CSF-1 Protein Research Articles

Expression of Macrophage Colony-Stimulating Factor CSF-1 in Spinal Cord Neurons in Mice with Experimental Autoimmune Encephalomyelitis.

In mice with acute and chronic models of experimental autoimmune encephalomyelitis (EAE), a quantitative study of the dynamics of CSF-1 and IL-34 protein levels in the spinal cord and blood plasma was conducted by ELISA and the specificity of CSF-1 expression in spinal cord cells was examined by immunohistological methods. A significant increase in the level of CSF-1 in the spinal cord was detected and populations of motoneurons with intense CSF-1 immunoreactivity were identified in mice with acute and chronic EAE. The obtained results suggest a possible role of CSF-1 in the pathogenesis and progression of EAE/multiple sclerosis.

Screening out serum protein biomarkers from both groups of asthma and ABPA patients

Clinical manifestations of asthma and allergic bronchopulmonary aspergillosis (ABPA) are very similar, so that they are easy to be misdiagnosed. To improve the diagnostic accuracy of these two diseases, the protein array was firstly applied for measuring serum proteins from asthma and ABPA patients,compared with normal control subjects in this study. Protein biomarkers associated to the disease processes and with diagnostic value were analyzed by bioinformatics. The multiple reaction monitoring mass spectrometry (MRM-MS) and enzyme linked immunosorbent assay (ELISA) were then used to verify the candidate biomarkers with enlarged size of samples. The diagnostic value of candidate proteins was additionally evaluated with the receiver operating characteristic (ROC) curve. Via the comparison analysis, the disease related-candidates were identified, including 7 differentially expressed proteins(DEPs) between asthma and normal groups, 15 DEPs between ABPA and normal groups, and 15 DEPs between ABPA and asthma groups. Using MRM method, SERPINF2, C6 and HABP2 proteins were showed differential expression significantly between asthma and normal groups,SERPINF2 and F10 were represented differential expression significantly between ABPA and normal control groups, and CPN2 and IGLL5 were displayed differential expression significantly between ABPA and asthma groups. Furthermore, ELISA validation data were also showed that serum CSF1 protein levels in ABPA patients were significantly lower than those in asthma patients, while TNFRSF10C levels in ABPA patients were significantly higher than those in asthma patients. ROC curve analysis revealed that the AUC of C6 were 0.9 with the sensitivity of 73.3% and the specificity of 99.9% in comparison between asthma and normal groups. The AUC of F10 was 0.871, with the sensitivity of 80.0% and the specificity of 85.7% in comparison between ABPA and normal groups. And the AUC of the combined diagnosis of CPN2 and IGLL5 was 0.867, with more sensitivity of 86.7% and the specificity of 80.0% in comparison those between ABPA and asthma groups. In conclusion, novel protein biomarkers in this study were closely related to the occurrence and development of asthma or ABPA, and might have potentials for assistant diagnostic and differential diagnosis of those two diseases in clinical application.

Abstract 1648: Evaluation of therapeutic drug candidates using a novel humanized B-hCSF1/hCSF1R mouse model

Abstract An often rate-limiting step for cancer immunotherapy is drug development. The generation of humanized mouse models can be used to assess the clinical utility of new drugs earlier in the discovery process. Tumor-associated macrophages (TAMs) represent an emerging therapeutic target, specifically high expression of the colony-stimulating factor 1 receptor (CSF1R) protein on tumors and TAMs facilitate a tumor permissive environment. Previous reports have demonstrated that blockade of the CSF1/CSF1R pathway reduced and reprogrammed intra-tumoral CSF1R+ TAMs, altogether alleviating immune suppression. To evaluate preclinical efficacy and toxicity of CSF1R targeted therapeutic candidates, Biocytogen has successfully generated a double humanized B-hCSF1/hCSF1R mouse model. The full-length encoding sequence of mouse Csf1 and the extracellular domain of Csf1r were replaced by human CSF1 and CSF1R genes, respectively. Evaluation of CSF1 and CSF1R protein expression in homozygous B-hCSF1/hCSF1R mice (H/H) was confirmed by ELISA and flow cytometry analysis. We next performed an in vivo efficacy screening study using genetically modified hCSF1 or wild-type MC38 cells inoculated into B-hCSF1/hCSF1R (H/H) mice and observed that anti-human CSF1R antibodies or combination therapy of anti-mouse PD-L1 and anti-human CSF1R antibodies were effective in controlling tumor growth. Our combined studies illustrate that Biocytogen’s novel B-hCSF1/hCSF1R mice offer a promising model for in vivo validation of CSF1 and CSF1R antibody therapeutics. Citation Format: Ruili Lyu, Shujin Zhang, Rebecca Soto, Suman Zhao, Chonghui Liu, Luke (Zhaoxue) Yu. Evaluation of therapeutic drug candidates using a novel humanized B-hCSF1/hCSF1R mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1648.

