acid-phosphatase and Osteopetrosis

Numerous experimental and clinical observations suggest that overall changes in bone resorption during menopause or treatment with hormone replacement therapy (HRT) are combined effects of changes in osteoclast number and function. Moreover, due to a coupling between osteoclastic bone resorption and osteoblastic bone formation, pronounced alteration of osteoclast number will eventually lead to alteration of osteoblastic bone formation. Fragments of type I collagen, such as the C- and N-terminal telopeptides of collagen type I (CTX and NTX, respectively), are generated during bone resorption and hence can be used as surrogate markers of osteoclast function. Circulating levels of different enzymes in the serum, such as TRAP 5b and cathepsin K are proportional to the number of osteoclasts, and hence can be used as surrogate markers of osteoclast number. Since antiresorptive effects can be obtained in different ways, we felt it was timely to discuss the different scenarios, highlight differences specific to different pharmacological interventions with different mechanisms of action, and discuss how these bone markers can assist us in a deeper analysis of the pharmacodynamics and safety profile of existing and upcoming drug candidates.

Topics: Acid Phosphatase; Biomarkers; Bone Density Conservation Agents; Bone Resorption; Cathepsin K; Cathepsins; Cell Count; Cell Differentiation; Collagen Type I; Female; Humans; Isoenzymes; Osteoclasts; Osteopetrosis; Osteoporosis; Peptides; Tartrate-Resistant Acid Phosphatase

TRACP is a lysosomal enzyme found in diverse tissues, where it is expressed in dendritic cells as well as osteoclasts and macrophages. To investigate the function of TRACP in vivo, we have generated mice in which the gene-encoding TRACP has been selectively disrupted by targeted homologous recombination in murine embryonic stem cells. Homozygous TRACP "knockout" mice have progressive foreshortening and deformity of the long bones and axial skeleton suggesting a role for TRACP in endochondral ossification. There is increased mineralization reflecting a mild osteopetrosis caused by reduced osteoclast modeling activity. These knockout mice also display an impairment of macrophage function with abnormal immunomodulatory cytokine responses. Superoxide formation and nitrite production were enhanced in stimulated macrophages lacking TRACP as was the secretion of the proinflammatory cytokines TNF-alpha, interleukin (IL)-1beta, and IL-12. TRACP knockout mice showed delayed clearance of the microbial pathogen Staphylococcus aureus after sublethal intraperitoneal inoculation. The macrophages lacking TRACP showed an increase in tartrate-sensitive lysosomal acid phosphatase activity (LAP). The TRACP knockout mice were bred with mice lacking LAP. Mice lacking both TRACP and LAP had even shorter bones than the TRACP single knockouts. Osteopontin, identical to the T-cell cytokine eta-1, accumulated adjacent to actively resorbing osteoclasts suggesting that both phosphatases are important for processing this protein. We propose that TRACP may be an important regulator of osteopontin/eta-1 activity common to both the immune system and skeleton.

Topics: Acid Phosphatase; Animals; Bone and Bones; Dendritic Cells; Homozygote; Interleukin-1; Interleukin-12; Isoenzymes; Lysosomes; Macrophages; Mice; Mice, Knockout; Osteopetrosis; Phenotype; Staphylococcus aureus; Superoxides; Tartrate-Resistant Acid Phosphatase; Time Factors; Tumor Necrosis Factor-alpha

Osteoclasts play important roles during bone growth and in maintaining bone health and bone homeostasis. Dysfunction or lack of osteoclasts leads to increased bone mass and osteopetrosis phenotypes in mouse and human. Here we report a severe osteopetrosis-like phenotype in transgenic medaka fish, in which membrane bound EGFP (mEGFP) was expressed in osteoclasts under control of the cathepsin K promoter (ctsk:mEGFP). In contrast to reporter lines with GFP expression in the cytoplasm of osteoclasts, adult fish of the mEGFP line developed bone defects indicative for an osteoclast dysfunction. Activity of tartrate-resistant acid phosphatase (TRAP) was down-regulated and excess bone was observed in most parts of the skeleton. The osteopetrotic phenotype was particularly obvious at the neural and haemal arches that failed to increase their volume in growing fish. Excess bone caused severe constriction of the spinal cord and the ventral aorta. The continuation of tooth development and the failure to shed teeth resulted in severe hyperdontia. Interestingly, at the vertebral column vertebral body arches displayed a severe osteopetrosis, while vertebral centra had no or only a mild osteopetrotic phenotype. This confirms previous reports from cichlids that, different from the arches, allometric growth of fish vertebral centra initially does not depend on the action of osteoclasts. Independent developmental mechanism that shapes arches and vertebral centra can also lend support to the hypothesis that vertebral centra and arches function as independent developmental modules. Together, this medaka osteopetrosis model confirms the importance of proper osteoclast function during normal skeletal development in teleost fish that requires bone modeling and remodeling.

Topics: Acid Phosphatase; Animals; Animals, Genetically Modified; Bone Remodeling; Cell Differentiation; Female; Isoenzymes; Male; Oryzias; Osteoclasts; Osteopetrosis; Phenotype; Tartrate-Resistant Acid Phosphatase

To assess the roles of Lrrk1 and Lrrk2, we examined skeletal phenotypes in Lrrk1 and Lrrk2 knockout (KO) mice. Lrrk1 KO mice exhibit severe osteopetrosis caused by dysfunction of multinucleated osteoclasts, reduced bone resorption in endocortical and trabecular regions, and increased bone mineralization. Lrrk1 KO mice have lifelong accumulation of bone and respond normally to the anabolic actions of teriparatide treatment, but are resistant to ovariectomy-induced bone boss. Precursors derived from Lrrk1 KO mice differentiate into multinucleated cells in response to macrophage colony-stimulating factor (M-CSF)/receptor activator of NF-κB ligand (RANKL) treatment, but these cells fail to form peripheral sealing zones and ruffled borders, and fail to resorb bone. The phosphorylation of cellular Rous sarcoma oncogene (c-Src) at Tyr-527 is significantly elevated whereas at Tyr-416 is decreased in Lrrk1-deficient osteoclasts. The defective osteoclast function is partially rescued by overexpression of the constitutively active form of Y527F c-Src. Immunoprecipitation assays in osteoclasts detected a physical interaction of Lrrk1 with C-terminal Src kinase (Csk). Lrrk2 KO mice do not show obvious bone phenotypes. Precursors derived from Lrrk2 KO mice differentiate into functional multinucleated osteoclasts. Our finding of osteopetrosis in Lrrk1 KO mice provides convincing evidence that Lrrk1 plays a critical role in negative regulation of bone mass in part through modulating the c-Src signaling pathway in mice.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Bone and Bones; Bone Density; Bone Resorption; CSK Tyrosine-Protein Kinase; Female; Gene Targeting; Isoenzymes; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Mice; Mice, Knockout; Mutant Proteins; Osteoclasts; Osteogenesis; Osteopetrosis; Ovariectomy; Peptide Fragments; Phenotype; Phosphorylation; Procollagen; Protein Binding; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins pp60(c-src); src-Family Kinases; Tartrate-Resistant Acid Phosphatase; X-Ray Microtomography

Receptor activator of nuclear factor-κB ligand (RANKL) inhibitors are being considered for use in children with osteogenesis imperfecta (OI). We sought to assess efficacy of two doses of a RANKL inhibitor, osteoprotegerin-immunoglobulin Fc segment complex (OPG-Fc), in a growing animal model of OI, the col1α2-deficient mouse (oim/oim) and its wild-type controls (+/+).. Treated mice showed runting and radiographic evidence of osteopetrosis with either high- (20 mg/kg twice weekly) or low-dose (1 mg/kg/week) OPG-Fc. Because of this adverse event, OPG-Fc treatment was halted, and the mice were killed or monitored for recovery with monthly radiographs and assessment of serum osteoclast activity (tartrate-resistant acid phosphatase 5b, TRACP-5b) until 25 wk of age.. Twelve weeks of OPG-Fc treatment resulted in radiographic and histologic osteopetrosis with no evidence of bone modeling and negative tartrate-resistant acid phosphatase staining, root dentin abnormalities, and TRACP-5b activity suppression. Signs of recovery appeared 4-8 wk post-treatment.. Both high- and low-dose OPG-Fc treatment resulted in osteopetrotic changes in infant mice, an outcome that was not seen in studies with the RANKL inhibitor RANK-immunoglobulin Fc segment complex (RANK-Fc) or in studies with older animals. Further investigations of RANKL inhibitors are necessary before their consideration for use in children.

