The muscle regulators MyoD and Myf-5 control cell cycle withdrawal and induction of differentiation in skeletal muscle cells. By immunofluorescence analysis, we show that MyoD and Myf-5 expression patterns become mutually exclusive when C2 cells are induced to differentiate with Myf-5 staining present in cells which fail to differentiate. Isolation of these undifferentiated cells reveals that upon serum stimulation they reenter the cell cycle, express MyoD and downregulate Myf-5. Similar regulations of MyoD and Myf-5 were observed using cultured primary myoblasts derived from satellite cells. To further analyze these regulations of MyoD and Myf-5 expression, we synchronized proliferating myoblasts. Analysis of MyoD and Myf-5 expression during cell cycle progression revealed distinct and contrasting profiles of expression. MyoD is absent in G0, peaks in mid-G1, falls to its minimum level at G1/S and reaugments from S to M. In contrast, Myf-5 protein is high in G0, decreases during G1 and reappears at the end of G1 to remain stable until mitosis. These data demonstrate that the two myogenic factors MyoD and Myf-5 undergo specific and distinct cell cycle–dependent regulation, thus establishing a correlation between the cell cycle–specific ratios of MyoD and Myf-5 and the capacity of cells to differentiate: (a) in G1, when cells express high levels of MyoD and enter differentiation; (b) in G0, when cells express high levels of Myf-5 and fail to differentiate.
During the early process of skeletal muscle differentiation, myogenic factors are not only involved in muscle-specific gene induction but also in regulating the transition from the proliferative stage, when MyoD and Myf5 are already expressed, to the orderly exit from the cell division cycle. This key step in skeletal muscle differentiation involves the down-regulation of cell cycle activators such as cyclins and cdks, and up-regulation of cell cycle inhibitors such as Rb, p21, p27, and p57. In particular, Rb and p21 have been shown to play an important role in the growth arrest of differentiating myoblasts. Their level and/or activity, while being negatively controlled by growth factors, appear to be positively linked with the myogenic factor MyoD, which plays a cooperative role in the induction of growth arrest. MyoD can block proliferation independently of its transcriptional activity. Therefore, the interplay between G1 cyclins and cdk inhibitors, on the one hand, and MyoD and its co-factors, on the other, plays a critical role in myoblast cell cycle withdrawal. Accurate synchronization of dividing myoblasts revealed that MyoD and Myf5 are themselves subject to specific cell cycle-dependent regulation, with MyoD at its highest level in early G1 and its lowest level at the G1 to S phase transition. The time-window when cells exit their cycle into differentiation is in G1, when MyoD is maximal and Myf5 is down. In contrast, quiescent non-differentiating myoblasts (i. e., in G0) present an opposite pattern for the two factors: high Myf5 and no MyoD. Several recent studies have focused on MyoD phosphorylation and its potential role in ubiquitination-mediated degradation of the protein. Linking this phosphorylation to the cell cycle-dependent drop in MyoD protein before S phase leads, to a mechanism implying cdk2-cyclin E and its inhibitors (p57kip and p21cip) in the tight control of MyoD levels and subsequent myoblast cell cycle progression or exit into differentiation.
The molecular mechanisms underlying the developmental regulation of L-type voltage-dependent Ca 2؉ channels (VDCCs) are still unknown. In this study, we have characterized the expression patterns of skeletal (␣ 1S ) and cardiac (␣ 1C ) L-type VDCCs during cardiogenic differentiation in H9C2 cells that derived from embryonic rat heart. We report that chronic treatment of H9C2 cells with 10 nM all-trans-retinoic acid (all-trans-RA) enhanced cardiac Ca 2؉ channel expression, as demonstrated by reverse transcription-polymerase chain reaction, immunoblotting, and indirect immunofluorescence studies, as well as patch-clamp experiments. In addition, RA treatment prevented expression of functional skeletal L-type VDCCs, which were restricted to myotubes that spontaneously appear in control H9C2 cultures undergoing myogenic transdifferentiation. The use of specific skeletal and cardiac markers indicated that RA, by preventing myogenic transdifferentiation, preserves cardiac differentiation of this cell line. Altogether, we provide evidence that cardiac and skeletal subtype-specific L-type Ca 2؉ channels are relevant functional markers of differentiated cardiac and skeletal myocytes, respectively. In conclusion, our data demonstrate that in vitro RA stimulates cardiac (␣ 1C ) L-type Ca 2؉ channel expression, therefore supporting the hypothesis that the RA pathway might be involved in the tissue specific expression of Ca 2؉ channels in mature cardiac cells.
MyoD and Myf5 belong to the family of basic helix-loop-helix transcription factors that are key operators in skeletal muscle differentiation. MyoD and Myf5 genes are selectively activated during development in a time and region-specific manner and in response to different stimuli. However, molecules that specifically regulate the expression of these two genes and the pathways involved remain to be determined. We have recently shown that the serum response factor (SRF), a transcription factor involved in activation of both mitogenic response and muscle differentiation, is required for MyoD gene expression. We have investigated here whether SRF is also involved in the control of Myf5 gene expression, and the potential role of upstream regulators of SRF activity, the Rho family G-proteins including Rho, Rac, and CDC42, in the regulation of MyoD and Myf5. We show that inactivation of SRF does not alter Myf5 gene expression, whereas it causes a rapid extinction of MyoD gene expression. Furthermore, we show that RhoA, but not Rac or CDC42, is also required for the expression of MyoD. Indeed, blocking the activity of G-proteins using the general inhibitor lovastatin, or more specific antagonists of Rho proteins such as C3-transferase or dominant negative RhoA protein, resulted in a dramatic decrease of MyoD protein levels and promoter activity without any effects on Myf5 expression. We further show that RhoA-dependent transcriptional activation required functional SRF in C2 muscle cells. These data illustrate that MyoD and Myf5 are regulated by different upstream activation pathways in which MyoD expression is specifically modulated by a RhoA/SRF signaling cascade. In addition, our results establish the first link between RhoA protein activity and the expression of a key muscle regulator.
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