Nicotinamide Improves Delayed Tooth Eruption in Runx2 +/− Mice

Patients with cleidocranial dysplasia (CCD) caused by mutations in RUNX2 have severe dental anomalies, including delayed or absent eruption of permanent teeth. This requires painful and expensive surgical/orthodontic intervention because of the absence of medicine for this condition. Here, we demonstrate that nicotinamide, a vitamin B3 and class III histone deacetylase inhibitor, significantly improves delayed tooth eruption in Runx2 +/− mice, a well-known CCD animal model, through the restoration of decreased osteoclastogenesis. We also found that Csf1 mRNA and protein levels were significantly reduced in Runx2 +/− osteoblasts as compared with wild type whereas RANKL and OPG levels had no significant difference between wild type and Runx2 +/− osteoblasts. The nicotinamide-induced restoration of osteoclastogenesis of bone marrow–derived macrophages in Runx2 +/− mice was due to the increased expression of RUNX2 and CSF1 and increased RANKL/OPG ratio. RUNX2 directly regulated Csf1 mRNA expression via binding to the promoter region of the Csf1 gene. In addition, nicotinamide enhanced the RUNX2 protein level and transacting activity posttranslationally with Sirt2 inhibition. Taken together, our study shows the potential and underlying molecular mechanism of nicotinamide for the treatment of delayed tooth eruption by using the Runx2 +/− murine model, suggesting nicotinamide as a candidate therapeutic drug for dental abnormalities in patients with CCD.

Pharmacological effects of vinorelbine in combination with lenvatinib in anaplastic thyroid cancer

Anaplastic thyroid cancer (ATC) is a rare neoplasia with a poor prognosis. Proliferation and apoptosis assays were performed on ATC cell lines (8305C, 8505C) exposed to vinorelbine, lenvatinib, as well as to concomitant combinations. ABCB1, ABCG2 and CSF-1 mRNA expression was evaluated by real time PCR. The relative levels of pospho Akt were investigated as part of a human phospho-kinase array analysis, and CSF-1 and VEGFR-2 protein levels were measured by ELISA. The intracellular concentration of lenvatinib in ATC cells was measured by combined reversed-phase liquid chromatography-tandem mass spectrometry. An ATC subcutaneous xenograft tumor model in nude mice was treated with vinorelbine, lenvatinib, or vinorelbine plus lenvatinib.After treatment with vinorelbine, lenvatinib, a significant antiproliferative effect in ATC cell lines was observed. The concomitant treatment of vinorelbine and lenvatinib revealed synergism for all the fractions of affected cells. A decrease in ABCB1 expression was reported in both ATC cell lines treated with the lenvatinib plus vinorelbine combination, as was an increase in the intracellular concentration of lenvatinib. The combination caused a decrease in Akt, GSK3α/β, PRAS40 and Src phosphorylation, and in both CSF-1 mRNA and protein levels. In the subcutaneous tumor model, the combination reduced the tumor volume during the treatment period.Our results establish the synergistic ATC antitumor activity of a vinorelbine and lenvatinib combination.

