(Received for publication, January 11, 1996; and in revised form, February 29, 1996)
From the
We have previously shown that c-Erb A and v-Erb A display a
cell-specific activity in avian myoblasts. In this work, we have
compared the molecular basis of thyroid hormone action in HeLa cells
and in QM7 myoblasts. The transcriptional activity of c-Erb A1
through a palindromic thyroid hormone response element (TRE) was
similar in both cell types. However, c-Erb A did not activate gene
transcription through a direct repeat sequence (DR) 4 TRE in myoblasts
in contrast to results obtained in HeLa cells. Moreover, whereas
retinoic acid receptor-AP-1 interactions were functional in both cell
types, thyroid hormone receptor (T3R)-AP-1 interactions were only
functional in HeLa cells. Using electrophoretic mobility shift assays,
functional tests, and Northern blot experiments, we observed that RXR
isoforms are not expressed in proliferating myoblasts. Expression of
RXR
in these cells did not influence T3R transcriptional activity
through a palindromic TRE but induced such an activity through a DR4
TRE. Moreover, it restored c-Erb A-AP-1 functionality in QM7 myoblasts
and enhanced the myogenic influence of T3. We also observed that c-Jun
overexpression in proliferating QM7 cells restored T3R transcriptional
activity through a DR4 TRE. Therefore, alternative mechanisms are
involved in the induction of T3R transcriptional activity according to
the cell status (proliferation: c-Jun; differentiation: RXR). In
addition we provide the first evidence that RXR is required to allow
inhibition of AP-1 activity by ligand-activated T3R. Lastly, we
demonstrate the importance of RXR in the regulation of myoblast
differentiation by T3.
Thyroid hormones exert critical effects on development, as well
as a variety of metabolic pathways. They bind to thyroid hormone
receptors (T3Rs), ()closely related to steroid hormone
receptors, and control the expression of specific target genes in a
ligand-dependent manner. There are two classes of T3Rs, TR
and
TR
, which are encoded on two separate genes. They bind to specific
regulatory sequences (thyroid hormone response element (TRE)) usually
found in the promoter area of T3 responsive genes. TREs are composed of
hexamer half-sites (AGGTCA) with degeneracy in sequence and
orientation. In the absence of T3, c-Erb A proteins generally repress
basal transcription. In the presence of the hormone, they positively or
negatively modulate transcription. In vitro, T3Rs bind to DNA
as monomer, homodimer, and heterodimer with members of the nuclear
receptor superfamily, such as retinoic acid receptors
(RARs)(1, 2) ,
RXR(3, 4, 5) , vitamin D3
receptor(6) , PPAR(7) , or COUP-Tf(8) . It is
generally assumed that the T3R-RXR heterodimer is a major transcription
complex, at least through a DR4 TRE, whereas the T3R homodimer is
probably not a significant transcription
factor(9, 10) .
In addition, as previously shown for ligand-activated glucocorticoid receptors (11) and retinoic acid receptors(12) , liganded T3Rs repress AP-1 activity(13, 14) . Conversely, stimulation of AP-1 activity by TPA or c-Jun overexpression inhibits T3Rs transcriptional activity(14) . It was proposed that a direct physical interaction between the AP-1 complex and T3Rs leads to a subsequent loss of activity through TRE or AP-1 responsive elements(14, 15) . The involvement of a third partner in stabilization of the T3R-AP-1 complex was also postulated(13, 16) . Therefore, although liganded c-Erb A proteins directly regulate T3 target gene expression, they repress transcription of AP-1-regulated genes. This dual pathway might regulate the expression of two different sets of genes respectively involved in cell proliferation and differentiation(13) .
We
have previously shown that T3 stimulation of quail myoblast
differentiation was enhanced by T3R
overexpression(17, 18, 19) . In this work, we
compared two T3-regulated mechanisms in QM7 myoblasts and in HeLa
cells. We report that QM7 cells do not significantly express RXR,
leading to a cell-specific activity of T3R
. In contrast to its
action in HeLa cells, c-Erb A
1 does not inhibit AP-1 activity in
QM7 cells, but RXR transfection induced functionality of T3R-AP-1
interactions. Last, whereas RXR does not affect T3R transcriptional
activity through a TRE
, it induces such an activity
through a DR4 TRE. In agreement with these data, RXR expression
potentiates the T3 stimulation of myoblast differentiation. These
findings suggest a crucial role of RXR for the regulation of cell
differentiation through interactions with T3R and AP-1 activity.
