RESUMO
Sense-antisense mRNA pairs generated by convergent transcription is a way of gene regulation. c-fms gene is closely juxtaposed to the HMGXB3 gene in the opposite orientation, in chromosome 5. The intergenic region (IR) between c-fms and HMGXB3 genes is 162 bp. We found that a small portion (â¼4.18%) of HMGXB3 mRNA is transcribed further downstream, including the end of the c-fms gene generating antisense mRNA against c-fms mRNA. Similarly, a small portion (â¼1.1%) of c-fms mRNA is transcribed further downstream, including the end of the HMGXB3 gene generating antisense mRNA against the HMGXB3 mRNA. Insertion of the strong poly(A) signal sequence in the IR results in decreased c-fms and HMGXB3 antisense mRNAs, resulting in up-regulation of both c-fms and HMGXB3 mRNA expression. miR-324-5p targets HMGXB3 mRNA 3' UTR, and as a result, regulates c-fms mRNA expression. HuR stabilizes c-fms mRNA, and as a result, down-regulates HMGXB3 mRNA expression. UALCAN analysis indicates that the expression pattern between c-fms and HMGXB3 proteins are opposite in vivo in breast cancer tissues. Together, our results indicate that the mRNA encoded by the HMGXB3 gene can influence the expression of adjacent c-fms mRNA, or vice versa.
Assuntos
Proteína Semelhante a ELAV 1/genética , Regulação Neoplásica da Expressão Gênica , Genes fms , Proteínas de Grupo de Alta Mobilidade/genética , MicroRNAs/genética , Regiões 3' não Traduzidas , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Cromossomos Humanos Par 5 , DNA Intergênico/genética , DNA Intergênico/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Feminino , Edição de Genes , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , MicroRNAs/metabolismo , Polimorfismo Genético , Proto-Oncogene Mas , RNA Antissenso/genética , RNA Antissenso/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
Models of macrophage subtypes require molecular characterization to reliably facilitate their differentiation. Although CD16+ (Fc-gamma III receptor) monocytes that express CD163 (a hemoglobin/haptoglobin receptor) have been implicated in a variety of disease states, the conditions necessary to establish lineages of these cell subtypes remains unknown. The current investigations utilize a cell line derived from acute myelogenous leukemia lineage, MonoMac-1, to interrogate the factors that promote the development of CD16+ macrophages that express CD163. Results implicate the glucocorticoid pathway as well as c-fms signaling based on the action of dexamethasone and macrophage colony-stimulating factor-1 in promoting CD16+ expression, in addition to phorbol myristate acetate and lipopolysaccharides treatment. The ability of glucocorticoid and c-fms receptor antagonists to inhibit CD16+ cell formation further establishes the role of these pathways in CD16 expression in this cell line. In view of the inherent difficulty in working with primary cells as well as donor variation, cell lines may be preferable to primary cells for their consistency. We envision that the process we use to induce CD16 expression in this cell type will be useful for screening and identification of drug candidates potentially useful for the treatment of diseases where the etiology involves the expansion of the CD16+ monocytes subset or the accumulation of CD163+ tissue macrophages.
Assuntos
Diferenciação Celular/fisiologia , Glucocorticoides/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Transdução de Sinais/fisiologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/fisiologia , Genes fms/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de IgG/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
This paper reports on the modification of magnetic beads with oligonucleotide capture probes with a specially designed pendant toehold (overhang) aimed specifically to capture double-stranded PCR products. After capture, the PCR products were selectively released from the magnetic beads by means of a toehold-mediated strand displacement reaction using short artificial oligonucleotide triggers and analysed using capillary electrophoresis. The approach was successfully shown on two genes widely used in human DNA genotyping, namely human c-fms (macrophage colony-stimulating factor) proto-oncogene for the CSF-1 receptor (CSF1PO) and amelogenin.
