The synergistic role of Pu.1 and Fms in zebrafish osteoclast-reducing osteopetrosis and possible therapeutic strategies

Osteoclasts are bone resorption cells of myeloid origin. Osteoclast defects can lead to osteopetrosis, a genetic disorder characterized by bone sclerosis for which there is no effective drug treatment. It is known that Pu.1 and Fms are key regulators in myelopoiesis, and their defects in mice can lead to reduced osteoclast numbers and consequent osteopetrosis. Yet how Pu.1 and Fms genetically interact in the development of osteoclasts and the pathogenesis of osteopetrosis is still unclear. Here, we characterized pu.1^G242D;fms^j4e1 double-deficient zebrafish, which exhibited a greater deficiency of functional osteoclasts and displayed more severe osteopetrotic symptoms than the pu.1^G242D or fms^j4e1 single mutants, suggesting a synergistic function of Pu.1 and Fms in the regulation of osteoclast development. We further demonstrated that Pu.1 plays a dominant role in osteoclastogenesis, whereas Fms plays a dominant role in osteoclast maturation. Importantly, treatment with the drug retinoic acid significantly relieved the different degrees of osteopetrosis symptoms in these models by increasing the number of functional osteoclasts. Thus, we report the development of valuable animal models of osteopetrosis, and our results shed light on drug development for antiosteopetrosis therapy.     

Secretion of a Truncated Osteopetrosis-associated Transmembrane Protein 1 (OSTM1) Mutant Inhibits Osteoclastogenesis through Down-regulation of the B Lymphocyte-induced Maturation Protein 1 (BLIMP1)-Nuclear Factor of Activated T Cells c1 (NFATc1) Axis

Genetic mutations in osteoclastogenic genes are closely associated with osteopetrotic bone diseases. Genetic defects in OSTM1 (osteopetrosis-associated transmembrane protein 1) cause autosomal recessive osteopetrosis in humans. In particular, OSTM1 mutations that exclude the transmembrane domain might lead to the production of a secreted form of truncated OSTM1. However, the precise role of the secreted form of truncated OSTM1 remains unknown. In this study, we analyzed the functional role of truncated OSTM1 in osteoclastogenesis. Here, we showed that a secreted form of truncated OSTM1 binds to the cell surface of osteoclast (OC) precursors and inhibits the formation of multinucleated OCs through the reduction of cell fusion and survival. Truncated OSTM1 significantly inhibited the expression of OC marker genes through the down-regulation of the BLIMP1 (B lymphocyte-induced maturation protein 1)-NFATc1 (nuclear factor of activated T cells c1) axis. Finally, we demonstrated that truncated OSTM1 reduces lipopolysaccharide-induced bone destruction in vivo. Thus, these findings suggest that autosomal recessive osteopetrosis patients with an OSTM1 gene mutation lacking the transmembrane domain produce a secreted form of truncated OSTM1 that inhibits osteoclastogenesis.     

Untersuchungen zur Mineralisierungsdichte im Hartgewebe mit Protein-Polysaccharid bzw. mit Kollagen als Hauptbestandteil der Matrix

Zusammenfassung Ultradünnschnitte von schmelznahem Dentin sowie von Osteopetroseknochen-Partien zeigten im Elektronenmikroskop, daß die proteinpolysaccharid-reichen Zonen des peritubulären Dentins sowie der sog. dichten Zonen des Osteopetrose-Knochens punktartige Calzium-Phosphat-Keime bzw. Ketten von Punktkeimen in dichter Zusammenlagerung besitzen, während in den kollagenreichen Zonen neben diesen Ketten und Nadeln größere Blättchen-Kristallite auftreten. Durch Entmineralisierung-Kontrastierung der Ultradünnschnitte mit Phosphorwolframsäure wurde gezeigt, daß im peritubulären Dentin und den dichten Partien des Osteopetroseknochens elektronenmikroskopisch keine Kollagenfasern mit Querstreifen vorkommen; diese sind aber im intertubulären Dentin und den normalen Partien des Osteopetroseknochens verbreitet. Durch Registrierung der Calzium- und Phosphor-Röntgenimpulse mit der elektronenmikroskopischen Mikrosonde (Cameca) konnte — bei Annahme angenähert gleicher Schnitt-Dicke in benachbarten Zonen — gefunden werden, daß der Calzium-Gehalt pro Volumeneinheit im Mittel von 100 Einzelmessungen im peritubulären Dentin um etwa 40 % höher liegt als im kollagenreichen intertubulären Dentin und in den proteinpolysaccharid-reichen Zonen der Osteopetrose-Proben um 65 % höher als in den normalen kollagenreichen Bezirken. Da entsprechende Werte für Phosphor gefunden wurden, bedeuten diese Prozentangaben zugleich dieselben Unterschiede in der Mineralisierungsdichte.

