Structure-function studies on human macrophage colony-stimulating factor (M-CSF)

M-CSF (CSF-1) can be produced in a variety of structural forms that may affect function in vivo. Truncated, nonglycosylated forms of recombinant M-CSF (rM-CSF) from E. coli have been refolded in vitro in high yield and shown to be functionally equivalent in vitro to glycosylated rM-CSF secreted from mammalian cells. An N-terminal domain of 149 amino acids is produced by all of the known M-CSF mRNA splice variants and is the region responsible for bioactivity observed in vitro. Heterodimeric rM-CSFs from different splice variants containing this domain were produced in pure form by refolding in vitro, and are fully active, but have yet to be observed in vivo. The circulating half-life of truncated M-CSF forms injected intravenously into rats increased with the MW of the M-CSF used. Large increases in half-life in vivo were observed following chemical addition of a single molecule of 10 kD polyethylene glycol to rM-CSF in vitro. The crystal structure of rM-CSF revealed that M-CSF is a member of a family of molecules related by having a distinctive four-helical-bundle structural core. Site-directed mutagenesis showed that residues in or near helix A and helix C are involved in receptor binding, as reflected by decreased bioactivity and receptor binding of certain mutants. A soluble form of the M-CSF receptor, c-fms, was produced in a baculovirus/Sf9 expression system and purified to homogeneity. The MW of rM-CSF saturated with this soluble receptor was determined by molecular sieve chromatography and light scattering. Each dimeric M-CSF molecule appears to bind two soluble receptor molecules in vitro, supporting the observation that M-CSF signaling is linked to receptor dimerization. Mol Reprod Dev 46:31–38, 1997. © 1997 Wiley-Liss, Inc.     

Differentiation of the IL-3-dependent NFS-60 cell line and adaption to growth in macrophage colony-stimulating factor

Macrophage CSF (M-CSF) induces responsive bone marrow precursors into rapid growth and differentiation to mature macrophages. Available cell lines that depend on M-CSF for growth are well differentiated and rather adherent. We investigated the effects of M-CSF on immature myeloid cell lines as models of the marrow precursors. The murine line NFS-60 requires IL-3 for growth and also responds to granulocyte-CSF and granulocyte-macrophage-CSF. Cultures of one NFS-60 subline, when switched from IL-3 to 10% L cell conditioned media, a source of M-CSF, or purified M-CSF, frequently acquired large, adherent cells. The adherent cells grew slowly in the presence of M-CSF, in contrast to the majority population of small, round, rapidly growing cells. The large cells had properties of differentiated macrophages that were absent in the nonadherent cells. Cells with macrophage phenotype were not observed in IL-3-supported cultures over many passages. A subline was derived from NFS-60 that grew rapidly and continuously in human or murine M-CSF as round, nonadherent cells. The line, called M-NFS-60, responded well to M-CSF and IL-3, weakly to granulocyte-CSF and not at all to murine granulocyte-macrophage-CSF, IL-4, or human IL-1. A mAb to human M-CSF specifically inhibited only M-NFS-60 proliferation induced by the human growth factor, whether produced by mammalian or bacterial cells. This study shows two effects of M-CSF on the IL-3-dependent NFS-60 line. Upon first exposure to M-CSF, cells may undergo global differentiation to slowly replicating macrophages in conditions we have not been able to define. The more common effect is rapid growth of immature myeloid cells like the bone marrow precursors, but with a block to differentiation. Thus, these cells may be useful as models of M-CSF-induced differentiation, and of permanently maintained macrophage precursors.     

Macrophage colony-stimulating factor pretreatment of bone marrow progenitor cells regulates osteoclast differentiation based upon the stage of myeloid development