Assuntos
DNA/análise , Genes fms , Imãs/química , Sondas de Oligonucleotídeos/química , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Sequência de Bases , DNA/genética , Eletroforese Capilar , Técnicas de Genotipagem , Humanos , Fenômenos Magnéticos , Reação em Cadeia da Polimerase , Proto-Oncogene MasRESUMO
FMS, first discovered as the oncogene responsible for Feline McDonough Sarcoma, is a type III receptor tyrosine kinase that binds to the macrophage or monocyte colony-stimulating factor (M-CSF or CSF-1). Signal transduction through that binding results in survival, proliferation, and differentiation of monocyte/macrophage lineage. Overexpression of CSF-1 and/or FMS has been implicated in a number of disease states such as the growth of metastasis of certain types of cancer, in promoting osteoclast proliferation in bone osteolysis, and many inflammatory disorders. Inhibition of CSF-1 and/or FMS may help treat these pathological conditions. This article reviews FMS gene, FMS kinase, CSF-1, IL-34, and their roles in bone osteolysis, cancer biology, and inflammation. Monoclonal antibodies, FMS crystal structure, and small molecule FMS kinase inhibitors of different chemical scaffolds are also reviewed.
Assuntos
Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Cristalização , Genes fms/fisiologia , Humanos , Inflamação/fisiopatologia , Interleucinas/fisiologia , Fator Estimulador de Colônias de Macrófagos/imunologia , Fator Estimulador de Colônias de Macrófagos/fisiologia , Neoplasias/fisiopatologia , Osteólise/fisiopatologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/classificação , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/imunologia , Receptor de Fator Estimulador de Colônias de Macrófagos/fisiologiaRESUMO
Background: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a common sinonasal inflammatory disorder with high heterogeneity. Increasing evidence have indicated that the infiltration of macrophages especially M2 macrophages play pivotal roles in the pathogenesis of CRSwNP, but the underlying mechanisms remain undetermined. This study sought to identify potential biomarkers related to M2 macrophages in CRSwNP. Methods: The expression datasets of GSE136825 and GSE179265 were download from Gene Expression Omnibus (GEO) database and merged. Then, CIBERSORT and weighted gene co-expression network analysis (WGCNA) algorithms were applied to identify M2 macrophage-related gene modules. Thereafter, differentially expressed genes (DEGs) related to M2 macrophages were selected to perform functional enrichment analyses. A protein-protein interaction (PPI) network was built to identify hub genes and quantitative real-time reverse transcriptions PCR was used to verify the bioinformatics results. Results: A total of 92 DEGs associated with M2 macrophages were identified for further analysis. The results of Gene ontology (GO) and Kyoto Encyclopedia of genes and genomes (KEGG) analyses illustrated that M2 macrophage-associated DEGs primarily enriched in immune responses and extracellular matrix structure. PPI network analysis identified 18 hub genes related to M2 macrophages that might be pivotal in the pathogenesis of CRSwNP. After verification, AIF1, C1QA, C1QB, C3AR1, CCR1, CD163, CD4, CD53, CD86, CSF1R, CYBB, FCER1G, FCGR3A, IL10RA, ITGB2, LAPTM5, PLEK, TYROBP were identified as potential M2 macrophage-related biomarkers for CRSwNP. Conclusion: These findings yield new insights into the hub genes and mechanisms related to M2 macrophages in the pathogenesis of CRSwNP. Further studies of these hub genes would help better understand the disease progression and identify potential treatment targets.
Assuntos
Pólipos Nasais , Sinusite , Humanos , Pólipos Nasais/genética , Sinusite/genética , Genes fms , Doença Crônica , MacrófagosRESUMO
Tumor microenvironment-targeted therapies are emerging as promising treatment options for different cancer types. Tumor-associated macrophages and microglia (TAMs) represent an abundant nonmalignant cell type in brain metastases and have been proposed to modulate metastatic colonization and outgrowth. Here we demonstrate that targeting TAMs at distinct stages of the metastatic cascade using an inhibitor of colony-stimulating factor 1 receptor (CSF1R), BLZ945, in murine breast-to-brain metastasis models leads to antitumor responses in prevention and intervention preclinical trials. However, in established brain metastases, compensatory CSF2Rb-STAT5-mediated pro-inflammatory TAM activation blunted the ultimate efficacy of CSF1R inhibition by inducing neuroinflammation gene signatures in association with wound repair responses that fostered tumor recurrence. Consequently, blockade of CSF1R combined with inhibition of STAT5 signaling via AC4-130 led to sustained tumor control, a normalization of microglial activation states and amelioration of neuronal damage.
