Citrate metabolism in the osteopetrotic bone of the grey-lethal mouse

The osteopetrotic bone of the grey-lethal mouse has been shown to have an abnormal citrate metabolism. Increased levels of this metabolite are found in bone and in the circulation. High bone values may be due in part to apparent decreased amounts of NADP available for the oxidative decarboxylation of citrate. However, an increased O₂ uptake by osteopetrotic bone fragments suggests that some at least of the increased citrate is being further metabolised along an oxidative pathway. Increased stimulation of parathyroid hormone secretion in response to overproduction of thyrocalcitonin has been suggested to account for the findings.

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Zusammenfassung Der osteopetrotische Knochen der grey-lethal Maus hat einen abnormen Zitratstoffwechsel; hohe Werte dieses Stoffwechselproduktes wurden sowohl im Knochen als auch im Kreislauf festgestellt. Die Zitratzunahme des Knochens mag teilweise auf eine Erniedrigung des NADP, der das Zitrat oxidativ dekarboxyliert, zurückgeführt werden. Inkubierte osteopetrotische Knochenfragmente zeigen eine als normale größere Sauerstoffaufnahme, was darauf hindeutet, daß das angesammelte Zitrat wenigstens zum Teil auf oxidativem Wege abgebaut wird. Als Ursache obiger Befunde wird eine erhöhte Parathormonsekretion, die durch eine Überproduktion von Thyrokalzitonin verursacht wird, vorgeschlagen.

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Résumé On a démontré que l'os osteopetrotique de la souris grey-lethal a une métabolisme irrégulière pour la citrate. On en trouve des quantités élevées dans l'os et dans la circulation. Il est possible que les quantités élevées dans l'os sont occasionnées en partie par des quantités diminuées de NADP utilisées pour le decarboxylation oxydative de la citrate. Cependant une absorption élevée de l'oxygène par des fragments de l'os osteopetrotique fait l'idée qu'une partie de la citrate élevée est metabolisée de plus par le chemin oxydative. On a suggéré qu'une stimulation élevée de la parathyroid en réponse d'une production trop grand de thyrocalcitonine offert une explication pour les résultats.

     

Fusion disability of embryonic osteoclast precursor cells and macrophages in the microphthalmic osteopetrotic mouse

Osteoclast formation in the microphthalmic osteopetrotic (mi) mouse was studied from very early embryonic to newborn stages. Embryonic and fetal milmi osteoclasts, generated during the period before bone marrow is formed in the long bones, were predominantly mononuclear and lacked ruffled borders. These cells did, however, show many osteoclastic morphologic and functional properties, such as an abundance of mitochondria, positive succinic dehydrogenase and acid phosphatase reactions, and close contact with and resorption of the calcified cartilage matrix (though diminished). These osteoclastic mononuclear cells appeared in vivo as well as in organ cultures of fetal metatarsal bones with their intact periostea. They also were observed in cocultures of periosteum-free fetal metatarsal bones, with several extraneous sources of osteoclast precursors: yolk sacs and abdominal regions of 9- and 11-day-old embryos, fetal livers, and precultured mononuclear phagocytes isolated from the fetal liver. In contrast, +/+ osteoclasts were always multinuclear, functioned normally in resorbing the calcified cartilage matrix, and had ruffled borders in vivo as well as when derived from the above-mentioned sources. Fetal liver-derived milmi macrophages also failed to form multinuclear foreign body giant cells as opposed to +/+ macrophages in granulomas on implanted pieces of Melinex. The fusion failure of cells derived from embryonic and fetal extramedullary milmi monocyte/macrophage sources contrasted with the occurrence of multinuclear osteoclasts and foreign body giant cells derived from precursors from the bone marrow in young milmi mice. We conclude that the fusion defect of milmi osteoclast precursor cells is already present in their ancestry in blood cell-forming organs of very young embryos and that these cells differentiate into mononuclear osteoclasts that function inefficiently in prenatal bone. We presume that in fully developed bone marrow, local factors are favorable for abolishing the fusion defect.     

