ß-Catenin-TCF/LEF signaling promotes steady-state and emergency granulopoiesis via G-CSF receptor upregulation.

The canonical Wnt signaling pathway is mediated by interaction of ß-catenin with the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors and subsequent transcription activation of Wnt-target genes. In the hematopoietic system, the function of the pathway has been mainly investigated by rather unspecific genetic manipulations of ß-catenin that yielded contradictory results. Here, we used a mouse expressing a truncated dominant negative form of the human TCF4 transcription factor (dnTCF4) that specifically abrogates ß-catenin-TCF/LEF interaction. Disruption of the ß-catenin-TCF/LEF interaction resulted in the accumulation of immature cells and reduced granulocytic differentiation. Mechanistically, dnTCF4 progenitors exhibited downregulation of the Csf3r gene, reduced granulocyte colony-stimulating factor (G-CSF) receptor levels, attenuation of downstream Stat3 phosphorylation after G-CSF treatment, and impaired G-CSF-mediated differentiation. Chromatin immunoprecipitation assays confirmed direct binding of TCF/LEF factors to the promoter and putative enhancer regions of CSF3R. Inhibition of ß-catenin signaling compromised activation of the emergency granulopoiesis program, which requires maintenance and expansion of myeloid progenitors. Consequently, dnTCF4 mice were more susceptible to Candida albicans infection and more sensitive to 5-fluorouracil-induced granulocytic regeneration. Importantly, genetic and chemical inhibition of ß-catenin-TCF/LEF signaling in human CD34+ cells reduced granulocytic differentiation, whereas its activation enhanced myelopoiesis. Altogether, our data indicate that the ß-catenin-TCF/LEF complex directly regulates G-CSF receptor levels, and consequently controls proper differentiation of myeloid progenitors into granulocytes in steady-state and emergency granulopoiesis. Our results uncover a role for the ß-catenin signaling pathway in fine tuning the granulocytic production, opening venues for clinical intervention that require enhanced or reduced production of neutrophils.

Endothelial CD36 Contributes to Postischemic Brain Injury by Promoting Neutrophil Activation via CSF3.

The scavenger receptor CD36 is a critical factor initiating ischemic brain injury, but the cell type(s) expressing CD36 and responsible for its harmful effects remain unknown. Using bone marrow (BM) chimeras subjected to transient middle cerebral artery occlusion, we found that CD36(-/-) mice transplanted with wild-type (WT) BM (WT→CD36(-/-)) have smaller infarcts (-67%), comparable with those of mice lacking CD36 both in brain and hematogenous cells (CD36(-/-) →CD36(-/-); - 72%). Conversely, WT mice receiving CD36(-/-) BM (CD36(-/-) →WT) have infarcts similar to WT→WT mice, suggesting that CD36 in the host brain (i.e., in microglia and endothelial cells), and not in hematogenous cells is involved in the damage. As anticipated, postischemic neutrophil infiltration in CD36(-/-) →CD36(-/-) mice was attenuated. Surprisingly, however, in WT→CD36(-/-) mice, in which infarcts were small, neutrophil infiltration was large and similar to that of CD36(-/-) →WT mice, in which infarcts were not reduced. Postischemic neutrophil free radical production was attenuated in WT→CD36(-/-) mice compared with CD36(-/-) →WT mice, whereas expression of the neutrophil activator colony-stimulating factor 3 (CSF3) was suppressed in CD36(-/-) cerebral endothelial cells, but not microglia. In CD36(-/-) cerebral endothelial cultures exposed to extracts from stroke brains, the upregulation of CSF3, but not neutrophil attractant chemokines, was suppressed. Intracerebroventricular administration of CSF3, 24 h after stroke, reconstituted neutrophil radical production and increased infarct volume in WT→CD36(-/-) mice. The findings identify endothelial cells as a key player in the deleterious effects of CD36 in stroke, and unveil a novel role of endothelial CD36 in enabling neutrophil neurotoxicity through CSF3. SIGNIFICANCE STATEMENT: Ischemic stroke is a leading cause of death and disability worldwide with limited therapeutic options. The inflammatory response initiated by cerebral ischemia-reperfusion contributes to ischemic brain injury and is a potential therapeutic target. Here we report that CD36, an innate immunity receptor involved in the initiation of postischemic inflammation, is a previously unrecognized regulator of neutrophil cytotoxicity. The effect is mediated by endothelial CD36 via upregulation of the neutrophil activator CSF3 in cerebral endothelial cells. Therefore, approaches to modulate cerebral endothelial CD36 signaling or to neutralize CSF3 may provide novel therapeutic opportunities to ameliorate postischemic inflammatory injury.