Assuntos
Neoplasias Encefálicas , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos , Animais , Neoplasias Encefálicas/secundário , Genes fms , Ativação de Macrófagos , Melanoma , Camundongos , Receptores de Fator Estimulador de Colônias/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Fator de Transcrição STAT5/genética , Neoplasias Cutâneas , Microambiente Tumoral , Melanoma Maligno CutâneoRESUMO
The effects of human immunodeficiency virus 1 (HIV-1) infection on cellular differentiation and NF-kappa B DNA binding activity have been investigated in a new model of myeloid differentiation. PLB-985 cells represent a bipotential myelomonoblastic cell population capable of either granulocytic or monocytic differentiation after induction with appropriate inducers. By virtue of the presence of CD4 on the cell surface, PLB-985 cells were chronically infected with HIV-1 strain IIIB. PLB-IIIB cells clearly possessed a more monocytic phenotype than the parental myeloblasts, as determined by differential staining, increased expression of the myeloid-specific surface markers, and transcription of the c-fms proto-oncogene. NF-kappa B binding activity was inducible by tumor necrosis factor and phorbol myristate acetate in PLB-985. However, in PLB-IIIB cells, constitutive expression of a novel NF-kappa B complex was detected, composed of proteins ranging between 70 and 110 kD. These proteins interacted specifically with the symmetric NF-kappa B site from the interferon beta (IFN-beta) promoter. Mutations affecting the 5' guanine residues of the kappa B site were unable to compete for these NF-kappa B-related proteins. Inducibility of endogenous IFN-beta and IFN-alpha RNA was also increased in PLB-IIIB cells. These studies indicate that HIV-1 infection of myelomonoblastic cells may select for a more mature monocytic phenotype and that unique subunit associations of NF-kappa B DNA binding proteins may contribute to differential NF-kappa B-mediated gene expression.
Assuntos
Síndrome da Imunodeficiência Adquirida/fisiopatologia , Células da Medula Óssea , HIV-1 , Monócitos/citologia , NF-kappa B/fisiologia , Sequência de Bases , Medula Óssea/imunologia , Medula Óssea/microbiologia , Antígenos CD4/análise , Diferenciação Celular , Expressão Gênica , Genes fms/genética , Genes myc/genética , Humanos , Interferon-alfa/genética , Interferon beta/genética , Modelos Biológicos , Dados de Sequência Molecular , Monócitos/imunologia , NF-kappa B/genética , Proto-Oncogene Mas , Proto-Oncogenes , Transcrição Gênica , Células Tumorais Cultivadas , Ativação ViralRESUMO
The receptor for macrophage colony stimulating factor (CSF-1), the c-fms gene product, is a key determinant in the differentiation of monocytic phagocytes. Dissection of the human and mouse c-fms proximal promoters revealed opposing roles for nuclear protooncogenes in the transcriptional regulation of this gene. On the one hand, c-ets-1, c-ets-2, and the macrophage-specific factor PU.1, but not the ets-factor PEA3, trans-activated the c-fms proximal promoter. On the other hand c-myb repressed proximal promoter activity in macrophages and blocked the action of c-ets-1 and c-ets-2. Basal c-fms promoter activity was almost undetectable in the M1 leukaemia line, which expressed high levels of c-myb, but was activated as cells differentiated in response to leukemia inhibitory factor and expressed c-fms mRNA. The repressor function of c-myb depended on the COOH-terminal domain of the protein. We propose that ets-factors are necessary for the tissue-restricted expression of c-fms and that c-myb acts to ensure correct temporal expression of c-fms during myeloid differentiation.
