We have previously shown that mitochondrial activity is an important regulator of myoblast differentiation, partly through processes targeting myogenin expression. Here, we investigated the possible involvement of c‐myc in these processes. Inhibition of mitochondrial activity by chloramphenicol abrogated the decrease in c‐myc mRNA and protein levels occurring at the onset of terminal differentiation. Conversely, stimulation of mitochondrial activity by overexpression of the T3 mitochondrial receptor (p43) down‐regulated c‐myc expression. In addition, c‐myc overexpression mimicked the influence of mitochondrial activity inhibition on myoblast differentiation. Moreover, like chloramphenicol, c‐myc overexpression strongly inhibited the myogenic influence of p43 overexpression. These data suggest that c‐Myc is an important target of mitochondrial activity involved in the myogenic influence of the organelle. Lastly, we found that chloramphenicol influence is negatively related to the frequency of post‐mitotic myoblasts in the culture at the onset of treatment, and cell cycle analyses demonstrated that the frequency of myoblasts in G0–G1 phase at cell confluence is increased by p43 overexpression and decreased by chloramphenicol or c‐myc overexpression. These results suggest that irreversible myoblast withdrawal from the cell cycle is a target of mitochondrial activity by control of c‐Myc expression. J. Cell. Physiol. 207: 75–86, 2006. © 2005 Wiley‐Liss, Inc.

Parallèlement à son rôle dans le métabolisme énergétique, l’activité mitochondriale intervient également dans l’induction de l’apoptose, ainsi que dans la régulation de la prolifération et de la différenciation cellulaires. Il existe en particulier une véritable régulation de la différenciation des myoblastes par l’activité mitochondriale, indépendante de la production d’ATP. Elle implique notamment le contrôle de l’expression de myogénine et de l’activité des facteurs myogéniques. Dans cette étude, nous démontrons que l’expression du proto-oncogène c-Myc est respectivement stimulée ou diminuée par une inhibition ou une stimulation de l’activité mitochondriale. Cette régulation s’effectue en grande partie au niveau de la stabilité du messager, et au niveau de la localisation cellulaire de la protéine dans les myoblastes aviaires. De plus, la surexpression de c-Myc reproduit très exactement les effets d’une inhibition de l’activité mitochondriale : i) abrogation de la différenciation terminale ; ii) inhibition de l’expression de Myogénine, sans altération de celle de MyoD ; iii) blocage de l’aptitude des facteurs myogéniques à induire la différenciation ; iv) inhibition de la sortie des myoblastes du cycle cellulaire. Ces résultats démontrent que c-Myc est une cible importante de l’activité mitochondriale, impliquée dans l’influence de l’organite sur la différenciation des myoblastes. Nous avons également mis en évidence l’existence d’un autre gène cible de l’organite qui code la phosphatase calcium dépendante Calcineurine. Son expression est respectivement inhibée ou stimulée par l’inhibition ou la stimulation de l’activité mitochondriale. De plus, l’expression d’une forme constitutivement active de Calcineurine stimule la différenciation des myoblastes et l’expression de Myogénine, alors que ces deux événements sont bloqués par l’expression d’un ARN antisens Calcineurine. Enfin, la stimulation de l’activité mitochondriale, comme l’expression d’une forme constitutivement active de Calcineurine stimule spécifiquement l’expression de l’isoforme lente des chaînes lourdes de myosine. Ces données démontrent donc que, notamment via l’expression de Calcineurine, l’activité mitochondriale régule non seulement la différenciation des myoblastes, mais détermine également le type contractile des fibres musculaires

Mitochondrial activity is a major regulator of myoblast differentiation and of the expression of myosin isoforms Besides their influence on fuel metabolism, mitochondrial activity is also involved in the induction of apoptosis and in the regulation of cell proliferation and differentiation. In particular, an actual regulation of myoblast differentiation by mitochondrial activity is well established, independently of ATP synthesis, through the control of myogenin expression and myogenic factor activity. In this study, we established that c-Myc expression is respectively up- or down-regulated by inhibition or stimulation of mitochondrial activity. This regulation essentially takes place at a post-transcriptional level and also concerns, in avian myoblast, the cellular localisation of the protein. In addition, c-Myc overexpression exactly mimics the influence of an inhibition of mitochondrial activity: i) inhibition of myoblast differentiation; ii) inhibition of myogenin expression; iii) inhibition of myogenic factor ability to induce differentiation; iv) inhibition of myoblast withdrawal from the cell cycle. These results demonstrate that c-Myc is an important target of mitochondrial activity involved in the myogenic influence of the organelle. We also identified another target gene of mitochondrial activity encoding the calcium-dependent phosphatase calcineurin. Its expression is respectively inhibited or stimulated by inhibition or stimulation of mitochondrial activity. In addition, expression of a "constitutively active form of calcineurin stimulates of myoblast differentiation and myosin isoform expression, whereas these two events are suppressed by expression of antisense calcineurin RNA. Lastly, stimulation of mitochondrial activity or overexpression of the constitutively active form of calcineurin specifically stimulates the expression of slow myosin heavy chain isoform. These data demonstrate that, partly via calcineurin expression, mitochondrial activity not only regulates myoblast differentiation, but also the contractile type of muscle fibres.

