Increased expression of the LAZ3 (BCL6) proto-oncogene accompanies murine skeletal myogenesis.

The structural alterations of the LAZ3 (BCL6) gene are one of the most frequent events found in non-Hodgkin lymphoma. LAZ3 encodes a transcriptional repressor with a POZ/zinc finger structure similar to several Drosophila development regulators and to the human promyelocytic leukemia-associated PLZF gene. Consistent with the origin of LAZ3-associated malignancies, LAZ3 is expressed in mature B-cells and required for germinal center formation. However, its ubiquitous expression, with predominant levels in skeletal muscle, suggests that it may act outside the lymphoid system. To study how LAZ3 could be involved in skeletal muscle differentiation, we examined its expression in the C2 muscle cells. We report here that LAZ3 is upregulated at both mRNA and protein levels during the differentiation of proliferating C2 myoblasts into post-mitotic myotubes. This rise in LAZ3 expression is both precocious and sustained, and is not reversed when myotubes are re-exposed to mitogen-rich medium, suggesting that irreversible evens occurring upon myogenic terminal differentiation contribute to lock LAZ3 upregulation. In addition, using two different models, we found that a "simple" growth-arrest upon serum starvation is not sufficient to induce LAZ3 upregulation which rather appears as a feature of myogenic commitment and/or differentiation. Finally, BrdU incorporation assays in C2 cells entering the differentiation pathway indicate that "high" LAZ3 expression strongly correlates with their exit from the cell cycle. Taken as a whole, these findings suggest that LAZ3 could play a role in muscle differentiation. Together with some results reported in other cell types, we propose that LAZ3 may contribute to events common to various differentiation processes, possibly the induction and stabilization of the withdrawal from the cell cycle.

9-cis-retinoic acid regulates the expression of the muscle determination gene Myf5.

Myf5 is a member of the MyoD family, a set of four helix-loop-helix transcription factors that controls myogenic differentiation. The Myf5 gene has both in vivo and in vitro expression patterns consistent with an involvement in the first events of myogenesis, such as acquisition and/or maintenance of myogenic "determined" phenotype. To date, very little is known about the mechanism underlying the tight regulation of Myf5 expression. We report here that retinoic acid (RA) reduces the level of Myf5 message in both mouse C2 and rat L6 cell lines, probably at the transcriptional level, because Myf5 mRNA stability is not affected by RA. This repression is dose dependent, starting at 0.1 microM of all-trans RA, and is not abrogated by cycloheximide, suggesting a direct involvement of RA receptors in the control of Myf5 expression. Furthermore, we compared the efficiency of natural (all-trans RA and 9-cis RA) or synthetic (TTNPB) retinoids that differentially activate the two families of RA receptors, RA receptors and retinoid X-receptors (9-cis RA). As 9-cis RA is about 10 times more efficient than all-trans RA in repressing Myf5, whereas TTNPB, which preferentially activates RA receptors, is far less potent, our data provide evidence for an important role of ligand-bound retinoid X-receptors in the mediation of this inhibition.

Overexpression of c-erbA proto-oncogene enhances myogenic differentiation.

Triiodothyronine (T3) positively regulates both the expression of the MyoD gene, a key myogenic regulator, and C2 muscle cell differentiation. To directly examine the role of its nuclear receptors in the control of myogenesis, we introduced a c-erbA expression vector into C2 muscle cells by transient or stable transfection. Our results show that c-erbA can play a potent role in the triggering of muscle terminal differentiation since its overexpression leads to: (1) a complete abrogation of the activity of the myogenesis inhibitor AP-1 (fos/jun) transcription factor; (2) an enhanced induction of MyoD expression upon T3 treatment; (3) the acquisition by T3 of the ability to trigger both growth arrest and terminal differentiation in the presence of large amounts of serum mitogens, a property that is otherwise specific to retinoic acid (RA). Thus, c-erbA is one of the two protooncogenes (with c-ski) that acts as positive regulator of muscle differentiation. Furthermore, the fact that c-erbA overexpression allows T3 to largely mimic the RA effects indicates that their biological differences in the modulation of myogenic program primarily rely on the differential expression of their receptors in C2 muscle cells rather than on an intrinsic specificity of their target genes.

Serum-induced inhibition of myogenesis is differentially relieved by retinoic acid and triiodothyronine in C2 murine muscle cells.

We recently reported that triiodothyronine (T3) enhances MyoD gene expression and accelerates terminal differentiation in murine C2 myoblasts. In this paper, we are interested in the effects of other hormones acting through related nuclear receptors. Retinoic acid (RA), but not estradiol or dexamethasone, is also able to enhance MyoD gene expression (about threefold). However, the effects of RA and T3 on myogenesis are quite distinct, with a much more potent RA action. Indeed, although T3 and RA positively regulate myogenesis with similar efficiency in poorly mitogenic conditions, in presence of high serum concentrations T3 can no longer trigger terminal differentiation whereas RA still remains efficient. Thus, serum concentration is a crucial parameter in discriminating between the effects of T3 and RA on myogenesis. The differential effects between these two hormone are likely to be related to the ability of RA-activated endogenous retinoic acid receptors (RARs) to induce C2 myoblasts growth-arrest and to extinguish AP1 activity (thought to act as an inhibitor of myogenesis) whereas T3-activated endogenous thyroid hormones receptors (THRs) are relatively inefficient. We propose that the much higher level of RARs in C2 cells versus THRs could to some extent account for the differential ability of T3 and RA to antagonize serum-regulated mitogenic pathways in myogenic cells. This study provides clear evidence for an important role of RA on MyoD gene expression and myogenesis and suggests that T3 and RA could play overlapping, but distinct, roles on muscle development.

3,5,3'-Triiodothyronine positively regulates both MyoD1 gene transcription and terminal differentiation in C2 myoblasts.

Thyroid hormones are among the positive regulators of muscle development in vivo, but little is known about the way they work. We demonstrate here that MyoD1, one of the master genes controlling myogenesis, is a target of T3. After proliferating C2 myoblasts have been treated with T3 for 15 h, we observed a rise in MyoD1 expression at both the mRNA and protein levels. This is the first positive hormonal control of MyoD1 gene expression reported so far. We also provide data which suggest that T3 nuclear receptor(s) have a direct role on MyoD1 gene transcription: 1) C2 cells express the alpha 1 form of T3 nuclear receptors; 2) T3 up-regulates MyoD1 gene transcription and does not affect MyoD1 mRNA stability, as demonstrated by run-on and actinomycin D chase experiments, respectively; and 3) this transcriptional activation does not need the synthesis of intermediate protein(s) since it is not abolished by simultaneous treatment with cycloheximide. Moreover, in presence of T3, the increase of MyoD1 transcripts is associated with a faster terminal differentiation. Indeed we observed an earlier expression of various markers of myogenesis including myogenin (a regulatory gene of the MyoD1 family mainly involved in the triggering of terminal differentiation), myosin light chain 1A, and troponin T in T3-treated cells vs. untreated cells. We suggest that the regulation of a pivotal myogenic gene could be an important step in the control exerted by T3 on muscle development in vivo.