Granulocyte colony-stimulating factor receptor signalling via Janus kinase 2/signal transducer and activator of transcription 3 in ovarian cancer.

BACKGROUND: Ovarian cancer remains a major cause of cancer mortality in women, with only limited understanding of disease aetiology at the molecular level. Granulocyte colony-stimulating factor (G-CSF) is a key regulator of both normal and emergency haematopoiesis, and is used clinically to aid haematopoietic recovery following ablative therapies for a variety of solid tumours including ovarian cancer. METHODS: The expression of G-CSF and its receptor, G-CSFR, was examined in primary ovarian cancer samples and a panel of ovarian cancer cell lines, and the effects of G-CSF treatment on proliferation, migration and survival were determined. RESULTS: G-CSFR was predominantly expressed in high-grade serous ovarian epithelial tumour samples and a subset of ovarian cancer cell lines. Stimulation of G-CSFR-expressing ovarian epithelial cancer cells with G-CSF led to increased migration and survival, including against chemotherapy-induced apoptosis. The effects of G-CSF were mediated by signalling via the downstream JAK2/STAT3 pathway. CONCLUSION: This study suggests that G-CSF has the potential to impact on ovarian cancer pathogenesis, and that G-CSFR expression status should be considered in determining appropriate therapy.

MiR-424 regulates monocytic differentiation of human leukemia U937 cells by directly targeting CDX2.

MiR-424 plays an important role via promoting the monocytic differentiation in many human leukemia cell lines. Here, we report that miR-424 decreased miR-125b expression to 36 % by directly targeting caudal type homeobox 2. However, miR-424 also decreased expression of Fes, PU.1 and colony-stimulating factor receptor (MCSFR). As Fes, PU.1 and MCSFR were down-regulated by over-expression of miR-125b (unpublished work), a similar effect of miR-424 and Fes siRNA on CD64, Egr-1, Egr-2 and CEBPA indicates that Fes may be an important downstream target of miR-424. We hypothesize that miR-424 promotes monocytic differentiation by regulating other critical factors and miR-424 has high affinity for these factors. For the first time, the molecular mechanism of miR-424 during monocytic differentiation of U937 cells has been elucidated in this study.

A highly phagocytic cell line TO from Atlantic salmon is CD83 positive and M-CSFR negative, indicating a dendritic-like cell type.

Leucocyte cell lines are valuable tools for immunological studies. In this study the TO cell line, originating from Atlantic salmon head kidney leucocytes, is described with respect to enzyme cytochemistry, functional studies, reactivity with leucocyte specific antibodies and immune gene expression. Pronounced characteristics of the TO cell line are the rapid adherence to the plastic growth surface, high phagocytic capacity and bactericidal functions. No respiratory burst activity, and little or no NO production were detected under the experimental conditions tested, and thus the TO cells appear to have other effective killing mechanisms. The cells are reactive with a leucocyte specific monoclonal antibody (MAb), but does not bind a neutrophil specific MAb or stain for myeloperoxidase. Real-time RT-PCR showed the expression in TO cells of several immune genes, some of which were significantly regulated following LPS stimulation. The expression of CD83 might indicate a dendritic cell (DC) origin of the TO cells, as this marker is considered a hallmark for DC. Expression of TCR-alpha or the macrophage marker M-CSFR was not detected. Based on the present analyses the TO cells display a mixture of known characteristics for macrophages and DCs. At the same time the TO cells lack some central functions of phagocytic/myeloid cells. As the TO cells are developed to a long-term culture one cannot exclude that some functions might have been lost in this process. Nevertheless, the features of the TO cells indicate their potential as a model system for immunological studies of salmon phagocytic cells.

Chotosan enhances macrophage colony-stimulating factor mRNA expression in the ischemic rat brain and C6Bu-1 glioma cells.

