Institute of Molecular Pathology Vienna Biocenter A 1030 Vienna, Austria
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ABSTRACT |
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INTRODUCTION |
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Terminal differentiation of progenitor cells is induced when self-renewal factors are removed and replaced by differentiation factors [erythropoietin (Epo) and insulin]. During this differentiation, the cells undergo four to five cell divisions, reduce their size by a factor of 34, and accumulate hemoglobin (2, 8). Proper regulation of the balance between self-renewal and terminal differentiation is of extreme importance to generate adequate numbers of mature erythrocytes in the blood stream during the entire lifetime of the organism.
Recent experiments showed that activation of both the GR and the estrogen receptor (ER) is required to induce self-renewal of avian erythroid progenitors. The two receptors cannot replace each others function but have to cooperate (7). The GR is expressed in many tissues including hematopoietic cells, and activation of the GR does influence erythropoiesis (9, 10, 11). Although the major function of the ER appears to be in the female reproductive system, a regulatory function of estrogen has been demonstrated in bone homeostasis (reviewed in Ref.12) and in neuronal tissue (Refs. 13 and 14 and references therein). Estrogens also increase the number of erythroid cells in mice in vivo (15, 16) and are required for proliferation of erythroid chicken cells in vitro (4).
Accumulating evidence suggests that the ER can be activated in the
absence of hormone through phosphorylation, e.g. by dopamine
or insulin-like growth factor I (IGF-I) in neuronal cells (13, 14, 17)
or by epidermal growth factor (EGF) or IGF-I in uterine tissue
(18, 19, 20). Also in the presence of estradiol, signal transduction from
activated receptor tyrosine kinases (IGF-I and EGF receptor) further
enhances ER activity through phosphorylation of N-terminal residues
(21, 22). Thus, the synergistic effect between TGF and estradiol in
avian erythroid progenitors may well be analogous to the synergistic
effects of EGF and estradiol in mammary epithelial cells and uterine
tissue (18, 19).
The cooperation of c-ErbB with the ER to cause self-renewal in normal erythroid progenitors closely resembles the cooperation between two retroviral oncoproteins, the avian erythroblastosis virus (AEV)-encoded proteins, v-ErbB and v-ErbA, in the induction of erythroleukemia (reviewed in Ref.2). V-ErbB is a mutated and constitutively active derivative of the avian EGF receptor, while V-ErbA is a mutated version of the thyroid hormone receptor. It was recently shown that v-ErbA has the ability to functionally replace both the ER and the GR in inducing sustained self-renewal of erythroid progenitors and to block terminal differentiation (23). Whereas investigations regarding the role of the GR in erythroid proliferation and differentiation are reported elsewhere (7), this paper concentrates on the role of the ER in erythropoiesis.
Earlier work indicated that the ER is able to arrest or retard erythroid differentiation, depending on the expression level (4). Activation of the ER inhibited the expression of several genes induced late in erythroid differentiation, such as the ion exchanger Band 3 and the globin genes. Blobel et al. (24) showed that the ER can form a protein complex with the erythroid-specific transcription factor Gata-1 in cells overexpressing the ER. This interaction was dependent on hormone and suppressed the transactivation of reporter genes by Gata-1. Gata-1 plays an essential role in erythroid development (25). Thus, this inactivation of Gata-1 by the E2-activated ER seemed difficult to reconcile with the essential role of the ER in erythroid self-renewal. Recently, however, it was shown that self-renewing avian erythroid progenitors express Gata-1 mainly as a cytoplasmic protein, essentially unable to bind DNA. Only upon induction of terminal differentiation in the presence of Epo, is Gata-1 translocated to the nucleus and acquires the capacity to bind DNA (26). It is possible that partial inactivation of Gata-1 by the ER can contribute to the differentiation arrest occurring during self-renewal.
In this paper, two complementary cell systems are used to demonstrate that the ER can affect erythroid differentiation at any stage and that the ER exhibits a dual role with respect to transcriptional control of genes up-regulated during erythroid differentiation. Gata-1, SCL-1, and the globin genes were repressed dependent on estradiol, while the expression of carbonic anhydrase II (CAII) and histone H5 was enhanced by estradiol. Repression and activation involved distinct mechanisms. Since the ER inhibits and the thyroid hormone receptor enhances erythroid differentiation, these receptors could interfere with each others function. However, activation of the ER had no effect on differentiation induction and target gene induction by the avian thyroid hormone receptor.
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RESULTS |
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Next, we sought to determine whether the activated receptor blocks entry into a terminal differentiation program, or whether the active ER is able to arrest ongoing differentiation at any stage. Therefore, we wanted to activate the ER in erythroid cells at various stages of differentiation and monitor its effect on the continuation of the differentiation program. Since normal progenitors down-regulate expression of the endogenous ER during terminal differentiation, we employed a clone of the erythroid cell line HD3 that lacks the endogenous chicken ER and constitutively expresses the human ER from a retroviral vector (clone 4; Ref.4). In this way, transcriptional regulation of the ER could be circumvented.
