Identification and characterization of Magmas, a novel mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction.

OBJECTIVE: The aim of this study was to identify granulocyte-macrophage colony-stimulating factor (GM-CSF) responsive genes. MATERIALS AND METHODS: Potential GM-CSF responsive genes were identified by comparing the mRNA expression pattern of the murine myeloid cell line PGMD1 grown in either interleukin-3 (IL-3) or GM-CSF by differential display. Human and murine cDNA clones of one of the bands having increased expression in GM-CSF were isolated. mRNA expression of the gene was examined by Northern blot. Immunohistochemistry and studies with a green fluorescent fusion protein were used to determine its intracellular location. Growth factor-stimulated proliferation of PGMD1 cells transfected with constitutively expressed sense and anti-sense cDNA constructs of the gene was measured by 3H-thymidine incorporation. RESULTS: A gene, named Magmas (mitochondria-associated granulocyte macrophage CSF signaling molecule), was shown to be rapidly induced when cells were switched from IL-3 to GM-CSF. Analysis of the amino acid sequence of Magmas showed it contained a mitochondrial signal peptide, but not any other known functional domains. The human and murine clones encode nearly identical 13-kDa proteins that localized to the mitochondria. Magmas mRNA expression was observed in all tissues examined. PGMD1 cells that overexpressed Magmas proliferated similarly to untransfected cells when cultured in IL-3 or GM-CSF. In contrast, cells with reduced protein levels grew normally in IL-3, but had impaired proliferation in GM-CSF. CONCLUSION: Magmas is a mitochondrial protein involved in GM-CSF signal transduction.

Demonstration of the low affinity alpha subunit of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSF-R alpha) on human trophoblast and uterine cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a classical haematopoietic cytokine which has also been implicated in placental growth and development. In this study we have performed a detailed immunohistological localization of the low affinity GM-CSF receptor (GM-CSF-R alpha) in human first trimester implantation site and non-pregnant endometrium. We have also investigated receptor expression and GM-CSF binding in vitro by normal first trimester trophoblast using flow cytometric analysis and compared this with JEG-3 and JAR choriocarcinoma cells. In the first trimester, the GM-CSF-R was found to be present on villous cytotrophoblast and all populations of extravillous trophoblast. Expression by villous syncytiotrophoblast was weak or absent, but this increased markedly by term. GM-CSF-R were also expressed by fetal Hofbauer cells within the mesenchyme of the chorionic villi and by uterine glandular epithelium and decidual macrophages within maternal decidua. GM-CSF-R was not expressed by glands in proliferative phase endometrium but began to appear during the secretory phase, suggesting hormonal regulation of the receptor on uterine glandular epithelium. Flow cytometric comparison of normal isolated first trimester trophoblast and JEG-3 and JAR choriocarcinoma cells revealed two- to threefold higher surface expression of GM-CSF-R by choriocarcinoma cells and higher binding capacity for rhGM-CSF than normal trophoblast. These results suggest that GM-CSF may regulate growth and development of human trophoblast. GM-CSF may also influence placental development and function by acting via decidual and fetal macrophages, and uterine glandular epithelium, which are the other cell populations to express the receptor.

Purification of hemopoietin 1: a multilineage hemopoietic growth factor.

Hemopoietin 1 (H-1) and the mononuclear phagocyte specific growth factor CSF-1 act synergistically on developmentally early bone marrow cells to generate primitive CSF-1 receptor-bearing cells. The H-1 activity of the serum-free medium conditioned by the human urinary bladder carcinoma cell line 5637 was shown to result from the sum of the activities of two charged species (pI approximately equal to 4.8, approximately equal to 85%; pI approximately equal to 5.3, approximately equal to 15%) of similar size. No qualitative difference in the biological activity of these two species was detected. A four-step procedure, involving batch DEAE-cellulose chromatography, chromatofocusing, gel filtration, and hydrophobic chromatography has been developed for the major (pI approximately equal to 4.8) species. H-1 was purified approximately 65,000-fold and recovered as 32% of the total activity of the starting material. The lowest concentration yielding maximal biological activity was approximately equal to 0.25 ng/ml. The 125I-labeled purified H-1, in either native or reduced form, behaved as a homogeneous single band that coelectrophoresed with the biological activity of purified H-1 on sodium dodecyl sulfate gel electrophoresis (NaDodSO4/PAGE). The molecular mass of the purified reduced H-1, determined by NaDodSO4/PAGE was approximately equal to 17 kDa. Recent studies indicate that the purified H-1 is a multilineage hemopoietic growth factor.

