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PubMed1997

Characterization and cellular distribution of the osteoclast ruffled membrane vacuolar H+-ATPase B-subunit using isoform-specific antibodies.

Acidification of the bone surface, leading to bone resorption, is accomplished by a vacuolar-type H+-ATPase present in a specialized domain of the plasma membrane of the osteoclast known as the ruffled membrane. Structure and function appears to be highly conserved within this class of multisubunit enzymes. However, cloning and sequencing of complementary DNA has shown that one of the subunits in the catalytic domain, the B-subunit, exists in at least two forms, B1 and B2. B1 messenger RNA has been found almost exclusively in the kidney, whereas messenger RNA for B2 has been found in all tissues studied, including the kidney. It has been speculated that the B1 isoform might be involved in targeting to the plasma membrane. In the present study, we have characterized the B-subunit of the chicken osteoclast H+-ATPase using antibodies directed against peptides with isoform-specific or conserved sequences of the B-subunit. Western analysis was performed on chicken osteoclast membrane vesicles and on partially purified chicken osteoclast H+-ATPase and was compared with similar analysis of H+-ATPase isolated from bovine kidney and brain. The B1-specific antibody reacted with a polypeptide of approximately 56 kD on immunoblots of the renal H+-ATPase, whereas no reaction could be detected against the osteoclast H+-ATPase or the osteoclast membrane vesicle preparation. In contrast, the antibody against a B2-specific sequence reacted with a peptide of approximately 56 kD on immunoblots of the osteoclast H+-ATPase, the renal H+-ATPase, and the clathrin-coated vesicle H+-ATPase. The antibody against a conserved region of the B-subunit did not generate any evidence for the presence of isoforms other than B2 in the osteoclast. Immunocytochemistry of rat osteoclasts on bovine bone slices using the B2 antibody showed intense polarized staining along the plasma membrane facing the bone surface in actively resorbing osteoclasts whereas nonresorbing osteoclasts were diffusely stained throughout the cytoplasm. By confocal microscopy, the B2 staining was located to the level of the ruffled membrane and appeared to be concentrated to the peripheral areas of the membrane adjacent to the sealing zone. We conclude that the osteoclast vacuolar H+-ATPase contains the B2 isoform and suggest that upon initiation of resorption the pump is translocated from the cell interior to a special domain of the ruffled membrane close to the sealing zone.

Amino Acid Sequence↗
PubMed2000

Fibroblastic stromal cells express receptor activator of NF-kappa B ligand and support osteoclast differentiation.

Osteoclast formation in bone is supported by osteoblasts expressing receptor activator of NF-kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) expression. Numerous osteotropic factors regulate expression levels of RANKL and the RANKL decoy receptor osteoprotegerin (OPG) in osteoblasts, thereby affecting osteoclast differentiation. However, not only in RANKL widely expressed in soft tissues, but osteoclasts have been noted in extraskeletal lesions. We found that cultured skin fibroblastic cells express RANKL, M-CSF, and OPG messenger (mRNA). Stimulation by 1 alpha,25 dihydroxyvitamin D3 [1,25(OH)2D3] plus dexamethasone (Dex) augmented RANKL and diminished OPG mRNA expression in fibroblastic cells and caused the formation of numerous osteoclasts in cocultures of skin fibroblastic cells with hemopoietic cells or monocytes. The osteoclasts thus formed expressed tartrate-resistant acid phosphatase (TRAP) and calcitonin (CT) receptors and formed resorption pits in cortical bone. Osteoclast formation also was stimulated (in the presence of Dex) by prostaglandin E2 (PGE2), interleukin-11 (IL-11), IL-1, tumor necrosis factor-alpha (TNF-alpha), and parathyroid hormone-related protein (PTHrP), factors which also stimulate osteoclast formation supported by osteoblasts. In addition, granulocyte-macrophage-CSF (GM-CSF), transforming growth factor-beta (TGF-beta), and OPG inhibited osteoclast formation in skin fibroblastic cell-hemopoietic cell cocultures; CT reduced only osteoclast nuclearity. Fibroblastic stromal cells from other tissues (lung, respiratory diaphragm, spleen, and tumor) also supported osteoclast formation. Thus, RANKL-positive fibroblastic cells in extraskeletal tissues can support osteoclastogenesis if osteolytic factors and osteoclast precursors are present. Such mesenchymally derived cells may play a role in pathological osteolysis and may be involved in osteoclast formation in extraskeletal tissues.