HuER-HD3 clone 4 cells were induced to differentiate for 64 h in
steroid-free medium containing anemic chicken serum and insulin. Since
synchrony of differentiation was limited, homogenous cell populations
at different stages of maturity were obtained by purification on
Percoll gradients (Fig. 1A; see
Materials and Methods). Two aliquots of these fractions were
reseeded in the same differentiation medium either containing estradiol
(E2) or the estrogen antagonist ICI 164,384. During the
next 4 days, cells were assayed for various differentiation parameters,
i.e. proliferation, hemoglobin accumulation, and cell
morphology.
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E2 clearly affected the progress of maturation in partially
differentiated cells. Upon E2 treatment, cells with a
density between 1.075 and 1.080 g/cm3 almost retained their
high thymidine incorporation level, while this decreased by a factor of
4 in the presence of the E2 antagonist (Fig. 1B).
Concurrently, hemoglobin levels increased only moderately in the
presence of E2, but strongly (5.5-fold) in the presence of
ICI 164,384 (Fig. 1C
). Together, this indicated that ongoing maturation
was arrested or heavily retarded in the presence of E2, but
that the cells continued to mature in the presence of ICI 164,384.
Morphological and histochemical analyses of cytospins confirmed these
results (Fig. 1D
). Before splitting, the cells consisted of late
reticulocytes, as shown by their small size, and light hemoglobin
staining (Fig. 1D
, left). Upon addition of ICI 164,384, the
cells progressed in differentiation and formed oval, well
hemoglobinized, mature erythrocytes (Fig. 1D
, right). Upon
addition of E2, however, the cells were mostly arrested at
the late reticulocyte state, with only a few being able to mature into
erythrocyte-like cells (Fig. 1D
, middle).
A result similar to that obtained for this cell fraction (density 1.0751.080 g/cm3) was obtained with all other fractions. Regardless of their initial stage of maturation, the cells were arrested at their particular stage of maturity when treated with E2, but continued to mature into erythrocytes in ICI. This indicated that addition of estrogen can block differentiation at any stage and that it does not merely affect the onset of the differentiation program. However, E2 was unable to cause retrodifferentiation of the partially mature cells and did not cause them to turn on a self-renewal program again.
The E2-Activated ER Suppresses
Transcription of Erythrocyte-Specific Genes
Activation of the erythroid differentiation program induces the
transcription of a number of genes whose products are required for
maturation of erythrocytes. A transient up-regulation of the
transcription factors Gata-1 and SCL-1 is followed by expression of the
erythrocyte-specific genes, histone H5, band 3, band 4.1, CAII, Ala-S,
and the globin genes (8). Previously, it was shown that cells arrested
in differentiation by the ER showed decreased mRNA levels of band 3,
band 4.1, -globin, and ß-globin (4), both in HD3 cells that
overexpress the human ER and in primary erythroid cells that express
the endogenous receptor. Since activation of the ER appeared to block
differentiation at any stage, it was of interest to determine whether
the ER could repress the above genes after they had been up-regulated
by differentiation induction.
Several of the late erythroid genes encode relatively stable mRNAs. Therefore, down-modulation of expression could not be studied using Northern blots. Rather, simplified run-on assays using the coding regions of the genes to be studied were performed. The initial studies were performed in HD3 cells that constitutively express the human ER and do not down-regulate the ER during differentiation. To rule out artefacts by the overexpressed ER, or by a possible residual activity of the v-ErbA oncoprotein in the HD3 cells, the experiments were repeated in primary cells that lack the v-ErbA oncoprotein and express the endogenous chicken ER.
HuER-HD3 clone 4 cells were induced to differentiate and were density
purified as described above. The partially mature cells obtained were
then treated with E2 or ICI 164,384 and processed for
run-on analysis. To determine suitable times for predifferentiation and
hormone treatment, cells were induced to differentiate for either 24 or
40 h and then subjected to estrogen/ICI treatment for 8 and
24 h. Figure 2A shows that the times
required for optimal differences between E2 and ICI varied
with the gene tested. After 24 h of predifferentiation, Gata-1 had
not yet reached maximal transcription levels. In the presence of
E2, the transcription rate remained constant, while it
increased 2-fold in a time-dependent fashion upon treatment with ICI
164,384. After 40 h of predifferentiation, Gata-1 transcription
was nearly maximal and thus only moderately reduced by subsequent
hormone treatment. In contrast, transcription of ß-globin was
submaximal both after 24 and 40 h of predifferentiation, allowing
the detection of a time-dependent, 5-fold increase upon ICI 164,384
treatment in both cases. Interestingly, E2 did not only
block the further increase in ß-globin transcription, but even caused
a moderate decrease in transcriptional rate after hormone treatment,
both after 24 and 40 h of predifferentiation.
