Calvarial Osteoclasts Express a Higher Level of Tartrate-Resistant Acid Phosphatase than Long Bone Osteoclasts and Activation Does not Depend on Cathepsin K or L Activity

Bone resorption by osteoclasts depends on the activity of various proteolytic enzymes, in particular those belonging to the group of cysteine proteinases. Next to these enzymes, tartrate-resistant acid phosphatase (TRAP) is considered to participate in this process. TRAP is synthesized as an inactive proenzyme, and in vitro studies have shown its activation by cysteine proteinases. In the present study, the possible involvement of the latter enzyme class in the in vivo modulation of TRAP was investigated using mice deficient for cathepsin K and/or L and in bones that express a high (long bone) or low (calvaria) level of cysteine proteinase activity. The results demonstrated, in mice lacking cathepsin K but not in those deficient for cathepsin L, significantly higher levels of TRAP activity in long bone. This higher activity was due to a higher number of osteoclasts. Next, we found considerable differences in TRAP activity between calvarial and long bones. Calvarial bones contained a 25-fold higher level of activity than long bones. This difference was seen in all mice, irrespective of genotype. Osteoclasts isolated from the two types of bone revealed that calvarial osteoclasts expressed higher enzyme activity as well as a higher level of mRNA for the enzyme. Analysis of TRAP-deficient mice revealed higher levels of nondigested bone matrix components in and around calvarial osteoclasts than in long bone osteoclasts. Finally, inhibition of cysteine proteinase activity by specific inhibitors resulted in increased TRAP activity. Our data suggest that neither cathepsin K nor L is essential in activating TRAP. The findings also point to functional differences between osteoclasts from different bone sites in terms of participation of TRAP in degradation of bone matrix. We propose that the higher level of TRAP activity in calvarial osteoclasts compared to that in long bone cells may partially compensate for the lower cysteine proteinase activity found in calvarial osteoclasts and TRAP may contribute to the degradation of noncollagenous proteins during the digestion of this type of bone. An erratum to this article is available at .     

Ultrastructural localization of a tartrate-resistant acid ATPase in bone

Osteoclasts are effector cells in bone breakdown, and the active bone resorption is confined to the ruffled border zone of these cells. An acid milieu is maintained in this zone which is probably a prerequisite for bone resorption. Tartrate-resistant acid phosphatase (TRAP) activity has been recognized as a characteristic property of osteoclasts and in several studies proposed as a cytochemical marker of osteoclasts. We have previously isolated and characterized a tartrate-resistant and iron-activated acid ATPase (TrATPase) from rat bone, the enzyme being a member of the TRAP family. In the present study the ultrastructural localization of this enzyme was delineated by employing immunogold technique on low temperature-embedded maxillar rat bone. Intensive immunolabeling was seen on the bone surfaces facing the ruffled border zone while lower amounts of marker were seen in adjacent bone areas, that is, on the bone surfaces facing the clear zone and deeper-into the bone. Within the osteoclasts gold markers were observed mainly in vesicular structures interpreted as lysosomes. Immunolabeling was also observed in the recently described endocytic cells located near osteoblasts and osteoclasts. Also in these cells, the marker was confined to lysosomelike structures. The amount of label in bone facing osteoblasts was low, as was the amount within osteoblasts. Our observation of extracellular localization, in particular accumulation of TrATPase in bone matrix facing the ruffled border area of the osteoclasts, favors the view that the enzyme is exported to areas of active bone resorption, thereby indicating a potential role for the enzyme in this process.     