Animals↗
PubMed2003

Role of osteoclast extracellular signal-regulated kinase (ERK) in cell survival and maintenance of cell polarity.

UNLABELLED: Morphological changes of osteoclasts by a MEK1 inhibitor, PD98059, were investigated to clarify a role of ERK. PD98059 promoted apoptosis of osteoclasts and the loss of ruffled borders. This study supports the importance of ERK in survival and polarity of osteoclasts. INTRODUCTION: Extracellular signal-regulated kinase (ERK) is a mitogen activated protein kinase (MAPK) that has been reported to play a role in the survival and apoptosis of osteoclasts. However, the precise signal transduction mechanism is not fully understood. The aim of this study was to clarify the role of ERK in osteoclasts by histological analysis. MATERIALS AND METHODS: Using a rat calvarial organ culture system, the inhibition of ERK phosphorylation by PD98059, a MAPK/ERK kinase 1 (MEK1) inhibitor, was assayed by immunoblotting. Morphological changes in osteoclasts induced by PD98059 were elucidated by light and electron microscopy. The cellular localization of ERK was also determined by immunoelectron microscopy. RESULTS: PD98059 inhibited phosphorylated ERK after a 1-h incubation. Ultrastructural study demonstrated that PD98059 induced the accumulation of vesicles and vacuoles in osteoclasts and the loss of ruffled border at 1 h. At 3 h, some osteoclasts showed apoptosis with nuclear condensation, and at 6 h after PD98059 treatment, many osteoclasts were detached from the bone surface and had lost their cell polarity. Electron microscopic immunohistochemistry revealed that ERK was mainly localized in the cytoplasm of clear zones in control osteoclasts, but apoptotic osteoclasts also showed immunoreactivity in clear zone-like structures in contact with osteoblast-lineage cells. CONCLUSION: These findings indicate that ERK in osteoclasts is involved in their survival and may be involved in the formation of a ruffled border and the maintenance of cell polarity.

Animals↗
PubMed2004

Recent advances in osteoclast biology and pathological bone resorption.

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.

Animals↗
PubMed2003

Distinct osteoclast precursors in the bone marrow and extramedullary organs characterized by responsiveness to Toll-like receptor ligands and TNF-alpha.

Osteoclasts are derived from hemopoietic stem cells and play critical roles in bone resorption and remodeling. Multinucleated osteoclasts are attached tightly to bone matrix, whereas precursor cells with the potential to differentiate into osteoclasts in culture are widely distributed. In this study, we assessed the characteristics of osteoclast precursors in bone marrow (BM) and in extramedullary organs as indicated by their responsiveness to ligands for Toll-like receptors (TLRs) and to TNF-alpha. Development of osteoclasts from precursor cells in the BM was inhibited by CpG oligonucleotides, a ligand for TLR9, but not by LPS, a ligand for TLR4. BM osteoclasts were induced by TNF-alpha as well as receptor activator of NF-kappaB ligand in the presence of M-CSF. Splenic osteoclast precursors, even in osteoclast-deficient osteopetrotic mice, differentiated into mature osteoclasts following exposure to TNF-alpha or receptor activator of NF-kappaB ligand. However, splenic osteoclastogenesis was inhibited by both LPS and CpG. Osteoclastogenesis from peritoneal precursors was inhibited by not only these TLR ligands but also TNF-alpha. The effects of peptidoglycan, a ligand for TLR2, were similar to those of LPS. BM cells precultured with M-CSF were characterized with intermediate characteristics between those of splenic and peritoneal cavity precursors. Taken together, these findings demonstrate that osteoclast precursors are not identical in the tissues examined. To address the question of why mature osteoclasts occur only in association with bone, we may characterize not only the microenvironment for osteoclastogenesis, but also the osteoclast precursor itself in intramedullary and extramedullary tissues.