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Next, we sought to confirm these data in primary, erythroid
progenitors. Although the endogenous ER is down-regulated during
progenitor differentiation, it is possible to delay this
down-regulation, if TGF plus ICI 164,384 is added together with the
differentiation factors (4). Under these conditions, differentiation is
induced but the ER is down-regulated only after 72 h. Primary
SCF/TGF
progenitors (5) were predifferentiated under these
conditions for 24 h, washed, split into two aliquots, incubated
with E2 or ICI 164,384 for another 24 h in the absence
of TGF
, and processed for run-on analysis.
The combined results from three independent experiments are shown in
Fig. 2C. They were highly reproducible with respect to which genes were
activated or repressed as a consequence of ER activation, but the
extent of the repression or activation was somewhat variable, perhaps
due to variations in the expression of the endogenous ER. Transcription
of the nuclear proteins, Gata-1 and SCL-1, was repressed
2-fold, as
was transcription of the erythrocyte genes
- and ß-globin. In
contrast to the HD3 cells, Gata-2 transcription was not affected by the
ER. In these run-on assays, the transcription rate of CAII was not
affected by E2 in HD3 cells, while transcription was
clearly enhanced in primary cells. Steady state levels of CAII are
increased by E2 in HD3 cells as judged on Northern blots
(Fig. 4
). The inability to see an up-regulation of CAII in these run-on
assays may be due to the choice of 24-h predifferentiation.
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Requirement of Functional Domains of the ER for Its Effects on
Erythroid Differentiation
To investigate by what mechanisms the ER may regulate its target
genes, we first determined which domains of the ER are required for the
observed differentiation arrest and for the repression or activation of
late erythroid genes. The ER has two transactivation domains, the
N-terminal AF-1 and the C-terminal AF-2. These domains were shown to
act independently and subject to both cell type and promoter context
(28, 29). In addition to regulating gene transcription via specific
binding to estrogen response elements (EREs), the ER also interferes
with the function of other transcription factors through
protein-protein interactions. Notably, the E2-activated
ER can interact with Gata-1 (24) or nuclear factor (NF)-B and AP-1
family members, which involves both N-terminal and C-terminal domains
of the ER that are different from the transactivation domains
(30, 31, 32, 33).
ER constructs mutated in one or both of the transcription activation
domains, or mutated in the DNA-binding domain (DBD) (Refs. 34 and 35;
Fig. 3A) were cloned into a retroviral
expression vector (pCRN, Ref.36) and transfected into HD3 cells.
Single-cell colonies were isolated, grown into clonal cultures, and
tested 1) for expression of the ER construct by Western blot (data not
shown) and 2) for its ability to terminally differentiate in the
presence of ICI under standard conditions as described above. For every
ER construct, a representative clone positive for ER expression by
Western blot was selected and induced to differentiate in the presence
of either E2 or ICI 164,384. During 4 days of
differentiation, hemoglobin levels, cell numbers, and cell volumes were
determined. As a representative parameter, the ability of the different
clones to differentiate in the presence of E2 was expressed
as percent reduction of hemoglobin per cell volume by E2,
relative to the ICI control at day 3 (Fig. 3B
). The wt mouse
ER (MOR) and mutant constructs carrying either a deletion in the AF-1
domain (amino acids 1121; Ref.34) or a mutation of the second zinc
finger of the DBD that disrupts DNA binding (C241A, C244A; Ref.35)
caused a distinct, E2 dependent arrest of differentiation
(Fig. 3B
). In contrast, mutant receptors carrying a mutated,
nonfunctional AF-2 domain (L543A, L544A; Ref.34) failed to affect
differentiation, either in the presence or absence of a functional AF-1
domain. These findings could be corroborated by evaluation of stained
cytospins from the same cell populations (data not shown).
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Avian bone marrow cells were cocultivated with an excess of
fibroblasts, which produced retroviruses expressing the various ER
mutants (Fig. 3A). Five days after infection of the bone marrow cells,
the resulting mass cultures were tested for expression of the mutant ER
proteins, using the monoclonal antibody H222, which recognizes the
mammalian, but not the avian, ER. All ER constructs were expressed at
comparable levels except for the DBD mutant (data not shown). We
repeatedly failed to obtain proliferating erythroid progenitors from
bone marrow infected with this mutant. The DBD mutant was already shown
to be toxic in HD3 cells upon E2 addition. However, HD3
cells can be grown in the presence of ICI, and activation of this
protein can be inhibited.