Altered Collagen in Tartrate-Resistant Acid Phosphatase (TRAP)-Deficient Mice: A Role for TRAP in Bone Collagen Metabolism

Tartrate-resistant acid phosphatase (TRAP) is an iron-containing protein that is highly expressed by osteoclasts, macrophages, and dendritic cells. The enzyme is secreted by osteoclasts during bone resorption, and serum TRAP activity correlates with resorptive activity in disorders of bone metabolism. TRAP is essential for normal skeletal development. In knockout mice lacking TRAP, bone shape and modeling is altered with increased mineral density. Here, we report the effect of TRAP on the biochemical and biomechanical properties of collagen, the major protein constituting the bone matrix, using these mice. Femurs from TRAP^-/- and wild-type mice were used in these studies. The biomechanical properties were investigated using a three-point bending technique. Collagen synthesis was determined by measuring cross-link content using high-performance liquid chromatography and amino acid analysis. Collagen degradation was determined by measuring matrix metalloproteinase-2 (MMP-2) activity. The rates of collagen synthesis and degradation were significantly greater in bones from TRAP^-/- mice compared with wild type. At 8 weeks, there was an increase in the intermediate cross-links but no significant difference in animals aged 6 months. There was a significant increase in mature cross-links at both ages. A significant increase in MMP-2 production both pro and active was observed. A significant increase in ultimate stress and Young’s modulus of elasticity was needed to fracture the bones from mice deficient in TRAP. We conclude that both synthesis as well as degradation of collagen are increased when TRAP is absent in mice at 8 weeks and 6 months of age, showing that TRAP has an important role in the metabolism of collagen.     

The relative contribution of cysteine proteinases and matrix metalloproteinases to the resorption process in osteoclasts derived from long bone and scapula

It has been suggested that functional heterogeneity exists between osteoclasts from different bone sites. This could be exploited to design therapeutics that would selectively inhibit bone resorption only at compromised sites. To further investigate the existence of functional differences between osteoclasts from different bone sites we assessed whether osteoclasts isolated from intramembranous bone differ from osteoclasts isolated from endochondral bone in the extent that they utilize cysteine proteinases and matrix metalloproteinases to degrade the organic matrix of bone. The differential involvement of the two classes of proteases was assessed by analyzing dose-dependent effects of the matrix metalloproteinase inhibitor, CT-1746, and of the cathepsin inhibitor, E64, on bone resorption. Osteoclasts isolated from the scapula (intramembranous) and long bones (endochondral) of newborn New Zealand white rabbits were seeded on cortical bovine bone slices in the presence or absence of inhibitors. Resorptive activity was evaluated by measuring the number and area of resorption pits and by measuring the release of collagen degradation products in the culture medium. In the absence of inhibitors, scapular osteoclasts and long bone osteoclasts had similar activity based on these criteria. The resorptive activity of scapular osteoclasts was inhibited to a greater extent by the MMP inhibitor CT-1746 than by the cysteine proteinase inhibitor E64. Conversely, resorption by osteoclasts derived from long bones was inhibited to a greater degree by the cysteine proteinase inhibitor. These results strongly suggest that there are functional differences between dispersed osteoclasts derived from the scapula and long bones, with scapular osteoclasts utilizing matrix metalloproteinases to a greater extent than cysteine proteinases and long bone osteoclasts using cysteine proteinases to a greater extent than matrix metalloproteinases.     

Transgenic mice overexpressing tartrate-resistant acid phosphatase exhibit an increased rate of bone turnover.

Tartrate-resistant acid phosphatase (TRAP) is a secreted product of osteoclasts and a lysosomal hydrolase of some tissue macrophages. To determine whether TRAP expression is rate-limiting in bone resorption, we overexpressed TRAP in transgenic mice by introducing additional copies of the TRAP gene that contained the SV40 enhancer. In multiple independent mouse lines, the transgene gave a copy number-dependent increase in TRAP mRNA levels and TRAP activity in osteoclasts, macrophages, serum, and other sites of normal low-level expression (notably, liver parenchymal cells, kidney mesangial cells, and pancreatic secretory acinar cells). Transgenic mice had decreased trabecular bone consistent with mild osteoporosis. Measurements of the bone formation rate suggest that the animals compensate for the increased resorption by increasing bone synthesis, which partly ameliorates the phenotype. These mice provide evidence that inclusion of an irrelevant enhancer does not necessarily override a tissue-specific promoter.     