Animals↗
PubMedDate not supplied

New knowledge on the origin, function and fate of osteoclasts.

The most recent findings on the origin, life-span and fate of the osteoclast can be summarized as follows. Osteoclasts originate from progenitor, mononuclear, lymphoid cells which reach the bone surface through the bloodstream. Osteoclasts resorb bone by secreting lysosomal enzymes and procollagenase in the osteoclast-bone interspace. The organic components of the matrix (first interfibrillary substance, then collagen fibrils) are digested in the extracellular space. Dislodged crystals and residual organic constituents are then phagocytosed and collected in cytoplasmic vacuoles where they are completely solubilized. The ruffled border and the adjacent "clear" zone constitute the resorbing apparatus, whose development is roughly proportional to osteoclast activity. Osteoclast nuclei are continuously incorporated and shed, so that individual cells are continuously renewed. This makes the life-span of the osteoclast extremely difficult to determine. The life of each individual osteoclast might theoretically continue as long as the stimulus to resorption persists and sufficient bone matrix is available. Primary abnormalities of the osteoclasts can induce pathologic skeletal changes, as in the case of osteopetrosis and Paget's disease of bone. Conversely, skeletal abnormalities may damage osteoclasts, as in the case of lead intoxication. When this happens, osteoclasts are essentially characterized by an underdeveloped ruffled border, pyknotic nuclei, detachment from the bone matrix and, finally, shrinkage and fragmentation. It is not yet known whether these changes only occur in pathologic conditions, or whether they are the alterations which lead senescent osteoclasts to death even in normal bone.

Animals↗
PubMed1996

Osteoclasts in normal and adjuvant arthritis bone tissues express the mRNA for both type I and II interleukin-1 receptors.

Osteoclasts are known to play a crucial role in both physiologic and pathologic bone resorption. Moreover, it is generally agreed that IL-1 has powerful effects on osteoclastic bone resorption, although the precise cellular sites and mechanisms by which IL-1 mediates osteoclastic bone resorption remain unclear. In particular, it is still controversial whether osteoclasts can respond to IL-1 directly. The expression of mRNA for type I IL-1 receptor (IL-1RI) and type II IL-1 receptor (IL-1RII) in osteoclasts was investigated in normal and inflammatory bone tissues by in situ hybridization to determine whether osteoclasts are the target cells for IL-1 and to elucidate the mechanism by which IL-1 induces osteoclastic bone resorption. For this study, normal tibiae were obtained from newborn, young, and adult mice and rats, and inflammatory bone tissues with bone destruction were obtained from adjuvant arthritis rat models. The results showed that (a) both IL-1 receptors (IL-1RI and -II) mRNA were expressed by osteoclasts in all tissue sections of normal tibiae; (b) at the stage of the adjuvant arthritis studied, the IL-1RI mRNA was the most predominant message in osteoclasts present in the area with serious cartilage and bone destruction, whereas the expression level of IL-1RII mRNA in these osteoclasts was weak; and (c) both IL-1RI and -II mRNA were expressed by osteoblasts, as well as by osteocytes localized in the osteoid. In addition, these messages were also expressed by chondrocytes, but the signals were not detected in the chondrocytes in the zones of hypertrophy and provisional calcification. Our present study demonstrates for the first time that mouse and rat osteoclasts express IL-1RI and -II mRNA, which suggest that a primary effect of IL-1 on osteoclasts may be one of the mechanisms by which IL-1 mediates normal bone remodeling and pathologic bone resorption in chronic inflammatory diseases.

Animals↗
PubMed1997

Osteoclasts constitutively express regulators of bone resorption: an immunohistochemical and in situ hybridization study.