At the same time expression of the ER constructs was examined, the mass
cultures were induced to differentiate in the presence of Epo/Insulin
plus either E2 or the antagonist ICI 164,384. During the
next 4 days, proliferation, hemoglobin accumulation, and cell volume
were monitored. Data obtained at day 3 were expressed as the
E2-induced percent decrease in hemoglobin per cell volume
(Fig. 3C; see above).
The primary cells infected with empty vector already showed an
E2-induced decrease in hemoglobin per cell volume, due to
the endogenous avian ER that is down-regulated during terminal
differentiation only after 2448 h (4). However, constitutive
expression of the wt ER resulted in a significantly stronger
inhibition of differentiation. In agreement with what was found in HD3
cells, constitutive expression of a mutant construct lacking AF-1
resulted in an arrest of differentiation comparable with the activity
of the wt ER (Fig. 3C). The AF-2/AF-1 mutant was inactive,
causing an E2 effect comparable to that of the control
cells. Expression of the AF-2 mutant, however, seemed to even stimulate
erythroid differentiation, which may be due to a dominant negative
effect of the mutant protein on the endogenous ER (Fig. 3C
). In line
with this, the inductive effect of AF-1 was not detected in the HD3
cell line lacking the endogenous ER.
In conclusion, the experiments in the HD3 cells combined with those in the primary cells show that an intact AF-2 domain, but neither the AF-1 domain nor the DBD, is required for the ER to arrest or delay erythroid differentiation.
Functional ER Domains Required for Regulation of Expression of
Erythrocyte-Specific Genes
Next, we wanted to determine which ER domains are required for
regulation of erythrocyte-specific genes. The results of the run-on
assays suggested that this regulation was complex, causing repression
of some erythroid target genes by the E2-activated ER, but
induction of others, e.g. CAII. It was therefore of interest
to determine whether genes found to be repressed by the ER (Fig. 2) in
both HD3 cells and primary cells required an intact AF-2, but not the
AF-1 or the DBD. Furthermore, such studies should reveal whether or not
the domains required for up-regulation of CAII were similar. Previous
experiments had established that the results on repression of Gata-1
and the globin genes obtained with HD3 clones closely matched those
obtained with primary cells (Figs. 2
and 3
). It therefore seemed safe
to use the HD3 clones expressing the various mutant ERs (see above) for
these experiments, since they do not show the variability seen in
infected mass cultures of primary cells (Fig. 2
). These HD3 clones also
have the advantage that they lack the endogenous ER.
In the previous run-on experiments we sought to identify target genes whose transcription could be reduced by the activated ER. Having established Gata-1 and globin genes as target genes for the ER, we employed Northern blots to test all mutant constructs for their effect on the accumulation of these and other erythrocyte genes, turned on at different times after differentiation induction. In these experiments, gene repression by the ER would be indicated as lower levels of accumulated mRNA induced by E2.
HD3 cells expressing the various constructs were induced to
differentiate in the presence of E2 or its antagonist ICI
164,384. RNA was isolated at the onset of differentiation and 1, 2, 4,
and 6 days later. Northern blots containing these RNAs were then probed
for the expression of a set of erythroid-specific genes (Fig. 4). In HD3 cells, as well as in primary
cells, terminal differentiation involves a transient up-regulation of
Gata-1 and SCL1, which is followed by expression of CAII, Band3, and
the globin genes (8). In HD3 cells expressing the wt MOR or
expressing the MOR with a deletion of AF-1, Gata-1 and SCL1 were
transiently up-regulated upon differentiation induction in the presence
of ICI. This transient up-regulation was largely inhibited in the
presence of E2, as can be seen when expression levels are
compared between E2 and ICI at days 1, 2, and 4. The reduction of gene
expression by E2 was similar in clones expressing
wt MOR or the AF-1 deletion mutant (Fig. 4A
and data not
shown; the data for the
AF-1 ER are shown rather than the
wt ER data, since the former are better comparable with the
AF-1/AF-2 double mutant in Fig. 4C
). In contrast, transient
up-regulation of another erythroid transcription factor, ECH, the
chicken homolog of NF-E2 (38), was similar in the presence
of E2 and ICI, thus hardly affected by the
E2-activated ER. As expected from previous results (8, 39),
CAII was up-regulated only at late stages of differentiation in the
presence of ICI 164,384. In contrast, E2 caused an
unexpected, premature activation of CAII.
In line with the biological data, mutation of the DBD did not interfere
at all with the repression of Gata-1 and SCL-1 by E2. Since
this mutant receptor interfered so severely with differentiation,
inhibition of mRNA accumulation by E2 was much stronger
than with the wt ER or the AF-1 ER, even affecting the expression
of ECH. Interestingly, however, the DBD mutation abrogated the
up-regulation of CAII by E2 (Fig. 4B
).
