RESUMO
Calcitonin is a thirty-two amino acid peptide that contains an N-terminal disulphide bridge and a C-terminal prolineamide residue. It is released from thyroid parafollicular C-cells and its direct actions on the osteoclast account for its physiological effects whether as a hypocalcaemic agent and a potent inhibitor of bone resorption. These effects likely reflect actions upon a number of specific osteoclast cell surface receptors that initiate intracellular signaling events through both cyclic AMP and calcium mediated second messenger pathways. Studies of its potent anti-resorptive effects have significant translational implications in the management of Paget's bone disease, osteoporosis, and hypercalcaemia. This chapter summarizes major concepts in the synthesis and structure of calcitonin and then proceeds to outline its cellular, molecular actions and therapeutic applications, whilst seeking to provide a reference source. More detailed accounts have been given on different aspects of calcitonin physiology and biochemistry in a number of recent reviews by ourselves and others (155,157, Zaidi et al., 1994; 2002).
Assuntos
Calcitonina/farmacologia , Calcitonina/fisiologia , Osteoclastos/fisiologia , Receptores da Calcitonina/metabolismo , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/tratamento farmacológico , Calcitonina/genética , Calcitonina/uso terapêutico , Humanos , Osteíte Deformante/tratamento farmacológico , Osteoclastos/metabolismo , Relação Estrutura-AtividadeRESUMO
The effects of the related cytokines interleukin-6 (IL-6), leukemia inhibitory factor (LIF) and oncostatin-M on bone resorption and cytosolic Ca(2+) signaling were compared in isolated rat osteoclasts. In the traditional disaggregated osteoclast (pit) assay, IL-6 and LIF, but not oncostatin-M, conserved the bone resorption otherwise inhibited by high extracellular [Ca(2+)] (15 mM). It produced a paradoxical, concentration-dependent stimulation of resorption by elevated extracellular Ca(2+). In the micro-isolated single osteoclast resorption assay, IL-6, high [Ca(2+)] or IL-6 plus high [Ca(2+)] all increased pit formation. In contrast, the IL-6 receptor (IL-6R)-specific agonist antibody MT-18 inhibited bone resorption in a concentration-dependent manner (1:500 to 1:500 000). MT-18 triggered cytosolic Ca(2+) signals in fura 2-loaded osteoclasts within approximately 10 min of application. Each cytosolic Ca(2+) transient began with a peak deflection that persisted in Ca(2+)-free, EGTA-containing extracellular medium, consistent with a release of intracellularly stored Ca(2+). This was followed by a sustained elevation of cytosolic [Ca(2+)] that was abolished in Ca(2+)-free medium, as expected from an entry of extracellular Ca(2+), and by the Ca(2+) channel antagonist Ni(2+). The inclusion of either IL-6 or soluble human (sh) IL-6R specifically reversed both the above effects of MT-18, confirming that both effects were specific for the IL-6R. The findings suggest that IL-6R activation by IL-6 stimulates osteoclastic bone resorption either by reversing the inhibitory effect of high extracellular Ca(2+) in stromal-containing systems or itself stimulating bone resorption along with Ca(2+) by micro-isolated osteoclasts. In contrast, activation of the IL-6R by an agonist antibody produces an inhibition of bone resorption and an associated triggering of the cytosolic Ca(2+) signals previously associated with regulation of bone resorptive function in other situations.