Macrophage colony stimulating factor (M-CSF) is a cytokine which has been recently reported to have a neuroprotective effect on ischemic rat brain. In this study, we investigated the effect of chotosan, an oriental medicine, which has been clinically demonstrated to be effective for the treatment of vascular dementia, on M-CSF gene expression in rats with permanent occlusion of bilateral common carotid arteries (P2VO) in vivo and in a C6Bu-1 glioma cell line in vitro. The expression level of M-CSF mRNA in the cerebral cortices of P2VO rats was significantly higher than that in the cerebral cortices of sham-operated animals. Repeated treatment of P2VO rats with chotosan (75 mg/kg per day) for 4 d after P2VO significantly increased the expression level of M-CSF mRNA in the cortex but it had no effect on the expression of beta-actin, granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF) mRNAs. Moreover, the present in vitro studies revealed that chotosan treatment (10-100 mug/ml) of C6Bu-1 glioma cells dose-dependently enhanced M-CSF mRNA expression without affecting the expression of G-CSF, GM-CSF, and inducible nitric oxide synthase mRNAs. The effect of chotosan was reversed by Ro 31-8220 (1 muM), a selective protein kinase C (PKC) inhibitor, but not by H-89 (10 muM), a selective protein kinase A (PKA) inhibitor. These findings suggest that the upregulatory effect of chotosan on M-CSF mRNA expression involves PKC and may play an important role in the anti-vascular dementia action of this formula.

Role of stem cell factor and its receptor in the pathogenesis of pediatric aplastic anemia.

In order to investigate the levels of stem cell factor (SCF) and its receptor c-kit protein and mRNA in pediatric aplastic anemia (AA) and their relevance to the pathogenesis, immunocytochemical and in situ hybridization were utilized to detect the expression of SCF and its receptor c-kit gene protein and mRNA, respectively in 59 children with AA and 51 normal controls. The relationship between SCF and c-kit and the pathogenesis of AA was analyzed subsequently. The results showed that the positive rate of SCF protein and mRNA expression in children with AA was significantly lower than that in healthy controls (P < 0.05). However, there was no significant difference in the positive rate of c-kit protein and mRNA expression between children with AA and control group (P > 0.05). It was concluded that the expression of SCF is significantly decreased in children with AA, which may be closely associated with the pathogenesis of the AA. c-kit may be unrelated to the development of pediatric AA. Therefore, AA in children may have abnormalities at SCF/c-kit signal transduction levels.

Role of stem cell factor and its receptor in the pathogenesis of pediatric aplastic anemia / 华中科技大学学报（医学）（英德文版）

In order to investigate the levels of stem cell factor (SCF) and its receptor c-kit protein and mRNA in pediatric aplastic anemia (AA) and their relevance to the pathogenesis, immunocytochemical and in situ hybridization were utilized to detect the expression of SCF and its receptor c-kit gene protein and mRNA, respectively in 59 children with AA and 51 normal controls. The relationship between SCF and c-kit and the pathogenesis of AA was analyzed subsequently. The results showed that the positive rate of SCF protein and mRNA expression in children with AA was significantly lower than that in healthy controls (P 0.05). It was concluded that the expression of SCF is significantly decreased in children with AA, which may be closely associated with the pathogenesis of the AA. c-kit may be unrelated to the development of pediatric AA. Therefore, AA in children may have abnormalities at SCF/c-kit signal transduction levels.

Gene expression in macrophage-rich inflammatory cell infiltrates in human atherosclerotic lesions as studied by laser microdissection and DNA array: overexpression of HMG-CoA reductase, colony stimulating factor receptors, CD11A/CD18 integrins, and interleukin receptors.

OBJECTIVE: Inflammatory cells play an important role in atherogenesis. However, more information is needed about their gene expression profiles in human lesions. METHODS AND RESULTS: We used laser microdissection (LMD) to isolate macrophage-rich shoulder areas from human lesions. Gene expression profiles in isolated cells were analyzed by cDNA array and compared with expression patterns in normal intima and THP-1 macrophages. Upregulation of 72 genes was detected with LMD and included 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, interferon regulatory factor-5 (IRF-5), colony stimulating factor (CSF) receptors, CD11a/CD18 integrins, interleukin receptors, CD43, calmodulin, nitric oxide synthase (NOS), and extracellular superoxide dismutase (SOD). Several of these changes were also present in PMA-stimulated THP-1 macrophages in vitro. On the other hand, expression of several genes, such as VEGF, tissue factor pathway inhibitor 2, and apolipoproteins C-I and C-II, decreased. CONCLUSIONS: Overexpression of HMG-CoA reductase in macrophage-rich lesion areas may explain some beneficial effects of statins, which can also modulate increased expression of CD11a/CD18 and CD43 found in microdissected cells. We also found increased expression of CSF receptors, IRF-5, and interleukin receptors, which could become useful therapeutic targets for the treatment of atherosclerotic diseases.