Factors affecting the growth and differentiation of haemopoietic cells in culture.

The proliferation and differentiation of haemopoietic cells are regulated by haemopoietic growth factors (HGFs). Lineage-specific HGFs regulate mature, developmentally late cells while multilineage HGFs regulate the developmentally early precursors. Several studies indicate that the pleiotropic response to HGFs is similar to the effects caused by other growth factor, such as epidermal growth factor and platelet-derived growth factor, on their respective target cells. The loss of regulation within the pathway of growth factor action may result in neoplastic transformation.

Solubilization and assay of a colony-stimulating factor receptor from murine macrophages.

The colony-stimulating factor, CSF-1, selectively stimulates the survival, proliferation, and differentiation of mononuclear phagocytes. The solubilization, assay, and characteristics of the CSF-1 receptor from the J774.2 murine macrophage cell line are described. The recovery of cell-surface receptor in the postnuclear supernatant membrane fraction of hypotonically disrupted cells was 76%. Recovery of the ligand binding activity of the receptor after solubilization of this fraction with 1% Triton X-100 was approximately 150%. The binding of 125I-CSF-1 to intact cells and membrane preparations was consistent with the existence of a single class of high-affinity receptor sites. In contrast, the equilibrium binding of 125I-CSF-1 to the solubilized postnuclear fraction indicated the existence of two distinct classes of binding site (apparent Kds 0.15 nM and 10 nM). A rapid assay was developed for the high-affinity sites, which were shown to be associated with the CSF-1 receptor. The function of the low-affinity sites, which have not been demonstrated on intact cells or cell membranes and which are 13 times more abundant than the high-affinity sites, is unknown. The solubilized high-affinity receptor-CSF-1 complex was stable on storage at 0 degrees C and -70 degrees C but dissociated at 37 degrees C. Dissociation also occurred at 0 degrees C in buffers of low pH (4.0) or high ionic strength (0.7 M NaCl).

A low-affinity human granulocyte-macrophage colony-stimulating factor/murine erythropoietin hybrid receptor functions in murine cell lines.

To identify domains in hematopoietic growth factor receptors that are important for signal transduction, a hybrid receptor (GMER) was constructed by splicing the DNA of the entire extracellular and transmembrane domains of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha 2 subunit (GMR) to the cytoplasmic domain of the murine erythropoietin receptor (mEpoR). The hybrid receptor was introduced into the interleukin-3 factor-dependent murine hematopoietic cell line Ba/F3. Cells that expressed high receptor numbers were selected by cell sorting using phycoerythrin-labeled human GM-CSF. Immunoprecipitation of GMER from Ba/F3 cells showed a band with an Mr of 105,000 daltons. Human GM-CSF binding to Ba/F3 cells that expressed GMER showed a kd of 3.0 nmol/L and 475 binding sites/cell, while the same cells that expressed GMR had 300 sites/cell and a kd of 3.5 nmol/L. The proliferative response to GM-CSF of Ba/F3 cells that expressed GMER showed 1/2 maximal cell growth (as measured by 3H-thymidine incorporation) at a GM-CSF concentration of 2.5 x 10(-8) mol/L. When cultured in human GM-CSF, Ba/F3-GMER cells expressed cell surface glycophorin. Similar results were obtained with Ba/F3 cells transfected with the mEpoR and cultured in erythropoietin. Expression of GMR plus the human GM-CSF receptor beta chain in the same cell line also resulted in human GM-CSF stimulated proliferation; however, cell surface glycophorin was not detected. These data show that a low-affinity GM-CSF/Epo hybrid receptor can promote GM-CSF-dependent proliferation and can induce the expression of glycophorin, an erythroid-specific protein.

Purification of the colony-stimulating factor 1 receptor and demonstration of its tyrosine kinase activity.

Colony-stimulating factor 1 (CSF-1) regulates the survival, proliferation, and differentiation of mononuclear phagocytes. The CSF-1 receptor was purified from cell membranes of the J774.2 mouse macrophage cell line by solubilization with Triton X-100, CSF-1 affinity chromatography, and gel filtration. The purified receptor is a protein or glycoprotein of 165 kDa comprising a single polypeptide chain that is not covalently associated, either as a homopolymer, or with any other protein. CSF-1 stimulated autophosphorylation of the purified receptor in tyrosine residues. Casein but not histone was shown to act as a substrate for the tyrosine protein kinase activity of purified receptor.