Assuntos
Reabsorção Óssea , Sinalização do Cálcio/efeitos dos fármacos , Citocinas/farmacologia , Citosol/metabolismo , Osteoclastos/metabolismo , Receptores de Interleucina-6/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Cálcio/metabolismo , Células Cultivadas , Citocinas/metabolismo , Inibidores do Crescimento/farmacologia , Interleucina-6/metabolismo , Interleucina-6/farmacologia , Fator Inibidor de Leucemia , Linfocinas/farmacologia , Oncostatina M , Osteoclastos/ultraestrutura , Peptídeos/farmacologia , Ratos , Ratos Wistar , Receptores de Interleucina-6/imunologiaRESUMO
Calcitonin was discovered as a hypocalcemic principal that was initially thought to originate from the parathyroid gland. This view was corrected subsequently, and an origin from the thyroid C cells was documented. The purification and sequencing of various calcitonins soon followed. Calcitonin is a 32-amino-acid-long peptide with an N-terminal disulfide bridge and a C-terminal prolineamide residue. The peptide was shown to potently inhibit bone resorption; however, a direct osteoclastic action of the peptide was confirmed only in the early 1980s. Several osteoclast calcitonin receptors have subsequently been cloned and sequenced. Specific regions of the receptor necessary for ligand binding and intracellular signaling through cyclic AMP and calcium have been identified through systematic deletion mutagenesis and chimeric receptor studies. Calcitonin's potent antiresorptive effect has led to its use in treating Paget's disease of bone, osteoporosis, and hypercalcemia. This review retraces key aspects of the synthesis and structure of calcitonin, its cellular and molecular actions, and its therapeutic uses as they have emerged over the 40 years since its discovery. The review also examines the implications of these findings for future clinical applications as a tribute to early workers to whom credit must be given for creation of an important and expanding field. Notable are the new approaches currently being used to enhance calcitonin action, including novel allosteric activators of the calcitonin receptor, modulation of the release of endogenous calcitonin by calcimimetic agents, as well as the development of oral calcitonins.
Assuntos
Remodelação Óssea/fisiologia , Osso e Ossos/fisiologia , Calcitonina/fisiologia , Doenças Ósseas/tratamento farmacológico , Doenças Ósseas/fisiopatologia , Calcitonina/uso terapêutico , Cálcio/fisiologia , HumanosRESUMO
Cathepsin K plays a key role in bone resorption. We provide the first evidence that osteoprotegerin ligand (OPGL), a critical pro-resorptive cytokine, acutely stimulates the expression of cathepsin K in osteoclasts. We used in situ RT-PCR and real time quantitative RT-PCR to analyze cathepsin K gene expression. OPGL enhanced cathepsin K mRNA levels in mature osteoclasts isolated from rat neonatal long bones. OPGL together with macrophage colony-stimulating factor (M-CSF) also stimulated cathepsin K gene expression in monocytic cells and multinucleate osteoclasts in bone marrow cultures. Real time quantitative RT-PCR demonstrated high levels of cathepsin K mRNA in bone marrow cultures, paralleling the degree of osteoclastogenesis. We therefore suggest that OPGL enhances bone resorption, at least in part, by inducing cathepsin K gene expression.
Assuntos
Proteínas de Transporte/farmacologia , Catepsinas/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Glicoproteínas de Membrana/farmacologia , Osteoclastos/enzimologia , Transcrição Gênica/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Células da Medula Óssea/citologia , Calcitriol/farmacologia , Catepsina K , Técnicas de Cultura de Células/métodos , Núcleo Celular/fisiologia , Células Cultivadas , Citocinas/farmacologia , Fêmur , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Cinética , Fator Estimulador de Colônias de Macrófagos/farmacologia , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Ligante RANK , RNA Mensageiro/genética , Ratos , Ratos Wistar , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tíbia , Fatores de TempoRESUMO
We provide the first demonstration for a Na+/Ca2+ exchanger, NCX-1, in the osteoclast. We speculate that by using Na+ exchange, NCX-1 couples H+ extrusion with Ca2+ fluxes during bone resorption. Microspectrofluorimetry of fura-2-loaded osteoclasts revealed a rapid and sustained, but reversible, cytosolic Ca2+ elevation upon Na+ withdrawal. This elevation was abolished by the cytosolic introduction (by gentle permeabilization) of a highly specific Na+/Ca2+ exchange inhibitor peptide, XIP, but not its inactive analogue, sXIP. Confocal microscopy revealed intense plasma membrane immunofluorescence with an isoform-specific monoclonal anti-NCX-1 antibody applied to gently permeabilized osteoclasts. Electrophysiological studies using excised outside-in membrane patches showed a low-conductance, Na+-selective, dichlorobenzamil-sensitive, amiloride-insensitive channel that we tentatively assigned as being an NCX. Finally, to examine for physiological relevance, an osteoclast resorption (pit) assay was performed. There was a dramatic reduction of bone resorption following NCX-1 inhibition by dichlorobenzamil and XIP (but not with S-XIP). Together, the results suggest that a functional NCX, likely NCX-1, is involved in the regulation of osteoclast cytosolic Ca2+ and bone resorption.