Inhibition of prostaglandin synthesis leads to a change in adherence of mouse osteoclasts from bone to periosteum

When mouse parietal bones were incubated for 1 day in medium containing indomethacin (Ind), the number of tartrate-resistant acid phosphatase-positive osteoclasts (TRAP+OC) counted on the bone surface was drastically reduced. This reduction did not occur with calcitonin or if the endocranial membrane (periosteum) was removed prior to incubation with Ind. The aim of this work was to determine the mechanism involved. TRAP+OC were found to be increased on the endocranial membrane adjacent to the resorbing surface after Ind treatment, compared with cultures supplemented with parathyroid hormone (PTH) or prostaglandin E2 (PGE2). However, this increase accounted for only half of those lost from the bone surface. TRAP negative osteoclasts were also seen on the membrane and, to a lesser extent, on the bone. Increased TRAP specific activity could be extracted from the endocranial membranes of bones incubated with Ind compared with PGE2 controls. When bones that had been exposed to Ind were then cultured for 1 day in PGE2, an increase in TRAP+OC occurred. This increase was blocked by the removal of the endocranial membrane prior to incubation with PGE2. We conclude that when prostaglandin production ceases, TRAP+OC become less adherent to bone and more adherent to the endocranial membrane. Stimulators of bone resorption appear to reverse this process. Received: 1 September 1995 / Accepted: 12 February 1996     

Multinucleate osteoclasts in medaka as evidence of active bone remodeling

Putative sites of bone resorption in the acellular bony skeleton of the medaka fish (Oryzias latipes) were investigated primarily by RNA in situ hybridization and histological analysis. Numerous cells that displayed intense enzymatic activity of tartrate-resistant acid phosphatase (TRAP), the main marker of osteoclasts, were distributed in the pharyngeal region of this fish. Moreover, these cells expressed cathepsin K, an osteoclast-specific gene, as well as the genes for TRAP and vacuolar-type proton ATPase (V-ATPase). Some of the TRAP-positive cells displayed all of the morphological characteristics equivalent to those of mammalian- and bird-type osteoclasts. These cells were associated primarily with the shedding teeth and their supporting bones (pedicles), where alkaline phosphatase (ALPase)-positive osteoblasts were also located, implying progressive bone remodeling associated with tooth replacement in these regions. In contrast, the inner aspects of the neural and hemal arches of the vertebral column, which were the only sites of bone resorption other than the tooth-bearing bones, showed sporadically aligned flat mononuclear TRAP-positive cells without a ruffled border, indicating a different mode of bone remodeling in these regions. These results suggest the feasibility of medaka as a model animal for the investigation of bone-related abnormalities and their genetic backgrounds.     

痩素对小鼠破骨细胞分化及功能的影响

目的 观察瘦素（leptin）对体外骨髓诱导培养的小鼠破骨细胞分化和功能的作用效应,探索leptin和骨吸收之间的关联.方法 建立由巨噬细胞集落刺激因子（M-CSF）和骨保护素配体（RANKL）为共同细胞因子的小鼠破骨细胞骨髓诱导培养体系,将不同浓度的leptin作用于破骨细胞.实验中根据培养液中是否加入M-CSF和RANKL并依据leptin浓度的不同分为：A组,M-CSF和RANKL;B组,M-CSF、RANKL和leptin（80 ng/ml）;C组,M-CSF、RANKL和leptin（160 ng/ml）;D组,M-CSF、RANKL和leptin（240 ng/ml）;E组,M-CSF、RANKL和leptin（320 ng/ml）;F组,M-CSF、RANKL和leptin（400 ng/ml）;同时设立空白对照组G组.于作用后第7天取细胞玻片进行抗酒石酸酸性磷酸酶（TRAP）染色,观察破骨细胞并计数;于第10天取出骨片进行甲苯胺蓝染液染色,在光镜和扫描电镜观察骨吸收陷窝形态.结果：诱导培养的小鼠破骨细胞形态特征明显;A组在破骨细胞数量与D、E、F组相比较有明显的统计学差异（P＜0.05）;A组骨吸收面积比与B、C、D、E、F组都有明显的统计学差异（P＜0.05）.结论：leptin抑制体外培养的破骨细胞的分化和骨吸收功能.     

