RESUMO
It is now firmly established that TSH may influence the physiology and patho-physiology of bone by activating osteoblasts and inhibiting osteoclast activity resulting in relative osteoprotection. Whether this influence is directly exerted by pituitary-derived TSH in vivo is less certain, because we have previously reported that the suppression of pituitary TSH does not remove such protection. Here, we have characterized the functional relevance of a novel form of the TSH-ß subunit, designated TSH-ßv, known to be produced by murine bone marrow cells. We found that fresh bone marrow-derived macrophages (MØs) preferentially produced TSH-ßv and, when cocultured with CHO cells engineered to overexpress the full-length TSH receptor, were able to generate the production of intracellular cAMP; a phenomenon not seen in control CHO cells, such results confirmed the bioactivity of the TSH variant. Furthermore, cocultures of MØs and osteoblasts were shown to enhance osteoblastogenesis, and this phenomenon was markedly reduced by antibody to TSH-ß, suggesting direct interaction between MØs and osteoblasts as observed under the electron microscope. These data suggest a new paradigm of local modulation of bone biology by a MØ-derived TSH-like molecule and raise the question of the relative contribution of local vs pituitary-derived TSH in osteoprotection.
Assuntos
Macrófagos/efeitos dos fármacos , Osteoblastos/metabolismo , Isoformas de Proteínas/farmacologia , Tireotropina Subunidade beta/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Técnicas de Cocultura , Cricetinae , Cricetulus , Macrófagos/fisiologia , Camundongos , Dados de Sequência Molecular , Conformação Proteica , Tireotropina Subunidade beta/genéticaRESUMO
Calcium transport and calcium signalling mechanisms in bone cells have, in many cases, been discovered by study of diseases with disordered bone metabolism. Calcium matrix deposition is driven primarily by phosphate production, and disorders in bone deposition include abnormalities in membrane phosphate transport such as in chondrocalcinosis, and defects in phosphate-producing enzymes such as in hypophosphatasia. Matrix removal is driven by acidification, which dissolves the mineral. Disorders in calcium removal from bone matrix by osteoclasts cause osteopetrosis. On the other hand, although bone is central to management of extracellular calcium, bone is not a major calcium sensing organ, although calcium sensing proteins are expressed in both osteoblasts and osteoclasts. Intracellular calcium signals are involved in secondary control including cellular motility and survival, but the relationship of these findings to specific diseases is not clear. Intracellular calcium signals may regulate the balance of cell survival versus proliferation or anabolic functional response as part of signalling cascades that integrate the response to primary signals via cell stretch, estrogen, tyrosine kinase, and tumor necrosis factor receptors.
Assuntos
Doenças Ósseas/fisiopatologia , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Animais , Transporte Biológico , Calcificação Fisiológica/fisiologia , Cálcio/fisiologia , Citosol/fisiologia , Humanos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Prótons , Receptores de Detecção de Cálcio/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , ATPases Vacuolares Próton-Translocadoras/fisiologiaRESUMO
How m-calpain is activated in cells has challenged investigators because in vitro activation requires near-millimolar calcium. Previously, we demonstrated that m-calpain activation by growth factors requires extracellular signal-regulated kinase (ERK); this enables tail deadhesion and allows productive motility. We now show that ERK directly phosphorylates and activates m-calpain both in vitro and in vivo. We identified serine 50 as required for epidermal growth factor (EGF)-induced calpain activation in vitro and in vivo. Replacing the serine with alanine limits activation by EGF and subsequent cell deadhesion and motility. A construct with the serine converted to glutamic acid displays constitutive activity in vivo; expression of an estrogen receptor fusion construct produces a tamoxifen-sensitive enzyme. Interestingly, EGF-induced m-calpain activation occurs in the absence of increased intracellular calcium levels; EGF triggers calpain even in the presence of intracellular calcium chelators and in calcium-free media. These data provide evidence that m-calpain can be activated through the ERK cascade via direct phosphorylation and that this activation may occur in the absence of cytosolic calcium fluxes.
