RHOA GTPASE AND SERUM RESPONSE FACTOR CONTROL SELECTIVELY THE EXPRESSION OF MYOD WITHOUT AFFECTING MYF5 IN MOUSE MYOBLASTS

MyoD and Myf5 belong to the family of basic helix-loop-helix transcrip tion 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 thes e 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 di fferentiation, is required for MyoD gene expression. We have investiga ted here whether SRF is also involved in the control of Myf5 gene expr ession, and the potential role of upstream regulators of SRF activity, the Rho family G-proteins including Rho, Rac, and CDC42, in the regul ation of MyoD and Myf5. We show that inactivation of SRF does not alte r Myf5 gene expression, whereas it causes a rapid extinction of MyoD g ene expression. Furthermore, we show that RhoA, but not Rac or CDC42, is also required for the expression of MyoD. Indeed, blocking the acti vity of G-proteins using the general inhibitor lovastatin, or more spe cific antagonists of Rho proteins such as C3-transferase or dominant n egative RhoA protein, resulted in a dramatic decrease of MyoD protein levels and promoter activity without any effects on Myf5 expression. W e further show that RhoA-dependent transcriptional activation required functional SRF in C2 muscle cells. These data illustrate that MyoD an d Myf5 are regulated by different upstream activation pathways in whic h MyoD expression is specifically modulated by a RhoA/SRF signaling ca scade. In addition, our results establish the first link between RhoA protein activity and the expression of a key muscle regulator.