Topics: Acid Phosphatase; Age Factors; Animals; Biomarkers; Bone Remodeling; Collagen Type I; Dentin; Disease Models, Animal; Female; Immunoconjugates; Immunoglobulin Fc Fragments; Isoenzymes; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Osteoclasts; Osteogenesis Imperfecta; Osteopetrosis; Osteoprotegerin; Radiography; RANK Ligand; Risk Assessment; Tartrate-Resistant Acid Phosphatase; Time Factors; Tooth Eruption; Weight Gain

Controlling osteoclastogenesis is critical to maintain physiological bone homeostasis and prevent skeletal disorders. Although signaling activating nuclear factor of activated T cells 1 (NFATc1), a transcription factor essential for osteoclastogenesis, has been intensively investigated, factors antagonistic to NFATc1 in osteoclasts have not been characterized. Here, we describe a novel pathway that maintains bone homeostasis via two transcriptional repressors, B cell lymphoma 6 (Bcl6) and B lymphocyte-induced maturation protein-1 (Blimp1). We show that Bcl6 directly targets 'osteoclastic' molecules such as NFATc1, cathepsin K, and dendritic cell-specific transmembrane protein (DC-STAMP), all of which are targets of NFATc1. Bcl6-overexpression inhibited osteoclastogenesis in vitro, whereas Bcl6-deficient mice showed accelerated osteoclast differentiation and severe osteoporosis. We report that Bcl6 is a direct target of Blimp1 and that mice lacking Blimp1 in osteoclasts exhibit osteopetrosis caused by impaired osteoclastogenesis resulting from Bcl6 up-regulation. Indeed, mice doubly mutant in Blimp1 and Bcl6 in osteoclasts exhibited decreased bone mass with increased osteoclastogenesis relative to osteoclast-specific Blimp1-deficient mice. These results reveal a Blimp1-Bcl6-osteoclastic molecule axis, which critically regulates bone homeostasis by controlling osteoclastogenesis and may provide a molecular basis for novel therapeutic strategies.

Topics: Acid Phosphatase; Animals; Bone and Bones; Bone Remodeling; Bone Resorption; Cathepsin K; Cell Count; Cell Differentiation; DNA-Binding Proteins; Down-Regulation; Gene Expression; Gene Expression Regulation; Growth Plate; Isoenzymes; Macrophages; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Nerve Tissue Proteins; NFATC Transcription Factors; Osteoblasts; Osteoclasts; Osteopetrosis; Positive Regulatory Domain I-Binding Factor 1; Promoter Regions, Genetic; Proto-Oncogene Proteins c-bcl-6; RANK Ligand; Signal Transduction; Tartrate-Resistant Acid Phosphatase; Transcription Factors

The three basic DNA-binding domain mutations of the microphthalmia-associated transcription factor (Mitf), Mitf(mi/mi), Mitf(or/or), and Mitf(wh/wh) affect osteoclast differentiation with variable penetrance while completely impairing melanocyte development. Mitf(or/or) mice exhibit osteopetrosis that improves with age and their osteoclasts form functional multinuclear osteoclasts, raising the question as to why the Mitf(or/or) mutation results in osteopetrosis. Here we show that Mitf(or/or) osteoclasts express normal levels of acid phosphatase 5 (Acp5) mRNA and significantly lower levels of Cathepsin K (Ctsk) mRNA during receptor activator of nuclear factor kappa B (NFkappaB) ligand (RANKL)-mediated differentiation. Studies using chromatin immunoprecipitation (ChIP) analysis indicate that low levels of Mitf(or/or) protein are recruited to the Ctsk promoter. However, enrichment of Mitf-transcriptional co-activators PU.1 and Brahma-related gene 1 (Brg1) are severely impaired at the Ctsk promoter of Mitf(or/or) osteoclast precursors, indicating that defective recruitment of co-activators by the mutant Mitf(or/or) results in impaired Ctsk expression in osteoclasts. Cathepsin K may thus represent a unique class of Mitf-regulated osteoclast-specific genes that are important for osteoclast function.

Topics: Acid Phosphatase; Age Factors; Aging; Animals; Animals, Newborn; Cathepsin K; Cathepsins; Cell Differentiation; Cells, Cultured; DNA Helicases; Gene Expression Regulation; Isoenzymes; Mice; Mice, Mutant Strains; Microphthalmia-Associated Transcription Factor; Mutation; NF-kappa B; Nuclear Proteins; Osteoclasts; Osteogenesis; Osteopetrosis; Promoter Regions, Genetic; Proto-Oncogene Proteins; RANK Ligand; RNA, Messenger; Tartrate-Resistant Acid Phosphatase; Trans-Activators; Transcription Factors

In vitro differentiated monocytes were used to characterize the cellular defect in a type of osteopetrosis with minimally functional osteoclasts, in which defects associated with common causes of osteopetrosis were excluded by gene sequencing. Monocytes from the blood of a 28-year-old patient were differentiated in media with RANKL and CSF-1. Cell fusion, acid compartments within cells, and tartrate resistant acid phosphatase (TRAP) activity were normal. However, the osteoclasts made abnormally small pits on the dentine. Phalloidin labeling showed that the cell attachments lacked the peripheral ring structure that supports lacunar resorption. Instead, the osteoclasts had clusters of podosomes near the center of cell attachments. Antibody to the alphavbeta3 integrin pair or to the C-terminal of beta3 did not label podosomes, but antibody to alphav labeled them. Western blots using antibody to the N-terminal of beta3 showed a protein of reduced size. Integrins beta1 and beta5 were upregulated, but, in contrast to observations in beta3 defects, alpha2 had not increased. The rho-GTP exchange protein Vav3, a key attachment organizing protein, did not localize normally with peripheral attachment structures. Vav3 forms of 70 kD and 90 kD were identified on western blots. However, the proteins beta3 integrin, Vav3, Plekhm1, and Src, implicated in attachment defects, had normal exon sequences. In this new type of osteopetrosis, the integrin-organizing complex is dysfunctional, and at least two attachment proteins may be partially degraded.

Topics: Acid Phosphatase; Adult; Blood Proteins; Blotting, Western; Bone Resorption; Cell Adhesion Molecules; Cell Differentiation; Cells, Cultured; Focal Adhesions; Humans; Integrins; Isoenzymes; Leukocytes, Mononuclear; Osteoclasts; Osteopetrosis; Phosphoproteins; Proto-Oncogene Proteins c-vav; Sequence Analysis, DNA; src-Family Kinases; Tartrate-Resistant Acid Phosphatase

A new spontaneous mouse mutant (ntl) with autosomal-recessive osteopetrosis was characterized. These mice formed tartrate-resistant acid phosphate (TRAP)-positive osteoclasts but their osteoclasts had no ruffled border and did not resorb bone. These mice displayed no tooth eruption or tooth root formation. Adult mutant mice developed odontoma-like proliferations near the proximal ends of the incisors. Intraperitoneal injection of progenitor cells from the liver of 16.5 days postcoitum wild-type embryos into newborn mutants rescued the osteopetrosis phenotype, indicating that the defects were intrinsic to the osteoclasts. Our findings not only provide further support for a critical role of osteoclasts in tooth eruption and tooth root development, but also suggest that the perturbation of the homeostasis of the odontogenic precursors of the incisors is primarily responsible for the development of the odontoma-like proliferations in this osteopetrosis mutant. Genetic mapping has narrowed down the location of the mutant allele to a genetic interval of 3.2 cM on mouse chromosome 17.

