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PubMed1999

Expression of estrogen receptor-alpha in cells of the osteoclastic lineage.

Estrogen deficiency at the menopause is associated with an increased rate of bone loss and subsequent risk of skeletal fracture. Whilst cells of the osteoblastic lineage are known to express estrogen receptors, the presence of estrogen receptors in osteoclasts remains controversial. We have examined expression of the classic estrogen receptor, estrogen receptor-alpha (ERalpha), during osteoclast differentiation. In situ mRNA hybridisation with a digoxygenin-labelled riboprobe to ERalpha mRNA, together with immunocytochemical analysis using a human ERalpha-specific monoclonal antibody demonstrated similar findings and confirmed the expression of ERalpha in chondroblasts and osteoblasts from human fetal bone and mineralising human bone marrow cultures. ERalpha expression was detected in human bone marrow cultures treated with 1,25(OH)2D3 and macrophage colony-stimulating factor and in macrophage cultures treated with 1,25(OH)2D3. However, in an in vitro model of human osteoclast formation, no ERalpha expression was observed in the osteoclasts that developed. The human preosteoclast TCG 51 cell line showed strong expression of ERalpha in contrast to the low levels observed in the more mature bone resorptive TCG 23 cell line. No expression was detectable in osteoclasts cultured from giant cell tumour of bone (GCTB) tissue or in osteoclasts in Pagetic, GCTB, or hyperparathyroid bone tissues. In conclusion, preosteoclasts express detectable levels of ERalpha, but osteoclast maturation and bone resorption is associated with loss of ERalpha expression. This indicates that ERalpha expression and regulation may play a role in osteoclast formation.

Bone Diseases↗

PubMed1999

Limited and selective localization of the lysosomal membrane glycoproteins LGP85 and LGP96 in rat osteoclasts.

Monospecific antibodies against two major glycoproteins of rat lysosomal membranes with apparent molecular masses of 96 and 85 kDa, termed LGP96 and LGP85, respectively, were used as probes to determine the expression and distribution of lysosomal membranes in rat osteoclasts. At the light microscopic level, the preferential immunoreactivity for both proteins was found at high levels at the side facing bone of actively bone-resorbing osteoclasts. Osteoclasts detached from bone surface were devoid of immunoreactivity for each protein. At the electron microscopic level, both proteins were exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts with well-developed ruffled border membrane. No immunolabeling for both proteins was observed in the basolateral membrane and the clear zone of bone-resorbing osteoclasts. The plasma membrane of preosteoclasts and post- and/or resting osteoclasts showed little or no reactivity against these two antibodies. The results indicate that lysosomal membrane glycoproteins are actively synthesized in active osteoclasts, rapidly transported to the ruffled border area, and contribute to the formation and maintenance of the acidic resorption lacuna of osteoclasts.

Animals↗

PubMed1999

Visualization of acidic compartments in cultured osteoclasts by use of an acidotrophic amine as a marker for low pH.

Using the acidotrophic amine 3-(2,4-dinitroanillino)-3'-amino-N-methyldipropylamine (DAMP) as a marker for low pH and immunofluorescence cytochemistry, we examined acidic compartments of osteoclasts cultured on cover glasses or bone slices, where they could resorb the bone surface, forming resorptive lacunae. DAMP-positive structures were seen as vesicular and tubular forms in the cytoplasm, indicating lysosomes and endosomes. Not only the osteoclastic cytoplasm but also the extracellular area around the ruffled border and resorptive lacunae were stained with DAMP, suggesting acidic regions. Immunofluorescence was localized predominantly on the substratum side of actively resorbing osteoclasts, whereas an evenly distributed staining pattern was seen in the nonactive cell. The most intensive reaction was seen at the advancing front of resorptive lacunae within the actively resorbing osteoclasts. The distribution pattern of DAMP seemed to be correlated with the osteoclastic activity, since osteoclasts exhibit alternating resorption and migration phases during the bone-remodeling cycle. In this culture system, the resorptive lacunae were left behind after the osteoclasts had completed resorption and migrated along the bone surface. These exposed resorptive lacunae were also stained with DAMP, which were presumably kept at an acidic pH. The effect of treatment with monensin, chloroquine, ammonium chloride, or nigericin was varied in terms of the immunoreactivity for DAMP, but not complete abolition of the staining was obtained. Weak bases such as chloroquine or ammonium chloride inhibited both intra- and extracellular immunoreactivity. Immunoreactivity for the vacuolar type of proton ATPase (V-ATPase) was demonstrable in the cytoplasm of the osteoclasts but was weakened by the addition of bafilomycin. Immunofluorescence of the resorptive lacunae was still retained even after the treatment with bafilomycin and acetazolamide. Besides, both bafilomycin and acetazolamide reversibly inhibited cellular acidity as judged by DAMP immunocytochemistry, which agrees with the fact that ostoeclastic acidification results from the action of vacuolar proton-pump ATPase coupled with carbonic anhydrase.

