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.

Characterization of promoter elements directing Mona/Gads molecular adapter expression in T and myelomonocytic cells: involvement of the AML-1 transcription factor.

Monocytic adaptor (Mona, also called Gads) is a molecular adaptor implicated in T cell activation and macrophage differentiation. The objective of this study was to identify elements regulating specific expression of Mona/Gads in human T cell and myelomonocytic cell lines. We first confirmed that the -2000 to +150 genomic region relative to the Mona gene transcription start site is sufficient to direct specific reporter gene expression in T cell lines, Jurkat, and MOLT-4 and in the immature myeloid cell lines, KG1a and RC2A. Deletion analysis and electrophoresis mobility shift assay identified several cis regulatory elements: overlapping initiator sequences, one interferon response factor-2 (IRF-2)-binding site at position -154, one GC box recognized by Sp1 and Sp3 at position -52, and two acute myeloid leukemia (AML)-1 binding sites at positions -70 and -13. Site-directed mutagenesis experiments indicated a key role of AML-1 for driving Mona expression in T cells and myeloid cells, and involvement of Sp1/Sp3 and IRF-2 transcription factors to modulate Mona expression in a cell-specific manner.

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.

Suppressor of cytokine signaling 1 interacts with the macrophage colony-stimulating factor receptor and negatively regulates its proliferation signal.

Macrophage colony-stimulating factor receptor (M-CSF-R) is a tyrosine kinase that regulates proliferation, differentiation, and cell survival during monocytic lineage development. Upon activation, M-CSF-R dimerizes and autophosphorylates on specific tyrosines, creating binding sites for several cytoplasmic SH2-containing signaling molecules that relay and modulate the M-CSF signal. Here we show that M-CSF-R interacts with suppressor of cytokine signaling 1 (Socs1), a negative regulator of various cytokine and growth factor signaling pathways. Using the yeast two-hybrid system, in vitro glutathione S-transferase-M-CSF-R pull-down, and in vivo coimmunoprecipitation experiments, we demonstrated a direct interaction between the SH2 domain of Socs1 and phosphorylated tyrosines 697 or 721 of the M-CSF-R kinase insert region. Moreover, Socs1 is tyrosine-phosphorylated in response to M-CSF. Ectopic expression of Socs1 in FDC-P1/MAC and EML hematopoietic cell lines decreased their growth rates in the presence of limiting concentrations of M-CSF. However, Socs1 expression did not totally suppress long term cell growth in the presence of saturating M-CSF concentrations, in contrast to other cytokines such as stem cell factor and interleukin 3. Taken together, these results suggest that Socs1 is an M-CSF-R-binding partner involved in negative regulation of proliferation signaling and that it differentially affects cytokine receptor signals.

Expression of Mona (monocytic adapter) in myeloid progenitor cells results in increased and prolonged MAP kinase activation upon macrophage colony-stimulating factor stimulation.

Mona is an SH3 and SH2 domain-containing adapter molecule that is induced during monocytic differentiation. Here we have first shown that M-CSFR is the major Mona partner in M-CSF signaling, the interaction being mediated through tyrosine 697 of the receptor. Next we asked whether Mona expression would alter the Ras/MAP kinase pathway since Mona is a likely competitor of Grb2 for binding to M-CSFR. We found that M-CSF induced late and massive phosphorylation of ERK molecules in Mona-expressing myeloid cells compared to non-expressing cells. These results suggest that Mona expression might modify M-CSF signaling during monocytic differentiation.

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.

Role of the membrane form of human colony-stimulating factor-1 (CSF-1) in proliferation of multipotent hematopoietic FDCP-mix cells expressing human CSF-1 receptor.

