Isolated Rat Osteoclasts Research Articles

Extracellular nucleotides activate non-selective cation and Ca(2+)-dependent K+ channels in rat osteoclasts.

1. Extracellular ATP elevates cytosolic free Ca2+ concentration ([Ca2+]i) in osteoclasts, but its effects on ion channels have not been reported previously. Membrane currents and [Ca2+]i were recorded in isolated rat osteoclasts using patch clamp and fluorescence techniques. 2. At negative membrane potentials, ATP (1-100 microM) activated an inward current that peaked rapidly and then declined. A later current was outward at potentials positive to the equilibrium potential for K+ (EK) and showed oscillations. 3. The initial inward current, studied in isolation using Cs+ in the electrode solution, showed rapid activation, inward rectification and reversal at +3 +/- 4 mV. Reduction of [Na+]o to 10 mM shifted the reversal potential to -21 +/- 3 mV, indicating that ATP activates a non-selective cation current, consistent with involvement of P2X receptors. 4. The later current activated by ATP, studied with K+ in the electrode solution, exhibited a linear I-V relationship, and reversed at -71 +/- 4 mV. The reversal potential shifted 51 mV per 10-fold change of [K+]o, indicating that ATP activates a K+ current (IK). 5. In fura-2-loaded cells, ATP caused elevation of [Ca2+]i that persisted in Ca(2+)-free solution, indicating that ATP induced release of Ca2+ from intracellular stores, consistent with involvement of P2Y receptors. Simultaneous patch clamp and fluorescence recordings revealed that IK was associated with the elevation of [Ca2+]i. Using a Ca2+ ionophore (4Br-A23187) to elevate [Ca2+]i, IK activated when [Ca2+]i exceeded approximately 400 nM, with half-maximal activation at 580 +/- 50 nM. 6. In cell-attached patches, ATP activated a channel with a conductance of 48 +/- 6 pS, that reversed director, near EK. Channel open probability increased with elevation of [Ca2+]i, indicating the Ca2+ dependence of this channel. 7. These results demonstrate that rat osteoclasts express two types of purinoceptors. P2X receptors give rise to non-selective cation current. P2Y receptors mediate Ca2+ release from stores, causing activation of a Ca(2+)-dependent K+ channel.

Glucocorticoids inhibit bone resorption by isolated rat osteoclasts by enhancing apoptosis.

We have studied the effects of glucocorticoids on the activity and viability of neonatal rat osteoclasts in vitro. In the bone slice assay, glucocorticoids caused a dose-dependent decrease in the amount of bone resorbed, which was accompanied by a parallel decrease in osteoclast number. Loss of osteoclasts was due to their death, which occurred by the process of apoptosis. Evidence for the latter was obtained by a range of techniques, including time-lapse video microscopy, acridine orange staining, DNA fragment detection and transmission electron microscopy. Immunocytochemistry revealed the presence of glucocorticoid receptors in osteoclasts, and glucocorticoid-induced cell death could be prevented by the glucocorticoid receptor antagonist, RU486. These observations suggest that glucocorticoids promote receptor-mediated apoptosis of rat osteoclasts in vitro. This finding may help to explain recent data indicating that, in sharp contrast with their effects on the human skeleton, glucocorticoids inhibit bone resorption and increase bone mass in rats in vivo.

Regulation of osteoclast function.

Regulation of osteoclast function.

P-679 - Na+/H+ exchanger regulates the intracellular pH of isolated rat osteoclasts in concert with H+-ATPase

P-679 - Na+/H+ exchanger regulates the intracellular pH of isolated rat osteoclasts in concert with H+-ATPase

Recording of mitochondrial transmembrane potential and volume in cultured rat osteoclasts by confocal laser scanning microscopy.

