Gérard Cabello

Mechanisms involved in the inhibition of myoblast proliferation and differentiation by myostatin

Muscle growth results from a set of complex processes including myogenic transcription factors expression and activity, cell cycle withdrawal, myoblast fusion in myotubes, and acquisition of an apoptosis-resistant phenotype. Myostatin, a member of the TGFbeta family, described as a strong regulator of myogenesis in vivo Nature 387 (1997), 83; FEBS Lett. 474 (2000), 71 is upregulated during in vitro differentiation Biochem. Biophys. Res. Commun. 280 (2001), 561. To improve characterization of myostatins myogenic influence, we stably transfected vectors expressing myostatin and myostatin antisense in C2C12 myoblasts. Here, we found that myostatin inhibits cell proliferation and differentiation. Our results also indicate that myogenin is an important target of myostatin. In addition, overexpressed but not endogenous myostatin decreases MyoD protein levels and induces changes in its phosphorylation pattern. We also established that myostatin overexpression reduces the frequency of G0/G1-arrested cells during differentiation. Conversely, inhibition of myostatin synthesis leads to enhanced cell cycle withdrawal and consequently stimulates myoblast differentiation. We examined the expression patterns of the pRb, E2F1, p53, and p21 proteins involved in cell cycle withdrawal. We found that myostatin overexpression increases p21 and p53 expression, as it does accumulation of hypophosphorylated Rb. Interestingly, myostatin overexpression strongly reduced low-mitogen-induced apoptosis, whereas antisense expression induced contrary changes. In conclusion, these data show the influence of overexpressed myostatin on myoblast proliferation, differentiation, and apoptosis is extended to endogenous myostatin. Though some differences in overexpression or inhibition of endogenous myostatin were observed, it appears that myogenin and p21 are essential targets of this growth factor.

A Variant Form of the Nuclear Triiodothyronine Receptor c-ErbAα1 Plays a Direct Role in Regulation of Mitochondrial RNA Synthesis

ABSTRACT In earlier research, we identified a 43-kDa c-ErbAα1 protein (p43) in the mitochondrial matrix of rat liver. In the present work, binding experiments indicate that p43 displays an affinity for triiodothyronine (T3) similar to that of the T3 nuclear receptor. Using in organello import experiments, we found that p43 is targeted to the organelle by an unusual process similar to that previously reported for MTF1, a yeast mitochondrial transcription factor. DNA-binding experiments demonstrated that p43 specifically binds to four mitochondrial DNA sequences with a high similarity to nuclear T3 response elements (mt-T3REs). Using in organello transcription experiments, we observed that p43 increases the levels of both precursor and mature mitochondrial transcripts and the ratio of mRNA to rRNA in a T3-dependent manner. These events lead to stimulation of mitochondrial protein synthesis. In transient-transfection assays with reporter genes driven by the mitochondrial D loop or two mt-T3REs located in the D loop, p43 stimulated reporter gene activity only in the presence of T3. All these effects were abolished by deletion of the DNA-binding domain of p43. Finally, p43 overexpression in QM7 cells increased the levels of mitochondrial mRNAs, thus indicating that the in organello influence of p43 was physiologically relevant. These data reveal a novel hormonal pathway functioning within the mitochondrion, involving a truncated form of a nuclear receptor acting as a potent mitochondrial T3-dependent transcription factor.

Mitochondrial Activity Is Involved in the Regulation of Myoblast Differentiation through Myogenin Expression and Activity of Myogenic Factors