Acetazolamide↗

PubMed2002

A selective inhibitor of matrix metalloproteinases inhibits the migration of isolated osteoclasts by increasing the life span of podosomes.

The osteoclast is a unique cell that cycles between bone resorption and migration. In this study, we used KB-R7785, an inhibitor of matrix metalloproteinases (MMPs), to investigate the role of MMPs in this cycle. Osteoclasts prepared from neonatal rabbits were processed to measure: (1) migration on hydroxyapatite (HA)-coated dishes, (2) the formation of an actin band, which is a large collection of podosomes, (3) the life span of podosomes, and (4) lacunar resorption on ivory slices. KB-R7785 significantly decreased the area of the tracks formed by osteoclasts on HA-coated dishes. The percentage of actin/podosome band formation, expressed as: (total length of the actin band/circumference of the osteoclast) x 100, was significantly increased after the addition of KB-R7785, which implies that podosome disassembly in osteoclasts was decreased by the inhibition of MMPs. Time-lapse cinemicrography demonstrated that the addition of KB-R7785 increased the life span of podosomes. Several indexes (number of resorption pits, total resorption area, and mean resorption area) of osteoclast resorption activity were significantly decreased by KB-R7785, while the average depth of resorption was increased. These results indicate that decreased podosome disassembly caused by an MMP inhibitor suppresses the migration of osteoclasts and, ultimately, inhibits osteoclastic bone resorption.

Actins↗

PubMed2006

Molecular regulation of osteoclast activity.

Osteoclasts are multinucleated cells derived from hematopoietic precursors that are primarily responsible for the degradation of mineralized bone during bone development, homeostasis and repair. In various skeletal disorders such as osteoporosis, hypercalcemia of malignancy, tumor metastases and Paget's disease, bone resorption by osteoclasts exceeds bone formation by osteoblasts leading to decreased bone mass, skeletal fragility and bone fracture. The overall rate of osteoclastic bone resorption is regulated either at the level of differentiation of osteoclasts from their monocytic/macrophage precursor pool or through the regulation of key functional proteins whose specific activities in the mature osteoclast control its attachment, migration and resorption. Thus, reducing osteoclast numbers and/or decreasing the bone resorbing activity of osteoclasts are two common therapeutic approaches for the treatment of hyper-resorptive skeletal diseases. In this review, several of the key functional players involved in the regulation of osteoclast activity will be discussed.

Animals↗

PubMed2006

Osteoclastic differentiation and function regulated by old and new pathways.

