Oncogenic Ras-induced secretion of a novel inhibitor of skeletal myoblast differentiation

Abstract

Expression of oncogenic H-Ras in 23A2 myoblasts (A2:H-Ras cells) is sufficient to induce both a transformed phenotype and a differentiation-defective phenotype. Because oncogenic Ras is known to induce the secretion of several different growth factors involved in maintaining the transformed phenotype of both fibroblast and epithelial cells, we explored the possibility that expression of oncogenic Ras in 23A2 myoblasts might lead to the secretion of a factor which inhibits differentiation. The differentiation of 23A2 myoblasts was inhibited (i) by coculture with an equal number of A2:H-Ras cells, (ii) by culture with an equal number of A2:H-Ras cells in the same tissue culture medium on an insert which allowed equilibration of molecules smaller than 1 micron, and (iii) by culture in media previously conditioned by A2:H-Ras cells. Similar results were obtained when 23A2 myoblasts expressing oncogenic N-Ras were substituted for A2:H-Ras cells in each assay. No inhibition of differentiation was observed, however, when differentiation-defective E1A-expressing 23A2 cells or C3H10T1/2 fibroblasts were substituted for A2:H-Ras cells. The differentiation inhibitor(s) in media conditioned by A2:H-Ras cells is heat stable, larger than 3 kD, and sensitive to the non-specific growth factor antagonist, suramin. Western analyses failed to detect either FGF-2 or TGFβ (the known inhibitors of myoblast differentiation) in media conditioned by A2:H-Ras cells. Furthermore, while FGF-2 is a potent activator of MAP kinase and TGFβ is a potent inhibitor of mink lung epithelial cell (CCL64) growth, conditioned media from A2:H-Ras cells does not activate MAP kinase and does not inhibit the growth of CCL64 cells. These results indicate that expression of oncogenic Ras induces the secretion of a novel inhibitor of skeletal myoblast differentiation. Furthermore, these results are the first to implicate an autocrine/paracrine mechanism in the inhibition of differentiation by oncogenic Ras.