RESUMO
To investigate the potential of ginsenosides in treating osteoporosis, ginsenoside compound K (GCK) was selected to explore the potential targets and mechanism based on network pharmacology (NP). Based on text mining from public databases, 206 and 6590 targets were obtained for GCK and osteoporosis, respectively, in which 138 targets were identified as co-targets of GCK and osteoporosis using intersection analysis. Five central gene clusters and key genes (STAT3, PIK3R1, VEGFA, JAK2 and MAP2K1) were identified based on Molecular Complex Detection (MCODE) analysis through constructing a protein-protein interaction network using the STRING database. Gene Ontology (GO) analysis implied that phosphatidylinositol-related biological process, molecular modification and function may play an important role for GCK in the treatment of osteoporosis. Function and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that the c-Fms-mediated osteoclast differentiation pathway was one of the most important mechanisms for GCK in treating osteoporosis. Meanwhile, except for being identified as key targets based on cytoHubba analysis using Cytoscape software, MAPK and PI3K-related proteins were enriched in the downstream of the c-Fms-mediated osteoclast differentiation pathway. Molecular docking further confirmed that GCK could interact with the cavity on the surface of a c-Fms protein with the lowest binding energy (-8.27 Kcal/moL), and their complex was stabilized by hydrogen bonds (Thr578 (1.97 Å), Leu588 (2.02 Å, 2.18 Å), Ala590 (2.16 Å, 2.84 Å) and Cys 666 (1.93 Å)), van der Waals and alkyl hydrophobic interactions. Summarily, GCK could interfere with the occurrence and progress of osteoporosis through the c-Fms-mediated MAPK and PI3K signaling axis regulating osteoclast differentiation.
Assuntos
Ginsenosídeos , Osteoporose , Humanos , Ginsenosídeos/farmacologia , Ginsenosídeos/uso terapêutico , Simulação de Acoplamento Molecular , Fosfatidilinositol 3-Quinases , Farmacologia em Rede , Osteoporose/tratamento farmacológicoRESUMO
Osteoclasts are bone resorbing cells that are responsible for physiological and pathological bone resorption. Macrophage colony stimulating factor (M-CSF) binds to the M-CSF receptor (c-FMS) and plays a key role in the differentiation and survival of macrophages and osteoclasts. THOC5, a member of the THO complex, has been shown to regulate hematopoiesis and M-CSF-induced macrophage differentiation. However, the role of THOC5 in osteoclasts remains unclear. Here, our study reveals a new role of THOC5 in osteoclast formation. We found that THOC5 shuttles between nucleus and cytoplasm in an M-CSF signaling dependent manner. THOC5 bound to FICD, a proteolytic cleavage product of c-FMS, and THOC5 facilitates the nuclear translocations of FICD. Decreased expression of THOC5 by siRNA-mediated knock down suppressed osteoclast differentiation, in part, by regulating RANK, a key receptor of osteoclasts. Mechanistically, knock down of THOC5 inhibited the expression of RANKL-induced FOS and NFATc1. Our findings highlight THOC5's function as a positive regulator of osteoclasts.
Assuntos
Fator Estimulador de Colônias de Macrófagos , Proteínas Nucleares , Osteoclastos , Osteogênese , Reabsorção Óssea , Diferenciação Celular , Humanos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Proteínas Nucleares/metabolismo , Osteoclastos/metabolismoRESUMO
Vitronectin is an abundant multifunctional glycoprotein found in serum, the extracellular matrix, and bone, and is involved in diverse physiological processes. Here, we developed a new bioactive dimeric peptide (VnP-8-DN1 dimer) from a human vitronectin-derived motif (IDAAFTRINCQG; residues 206-217; VnP-8) via removal of an isoleucine residue at the N-terminus of VnP-8 and spontaneous air oxidation. The VnP-8-DN1 dimer potently enhanced cell attachment activity, and this activity was mediated by binding to cellular heparan sulfate proteoglycan receptors. Moreover, the VnP-8-DN1 dimer suppressed osteoclast differentiation by blocking the early stage of osteoclastogenesis induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). Furthermore, the VnP-8-DN1 dimer decreased the bone-resorbing activity of osteoclasts and increased the survival of osteoclast precursor cells by decreasing the cellular level of c-Fms and reducing RANK expression. Taken together, these results demonstrate that the VnP-8-DN1 dimer inhibits the early stages of M-CSF- and RANK-induced osteoclast differentiation by binding to c-Fms and inhibiting M-CSF signaling.