In both cell types,
co-transfection of the pRS c-Erb A1 expression vector induced a
T3-dependent transcriptional activation of the
TRE
-glo-CAT construct (up to 10-fold, p <
0.001; Fig. 1, A and B). Therefore T3R
displayed a similar transcriptional activity in QM7 myoblasts and in
HeLa cells through a TRE
.
Figure 1:
Differences in c-Erb A1
transcriptional activity assessed in HeLa cells and in QM7 myoblasts
when using a TRE
or a DR4 TRE. Cells were transfected
with 1 µg/dish of the TRE
-glo-CAT (A and B) or the DR4-tk-CAT reporter gene (C and D)
together with 2 µg of c-Erb A
1 expression vector. T3
(10
M) was added in the culture medium when
indicated. The results are normalized to
-galactosidase activities
and expressed as percentages of control cells CAT activity. A and C, HeLa cells. B and D, QM7
myoblasts. Three independent transfection experiments were performed in
each case.
Using the DR4-tk-CAT
reporter gene, we observed striking differences. A significant
induction of CAT activity by liganded c-Erb A1 was recorded in
HeLa cells (3-fold induction, p < 0.005; Fig. 1C). In QM7 myoblasts, T3R
significantly
decreased basal CAT activity in the absence of T3 (p <
0.025). The addition of the hormone abrogated this inhibition but did
not induce any stimulation of CAT activity (Fig. 1D).
Therefore, as expected, the liganded T3R was able to stimulate the
expression of genes under the control of a DR4 TRE in HeLa cells.
However, in QM7 myoblasts, T3R
acts only as a transcriptional
repressor of gene transcription through a DR4 TRE, and T3 abrogates
this activity.
As expected, T3R and RAR
activated by their cognate
ligands (10
M T3; 10
M RA) strongly inhibited the TPA-stimulated AP-1
activity in HeLa cells (in both cases: -80%, p <
0.005; Fig. 2A). However, liganded T3R
did not
repress the TPA-induced AP-1 activity in QM7 cells (Fig. 2B) or in secondary quail myoblasts (Fig. 2C). Similar results were obtained when AP-1
activity was stimulated by chicken c-Jun overexpression using the
-73 col-CAT reporter gene in QM7 cells (Fig. 2D).
Figure 2:
In contrast to RAR , ligand-activated
c-Erb A
1 does not repress AP-1 activity in quail myoblasts. Cells
were transfected with 1 µg/dish of the (AP-1)
-tk-CAT (A, B, C, and the left panel of D) or the -reporter gene (right panel of D) together with 2 µg of c-Erb A
1 expression vector
or 2 µg of RAR
expression vector. TPA (50 ng/ml), T3
(10
M), or retinoic acid (RA,
10
M) were added in the culture medium when
indicated. Stimulation of AP-1 activity was also achieved by
transfection of 2 µg of c-Jun expression vector (C). The
results are normalized to
-galactosidase activities and expressed
as percentages of TPA- or c-Jun-stimulated activity in control cells. A, HeLa cells. B, C, and D, QM7
myoblasts. Three independent transfection experiments were performed in
each case.
However, in QM7 myoblasts the
ligand-dependent repression by endogenous or exogenous RAR occurred as
observed in HeLa cells (Fig. 2, A and B).
Therefore, activated T3R and RAR
do not display a similar
activity in myoblasts, whereas no difference could be noted in HeLa
cells. These results suggest that interactions of these receptors with
the AP-1 complex might not be mediated through strictly identical
pathways.
Figure 3:
Stimulation of AP-1 activity differently
affects c-Erb A1 transcriptional activity according to the cell
type and the studied TRE. Cells were transfected with 1 µg/dish of
the TRE
-glo-CAT (A and B) or the
DR4-tk-CAT reporter gene (C and D) together with 2
µg of c-Erb A
1 expression vector. AP-1 activity was
stimulated by TPA treatment (50 ng/ml, A, B, C, and D) or transfection of 2 µg of c-Jun
expression vector (D). T3 (10
M)
was added in the culture medium when indicated. The results are
normalized to
-galactosidase activities and expressed as
percentages of control cells CAT activity. A and C,
HeLa cells. B and D, QM7 myoblasts. Three independent
transfection experiments were performed in each
case.