Proteolytic processing and polarized secretion of tartrate-resistant acid phosphatase is altered in a subpopulation of metaphyseal osteoclasts in cathepsin K-deficient mice

Tartrate-resistant acid phosphatase (TRAP) is an enzyme highly expressed in osteoclasts and thought to participate in osteoclast-mediated bone turnover. Cathepsin K (Ctsk) is the major collagenolytic cysteine proteinase expressed in osteoclasts and has recently been shown to be able to proteolytically process and activate TRAP in vitro. In this study, 4-week-old Ctsk(-/-) mice were analysed for TRAP expression at the mRNA, protein and enzyme activity levels to delineate a role of cathepsin K in TRAP processing in osteoclasts in vivo. The absence of cathepsin K in osteoclasts was associated with increased expression of TRAP mRNA, monomeric TRAP protein and total TRAP activity. Proteolytic processing of TRAP was not abolished but prematurely arrested at an intermediate stage without changing enzyme activity, a finding confirmed with RANKL-differentiated osteoclast-like cell line RAW264.7 treated with the cysteine proteinase inhibitor E-64. Thus, the increase in total TRAP activity was mainly due to increased cellular content of monomeric TRAP. The increase in monomeric TRAP expression was more pronounced in osteoclasts of the distal compared to the proximal part of the metaphyseal trabecular bone, suggesting a site-dependent role for cathepsin K in TRAP processing. Moreover, intracellular localization of monomeric TRAP was altered in distal metaphyseal osteoclasts from Ctsk(-/-) mice. Additionally, TRAP was secreted into the ruffled border as the processed form in osteoclasts of Ctsk(-/-) mice, unlike in osteoclasts from wild-type mice which secreted TRAP to the resorption lacuna as the monomeric form. The results demonstrate that cathepsin K is not only involved in proteolytic processing but also affects the intracellular trafficking of TRAP, particularly in osteoclasts of the distal metaphysis. However, contribution by other yet unidentified protease(s) to TRAP processing must also be invoked since proteolytic cleavage of TRAP is not abolished in Ctsk(-/-) mice. Importantly, this study highlights functional differences between bone-resorbing clasts within the trabecular metaphyseal bone, suggesting potentially important differences in the regulation of differentiation and activation depending on the precise anatomical localization of the clast population.     

Osteoclastic bone degradation and the role of different cysteine proteinases and matrix metalloproteinases: differences between calvaria and long bone.

