in the mutant EBs did not differentiate fully to the
hemoglobinized cells, as in the wild-type EBs.
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| Fig 2.
Lack of ALAS-E affects erythroid differentiation of ES
cells at late stage. EBs formed from the wild-type (a) and ALAS-E(-) mutant ES cells (b) were dissociated with collagenase and stained with
benzidine. Note the presence of dark blue cells in the wild-type EBs,
which are positive to the benzidine staining. The wild-type (c) and
mutant ES cells (d) were also cocultured with OP9 stroma cells and
colonies formed were stained with benzidine. Hematopoietic cells
obtained from the two-step coculture culture system with OP9 cells were
then analyzed. The expression of TER119 was analyzed by FACS.
Comparable numbers of TER119-positive cells were floated from ALAS-E(-)
mutant ES cell culture (f) as does from the wild ES cell culture (e).
However, no benzidine staining-positive cells were observed in the
hematopoietic cells from the mutant ES cells (h), while a number of
benzidine-positive cells were observed in the floating hematopoietic
cells from the wild-type ES cells (g).
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This conclusion was also confirmed by using another recently developed
in vitro differentiation system, which uses coculture of ES cells with
OP9 stromal cells.15 OP9 is a cell line that lacks a
macrophage colony-stimulating factor (M-CSF) receptor and
is known to permit efficient hematopoietic cell differentiation of ES
cells, without forming EBs.15 Using this system, both the
wild-type and the mutant ES cells were cocultured on a layer of OP9
cells for 5 days and then transferred to a new layer of OP9 cells after
dissociation with trypsin. Development of hematopoietic cell colonies
was observed in association with the OP9 layer. Figure 2C and
D show the benzidine-positive colonies from the wild-type
and mutant ES cells, respectively, on the day 14 culture. In the
wild-type ES cell culture, there were a number of
benzidine-positive cell aggregates (Fig 2C), indicating
that these colonies contain hemoglobinized cells. In contrast, there
were no benzidine-positive colonies in ALAS-E(-) cells (Fig
2D).
The colonies formed in this culture were mixed colonies, rather than
pure hematopoietic colonies. Thus, we selected hematopoietic cells
according to the procedure of Nakano et al.15 In this method, it was shown that cells floated after the second passage of ES
cells contain cells of various hematopoietic lineages. Floating cells
were harvested from day 14 culture and stained for TER119 erythroid-specific marker, which is known to be exclusively expressed in mature erythrocytes.
Approximately 5% of both the wild-type and ALAS-E(-) ES cells were
found to be positive for TER119 (Fig 2E and F). This finding thus
suggests that both the wild-type and ALAS-E(-) cells have developed to
a stage beyond colony-forming unit-erythroid
(CFU-E).21 The percentages of cells positive
for other cell lineage-specific markers, such as B220 for B-cell
lineage, GR1 for the granulocyte lineage, and Mac1 for the monocyte
lineage, were similar for both the wild-type and ALAS-E(-) ES cells
(data not shown). There were cells that were positive for hemoglobin
synthesis in the wild-type ES culture as judged by positive stain with
benzidine (Fig 2G), while there were no cells that were
positive for benzidine in ALAS-E(-) ES cells (Fig 2H). A
summary of repeated experiments is shown in
Table 1. These findings clearly show that
while ALAS-E(-) ES cells develop to the TER119-positive stage, these
cells are not capable of hemoglobin synthesis.
Expression of mRNAs for ALAS-E and erythroid transcription factors in
ALAS-E(-) EBs.
To examine the effect of ALAS-E gene disruption in the early stage of
erythropoiesis, we examined expression of mRNAs for ALAS-E and
erythroid transcription factors in the wild-type and ALAS-E(-) EBs. EBs
were harvested at various time points, and RT-PCR was performed using
total RNA to determine the levels of mRNAs. As shown in
Fig 3, the normal and mutated ALAS-E mRNAs were detectable by day 4 in the wild-type and ALAS-E(-) EBs,
respectively. By Southern blot analysis of the PCR products, expression
of normal ALAS-E mRNA and the mutant mRNA lacking exon 8 through exon
10, in the wild-type and ALAS-E(-) cells, respectively, was
demonstrated (data not shown).
