Retinoid X Receptor and c-erbA/Thyroid Hormone Receptor Regulate Erythroid Cell Growth and Differentiation
Petr Bart
n
k and
Martin Zenke
Max-Delbrück-Center for Molecular Medicine D-13122
Berlin, Germany
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ABSTRACT
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Nuclear receptors are important regulators of
erythroid cell development. Here we investigated the impact of retinoid
X receptor (RXR), retinoic acid receptor (RAR), and of the
c-erbA/thyroid hormone (T3) receptor
(c-erbA/TR) on growth and differentiation of erythroid cells using an
in vitro culture system of stem cell
factor-dependent erythroid progenitors. RXR, RAR, and
c-erbA/TR-specific ligands were found to induce erythroid-specific gene
expression and to accelerate erythroid differentiation in culture, with
T3 being most effective. Furthermore, while
ligand-activated c-erbA/TR accelerated differentiation, unliganded
c-erbA/TR effectively blocked differentiation and supported sustained
progenitor growth in culture. Thus, c-erbA/TR appears to act as a
binary switch affecting erythroid cell fate: unliganded c-erbA/TR
supports growth while ligand-activated c-erbA/TR induces
differentiation. Additionally, to determine the impact of RXR for
erythroid cell development, dominant interfering mutant RXRs, lacking
the transcriptional activator functions AF-1 and AF-2, or AF-2 only, or
the entire DNA-binding domain, were introduced into erythroid
progenitor cells via recombinant retrovirus vectors and analyzed for
RXR-specific effects. It was found that expression of wild-type RXR and
of the RXR mutants devoid of AF-1 and/or AF-2 supported a transient
outgrowth of erythroid cells. In marked contrast, expression of the
dominant interfering
DNA-binding domain RXR, containing a deletion
of the entire DNA-binding domain, was incompatible with erythroid cell
growth in vitro, suggesting a pivotal role of RXR for
erythroid cell development.
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INTRODUCTION
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The c-erbA thyroid hormone (T3) receptor (c-erbA/TR),
retinoic acid (RA) receptor (RAR), and the retinoid X receptor (RXR)
are members of the large family of nuclear receptors (NRs) that
effectively induce gene transcription when activated by cognate ligand
(1, 2, 3, 4, 5, 6). Both c-erbA/TR and RAR require heterodimerization with RXR for
high-affinity binding to their cognate response elements (4, 5).
Furthermore, in addition to ligand-dependent activation of
transcription, selected NRs including the c-erbA/TR and RAR repress
basal transcription in the absence of ligand (see Refs. 5, 6, 7, 8 for
references). Most significantly, both gene activation and repression by
these factors occur through recruitment of specific coactivators and
corepressors, respectively (Refs. 8, 9, 10, 11, 12, 13 and reviews of 57, 14,
15). This way, the overall transcriptional activity of a given gene
depends on multiple signals determined by factors that either directly
bind to DNA and act as activators or repressors, or alternatively, by
ligand-dependent DNA-binding proteins, like the NRs, that in a
ligand-dependent fashion recruit specific coactivators or repressors to
activate or repress transcription. Additionally, more recent studies
demonstrated that NR coactivators and corepressors are components of
multimeric enzymatic complexes, containing histone acetyltransferases
and histone deacetylases, respectively, that might cause chromatin
remodeling at the promoter site (reviewed in Refs. 6, 16).
NRs regulate complex biological processes like cell growth and
differentiation, morphogenesis, and organogenesis by specifically
modulating gene transcription. Additionally, abnormal and/or mutated
NRs were found to lead to aberrant gene expression and oncogenic
transformation (17, 18, 19, 20). The v-erbA oncogene, a retrovirus-transduced
version of c-erbA/TR contained in the avian erythroblastosis virus AEV,
represents such an oncogenic version. V-erbA lacks hormone binding
activity and has suffered multiple point mutations and short N- and
C-terminal deletions (19, 20). Most significantly, v-erbA lost the
ligand-dependent transcriptional activator function AF-2, but still
contains functional silencing domains that bind corepressors (11, 12, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28). In erythroid cells v-erbA effectively suppresses expression of
erythroid specific genes [like band 3 and carbonic anhydrase II
(CAII)] and inhibits terminal differentiation (18, 19, 20, 29, 30, 31). In
marked contrast, ligand-activated c-erbA/TR was found to effectively
induce gene transcription and to accelerate erythroid cell
differentiation (19, 32, 33, 34).
Erythroid progenitor cells can be isolated from chicken and human bone
marrow, human cord blood preparations, or human CD34+
peripheral blood stem cells, and amplified in vitro in the
presence of stem cell factor (SCF) or transforming growth factor type
(TGF
), erythropoietin, and steroid hormones as homogenous
cell populations to large cell numbers (35, 36, 37, 38, 39, 40, 41, 42, 43). Additionally, cells
can be induced to undergo normal terminal differentiation in the
presence of specific differentiation factor, like erythropoietin and
insulin, and yield fully mature erythrocytes. Such in vitro
systems allow the study of NR function and of the activity of
NR-specific ligands within an ongoing differentiation program under
well defined culture conditions. Furthermore, a number of recombinant
retrovirus vectors are available for introduction and expression in
chicken erythroid cells of NRs or mutated versions thereof, to
determine their impact on erythroid cell development (19, 30, 31, 32, 33, 37, 41, 44, 45). These experimental systems therefore complement and extend
previously developed in vitro growth and differentiation
systems based on erythroid progenitor cells that were conditionally
transformed by temperature-sensitive (ts) tyrosine kinase oncogenes
(18, 19, 38, 39).