Osteoclasts are multinuclear bone-resorbing cells which contain abundant mitochondria. Morphological studies have suggested that a correlation may exist between mitochondrial concentration and bone resorption by osteoclasts. However, investigation of mitochondrial transmembrane potential (delta psi) and volume has been hampered by the difficulty in obtaining a sufficient number of osteoclasts for assessing these characteristics by flow cytometric analysis. In this study, we have used confocal laser scanning microscopy after loading the cells with Rhodamine 123 and 10-nonyl Acridine Orange to record mitochondrial delta psi and volume, respectively, in isolated rat osteoclasts cultured on bovine bone slices. Optimal staining conditions were found to be 10 micrograms ml-1 for 40 min for Rhodamine, and 1 microM for 10 min for the 10-nonyl Acridine Orange derivative. Two osteoclast populations, whose shape seemed to reflect bone resorption and migratory functions, were identified depending on their shape and on the distribution of the two dye probes. 'Round-shaped' osteoclasts had significantly higher mitochondrial delta psi and volume in the apical regions than in the basolateral portions (p < 0.00001). In contrast, mitochondrial delta psi and volume in 'irregular-shaped' osteoclasts were rather evenly distributed in both these regions (p > 0.05). Our results indicate that there is an apical polarization of mitochondria in osteoclasts corresponding to the energy demands associated with bone resorption.

P19. The effect of hepatocyte growth factor on the behaviour of isolated rat osteoclasts

P19. The effect of hepatocyte growth factor on the behaviour of isolated rat osteoclasts

Osteoblast-mediated effects of zinc on isolated rat osteoclasts: inhibition of bone resorption and enhancement of osteoclast number.

Zinc is an important element in biology yet little is understood of its role in bone cell metabolism and function. This study examined the effects of zinc on osteoclast (OC) function in cultures derived from neonatal rats and in cocultures of OC and UMR 106-01 osteoblast-like cells (UMR/OC cocultures). Treatment with zinc (10(-12)-10(-4) mol/L) had no effect on either bone resorption or the number of multinucleate cells positive for tartrate-resistant acid phosphatase (TRACP + ve MNC) in OC cultured for 24 h on bone slices. However, in UMR/OC cocultures, 10(-4) mol/L zinc (but not lower concentrations) decreased resorption pit formation by approximately 50% and increased TRACP + ve MNC number by approximately 40%. When osteoblast-like cells were pretreated with zinc prior to, but not during, coculture with OC, effects on TRACP + ve MNC and pit number persisted, although the effect was reduced. Zinc treatment also inhibited resorption and stimulated TRACP and calcitonin receptor (CTR) + ve MNC numbers in long-term (96-120 h) UMR/OC cocultures. Our results indicate that zinc increases TRACP + ve CTR + ve MNC numbers yet inhibits bone-resorbing activity, and that these effects are dependent on the presence of osteoblastic cells. Zinc is abundant in bone and may act as a local regulator of bone cells.

Bisphosphonates induce osteoblasts to secrete an inhibitor of osteoclast-mediated resorption.

Current knowledge indicates that osteoblasts play an integral role in osteoclastic bone resorption through an osteoclast-stimulating activity produced by osteoblasts in response to resorption-promoting osteotropic factors. Previously, we have shown that the inhibitory action of the bisphosphonates on bone resorption in part is mediated by osteoblasts. The aim of the present study was to investigate whether the bisphosphonate-generated inhibition is due to these compounds decreasing the synthesis of the osteoclast-stimulating activity or is the result of osteoblasts synthesizing an osteoclast resorption inhibitor. Using the osteoblastic cell line CRP 10/30, which produces osteoclast- stimulating activity, constitutively and employing isolated rat osteoclasts cultured on ivory, evidence was obtained indicating that the bisphosphonates ibandronate and alendronate at a concentration of 10(-7) M induce osteoblasts to synthesize an osteoclast inhibitor that reduces pit formation by more than 50%. The inhibitor is heat and proteinase labile and has a molecular mass between 1-10 kDa. The reduction of resorption pits is paralleled by a decrease in tartrate-resistant acid phosphatase-positive mono- and multinucleated cells, whereas the mean area resorbed per pit was not changed, suggesting that the inhibitor affects osteoclast formation and/or survival and probably not the osteoclast resorption activity. Rat preosteoblastic cells and rat dermal fibroblasts were found not to produce the inhibitor. In conclusion, osteoblasts aside from their role of mediating osteoclastic resorption promoters are also involved in inhibiting bone resorption through the synthesis of an osteoclast resorption inhibitor.

Extracellularly applied ruthenium red and cADP ribose elevate cytosolic Ca2+ in isolated rat osteoclasts.