MiR-423-5p may regulate ovarian response to ovulation induction via CSF1

BackgroundWe have previously shown that hsa-miR-423-5p expression in ovarian granulosa cells is decreased in high ovarian response populations. The objective of the present study was to find the target gene and mechanism for miR-423-5p involved in ovarian response regulation.Methods(a) TargetScan was used to predict the target gene of hsa-miR-423-5p. (b) A model for hsa-miR-423-5p overexpression or inhibition was constructed by transfecting KGN cells with lentivirus. CSF1 mRNA and protein expression and luciferase activity were measured. (c) The cell cycles of control and lentivirus treated KGN cells were analyzed. Western blot was used to measure the expression of CDKN1A in KGN cells. (d) The concentration of E2 in KGN cell culture medium were measured.Results(a) TargetScan revealed that the 3′ un-translated region of CSF1 matched 11 bases at the 5′ end of miR-423-5p, making it a likely target gene. (b) Overexpression or inhibition of miR-423-5p were associated with respective decreases or increases in CSF1 expression (both mRNA and protein) (p < 0.05) and luciferase activity (p < 0.05). (c) When miR-423-5p expression increased, the number of G0/G1 phase cells and the expression of CDKN1A protein increased while estradiol concentrations in the cell culture solution decreased (p < 0.05). However, when miR-423-5p expression decreased, the number of S phase cells increased and E2 concentrations increased while the expression of CDKN1A protein decreased (p < 0.05).ConclusionsColony stimulating factor 1 is a target gene of miR-423-5p and that it may regulate ovarian response to ovulation induction by affecting granulosa cells proliferation and estrogen secretion.

The alternative spliced 3′-UTR mediated differential secretion of macrophage colony stimulating factor in breast cancer cells

CSF-1 mRNA 3′UTR variants (var) are generated from alternative splicing. CSF-1 protein encoded by var-1 mRNA with long 3′UTR derived from exon-10 is rapidly secreted compared to the CSF-1 protein encoded by var-4 mRNA with short 3′UTR derived from exon-9. Secretion kinetics indicates that HuR, which binds the CSF-1 var-1 mRNA, but not var-4 mRNA, accelerates the secretion of CSF-1 protein. HuR overexpression increases the secretion rate of CSF-1 protein. In contrast, silencing of HuR does not have such an effect, suggesting other compensatory mechanisms. Effect of the CSF-1 mRNA variant 3′UTRs on cellular phenotype shows both CSF-1 var-1 or -4 mRNA is involved in the enhanced rates of migration and invasion observed by both in vitro in breast cancer cells. Our study indicates that the alternative splicing of CSF-1 mRNA 3′UTR can regulate differential secretion of CSF-1 protein.

Novel CSF1-S100A10 fusion gene and CSF1 transcript identified by RNA sequencing in tenosynovial giant cell tumors.

RNA-sequencing was performed on three tenosynovial giant cell tumors (TSGCT) in an attempt to elicit more information on the mechanisms of CSF1 expression in this tumor type. A novel CSF1-S100A10 fusion gene was found in a TSGCT that carried the translocation t(1;1)(q21;p11) as the sole karyotypic abnormality. In this fusion gene, the part of CSF1 coding for the CSF1 protein (exons 1–8 in sequences with accession nos. NM_000757 and NM_172212) is fused to the 3′-part of S100A10. Since the stop codon TAG of CSF1 is present in it, the CSF1-S100A10 fusion gene’s predominant consequence seems to be the replacement of the 3′-untranslated region (UTR) of CSF1 (exon 9; nt 2092–4234 in sequence with accession no. NM_000757 or nt 2092–2772 in NM_172212) by the 3′-end of S100A10 (exon 3; nt 641–1055 in sequence with accession no. NM_002966). In the other two TSGCT, a novel CSF1 transcript was detected, the same in both tumors. Similar to the occurrence in the CSF1-S100A10 fusion gene, the novel CSF1 transcript 3′-UTR is replaced by a new exon located ∼48 kb downstream of CSF1 and 11 kb upstream of AHCYL1. Although only 3 TSGCT were available for study, the finding in all of them of a novel CSF1-S100A10 fusion gene or CSF1 transcript indicates the existence of a common pathogenetic theme in this tumor type: the replacement of the 3′-UTR of CSF1 with other sequences.

Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA

CSF-1 mRNA 3'UTR contains multiple unique motifs, including a common microRNA (miRNA) target in close proximity to a noncanonical G-quadruplex and AU-rich elements (AREs). Using a luciferase reporter system fused to CSF-1 mRNA 3'UTR, disruption of the miRNA target region, G-quadruplex, and AREs together dramatically increased reporter RNA levels, suggesting important roles for these cis-acting regulatory elements in the down-regulation of CSF-1 mRNA. We find that nucleolin, which binds both G-quadruplex and AREs, enhances deadenylation of CSF-1 mRNA, promoting CSF-1 mRNA decay, while having the capacity to increase translation of CSF-1 mRNA. Through interaction with the CSF-1 3'UTR miRNA common target, we find that miR-130a and miR-301a inhibit CSF-1 expression by enhancing mRNA decay. Silencing of nucleolin prevents the miRNA-directed mRNA decay, indicating a requirement for nucleolin in miRNA activity on CSF-1 mRNA. Downstream effects followed by miR-130a and miR-301a inhibition of directed cellular motility of ovarian cancer cells were found to be dependent on nucleolin. The paradoxical effects of nucleolin on miRNA-directed CSF-1 mRNA deadenylation and on translational activation were explored further. The nucleolin protein contains four acidic stretches, four RNA recognition motifs (RRMs), and nine RGG repeats. All three domains in nucleolin regulate CSF-1 mRNA and protein levels. RRMs increase CSF-1 mRNA, whereas the acidic and RGG domains decrease CSF-1 protein levels. This suggests that nucleolin has the capacity to differentially regulate both CSF-1 RNA and protein levels. Our finding that nucleolin interacts with Ago2 indirectly via RNA and with poly(A)-binding protein C (PABPC) directly suggests a nucleolin-Ago2-PABPC complex formation on mRNA. This complex is in keeping with our suggestion that nucleolin may work with PABPC as a double-edged sword on both mRNA deadenylation and translational activation. Our findings underscore the complexity of nucleolin's actions on CSF-1 mRNA and describe the dependence of miR-130a- and miR-301a-directed CSF-1 mRNA decay and inhibition of ovarian cancer cell motility on nucleolin.

CALGB 30601: A phase II study of dasatinib (D) in patients (pts) with previously treated malignant mesothelioma (MM).

7037 Background: SRC is commonly over-expressed in MM. D is a potent inhibitor of SRC family kinases, EphA2 and PDGFRβ. There are no approved therapies for MM pts who progress on pemetrexed. We therefore conducted a phase II trial of D in MM pts who had received 1 prior pemetrexed-based regimen. Methods: Single arm phase II. Eligible pts had unresectable MM, PS 0-1, measurable disease, and no symptomatic effusions. Primary endpoint: Progression-free survival (PFS) at 24 weeks (wks). D 70 mg BID was given orally. CT scans were obtained every 2 months. Pre- and posttreatment plasma VEGF, PDGFβ, and serum CSF-1 and mesothelin-related protein were collected. Tumor was evaluated for expression of EphA2 and PDGFRβ. Results: 46 pts enrolled at 12 sites 9/07-8/09, 35 are evaluable for PFS, 22 for response, and 46 for toxicity. Pt characteristics: Male 72%; median age 68 (range 35, 81); PS 0 41%; epithelial histology 72%, pleural 76%. Median cycles given 2 (range 1-8). The starting dose was reduced to 50 mg BID after the first 23 pts enrolled because 50% of the first 12 pts enrolled had AE ≥ grade 3. Grade 3/4 toxicities (% pts): anemia 2%, fatigue 11%, pleural effusion 9%, pericardial effusion 2%, pneumonitis 2%, hypoxia 2%, nausea 4%, hyponatremia 7%, hypophosphatemia 2%.There were 3 grade 5 toxicities: 1 ARDS, 1 respiratory failure, 1 unknown. Efficacy: 24-week PFS rate 15% (95% CI 6%, 29%), median PFS 8.3 wks (7.7, 10.3), median overall survival 20.7 wks (11.4, 28.3), partial response 0%; stable disease 20%. Conclusions: Dasatinib is inactive in previously-treated MM; the 70 mg dose is poorly tolerated. Further data on biologic correlates will be presented. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Bristol-Myers Squibb