Bone resorption is controlled by the local production of soluble regulatory molecules within the marrow microenvironment that mediate osteoclast recruitment, differentiation, and activation. Under normal conditions osteoclasts are rarely seen; in many pathologic states, however, the number of osteoclasts is dramatically increased, resulting in a net-loss of bone mass. The role of the osteoclasts as autocrine regulators of bone resorption in either normal or pathologic conditions has not been extensively investigated. The expression of IL-1 beta, IL-6, and TNF-alpha was examined in osteoclasts by immunohistochemistry under conditions of normal, reactive, and pathologic bone resorption, including growth plate (3 cases), fracture callus (5 cases), osteomyelitis (3 cases), Paget's disease (6 cases), giant-cell tumor of bone (14 cases), and brown tumor of hyperparathyroidism (2 cases). In each case, osteoclasts demonstrated immunoreactivity for IL-1 beta, IL-6, and TNF-alpha. In areas of active bone resorption, the intensity and uniformity of staining among the various conditions were similar, suggesting constitutive expression of these cytokines by activated osteoclasts. Giant-cell tumors of bone showed cytokine reactivity in over half of the giant cells, whereas stromal cells showed scattered staining. In acute osteomyelitis, inflammatory cells (mainly macrophages) and osteoclasts were intensely positive for all three cytokines. The immunohistochemical findings were confirmed by in situ hybridization using probes specific for IL-6 and TNF-alpha, the pattern of mRNA expression paralleled that of immunoreactivity for these cytokines. These findings support the notion of autocrine/paracrine regulation of bone remodeling by osteoclasts. Because overproduction of these cytokines may enhance bone resorption through the stimulation of osteoclast progenitor cells as well as mature osteoclasts, pathologic bone lesions with a large increase in the number of osteoclasts may be self-perpetuating. Alteration in the synthesis, secretion, or activity of these important regulatory molecules may in turn alter bone remodeling and loss.

Bone Diseases↗
PubMed1997

Fimbrin in podosomes of monocyte-derived osteoclasts.

Fimbrin, an actin-bundling protein, is a component of the osteoclast adhesion complexes called podosomes. In this study, we (1) determined the localization of fimbrin in the mature rabbit osteoclast as well as in differentiating osteoclasts using the avian monocyte-derived osteoclast differentiation model, (2) characterized the distribution and accumulation of three fimbrin isotypes (T, L, and I) in avian monocytes as they fused to form multinucleate osteoclast-like cells, and (3) report for the first time, a close spatial relationship between podosomes and microtubules using fimbrin as a marker of the podosome. Immunofluorescence using anti-T-fimbrin, anti-L-fimbrin, and pan-isotype-anti-fimbrin antibodies, showed that fimbrin is an integral component of the podosome core in the mature rabbit osteoclast and in the monocyte-derived osteoclast throughout differentiation. Anti-I-fimbrin, however, did not show immunoreactivity in these cultures. These studies also show that in the avian model of monocyte-derived osteoclast differentiation, day 2 cells (D2) are predominantly mononucleate and have few podosomes. By days 4 and 6 in culture (D4 and D6), many cells have fused and punctate rows of podosomes are commonly observed at cell margins. Analysis by Western blot of protein accumulation showed that after an initial small rise from D2 to D4, L-fimbrin levels remained relatively constant from D4 to D6. However, T-fimbrin protein levels increase steadily from D2 to D6, suggesting that it may be related to the increase in podosome formation as monocytes fuse to form osteoclasts. Finally, we examined the distribution of podosomes relative to other cytoskeletal elements such as microtubules and intermediate filaments. Double immunofluorescence labeling using anti-fimbrin and anti-tubulin showed podosomes lying adjacent to microtubules at cell margins. When osteoclasts were treated with nocodazole (1 X 10(-6) M) to disrupt microtubules, the distribution of podosomes became more random and was no longer confined to the cell periphery. These results suggest that microtubule-podosome interactions may play a role in osteoclast adhesion.