Assuntos
Reabsorção Óssea , Osteoclastos/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Células Cultivadas , Imuno-Histoquímica , Potenciais da Membrana , Osteoclastos/fisiologia , Ratos , Trocador de Sódio e Cálcio/químicaRESUMO
The causes for the propensity of metastasized prostate cancer cells to grow in bone and to induce osteoblastic lesions remain unresolved. Co-culture of human prostate cancer cell lines with bone slices was determined to increase the level of endothelin-1 (ET-1) mRNA and its production. ET-1 is an ejaculate protein that also stimulates osteoblasts. Osteoclastic bone resorption was significantly blocked by the presence of androgen-independent prostate cancer cells in a dose-dependent manner as that of synthetic ET-1. The inhibition could be neutralized by specific ET-1 antibody, indicating the association of prostate cancer-derived ET-1 with inhibition of bone resorption. The combined ET-1 activity on osteoclasts and osteoblasts disrupts bone remodelling. ET-1 production is also elevated in the presence of prostate-specific antigen (PSA). ET-1 in turn enhances DNA synthesis of prostate cancer cells. Interactions among cancer cells, bone, ET-1 and PSA may be critical in cancer growth and lesions in bone.
Assuntos
Reabsorção Óssea/etiologia , Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Endotelina-1/metabolismo , Neoplasias da Próstata/complicações , Neoplasias da Próstata/metabolismo , Análise de Variância , Northern Blotting , Divisão Celular , Endotelina-1/genética , Humanos , Masculino , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
We report the molecular cloning and functional characterization of a novel member of the CD38 family of cyclic ADP-ribose (cADPr)-generating cyclases. We cloned a cDNA insert that encoded a 298-amino-acid-long protein (M(w) approximately 39 kDa). The predicted protein displayed 69, 61, and 58% similarity, respectively, to mouse, rat, and human CD38. Rabbit CD38 was also 28% homologous to Aplysia ADP-ribosyl cyclase and leukocyte CD157 (another ADP-ribosyl cyclase); the three cyclases shared 10 cysteine and 2 adjacent proline residues. We then transfected CD38-negative NIH3T3 cells with cDNA encoding a CD38-EGFP fusion protein. Epifluorescence microscopy showed intense EGFP fluorescence confirming CD38 expression. We finally confirmed the ADP-ribosyl cyclase activity of the expressed CD38 by measuring its ability to catalyze the cyclization of the nicotinamide adenine dinucleotide (NAD(+)) surrogate, NGD(+), to its fluorescent nonhydrolyzable derivative, cGDPr.