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Investigations of mineralization in hard tissues with protein-polysaccharides or collagen as main constituents of the matrix

Summary Ultrathin sections of mature dentine in the vicinity of enamel and sections of osteopetrotic bone were examined. In the protein-polysaccharide-rich regions of the peritubular dentine, as well as in the so-called dense zones of the osteopetrotic bone, dot-like nuclei, or short chains of them, in close packing were found, whereas in the intertubular collagen-rich dentine zones, as well as the normal regions of osteopetrotic bone, many platelike crystals were found among the chains of dots. By demineralization-staining experiments with phosphotungstic acid, it was found that in the peritubular dentine as well as the so-called dense zones of the osteopetrotic bone there were no collagen fibres visible, while they dominated in the intertubular dentine as well as the normal regions of the osteopetrotic bone. The calcium and phosphorous concentrations in the sections were examined in the Cameca electronmicroscopical probe. Assuming that neighbouring regions were of nearly equal thickness, the mean calcium content per unit volume from 100 single point measurements for each region was found to be about 40 % higher in the peritubular region than in the collagen-rich intertubular region, and about 65 % higher in the so-called dense regions of the osteopetrose bone than in the normal collagen-rich zones. The same was found for phosphorous.

Wir danken Frl. Ch. Lippert, Frl. I. Schütte und Herrn J. Schreiber für wertvolle Hilfe bei der technischen Durchführung der Untersuchungen. Der Deutschen Forschungsgemeinschaft danken wir für die Unterstützung unserer Arbeit.     

Low-density lipoprotein receptor deficiency causes impaired osteoclastogenesis and increased bone mass in mice because of defect in osteoclastic cell-cell fusion

Osteoporosis is associated with both atherosclerosis and vascular calcification attributed to hyperlipidemia. However, the cellular and molecular mechanisms explaining the parallel progression of these diseases remain unclear. Here, we used low-density lipoprotein receptor knockout (LDLR(-/-)) mice to elucidate the role of LDLR in regulating the differentiation of osteoclasts, which are responsible for bone resorption. Culturing wild-type osteoclast precursors in medium containing LDL-depleted serum decreased receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation, and this defect was additively rescued by simultaneous treatment with native and oxidized LDLs. Osteoclast precursors constitutively expressed LDLR in a RANKL-independent manner. Osteoclast formation from LDLR(-/-) osteoclast precursors was delayed, and the multinucleated cells formed in culture were smaller and contained fewer nuclei than wild-type cells, implying impaired cell-cell fusion. Despite these findings, RANK signaling, including the activation of Erk and Akt, was normal in LDLR(-/-) preosteoclasts, and RANKL-induced expression of NFATc1 (a master regulator of osteoclastogenesis), cathepsin K, and tartrate-resistant acid phosphatase was equivalent in LDLR-null and wild-type cells. In contrast, the amounts of the osteoclast fusion-related proteins v-ATPase V(0) subunit d2 and dendritic cell-specific transmembrane protein in LDLR(-/-) plasma membranes were reduced when compared with the wild type, suggesting a correlation with impaired cell-cell fusion, which occurs on the plasma membrane. LDLR(-/-) mice consistently exhibited increased bone mass in vivo. This change was accompanied by decreases in bone resorption parameters, with no changes in bone formation parameters. These findings provide a novel mechanism for osteoclast differentiation and improve the understanding of the correlation between osteoclast formation and lipids.     