Assuntos
Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Proto-Oncogenes , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Células 3T3 , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Chlorocebus aethiops , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Genes fms , Humanos , Camundongos , Dados de Sequência Molecular , Oncogenes , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-ets , Proteínas Proto-Oncogênicas c-myb , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
Hematopoietic stem cells and multipotent progenitors exhibit low-level transcription and partial chromatin reorganization of myeloid cell-specific genes including the c-fms (csf1R) locus. Expression of the c-fms gene is dependent on the Ets family transcription factor PU.1 and is upregulated during myeloid differentiation, enabling committed macrophage precursors to respond to colony-stimulating factor 1. To analyze molecular mechanisms underlying the transcriptional priming and developmental upregulation of the c-fms gene, we have utilized myeloid progenitors lacking the transcription factor PU.1. PU.1 can bind to sites in both the c-fms promoter and the c-fms intronic regulatory element (FIRE enhancer). Unlike wild-type progenitors, the PU.1(-/-) cells are unable to express c-fms or initiate macrophage differentiation. When PU.1 was reexpressed in mutant progenitors, the chromatin structure of the c-fms promoter was rapidly reorganized. In contrast, assembly of transcription factors at FIRE, acquisition of active histone marks, and high levels of c-fms transcription occurred with significantly slower kinetics. We demonstrate that the reason for this differential activation was that PU.1 was required to promote induction and binding of a secondary transcription factor, Egr-2, which is important for FIRE enhancer activity. These data suggest that the c-fms promoter is maintained in a primed state by PU.1 in progenitor cells and that at FIRE PU.1 functions with another transcription factor to direct full activation of the c-fms locus in differentiated myeloid cells. The two-step mechanism of developmental gene activation that we describe here may be utilized to regulate gene activity in a variety of developmental pathways.
Assuntos
Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica no Desenvolvimento , Genes fms/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Transcrição Gênica/genética , Animais , Sequência de Bases , Montagem e Desmontagem da Cromatina/genética , Desoxirribonuclease I/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Elementos Facilitadores Genéticos , Histonas/metabolismo , Cinética , Metilação , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Células NIH 3T3 , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas/deficiência , RNA Polimerase II/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Transativadores/deficiência , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
The proliferation and activation of microglia, the resident macrophages in the brain, is a hallmark of many neurodegenerative diseases such as Alzheimer's disease (AD) and prion disease. Colony stimulating factor 1 receptor (CSF1R) is critically involved in regulating microglial proliferation, and CSF1R blocking strategies have been recently used to modulate microglia in neurodegenerative diseases. However, CSF1R is broadly expressed by many cell types and the impact of its inhibition on the innate immune system is still unclear. CSF1R can be activated by two independent ligands, CSF-1 and interleukin 34 (IL-34). Recently, it has been reported that microglia development and maintenance depend on IL-34 signaling. In this study, we evaluate the inhibition of IL-34 as a novel strategy to reduce microglial proliferation in the ME7 model of prion disease. Selective inhibition of IL-34 showed no effects on peripheral macrophage populations in healthy mice, avoiding the side effects observed after CSF1R inhibition on the systemic compartment. However, we observed a reduction in microglial proliferation after IL-34 inhibition in prion-diseased mice, indicating that microglia could be more specifically targeted by reducing IL-34. Overall, our results highlight the challenges of targeting the CSF1R/IL34 axis in the systemic and central compartments, important for framing any therapeutic effort to tackle microglia/macrophage numbers during brain disease.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Encéfalo/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Interleucinas/antagonistas & inibidores , Microglia/efeitos dos fármacos , Degeneração Neural , Doenças Priônicas/tratamento farmacológico , Animais , Anticorpos Monoclonais/toxicidade , Anticorpos Neutralizantes/toxicidade , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Genes fms , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interleucinas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Transdução de SinaisRESUMO
We generated double-transgenic mice carrying cointegrated tissue-specific Gal4 and Gal4 reporter transgenes to direct transgene overexpression in the mononuclear phagocyte system (MPS). A modified promoter of the Csf1r (c-fms) gene, containing a deletion of the trophoblast-specific promoter, was used to drive the expression of Gal4VP16 transcriptional activator specifically in macrophages. This module was cointegrated with a fluorescent reporter, enhanced cyan fluorescent protein (ECFP), driven by a Gal4-dependent promoter. ECFP fluorescence was first detected in forming blood islands of the yolk sac at 8 dpc, then in macrophages in the yolk sac and the embryo proper. In adult mice ECFP was detected primarily in monocytes, tissue macrophages, microglia, and dendritic cells, including Langerhans cells of the skin. Crossing of these mice to transgenics containing tagged protein under control of a Gal4-dependent promoter directed expression of that protein in mononuclear phagocytes of double-transgenic animals. The new mouse line provides a useful tool for overexpression of transgenes in cells of the myeloid lineage, while simultaneously labeling them by ECFP expression.