Assuntos
Reabsorção Óssea , Fator Estimulador de Colônias de Macrófagos , Reabsorção Óssea/metabolismo , Diferenciação Celular , Humanos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Glicoproteínas de Membrana/metabolismo , Osteoclastos/metabolismo , Osteogênese , Ligante RANK/metabolismo , Ligante RANK/farmacologia , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Vitronectina/metabolismo , Vitronectina/farmacologiaRESUMO
Accumulating evidence suggests that Rab GTPases representing the largest branch of Ras superfamily have recently emerged as the core factors for the regulation of osteoclastogenesis through modulating vesicular transport amongst specific subcellular compartments. Among these, Rab34 GTPase has been identified to be important for the post-Golgi secretory pathway and for phagocytosis; nevertheless, its specific role in osteoclastogenesis has been completely obscure. Here, upon the in vitro model of osteoclast formation derived from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we reveal that Rab34 regulates osteoclastogenesis bidirectionally. More specifically, Rab34 serves as a negative regulator of osteoclast differentiation by promoting the lysosome-induced proteolysis of two osteoclastogenic surface receptors, c-fms and RANK, via the axis of early endosomes-late endosomes-lysosomes, leading to alleviate the transcriptional activity of two of the master regulator of osteoclast differentiation, c-fos and NFATc-1, eventually attenuating osteoclast differentiation and bone resorption. Besides, Rab34 plays a crucial role in modulating the secretory network of lysosome-related proteases including matrix metalloprotease 9 and Cathepsin K across the ruffled borders of osteoclasts, contributing to the regulation of bone resorption.
Assuntos
Reabsorção Óssea , Osteogênese , Animais , Reabsorção Óssea/metabolismo , Diferenciação Celular , Camundongos , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
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
Rab11b, abundantly enriched in endocytic recycling compartments, is required for the establishment of the machinery of vesicle trafficking. Yet, no report has so far characterized the biological function of Rab11b in osteoclastogenesis. Using in vitro model of osteoclasts differentiated from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we elucidated that Rab11b served as an inhibitory regulator of osteoclast differentiation sequentially via (i) abolishing surface abundance of RANK and c-Fms receptors; and (ii) attenuating nuclear factor of activated T-cells c1 (NFATc-1) upstream signaling cascades, following RANKL stimulation. Rab11b was localized in early and late endosomes, Golgi complex, and endoplasmic reticulum; moreover, its overexpression enlarged early and late endosomes. Upon inhibition of lysosomal function by a specific blocker, chloroquine (CLQ), we comprehensively clarified a novel function of lysosomes on mediating proteolytic degradation of c-Fms and RANK surface receptors, drastically ameliorated by Rab11b overexpression in RAW-D cell-derived osteoclasts. These findings highlight the key role of Rab11b as an inhibitor of osteoclastogenesis by directing the transport of c-Fms and RANK surface receptors to lysosomes for degradation via the axis of early endosomes-late endosomes-lysosomes, thereby contributing towards the systemic equilibrium of the bone resorption phase.