Calcitonin receptor binding as a marker of osteoclast heterogeneity in osteopetrotic rodents

We have employed a radioautographic technique to examine in vivo receptor binding of calcitonin to osteoclasts in four rodent mutants with osteopetrosis. 125I-Labeled calcitonin was injected intravenously alone or with excess unlabeled calcitonin to osteopetrotic (op/op), osteosclerotic (oc/oc), and microphthalmic (mi/mi) mice and to incisor absent (ia/ia) rats. Similar experiments were performed simultaneously in phenotypically normal littermates. Specific binding of calcitonin to receptors on osteoclasts and osteoclast morphology were then examined by light and electron microscope radioautography. Calcitonin binding was increased in mi/mi mice, where osteoclasts were abundant but reduced in size, and was also increased in op/op mice in association with an undulated and redundant osteoclast cell membrane. Binding of the hormone was markedly diminished on osteoclasts of oc/oc mice and ia/ia rats. Thus, in these rodent models of osteopetrosis all of which manifest reduced skeletal remodeling and share a recessive pattern of inheritance, considerable heterogeneity of osteoclast characteristics was demonstrable. Although calcitonin may play no primary pathogenetic role in most forms of this disease, calcitonin receptor binding is a morphological and functional marker of osteoclasts that can be used in assessing the pathophysiology of disorders of bone remodeling.     

The osteopetrotic syndrome in the microphthalmic mutant mouse

A histological and biochemical study of the osteopetrosis in the microphthalmic mutant mouse has been undertaken. All the classical features of the disease previously described in the grey-lethal mouse are found here. The microphthalmic mouse has a mild hypocalcaemia, hypophosphatemia, an increased serum alkaline phosphatase and greatly increased bone and serum citrate. Some mice are found with normal levels of all these parameters but histologically the severe osteopetrotic lesion is always present. In spite of this, 28% of the mice have a life expectancy in excess of three months. In this respect they differ from the grey-lethal mouse. A common aetiology is considered for the syndrome in both mutant mice.     

Osteocalcin fragment in bone matrix enhances osteoclast maturation at a late stage of osteoclast differentiation

Although 14-day-old mouse embryonic calvarial cells cultured in plastic culture dishes in the presence of 1,25-dihydroxyvitamin D₃[1,25-(OH)₂D₃] for 7 days could barely resorb bone slices, the same calvarial cells cultured with an ethylenediaminetetraacetic acid (EDTA) extract from bovine bone powder under the same conditions stimulated pit formation on bone slices in a dose-dependent manner. Therefore, the present study was conducted to purify and characterize this osteoclast maturation-inducing factor(s) from the bone matrix. The protein having osteoclast maturation-inducing activity in the EDTA extract was purified by gel filtration over Superdex 75 preparation grade and chromatography on hydroxyapatite, Mono Q, and C8 reversed-phase HPLC by monitoring the ability of the eluted fractions to elicit pit formation on bone slices. The molecular weight of the purified protein estimated by high-resolution polyacrylamide gel electrophoresis was 5.7kDa and 6.8kDa in the respective absence and presence of 2-mercaptoethanol. The sequence of the 30-amino-acid purified protein corresponded to the 7th to 36th residues of bovine osteocalcin. The osteocalcin fragment, missing the initial 6 residues at the N-terminal region, exhibited higher osteoclast maturation-inducing ability than bovine intact osteocalcin on a per weight basis. The osteocalcin fragment had no effect on the expression of receptor activator of nuclear factor (NF)-B ligand (RANKL) and osteoprotegerin (OPG) genes in calvarial cells, nor did it enhance the bone-resorbing activity of mature osteoclasts. When the osteocalcin fragment was added to late-stage (days 4–7) or to early-stage (days 0–3) cultures of calvarial cells pretreated with 1,25-(OH)₂D₃, its stimulatory effect was observed in the late-stage cultures rather than in the early-stage ones. In addition, the osteocalcin fragment directly enhanced the formation of osteoclasts with bone-resorbing ability from Mac-1⁺ c-Fms⁺ cells in the presence of macrophage colony-stimulating factor (MCSF) and RANKL. These results suggest that the osteocalcin fragment in bone matrix is involved in osteoclast maturation, especially at a late stage of osteoclast differentiation.     

