Retinoic acid receptors and muscle b-HLH proteins: partners in retinoid-induced myogenesis.

The results reported here indicate that retinoic acid (RA) induces growth arrest and differentiation only in MyoD-expressing muscle cells. Transient transfection assays reveal a functional interaction between MyoD, a key myogenic regulator and RA-receptors, principal mediators of RA actions. Interestingly, we demonstrate that RXR-MyoD-containing complexes are recruited at specific MyoD DNA-binding sites in muscle cells. Furthermore, we also demonstrate that RA-receptors and the muscle basic helix-loop-helix (b-HLH) proteins interact physically. Mutational analysis suggests that this interaction occurs via the basic region of muscle b-HLH proteins and the DNA-binding domain of RA-receptors and is important for functional interactions between these two families of transcription factors. In conclusion, these results highlight novel interactions between two distinct groups of regulatory proteins that influence cell growth and differentiation.

Functional specificity of the two retinoic acid receptor RAR and RXR families in myogenesis.

In C2 myoblasts, retinoic acid (RA) is an efficient inducer of both growth arrest and differentiation. These RA effects are mediated through at least two classes of retinoic acid receptors (RARs and RXRs), which belong to the nuclear receptor superfamily. To determine the role played by each RAR or RXR family in this model system, we have analysed the effects of RA in C2 myoblasts expressing a dominant negative RAR (dnRAR) or a dominant negative RXR (dnRXR). The stable expression of dnRAR or dnRXR in C2 cells delays the RA-induced growth arrest and differentiation, an effect which is more pronounced in C2-dnRXR myoblasts. Furthermore, the RA-inducible expression of MyoD gene is lost in C2-dnRXR but not in C2-dnRAR cells, indicating that each family of retinoid receptors RAR and RXR may regulate distinct subsets of RA-responsive genes. Finally, using C2 cell lines with different retinoid responsiveness, we provided evidence for a link between the RXR and MyoD families in the process of myogenic differentiation. These results illustrate a critical role for RA-receptors in RA-control of C2 myogenesis and provide tools for studying the function of RA and its receptors during vertebrate development.

Expression of the estrogen-related receptor 1 (ERR-1) orphan receptor during mouse development.

We studied the expression of the estrogen-related receptor 1 (ERR-1) during mouse embryonic development. ERR-1 is expressed at very early stages in ES cells and at E8.5 in the mesodermal cells of the visceral yolk sac. ERR-1 continues to be expressed later in mesodermal tissues and particularly in heart and in skeletal muscles. This expression persists during all the embryonic development and in adult stage. ERR-1 transcripts level increases during muscle differentiation. Accordingly, we show that ERR-1 expression increases during the myoblast to myotube transition in differentiating C2 myoblastic cells. ERR-1 has also been detected in the nervous system during embryonic development. At E10.5, a high level of ERR-1 transcripts can be observed in differentiated cells of the intermediate zone of the spinal cord which also suggests a role of ERR-1 in the differentiation of the nervous system. The same is observed in the telencephalon vesicules at E13.5. Later, at E15.5 and E17.5, expression persists in the spinal cord but decreases dramatically in the central nervous system. Moreover, ERR-1 expression increases during skin formation and is detected in the stratum spinosum which contains differentiated Malpighian cells. Finally, we also observed ERR-1 in endodermal derivatives such as the epithelium of intestine and urogenital system. The DNA target of ERR-1 has been identified to be the SF-1/FTZ-F1 responsive element (SFRE) and we show in this paper that SF-1/FTZ-F1 and ERR-1 bind to and activate transcription independently through the SFRE element. Our study suggests that ERR-1 may be implicated in numerous physiological or developmental functions, particularly in the muscle, the central and peripheral nervous system and the epidermis. Interestingly, in these various systems ERR-1 expression is correlated with post-mitotic cells stage, suggesting that ERR-1 may play a role in the differentiation process.

RXR alpha is essential for mediating the all-trans retinoic acid-induced growth arrest of C2 myogenic cells.

In C2 muscle cells, retinoic acid (RA) induces growth arrest associated with terminal differentiation. These RA actions are presumed to be mediated through nuclear receptors (RARs and RXRs) that belong to the superfamily of ligand-dependent transcription factors. In this study, we have characterized a myogenic C2 subclone, that unlike parental cells, is resistant to growth inhibition and differentiation by RA. Examination of these RA-sensitive and resistant C2 cells for the expression of retinoid acid receptors revealed a lack of RXR alpha expression at the myoblast stage in resistant C2 cells. To determine the functions of RXR alpha, we introduced an RXR alpha expression vector into RA-resistant C2 cells by transient or stable transfections. Our results show that RXR alpha restores the response to RA in this subclone with respect to AP1 inhibition and growth arrest. These observations indicate that RXR alpha plays a crucial role in mediating RA induced growth arrest of C2 myogenic cells.