More
striking is the observation that in QM7 myoblasts, TPA induced a strong
transcriptional activity of ligand-activated T3R (7-fold
induction, p < 0.001) when using a DR4-tk-CAT gene reporter (Fig. 3D). Similar results were obtained using
c-jun transfection (Fig. 3D). In contrast,
using the same reporter gene, TPA treatment inhibited T3R
transcriptional activity in HeLa cells, with an efficiency similar to
that recorded using a TRE
-glo-CAT gene reporter (Fig. 3C, p < 0.005).
These data suggest that TPA influence upon T3R transcriptional activity depends on the TRE and on the cell type. In particular, an elevated AP-1 activity induces transcriptional functionality of liganded T3R through a DR4 TRE in QM7 myoblasts.
When T3R
overexpressing HeLa cell extracts were incubated with a direct repeat
sequence (DR4) probe, three complexes displaying different binding
intensities were detected with complex II on the brink of detection (Fig. 4A, lanes 1 and 3). Using a
TRE probe (Fig. 4B, lanes 3 and 5), no significant differences were observed in the binding
ability of these three complexes. Using extracts of T3R-overexpressing
QM7 myoblasts and a DR4 probe, only two fast mobility complexes (II and
III) were observed (Fig. 4A, lanes 2 and 4). However, binding of complex III was strongly reduced when
using a TRE
probe (Fig. 4B, lanes 2 and 4).
Figure 4:
C-Erb
A 1 binding on DR4 or palindromic TREs differs in quail myoblasts
and in HeLa cells. Gel retardation assays were performed using a DR4 (A) or a palindromic TRE (B). 10 µg of protein of
whole extracts of c-Erb A- or poly(A)-expressing cells were used in
each case. A, DR4 TRE. Lanes 1 and 3, c-Erb
A
1 HeLa extracts. Lanes 2 and 4, c-Erb A
1
QM7 extracts. B, palindromic TRE. Lane 1, poly(A)
HeLa extracts. Lanes 2 and 4, c-Erb A
1 QM7
extracts. Lanes 3 and 5, c-Erb A
1 HeLa
extracts. Lane 6, poly(A) QM7
extracts.
When a 5-fold protein excess of control HeLa
cellular extract was mixed with T3R-expressing QM7 cellular extract,
binding of the receptor as three complexes was observed (Fig. 5). Formation of complex I was associated with a decrease
of complexes II and III (Fig. 5, lane 2). Preincubation
with an antibody raised against c-Erb A1 confirmed that T3R was a
component of these three complexes (Fig. 5, lane 6). In
agreement with this last observation, an excess of cold DR4 was found
to compete binding of complexes I, II, and III to the probe (Fig. 5, lane 5). Interestingly, binding of complex I
was also efficiently competed by molar excess of cold DR5 and DR1
probes (Fig. 5, lanes 3 and 4). These data
suggest that a T3R partner able to bind to DR5 and DR1 responsive
elements is expressed in HeLa but not in QM7 cells.
Figure 5:
HeLa complementation restores a c-Erb A
heterodimeric binding in HeLa cells competed by an excess of cold DR1
and DR5 probes. Gel retardation assays were performed using a
TRE probe as described in the legend to Fig. 4. Lane 1, c-Erb A
1 QM7 extracts. Lane 2, c-Erb A
1 QM7 extracts (1 vol) + nontransfected HeLa extracts (5
vol). Lane 3, c-Erb A
1 QM7 extracts (1 vol) +
nontransfected HeLa extracts (5 vol) + 200 ng cold DR1
oligonucleotide. Lane 4, c-Erb A
1 QM7 extracts (1 vol)
+ nontransfected HeLa extracts (5 vol) + 200 ng cold DR5
oligonucleotide. Lane 5, c-Erb A
1 QM7 extracts (1 vol)
+ nontransfected HeLa extracts (5 vol) + 200 ng cold DR4
oligonucleotide. Lane 6, c-Erb A
1 QM7 extracts (1 vol)
+ nontransfected HeLa extracts (5 vol) + c-Erb A
1
antiserum used at a final 1:5 dilution. Ab,
antibody.
When RXR
and T3R
were co-expressed in QM7 myoblasts, a third complex was
detected (Fig. 6, lane 7). It displayed the same
mobility as complex I in QM7 expressing T3R extracts mixed with control
extracts of HeLa cells (Fig. 6, lanes 7 and 8). In addition, preincubation of cell extracts with an
antibody raised against all RXR isoforms suppressed the slow mobility
signal (complex I) both in T3R-RXR-expressing myoblasts and in
T3R-expressing HeLa cells (Fig. 6, lanes 4 and 5). In addition, binding of complexes II and III was not
affected in these two cell types, thus demonstrating absence of RXR in
these fast mobility complexes.