Previous studies addressed the question of the impact of
hormone-activated c-erbA/TR and RAR for induction of erythroid cell
differentiation (19, 32, 33, 34). It was found that ligand-activated
c-erbA/TR effectively induced erythroid gene transcription (like CAII)
and accelerated erythroid cell differentiation in vitro.
Here we show that additionally unliganded c-erbA/TR affects erythroid
cell development. In the absence of hormone, c-erbA/TR inhibits
differentiation and supports sustained growth of normal untransformed
SCF- dependent erythroid progenitor cells in culture and therefore
exerts an activity similar to v-erbA. Furthermore, we addressed the
question of the impact of RXR, the obligate heterodimeric partner of
c-erbA/TR, for erythroid cell growth and differentiation by ectopic
expression of dominant interfering RXR mutants. Our results support the
idea that RXR is a pivotal factor for erythroid cell growth and
differentiation.
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RESULTS
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Unliganded c-erbA/TR Supports Sustained Growth of Erythroid
Progenitor Cells in Vitro
Ligand-activated c-erbA/TR effectively induces erythroid
cell-specific gene transcription and accelerates erythroid
differentiation in vitro (19, 32, 33, 34). A number of more
recent studies demonstrated that unliganded c-erbA/TR affects gene
transcription by binding transcriptional corepressors, like N-CoR/SMRT
(nuclear receptor corepressor/silencing mediator of retinoic acid and
thyroid hormone receptor), that silence expression of c-erbA/TR
responsive promoters (8, 9, 1113, 28 and reviewed in Refs. 5, 6, 7, 14).
Thus, additionally unliganded c-erbA/TR can be expected to contain a
specific activity and to affect growth and/or differentiation of
erythroid cells. Such an idea is conceivable in light of the fact that
N-CoR/SMRT corepressors are widely expressed in various tissues and
cell lines (14).
To test this hypothesis, SCF-dependent erythroid progenitor cells were
prepared from chicken bone marrow and infected with a c-erbA/TR
containing recombinant retrovirus to augment c-erbA/TR levels in these
cells and thereby achieve a more pronounced biological effect. As a
control, cells were infected with v-erbA virus, or with neo virus only
or left uninfected. Cells were then cultured in medium (that was
extensively deprived of T3 and retinoids; see
Materials and Methods) and SCF, and cell numbers were
determined in regular time intervals (Fig. 1A
). An outgrowth of c-erbA/TR and v-erbA
retrovirus-infected cells was readily obtained. Additionally, by
Western blot analysis we show that cells expressed the c-erbA/TR and
v-erbA proteins, respectively (Fig. 1B
). Cells also expressed the
erythroid-specific transcription factor GATA-1, demonstrating that they
were of erythroid origin. As expected, control cells infected with neo
virus only or left uninfected exhibited a restricted growth potential
and ceased proliferation after 810 days in culture (Fig. 1A
and data
not shown). However, most significantly c-erbA/TR in the absence of
T3 blocked erythroid differentiation and effectively
supported sustained growth of erythroid progenitors (Fig. 1
, A and C)
yielding at day 8 of culture up to 10-fold higher cumulative cell
numbers than control; v-erbA cells grew, as expected, faster and
reached about 30-fold higher cumulative cell numbers than control (day
8). Cell numbers further increased with time in culture resulting at
day 12 in a more than 500-fold (c-erbA/TR) or 3500-fold (v-erbA)
amplification of erythroid progenitor cells over control; c-erbA/TR
cells grew for at least 30 days without signs of senescence similar to
v-erbA control. In addition, td359 v-erbA that contains a specific
point mutation in the N-CoR/SMRT binding site (11, 27, 46) was
essentially inactive in supporting erythroid progenitor cell growth
(Fig. 1A
). Interestingly, unliganded RAR and vitamin D3
receptor, which belong to the same NR family as c-erbA/TR and
also bind corepressors, were deficient in inducing sustained progenitor
cell growth, thus pointing to a specific role of c-erbA/TR in erythroid
cells (P. Bartunek and M. Zenke, unpublished).

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Figure 1. Unliganded c-erbA/TR Supports Sustained Growth of
SCF-Dependent Erythroid Progenitors
A, Growth of c-erbA/TR, wild-type v-erbA, and td359 mutant v-erbA
expressing SCF-dependent erythroid progenitor cells (c-erbA/TR, v-erbA,
and td359, respectively) was analyzed by counting cells in regular time
intervals. Cumulative cell numbers are shown. Control, Cells expressing
neo virus only. B, SCF-dependent erythroid progenitor cells expressing
c-erbA/TR or v-erbA (same cells as in panel A, day 7) were analyzed by
Western blotting using an erbA-specific antibody. GATA-1 protein
expression is also shown. C, Cells at day 10 of culture (c-erbA/TR and
v-erbA cells of panel A, as indicated) were centrifuged onto slides and
stained with neutral benzidine and histological dyes.
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Finally, when aliquots of the cultures were seeded in colony assays,
unliganded c-erbA/TR clearly supported colony formation, and the number
of colonies obtained was, as expected from the results shown above,
lower than that for v-erbA control cells (data not shown). We also note
that in some experiments (like the one shown in Fig. 1A
) growth of
c-erbA/TR cells was somewhat delayed at day 810 of culture as
compared with v-erbA cells, which is probably due to selection of cells
with higher proliferative potential and/or higher c-erbA/TR
levels. Additionally, c-erbA/TR cells were fully competent in
differentiating terminally into mature erythrocytes and differentiation
was, as expected, effectively accelerated by T3 treatment
(data not shown).