Osteoclasts (OCs) are large, multinucleated bone resorbing cells originating from the bone marrow myeloid lineage, and share a common progenitor with macrophages and dendritic cells. Bone marrow cells (BMCs) are a common source for in vitro osteoclastogenesis assays but are a highly heterogeneous mixture of cells. Protocols for in vitro osteoclastogenesis vary considerably thus hindering interpretation and comparison of results between studies. Macrophage colony-stimulating factor (M-CSF) pretreatment is commonly used to expand OC progenitors (OCPs) in BMC cultures before in vitro differentiation. However, the failure of osteoclastogenesis of M-CSF primed bone marrow myeloid blasts has been reported. In this study, we used a simple method of differential adherence to plastic to enrich OCP from mouse BMCs. We found that M-CSF pretreatment of plastic-adherent BMCs (adBMCs) increased the number of CD11b-F4/80+ macrophages and decreased the number of CD11b+ monocytes resulting in decreased OC formation. M-CSF pretreatment of purified c-Kit+ progenitors weakly inhibited OC formation, whereas M-CSF pretreatment of purified c-Kit-CD11b+ progenitors promoted the formation of large OC. M-CSF pretreatment increased the proliferation of both purified c-Kit+ and c-Kit-CD11b+ cells and increased the percentage of CD11b-F4/80+ cells from c-Kit+ progenitors. In addition, M-CSF pretreatment increased the percentage of CD11b+ F4/80− cells from purified c-Kit-CD11b+ cells. M-CSF pretreatment increased the percentage of CD14 + CD16 + intermediate monocytes and subsequent OC formation from human 2adBMCs, and increased OC formation of purified CD14 + cells. Together, these results indicate that in vitro OCP expansion in the presence of M-CSF and bone marrow stromal cells is dependent upon the developmental stage of myeloid cells, in which M-CSF favors macrophage differentiation of multipotent progenitors, promotes monocyte maturation and supports differentiation of late-stage OCP cells.     

Regulation of murine mononuclear phagocyte inflammatory products by macrophage colony-stimulating factor. Lack of IL-1 and prostaglandin E2 production and generation of a specific IL-1 inhibitor

The influence of macrophage (M)-CSF on the production of inflammatory mediators has been examined in murine peritoneal macrophages. Cultures of macrophages treated with up to 30,000 U/ml of human rM-CSF or with 10,000 U/ml of L929-derived M-CSF did not reveal either PGE2, IL-1, or IL-6 secretion. In contrast, LPS, which served as a positive control, stimulated production of significant levels of PGE2, IL-1, and IL-6. Furthermore, Northern blot analysis of macrophage RNA revealed a strong induction of IL-1 alpha and IL-6 mRNA by LPS but not by M-CSF. Conditioned medium from macrophage cultures treated with purified L929 or human rM-CSF in combination with LPS exhibited a significant reduction of IL-1 bioactivity as compared with an LPS challenge alone. To investigate the mechanism involved in this M-CSF-dependent reduction of IL-1 bioactivity, we measured IL-1 alpha gene expression. The addition of M-CSF to LPS-treated macrophages did not affect IL-1 alpha mRNA levels suggesting that M-CSF may regulate production of an IL-1 inhibitor. This hypothesis was shown to be valid because removal of IL-1 alpha from conditioned medium of LPS plus M-CSF-treated cells allowed the detection of a nondialyzable factor that blocked IL-1-dependent thymocyte proliferation. The inhibitor appeared to be specific because it did not inhibit IL-2 and TNF bioactivities. Furthermore, this IL-1 inhibitor, which binds to cells and not to IL-1, competed with the binding of radioactive IL-1 alpha or beta to EL-4.6.1 cells. The results demonstrate that M-CSF alone does not induce proinflammatory mediators and PGE2 as was previously published. The data also suggest that M-CSF may play a role in the down-regulation of inflammatory responses.     

Molecular cloning,expression in Escherichia coli and production of bioactive homogeneous recombinant human granulocyte and macrophage colony stimulating factor

Human granulocyte and macrophage colony stimulating factor (hGM-CSF) is a glycoprotein that activates and enhances the differentiation and survival of neutrophils, eosinophils and macrophages, which play a key role in the innate immune response. Here we describe the construction of the hGM-CSF encoding gene, cloning, expression in Escherichia coli, purification of recombinant hGM-CSF, N-terminal amino acid sequencing, and biological activity assay using TF-1 cells. The results presented show that the combination of experimental strategies employed to obtain recombinant hGM-CSF can yield biologically active protein, and may be useful to scaling-up production of biosimilar protein.     