We demonstrated recently that the divalent cation-sensing receptor on the osteoclast, the Ca2+ receptor (CaR), is a functional component of a cell surface-expressed ryanodine receptor-like molecule (RyR). The objective of the present study was to further characterize this putative RyR by use of the two well-known cell-impermeant RyR modulators, ruthenium red and adenosine 3',5'-cyclic diphosphate ribose (cADPr). We found that, when applied extracellularly, ruthenium red (5 x 10(-8)-10(-4) M) and cADPr (5 x 10(-6) M) triggered an elevation of cytosolic [Ca2+]. Depolarization of the cell membrane by the application of 0.1 M K+ in the presence of 5 x 10(-6) M. valinomycin resulted in a concentration-dependent increase in the magnitude of the cytosolic Ca2+ response to extracellular ruthenium red (5 x 10(-9) and 5 x 10(-5) M), a phenomenon that was not seen when osteoclasts were hyperpolarized using 5 x 10(-3) M K+ with 5 x 10(-6) M valinomycin. In the presence of an intact nonleaky cell membrane, these results would favor a plasma membrane locus of action for the two modulators. Furthermore, pretreatment of osteoclasts with either modulator resulted in a markedly attenuated cytosolic Ca2+ transient elicited in response to the CaR agonist Ni2+, thus confirming an interaction between the cADPr- and ruthenium red-sensitive sites and the osteoclast CaR. The inhibition of the cytosolic Ca2+ response to Ni2+ induced by ruthenium red remained unchanged in the face of membrane potential changes. Finally, the cytosolic Ca2+ response to caffeine (5 x 10(-4) M), another RyR modulator, was also strongly attenuated by pretreatment with 5 x 10(-9) M ruthenium red. We conclude that ruthenium red and cADPr act on plasma membrane-resident sites and that both these sites interact with the process of divalent cation sensing.

P-259 - Isolated rat osteoclast adherence and differentiation induced by CSF-1 shifts in extracellular pH.

P-259 - Isolated rat osteoclast adherence and differentiation induced by CSF-1 shifts in extracellular pH.

Downregulation of colony-stimulating factor-1 (CSF-1) binding by CSF-1 in isolated osteoclasts.

Colony-stimulating factor-1 (CSF-1), also called macrophage colony-stimulating factor, is the growth factor for the cells of the mononuclear phagocytic system. Furthermore, CSF-1 is essential in osteoclastogenesis and also affects mature osteoclasts. The receptor for CSF-1 was demonstrated on cells of the osteoclast lineage, with highest levels on the mature cells. This study investigated whether the binding of CSF-1 to isolated rat osteoclasts is modulated by the growth factor itself. Exposure of osteoclasts to CSF-1 for 1 hour virtually abolished binding of the growth factor. After removal of CSF-1, binding sites were restored within 4 hours. This recovery was blocked by cycloheximide, indicating the dependence on new protein synthesis for reexpression of receptors on the cell surface. The observed downregulation of CSF-1 binding sites might be a mechanism to control the effects of the growth factor on mature osteoclasts.

Translocation of protein kinase C isoenzymes by elevated extracellular Ca 2+ concentration in cells from a human giant cell tumor of bone

In this study we investigated the protein kinase C isoenzymes expressed by human osteoclast-like cells harvested from a giant cell tumor of bone (GCT23 cells), and by freshly isolated rat osteoclasts. Immunoblotting analysis revealed that the -α, -δ, and -ϵ, PKC isoforms, but not the -β isoenzyme, are expressed by GCT23 cells. Immunofluorescence studies demonstrated that PKC-α, -δ, and -ϵ are homogeneously expressed by both mononuclear and multinucleated GCT23 cells, as well as by rat osteoclasts. Similar to authentic osteoclasts, GCT23 cells responded to an increase of extracellular Ca 2+ concentration ([Ca 2+] 0) with a dose-dependent elevation of the cytosolic free Ca 2+ concentration ([Ca 2+] i). An increase of [Ca 2+] 0 stimulated the translocation of PKC-α from the cytosolic to the particulate fraction, suggesting the involvement of this isoenzyme in the signal transduction mechanism prompted by stimulation of the [Ca 2+] 0 sensing. By contrast, PKC-δ was not altered by exposure to elevated [Ca 2+] 0, whereas PKC-ϵ underwent reciprocal translocation, disappearing from the insoluble fraction and increasing in the cytosol. The effects of PKC on GCT23 cell functions were investigated by treatment with phorbol 12-myristate, 13-acetate (PMA). We observed that activation of PKC by PMA failed to affect adhesion onto the substrate, but down-regulated the [Ca 2+] 0-induced [Ca 2+] i increases. The latter effect was specific, since it was reversed by treatment with the PKC inhibitors staurosporine and chelerythrine.