Because IL-3-dependent multipotential FDCP-Mix cells expressing human colony-stimulating factor-1 (CSF-1) receptor did not proliferate in response to soluble CSF-1, we investigated whether their proliferation would be induced in co-culture with adherent cells expressing the membrane form of CSF-1 (MemCSF-1). FDCP-Mix cells with high CSF-1R expression (NAF21 cells) were placed on stromal MS-5 cells or STO fibroblasts expressing MemCSF-1 (2M-1 cells and STO-M2 cells, respectively), in absence of IL-3. NAF21 cells bound significantly to 2M-1 cells as compared to control FDCP-Mix cells. Adhesion of NAF21 cells was inhibited by anti-huCSF-1 antibodies, as well as anti-huCSF-1R antibodies. Interestingly, NAF21 cells proliferated on both 2M-1 and STO-M2 cells but with very different kinetics. Moreover, NAF21 cell proliferation was also supported by glutaraldehyde-fixed 2M-1 cells or highly concentrated MS-5 cell culture supernatant, but not by CSF-1 coated on culture dishes. These results strongly suggest that MemCSF-1/CSF-1R interaction mediates a specific adhesion of NAF21 cells to stromal cells and allows stimulation of hematopoietic cells by stromal cell-derived factors expressed in a membrane-bound form or concentrated within the extracellular matrix. Thus, cytokine receptors deficient in mitogenic signalling may nevertheless have a regulatory role in hematopoietic progenitor cell proliferation by acting as adhesion molecules.

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.

Colony-stimulating factor-1 impairs both proliferation and differentiation signals of erythropoietin during the commitment of bipotential NFS-60 cell line to the monocytic lineage.

The interleukin-3 (IL-3) dependent cell line NFS-60 contains bipotential progenitors that exhibit both erythroid and myelomonocytic potentials. In order to study their commitment to the monocytic lineage, NFS-60 cells were retrovirally transduced with mouse c-fms cDNA, which encodes the colony-stimulating factor-1 receptor (CSF-1R), resulting in the N-Fms cell line. N-Fms cells proliferated in response to CSF-1 with a growth rate similar to that obtained in response to IL-3 and progressively differentiated from myeloid blasts to monocytic cells within 3 days of culture. When maintained in IL-3, about 3% of N-Fms cells formed large hemoglobinized colonies in semisolid cultures supplemented with erythropoietin (EPO). However, this property was lost after a 24-hour cultivation in the presence of CSF-1 or, interestingly, both CSF-1 and IL-3. This loss of response to EPO was reverted following a brief passage (24 hours) in IL-3, but the rescued colonies did not undergo terminal erythrocytic differentiation. Furthermore, CSF-1 also affected proliferative response to EPO of N-Fms cells constitutively expressing EPO receptors. Our data strongly suggest that CSF-1 can suppress erythroid potential in bipotential N-Fms cells by altering proliferative and differentiation signal of EPO.

Unexpected and coordinated expression of Spi-1, Fli-1, and megakaryocytic genes in four Epo-dependent cell lines established from transgenic mice displaying erythroid-specific expression of a thermosensitive SV40 T antigen.

Most erythroleukemic cell lines established in vitro coexpress erythrocytic and megakaryocytic markers that often are associated with expression of Spi-1 and/or Fli-1 transcription factors known as transactivators of megakaryocyte-specific promoters. In the present study, we examined the possibility of establishing new cell lines keeping strictly erythroid-specific properties in vitro through the targeted and conditional immortalization of erythrocytic progenitors. For that purpose, we established several lines of transgenic mice displaying erythroid-specific expression of a thermosensitive SV40 T antigen. As expected, these transgenic mice developed splenomegaly due to the massive amplification of Ter 119 positive erythroid nucleated cells expressing T antigen. Despite this drastic effect in vivo, the in vitro immortalization of erythropoietin-dependent erythroid progenitors unexpectedly occurred at low frequency, and all four cell lines established expressed both erythrocytic (globins) and megakaryocytic markers (glycoprotein IIb, platelet factor 4) as well as Spi-1 and Fli-1 transcripts at permissive temperature. Switching the cells to the nonpermissive temperature led to a marked increase in globin gene expression and concomitant decrease in expression of Spi-1, Fli-1, and megakaryocytic genes in an erythropoietin-dependent manner. Interestingly, enhanced expression of Spi-1 and Fli-1 genes already was detected in the Ter 119 positive cell population of transgenic mice spleen in vivo. However, like normal Ter 119 erythroid cells, these Ter 119 positive cells from transgenic mice still expressed high levels of beta-globin and very low or undetectable glycoprotein IIb and platelet factor 4 megakaryocytic transcripts. Taken together, these data indicate that the unexpected expression of megakaryocytic genes is a specific property of immortalized cells that cannot be explained only by enhanced expression of Spi-1 and/or Fli-1 genes.

