1,25-Dihydroxyvitamin D(3) and prostaglandin E(2) act directly on circulating human osteoclast precursors.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and prostaglandin E(2) (PGE(2)) are known to influence osteoclast formation indirectly through their effects on osteoblasts. To determine whether 1, 25(OH)(2)D(3) and PGE(2) also have a direct effect on circulating osteoclast precursors, these factors were added to long-term cultures of human peripheral blood mononuclear cells (PBMCs) in the presence of osteoprotegerin ligand and macrophage colony-stimulating factor (M-CSF) (+/-dexamethasone). The number of TRAP(+) and VNR(+) multinucleated cells and the area of lacunar resorption were decreased when 1,25(OH)(2)D(3) alone was added. A marked increase in resorption pit formation was noted when the combination of 1, 25(OH)(2)D(3) and dexamethasone was added to PBMC cultures. Dose-dependent inhibition of osteoclast formation and lacunar resorption was seen when PGE(2) was added to PBMC cultures in both the presence and the absence of dexamethasone. Thus, 1,25(OH)(2)D(3) and PGE(2) not only influence osteoclast formation in the presence of bone stromal cells but also act directly on circulating osteoclast precursors to influence osteoclast differentiation.     

Osteoprotegerin and osteoprotegerin ligand effects on osteoclast formation from human peripheral blood mononuclear cell precursors

Osteoprotegerin (OPG) and its ligand (OPGL) negatively and positively regulate osteoclastogenesis in the mouse. OPG inhibits osteoclastogenesis by sequestering its ligand, OPGL, the osteoclast differentiation and activation factor. This study demonstrates the effects of soluble muOPGL and huOPG on the developing human osteoclast phenotype, on bone slices, using peripheral blood mononuclear cells (PBMCs), cultured for 2 weeks, without stromal cells. OPGL (2-50 ng/ml), in combination with CSF-1, hydrocortisone (HC), and 1,25(OH)2D3, increases the size of osteoclast-like cells on bone, as defined by the acquisition of osteoclast markers: vitronectin receptor (VR), tartrate-resistant acid phosphatase (TRAP), multinuclearity, and bone resorption. By 14 days, with 20 ng/ml OPGL, the largest cells/10x field have achieved an average diameter of 163+/-38 microm, but only approximately 10-20 microm in its absence and the number of osteoclast-like cells/mm2 bone surface is about 128. By scanning electron microscopy, OPGL-treated (20-ng/ml) cultures contain small osteoclast-like cells on bone with ruffled "apical" surfaces by day 7; by day 15, large osteoclast-like cells are spread over resorption lacunae. At 15 ng/ml OPGL, about 37% of the bone slice area is covered by resorption lacunae. OPG (5-250 ng/ml) antagonizes the effects of OPGL on the morphology of the osteoclast-like cells that form, as well as bone erosion. For cells grown on plastic, Cathepsin K mRNA levels, which are barely detectable at plating, are elevated 7-fold, by 5 days, in the presence, not the absence, of OPGL (20 ng/ml) + CSF-1 (25 ng/ml). Similar findings are observed in experiments performed in the absence of HC and 1,25(OH)2D3, indicating that HC and 1,25(OH)2D3 are not needed for OPGL-induced osteoclast differentiation. In conclusion, this study confirms a pivotal role for OPGL and OPG in the modulation of human osteoclast differentiation and function, suggesting a use for OPG for treating osteoclast-mediated bone disease in humans.     

Regulation of osteoclast activity.

Osteoclasts are the primary cell type responsible for bone resorption. This paper reviews many of the known regulators of osteoclast activity, including hormones, cytokines, ions, and arachidonic acid metabolites. Most of the hormones and cytokines that inhibit osteoclast activity act directly on the osteoclasts. In contrast, most of the hormones and cytokines that stimulate osteoclast activity act indirectly through osteoblasts. Particularly interesting in this regard are agents that directly inhibit activity of highly purified osteoclasts yet stimulate activity of osteoclasts that are co-cultured with osteoblasts. Recent studies have demonstrated that the primary mechanism by which bone resorptive agents stimulate osteoclast activity indirectly is likely to be up-regulation of production of osteoclast differentiation factor/osteoprotegerin ligand (ODF/OPGL) by the osteoblasts. In addition to discussing regulators of osteoclast activity per se, this paper also reviews the role of osteoclast apoptosis to limit the extent of bone resorption.     