Assuntos
Calpaína/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Substituição de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Cálcio/metabolismo , Calpaína/química , Calpaína/genética , Linhagem Celular , Movimento Celular , DNA Complementar/genética , Ativação Enzimática/efeitos dos fármacos , Humanos , Técnicas In Vitro , Sistema de Sinalização das MAP Quinases , Camundongos , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/químicaRESUMO
The osteoclast is a bone-degrading polykaryon. Recent studies have clarified the differentiation of this cell and the biochemical mechanisms it uses to resorb bone. The osteoclast derives from a monocyte/macrophage precursor. Osteoclast formation requires permissive concentrations of M-CSF and is driven by contact with mesenchymal cells in bone that bear the TNF-family ligand RANKL. Osteoclast precursors express RANK, and the interaction between RANKL and RANK (which is inhibited by OPG) is the major determinant of osteoclast formation. Hormones, such as PTH/PTHrP, glucocorticoids and 1,25(OH)2D3, and humoral factors, including TNFalpha, interleukin-1, TGFss and prostaglandins, influence osteoclast formation by altering expression of these molecular factors. TNFalpha, IL-6 and IL-11 have also been shown to promote osteoclast formation by RANKL-independent processes. RANKL-dependent/independent osteoclast formation is likely to play an important role in conditions where there is pathological bone resorption such as inflammatory arthritis and malignant bone resorption. Osteoclast functional defects cause sclerotic bone disorders, many of which have recently been identified as specific genetic defects. Osteoclasts express specialized proteins including a vacuolar-type H+-ATPase that drives HCl secretion for dissolution of bone mineral. One v-ATPase component, the 116 kD V0 subunit, has several isoforms. Only one isoform, TCIRG1, is up-regulated in osteoclasts. Defects in TCIRG1 are common causes of osteopetrosis. HCl secretion is dependent on chloride channels; a chloride channel homologue, CLCN7, is another common defect in osteopetrosis. Humans who are deficient in carbonic anhydrase II or who have defects in phagocytosis also have variable defects in bone remodelling. Organic bone matrix is degraded by thiol proteinases, principally cathepsin K, and abnormalities in cathepsin K cause another sclerotic bone disorder, pycnodysostosis. Thus, bone turnover in normal subjects depends on relative expression of key cytokines, and defects in osteoclastic turnover usually reflect defects in specific ion transporters or enzymes that play essential roles in bone degradation.
Assuntos
Reabsorção Óssea/patologia , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteoclastos/fisiologia , Animais , Calcitriol/metabolismo , Calcitriol/farmacologia , Diferenciação Celular , Citocinas/metabolismo , Citocinas/farmacologia , Glicoproteínas/metabolismo , Glicoproteínas/farmacologia , Humanos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Modelos Biológicos , Osteoclastos/efeitos dos fármacos , Osteoprotegerina , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Fator de Necrose TumoralRESUMO
The thyrotropin (TSH) receptor (TSHR) is a member of the heterotrimeric G protein-coupled family of receptors whose main function is to regulate thyroid cell proliferation as well as thyroid hormone synthesis and release. In this study, we generated a TSHR knockout (TSHR-KO) mouse by homologous recombination for use as a model to study TSHR function. TSHR-KO mice presented with developmental and growth delays and were profoundly hypothyroid, with no detectable thyroid hormone and elevated TSH. Heterozygotes were apparently unaffected. Knockout mice died within 1 week of weaning unless fed a diet supplemented with thyroid powder. Mature mice were fertile on the thyroid-supplemented diet. Thyroid glands of TSHR-KO mice produced uniodinated thyroglobulin, but the ability to concentrate and organify iodide could be restored to TSHR-KO thyroids when cultured in the presence of the adenylate cyclase agonist forskolin. Consistent with this observation was the lack of detectable sodium-iodide symporter expression in TSHR-KO thyroid glands. Hence, by using the TSHR-KO mouse, we provided in vivo evidence, demonstrating that TSHR expression was required for expression of sodium-iodide symporter but was not required for thyroglobulin expression, suggesting that the thyroid hormone synthetic pathway of the mouse could be dissociated into TSHR-dependent and -independent steps.
Assuntos
Receptores da Tireotropina/fisiologia , Simportadores/biossíntese , Hormônios Tireóideos/biossíntese , Tireotropina/fisiologia , Inibidores de Adenilil Ciclases , Adenilil Ciclases/fisiologia , Animais , Bovinos , Colforsina/farmacologia , AMP Cíclico/fisiologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Marcação de Genes , Genes Letais , Genes Reporter , Terapia de Reposição Hormonal , Hipotireoidismo/genética , Hipotireoidismo/metabolismo , Transporte de Íons , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores da Tireotropina/deficiência , Receptores da Tireotropina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Iodeto de Sódio/farmacocinética , Simportadores/deficiência , Simportadores/genética , Tireoglobulina/biossíntese , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Hormônios Tireóideos/administração & dosagem , Tireotropina/sangue , Tireotropina/farmacologia , Extratos de Tecidos/uso terapêuticoRESUMO
Osteoclasts are terminally differentiated, multinucleated cells of monocytic origin. In this study, we report that osteoclasts secrete a 35 kD protein and that phorbol myristate acetate treatment stimulates secretion dramatically. Peptide digests of the protein were analyzed by mass spectroscopy. The protein was identified as myb induced myeloid protein-1 precursor (mim-1 protein). Mim-1 is expressed specifically by hematopoietic cells and has no known function. It is homologous with the neutrophil chemokine, chondromodulin II, which stimulates proliferation of osteoblasts and chondrocytes. Western analysis showed that osteoclasts secrete mim-1 into culture media. Immunofluorescence studies demonstrated a cytoplasmic and perinuclear distribution of mim-1 in both avian osteoclasts and human osteoclast-like cells. Expression and secretion of a chemokine-like protein by osteoclasts suggests a novel signaling pathway in the bone microenvironment that may be involved in coordinating bone remodeling.
