Verbatim): Myeloid cells are important in many disease processes, including leukemia, marrow failure, auto-immune diseases, and emphysema. C/EBPalpha (C/EBPa) and PU.1 are key regulators of myelopoiesis. C/EBPa null mice lack granulocytes, and PU.1 null mice lack B cells, granulocytes, and monocytes. 32D cl3 myeloid cells were developed expressing C/EBPa-ER or PU.1-ER. Activation of C/EBPa-ER increased MPO, lactoferrin, and G-CSF receptor mRNA expression and induced a neutrophilic morphology and a G1 cell cycle arrest. In contrast, activation of PU.1-ER only induced MPO RNA. PU.1 mRNA was rapidly induced by C/EBPa-ER, even in the presence of cycloheximide. Thus, the central hypothesis of aim1 is that C/EBPa activates the PU.1 gene in normal hematopoietic cells. Cis elements in the PU.1 gene which mediate this activation will be identified. The effect of retroviral expression of C/EBPa-ER in C/EBPa-null cells and of transgenic expression of C/EBPa in B cells on PU.1 levels will be determined.
Aim 2 will test the hypothesis that PU.1 is not as effective as C/EBPa for induction of myeloid genes in normal cells. PU.1 will be expressed in C/EBPa-null cells using a retroviral vector and in B cells using the Ig u enhancer. The effect of PU.1 on myeloid genes and on C/EBPa expression will be assessed. Other C/EBPs may compensate for the lack of C/EBPa in C/EBPa null mice. 32D cl3 lines were characterized expressing KRAB-C/EBPa-ER, containing the C/EBPa DNA-binding domain and the KRAB repression domain. Activation of this protein potently inhibits global C/EBP activities, and consequently G-CSF receptor RNA expression is reduced. 32D cl3 cells expressing activated KRAB-C/EBPa-ER and exogenous G-CSF receptor do not differentiate to neutrophils or express MPO RNA, but develop an immature, monocytic morphology. If C/EBP inhibition is relieved after 24 hrs, the cells no longer can mature to neutrophils.
Aims 3 will test the hypotheses that C/EBPs are required for both monopoiesis and granulopoiesis, but that a modest decrease in C/EBP activities is permissive for monopoiesis if exogenous M-CSF receptor is provided. KRAB-C/EBPa-ER will be expressed in 32D cl3 cells or in normal hematopoietic cells, as a transgene or using retroviral vectors, alone, with the G-CSF receptor, or with the M-CSF receptor. Myelopoiesis at various levels of C/EBP activity and progenitor fate after various times of C/EBP inhibition will be assessed.