Statins modulate the levels of osteoprotegerin/receptor activator of NFkappaB ligand mRNA in mouse bone-cell cultures.

Statins stimulate bone formation partly by inducing osteoblast differentiation, although there is controversy about the effects of statins on bone mineral density and fracture risk. Several studies have revealed that statins suppress bone resorption. However, the mechanism by which statins inhibit bone resorption is still unclear. The present study was performed to clarify the effects of statins on osteoclast formation as well as the levels of osteoprotegerin (OPG) and receptor activator of NFkappaB ligand (RANKL) mRNA in mouse bone-cell cultures by semiquantitative RT-PCR. 10(-8) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] significantly stimulated osteoclast formation and 10(-6) M statins (mevastatin and simvastatin) significantly antagonized osteoclast formation stimulated by 1,25(OH)2D3 in mouse bone-cell cultures, including both osteoblasts and osteoclasts. 10(-6) M mevastatin and simvastatin increased the level of OPG mRNA in mouse bone-cell cultures. On the other hand, 10(-6) M mevastatin and simvastatin inhibited the level of RANKL mRNA in these cultures. In conclusion, the present study demonstrates that statins inhibit osteoclast formation in mouse bone-cell cultures. Moreover, statins also increased and decreased the levels of OPG and RANKL mRNA expression in these cultures, respectively. The modulation of OPG/RANKL may be involved in the inhibition of osteoclast formation by statins.     

Effect of high phosphate concentration on osteoclast differentiation as well as bone-resorbing activity

Although high inorganic phosphate (Pi) concentration in culture media directly inhibits generation of new osteoclasts and also inhibits bone resorption by mature osteoclasts, its precise mechanism and the physiological role have not been elucidated. The present study was performed to investigate these issues. Increase in extracellular Pi concentration ([Pi](e)) (2.5-4 mM) concentration dependently inhibited 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] or parathyroid hormone (PTH)-(1-34)-induced osteoclast-like cell formation from unfractionated bone cells in the presence of stromal cells. Increase in [Pi](e) (2.5-4 mM) concentration dependently inhibited 1,25(OH)(2)D(3)-, PTH-(1-34)-, or receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF)-induced osteoclast-like cell formation from hemopoietic blast cells in the absence of stromal cells. Increase in [Pi](e) (2.5-4 mM) dose dependently stimulated the expression of osteoprotegerin (OPG) mRNA and increased the expression of OPG mRNA suppressed by PTH-(1-34) or 1,25(OH)(2)D(3) in unfractionated bone cells, while it did not affect RANKL mRNA. Increase in [Pi](e) (2.5-4 mM) concentration dependently inhibited the bone-resorbing activity of isolated rabbit osteoclasts. Increase in [Pi](e) (4 mM) induced the apoptosis of isolated rabbit osteoclasts while it did not affect the apoptosis of osteoclast precursor cells and mouse macrophage-like cell line C7 cells that can differentiate into osteoclasts in the presence of RANKL and M-CSF. These results indicate that increase in [Pi](e) inhibits osteoclast differentiation both by up-regulating OPG expression and by direct action on osteoclast precursor cells. It is also indicated that increase in [Pi](e) inhibits osteoclastic activity at least in part by the direct induction of apoptosis of osteoclasts.     