The beta chain of the interleukin-3 receptor functionally associates with the erythropoietin receptor.

Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors share a common beta chain (beta(c)), and both cytokines enhance erythropoietin (Epo)-dependent in vitro erythropoiesis by primary hematopoietic progenitors and factor-dependent cells. These data suggest that the Epo receptor and beta(c) may functionally interact. To determine whether such interactions can be documented, we studied a murine factor-dependent cell line (Ba/F3), which endogenously expresses IL-3R. First, Ba/F3 cells were transfected with murine EpoR, making them responsive to both IL-3 and Epo. Next, the EpoR expressing cells were transfected with murine beta(c). This resulted in an enhanced sensitivity of these cells to Epo, which was especially pronounced at low Epo concentrations. Ba/F3-EpoR were then treated with antisense oligodeoxynucleotides to the murine beta. Control sense and nonsense had no effect on Epo-dependent growth, but the antisense markedly and specifically inhibited Epo-dependent growth. In contrast, the antisense did not affect beta-globin message levels (another Epo-responsive effect in these cells) detectable by Northern blot. Finally, Western blot analysis of proteins immunoprecipitated from cells expressing both receptors with antibody against beta and blotted with antibody against EpoR, or immunoprecipitated with antibody against EpoR and blotted with antibody against beta, showed that EpoR and beta coimmunoprecipitate. These data show that the beta chain functionally and physically associates with the EpoR. This suggests that these cytokine receptors exist as a large supercomplex and offers the first molecular explanation for the synergistic effects of IL-3 and GM-CSF with Epo during erythropoiesis.

Neutrophil-specific reduction in the expression of granulocyte--macrophage colony-stimulating factor receptor subunits in myelodysplastic syndromes.

The proliferative and differentiative response of neutrophils to granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to be impaired in patients with myelodysplastic syndromes (MDS). To investigate the mechanisms of the defective response in MDS, we examined expression levels of GM-CSF receptor alpha (GMR alpha) and common beta (beta c) subunits on CD16(+) neutrophils, CD14(+) monocytes and CD3(+) T cells from 26 MDS patients and 10 healthy controls using flow cytometry. Expression of GMR alpha was significantly decreased on the neutrophils of five out of 26 patients and was not specific for any FAB subtype. In contrast, beta c expression on neutrophils was significantly reduced in 14 out of 26 patients with a higher proportion occurring in the advanced stages of MDS including refractory anaemia with excess of blasts (RAEB), RAEB in transformation (RAEBt) and overt leukaemia compared with refractory anaemia (RA)/RA with ringed sideroblasts (RARS) or healthy controls. Decreased beta c also correlated with the degree of hypogranular neutrophil morphology and increased infection. Expression of both subunits on T cells and monocytes in MDS was similar to normal controls. Polymerase chain reaction amplification of reverse-transcribed mRNA isolated from the affected neutrophils suggests that the reduction of beta c may result from decreased message levels. The observed reduction in GM-CSF receptor expression could account for the impaired proliferative and maturational responses in MDS.

A low affinity chimeric human alpha/beta-granulocyte-macrophage colony-stimulating factor receptor induces ligand-dependent proliferation in a murine cell line.

The high affinity receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) is composed of at least two subunits, an 85-kDa low affinity GM-CSF-binding protein (alpha-GMR) and a 120-kDa beta-subunit (beta-GMR) necessary for high affinity binding and signal transduction. Previous studies have shown that deletion of the intracellular domain of alpha-GMR inactivates the receptor's ability to support proliferation, but has no effect on GM-CSF binding. Using anti-alpha-GMR- and anti-beta-GMR-specific antibodies, we show that alpha-GMR and beta-GMR coprecipitate only after GM-CSF binding, suggesting that binding of GM-CSF induces stabilization or assembly of an activated receptor complex involving recruitment of beta-GMR chains. To understand the contribution of each subunit of this receptor to the generation of an activated receptor complex, we attempted to construct minimal receptors with some or all of the functions of the wild-type heterodimer. We found that a hybrid human alpha/beta-GMR molecule in which the extracellular and transmembrane segments are composed of alpha-GMR sequences and the intracellular segment is composed of beta-GMR bound GM-CSF with low affinity, but activated tyrosine kinase activity, induced receptor internalization, and supported short- and long-term proliferation of transfected Ba/F3 cells. At least 1 ng/ml human GM-CSF was required for growth stimulation, and maximal proliferation occurred at a concentration of 10 ng/ml. This was 10-100-fold more than needed to stimulate growth of Ba/F3 cells expressing both full-length human alpha-GMR and beta-GMR and 1000-fold less than needed to stimulate growth of Ba/F3 cells expressing only human alpha-GMR. These results indicate that the cytoplasmic domain of alpha-GMR is not required to initiate a unique signaling event for proliferation in Ba/F3 cells, but can be functionally replaced by the cytoplasmic domain of beta-GMR.