Assuntos
Antígenos CD , Antígenos de Diferenciação/genética , NAD+ Nucleosidase/genética , Células 3T3 , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação/química , Antígenos de Diferenciação/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Complementar , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Glicoproteínas de Membrana , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , NAD+ Nucleosidase/química , NAD+ Nucleosidase/metabolismo , Coelhos , Ratos , Homologia de Sequência de AminoácidosRESUMO
We report the first biochemical and functional characterization of inositol trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) in the nuclear membrane of bone-forming (MC3T3-E1) osteoblasts. Intact nuclei fluoresced intensely with anti-RyR (Ab(34)) and anti-IP(3)R (Ab(40)) antisera in a typically peripheral nuclear membrane pattern. Isolated nuclear membranes were next subjected to SDS-PAGE and blotted with isoform-specific anti-receptor antisera, notably Ab(40), anti-RyR-1, anti-RyR-2 (Ab(129)), and anti-RyR-3 (Ab(180)). Only anti-RyR-1 and Ab(40) showed bands corresponding, respectively, to full-length RyR-1 ( approximately 500 kDa) and IP(3)R-1 (approximately 250 kDa). Band intensity was reduced by just approximately 20% after brief tryptic proteolysis of intact nuclei; this confirmed that isolated nuclear membranes were mostly free of endoplasmic reticular contaminants. Finally, the nucleoplasmic Ca(2+) concentration ([Ca(2+)](np)) was measured in single nuclei by using fura-dextran. The nuclear envelope was initially loaded with Ca(2+) via Ca(2+)-ATPase activation (1 mM ATP and approximately 100 nM Ca(2+)). Adequate Ca(2+) loading was next confirmed by imaging the nuclear envelope (and nucleoplasm). Exposure of Ca(2+)-loaded nuclei to IP(3) or cADP ribose resulted in a rapid and sustained [Ca(2+)](np) elevation. Taken together, the results provide complementary evidence for nucleoplasmic Ca(2+) influx in osteoblasts through nuclear membrane-resident IP(3)Rs and RyRs. Our findings may conceivably explain the direct regulation of osteoblastic gene expression by hormones that use the IP(3)-Ca(2+) pathway.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Núcleo Celular/metabolismo , Osteoblastos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Células 3T3 , Animais , Imuno-Histoquímica , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Camundongos , Membrana Nuclear/metabolismo , Isoformas de Proteínas/metabolismoRESUMO
Herein we demonstrate that replicative cellular senescence in vitro results in sharply reduced inositol 1,4,5-trisphosphate (IP(3)) receptor levels, reduced mitogen-evoked IP(3) formation and Ca(2+) release, and Ca(2+) store depletion. Human diploid fibroblasts (HDFs) underwent either 30 mean population doublings [mean population doublings (MPDs) thymidine labeling index (TI) >92% ("young") or between 53 and 58 MPDs (TI < 28%; "senescent")]. We found that the cytosolic Ca(2+) release triggered by either ionomycin or by several IP(3)-generating mitogens, namely bradykinin, thrombin, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), was attenuated markedly in senescent HDFs. Notably, the triggered cytosolic Ca(2+) transients were of a smaller magnitude in senescent HDFs. However, the response latency seen with both PDGF and EGF was greater for senescent cells. Finally, a smaller proportion of senescent HDFs showed oscillations. In parallel, IP(3) formation in response to bradykinin or EGF was also attenuated in senescent HDFs. Furthermore, senescent HDFs displayed a sharply diminished Ca(2+) release response to intracellularly applied IP(3). Finally, to compare IP(3) receptor protein levels directly in young and senescent HDFs, their microsomal membranes were probed in Western blots with a highly specific anti-IP(3) receptor antiserum, Ab(40). A approximately 260-kDa band corresponding to the IP(3) receptor protein was noted; its intensity was reduced by approximately 50% in senescent cells. Thus, we suggest that reduced IP(3) receptor expression, lowered IP(3) formation, and Ca(2+) release, as well as Ca(2+) store depletion, all contribute to the deficient Ca(2+) signaling seen in HDFs undergoing replicative senescence.
Assuntos
Canais de Cálcio/metabolismo , Fibroblastos/fisiologia , Inositol 1,4,5-Trifosfato/biossíntese , Receptores Citoplasmáticos e Nucleares/metabolismo , Bradicinina/farmacologia , Cálcio/metabolismo , Células Cultivadas , Senescência Celular/fisiologia , Criança , Citosol/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Substâncias de Crescimento/farmacologia , Humanos , Inositol 1,4,5-Trifosfato/farmacologia , Receptores de Inositol 1,4,5-Trifosfato , Mitógenos/farmacologia , Trombina/farmacologiaRESUMO
Nucleoplasmic calcium ions (Ca2+) influence nuclear functions as critical as gene transcription, apoptosis, DNA repair, topoisomerase activation and polymerase unfolding. Although both inositol trisphosphate receptors and ryanodine receptors, types of Ca2+ channel, are present in the nuclear membrane, their role in the homeostasis of nuclear Ca2+ remains unclear. Here we report the existence in the inner nuclear membrane of a functionally active CD38/ADP-ribosyl cyclase that has its catalytic site within the nucleoplasm. We propose that the enzyme catalyses the intranuclear cyclization of nicotinamide adenine dinucleotide to cyclic adenosine diphosphate ribose. The latter activates ryanodine receptors of the inner nuclear membrane to trigger nucleoplasmic Ca2+ release.