Inhibition of glycogen phosphorylase (GP) by CP-91,149 induces growth inhibition correlating with brain GP expression.

The role of glycogenolysis in normal and cancer cells was investigated by inhibiting glycogen phosphorylase (GP) with the synthetic inhibitor CP-91,149. A549 non-small cell lung carcinoma (NSCLC) cells express solely the brain isozyme of GP, which was inhibited by CP-91,149 with an IC(50) of 0.5 microM. When treated with CP-91,149, A549 cells accumulated glycogen with associated growth retardation. Treated normal skin fibroblasts also accumulated glycogen with G1-cell cycle arrest that was associated with inhibition of cyclin E-CDK2 activity. Overall, cells expressing high levels of brain GP were growth inhibited by CP-91,149 correlating with glycogen accumulation whereas cells expressing low levels of brain GP were not affected by the drug. Analyses of 59 tumor cell lines represented in the NCI drug screen identified that every cell line expressed brain GP but the profile was dominated by a few highly GP expressing cell lines with lower than mean GP-a enzymatic activities. The correlation program, COMPARE, identified that the brain GP protein measured in the NCI cell lines corresponded with brain GP mRNA expression, ADP-ribosyltransferase 3, and colony stimulating factor 2 receptor alpha in the 10,000 gene microarray database with similar correlation coefficients. These results suggest that brain GP is present in proliferating cells and that high protein levels correspond with the ability of CP-91,149 to inhibit cell growth.

Differential expression of transforming growth factor-alpha and macrophage colony-stimulating factor/colony-stimulating factor-1R (c-fins) by multinucleated giant cells involved in pathological bone resorption at the site of orthopaedic implants.

The immunologic response to prosthetic biomaterial particles is characterized by macrophage-rich inflammatory infiltrate, formation of multinucleated giant cells, and aseptic loosening at the site of arthroplasty. We investigated the in vivo expression and tissue distribution of transforming growth factor alpha, macrophage colony-stimulating factor, and the receptor for colony-stimulating factor-1 at the site of bone erosion in patients with clinically failed orthopaedic implants (n = 30). The expression was further compared with that detected in the inflamed synovial membranes from patients with rheumatoid arthritis or osteoarthritis (n = 15) and one patient with osteoclastoma (giant cell tumour of bone). Immunostaining of the tissue demonstrated positivity for transforming growth factor alpha within the inflammatory macrophage and multinucleated giant cell infiltrate in the diseased synovial membrane and the bone-implant interface. A comparative analysis between the synovium and retrieval interface membranes (pseudosynovium) revealed a high level of expression of transforming growth factor alpha, with intense membrane staining on multinucleated giant cells in all failed arthroplasties with pseudosynovium. In addition, the frequency, antigenic phenotype, and pattern of transforming growth factor alpha expression on multinucleated giant cells in the interface were markedly similar to those observed for multinucleated giant cells in osteoclastoma. Multinucleated giant cells within the interface lacked the expression of macrophage colony-stimulating factor and colony-stimulating factor-1 receptor, whereas those at the bone surfaces exhibited strong immunoreactivity. The predominant expression of transforming growth factor alpha by multinucleated giant cells in the bone-implant interface and its similarity to osteoclastoma highlight the importance of assessing transforming growth factor alpha as a possible contributor to the development of bone-resorbing giant cells at the site of failed orthopaedic implants.

Direct comparison of the effects of CSF-1 (M-CSF) and GM-CSF on human monocyte DNA synthesis and CSF receptor expression.

There is evidence that a proportion of human monocytes can proliferate in vitro in response to colony-stimulating factor-1 (CSF-1, also known as M-CSF) and granulocyte-macrophage CSF (GM-CSF). To determine whether there are differences in DNA synthesis responses to these CSF, a large study using purified human peripheral blood monocytes from 45 donors was performed under optimized culture conditions. In contrast to the consistent response to CSF-1, approximately 20% of donors have monocytes that do not respond or have a minimal DNA synthesis response to GM-CSF stimulation. However, analysis demonstrated that no statistically significant differences exist in the levels of CSF-1 and GM-CSF-stimulated proliferation in monocytes. In addition, CSF-1 receptor (CSF-1R) blocking experiments indicated that a proportion of the GM-CSF-induced DNA synthesis is due to endogenous levels of CSF-1. As a further comparison of the actions of the two CSFs, CSF-1R and GM-CSFR levels were measured by flow cytometry, and it was shown that GM-CSFR levels decreased within 5 days of culture, independent of the conditions examined. In contrast, CSF-1R levels at day 5 approximated those measured in uncultured monocytes. Whether the proliferating subpopulation(s) express one or both CSF receptors at the beginning or at the end of culture is as yet unknown. The information obtained in this study will be useful for the design of strategies to enrich for the subpopulation in question based on CSF receptor expression.