Expression of a novel recombinant stem cell factor/macrophage colony-stimulating factor fusion protein in baculovirus-infected insect cells

A novel human stem cell factor (SCF)/macrophage colony-stimulating factor (M-CSF) fusion protein gene was constructed, in which the coding regions of human SCF cDNA (1-165aa) and the truncated M-CSF cDNA (1-149aa) were connected by a linker sequence encoding a short peptide GGGGSGGGGSGG. The SCF/M-CSF gene was cloned into baculovirus transfer vector pVL1392 under the control of polyhedrin promoter and expressed in the Sf9 cells (Spodoptera frugiperda). SDS-PAGE and Western blot analysis showed that the purified fusion protein was a homodimer with a molecular weight about 84kDa under non-reducing conditions or a monomer about 42kDa under reducing conditions. The specific activity of rhSCF/M-CSF was 17 times as high as that of monomeric rhSCF to stimulate the proliferation of TF-1 cell. The results of macrophages colony-forming (CFU-M) assay performed with human bone marrow mononuclear cells demonstrated that rhSCF/M-CSF was more potent in promoting CFU-M than the equimolar of SCF, M-CSF or that of two cytokines mixture.     

Properties of primary murine stroma induced by macrophage colony-stimulating factor

The ability of purified human macrophage colony-stimulating factor (M-CSF) to accelerate the formation of stromal cells from murine bone marrow cells was investigated. The liquid culture of the marrow cells with M-CSF resulted in the formation of monolayers of macrophages on day 7. When the M-CSF was removed on that day and the residual adherent cells were cultured in the absence of M-CSF for an additional 7 days, many colonies appeared with cells that were morphologically distinguishable from M-CSF-derived macrophages. The appearance of the colonies was dependent on the concentration of M-CSF used at the beginning of the culture. Each colony was isolated as a single clone and analyzed. All clones were negative for esterase staining. These cells did not express M-CSF receptor mRNA and did not show a mitogenic response to M-CSF. On the contrary, these cells could be stimulated to proliferate by fibroblast growth factor and platelet-derived growth factor. The polymerase chain reaction analysis of these cells demonstrated constitutive expression of mRNA for M-CSF, stem cell factor, and interleukin (IL)-1, but not IL-3. Some clones expressed mRNA for granulocyte/M-CSF and IL-6. We also examined the ability of the cells to maintain murine bone marrow high proliferative potential colony-forming cells (HPP-CFC) in a coculture system. Most of the clones showed a significant increase in total HPP-CFC numbers after 2 weeks of coculture, although the extent of stimulation differed among clones. These results suggested that the colonies established by M-CSF were composed of functional stromal cells that were phenotypically different from macrophages. J. Cell. Physiol. 173:1–9, 1997. © 1997 Wiley-Liss, Inc.     

Chicken myeloid stem cells infected by retroviruses carrying the v-fps oncogene do not require exogenous growth factors to differentiate in vitro

To determine the function of c-fps in chicken macrophages and granulocytic cells we have infected chicken bone marrow cells with retroviruses containing the v-fps oncogene. Normal chicken macrophage progenitors, M-CFCs, give rise to macrophage colonies in semisolid cultures when macrophage colony stimulating factor (M-CSF) is added into the culture medium. Upon infection with v-fps bearing retroviruses, we observed that M-CFCs were induced to develop macrophage colonies in vitro without exogenous M-CSF. This activation results from a direct effect of v-fps on the M-CFCs. No leukemic transformation was observed in the infected colonies. By comparing the effects of several retroviruses, we showed that the induction of M-CFC development is specific to v-fps containing viruses and mediated by the v-fps protein. These observations support the hypothesis that the c-fps gene is involved in the control of proliferation and/or differentiation of myeloid cells.     

Down-regulation of osteoprotegerin production in bone marrow macrophages by macrophage colony-stimulating factor

Macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) induce the differentiation of bone marrow macrophages (BMMs) into osteoclasts. To delineate mechanisms involved, the effect of M-CSF on the production of osteoprotegerin (OPG), decoy receptor of RANKL, in BMMs was investigated. Mouse bone marrow cells were cultured with M-CSF for 4 days and adherent cells formed were used as BMMs. BMMs were cultured with or without M-CSF, and analyzed for expression of OPG and receptor activator of NF-kappaB (RANK; receptor for RANKL) mRNAs by real-time polymerase chain reaction and secretion of OPG by enzyme-linked immunosorbent assay. BMMs expressed macrophage markers, CD115 (c-fms), Mac-1 and F4/80, and showed phagocytotic activity. In addition, BMMs expressed OPG mRNA and secreted OPG into medium. M-CSF inhibited both the OPG mRNA expression and the OPG secretion dose-dependently and reversibly. The expression of RANK mRNA was not significantly affected by M-CSF. The results showed that M-CSF suppresses the OPG production in BMMs, which may increase the sensitivity of BMMs to RANKL.     