Osteoclastic bone degradation involves the activity of cathepsin K. We found that in addition to this enzyme other, yet unknown, cysteine proteinases participate in digestion. The results support the notion that osteoclasts from different bone sites use different enzymes to degrade the collagenous bone matrix. INTRODUCTION: The osteoclast resorbs bone by lowering the pH in the resorption lacuna, which is followed by secretion of proteolytic enzymes. One of the enzymes taken to be essential in resorption is the cysteine proteinase, cathepsin K. Some immunolabeling and enzyme inhibitor data, however, suggest that other cysteine proteinases and/or proteolytic enzymes belonging to the group of matrix metalloproteinases (MMPs) may participate in the degradation. In this study, we investigated whether, in addition to cathepsin K, other enzymes participate in osteoclastic bone degradation. MATERIALS AND METHODS: In bones obtained from mice deficient for cathepsin K, B, or L or a combination of K and L, the bone-resorbing activity of osteoclasts was analyzed at the electron microscopic level. In addition, bone explants were cultured in the presence of different selective cysteine proteinase inhibitors and an MMP inhibitor, and the effect on resorption was assessed. Because previous studies showed differences in resorption by calvarial osteoclasts compared with those present in long bones, in all experiments, the two types of bone were compared. Finally, bone extracts were analyzed for the level of activity of cysteine proteinases and the effect of inhibitors hereupon. RESULTS: The analyses of the cathepsin-deficient bone explants showed that, in addition to cathepsin K, calvarial osteoclasts use other cysteine proteinases to degrade bone matrix. It was also shown that, in the absence of cathepsin K, long bone osteoclasts use MMPs for resorption. Cathepsin L proved to be involved in the MMP-mediated resorption of bone by calvarial osteoclasts; in the absence of this cathepsin, calvarial osteoclasts do not use MMPs for resorption. Selective inhibitors of cathepsin K and other cysteine proteinases showed a stronger effect on calvarial resorption than on long bone resorption. CONCLUSIONS: Our findings suggest that (1) cathepsin K-deficient long bone osteoclasts compensate the lack of this enzyme by using MMPs in the resorption of bone matrix; (2) cathepsin L is involved in MMP-mediated resorption by calvarial osteoclasts; (3) in addition to cathepsin K, other, yet unknown, cysteine proteinases are likely to participate in skull bone degradation; and finally, (4) the data provide strong additional support for the existence of functionally different bone-site specific osteoclasts.     

Expression of bone resorption genes in osteoarthritis and in osteoporosis

Cathepsin K and MMP-9 are considered to be the most abundant proteases in osteoclasts. TRAP is a marker for osteoclasts, and there is increasing evidence of its proteolytic role in bone resorption. RANKL is a recently discovered regulator of osteoclast maturation and activity and induces expression of many genes. This study compared cathepsin K, MMP-9, TRAP, RANKL, OPG, and osteocalcin gene expression in the proximal femur of patients with osteoarthritis with that of patients with femoral neck fracture. Fifty-six patients undergoing arthroplasty because of osteoarthritis or femoral neck fracture were included in the study. Total mRNA was extracted from the bone samples obtained from the intertrochanteric region of the proximal femur. Real-time RT-PCR was used to quantify CTSK (cathepsin K), MMP-9 (matrix metalloproteinase 9), ACP5 (TRAP), TNFSF11 (RANKL), TNFRSF11B (OPG), and BGLAP (osteocalcin) mRNAs. The levels of mRNAs coding for MMP-9 and osteocalcin indicated higher expression in the osteoarthritic group (P = 0.011, P = 0.001, respectively), whereas RANKL expression and the ratio RANKL/OPG were both significantly lower in the osteoarthritic group than in the fracture group. Expression of cathepsin K, MMP-9, and TRAP relative to RANKL was significantly higher in the osteoarthritic group. Ratios of all three proteolytic enzymes relative to formation marker osteocalcin were higher in the fracture group. Gene expression of cathepsin K, MMP-9, TRAP, RANKL, OPG, and osteocalcin and the association between their mRNA levels pointed to higher bone resorption and bone formation in osteoarthritis, differences in balance between them, and differences in regulation of bone resorption in osteoarthritic and osteoporotic bone.     