Sleeping beauty-mediated somatic mutagenesis implicates CSF1 in the formation of high-grade astrocytomas.

The Sleeping Beauty (SB) transposon system has been used as an insertional mutagenesis tool to identify novel cancer genes. To identify glioma-associated genes, we evaluated tumor formation in the brain tissue from 117 transgenic mice that had undergone constitutive SB-mediated transposition. Upon analysis, 21 samples (18%) contained neoplastic tissue with features of high-grade astrocytomas. These tumors expressed glial markers and were histologically similar to human glioma. Genomic DNA from SB-induced astrocytoma tissue was extracted and transposon insertion sites were identified. Insertions in the growth factor gene Csf1 were found in 13 of the 21 tumors (62%), clustered in introns 5 and 8. Using reverse transcription-PCR, we documented increased Csf1 RNAs in tumor versus adjacent normal tissue, with the identification of transposon-terminated Csf1 mRNAs in astrocytomas with SB insertions in intron 8. Analysis of human glioblastomas revealed increased levels of Csf1 RNA and protein. Together, these results indicate that SB-insertional mutagenesis can identify high-grade astrocytoma-associated genes and they imply an important role for CSF1 in the development of these tumors.

Enhanced Ovarian Cancer Tumorigenesis and Metastasis by the Macrophage Colony-Stimulating Factor

Coexpression of the macrophage colony-stimulating factor (CSF-1) and its receptor (CSF-1R) in metastatic ovarian cancer specimens is a predictor of poor outcome in epithelial ovarian cancer. This suggests that an autocrine loop is produced by which ovarian tumors can secrete CSF-1 stimulating the CSF-1R resulting in a more aggressive phenotype. Our current work sought to validate this autocrine stimulation model using stable transfection of a 4-kb CSF-1 construct into otherwise nonvirulent Bix3 ovarian cancer cells. A representative clone, Bix3T8.2, produced a 72-fold increase in CSF-1 gene transcription rate (by nuclear run-off assays) and a 57-fold increase in secreted CSF-1 protein (by sandwich ELISA), compared to parent cells. Comparison of Bix3T8.2 invasion, adhesion, and motility in vitro and metastasis in vivo were made to parental and transfectant controls. Up to 12-fold higher invasiveness was seen with Bix3T8.2 and 2- and 6-fold higher adhesion and motility, respectively, over controls in vitro. In nude mice, i.p. injection of Bix3T8.2 produced a wide array of visceral, nodal, and distant metastasis with a degree of enhanced tumor burden not seen in any of the 10 mice inoculated with transfectant control cells. Complete absence of tumor take distinguished 40% of mice implanted with transfectant control cells. Disruption of this autocrine loop using antisense oligomer therapy against CSF-1R and 3′ untranslated region knockdown of CSF-1 protein resulted in reversal of in vitro and in vivo tumor phenotypes. This CSF-1 feedback loop offers a model by which novel biologic therapies can potentially target multiple levels of this pathway.