Mutation of macrophage colony stimulating factor (Csf1) causes osteopetrosis in the tl rat

Osteopetrosis results from a heterogeneous group of congenital bone diseases that display inadequate osteoclastic bone resorption. We recently mapped tl (toothless), a mutation that causes osteopetrosis in rats, to a genetic region predicted to include the rat Csf1 gene. In this study, we sequenced the coding sequence of the rat Csf1 gene to determine if a mutation in Csf1 could be responsible for the tl phenotype. Sequencing revealed a 10-base insertion in the coding sequence of mutant animals that produces a frameshift and generates a stop codon early in the mutant Csf1 coding sequence. The 41 amino acid polypeptide predicted to be produced from the Csf1 promoter would have only the first nine amino acids of the wild-type rat protein. These data suggest that osteopetrosis develops in tl/tl rats because they cannot produce functional mCsf, a growth factor required for osteoclast differentiation and activation.     

Severe developmental bone phenotype in ClC-7 deficient mice

Bone development is dependent on the functionality of three essential cell types: chondrocytes, osteoclasts and osteoblasts. If any of these cell types is dysfunctional, a developmental bone phenotype can result.The bone disease osteopetrosis is caused by osteoclast dysfunction or impaired osteoclastogenesis, leading to increased bone mass. In ClC-7 deficient mice, which display severe osteopetrosis, the osteoclast malfunction is due to abrogated acidification of the resorption lacuna. This study sought to investigate the consequences of osteoclast malfunction on bone development, bone structure and bone modeling/remodeling in ClC-7 deficient mice. Bones from wildtype, heterozygous and ClC-7 deficient mice were examined by bone histomorphometry and immunohistochemistry.ClC-7 deficient mice were found to have a severe developmental bone phenotype, characterized by dramatically increased bone mass, a high content of cartilage remnants, impaired longitudinal and radial growth, as well as lack of compact cortical bone development. Indices of bone formation were reduced in ClC-7 deficient mice; however, calcein labeling indicated that mineralization occurred on most trabecular bone surfaces. Osteoid deposition had great regional variance, but an osteopetrorickets phenotype, as observed in oc/oc mice, was not apparent in the ClC-7 deficient mice. A striking finding was the presence of very large abnormal osteoclasts, which filled the bone marrow space within the ClC-7 deficient bones. The development of these giant osteoclasts could be due to altered cell fate of the ClC-7 deficient osteoclasts, caused by increased cellular fusion and/or prolonged osteoclast survival.In summary, malfunctional ClC-7 deficient osteoclasts led to a severe developmental bone phenotype including abnormally large and non-functional osteoclasts. Bone formation paremeters were reduced; however, bone formation and mineralization were found to be heterogenous and continuing.     

Osteoclasts secrete non-bone derived signals that induce bone formation

Bone turnover is a highly regulated process, where bone resorption in the normal healthy individual always is followed by bone formation in a manner referred to as coupling. Patients with osteopetrosis caused by defective acidification of the resorption lacuna have severely decreased resorption, in face of normal or even increased bone formation. This suggests that osteoclasts, not their resorptive activity, are important for sustaining bone formation. To investigate whether osteoclasts mediate control of bone formation by production of bone anabolic signals, we collected conditioned media (CM) from human osteoclasts cultured on either bone or plastic, and tested their effects on bone nodule formation by osteoblasts. Both types of CM were shown to dose-dependently induce bone nodule formation, whereas non-conditioned osteoclast culture medium had no effects. These data show that osteoclasts secrete non-bone derived factors, which induce preosteoblasts to form bone-like nodules, potentially explaining the imbalanced coupling seen in osteopetrotic patients.     