In summary, it appears that unliganded c-erbA/TR behaves very similarly
to v-erbA in supporting sustained proliferation of SCF-dependent
erythroid progenitor cells in vitro and was only slightly
less efficient than v-erbA. This might be because the differentiation
block imposed by unliganded c-erbA/TR is less effective as compared
with that of v-erbA (Fig. 1C
; see also Discussion).
Alternatively, some residual T3 that could not be removed
from serum might compromise c-erbA/TR repressor function.
Ligand-Activated RXR, RAR, and c-erbA/TR Accelerate Erythroid Cell
Differentiation and Induce Erythroid Gene Transcription
RXR subserves the role of an obligate heterodimeric partner to
several NRs including c-erbA/TR and RAR and can also function as a
homodimer (4, 5). RXR expression is found in both tyrosine kinase
oncogene-transformed and also normal untransformed erythroid progenitor
cells of chicken (data not shown). Furthermore, while previous studies
demonstrated the importance of c-erbA/TR and RAR for erythroid
cell-specific gene expression and differentiation (19, 32, 33, 34), the
impact of RXR for proper erythroid cell development has so far not been
studied. To address this question, erythroid cells were, first, treated
with specific ligands that activate RXR, RAR, or c-erbA/TR and then
analyzed for ligand-specific effects on differentiation and
erythroid-specific gene expression. Second, since erythroid progenitor
cells express at least two RXR isoforms (RXR
and RXR
, Ref. 47 ; N.
Koritschoner, P. Bartunek, and M. Zenke, in preparation)
dominant-negative RXR versions were introduced and expressed in
erythroid cells via recombinant retroviral vectors to interfere with
RXR function; cells were then analyzed for RXR-specific effects.
First we determined the individual contribution of activated RXR, RAR,
and c-erbA/TR on erythroid cell differentiation by administration of
specific ligands. SCF-dependent erythroid progenitor cells were
prepared and treated with T3, all-trans-RA
(atRA), 9-cis-RA (9cRA), and RXR agonist SR11237, and
various combinations thereof. Such progenitor cells can be induced to
undergo normal terminal differentiation in vitro in the
presence of anemic chicken serum (as a source of erythropoietin) and
insulin (36, 39, 40, 41). After induction of differentiation, cells begin
to accumulate hemoglobin and gradually acquire the morphology of normal
avian erythrocytes (Fig. 2A
). During
differentiation, cells undergo four to five cell divisions which are
accompanied by a decrease in cell size and followed by cell cycle
arrest (Fig. 2B
and data not shown). Under these conditions such
erythroid progenitors differentiate into fully mature erythrocytes
after 4 days in culture (40). Interestingly, administration of
T3, atRA, and 9cRA, or various combinations thereof
effectively accelerated the differentiation process. While untreated
cells at day 2 of differentiation were only partially hemoglobinized,
T3-, atRA-, and 9cRA-treated cells had already further
progressed in differentiation, T3 being the most effective
factor (Fig. 2C
). The RXR agonist SR11237 was as effective as atRA
(data not shown). Additionally, simultaneous administration of
T3 plus atRA, 9cRA, or SR11237 was more efficient in
accelerating differentiation than any treatment with single factor
(Fig. 2C
and data not shown). For example, at day 2 of culture,
T3 plus 9cRA-treated cells had reached a differentiation
stage that was equivalent to day 3 of differentiation of untreated
cells. The ligand-specific effects on differentiation were also evident
when analyzing reduction in cell size in response to factor and loss of
proliferative potential in [3H]thymidine incorporation
assays (Fig. 2D
and data not shown).

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Figure 2. T3, atRA, and 9cRA Accelerate Erythroid
Cell Differentiation
A, Cytospin preparation of SCF-dependent erythroid progenitors before
(d0) and 2 and 3 days after differentiation (2d and 3d, respectively)
were stained with neutral benzidine and histological dyes as in Fig. 1C . B, Cell profiles of the cells shown in panel A demonstrate the
reduction in cell size during differentiation; undifferentiated cells
(dark), differentiated cells at day 2
(gray) and at day 3 (white). C,
SCF-dependent erythroid progenitor cells were differentiated in the
presence of T3, atRA, 9cRA, T3+atRA, or
T3+9cRA or left untreated (no) as indicated. Cytospin
preparations at day 2 of differentiation stained with neutral benzidine
and histological dyes are shown. D, Cell profiles of the cells shown in
panel C demonstrate the effect of T3+9cRA on reduction of
cell size (gray); untreated cells
(white).
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In summary, these results demonstrate that, when individual compounds
were administered, T3 was most effective in accelerating
erythroid cell differentiation as judged 1) by morphological changes
and accumulation of hemoglobin that were assessed histologically and 2)
by reduction in cell size and proliferative potential. Simultaneous
administration of T3 and 9cRA, which activate multiple
receptors, was the most effective factor combination. Essentially the
same result was obtained when clonal cell populations of
ts-v-myb + epidermal growth factor
receptor-transformed erythroblasts (48) were induced to
differentiate by temperature shift to 42 C minus EGF (to inactivate
ts-v-myb and epidermal growth factor receptor,
respectively) in the presence of T3, atRA, 9cRA, or
T3 plus 9cRA (data not shown).