PDGF Up-regulates CSF-1 Gene Transcription in Ameloblast-like Cells

Macrophage colony-stimulating factor (CSF-1) is a key regulatory cytokine for amelogenesis, and ameloblasts synthesize CSF-1. We hypothesized that PDGF stimulates DNA synthesis and regulates CSF-1 in these cells. We determined the effect of PDGF on CSF-1 expression using MEOE-3M ameloblasts as a model. By RT-PCR, MEOE-3M expressed PDGFRs and PDGF A- and B-chain mRNAs. PDGF-BB increased DNA synthesis and up-regulated CSF-1 mRNA and protein in MEOE-3M. Cells transfected with CSF-1 promoter deletion constructs were analyzed. A PDGF-responsive region between -1.7 and -0.795 kb, containing a consensus Pea3 binding motif, was identified. Electrophoretic mobility shift assay (EMSA) showed that PDGF-BB stimulated protein binding to this motif that was inhibited in the presence of anti-Pea3 antibody. Analysis of these data provides the first evidence that PDGF-BB is a mitogen for MEOE-3M and increases CSF-1 protein levels, predominantly by transcription. Elucidation of the cellular pathways that control CSF-1 expression may provide novel strategies for the regulation of enamel matrix formation.

Translocation and Expression of CSF1 in Pigmented Villonodular Synovitis, Tenosynovial Giant Cell Tumor, Rheumatoid Arthritis and Other Reactive Synovitides

We recently demonstrated that CSF1, the ligand of the tyrosine kinase receptor, CSF1R, can be translocated in pigmented villonodular synovitis (PVNS) and tenosynovial giant cell tumor (TGCT). In this study, we evaluated the staining characteristics of PVNS/TGCT and reactive synovitides for CSF1 and CSF1R by in situ hybridization and immunohistochemistry on tissue microarrays and correlated these findings with the recently described translocation. We collected specimens of TGCT/PVNS from 60 patients and of rheumatoid arthritis and other reactive synovitides from 74 patients. We identify 2 groups of PVNS and TGCT cases by the presence of CSF1 translocation and CSF1 expression. The first group (35 of 57 cases; 61%) had both the CSF1 translocation and high expression of CSF1 RNA, confirming our previous findings. Interestingly, a second group (22 of 57 cases; 39%) was identified that showed high expression of CSF1 RNA or CSF1 protein but did not have the translocation. The rheumatoid arthritis and reactive synovitis specimens showed localization of CSF1 RNA and protein to the synovial lining cells, implying a possible role for CSF1 in the pathogenesis of these lesions. As the CSF1 translocation is postulated to play an important role in the biology of PVNS/TGCT, the consistent presence of CSF1 expression in translocation-negative cases implies that other mechanisms can lead to CSF1 up-regulation. The consistent presence of CSF1 overexpression in all cases of PVNS/TGCT and reactive synovitides suggests both an important role for CSF1 in the spectrum of synovial pathologies and the possibility of targeting the CSF1/CSF1R interaction therapeutically.

Differential Regulation of Colony Stimulating Factor 1 and Macrophage Migration Inhibitory Factor Expression by Inflammatory Cytokines in Term Human Decidua: Implications for Macrophage Trafficking at the Fetal-Maternal Interface1

Macrophages are a major component of the leukocyte population of human pregnant endometrium. Although several crucial functions have been ascribed to these cells, the mechanisms underlying macrophage trafficking in the placental bed are poorly understood. The aim of this study was to evaluate the in vivo expression of two potentially antagonistic macrophage-targeting chemokines, colony stimulating factor 1 (CSF1, also known as M-CSF) and macrophage migration inhibitory factor (MIF), in term decidua, and to examine the effects of the inflammatory cytokines tumor necrosis factor (TNF, also known as TNF alpha) and interleukin 1beta (IL1B) on CSF1 and MIF expression in cultured decidual cells. The expression of CSF1 and MIF in term decidua was evaluated by immunohistochemistry. Cultured decidual cells were primed with estradiol (E2) or with E2+medroxyprogesterone acetate (MPA), and then incubated with corresponding steroid(s) with or without TNF or IL1B. The levels of CSF1 and MIF protein and mRNA were assessed by ELISA and quantitative RT-PCR, respectively. Immunostaining for CSF1 and MIF was observed in term decidua. The levels of secreted CSF1 and MIF were similarly unchanged whether the decidual cells were incubated with E2 or with E2+MPA. The CSF1 levels significantly increased in cultures exposed to E2 or E2+MPA plus TNF or IL1B. In contrast, the MIF levels in TNF- and IL1B-treated cells were not changed significantly from the control cultures. The ELISA data were confirmed by quantitative RT-PCR analysis. These results indicate that CSF1 and MIF are involved in regulating macrophage trafficking at the fetal-maternal interface, and suggest a mechanism by which inflammatory cytokines influence pregnancy by regulating decidual macrophage infiltration.