Effects of interleukin 3 and of granulocyte-macrophage and macrophage colony stimulating factors on osteoclast differentiation from mouse hemopoietic tissue

The effects of granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 3 (IL3) on osteoclast formation were tested by incubation of murine hemopoietic cells on plastic coverslips and bone slices with GM-CSF, M-CSF, or IL3, with or without 1,25(OH)2 vitamin D3 (1,25(OH)2D3). Osteoclastic differentiation was detected after incubation by scanning electron microscopical examination of bone slices for evidence of osteoclastic excavations, and by autoradiographic assessment of cells for 1,25(OH)2D3-calcitonin (CT) binding. The differentiation of CT-receptor-positive cells preceded bone resorption, but the number that developed correlated with the extent of bone resorption (r = 0.88). M-CSF and GM-CSF substantially reduced bone resorption and CT-receptor-positive cell formation. The degree of inhibition of bone resorption could not be attributed to effects on the function of mature cells, since M-CSF inhibits resorption by such cells only by 50%, and GM-CSF has no effect. GM-CSF inhibited the development of mature function (bone resorption) to a greater extent than it inhibited CT-receptor-positive cell formation. Since CT-receptor expression antedated resorptive function, this suggests that GM-CSF resulted in the formation of reduced numbers of relatively immature osteoclasts. This suggests that it may exert a restraining effect on the maturation of cells undergoing osteoclastic differentiation in response to 1,25(OH)2D3. Conversely, IL3, which also has no effect on mature osteoclasts, by itself induced CT-receptor expression but not bone resorption; in combination with 1,25(OH)2D3 it induced a threefold increase in bone resorption and CT-receptor-positive cells compared with cultures incubated with 1,25(OH)2D3 alone. IL3 did not induce CT-receptors in peritoneal macrophages, blood monocytes, or J 774 cells. The results suggest that IL3 induces only partial maturation of osteoclasts, which is augmented or completed by additional factors such as 1,25(OH)2D3.     

Echistatin is a potent inhibitor of bone resorption in culture

The venom protein, s-echistatin, originally derived from the saw-scaled viper Echis carinatus, was found to be a potent inhibitor of bone resorption by isolated osteoclasts. This Arg24-Gly25-Asp26-(RGD)-containing protein inhibited the excavation of bone slices by rat osteoclasts (IC50 = 0.1 nM). It also inhibited the release of [3H]proline from labeled bone particles by chicken osteoclasts (IC50 = 100 nM). By comparison, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibited resorption by rat or chicken osteoclasts with an IC50 of 0.1 mM while ala24-echistatin was inactive. Video microscopy showed that rat osteoclast attachment to substrate was more sensitive to s-echistatin than was the attachment of mononuclear cells or chicken osteoclasts. The difference in sensitivity of rat and chicken osteoclasts to s-echistatin may be due to differences between receptors on rat and chicken osteoclasts for s-echistatin. Antibody localization of echistatin on these cells showed much greater echistatin binding to rat osteoclasts than to chicken osteoclasts. Laser scanning confocal microscopy after immunohistochemical staining showed that s-echistatin binds to osteoclasts, that s-echistatin receptors are most abundant at the osteoclast/glass interface, and that s-echistatin colocalizes with vinculin. Confocal interference reflection microscopy of osteoclasts incubated with s-echistatin, demonstrated colocalization of s-echistatin with the outer edges of clusters of grey contacts at the tips of some lamellipodia. Identification of the echistatin receptor as an integrin was confirmed by colocalization of echistatin fluorescence with staining for an alpha-like subunit. Attachment of bone particles labeled with [3H]proline to chicken osteoclasts confirmed that the mechanism of action of echistatin was to inhibit osteoclast binding to bone presumably by disrupting adhesion structures. These data demonstrate that osteoclasts bind to bone via an RGD-sequence as an obligatory step in bone resorption, that this RGD-binding integrin is at adhesion structures, and that it colocalizes with vinculin and has an alpha-like subunit.     

Effects of 1,25 dihydroxyvitamin D on osteoclast number and cytochemistry in normal and osteopetrotic (os) rabbits