Second, to assess T3 and RA-specific effects on erythroid
cell differentiation quantitatively, RNA was prepared and analyzed for
expression of the erbA target gene CAII (19, 32). SCF-dependent
erythroid progenitor cells were treated with ligand (Fig. 2
, C and D)
and 24 h later analyzed for CAII expression in Northern blots
(Fig. 3
, A and B). CAII mRNA levels were
low in untreated cells and induced by T3, atRA, 9cRA, and
SR11237, with T3 and 9cRA being most effective. Simultaneous treatment
with multiple ligands (to activate multiple receptor complexes) further
increased CAII expression which was highest for T3 plus
atRA and T3 plus 9cRA (
6- to 7.3-fold higher than in
untreated control; Fig. 3
, A and B, and data not shown). Interestingly,
RXR agonist SR11237 exhibited some activity on its own (Fig. 3
, A and
B), which is surprising in light of the concept that RXR complexed with
unliganded c-erbA/TR or RAR should be transcriptionally silent (Ref. 49
and references therein). Some residual T3 that could not be
removed from serum and that synergizes with SR11237 might account for
the activity observed.

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Figure 3. T3, atRA, 9cRA, and SR11237 (SR) induce
CAII Transcription in SCF-Dependent Erythroid Progenitor Cells
A, SCF-dependent erythroid progenitor cells were differentiated in the
presence of various ligands and combinations thereof, as indicated, or
left untreated (no). After 24 h, RNA was isolated and analyzed for
CAII expression by Northern blotting (10 µg total RNA/lane). The blot
was subsequently hybridized with a 18S rRNA-specific probe to
demonstrate equal RNA loading per lane. B, CAII mRNA levels of panel A
were quantified by PhosphorImager and are plotted as fold induction of
control (untreated cells, no) set arbitrarily at 1. Average values of
three independent experiments are shown. Standard deviations are
indicated.
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Finally, the ligand-specific effects measured on CAII transcription
were also observed in undifferentiated proliferating cells.
SCF/TGF
-dependent erythroid progenitor cells were grown in the
presence of SCF and TGF
(36, 37), treated with ligand for 3 h,
and analyzed for specific effects on CAII transcription by Northern
blotting. Again, when single compounds were administered T3
was found to be the most effective factor; simultaneous treatment with
T3 and 9cRA yielded maximum CAII transcription (Fig. 4A
). We also emphasize that in these
experiments activation of endogenous nuclear receptors rapidly induced
transcription of an endogenous gene (CAII) within 3 h of ligand
treatment which, most importantly, occurred uncoupled from
differentiation. While these results are in line with our previous
studies, which identified CAII as a direct target gene of
hormone-activated c-erbA/TR, so far only an erbA-binding site has been
identified within this gene (32); the identity of additional NR
response elements remains to be determined.

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Figure 4. T3, atRA, and 9cRA Induce CAII
Transcription in Proliferating Erythroid Cells
A, Proliferating erythroid progenitor cells (grown in the presence of
SCF, TGF , and steroid hormones; see Materials and
Methods) were treated with T3, atRA, 9cRA, or
T3+9cRA for 3 h or left untreated (no) as indicated.
Total RNA (10 µg per lane) was analyzed for CAII and c-myb
mRNA levels by Northern blot hybridization (CAII and c-myb,
respectively). 18S rRNA stained with methylene blue is shown (18S). B,
HD3 erythroblasts overexpressing c-erbA/TR (32 ) were treated with
T3 for 2, 4, and 6 h or left untreated (0). RNA was
analyzed for CAII and c-myb expression by Northern
blotting. 18S rRNA stained with methylene blue shows equal RNA loading
per lane (18S).
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Interestingly, in such cells there was an inverse relationship between
the induction of CAII transcription and c-myb mRNA
levels. Expression of c-myb was highest in untreated
cells and reduced by 2-fold within 3 h of treatment with
T3, atRA, 9cRA, and T3 plus 9cRA (Fig. 4A
).
Essentially the same result was obtained with HD3 erythroblasts that
ectopically expressed c-erbA/TR (32); since these cells contained
higher c-erbA/TR levels, the magnitude of c-myb
down-regulation after 2, 4, and 6 h T3 treatment was
even more pronounced (Fig. 4B
). This observation is interesting since
in erythroid progenitor cells c-myb expression correlates
with their proliferative potential and declines when cells
differentiate (19, 31, 32, 40, 45). Most importantly, in these
experiments hormone treatment led to c-myb down-regulation
uncoupled from differentiation, suggesting that ligand-activated NRs
might cause and/or contribute to growth arrest by down-regulating
c-myb expression. Whether ligand-activated RXR, RAR, or
c-erbA/TR directly effect c-myb transcription by binding to
c-myb regulatory elements still remains to be determined.
Our results also indicate that ligand-activated NRs are dominant over
c-kit/SCF or TGF
receptor in regulating
c-myb expression. In summary, all these data point to an
important function of ligand-activated RXR, RAR, and c-erbA/TR in
induction of erythroid cell differentiation and gene transcription,
with, among the NRs analyzed, T3-activated c-erbA/TR being
most effective.
Dominant-Negative RXR Receptors Affect Erythroid Cell Growth
While the experiments described above assessed RXR activity on
differentiation, we were also interested in determining whether RXR in
the absence of ligand would, similar to unliganded c-erbA/TR, affect
growth of erythroid progenitor cells. Since erythroid progenitor cells
express at least two RXR isoforms (N. Koritschoner, P. Bartunek, and M.