Osteoclasts prefer aged bone

Summary We investigated whether the age of the bones endogenously exerts control over the bone resorption ability of the osteoclasts, and found that osteoclasts preferentially develop and resorb bone on aged bone. These findings indicate that the bone matrix itself plays a role in targeted remodeling of aged bones. Introduction Osteoclasts resorb aging bone in order to repair damage and maintain the quality of bone. The mechanism behind the targeting of aged bone for remodeling is not clear. We investigated whether bones endogenously possess the ability to control osteoclastic resorption. Methods To biochemically distinguish aged and young bones; we measured the ratio between the age-isomerized βCTX fragment and the non-isomerized αCTX fragment. By measurement of TRACP activity, CTX release, number of TRACP positive cells and pit area/pit number, we evaluated osteoclastogenesis as well as osteoclast resorption on aged and young bones. Results We found that the αCTX / βCTX ratio is 3:1 in young compared to aged bones, and we found that both α and βCTX are released by osteoclasts during resorption. Osteoclastogenesis was augmented on aged compared to young bones, and the difference was enhanced under low serum conditions. We found that mature osteoclasts resorb more on aged than on young bone, despite unchanged adhesion and morphology. Conclusions These data indicate that the age of the bone plays an important role in controlling osteoclast-mediated resorption, with significantly higher levels of osteoclast differentiation and resorption on aged bones when compared to young bones. Kim Henriksen and Diana J. Leeming contributed equally. Financial disclosure: Morten A. Karsdal, Per Qvist and Claus Christiansen own stock options in Nordic Bioscience A/S     

Identification of angiogenin as the osteoclastic bone resorption-inhibitory factor in bovine milk

We identified, for the first time, the factor responsible for inhibiting osteoclast-mediated bone resorption in the basic protein fraction of bovine milk (milk basic protein, MBP). The protein was purified by a combination of ion and gel column chromatography from MBP, based on its activity to prevent unfractionated rabbit bone cells from forming pits on dentine slices. It was found to have a molecular weight of 15 kDa on SDS-PAGE, and the sequence of the N-terminal 25 amino acid residues was identical to that of bovine angiogenin. The purified bovine angiogenin inhibited the pit-forming activity of both unfractionated bone cells and purified osteoclasts in a dose-dependent manner, and the inhibitory activity was markedly suppressed by treatment with anti-bovine angiogenin antibody. The inhibitory activity was confirmed in mice both in vitro and in vivo. Treatment of osteoclasts with bovine angiogenin resulted in an impairment of the formation F-actin ring and a reduction in the mRNA levels of TRAP and cathepsin K, both known to be essential for the bone resorption activity of osteoclasts. These results suggest that bovine angiogenin is the substance mainly responsible for the inhibitory effect of bovine milk on osteoclast-mediated bone resorption, and that it exerts its activity by acting directly on the osteoclasts.     

Parathyroid hormone-related protein is required for normal intramembranous bone development.

It is well established that parathyroid hormone-related protein (PTHrP) regulates chondrocytic differentiation and endochondral bone formation. Besides its effect on cartilage, PTHrP and its major receptor (type I PTH/PTHrP receptor) have been found in osteoblasts, suggesting an important role of PTHrP during the process of intramembranous bone formation. To clarify this issue, we examined intramembranous ossification in homozygous PTHrP-knockout mice histologically. We also analyzed phenotypic markers of osteoblasts and osteoclasts in vitro and in vivo. A well-organized branching and anastomosing pattern was seen in the wild-type mice. In contrast, marked disorganization of the branching pattern of bone trabeculae and irregularly aligned osteoblasts were recognized in the mandible and in the bone collar of the femur of neonatal homozygous mutant mice. In situ hybridization showed that most of the osteoblasts along the bone surfaces of the wild-type mice and some of the irregularly aligned osteoblastic cells in the homozygous mice expressed osteocalcin. Alkaline phosphatase (ALP) activity and expression of osteopontin messenger RNA (mRNA) in primary osteoblastic cells did not show significant differences between cultures derived from the mixture of heterozygous mutant and wild-type mice (+/? mice) and those from homozygous mutant mice. However, both mRNA and protein levels of osteocalcin in the osteoblastic cells of homozygous mutant mice were lower than those of +/? mice, and exogenous PTHrP treatment corrected this suppression. Immunohistochemical localization of characteristic markers of osteoclasts and ruffled border formation did not differ between genotypes. Cocultures of calvarial osteoblastic cells and spleen cells of homozygous mutant mice generated an equivalent number of tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear and multinucleated cells and of pit formation to that of +/? mice, suggesting that osteoclast differentiation is not impaired in the homozygous mutant mice. These results suggest that PTHrP is required not only for the regulation of cartilage formation but also for the normal intramembranous bone development.     