Transforming growth factor-beta induces osteoclast ruffling and chemotaxis: potential role in osteoclast recruitment.

Transforming growth factor-beta (TGF-beta) is released from the matrix during bone resorption and has been implicated in the pathogenesis of giant cell tumors of bone and the expansion of breast cancer metastases in bone. Because osteoclasts mediate tumor-induced osteolysis, we investigated whether TGF-beta stimulates osteoclast recruitment. Osteoclasts were isolated from rat long bones and time-lapse video microscopy was used to monitor their morphology and motility. Within 5 minutes, TGF-beta (0.1 nM) induced dynamic ruffling, with 65% of osteoclasts displaying membrane ruffles compared with 35% in untreated controls. Over a 2-h period, osteoclasts exhibited significant directed migration toward a source of TGF-beta, indicating chemotaxis. echistatin, an alphavbeta3 integrin blocker that inhibits macrophage colony-stimulating factor (M-CSF)-induced osteoclast migration, did not prevent the migration of osteoclasts toward TGF-beta. In contrast, a beta1 integrin blocking antibody inhibited osteoclast chemotaxis toward TGF-beta but not M-CSF. These data indicate the selective use of integrins by osteoclasts migrating in response to different chemotaxins. In addition, wortmannin and U0126 inhibited TGF-beta-induced chemotaxis, suggesting involvement of the phosphatidylinositol 3 (PI 3) kinase and mitogen-activated protein (MAP) kinase signaling pathways. Physiologically, TGF-beta, may coordinate osteoclast activity by recruiting osteoclasts to existing sites of resorption. Pathologically, TGF-beta-induced osteoclast recruitment may be critical for expansion of primary and metastatic tumors in bone.     

Alpha9beta1: a novel osteoclast integrin that regulates osteoclast formation and function.

We identified a previously unknown integrin, alpha(9)beta(1), on OCLs and their precursors. Antibody to alpha(9) inhibited OCL formation in human marrow cultures, and OCLs from alpha(9) knockout mice had a defect in actin ring reorganization and an impaired bone resorption capacity. INTRODUCTION: Integrins play important roles in osteoclast (OCL) formation and function. Mature OCLs mainly express alpha(v)beta(3) integrin, a heterodimer adhesion receptor that has been implicated in osteoclastic bone resorption. We identified ADAM8, a disintegrin and metalloproteinase, as a novel stimulator of OCL differentiation and showed that the disintegrin domain of ADAM8 mediated its effects on OCL formation. Because the disintegrin domain of ADAM8 does not bind Arg-Gly-Asp (RGD) sequences, we determined which integrin bound ADAM8 and characterized its role in OCL formation and activity. MATERIALS AND METHODS: Chinese hamster ovary cells (CHO) expressing different integrin subunits were tested for their capacity to bind the disintegrin domain of ADAM8. Mouse or human bone marrow cells and purified OCL precursors were tested for alpha(9)beta(1) integrin expression by Western blot, immunocytochemistry, and real-time RT-PCR. A monoclonal antibody to human alpha(9) was used to block alpha(9)beta(1) on OCL precursors stimulated by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] or RANKL. Vertebrae of 7-day-old alpha(9)(-/-) mice and wildtype (WT) littermates were compared using bone histomorphometry and 3D microCT analysis. RESULTS: Alpha(9) integrin was expressed by mouse and human bone marrow-derived OCLs and their precursors. Importantly, the anti-alpha(9) antibody inhibited human OCL formation stimulated by 1alpha,25(OH)(2)D(3) or RANKL dose-dependently. Furthermore, analysis of OCLs formed in marrow cultures from alpha(9)(-/-) mice showed that the OCLs formed were more contracted and formed significantly less bone resorption pits on dentin slices. Histologic analysis of alpha(9)(-/-) vertebrae showed thickened trabecular regions and retained cartilage within vertebral bodies of alpha(9)(-/-) mice. 3D microCT analysis of alpha(9)(-/-) vertebrae also showed a significant increase in trabecular bone volume/total tissue volume and a tendency for decreased trabecular separation compared with WT mice. CONCLUSIONS: These results support a previously unknown role for alpha(9)beta(1) integrin in OCL formation and function.     