Zenke, in preparation) we sought to subvert endogenous RXR activity in
such cells by ectopic expression of dominant interfering RXR
versions.
To this end, retroviral vectors were constructed that contained mutated
RXRs devoid of AF-1 and AF-2, or of AF-2 only, or that had the
DNA-binding domain (DBD) deleted (140448 RXR, 1445 RXR and
DBD
RXR, respectively; Fig. 5A
). 1445 RXR
and 140448 RXR were expected to target RXR/RXR homodimers and also
heterodimeric RXR complexes where RXR is the transcriptionally active
partner and to render them transcriptionally inactive. Additionally,
both 1445 RXR and 140448 RXR were still able to form heterodimers
with c-erbA/TR since such heterodimers did respond to T3
and 9cRA in tyrosine kinase oncogene-transformed erythroblasts
expressing these mutants (P. Bartunek and M. Zenke, unpublished).
Furthermore, both RXR mutants effectively heterodim-erized with RAR
and supported RAR-specific transactivation in response to ligand (50).
The
DBD RXR, however, should target both homodimeric and
heterodimeric RXR complexes and render them nonfunctional for DNA
binding and thus should generate a functional knock-out of all RXR
complexes within the respective cell.

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Figure 5. Transcriptional Activity and Expression of
Wild-Type and Mutant RXR
A, Schematic representation of human RXR , human 1445 RXR , mouse
140448 RXR , and human DBD RXR (RXR, 1445, 140448, and
DBD, respectively). The DBD is shown in black. B,
RXR, 1445 RXR, 140448 RXR, and DBD RXR contained in recombinant
retrovirus vectors were analyzed for transcriptional activity in
response to 9cRA in transient cotransfection experiments in CEF. The
luciferase reporter construct contains the rat CRBPII RXRE (RXRE).
Control, No RXR expression plasmid transfected. C, CEF stably
expressing RXR, 1445 RXR, 140448 RXR, and DBD RXR were analyzed
by Western blotting using an RXR-specific antibody as indicated.
Control, CEF containing empty vector only.
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As expected 1445 RXR, 140448 RXR, and
DBD RXR were
transcriptionally inactive in inducing 9cRA-dependent expression of a
RXRE reporter plasmid in transient cotransfection experiments in
chicken embryo fibroblasts (CEF);
DBD RXR also suppressed endogenous
RXR activity (Fig. 5B
). Additionally,
DBD RXR effectively silenced
c-erbA/TR- and T3-dependent transcription from a TRE-driven
reporter construct (data not shown). Activities of 1445 RXR and
140448 RXR were described previously (50, 51). Recombinant retroviral
vectors containing wild-type or mutant RXRs were then transfected into
CEF together with RCAN helper virus DNA and virus producing
neomycin-resistant cells were selected. Cells effectively expressed
wild-type and mutant RXR proteins as determined by Western blot
analysis (Fig. 5C
).
The effect of wild-type and mutant RXRs on the proliferative potential
of erythroid progenitor cells was then tested, using a strategy
employed before for studying c-erbA/TR function (see above).
SCF-dependent erythroid progenitor cells were prepared from chicken
bone marrow and infected with recombinant retroviruses containing
1445 RXR, 140448 RXR,
DBD RXR, and wild-type RXR. As controls,
cells were infected with v-erbA virus or neo control virus only or left
untreated. Growth of retrovirus-infected cells was then monitored by
determining cell numbers in regular time intervals (Fig. 6
, A and C). At day 7 post infection,
expression of the various RXR proteins was readily detectable in
Western blots (Fig. 6B
and data not shown). Control cells infected with
neo virus only or uninfected cells showed the expected growth potential
and ceased proliferation at day 78 of culture (Fig. 6
, A and C, and
data not shown). Additionally, cells containing either wild-type RXR,
1445 RXR, or 140448 RXR behaved very similarly and grew only
marginally faster than control, achieving 2- to 3-fold higher
cumulative cell numbers at day 78 of culture (Fig. 6
, A and C). Thus,
both wild-type and the 1445 or 140448 mutant RXRs were found to
support a transient outgrowth of erythroid cells. In marked contrast,
DBD RXR cells were severely compromised in growth, and there was no
outgrowth of erythroid progenitor cells in such cultures (Fig. 6
, A and
C). In some experiments, nonerythroid cells were eventually obtained
that expressed
DBD RXR and myeloid- specific markers (data not
shown). v-erbA control cells showed, as expected, maximal growth rates
(Fig. 6
, A and C).

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Figure 6. RXR, 1445 RXR, and 140448 RXR Transiently
Enhance Growth Rates of SCF-Dependent Erythroid Progenitor Cells
in Vitro, while DBD RXR is Deleterious to Growth
A, Growth of erythroid progenitor cells expressing RXR, DBD RXR or,
as a positive control, v-erbA was monitored as in Fig. 1A . Control,
Cells containing empty vector only. B, Progenitor cells express RXR and
DBD RXR (day 7) as demonstrated by Western blotting using a
RXR -specific antibody. Control, Progenitor cells containing empty
vector only. C, Growth properties of erythroid progenitor cells
expressing RXR, 1445 RXR, 140448 RXR, DBD RXR, c-erbA/TR, and
v-erbA. Cumulative cell numbers at day 7 of culture (average values of
three independent experiments) are shown. Control, Progenitor cells
containing empty vector only.
