RESUMO
Osteocalcin, also called Bone Gla Protein (BGP), is the most abundant of the non-collagenous proteins of bone produced by osteoblasts. It consists of a single chain of 46-50 amino acids, according to the species, and contains three vitamin K-dependent gamma-carboxyglutamic acid residues (GLA), involved in its binding to calcium and hydroxylapatite. Accumulating evidences suggest its involvement in bone remodeling, its physiological role, however, is still unclear. In this study the adhesion properties and the biological effects of osteocalcin on osteoclasts have been analyzed using as an experimental model, human osteoclast-like cells derived from giant cell tumors of bone (GCT). Osteocalcin promoted adhesion and spreading of these cells, triggering the release of bone sialoprotein (BSP), osteopontin (OPN) and fibronectin (FN), that in turn induced the clustering in focal adhesions of beta 1 and beta 3 integrin chains. Spreading was dependent upon the synthesis of these proteins. In fact, when the cells were incubated in the presence of monensin during the adhesion assay, they still adhered but spreading did not occur, focal adhesions disappeared and BSP, OPN, and FN were accumulated in intracellular granules. Furthermore osteocalcin induced chemotaxis in a dose-dependent manner. The action of BGP on osteoclasts was mediated by an intracellular calcium increase due to release from thapsigargin-sensitive stores. These results provide evidences that BGP exerts a role in the resorption process, inducing intracellular signaling, migration and adhesion, followed by synthesis and secretion of endogenous proteins.
Assuntos
Cálcio/metabolismo , Quimiotaxia/efeitos dos fármacos , Proteínas da Matriz Extracelular/biossíntese , Osteocalcina/farmacologia , Osteoclastos/fisiologia , Transdução de Sinais/fisiologia , Neoplasias Ósseas , Adesão Celular/efeitos dos fármacos , Citosol/metabolismo , Fibronectinas/biossíntese , Tumores de Células Gigantes , Humanos , Sialoproteína de Ligação à Integrina , Integrinas/fisiologia , Cinética , Osteoclastos/efeitos dos fármacos , Osteopontina , Sialoglicoproteínas/biossíntese , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The effects of retinol (vitamin A) and retinoic acid on primary cultures of isolated chicken osteoclasts have been studied. The experiments were performed to establish the direct actions of these two agents on the organization of cytoskeletal structures, on the acid phosphatase contents, and on the bone resorption activities of these cells. The results showed that by treating the cultures with retinol or retinoic acid, from 10(-8) to 10(-5) M, there were dose-related responses of the osteoclasts. Adhesion to the substratum was stimulated by increasing the number of cells exhibiting the specialized dot-like adhesion structures, or podosomes, which represent the active part of the sealing zone. The treatments also induced rearrangement of the microtubular patterns with reversible depolymerization of microtubules. Acid phosphatase activity was significantly higher both in vitamin A-treated osteoclasts and in their media. When [3H]proline-labeled bone particles were added to the retinoid-treated osteoclasts, the release of [3H]proline was increased significantly compared to controls. These results suggest that the two vitamin A metabolites cause several modifications of the metabolic status of isolated osteoclasts that result in augmented rates of bone resorption.
Assuntos
Reabsorção Óssea/efeitos dos fármacos , Osteoclastos/citologia , Tretinoína/farmacologia , Vitamina A/farmacologia , Fosfatase Ácida/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Galinhas , Imunofluorescência , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Osteoclastos/efeitos dos fármacos , Osteoclastos/enzimologia , Valores de Referência , Tubulina (Proteína)/análiseRESUMO
Recent studies reported an increased expression of osteopontin (OPN) in metastatic breast cancer cells, but the mechanisms modulating OPN production and the interaction of the cells with the secreted protein are far from clear. In this work, we utilized as an experimental system the cell line MDA-231 and we showed that HGF and M-CSF significantly enhance their adhesion onto OPN. Furthermore, in the presence of HGF and M-CSF, MDA-231 cells can adhere when plated onto BSA via increased OPN secretion. Moreover HGF and M-CSF induce de novo synthesis of OPN. In conclusion, these data suggest that HGF and M-CSF stimulate OPN production by MDA-231 cells, and that OPN is subsequently used as a substrate for cell adhesion.