The Effect of Hepatocyte Growth Factor on the Behavior of Osteoclasts

Hepatocyte growth factor (HGF) stimulates the growth, motility and morphogenesis of a variety of cell types, including hemopoietic progenitors. We found that HGF is a potent inhibitor of bone resorption by isolated rat osteoclasts. However, in the presence of the osteoblastic cell line UMR 106, it stimulated osteocIastic resorption. HGF also increased osteoclastic motility and spread area, over a similar concentration range. We detected no effect on osteoclast formation or survival. Our data suggest that HGF may be involved in the recruitment of osteoclasts to sites of bone resorption, but that during migration resorptive functions are suppressed. Once on resorptive sites, the osteoclast response to HGF is modulated by osteoblastic cells and the bone resorptive activity of osteoclasts may be stimulated accordingly.

Macrophage inflammatory protein-1 alpha and IL-8 stimulate the motility but suppress the resorption of isolated rat osteoclasts.

Cells of the osteoblastic lineage play a major role in the regulation of osteoclastic bone resorption. Recent studies have demonstrated production of chemokines by osteoblastic cells. Although these phagocyte-stimulating and proinflammatory cytokines act as chemoattractants and activators for other members of the hemopoietic lineage, their actions on osteoclasts have not been characterized. We found that macrophage inflammatory protein-1 alpha (MIP-1 alpha) and IL-8 inhibited bone resorption by rat osteoclasts, primarily through reduction in the proportion of osteoclasts resorbing bone, a pattern of inhibition previously observed in response to macrophage CSF (M-CSF). MIP-2, RANTES, MIP-1 beta, and monocyte chemotactic protein-1 were without effect on resorption. MIP-1 alpha and IL-8, but not the other chemokines, also stimulated osteoclastic motility and increased the osteoclast spread area in a dose-dependent manner, over the same concentration range as that which inhibited bone resorption. In addition, MIP-1 alpha induced osteoclast orientation in a gradient of the chemokine, and stimulated osteoclast migration. We detected no effect of chemokines on osteoclast formation or survival. Our data suggest that chemokines can promote osteoclast orientation and migration, processes that might be involved in chemotaxis; it seems appropriate that resorptive functions should be suppressed during migration. Because chemokines are proinflammatory, their actions on osteoclasts might represent mechanisms by which bone resorption is modulated by the inflammatory process when this occurs in bone. However, given that chemokines are increasingly recognized to be multifunctional and that they are produced by cells of the osteoblastic lineage, they may also be components of the physiologic regulation of bone resorption.

Inhibition of bone resorption in vitro by selective inhibitors of gelatinase and collagenase