Assuntos
Acetiltransferases , Peptídeos e Proteínas de Sinalização Intercelular , Osteoclastos/metabolismo , Biossíntese de Proteínas , Sequência de Aminoácidos , Animais , Western Blotting , Reabsorção Óssea , Carcinógenos , Diferenciação Celular , Divisão Celular , Núcleo Celular/metabolismo , Células Cultivadas , Galinhas , Condrócitos/metabolismo , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Substâncias de Crescimento/química , Humanos , Espectrometria de Massas , Microscopia de Fluorescência , Dados de Sequência Molecular , Osteoblastos/metabolismo , Proteína Quinase C/metabolismo , Proteínas/química , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Acetato de Tetradecanoilforbol , Fatores de TempoRESUMO
Osteoclasts are macrophage-derived polykaryons that degrade bone in an acidic extracellular space. This differentiation includes expression of proteinases and acid transport proteins, cell fusion, and bone attachment, but the sequence of events is unclear. We studied two proteins expressed at high levels only in the osteoclast, cathepsin K, a thiol proteinase, and tartrate-resistant acid phosphatase (TRAP), and compared this expression with acid transport and bone degradation. Osteoclastic differentiation was studied using human apheresis macrophages cocultured with MG63 osteosarcoma cells, which produce cytokines including RANKL and CSF-1 that mediate efficient osteoclast formation. Immunoreactive cathepsin K appeared at 3-5 days. Cathepsin K activity was seen on bone substrate but not within cells, and cathepsin K increased severalfold during further differentiation and multinucleation from 7 to 14 days. TRAP also appeared at 3-5 d, independently of cell fusion or bone attachment, and TRAP activity reached much higher levels in osteoclasts attached to bone fragments. Two proteinases that occur in the precursor macrophages, cathepsin B, a thiol proteinase related to cathepsin K, and an unrelated lysosomal aspartate proteinase, cathepsin D, were also studied to determine the specificity of the differentiation events. Cathepsin B occurred at all times, but increased two- to threefold in parallel with cathepsin K. Cathepsin D activity did not change with differentiation, and secreted activity was not significant. In situ acid transport measurements showed increased acid accumulation after 7 days either in cells on osteosarcoma matrix or attached to bone, but bone pit activity and maximal acid uptake required 10-14 days. We conclude that TRAP and thiol proteinase expression begin at essentially the same time, and precede cell fusion and bone attachment. However, major increases in acid secretion and proteinases expression continue during cell fusion and bone attachment from 7 to 14 days.
Assuntos
Endopeptidases/biossíntese , Osteoclastos/citologia , Osteoclastos/enzimologia , Fosfatase Ácida/biossíntese , Ácidos/metabolismo , Androstadienos/farmacologia , Western Blotting , Catepsina B/biossíntese , Catepsina D/biossíntese , Catepsina K , Catepsinas/biossíntese , Diferenciação Celular , Fusão Celular , Células Cultivadas , Técnicas de Cocultura , Inibidores Enzimáticos/farmacologia , Humanos , Isoenzimas/biossíntese , Macrófagos/metabolismo , Fosfatase Ácida Resistente a Tartarato , Fatores de Tempo , WortmaninaRESUMO
Bone marrow stromal stem cells differentiate into many different types of cells including osteoblasts and adipocytes. Long-term glucocorticoid treatment decreases osteoblastic activity but increases adipocytes. We investigated the mechanism of glucocorticoid-induced PPARgamma2 transcription. Treatment of human bone marrow stromal cells with dexamethasone induced the differentiation of these cells into adipocytes as measured by oil-red O staining, and Northern blot analysis showed that dexamethasone strongly induced PPARgamma2 mRNA expression in cells cultured in adipocyte induction medium. Moreover, the mRNA of C/EBPdelta, an adipocyte-promoting transcription factor, was also induced by dexamethasone in the presence of induction medium. Gel mobility shift assays using purified GST-C/EBPdelta fusion protein showed that C/EBPdelta specifically binds to a 40-base pair DNA element from PPARgamma2 promoter, which was found to contain a tandem repeat of C/EBP binding sites. Transfection studies in mouse mesenchymal C3H10T1/2 cells showed that it is the tandem repeat of the C/EBP binding site in PPARgamma2 promoter region that regulates dexamethasone-mediated PPARgamma2 gene activation. We conclude that glucocorticoid-induced adipogenesis from bone marrow stromal cells is mediated through a reaction cascade in which dexamethasone transcriptionally activates C/EBPdelta; C/EBPdelta then binds to PPARgamma2 promoter and transactivates PPARgamma2 gene expression. This activated master regulator, in turn, initiates the adipocyte differentiation.