Assuntos
Osteoclastos/metabolismo , Osteogênese , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Proteólise , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Osteoclast differentiation and activity are controlled by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor-κB ligand (RANKL). Rab11A GTPase, belonging to Rab11 subfamily representing the largest branch of Ras superfamily of small GTPases, has been identified as one of the crucial regulators of cell surface receptor recycling. Nevertheless, the regulatory role of Rab11A in osteoclast differentiation has been completely unknown. In this study, we found that Rab11A was strongly upregulated at a late stage of osteoclast differentiation derived from bone marrow-derived macrophages (BMMs) or RAW-D murine osteoclast precursor cells. Rab11A silencing promoted osteoclast formation and significantly increased the surface levels of c-fms and receptor activator of nuclear factor-κB (RANK) while its overexpression attenuated osteoclast formation and the surface levels of c-fms and RANK. Using immunocytochemical staining for tracking Rab11A vesicular localization, we observed that Rab11A was localized in early and late endosomes, but not lysosomes. Intriguingly, Rab11A overexpression caused the enhancement of fluorescent intensity and size-based enlargement of early endosomes. Besides, Rab11A overexpression promoted lysosomal activity via elevating the endogenous levels of a specific lysosomal protein, LAMP1, and two key lysosomal enzymes, cathepsins B and D in osteoclasts. More importantly, inhibition of the lysosomal activity by chloroquine, we found that the endogenous levels of c-fms and RANK proteins were enhanced in osteoclasts. From these observations, we suggest a novel function of Rab11A as a negative regulator of osteoclastogenesis mainly through (i) abolishing the surface abundance of c-fms and RANK receptors, and (ii) upregulating lysosomal activity, subsequently augmenting the degradation of c-fms and RANK receptors, probably via the axis of early endosomes-late endosomes-lysosomes in osteoclasts.
Assuntos
Fator Estimulador de Colônias de Macrófagos/metabolismo , Osteogênese/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Endossomos/metabolismo , Regulação da Expressão Gênica , Inativação Gênica , Células HEK293 , Humanos , Lisossomos/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/metabolismo , Proteólise , Ligante RANK/metabolismoRESUMO
Osteoporosis morphology is characterized by bone resorption and decreases in micro-architecture parameters. Anti-osteoporosis therapy targets osteoclasts because bone resorption is a unique function of osteoclasts. Anti-c-fms antibodies against the receptor for macrophage colony-stimulating factor (M-CSF) inhibit osteoclast formation and bone resorption in vitro and in vivo. However, the effect of anti-c-fms antibodies on bone resorption in ovariectomized (OVX) mice is unknown. In this study, we evaluated the effect of anti-c-fms antibodies on osteoclast formation and bone resorption in osteoblast-osteoclast precursor co-culture in vitro and in OVX mice. Osteoblast and osteoclast precursor co-cultures treated with anti-c-fms antibodies showed significantly inhibited osteoclast formation, while cultures without anti-c-fms antibody treatment showed osteoclast formation. However, anti-c-fms antibodies did not change the receptor activator of nuclear factor kappa-B ligand (RANKL) or osteoprotegrin (OPG) expression during osteoblast and osteoclast differentiation in vitro. These results indicate that anti-c-fms antibodies directly affected osteoclast formation from osteoclast precursors in co-culture. OVX mice were treated with intraperitoneal injections of anti-c-fms antibody. The trabecular bone structure of the femur was assessed by micro-computer tomography. The anti-c-fms antibody inhibited osteoclast formation and bone loss compared with PBS-treated OVX mice. These results indicate potential for the therapeutic application of anti-c-fms antibodies for postmenopausal osteoporosis.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Reabsorção Óssea/prevenção & controle , Osteoblastos/citologia , Osteoclastos/citologia , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacologia , Reabsorção Óssea/diagnóstico por imagem , Reabsorção Óssea/etiologia , Reabsorção Óssea/metabolismo , Osso Esponjoso/diagnóstico por imagem , Osso Esponjoso/efeitos dos fármacos , Osso Esponjoso/metabolismo , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cocultura , Modelos Animais de Doenças , Feminino , Injeções Intraperitoneais , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteoprotegerina/metabolismo , Ovariectomia , Ligante RANK/metabolismo , Microtomografia por Raio-XRESUMO
