RESUMO
Wilms' tumor 1 (WT1), a zinc-finger transcription regulator of the early growth response family, identified as the product of a tumor suppressor gene of Wilms' tumors, bears potential ability to induce macrophage differentiation in blood cell differentiation. Herein, we examined the involvement of WT1 in the regulation of osteoclastogenesis. We detected a high level of WT1 protein expression in osteoclast precursors; however, WT1 expression was markedly suppressed during osteoclastogenesis. We examined expression of WT1 transcripts in bone tissue by RNA in situ hybridization. We found a high level of antisense transcripts in osteoclasts actively resorbing bone in mandible of newborn rats. Expression of antisense WT1 RNA in mandible was also confirmed by Northern blot analysis and strand-specific RT-PCR. Overexpression of antisense WT1 RNA in RAW-D cells, an osteoclast precursor cell line, resulted in a marked enhancement of osteoclastogenesis, suggesting that antisense WT1 RNA functions to suppress expression of WT1 protein in osteoclastogenesis. High level expression of antisense WT1 RNA may contribute to commitment to osteoclastogenesis, and may allow osteoclasts to maintain or stabilize their differentiation state.
Assuntos
Diferenciação Celular/genética , Osteoclastos/citologia , Osteogênese/genética , RNA Antissenso/biossíntese , Proteínas WT1/biossíntese , Animais , Northern Blotting , Linhagem Celular , Regulação da Expressão Gênica , Hibridização In Situ , Masculino , Camundongos , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Proteínas WT1/genéticaRESUMO
Stimulated emission pumping with fluorescence depletion spectroscopy is used to determine the NaD X 1Σ+ ground-state dissociation energy and its isotopic shift. A total of 230 rovibrational levels in the range 9 ≤ vâ³ ≤ 29 and 1 ≤ Jâ³ ≤ 11 are observed, where vâ³ = 29 is about 50 cm-1 below the dissociation limit. Analysis of the highest five vibrational levels yields the dissociation energy De = 15 822 ± 5 cm-1 with a vibrational quantum number at dissociation vD = 31.2 ± 0.1. The energy difference in the well depth of this isotopologue with respect to that of NaH is δDe = De(NaH) - De(NaD) = -7 cm-1. A new set of Dunham coefficients is derived to fit all the observed energy levels to within the experimental uncertainty.
RESUMO
Galectins are a unique family of lectins bearing one or two carbohydrate recognition domains (CRDs) that have the ability to bind molecules with ß-galactoside-containing carbohydrates. It has been shown that galectins regulate not only cell growth and differentiation but also immune responses, as well as inflammation. Galectin-9, a tandem repeat type of galectin, was originally identified as a chemotactic factor for eosinophils, and is also involved in the regulatory process of inflammation. Here, we examined the involvement of galectin-9 and its receptor, T-cell immunoglobulin- and mucin-domain-containing molecule 3 (Tim-3), in the control of osteoclastogenesis and inflammatory bone destruction. Expression of Tim-3 was detected in osteoclasts and its mononuclear precursors in vivo and in vitro. Galectin-9 markedly inhibited osteoclastogenesis as evaluated in osteoclast precursor cell line RAW-D cells and primary bone marrow cells of mice and rats. The inhibitory effects of galectin-9 on osteoclastogenesis was negated by the addition of ß-lactose, an antagonist for galectin binding, suggesting that the inhibitory effect of galectin-9 was mediated through CRD. When galectin-9 was injected into rats with adjuvant-induced arthritis, marked suppression of bone destruction was observed. Inflammatory bone destruction could be efficiently ameliorated by controlling the Tim-3/galectin-9 system in rheumatoid arthritis.