Hierarchical down-modulation of hemopoietic growth factor receptors

Granulocytes and macrophages can be produced in vitro when progenitor cells from mouse bone marrow are stimulated by any of four distinct colony stimulating factors, Multi-CSF (IL-3), GM-CSF, G-CSF, and M-CSF (CSF-1). At 0 degrees C the four CSFs do not cross-compete for binding to bone marrow cells, indicating that each has a specific cell surface receptor. However, at 21 degrees C or 37 degrees C, Multi-CSF inhibits binding of the other three CSFs and GM-CSF inhibits binding of G-CSF and M-CSF. Rather than competing directly for receptor binding, the binding of Multi-CSF, GM-CSF, or G-CSF to their own receptor induces the down-modulation (and thus activation) of other CSF receptors at 37 degrees C. The pattern and potency of down-modulation activity exhibited by each type of CSF parallels the pattern and potency of its biological activity. We propose a model in which the biological interactions of the four CSFs are explained by their ability to down-modulate and activate lineage-specific receptors.     

Prostaglandin E2 regulates macrophage colony stimulating factor secretion by human bone marrow stromal cells.

Bone marrow stromal cells regulate marrow haematopoiesis by secreting growth factors such as macrophage colony stimulating factor (M-CSF) that regulates the proliferation, differentiation and several functions of cells of the mononuclear-phagocytic lineage. By using a specific ELISA we found that their constitutive secretion of M-CSF is enhanced by tumour necrosis factor-alpha (TNF-alpha). The lipid mediator prostaglandin E2 (PGE2) markedly reduces in a time- and dose-dependent manner the constitutive and TNF-alpha-induced M-CSF synthesis by bone marrow stromal cells. In contrast, other lipid mediators such as 12-HETE, 15-HETE, leukotriene B4, leukotriene C4 and lipoxin A4 have no effect. EP2/EP4 selective agonists (11-deoxy PGE1 and 1-OH PGE1) and EP2 agonist (19-OH PGE2) inhibit M-CSF synthesis by bone marrow stromal cells while an EP1/EP3 agonist (sulprostone) has no effect. Stimulation with PGE2 induces an increase of intracellular cAMP levels in bone marrow stromal cells. cAMP elevating agents (forskolin and cholera toxin) mimic the PGE2-induced inhibition of M-CSF production. In conclusion, PGE2 is a potent regulator of M-CSF production by human bone marrow stromal cells, its effects being mediated via cAMP and PGE receptor EP2/EP4 subtypes.     

A recombinant human G-CSF/GM-CSF fusion protein from E. coli showing colony stimulating activity on human bone marrow cells

Granulocyte-Macrophage colony stimulating factor (GM-CSF) and Granulocyte colony stimulating factor (G-CSF) are cytokines involved in the differentiation of bone marrow progenitor cells into myeloid cells. They also activate mature myeloid cells to mediate a variety of antimicrobial activities and inflammatory responses. Recombinant GM-CSF and G-CSF proteins have been used to treat various diseases including cancer and hematopoietic diseases and to isolate peripheral blood progenitor cells for bone marrow transplantation. A plasmid construct expressing recombinant human G-CSF/GM-CSF fusion protein has now been prepared by linking the human G-CSF and GM-CSF coding regions and the recombinant fusion protein has been successfully expressed in E. coli. The recombinant human G-CSF/GM-CSF fusion protein was extracted and purified from the cellular inclusion and refolded into the biologically active form to show colony stimulating activity. The recombinant fusion protein exhibited colony stimulating activity on human bone marrow cell cultures, indicating that the linkage of GM-CSF and G-CSF by a linker peptide may not interrupt activities of the cytokines in the fusion protein. The colony forming unit of the fusion protein was also higher than those of the cultures treated with the same molar numbers of the recombinant human GM-CSF and G-CSF separately, which suggests that the fusion protein presumably retains both G-CSF and GM-CSF activities.     