Osteoclast-mediated bone resorption is increased in response to 1,25 dihydroxyvitamin D (1,25[OH]2D or calcitriol). Osteopetrosis is a metabolic bone disease characterized by defective, osteoclast-mediated bone resorption, which co-exists with elevated serum 1,25-(OH)2D levels in some osteopetrotic children and animals. We examined the effects of high doses of calcitriol on osteoclast number and cytochemistry in both normal and osteopetrotic (os) rabbits. Calcitriol was continuously infused at doses of 0.5, 2.5, or 25 micrograms/kg/day via subcutaneously implanted osmotic minipumps for a period of 7 days. Following treatment, the proximal tibial metaphyses were processed for histomorphometric and cytochemical analyses. Sections were stained for tartrate-resistant acid phosphatase (TrAP) or acid ATPase (TraATPase). Osteoclasts were significantly reduced in untreated os rabbits compared with age-matched normal littermates between birth and 3 weeks of age (41-46% of normal). Whereas most normal osteoclasts (85%) stained heavily for TrAP or TraATPase, less than half of os osteoclasts were heavily stained for these acid hydrolases. Infusions of 1,25(OH)2D resulted in elevations of osteoclast numbers in both normal and os rabbits, but the number of osteoclasts remained significantly lower in mutants than in normal littermates at any given dose. Calcitriol infusions also resulted in a significant increase in the percentage of os osteoclasts staining heavily for TrAP and TraATPase. These results suggest that in response to 1,25(OH)2D normal osteoclasts increase their production of acid hydrolases before increasing cell numbers and that, in spite of high levels of endogenous calcitriol, os rabbits can respond to exogenous 1,25(OH)2D as evidenced by increased osteoclast number and cytochemical staining, even though these osteoclasts fail to resorb the excess skeletal matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of 24,25-dihydroxyvitamin D3 in osteoclasts.

Previous results demonstrated that the administration of pharmacological doses of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) to animals reduces bone resorption and increases bone volume with a decrease in osteoclast number. In order to clarify whether 24,25(OH)2D3 has an effect to inhibit osteoclastic bone resorption, the effect of 24,25(OH)2D3 on the formation and function of osteoclastic cells was examined in vitro. Treatment of hemopoietic blast cells, which are progenitors of osteoclasts, with parathyroid hormone (PTH) or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) stimulated the formation of osteoclast-like multinucleated cells in a dose-dependent manner. Although 24,25(OH)2D3 in itself had little effect on osteoclast-like multinucleated cells formation, it inhibited the stimulatory effect of PTH on the formation of osteoclastic cells. In addition, 24,25(OH)2D3 also inhibited the stimulation of resorption pit formation by osteoclasts under stimulation with PTH. In contrast, 1,25(OH)2D3 stimulated the formation and function of osteoclastic cells even at low concentrations, and the effect was additive to PTH. These results could not be explained by either an agonistic or antagonistic effect of 24,25(OH)2D3 on 1,25(OH)2D3, and are consistent with the assumption that 24,25(OH)2D3 has a unique inhibitory effect on the formation and function of osteoclasts. Because 24,25(OH)2D3 is shown to stimulate the degradation of 1,25(OH)2D3 and because the formation of 24,25(OH)2D3 is stimulated by 1,25(OH)2D3 not only in the kidney but also in many of its target tissues, including bone, the inhibitory effect of 24,25(OH)2D3 on osteoclastic bone resorption may play a role in the local modulation of the actions of osteotropic hormones in bone.     

Blockade of osteoclast-mediated bone resorption through occupancy of the integrin receptor: A potential approach to the therapy of osteoporosis

Bone resorption requires the tight attachment of the bone-resorbing cells, the osteoclasts, to the bone mineralized matric. Integrins, a class of cell surface adhesion glycoproteins, play a key role in the attachment process. Most integrins bind to their ligands via the arginyl-glycyl-aspartyl (R-G-D) tripeptide present within the ligand sequence. The interaction between integrins and ligaands results in bidirectional transfer of signals across the plasma Membrane. Tyrosine phosphorylaation occurs within cells as a result of integrin binding to ligaands and probably plays a role in the formation of the osteoclast clear zone, a specialized region of the osteoclast membraane maintained by cytoskeletal structure and involved in bone resorption. Human osteoclasts express α₂β₃ and α_vβ₃ integrins on their surface. Such signaling may also lead to “inside-out” effects, like increased expression of integrin receptors on the cell surface, or increased affinity of the integrin to its ligand. The α_vβ₃ integrin, a vitronectin receptor, plays an essential role in bone resorption. Antibodies to this integrin and short synthetic RGD-containing peptides are able to block bone resorption in vitro. Echistatin, an RGD-containing protein from a snake venom, binds to the α_vβ₃ integrin and blocks bone resssorption both in vitro and in vivo. Peptides containing the RGD motif are potential competitive “antagonists” a of the osteoclast integrins and may have utility in the blockage of bone resorption. Agonists may be identified by stimulation of intracellularsignaling. In theory, tissue spacificity can be achieved by (1) introducing specific amino acids in positions adjacent to the RGD sequence, (2) identifying non-RGD integrin binding domains, or (3) modulating the affinity of integrins for their endogenous ligands.     