Assuntos
Artrite/complicações , Reabsorção Óssea/etiologia , Galectinas/metabolismo , Osteoclastos/fisiologia , Receptores de Superfície Celular/metabolismo , Animais , Artrite/induzido quimicamente , Artrite/metabolismo , Reabsorção Óssea/metabolismo , Feminino , Humanos , Lactose , Masculino , Camundongos Endogâmicos C57BL , Ratos Sprague-DawleyRESUMO
Mesenchymal stem cells (MSCs) have potential to differentiate into multiple cell lineages. Recently, it was shown that MSCs also have anti-inflammatory and immunomodulatory functions. In this report, we investigated the regulatory function of MSCs in the development of inflammatory bone destruction in rats with adjuvant-induced arthritis (AA rats). MSCs were isolated from rat bone marrow tissues, expanded in the presence of basic FGF, and intraperitoneally injected into AA rats. MSC administration significantly suppressed inflammatory parameters: swelling score, swelling width, and thickness of hind paw. Radiographic evaluation indicated that MSC significantly suppressed bone destruction. Histological analysis showed that administration of MSCs markedly suppressed osteoclastogenesis in AA rats. To further delineate their effects on osteoclastogenesis, MSCs were added to in vitro bone marrow cultures undergoing osteoclastogenesis. MSCs significantly suppressed osteoclastogenesis in this system. Chemokine receptor expression in MSCs was assessed by RT-PCR, and a chemotactic assay was performed using a transwell culture system. MSCs showed significant chemotaxis to MIP-1α (CCL3) and SDF-1α (CXCL12), chemokines preferentially expressed in the area of inflammatory bone destruction. Furthermore, MSCs expressed IL-10 and osteoprotegerin, cytokines that suppress osteoclastogenesis. These data suggest that recruitment of MSC to the area of bone destruction in AA rats could suppress inflammatory bone destruction and raise the possibility that MSCs may have potential for the treatment of inflammatory bone destruction in arthritis.
Assuntos
Artrite Experimental/imunologia , Reabsorção Óssea/prevenção & controle , Células-Tronco Mesenquimais/imunologia , Animais , Artrite Experimental/complicações , Artrite Experimental/terapia , Reabsorção Óssea/etiologia , Reabsorção Óssea/imunologia , Diferenciação Celular , Quimiocina CCL3/metabolismo , Quimiocina CXCL12/metabolismo , Quimiocinas/metabolismo , Quimiotaxia , Citocinas/metabolismo , Feminino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/patologia , Osteoclastos/imunologia , Osteoclastos/patologia , Ratos , Ratos Endogâmicos Lew , Receptores de Quimiocinas/metabolismoRESUMO
Osteoclasts are the multinucleated giant cells formed by cell fusion of mononuclear osteoclast precursors. Despite the finding of several membrane proteins involving DC-STAMP as regulatory proteins required for fusion among osteoclast precursors, cellular and molecular events concerning this process are still ambiguous. Here we identified Tunneling Nanotubes (TNTs), long intercellular bridges with small diameters, as the essential cellular structure for intercellular communication among osteoclast precursors in prior to cell fusion. Formation of TNTs was highly associated with osteoclastogenesis and it was accompanied with the significant induction of the M-Sec gene, an essential gene for TNT formation. M-Sec gene expression was significantly upregulated by RANKL-treatment in osteoclast precursor cell line. Blockage of TNT formation by Latrunclin B or by M-Sec siRNA significantly suppressed osteoclastogenesis. We have detected the rapid intercellular transport of not only the membrane phospholipids labeled with DiI but also the DC-STAMP-GFP fusion protein through TNTs formed among osteoclast precursors during osteoclastogenesis. Transportation of such regulatory molecules through TNTs would be essential for the process of the specific cell fusion among osteoclast precursors.
Assuntos
Diferenciação Celular , Extensões da Superfície Celular/ultraestrutura , Osteoclastos/fisiologia , Animais , Transporte Biológico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Extensões da Superfície Celular/metabolismo , Células Cultivadas , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Osteoclastos/ultraestrutura , Fosfolipídeos/metabolismo , Ratos , Tiazolidinas/farmacologia , Fatores de Necrose Tumoral/genética , Fatores de Necrose Tumoral/metabolismo , Regulação para CimaRESUMO
To search cell surface molecules involved in the regulation of osteoclastogenesis, especially in fusion process, it is one powerful approach to obtain monoclonal antibodies bearing ability to block formation of multinucleated osteoclasts. Ideally, direct bio-assay of hybridoma supernatants is quite convenient to screen monoclonal antibodies of interest from numerous culture wells. However, addition of hybridoma supernatant containing hypoxanthine-aminopterin-thymidine (HAT), components of the selection medium, to whole bone marrow cultures strikingly suppressed osteoclastogenesis. Here we clarified aminopterin is the responsible component in HAT medium to inhibit osteoclastogenesis. Methotrexate (MTX), mono-methylated aminopterin, showed similar suppressive effect on osteoclastogenesis. When bone marrow cells were cultured in the presence of all nucleosides, aminopterin and MTX-induced suppression of osteoclastogenesis was abrogated. Among four nucleosides only adenosine canceled aminopterin-induced suppression of osteoclastogenesis. Direct bio-assay of hybridoma supernatant containing HAT selection medium is now available to screen monoclonal antibodies if adenosine-containing culture medium was utilized for evaluating osteoclastogenesis.