A novel growth-factor-dependent myeloid cell line derived from mouse bone marrow cells contains progenitors endowed with high proliferative potential.

Constitutive expression of human colony-stimulating factor-1 receptor (CSF-1R) confers long-lasting CSF-1-dependent proliferation to mouse myeloid cell lines. We developed mice transgenic for human CSF-1R because mouse CSF-1 cannot activate human CSF-1R. Then bone marrow cells from transgenic mice were plated onto MS-5 stromal cells expressing the membrane form of human CSF-1 (2M-1 cells) in order to combine the hematopoietic supporting properties of stromal cells and the proliferative effects of CSF-1. Thus, we were able to derive a hematopoietic cell line, called 47.10, that grew indefinitely under these conditions, whereas no cell line could be developed from nontransgenic mice. Proliferation of 47.10 cells is severely affected by neutralizing anti-CSF-1R monoclonal antibodies. Morphologic and cytofluorometry analysis established that most 47.10 cells are immature myelomonocytic cells. Consistent with this phenotype, the myeloid transcription factor PU.1, but not the erythroid transcription factor GATA-1, is expressed in 47.10 cells. A few 47.10 cells (3-5%) do not express lineage specific markers; they differentiate spontaneously to lineage-positive cells after replating on 2M-1 cells. In agar cultures, 47.10 cells form 7- and 14-day colonies in response to a cocktail of granulocyte/macrophage colony-stimulating factor (2.5 ng/mL), interleukin-3 (1 ng/mL), and mouse CSF-1 (10 ng/mL). Under these conditions, about 0.5% of 47.10 cells formed large 14-day colonies (>1 mm) composed of mature monocytes and granulocytes, reflecting the presence of progenitors endowed with high proliferative potential (HPP-47.10 cells). In conclusion, we have characterized a novel continuous myeloid cell line presenting a hierarchical structure similar to that of the bone marrow progenitor cell compartment.

Mona, a novel hematopoietic-specific adaptor interacting with the macrophage colony-stimulating factor receptor, is implicated in monocyte/macrophage development.

The production, survival and function of monocytes and macrophages are regulated by the macrophage colony-stimulating factor (M-CSF or CSF-1) through its tyrosine kinase receptor Fms. Binding of M-CSF results in Fms autophosphorylation on specific tyrosines that act as docking sites for intracellular signaling molecules containing SH2 domains. Using a yeast two-hybrid screen, we cloned a novel adaptor protein which we called 'Mona' for monocytic adaptor. Mona contains one SH2 domain and two SH3 domains related to the Grb2 adaptor. Accordingly, Mona interacts with activated Fms on phosphorylated Tyr697, which is also the Grb2-binding site. Furthermore, Mona contains a unique proline-rich region located between the SH2 domain and the C-terminal SH3 domain, and is apparently devoid of any catalytic domain. Mona expression is restricted to two hematopoietic tissues: the spleen and the peripheral blood mononuclear cells, and is induced rapidly during monocytic differentiation of the myeloid NFS-60 cell line in response to M-CSF. Strikingly, overexpression of Mona in bone marrow cells results in strong reduction of M-CSF-dependent macrophage production in vitro. Taken together, our results suggest an important role for Mona in the regulation of monocyte/macrophage development as controlled by M-CSF.

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).

Modulation of human and mouse erythropoiesis by thyroid hormone and retinoic acid: evidence for specific effects at different steps of the erythroid pathway.