Prognostic significance of colony-stimulating factor receptor expression in ipsilateral breast cancer recurrence.

The macrophage colony-stimulating factor receptor (CSF-1R), the product of the c-fms proto-oncogene, regulates normal proliferation and differentiation of macrophages and trophoblasts. Recent research found abnormal expression of CSF-1R in human carcinomas of the breast, endometrium, and ovary. Furthermore, activation of CSF-1R by its ligand has been shown to regulate invasiveness and anchorage-independent growth in breast carcinoma cells. To study the significance of CSF-1R expression in breast cancer, we designed a case-controlled immunohistochemical study. We chose 80 patients from a database of 1200 early stage I or II breast cancer patients treated with conservative surgery and radiation therapy. Expression of CSF-1R in the tumors of 40 patients who experienced an ipsilateral breast tumor recurrence (IBTR) as a primary site of relapse were compared with 40 patients who had not experienced an IBTR. The index and control patients were matched by age, clinical stage, nodal status, and follow-up. Paraffin-embedded sections were immunostained with antibodies directed toward CSF-1R. For the CSF-1R antibody, a total of 28 index cases (70%) demonstrated strong staining, whereas only 16 control cases (40%) demonstrated high immunoreactivity (P = 0.007). The CSF-1R antibody showed a positive correlation for local relapse, but no correlation was found between CSF-1R expression and distant metastasis. In summary, our findings provide evidence for the poor prognostic role of CSF-1R in IBTR.

Expression and function of colony-stimulating factors and their receptors in human prostate carcinoma cell lines.

BACKGROUND: The predeliction for prostate carcinoma cells to metastasize to bone suggests the hypothesis that bone and/or bone marrow-derived factors may promote prostate carcinoma cell growth or survival, or serve as chemoattractants for these cells. METHODS: We screened three prostate carcinoma cell lines, DU-145, PC-3, and LNCaP, for the expression of several hematopoiesis-associated colony-stimulating factors (CSFs) and their receptors using RT-PCR (reverse transcriptase-polymerase chain reaction) and immunohistochemical methods, and examined their functional effects. RESULTS: All of these cell lines express granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), and the DU-145 and PC-3 lines express stem-cell factor (SCF), as determined by RT-PCR and ELISA. Each of these cell lines expresses the receptors for SCF, GM-CSF, M-CSF, and granulocyte colony-stimulating factor (G-CSF). M-CSF enhanced the soft-agar clonogenicity of PC-3 and DU-145 cells, and GM-CSF stimulated all three cell lines. SCF stimulated the clonogenic growth of DU-145 cells. G-CSF marginally abrogated the induction of cell death in the PC-3 and LNCaP cell lines under serum-free conditions. GM-CSF and M-CSF stimulated modest chemotaxis of PC-3, DU-145, and LNCaP cells (most prominently in PC-3 cells). CONCLUSIONS: These data suggest that 1) CSFs may be part of a network of paracrine and autocrine loops that modulate prostate carcinoma cell activity, and 2) the growth-stimulatory, survival-enhancing, and/or chemotactic actions of bone marrow-derived CSFs on prostate carcinoma cells may explain in part why bone is a preferential site of prostatic carcinoma metastases.

Localization and expression of CSF-1 receptor in rat dental follicle cells.