Assuntos
Fator de Crescimento de Hepatócito/fisiologia , Fator Estimulador de Colônias de Macrófagos/fisiologia , Sialoglicoproteínas/metabolismo , Western Blotting , Matriz Óssea/metabolismo , Adesão Celular , Humanos , Osteopontina , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The structure of the osteoblastic layer on trabecular bone surfaces showing different accretion rates was examined. Volume and secretory surface of the cells and thickness of the underlying osteoid border were measured within serial sections. Shape, size and arrangement of osteoblasts varied greatly on the different trabeculae examined, but were relatively homogeneous over each surface. A correlation was found between the volume-secretory surface ration (i.e. the relative amount of protoplasmatic volume involved in osteoid production) and the thickness of the underlying osteoid seam measured separately for each osteoblast.
Assuntos
Desenvolvimento Ósseo , Osteoblastos , Animais , Osso e Ossos/citologia , Contagem de Células , Cães , Tamanho do Órgão , Propriedades de Superfície , Tíbia/anatomia & histologiaRESUMO
Two patients with spontaneous brain herniation into the middle ear have been operated on with a combined otoneurological approach. In case No. 1, two 2 x 3 mm arachnoid tissue herniations were found in the tegmen antri of the left ear. Six years later, a 8 x 9 mm mass consisting of prolapsed brain was removed from the right ear. The histological examination showed normal but disorganized nervous tissue. The surface consisted of middle ear mucosa or modified glial cells. More deeply numerous well preserved neurons and synapses were observed. In case No. 2, a 2 x 1 cm herniation was found in contact with the ossicles and the bony walls of the middle ear. The herniation consisted of partly degenerated nervous tissue which could explain the episode of temporal lobe seizure the patient experienced 8 years before surgery. In the world literature during the last 40 years, 29 cases of spontaneous or idiopathic brain herniation into the middle ear and mastoid have been reported. In 10, the herniations were multiple, as in our case No. 1. Case No. 1 is interesting also because the spontaneous brain herniation was bilateral.
Assuntos
Otorreia de Líquido Cefalorraquidiano/patologia , Orelha Média/patologia , Encefalocele/patologia , Otorreia de Líquido Cefalorraquidiano/cirurgia , Dendritos/ultraestrutura , Orelha Média/cirurgia , Encefalocele/cirurgia , Feminino , Humanos , Microscopia Eletrônica , Pessoa de Meia-Idade , Neurônios/patologia , RecidivaRESUMO
The pattern of the cortical vascular network and the changes occurring in it during the formation of Haversian systems have been studied in the diaphyses of the tibiae of dogs of various ages. The number, area, perimeter and microscopic structure of the vessels have been investigated with respect to the size of the Haversian canals and length of the osteoblastic layer, using India ink perfusion of the vascular network. The vascular network in compact bone undergoes conspicuous changes as a result of bone growth and turnover processes. The vessels proliferate during the formation of resorption cavities, eventually forming small plexuses of capillaries and tortuous venules. As osteons are laid down and their canals become more narrow, the progressive disappearance of the largest vessels leads to a reduction in the number of vessels in each canal. Morphometric analysis reveals a close correlation between the rate of osteonic bone deposition, the size of osteoblasts and the surface area of the vessels.
Assuntos
Ósteon/irrigação sanguínea , Animais , Artérias/anatomia & histologia , Cães , Ósteon/anatomia & histologia , Periósteo/irrigação sanguínea , Tíbia/anatomia & histologia , Tíbia/irrigação sanguínea , Tíbia/crescimento & desenvolvimento , Veias/anatomia & histologiaRESUMO
Because of the well-known technical difficulties in investigating the histology and physiology of the skeleton, bone physiology is not yet completely understood. Much new information has been obtained in recent years with in vitro investigations, but animal models are widely used when the overall effects of hormones or therapeutic agents or both need to be investigated. In this review, we discuss results obtained over the past year on several not yet completely understood topics. The effect of exercise on bone mass has been stressed by several authors, and at the cellular level, it has been suggested that the inositol cascade is the intracellular transduction pathway for mechanical stimuli. Ovariectomized rat systems have been widely used to demonstrate the protective effect of estrogen against bone resorption, and interesting indications of an estrogen-dependent stimulation of bone formation have been presented. The anabolic effect of low doses of fluoride has also been assessed.