Two low-molecular-mass inhibitors of matrix metalloproteinases (MMPs), CT1166, a concentration-dependent selective inhibitor of gelatinases A and B, and Ro 31-7467, a concentration-dependent selective inhibitor of collagenase, were examined for their effects on bone resorption and type-I collagenolysis. The test systems consisted of measuring (1) the release of [3H]proline from prelabelled mouse calvarial explants; (2) the release of 14C from prelabelled type-I collagen films by mouse calvarial osteoblasts; and (3) lacunar resorption by isolated rat osteoclasts cultured on ivory slices. In 24 h cultures, CT1166 and Ro 31-7467 inhibited both interleukin-1 alpha- (IL-1 alpha; 10(-10) M) and 1,25-dihydroxyvitamin D3 (10(-8) M)-stimulated bone resorption in cultured neonatal mouse calvariae at concentration selective for the inhibition of gelatinase (10(-9) M for CT1166) and collagenase (10(-8) M for Ro 31-7467) respectively. For each compound the inhibition was dose-dependent, reversible, and complete at a 10(-7) M concentration. However, CT1166 (10(-9) M) and Ro 31-7467 (10(-8) M) in combination were required to completely abolish IL-1 alpha-stimulated bone resorption in mouse calvariae throughout a 96 h culture period. Neither of the inhibitors affected protein synthesis, DNA synthesis nor the IL-1 alpha-stimulated secretion of the lysosomal enzyme, beta-glucuronidase. Both CT1166 and Ro 31-7467 partially inhibited IL-1 alpha-stimulated lacunar resorption by isolated osteoclasts, but were without effect on unstimulated lacunar resorption. Rodent osteoclasts produced collagenase and gelatinases-A and -B activity. In contrast the substrate used to assess osteoclast lacunar resorption contained no detectable collagenase or gelatinase activity. Both compounds dose-dependently inhibited 1,25-dihydroxyvitamin D3 (10(-8) M)-stimulated degradation of type-I collagen by mouse calvarial osteoblasts; however, complete inhibition of collagenolysis was only achieved at concentrations at which CT1166 and Ro 31-7467 act as general MMP inhibitors. This study demonstrates that collagenase and gelatinases A and/or B participate in bone resorption. While these MMPs may be primarily involved in osteoid removal, we conclude that they may also be released by osteoclasts, where they participate in bone collagen degradation within the resorption lacunae.

Wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, inhibits osteoclastic bone resorption in vitro

Phosphatidylinositol 3-kinase (Pl3-k) is involved in cellular signaling via the phosphoinositol pathway leading to mitogenesis in response to growth factors in proliferating cells, as well as cytoskeletal changes and secretory responses in terminally differentiated cells. The fungal metabolite, wortmannin, is a potent and selective inhibitor of Pl3-k at nanomolar concentrations. We show that wortmannin dose-dependently (0.001-1 microM) inhibits bone resorption by isolated rat osteoclasts in the bone slice pit assay with an IC50 of approximately 5 nM. Wortmannin was not cytotoxic since osteoclast morphology and survival on bone slices was unaffected by concentrations up to 1 microM. Since primary osteoclasts are terminally differentiated cells and osteoclast cytoplasmic spreading and morphology was unaffected by wortmannin, we suggest that Pl3-k signaling is involved in vesicle exocytosis and ruffled border membrane formation that are required for osteoclastic bone resorption to take place.

Zinc is a potent inhibitor of osteoclastic bone resorption in vitro

It is well established that zinc, an essential trace element, plays an important role in growth and stimulates bone formation. However, the effects of zinc on bone resorption have received little attention. We studied its effects on isolated rat osteoclasts. Unexpectedly, osteoclasts were exquisitely sensitive to zinc, with a significant decrease in bone resorption occurring at concentrations as low as 10(-14) M. This effect was specific for zinc and was not observed with the other transitional or alkaline metals studied. There was no evidence of toxicity at concentrations up to 10(-4) M. Zinc also completely abolished the stimulatory effect of parathyroid hormone. Zinc is therefore a highly potent and selective inhibitor of osteoclastic bone resorption in vitro. The mode of action remains to be established and may represent a novel inhibitory mechanism in the osteoclast.

Caffeine modulates Ca2+ receptor activation in isolated rat osteoclasts and induces intracellular Ca2+ release

A ryanodine-sensitive pathway is involved in intracellular Ca2+ release in response to activation of the osteoclast cell surface Ca2+ receptor. We now report that the ryanodine-receptor modulator, caffeine itself released intracellularly stored Ca2+ and, strongly inhibited Ca2+ release triggered in response to Ca(2+)-receptor activation by Ni2+, a surrogate cation agonist. Caffeine yielded a bell-shaped concentration-response curve (0.005-2 mM) and displayed use-dependent inactivation. Furthermore, responses to caffeine were abolished on prior application of Ni2+ (5 mM). Subthreshold (0.005 mM) caffeine concentrations abolished Ni(2+)-induced elevations in the cytosolic Ca2+ concentration ([Ca2+]). However, in a Ca(2+)-free, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-containing solution (extracellular [Ca2+] < 10 nM), caffeine (0.5 mM) neither elevated [Ca2+] nor inhibited the response to Ni2+. Finally, when caffeine was applied to intercept the plateau phase of the cytosolic Ca2+ signal triggered by extracellular Ca2+ elevation (10 mM), a rapid but reversible inactivation followed. These studies strongly indicate the existence of a caffeine-sensitive mechanism for the release of intracellularly stored Ca2+ in the osteoclast.