Animals↗
PubMed1996

Different calcium sensitivity in osteoclasts on glass and on bone and maintenance of cytoskeletal structures on bone in the presence of high extracellular calcium.

The sensitivity of rat osteoclasts to increased extracellular calcium concentrations ([Ca2+]e) was investigated by single cell measurements of free cytosolic calcium concentrations ([Ca2+]i), by changes in microfilament organization of resorbing osteoclasts, and by in vitro bone resorption assays. Osteoclasts cultured on glass and on bone showed clear differences in their responses, as in 44% and 52% of osteoclasts on glass but in only 21% and 25% of osteoclasts on bone [Ca2+]i increased when [Ca2+]e was increased from 2 mM to 6 or 10 mM via perfusion, respectively. Bone resorption was inhibited without changes in the osteoclast numbers only by 10 mM [Ca2+]e in 2 day cultures. Furthermore, there were no changes in the organization of microfilament structures in resorbing osteoclasts after increased [Ca2+]e (up to 20 mM [Ca2+]e, 30 min incubation). These results suggest that the sensitivity of osteoclasts to increased [Ca2+]e is dependent on their activation phase (resting/migrating vs. resorbing) and that resorbing osteoclasts are not sensitive to increased [Ca2+]e or that the sensing system cannot be reached in polarized resorbing osteoclasts. In contrast, increasing [Ca2+]i through the use of calcium ionophores dispersed specific microfilament structures at the sealing zone transiently in a few minutes. This shows that [Ca2+]i is used as a signaling mechanism to inactivate osteoclasts, with a similar end result on microfilament structures at the sealing zone as caused by increased concentration of cAMP and activation of protein kinase C.

Actin Cytoskeleton↗
PubMed1997

Role of c-Src in cellular events associated with colony-stimulating factor-1-induced spreading in osteoclasts.

We and others have observed that in response to treatment with Colony Stimulating Factor-1 (CSF-1) neonatal rat osteoclasts demonstrate rapid cytoplasmic spreading. The receptor for CSF-1, c-Fms, is expressed in osteoclasts, possesses intrinsic tyrosine-kinase activity, and signals via rapid phosphorylation of selected proteins. It has been reported previously that c-Src becomes tyrosine phosphorylated following CSF-1 treatment of fibroblasts overexpressing c-Fms. We therefore examined the cellular events associated with CSF-1-induced spreading in osteoclasts and what role, if any, c-Src played in these processes. Confocal microscopic studies using phosphotyrosine (P-tyr) monoclonal antibodies demonstrated that CSF-1 induced a significant dose- and time-dependent increase in P-tyr labeling of neonatal rat osteoclasts. Phalloidin staining was consistent with partial to complete disassembly of the actin attachment ring with redistribution of actin to the spreading cytoplasmic edge of the cell. Quantitation of cellular F-actin using NBD-phallicidin confirmed a decrease in polymerized actin following exposure to CSF-1. In contrast, CSF-1 failed to induce any cytoplasmic spreading in osteoclasts isolated from mice with targeted disruption of the src gene. Further, in src- osteoclasts no well defined attachment ring could be identified. To investigate cell-signaling events associated with osteoclast spreading, detergent lysates were made from purified multinucleated osteoclast-like cells (OCLs) obtained by coculturing murine bone marrow and osteoblasts with calcitriol. Western blot analyses of lysates from control and CSF-1-treated normal cells indicated that several proteins were specifically phosphorylated in response to CSF-1, most notably proteins of 165, 60, and 85-90 kDa. Immunoprecipitation studies revealed that the 165 and 60 kDa proteins were, respectively, c-Fms and c-Src. The c-Src kinase activity was increased 2.9-fold following CSF-1 treatment. The 85-90 kDa protein is as yet unidentified. Since activated receptor tyrosine kinases may induce spreading in part by reducing phosphoinositol 4,5-bisphosphate (PIP2) binding to actin-associated proteins, a monoclonal antibody to PIP2 was used to assess the nature of PIP2 binding proteins in OCLs. Proteins of 85-90 kDa, 43 kDa, and 30 kDa were consistently demonstrated to bind PIP2. Further, the PIP2 content of the 85-90 kDa protein appeared to decrease with CSF-1 treatment. Whether this protein represents the phosphoprotein of the same M.W. is unclear. We also examined the effect of CSF-1 on the PIP2 content of alpha-actinin. Alpha-actinin showed low-level PIP2 binding, which was demonstrable only after immuno-precipitation and did not change with CSF-1 treatment. However, CSF-1 did cause a significant decline in the phosphotyrosine content of alpha-actinin. In contrast, in src- OCLs, CSF-1 induced more prolonged phosphorylation of c-Fms, and the 85-90 kDa protein was markedly hypophosphorylated. Further, alpha-actinin did not dephosphorylate in src- cells. We conclude that CSF-1-induced osteoclast spreading is accompanied by rapid reorganization of the actin cytoskeleton and phosphorylation of several cellular substrates, including c-Fms and c-Src. PIP2 binding to at least one protein appears to decrease with CSF-1 treatment, which may favor actin depolymerization. The reduced tyrosine phosphorylation of alpha-actinin could effect its ability to bind to actin. Thus c-Src may play an important role in these cellular events since in its absence, osteoclasts do not spread and signaling events downstream are altered. Whether these changes relate in part to the basal abnormalities in the cytoskeletal organization of src- osteoclasts remains to be determined.