The signaling pathways of growth factors, including platelet-derived growth factor, can be considered specific targets for overcoming the poor prognosis of idiopathic pulmonary fibrosis. Nintedanib, the recently approved multiple kinase inhibitor, has shown promising antifibrotic effects in patients with idiopathic pulmonary fibrosis; however, its efficacy is still limited, and in some cases, treatment discontinuation is necessary owing to toxicities such as gastrointestinal disorders. Therefore, more effective agents with less toxicity are still needed. TAS-115 is a novel multiple tyrosine kinase inhibitor that preferably targets platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor, and c-FMS in addition to other molecules. In this study, we evaluated the antifibrotic effect of TAS-115 on pulmonary fibrosis in vitro and in vivo. TAS-115 inhibited the phosphorylation of PDGFR on human lung fibroblast cell line MRC-5 cells and suppressed their platelet-derived growth factor-induced proliferation and migration. Furthermore, TAS-115 inhibited the phosphorylation of c-FMS, a receptor of macrophage colony-stimulating factor, in murine bone marrow-derived macrophages and decreased the production of CCL2, another key molecule for inducing pulmonary fibrosis, under the stimulation of macrophage colony-stimulating factor. Importantly, the inhibitory effects of TAS-115 on both PDGFR and c-FMS were 3- to 10-fold higher than those of nintedanib. In a mouse model of bleomycin-induced pulmonary fibrosis, TAS-115 significantly inhibited the development of pulmonary fibrosis and the collagen deposition in bleomycin-treated lungs. These data suggest that strong inhibition of PDGFR and c-FMS by TAS-115 may be a promising strategy for overcoming the intractable pathogenesis of pulmonary fibrosis.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Fibrose Pulmonar/tratamento farmacológico , Quinolinas/farmacologia , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Tioureia/análogos & derivados , Animais , Bleomicina/toxicidade , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Tioureia/farmacologiaRESUMO
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
Activation of the RAS/ERK and its downstream signaling components is essential for growth factor-induced cell survival, proliferation, and differentiation. The Src homology-2 domain containing protein tyrosine phosphatase 2 (SHP2), encoded by protein tyrosine phosphatase, non-receptor type 11 ( Ptpn11), is a positive mediator required for most, if not all, receptor tyrosine kinase-evoked RAS/ERK activation, but differentially regulates the PI3K/AKT signaling cascade in various cellular contexts. The precise mechanisms underlying the differential effects of SHP2 deficiency on the PI3K pathway remain unclear. We found that mice with myelomonocytic cell-specific [ Tg(LysM-Cre); Ptpn11fl/fl mice] Ptpn11 deficiency exhibit mild osteopetrosis. SHP2-deficient bone marrow macrophages (BMMs) showed decreased proliferation in response to M-CSF and decreased osteoclast generation. M-CSF-evoked ERK1/2 activation was decreased, whereas AKT activation was enhanced in SHP2-deficient BMMs. ERK1/2, via its downstream target RSK2, mediates this negative feedback by negatively regulating phosphorylation of M-CSF receptor at Tyr721 and, consequently, its binding to p85 subunit of PI3K and PI3K activation. Pharmacologic inhibition of RSK or ERK phenotypically mimics the signaling defects observed in SHP2-deficient BMMs. Furthermore, this increase in PI3K/AKT activation enables BMM survival in the setting of SHP2 deficiency.-Wang, L., Iorio, C., Yan, K., Yang, H., Takeshita, S., Kang, S., Neel, B.G., Yang, W. An ERK/RSK-mediated negative feedback loop regulates M-CSF-evoked PI3K/AKT activation in macrophages.