Adenovirus-mediated BMP2 expression in human bone marrow stromal cells

Recombinant adenoviral vectors have been shown to be potential new tools for a variety of musculoskeletal defects. Much emphasis in the field of orthopedic research has been placed on developing systems for the production of bone. This study aims to determine the necessary conditions for sustained production of high levels of active bone morphogenetic protein 2 (BMP2) using a recombinant adenovirus type 5 (Ad5BMP2) capable of eliciting BMP2 synthesis upon infection and to evaluate the consequences for osteoprogenitor cells. The results indicate that high levels (144 ng/ml) of BMP2 can be produced in non-osteoprogenitor cells (A549 cell line) by this method and the resultant protein appears to be three times more biologically active than the recombinant protein. Surprisingly, similar levels of BMP2 expression could not be achieved after transduction with Ad5BMP2 of either human bone marrow stromal cells or the mouse bone marrow stromal cell line W20-17. However, human bone marrow stromal cells cultured with 1 microM dexamethasone for four days, or further stimulated to become osteoblast-like cells with 50 microg/ml ascorbic acid, produced high levels of BMP2 upon Ad5BMP2 infection as compared to the undifferentiated cells. The increased production of BMP2 in adenovirus transduced cells following exposure to 1 microM dexamethasone was reduced if the cells were not given 50 microg/ml ascorbic acid. When bone marrow stromal cells were allowed to become confluent in culture prior to differentiation, BMP2 production in response to Ad5BMP2 infection was lost entirely. Furthermore, the increase in BMP2 synthesis seen during differentiation was greatly decreased when Ad5BMP2 was administered prior to dexamethasone treatment. In short, the efficiency of adenovirus mediated expression of BMP2 in bone marrow stromal cells appears to be dependent on the differentiation state of these cells.     

Effect of cytokines on the proliferation/differentiation of stroma-initiating cells

A culture system that identifies the precursor of murine bone marrow fibroblastic stromal cells (stroma-initiating cells, SIC) has been developed. In this system, mature fibroblasts are depleted by adherence to plastic dishes and the nonadherent cells are seeded at a low density, which results in the formation of colonies composed of fibroblastic cells. Macrophage colony-stimulating factor (M-CSF) has been shown to accelerate the colony formation in the system. In this study, we examined the stroma-inducing activity of a number of cytokines. Neither granulocyte-CSF, stem cell factor, interleukin (IL)-1, IL-6, transforming growth factor, epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, nor fibroblast growth factor showed the activity. Similarly, tumor necrosis factor (TNF) did not show any stroma-inducing activity, but the factor inhibited the stromal colony formation induced by M-CSF. In this study, we found that granulocyte/macrophage-CSF (GM-CSF) and IL-3, as well as M-CSF had the stroma-inducing activity. Neither an additive nor synergistic effect was observed when the three factors were assayed in various combinations. The stroma-inducing activity of M-CSF, GM-CSF and IL-3 was observed even if lineage-negative bone marrow cells were used as target cells, suggesting that mature hematopoietic cells such as macrophages and granulocytes were not involved in the induction of stromal colony formation by these factors. Our results raise the possibility that GM-CSF and IL-3 as well as M-CSF stimulate the proliferation or differentiation of the precursor of bone marrow fibroblastic stromal cells.     

Macrophage colony-stimulating factor induces the expression of mitogen-activated protein kinase phosphatase-1 through a protein kinase C-dependent pathway.

M-CSF triggers the activation of extracellular signal-regulated protein kinases (ERK)-1/2. We show that inhibition of this pathway leads to the arrest of bone marrow macrophages at the G0/G1 phase of the cell cycle without inducing apoptosis. M-CSF induces the transient expression of mitogen-activated protein kinase phosphatase-1 (MKP-1), which correlates with the inactivation of ERK-1/2. Because the time course of ERK activation must be finely controlled to induce cell proliferation, we studied the mechanisms involved in the induction of MKP-1 by M-CSF. Activation of ERK-1/2 is not required for this event. Therefore, M-CSF activates ERK-1/2 and induces MKP-1 expression through different pathways. The use of two protein kinase C (PKC) inhibitors (GF109203X and calphostin C) revealed that M-CSF induces MKP-1 expression through a PKC-dependent pathway. We analyzed the expression of different PKC isoforms in bone marrow macrophages, and we only detected PKCbetaI, PKCepsilon, and PKCzeta. PKCzeta is not inhibited by GF109203X/calphostin C. Of the other two isoforms, PKCepsilon is the best candidate to mediate MKP-1 induction. Prolonged exposure to PMA slightly inhibits MKP-1 expression in response to M-CSF. In bone marrow macrophages, this treatment leads to a complete depletion of PKCbetaI, but only a partial down-regulation of PKCepsilon. Moreover, no translocation of PKCbetaI or PKCzeta from the cytosol to particulate fractions was detected in response to M-CSF, whereas PKCepsilon was constitutively present at the membrane and underwent significant activation in M-CSF-stimulated macrophages. In conclusion, we remark the role of PKC, probably isoform epsilon, in the negative control of ERK-1/2 through the induction of their specific phosphatase.     