Osteoblast-specific targeting of soluble colony-stimulating factor-1 increases cortical bone thickness in mice.

The soluble and membrane-bound forms of CSF-1 are synthesized by osteoblasts and stromal cells in the bone microenvironment. Transgenic mice, generated to selectively express sCSF-1 in bone, showed increased cortical thickness in the femoral diaphysis caused by new bone formation along the endosteal surface. The ability of sCSF-1 to enhance bone cell activity in vivo is potentially relevant for increasing cortical bone in a variety of disorders. The soluble form of colony-stimulating factor-1 (sCSF-1) and the membrane-bound form of CSF-1 (mCSF-1) have been shown to support osteoclastogenesis in vitro; however, the effect of each peptide on bone remodeling in vivo is unclear. To determine the effect of sCSF-1, selectively expressed in bone, the skeletal phenotype of transgenic mice harboring the human sCSF-1 cDNA under the control of the osteocalcin promoter was assessed. At 5 and 14 weeks, mice were analyzed for CSF-1 protein levels, weighed, and X-rayed, and femurs were removed for peripheral quantitative computed tomography, histology, and histomorphometry. High levels of human sCSF-1 were detected in bone extracts and, to a lesser extent, in plasma. Adult transgenic mice showed normal body weight and increased circulating monocytic cells. At 5 weeks, the femoral diaphysis was similar in CSF-1T and wt/wt littermates. However, by 14 weeks, the femoral diaphysis in CSF-1T mice showed increased cortical thickness and bone mineral density. In contrast to the diaphysis, the femoral metaphysis of CSF-1T mice showed normal cancellous bone comparable with wt/wt littermates at each time point. Histological sections demonstrated increased woven bone along the endosteal surface of the diaphysis and intracortical remodeling. Fluorochrome-labeling analysis confirmed endocortical bone formation in CSF-1T, with a 3.1-fold increase in the percentage of double-labeled surfaces and a 3.6-fold increase in the bone formation rate compared with wt/wt mice. Although remodeling resulted in a slightly porous cortex, sCSF-1 preferentially stimulated endocortical bone formation, leading to increased cortical thickness. These findings indicate that sCSF-1 is a key determinant of bone cell activity in the corticoendosteal envelope.

Cloning and characterization of the CSF1 gene of Saccharomyces cerevisiae, which is required for nutrient uptake at low temperature.

We have isolated cold-sensitive fermentation mutants (Csf mutants) of a commercial baker's yeast that have practically no fermentation capacity at 5 degrees C and return to their normal capacity at 25 to 40 degrees C. CSF1 was cloned by functional complementation of the Csf phenotype. CSF1 contain an open reading frame of 8,874 nucleotides, encoding a protein of 2,958 amino acids. The nucleotide sequence was identical to that of the YLR087C gene in the Saccharomyces genome database, but there was no information about the function of the predicted CSF1 (YLR087C) protein. Gene disruption shows that CSF1 is required for growth and fermentation only at low temperatures. Permeabilized cells of the disruptant showed nearly the same ethanol production rate as those of the parent strain, even at 10 degrees C. The disruptant cells had the same glucose uptake rates as the parental cells at 30 degrees C, but three- to fivefold-lower rates than the parental cells at 10 degrees C. These findings suggest that CSF1 associates with a new nutrient transport system which exists on the plasma membrane and is required only at low temperature.

Effects of colony stimulating factor-1 on human extravillous trophoblast growth and invasion.