Assuntos
Células da Medula Óssea/enzimologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/enzimologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Proteína Quinase 3 Ativada por Mitógeno/genética , Fosfatidilinositol 3-Quinases/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/deficiência , Proteínas Proto-Oncogênicas c-akt/genética , Células RAW 264.7 , Proteínas Quinases S6 Ribossômicas 90-kDa/genéticaRESUMO
Anti-osteoporotic activity of a blocker of the ubiquitin-proteasome system, bortezomib, has known to be achieved by directly opposed action in increased bone formation by osteoblasts and in decreased bone destruction by osteoclasts. However, the mechanisms underlying the proteasome blocker inhibition of osteoclast differentiation and function are not fully understood. Here, we observed that proteasome inhibitors, such as MG132 and bortezomib, in osteoclasts accelerated the degradation of c-Fms, a cognate receptor of macrophage colony-stimulating factor (M-CSF), and did not affect the amount of receptor activator of nuclear factor kappa-B (RANK), a receptor of receptor activator of nuclear factor kappa-B ligand (RANKL). c-Fms degradation induced by proteasome inhibitors was controlled by the activation of p38/tumor necrosis factor-alpha converting enzyme (TACE)-mediated regulated intramembrane proteolysis (RIPping). This was validated through the restoration of c-Fms using specific inhibitors of p38 and TACE, and a stimulation of p38-dependent TACE. In addition, c-Fms degradation by proteasome inhibition completely blocked M-CSF-mediated intrinsic signalling and led to the suppression of osteoclast differentiation and bone resorption. In a mouse model with intraperitoneal administration of lipopolysaccharide (LPS) that stimulates osteoclast formation and leads to bone loss, proteasome blockers prevented LPS-induced inflammatory bone resorption due to a decrease in the number of c-Fms-positive osteoclasts. Our study showed that accelerating c-Fms proteolysis by proteasome inhibitors may be a therapeutic option for inflammation-induced bone loss.
Assuntos
Reabsorção Óssea/etiologia , Reabsorção Óssea/metabolismo , Inflamação/complicações , Osteoclastos/citologia , Osteoclastos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Ubiquitina/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Reabsorção Óssea/patologia , Reabsorção Óssea/prevenção & controle , Bortezomib/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Masculino , Camundongos , Inibidores de Proteassoma/farmacologia , Proteólise , Receptor de Fator Estimulador de Colônias de Macrófagos/genéticaRESUMO
Neuromedin B (NMB), a mammalian bombesin-like peptide, regulates diverse physiological processes, such as energy metabolism, memory and fear behavior, and cellular growth, through its cognate receptor, NMBR. In this study, we report that NMB expression was upregulated during osteoclast development and that silencing NMB or NMBR attenuated osteoclast generation mediated by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). We found that knockdown of NMB or NMBR using a small hairpin RNA suppressed M-CSF-induced proliferation of osteoclast precursor cells without altering osteoclast differentiation. Interestingly, NMB or NMBR knockdown reduced the expression of the M-CSF receptor, c-Fms, which is an important modulator of osteoclast development. Consequently, NMB or NMBR silencing inhibited M-CSF/c-Fms-mediated downstream signaling pathways like activation of ERK and Akt and induction of D-type cyclins, cyclin D1 and D2. Moreover, knockdown of NMB or NMBR accelerated apoptosis in osteoclast lineage cells by inducing caspase-3, caspase-9, and Bim expression. In summary, our study demonstrates that the NMB/NMBR axis plays a pivotal role in osteoclast generation by modulating the proliferation and survival of osteoclast lineage cells.