注射紫杉醇的小鼠骨髓细胞体外分化巨噬细胞增强

目的研究小鼠腹腔注射紫杉醇对体外骨髓细胞诱导分化巨噬细胞的影响。方法小鼠连续5d腹腔注射紫杉醇,无菌制备骨髓细胞,用含巨噬细胞集落刺激因子（M-CSF）的RPMI1640培养液培养骨髓细胞,通过流式细胞仪对其诱导分化的巨噬细胞表面分子、吞噬功能进行分析。结果紫杉醇明显降低小鼠骨髓细胞数量,但骨髓细胞体外诱导分化成巨噬细胞的数量明显增加;F4/80^＋巨噬细胞中CD80、CD14表面分子表达升高,而I-A^d表达降低;紫杉醇处理组诱导分化的巨噬细胞吞噬鸡红细胞的能力提高。结论结果提示紫杉醇可能具有调节巨噬细胞表面分子的表达和吞噬功能。     

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.     

Effect of cytokines and growth factors on the macrophage colony-stimulating factor secretion by human bone marrow stromal cells

We have investigated the effect of growth factors, inflammatory and anti-inflammatory cytokines on the macrophage colony-stimulating factor (M-CSF) secretion by cultured human bone marrow stromal cells. Their production of M-CSF cultured in serum-free medium is enhanced in a time-dependent manner in response to tumour necrosis factor (TNF-)alpha and interleukin (IL-)4 but not to IL-1, IL-3, IL-6, IL-7, IL-10, SCF, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, bFGF and transforming growth factor (TGF-)beta. The co-addition of IL-4 and TNF-alpha has a greater than additive effect on the secretion of M-CSF suggesting that they act synergistically. The anti-inflammatory molecules IL-10 and TGF-beta have no effect on the TNF-alpha-induced M-CSF synthesis by marrow stromal cells. In conclusion TNF-alpha and IL-4 are potent stimulators of the M-CSF synthesis by human bone marrow stromal cells, a result of importance regarding the role of M-CSF in the proliferation/differentiation of mononuclear-phagocytic cells and the role of marrow stromal cells as regulators of marrow haematopoiesis.     

Requirement of N-myristoyltransferase 1 in the development of monocytic lineage

N-myristoyltransferase (NMT) exists in two isoforms, NMT1 and NMT2, that catalyze myristoylation of various proteins crucial in signal transduction, cellular transformation, and oncogenesis. We have recently demonstrated that NMT1 is essential for the early development of mouse embryo. In this report, we have demonstrated that an invariant consequence of NMT1 knock out is defective myelopoesis. Suppressed macrophage colony forming units were observed in M-CSF-stimulated bone marrow cells from heterozygous (+/-) Nmt1-deficient mice. Homozygous (-/-) Nmt1-deficient mouse embryonic stem cells resulted in drastic reduction of macrophages when stimulated to differentiate by M-CSF. Furthermore, to understand the requirement of NMT1 in the monocytic differentiation we investigated the role of NMT, pp60c-Src (NMT substrate) and heat shock cognate protein 70 (inhibitor of NMT), during PMA-induced differentiation of U937 cells. Src kinase activity and protein expression increased during the differentiation process along with regulation of NMT activity by hsc70. NMT1 knock down in PMA treated U937 cells showed defective monocytic differentiation. We report in this study novel observation that regulated total NMT activity and NMT1 is essential for proper monocytic differentiation of the mouse bone marrow cells.