Topics: Acid Phosphatase; Alleles; Animals; Biomarkers; Bone Resorption; Cells, Cultured; Chloride Channels; Chromosomes, Mammalian; Disease Models, Animal; Genes, Recessive; Genetic Linkage; Genotype; Homeostasis; Incisor; Isoenzymes; Liver; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Mutant Strains; Mutation; Odontogenesis; Odontoma; Osteoclasts; Osteopetrosis; Phenotype; Stem Cells; Tartrate-Resistant Acid Phosphatase; Tooth Eruption; Tooth Root

Autosomal-Recessive Osteopetrosis (ARO) comprises a heterogeneous group of bone diseases for which mutations in five genes are known as causative. Most ARO are classified as osteoclast-rich, but recently a subset of osteoclast-poor ARO has been recognized as due to a defect in TNFSF11 (also called RANKL or TRANCE, coding for the RANKL protein), a master gene driving osteoclast differentiation along the RANKL-RANK axis. RANKL and RANK (coded for by the TNFRSF11A gene) also play a role in the immune system, which raises the possibility that defects in this pathway might cause osteopetrosis with immunodeficiency. From a large series of ARO patients we selected a Turkish consanguineous family with two siblings affected by ARO and hypogammaglobulinemia with no defects in known osteopetrosis genes. Sequencing of genes involved in the RANKL downstream pathway identified a homozygous mutation in the TNFRSF11A gene in both siblings. Their monocytes failed to differentiate in vitro into osteoclasts upon exposure to M-CSF and RANKL, in keeping with an osteoclast-intrinsic defect. Immunological analysis showed that their hypogammaglobulinemia was associated with impairment in immunoglobulin-secreting B cells. Investigation of other patients revealed a defect in both TNFRSF11A alleles in six additional, unrelated families. Our results indicate that TNFRSF11A mutations can cause a clinical condition in which severe ARO is associated with an immunoglobulin-production defect.

Topics: Acid Phosphatase; Actins; Agammaglobulinemia; Amino Acid Sequence; Amino Acid Substitution; Argentina; Arginine; Biopsy; Case-Control Studies; Cell Line, Transformed; Cell Proliferation; Cell Transformation, Viral; Cells, Cultured; Cohort Studies; Consanguinity; Cysteine; Dendrites; DNA Mutational Analysis; Female; Genes, Recessive; Herpesvirus 4, Human; Heterozygote; Homozygote; Humans; Ilium; Isoenzymes; Leukocyte Common Antigens; Leukocytes, Mononuclear; Lipopolysaccharides; Macrophage Colony-Stimulating Factor; Male; Models, Immunological; Molecular Sequence Data; Mutation, Missense; Osteoclasts; Osteopetrosis; Osteoprotegerin; Pakistan; Pedigree; Polymorphism, Genetic; Protein Structure, Tertiary; Radiography, Thoracic; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Vitronectin; Sequence Homology, Amino Acid; Tartrate-Resistant Acid Phosphatase; Turkey

We studied phenotypic and cellular aspects in a patient with a heterozygous mutation of the PLEKHM1 gene and obtained some indications regarding the role of the protein in bone cell function. Plekhm1 is involved in osteoclast endosomal vesicle acidification and TRACP exocytosis, contributing to events involved in osteoclast-osteoblast cross-talk.. The gene PLEKHM1 encodes a nonsecretory adaptor protein that localizes to endosomal vesicles. A highly truncated Plekhm1 protein was previously found in a patient with intermediate autosomal recessive osteopetrosis.. We describe a new heterozygous mutation in the PLEKHM1 gene in a patient presenting with low vertebral and femoral T-scores and areas of focal sclerosis. Clinical evaluation, mutational analysis, assessment of in vitro osteoclast morphology and activity, transfection studies, and evaluation of osteoclast-osteoblast cross-talk were carried out.. Direct DNA sequencing showed a heterozygous C to T substitution on cDNA position 2140 of the PLEKHM1 gene, predicted to lead to an R714C mutant protein. The mutation was not found in 104 control chromosomes. In vitro, patient's osteoclasts showed normal formation rate, morphology, number of nuclei, and actin rings but lower TRACP activity and higher endosomal pH than control osteoclasts. The patient had high serum PTH and TRACP, despite low TRACP activity in osteoclasts in vitro. HEK293 cells overexpressing either wildtype or Plekhm1-R714C showed no difference in localization of the variants, and co-transfection with a TRACP vector confirmed low TRACP activity in cells carrying the R714C mutation. RAW 264.7 osteoclast-like cells expressing the Plekhm1-R714C variant also showed low TRACP activity and reduced ability to acidify endosomal compartments compared with cells expressing the wildtype protein. Reduced intracellular TRACP was caused by increased protein secretion rather than reduced expression. TRACP-containing conditioned medium was able to increase osteoblast alkaline phosphatase, suggesting the focal osteosclerosis is a result of increased osteoclast-osteoblast coupling.. We provide further evidence for a role of Plekhm-1 in osteoclasts by showing that a novel mutation in PLEKHM1 is associated with a complex bone phenotype of generalized osteopenia combined with "focal osteosclerosis." Our data suggest that the mutation affects endosomal acidification/maturation and TRACP exocytosis, with implications for osteoclast-osteoblast cross-talk.

Topics: Acid Phosphatase; Adaptor Proteins, Signal Transducing; Adult; Amino Acid Substitution; Autophagy-Related Proteins; Cell Communication; Cell Line; DNA Mutational Analysis; Endosomes; Exocytosis; Female; Heterozygote; Humans; Isoenzymes; Membrane Glycoproteins; Osteoclasts; Osteopetrosis; Parathyroid Hormone; Point Mutation; Tartrate-Resistant Acid Phosphatase

Topics: Absorptiometry, Photon; Acid Phosphatase; Adult; Anti-Inflammatory Agents, Non-Steroidal; Bone Density; Chronic Kidney Disease-Mineral and Bone Disorder; Diagnosis, Differential; Female; Humans; Isoenzymes; Low Back Pain; Lumbar Vertebrae; Magnetic Resonance Imaging; Osteopetrosis; Physical Therapy Modalities; Tartrate-Resistant Acid Phosphatase

Bone resorption by osteoclasts stimulates bone formation by osteoblasts. To isolate osteoblastic factors coupled with osteoclast activity, we performed microarray and cluster analysis of 8 tissues including bone, and found that among 10,490 genes, osteomodulin (OMD), an extracellular matrix keratan sulfate proteoglycan, was simultaneously induced with osteoclast-specific markers such as MMP9 and Acp5. OMD expression was detected in osteoblasts and upregulated during osteoblast maturation. OMD expression in osteoblasts was also detected immunohistochemically using a specific antibody against OMD. The immunoreactivity against OMD decreased in op/op mice, which lack functional macrophage colony stimulating factor (M-CSF) and are therefore defective in osteoclast formation, when compared to wild-type littermates. OMD expression in op/op mice was upregulated by M-CSF treatment. Since the M-CSF receptor c-Fms was not expressed in osteoblasts, it is likely that OMD is an osteoblast maturation marker that is induced by osteoclast activity.

Topics: 3T3 Cells; Acid Phosphatase; Animals; Cell Line; Cells, Cultured; Extracellular Matrix Proteins; Fluorescent Antibody Technique; Gene Expression; Gene Expression Profiling; Humans; Isoenzymes; Macrophage Colony-Stimulating Factor; Male; Matrix Metalloproteinase 9; Mice; Mice, Mutant Strains; Oligonucleotide Array Sequence Analysis; Osteoblasts; Osteoclasts; Osteopetrosis; Proteoglycans; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Tartrate-Resistant Acid Phosphatase

Asymptomatic gene carriers and clinically affected ADO2 subjects have the same ClCN7 mutation. We examined osteoclastic bone resorption in vitro as well as osteoclast formation, several markers, acid secretion, and cytoskeletal structure. We found that ADO2 expression results from osteoclast specific properties.. Autosomal dominant osteopetrosis type II (ADO2) is a heritable osteosclerotic disorder that results from heterozygous mutations in the ClCN7 gene. However, of those individuals with a ClCN7 mutation, one third are asymptomatic gene carriers who have no clinical, biochemical, or radiological manifestations. Disease severity in the remaining two thirds is highly variable.. Human peripheral blood mononuclear cells were isolated and differentiated into osteoclasts by stimulation with hRANKL and human macrophage-colony stimulating factor (hM-CSF). Study subjects were clinically affected subjects, unaffected gene carriers, and normal controls (n = 6 in each group). Pit formation, TRACP staining, RANKL dose response, osteoclast markers, acid secretion, F-actin ring, and integrin alpha(v)beta3 expression and co-localization were studied.. Osteoclasts from clinically affected subjects had severely attenuated bone resorption compared with those from normal controls. However, osteoclasts from unaffected gene carriers displayed similar bone resorption to those from normal controls. In addition, the resorption lacunae from both unaffected gene carriers and normal controls appeared much earlier and spread much more rapidly than those from clinically affected subjects. As time progressed, the distinction between clinically affected subjects and the other two groups increased. No significant difference was found in acidic secretion or osteoclast formation between the three groups. Osteoclast cytoskeletal organization showed no difference between the three groups but there was low cellular motility in clinically affected subjects.. Osteoclasts from the unaffected gene carriers, in contrast to those from the clinically affected subjects, functioned normally in cell culture. This finding supports the hypothesis that intrinsic osteoclast factors determine disease expression in ADO2. Further understanding of this mechanism is likely to lead to the development of new approaches to the treatment of clinically affected patients.