Assuntos
Ciclina D/metabolismo , Inativação Gênica , Fator Estimulador de Colônias de Macrófagos/metabolismo , Neurocinina B/análogos & derivados , Osteoclastos/citologia , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Receptores da Bombesina/metabolismo , Células-Tronco/citologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Camundongos , Neurocinina B/genética , Neurocinina B/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Ligante RANK/farmacologia , Receptores da Bombesina/antagonistas & inibidores , Receptores da Bombesina/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
Evidence suggests that macrophage colony-stimulating factor (M-CSF) participates critically in atherosclerosis; little is known about the role of M-CSF in the development of neointimal hyperplasia following mechanical vascular injury. We examined the expression of M-CSF and its receptor, c-fms, in rodent and rabbit models of arterial injury. Injured rat carotid arteries expressed 3- to 10-fold higher levels of M-CSF and c-fms mRNA and protein following balloon injury as compared to uninjured arteries. In the rabbit, M-CSF protein expression was greatest in neointimal smooth muscle cells (SMCs) postinjury, with some expression in medial SMCs. M-CSF-positive SMCs exhibited markers of proliferation. At 30days postinjury, neointimal SMCs in the adjacent healed area near the border between injured and uninjured zone lost both proliferative activity and overexpression of M-CSF. The presence of induced M-CSF and c-fms expression correlated with the initiation of SMCs proliferation. M-CSF stimulated incorporation of [(3)H] thymidine in human aortic smooth muscle cells in a concentration-dependent manner. Serum-free conditioned medium from aortic SMCs also promoted DNA synthesis, and this effect was blocked by M-CSF specific antibody. To test further the role of M-CSF in vivo, we induced arterial injury by placing a periadventitial collar around the carotid arteries in compound mutant mice lacking apolipoprotein apoE (apoE(-/-)) and M-CSF. Loss of M-CSF abolished the neointimal hyperplastic response to arterial injury in apoE(-/-) mice. Local delivery of M-CSF to the injured artery restored neointimal proliferation, suggesting a critical role of M-CSF for the development of neointimal thickening following arterial injury.
Assuntos
Lesões das Artérias Carótidas/patologia , Fator Estimulador de Colônias de Macrófagos/biossíntese , Neointima/patologia , Animais , Lesões das Artérias Carótidas/metabolismo , Modelos Animais de Doenças , Immunoblotting , Fator Estimulador de Colônias de Macrófagos/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Neointima/metabolismo , Coelhos , Ratos , Ratos Sprague-DawleyRESUMO
Cancer cells employ a variety of mechanisms to evade apoptosis and senescence. Pre-eminent among these is the aberrant co-expression of growth factors and their ligands, forming an autocrine growth loop that promotes tumour formation and progression. One growth loop whose transforming potential has been repeatedly demonstrated is the CSF-1/CSF-1R axis. Expression of CSF-1 and/or CSF-1R has been documented in a number of human malignancies, including breast, prostate and ovarian cancer and classical Hodgkin's lymphoma (cHL). This review summarizes the large body of work undertaken to study the role of this cytokine receptor system in malignant transformation. These studies have attributed a key role to the CSF-1/CSF-1R axis in supporting tumour cell survival, proliferation and enhanced motility. Moreover, increasing evidence implicates paracrine interactions between CSF-1 and its receptor in defining a tumour-permissive and immunosuppressive tumour-associated stroma. Against this background, we briefly consider the prospects for therapeutic targeting of this system in malignant disease.
Assuntos
Fatores Estimuladores de Colônias/fisiologia , Neoplasias/fisiopatologia , Receptor de Fator Estimulador de Colônias de Macrófagos/fisiologia , Humanos , Ligantes , Receptor de Fator Estimulador de Colônias de Macrófagos/químicaRESUMO
BACKGROUND: Lipocalin-2 (LCN2), a small glycoprotein, has a pivotal role in diverse biological processes such as cellular proliferation and differentiation. We previously reported that LCN2 is implicated in osteoclast formation induced by receptor activator of nuclear factor-kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). In the present study, we used a knockout mouse model to further investigate the role of LCN2 in osteoclast development. METHODS: Osteoclastogenesis was assessed using primary bone marrow-derived macrophages. RANKL and M-CSF signaling was determined by immunoblotting, cell proliferation by bromodeoxyuridine (BrdU) enzyme-linked immunosorbent assay (ELISA), and apoptosis by cell death detection ELISA. Bone morphometric parameters were determined using a micro-computed tomography system. RESULTS: Our results showed that LCN2 deficiency increases tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclast formation in vitro, a finding that reflects enhanced proliferation and differentiation of osteoclast lineage cells. LCN2 deficiency promotes M-CSF-induced proliferation of bone marrow macrophages (BMMs), osteoclast precursors, without altering their survival. The accelerated proliferation of LCN2-deficient precursors is associated with enhanced expression and activation of the M-CSF receptor, c-Fms. Furthermore, LCN2 deficiency stimulates the induction of c-Fos and nuclear factor of activated T cells c1 (NFATc1), key transcription factors for osteoclastogenesis, and promotes RANKL-induced inhibitor of kappa B (IκBα) phosphorylation. Interestingly, LCN2 deficiency does not affect basal osteoclast formation in vivo, suggesting that LCN2 might play a role in the enhanced osteoclast development that occurs under some pathological conditions. CONCLUSIONS: Our study establishes LCN2 as a negative modulator of osteoclast formation, results that are in accordance with our previous findings.