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| Fig 3.
RT-PCR analysis of the expression of erythroid
transcription factors in EBs. Expression of mRNAs coding for ALAS-E and
erythroid transcription factors GATA-1, p45, and EKLF were examined by
RT-PCR. RNA samples were obtained from day 2 through day 12 EBs in
culture. WT, wild-type EBs; ALAS-E(-), ALAS-E(-) EBs.
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Expression of GATA-1, p45 NF-E2, and EKLF mRNAs was also examined using
RT-PCR of RNAs isolated from EBs. Similar to ALAS-E mRNA, levels of
these mRNAs increased rapidly in the wild-type EBs and reached a
maximum on day 8 (Fig 3). The level and the time course of the
expression of these mRNAs in ALAS-E(-) EBs was very similar to those
that were observed in the wild-type EBs, indicating that heme
deficiency per se does not affect the early stage of erythropoiesis.
Expression of globin mRNA and protein in ALAS-E(-) EBs.
We next examined globin gene expression by using RT-PCR. h1-globin
mRNA was found increased by day 4, but that of the -major globin
became detectable for the first time on day 6 in the wild-type EBs
(Fig 4). In addition, -major globin mRNA levels were
lower in ALAS-E(-) EBs than in the wild-type EBs, while h1-globin
mRNA expression in the mutant EBs showed a similar time course and levels to those in the wild-type EBs (Fig 4). Specifically, after day
8, the -major globin mRNA level declined more rapidly in ALAS-E(-)
EBs than in the wild-type EBs. This finding suggests that the
transcription and/or stability of the -major globin mRNA,
but not of h1-globin, was suppressed by the lack of ALAS-E expression.
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| Fig 4.
Expression of globin mRNAs in differentiating EBs. The
levels of mRNAs coding for h1 and -major globin were examined by RT-PCR. RNA samples were obtained from day 2 through day 12 EBs in
culture. The top panel shows the photograph, while the bottom panel is
a graphic presentation of the data.
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We next examined the expression of globin protein by immunoblot
analysis using an antibody raised against adult mouse hemoglobin. As
shown in Fig 5, adult hemoglobin levels were very much
lower in ALAS-E(-) EBs than in the wild-type EBs, despite the fact that a substantial amount of -major globin mRNA was detectable in these
cells (see Fig 4). In contrast to -major globin protein, GATA-1
protein was significantly expressed, which is consistent with the
result of its mRNA expression (see Fig 3A ), both in ALAS-E(-) and
wild-type EBs (data not shown). These findings suggest that decreased
-major globin in ALAS-E(-) EBs is not only due to suppressed
expression of its mRNA, but also to an additional posttranscriptional
mechanism.
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| Fig 5.
Expression of adult globin protein in differentiating
EBs. Adult globin proteins were analyzed by Western blot analysis.
Proteins were isolated from EBs of day 6, 8, and 10 in culture.
Immunoquantitation was performed using a rabbit antimouse hemoglobin
serum and a goat antirabbit-IgG coupled with horseradish peroxidase in
an ECL assay system. In the top panel, lane P shows the analysis protein sample from DMSO-treated MEL cells as a positive control, lanes
WT show the analysis of proteins from wild-type EBs in culture of day
6, 8, and 10. Lanes ASE(-) show the analysis of proteins from ALAS-E(-)
EBs in culture of day 6, 8, and 10 and lanes ASE (-) + ALA show the
analysis of proteins from ALAS-E(-) EBs in culture with the addition of
ALA of day 6, 8, and 10. The bottom panel is a graphic presentation of
the data.