A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function

Osteoclasts, the multinucleated giant cells that resorb bone, develop from monocyte-macrophage lineage cells. Osteoblasts or bone marrow stromal cells have been suggested to be involved in osteoclastic bone resorption. The recent discovery of new members of the tumor necrosis factor (TNF) receptor-ligand family has elucidated the precise mechanism by which osteoblasts/stromal cells regulate osteoclast differentiation and function. Osteoblasts/stromal cells express a new member of the TNF-ligand family "osteoclast differentiation factor(ODF)/osteoprotegerin ligand (OPGL)/TNF-related activation-induced cytokine (TRANCE)/receptor activator of NF-kB ligand (RANKL)" as a membrane associated factor. Osteoclast precursors which possess RANK, a TNF receptor family member, recognize ODF/OPGL/TRANCE/RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of macrophage colony-stimulating factor. Mature osteoclasts also express RANK, and their bone-resorbingactivity is also induced by ODF/OPGL/TRANCE/RANKL which osteoblasts/stromal cells possess. Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF)/TNF receptor-like molecule 1 (TR1) is a soluble decoy receptor for ODF/OPGL/TRANCE/RANKL. Activation of NF-kB and c-Jun N-terminal kinase through the RANK-mediated signaling system appears to be involved in differentiation and activation of osteoclasts.     

The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts

Osteoprotegerin (OPG) and OPG-ligand (OPGL) potently inhibit and stimulate, respectively, osteoclast differentiation (Simonet, W.S., D.L. Lacey, C.R. Dunstan, M. Kelley, M.-S. Chang, R. Luethy, H.Q. Nguyen, S. Wooden, L. Bennett, T. Boone, et al. 1997. Cell. 89:309-319; Lacey, D.L., E. Timms, H.-L. Tan, M.J. Kelley, C.R. Dunstan, T. Burgess, R. Elliott, A. Colombero, G. Elliott, S. Scully, et al. 1998. Cell. 93: 165-176), but their effects on mature osteoclasts are not well understood. Using primary cultures of rat osteoclasts on bone slices, we find that OPGL causes approximately sevenfold increase in total bone surface erosion. By scanning electron microscopy, OPGL-treated osteoclasts generate more clusters of lacunae on bone suggesting that multiple, spatially associated cycles of resorption have occurred. However, the size of individual resorption events are unchanged by OPGL treatment. Mechanistically, OPGL binds specifically to mature OCs and rapidly (within 30 min) induces actin ring formation; a marked cytoskeletal rearrangement that necessarily precedes bone resorption. Furthermore, we show that antibodies raised against the OPGL receptor, RANK, also induce actin ring formation. OPGL-treated mice exhibit increases in blood ionized Ca++ within 1 h after injections, consistent with immediate OC activation in vivo. Finally, we find that OPG blocks OPGL's effects on both actin ring formation and bone resorption. Together, these findings indicate that, in addition to their effects on OC precursors, OPGL and OPG have profound and direct effects on mature OCs and indicate that the OC receptor, RANK, mediates OPGL's effects.     

Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression

Osteopontin (OPN) was expressed in murine wild-type osteoclasts, localized to the basolateral, clear zone, and ruffled border membranes, and deposited in the resorption pits during bone resorption. The lack of OPN secretion into the resorption bay of avian osteoclasts may be a component of their functional resorption deficiency in vitro. Osteoclasts deficient in OPN were hypomotile and exhibited decreased capacity for bone resorption in vitro. OPN stimulated CD44 expression on the osteoclast surface, and CD44 was shown to be required for osteoclast motility and bone resorption. Exogenous addition of OPN to OPN-/- osteoclasts increased the surface expression of CD44, and it rescued osteoclast motility due to activation of the alpha(v)beta(3) integrin. Exogenous OPN only partially restored bone resorption because addition of OPN failed to produce OPN secretion into resorption bays as seen in wild-type osteoclasts. As expected with these in vitro findings of osteoclast dysfunction, a bone phenotype, heretofore unappreciated, was characterized in OPN-deficient mice. Delayed bone resorption in metaphyseal trabeculae and diminished eroded perimeters despite an increase in osteoclast number were observed in histomorphometric measurements of tibiae isolated from OPN-deficient mice. The histomorphometric findings correlated with an increase in bone rigidity and moment of inertia revealed by load-to-failure testing of femurs. These findings demonstrate the role of OPN in osteoclast function and the requirement for OPN as an osteoclast autocrine factor during bone remodeling.     

Increasing membrane-bound MCSF does not enhance OPGL-driven osteoclastogenesis from marrow cells

Macrophage colony-stimulating factor (MCSF) and osteoprotegerin ligand (OPGL), both produced by osteoblasts/stromal cells, are essential factors for osteoclastogenesis. Whether local MCSF levels regulate the amount of osteoclast formation is unclear. Two culture systems, ST-2 and Chinese hamster ovary-membrane-bound MCSF (CHO-mMCSF)-Tet-OFF cells, were used to study the role of mMCSF in osteoclast formation. Cells from bone marrow (BMM) or spleen were cultured with soluble OPGL on glutaraldehyde-fixed cell layers; osteoclasts formed after 7 days. Osteoclast number was proportional to the amount of soluble OPGL added. In contrast, varying mMCSF levels in the ST-2 or CHO-mMCSF-Tet-OFF cell layers, respectively by variable plating or by addition of doxycycline, did not affect BMM osteoclastogenesis: 20-450 U of mMCSF per well generated similar osteoclast numbers. In contrast, spleen cells were resistant to mMCSF: osteoclastogenesis required > or = 250 U per well and further increased as mMCSF rose higher. Our results demonstrate that osteoclast formation in the local bone environment is dominated by OPGL. Increasing mMCSF above basal levels does not further enhance osteoclast formation from BMMs, indicating that mMCSF does not play a dominant regulatory role in the bone marrow.     

Integrin subunit β3 plays a crucial role in the movement of osteoclasts from the periosteum to the bone surface

We have shown that, when mouse parietal bones were incubated in culture medium containing indomethacin, the number of tartrate-resistant acid phosphatase-positive osteoclasts (TRAP + OCs) on the bone surface was drastically reduced (down-regulation), and the number on the periosteal membrane adjacent to the resorbing surface was increased. Subsequent incubation of bones with prostaglandin E2 (PGE2) rapidly reversed these changes (up-regulation). In the work reported here, the osteoclast-associated integrin subunit β3 was stained by immunohistochemistry. The β3-positive osteoclast (β3 + OC) population on freshly isolated bone was comprised of about 67% TRAP + OCs and 33% TRAP − OCs. Like TRAP + OCs, β3 + OCs were reduced in number on the surface of bones incubated with indomethacin, but, in contrast to the TRAP + OCs, β3 + OCs were not seen on the periosteal membrane. Following up-regulation of TRAP + OCs with PGE2, large numbers of β3 + OCs appeared on the bone surface and, again, were not seen on the periosteal membrane. Echistatin, a peptide that binds to the αvβ3 integrin on osteoclasts, was found to inhibit the up-regulation of TRAP + OCs in a dose-dependent manner but had no effect on the down-regulation of TRAP + OCs. Similarly, echistatin inhibited the up-regulation of β3 + OCs on the bone surface, and, under these conditions, β3 + OCs were observed on the periosteal membrane. The addition of anti-β3 antibody also inhibited the up-regulation of TRAP + OCs in response to PGE2. The association of β3 protein expression with the up-regulated osteoclast and the inhibition of up-regulation by echistatin and by anti-β3 antibody provide strong evidence that β3 plays an essential role in the movement of osteoclasts from the membrane to the bone. J. Cell. Physiol. 175:1–9, 1998. © 1998 Wiley-Liss, Inc.     