Topics: Acid Phosphatase; Adolescent; Adult; Aged; Biomarkers; Bone Resorption; Carrier Proteins; Cell Differentiation; Cells, Cultured; Child; Chloride Channels; Female; Genes, Dominant; Heterozygote; Humans; Isoenzymes; Leukocytes, Mononuclear; Male; Membrane Glycoproteins; Middle Aged; Osteoclasts; Osteopetrosis; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Tartrate-Resistant Acid Phosphatase

Patients with defective osteoclastic acidification have increased numbers of osteoclasts, with decreased resorption, but bone formation that remains unchanged. We demonstrate that osteoclast survival is increased when acidification is impaired, and that impairment of acidification results in inhibition of bone resorption without inhibition of bone formation. We investigated the role of acidification in human osteoclastic resorption and life span in vitro using inhibitors of chloride channels (NS5818/NS3696), the proton pump (bafilomycin) and cathepsin K. We found that bafilomycin and NS5818 dose dependently inhibited acidification of the osteoclastic resorption compartment and bone resorption. Inhibition of bone resorption by inhibition of acidification, but not cathepsin K inhibition, augmented osteoclast survival, which resulted in a 150 to 300% increase in osteoclasts compared to controls. We investigated the effect of inhibition of osteoclastic acidification in vivo by using the rat ovariectomy model with twice daily oral dosing of NS3696 at 50 mg/kg for 6 weeks. We observed a 60% decrease in resorption (DPYR), increased tartrate-resistant acid phosphatase levels, and no effect on bone formation evaluated by osteocalcin. We speculate that attenuated acidification inhibits dissolution of the inorganic phase of bone and results in an increased number of nonresorbing osteoclasts that are responsible for the coupling to normal bone formation. Thus, we suggest that acidification is essential for normal bone remodeling and that attenuated acidification leads to uncoupling with decreased bone resorption and unaffected bone formation.

Topics: Acid Phosphatase; Adult; Animals; Bone and Bones; Bone Density; Bone Resorption; Cathepsin K; Cathepsins; Cells, Cultured; Chloride Channels; Dose-Response Relationship, Drug; Family Health; Female; Humans; Immunohistochemistry; Indicators and Reagents; Isoenzymes; Macrolides; Macrophage Colony-Stimulating Factor; Macrophages; Male; Middle Aged; Models, Biological; Mutation; Osteoclasts; Osteopetrosis; Ovary; Oxazines; Phenotype; Phenylurea Compounds; Rats; Rats, Sprague-Dawley; Tartrate-Resistant Acid Phosphatase; Tetrazoles; Time Factors; Xanthenes

We report on a case of osteoclast-poor osteopetrosis who received a hematopoietic stem cell graft and, despite hematological engraftment, showed little signs of response in the skeletal defect. Clinical and laboratory studies supported the concept that the bone microenvironment remained abnormal, thus reducing the clinical response to transplantation.. Osteopetrosis is a rare genetic disorder characterized by severely reduced bone resorption resulting from a defect in either osteoclast development (osteoclast-poor osteopetrosis) or activation (osteoclast-rich osteopetrosis). Patients with osteoclast-rich osteopetrosis can be rescued by allogenic hematopoietic stem cell transplantation; however, little information exists concerning the success of transplantation as a treatment for osteoclast-poor osteopetrosis. We report on a child with osteoclast-poor osteopetrosis whose diagnosis was delayed, consequently receiving a cord blood transplant from an unrelated donor at the age of 8 years. Engraftment was deemed successful by peripheral blood genotyping, although >3 years after transplantation there was little rescue of the skeletal defect and anemia, and extramedullary hematopoiesis persisted.. Peripheral blood mononuclear cells from the osteopetrosis patient, before and after transplantation, were used to generate osteoclasts in vitro in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL.. Before transplantation few, small mononuclear osteoclasts formed (F-actin ring-positive cells, co-localizing with vitronectin receptor [alphavbeta3 integrin] and TRACP) associated with occasional, small resorption lacunae. Low levels of collagen C-terminal telopeptide (CTx) fragments were released from these cultures as assessed by ELISA (CrossLaps; patient, 12.85 nM; control, 448.6 nM). In contrast, osteoclasts formed in cultures after transplantation formed to a similar degree to control cultures from healthy individuals: large numbers of osteoclasts containing numerous nuclei were present, and approximately 50% of the surface of bone slices was resorbed, associated with intermediate levels of collagen fragment release (116.48 nM). The culture data reflect the histopathology and radiological findings and also support previous studies showing that neither M-CSF nor RANKL rescues osteoclast-poor osteopetrosis.. This is the first case reported in which a successful hematopoietic engraftment failed to correct an osteopetrotic skeletal defect, and this finding may be credited to the age at which the child was transplanted.

Topics: Acid Phosphatase; Biopsy; Carrier Proteins; Cartilage; CD11c Antigen; CD18 Antigens; Cell Differentiation; Child; Collagen; Collagen Type I; Cord Blood Stem Cell Transplantation; DNA Mutational Analysis; Female; Femur; Glycoproteins; Hematologic Diseases; Humans; Humerus; Integrin alphaVbeta3; Isoenzymes; Leukocytes, Mononuclear; Macrophage Colony-Stimulating Factor; Membrane Glycoproteins; Osteoclasts; Osteopetrosis; Osteoprotegerin; Peptides; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tartrate-Resistant Acid Phosphatase; Transplantation, Homologous; Treatment Outcome

The authors treated a 6-month-old boy with malignant infantile osteopetrosis using bone marrow transplantation. The patient's clinical response was compared with his biochemical response for bone metabolic markers such as tartrate-resistant acid phosphatase 5b (TRAcP 5b) and bone-specific alkaline phosphatase (BAP). Treatment was successful, resulting in a decrease in the serum TRAcP 5b level. These bone-specific markers may be useful for the early assessment of malignant infantile osteopetrosis patients with stem cell transplantation.

Topics: Acid Phosphatase; Alkaline Phosphatase; Biomarkers; Bone Marrow Transplantation; Bone Neoplasms; Graft Survival; Humans; Infant; Isoenzymes; Male; Osteoblasts; Osteoclasts; Osteopetrosis; Tartrate-Resistant Acid Phosphatase; Treatment Outcome

Albers-Schönberg disease, or autosomal dominant osteopetrosis type II (ADO2), is caused by ineffective osteoclastic bone resorption resulting from mutations in the chloride channel 7 (ClCN7) gene. Individuals with ADO2 have increased numbers of large ineffective osteoclasts in addition to increased serum total tartrate-resistant acid phosphatase (TRACP) activity.. We investigated the serum activity of the osteoclast-derived 5b isoform of TRACP (TRACP 5b) and concentrations of the bone formation marker osteocalcin in clinically affected individuals, unaffected gene carriers, and healthy controls from 10 ADO2 families with known ClCN7 gene mutations. Bone fracture prevalence was studied in association with the serum markers.. Similar to total TRACP, TRACP 5b was significantly increased in clinically affected individuals compared with age-matched controls. TRACP 5b correlated significantly with total TRACP (r = 0.833; P <0.001), suggesting that most of the TRACP in the serum of ADO2 patients is osteoclast-derived TRACP 5b. Osteocalcin was significantly increased in affected adults and slightly decreased in affected children. TRACP 5b and total TRACP were significantly increased in clinically affected individuals with severe fractures (P <0.05).. The results indicate that in ADO2, serum TRACP 5b reflects the number of osteoclasts and that the extremely high serum TRACP 5b activity is a specific indicator of the disease. Similar to total TRACP, TRACP 5b appears to be a potentially useful marker to stratify individuals with ClCN7 gene mutations into clinically affected and unaffected gene carriers. It may also have a prognostic value in the prediction of fractures in patients with a ClCN7 gene mutation.