Figure 6:
Evidence that RXR is the
heterodimerization partner of c-Erb A in HeLa cells. Gel retardation
assays were performed using a TRE probe as described in
the legend to Fig. 4. Lane 1, extracts of c-Erb A
1 overexpressing QM7 + c-Erb A
1 antiserum at a final
1:5 dilution. Lane 2, extracts of c-Erb A
1 and RXR
overexpressing QM7 + c-Erb A
1 antiserum used at a final 1:5
dilution. Lane 3, c-Erb A
1 QM7 extracts (1 vol) +
nontransfected HeLa extracts (5 vol) and c-Erb A
1 antiserum used
at a final 1:5 dilution. Lane 4, c-Erb A
1 QM7 extracts
+ RXR antiserum used at a final 1:5 dilution. Lane 5,
c-Erb A
1 HeLa extracts + RXR antiserum used at a final 1:5
dilution. Lane 6, c-Erb A
1 QM7 extracts. Lane
7, extracts of c-Erb A
1- and RXR
-overexpressing QM7
cells. Lane 8, c-Erb A
1 QM7 extracts (1 vol) +
nontransfected HeLa extracts (5 vol). Ab,
antibody.
Because only two RXR isoforms (
and
) are characterized in avian
species(24, 30) , expression of theses receptors was
assessed by Northern blot in proliferative QM7 myoblasts. Whereas the
two transcripts were easily detected in 4.5- and 5.5-day-old quail
embryo in agreement with previous data(24, 30) , we
failed to detect them in QM7 extracts (Fig. 7), in agreement
with our EMSA data.
Figure 7:
RXR isoforms are not expressed in
proliferating QM7 myoblasts. RXR (5 kilobases) and
(2.5
kilobases) RNAs were detected by Northern blot analysis using
homologous probe in chicken embryos but not in QM7 cells. Lane
1, total RNA from stage 25 chicken embryos (4.5 days in
ovo). Lane 2, total RNA from stage 27 chicken embryos
(5.5 days in ovo). Lane 3, total RNA from QM7 cells
48 h after seeding (T3 depleted culture medium). Lane 4, total
RNA from QM7 cells 48 h after seeding (0.6 nM T3 supplemented
medium). 15 and 30 µg were loaded in lanes 1 and 2 and in lanes 3 and 4,
respectively.
Figure 8:
RXR expression affects c-Erb A
transcriptional activity when using a DR4 TRE but not a TRE
construct. Cells were transfected with 1 µg/dish of the
TRE
-glo-CAT (A and B) or the DR4-tk-CAT
reporter gene (C and D) together with 2 µg of
c-Erb A
1 expression vector. A and C, HeLa cells
were transfected with 2 µg of RXR
expression vector when
indicated. B and D, QM7 myoblasts were stably
transfected using the RXR
(+RXR) or the corresponding
``empty'' expression vector (control cells). E, RXR
expression was assessed by comparison of the activation of a
DR1-tk-CAT reporter gene in control and RXR-expressing myoblasts (1
µg of reporter gene was transiently transfected in cells grown
without or with 9-cis-RA). T3 (10
M) was
added in the culture medium when indicated. The results are normalized
to
-galactosidase activities and expressed as percentages of
control cells CAT activity. Three (A, B, C,
and D) or five (E) independent transfection
experiments were performed.
QM7 myoblasts were
transfected with pRS c-RXR and pSV2-neo
expression
plasmids. Control myoblasts were obtained by co-transfecting pRS
poly(A) vector with pSV2-neo
plasmid. Stable expression of
RXR
was tested using a DR1 CAT reporter gene (Fig. 8E). Using a TRE
-glo-CAT gene
reporter, we observed that RXR
expression did not significantly
influence the liganded T3R
transcriptional activity (Fig. 8B) but restored a transcriptional activity of
liganded c-Erb A through a DR4 TRE in QM7 myoblasts (about 4-fold
induction, p < 0.005; Fig. 8D). Therefore,
this set of data brings evidence that T3R could be fully active through
a synthetic TRE
in the absence of RXR, whereas the
T3R-RXR heterodimer is a major transcription complex on a DR4 TRE.