Expression and function of the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit.

To determine the expression and function of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha chain (GMR alpha) during hematopoiesis and on leukemic cells, monoclonal antibodies were raised by immunizing mice with cells expressing high levels of human GMR alpha. A pool of five antibodies isolated from three different mice was used to characterize GMR alpha. This antibody pool (anti-GMR alpha) immunoprecipitated a protein with the expected molecular weight of GMR alpha from COS cells transiently transfected with the GMR alpha gene. In factor-dependent cells, GMR alpha existed as a phosphoprotein. However, its phosphorylation was not stimulated by the presence of GM-CSF. Anti-GMR alpha inhibited the GM-CSF-dependent growth of cell lines and normal bone marrow cells and inhibited the binding of iodinated GM-CSF to its receptor. Cell surface expression of GMR alpha was examined using anti-GMR alpha and flow cytometry. GMR alpha was readily detectable on both blood monocytes and neutrophils. In adherence-depleted normal bone marrow, two separate populations expressed GMR alpha. The most positive cells were predominantly macrophages, whereas the cells that expressed less GMR alpha were largely myelocytes and metamyelocytes. A small population of lin-CD34+ or CD34+CD38- cells also expressed GMR alpha, but they were not capable of significant growth in colony-forming assays. In contrast, the majority of lin-CD34+ and CD34+CD38- cells were GMR alpha-, yet they produced large numbers of myeloid and erythroid colonies in the same assay. Malignant cells from patients with leukemia were also tested for GMR alpha expression. All of the myeloid leukemias and only rare lymphoid leukemias surveyed tested positive for GMR alpha. These results show that anti-GMR alpha is useful for the functional characterization of the GMR alpha and for the detection of myeloid leukemia and that GMR alpha is expressed on certain lineages throughout hematopoietic development; however, progenitors that express the receptor may have a reduced capacity to proliferate in response to hematopoietic growth factors.

The beta c component of the granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin 3 (IL-3)/IL-5 receptor interacts with a hybrid GM-CSF/erythropoietin receptor to influence proliferation and beta-globin mRNA expression.

BACKGROUND: The interaction of different members of the hematopoietic growth factor receptor family may be relevant to the increased proliferation and the failure of differentiation that characterizes the myeloid leukemias. We recently demonstrated that a chimeric receptor (GMER) that is composed of the extracellular and transmembrane domains of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain (GMR alpha) and the cytoplasmic domain of the murine erythropoietin receptor mEpoR binds hGM-CSF with low affinity (3 nM) and confers both proliferative and differentiation signals to stably transfected murine Ba/F3 cells. MATERIALS AND METHODS: To investigate whether the common beta-subunit of the GM-CSF receptor (beta c) can interact with GMER, either the entire beta-subunit or a mutant, truncated beta-subunit that completely lacks the cytoplasmic domain (beta tr) was introduced into Ba/F3 cells that express GMER, and the binding of GM-CSF as well as proliferation and differentiation responses were measured. RESULTS: Scatchard analysis showed that both GMER + beta c and GMER + beta tr bound hGM-CSF with high affinity (Kd 40 pM to 65 pM). Proliferation assays showed that the maximum growth of cells expressing GMER + beta c was identical to that of cells with GMER alone. However, proliferation of the cells that expressed GMER + beta tr was reduced by 80-95% of GMER. Dose-response curves showed that the concentration of GM-CSF required for half-maximal growth was 0.5-5.0 pM for GMER + beta c and 0.5-5 nM for GMER and GMER + beta tr. The EpoR cytoplasmic domain of GMER also undergoes ligandinducible tyrosine phosphorylation. However, the tyrosine phosphorylation did not correlate with growth in cells expressing beta tr. Coexpression of beta c with GMER in Ba/F3 cells grown in hGM-CSF markedly enhanced beta-globin mRNA expression. CONCLUSIONS: These results indicate that beta c can transduce a unique signal in association with GMER to influence both proliferative and differentiation signal pathways.