The osteoclast is a specialized multinucleated variant of the macrophage family. It degrades mineralized tissue, and is required for modeling and remodeling of bone. The osteoclast has long been known to require vitamin D for its differentiation and to be regulated by parathyroid hormone via circulating Ca(2+) levels. Two local signals important in osteoclast survival and differentiation, CSF-1 and RANKL, were characterized by the mid-1990 s. A basic framework of specialized cell attachment and resorption molecules was also clear by that time, including the alpha(v)beta(3) integrin, the key adhesion molecule of the mature osteoclast, the highly expressed vacuolar-type H(+)-ATPase that drives acid secretion to dissolve mineral, and cathepsin K, the predominant acid proteinase for collagenolysis. Recently, additional detail has been added to this framework, showing that the osteoclast has more complex regulation than was previously believed. These include the findings that one component of the V-H(+)-ATPase is unique to the osteoclast, that chloride transport and probably Cl(-)/H(+) exchange are also required for mineral degradation, and that additional receptors besides RANK and Fms regulate osteoclast formation and survival. Additional receptors include estrogen receptor-alpha, TNF-family receptors other than RANK, and, at least in some cases, glycoprotein hormone receptors including the TSH-R and the FSH-R. Challenges in understanding osteoclast biology include how the signalling mechanisms function cooperatively. Recent findings suggest that there is a network of cytoplasmic adapters, including Gab-2 and BCAR1, which are modified by multiple signalling mechanisms and which serve to integrate the signalling pathways.

Animals↗

PubMed1991

Macrophage colony stimulating factor (M-CSF) is essential for osteoclast formation in vitro.

The op/op mouse, in which the M-CSF gene is mutated, has greatly reduced numbers of macrophages and osteoclasts. We assessed the ability of M-CSF to induce osteoclast and macrophage formation in op/op hemopoietic cells in vitro. Osteoclast production was undetectable in op/op cell cultures, but was restored by M-CSF at concentrations approximately an order of magnitude higher than those that induced macrophages. In normal hemopoietic tissue M-CSF similarly increased macrophage numbers, but inhibited osteoclast formation. Despite cure of the macrophage defect, neither interleukin 3 nor granulocyte-macrophage CSF were able to induce osteoclastic differentiation in op/op cells. The results suggest that M-CSF induces osteoclastic differentiation but that macrophages, which are also induced by M-CSF, suppress osteoclast differentiation. Macrophages induced by other cytokines seem unable to contribute to osteoclast-formation.

Animals↗

PubMed1986

Formation sites and distribution of osteoclast progenitor cells during the ontogeny of the mouse.

The presence of osteoclast progenitor cells in embryonic, fetal, young growing, and adult murine tissues and organs was investigated in a coculture system with fetal metatarsal bones stripped of periosteum and not yet invaded by osteoclasts. Osteoclasts were found to originate from the early yolk sac and from every tissue tested in the fetus and young mouse. In the adult mouse they were formed only from tissues with a large mononuclear phagocyte population. No osteoclasts could be generated from the young embryo proper, prior to establishment of the vascular connection with the yolk sac. Progenitors of osteoclasts or their stem cells therefore do not develop from undifferentiated mesenchyme outside the yolk sac, but are distributed from the yolk sac to embryonic tissues and hematopoietic organs through the vascular circulation. The embryonic distribution of osteoclast progenitors coincides with the distribution of immature macrophages. Furthermore, they are present before the formation of monocytes in the fetus. The results also indicate that osteoclast precursor cells are not identical with mature, differentiated macrophages, but are cells with little capacity to phagocytose and therefore are, at the most immature progenitors of macrophages or cells of an early diverging lineage. In view of these results the derivation of osteoclasts is discussed.

Age Factors↗

PubMed1992

Evidence that protein kinase-A, calcium-calmodulin kinase and cytoskeletal proteins are involved in osteoclast retraction induced by calcitonin.

Calcitonin is a direct inhibitor of osteoclastic activity. Osteoclast retraction is readily induced by calcitonin and it is possible that calcitonin-induced inhibition of bone resorption is in part due to this effect. However, little is known of the mechanisms of this action. In these studies, we have investigated the intracellular signalling pathway of calcitonin-induced osteoclast retraction using cultures of freshly isolated rat osteoclasts. The spread area occupied by single Giemsa-stained rat osteoclasts was measured in vitro by a computer imaging analysis system and used as a quantitative parameter for calculating the degree of osteoclast retraction in response to various agents. Our results show that cAMP may be an important second messenger in the reaction of osteoclasts to calcitonin. Moreover, both protein kinase-A and calcium/calmodulin-dependent protein kinase are involved in the osteoclast retraction induced by this hormone, while cytoskeletal proteins are required for the process to occur.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1993

Involvement of adhesion molecules LFA-1 and ICAM-1 in osteoclast development.