Assuntos
Adipócitos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Sequência de Bases , Sítios de Ligação/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , DNA/genética , DNA/metabolismo , Primers do DNA/genética , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Humanos , Camundongos , Sequências de Repetição em Tandem , Transcrição GênicaRESUMO
We studied intracellular calcium ([Ca(2+)](i)) in acid-secreting bone-attached osteoclasts, which produce a high-calcium acidic extracellular compartment. Acid secretion and [Ca(2+)](i) were followed using H(+)-restricted dyes and fura-2 or fluo-3. Whole cell calcium of acid-secreting osteoclasts was approximately 100 nM, similar to cells on inert substrate that do not secrete acid. However, measurements in restricted areas of the cell showed [Ca(2+)](i) transients to 500-1000 nM consistent with calcium puffs, transient (millisecond) localized calcium elevations reported in other cells. Spot measurements at 50-ms intervals indicated that puffs were typically less than 400 ms. Transients did not propagate in waves across the cell in scanning confocal measurements. Calcium puffs occurred mainly over regions of acid secretion as determined using lysotracker red DND99 and occurred at irregular periods averaging 5-15 s in acid secreting cells, but were rare in lysotracker-negative nonsecretory cells. The calmodulin antagonist trifluoperazine, cell-surface calcium transport inhibitors lanthanum or barium, and the endoplasmic reticulum ATPase inhibitor thapsigargin had variable acute effects on the mean [Ca(2+)](i) and puff frequency. However, none of these agents prevented calcium puff activity, suggesting that the mechanism producing the puffs is independent of these processes. We conclude that [Ca(2+)](i) transients in osteoclasts are increased in acid-secreting osteoclasts, and that the puffs occur mainly near the acid-transporting membrane. Cell membrane acid transport requires calcium, suggesting that calcium puffs function to maintain acid secretion. However, membrane H(+)-ATPase activity was insensitive to calcium in the 100 nM-1 microM range. Thus, any effects of calcium puffs on osteoclastic acid transport must be indirect.
Assuntos
Cálcio/análise , Cálcio/metabolismo , Osteoclastos/química , Osteoclastos/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Compostos de Bário/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Cálcio/farmacologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/metabolismo , Calmodulina/antagonistas & inibidores , Membrana Celular/metabolismo , Células Cultivadas , Galinhas , Cloretos/farmacologia , Antagonistas de Dopamina/farmacologia , Inibidores Enzimáticos/farmacologia , Vidro , Ácido Clorídrico/metabolismo , Lantânio/farmacologia , Osteoclastos/citologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/metabolismo , Tapsigargina/farmacologia , Trifluoperazina/farmacologiaRESUMO
Mast cells accumulate in hyperparathyroid bone, but the reason is not clear. We compared the distribution of mast cells and related growth factors in normal and hyperparathyroid bone. Mast cell formation was strongly affected by proximity to bone-forming surfaces of hyperparathyroid bone. Hyperparathyroidism greatly increased the production by active, bone-synthesizing osteoblasts of stem cell factor (SCF) but not of IL-3. Osteoblast SCF was distributed to the basolateral cell membranes, and its cDNA sequence (GenBank AF119835) is homologous to the murine membrane-bound SCF. Quiescent osteoblasts did not produce detectable SCF. Synthetic osteoblasts in normal bone were SCF positive, but comprised a much smaller population of cells, in keeping with the slow turnover of normal bone. Major SCF isoforms on immunoblot analysis of osteoblast-fraction proteins from high-turnover bone had M(r)s of about 48 and 40 kDa. Similar SCF isoforms were produced by MG63 osteoblast-derived cells and were identified by several anti-SCF antibodies. SCF is expressed in several mesenchymal cell types in a complementary fashion with cells bearing its receptor. SCF potently facilitates differentiation of mast cells, so the increase in paratrabecular mast cells in hyperparathyroid bone is probably driven by osteoblastic SCF. However, since mast cells are not normal components of bone, osteoblastic SCF probably regulates other cells, with mast cell differentiation occurring as a side effect greatly increased osteoblastic activity.
Assuntos
Osso e Ossos/metabolismo , Hiperparatireoidismo/metabolismo , Mastócitos/patologia , Osteoblastos/metabolismo , Fator de Células-Tronco/biossíntese , Osso e Ossos/citologia , Diferenciação Celular , Feminino , Humanos , Hiperparatireoidismo/patologia , Immunoblotting , Imuno-Histoquímica , Interleucina-3/metabolismo , Pessoa de Meia-Idade , Isoformas de Proteínas/metabolismo , Células Tumorais CultivadasRESUMO
Bone resorption by osteoclasts is modified by agents that affect cyclic guanosine monophosphate (cGMP), but their relative physiological roles, and what components of the process are present in osteoclasts or require accessory cells such as osteoblasts, are unclear. We studied cGMP regulation in avian osteoclasts, and in particular the roles of nitric oxide and natriuretic peptides, to clarify the mechanisms involved. C-type natriuretic peptide drives a membrane guanylate cyclase, and increased cGMP production in mixed bone cells. However, C-type natriuretic peptide did not increase cGMP in purified osteoclasts. By contrast, osteoclasts did produce cGMP in response to nitric oxide (NO) generators, sodium nitroprusside or 1-hydroxy-2-oxo-3,3-bis(3-aminoethyl)-1-triazene. These findings indicate that C-type natriuretic peptide and NO modulate cGMP in different types of bone cells. The activity of the osteoclast centers on HCI secretion that dissolves bone mineral, and both NO generators and hydrolysis-resistant cGMP analogues reduced bone degradation, while cGMP antagonists increased activity. NO synthase agonists did not affect activity, arguing against autocrine NO production. Osteoclasts express NO-activated guanylate cyclase and cGMP-dependent protein kinase (G-kinase). G-kinase reduced membrane HCI transport activity in a concentration-dependent manner, and phosphorylated a 60-kD osteoclast membrane protein, which immunoprecipitation showed is not an H+-ATPase subunit. We conclude that cGMP is a negative regulator of osteoclast activity. cGMP is produced in response to NO made by other cells, but not in response to C-type natriuretic peptide. G-kinase modulates osteoclast membrane HCI transport via intermediate protein(s) and may mediate cGMP effects in osteoclasts.