Assuntos
Antígenos CD , Antígenos de Diferenciação/metabolismo , Cálcio/metabolismo , Núcleo Celular/metabolismo , NAD+ Nucleosidase/metabolismo , Membrana Nuclear/metabolismo , Células 3T3 , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Adenosina Difosfato Ribose/análogos & derivados , Adenosina Difosfato Ribose/farmacologia , Animais , Fracionamento Celular/métodos , ADP-Ribose Cíclica , Genes Reporter/genética , Immunoblotting , Inositol 1,4,5-Trifosfato/farmacologia , Glicoproteínas de Membrana , Camundongos , Microscopia Confocal , Complexos Multienzimáticos , NAD/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
The multifunctional ADP-ribosyl cyclase, CD38, catalyzes the cyclization of NAD(+) to cyclic ADP-ribose (cADPr). The latter gates Ca(2+) release through microsomal membrane-resident ryanodine receptors (RyRs). We first cloned and sequenced full-length CD38 cDNA from a rabbit osteoclast cDNA library. The predicted amino acid sequence displayed 59, 59, and 50% similarity, respectively, to the mouse, rat, and human CD38. In situ RT-PCR revealed intense cytoplasmic staining of osteoclasts, confirming CD38 mRNA expression. Both confocal microscopy and Western blotting confirmed the plasma membrane localization of the CD38 protein. The ADP-ribosyl cyclase activity of osteoclastic CD38 was next demonstrated by its ability to cyclize the NAD(+) surrogate, NGD(+), to its fluorescent derivative cGDP-ribose. We then examined the effects of CD38 on osteoclast function. CD38 activation by an agonist antibody (A10) in the presence of substrate (NAD(+)) triggered a cytosolic Ca(2+) signal. Both ryanodine receptor modulators, ryanodine, and caffeine, markedly attenuated this cytosolic Ca(2+) change. Furthermore, the anti-CD38 agonist antibody expectedly inhibited bone resorption in the pit assay and elevated interleukin-6 (IL-6) secretion. IL-6, in turn, enhanced CD38 mRNA expression. Taken together, the results provide compelling evidence for a new role for CD38/ADP-ribosyl cyclase in the control of bone resorption, most likely exerted via cADPr.
Assuntos
Antígenos CD , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Reabsorção Óssea , NAD+ Nucleosidase/genética , NAD+ Nucleosidase/metabolismo , Osteoclastos/metabolismo , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Adenosina Difosfato Ribose/análogos & derivados , Adenosina Difosfato Ribose/metabolismo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Antígenos de Diferenciação/química , Sequência de Bases , Sinalização do Cálcio , Membrana Celular/enzimologia , Células Cultivadas , Clonagem Molecular , ADP-Ribose Cíclica , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Interleucina-6/farmacologia , Glicoproteínas de Membrana , Dados de Sequência Molecular , NAD/análogos & derivados , NAD/metabolismo , NAD+ Nucleosidase/química , Osteoclastos/citologia , Osteoclastos/enzimologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Coelhos , Ratos , Ratos Wistar , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The osteoclast is a cell that is unique in its ability to resorb bone and, in doing so, becomes exposed to unusually high millimolar Ca2+ concentrations. It is generally accepted that, during resorption, osteoclasts can "sense" changes in their ambient Ca2+ concentration. This triggers a sharp cytosolic Ca2+ increase through both Ca2+ release and Ca2+ influx. The change in cytosolic Ca2+ is transduced finally into inhibition of bone resorption. It has been shown that a type 2 ryanodine receptor isoform, expressed uniquely in the plasma membrane, functions as a Ca2+ influx channel and possibly as a Ca2+ sensor. Ryanodine receptors are ordinarily Ca2+ release channels that have a microsomal membrane location in a wide variety of eukaryotic cells, including the osteoclasts. However, only recently has it become obvious that ryanodine receptors are also expressed in osteoclast nuclear membranes, at which site they probably gate nucleoplasmic Ca2+ influx. Nucleoplasmic Ca2+ in turn regulates key nuclear processes, including gene expression and apoptosis. Here, we review the potential mechanisms underlying the recognition, movement, and effects of Ca2+ in the osteoclast. We will also speculate on the general biological significance of the unique processes used by the osteoclast to handle high Ca2+ loads during bone resorption.