Assuntos
Adenosina/farmacologia , Aminopterina/farmacologia , Analgésicos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Osteoclastos/metabolismo , Animais , Diferenciação Celular , Hibridomas/citologia , Hibridomas/metabolismo , Masculino , Camundongos , Osteoclastos/citologia , Ratos , Ratos Sprague-DawleyRESUMO
Nordihydroguaiaretic acid (NDGA) is known to have prominent anticancer activity against several cancers, and is also known to be an inhibitor of 5-lipoxygenase (5-LO). In this study, we investigated the regulatory function of NDGA on inflammatory bone destruction mediated by osteoclasts. NDGA markedly inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced formation of osteoclasts in cultures of murine osteoclast precursor cell line RAW-D cells and primary bone marrow-derived macrophages culture systems. The inhibitory effect of NDGA on osteoclastogenesis did not arise from the inhibition of 5-LO activity. NDGA did not affect MAPKs, such as p38, JNK, and NF-κB, but significantly inhibited the induction of NFATc1, a key transcription factor for osteoclastogenesis. NDGA also suppressed activation of ERK in osteoclast precursors. RANKL-induced calcium oscillation observed in osteoclast precursors was completely diminished by the addition of NDGA. In mature osteoclasts, RANKL-induced nuclear translocation of NFATc1 was clearly inhibited by NDGA treatment. Finally, in vivo studies demonstrated that administration of NDGA significantly reduced severe bone destruction and osteoclast recruitment in the ankle joint of rats with adjuvant-induced arthritis. These results indicate the potential utility of NDGA as a therapeutic agent for ameliorating inflammatory bone destruction in rheumatoid arthritis.
Assuntos
Artrite Experimental/tratamento farmacológico , Reabsorção Óssea/tratamento farmacológico , Masoprocol/farmacologia , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Reabsorção Óssea/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/genética , Osteíte/tratamento farmacológico , Osteíte/metabolismo , Ligante RANK/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: Since transcription factors expressed in osteoclasts are possible targets for regulation of bone destruction in bone disorders, we investigated the expression of the transcription factor FBI-1/OCZF/LRF (in humans, factor that binds to inducer of short transcripts of human immunodeficiency virus type 1; in rats, osteoclast-derived zinc finger; in mice, leukemia/lymphoma-related factor) in patients with rheumatoid arthritis (RA), and assessed its role in osteoclastogenesis in vivo. METHODS: Expression of FBI-1/OCZF was investigated in subchondral osteoclasts in human RA and in rat adjuvant-induced arthritis (AIA) using immunostaining and in situ hybridization, respectively. Transgenic mice overexpressing OCZF (OCZF-Tg) under the control of the cathepsin K promoter were generated, and bone mineral density and bone histomorphometric features were determined by peripheral quantitative computed tomography, calcein double-labeling, and specific staining for osteoclasts and osteoblasts. LRF/OCZF expression and the consequence of LRF inhibition were assessed in vitro with RANKL-induced osteoclast differentiation. RESULTS: FBI-1/OCZF was detected in the nuclei of osteoclasts in rat AIA and human RA. RANKL increased the levels of LRF messenger RNA and nuclear-localized LRF protein in primary macrophages. In OCZF-Tg mice, bone volume was significantly decreased, the number of osteoclasts, but not osteoblasts, was increased in long bones, and osteoclast survival was promoted. Conversely, inhibition of LRF expression suppressed the formation of osteoclasts from macrophages in vitro. CONCLUSION: FBI-1/OCZF/LRF regulates osteoclast formation and apoptosis in vivo, and may become a useful marker and target in treating disorders leading to reduced bone density, including chronic arthritis.