Actinin↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed1988

The regulation of osteoclastic development and function.

Cells of the osteoblastic lineage exert a dominant influence on osteoclastic bone resorption. They form a communicating network of osteocytes, surface osteocytes and osteoblasts that seems well placed to monitor the structure and performance of bone and to judge where bone formation or resorption is appropriate. Osteoblasts produce prostaglandins (PGs) which strongly inhibit osteoclastic resorption. None of the agents that stimulate resorption in intact bone, such as parathyroid hormone (PTH), interleukin 1 (IL-1), 1,25-(OH)2 vitamin D3 (1,25-(OH)2D3) or tumour necrosis factors, affects isolated osteoclasts, but all induce osteoblastic cells to produce osteoclastic resorption stimulatory activity (ORSA) that acts directly on osteoclasts. Osteoblasts seem to initiate resorption as well as stimulating or inhibiting it. Contact with bone mineral appears to be necessary: osteoclasts resorb mineralized but not unmineralized bone. All bone surfaces are lined by unmineralized organic material. Osteoblastic cells secrete neutral proteases, including collagenase, in response to hormonal stimulators of bone resorption. Incubation of osteoblasts, in the presence of PTH, on such surfaces or preincubation of the bone with collagenase predisposes bone to osteoclastic resorption. Agents that stimulate resorption in organ cultures seem to share these osteoblast-mediated mechanisms for induction and stimulation of resorption but 1,25-(OH)2D3 stimulates it through an additional mechanism. We have found that osteoclasts can be induced from haemopoietic tissue (including haemopoietic spleen cells) in the presence of 1,25-(OH)2D3--PTH and IL-1 have no effect in this system. Because osteoclasts lack receptors for 1,25-(OH)2D3 these results suggest either that osteoclast precursors lose 1,25-(OH)2D3 receptors during differentiation, or that a 1,25-(OH)2D3-responsive accessory cell in bone marrow induces osteoclastic differentiation in the presence of 1,25-(OH)2D3.

Animals↗
PubMed2005

Tumor necrosis factor prevents alendronate-induced osteoclast apoptosis in vivo by stimulating Bcl-xL expression through Ets-2.