Assuntos
Células Dendríticas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Genes Sintéticos/genética , Genes fms , Proteínas de Fluorescência Verde/genética , Macrófagos/metabolismo , Camundongos Transgênicos/genética , Microglia/metabolismo , Monócitos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transgenes , Animais , Linhagem Celular/metabolismo , Linhagem da Célula , Cruzamentos Genéticos , Proteínas de Ligação a DNA , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos/embriologia , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Ativação Transcricional , Transgenes/genética , Saco Vitelino/citologiaRESUMO
The proliferation, differentiation and survival of mononuclear phagocytes depend on signals from the receptor for macrophage colony-stimulating factor, CSF1R. The mammalian Csf1r locus contains a highly conserved super-enhancer, the fms-intronic regulatory element (FIRE). Here we show that genomic deletion of FIRE in mice selectively impacts CSF1R expression and tissue macrophage development in specific tissues. Deletion of FIRE ablates macrophage development from murine embryonic stem cells. Csf1rΔFIRE/ΔFIRE mice lack macrophages in the embryo, brain microglia and resident macrophages in the skin, kidney, heart and peritoneum. The homeostasis of other macrophage populations and monocytes is unaffected, but monocytes and their progenitors in bone marrow lack surface CSF1R. Finally, Csf1rΔFIRE/ΔFIRE mice are healthy and fertile without the growth, neurological or developmental abnormalities reported in Csf1r-/- rodents. Csf1rΔFIRE/ΔFIRE mice thus provide a model to explore the homeostatic, physiological and immunological functions of tissue-specific macrophage populations in adult animals.
Assuntos
Genes fms/genética , Macrófagos/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Deleção de Sequência , Animais , Sequência de Bases , Diferenciação Celular , Proliferação de Células , Modelos Animais de Doenças , Células-Tronco Embrionárias/patologia , Fator de Crescimento Epidérmico , Feminino , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Macrófagos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Monócitos/metabolismo , Fagocitose , Células RAW 264.7 , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
E2a/Pbx1 is a fusion oncoprotein resulting from the t(1;19) translocation found in human pre-B acute lymphocytic leukemia and in a small number of acute T-lymphoid and myeloid leukemias. It was previously suggested that E2a/Pbx1 could cooperate with normal or oncogenic signaling pathways to immortalize myeloid and lymphoid progenitor cells. To address this question, we introduced the receptor of the macrophage-colony-stimulating factor (M-CSF-R) in pro-T cells immortalized by a conditional, estradiol-dependent, E2a/Pbx1-protein, and continuously proliferating in response to stem cell factor and interleukin-7. We asked whether M-CSF-R would be functional in an early T progenitor cell and influence the fate of E2a/Pbx1-immortalized cells. E2a-Pbx1 immortalized pro-T cells could proliferate and shifted from lymphoid to myeloid lineage after signaling through exogenously expressed M-CSF-R, irrespective of the presence of estradiol. However, terminal macrophage differentiation of the cells was obtained only when estradiol was withdrawn from cultures. This demonstrated that M-CSF-R is functional for proliferation and differentiation signaling in a T-lymphoid progenitor cell, which, in addition, unveiled myeloid potential of pro-T progenitors. Moreover, the block of differentiation induced by the E2a/Pbx1 oncogene could be modulated by hematopoietic cytokines such as M-CSF, suggesting plasticity of leukemic progenitor cells. Finally, additional experiments suggested that PU.1 and eight twenty-one transcriptional regulators might be implicated in the mechanisms of oncogenesis by E2a/Pbx1.