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Taken together, these studies demonstrate that a functional knock-out
of RXR is incompatible with erythroid cell growth in vitro.
We emphasize, however, that
DBD RXR is not cytotoxic per
se, since the myeloid cells eventually obtained expressed
DBD
RXR protein (data not shown).
DBD RXR was also efficiently expressed
in CEF (Fig. 5C
).
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DISCUSSION
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The activities of the c-erbA/TR, RAR, and RXR NRs and their
functional domains have been extensively studied in transient
transfection systems and more recently in whole animals by targeted
gene inactivation experiments (1, 2, 3, 4, 5, 6, 7). However, there is clearly a
paucity of in vitro differentiation systems with primary
cells, where the activities of NR-specific ligands and by ectopic
and/or overexpression the impact of individual NRs on growth and
differentiation can be faithfully investigated. In vitro
systems of primary erythroid progenitor cells, like the one described
in this paper, provide such opportunities. Obviously NRs in such
culture systems will affect the expression of multiple genes, both
directly and indirectly, as well as positively and negatively. While it
is well established that ligand-activated c-erbA/TR induces red
cell-specific gene expression and promotes erythroid differentiation,
we demonstrate in this study that additionally unliganded c-erbA/TR
affects erythroid cell development. Furthermore, we have addressed the
question of the impact of RXR, the obligate heterodimeric partner of
c-erbA/TR, on erythroid cell growth and differentiation.
Here we show that unliganded c-erbA/TR represses erythroid cell
differentiation and supports sustained growth of SCF-dependent
erythroid progenitor cells in vitro. This finding extends
our previous observation in tyrosine kinase oncogene-transformed
erythroblasts where overexpressed gag-c-erbA/TR and chimeric
v-erbA/c-erbA/TRs in the absence of ligand suppressed gene expression
and differentiation (19). Thus, c-erbA/TR appears to act as a binary
switch affecting erythroid cell fate: unliganded c-erbA/TR supports
growth while ligand-activated c-erbA/TR induces differentiation. This
dual activity of c-erbA/TR appears to be due to its ability to repress
and activate gene expression, such as CAII, by recruitment of specific
corepressors and coactivators, respectively, in response to hormone
(Fig. 7
; Refs. 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 19).

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|
Figure 7. Schematic Representation of c-erbA/TR and RXR
Action during Erythroid Cell Growth and Differentiation
In the absence of hormone c-erbA/TR blocks differentiation and supports
growth of erythroid progenitors (A) while a hormone-activated c-erbA/TR
promotes differentiation (B). In the absence of hormone, c-erbA/TR
recruits corepressors that silence gene transcription of gene X
(e.g. CAII). Alternatively and depending on the target
gene (Y) corepressor binding might also lead to gene activation (54 ).
Ligand-activated c-erbA/TR induces transcription of gene X
(e.g. CAII) via coactivators and simultaneously might
suppress transcription of other genes Y (e.g.
c-myb).
|
|
Furthermore, in its unliganded state c-erbA/TR exhibits an activity
very similar to its oncogenic variant v-erbA. This observation is most
interesting, since both unliganded c-erbA/TR and v-erbA appear to
recruit a similar or identical set of transcriptional corepressors
through the N-CoR/SMRT-binding site residing in the hinge region of the
erbA molecules (9, 11, 12, 13, 28). Thus, the experiments described in this
paper would be in accord with a model where the v-erbA oncoprotein is
locked in an inactive receptor conformation resembling unliganded
c-erbA/TR and due to its association with corepressor proteins acts as
a constitutive repressor of transcription. Such a concept would also be
in line with studies in which a specific point mutation contained in
the N-CoR/SMRT binding site present in td359 v-erbA, which abrogates
corepressor binding if put in the context of c-erbA/TR, affects the
silencing function of v-erbA and renders it nontransforming (11, 27, 28, 46). There are, however, differences between unliganded c-erbA/TR
and v-erbA, which are also revealed in the present study; v-erbA is,
for example, more effective in supporting sustained progenitor cell
growth than unliganded c-erbA/TR, which might be due to the
specific point mutations both within and outside of the DBD that affect
sequence-specific DNA binding (52, 53). Thus, gene regulation in
erythroid cells by v-erbA might comprise a more broad set of target
genes including those regulated by other NRs and/or transcription
factors. Our recent finding that erythroid cells express a number of
other NRs and orphan receptors, which might be important for red blood
cell development (N. Koritschoner, P. Bartunek, and M. Zenke,
unpublished), would be in line with such an idea. Alternatively, v-erbA
might be more restricted in its DNA-binding specificity than c-erbA/TR,
as suggested before (47, 52, 53), or even have a specific set of
v-erbA-specific sites that are not shared with other transcription
factors. Furthermore, other studies proposed that v-erbA mimics
activities of hormone-activated estrogen receptor and glucocorticoid
receptor (37, 42, 45) indicating that v-erbA might act through
more than one mechanism.
