Src-family kinases play an essential role in differentiation signaling downstream of macrophage colony-stimulating factor receptors mediating persistent phosphorylation of phospholipase C-gamma2 and MAP kinases ERK1 and ERK2.

Macrophage colony-stimulating factor (M-CSF) has been found to be involved in multiple developmental processes, especially production of cells belonging to the mononuclear phagocyte system. The decision of myeloid progenitor cells to commit to differentiation depends on activation levels of the mitogen-activated protein kinases (MAPK), ERK1 and ERK2. Using the murine myeloid progenitor cell line FD-Fms, we show here that persistent activity of Src-family kinases (SFK) is necessary for FD-Fms cell differentiation to macrophages in response to M-CSF. Chemical inhibition of SFK blocked FD-Fms cell differentiation while it caused strong inhibition of the late phosphorylation of phospholipase C (PLC)-gamma2 and MAPK. The PLC inhibitor U73122, previously shown to block M-CSF-induced differentiation, strongly decreased long-term MAPK phosphorylation. Interestingly, inhibiting SFK with SU6656 or the MAPK kinases MEK with U0126 significantly impaired development of mononuclear phagocytes in cultures of mouse bone marrow cells stimulated with M-CSF. Collectively, results support a model in which SFK are required for sustained PLC activity and MAPK activation above threshold required for commitment of myeloid progenitors to macrophage differentiation.

E2a/Pbx1 oncogene inhibits terminal differentiation but not myeloid potential of pro-T cells.

E2a/Pbx1 is a fusion oncoprotein resulting from the t(1;19) translocation found in human pre-B acute lymphocytic leukemia and in a small number of acute T-lymphoid and myeloid leukemias. It was previously suggested that E2a/Pbx1 could cooperate with normal or oncogenic signaling pathways to immortalize myeloid and lymphoid progenitor cells. To address this question, we introduced the receptor of the macrophage-colony-stimulating factor (M-CSF-R) in pro-T cells immortalized by a conditional, estradiol-dependent, E2a/Pbx1-protein, and continuously proliferating in response to stem cell factor and interleukin-7. We asked whether M-CSF-R would be functional in an early T progenitor cell and influence the fate of E2a/Pbx1-immortalized cells. E2a-Pbx1 immortalized pro-T cells could proliferate and shifted from lymphoid to myeloid lineage after signaling through exogenously expressed M-CSF-R, irrespective of the presence of estradiol. However, terminal macrophage differentiation of the cells was obtained only when estradiol was withdrawn from cultures. This demonstrated that M-CSF-R is functional for proliferation and differentiation signaling in a T-lymphoid progenitor cell, which, in addition, unveiled myeloid potential of pro-T progenitors. Moreover, the block of differentiation induced by the E2a/Pbx1 oncogene could be modulated by hematopoietic cytokines such as M-CSF, suggesting plasticity of leukemic progenitor cells. Finally, additional experiments suggested that PU.1 and eight twenty-one transcriptional regulators might be implicated in the mechanisms of oncogenesis by E2a/Pbx1.

Genomic organization and restricted expression of the human Mona/Gads gene suggests regulation by two specific promoters.

Monocytic adaptor (Mona) also known as Gads is a Grb2-related adaptor whose expression is restricted to hematopoietic cells. It plays an important role in intracellular signaling in T cells, monocytic cells, and platelets. Here we investigated the regulatory aspects of Mona expression in human hematopoietic cells. This was carried out by combining nucleotide sequence analyzes and experimental approaches. We confirmed that Mona expression is restricted to T-cell, myeloid and platelet lineages. In the various cells examined, we detected two major Mona transcripts (1.9 and 4 kb), likely resulting from the alternative use of two polyadenylation sites. Consequently, Mona transcripts of the same size have identical 3' untranslated region (UTR), irrespective of the cell type. In contrast, Mona transcripts contain either 5' UTR-1A or -1B exons, that were detected in a cell-lineage specific manner. Thus, T cells and several myeloid cell lines express 5' UTR-1A-containing transcripts, whereas platelets and cell lines exhibiting megakaryocytic potential express 5' UTR-1B-containing transcripts. Interestingly, 5' UTR-1A is generated from an exon located approximately 45 kb upstream of exon 1B. This suggested that lineage-restricted transcription of the Mona gene is controlled by specific promoters. Indeed, 2-kb genomic fragments upstream of each 5'-UTR showed lineage-restricted ability to drive expression of luc reporter gene.

Receptor for macrophage colony-stimulating factor transduces a signal decreasing erythroid potential in the multipotent hematopoietic EML cell line.