We report here that leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) are involved in osteoclast development. Osteoclast development was observed on co-culture of mouse spleen cells and mouse bone marrow derived clonal stromal cells, TMS-14, in the presence of 1 alpha, 25-dihydroxyvitamin D-3 (1 alpha, 25-(OH)2D3) for 8 days, and quantified with respect to tartrate-resistant acid phosphatase (TRACP) activity. When either one of the monoclonal antibodies (MAbs) to mouse LFA-1 and mouse ICAM-1 was added to the co-culture system, the TRACP activity was significantly inhibited. The experiment in which one-day treatment with each of these MAbs was performed during the 8 days of cultivation showed that the inhibitory effects of both MAbs on the TRACP activity at 8 days were observed from an early stage of the culture, but were more notable at a later stage (days 4-6). As the expression of ICAM-1 was observed on both spleen cells and TMS-14, we next examined whether the interaction between stromal cells and osteoclast progenitors or among osteoclast progenitors was more important for osteoclast development. To determine this, rat spleen cells and a MAb to rat ICAM-1 were used instead of those of mouse. When MAb to rat ICAM-1 or mouse ICAM-1 was added to the co-culture system of rat spleen cells and TMS-14, the inhibitory effect of the MAb to rat ICAM-1 was mainly observed at a later stage of the culture period and that of anti-mouse ICAM-1 antibody was only observed at an earlier stage. These results indicate that adhesion molecules LFA-1 and ICAM-1 may play a role in osteoclast development via interaction between stromal cells and osteoclast progenitors as well as among osteoclast progenitors.

Acid Phosphatase↗

PubMed1991

Calcitonin receptor expression and its regulation by 1 alpha-25-dihydroxyvitamin D3 during de novo osteoclast formation in organ cultures of fetal mouse metatarsals.

Organ cultures of 15-day embryonic mouse metatarsals cultured in serumless, chemically-defined medium were used to investigate the influence of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) on calcitonin receptor (CTR) expression in osteoclasts formed from in situ progenitors. CTR expression was demonstrated in tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts and their precursors. The expression of TRAP preceded that of CTR and was first observed in cells identified as osteoclast precursors. Not all TRAP+ precursors were CTR+ and from 40 to 60% of newly formed osteoclasts lacked CTR. Treatment with 1,25(OH)2D3 increased precursor numbers without affecting CTR expression, whereas both the numbers and the percentage of newly formed osteoclasts expressing CTR was increased by 1,25(OH)2D3. Grain count analysis revealed that this increased percentage of newly formed CTR+ osteoclasts were cells with low and medium levels of CTR expression. This apparent down-regulation of CTR expression in newly formed osteoclasts may help to explain the 'escape' of osteoclastic bone resorption from the inhibitory effects of calcitonin.

Acid Phosphatase↗

PubMed1995

Inhibitory effect of zinc-chelating dipeptide on parathyroid hormone-stimulated osteoclast-like cell formation in mouse marrow cultures: involvement of calcium signaling.

A possible mechanism of zinc action inhibiting the PTH-induced osteoclast-like cell formation in mouse marrow culture system in vitro was investigated. Bone marrow cells were cultured for 7 days in alpha-minimal essential medium containing a well-known bone-resorbing agent parathyroid hormone (1-34) (PTH). Osteoclast-like cell formation was estimated with staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The effect of zinc sulfate (10(-6) M) or beta-alanyl-L-histidinato zinc (AHZ; 10(-6) M) inhibiting the PTH (10(-8) M)-induced osteoclast-like cell formation was clearly seen in the absence or presence of theophylline (10(-4) M). However, zinc compounds did not inhibit the stimulatory effect of dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP; 10(-4) M) on osteoclast-like cell formation. The stimulating effect of PTH (10(-8) M) on osteoclast-like cell formation was clearly weakened (about 50%) in the presence of EGTA (1.0 mM) or dibucaine (10(-5) M). Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator (10(-8) to 10(-6) M), clearly stimulated osteoclast-like cell formation. PMA effect was inhibited by the presence of AHZ (10(-6) M) or zinc sulfate (10(-8) M). However, the inhibitory effect of zinc compounds was not seen in the presence of both PTH (10(-8) M) and PMA (10(-6) M). The present findings suggest that zinc compounds inhibit PTH-stimulated osteoclast-like cell formation mediated through the Ca(2+)-dependent activation of protein kinase C.