Receptors for L-triiodothyronine (T3) and alltrans retinoic acid (ATRA) are DNA-binding proteins that can form transcriptionally active heterodimers. In this study, we sought whether T3 and ATRA could cooperate to modulate human and mouse erythropoiesis in vitro. Effects of T3 and ATRA were first assessed on burst forming unit-erythroid (BFU-E) proliferation and differentiation in semi-solid cultures. T3 did not alter the cloning efficiency of BFU-E but it decreased the production of colony forming unit-erythroid (CFU-E) during the course of BFU-E development. In contrast to T3, ATRA inhibited the early steps of BFU-E proliferation. ATRA and T3 acted in a dose-dependent manner with optimal effects at 10(-6) M and 10(-8) M, respectively. Furthermore, T3 and ATRA used in combination had more pronounced effects than when used alone, but only at their respective optimal concentrations, indicating that these effects were additive rather than synergistic. Similar results were obtained with unfractionated mouse bone marrow cells or human CD34+ bone marrow cells, suggesting that the effects of T3 or ATRA were not mediated by accessory cells. This study was extended to the mouse IL-3-dependent NFS-60 cells that can differentiate in vitro into mature erythroid cells in response to erythropoietin (Epo). When used alone, neither T3 nor ATRA could affect NFS-60 cell proliferation in response to Epo; however, T3 and ATRA had an anti-proliferative effect when used together. In addition, T3-dramatically reduced the proportion of hemoglobinized colonies in Epo-stimulated cultures of NFS-60 cells. Furthermore, ATRA, but not T3, could inhibit the IL-3-dependent proliferation of NFS-60 cells. Altogether these data suggest that T3 and ATRA can cooperate in modulating in vitro erythropoiesis although having individual effects at different but overlapping steps along the erythroid pathway.

Isolation and characterization of c-fos-expressing murine bone marrow stromal cell lines supporting myeloid differentiation.

We have previously reported that constitutive expression of c-fos oncogene allows long-term proliferation of primary mouse bone marrow stromal cells favoring the granulocytic differentiation of myeloid precursors in an in vitro assay. Retrovirus-mediated gene transfer of the human c-fos gene was used here for immortalizing nine mouse bone marrow cell lines which were studied in detail. However, due to low expression of the ectopic c-fos gene, none of them showed characteristics of transformation as assayed by dependence upon serum for growth, the inability to form colonies in agar and contact inhibition. All of them displayed a fibroblastoid phenotype, as deduced from morphological observation and analysis of several differentiation markers. They mostly supported the granulocytic differentiation of bone marrow myeloid precursors in a GM-assay, as did c-fos-expressing primary stromal cells. Their potential for supporting myeloid progenitor proliferation was however significantly lower than that of the whole adherent layer of the Dexter-type long-term bone marrow culture they derived from (STNT cells). They showed significant variations with respect to their cytokine gene expression analyzed at the RNA level in keeping with the notion of stromal cell heterogeneity in the bone marrow. Interestingly, none of them secreted GM-CSF, SCF or IL-3, which are cytokines reputed for their ability to stimulate hematopoietic progenitors, and strikingly, only two of them were able to produce detectable levels of G-CSF in culture supernatants despite the propensity of all of them to favor granulocyte differentiation. Finally, in coculture assay, bone marrow cells were able to diminish the expression of several cytokine genes albeit at a much lower degree than in the original STNT cells.

Expression of functional human CSF-1 receptors on mouse bone marrow erythroid progenitor cells.