Assuntos
Diferenciação Celular , Linhagem da Célula , Transformação Celular Neoplásica , Proteínas de Homeodomínio/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Linfócitos T/metabolismo , Animais , Western Blotting , Proliferação de Células , Estradiol/farmacologia , Feminino , Citometria de Fluxo , Genes fms/genética , Genes fms/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/genética , Interleucina-7/farmacologia , Linfócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Células Mieloides/metabolismo , Proteínas de Fusão Oncogênica/genética , Fagocitose , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fator de Células-Tronco/farmacologia , Células-Tronco/metabolismo , Células-Tronco/patologiaRESUMO
OBJECTIVES: Granulosa cell tumors of the ovary (GCT) represent approximately 5% of malignant ovarian tumors. Surgery remains the primary modality of therapy and treatment options for advanced disease are limited. The molecular pathogenesis of GCT is not known but is likely to involve activation of tyrosine kinase-mediated cell signaling pathways. A recent case report of a patient with advanced recurrent GCT responding to the tyrosine kinase inhibitor, imatinib mesylate prompted us to explore a role for these therapies in GCT. METHODS: The expression of the imatinib-sensitive tyrosine kinases, c-kit, c-Abl, PDGFR-alpha and PDGFR-beta, was determined using RT-PCR in a panel of GCT. Activating mutations of c-kit and PDGFR-alpha were also sought. The functional response was examined in two human-derived GCT cell lines. RESULTS: All four kinases were expressed but at levels lower than those observed in pre-menopausal ovarian samples. Mutations in c-kit and PDGFR-alpha were not found. Both cell lines responded to imatinib and to the second generation, tyrosine kinase inhibitor, nilotinib, with dose-dependent decreases in cell proliferation and viability. These responses paralleled the imatinib-sensitive, K562 cell line but at approximately 240- and approximately 1000-fold higher concentrations of imatinib and nilotinib, respectively. CONCLUSIONS: Our study suggests that human GCT, in general, are unlikely to respond to imatinib or nilotinib therapy. The response of the cell lines at high concentrations implies an "off-target" effect, which suggests that a tyrosine kinase inhibitor, of appropriate specificity, may represent a therapeutic option in GCT.
Assuntos
Tumor de Células da Granulosa/tratamento farmacológico , Tumor de Células da Granulosa/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Piperazinas/farmacologia , Pirimidinas/farmacologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzamidas , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Genes fms , Tumor de Células da Granulosa/metabolismo , Tumor de Células da Granulosa/patologia , Humanos , Mesilato de Imatinib , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-abl/biossíntese , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-kit/biossíntese , Proteínas Proto-Oncogênicas c-kit/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/biossíntese , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/biossíntese , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Colony stimulating factor-1 (CSF-1) is one of the most common proinflammatory cytokine responsible for various inflammatory disorders. It has a remarkable role in the development and progression of osteoarthritis, cancer and other autoimmune disease conditions. The CSF-1 acts by binding to the receptor, called colony stimulating factor-1 receptor (CSF-1R) also known as c-FMS resulting in the cascade of signalling pathway causing cell proliferation and differentiation. Interleukin-34 (IL-34), recently identified as another ligand for CSF-IR, is a cytokine protein. Both, CSF-1 and IL-34, although two distinct cytokines, follow the similar signalling pathway on binding to the same receptor, CSF-1R. Like CSF-1, IL-34 promotes the differentiation and survival of monocyte, macrophages and osteoclasts. This CSF-1R/c-FMS is over expressed in many cancers and on tumour associated macrophages, consequently, have been exploited as a drug target for promising treatment for cancer and inflammatory diseases. Some CSF-1R/c-FMS inhibitors such as ABT-869, Imatinib, AG013736, JNJ-40346527, PLX3397, DCC-3014 and Ki20227 have been successfully used in these disease conditions. Many c-FMS inhibitors have been the candidates of clinical trials, but suffer from some side effects like cardiotoxicity, vomiting, swollen eyes, diarrhoea, etc. If selectivity of cFMS inhibition is achieved successfully, side effects can be overruled and this approach may become a novel therapy for treatment of various therapeutic interventions. Thus, successful targeting of c-FMS may result in multifunctional therapy. With this background of information, the present review focuses on the recent developments in the area of CSF-1R/c-FMS inhibitors with emphasis on crystal structure, mechanism of action and various therapeutic implications in which c-FMS plays a pivotal role. The review on structure activity relationship of various compounds acting as the inhibitors of c-FMS which gives the selection criteria for the development of novel molecules is also being presented.