Animals↗

PubMed1984

Osteoclast populations in congenital osteopetrosis: additional evidence of heterogeneity.

Osteopetrosis is a metabolic bone disease characterized by excessive accumulation of skeletal mass due to a reduction in bone resorption. The pathogenesis of osteopetrosis is reduced osteoclast function. Reports of osteoclast numbers in several mammalian mutations exhibiting osteopetrosis have shown them to be increased, decreased, or normal in numbers. The present investigation quantitated the osteoclast populations and examined the cytology of osteoclasts by light microscopy in calvarial and tibial sites in one rabbit and two mouse mutations and compared them with their normal littermates. Our observations show that osteoclast numbers are, depending on the particular mutation, increased, decreased, or comparable to those found in normal littermates. In each mutation, however, osteoclasts fail to exhibit the cytoplasmic vacuolization next to bone surfaces seen in normal osteoclasts. These data provide additional evidence of heterogeneity in the congenital mammalian osteopetrosis and suggest that these mutations may be significant sources of new information about the biology of osteoclasts.

Animals↗

PubMed1985

The lifespan of osteoclasts: experimental studies using the giant granule cytoplasmic marker characteristic of beige mice.

Osteoclasts are large multinucleated skeletal cells that form by fusion of bloodborne mononuclear precursors. Fusion with mononuclear precursors occurs throughout life, and survival of osteoclasts is believed to be dependent upon continued replenishment by fusion. This study examined osteoclast lifespan, defined as maximal survival without fusion, in normal mice irradiated to eliminate host stem cells and rescued with stem cells from beige (bg) mice whose osteoclasts have a distinctive phenotype. Osteoclasts of donor phenotype appeared during the second week and progressively increased so that by the sixth week no osteoclasts of host phenotype were present. Radiation alone did not produce any change in osteoclast phenotype. These data are interpreted to indicate that the maximal survival of osteoclasts without fusion of precursors is less than 6 weeks.

Animals↗

PubMedDate not supplied

Role of the endothelial cell in osteoclast control: new perspectives.

The osteoclast is of central importance in the process of bone remodeling. Its function is regulated by hormones and locally produced factors. Endothelial cells occur in close proximity to the osteoclast. Some endothelial cell-derived products, including endothelins, nitric oxide, and reactive oxygen species, have been recently implicated as modulators of osteoclast function. Endothelins inhibit bone resorption and osteoclast margin ruffling (quiescence or Q effect) at concentrations similar to those effective for their primary vasoconstrictive action. Contrary to expectations, however, it has been shown that endothelin action on the osteoclast is not mediated through an elevation of cytosolic Ca2+. Nitric oxide (NO) produces marked cell retraction (retraction or R effect), but its detailed mode of action is unknown. However, it is clear that the effects of this autocoid are not due to enhanced cyclic guanosine monophosphate (cGMP) production, a transduction system commonly used by NO. Finally, the reactive oxygen species H2O2 has been shown recently to enhance osteoclastic activity. Thus, the reported effects of the endothelial cell-derived products on the osteoclast are generally consistent with a regulatory role for endothelial cells in osteoclast control and suggest the existence of unique activation pathways, well worth exploring further. Unravelling the responsible mechanisms may also help understand the pathophysiology of a range of bone and joint diseases. For example, in rheumatoid arthritis, there is increased H2O2 production from activated neutrophils, and bone resorption is a major pathophysiological feature.