Chondroclasts and Osteoclasts in Bones of Young Rats: Comparison of Ultrastructural and Functional Features

The aim of the present study was to characterize cells involved in resorption during endochondral bone formation. We investigated whether the cells involved in cartilage breakdown at the epiphyseal/metaphyseal border, i.e., chondroclasts, share the characteristics of bone/cartilage-resorbing osteoclasts at the metaphyseal/diaphyseal border regarding ultrastructural features and functional activity. Morphometric evaluation showed that chondroclasts do not form ruffled borders and clear zones, i.e., well-known resorption characteristics, to the same extent as osteoclasts, present at the lower metaphysis. Instead, chondroclasts tend to express an undifferentiated surface adjacent to the matrix, not structurally different from the basolateral plasma membrane. Tartrate-resistant acid phosphatase (TRAP) was used as a marker for functional activity. Immunohistochemical staining by light microscopy was strong in both chondroclasts and in osteoclasts. Furthermore, in situ hybridization revealed large amounts of TRAP mRNA in chondroclasts as well as in osteoclasts. Ultrastructural immunohistochemistry suggests extensive secretion of the TRAP enzyme in the ruffled border area of both chondroclasts and osteoclasts. Intracellular accumulation was seen particularly in chondroclasts, possibly as a consequence of a relative disinclination to develop a ruffled border. Thus, semiquantitative estimation of TRAP distribution showed an inverse relationship between extracellular and intracellular TRAP in chondroclasts and osteoclasts. These results indicate that chondroclasts and osteoclasts differ, not only with respect to location but possibly also by mode of action. The observed differences may reflect the maturation sequence of these multinucleated cells when associated with different metaphyseal trabecular surfaces. Received: 22 January 1998 / Accepted 8 April 1998     

Human osteoclast cathepsin K is processed intracellularly prior to attachment and bone resorption.

Cathepsin K is a member of the papain superfamily of cysteine proteases and has been proposed to play a pivotal role in osteoclast-mediated bone resorption. We have developed a sensitive cytochemical assay to localize and quantify osteoclast cathepsin K activity in sections of osteoclastoma and human bone. In tissue sections, osteoclasts that are distant from bone express high levels of cathepsin K messenger RNA (mRNA) and protein. However, the majority of the cathepsin K in these cells is in an inactive zymogen form, as assessed using both the cytochemical assay and specific immunostaining. In contrast, osteoclasts that are closer to bone contain high levels of immunoreactive mature cathepsin K that codistributes with enzyme activity in a polarized fashion toward the bone surface. Polarization of active enzyme was clearly evident in osteoclasts in the vicinity of bone. The osteoclasts apposed to the bone surface were almost exclusively expressing the mature form of cathepsin K. These cells showed intense enzyme activity, which was polarized at the ruffled border. These results suggest that the in vivo activation of cathepsin K occurs intracellularly, before secretion into the resorption lacunae and the onset of bone resorption. The processing of procathepsin K to mature cathepsin K occurs as the osteoclast approaches bone, suggesting that local factors may regulate this process.     

Inhibiting and stimulating effects of TGF-beta 1 on osteoclastic bone resorption in fetal mouse bone organ cultures