To characterize the regulatory pathways involved in the inhibition of cell differentiation induced by the impairment of mitochondrial activity, we investigated the relationships occurring between organelle activity and myogenesis using an avian myoblast cell line (QM7). The inhibition of mitochondrial translation by chloramphenicol led to a potent block of myoblast differentiation. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone and oligomycin, which affect the organelle at different levels, exerted a similar influence. In addition, we provided evidence that this phenomenon was not the result of an alteration in cell viability. Conversely, overexpression of the mitochondrial T3 receptor (p43) stimulated organelle activity and strongly potentiated myoblast differentiation. The involvement of mitochondrial activity in an actual regulation of myogenesis is further supported by results demonstrating that the muscle regulatory gene myogenin, in contrast toCMD1 (chicken MyoD) and myf5, is a specific transcriptional target of mitochondrial activity. Whereas myogenin mRNA and protein levels were down-regulated by chloramphenicol treatment, they were up-regulated by p43 overexpression, in a positive relationship with the expression level of the transgene. We also found that myogenin or CMD1 overexpression in chloramphenicol-treated myoblasts did not restore differentiation, thus indicating that an alteration in mitochondrial activity interferes with the ability of myogenic factors to induce terminal differentiation.

Oxidative stress in rats fed a high-fat high-sucrose diet and preventive effect of polyphenols: Involvement of mitochondrial and NAD(P)H oxidase systems

Mitochondrial and NADPH oxidase systems and oxidative stress were investigated in 12 week high-fat high-sucrose (HFHS) diet-fed rats. A protective effect of wine polyphenol (PP) extract was also examined. In liver, maximal activities of CII and CII+III mitochondrial complexes were decreased but NADPH oxidase expression (p22(phox) and p47(phox)) and NADPH oxidase-dependent superoxide anion production were not modified, whereas oxidative stress (lipid and protein oxidation products and antioxidant systems) was increased with HFHS diet. In muscle, anion superoxide production was slightly increased while mitochondrial complex activities and lipid and protein oxidation products were not modified with HFHS diet. In heart, NADPH oxidase expression and superoxide anion production were increased, and maximal activity of mitochondrial respiratory chain complexes or oxidative stress parameters were not modified. Wine polyphenol extract had an inhibiting effect on liver oxidative stress and on heart NADPH oxidase expression and superoxide anion production, and on induction of hepatic steatosis with HFHS diet. Induction of mitochondrial dysfunction could be a primary event in the development of oxidative stress in liver, while in skeletal muscle and in heart the NADPH oxidase system seems to be mainly involved in oxidative stress. Wine polyphenol extract was shown to partially prevent oxidative stress in liver and heart tissues and to nearly completely prevent steatosis development in liver.

Endocrine regulation of mitochondrial activity: involvement of truncated RXRα and c-Erb Aα1 proteins

The importance of mitochondrial activity has recently been extended to the regulation of developmental processes. Numerous pathologies associated with organelles dysfunctions emphasize their physiological importance. However, regulation of mitochondrial genome transcription, a key element for organelles 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 RXRα (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 RXRα 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.—Casas, F., Daury, L., Grandemange, S., Busson, M., Seyer, P., Hatier, R., Carazo, A., Cabello, G., Wrutniak‐Cabello, C. Endocrine regulation of mitochondrial activity: involvement of truncated RXRα and c‐Erb Aαl proteins. FASEB J. 17, 426–436 (2003)

How to boost antioxidants by lipophilization

Covalent modification of antioxidants through lipophilization is an important field of research aiming at developing antioxidants with improved efficacy. However, due to insufficient knowledge on how hydrophobicity affects antioxidant activity, lipophilization strategies have been largely based on empirism. Often, the resulting lipophilized antioxidants were not optimal. Here we described how the body of knowledge regarding hydrophobicity has been dramatically redefined as unexpected results were recently published. Using a broad range of lipophilized antioxidants assessed in dispersed lipids models and cultured cells, it has been demonstrated that the antioxidant activity increases progressively with increasing chain length up to a critical point, beyond which the activity of the compounds dramatically decreases. Taking into account this nonlinear phenomenon, also known as cut-off effect, antioxidant drug designers now have to seek the critical chain length to synthesize the optimal drug in a rational manner. Here, we briefly presented three putative mechanisms of action to try to account for the cut-off effect.