Assuntos
GMP Cíclico/biossíntese , Óxido Nítrico/fisiologia , Osteoclastos/metabolismo , Ácidos/antagonistas & inibidores , Animais , Células da Medula Óssea/metabolismo , Células Cultivadas , Galinhas , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Concentração de Íons de Hidrogênio , Proteínas de Membrana/metabolismo , Peptídeo Natriurético Tipo C/fisiologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Osteoclastos/enzimologia , FosforilaçãoRESUMO
Osteoclasts are multinucleated monocyte-macrophage derivatives that degrade bone. Their specialized role is central to a process that continuously removes and replaces segments of the skeleton in the higher vertebrates. Osteoclasts allow skeletal mineral to be used to manage extracellular calcium activity, which is an important adaptation for life on land, and solid skeletal structure to be replaced by hollow architecture that has a superior strength-to-weight ratio. Degrading bone also allows periodic repair and remodeling for ordered growth and efficient response to mechanical loads. A fairly comprehensive view of osteoclastic ontogeny and function is emerging from recent studies. Osteoclasts dissolve bone mineral by massive acid secretion and secrete specialized proteinases that degrade the organic matrix, mainly type I collagen, in this acidic milieu. The site of bone dissolution is a high-calcium environment; removal of degradation products by transcytosis of membrane vesicles allows the osteoclast to maintain a normal intracellular calcium. Osteoclastic differentiation is normally balanced with bone formation, although bone formation is the function of unrelated stromal cell-derived osteoblasts. Interactions between osteoclast precursors and bone-forming cells are believed to control osteoclast differentiation under most circumstances, preserving bone architecture over many cycles of bone replacement.
Assuntos
Remodelação Óssea/fisiologia , Reabsorção Óssea/patologia , Osteoclastos/fisiologia , Animais , Densidade Óssea , Osso e Ossos/citologia , Calcificação Fisiológica , Cálcio/metabolismo , Anidrases Carbônicas/metabolismo , Cartilagem/metabolismo , Diferenciação Celular , Citocinas/fisiologia , Durapatita/metabolismo , Homeostase , Humanos , Ácido Clorídrico/metabolismo , Modelos Biológicos , Fosforilação Oxidativa , Hormônio Paratireóideo/fisiologia , Fósforo/metabolismo , ATPases Translocadoras de Prótons/fisiologia , Ratos , Células Estromais/fisiologia , Vertebrados/fisiologiaRESUMO
We studied effects of calmodulin antagonists on osteoclastic activity and calmodulin-dependent HCl transport. The results were compared to effects on the calmodulin-dependent phosphodiesterase and antagonist-calmodulin binding affinity. Avian osteoclast degradation of labeled bone was inhibited approximately 40% by trifluoperazine or tamoxifen with half-maximal effects at 1-3 microM. Four benzopyrans structurally resembling tamoxifen were compared: d-centchroman inhibited resorption 30%, with half-maximal effect at approximately 100 nM, cischroman and CDRI 85/287 gave 15-20% inhibition, and l-centchroman was ineffective. No benzopyran inhibited cell attachment or protein synthesis below 10 microM. However, ATP-dependent membrane vesicle acridine transport showed that H(+)-ATPase activity was abolished by all compounds with 50% effects at 0.25-1 microM. All compounds also inhibited calmodulin-dependent cyclic nucleotide phosphodiesterase at micromolar calcium. Relative potency varied with assay type, but d- and l-centchroman, surprisingly, inhibited both H(+)-ATPase and phosphodiesterase activity at similar concentrations. However, d- and l-centchroman effects in either assay diverged at nanomolar calcium. Of benzopyrans tested, only the d-centchroman effects were calcium-dependent. Interaction of compounds with calmodulin at similar concentrations were confirmed by displacement of labeled calmodulin from immobilized trifluoperazine. Thus, the compounds tested all interact with calmodulin directly to varying degrees, and the observed osteoclast inhibition is consistent with calmodulin-mediated effects. However, calmodulin antagonist activity varies between specific reactions, and free calcium regulates specificity of some interactions. Effects on whole cells probably also reflect other properties, including transport into cells.