Colony-stimulating factor-1 (CSF-1) accelerates tooth eruption in rats and is localized in the dental follicle, a loose connective tissue sac that is necessary for eruption to occur. CSF-1 enhances the cellular events that occur in the follicle prior to eruption--namely, an influx of monocytes into the follicle early post-natally to form the osteoclasts needed to resorb bone for the eruption pathway. Because CSF-1 levels are at a peak at day 3 post-natally, and because CSF-1 has an autocrine effect on its own gene expression, the question remains as to what causes the subsequent decline in CSF-1 protein and mRNA after day 3 post-natally. To determine if the autocrine effect is inhibited through the CSF-1 receptor, analysis of the CSF-1 receptor mRNA levels in cultured dental follicle cells reveals that high concentrations of CSF-1 reduce the gene expression of the CSF-1 receptor. Interleukin 1 alpha, a molecule that enhances CSF-1 gene expression, has no effect on CSF-1 receptor mRNA levels. Immunostaining for the CSF-1 receptor protein shows that it is present in the dental follicle early post-natally and is either absent or greatly reduced by day 10 post-natally. Earlier studies showed that the mRNA levels of the CSF-1 receptor also parallel this time course. Thus, the above results suggest that the feedback inhibition of the autocrine effect of CSF-1 on its own expression is through the effect of CSF-1 inhibiting the translation and transcription of its receptor. In turn, these molecular interactions possibly regulate the cellular events that occur in the follicle prior to and during eruption.

Increasing c-FMS (CSF-1 receptor) expression decreases retinoic acid concentration needed to cause cell differentiation and retinoblastoma protein hypophosphorylation.

Increasing the expression of c-FMS (colony-stimulating factor 1 receptor) by introduction of a transgene reduced the concentration of retinoic acid or 1,25-dihydroxy vitamin D3 needed to cause myeloid or monocytic cell differentiation and hypophosphorylation of the retinoblastoma tumor suppressor protein (RB) typically associated with cell cycle G0 arrest and differentiation of HL-60 human myelo-monoblastic precursor cells. The data are consistent with a model in which signals originating with retinoic acid and c-FMS integrate to cause differentiation, RB hypophosphorylation, and G0 arrest. Furthermore, these two signals can compensate for each other. Three HL-60 sublines described previously (A. Yen et al., Exp. Cell Res., 229: 111-125, 1996) expressing low (wild-type HL-60), intermediate, and high cell surface c-FMS were treated with various concentrations of retinoic acid. The lowest concentration tested, 10(-8) M, induced significant differentiation of only the high c-FMS-expressing cells, with no accompanying hypophosphorylated RB or G0 arrest. The low and intermediate c-FMS expressing cells showed no induced differentiation, hypophosphorylation of RB, or G0 arrest. A 10-fold higher retinoic acid concentration, 10(-1) M, induced significant differentiation of both intermediate and high c-FMS-expressing cells. It induced RB hypophosphorylation only in high c-FMS-expressing cells but with no accompanying G0 arrest in any of the cells. The highest retinoic acid concentration, 10(-6) M, elicited differentiation, hypophosphorylation of RB, and G0 arrest in low, intermediate, and high c-FMS-expressing cells. As the concentration of retinoic acid increased, cell differentiation, hypophosphorylation of RB, and G0 arrest were progressively elicited within this ensemble of cells with different c-FMS expression levels. Thus, for example, at the lowest concentration of retinoic acid, expression of high enough c-FMS still allowed differentiation. At higher concentrations, progressively less c-FMS was needed for differentiation. The apparent threshold for the sum of the retinoic acid plus c-FMS originated signals to elicit differentiation, hypophosphorylation of RB, and G0 arrest increased, in that order. Thus retinoic acid-induced cell differentiation, RB hypophosphorylation, and G0 arrest have different signal threshold requirements. 1,25-Dihydroxy vitamin D3, also a ligand for a member of the steroid thyroid hormone receptor superfamily, caused monocytic differentiation with a similar c-FMS dependency, indicating that these effects characterize both myeloid and monocytic differentiation.

CSF-1 receptor/insulin receptor chimera permits CSF-1-dependent differentiation of 3T3-L1 preadipocytes.

A chimeric growth factor receptor (CSF1R/IR) was constructed by splicing cDNA sequences encoding the extracellular ligand binding domain of the human colony stimulating factor-1 (CSF-1) receptor to sequences encoding the transmembrane and cytoplasmic domains of the human insulin receptor. The addition of CSF-1 to cells transfected with the CSF1R/IR chimera cDNA stimulated the tyrosine phosphorylation of a protein that was immunoprecipitated by an antibody directed against the carboxyl terminus of the insulin receptor. Phosphopeptide maps of the 32P-labeled CSF1R/IR protein revealed the same pattern of phosphorylation observed in 32P-labeled insulin receptor beta subunits. CSF-1 stimulated the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and Shc in cells expressing the CSF1R/IR chimera. Lipid accumulation and the expression of a differentiation-specific marker demonstrated that 3T3-L1 preadipocytes undergo CSF-1-dependent differentiation when transfected with the CSF1R/IR chimera cDNA but not when transfected with the expression vector alone. A 12-amino acid deletion within the juxtamembrane region of the CSF1R/IR (CSF1R/IRDelta960) blocked CSF-1-stimulated phosphorylation of IRS-1 and Shc but did not inhibit CSF-1-mediated differentiation of 3T3-L1 preadipocytes. These observations indicate that adipocyte differentiation can be initiated by intracellular pathways that do not require tyrosine phosphorylation of IRS-1 or Shc.