Ribosome inactivating protein B-chain induces osteoclast differentiation from monocyte/macrophage lineage precursor cells

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between lectins and their receptors. A type-2 ribosome inactivating protein consists of an A chain and a B chain. The glycosylated B chain binds specifically to galactose moieties of sugar molecules. In this study we showed that the recombinant ribosome inactivating protein B-chain (rRBC) could induce osteoclast formation from human monocytes and murine RAW264.7 macrophages. Tartrate-resistant acid phosphatase (TRAP) staining and bone resorption assays demonstrated that differentiation of osteoclast-like cells was induced in the presence of rRBC in a dose-dependent manner. The rRBC-induced osteoclast differentiation was independent of caspase activation and apoptosis induction activity; however, rRBC-induced osteoclastogenesis was dependent on activation of NF-κB, ERK1/2, and p38 MAP kinase. Thus, our data demonstrated that rRBC induced osteoclast differentiation through a non-apoptotic signaling pathway. In addition to triggering apoptosis, the rRBC also induced osteoclast differentiation. According to this study, a novel role is proposed for rRBC in regulating osteoclast differentiation and in osteoimmunology.     

Qualitative defects in natural killer cell function in ia osteopetrotic rats.

Recent studies have provided evidence that cells of the immune system and their associated cytokines function in the regulation of bone turnover. The incisors absent (ia) osteopetrotic rat represents a model in which a defect in the immune system and bone resorption can be studied. Osteopetrosis in the ia rat is characterized by a generalized excess accumulation of bone as a result of reduced bone resorption by defective osteoclasts that lack a ruffled border and the ability to exocytose their osteolytic enzymes. Previous attempts to identify associated defects in the ia immune system have proven unsuccessful; ia rats demonstrate normal delayed hypersensitivity, mitogenic activity, and macrophage function. Inasmuch as the skeletal manifestations of the ia mutation may be the result of a defect in exocytosis, related defects may be evident in immune cells utilizing exocytosis of granules or enzymes for their cytolytic function. Natural killer (NK) cells function by such a mechanism. Therefore, these studies were undertaken to evaluate the natural immune system in ia rats. NK activity assessed by 51Cr release assays was significantly reduced in ia animals compared to normal littermates. Mononuclear cells isolated from the peripheral blood of ia rats revealed a significantly greater percentage of large granular lymphocytes than normal littermates. Comparison of NK cell phenotypes using two phenotypic parameters for NK cells (OX8+, OX19- cells and 3.2.3+ cells) revealed that the mononuclear isolates of spleen and peripheral blood of mutant animals had significantly greater percentages of OX8+, OX19- and 3.2.3+ cells than normal controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transforming growth factor-beta induces osteoclast formation in the absence of RANKL