Figure 9:
RXR expression induces functionality
of AP-1-c-Erb A interactions in QM7 myoblasts. QM7 myoblasts were
stably transfected using the RXR
(+RXR) or the corresponding
empty expression vector (control cells). Thereafter, cells were
transfected with 1 µg/dish of the
[AP-1]
-tk-CAT (A),
TRE
-glo-CAT (B), or DR4-tk-CAT reporter gene (C) together with 2 µg of c-Erb A
1 expression vector
when indicated. TPA (50 ng/ml) and/or T3 (10
M) were added in the culture medium when indicated. A, ligand-activated c-Erb A represses AP-1 activity in RXR
-expressing myoblasts. B and C, stimulation of
AP-1 activity inhibits c-Erb A transcriptional activity in RXR
-expressing myoblasts when using a TRE
glo-CAT (B) or a DR4-tk-CAT reporter gene (C). Three
independent experiments were performed. Similar results were obtained
when RXR
expression was obtained by transient transfections
experiments.
Conversely, we demonstrated that
RXR expression restored the inhibition of T3R transcriptional
activity by AP-1 in QM7 myoblasts, whatever the TRE. Using a
TRE
, TPA stimulation of AP-1 activity strongly inhibited
CAT induction by liganded T3R in RXR
-expressing myoblasts
(-85%, p < 0.005; Fig. 9B). Similar
data were obtained using a DR4 TRE (-55%, p < 0.025; Fig. 9C), but a significant induction of CAT activity
remained (3-fold induction, p < 0.01; Fig. 9C), suggesting that RXR expression partly
preserved the transcriptional T3R activity through a DR4 TRE, even when
AP-1 activity was elevated.
Figure 10:
RXR expression strongly enhances
the stimulation of QM7 myoblast differentiation induced by T3 in
control or c-Erb A
1 overexpressing cells. Connectin expression
was assessed by cytoimmunofluorescence 2 days after the induction of
differentiation with an antibody raised against connectin and a
fluorescein-conjugated antibody raised against mouse immunoglobulins
(
100). When indicated, 0.6 nM T3 was added in the
culture medium. A, control cells. B, control cells
+ T3. C, T3R-expressing cells. D, T3R-expressing
cells + T3. E, RXR
-expressing cells. F,
RXR
-expressing cells + T3. G, T3R + RXR
-expressing cells. H, T3R + RXR
-expressing
cells + T3. These microphotographs are representative of three
independent experiments.
Because an excess of cold DR1 or DR5
probes efficiently competed complex I binding to a TRE,
RXR, PPAR, or COUP-Tf I and II, which are able to bind to these
response
elements(31, 32, 33, 34, 35, 36, 37, 38, 39) ,
could be possible partners of T3R
1 in HeLa cells. In EMSA
experiments, using 4RX-1D12 antibody reacting against all RXR
isoforms(40) , we identified RXR as the partner of T3R in
complex I of HeLa cells. Furthermore, RXR expression induced formation
of an additional complex in QM7 myoblasts with the same mobility as
complex I.
These results were in line with some previous data suggesting that RXR isoforms are not expressed in proliferating myoblasts(41) . The present study also clearly indicates that RXR isoforms are weakly or not expressed in proliferative quail myoblasts: (i) transcriptional activity of 9-cis-RA from a DR1-tk-CAT reporter gene is not significant (Fig. 7); (ii) we failed to detect any T3R-RXR heterodimers in avian myoblasts (Fig. 6); and (iii) we failed to detect RXR mRNAs in our cell extracts.
However, T3-activated c-Erb A is devoided of transcriptional activity through a DR4 TRE in the absence of RXR: (i) T3R represses the basal expression level in absence of T3; (ii) the addition of the hormone abrogates this inhibition but does not stimulate transcription; (iii) in cells expressing RXR such as HeLa cells, liganded T3R displays a significant transcriptional activity through a DR4 TRE; and (iv) a similar activity is restored in myoblasts after RXR expression. Therefore these data clearly indicate that the T3R-RXR heterodimer is a major transcription complex on a DR4 TRE.