Assuntos
Reabsorção Óssea/metabolismo , Cálcio/metabolismo , Modelos Biológicos , Osteoclastos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Animais , Cafeína/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio , Cátions Bivalentes/metabolismo , Células Cultivadas , Espaço Extracelular/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Transporte de Íons , Microssomos/metabolismo , Membrana Nuclear/metabolismo , Ratos , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacosRESUMO
Osteoclasts are exposed to unusually high, millimolar, Ca2+ concentrations and can "sense" changes in their ambient Ca2+ concentration during resorption. This results in a sharp cystolic Ca2+ increase through both Ca2+ release and Ca2+ influx. The rise in cystolic Ca2+ is transduced finally into an inhibition of bone resorption. We have shown that a type 2 ryanodine receptor isoform, expressed uniquely in the osteoblast plasma membrane, functions as a Ca2+ influx channel, and possibly as a Ca2+ sensor. Ryanodine receptors are ordinarily microsomal membrane Ca2+ release channels. They have only recently been shown to be expressed a other sites, including nuclear membranes. At the latter site, ryanodine receptors gate nucleoplasmic Ca2+ influx. Nucleoplasmic Ca2+, in turn, regulates key nuclear processes, including gene expression and apoptosis. Here, we review potential mechanisms underlying the recognition, movement, and actions of Ca2+ in the osteoclast.
Assuntos
Cálcio/fisiologia , Osteoclastos/fisiologia , Animais , Calcitonina/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Interleucina-6/fisiologia , Potenciais da Membrana , Transdução de SinaisRESUMO
Nitric oxide (NO) triggers marked osteoclast retraction which closely resembles that due to Ca2+. The effect of Ca2+ has been attributed to a stimulated release of NO. Here, we show for the first time, by direct measurement with a microsensor, that osteoclasts do indeed produce NO and that this production is enhanced by a high Ca2+. We also show that the Ca2+ ionophore, A23187, mimics the latter. Furthermore, osteoclasts on dentine produce more NO than osteoclasts on glass and NO release from dentine-plated osteoclasts is much less sensitive to stimulation by Ca2+. Finally, the microsomal Ca2+ store-depleting agent, thapsigargin, attenuates NO release only from osteoclasts on glass, suggesting that stored Ca2+ has the dominant effect in modulating NO release from non-resorbing cells. NO is a powerful inhibitor of bone resorption: a direct demonstration of its production is therefore strong evidence for a role in modulating osteoclast function.
Assuntos
Óxido Nítrico/metabolismo , Osteoclastos/metabolismo , Animais , Técnicas Biossensoriais , Calcimicina/farmacologia , Cálcio/farmacologia , Galinhas , Ionóforos/farmacologia , NADP/farmacologia , Óxido Nítrico/análise , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Tapsigargina/farmacologiaRESUMO
We provide the first molecular evidence for the presence of a functional serine/threonine phosphatase, calcineurin-A (CN-A), in the osteoclast. Polymerase chain reaction (PCR) of an osteoclast cDNA library, together with restriction mapping, revealed two isoform sequences, alpha and beta. We then examined the functionality of the detected CN-A by assessing the effect of a classical antagonist, cyclosporin A (CsA), in the osteoclast resorption (pit) assay. CsA (0.1 and 1 microg ml-1) potently inhibited bone resorption. The presence of lymphocytes, with or without prior exposure to CsA in vivo, failed to reverse the CsA-induced resorption-inhibition. Expectedly, CsA had no direct effect on cytosolic Ca2+ levels in fura-2-loaded osteoclasts. These studies are a prelude to further investigations into the possible role of CN-A in osteoclast regulation. Finally, mechanistic studies on the bone effects of CsA, a widely used immunosupressant, should proceed from these observations.