RESUMO
BACKGROUND: Lipocalin-2 (LCN2), a small glycoprotein, has a pivotal role in diverse biological processes such as cellular proliferation and differentiation. We previously reported that LCN2 is implicated in osteoclast formation induced by receptor activator of nuclear factor-kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). In the present study, we used a knockout mouse model to further investigate the role of LCN2 in osteoclast development. METHODS: Osteoclastogenesis was assessed using primary bone marrow-derived macrophages. RANKL and M-CSF signaling was determined by immunoblotting, cell proliferation by bromodeoxyuridine (BrdU) enzyme-linked immunosorbent assay (ELISA), and apoptosis by cell death detection ELISA. Bone morphometric parameters were determined using a micro-computed tomography system. RESULTS: Our results showed that LCN2 deficiency increases tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclast formation in vitro, a finding that reflects enhanced proliferation and differentiation of osteoclast lineage cells. LCN2 deficiency promotes M-CSF-induced proliferation of bone marrow macrophages (BMMs), osteoclast precursors, without altering their survival. The accelerated proliferation of LCN2-deficient precursors is associated with enhanced expression and activation of the M-CSF receptor, c-Fms. Furthermore, LCN2 deficiency stimulates the induction of c-Fos and nuclear factor of activated T cells c1 (NFATc1), key transcription factors for osteoclastogenesis, and promotes RANKL-induced inhibitor of kappa B (IkappaBalpha) phosphorylation. Interestingly, LCN2 deficiency does not affect basal osteoclast formation in vivo, suggesting that LCN2 might play a role in the enhanced osteoclast development that occurs under some pathological conditions. CONCLUSIONS: Our study establishes LCN2 as a negative modulator of osteoclast formation, results that are in accordance with our previous findings.
Assuntos
Animais , Camundongos , Fosfatase Ácida , Apoptose , Fenômenos Biológicos , Medula Óssea , Bromodesoxiuridina , Morte Celular , Proliferação de Células , Ensaio de Imunoadsorção Enzimática , Glicoproteínas , Immunoblotting , Fator Estimulador de Colônias de Macrófagos , Macrófagos , Camundongos Knockout , NF-kappa B , Osteoclastos , Fosforilação , Ligante RANK , Linfócitos T , Fatores de TranscriçãoRESUMO
Lipocalin-2 (LCN2) is a member of the lipocalin superfamily and plays a critical role in the regulation of various physiological processes, such as inflammation and obesity. In this study, we report that LCN2 negatively modulates the proliferation and differentiation of osteoclast precursors, resulting in impaired osteoclast formation. The overexpression of LCN2 in bone marrow-derived macrophages or the addition of recombinant LCN2 protein inhibits the formation of multinuclear osteoclasts. LCN2 suppresses macrophage colony-stimulating factor (M-CSF)-induced proliferation of osteoclast precursor cells without affecting their apoptotic cell death. Interestingly, LCN2 decreases the expression of the M-CSF receptor, c-Fms, and subsequently blocks its downstream signaling cascades. In addition, LCN2 inhibits RANKL-induced osteoclast differentiation and attenuates the expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), which are important modulators in osteoclastogenesis. Mechanistically, LCN2 inhibits NF-κB signaling pathways, as demonstrated by the suppression of IκBα phosphorylation, nuclear translocation of p65, and NF-κB transcriptional activity. Thus, LCN2 is an anti-osteoclastogenic molecule that exerts its effects by retarding the proliferation and differentiation of osteoclast lineage cells.