OBJECTIVE: To test the hypothesis that hematopoietic growth factors may influence lineage choice in pluripotent progenitor cells, we investigated the effects of macrophage colony-stimulating factor (M-CSF) on erythroid and myeloid potentials of multipotent EML cells ectopically expressing M-CSF receptor (M-CSFR). METHODS: EML cells are stem cell factor (SCF)-dependent murine cells that give rise spontaneously to pre-B cells, burst-forming unit erythroid (BFU-E), and colony-forming unit granulocyte macrophage (CFU-GM). We determined BFU-E and CFU-GM frequencies among EML cells transduced with murine M-CSFR, human M-CSFR, or chimeric receptors, and cultivated in the presence of SCF, M-CSF, or both growth factors. Effects of specific inhibitors of signaling molecules were investigated. RESULTS: EML cells transduced with murine M-CSFR proliferated in response to M-CSF but also exhibited a sharp and rapid decrease in BFU-E frequency associated with an increase in CFU-GM frequency. In contrast, EML cells expressing human M-CSFR proliferated in response to M-CSF without any changes in erythroid or myeloid potential. Using chimeric receptors between human and murine M-CSFR, we showed that the effects of M-CSF on EML cell differentiation potential are mediated by a large region in the intracellular domain of murine M-CSFR. Furthermore, phospholipase C (PLC) inhibitor U73122 interfered with the negative effects of ligand-activated murine M-CSFR on EML cell erythroid potential. CONCLUSION: We propose that signaling pathways activated by tyrosine kinase receptors may regulate erythroid potential and commitment decisions in multipotent progenitor cells and that PLC may play a key role in this process.

Differential effects of human erythroid burst stimulating activity (HuEBSA) on human cord blood burst forming units-erythroid (BFU-Es) as a function of their differentiation state.

An erythroid stimulating activity which promotes the growth of small bursts probably arising from mature burst forming units-erythroid (BFU-Es) of adult human bone marrow cells and called human erythroid burst stimulating activity (HuEBSA), was previously found in media conditioned by a fetal human kidney cell line. In the present work we report that adding HuEBSA to cultures did not increase the burst number but increased the size of bursts from cord blood (CB) cells. A similar observation was made using stem cell factor (SCF). However, a synergistic effect on the burst number was noted when both HuEBSA and SCF were introduced to cultures. We also noticed that CB erythroid progenitors pre-cultured with 5637-Conditioned Medium [as a source of burst promoting activity (BPA)] and erythopoietin (Epo) for 3 days could be stimulated by HuEBSA but not by SCF. Similar results were obtained when interleukin 3 (IL-3) was introduced with Epo to the pre-cultures. These results suggest that two different populations of erythroid progenitors coexist in cord blood, one is Epo- and IL-3-sensitive, the other solely Epo-sensitive. It also seems probable that HuEBSA acts on erythroid progenitors arising from the more immature erythroid population, since its stimulating activity was evident after a 3-day pre-culture of cord blood cells in Epo and IL-3.

A human cell line isolated from fetal kidney produces an apparently erythroid-specific stimulating activity.

The burst formation from human and murine burst forming unit-erythroid (BFU-E) requires the presence of erythropoietin (Epo) in semi-solid cultures of bone marrow cells. A number of haematopoietic factors are described that increase the burst number: interleukin 3 (IL-3), stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-9, IL-11, insulin-like growth factor I, and erythroid potentiating activity (EPA). The authors now show that another activity present in medium conditioned from adult or fetal human kidney cells specifically stimulates the proliferation of BFU-E. A cell line derived from fetal kidney produced such an activity, which was shown to be different from the previously cited haematopoietins, acted on CD34(+)-enriched BFU-E and promoted an increase in CFU-E number in the bone marrow of injected animals, could be precipitated using 40% ammonium sulfate, was destroyed by proteolytic enzymes and was shown to be a glycoprotein by its retention on ConA-Sepharose. The authors propose to call this apparently novel activity, which influences only the number of bursts, human erythroid burst-stimulating activity (hEBSA).

Erythropoiesis in long term cultures of foetal liver cells is transiently obtained on adult but not on foetal adherent cell layers.

We have previously reported long term erythroid differentiation of adult bone marrow cells seeded onto adherent cells derived from adult bone marrow. In this paper, we show that the adherent cells obtained from foetal liver do not support the erythroid differentiation of either adult bone marrow cells or foetal liver cells. Adherent layers derived from bone marrow of adult W/Wv mice supported differentiation of adult bone marrow precursors, but foetal liver progenitors only produced erythrocytes for a few weeks and the foetal origin of these red cells was confirmed by haemoglobin typing. The duration and extent of erythropoiesis was generally inversely proportional to the cell dose. Foetal progenitors were as sensitive to erythropoietin as adult cells, but were optimally stimulated at a lower plateau concentration. These results suggest that inhibitory cells present in foetal liver may block erythropoiesis and their growing importance with age may provide an explanation for the arrest of erythropoiesis in the liver at late developmental stages.

Erythroid precursors cultured from adult mice are sensitive to H2O2 toxicity.

The growth of late erythroid precursors (CFU-Es) from adult bone marrow is inhibited when Iscove 's modified Dulbecco's medium supplied in liquid form is used. Catalase and other H2O2 destroying compounds restore the capacity of culture medium to support colony development. However early precursors from adult bone marrow and fetal liver CFU-Es were resistant to H2O2.

Detection of a fetal antigen on mouse erythroleukemic Friend cells.

A fetal antigen, expressed on all fetal erythrocytes during normal ontogeny, was detected on Friend erythroleukemic cells but not on circulating erythrocytes from leukemic mice. Friend cells were shown to express the fetal antigen both by immunofluorescence and antiserum absorption. The fetal antigen thus allows a clearcut distinction between the tumoral step from which emerge the Friend and erythro-proliferative cells.