Topics: Acid Phosphatase; Adolescent; Adult; Biomarkers; Child; Chloride Channels; Female; Fractures, Bone; Heterozygote; Humans; Isoenzymes; Male; Middle Aged; Osteoclasts; Osteopetrosis; Tartrate-Resistant Acid Phosphatase

Osteoclasts in osteopetrotic (op/op) mice are substantially reduced by the absence of functional activities of macrophage colony-stimulating factor (M-CSF). However, it is known that osteoclasts appear in op/op skeletal bones with aging, although the molecular mechanism for this is unknown. In order to investigate osteoclastic recruitment in the jaw bones of op/op mice, osteoclastic distribution was analysed for 2 yr after birth by histochemistry for tartrate-resistant acid phosphatase activity and immunohistochemistry for cathepsin K. Osteoclasts in op/op mandibular bones decreased rapidly in number after birth and disappeared by 3 d, although there was no difference in the osteoclastic distribution between op/op and normal littermates at birth. At 2 wk, osteoclasts began to reappear around op/op tooth germs, where no apparent connective tissue layer intervened between tooth germs and bone trabeculae. They increased in number and were scattered over the mandible, reaching a maximum at 8 wk, when periodontal ligament-like structures were recognizable around incisor germs. Osteoclasts then again decreased gradually, and after 62 wk few osteoclasts were seen in op/op mandibular bones, whose marrow space disappeared. These findings suggest that osteoclasts are recruited in an M-CSF-independent manner in op/op mandibles, especially in areas around tooth germs.

Topics: Acid Phosphatase; Aging; Animals; Biomarkers; Bone Marrow; Cathepsin K; Cathepsins; Cell Count; Cysteine Endopeptidases; Incisor; Isoenzymes; Mandible; Mice; Mice, Inbred Strains; Mutation; Osteoclasts; Osteopetrosis; Periodontal Ligament; Polymorphism, Single-Stranded Conformational; Tartrate-Resistant Acid Phosphatase; Tooth Germ

Autosomal dominant osteopetrosis II (ADOII) is a relatively benign disorder caused by a missense mutation in the ClCN7 gene. In this study, we characterize the osteoclasts from patients with ADOII, caused by a G215R mutation, and investigate the effect on osteoclast function in vitro. Osteoclasts from ADOII patients and healthy age- and sex-matched controls, were used to evaluate osteoclastogenesis, cell fusion, acidification, and resorptive activity. ADOII osteoclasts in vivo have increased number and size. However, in vitro we observed no significant changes in the osteoclast formation rate, the morphology, and the expression of markers, such as cathepsin K and tartrate-resistant acid phosphatase. When mature ADOII osteoclasts were investigated on mineralized bone, they degraded the bone material, however only to 10 to 20% of the level in controls. We show by acridine orange, that the reduced chloride transport leads to reduced acidification. We show that the residual activity is sensitive to inhibitors of cathepsins and chloride channels, confirming that resorption is reduced but present. In conclusion, this is the first functional in vitro study of human ADOII osteoclasts. We show normal osteoclastogenesis in ADOII osteoclasts. However, the residual activity of the ClC-7 channel in ADOII osteoclasts does not allow sufficient acidification and thereby resorption.

Topics: Acid Phosphatase; Acridine Orange; Age Factors; Biological Transport; Bone Resorption; Calcium Phosphates; Cathepsin K; Cathepsins; Cell Differentiation; Cell Fusion; Chloride Channels; Chlorides; Enzyme Inhibitors; Humans; Immunoblotting; Immunohistochemistry; Isoenzymes; Lipopolysaccharide Receptors; Macrolides; Monocytes; Mutation; Osteoclasts; Osteopetrosis; Sex Factors; Tartrate-Resistant Acid Phosphatase; Time Factors

We studied osteoclastic differentiation from normal and osteopetrotic human CD14 cells in vitro. Defects in acid transport, organic matrix removal, and cell fusion with deficient attachment were found. Analysis of genotypes showed that TCIRG1 anomalies correlated with acid transport defects, but surprisingly, organic matrix removal failure correlated with CLCN7 defects; an attachment defect had normal TCIRG1 and CLCN7.. Osteopetrotic subjects usually have normal macrophage activity, and despite identification of genetic defects associated with osteopetrosis, the specific developmental and biochemical defects in most cases are unclear. Indeed, patients with identical genotypes often have different clinical courses. We classified defects in osteoclast differentiation in vitro using four osteopetrotic subjects without immune or platelet defects, three of them severe infantile cases, compared with normals.. Osteoclast differentiation used isolated CD14 cells; results were correlated with independent analysis of two key genes, CLCN7 and TCIRG1. CD14 cell attachment and cell surface markers and extent of differentiation in RANKL and colony-stimulating factor (CSF)-1 were studied using acid secretion, bone pitting, enzyme, and attachment proteins assays.. CD14 cells from all subjects had similar lysosomal and nonspecific esterase activity. With the exception of cells from one osteopetrotic subject, CD14 cells from osteopetrotic and control monocytes attached similarly to bone or tissue culture substrate. Cells from one osteopetrotic subject, with normal CLCN7 and TCIRG1, did not attach to bone, did not multinucleate, and formed no podosomes or actin rings in RANKL and CSF-1. Attachment defects are described in osteopetrosis, most commonly mild osteopetrosis with Glantzman's thrombasthenia. However, this case, with abnormal integrin alphavbeta3 aggregates and no osteoclasts, seems to be unique. Two subjects were compound heterozygotes for TCIRG1 defects; both had CD14 cells that attached to bone but did not acidify attachments; cell fusion and attachment occurred, however, in RANKL and CSF-1. This is consistent with TCIRG1, essential for H+-ATPase assembly at the ruffled border. A compound heterozygote for CLCN7 defects had CD14 cells that fused in vitro, attached to bone, and secreted acid, TRACP, and cathepsin K. However, lacunae were shallow and retained demineralized matrix. This suggests that CLCN7 may not limit H+-ATPase activity as hypothesized, but may be involved in control of organic matrix degradation or removal.

Topics: Acid Phosphatase; Acids; Adult; Antigens, CD; Bone and Bones; Bone Resorption; Carrier Proteins; Cathepsin K; Cathepsins; Cell Adhesion; Cell Differentiation; Cell Separation; Cells, Cultured; Chloride Channels; Female; Flow Cytometry; Genotype; Giant Cells; Humans; Infant; Integrin alphaVbeta3; Interleukins; Isoenzymes; Leukocytes, Mononuclear; Lipopolysaccharide Receptors; Macrophage Colony-Stimulating Factor; Male; Membrane Glycoproteins; Monocytes; Mutation; Naphthol AS D Esterase; Osteoclasts; Osteopetrosis; Protein Subunits; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Stem Cell Factor; Tartrate-Resistant Acid Phosphatase; Vacuolar Proton-Translocating ATPases

The coordination of cell cycle progression and osteoclast differentiation by RANKL signaling was studied. Experiments with mouse genetic models revealed that RANKL promoted cell cycle withdrawal of osteoclast precursors dependent on the cyclin kinase inhibitor p27-KIP1, but that both p27-KIP1 and p21-CIP1 were required for osteoclast differentiation. These cyclin inhibitors may directly regulate osteoclast differentiation in addition to regulating cell cycle withdrawal.. RANKL stimulates mononuclear precursor cells of the myeloid lineage to differentiate into multinuclear osteoclasts, thus providing a system to study the fundamental problem of coordination of cell cycle progression with cell differentiation.. Mice that lack expression of functional cyclin inhibitors p27KIP1and p21CIP1 were used to study cell cycle progression and differentiation of osteoclast precursors in vitro and in vivo.. Experiments with cells derived from p27KIP1- and p21CIP1-deficient mice indicated that p27KIP1 function alone was necessary for RANKL-mediated cell cycle withdrawal by osteoclast precursors, but osteoclasts from mice with single mutations in either of these two genes differentiated normally. In contrast, p21/p27 double knockout mice developed osteopetrosis, with fewer osteoclasts that exhibited lower TRACP activity and abnormal cell morphology present in long bone. Moreover, isolated osteoclast progenitors from p21/p27 double knockout mice were defective in RANKL-mediated differentiation in vitro, expressing low levels of osteoclast-specific genes like TRACP and cathepsin K. Taken together, these data suggest p27KIP1 and p21CIP1 play roles in osteoclast differentiation in response to RANKL signaling distinct from their roles in promoting cell cycle withdrawal.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Blotting, Western; Bone Marrow Cells; Carrier Proteins; Cathepsin K; Cathepsins; CDC2-CDC28 Kinases; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cell Proliferation; Cells, Cultured; Chromones; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p18; Cyclin-Dependent Kinase Inhibitor p19; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Enzyme Inhibitors; Femur; Flavonoids; Gene Expression; Imidazoles; Isoenzymes; Macrophage Colony-Stimulating Factor; Macrophages; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Morpholines; Osteoclasts; Osteopetrosis; Pyridines; Radiography; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Reverse Transcriptase Polymerase Chain Reaction; Tartrate-Resistant Acid Phosphatase; Tumor Suppressor Proteins