Osteoclast signalling pathways

The osteoclast is a monocyte-derived cell with complex regulatory control due to its role, balancing calcium homeostasis with skeletal modelling and repair. Normal differentiation requires tyrosine kinase- and tumor necrosis-family receptors, normally fms and RANK. Ligands for these receptors plus unidentified serum or cell-presented factor(s) are needed for in vitro differentiation, possibly signalling via an immune-like tyrosine kinase acceptor molecule. Osteoclast development and activity are increased by cytokines signalling through GP130, such as IL-6, by TGF-beta, and by IL-1, although these cannot replace serum. Other tyrosine kinase receptors including kit and met can augment fms signalling, and TNFs other than RANKL, including TNFalpha and TRAIL, modify RANK signalling, which is also susceptible to interference by interferons. The situation is further complicated by G-protein coupled receptors including the calcitonin receptor, by integrin or calcium-mediated signals, and by estrogen receptors, which operate in bone largely via NO downstream signals. Differentiation, activity, and survival signals merge in intracellular second messengers. These include cytoplasmic kinases of several families; differentiation pathways often terminate in Erk/Jun kinases or NF-kappaB. Key regulatory intermediates include TRAF6, src, Smad3, phosphatidylinositol-3-kinase, Jak/Stat, and the cGMP-dependent protein kinase I. There are substantial uncertainties regarding how intracellular agents connect to primary signals. The frontier includes characterization of how scaffolding/adapter proteins, such as cbl, gab, grb, p130Cas, and shc, as well as itam-containing proteins and nonreceptor tyrosine kinase adapters of the src and syk families, delimit and integrate signals of multiple receptors to bring about specific outcomes.     

Cellular response to ectopically implanted silk sutures and osteopetrotic bone

Summary Faulty osteoclasts, characteristic of the incisors-absent (ia) rat mutation of osteopetrosis, cause a resorptive defect which results in the persistence of immature, highly mineralized bone matrix. We implanted osteopetrotic bone subcutaneously into normal andia rats to determine ifia bone could induce functionally active and morphologically identifiable osteoclasts at the implant surface. Assays of⁴⁵Ca released from the preparations showed that normal andia recipients were capable of equivalent cell-mediated release of Ca over a 2-week implant period, indicating that theia resorptive defect was not reproduced at the subcutaneous site. Freeze-thawed osteopetrotic bone released twice as much⁴⁵Ca as normal bone. This difference was eliminated by collagenase treatment. Cellular profiles were similar in both normal andia animals regardless of the implant preparation. At 3 days after implantation, both bone and suture were surrounded by mononuclear cells. By 14 days, multinucleated cells appeared at the implant surfaces. Morphological comparison of implant-induced multinucleated cells and tibial osteoclasts indicated that bone-elicited multinucleated cells lacked the ruffled borders characteristic of normal osteoclasts or the extensive clear zones typical ofia osteoclasts, but more closely resembled suture-induced macrophage-polykaryons. We conclude that ectopically implantedia bone as compared to normal bone elicits a different functional response from structurally similar cell populations. Bone-elicited multinucleated cells could not be classified as active osteoclasts despite evidence of release of⁴⁵Ca. Release of labeled Ca was probably due to the action of mononuclear phagocytes and macrophage-polykaryons rather than to osteoclastic resorption.     

Bone scintigraphy leading to diagnosis of malignant osteopetrosis in a six-year-old boy

IntroductionOsteopetrosis is a rare disease but early detection is essential for future improvement of quality of life. Despite X-ray can be a diagnostic examination itself, bone scintigraphy can be helpful when the radiographic imaging is not typical.Case reportWe report a case of a 6-year-old boy with pain and limping of the left lower limb persisting 4 months after a left ankle sprain. He underwent a whole-body bone scintigraphy with HDP-99mTc and overlapping image focused on the hip, which showed abnormal increased diffuse uptake in proximal and distal epiphysis of femurs and tibias showing higher uptake in femurs. It also showed, involvement of proximal epiphysis of humerus. Kidneys were not visible, suggesting bone superscan. Differential diagnosis was recommended with osteopetrosis, osteomalacy.ConclusionBone scintigraphy can contribute for extensive skeletal evaluation and patients follow-up. Thus, bone scintigraphy may be useful in symptomatic and in asymptomatic patients with OP.