Assuntos
Reabsorção Óssea , Calcineurina/biossíntese , Ciclosporina/metabolismo , Osteoclastos/metabolismo , Animais , Calcineurina/genética , Inibidores de Calcineurina , Ciclosporina/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , RatosRESUMO
The intracellular mechanisms responsible for inhibition of osteoclast activity are of significant interest in the search for more effective ways of managing bone diseases associated with enhanced bone resorption. Previous studies have suggested that the protein kinase C (PKC) pathway is an important inhibitory second messenger in osteoclasts. We, therefore, investigated the effects of the synthetic peptide fragments, PKC(530-558) and (19-36), which correspond to parts of the catalytic and regulatory domains of PKC, on the activity of isolated osteoclasts. These fragments have been shown to activate and inhibit PKC, respectively, in biochemical studies employing isolated rat brain PKC, but have rarely been employed in studies of cellular activity. PKC(19-36), an enzyme inhibitor (PKC-I), had no effect by itself on osteoclastic bone resorption. However, PKC(530-558), a PKC activator (PKC-A), caused a dose-responsive inhibition of bone resorption, which was accompanied by a rapid and distinctive change in osteoclast morphology. This effect was reversible: (a) upon removal of PKC-A, (b) upon continuous exposure to this fragment for more than 36 h, or (c) in the presence of PKC-I. In conclusion, a short synthetic peptide fragment of PKC (PKC-A) significantly inhibits osteoclastic bone resorption; this, together with the fact that the inhibitory effect is abolished in the presence of PKC-I, provides further evidence for an important physiological role for the PKC pathway in the regulation of osteoclast activity. Selective activation of this pathway may have important therapeutic implications for the management of bone diseases associated with enhanced resorption.
Assuntos
Osteoclastos/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Proteína Quinase C/química , Animais , Reabsorção Óssea/fisiopatologia , Permeabilidade da Membrana Celular/fisiologia , Sobrevivência Celular/fisiologia , Técnicas In Vitro , Fragmentos de Peptídeos/química , RatosRESUMO
We describe a physiologically significant mechanism through which interleukin-6 (IL-6) and a rising ambient Ca2+ interact to regulate osteoclastic bone resorption. VOXEL-based confocal microscopy of nonpermeabilized osteoclasts incubated with anti- IL-6 receptor antibodies revealed intense, strictly peripheral plasma membrane fluorescence. IL-6 receptor expression in single osteoclasts was confirmed by in situ reverse transcriptase PCR histochemistry. IL-6 (5 ng/l to 10 microg/l), but not IL-11 (10 and 100 microg/l), reversed the inhibition of osteoclastic bone resorption induced by high extracellular Ca2+ (15 mM). The IL-6 effect was abrogated by excess soluble IL-6 receptor (500 microg/l). Additionally, IL-6 (5 pg/l to 10 microg/l) inhibited cytosolic Ca2+ signals triggered by high Ca2+ or Ni2+. In separate experiments, osteoclasts incubated in 10 mM Ca2+ or on bone released more IL-6 than those in 1.25 mM Ca2+. Furthermore, IL-6 mRNA histostaining was more intense in osteoclasts in 10 or 20 mM Ca2+ than cells in 1.25 mM Ca2+. Similarly, IL-6 receptor mRNA histostaining was increased in osteoclasts incubated in 5 or 10 mM Ca2+. Thus, while high Ca2+ enhances IL-6 secretion, the released IL-6 attenuates Ca2+ sensing and reverses inhibition of resorption by Ca2+. Such an autocrine-paracrine loop may sustain osteoclastic activity in the face of an inhibitory Ca2+ level generated locally during resorption.