Assuntos
Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Densidade Óssea/fisiologia , Proteínas de Ligação a DNA/metabolismo , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Fatores de Transcrição/metabolismo , Animais , Artrite Experimental/genética , Artrite Reumatoide/genética , Osso e Ossos/metabolismo , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Osteoblastos/metabolismo , Ligante RANK/genética , Ratos , Fatores de Transcrição/genéticaRESUMO
Methotrexate (MTX) is widely utilized for the treatment of patients with rheumatoid arthritis (RA); however, recent observation of the MTX-resistant patients proposed some difficulty in MTX-dependent therapeutic approach for RA. To access cellular events related to MTX resistance in RA in respect to inflammatory bone destruction, we investigated on an involvement of the potent inflammatory mediator adenosine in the regulation of osteoclastogenesis and inflammatory bone destruction. In rats with adjuvant-induced arthritis (AA rats), MTX efficiently suppressed bone destruction when it was administrated within 3 days after adjuvant injection, while it could not suppress inflammatory bone destruction if MTX was injected at the time of onset of inflammation (at day 10 after adjuvant injection). Time-course change in the level of plasma adenosine of AA rats was estimated by use of high-performance liquid chromatography and elucidated that adenosine level was markedly elevated till 10 days after adjuvant injection. In vitro bone marrow culture system for evaluating osteoclastogenesis, MTX markedly suppressed osteoclastogenesis in a stromal cell-dependent manner. This MTX-induced suppression of osteoclastogenesis was abrogated by the addition of adenosine. MTX suppressed the expression of mRNA for the receptor activator NF-κB ligand (RANKL), but it did not suppress the expression of osteoprotegerin (OPG). The addition of MTX and adenosine together markedly suppressed the level of OPG expression. Abolishment of MTX action by adenosine was significantly blocked by MRS1754, a highly selective antagonist for the A(2b) adenosine receptor (A(2b)AR), but not by caffeine, an antagonist for A1, A(2a), A3 AR (A1AR, A(2a)AR, and A3AR), which suggests that adenosine acts through A(2b)AR. Immunohistochemical studies showed abundant expression of A(2b)AR in cells localized in the bone-bone marrow boundary of the distal tibia in AA rats but not in control rats. When adenosine was injected in the ankle joints of MTX-treated AA rats, the suppressive effects of MTX on bone destruction was abolished. The current data therefore suggest that upregulation of adenosine production abolished the suppressive effect of MTX on osteoclastic bone destruction. Involvement of the adenosine-A(2b)AR system may explain MTX resistance in RA.
Assuntos
Adenosina/fisiologia , Antirreumáticos/antagonistas & inibidores , Artrite Experimental/tratamento farmacológico , Osso e Ossos/patologia , Metotrexato/antagonistas & inibidores , Osteoclastos/efeitos dos fármacos , Adenosina/sangue , Animais , Antirreumáticos/farmacologia , Antirreumáticos/uso terapêutico , Artrite Experimental/patologia , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Primers do DNA , Imuno-Histoquímica , Metotrexato/farmacologia , Metotrexato/uso terapêutico , Osteoclastos/citologia , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Galectin-3 is a beta-galactoside-binding animal lectin having pleiotropic effects on cell growth, differentiation, and apoptosis. This lectin has been shown to be involved in phagocytosis by macrophages and in inflammation. Here we investigated an involvement of galectin-3 in the regulatory process of inflammatory bone resorption in rats with adjuvant-induced arthritis (AA rats) accompanying severe bone destruction in the ankle joints. The protein level of galectin-3 in the ankle-joint extracts was markedly augmented at week 3 after adjuvant injection, at the time when severe bone destruction was observed. Immunohistochemical analysis revealed an extremely high expression of galectin-3 in macrophages and granulocytes infiltrated in the area of severe bone destruction. To estimate the role of galectin-3 in osteoclastogenesis and osteoclastic bone resorption, recombinant galectin-3 was added to in vitro culture systems. Galectin-3 markedly inhibited the formation of osteoclasts in cultures of murine osteoclast precursor cell line as well as in rat bone marrow culture systems. This inhibition was not observed by heat-inactivated galectin-3 or by galectin-7. Although recombinant galectin-3 did not affect signaling through mitogen-activated protein kinase (MAPK) or nuclear factor-kappaB (NF-kappaB), it specifically suppressed the induction of nuclear factor of activated T-cells c1 (NFATc1). Galectin-3 significantly inhibited dentine resorption by mature osteoclasts in vitro. Furthermore, in vivo studies clearly showed a significant suppression of bone destruction and osteoclast recruitment accompanying arthritis, when galectin-3 was injected into the cavity of ankle joint of AA rats. Thus, abundant galectin-3 observed in the area of severe bone destruction may act as a negative regulator for the upregulated osteoclastogenesis accompanying inflammation to prevent excess bone destruction.