OBJECTIVE: To investigate why bisphosphonates are less effective at preventing focal bone loss in rheumatoid arthritis (RA) patients than in those with generalized osteoporosis, and the mechanisms involved. METHODS: The response of osteoclasts to alendronate (ALN) in tumor necrosis factor-transgenic (TNF-Tg) mice that develop erosive arthritis and in wild-type littermates was studied. TNF-Tg and wild-type mice were given ALN, and the osteoclast numbers in the inflamed joints and in the long bones were compared. The expression levels of Bcl-xL in the osteoclasts of TNF-Tg and wild-type mice were examined by immunostaining. The effect of overexpression of Bcl-xL and Ets-2 proteins on ALN-induced osteoclast apoptosis was determined using an in vitro osteoclast survival assay and retrovirus transfer approach. RESULTS: ALN reduced osteoclast numbers in the metaphyses by 97%, but by only 46% in the adjacent inflamed joints. Bcl-xL expression was markedly higher in osteoclasts in the joints than in those in the metaphyses of TNF-Tg mice. Bcl-xL or Ets-2 overexpression protected osteoclasts from ALN-induced apoptosis, and TNF stimulated Bcl-xL and Ets-2 expression in osteoclasts. Overexpression of Ets-2 increased Bcl-xL messenger RNA in osteoclasts, while a dominant-negative form of the Ets-2 blocked the protective effect of Bcl-xL or TNF on ALN-induced apoptosis. CONCLUSION: The reduced efficacy of bisphosphonates to stop bone erosion in the inflamed joints of RA patients may result from local high levels of TNF up-regulating Ets-2 expression in osteoclasts, which in turn stimulates Bcl-xL expression in them and reduces their susceptibility to bisphosphonate-induced apoptosis.

Alendronate↗
PubMed1991

Kinetics of the osteoclast cytoskeleton during the resorption cycle in vitro.

Resorption and migration phases alternate in the life of the osteoclast. We have previously described a specific microfilament structure at the attachment sites in resorbing osteoclasts. In the present study we have examined microfilaments and microtubules in both resorbing and migrating rat osteoclasts cultured on bone slices. In migrating osteoclasts microfilaments form so-called podosome structures containing vinculin, talin, and F-actin at the paramarginal area of the cell. When the osteoclast prepares itself for resorption, the podosomes gather to a certain area and form a broad ring around the area, which is then resorbed. In the resorbing osteoclast, vinculin and talin form a continuous double circle, which may be partially formed by podosomes, and between these double circles a broad zone is formed by F-actin. Narrow vinculin and F-actin rings were found in osteoclasts at the end of the resorption phase. The different configurations of microfilaments in 1 and 2 day cultures were correlated in terms of their relationship to the resorption lacunae. The vitamin A derivative isotretinoin significantly stimulated resorption and increased the number of microfilament configurations associated with the resorption pits. On the other hand, Bt2cAMP abolished resorption and prevented the formation of a specific ring structure of microfilaments. Based on these data, a kinetic model of the whole migration-resorption cycle of the osteoclast cultured on the bone slice is presented. With alpha-tubulin stainings of microtubules two different cytoskeletal organizations were observed. In migrating osteoclasts, microtubules were evenly distributed over the whole cell. In the resorbing osteoclast, there was a noticeable concentration of these cytoskeletal structures at cytoplasmic sites closest to the resorption lacuna. This orientation of microtubules may reflect the active secretory function of the resorbing osteoclast.

Actin Cytoskeleton↗
PubMed1992

An assay system utilizing devitalized bone for assessment of differentiation of osteoclast progenitors.

The present study provides a novel assay system to examine the differentiation of osteoclast progenitors on devitalized bone slices. We used the population of bone cells liberated enzymatically from 14-day-old mouse embryonal calvariae as a source of osteoclast progenitors. The analysis of differentiation of osteoclast progenitors into preosteoclasts and mature osteoclasts was assessed in terms of the formation of TRAP-positive cells and pits or resorption lacunae, respectively, on devitalized bone slices. Osteoclasts having bone-resorbing activity appeared when the calvarial cell population was cultured in the presence of 1 alpha,25-(OH)2D3 on devitalized bone slices. The resorbing activity increased in a 1 alpha,25-(OH)2D3 dose-related manner. However, calcitonin, a potent inhibitor of differentiation and activation of osteoclast lineage cells, reduced the area of the resorption lacunae in a dose-dependent fashion. The bone-resorbing cells on the bone slices expressed an obvious ruffled border and clear zone, structures specific to mature osteoclasts. These results suggest that osteoclast progenitors in the mouse calvarial population examined differentiated into mature osteoclasts in the presence of 1 alpha,25-(OH)2D3 on devitalized bone slices. Further, using this assay system we assessed the effect of some other osteotropic factors on the differentiation of osteoclast progenitors to mature osteoclasts. IL-1, IL-6, and PTH increased the formation of TRAP-positive cells and pits and the area of resorption lacunae in a dose-dependent fashion. However, prostaglandin E2 was unable to induce the formation of resorption lacunae, although a significant appearance of TRAP-positive cells was observed at a concentration of 200 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗
PubMed1992

