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.(ABSTRACT TRUNCATED AT 250 WORDS)
Krosl
J
J
Terry Fox Laboratory, BC Cancer Research Centre, Vancouver, Canada.
Damen
J E
JE
Krystal
G
G
Humphries
R K
RK
D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D050981:Erythroid-Specific DNA-Binding Factors; D004921:Erythropoietin / Q000494:pharmacology*; D018014:Gene Transfer Techniques; D019833:Genistein; D005914:Globins / Q000235:genetics; D007377:Interleukin-3 / Q000494:pharmacology*; D007529:Isoflavones / Q000494:pharmacology; D007700:Kinetics; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000378:metabolism; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D007377:Interleukin-3; D007529:Isoflavones; D012333:RNA, Messenger; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D014157:Transcription Factors; D004921:Erythropoietin; D005914:Globins; D019833:Genistein; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D004121:Dimethyl Sulfoxide