Autosomal-recessive osteopetrosis is a severe genetic disease caused by osteoclast failure. Approximately 50% of the patients harbor mutations of the ATP6i gene, encoding for the osteoclast-specific a3 subunit of V-ATPase. We found inactivating ATP6i mutations in four patients, and three of these were novel. Patients shared macrocephaly, growth retardation and optic nerve alteration, osteosclerotic and endobone patterns, and high alkaline phosphatase and parathyroid hormone levels. Bone biopsies revealed primary spongiosa lined with active osteoblasts and high numbers of tartrate-resistant acid phosphatase (TRAP)-positive, a3 subunit-negative, morphologically unremarkable osteoclasts, some of which located in shallow Howship lacunae. Scarce hematopoietic cells and abundant fibrous tissue containing TRAP-positive putative osteoclast precursors were noted. In vitro osteoclasts were a3-negative, morphologically normal, with prominent clear zones and actin rings, and TRAP activity more elevated than in control patients. Podosomes, alphaVbeta3 receptor, c-Src, and PYK2 were unremarkable. Consistent with the finding in the bone biopsies, these cells excavated pits faintly stained with toluidine blue, indicating inefficient bone resorption. Bone marrow transplantation was successful in all patients, and posttransplant osteoclasts showed rescue of a3 subunit immunoreactivity.

Topics: Acid Phosphatase; Biopsy; Bone and Bones; Bone Marrow Transplantation; Cells, Cultured; Disease Progression; DNA Mutational Analysis; Exons; Female; Genes, Recessive; Genotype; Humans; Ilium; Infant; Infant, Newborn; Introns; Isoenzymes; Male; Mutation; Osteoclasts; Osteopetrosis; Phenotype; Protein Subunits; Radiography; Tartrate-Resistant Acid Phosphatase; Vacuolar Proton-Translocating ATPases

The activity of the TRACP promoter has been investigated as a model of gene regulation in osteoclasts. The murine TRACP gene promoter contains potential binding sites for a number of transcription factors in particular, candidate sites for the Ets factor PU.1 and for the microphthalmia transcription factor (MiTF). These are of relevance to osteoclast biology because the PU.1 knockout mouse has an osteopetrotic phenotype, and MiTF, when mutated in the mi/mi mouse, also results in osteopetrosis. The binding sites for both of these factors have been identified, and they have been determined to be functional in regulating TRACP expression. A novel assay system using the highly osteoclastogenic RAW/C4 subclone of the murine macrophage cell line RAW264.7 was used to perform gene expression experiments on macrophage and osteoclast cell backgrounds. We have shown that TRACP expression is a target for regulation by the macrophage/osteoclast transcription factor PU.1 and the osteoclast commitment factor MiTF and that these factors act synergistically in regulating this promoter. This directly links two controlling factors of osteoclast differentiation to the expression of an effector of cell function.

Topics: Acid Phosphatase; Animals; Binding Sites; Cell Line; DNA-Binding Proteins; Gene Expression Regulation; Genes, Reporter; Isoenzymes; Luciferases; Mice; Mice, Knockout; Microphthalmia-Associated Transcription Factor; Osteopetrosis; Phenotype; Promoter Regions, Genetic; Proto-Oncogene Proteins; Tartrate-Resistant Acid Phosphatase; Trans-Activators; Transcription Factors

First molars fail to erupt in the incisor-absent (ia/ia) rat because of a defect in osteoclast function. Growth factors that regulate local bone metabolism include growth hormone (GH), insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF) and interleukin-1 alpha (IL-1alpha). Since osteoclast function may be affected by these factors, the aim of this study was to determine the distribution of GH receptor (GHr), IGF-I, EGF and IL-1alpha, in osteoclasts located occlusal to the erupting first molar, in the 'eruption pathway', in normal and ia/ia rats. Sagittal sections of the first molar and adjacent bone from 3- and 9-d-old animals were examined. Osteoclasts were identified using tartrate-resistant acid phosphatase (TRAP). The TRAP-positive osteoclast cell numbers were higher in ia/ia animals at 3 and 9 days-of-age. In the ia/ia group, fewer osteoclasts were GHr- and IGF-I-positive at 3 d of age, and at 9 d of age fewer osteoclasts were GHr-positive. In the ia/ia rat, defective osteoclast function failed to resorb bone to provide an eruption pathway for the lower first molar. The expression of GHr, and to some degree IGF-I, by these osteoclasts was reduced, which may be related to their ability to differentiate and function.

Topics: Acid Phosphatase; Animals; Disease Models, Animal; Immunohistochemistry; Incisor; Isoenzymes; Mandible; Molar; Osteoclasts; Osteopetrosis; Rats; Rats, Mutant Strains; Receptors, Somatotropin; Tartrate-Resistant Acid Phosphatase; Tissue Distribution; Tooth Eruption; Tooth Germ

In the toothless (tl/tl) osteopetrotic rat, teeth form but fail to erupt. Treatment of tl/tl rats with colony-stimulating factor-1 (CSF-1) activates bone resorption by osteoclasts, permits tooth eruption, and up-regulates the immunoreactivity of bone marrow mononuclear cells to growth hormone receptor (GHr) and insulin-like growth factor (IGF)-I. This study examined the distribution of tartrate-resistant acid phosphatase (TRAP) and immunoreactivity for GHr and IGF-I in osteoclast-like cells located on the alveolar bone margin, adjacent to the lower first molar crown, in 14-day-old normal and tl/tl rats, following treatment with CSF-1. Osteoclast-like cells demonstrated a positive reaction for TRAP, GHr, and IGF-I in all groups. However, in tl/tl tissue, osteoclast-like cells were generally negative for GHr. There was no significant difference in the total number of TRAP-, GHr-, and IGF-I-positive osteoclast-like cells on the adjacent bone margin in normal, normal treated with CSF-1, and tl/tl rats. CSF-1 treatment of the tl/tl rat significantly increased the total number of osteoclast-like cells, which were positive for TRAP (p < 0.001), GHr (p < 0.05) and IGF-I (p < 0.01).

Topics: Acid Phosphatase; Animals; Animals, Genetically Modified; Antibodies, Monoclonal; Bone Resorption; Heterozygote; Homozygote; Humans; Immunohistochemistry; Insulin-Like Growth Factor I; Isoenzymes; Leukocytes, Mononuclear; Macrophage Colony-Stimulating Factor; Mutation; Osteoclasts; Osteopetrosis; Rats; Receptors, Somatotropin; Tartrate-Resistant Acid Phosphatase; Tooth Eruption

Soluble colony-stimulating factor-1 (sCSF-1) and membrane bound CSF-1 are synthesized by osteoblasts and stromal cells. However, the precise role of each form in osteoclastogenesis is unclear. In the op/op mouse, absence of osteoblast-derived CSF-1 leads to decreased osteoclasts and osteopetrosis. To determine whether sCSF-1 gene replacement can cure the osteopetrotic defect, we took advantage of the osteoblast specificity of the osteocalcin promoter to selectively express sCSF-1 in the bone of op/op mice. Transgenic mice harboring the human sCSF-1 cDNA under the control of the osteocalcin promoter were generated and cross-bred with heterozygous op/wt mice to establish op/op mutants expressing the transgene (op/opT). The op/op genotype and transgene expression were confirmed by PCR and Southern blot analysis, respectively. High levels of human sCSF-1 protein were selectively expressed in bone. At 2(1/2) wk, op/opT mice showed normal growth and tooth eruption. Femurs removed at 5 and 14 wk were analyzed by peripheral quantitative computed tomography and histomorphometry. The abnormal bone mineral density, cancellous bone volume, and growth plate width observed in op/op mice was completely reversed in op/opT mice by 5 wk, and this effect persisted at 14 wk, with measurements comparable with wt/wt mice at each time point. Correction of the skeletal abnormalities in the 5-wk-old op/opT mice correlated with a marked increase in the total osteoclast number, and their number per millimeter of bone surface compared with that of op/op mutants. Osteoclast number was maintained at 14 wk in op/opT mice and morphologically resembled wt/wt osteoclasts. These results indicate that sCSF-1 is sufficient to drive normal osteoclast development and that the osteocalcin promoter provides an efficient tool for delivery of exogenous genes to the bone. Moreover, targeting sCSF-1 to osteoblasts in the bone microenvironment may be a potentially useful therapeutic modality for treating bone disorders.