Both c-erbA/TR and v-erbA associate with RXR, an obligate heterodimeric
partner of a number of NRs (4, 5, 47). Erythroid cells express RXR
and RXR
(N. Koritschoner, P. Bartunek, and M. Zenke, in preparation)
and in the present study we have addressed the question of the impact
of RXR for erythroid cell growth and differentiation. It was found that
selective ligands, which activate RXR or the c-erbA/TR and RAR proteins
contained in the heterodimeric RXR/c-erbA/TR and RXR/RAR complex,
accelerate erythroid differentiation and induce gene transcription
(like CAII; Fig. 7
). When individual compounds were administered
T3 was most effective, supporting the view that c-erbA/TR
is one of the major ligand-dependent transcription factors in erythroid
cells. Additionally, at the same time ligand-activated NRs, such as
c-erbA/TR, down-regulate the expression of other genes like
c-myb (Fig. 7
). Whether this involves a direct interaction
of NRs with c-myb regulatory sequences remains to be
determined. Furthermore, GATA-1 and GATA-2, which play decisive roles
in red blood cell development (Refs. 41, 44 and references therein)
are not directly regulated by c-erbA/TR, RAR, and RXR (K. Briegel, P.
Bartunek, and M. Zenke, unpublished).
Finally to determine the impact of RXR on erythroid cell growth,
dominant interfering mutant RXRs lacking the transcriptional activator
functions AF-1 and AF-2, or AF-2 only, or the entire DBD (140448 RXR,
1445 RXR,
DBD RXR, respectively) were ectopically expressed in
erythroid cells. Interestingly, expression of wild-type RXR, 140448
RXR, or 1445 RXR exhibited activities that supported a transient
outgrowth of erythroid cell, probably by enhancing the transcriptional
repressor function of unliganded endogenous c-erbA/TR. In support of
this idea, both wild-type and 140448 and 1445 mutant RXRs contain
heterodimerization and DNA-binding functions (50) and bind c-erbA/TR
and thereby might target such hereodimeric complexes to specific genes
that are silenced. Alternatively, unliganded RXR might have, similar to
unliganded c-erbA/TR, some activity on its own in supporting erythroid
progenitor cell growth, potentially through N-CoR/SRMT binding. The
observation that RXR binds N-CoR/SMRT less efficiently than c-erbA/TR
(11) might explain why only a transient outgrowth is observed. Most
importantly, forced expression of
DBD RXR severely compromised
erythroid cell growth, while its expression was not deleterious for
myeloid cells and CEF.
DBD RXR targets both homodimeric and
heterodimeric RXR complexes and renders them nonfunctional for DNA
binding and thus generates a functional knock-out of all RXR complexes
within the cell. Thus, our studies demonstrate an important role of RXR
expression in erythroid cell development.
 |
MATERIALS AND METHODS
|
---|
Cells and Tissue Culture
SCF and TGF
-dependent erythroid progenitor cells were
prepared from bone marrow of 3- to 10-day-old SPAFAS chicks essentially
as described (36, 39, 41). Briefly, after Ficoll-Hypaque centrifugation
(density 1.077 g/cm3; Eurobio, Paris, France) bone marrow
cells (24 x 106 cells/ml) were cultured in modified
CFU-E medium (18, 39) containing 100 ng/ml avian SCF (55). Tested
batches of FCS (Boehringer Mannheim, Mannheim, Germany), chicken serum
(ChS, Sigma, St. Louis, MO), and detoxified, delipidated BSA (fraction
V, Sigma) were used. Recombinant chicken myelomonocytic growth factor
[cMGF, 40 ng/ml (56, 57)] was present during the initial phase of
culture (days 1 and 2) to induce differentiation and adherence of
macrophages and macrophage precursor cells. At day 2, nonadherent cells
were recovered and grown in CFU-E medium plus 100 ng/ml avian SCF.
Routinely, homogenous cultures of SCF-dependent erythroid progenitor
cells were obtained after 34 days of culture at 37 C in 5%
CO2 atmosphere and high humidity (95%).
To prepare TGF
-dependent progenitors, chicken bone marrow cells were
prepared and cultured in modified CFU-E medium essentially as described
above, but containing 100 ng/ml SCF, 5 ng/ml TGF
(Promega, Madison,
WI), 10-6 M estrogen (Sigma), and
10-6 M dexamethasone (Sigma; Refs. 36, 37, 42). Homogenous populations of TGF
-dependent erythroid progenitor
cells were obtained at days 1214. Cell growth was monitored by
measuring cell numbers in regular time intervals by the CASY-1 Cell
Counter and Analyser System (Schàrfe, Reutlingen, Germany).
For transformation of CEF with recombinant retroviral vectors (see
below), cells were grown in DMEM supplemented with 8% FCS (Boehringer
Mannheim), 2% ChS (Sigma), 10 mM HEPES, pH 7.0, and 100
U/ml penicillin and streptomycin (in the following referred to as
standard growth medium). CEF were transfected with 10 µg retroviral
vector DNA and 1 µg RCAN helper virus DNA as described previously
(41, 44). After G418 selection, these virus-producing CEF were then
used for infection of chicken bone marrow progenitor cells in modified
CFU-E medium [containing FCS and ChS that were extensively deprived of
T3 and retinoids (58, 59)]. Briefly, chicken bone marrow
was prepared (see above) and cocultured with virus-releasing, mitomycin
C-treated CEF in the presence of SCF for 2 days. At day 2, nonadherent
cells infected with v-erbA, td359 v-erbA, c-erbA/TR, RXR, 1445 RXR,
140448 RXR, or
DBD RXR viruses were collected and cultured in
modified CFU-E medium (deprived of T3 and retinoids; see
above) plus 100 ng/ml SCF and 40 ng/ml recombinant human insulin-like
growth factor type 1 (long-R3 IGF-I; Sigma). CEF producing
empty neo vector pSFCV-LE (60) served as control and were used for
infection of bone marrow cells accordingly. Cell growth was assessed by
counting cells in regular time intervals (see above).