The effects of bisphosphonates on the resorption cycle of isolated osteoclasts

Binding sites for wheat germ agglutinin (WGA)-lectin have been shown to become revealed in the demineralized resorption lacunae that osteoclasts excavate on bone substrate. Peroxidase-conjugated WGA-lectin, which binds to bone matrix glycoconjugates and proteoglycans, was used in pit formation assays to assess the activity of isolated osteoclasts cultured on either 3-amino-1,1-hydroxypropylidene-bisphosphonate (APD)-or dichloromethylene bisphosphonate (Cl2MBP)-covered bone slices. Immunofluorescence and histochemical techniques were also used to study the effects of bone-bound bisphosphonates on isolated rat osteoclasts. Neither APD nor Cl2MBP interfered with the special organization of actin or vinculin in osteoclasts when the cells were initializing their resorption cycle. After 24 hours of culture, the number of resorbing osteoclasts increased strongly on control slices, but remained low on either APD- or Cl2MBP-treated slices. At this time, the actin and vinculin rings in osteoclasts also started to exhibit abnormal, more diffuse staining. Both bisphosphonates studied resulted in signs of cytotoxicity: the number of osteoclasts decreased on APD- or Cl2MBP-covered bone during the course of the study and those remaining attached exhibited severe cytoplasmic retractions. The total areas of resorption remained at significantly lower levels in both experimental groups studied, and this was due to decreases in both the number and sizes of individual resorption pits. The size of the most extensive lacunae detected on the Cl2MBP slices did not exceed 5 x 10(3) microns 2, whereas on the control slices, resorption pits bigger than 15 x 10(3) microns 2 were frequently discovered.

The effects of selective inhibitors of matrix metalloproteinases (MMPs) on bone resorption and the identification of MMPs and TIMP-1 in isolated osteoclasts.

We have compared the effects of a general matrix metalloproteinase (MMP) inhibitor (CT435) with those of a concentration-dependent specific gelatinase inhibitor (CT543; Ki < 20 nM) on bone resorption in vitro. The test systems consisted of measuring: (i) the release of 45Ca2+ from prelabelled mouse calvarial explants; (ii) the release of 45Ca2+ from prelabelled osteoid-free calvarial explants co-cultured with purified chicken osteoclasts; and (iii) lacunar resorption by isolated rat osteoclasts cultured on ivory slices. Both CT435 and CT543 dose-dependently inhibited the release of 45Ca2+ from neonatal calvarial bones stimulated by either parathyroid hormone or 1,25-dihydroxyvitamin D3. Moreover, CT543 produced a 40% inhibition at a concentration (10(-8) M) selective for the inhibition of human gelatinases A and B. CT435 (10(-5) M) and CT543 (10(-5) M) partially inhibited the release of 45Ca2+ from osteoid-free calvarial explants by chicken osteoclasts with a maximum of approximately 25% for unstimulated cultures, and approximately 36% for cultures stimulated by interleukin-1 alpha (IL-1 alpha; 10(-10) M). Neither inhibitor prevented lacunar resorption on ivory by unstimulated rat osteoclasts, but the compounds produced a partial reduction in both the number and total surface area of lacunae in IL-1 alpha-stimulated cultures, with maximal action at 10(-5) M. Neither of the inhibitors affected protein or DNA synthesis, nor the IL-1 alpha-stimulated secretion of the lysosomal enzyme beta-glucuronidase. Immunocytochemistry demonstrated that isolated rabbit osteoclasts constitutively expressed gelatinase A and synthesized gelatinase B, collagenase and stromelysin, as well as the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) following IL-1 alpha stimulation. These experiments have shown that in addition to collagenase, gelatinases A and B are likely to play a significant role in bone resorption. They further suggest that MMPs produced by osteoclasts are released into the sub-osteoclastic resorption zone where they participate in bone collagen degradation.