Topics: Acid Phosphatase; Animals; Body Weight; Bone Density; Femur; Genetic Therapy; Humans; Isoenzymes; Macrophage Colony-Stimulating Factor; Mice; Mice, Transgenic; Osteoblasts; Osteocalcin; Osteopetrosis; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Tartrate-Resistant Acid Phosphatase; Tomography, X-Ray Computed; Transgenes

Autosomal dominant osteopetrosis type II (ADO2) is typically diagnosed from radiographs, which demonstrate the pathognomonic findings of osteosclerosis and endobone formation. Individuals with ADO2 also have elevated serum levels of tartrate-resistant acid phosphatase (TRAP) and the BB isoenzyme of creatine kinase (CK-BB). In the current study, we tested the utility of these enzymes in making or refuting a diagnosis of ADO2. Furthermore, because ADO2 has incomplete penetrance, we examined whether TRAP and CK-BB were helpful in identifying gene carriers. We studied eight families, measured serum levels of TRAP and CK-BB in 52 affected individuals and 12 obligate gene carriers, and compared their values with age-matched controls. Our results demonstrate that affected patients have significantly elevated levels of both TRAP and CK-BB. In contrast, gene carriers have values that are not different from controls. Furthermore, in our study population, TRAP and CK-BB have a high diagnostic sensitivity and specificity, particularly in children. From this large study of ADO2 patients and carriers, we conclude that: 1) TRAP and CK-BB are significantly elevated in patients with ADO2, 2) obligate carriers cannot be adequately identified by measurement of these analytes, and 3) TRAP and CK-BB are highly sensitive and specific diagnostic tests that can efficiently and effectively screen high-risk individuals who have not had previous radiographic assessment.

Topics: Acid Phosphatase; Adolescent; Adult; Brain; Child; Child, Preschool; Creatine Kinase; Creatine Kinase, BB Form; Female; Genes, Dominant; Genetic Carrier Screening; Humans; Isoenzymes; Male; Middle Aged; Osteopetrosis; Radiography; Tartrate-Resistant Acid Phosphatase

The osteopetrotic grey-lethal (gl) mouse mutant displays many similarities to the human malignant autosomal-recessive form of osteopetrosis. In this study, we show that the gl osteopetrotic bone phenotype is characterized by the presence of numerous differentiated multinucleated osteoclasts. A significant increase in the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts was detected in vivo, suggesting induction of differentiation in the osteoclast lineage as a compensatory mechanism. These gl osteoclast cells demonstrated a defective cytoskeletal reorganization and an underdeveloped ruffled border, a membrane structure essential for active bone resorption. Accordingly, resorption activity of these cells is markedly impaired by four- to tenfold as evaluated with the pit formation assay. This low bone resorption in gl osteoclasts is highly reminiscent of the loss in key enzymes, V-ATPase or cathepsin-K, and in signaling factors, Src or TRAF-6, which were shown not to be significantly altered in gl osteoclasts. Thus, independently of a deficiency in V-ATPase, Src, cathepsin-K, and TRAF-6, the gl mutation results in increased number of osteoclasts, characterized by a disrupted cytoskeleton and an underdeveloped ruffled border.

Topics: Acid Phosphatase; Animals; Bone and Bones; Bone Resorption; Cell Differentiation; Cells, Cultured; Coculture Techniques; Cytoskeletal Proteins; Cytoskeleton; Disease Models, Animal; Genes, Lethal; Immunohistochemistry; Isoenzymes; Mice; Mice, Mutant Strains; Microscopy, Electron; Mutation; Osteoclasts; Osteopetrosis; Phenotype; Proteins; Proto-Oncogene Proteins pp60(c-src); Proton-Translocating ATPases; Tartrate-Resistant Acid Phosphatase; TNF Receptor-Associated Factor 6; Vacuolar Proton-Translocating ATPases

Osteopetrotic (op/op) mice do not exhibit bone remodeling because of defective osteoclast formation caused by the depletion of macrophage colony-stimulating factor (M-CSF). In the present study, we investigated tibial bones of op/op mice with or without prior injections of M-CSF to determine whether osteoclast formation and subsequent bone resorption could activate osteoblasts, which is known as a "coupling" phenomenon. In op/op mice, no osteoclasts were present, but the metaphyseal osteoblasts adjacent to the growth plate cartilage seemed to be active, revealing an intense alkaline phosphatase (ALPase) immunoreactivity. Consequently, primary trabecular bones were extended continuously to the diaphysis, indicating that bone modeling is well achieved in op/op mice. In contrast with the metaphysis, most of the diaphyseal osteoblasts were flattened and showed weak ALPase activity, and, as a result, they seemed to be less active. Osteopontin (OPN) was localized slightly at the interface between bone and cartilage matrices of the primary trabeculae. In contrast, in op/op mice injected with M-CSF, tartrate-resistant acid phosphatase-positive osteoclasts appeared, resorbing trabecular bones of the diaphyseal region. The diaphyseal osteoblasts in the vicinity of the active osteoclasts were cuboidal and exhibited strong ALPase immunoreactivity. OPN was observed not only at the bone-cartilage interface, but also significantly on the resorption lacunae beneath the bone-resorbing osteoclasts. These observations indicate that the activation of diaphyseal osteoblasts appears to be coupled with osteoclast formation and subsequent osteoclastic bone resorption. Alternatively, the metaphyseal osteoblasts at the chondro-osseous junction seemed to be less affected by osteoclastic activity.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Injections; Isoenzymes; Macrophage Colony-Stimulating Factor; Mice; Mice, Inbred Strains; Mice, Mutant Strains; Osteoblasts; Osteoclasts; Osteopetrosis; Osteopontin; Recombinant Proteins; Sialoglycoproteins; Tartrate-Resistant Acid Phosphatase; Tibia

Interferon-gamma (IFN-gamma) treatment increases osteoclastic bone resorption in vivo in patients with malignant osteopetrosis (OP). The treatment effect was studied in vitro in osteoclasts generated by culturing peripheral white blood cells (PWBC) from OP patients and normal human control subjects. Osteoclasts were treated with or without IFN-gamma prior to the end of the culture period. Osteoclasts from normal subjects were large in size (161 +/- 18 microm in diameter) with >10 nuclei per osteoclast. These cells showed intense staining for tartrate-resistant acid phosphatase (TRAP), expressed abundant calcitonin receptors (CTR), and formed numerous resorption pits on bovine bone slices, indicative of authentic osteoclasts. In contrast, similarly cultured osteoclasts from OP patients were smaller in size (18 +/- 3 microm in diameter), with 2-3 nuclei per osteoclast, and stained lightly for TRAP. However, IFN-gamma treatment of osteoclasts from OP patients resulted in the formation of larger osteoclasts (171 +/- 33 microm in diameter) with >10 nuclei per cell, similar in appearance to osteoclasts from normal subjects. IFN-gamma stimulation increased the intensity of TRAP staining (p < 0.0001) to levels near that of the normal osteoclasts. Unstimulated osteoclasts from 6 OP patients had a significantly lower baseline level of superoxide production, as measured by nitroblue tetrazolium reduction (p < 0.0001), compared with normal osteoclasts. IFN-gamma markedly increased (p < 0.0001) superoxide production. Whereas there was a 3-fold increase in superoxide generation in OP patients' osteoclasts, osteoclasts from control subjects had only a small and insignificant increase in superoxide production after IFN-gamma treatment.