A 45 kDa protein related to PPARγ2, induced by peroxisome proliferators, is located in the mitochondrial matrix

Besides their involvement in the control of nuclear gene expression by activating several peroxisome proliferator‐activated receptors (PPARs), peroxisome proliferators influence mitochondrial activity. By analogy with the previous characterization of a mitochondrial T3 receptor (p43), we searched for the presence of a peroxisome proliferator target in the organelle. Using several antisera raised against different domains of PPARs, we demonstrated by Western blotting, immunoprecipitation and electron microscopy experiments, that a 45 kDa protein related to PPARγ2 (mt‐PPAR) is located in the matrix of rat liver mitochondria. In addition, we found that the amounts of mt‐PPAR are increased by clofibrate treatment. Moreover, in EMSA experiments mt‐PPAR bound to a DR2 sequence located in the mitochondrial D‐loop, by forming a complex with p43. Last, studies of tissue‐specific expression indicated that mt‐PPAR is detected in mitochondria of all tissues tested except the brain in amounts positively related to p43 abundance.

Mitochondrial activity regulates myoblast differentiation by control of c‐Myc expression

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.

Coactivation of nuclear receptors and myogenic factors induces the major BTG1 influence on muscle differentiation

The btg1 (B-cell translocation gene 1) gene coding sequence was isolated from a translocation break point in a case of B-cell chronic lymphocytic leukaemia. We have already shown that BTG1, considered as an antiproliferative protein, strongly stimulates myoblast differentiation. However, the mechanisms involved in this influence remained unknown. In cultured myoblasts, we found that BTG1 stimulates the transcriptional activity of nuclear receptors (T3 and all-trans retinoic acid receptors but not RXRα and PPARγ), c-Jun and myogenic factors (CMD1, Myf5, myogenin). Immunoprecipitation experiments performed in cells or using in vitro-synthesized proteins and GST pull-down assays established that BTG1 directly interacts with T3 and all-trans retinoic acid receptors and with avian MyoD (CMD1). These interactions are mediated by the transactivation domain of each transcription factor and the A box and C-terminal part of BTG1. NCoR presence induces the ligand dependency of the interaction with nuclear receptors. Lastly, deletion of BTG1 interacting domains abrogates its ability to stimulate nuclear receptors and CMD1 activity, and its myogenic influence. In conclusion, BTG1 is a novel important coactivator involved in the regulation of myoblast differentiation. It not only stimulates the activity of myogenic factors, but also of nuclear receptors already known as positive myogenic regulators.

Overexpression of the Mitochondrial T3 Receptor Induces Skeletal Muscle Atrophy during Aging

In previous studies, we characterized a new hormonal pathway involving a mitochondrial T3 receptor (p43) acting as a mitochondrial transcription factor. In in vitro and in vivo studies, we have shown that p43 increases mitochondrial transcription and mitochondrial biogenesis. In addition, p43 overexpression in skeletal muscle stimulates mitochondrial respiration and induces a shift in metabolic and contractile features of muscle fibers which became more oxidative. Here we have studied the influence of p43 overexpression in skeletal muscle of mice during aging. We report that p43 overexpression initially increased mitochondrial mass. However, after the early rise in mitochondrial DNA occurring at 2 months of age in transgenic mice, we observed a progressive decrease of mitochondrial DNA content which became 2-fold lower at 23 months of age relatively to control animals. Moreover, p43 overexpression induced an oxidative stress characterized by a strong increase of lipid peroxidation and protein oxidation in quadriceps muscle, although antioxidant enzyme activities (catalase and superoxide dismutase) were stimulated. In addition, muscle atrophy became detectable at 6 months of age, probably through a stimulation of the ubiquitin proteasome pathway via two muscle-specific ubiquitin ligases E3, Atrogin-1/MAFbx and MuRF1. Taken together, these results demonstrate that a prolonged stimulation of mitochondrial activity induces muscle atrophy. In addition, these data underline the importance of a tight control of p43 expression and suggest that a deregulation of the direct T3 mitochondrial pathway could be one of the parameters involved in the occurrence of sarcopenia.