AUTONOMOUS DIFFERENTIATION IN THE MOUSE MYOGENIC CELL-LINE, C2, INVOLVES A MUTUAL POSITIVE CONTROL BETWEEN INSULIN-LIKE GROWTH-FACTOR-II AND MYOD, OPERATING AS EARLY AS AT THE MYOBLAST STAGE

We have studied the contribution of the endogenous production of insul in-like growth factor II (IGFII) and of the muscle regulatory factor, MyoD, to the autonomy of differentiation in isolated skeletal myoblast s. Inhibition of MyoD and IGFII gene expression in myoblasts of the mo use myogenic cell line, C2, was achieved by transfection and selection of stably transfected cells (anti-MyoD and anti-IGFII cells) with vec tors producing MyoD or IGFII antisense RNA. We observed that inhibitin g either MyoD or IGFII has multiple and similar consequences. In addit ion to the inhibition of the target gene, expression of MyoD transcrip ts in anti-IGFII myoblasts and expression of IGFII in anti-MyoD myobla sts were also abolished, whereas accumulation of transcripts for the m uscle regulatory factor, Myf5, was markedly increased in both cell typ es. However, despite this Myf5 up-regulation, both anti-IGFII and anti -MyoD myoblasts lost the ability to undergo autonomous differentiation (differentiation in the absence of added IGF), further indicating tha t Myf5 and MyoD are not strictly interchangeable. Additional evidence of a link between MyoD and IGFII was obtained: (1) forced expression o f the MyoD cDNA stimulated IGFII gene expression, and (2) treatment of C2 myoblasts with fibroblast growth factor, not only diminished MyoD expression and compromised differentiation as previously shown by othe rs, but also abolished IGFII expression. These experiments showing los s or gain of function argue in favor of a mutual positive control betw een IGFII and MyoD operating as early as the myoblast stage.