Prostaglandin E2 receptors EP2 and EP4 are down-regulated during differentiation of mouse osteoclasts from their precursors

Prostaglandin E2 (PGE2) has been proposed to be a potent stimulator of bone resorption. However, PGE2 itself has been shown to directly inhibit bone-resorbing activity of osteoclasts. We examined the role of PGE2 in the function of mouse osteoclasts formed in vitro. Bone marrow macrophage osteoclast precursors expressed PGE2 receptors EP1, EP2, EP3beta, and EP4, and the expression of EP2 and EP4 was down-regulated during osteoclastic differentiation induced by receptor activator of NF-kappaB ligand and macrophage colony-stimulating factor. In contrast, functional EP1 was continuously expressed in mature osteoclasts. PGE2 as well as calcitonin caused intracellular Ca2+ influx in osteoclasts. However, PGE2 and 17-phenyltrinol-PGE2 (an EP1 agonist) failed to inhibit actin-ring formation and pit formation by osteoclasts cultured on dentine slices. When EP4 was expressed in osteoclasts using an adenovirus carrying EP4 cDNA, both actin-ring and pit-forming activities of osteoclasts were inhibited in an infectious unit-dependent manner. Treatment of EP4-expressing osteoclasts with PGE2 further inhibited their actin-ring and pit-forming activities. Such inhibitory effects of EP4-mediated signals on osteoclast function are similar to those that are calcitonin receptor-mediated. Thus, osteoclast precursors down-regulate their own EP2 and EP4 levels during their differentiation into osteoclasts to escape inhibitory effects of PGE2 on bone resorption.     

Low calcium environment effects osteoprotegerin ligand/osteoclast differentiation factor

In coculture with osteoblastic cell line MC3T3-E1 (E1) and mouse bone marrow cells, we reported that numbers of osteoclasts rose significantly on exposure to a low-calcium environment. Here we examined how osteoblasts influence osteoclastogenesis under a low-calcium environment. Comparing low extracellular calcium with a regular calcium environment, osteoprotegerin ligand (OPGL)/osteoclast differentiation factor (ODF) mRNA expression show more increase in the culture of low-calcium environment than in that of a regular calcium environment. Calcium-sensing receptor (CaSR), which was supposed as one of the mechanisms of recognizing extracellular calcium, existedon the surface of E1 cells. When E1 cells stimulated with agonists of CaSR, gadolinium, and neomycin, OPGL/ODF mRNA expression decreased. Moreover, these agonists reduced osteoclast formation in coculture. Taken together, it is possible that osteoblasts may recognize extracellular calcium via CaSR and regulate osteoclastogenesis.     

Baculovirus-expressed vitamin D-binding protein-macrophage activating factor (DBP-maf) activates osteoclasts and binding of 25-hydroxyvitamin D(3) does not influence this activity