In cells expressing the AF-2 mutant protein, either alone or in
addition to a deletion of AF-1, gene expression was not affected by
E2 as compared with ICI, suggesting that these mutant
receptors were inactive. Both the repression of Gata-1 and SCL-1 by
E2 and the E2-induced up-regulation of CAII
were abrogated (Fig. 4C and data not shown). A small repression by
E2 is visible at day 1, but ethidium bromide staining and
additional experiments strongly suggest that this is due to small RNA
loading differences.
Thus, the AF-2, but not AF-1 or the DBD, is required for
E2-dependent repression of the transcription factors Gata-1
and SCL-1. The same was true for the - and ß-globin genes (data
not shown). However, the premature up-regulation of CAII requires an
intact AF-2 and an intact DBD.
The ER Does Not Inhibit Acceleration of Erythroid Differentiation
by the Thyroid Hormone Receptor TR-
Phenotypically, the arrest of differentiation and the repression
of erythrocyte-specific genes resemble respective functions of the
oncoprotein v-ErbA. However, v-ErbA lacks a functional AF-2 domain and
seems to require specific DNA binding for its function (40) while the
ER requires an intact AF-2 domain but no intact DBD for differentiation
arrest and gene repression (see above). As a part of its multiple
functions (23) v-ErbA suppresses the acceleration of erythroid
differentiation by the endogenous, T3-activated thyroid
hormone receptor (TR-/c-ErbA, (41). It was therefore of interest to
study whether the ER would also interfere with TR-
/c-ErbA function.
This appeared possible since the AF-2 domain is well conserved among
all nuclear hormone receptors (31, 42, 43), and specific putative
coactivators interacting with the AF-2 domain have recently been
described (44, 45, 46, 47, 48, 49, 50, 51, 52).
To test whether retardation of erythroid differentiation by
E2 was dominant over the acceleration of maturation by
thyroid hormone (T3), erythroid progenitors (grown in
TGF and E2) were induced to differentiate in the
presence of either E2, T3, or combinations
thereof. Two days later, hemoglobin content, cell numbers, and cell
volumes were determined. Figure 5A
shows
that T3-enhanced hemoglobin levels normalized to cell
volume, whereas addition of E2 resulted in a retardation of
hemoglobin accumulation. In the presence of E2,
T3 caused a similar or even stronger acceleration of
differentiation, suggesting that the ER is completely unable to
interfere with TR-
/c-ErbA action. This result was obtained both with
normal progenitors expressing the endogenous ER and with cells
constitutively expressing the human ER from a retroviral vector. Thus,
the apparent inability of the ER to interfere with T3
action cannot be due to down-regulation of the ER during
differentiation.
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DISCUSSION |
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Repression of Gene Expression by Nuclear Hormone Receptors
Several nuclear hormone receptors are known to repress
transcription of specific genes. The thyroid hormone receptor (TR) is
present in the nucleus and binds to thyroid hormone response elements
both in the absence and presence of hormone. In the absence of ligand,
however, the TR represses transcription (54, 55, 56), which is mediated by
a transferable repression domain (57). Repression occurs independently
of the conserved AF-2 domain. Instead, the repression domain is located
in the hinge region and binds a corepressor, N-CoR (nuclear receptor
corepressor) or SMRT (silencing mediator for retinoid and thyroid
receptors) (47, 58, 59).
Repression of erythroid differentiation by the ER specifically required
an intact AF-2 domain. Therefore, it is unlikely that a corepressor, as
described to bind the TR and v-ErbA, is involved in repression of
differentiation by the ER. Moreover, repression by v-ErbA or the TR
requires DNA binding to allow interaction of the corepressor with the
target genes. In contrast, repression of erythroid differentiation and
erythroid gene transcription by the ER is independent of DNA
binding.
Repression of gene transcription by steroid hormone receptors is
commonly due to protein-protein interactions between the steroid
hormone receptors and other transcription factors among which AP-1 or
NF-B (reviewed in Ref.60). Since the result of AP-1/receptor
interactions is largely cell type and promoter dependent and since
several domains of the hormone receptors were reported to be involved,
distinct mechanisms may underly repression of gene transcription by the
ER and the GR (31, 32, 33). Mutation alanalysis and the use of steroid
analogs such as Tamoxifen or RU38486, which block transactivation via
the AF-2 domain of the ER and the GR, respectively, showed that
repression is mainly independent of homodimerization, DNA recognition,
and the transactivating AF-2 domain (31, 61, 62). Similarly, both the
ER and the GR were reported to interfere with transcriptional
activation by NF-
B (30, 63), but again independently of an active
AF-2 domain. Since repression of differentiation in avian erythroblasts
required an intact AF-2, the underlying mechanism of repression is most
likely different from the ones described above. In addition,
transcriptional activation of a downstream repressor by the ER is
excluded, since no intact DBD is required.