Bone Remodeling↗

PubMed1995

Colony-stimulating factor 1 when combined with parathyroid hormone or 1,25-dihydroxyvitamin D can produce osteoclasts in cultured neonatal metatarsals from toothless (tl-osteopetrotic) rats.

Toothless (tl-osteopetrotic) rats have little or no endogenous bone resorption, no marrow spaces, and very few osteoclasts and macrophages, and their live metatarsal rudiments cannot support the development of normal osteoclasts in vitro. The recent demonstration that exogenous colony-stimulating factor 1 (CSF-1) improves skeletal sclerosis and increases osteoclasts in tl rats in vivo, prompted us to explore conditions that enable osteoclasts to be formed in tl metatarsals in vitro. Coculture of neonatal tl metatarsals with CSF-1 alone produced no osteoclasts, but the addition of normal spleen and bone marrow cells and parathyroid hormone or 1,25-dihydroxyvitamin D produced osteoclasts in most cultures. Identical cultures of metatarsals from the CSF-1 deficient op/op mouse produced similar results. Within the contexts of the role of CSF-1 in osteoclastogenesis and the different biologic manifestations of osteopetrosis in these two mutations, we interpret these results to mean that other factors are required to restore osteoclast function completely in tl rats and op mice. Thus, experimental studies of these mutations are likely to provide new insights on both osteopetrosis and osteoclast biology.

Animals↗

PubMed1996

Paget's disease and osteoclast biology.

Paget's disease is the most exaggerated example of bone remodeling in which abnormal osteoclastic bone resorption remains coupled to new bone formation. There are abnormalities in the stages of osteoclast development, and studies in Paget's disease have suggested a major role for IL-6 in human osteoclast activity. The pathophysiologic basis for these abnormalities is not clearly defined, except that the primary cellular abnormality resides in the osteoclast. Many important questions about the pathophysiology of Paget's disease still remain to be answered, including: (1) What is the identity of the virus in pagetic osteoclasts?; (2) Are Paget's patients in different geographical locales harboring a similar virus in their osteoclasts, or can different paramyxoviruses induce Paget's disease?; (3) How is the virus maintained and propagated for many years, so that it can be expressed in the osteoclast, a cell with a finite lifespan?; and (4) Since Paget's disease has a very high familial tendency, with up to 40% of patients having an affected relative, what is the genetic locus associated with Paget's disease, and does this genotype result in an increased propensity for hematopoietic cells such as the osteoclast to harbor paramyxoviruses? The application of the techniques of molecular and cell biology to Paget's disease should provide answers to some of these questions and give important insights into the normal bone remodeling process.

Biological Factors↗

PubMed2005

Immunohistochemical evaluation of osteoclast recruitment during experimental tooth movement in young and adult rats.

OBJECTIVE: Orthodontic tooth movement starts slower in adults than in juveniles, but the rate of tooth movement in later phases is the same in both age groups. The hypotheses to be tested are that these phenomena are related to slower osteoclast recruitment in adults than in juveniles, but that in later phases the osteoclast numbers are the same in both age groups. DESIGN: Standardized orthodontic tooth movement was performed in two groups of 30 rats, aged 6 weeks and 9-12 months, respectively. All maxillary molars at one side were together moved mesially by a continuous force of 10 cN. The other side served as a control. After 1, 2, 4, 8 and 12 weeks, groups of animals were killed. After ED1 staining osteoclast numbers at the mesial and distal sides of selected roots were counted. RESULTS: At the compression sides osteoclast numbers increased in both age groups. In young rats, a maximum was reached at 2 weeks, in adults at 4 weeks. In later phases of tooth movement, the number of osteoclasts in the adults was approximately twice as high as in the juveniles, while the rate of tooth movement was the same. A positive correlation between the rate of tooth movement and osteoclast numbers was found only in young rats. CONCLUSIONS: Orthodontic forces induce faster osteoclast recruitment in young than in adult rats and more osteoclasts are needed to achieve a certain rate of tooth movement in adult than in young rats.

Age Factors↗