We report a striking exception to this rule. In QM7 cells, TPA stimulation or c-Jun overexpression induces a strong transcriptional activity to liganded T3R through a DR4 TRE. Disruption by T3 of the c-Erb A homodimer binding to a DR4 TRE (10, 42) probably explains the inability of T3R to increase gene transcription by itself. Therefore, it could be proposed that c-Jun acts by stabilizating homodimer binding to DNA. In addition, according to the hypothesis of Pfahl(16) , Jun could function as a bridging molecule between c-Erb A and the transcriptional machinery. However, because we have not observed a similar T3R-Jun interaction in HeLa cells, a muscle-specific protein could be involved in this bridging, as already proposed ( (16) and Fig. 11).
Figure 11: Hypothetic scheme involving AP-1 and RXR in the regulation of myoblast differentiation by T3. This hypothesis only considers results obtained using a DR4 TRE, closely related to natural TREs. It is based on the original proposition of Pfahl(16) . A, in proliferating myoblasts, a high AP-1 activity is recorded, thus repressing differentiation. In these conditions, according to the hypothesis of this study, c-Jun could function as a bridging molecule between T3R bound to a DR4 TRE and the transcriptional machinery. However, our data demonstrate that c-Jun induces a c-Erb A transcriptional activity in myoblasts but not in HeLa cells. These data suggest that another molecule, which may differ from cell type to cell type, is necessary to stabilize Jun binding to the receptor, in agreement with the proposition of Pfahl(16) . We propose that a muscle-specific factor (MSF) expressed in proliferative myoblasts plays this role. Such a mechanism could induce the activation of a set of genes involved in myogenic differentiation by T3. B, RXR expression induces formation of a T3R-AP-1 inactive complex either through DR4 or TPA responsive elements. Molecules involved in the bridging between the T3R homodimer and the transcriptional machinery could be directly released by RXR (inducing inactivity of the transcriptional complex) or indirectly (as a consequence of a disruption of the interaction of the transcription complex with DNA induced by RXR). Consequently, AP-1 activity is strongly inhibited, thus derepressing terminal differentiation. In these conditions, T3 responsive proteins synthetized in A could induce terminal differentiation. In differentiated cells, AP-1 activity remains depressed; T3-regulated gene expression is activated by RXR/T3R heterodimer. In this scheme, the T3 transcriptional pathway is always functional in relation to c-Jun (proliferation) or RXR (differentiation) expression. In conjunction with T3R (but probably with other nuclear receptors such as RARs), RXR represses AP-1 activity and overcomes the differentiation block.
Furthermore, we have obtained original data
establishing a major role of RXR in of T3R-AP-1 functionality. In
contrast to RAR , T3R does not repress AP-1 activity in quail
myoblasts. In addition, TPA stimulation of endogenous AP-1 activity
does not inhibit the ligand-dependent transcriptional activity of T3R
in these cells. Interestingly, in contrast to COUP-Tf I, PPAR
, or
RAR
, RXR
expression restored functionality of T3R-AP-1
interactions in quail myoblasts. Similar data were obtained with RXR
expression, thus suggesting that such an activity is specific of
RXR isoforms. Functional interactions between RXR and T3Rs are well
documented. However, until this work, it was assumed that RXR
interaction with c-Erb A only affected the transcriptional activity of
T3R. Our data extend the importance of this interaction to the
functionality of T3R-AP-1 interactions. Consequently, they suggest that
RXR affects all pathways of T3 action.
The physiological relevance of these data is well illustrated by the observation that RXR strongly potentiates the stimulation of differentiation induced by T3 in control or in c-Erb A overexpressing myoblasts. Because myoblast withdrawal from the cell cycle is the first event of terminal differentiation, an anticipated differentiation would probably result in a reduced number of muscle fibers and consequently an important impairment of muscle development. As previously observed, RXR is not expressed before induction of terminal differentiation in murine myoblasts(33) . Our data also indicate that in QM7 cells, RXR is not significantly expressed in proliferating cells. Therefore, RXR absence during the earliest steps of muscle development could provide a protection against such a precocious differentiation.
T3 regulates the expression of a large set of genes, involved in developmental processes and in cell metabolism regulation. A lack of RXR expression in proliferating myoblasts inducing a T3R transcriptional inefficiency would probably severely impair cell metabolism and viability if we consider that direct repeats are the most frequently described TREs. Interestingly, AP-1 activity could restore the T3R transcriptional activity. Therefore, our data indicate that alternative mechanisms are involved in the preservation of T3R activity: first, via AP-1 activity, which also inhibits differentiation, and second via RXR expression, which also inhibits AP-1 activity through a T3R-related mechanism and probably derepresses differentiation (Fig. 11).