Instead, the specific requirement for an intact AF-2 suggests that this transactivation domain may bind ("squelch") coactivators that mediate contacts between specific transcription factors and the basic transcription machinery (for reviews see Refs. 64 and 65). As a result, these coactivators would be less available for other transcription factors that enhance erythroid differentiation. The AF-2 domains of the ER and the thyroid/retinoid receptors are highly homologous, and squelching of factors bound to the AF-2 domain have been described between the ER and the TR (43). The E2-bound ER inhibits differentiation markedly, while both retinoids and thyroid hormone enhance erythroid differentiation (41, 66).
Several proteins were recently isolated that interact with the AF-2 domain of E2-activated nuclear hormone receptors, namely RIP (receptor-interacting protein)-140 and RIP-160 (45, 67), ERAP (estrogen receptor-associated protein)-140 and ERAP-160 (44), TIF (transcriptional intermediary factor)-1 (46), a number of isoforms of SRC-1 (steroid receptor coactivator-1) (49, 50), TRIP1 (thyroid hormone receptor interacting protein 1)/SUG-1 (48, 51), TIF2 (52), AIB1 (amplified in breast cancer) (68), and ACTR (69). SRC-1, TIF2, ALB1, and ACTR have been shown to stimulate the transcriptional activity of nuclear receptors and warrant the name coactivators. The roles of the remaining receptor-interacting proteins are still unclear.
We showed that the ER was not effectively competing with T3-activated TR in the erythroid progenitors. In contrast, the activated ER and TR cooperated in the induction of CAII and histone H5 expression in primary cells. This does not necessarily exclude the possibility that common coactivators are used by the TR and the ER. On promoters harboring binding sites for both receptors, the joint binding of certain coactivators may result in synergism rather than competition. Yet, an antagonistic effect may be seen on promoters that harbor a thyroid hormone response element, but not an estrogen response element (ERE).
Does the ER Directly Interact with Gata-1?
Recently it was shown by Blobel et al. (24) that the ER
and Gata-1 form a complex both in vitro and upon
overexpression of the ER in MEL cells. Upon activation of Gata-1,
Gata-1 expression is increased via an autoregulatory loop. Since most
erythroid-specific genes contain Gata-1 response elements in their
promoter, partial inactivation of Gata-1 could be responsible for the
observed repression of differentiation. Both in primary cells and in
the cell line HD3 we observed lower Gata-1 levels in the presence of
E2. Our data showing that the AF-2- but not the DBD domains
of the ER are essential for Gata-1 repression is in line with Blobels
observation that tamoxifen acts as a full antagonist of the ER in
transient transactivation assays using a part of the EKL-F promoter
that has a Gata site but no ERE (24).
Gata-1 has an essential function in erythropoiesis, and total inactivation of the gene in mice is lethal due to the absence of mature erythrocytes in the embryo (25). However, regulation of Gata-1 function during differentiation is unexpectedly complex. In self-renewing, primary avian erythroid cells, Gata-1 was shown to be located mainly in the cytoplasm. Only induction of terminal differentiation by Epo resulted in translocation to the nucleus, DNA binding, and up-regulation of expression (26). Gata-1 controls maturation of erythrocytes, but also survival of erythroid progenitors (70). Blobel and Orkin (71) showed that MEL cells overexpressing the ER become apoptotic in the presence of estrogen. However, we found that activation of the ER is essential to induce self-renewal of avian erythroid progenitors. At the expression levels seen in our cells, the ER does not completely suppress Gata-1 expression, but rather reduces its up-regulation during terminal differentiation. Therefore, the ER may inhibit the up-regulation of late erythroid differentiation genes that are dependent on high levels of Gata-1 in the nucleus, while essential functions of Gata-1 in proliferating progenitors may not be impaired. The apoptotic effects seen by Blobel and Orkin (71) may well be the result of overexpression, since we also observed growth arrest and cell death in addition to a differentiation arrest in clones expressing very high levels of the ER (Ref. 4 and M. von Lindern, and H. Beug, unpublished observations). Another reported effect of E2 during erythroid differentiation, i.e. an E2-induced reduction of erythroid colony number determined in a BFU-E assay (24), is also not necessarily the result of an adverse effect on erythropoiesis. Rather, it could be due to repression of terminal differentiation, which would result in reduced or absent hemoglobinization of the growing colonies and thus in a highly reduced score of red colonies. The reverse, a higher score of colonies because of increased hemoglobinization, has been reported in the presence of retinoic acid, which enhances hemoglobin expression (72). In line with this, we never observed an adverse effect of E2 on cell proliferation in liquid cultures of human erythroid progenitors (M. von Lindern, submitted).
