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Principal
results and work in progress
MOLECULAR BASIS OF THE TRIIODOTHYRONINE MYOGENIC INFLUENCE.
IDENTIFICATION AND PHYSIOLOGICAL IMPORTANCE OF THE
DIRECT T3 MITOCHONDRIAL PATHWAY.
Molecular
basis of the triiodothyronine myogenic influence.
[Results] [Work in progress]
Results.
The importance
of thyroid hormone for muscle development is well established in vivo
. This hormone
stimulates the growth of this tissue by increasing the number and the
diameter of muscle fibres. In addition, it influences myosin heavy chain a and b expression
at the transcriptional level in cardiac muscle or indirectly in skeletal muscle.
As this hormone also increases mitochondriogenesis and mitochondrial activity,
it is also probably involved in the acquisition of metabolic features of muscle
fibres. However, at the beginning of our work, the molecular basis of this
myogenic influence was very poorly known.
|
QM7 Myoblasts. |
Our
experimental work has been performed using secondary cultures of embryonic
quail myoblasts or the QM7 myoblast line established from the same avian
specie. We found that T3 accelerates myoblast withdrawal from the cell cycle,
a crucial event for the induction of
differentiation, and stimulates their terminal differentiation. In
addition, we have demonstrated that inhibition of TPA inducible AP-1 activity
(c-Jun/c-Fos or c-Jun/Fra2 transcriptional activity) is an important
mechanism involved in the T3 myogenic influence. |
|
|
Secondary quail myoblast
culture: fusion of mononucleated cells. |
same
microscopic field, connectin detection (muscle specific protein). |
Interestingly, this
mechanism is only functional at a particular stage of myoblast progression in
the differentiation programme; i) in the T3 presence, the T3 nuclear receptor
c-Erb A a 1
represses TPA inducible AP-1 activity only after expression of RXR, its
heterodimerization partner; this event occurs at the induction of terminal
differentiation; ii) simultaneously, a transient rise in cell cAMP amounts,
potentiated by T3, increases the expression of the cAMP dependent transcription
factor CREB, which represses also AP-1 activity. These data led us to identify
the myogenic pathways triggered by this repression of TPA inducible AP-1
activity.
We have established that expression of btg1,
a gene encoding an antiproliferative protein, is strongly repressed by AP-1 activity
at the transcriptional level. Consequently AP-1 inhibition induces BTG1 protein
synthesis at the onset of myoblast differentiation. In addition, this protein
is submitted to a cellular traffic, and T3 potentiates the extent of its
nuclear localization. Furthermore, stable BTG1 overexpression in avian
myoblasts mimics the myogenic T3 influence, according to a pathway initiated at
the nuclear level. Therefore, BTG1 is both an indirect (through AP-1 activity
inhibition) and a direct T3 target (nuclear localization), involved in the
myogenic activity of this hormone.
We have recently elucidated the basis of the
myogenic influence of BTG1. We have shown that this protein is a
transcriptional coactivator of T3 and retinoic acid nuclear receptors, already
known to induce stimulation of myoblast differentiation. More interestingly, it
also plays the same function relatively to myogenic factors Myf5, MyoD and
Myogenin. As BTG1 is expressed at the onset of terminal differentiation, BTG1
has to be considered as a major myogenic protein. This conclusion is in
agreement with the observation that expression of a BTG1 mRNA antisens
considerably alters myoblast differentiation. In addition, although resulting
only from in vitro studies,
these data are well supported by the pattern of BTG1 expression studied by in
situ hybridisation during chicken
embryogenesis.
|
Furthermore, a diversity of AP-1 complexes
occurs in myoblasts, resulting from a competition between c-Fos, Fra2 and
ATF2 for binding to their common dimerisation partner, c-Jun. We found that
inhibition of TPA inducible AP-1 activity occurring at the onset of
differentiation favours froamtion of c-Jun/ATF2 complexes relatively to
c-Jun/c-Fos and c-Jun/Fra2 complexes. Interestingly, whereas c-Jun/c-Fos and
c-Jun/Fra2 complexes fully abrogates myoblast withdrawal from the cell cycle
and terminal differentiation, the c-Jun/ATF2 complex strongly potentiates
these two events, through a stimulation of myogenin expression. |
|
|
This
set of results, summarized in the figure on the left, underlines that T3 ant its nuclear
receptors are not able to inhibit TPA inducible AP-1 activity by themselves.