Vitamin D-binding protein (DBP) is a multi-functional serum protein that is converted to vitamin D-binding protein-macrophage activating factor (DBP-maf) by post-translational modification. DBP-maf is a new cytokine that mediates bone resorption by activating osteoclasts, which are responsible for resorption of bone. Defective osteoclast activation leads to disorders like osteopetrosis, characterized by excessive accumulation of bone mass. Previous studies demonstrated that two nonallelic mutations in the rat with osteopetrosis have independent defects in the cascade involved in the conversion of DBP to DBP-maf. The skeletal defects associated with osteopetrosis are corrected in these mutants with in vivo DBP-maf treatment. This study evaluates the effects of various forms of DBP-maf (native, recombinant, and 25-hydroxyvitamin D(3) bound) on osteoclast function in vitro in order to determine some of the structural requirements of this protein that relate to bone resorbing activities. Osteoclast activity was determined by evaluating pit formation using osteoclasts, isolated from the long bones of newborn rats, incubated on calcium phosphate coated, thin film, Ostologic MultiTest Slides. Incubation of osteoclasts with ex vivo generated native DBP-maf resulted in a dose dependent, statistically significant, activation of the osteoclasts. The activation was similar whether or not the vitamin D binding site of the DBP-maf was occupied. The level of activity in response to DBP-maf was greater than that elicited by optimal doses of other known stimulators (PTH and 1,25(OH(2)D(3)) of osteoclast function. Furthermore, another potent macrophage activating factor, interferon--gamma, had no effect on osteoclast activity. The activated form of a full length recombinant DBP, expressed in E. coli showed no activity in the in vitro assay. Contrary to this finding, baculovirus-expressed recombinant DBP-maf demonstrated significant osteoclast activating activity. The normal conversion of DBP to DBP-maf requires the selective removal of galactose and sialic acid from the third domain of the protein. Hence, the differential effects of the two recombinant forms of DBP-maf is most likely related to glycosylation; E. coli expressed recombinant DBP is non-glycosylated, whereas the baculovirus expressed form is glycosylated. These data support the essential role of glycosylation for the osteoclast activating property of DBP-maf.     

In vivo administration of 1,25-dihydroxyvitamin D3 suppresses the expression of RANKL mRNA in bone of thyroparathyroidectomized rats constantly infused with PTH

It is known that pharmacological or toxic doses of vitamin D induce bone resorption both in vivo and in vitro, whereas physiological doses of the vitamin have a protective effect on bone in vivo. To investigate the discrepancies of the dose-dependent effect of vitamin D on bone resorption, we examined the in vivo effect of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] on the expression of the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) and osteoprotegerin (OPG) mRNAs in bone of thyroparathyroidectomized (TPTX) rats infused with or without parathyroid hormone (PTH). Continuous infusion of 50 ng/h of PTH greatly increased the expression of RANKL mRNA in bone of TPTX rats. Expression of OPG mRNA was not altered by PTH infusion. When graded doses of 1,25(OH)(2)D(3) was daily administered orally for 14 days to normocalcemic TPTX rats constantly infused with PTH, 0.01 and 0.1 microg/kg of 1,25(OH)(2)D(3) inhibited the PTH-induced RANKL mRNA expression, but 0.5 microg/kg of the vitamin did not inhibit it. Regulator of G protein signaling-2 (RGS-2) gene expression was suppressed by 1,25(OH)(2)D(3) dose-dependently, but PTH/PTHrP receptor mRNA expression was not altered. Bone morphometric analyses revealed that 1,25(OH)(2)D(3) suppressed PTH-induced osteoclast number in vivo. These results suggest that pharmacological or toxic doses of 1,25(OH)(2)D(3) stimulate bone resorption by inducing RANKL, but a certain range of physiological doses of the vitamin inhibit PTH-induced bone resorption, the latter mechanism appeared to be mediated, at least in part, by the suppression of the PTH/PTHrP receptor-mediated signaling.     

Integrins and signaling in osteoclast function

Integrins are heterodimeric adhesion receptors that mediate cell–matrix and cell–cell interactions. Osteoclasts highly express the αvβ3 integrin, which binds to a variety of extracellular matrix proteins including vitronectin, osteopontin and bone sialoprotein. RGD-containing peptides, RGD-mimetics and αvβ3 blocking antibodies inhibit bone resorption in vitro and in vivo, suggesting that this integrin plays an important role in osteoclast function. RGD-containing peptides were shown to raise cytosolic calcium in osteoclasts. Furthermore, several signaling and adaptor molecules were found to be involved in αvβ3 integrin-dependent signaling pathways, including phosphatidylinositol 3-kinase, c-Src, PYK2 and p130^cas. In addition, cytoskeletal molecules such as paxillin, vinculin, gelsolin and F-actin are recruited to adhesion contacts upon integrin activation. Many of these molecules signaling and cytoskeletal localize to the sealing zone of actively resorbing osteoclasts, suggesting that they play a role in linking the adhesion of osteoclasts to the bone matrix with the cytoskeletal organization and the polarization and activation of these cells for bone resorption.