Assuntos
Genes fms/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Fator Estimulador de Colônias de Macrófagos/metabolismo , Sequência de Aminoácidos , Animais , Axitinibe , Genes fms/fisiologia , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Indazóis/farmacologia , Indazóis/uso terapêutico , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Fator Estimulador de Colônias de Macrófagos/genética , Estrutura Secundária de Proteína , Piridinas/farmacologia , Piridinas/uso terapêutico , Relação Estrutura-AtividadeRESUMO
Vascular medial smooth muscle cells migrate, proliferate and transform to foam cells in the process of atherosclerosis. We have reported that the intimal smooth muscle cells express proto-oncogene c-fms, a characteristic gene of monocyte-macrophages, which is not normally expressed in medial smooth muscle cells. In the present study, we demonstrated that combinations of platelet-derived growth factor (PDGF)-BB and either epidermal growth factor (EGF) or fibroblast growth factor (FGF) induced high expression of c-fms in normal human medial smooth muscle cells to the level of intimal smooth muscle cells or monocyte-derived macrophages, whereas c-fms expression by PDGF-BB alone was 1/10 and both EGF and FGF had no independent effect on c-fms expression. By contrast, interferon (IFN)-gamma and macrophage colony-stimulating factor (M-CSF) suppressed the induction of c-fms expression. These results indicate that multiple growth factors and cytokines may play a role in the phenotypic transformation of medial smooth muscle cells to intimal smooth muscle cells in atherosclerotic lesions by altering c-fms expression.
Assuntos
Citocinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes fms/genética , Proteínas de Membrana , Músculo Liso Vascular/fisiologia , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Receptores de Lipoproteínas , Aorta/citologia , Arteriosclerose/etiologia , Células Cultivadas , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Interferon gama/farmacologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Desenvolvimento Muscular , Músculo Liso Vascular/crescimento & desenvolvimento , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proto-Oncogene Mas , RNA Mensageiro/análise , Receptores Imunológicos/análise , Receptores Depuradores , Receptores Depuradores Classe B , Túnica Íntima/citologia , Túnica Íntima/crescimento & desenvolvimento , Túnica Média/citologia , Túnica Média/crescimento & desenvolvimentoRESUMO
The mechanism of action of macrophage colony-stimulating factor (M-CSF) in osteoclast development was examined in a co-culture system of mouse osteoblastic cells and spleen cells. In this co-culture, osteoclast-like multinucleated cells (MNCs) were formed within 6 d in response to 10 nM 1 alpha,25(OH)2D3 added only for the final 2 d of culture. Simultaneously adding hydroxyurea for the final 2 d completely inhibited proliferation of cultured cells without affecting 1 alpha,25(OH)2D3-stimulated MNC formation. Autoradiographic examination using [3H]-thymidine revealed that osteoclast progenitors primarily proliferated during the first 4 d, whereas their differentiation into MNCs occurred predominantly during the final 2 d of culture in response to 1 alpha,25(OH)2D3. When anti-M-CSF antibody or anti-M-CSF receptor antibody was added either for the first 4 d or for the final 2 d, the MNC formation was similarly inhibited. In co-cultures of normal spleen cells and osteoblastic cells obtained from op/op mice, which cannot produce functionally active M-CSF, the lack of M-CSF either for the first 4 d or for the final 2 d failed to form MNCs in response to 1 alpha,25(OH)2D3 added for the last 2 d. These results clearly indicate that M-CSF is indispensable for both proliferation of osteoclast progenitors and their differentiation into mature osteoclasts.
Assuntos
Fator Estimulador de Colônias de Macrófagos/farmacologia , Osteoclastos/citologia , Células-Tronco/citologia , Animais , Animais Recém-Nascidos , Anticorpos/farmacologia , Calcitriol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , DNA/biossíntese , Feminino , Genes fms , Hidroxiureia/farmacologia , Cinética , Fator Estimulador de Colônias de Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos , Osteoclastos/efeitos dos fármacos , Receptor de Fator Estimulador de Colônias de Macrófagos/imunologia , Receptor de Fator Estimulador de Colônias de Macrófagos/fisiologia , Baço/citologia , Baço/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Timidina/metabolismo , Fatores de TempoRESUMO
To address the role of ras signaling in monocytic phagocytes in vivo, the expression of two dominant suppressors of in vitro ras signaling pathways, the carboxyl-terminal region of the GTPase-activating protein (GAP-C) and the DNA binding domain of the transcription factor ets-2, were targeted to this cell compartment. A 5-kb portion of the human c-fms proximal promoter was shown to direct expression of the transgenes to the monocytic lineage. As a result of the GAP-C transgene expression, ras-GTP levels were reduced in mature peritoneal macrophages by 70%. The terminal differentiation of monocytes was altered, as evidence by the accumulation of atypical monocytic cells in the blood. Mature peritoneal macrophages exhibited changes in colony-stimulating factor 1-dependent survival and structure. Further, expression of the colony-stimulating factor 1-stimulated gene urokinase plasminogen activator was inhibited in peritoneal macrophages. The results indicate that ras action is critical in monocytic cells after these cells have lost the capacity to traverse the cell cycle.