The effects of TGF-beta 1 on osteoclastic resorption of fetal mouse calvaria and long bones at various stages of development was studied in organ culture. In resorbing calvariae and long bones with an established marrow cavity TGF-beta 1 (4-10 ng/ml) had a stimulating effect on 45Ca release that was partially inhibited by indomethacin. In primitive long bones, however, which were explanted before osteoclast invasion and excavation of a marrow cavity had started, TGF-beta 1 (1-4 ng/ml) inhibited 45Ca release by an indomethacin-insensitive mechanism. Histomorphometry of long bones after staining for tartrate-resistant acid phosphatase (TRAP) revealed that TGF-beta 1 treatment inhibited the migration of TRAP-positive cells from periosteum to developing marrow cavity and inhibited cell fusion. However, the formation of (mononuclear) TRAP-positive cells in the periosteum-perichondrium was strongly enhanced. These data suggest that TGF-beta 1 modulates various steps in the cascade of osteoclast development, recruitment, and activation in different ways, involving both prostaglandin-mediated and prostaglandin-independent pathways. Therefore the net effect of exogenous TGF-beta 1 on osteoclastic resorption in bone organ cultures depends on the relative prevalence of osteoclast progenitors, precursors, and mature osteoclasts in the tissue under study.     

BM210955对破骨细胞骨吸收的抑制作用

细胞水平研究国产双磷酸盐药物－ＢＭ２１０９５５的骨吸收抑制作用。由１日龄ＳＤ大鼠四肢长骨分离破骨细胞（ＯＣ）并接种于牛皮质骨薄切片上,在不同浓度ＢＭ２１０９５５作用下培养,定时取骨片作ＴＲＡＰ免疫组化染色,计数阳性多核细胞后,经超声去除细胞后作甲苯胺蓝染色,光镜下作吸收陷窝计数,数据以ｘ±ｓ表示,并与对照比较作ｔ检验。结果显示：①ＢＭ２１０９５５能降低体外培养ＯＣ的数目,１０－８ｍｏｌ／Ｌ组的ＴＲＡＰ阳性多核细胞较对照组减少７３％,差异具有非常显著性,Ｐ＜０．０１。②ＢＭ２１０９５５抑制ＯＣ的陷窝形成能力,１０－１０、１０－８ｍｏｌ／Ｌ组的抑制率分别为７６．３２％和８７．９９％,均与对照有明显差异,Ｐ＜０．０１。③上述结果与剂量有关,剂量越高,抑制效应越明显     

Ipriflavone inhibits osteoclast differentiation in parathyroid transplanted parietal bone of rats

Summary Ipriflavone, a synthetic isoflavone-derived flavonoid, was shown to have inhibitory effect on bone resorption. In order to study its mechanism of action directly on bone, 46 female Wistar rats were divided into six groups and medicated orally for 25 days as follows: groups 1 and 2 were given 1% carboxymethylcellulose solution (vehicle), groups 3, 4, 5, and 6 were administered ipriflavone at doses of 0.178, 0.356, 0.712, and 1.424 mmol/kg/day (suspended in vehicle), respectively. On the 22nd day, parathyroid glands, taken from donor rats, were transplanted in contact with the outer surface of the periosteum of both the right and the left parietal bones of rats from groups 2, 3, 4, 5, and 6. The group 1 rats underwent sham operation. Bone histomorphometry, performed on the ectocranial periosteum of parietal bones, showed that absolute erosion boundary, absolute eroded area, absolute erosion depth, number of tartrate-resistant acid phosphatase (TRAP)-positive polinucleated osteoclasts, and number of TRAP-positive mononucleated cells decreased in ipriflavone-treated rats compared with group 2 rats. The reduction was roughly proportional to the increase of drug dosage and reached statistical significance in rats of groups 5 and 6. The same parameters were extremely low in group 1 rats. Mineral apposition rate did not differ in any of the groups. Significant increase of serum calcium and significant decrease of serum phosphate were found in group 2 rats compared with group 1 rats, whereas no differences from controls were detected in ipriflavone-treated animals.The results demonstrate that ipriflavone has a direct inhibitory effect upon bone resorption, probably by reducing recruitment or differentiation of osteoclasts, rather than by inhibiting the resorption activity of differentiated osteoclasts. Ipriflavone also seems to exert a protective action against parathyroid hormone (PTH) diffusion from the site of parathyroid gland transplantation.