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If ALA is the rate-limiting factor for erythroid heme synthesis and
expression of -major globin, an addition of ALA to ALAS-E(-) EBs is
expected to correct these problems. When ALA (100 µmol/L) was added
to the culture, heme content in ALAS-E(-) EBs was found to increase and
eventually reached the level found in the wild-type EBs (data not
shown), showing that the defect in heme synthesis in ALAS-E(-) cells
was due to ALA deficiency by the lack of ALAS-E expression. The level
of adult hemoglobin was also significantly restored, although not
complete, by ALA treatment (see Fig 5). These results indicate that the
suppression of -major globin synthesis is due to heme deficiency by
the lack of ALA formation and show the critical role of heme synthesis
in -major globin synthesis.
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DISCUSSION |
Erythroid cells synthesize a large amount of heme, which is several
orders of magnitude greater than other cells,1 and yet in
normal erythroid cells, the amount of heme and globin is stoichiometrically matched. Thus, there must be an extremely fine control to maintain a balance between the synthesis of heme and globin
in developing erythroid cells. In this study, we disrupted genetically
the expression of ALAS-E that catalyzes the first step of heme
biosynthesis and studied its effect on erythroid cell differentiation
by using two in vitro differentiation systems of ES cells. The results
clearly showed that ALAS-E is indispensable for the development of
hemoglobinized mature erythroid cells and suggest that the rate of heme
synthesis in erythroid cells plays a critical role in hemoglobin
formation in these cells.
No hemoglobinized cells were found in cultures of ALAS-E(-) ES cells in
both ES cell differentiation systems. Because the genes encoding
erythroid transcription factors were expressed in ALAS-E(-) cells
similarly as in the wild-type cells, it is unlikely that the number of
erythroid precursors may be decreased in ALAS-E(-) EBs. Thus, the
observed difference in the phenotype in ALAS-E(-) cells is more likely
to be due to the lack of heme supply, which principally affected the
late stage development of erythroid cells.
A positive role of heme on erythroid differentiation is well
documented, while its exact mechanism of action is yet to be defined.
35-37 Heme may have several effects in erythroid
development, as it is known to upregulate heme pathway
enzyme,5,36 as well as globin genes in MEL
cells,38,39 and to stimulate erythroid colony formation in
primary bone marrow cultures.40 These effects of heme in
erythroid cells are in marked contrast to its effect on its own
synthesis in the liver, which is exclusively suppressive. The
difference in the action of heme between the erythroid and the
nonerythroid tissues may importantly be related to the tissue-specific expression of the ALAS gene.
Our findings suggest that -major globin expression is suppressed not
only at the transcriptional level, but also at the translational level
in ALAS-E(-) EBs. Using ALAS-E(-) cells, it is now possible for the
first time to define the significance of the entire lack of erythroid
heme formation in these cells. For example, heme is known to be
necessary for translation of globin mRNA through inhibiting the
activity of eIF-2 kinase (HRI, reviewed in Chen and
London8). During the initiation of translation, eIF-2 forms a ternary complex with initiator tRNA charged with methionine and
guanosine triphosphate (GTP). Phosphorylation of the
eIF-2 at Ser 51 reduces its activity by impairing the rate of
eIF-2B-dependent guanosine diphosphate (GDP)-GTP
exchange reaction. Heme deficiency in ALAS-E(-) EBs may thus interfere
with translation of globin from its mRNA by inhibiting the ternary
complex formation.
Alternatively, the lack of heme may also affect the stability of
-major globin mRNA. Because the extent of a decrease in -major
globin synthesis was far greater than that of a decrease in its mRNA
level, it is also possible that there is an additional posttranscriptional effect in globin synthesis due to heme deficiency, which may decrease the steady state level of -major globin mRNA in
ALAS-E(-) EBs. The significance of heme deficiency in -major globin
synthesis is also corroborated by the fact that globin protein
synthesis was partially restored by treatment of ALAS-E(-) ES cells
with exogenous ALA.