Assuntos
Osso e Ossos/fisiologia , Animais , Fenômenos Biomecânicos , Osso e Ossos/irrigação sanguínea , Osso e Ossos/efeitos dos fármacos , Modelos Animais de Doenças , Estrogênios/farmacologia , Feminino , Fluoretos/farmacologia , Ovariectomia , Estimulação Física , Prostaglandinas/fisiologia , Fluxo Sanguíneo RegionalRESUMO
Single cell [Ca2+], studies were performed in chicken and rat osteoclasts loaded with fura-2 and exposed to a variety of treatments. Under resting conditions, basal [Ca2+]i, was 79.2 +/- 47.3 and 84.3 +/- 65.7 nM (averages +/- S.D.; n = 141 and 126) in the osteoclasts of the two species, respectively. Basal [Ca2+]i was stable in all rat and in approximately 80% of chicken osteoclasts. In the remaining 20%, spontaneous, irregular [Ca2+], fluctuations were observed (amplitude range: 50-200 nm over basal values). Increase of [Ca2+]o over the concentration of the Krebs-Ringer incubation medium (2 mM) induced rises of [Ca2+] in almost all cells investigated. [Ca2+] rises were already appreciable with 0.5 mM [Ca2+]o additions and reached high values with 4 mM additions: 390 +/- 113 and 364 +/- 214 nM [Ca2+], in rat and chicken osteoclasts, respectively (n = 122 and 101). Qualitatively, the responses to [Ca2+]o additions consisted of discrete [Ca2+]i transients, biphasic (an initial spike followed by a plateau), or monophasic (either the spike or the plateau). In a few chicken osteoclasts, the [Ca2+]i increase occurring after [Ca2+]o addition consisted of multiple, irregular fluctuations, similar to those observed in 20% of these cells under resting conditions. In individual osteoclasts subsequently exposed to multiple [Ca2+]o increase pulses, the type of the [Ca2+]i transient (mono- or biphasic) was maintained, and the size was dependent on the magnitude of the [Ca2+]o additions. Effects similar to those of [Ca2+]o were induced by the addition of Cd2+ or Ba2+ (but not La3+ or Mg2+) into the medium. The Cd2+ effect was maintained in part even in a Ca2+-free medium. Of various hormones and factors, parathormone, 1,25-dihydroxyvitamin D3, and prostaglandin E2 were inactive. In contrast, calcitonin was active in rat osteoclasts (which express numerous receptors). [Ca2+]i increases were small (19 +/- 17.9 nM; n = 21) when the hormone was administered alone; they were synergistic (severalfold potentiation) when the hormone was administered before or after [Ca2+]o. The [Ca2+]i effects of calcitonin were mimicked by 8Br-cAMP (31 +/- 26 nM; n = 12) when the nucleotide was administered alone; marked synergism when it was administered in combination with [Ca2+]o. This paper demonstrates for the first time that changes of [Ca2+]i are induced in osteoclasts by treatments with [Ca2+]o and calcitonin and can therefore be involved in intracellular mediation of the physiological effects of these two extracellular signals.
Assuntos
Calcitonina/fisiologia , Cálcio/metabolismo , Citosol/metabolismo , Espaço Extracelular/fisiologia , Fura-2/análogos & derivados , Osteoclastos/fisiologia , Animais , Benzofuranos , Cádmio , Cálcio/fisiologia , Galinhas , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Citosol/fisiologia , Sinergismo Farmacológico , Espaço Extracelular/metabolismo , Feminino , Hormônios/farmacologia , Lantânio , Magnésio , Osteoclastos/metabolismo , Ratos , Ratos EndogâmicosRESUMO
The adhesion of osteoclasts to the bone matrix is mandatory for bone resorption. Contact of the osteoclast with bone surface induces, in fact, cell polarization and organization of the resorbing apparatus, the so-called "ruffled border." Cell-matrix interaction in osteoclasts is a complex phenomenon resulting from formation of the "clear zone," a cytoplasmic area presenting the adhering plasma membrane, or "sealing membrane." The sealing membrane surrounds the ruffled border and seals the resorbing compartment, namely the extracellular space in which bone resorption takes place. Adhesion at this level occurs via specialized discrete structures, the "podosomes." Podosomes present most of the protein commonly found in focal adhesions, but with a peculiar organization. They are dynamic elements suitable for regulation, according with the functional demand of the cell. Their assembly increases during bone resorption and is regulated by the cytosolic free calcium concentration and the activity of protein kinase C.