Assuntos
Artrite Experimental/metabolismo , Artrite Experimental/patologia , Galectina 3/metabolismo , Osteoclastos/patologia , Animais , Células da Medula Óssea/citologia , Reabsorção Óssea/prevenção & controle , Metabolismo dos Carboidratos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Galectina 3/administração & dosagem , Galectina 3/antagonistas & inibidores , Galectina 3/farmacologia , Injeções Intra-Articulares , Masculino , Fatores de Transcrição NFATC/antagonistas & inibidores , Fatores de Transcrição NFATC/biossíntese , Ligante RANK/metabolismo , Ligante RANK/farmacologia , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Índice de Gravidade de Doença , Células-Tronco/citologia , Tíbia/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
We investigated here whether adiponectin can exhibit an inhibitory effect on tumor necrosis factor-alpha (TNF-alpha)- and receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclastogenesis by using RAW264 cell D clone with a high efficiency to form osteoclasts. Globular adiponectin (gAd) strongly inhibited TNF-alpha/RANKL-induced differentiation of osteoclasts by interfering with TNF receptor-associated factor 6 production and calcium signaling; consequently, the induction of nuclear factor of activated T cells c1 (NFATc1) was strongly inhibited. Moreover, we observed that inhibition of AMP-activated protein kinase abrogated gAd inhibition for TNF-alpha/RANKL-induced NFATc1 expression. Our data suggest that adiponectin acts as a potent regulator of bone resorption observed in diseases associated with cytokine activation.
Assuntos
Complexos Multienzimáticos/fisiologia , Fatores de Transcrição NFATC/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Ligante RANK/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases Ativadas por AMP , Adiponectina/farmacologia , Adiponectina/fisiologia , Animais , Doenças Ósseas/enzimologia , Reabsorção Óssea/enzimologia , Regulação para Baixo/efeitos dos fármacos , Combinação de Medicamentos , Ativação Enzimática , Camundongos , Complexos Multienzimáticos/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Adiponectina/metabolismo , Receptores de Adiponectina/fisiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Previous epidemiologic studies have suggested that periodontal disease is closely related to obesity and glucose tolerance. As the level of adiponectin, an adipocyte-derived cytokine, in plasma had been reported to decrease in obese and type 2 diabetes patients, we explored the role of adiponectin in the etiology of periodontitis using the D clone of RAW264, a clone that exhibits highly efficient osteoclast formation, to determine whether adiponectin acts as a regulatory molecule in osteoclast formation stimulated by lipopolysaccharide of periodontopathic bacteria. We observed that adiponectin acted as a potent inhibitor of osteoclast formation stimulated by Toll-like receptor 4 (TLR4) ligand and receptor activator of NF-kappaB ligand (RANKL). Because NF-kappaB is an important transcription factor in osteoclast formation, we examined the effect of adiponectin on its transcriptional activity. A luciferase assay showed that adiponectin was able to inhibit the TLR4-mediated NF-kappaB activity in RAW264 cells. In addition, we observed that the cytokine was actually able to inhibit TLR4-mediated expression of the gene for inducible nitric oxide synthase and production of nitric oxide in the cells. These observations strongly suggest that adiponectin may function as a negative regulator of lipopolysaccharide/RANKL-mediated osteoclast formation in periodontal disease.
Assuntos
Adiponectina/farmacologia , Aggregatibacter actinomycetemcomitans/química , Lipopolissacarídeos/farmacologia , Osteoclastos/efeitos dos fármacos , Periodontite/etiologia , Infecções por Actinobacillus , Animais , Linhagem Celular , Expressão Gênica , Lipopolissacarídeos/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Osteoclastos/citologia , Osteoclastos/metabolismo , Periodontite/microbiologia , Ligante RANK/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismoRESUMO
It has been reported that deer antler extract has anti-bone resorptive activity in vivo. However, little is known about the cellular and molecular mechanism of this effect. In this study, we investigated the effects of deer antler extracts on osteoclast differentiation and bone-resorption in vitro. Chloroform extract (CE-C) of deer antler inhibited osteoclast differentiation in mouse bone marrow cultures stimulated by receptor activator of NF-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF). CE-C suppressed the activation of extracellular signal-regulated kinase (ERK), protein kinase B (PKB/Akt) and inhibitor of kappa B (I-kappaB) by RANKL in osteoclast precursor cells. It also inhibited the bone resorptive activity of differentiated osteoclasts that was accompanied by disruption of actin rings and induction of the apoptosis. These results demonstrate deer antler extract may be a useful remedy for the treatment of bone-resorption diseases such as osteoporosis.