Assuntos
Calmodulina/metabolismo , Osteoclastos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Tamoxifeno/farmacologia , Animais , Anticarcinógenos/farmacologia , Benzopiranos/farmacologia , Reabsorção Óssea , Calmodulina/efeitos dos fármacos , Centocromano/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1 , Relação Dose-Resposta a Droga , Feminino , Osteoclastos/efeitos dos fármacos , Diester Fosfórico Hidrolases/efeitos dos fármacos , Piperidinas/farmacologia , ATPases Translocadoras de Prótons/efeitos dos fármacos , Trifluoperazina/farmacologiaRESUMO
Osteoclasts mediate bone resorption by secretion at the site of bone attachment. This process depends on calmodulin concentrated at a specialized acid-secreting membrane. We hypothesized that increased calmodulin and bone attachment were required for acid secretion. We tested this by studying calmodulin, bone attachment, and membrane acid transport in osteoclasts and their precursor mononuclear cells. Osteoclasts and macrophages were isolated from medullary bone of hens; cell fractions were prepared after culturing cells with or without bone. Calmodulin was visualized by Western analysis; calmodulin mRNA was determined by Northern hybridization, and ATP-dependent membrane acid transport was assayed by acridine orange uptake. Calmodulin decreased in osteoclasts cultured without bone. Calmodulin in isolated macrophages was approximately 25% of osteoclast levels, but increased several fold by 5 days. Bone had no effect. Calmodulin mRNA was similar in osteoclasts with or without bone. However, only osteoclasts cultured with bone retained acid transport capacity. Macrophage calmodulin mRNA was not affected by bone, but increased three fold by day 5, paralleling protein production. Macrophages developed acid transport capacity at 3-5 days, but at lower levels than osteoclasts, and bone had no measurable effect. Chicken cells express 1.6 kb and inducible 1.9 kb calmodulin transcripts; in macrophages and osteoclasts, the 1.9 kb transcript predominated. We conclude that, following isolation, calmodulin levels decline in osteoclasts via a post-transcriptional mechanism. In cultured macrophages, by contrast, calmodulin mRNA, protein, and acid secretion increase with time independently of bone substrate, possibly reflecting differentiation in vitro. Increased calmodulin correlated with membrane acid transport capacity in both cell types. The macrophage findings indicate that stimuli other than bone influence acid transport capacity in this family of cells.
Assuntos
Matriz Óssea/metabolismo , Osso e Ossos/citologia , Calmodulina/metabolismo , Membrana Celular/metabolismo , Macrófagos/metabolismo , Osteoclastos/metabolismo , ATPases Translocadoras de Prótons/metabolismo , ATPases Vacuolares Próton-Translocadoras , Animais , Transporte Biológico Ativo , Osso e Ossos/metabolismo , Adesão Celular , Células Cultivadas , Galinhas , Feminino , Expressão Gênica , Concentração de Íons de Hidrogênio , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
We used highly purified avian osteoclasts and isolated membranes from osteoclasts to study effects of tamoxifen, 4-hydroxytamoxifen, calmodulin antagonists, estrogen, diethylstilbestrol, and the anti-estrogen ICI 182780 on cellular degradation of 3H-labeled bone in vitro and on membrane HCl transport. Bone resorption was reversibly inhibited by tamoxifen, 4-hydroxytamoxifen, and trifluoperazine with IC50 values of approximately 1 microM. Diethylstilbestrol and 17-beta-stradiol had no effects on bone resorption at receptor-saturating concentrations, while ICI 182780 inhibited bone resorption at concentrations greater than 1 microM. At these concentrations ICI 182780, like tamoxifen, inhibits calmodulin-stimulated cyclic nucleotide phosphodiesterase activity. Membrane HCl transport, assessed by ATP-dependent acridine orange uptake, was unaffected by 17-beta-estradiol and diethylstilbestrol at concentrations up to 10 microM, while ICI greater than 1 microM. In contrast HCl transport was inhibited by tamoxifen, 4-hydroxytamoxifen, and the calmodulin antagonists, trifluoperazine and the calmidazolium, with IC50 values of 0.25-1.5 microM. These results suggested the presence of a membrane-associated non-steroid receptor for tamoxifen in osteoclasts. Tamoxifen binding studies demonstrated saturable binding in the osteoclast particulate fraction, but not in the nuclear or cytosolic fractions. Membranes enriched in ruffled border by differential centrifugation following nitrogen cavitation showed binding consistent with one site, Kd approximately microM. Our findings indicate that tamoxifen inhibits osteoclastic HCl transport by binding membrane-associated target(s), probably similar or related to calmodulin antagonist targets. Further, effects of estrogens or highly specific anti-estrogens on bone turnover do not support the hypothesis of a direct effect on osteoclasts by these compounds in this species.