Mitochondrial dysfunctions are frequently reported in cancer cells, but their direct involvement in tumorigenesis remains unclear. To understand this relation, we stimulated mitochondrial activity by overexpression of the mitochondrial triiodothyronine receptor (p43) in human dermal fibroblasts. In all clones, this stimulation induced morphologic changes and cell fusion in myotube-like structures associated with the expression of several muscle-specific genes (Myf5, desmin, connectin, myosin, AchRalpha). In addition, these clones displayed all the in vivo and in vitro features of cell transformation. This phenotype was related to an increase in c-Jun and c-Fos expression and extinction of tumor suppressor gene expression (p53, p21WAF1, Rb3). Lastly, reactive oxygen species (ROS) production was increased in positive correlation to the stimulation of mitochondrial activity. The direct involvement of mitochondrial activity in this cell behavior was studied by adding chloramphenicol, an inhibitor of mitochondrial protein synthesis, to the culture medium. This inhibition resulted in partial restoration of the normal phenotype, with the loss of the ability to fuse, a strong decrease in muscle-specific gene expression, and potent inhibition of the transformed phenotype. However, expression of tumor suppressor genes was not restored. Similar results were obtained by using N-acetylcysteine, an inhibitor of ROS production. These data indicate that stimulation of mitochondrial activity in human dermal fibroblasts induces cell transformation through events involving ROS production.

The regulation of gene expression by thyroid hormone (T-3) involves binding of the hormone to nuclear receptors [ thyroid hormone receptor (TR)] acting as T-3-dependent transcription factors encoded by TR alpha (NR1A1) and TR beta (NR1A2) genes. Several TR alpha variants have already been characterized, but only some of them display T-3 binding activity. In this study, we have identified another transcript, TR alpha-Delta E6, produced by alternative splicing with microexon 6b instead of exon 6. This splicing leads to the synthesis of a protein devoid of a hinge domain. The TR alpha-Delta E6 transcript is detected in all mouse tissues tested. Although TR alpha-Delta E6 did not bind DNA, its expression induced a TR alpha 1 sequestration in the cytoplasm. Functional studies demonstrated that TR alpha-Delta E6 inhibits the transcriptional activity of TR alpha 1 and retinoic X receptor-alpha, but not of retinoic acid receptor-alpha. We also found that TR alpha-Delta E6 efficiently decreased the ability of TR alpha to inhibit MyoD transcriptional activity during myoblast proliferation. Consequently, when overexpressed in myoblasts, it stimulated terminal differentiation. We suggest that this novel TR alpha variant may act as down regulator of overall T-3 receptor activity, including its ability to repress MyoD transcriptional activity during myoblast proliferation.

The importance of mitochondrial activity has recently been extended to the regulation of developmental processes. Numerous pathologies associated with organelle's dysfunctions emphasize their physiological importance. However, regulation of mitochondrial genome transcription, a key element for organelle's function, remains poorly understood. After characterization in the organelle of a truncated form of the triiodothyronine nuclear receptor (p43), a T3-dependent transcription factor of the mitochondrial genome, our purpose was to search for other mitochondrial receptors involved in the regulation of organelle transcription. We show that a 44 kDa protein related to RXRalpha (mt-RXR), another nuclear receptor, is located in the mitochondrial matrix. We found that mt-RXR is produced after cytosolic or intramitochondrial enzymatic cleavage of the RXRalpha nuclear receptor. After mitochondrial import and binding to specific sequences of the organelle genome, mt-RXR induces a ligand-dependent increase in mitochondrial RNA levels. mt-RXR physically interacts with p43 and acts alone or through a heterodimerical complex activated by 9-cis-retinoic acid and T3 to increase RNA levels. These data indicate that hormonal regulation of mitochondrial transcription occurs through pathways similar to those that take place in the nucleus and open a new way to better understand hormone and vitamin action at the cellular level.