Assuntos
Reabsorção Óssea , Osteoclastos/fisiologia , Animais , Matriz Óssea/fisiologia , Adesão Celular , Membrana Celular/fisiologia , Humanos , Osteoclastos/ultraestrutura , Transdução de SinaisRESUMO
A procedure for the isolation of osteoclasts from the heterogeneus population of medullary bone and marrow cells is presented. Cell suspensions were prepared from medullary bone of laying hens by mechanical dispersion. Following an osmotic shock and two 90 min unit gravity sedimentations, fractions highly enriched in osteoclasts were collected and subsequently cultivated in MEM with FCS. Ara C was added to the cultures for 48h to remove all the cells entering the mitotic cycle. The advantages and the possible applications of the method are discussed.
Assuntos
Osteoclastos/análise , Animais , Medula Óssea/análise , Separação Celular/métodos , Precipitação Química , Galinhas , Citarabina/farmacologia , Feminino , Pressão OsmóticaRESUMO
We tested whether recognition of bone-related peptides regulates intracellular Ca2+ concentration ([Ca2+]i) of giant cell tumor of bone (GCT). [Ca2+]i was measured in single cells by fura 2 fluorometry. GCT cells were sensitive to bone sialoprotein-II (BSP-II), osteopontin (OPN), and related fragments. Responses consisted of a prompt increase of [Ca2+]i, mostly transient, with a peak followed by a rapid return toward baseline. Responses were not mimicked by bovine plasma fibronectin. Sensitivity of GCT cells to bone peptides was specific, since BALB/3T3 fibroblasts and U-937 histiocytic lymphoma cells with monocytic phenotype failed to respond to BSP-II and OPN fragments. GRGDSP synthetic esapeptide, carrying the Arg-Gly-Asp adhesive motif, and GRGESP (Asp replaced by Glu), but not the GRADSP (Gly replaced by Ala), were active in inducing [Ca2+]i transients as well. Responses were observed also in cells treated with the BSP-II 1C fragment, lacking any known adhesive sequence, indicating that the active peptides inducing [Ca2+]i increments may be multiple. Sensitivity to extracellular matrix peptides was present in a variable fraction of the cells and was downregulated on long-term culture. The mechanism inducing [Ca2+]i elevations was mostly related to Ca2+ release from thapsigargin-sensitive intracellular pools.
Assuntos
Cálcio/metabolismo , Oligopeptídeos/farmacologia , Osteoclastos/fisiologia , Fragmentos de Peptídeos/farmacologia , Sialoglicoproteínas/farmacologia , Transdução de Sinais/fisiologia , Células 3T3 , Sequência de Aminoácidos , Animais , Neoplasias Ósseas , Reabsorção Óssea , Calcitonina/farmacologia , Bovinos , Linhagem Celular , AMP Cíclico/metabolismo , Ácido Egtázico/farmacologia , Tumor de Células Gigantes do Osso , Humanos , Sialoproteína de Ligação à Integrina , Cinética , Linfoma Difuso de Grandes Células B , Camundongos , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Osteoclastos/efeitos dos fármacos , Osteopontina , Osteossarcoma , Fosfoproteínas , Prolina/análise , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Osteoclast precursors are chemotactically attracted to sites of bone resorption via migration pathways that include transendothelial crossing in blood capillaries. Transendothelial migration involves poorly understood interactions with basal lamina molecules, including laminins. To investigate osteoclast-laminin interactions, we used human osteoclast-like cell lines obtained from giant cell tumors of bone (GCT 23 and GCT 24). These cell lines are a well-characterized model for osteoclast functions, such as bone resorption and the behaviour of osteoclast precursors. Both GCT cell lines adhered to laminin-2 (merosin) coated wells in standard adhesion assays, but failed to adhere to laminin-1 (EHS-laminin). By light microscopy, GCT cells on laminin-2 were partially spread, with a motile morphology. None of the anti-integrin antibodies tested inhibited GCT cells adhesion to laminin-2. Peptides containing the integrin adhesion site RGD or the laminin adhesion sequence IKVAV did not inhibit GCT cell adhesion to laminin-2. By immunofluorescence, beta 1 integrins were organized in focal adhesions. However, in the presence of monensin this reorganization of beta 1 integrins was abolished, indicating that it was probably due to secretion of fibronectin by GCT cells subsequent to adhesion to laminin-2. GCT cells transmigrated through membranes