Assuntos
Reabsorção Óssea/prevenção & controle , ATPases Translocadoras de Prótons/efeitos dos fármacos , Tamoxifeno/farmacologia , Trifluoperazina/farmacologia , Animais , Transporte Biológico , Calmodulina/antagonistas & inibidores , Membrana Celular/metabolismo , Células Cultivadas , Galinhas , Dietilestilbestrol/farmacologia , Sinergismo Farmacológico , Estradiol/análogos & derivados , Estradiol/farmacologia , Feminino , Fulvestranto , Ácido Clorídrico/metabolismo , Osteoclastos/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/metabolismoRESUMO
Osteoclasts develop from precursor cells of the monocyte series. However, specialized differentiation for efficient bone degradation separates the osteoclast from the macrophage. The physical reasons for these differences are emerging from the study of osteoclastic physiology and biochemistry. Key osteoclast specializations are multinucleation, formation of a tightly sealed extracellular compartment on bone, and high-capacity secretion of HCl and acid proteases into this extracellular site. Multinucleation increases efficiency of extracellular attachment processes. The attachment process is mediated by cell membrane integrins, and is sensitive to changes in intracellular or extracellular calcium. Acid production exploits carbonic acid as the source of acid and conjugate base equivalents, reflected in abundant osteoclastic carbonic anhydrase type II expression. Secretion of acid involves extremely high expression of vacuolar-type H(+)-ATPase and a chloride channel in the cell's specialized acid secreting organelle, the ruffled membrane, which is polarized to the osteoclast's bone attachment. Acid secretion is balanced by chloride-bicarbonate exchange in the cell's nonbone attached membranes; this functionally resembles the band 3 chloride-bicarbonate exchanger of the red cell carbon dioxide transport system. Bone collagen is degraded by acid proteases secreted into the acid degradation site via the mannose-6-phosphate receptor system, which is targeted to lysosomes in other cells. Functional deficits, as in osteopetrosis, may affect any of the elements involved in osteoclast differentiation. Furthermore, new antiosteoclastic therapeutic agents may inhibit osteoclast biochemistry intentionally, such as for the control of hypercalcemia of malignancy.
Assuntos
Osteoclastos/fisiologia , Bicarbonatos/metabolismo , Transporte Biológico Ativo , Reabsorção Óssea , Cálcio/metabolismo , Ácido Carbônico/metabolismo , Núcleo Celular/fisiologia , Colágeno/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hidroxiapatitas/metabolismo , Integrinas/fisiologia , Osteoclastos/química , Osteoclastos/ultraestrutura , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
The antineoplastic properties of suramin, a polyanionic agent with demonstrated antigrowth factor activity, are under evaluation in vitro, in vivo, and in clinical trials. Suramin has been shown to have antitumor activity in patients with advanced, hormone refractory prostate cancer. During these trials, significant resolution of osseous pain was observed in nearly three quarters of the patients treated with suramin. To evaluate the effect of suramin on bone cells, we studied the effect of suramin on bone resorption in a neonatal mouse calvarial assay. Suramin inhibited bone-resorbing activity in a dose-related fashion and had an additive effect with calcitonin. Calvaria pretreated with suramin had less bone-resorbing activity, fewer attached osteoblasts, and less medium alkaline phosphatase activity than control calvaria. Suramin also inhibited osteoclastic release of tritiated proline from labeled bone in a dose-dependent fashion. The effect of metastatic prostate carcinoma on bone is incompletely understood, but may be moderated by tumor-produced factors and/or cytokines. The effects of several such agents, therefore, were examined in combination with suramin. Bone resorption induced by PTH, epidermal growth factor, tumor necrosis factor, and a tumor-produced factor, PTH related-protein, was blocked by suramin. The ability of suramin to inhibit the bone-resorbing effects of several cytokines suggests that its mechanism may involve direct action on bone metabolism. Autoradiography performed on calvaria treated with labeled suramin demonstrated heavy deposition of suramin on the outer surface of the matrix, adjacent to osteoblasts and osteoclasts lining the outer table, suggesting that bone cells may be subject to high local concentrations of the drug, in keeping with this hypothesis.