Rat osteoclasts adhere to a wide range of RGD (Arg-Gly-Asp) peptide-containing proteins, including the bone sialoproteins and fibronectin, via a beta 3 integrin.

The ligand binding ability of rat osteoclast adhesion receptors was investigated in an attachment assay using osteoclasts disaggregated from bone. Osteoclasts adhered well to the Arg-Gly-Asp (RGD)-containing proteins osteopontin (bone sialoprotein I) and BSP (bone sialoprotein II), vitronectin, fibrinogen, von Willebrand factor, and fibronectin. Osteoclasts also adhered, but less strongly, to type I collagen. No attachment of osteoclasts was observed to thrombospondin, tenascin, laminin, or a range of non-RGD-containing bone proteins and proteins from other sources. The attachment of osteoclasts to all ligands was abolished in the presence of GRGDSP peptide, indicating the involvement of the RGD cell binding sequence in ligand binding. Attachment of osteoclasts to all substrates, with the exception of type I collagen, was also strongly inhibited by the addition of monoclonal antibody F11 to the beta 3 integrin subunit, indicating that a beta 3 integrin, probably the vitronectin receptor, was involved. Attachment to type I collagen was blocked by EDTA chelation of divalent cations and was not significantly affected by anti-beta 3 or anti-beta 1 antibodies; when taken with the inhibition by RGD peptide, this suggests the involvement of various receptors, possibly including nonintegrin collagen receptors, in the binding of osteoclasts to this protein. These results define the wide range of ligands for extracellular matrix receptors in osteoclasts in vitro. It remains to be established which of these proteins are important in osteoclast adhesion and osteoclastic bone resorption in vivo.

Amino Acid Sequence↗
PubMed1993

Role of bone matrix in osteoclast recruitment in cultured fetal rat calvariae.

In cultured 19 day fetal rat calvaria, osteoclasts first appear after 48 h, more rapidly than with other cultured embryonic long bone rudiments. This may be because the calvarial osteoclast precursors are more differentiated or intramembraneous bone is a more powerful stimulus for osteoclast maturation than endochondral bone. To investigate this further, 19 day calvariae were stripped of their endocranial membranes, devoiding them of osteoclast precursors, and cocultured with the membranes or with other sources of these cells, such as bone marrow, fetal liver, spleen, and blood. There was similar recruitment of mature osteoclasts onto the surface of the "stripped" calvariae from the endocranial membranes and from the hematopoietic tissues after 48 h culture. Intact 19 day fetal calvariae were cultured with human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) or with 1,25-dihydroxyvitamin D3, [1,25-(OH)2D3], each thought to influence different stages of osteoclast maturation. They stimulated osteoclast recruitment, although 1,25-(OH)2D3 was effective only in the first 24 h of culture. They also increased osteoclast recruitment from fetal liver onto stripped calvariae. When intact bones were cultured with hrGM-CSF and 1,25-(OH)2D3 together, osteoclast number decreased but their area increased. Calvariae therefore appear to contain osteoclast precursors at earlier (GM-CSF-sensitive) and later [1,25-(OH)2D3-sensitive] stages. As recruitment onto stripped calvariae was similar whichever source of precursors was used, it is likely that calvarial bone matrix is an important influence on rapid osteoclast maturation in these bones in vitro.

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