Apoptosis plays an important role in the regulation of bone turnover. Previously, we showed that 1,25(OH)2D3, the active form of vitamin D, may increase osteoblast survival by inhibiting apoptosis induced by serum deprivation. Human osteoblasts express the Fas receptor on their surface and its interaction with Fas ligand has been closely associated with human osteoblast apoptosis. To investigate the mechanism of 1,25(OH)2D3 inhibition of apoptosis in osteoblasts isolated from human calvaria, cells were exposed to Fas antibody. Visualization of apoptotic cells using annexin V revealed a significant decrease in apoptosis at 48 h in the presence of 1,25(OH)2D3 (14 +/- 4%, P < 0.04) compared with non-treated cells (52 +/- 4%). Furthermore, flow cytometric analysis of TUNEL-labeled osteoblasts showed a significant decrease in apoptotic cells in 1,25(OH)2D3-treated cultures (12 +/- 2%) at 48 h compared with non-treated cultures (44 +/- 3%, P < 0.04). Additionally, cells treated with 1,25(OH)2D3 survived longer as found by MTS analysis. To further explore the mechanism of 1,25(OH)2D3-mediated inhibition of apoptosis, we examined the changes in activation of death domain proteins, cleavage of caspases and mitochondrial regulators of apoptosis by Western blot analysis. A significant inhibition of caspase-8 cleavage and activity in 1,25(OH)2D3-treated cells was observed in conjunction with a decrease in the expression of the proapoptotic protein Bax with a significant increase in the expression of antiapoptotic protein Bcl-2. Furthermore, the levels of p21Cip1/WAF1, which inhibits the cleavage of caspase-8, was found to be highly induced in 1,25(OH)2D3-treated cells. In summary, these results demonstrate that the anti-apoptotic effect of 1,25(OH)2D3 in human osteoblasts after the activation of Fas-ligand is mediated by the regulation of components of both the mitochondrial and Fas-related pathways.     

Impairment of macrophage colony-stimulating factor production and lack of resident bone marrow macrophages in the osteopetrotic op/op mouse

Mouse calvaria-derived osteoblastlike cells have been shown to produce macrophage colony-stimulating factor (M-CSF). This factor may be involved in osteoclastogenesis and thus in bone resorption. In the present study we investigated whether the production of M-CSF was altered in the osteopetrotic mouse mutant strain op/op, characterized by a decrease in osteoclast number and an impairment of bone resorption. Whole calvariae and cells, as well as skin and lung fibroblasts, of the op/op mouse were found to produce no measurable M-CSF, in contrast to tissue and cells derived from normal littermates. M-CSF was identified by colony assay in semisolid media and by inhibition of the biologic activity with antiserum against M-CSF. Furthermore, the number of resident macrophages, identified by F4/80 antigen (F4/80 Ag) immunohistochemistry, was drastically decreased in bone and bone marrow of the op/op mouse, but in skin these cells were normal in number and morphology. These findings suggest that both M-CSF and resident macrophages play a role in the mechanism of bone resorption. The op/op mouse appears to be a valuable model to further investigate such a hypothesis.     

Expression of calcitonin receptors during osteoclast differentiation in mouse metatarsals

Metatarsal bones of 15-day-old mouse embryos contain proliferative tartrate-resistant acid phosphatase (TRAP) negative (-) osteoclast progenitors that progressively differentiate into multinucleated TRAP positive (+) osteoclasts. Using histochemical and autoradiographic techniques, we have examined the expression of calcitonin receptors during osteoclast differentiation in mouse metatarsals. Fresh mouse metatarsals from embryos aged 14-17 days and metatarsals from 15-day-old embryos cultured for 1, 2, 3, and 6 days were stained for TRAP. Calcitonin binding to osteoclasts and their precursors was studied by incubating metatarsals with [125I]salmon calcitonin (sCT) and quantitating grain counts from autoradiographs of tissue sections. Calcitonin receptors first appear on nonproliferating osteoclast precursors, most often just after or simultaneously with the development of TRAP activity. The effect of sCT on the development of TRAP+ mononuclear preosteoclasts was examined by culturing 15-day-old metatarsals in the continuous presence of 5 mU sCT for periods of up to 3 days and quantitating the number of TRAP+ mononuclear preosteoclasts that develop. Calcitonin did not affect the differentiation of osteoclasts up to the stage of the TRAP+ mononuclear preosteoclast.     

