Expression of C/EBPε Restores G-CSF-Induced Granulocytic Differentiation of Myeloid Cells Expressing the Carboxy Truncated G-CSF Receptor Associated with AML/SCN.

Mutations in the G-CSF receptor gene causing carboxy terminal truncation of the G-CSF receptor have been associated with the development of acute myelogenous leukemia (AML) in patients with severe congenital neutropenia (SCN). The truncated G-CSF receptors mediate augmented cell proliferation and survival, but are defective in inducing granulocytic differentiation in myeloid cell lines and transgenic mice. Little is known about the mechanism by which the carboxy terminus of the G-CSF receptor regulates granulocytic differentiation. It has been shown that myeloid transcription factors including C/EBPα, C/EBPε and PU.1 are critically involved in granulopoiesis. Of the three transcription factors, expression of C/EBPε was dramatically upregulated upon G-CSF treatment of myeloid 32D cells expressing the wild type G-CSF receptor (32D/WT). C/EBPε upregulation by G-CSF was abolished in 32D cells expressing the G-CSF receptor mutant D715 (32D/D715), which lacked a region of 98 amino acids in the carboxy terminus. Forced expression of C/EBPε in 32D/D715 cells at levels comparable to those in G-CSF-treated 32D/WT cells resulted in markedly attenuated cell proliferation and survival, and rapid terminal granulocytic differentiation in response to G-CSF. These data indicated that the functional defect of the D715 mutant in mediating granulocytic differentiation may arise from its inability to upregulate C/EBPε expression. Notably, forced expression of C/EBPε failed to induce terminal granulocytic differentiation of 32D/D715 cells cultured in IL-3, suggesting that the D715 receptor acted in collaboration with C/EBPε to drive granulocytic differentiation. Of the four cytoplasmic tyrosine (Tyr) residues of the G-CSF receptor, the D715 mutant contains only Tyr 704 that is involved in Stat3 activation. Because Stat3 is an important regulator of granulopoiesis, experiments are under way to address whether Tyr 704 in the D715 mutant is required for granulocytic differentiation driven by C/EBPε.