Assuntos
Genes ras , Monócitos/citologia , Monócitos/fisiologia , Transdução de Sinais , Animais , Diferenciação Celular , Feminino , Imunofluorescência , Expressão Gênica , Genes fms , Guanosina Trifosfato/metabolismo , Humanos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Proteínas ras/biossínteseRESUMO
The gene encoding the receptor for macrophage colony-stimulating factor 1 (CSF-1), the c-fms protooncogene, is selectively expressed in immature and mature mononuclear phagocytes and trophoblasts. Exon 1 is expressed only in trophoblasts. Isolation and sequencing of genomic DNA flanking exon 2 of the murine c-fms gene revealed a TATA-less promoter with significant homology to human c-fms. Reverse transcriptase primer extension analysis using exon 2 primers identified multiple clustered transcription initiation sites. Their position was confirmed by RNase protection. The same primer extension products were detected in equal abundance from macrophage or nonmacrophage sources of RNA. c-fms mRNA is acutely down-regulated in primary macrophages by CSF-1, bacterial lipopolysaccharide (LPS), and phorbol myristate acetate (PMA). Each of these agents reduced the abundance of c-fms RNA detectable by primer extension using an exon 3 primer without altering the abundance of presumptive short c-fms transcripts detected with exon 2 primers. Primer extension analysis with an intron 2 primer detected products at greater abundance in nonmacrophages. Templates detected with the intronic primer were induced in macrophages by LPS, PMA, and CSF-1, suggesting that each of the agents caused a shift from full-length c-fms mRNA production to production of unspliced, truncated transcripts. The c-fms promoter functioned constitutively in the RAW264 macrophage cell line, the B-cell line MOPC.31C, and several nonhematopoietic cell lines. Macrophage-specific expression and responsiveness to selective repression by LPS and PMA was achieved by the incorporation of intron 2 into the c-fms promoter-reporter construct. The results suggest that expression of the c-fms gene in macrophages is controlled by sequences in intron 2 that act by regulating transcription elongation.
Assuntos
Regulação da Expressão Gênica , Genes fms , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA/química , DNA/genética , DNA/isolamento & purificação , Regulação para Baixo , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Ratos , TATA Box , Células Tumorais CultivadasRESUMO
The macrophage colony-stimulating factor receptor encoded by the c-fms gene is expressed in vascular intimal smooth muscle cells isolated from atherosclerotic lesions. A combination of platelet-derived growth factor-BB and epidermal growth factor induces stable expression of c-fms in normal vascular medial smooth muscle cells. The mechanism by which these growth factors induce c-fms expression has now been investigated in an attempt to gain insight into the events that underlie the phenotypic conversion of vascular smooth muscle cells in atherosclerosis. Deletion analysis of the c-fms promoter revealed that the region including a binding site for transcription factor PU.1 was required for transcriptional activity in human aortic medial smooth muscle cells. Mutation in the PU.1 binding site markedly reduced promoter activity. Northern (RNA) blot analysis demonstrated that growth factors induced the expression of PU.1 mRNA in vascular medial smooth muscle cells and that PU.1 mRNA was expressed in vascular intimal smooth muscle cells. PU.1 antisense oligonucleotides inhibited growth factor-induced c-fms expression and foam cell formation. These results suggest that transcription factor PU.1 plays an essential role in the phenotypic conversion of vascular smooth muscle cells to macrophagelike cells by mediating the induction of c-fms.