coated with laminin-2, much more efficiently than through membranes coated with collagen. Migration was induced by osteocalcin, as a chemoattractant, in a dose-dependent manner. At low osteocalcin concentrations, transmigration was detectable on laminin-2 but not collagen. In cells loaded with fura-2, a sharp increase in intracellular Ca2+ was detected upon addition of soluble laminin-2, but not laminin-1, due to release from thapsigargin-dependent intracellular stores. In summary, osteoclasts may recognize laminin isoforms differentially. Initial adhesion to laminin-2 appears to be due to integrin-independent mechanisms. Such adhesion, though, may trigger secretion of fibronectin that could then support spreading and efficient chemotactic migration. These mechanisms may play an important role in facilitating chemotactic migration of osteoclast precursors toward the bone surface.
Assuntos
Cálcio/metabolismo , Laminina/metabolismo , Osteoclastos/fisiologia , Adesão Celular/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Quimiotaxia/fisiologia , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Humanos , Integrinas/metabolismo , Laminina/farmacologia , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologiaRESUMO
Breast cancers commonly cause osteolytic metastases in bone, a process that is dependent upon osteoclast-mediated bone resorption, but the mechanism responsible for tumor-mediated osteoclast activation has not yet been clarified. In the present study we utilized a well-known human breast cancer cell line (MDA-231) in order to assess its capability to influence osteoclastogenesis in human bone marrow cultures and bone resorption in fully differentiated osteoclasts. We demonstrated that conditioned medium (CM) harvested from MDA-231 increased the formation of multinucleated TRAP-positive cells in bone marrow cultures. Bone resorption activity of fully differentiated human osteoclasts and of osteoclast-like cell lines, from giant cell tumors of bone (GCT), was highly increased by the presence of MDA-231 CM. Moreover, while MDA-231 by themselves did not produce IL-6 tumor cell, CM increased the secretion of IL-6 by primary human osteoclasts and GCT cell lines compared to untreated controls. These data suggest that MDA-231 produce osteoclastic activating factor(s) that increase both osteoclast formation in bone marrow culture and bone resorption activity by mature cells. Moreover, breast cancer cells stimulate IL-6 secretion by osteoclasts that is one of the factors known to supports osteoclastogenesis.
Assuntos
Células da Medula Óssea/citologia , Reabsorção Óssea , Meios de Cultivo Condicionados/farmacologia , Osteoclastos/citologia , Fosfatase Ácida/metabolismo , Biomarcadores , Neoplasias Ósseas , Neoplasias da Mama , Carcinoma Hepatocelular , Diferenciação Celular , Células Cultivadas , Feminino , Tumores de Células Gigantes , Humanos , Isoenzimas/metabolismo , Neoplasias Hepáticas , Osteogênese , Fosfatase Ácida Resistente a Tartarato , Células Tumorais CultivadasRESUMO
Integrins mediate cell adhesion and can induce different cellular responses, including changes in intracellular pH, changes and oscillation in intracellular free calcium, and protein phosphorylation on tyrosine. During bone resorption, the integrin alphav beta3 regulates adhesion of osteoclasts to bone extracellular matrix proteins, such us osteopontin (Opn). Adhesion via alphav beta3 is followed by osteoclast polarization onto the bone surface and by the onset of bone resorption. To characterize these events at the molecular level, we investigated the state of activation of alphav beta3 on the human osteoclast-like cell line GCT23 using the monoclonal antibody AP5 which binds to and can induce, under low calcium conditions, activated alphav beta3. By flow cytometry, approximately 50% of alphav beta3 on the surface of the osteoclast-like cell line GCT23 was reactive with AP5 and was therefore in the activated state. Incubation with AP5 in the presence of low calcium concentrations increased activated alphav beta3 to 90-100%. Activation of alphav beta3 increased the efficiency of GCT23 adhesion to Opn by 2-fold. Furthermore, haptotactic migration on Opn was also enhanced about 40% compared to control. We propose that changes in the activation state of alphav beta3 may be a regulation point for osteoclasts during bone resorption.