Suppression or overexpression of genes encoding myeloid growth factors or their receptors.

Gene inactivation data have documented that erythropoietin, G-CSF, M-CSF and thrombopoietin are major regulators in vivo respectively of red cell, neutrophil, macrophage and platelet production. Transgenic mice can provide valuable models for observing the consequences of excessive stimulation by a particular growth factor but are subject to variation based on the use of differing promoters for the inserted gene.

Effects of human trophoblast-induced interferons on the expression of proto-oncogenes c-fms/CSF-1R, EGF-R and c-erbB2 in invasive and non-invasive trophoblast.

The human cytotrophoblast is the first fetal cell type to arise during embryogenesis and differentiate along two pathways to the invasive (extravillous) and non-invasive (villous) populations. The non-invasive villous trophoblast differentiate morphologically and biochemically to form terminally differentiated multinucleated syncytial trophoblast. First trimester invasive and non-invasive trophoblast were isolated from human placentae (5-12 weeks) and were cultured in vitro. The villous trophoblast cells differentiated in vitro to form aggregated syncytial cells which was associated with increased expression of epidermal growth factor receptor (EGF-R). The invasive trophoblast cells expressed colony-stimulating factor receptor (c-fms/CSF-1R) and c-erbB2 proteins but low levels of EGF-R. We studied the effects of human trophoblast-induced interferon (IFN)-alpha/beta on the expression of c-fms/CSF-1R, EGF-R and c-erbB2 whose ligands are reported to be involved in the regulation of growth and differentiation of normal invasive and non-invasive trophoblast cells. Human trophoblast-induced IFN-alpha/beta (100 IU/ml) reduced the expression of EGF-R in both invasive and non-invasive trophoblast cells as determined by quantitative enzyme-linked immunosorbant assay ('ELISA') and western immunoblot methods. The same amount of IFN activity reduced the expression of c-fms/CSF-1R and c-erbB2 proto-oncogene products in invasive trophoblast cells. These results may suggest a possible role of trophoblast-induced IFNs in the regulation of normal trophoblast growth, differentiation and function.

T cell-dependent loss of proliferative responsiveness to colony-stimulating factor-1 by a murine epidermal-derived dendritic cell line, XS52.

We have reported previously that XS52 cells, a long-term dendritic cell (DC) line established from mouse epidermis, proliferate maximally in response to CSF-1, and that XS52 cells expanded in this manner induce brisk proliferation of HDK-1 T cells (KLH-specific Th1 clone) and 5S8 T cells (DNBS-specific Th0 clone) in the presence of Ag. Our purpose was to determine whether CSF-1-dependent mitotic potential of XS52 cells might be affected upon Ag-dependent interaction with these T cell clones. Both surface CSF-1R expression and mitotic responsiveness to CSF-1 became undetectable within 24 h after incubation with each T cell clone in the presence of relevant Ag. By contrast, incubation with T cells alone or Ag alone had minimal effect, indicating a requirement for both T cells and Ag. Exposure of fresh XS52 cells to the supernatant collected from complete XS52/HDK-1/KLH or XS52/5S8/DNBS coculture was sufficient to abrogate both CSF-1R expression and CSF-1 responsiveness. Importantly, both were restored by mAb against IFN-gamma, and both were diminished by rIFN-gamma in the absence of T cells or Ag. Thus, IFN-gamma, which was detected in relatively large amounts in the above supernatants, serves as a major mediator. rIFN-gamma reduced the number of CSF-1 binding sites on XS52 cell surface, without affecting CSF-1R mRNA expression. Thus, it appears that IFN-gamma down-regulates CSF-1R by a post-transcriptional mechanism. We interpret these results to document a novel, bi-directional signaling event in which Ag-dependent DC-T cell interaction promotes the growth of T cells, but inhibits the growth of DC.