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 to CMD1 (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.Recent studies emphasize that mitochondria, in addition to their well known involvement in the regulation of energy metabolism, are implicated in the regulation of cell growth and differentiation. In particular, mitochondrial events are involved in the preliminary steps of apoptosis (1), and inhibition of mitochondrial activity, either by deleting mtDNA (rho°cells) or by blocking translation in the organelle, has been shown to stop or decrease the proliferation of different cell lines (2-4). Furthermore, the general activity of the organelle, not restricted to energy production, is implicated in such regulation (5, 6). In addition, mitochondrial protein synthesis inhibition is associated with the impairment of differentiation of different cell lines, such as mouse erythroleukemia (7) and mastocytoma cells (8), neurons (9), and human (10), avian (11) or murine myoblasts (12). In agreement with these data, several pathologies are associated with mitochondrial disorders, even if the links between mitochondrial genome rearrangements or activity and pathological symptoms are not always clearly established. Despite these reports, little is known about the molecular mechanisms involved in these regulations. First, the exclusive use of inhibitors of mitochondrial function in previous reports was not fully adapted to demonstrating the occurrence of an actual regulatory pathway involving mitochondrial activity in the regulation of cell differentiation. Second, the nature of the molecular signals underlying the...

In earlier research, we identified a 43-kDa c-ErbAalpha1 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.

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Vasopressin (AVP) receptors present in In-R1-G9 cells, a hamster glucagon-secreting ␣-pancreatic cell line, were characterized using SSR-149415, a selective nonpeptide V 1b receptor antagonist, and reference AVP compounds. Binding experiments, using [ 3 H]AVP as a ligand, identified a single population of high-affinity binding sites. SSR-149415 competitively inhibited this binding and exhibited nanomolar and stereospecific affinity for these sites. The affinity of various AVP/oxytocin ligands confirmed a V 1b binding profile. In functional studies, AVP was a potent stimulant in inducing intracellular Ca 2ϩ increase, glucagon secretion, and cell proliferation. These effects were fully antagonized by SSR-149415 with a nanomolar potency, whereas its diasteroisomer as well as two selective V 1a and V2 receptor antagonists were much less potent. Additionally, the order of potency of AVP agonists and antagonists was in agreement with V 1b-mediated effects. By RT-PCR, we confirmed the presence of V 1b receptor mRNA in both In-R1-G9 cells and in human pancreas. The distribution pattern of V 1b receptors investigated in human pancreas by immunohistochemistry showed strong labeling in islets of Langerhans, and colocalization studies indicated that this receptor was expressed in ␣-glucagon, ␤-insulin, and somatostatin pancreatic cells. Thus, in In-R1-G9 cells, AVP mediates intracellular Ca 2ϩ increase, glucagon secretion, and cell proliferation by activating V 1b receptors, and these effects are potently antagonized by SSR-149415. Moreover, the presence of V 1b receptors also found in human Langerhans islets could suggest hormonal control of AVP in human pancreas.arginine vasopressin V 1b receptors; glucagon; SSR-149415 VASOPRESSIN (AVP) exerts a number of central and peripheral functions in mammals. Among them, it controls water excretion by the kidney, vascular smooth muscle cell and uterine contraction, platelet aggregation, clotting factor release, liver glycogenolysis, mitogenesis, and hormonal secretion (i.e., release of aldosterone by adrenals, glucagon, and insulin in pancreas and corticotropin release by the adenohypophysis). In concert with oxytocin (OT), a structurally related neurohypophysial nonapeptide, AVP also controls several behavioral and memory processes (4, 9). These actions are mediated by specific G protein-coupled receptors, named V 1a , V 1b , V 2 , and OT, which have been cloned and characterized in animals and in humans. The V 2 receptor is positively coupled to adenylyl cyclase, whereas the others are linked to phospholipase C activation and intracellular Ca 2ϩ mobilization (29). The V 1b (or V 3 ) receptor has been cloned in mice, rats, and humans (7,14,20,27,30). This receptor is involved mainly in the stimulating effect of AVP on corticotropin secretion in the pituitary, and its role in stress and emotional situations is now well documented (1). As recently demonstrated by RT-PCR, in situ hybridization, or immunohistochemistry, the V 1b receptor gene/protein is expressed not only in anterior ...

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