Assuntos
Reabsorção Óssea/metabolismo , Cálcio/metabolismo , Interleucina-6/farmacologia , Osteoclastos/fisiologia , Animais , Imunofluorescência , Hibridização in Situ Fluorescente , Interleucina-11/farmacologia , Interleucina-6/metabolismo , Proteínas de Membrana/análise , Níquel/farmacologia , RNA Mensageiro/metabolismo , Ratos , Receptores de Interleucina-6/análiseRESUMO
Upon removal of its sialic acid or galactose residue, vitamin D-binding protein (DBP) becomes a potent macrophage-activating factor, DBP-MAF. Here we document a new function of DBP-MAF and its parent molecule, DBP, in osteoclast control. We show that all DBPs potently inhibit extracellular Ca2+ (cation) sensing at low nanomolar concentrations with the following rank order of potency: native DBP = sialidase-treated DBP > beta-galactosidase-treated DBP. This attenuation remains unaffected despite co-incubation either with the native DBP ligand, 1,25-dihydroxyvitamin D3, or with an asialoglycoprotein receptor modulator, asialoorosomucoid. Taken together, the results suggest that circulating DBP may play a role in the systemic control of osteoclastic bone resorption, a hitherto unrecognized action of the protein.
Assuntos
Cálcio/metabolismo , Osteoclastos/metabolismo , Proteína de Ligação a Vitamina D/metabolismo , Animais , Receptor de Asialoglicoproteína , Assialoglicoproteínas/farmacologia , Reabsorção Óssea/etiologia , Reabsorção Óssea/metabolismo , Calcitriol/farmacologia , Espaço Extracelular/metabolismo , Técnicas In Vitro , Fatores Ativadores de Macrófagos/química , Fatores Ativadores de Macrófagos/metabolismo , Neuraminidase , Orosomucoide/análogos & derivados , Orosomucoide/farmacologia , Osteoclastos/efeitos dos fármacos , Ratos , Receptores de Superfície Celular/efeitos dos fármacos , Proteína de Ligação a Vitamina D/química , beta-GalactosidaseRESUMO
We report the effects of pulsed electromagnetic fields (PEMFs) on the responsiveness of osteoclasts to cellular, hormonal, and ionic signals. Osteoclasts isolated from neonatal rat long bones were dispersed onto either slices of devitalised cortical bone (for the measurement of resorptive activity) or glass coverslips (for the determination of the cytosolic free Ca2+ concentration, [Ca2+]). Osteoclasts were also cocultured on bone with osteoblastlike, UMR-106 cells. Bone resorption was quantitated by scanning electron microscopy and computer-assisted morphometry. PEMF application to osteoblast-osteoclast cocultures for 18 hr resulted in a twofold stimulation of bone resorption. In contrast, resorption by isolated osteoclasts remained unchanged in the presence of PEMFs, suggesting that osteoblasts were necessary for the PEMF-induced resorption simulation seen in osteoblast-osteoclast cocultures. Furthermore, the potent inhibitory action of the hormone calcitonin on bone resorption was unaffected by PEMF application. However, PEMFs completely reversed another quite distinct action of calcitonin on the osteoclast: its potent inhibitory effect on the activation of the divalent cation-sensing (or Ca2+) receptor. For these experiments, we made fura 2-based measurements of cytosolic [Ca2+] in single osteoclasts in response to the application of a known Ca2+ receptor agonist, Ni2+. We first confirmed that activation of the osteoclast Ca2+ receptor by Ni2+ (5 mM) resulted in a characteristic monophasic elevation of cytosolic [Ca2+]. As shown previously, this response was attenuated strongly by calcitonin at concentrations between 0.03 and 3 nM but remained intact in response to PEMFs. PEMF application, however, prevented the inhibitory effect of calcitonin on Ni2+-induced cytosolic Ca2+ elevation. This suggested that the fields disrupted the interaction between the calcitonin and Ca2+ receptor systems. In conclusion, we have shown that electromagnetic fields stimulate bone resorption through an action on the osteoblast and, by abolishing the inhibitory effects of calcitonin, also restore the responsiveness of osteoclasts to divalent cations.