Xenogeneic skin graft rejection in M-CSF/macrophage deficient osteopetrotic mice

Zhao Y, Xiong W, Yang T, Prall A, Baxter BT, Langnas AN. Xenogeneic skin graft rejection in M‐CSF/macrophage deficient osteopetrotic mice. Xenotransplantation 2003; 10: 232–239. © Blackwell Munksgaard, 2003 Background: The cellular infiltrate in xenografts suggests that macrophages may be involved in xenograft rejection. However, the precise role of macrophages in xenograft rejection has not yet been fully addressed. Methods: Xenogeneic rat skin grafts were transplanted to macrophage colony stimulating factor (M‐CSF)/macrophage‐deficient osteopetrotic ([OP]^–/–) and wild‐type control mice. Skin graft survival and antidonor rat humoral responses were quantified. Results: Xenogeneic rat skin grafts survived 13 days in wild‐type control mice, survival of rat skin grafts was significantly prolonged to 24 days in [OP]^–/– mice (P<0.01). Similar results were observed in sensitized [OP]^–/– and control mouse recipients, showing markedly prolonged rat skin graft survival in [OP]^–/– mice. Levels of T‐cell‐dependent antirat antibodies [immunoglobulin G (IgG)2a and IgG3] in sera of [OP]^–/– mice were significantly lower than that of control mice 2 weeks post‐rat skin grafting. The proliferative responses to xenogeneic rats not to allogeneic mouse stimulation of T cells from [OP]^–/– mice were significantly lower than that of wild‐type mice. However, neutrilization of M‐CSF by anti‐M‐CSF monoclonal antibody (mAb) or the addition of M‐CSF to the in vitro culture systems of wild‐type or [OP]^–/– mouse T‐responder cells, respectively, did not significantly change proliferative responses and cytolytic function against xenogeneic rat targets of wild‐type or [OP]^–/– mouse T‐responder cells. Conclusions: The in vitro data indicate that M‐CSF does not directly regulate cellular immune responses to xenoantigens. The present studies indicate that macrophages may play an important role in immune rejection of xenografts. The precise role of macrophages in xenograft rejection should be further investigated.     

Altered osteoclast development and function in osteopontin deficient mice

The role of osteopontin in bone resorption was elucidated by studies of mice with knock out of the osteopontin gene generated by a different approach compared to previous models. Thus, a targeting vector with the promoter region as well as exons 1, 2, and 3 of the osteopontin gene was replaced by a loxP‐flanked Neo‐TK cassette, and this cassette was eliminated through transient expression of Cre recombinase. The recombined ES cells were used to create mice lacking expression of the osteopontin gene. Tissues from these mice were subjected structural and molecular analyses including morphometry and proteomics. The bone of the null mice contained no osteopontin but showed no significant alterations with regard to other bone proteins. The bone volume was normal in young null animals but in the lower metaphysis, the volume and number of osteoclasts were increased. Notably, the volume and length of the osteoclast ruffled border was several folds lower, indicating a lower resorptive capacity. The null mice did not develop the bone loss characteristic for osteoporosis demonstrated in old wild‐type female animals. This quantitative study demonstrates a bone phenotype in the osteopontin null mice of all ages. The data provides further evidence for a role of osteopontin in osteoclast activity. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:721–728, 2008     

Hydrocortisone rapidly induces aortic rupture in a genetically susceptible mouse

The Blotchy mouse has an X-linked trait that leads to aortic aneurysms and subsequent fatal rupture in nearly all affected male mice. Heterozygous female mice occasionally develop aneurysms, but they rarely rupture. Ten heterozygous female mice received 0.45 mg/mL of hydrocortisone acetate in drinking water. Within 2 weeks, 9 of 10 mice were dead (6 with proved aortic rupture, 3 with presumed rupture). The 10th mouse was documented to have an aortic aneurysm. A dose-response curve was generated. Hydrocortisone's effect was shown to be dose-dependent. In another experiment, normal female mice received 0.10 mg/mL of hydrocortisone acetate for 14 days. Two mice developed aneurysms, and the others developed aortic ectasia. These experiments establish the role of hydrocortisone in the induction of aortic rupture in a mouse with genetic susceptibility and the induction of aneurysms and ectasia in normal mice.