Erythropoietin receptor and STAT5-specific pathways promote SKT6 cell hemoglobinization.
Erythrocyte production in mammals is known to depend on the exposure of committed progenitor cells to the glycoprotein hormone erythropoietin (Epo). In chimeric mice, gene disruption experiments have demonstrated a critical role for Epo signaling in development beyond the erythroid colony-forming unit (CFU-e) stage. However, whether this might include the possible Epo-specific induction of red blood cell differentiation events is largely unresolved. To address this issue, mechanisms of induced globin expression in Epo-responsive SKT6 cells have been investigated. Chimeric receptors containing an epidermal growth factor (EGF) receptor extracellular domain and varied Epo receptor cytoplasmic domains first were expressed stably at physiological levels in SKT6 cells, and their activities in mediating induced hemoglobinization were assayed. While activity was exerted by a full-length chimera (EE483), truncation to remove 7 of 8 carboxyl-terminal tyrosine sites (EE372) markedly enhanced differentiation signaling. Moreover, mutation of a STAT5 binding site in this construct (EE372-Y343F) inhibited induced globin expression and SKT6 cell hemoglobinization, as did the ectopic expression of dominant-negative forms of STAT5 in parental SKT6 cells. As in normal CFU-e, SKT6 cells also were shown to express functional receptors for stem cell factor (SCF). To further define possible specific requirements for differentiation signaling, effects of SCF on SKT6 cell hemoglobinization were tested. Interestingly, SCF not only failed to promote globin expression but inhibited this Epo-induced event in a dose-dependent, STAT5-independent fashion. Thus, effects of Epo on globin expression may depend specifically on STAT5-dependent events, and SCF normally may function to attenuate terminal differentiation while promoting CFU-e expansion.
Department of Biochemistry and Molecular Biology, the Center for Gene Regulation, The Pennsylvania State Univeristy, University Park, PA 16802, USA.
R C
RC
Gregory
N
N
Jiang
K
K
Todokoro
J
J
Crouse
R E
RE
Pacifici
D M
DM
Wojchowski
D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division; D004268:DNA-Binding Proteins / Q000502:physiology*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000096:biosynthesis / Q000235:genetics; D004920:Erythropoiesis / Q000502:physiology*; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects; D005914:Globins / Q000096:biosynthesis*; D006454:Hemoglobins / Q000096:biosynthesis*; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology; D051379:Mice; D008894:Milk Proteins; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D019009:Proto-Oncogene Proteins c-kit / Q000502:physiology; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D050799:STAT5 Transcription Factor; D017384:Sequence Deletion; D015398:Signal Transduction; D015534:Trans-Activators / Q000502:physiology*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D006454:Hemoglobins; D008894:Milk Proteins; D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D004815:Epidermal Growth Factor; D005914:Globins; D066246:ErbB Receptors; D019009:Proto-Oncogene Proteins c-kit