Assuntos
Animais Recém-Nascidos/fisiologia , Reabsorção Óssea/fisiopatologia , Osteoblastos/citologia , Crânio/citologia , Suramina/farmacologia , Fosfatase Alcalina/análise , Animais , Animais Recém-Nascidos/metabolismo , Autorradiografia , Calcitonina/farmacologia , Cálcio/análise , Células Cultivadas , Meios de Cultura/química , Relação Dose-Resposta a Droga , Fator de Crescimento Epidérmico/farmacologia , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteoclastos/fisiologia , Hormônio Paratireóideo/farmacologia , Proteína Relacionada ao Hormônio Paratireóideo , Prolina/metabolismo , Proteínas/farmacologia , Crânio/efeitos dos fármacos , Crânio/metabolismo , Trítio , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
We prepared proton-transporting membrane vesicles from the avian osteoclast's ruffled membrane, a specialized region of the cell surface that acidifies the bone resorption space. We demonstrated a unique conductive Cl- permeability that is charge coupled to the vesicle H(+)-ATPase and is required for acidification. Ion replacement indicated an anion selectivity of Br- approximately Cl- greater than SO4(2-) greater than NO3- approximately SCN- in supporting acidification. The anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (10 microM) was a competitive inhibitor of acidification and raised the Michaelis constant for ATP of the proton pump approximately 11-fold in 120 mM KCl. Inhibition was reversed by valinomycin, which provides an alternate path for charge neutralization. The Cl- dependence of acidification was nonlinear and yielded a Hill coefficient of 3-4, showing that it is distinct from a linear Cl- dependence reported for acidification of renal cortical endosomes. The K+ ionophore valinomycin augmented H+ transport in K2SO4, and not in KCl. Dependence of Cl- transport on membrane potential was confirmed by direct measurement of 36Cl- transport. We uncoupled charge transport from proton transport with a large excess of ammonia, which had no effect on 36Cl- accumulation in vesicles, and by measuring 36Cl- accumulation in response to a membrane diffusion potential, produced with a [K+] gradient and valinomycin in the absence of ATP. These experiments demonstrate that the electrogenic proton pump of the osteoclast ruffled membrane is charge coupled to a passive Cl- permeability in the same membrane.
Assuntos
Permeabilidade da Membrana Celular , Membrana Celular/metabolismo , Cloretos/metabolismo , Osteoclastos/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Laranja de Acridina , Trifosfato de Adenosina/metabolismo , Animais , Ânions , Transporte Biológico , Membrana Celular/ultraestrutura , Galinhas , Feminino , Concentração de Íons de Hidrogênio , Cinética , Microscopia Eletrônica , Modelos Biológicos , Osteoclastos/ultraestruturaRESUMO
Several lines of indirect evidence suggest that a monocyte family precursor gives rise to the osteoclast, although this hypothesis is controversial. Starting with a uniform population of nonspecific esterase positive, tartrate-sensitive, acid phosphatase-producing, mannose receptor-bearing mononuclear cells, prepared from dispersed marrow of calcium-deprived laying hens by cell density separation and selective cellular adherence, we generated multinucleated cells in vitro. When cultured with devitalized bone, these cells show, by electron microscopy, the characteristic osteoclast morphology in that they are mitochondria-rich, multinucleated, and, most importantly, develop characteristic ruffled membranes at the matrix attachment site. Moreover, as documented by scanning electron microscopy, these cells pit bone slices in a manner identical to freshly isolated osteoclasts. In addition, isoenzymes of acid phosphatase from generated osteoclasts, separated by 7.5% polyacrylamide gel electrophoresis at pH 4, are identical to those of mature osteoclasts in migration pattern and tartrate resistance, although the precursor cells from which the osteoclasts are generated produce an entirely different isoenzyme, which is tartrate-sensitive and migrates less rapidly at pH 4. The fused cells also exhibit a cAMP response to prostaglandin E2. Therefore, osteoclast-like cells can be derived by in vitro culture of a marrow-derived monocyte cell population.
Assuntos
Galinhas/fisiologia , Osteoclastos/fisiologia , Fagócitos/fisiologia , Animais , Células da Medula Óssea , Fusão Celular , Linhagem Celular , AMP Cíclico/biossíntese , Histocitoquímica , Microscopia Eletrônica , Monócitos/metabolismo , Osteoclastos/metabolismo , Osteoclastos/ultraestruturaRESUMO
We demonstrated the cells producing collagenase and the time course of collagenase-production at early stages of wound healing, using histology and two immunohistochemical procedures on cross sections of rat skin harvested 0, 3, 5, 7 and 12 days after full-thickness incisions. A monospecific rabbit polyclonal antibody to neutral collagenase purified from rat myometrial cells was used to demonstrate collagenase production. Specificity of this reaction was confirmed by blocking the reaction with excess homogeneously purified antigen. Macrophages were simultaneously labelled using a mouse anti-rat monoclonal antibody recognizing exclusively mature macrophages. Intracellular collagenase was not reliably detectable at day 0, but was prominent at days 3 and 5 and thereafter declined. Double labeling technique showed occasional macrophages producing collagenase in the developing granulation tissue, but most cells labeled as macrophages were negative for collagenase. Most activity was found in fibroblasts adjacent to granulation tissue elements. Since the granulation tissue parallels revascularization in a dendritic pattern, a cross section at three days typically shows an annulus of collagenase-positive cells surrounding a branch of the active granulation tissue. At days 5, 7 and 12 after wounding the pattern of collagenase expression became indistinct as more tissue was involved in the granulation process. However, double-labelling for macrophages and collagenase showed the dichotomy between collagenase expression and presence of macrophages to persist. The finding that collagenase is produced in connective tissue adjacent to granulation tissue suggests an inductive process, possibly due to diffusion of cytokines produced by granulation tissue elements.