Rapid and Irreversible Activation of MAPK Kinase Pathway during Myelopoiesis.

Mononuclear phagocytes are essential for innate immunity as well as wound healing, tissue remodeling and angiogenesis. We hypothesize that the state of macrophage maturation may prolong chronic inflammation. Despite finding that normal macrophage maturation is disrupted in some myeloid leukemias and the identification of several signal transduction pathways involved in myelopoiesis, little is known about steps involved in this maturational pathway. We examined the pathways responsible for macrophage differentiation in the myeloid leukemic THP-1 cell line either treated with PMA (to induce differentiation) or with DMSO (vehicle) as a model. We found that treatment of THP-1 cells with PMA resulted in cells becoming macrophage-like as indicated by cellular adherence and expression of cell surface antigens. To correspond with surface expression of CD68, MHC II, CD11b, mannose receptor and CD14, we further characterized macrophage differentiation at the molecular level. Based on microarray and proteomics data comparing human monocytes to macrophages, we selected several genes to monitor during the process of myeloid differentiation. By RT-PCR analysis, we found increase expression of cathespins B and H, metalloproteinases (MMP9 and MMP2), and interleukin 4 inducible gene 1 (IL4-i1), whereas expression of leukocyte inhibitory factor (LEI) and calgranulin B decreased during differentiation. Furthermore, PMA-induced differentiation of THP-1 cells required only 15 min stimulation. We next examined the intracellular pathways mediating differentiation of THP1 cells. In the presence of PMA and the Mek inhibitor, U0126, THP-1 cells failed to become adherent, did not express macrophage surface antigens, did not upregulate expression of MMP9 or IL4-il, and calgranulin B expression was maintained indicating that the cells were still immature. However, when cells were pretreated with PMA for 1 hr prior to adding U0126, the cells continued to differentiate. Cytosolic and nuclear fractions were isolated from cells treated with PMA in the absence or presence of U0126 and found that U0126 did not prevent the phosphorylation of Erk1/2 but inhibited their nuclear translocation. In the absence of pErk1/2 nuclear translocation in the U0126-treated cells, there was no increase in expression of p21 CIP1 , indicating that cell arrest did not occur. Since maturation of these cells was associated with the nuclear translocation of both Erk1/2, we were interested in determining which Erk isoform was essential for differentiation. THP-1 cells were transiently transfected with either DN-Erk1 or DN-Erk2 or both then treated with PMA. There was no change in cellular adherence when DN-Erk1 was expressed alone but there was a two-fold decrease in adherence in the DN-Erk2 transfected cells as measured by crystal violet staining. Since MAPK kinase pathway was critical in regulating THP-1 differentiation, we examined whether this was conserved in GM-CSF-induced human peripheral blood monocyte (PBM) maturation to macrophages. We found that GM-CSF-induced the phosphorylation of Erk1/2 within 15 min of treatment and was still detectable at d3 and d7. Treatment of PBM with U0126 inhibited GM-CSF-induced differentiation. Similar to that seen in THP-1 cells, U0126 failed to inhibit differentiation in PBM pretreated with GM-CSF for 1h. Our observations suggest that commitment to differentiate occurs very rapidly and is irreversible under certain conditions. Dissection of the role of Mek and Erk kinases in myeloid differentiation may lead to the identification of therapeutic targets in myeloid leukemia.