C/EBPalpha bypasses granulocyte colony-stimulating factor signals to rapidly induce PU.1 gene expression, stimulate granulocytic differentiation, and limit proliferation in 32D cl3 myeloblasts.
Within hematopoiesis, C/EBPalpha is expressed only in myeloid cells, and PU.1 is expressed mainly in myeloid and B-lymphoid cells. C/EBPalpha-deficient mice lack the neutrophil lineage and retain monocytes, whereas PU.1-deficient mice lack monocytes and have severely reduced neutrophils. We expressed a C/EBPalpha-estrogen receptor ligand-binding domain fusion protein, C/EBPalphaWT-ER, in 32D cl3 myeloblasts. 32D cl3 cells proliferate in interleukin-3 (IL-3) and differentiate to neutrophils in granulocyte colony-stimulating factor (G-CSF). In the presence of estradiol, C/EBPalphaWT-ER induced morphologic differentiation and the expression of the myeloperoxidase, lactoferrin, and G-CSF receptor mRNAs. C/EBPalphaWT-ER also induced a G1/S cell cycle block, with induction of p27 and Rb hypophosphorylation. bcr-ablp210 prevented 32D cl3 cell differentiation. Activation of C/EBPalpha-ER in 32D-bcr-ablp210 or Ba/F3 B-lymphoid cells induced cell cycle arrest independent of terminal differentiation. C/EBPalphaWT-ER induced endogenous PU.1 mRNA within 8 hours in both 32D cl3 and Ba/F3 cells, even in the presence of cycloheximide, indicating that C/EBPalpha directly activates the PU.1 gene. However, activation of a PU.1-ER fusion protein in 32D cl3 cells induced myeloperoxidase (MPO) RNA but not terminal differentiation. Thus, C/EBPalpha acts downstream of G-CSF and upstream of PU.1, p27, and potentially other factors to induce myeloblasts to undergo granulocytic differentiation and cell cycle arrest.
Johns Hopkins Oncology Center, Division of Pediatric Oncology, Baltimore, MD, USA.
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Wang
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Scott
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Sawyers
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Friedman
D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D006410:Hematopoiesis / Q000187:drug effects / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects*; D007377:Interleukin-3 / Q000494:pharmacology; D051379:Mice; D009504:Neutrophils / Q000166:cytology*; D009687:Nuclear Proteins / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D015534:Trans-Activators / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D000099232:Proto-Oncogene Protein Spi-1
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D016179:Granulocyte Colony-Stimulating Factor; D007377:Interleukin-3; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1