Clearly, not all genes harboring Gata-1 response elements in their promoter are repressed in the presence of E2. The E2-induced genes CAII and histone H5 are also transcriptionally regulated by Gata-1 (73). The affinity of Gata-1 for multiple interaction partners, possibly including the ER, is likely to determine what processes may be affected by activation of the ER.
Both the ER and v-ErbA Repress Erythroid Differentiation
The cooperation between v-ErbB and v-ErbA in avian erythroleukemia
resembles the cooperation between endogenous receptor kinases and
steroids in self-renewal of primary erythroid cells (7). The ER and
the GR were shown to have distinct functions in stimulating
proliferation and inhibiting differentiation, while v-ErbA abrogated
the requirement for both steroid ligands (23). Like the
ligand-activated GR, v-ErbA may enhance the expression of genes
involved in proliferation. The repression of erythroid differentiation
genes, however, seems to parallel the function of the
E2-activated ER.
However, the mechanisms employed by the two nuclear hormone receptors to repress differentiation are clearly distinct. In contrast to the ER, V-ErbA requires its DBD, while its AF-2 domain is deleted (see review in Ref.74). Binding of the co-repressor N-CoR or SMRT is essential for the function of v-ErbA (59). Instead, the requirement for an intact AF-2 domain in the ER suggests "squelching" of co-activators. It is possible that these two mechanisms could lead to the same biological effect and that the two receptors even interact with the same set of target genes. V-ErbA needs DNA binding in order to target the corepressor to a certain promoter. In contrast, the ER may bind coactivators required by other transcription factors that could activate that gene. To deplete these other transcription factors from their coactivators, the ER does not have to bind DNA, but its AF-2 domain must be available.
v-ErbA expressing HD3 cells may constitute a less suitable system,
since v-ErbA and ER action may interfere. The observation that the
effect of E2 on the expression of Gata-2 and histone H5 is
different in HD3 cells compared with primary cells (Fig. 2B)
necessitated further caution in the use of HD3 cells. However, it was
previously shown that in the presence of H7 and upon inactivation of
ts c-ErbB at 42 C, v-ErbA is inactive (75). Recent
experiments, in which v-ErbA is expressed in primary erythroid cells,
convincingly showed that v-ErbA effects are completely dependent on the
presence of activated c-Kit or c-ErbB (23). In the absence of c-Kit or
c-ErbB signaling, v-ErbA-expressing cells were unable to inhibit the
differentiation-associated up-regulation of CAII and histone H5.
As experiments using HD3 cells expressing the ER constructs were
performed under conditions rendering v-ErbA inactive, we are confident
that ER effects can be accurately measured in HD3 cells. The
discrepancies in expression of Gata-2, CAII, and histone H5 in primary
vs. HD3 cells may be due to differences in the stage of
erythroid development, represented by these different cells. When the
effect of E2 on Gata-1 is measured at subsequent stages of
differentiation (Fig. 2B
), the inhibiting effect of the ER is obvious
when Gata-1 expression is increasing 24 h after differentiation
induction. However, once Gata-1 expression is at its maximum 48 h
after differentiation induction, E2 has no significant
effect anymore. HD3 cells express clearly detectable levels of Gata-2,
which can be repressed by E2. In primary SCF cells,
however, Gata-2 expression is hardly detectable in a run-on experiment,
which explains why an inhibitory effect is not detected either.
Similarly, histone H5 is expressed in HD3 cells and can be repressed by
E2, while it is hardly expressed in self-renewing primary
cells, where E2 enhances its expression. The function of
the ER in the development of mature erythroid cells may not simply be
to support self-renewal of immature cells through inhibition of
differentiation, but the ER may have a role in the timing of the
proliferation and differentiation program. Dependent on the stage of
the cells, dependent on other transcription factors present, expression
of some genes is inhibited by a mechanism independent of DNA binding,
while others can be activated by a mechanism requiring binding of the
ER to EREs.
In conclusion, the E2-activated ER has an important, though possibly subtle, role in regulating the balance between erythroid progenitor proliferation and differentiation. Since the ER is down-regulated at the onset of terminal differentiation, it may contribute to a proper timing of terminal differentiation in primary erythroid cells.