Colony stimulating factor (CSF)-1 has been localized in a variety of tissues and shown to influence proliferation and differentiation of numerous cell types. Messenger RNA and protein products of CSF-1 and its receptor (c-fms) have been identified in the human placenta and decidua. We examined whether CSF-1 and c-fms mRNA and protein are expressed by normal human first trimester invasive extravillous trophoblast (EVT) cells propagated in culture and whether CSF-1 influences proliferation and/or invasion of these cells. CSF-1 mRNA and protein expression was determined by RT-PCR and immunofluorescence microscopy. Proliferation was assessed by the cellular uptake of tritiated thymidine and invasion was evaluated by Matrigel invasion assay as well as Northern blot analysis of mRNA expression for invasion-associated enzymes and their inhibitors. Results revealed that normal invasive EVT cells in culture express both CSF-1 and c-fms mRNA and protein. Under serum-free conditions, exogenous CSF-1 greatly stimulated the proliferation of these cells. CSF-1 neutralizing and c-fms receptor blocking antibody (Ab) each abolished the growth stimulatory effects of CSF-1, indicating that CSF-1 and c-fms interaction was responsible for these effects. In fact, c-fms Ab alone reduced proliferation to below background levels. While exogenous CSF-1 failed to influence EVT cell invasiveness, Northern blot analysis of mRNA indicated a slight upregulation of the invasion-associated enzyme 72 kDa type IV collagenase as well as its natural inhibitor tissue inhibitor of metalloprotease (TIMP)-1, so that the balance between the two remained unaltered. These findings suggest that CSF-1 may represent an autocrine (and possibly paracrine) growth stimulatory factor for the invasive trophoblast cells in situ with no net effect on their invasiveness.

Colony stimulating factor-1 expression is developmentally regulated in the mouse.

Colony stimulating factor-1 (CSF-1) regulates the survival, proliferation, and differentiation of mononuclear phagocytes. To determine whether CSF-1 plays a role in the perinatal development of these cells, CSF-1 protein and mRNA expression in tissues and serum from fetal/neonatal mice and their mothers was analyzed. As fetal/neonatal age increased, CSF-1 concentrations rose in liver, kidney, and lung, declined in brain and serum, and did not change in intestine and heart. Concurrently, fetal/neonatal CSF-1 concentrations were higher in liver, kidney, and serum and lower in lung, brain, intestine, and heart than maternal tissue/serum concentrations, which showed no correlations with gestational or postpartum stage. CSF-1 mRNA was detected in all tissues examined and its expression increased in lung and heart and decreased in brain with increasing fetal/neonatal age. The developmental regulation of mouse CSF-1 expression appears to he important for mononuclear phagocyte development during this period.

Expression of CSF-1, c-fms, and MCP-1 in the central nervous system of rats with experimental allergic encephalomyelitis.

We have examined the expression of factors associated with the growth, differentiation, and chemotaxis of cells of the monocyte/macrophage series in the central nervous system of Lewis rats sensitized to develop experimental allergic encephalomyelitis. CSF-1 mRNA increased significantly over that found in control animals (sensitized with OVA in CFA or CFA alone). The elevation in the levels of this growth factor commenced immediately before the onset of early clinical signs and peaked immediately before maximal clinical incidence of disease. Expression of CSF-1 message declined to base-line values with resolution of the disease process. CSF-1 protein was also detected in the central nervous system at the height of clinical disease. Expression of the receptor for CSF-1, the proto-oncogene c-fms, also paralleled the early disease process. Elevated levels of c-fms mRNA were detected immediately before the onset and peaked at the height of clinical signs of disease. In contrast to CSF-1 levels, elevated c-fms message expression persisted after resolution of the acute phase of experimental allergic encephalomyelitis. Levels of macrophage chemotactic factor-1 message were also elevated immediately before the onset of clinical signs, peaked with the height of clinical disease, and declined with resolution of the disease. Unlike CSF-1 or c-fms, no endogenous macrophage chemotactic factor-1 message was detected in control animals. Macrophage chemotactic factor-1 protein was demonstrated by Western blot in the central nervous system at the height of clinical disease. The results support the conclusion that expression of factors that specifically target cells of the monocyte/macrophage series are an important component of the disease process in experimental allergic encephalomyelitis.