Therefore, the stimulation of terminal differentiation by T3 is only
effective at a particular step of the myogenic programme. Consequently, such
a mechanism conferes a protection against a precocious induction of myoblast
differentiation able to induce a significant reduction in the duration of the
proliferation period, and consequently an alteration of muscle development. |
In parallel to this
work, we have established a surprising myogenic activity of the v-ErbA
oncogene, viral homologue of the c-ErbA a 1 gene encoding a T3 nuclear
receptor. Whereas this oncogene is generally considered as a potent repressor
of cell differentiation, we have shown that it efficiently potentiates avian
myoblast differentiation. A study of the mechanisms involved in this myogenic
influence led to the observation that, in RXR absence, v-ErbA is not an
antagonist of T3 nuclear receptors, but, in contrast, that it induces their
activity in the absence of T3. These myogenic and transcriptional activities
disappear in myoblasts stably expressing RXR.
[Up]
Work in progress.
Our present studies
are focused on the following aspects :
-
Since 1996, we have shown that T3 nuclear receptors transcriptional activity is
mediated by different complexes in proliferating (interaction of T3 receptors
with a c-Jun/MyoD complex) or in differentiating myoblasts (T3 receptor/RXR).
Moreover, as the responsive sequences recognized by these two complexes are not
identical, we suggest that the set of T3 target genes differs during
proliferation and differentiation. The present work is performed with the aim
to characterize, using microarrays, the set of genes differentially regulated
by T3 during these two periods.
-In order to better know
other developmental events regulated by T3 during the embryonic life, the
hormone influence on earlier myogenic processes will be investigated by
studying its influence on determination of pluripotent precursor cells toward
the myogenic lineage, using a human mesenchymatous cell line.
[Up]
IDENTIFICATION AND PHYSIOLOGICAL
IMPORTANCE OF THE DIRECT T3 MITOCHONDRIAL PATHWAY.
Results.
T3 is known to play an important regulatory influence on the acquisition
of metabolic and contractile features of muscle fibres. This hormone is also
considered as a major regulator of mitochondriogenesis and mitochondrial
activity, important parameters involved in the definition these features.
Numerous studies have established that T3 increases mitochondrial oxygen
consumption and transcription of the organelle genome, in part independently of
its nuclear pathway. These data led us to search for the presence of T3
receptors inside mitochondria.
Using
highly purified rat liver mitochondria, we have characterized a 43
kDa-truncated form of the T3 c-Erb A a 1 nuclear receptor located in the mitochondrial matrix (p43). This
protein binds to specific sequences of the mitochondrial genome simlar to that
recognized by T3 nuclear receptors. Within few minutes of the T3 presence, this
binding stimulates mitochondrial transcription and consequently mitochondrial
protein synthesis. Stable p43 overexpression experiments using different cell
types established that this pathway is involved in the regulation of
mitochondriogenesis and mitochondrial activity, and therefore controls cell
energy metabolism.
|
The discovery of this
receptor led us to identify two other receptors also belonging from the
nuclear receptor superfamily. We brought evidence of the occurrence of
truncated forms of PPAR g 2 and RXR a in the
mitochondrial matrix, which heterodimerize with p43 and bind to the organelle
DNA. These data indicate that the hormonal regulation of mitochondrial genome
transcription shares unexpected similarities with that reported at the
nuclear level: T3 receptor heterodimerization with PPAR or RXR, binding to
specific responsive elements, transcriptional activity only in the T3
presence. |
|
Besides to its
metabolic activity, we established that the direct T3 mitochondrial pathway
involved in the processes of mitochondriogenesis, is also implicated in the
regulation of myoblast differentiation. Indeed, p43 overexpression induces
myoblast differentiation, even when it is strongly repressed by the presence of
high serum concentrations in the culture medium. Conversely, drugs antagonizing
p43 mitochondrial influence fully abrogates myoblast differentiation. Study of
the molecular basis of this myogenic influence led us to conclude that myogenin
is a crucial target of this pathway which also interferes with the ability of
endogenous or overexpressed myogenic factors to induce myoblast
differentiation.