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MATERIALS AND METHODS |
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Viruses, Transfections, and Infections
DNA fragments containing the open reading frame of the
wt mouse estrogen receptor (MOR) (77); MOR 121599 (AF-1
deleted); MOR L543A/L544A (mutated AF-2); MOR 121599,L543A/L544A
[AF-1 deleted and AF-2 mutated (34)]; and MOR C241A,C244A [no DNA
binding (35)] were excised from the expression vector pJ3 and inserted
behind the CMV promoter of the avian retroviral expression vector
pCRNCM (36). The retroviral constructs were transfected together with
RCAN helper virus DNA in HD3 cells using diethylaminoethyl-dextran, and
single cell derived colonies were selected in G418-containing methocell
(36). The same retroviral constructs plus RCAN DNA were also
transfected into CEFs using calcium phosphate precipitates, and
G418-resistent cultures were selected (36). To generate mass cultures
of SCF progenitors expressing the various MOR constructs, 40 x
106 Ficoll purified bone marrow cells were cocultivated
with 12 x 106 virus producing CEFs for 1 day in 10
ml modified CFU-E medium supplemented with 100 ng/ml avian recombinant
SCF. Next day nonadherent bone marrow cells were transferred to a fresh
Petri dish and grown for 4 days in the same medium. The cells were kept
at a density of 24 x 106/ml, daily transferred to a
fresh dish to remove adherent cells, and daily provided with fresh
medium and factors.
Measurement of Differentiation Parameters
Cell numbers and cell volume were measured with an electronic
cell counter (CASY-1, Schärfe-System, Reutlingen, Germany).
Aliquots of 12 x 105 cells (50 µl of an average
culture) were transferred to 96-well plates to assay for
[3H]thymidine incorporation, hemoglobin determination, or
cytocentrifugation (see below).
Analysis of Differentiation by Cell Morphology and Staining
Cells were cytocentrifuged and subsequently stained with
histological dyes and neutral benzidine for hemoglobin as described by
Beug et al. (27). Images were taken using a CCD camera
(Photometrics, Tucson, AZ) and a blue filter (480 nm), so that mature
cells (stained yellow to brownish) appear darkly stained. Images were
processed with Adobe Photoshop (Adobe Systems, Inc., Mountain View,
CA).
[3H]Thymidine Incorporation
Aliquots (3 x 50 ml) of the cultures were dispensed into
the wells of a 96-well plate and labeled for 2 h with 0.8 mCi
[3H]thymidine per well ([3H]-TdR, Amersham,
Arlington heights, IL; 30 Ci/mmol). Labeled cells were harvested
onto glass fiber filters using an automated cell harvester (Tomtec,
Orange, CT), and the cell-bound radioactivity was determined in an
96-well scintillation counter (Microbeta 1450; Wallac, Gaithersburg,
MD) essentially as described earlier (37, 78).
Photometric Hemoglobin Assay
Aliquots (3 x 50 ml) of the cultures were removed and
processed for photometric determination of hemoglobin as described
(79).
Northern Blot
RNA was isolated from HD3 cells or erythroid progenitor cells
using the method of Chomczynski and Sacchi (80) with minor
modifications. Cells were lysed in guanidinium isothiocyanate buffer
and NaAc, pH 4.0, was added to 25 mM. The solution was
extracted with H2O-saturated phenol, chloroform, and
isoamylalcohol. RNA was precipitated from the aqueous phase with
isopropanol, all as described. Subsequently, the pellet was dissolved
in 10 mM Tris, pH 71 mM EDTA-0.2% SDS, and
proteinase K was added to 200 µg/ml. After 30 min incubation at 37 C,
the solution was extracted with phenol-chloroform-isoamylalcohol
(25:24:1), pH 7, and precipitated with ethanol. RNA (1020 µg) was
run on a formaldehyde-containing agarose gel and transferred to nylon
filters (Gene Screen, Life Science Products, Boston, MA) using
conventional procedures (81). The cDNA probes used have been described
previously described (8, 82)
Run-on Analysis
HD3 cells or primary erythroid progenitors were differentiated
in medium with freon-stripped serum to reduce serum levels of
estradiol, 2% AS, and insulin as described above. HD3 cells were
differentiated in the presence of 25 µM H7 at 42 C. To
the primary cells 2 ng/ml TGF were added. After differentiation for
the times indicated in the text, the cells were split in two aliquots,
and either estradiol or ICI 164,384 was added to 106
M. At times indicated, the cells were harvested and nuclei
were isolated and processed for run-on analysis as described (40).
Radioactively labeled RNA was hybridized to filters on which total
plasmid DNA containing the probes mentioned above (8) was blotted.
Radioactive signals were measured on a PhosphoImager (Molecular
Dynamics, Sunnyvale, CA) using the Imagequant program (Molecular
Dynamics). Signals were corrected for small differences in the amount
of nuclei used using the transcriptional signals for the constitutively
expressed HEO (HD3 clone 4) or for rRNA.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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M. von Lindern and H. Beug were recipients of funding from the Austrian Research Promotion Fund.
1 Present adress: Department of Biological Chemistry, University of
California, Los Angelos, CA 90095-1737.
2 Imperial Cancer Research Fund, 44 Lincolns Inn Fields, London
WC2A-3PX U.K.
Received for publication April 18, 1997. Revision received November 17, 1997. Accepted for publication November 19, 1997.
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REFERENCES |
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