Assuntos
Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Osteoclastos/fisiologia , Receptores de Vitronectina/fisiologia , Sialoglicoproteínas/farmacologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Neoplasias Ósseas , Cálcio/farmacologia , Epitopos/imunologia , Humanos , Cinética , Osteoclastos/efeitos dos fármacos , Osteopontina , Receptores de Vitronectina/imunologia , Células Tumorais CultivadasRESUMO
The distribution of some cytoskeletal structures (microtubules, microfilaments, intermediate filaments) has been studied by indirect immunofluorescence microscopy and affinity purified antibodies in osteoclasts isolated from medullary bone of laying hens and in hen blood monocytes cultured in vitro. Both cell types show similar patterns of distribution of cytoskeletal structures and this further supports the concept that these cells are closely related. Osteoclasts and monocytes are also similar in their adhesion patterns, because they adhere to fibronectin-free areas and show closely comparable cell-to-substrate interactions when observed with interference reflection microscopy.
Assuntos
Monócitos/citologia , Osteoclastos/citologia , Animais , Adesão Celular , Células Cultivadas , Galinhas , Feminino , Fibronectinas/análise , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Microtúbulos , Monócitos/análise , Osteoclastos/análiseRESUMO
Osteoblast-osteoclast relationships in bone resorption are unclear. We investigated whether osteoblasts constitutively influence osteoclast activity. We employed a serum-free co-culture system in which chicken osteoclasts and chick calvaria or, alternatively, isolated chick osteoblasts were cultured in two different compartments separated by a 0.45 micron porous membrane permeable to soluble molecules. Osteoclastic bone resorption, evaluated by release of 3H-proline from prelabeled bone fragments, was significantly enhanced by bone cells resident in the calvaria, as well as by isolated osteoblasts. Stimulation was specific, since periosteal cells, or skin fibroblasts, failed to mimic osteoblast activity. Conditioned medium from osteoblast cultures stimulated osteoclast function in a similar manner, indicating that paracrine signals, capable of crossing the porous membrane separating the two compartments, are released by the bone forming cells.
Assuntos
Reabsorção Óssea , Comunicação Celular , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Fosfatase Alcalina/metabolismo , Animais , Células Cultivadas , Galinhas , Colágeno/biossíntese , Técnicas de Cultura/instrumentação , Técnicas de Cultura/métodos , AMP Cíclico/metabolismo , Modelos Biológicos , Osteoblastos/citologia , Osteocalcina/biossíntese , Osteoclastos/citologia , Prolina/metabolismoRESUMO
Osteoclasts are polarized cells with a basolateral and an apical membrane exposed to different extracellular Ca2+ ([Ca2+]o) and H+ (pHe) concentrations. Osteoclast bone resorption is inhibited in vitro by increases of [Ca2+]o slightly above physiological levels, detected by a [Ca2+]o sensing causing elevations of the intracellular signal, [Ca2+]i. Nevertheless, during bone resorption the apical membrane is exposed to [Ca2+]o severalfold higher than physiological without apparent inhibition of osteoclast functions. Because pHe facing the apical membrane is acidic, in this single-cell [Ca2+]i and intracellular pH study we addressed the question of whether the responses of human osteoclast-like cells from a giant cell tumor of bone to elevated [Ca2+]o are altered by reducing pHe. We first observed that low pHe stimulated Ca2+ efflux and cell acidification. We then demonstrated that the amplitude of the [Ca2+]o-dependent [Ca2+]i "spikes" is downregulated by low pHe, with approximately 70-fold higher [Ca2+]o required to induce significant responses at pHe 6.0 compared with pHe 7.4. Similar downregulation was observed in authentic freshly isolated rat osteoclasts. Finally, we observed that occupancy of the [Ca2+]o sensing by Ca2+ prompted rapid and transient cell acidification partially counteracted by a Na(+)-dependent amiloride derivative-sensitive H+ transport. These results demonstrate that the cascade of events triggered by activation of the [Ca2+]o sensing is affected by environmental pH and in turn influences cellular H+ transport. Such pH-related features of the [Ca2+] o sensing mechanism might be relevant for the regulation of osteoclast-like function.