Assuntos
Chifres de Veado/química , Reabsorção Óssea/prevenção & controle , Diferenciação Celular/efeitos dos fármacos , Cervos , Osteoclastos/efeitos dos fármacos , Extratos de Tecidos/farmacologia , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Clorofórmio/química , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ligante RANK/farmacologia , Extratos de Tecidos/química , Extratos de Tecidos/isolamento & purificaçãoRESUMO
Osteoclasts are multinucleated cells formed by multiple steps of cell differentiation from progenitor cells of hematopoietic origin. Intervention in osteoclast differentiation is considered as an effective therapeutic approach to the treatment for bone diseases involving osteoclasts. In this study, we found that the organic compound (S)-1-lyso-2-stearoylamino-2-deoxy-sn-glycero-3-phosphatidylcholine (SCOH) inhibited osteoclast differentiation. The inhibitory effect of SCOH was observed in mouse bone marrow cell cultures supported either by coculturing with osteoblasts or by adding macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL). M-CSF and RANKL activate the ERK, Akt, and NF-kappaB signal transduction pathways, and SCOH suppressed this activation. SCOH also inhibited the bone resorptive activity of differentiated osteoclasts. It attenuated bone resorption, actin ring formation, and survival of mature osteoclasts. Reduced activation of Akt and NF-kappaB and decreased induction of XIAP were observed in mature osteoclasts treated with SCOH. Thus, this novel phosphatidylcholine derivative may be useful for treating bone-resorption diseases.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Lisofosfatidilcolinas/farmacologia , Osteoclastos/efeitos dos fármacos , Proteínas Serina-Treonina Quinases , Actinas/metabolismo , Animais , Reabsorção Óssea/tratamento farmacológico , Proteínas de Transporte/farmacologia , Células Cultivadas , Interações Medicamentosas , Lisofosfatidilcolinas/química , Lisofosfatidilcolinas/uso terapêutico , Glicoproteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Transdução de Sinais , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo XRESUMO
To investigate the molecular mechanisms of lung cancer-induced bone metastasis, we established a bone-seeking subclone (HARA-B4) from a human squamous lung cancer cell line (HARA) using an in vivo selection method. We compared comprehensive gene expression profiles between HARA and HARA-B4, and identified the critical factors for the formation of bone metastasis using in vitro and in vivo assays. The number of bone metastatic colonies in the hind legs was significantly higher in HARA-B4-inoculated mice than in HARA-inoculated mice at 4 weeks after inoculation. In addition, visceral (adrenal) metastases were not found in HARA-B4-inoculated mice at autopsy, suggesting an increase in cancer cell tropism to bone in HARA-B4. Based on a comprehensive gene expression analysis, the expression level of CXC chemokine ligand 14 (CXCL14) was 5-fold greater in HARA-B4 than in HARA. Results of a soft agar colony formation assay showed that anchorage-independent growth ability was 4.5-fold higher with HARA-B4 than with HARA. The murine pre-osteoblast cell line MC3T3-E1 and the pre-osteoclast/macrophage cell line RAW264.7 migrated faster toward cultured HARA-B4 cells than toward HARA cells in a transwell cell migration assay. Interestingly, CXCL14 was shown to be involved in all events (enhancement of cancer cell tropism to the bone, anchorage-independent growth and/or recruitment of bone marrow cells) based on siRNA experiments in HARA-B4 cells. Furthermore, in clinical specimens of lung cancer-induced bone metastasis, expression of CXCL14 was observed in the tumor cells infiltrated in bone marrow in all specimens examined. CXCL14 was able to promote bone metastasis through enhancement of cancer cell tropism to the bone and/or recruitment of bone marrow cells around metastatic cancer cells.