The Raf-1 protein mediates insulin-like growth factor-induced proliferation of erythroid progenitor cells

Previous studies from this and other laboratories have shown that insulin-like growth factor-1 (IGF-I) and insulin-like growth factor-2 (IGF-II) support erythroid colony formation in cultures supplemented with serum substitute and recombinant erythropoietin. Subpopulations of IGF-I- and IGF-II-dependent, erythropoietin-independent colony-forming unit-erythroid (CFU-E)-derived colonies and BFU-E-derived colonies were identified under serum-substituted conditions for adult bone-marrow-derived erythroid progenitors which proliferate in the absence and presence of exogenous anti-erythropoietin receptor monoclonal antibody and in serum-substituted medium that was preadsorbed with anti-erythropoietin IgG. To assess whether Raf-1 is required for the formation of IGF-dependent, erythropoietin-independent human erythroid colonies, 5-15 microM sense or antisense oligomer to raf-1 were added to serum-substituted cultures containing either 2 U/ml recombinant human erythropoietin (rHuEpo) alone or 0-1,000 ng/ml IGF-I or IGF-II with/without 2 U/ml rHuEpo. Both erythropoietin-induced and IGF-induced erythroid colony formation were completely blocked by antisense (but not sense) oligomers to raf-1. Purified human CFU-Es were examined for Raf-1 message and protein. Total RNA was extracted, and raf-1 mRNA was detected on Northern blots. Furthermore, a 74 kD protein, corresponding to Raf-1, was also detected in CFU-Es purified from human adult sources. Together, these studies support the hypothesis that the Raf-1 protein mediates both erythropoietin-induced and IGF-induced signal transduction in human erythroid progenitor cells.