ERYTHROPOIETIN AND INTERLEUKIN-3 INDUCE DISTINCT EVENTS IN ERYTHROPOIETIN RECEPTOR-EXPRESSING BA/F3 CELLS

To compare the signal transduction pathways used by erythropoietin (Ep) and interleukin-3 (IL-3), the cDNA for the murine erythropoietin receptor (EpR) was introduced into the IL-3-responsive cell lines Ba/F3 and DA-3 using retrovirally mediated gene transfer. After selection in G418 and IL-3, clones expressing comparable levels of cell surface EpR were identified using biotinylated Ep and flow cytometry. A comparison of the effects of Ep and IL-3 on these cells showed that most EpR(+) Ba/F3 clones, when first exposed to Ep, dramatically increased their levels of beta-globin mRNA. The kinetics of appearance of this message after exposure to Ep varied considerably from clone to clone, with some clones showing a marked increase in beta-globin mRNA within 1 hour, while others required several days before an increase was observed. Interestingly, not only was this increase not seen with IL-3, but IL-3 prevented the Ep-induced appearance of beta-globin message. On the other hand, none of the EpR(+) DA-3 cell clones tested increased their levels of beta-globin mRNA in response to Ep. While the EpR(+) DA-3 clones showed identical proliferative responses to IL-3 and Ep, most EpR(+) Ba/F3 clones displayed a marked, albeit transient, proliferative lag when first exposed to Ep. This was manifested as both an increased doubling time in liquid culture and a decreased colony size in methylcellulose. Plating efficiencies of EpR(+) Ba/F3 cells in methylcellulose, however, were identical in response to IL-3 and Ep, suggesting that the Ep-induced lag in proliferation reflected a growth delay of the entire population of cells to Ep rather than a selection of an Ep-responsive subpopulation. Flow cytometric analysis established that this growth delay was due to a lengthening of the first G1 period after exposure to Ep. Interestingly, this Ep-induced delay in entry into the S phase was not detected in cells stimulated with both Ep and IL-3 nor in EpR(+) Ba/F3 cell clones that did not show an increase in beta-globin mRNA in response to Ep. Thymidine-induced growth arrest, however, showed that delaying entry into S phase alone was not sufficient to stimulate beta-globin mRNA in the absence of Ep. Further studies established that the Ep-induced increase in beta-globin mRNA could be inhibited by the tyrosine kinase inhibitor genistein and the protein kinase C inhibitor Compound 3. Taken together, these results confirm that Ep and IL-3 can trigger qualitatively different responses in EpR(+) Ba/F3 cells, that the Ep-induced increase in beta-globin transcription correlates with a lengthening of the first G1 period after exposure to Ep, and that protein phosphorylation events play a critical role in this Ep-induced partial differentiation. (C) 1995 by The American Society of Hematology.