Terminal differentiation of Sol 8 myoblasts is retarded by a transforming growth factor-β autocrine regulatory loop

Abstract

In DM (differentiation medium), Sol 8 myoblasts spontaneously form myotubes and express the βMHC (β-myosin heavy chain), their main marker of terminal differentiation. This marker is detectable at 24 h, and increases up to 72 h. Our aim was to define temporal effects of TGFβ (transforming growth factor β) on βMHC expression in Sol 8 cells. TGFβ1 (1 ng/ml) added at time zero to DM decreased MyoD expression and completely inhibited βMHC expression in Sol 8 cells. This inhibition of βMHC expression was progressively lost when TGFβ1 was added from 8 to 34 h. After 34 h, the cells were irreversibly differentiated, and TGFβ1 did not inhibit βMHC accumulation any longer. Two independent approaches showed that a TGFβ autocrine regulatory loop retarded and partially impaired Sol 8 cell terminal differentiation. First, permanent immunoneutralization of the active TGFβs released by the cells into DM increased βMHC levels at 72 h compared with controls. Secondly, a dominant-negative mutant of the TGFβ type II receptor was overexpressed in Sol 8 cells under the control of the βMHC promoter. Both the dominant-negative receptor and the βMHC gene were expressed after 24 h in DM. The delayed blocking of the TGFβ signalling pathway by the dominant-negative receptor was as effective as permanent immunoneutralization to promote βMHC expression. To conclude, TGFβ inhibits Sol 8 cell terminal differentiation within a narrow time interval (24–34 h) that coincides with the onset of βMHC expression.