We have previously shown that a murine multipotent hematopoietic cell line could proliferate in response to CSF-1 after retroviral transfer of human CSF-1 receptor (CSF-1R) gene (c-fms) without loss of the erythroid differentiation potential. In the light of these data, we asked whether ectopic expression of human c-fms gene would lead to the conditional immortalization of murine hematopoietic progenitor cells. In the present studies, murine bone marrow cells were infected with recombinant retroviruses containing the human c-fms cDNA. We found that CSF-1 could exert a stimulatory activity on erythroid progenitors in the presence of Epo only when bone marrow cells had been previously infected with c-fms retroviruses. In addition, expression of human CSF-1R in murine Epo-dependent, v-src-immortalized cells resulted in CSF-1-dependent proliferation of these cells in the absence of Epo. These data show that human CSF-1R (i) can stimulate early bone marrow erythroid progenitors, (ii) might require additional signals provided by oncogenes or cytokine receptors to transduce a mitogenic signal into mouse bone marrow erythroid progenitors.

Expression of human colony-stimulating factor-1 (CSF-1) receptor in murine pluripotent hematopoietic NFS-60 cells induces long-term proliferation in response to CSF-1 without loss of erythroid differentiation potential.

NFS-60 and FDCP-Mix cells are interleukin-3--dependent multipotent hematopoietic cells that can differentiate in vitro into mature myeloid and erythroid cells. Retrovirus-mediated transfer of the human colony-stimulating factor-1 (CSF-1) receptor gene (c-fms) enabled NFS-60 cells but not FDCP-Mix cells to proliferate in response to CSF-1. The phenotype of NFS-60 cells expressing the human CSF-1 receptor (CSF-1R) grown in CSF-1 did not grossly differ from that of original NFS-60 as assessed by cytochemical and surface markers. Importantly, these cells retained their erythroid potentiality. In contrast, a CSF-1-dependent variant of NFS-60, strongly expressing murine CSF-1R, differentiated into monocyte/macrophages upon CSF-1 stimulation and almost totally lost its erythroid potentiality. We also observed that NFS-60 but not FDCP-Mix cells could grow in response to stem cell factor, (SCF), although both cell lines express relatively high amounts of SCF receptors. This suggests that SCF-R and CSF-1R signalling pathways share at least one component that may be missing or insufficiently expressed in FDCP-Mix cells. Taken together, these results suggest that human CSF-1R can use the SCF-R signalling pathway in murine multipotent cells and thereby favor self-renewal versus differentiation.

Murine interleukin 9 stimulates the proliferation of mouse erythroid progenitor cells and favors the erythroid differentiation of multipotent FDCP-mix cells.

Murine interleukin 9 (mIL-9) is a T-cell-derived growth factor that stimulates erythroid burst-forming units (BFU-E) from murine bone marrow. We further investigated this activity using enriched mouse bone marrow progenitors and the multipotent interleukin 3 (IL-3)-dependent FDCP-Mix cell line. We report here that mIL-9 stimulates erythroid burst formation of total bone marrow cells and accessory cell-depleted bone marrow cells, even in serum-free cultures. On the other hand, we observed that although mIL-9 could not support proliferation of FDCP-Mix cells, it favors erythroid differentiation of these cells in the presence of both IL-3 and erythropoietin. These results strongly suggest that mIL-9 acts directly on mouse erythroid progenitor cells.

Insulin-like growth factor-1 stimulates proliferation of myeloid FDC-P1 cells overexpressing the human colony-stimulating factor-1 receptor.

Retrovirally expressed human CSF-1 receptor can induce CSF-1-dependent growth of IL-3-dependent hemopoietic cells FDC-P1. Here we show that expression of the human CSF-1 receptor also allowed FDC-P1 cells to grow in response to Insulin-like Growth Factor-1 (IGF-I). The authentic receptor for IGF-I was identified by affinity cross-linking and binding analysis on both control (infected with a neo vector) and CSF-1 receptor expressing FDC-P1 cells. DNA and RNA analysis of these cells and of five clones of IGF-I responsive cells demonstrated that the IGF-I receptor gene was not rearranged nor was it abnormally expressed in IGF-I responsive cells. These results suggest that myeloid cells over-expressing CSF-1R (c-fms protooncogene product) might have a proliferative advantage over normal myeloid cells in a physiological situation, independently of the presence of CSF-1 or the capacity of the cells to respond to CSF-1. This would indicate a possible role for c-fms in human neoplasia.