In addition, p43 overexpression in human dermal fibroblasts strongly
stimulates mitochondrial activity. In turn, this event induces an oxidative
stress associated to extinction of three tumour suppressor genes expression,
and an increase in c-Jun and c-Fos expression. This change in gene expression
induces the acquisition of a rhabdomyosarcome-like phenotype (cell
transformation associated to muscle specific gene expression) by dermal
fibroblasts. Using anitoxidant drugs, we demonstrate that oxidative stress
induced by p43 overexpression plays a major role in cell transformation.
These results indicate that the direct T3 mitochondrial pathway is
involved in important physiological processes including cell differentiation
and transformation. They provide a new explanation of the severity of
degenerative pathologies associated to mitochondrial deficiencies; they also
implicate the mitochondrial abnormalities often reported in tumour tissues in
the processes leading to the induction, the maintenance and the development of
oncogenic processes.
We have demonstrated the occurrence of a new
hormonal pathway mediated by receptors localized in mitochondria. The
innovative aspects of this finding open a new field of research raising a lot
of questions: understanding of the receptors import processes into the
organelle, molecular basis of their transcriptional activity, physiological
importance…
The aim of the present studies is to elucidate the import processes of
p43 and mt-RXR (truncated form of the nuclear receptor RXR a ). We have already obtained data indicating that these import pathways
are atypical. In addition, the nature of the p43 sequences involved in its
translocation into the organelle, recently identified (three different
sequences working together) clearly establishes that this pathway is different
than that classically described.
Another aim is to identify the transcriptional complex inducing the p43
stimulation of mitochondrial genome expression. In this purpose, mitochondrial
matrix proteins interacting with p43 have been characterized by a proteomic
approach. We are presently studying the physiological consequences of these
interactions
Besides to this
work, the identification of another 28 kDa truncated c-ErbA a 1 protein located in the mitochondrial inner membrane raises the
question of its function. Preliminary data indicates that this second
mitochondrial T3 receptor influences the organelle oxygen consumption.
In addition,
besides these mechanistic studies, work is in progress in order to improve our
knowledge of the actual physiological importance of this mitochondrial pathway.
In particular, its influence on myoblast differentiation is under study, and we
have characterized two other nuclear genes regulated by mitochondrial activity.
One of them is a upstream target involved in the regulation of myogenin
expression. The other led us to develop new studies concerning the influence of
the p43 pathway on the acquisition of contractile features by muscle fibres. Moreover,
a human nutrition programme supported by ANR is devloped, with the aim to characterize
the involvement of changes in mitochondrial activity induce by hypercaloric
diets in pathologies such as metabolic syndrome, diabetes, obesity or
cardiovascular diseases. Lastly, nuclear target genes of mitochondrial activity
in myoblasts will be identified, as exhaustively as possible, by using
microarrays. The signalling between mitochondria and the nucleus at the origin
of this genic regulation is under study with a particular interest fot Reactive
Oxygen species or calcium signalling.
In
parallel to these in vitro studies,
we are developing a transgenesis programme in order to validate in vivo results
obtained in cell cultures. Mice specifically overexpressing p43
in skeletal muscle are already obtained, and phenotypage is in progress. Mice
knock-outed for p43 are under obtention. Such animals could also constitute
experimental models for human mitochondrial pathologies. This part of the
programme is performed with the collaboration of the transgenesis unit of the
laboratory (Christelle Bertrand et Laurence Lepourry).