Our findings also show that while heme synthesis by normal expression
of ALAS-E is critical for the late stage in erythroid differentiation,
which involves hemoglobin synthesis, it may not be so essential for the
early stage, which involves expression of other early erythroid genes.
The reason(s) for different responses of h1 and
-major globin mRNAs to the supply of heme by ALAS-N in ALAS-E(-) cells is not understood at present. One of the technical
difficulties in dealing with this question is the fact that mouse
ALAS-N cDNA has not been cloned, thus prohibiting evaluation of this
question directly. It should be noted, however, that primitive and
definitive erythropoiesis are known to be differentially affected,
depending on which transcription factors are knocked out, eg,
c-Myb,41 EKLF,42,43 and GATA-1.44
Thus, h1 and -major globin mRNAs might be differently
regulated by the lack of ALAS-E expression. It is also
possible that ALAS-E may be more important for -major than
h1 globin mRNA expression, as -major globin synthesis
in MEL cells is exclusively dependent on ALAS-E, rather than
ALAS-N.5,45 For example, ALAS-E mRNA levels in untreated
MEL cells are  10-fold higher than ALAS-N mRNA, and the former is
increased more than ~30-fold at 72 hours as compared with untreated
cells,39 while the latter is downregulated during erythroid
differentiation of these cells.5,39 While
these findings do not provide a direct answer to the question, they
collectively suggest that expression of -major globin mRNAs may be
more critically dependent on the availability of erythroid heme, or
ALAS-E expression, than h1 globin mRNA.
Our findings show that the percentages of TER119-positive cells were
similar both in the wild-type and ALAS-E(-) ES cells. Because
TER119-antigen is expressed only in late stage erythroid cells
including mature erythrocytes, but not in erythroid progenitors such as
burst-forming unit-erythroid (BFU-E) or
CFU-E,21 it can be inferred that the ALAS(-) ES cells
differentiated to a stage similar to that of the wild-type cells, which
were TER119-positive. This finding is also consistent with the
observation in patients with x-linked sideroblastic anemia (XLSA), in
which a mutation of the ALAS-E gene has been documented,34
that erythroid cells usually differentiate to a late stage of erythroid
development, ie, mature erythrocytes, while these cells have a
decreased amount of hemoglobin.
In contrast to the bone marrow of patients with XLSA, however, ring
sideroblasts, which are the characteristic of this condition, were not
observed in ALAS-E(-) cells cultured in vitro. The reason(s) for this
discrepancy is unclear at present. It is possible that the in vitro
system may lack a certain important factor(s) for complete erythroid
differentiation, thus it may not permit the formation of ring
sideroblasts. Alternatively, unlike the mutation of ALAS-E gene in
patients with XLSA, which have residual enzyme activity, the null
mutation of ALAS-E in ALAS-E(-) cells may be too stringent for the
development of ring sideroblasts. To clarify this question, a new
knockdown targeting and an animal model of ALAS-E(-) is necessary, and
these experiments are under way in our laboratory.
| |
FOOTNOTES |
Submitted February 10, 1997;
accepted September 22, 1997.
Supported in part by Grant No. DK-32890 from the United States Public
Health Service (to S.S.), The Yamanouchi Molecular
Medicine Fund from the Yamanouchi USA Foundation (to S.S.), Japanese
Society for Promotion of Science and The Uehara Memorial Foundation (to M.Y.), and Grant-in-Aid from the Ministry of Education, Science and
Culture of Japan (to H.F. and M.Y.).
Address reprint requests to Shigeru Sassa, MD, PhD, The Rockefeller
University, 1230 York Ave, New York, NY 10021.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
We are grateful to Drs P. Pandolfi, Memorial Sloan-Kettering Cancer
Center, New York, NY, for his generous supply of CCE ES cells; T. Kina
for TER119; T. Nakano, Osaka University, Osaka, Japan, for his helpful
discussion; Kirin Brewery Co for rh erythropoietin; and to Luba
Garbaczewski for her technical assistance.
| |
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Abstract
Introduction
Abstract