Construction of Retroviral Vectors
Retroviral vectors containing RXR, 1445 RXR, 140448 RXR, or
DBD RXR were constructed by subcloning wild-type human RXR
cDNA
(61), human 1445 RXR
(51), mouse 140448 RXR
(dnRXR
AB in
50), and human
DBD RXR
into the EcoRI site of pSFCV-LE
(60); in
DBD RXR amino acid positions 46222 comprising the entire
DBD of human RXR
are deleted. Recombinant retrovirus vectors
containing v-erbA and c-erbA/TR, respectively, were described
previously (23, 32).
Differentiation Assays
To induce erythroid differentiation, cultures of SCF-dependent
erythroid progenitors were incubated in CFU-E medium without chicken
serum (2 x 106 cells/ml; Ref. 18) supplemented with
3% anemic ChS (as a source for erythropoietin) plus 10 ng/ml
recombinant human insulin (Novo Nordisk). Cells were treated with
receptor-specific ligands [1 x 10-7 M
T3, Sigma; 1 x 10-6 M atRA,
Sigma; 1 x 10-6 M 9cRA, Sigma; 1 x
10-6 M SR11237 (62)] that were applied
individually or in various combinations thereof. Erythroid
differentiation was assessed by determining hemoglobin accumulation in
cytospin preparations stained with neutral benzidine and Diff-Quik
[Baxter, Switzerland (39, 44)], by measuring reduction in cell size
with the CASY-1 Cell Counter and Analyser System (40, 41) and loss of
proliferative potential in [3H]thymidine incorporation
assays (39, 44). Photographs were taken with Axiophot II microscope
(Carl Zeiss, Jena, Germany) and Kontron ProgRes 3012 CCD camera
followed by image processing with Adobe Photoshop software.
RNA Analysis and Northern Blotting
Total RNA was prepared and analyzed by Northern blotting as
described previously (41, 44). Blots were stained with methylene blue
to demonstrate equal RNA loading per lane. Probes corresponding to cDNA
of human RXR
(61), chicken v-myb [EcoRI-XbaI
fragment (63)], CAII (19, 32), and to 18S rRNA were prepared by random
priming and used for hybridization. Blots were hybridized, washed, and
exposed to film and/or evaluated by PhosphorImager and ImageQuant
software (Molecular Dynamics, Sunnyvale, CA).
Transient Transfections
Calcium phosphate coprecipitation was used to transiently
transfect CEF with retroviral expression vectors containing RXR, 1445
RXR, 140448 RXR, or
DBD RXR (see above) together with a luciferase
reporter containing the rat CRBPII RXRE
(5'-GCTGTCACAGGTCACAGGTCACAGGTCACAGT TCA-3') between HindIII
and XhoI at position -109 of the thymidine kinase promoter
(32, 61). RSV-ß-gal was cotransfected to normalize for transfection
efficiencies. Transfected cells were grown in standard growth medium
containing FCS and ChS that were extensively deprived of T3
and retinoids (see above). 9cRA (10-6 M) was
added, and 2 days later cell extracts were prepared and analyzed for
luciferase and ß-galactosidase activity as described (32). Luciferase
values were normalized for ß-galactosidase activity.
Western Blot Analysis
Total cell extracts were separated on a 10% SDS-PAGE gel and
blotted onto nitrocellulose membranes (BA85, Schleicher & Schuell,
Dassel, Germany). After blocking membranes overnight in TBS (25
mM Tris-HCl, pH 7.4, 137 mM NaCl, 5
mM KCl, 0.7 mM CaCl2, 0.5
mM MgCl2, 0.6 mM
Na2HPO4) containing 3% BSA, 1 mM
EDTA, 0.05% Tween-20, and two washes with wash buffer (50
mM Tris-HCl, pH 8.0, 0.1 M NaCl, 0.1%
Tween-20), antibody was added and incubated in blocking buffer for
1 h. The primary antibodies used were erbA-specific rabbit
polyclonal antibody (a kind gift of J. Ghysdael, Orsay, France),
RXR
-specific mouse monoclonal antibody 4RX-3A2(DE) (Ref. 64 ; a kind
gift of M.-P. Gaub and P. Chambon, Strasbourg, France), and
GATA-1-specific rabbit polyclonal antipeptide antibody (41).
Subsequently, blots were washed three times with wash buffer and
incubated with the appropriate secondary antibodies [enhanced
chemiluminescence (ECL) kit, Amersham, Little Chalfont, UK] in TBS
supplemented with 5% nonfat milk powder for 45 min. All reactions and
incubations were performed at room temperature. Membranes were washed
another five times with wash buffer, developed in ECL reagents, and
exposed to film.
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank P. Chambon, H. Gronemeyer, and H.
Stunnenberg for recombinant plasmids, RXR-specific antibodies, and
SR11237; J. Ghysdael for anti-erbA-specific antiserum; and P. Chambon,
H. Gronemeyer, and N. Koritschoner for critical review of the
manuscript; G. Blendinger for expert technical assistance; and I.
Gallagher for typing the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Martin Zenke, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, D-13122 Berlin, Germany. E-mail: zenke{at}mdc-berlin.de
Received for publication February 18, 1998.
Revision received May 21, 1998.
Accepted for publication June 15, 1998.
 |
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