Assuntos
Cálcio/metabolismo , Células Quimiorreceptoras/fisiologia , Espaço Extracelular/metabolismo , Concentração de Íons de Hidrogênio , Osteoclastos/metabolismo , Fura-2 , Humanos , Membranas Intracelulares/metabolismo , Concentração Osmolar , Células Tumorais CultivadasRESUMO
The effect of Retinoic Acid (RA) on human osteoclast-like cell lines, obtained from Giant Cell tumors (GCT) of bone, has been investigated evaluating its action on bone resorption, cell proliferation, microtubular organization and gelatinases expression and activity. Increasing concentrations of RA significantly dose-dependently decreased GCTs bone resorption, while 10(-7) M RA promoted an increase of cell proliferation. By immunofluorescence we demonstrated that GCTs express A and B gelatinases and, by zymography, that their activity was enhanced in medium collected from GCTs cultured in the presence of 10(-7) M RA. These data indicate that RA increases cell proliferation and modulates metalloproteinases (MMPs) activity, crucial events during the migration of osteoclast precursors toward bone surfaces.
Assuntos
Divisão Celular/efeitos dos fármacos , Gelatinases/metabolismo , Osteoclastos/efeitos dos fármacos , Tretinoína/farmacologia , Reabsorção Óssea , Imunofluorescência , Humanos , Osteoclastos/enzimologia , Osteoclastos/patologia , Timidina/metabolismo , Trítio , Células Tumorais CultivadasRESUMO
Hepatocyte growth factor (HGF), also known as scatter factor, is a powerful motogen, mitogen, and morphogen produced by cells of mesodermal origin, acting on epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c-MET protooncogene. We show that the HGF receptor is expressed by human primary osteoclasts, by osteoclast-like cell lines, and by osteoblasts. In both cell lineages, HGF stimulation triggers the receptor kinase activity and autophosphorylation. In osteoclasts, HGF receptor activation is followed by increase in intracellular Ca2+ concentration and by activation of the pp60c-Src kinase. HGF induces changes in osteoclast shape and stimulates chemotactic migration and DNA replication. Osteoblasts respond to HGF by entering the cell cycle, as indicated by stimulation of DNA synthesis. Interestingly, osteoclasts were found to synthesize and secrete biologically active HGF. These data strongly suggest the possibility of an autocrine regulation of the osteoclast by HGF and a paracrine regulation of the osteoblast by the HGF produced by the osteoclast.
Assuntos
Fator de Crescimento de Hepatócito/farmacologia , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Neoplasias Ósseas , Cálcio/metabolismo , Comunicação Celular , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Replicação do DNA/efeitos dos fármacos , Ativação Enzimática , Tumores de Células Gigantes , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Cinética , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-met , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptores Proteína Tirosina Quinases/biossíntese , Fatores de TempoRESUMO
We have verified the hypothesis that multiple myeloma (MM) may be disseminated by circulating clonogenic cells that selectively home to the bone marrow (BM) to receive the signal(s) leading to proliferation, terminal differentiation, and production of the osteoclast activating factors. Long-term cultures of stromal cells have been developed from the BM of nine patients with MM. These cells were mostly fibroblast-like elements, interspersed with a proportion of scattered macrophages and rare osteoclasts. BM stromal cells were CD54+, produced high levels of interleukin-6 (IL-6) and measurable amounts of IL-1 beta, and were used as feeder layers for autologous peripheral blood mononuclear cells (PBMC). After 3 weeks of cocultures, monoclonal B lymphocytes and plasma cells, derived from PBMC, developed and the number of osteoclasts significantly increased. Both populations grew tightly adherent to the stromal cell layer and their expansion was matched by a sharp increase of IL-6 and by the appearance of IL-3 in the culture supernatant. These data attribute to BM stromal cells a critical role in supporting the growth of B lymphocytes, plasma cells, and osteoclasts and the in vivo dissemination of MM.