Assuntos
Proteínas de Fase Aguda/metabolismo , Diferenciação Celular , Linhagem da Célula , Lipocalinas/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Humanos , Lipocalina-2 , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Several studies have shown that blockade of colony stimulating factor-1 (CSF-1) or its receptor (CSF-1R) inhibits disease progression in rodent models of rheumatoid arthritis (RA); however, the role of the CSF-1/CSF-1R pathway in RA-induced pain and functional deficits has not been studied. Thus, we examined the effect of chronic intra-articular administration of a monoclonal anti-CSF-1R antibody (AFS98) on spontaneous pain, knee edema and functional disabilities in mice with arthritis. Unilateral arthritis was produced by multiple injections of complete Freund's adjuvant (CFA) into the right knee joint of adult male ICR mice. CFA-injected mice were then treated twice weekly from day 10 until day 25 with anti-CSF-1R antibody (3 and 10 µg/5 µL per joint), isotype control (rat IgG 10 µg/5 µL per joint) or PBS (5 µl/joint). Knee edema, spontaneous flinching, vertical rearing and horizontal exploratory activity were assessed at different days. Additionally, counts of peripheral leukocytes and body weight were measured to evaluate general health status. Intra-articular treatment with anti-CSF-1R antibody significantly increased horizontal exploratory activity and vertical rearing as well as reduced spontaneous flinching behavior and knee edema as compared to CFA-induced arthritis mice treated with PBS. Treatment with this antibody neither significantly affect mouse body weight nor the number of peripheral leukocytes. These results suggest that blockade of CSF-1R at the initial injury site (joint) could represent a therapeutic alternative for improving the functional disabilities and attenuating pain and inflammation in patients with RA.
Assuntos
Anticorpos Monoclonais/farmacologia , Artrite Experimental/fisiopatologia , Articulação do Joelho/fisiopatologia , Dor/fisiopatologia , Receptor de Fator Estimulador de Colônias de Macrófagos/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Artrite Experimental/tratamento farmacológico , Artrite Experimental/imunologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Artrite Reumatoide/fisiopatologia , Edema/tratamento farmacológico , Edema/patologia , Adjuvante de Freund , Inflamação/imunologia , Injeções Intra-Articulares , Articulação do Joelho/imunologia , Articulação do Joelho/patologia , Masculino , Camundongos Endogâmicos ICR , Dor/tratamento farmacológicoRESUMO
Breast cancer cells preferentially home to the bone microenvironment, which provides a unique niche with a network of multiple bidirectional communications between host and tumor, promoting survival and growth of bone metastases. In the bone microenvironment, the c-fms proto-oncogene that encodes for the CSF-1 receptor, along with CSF-1, serves as one critical cytokine/receptor pair, functioning in paracrine and autocrine fashion. Previous studies concentrated on the effect of inhibition of host (mouse) c-fms on bone metastasis, with resulting decrease in osteolysis and bone metastases as a paracrine effect. In this report, we assessed the role of c-fms inhibition within the tumor cells (autocrine effect) in the early establishment of breast cancer cells in bone and the effects of this early c-fms inhibition on subsequent bone metastases and destruction. This study exploited a multidisciplinary approach by employing two non-invasive, in vivo imaging methods to assess the progression of bone metastases and bone destruction, in addition to ex vivo analyses using RT-PCR and histopathology. Using a mouse model of bone homing human breast cancer cells, we showed that an early one-time application of anti-human c-fms antibody delayed growth of bone metastases and bone destruction for at least 31 days as quantitatively measured by bioluminescence imaging and computed tomography, compared to controls. Thus, neutralizing human c-fms in the breast cancer cell alone decreases extent of subsequent bone metastasis formation and osteolysis. Furthermore, we are the first to show that anti-c-fms antibodies can impact early establishment of breast cancer cells in bone.