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In order to characterize Sterling's triiodothyronine (T3) mitochondrial receptor using photoaffinity labeling, we observed two specific T3-binding proteins in the inner membrane (28 kDa) and in the matrix (43 kDa) of rat liver mitochondria. Western blots and immunoprecipitation using antibodies raised against the T3-binding domain of the T3 nuclear receptor c-Erb A alpha 1 indicated that at least the 43-kDa protein was c-Erb A alpha 1-related. In addition, gel mobility shift assays demonstrated the occurrence of a c-Erb A alpha 1-related mitochondrial protein that specifically binds to a natural or a palindromic thyroid-responsive element. Moreover, this protein specifically binds to a direct repeat 2 sequence located in the D-loop of the mitochondrial genome. Furthermore, electron microscopy studies allowed the direct observation of a c-Erb A-related protein in mitochondria. Lastly, the relative amounts of the 43-kDa protein related to c-Erb A alpha 1 were in good correlation with the known mitochondrial mass in three typical tissues. Interestingly, expression of a truncated form of the c-Erb A alpha 1 nuclear receptor in CV1 cells was associated with a mitochondrial localization and a stimulation of mitochondrial activity. These results supply evidence of the localization of a member of the nuclear receptor superfamily in the mitochondrial matrix involved in the regulation of mitochondrial activity that could act as a mitochondrial T3-dependent transcription factor.

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...

Triiodothyronine (T3) is considered a major regulator of mitochondrial activity. In this review, we show evidence of the existence of a direct T3 mitochondrial pathway, and try to clarify the respective importance of the nuclear and mitochondrial pathways for organelle activity. Numerous studies have reported short-term and delayed T3 stimulation of mitochondrial oxygen consumption. Convincing data indicate that an early influence occurs through an extra-nuclear mechanism insensitive to inhibitors of protein synthesis. Although it has been shown that diiodothyronines could actually be T3 mediators of this short-term influence, the detection of specific T3-binding sites, probably corresponding to a 28 kDa c-Erb A 1 protein of the inner membrane, also supports a direct T3 influence. The more delayed influence of thyroid hormone upon mitochondrial respiration probably results from mechanisms elicited at the nuclear level, including changes in phospholipid turnover and stimulation of uncoupling protein expression, leading to an increased inner membrane proton leak. However, the involvement of a direct mitochondrial T3 pathway leading to a rapid stimulation of mitochondrial protein synthesis has to be considered.Both pathways are obviously involved in the T3 stimulation of mitochondrial genome transcription. First, a 43 kDa c-Erb A 1 protein located in the mitochondrial matrix (p43), acting as a potent T3-dependent transcription factor of the mitochondrial genome, induces early stimulation of organelle transcription. In addition, T3 increases mitochondrial TFA expression, a mitochondrial transcription factor encoded by a nuclear gene. Similarly, the stimulation of mitochondriogenesis by thyroid hormone probably involves both pathways. In particular, the c-erb A gene simultaneously encodes a nuclear and a mitochondrial T3 receptor (p43), thus ensuring coordination of the expression of the mitochondrial genome and of nuclear genes encoding mitochondrial proteins.Recent studies concerning the physiological importance of the direct mitochondrial T3 pathway involving p43 led to the conclusion that it is not only involved in the regulation of fuel metabolism, but also in the regulation of cell differentiation. As the processes leading to or resulting from differentiation are energy-consuming, p43 coordination of metabolism and differentiation could be of significant importance in the regulation of development.

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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