Topics: Acid Phosphatase; Adult; Animals; Calcitonin; Calcitriol; Cattle; Cell Differentiation; Cell Size; Cells, Cultured; Child, Preschool; Culture Media; Dexamethasone; Humans; Infant; Interferon-gamma; Isoenzymes; Leukocytes, Mononuclear; Macrophage Colony-Stimulating Factor; Osteoclasts; Osteopetrosis; Receptors, Calcitonin; Reference Values; Superoxides; Tartrate-Resistant Acid Phosphatase

High systemic levels of osteoprotegerin (OPG) in OPG transgenic mice cause osteopetrosis with normal tooth eruption and bone elongation and inhibit the development and activity of endosteal, but not periosteal, osteoclasts. We demonstrate that both intravenous injection of recombinant OPG protein and transgenic overexpression of OPG in OPG(-/-) mice effectively rescue the osteoporotic bone phenotype observed in OPG-deficient mice. However, intravenous injection of recombinant OPG over a 4-wk period could not reverse the arterial calcification observed in OPG(-/-) mice. In contrast, transgenic OPG delivered from mid-gestation through adulthood does prevent the formation of arterial calcification in OPG(-/-) mice. Although OPG is normally expressed in arteries, OPG ligand (OPGL) and receptor activator of NF-kappaB (RANK) are not detected in the arterial walls of wild-type adult mice. Interestingly, OPGL and RANK transcripts are detected in the calcified arteries of OPG(-/-) mice. Furthermore, RANK transcript expression coincides with the presence of multinuclear osteoclast-like cells. These findings indicate that the OPG/OPGL/RANK signaling pathway may play an important role in both pathological and physiological calcification processes. Such findings may also explain the observed high clinical incidence of vascular calcification in the osteoporotic patient population.

Topics: Acid Phosphatase; Animals; Aorta; Blotting, Western; Bone Density; Calcinosis; Cathepsin K; Cathepsins; CHO Cells; Cricetinae; Femur; Glycoproteins; Humans; Immunohistochemistry; In Situ Hybridization; Isoenzymes; Mice; Mice, Knockout; Mice, Transgenic; NF-kappa B; Osteoclasts; Osteopetrosis; Osteoporosis; Osteoprotegerin; Radiography; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Fusion Proteins; Tartrate-Resistant Acid Phosphatase

Type II autosomal dominant osteopetrosis (ADO2) is an inherited disorder characterized by increased skeletal mass and characteristic abnormalities evident on radiography. Although previous investigators have described nonpenetrant individuals (carriers), it is not known whether carriers manifest subtle abnormalities. We hypothesized that ADO2 carriers would have an abnormality of osteoclast function that would lead to changes in bone mineral density (BMD), in serum tartrate-resistant acid phosphatase (TRAP), or in creatine kinase isoenzyme BB (CK-BB) levels that would permit carrier recognition. We identified a female carrier in a well-established ADO2 family and measured BMD, serum TRAP, and CK-BB concentrations. She had normal BMD, serum TRAP, and CK-BB concentrations. Thus, these measurements cannot be used to exclude carrier status in individuals who are seen for genetic counseling. However, measurements in other asymptotic carriers are necessary before concluding that these measurements are normal in all or most nonpenetrant individuals.

Topics: Absorptiometry, Photon; Acid Phosphatase; Adult; Bone Density; Child, Preschool; Creatine Kinase; Female; Genes, Dominant; Heterozygote; Humans; Isoenzymes; Middle Aged; Osteopetrosis; Pedigree

Insulin-like growth factor-I (IGF-I) plays a major role in regulating cell growth. This study examined the immunohistochemical distribution of IGF-I and IGF-I receptor (IGF-IR) in tibias from normal and osteopetrotic (toothless, tl/tl) rats, following treatment with colony stimulating factor-1 (CSF-1). In normal rats, immunoreactivity for IGF-I and IGF-IR was detected in cells of the articular and epiphyseal cartilage, secondary ossification centres, zones of resting and proliferating chondrocytes and bone marrow. Bone marrow cells immunoreactive for IGF-I and IGF-IR were significantly reduced in the tl/tl rat (p < 0.001) compared with normal animals. Treatment of tl/tl rats with CSF-1 increased immunoreactivity for IGF-I and IGF-IR in bone marrow cells as well as the number of TRAP positive osteoclasts. This increase was the result of recruitment of a range of hematopoietic cell types, including eosinophils, polymorphs and a substantial number of monocyte-like cells demonstrating strong immunoreactivity to IGF-I/IGF-IR. The differences in relative immunoreactivity for IGF-I/IGF-IR by bone marrow cells in untreated and CSF-1-treated tl/tl rats indicate a CSF-1-dependent recruitment of cells bearing surface IGF-IRs which may be mediated by an increase in local or systemic IGF-I.

Topics: Acid Phosphatase; Animals; Biomarkers; Bone Marrow Cells; Cartilage, Articular; Immunohistochemistry; Insulin-Like Growth Factor I; Isoenzymes; Macrophage Colony-Stimulating Factor; Monocytes; Osteoclasts; Osteopetrosis; Rats; Rats, Mutant Strains; Receptor, IGF Type 1; Tartrate-Resistant Acid Phosphatase; Tibia

The cellular distribution of osteoclast integrin subunits alpha(v) and beta(3), the tissue distribution, and level of the apparent ligand osteopontin (OPN) as well as of the putative regulatory enzyme tartrate-resistant acid phosphatase (TRAP) were studied along with the intracellular distribution of the activation marker c-src in osteopetrotic ia/ia (incisors-absent) mutant rats and their normal littermates. In ia/ia rats, the osteoclasts are incapable of bone matrix resorption. Ultrastructurally the cells exhibit extended clear zones at the expense of ordinary ruffled borders. A secretory dysfunction in the mutant is strongly suggested by the absence of detectable extracellular TRAP, concomitant with an accumulation of the enzyme in abundant small cytoplasmic vesicles. Moreover, TRAP mRNA, protein content, as well as enzymatic activity were elevated. Furthermore, increased levels of integrin subunits alpha(v) and beta(3) were detected at the clear zone of mutant osteoclasts. OPN mRNA levels were elevated in long bones from mutants. In ia/ia rats, immunolabeling for OPN was homogeneously distributed at the surface facing osteoclasts, while in normal littermates it was concentrated at the clear zone area and barely detectable at ruffled borders. The absence of OPN labeling in the abundant, putative intracellular secretory vesicles in mutant osteoclasts suggests that these cells do not produce OPN. The osteoclasts of ia/ia rats appeared to produce and translocate the c-src protein to the cell membrane. In ia/ia a defect ruffled border-formation is observed along with extensive clear zone formation and decreased secretory function. The lesion may be due to a signaling defect, but in that case the defect seems to be located downstream to or not involving the c-src pathway. Our results illustrate the close relationship between secretory function and ruffled border formation in osteoclasts, a relationship that appears to be necessary for proper resorptive function.

Topics: Acid Phosphatase; Animals; Antigens, CD; Bone Resorption; Cell Adhesion; Cell Membrane; Integrin alphaV; Integrin beta3; Isoenzymes; Osteoclasts; Osteopetrosis; Osteopontin; Platelet Membrane Glycoproteins; Proto-Oncogene Proteins pp60(c-src); Rats; Rats, Mutant Strains; RNA, Messenger; Sialoglycoproteins; Signal Transduction; Tartrate-Resistant Acid Phosphatase

Osteoclasts from a patient affected by osteopetrosis were examined in vivo and in vitro. Iliac crest biopsy revealed an osteosclerotic pattern, with prominent numbers of osteoclasts noted for hypernuclearity and incomplete adherence to the bone surface. A population comprising tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated and mononuclear cells, and alkaline phosphatase-positive stromal fibroblasts was obtained in vitro from bone marrow. Mononuclear TRAP-positive precursors spontaneously fused in culture to form giant osteoclast-like cells. These cells expressed the osteoclast marker MMP-9 and calcitonin receptor, and lacked the macrophage marker, Fc receptor. Expression and distribution of c-src, c-fms, and CD68, and response to steroid hormones relevant to osteoclast differentiation and function were apparently normal, whereas cell retraction in response to calcitonin was impaired. TRAP-positive multinucleated cells did not form osteoclast-specific adhesion structures (clear zone, podosomes, or actin rings). Bone resorption rate was severely reduced in vitro. Focal adhesions and stress fibers were observed en lieu of podosomes and actin rings. Adhesion structures contained low levels of immunoreactive vitronectin receptor, most of this integrin being retained in cytoplasmic vesicles. These data provide the first characterization of abnormal differentiation and function of human osteopetrotic osteoclast-like cells.

Topics: Acid Phosphatase; Alkaline Phosphatase; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Calcitonin; Cell Adhesion; Child; Female; Fluorescent Antibody Technique; Genes, src; Histocytochemistry; Humans; Isoenzymes; Microscopy, Electron; Osteoclasts; Osteopetrosis; Receptor, Macrophage Colony-Stimulating Factor; Receptors, Calcitonin; Receptors, Vitronectin; Tartrate-Resistant Acid Phosphatase

Dental innervation occurs concurrently with tooth development, eruption, and root formation and is suggested to interact with developing tissues. The purpose of the present study was to investigate dental innervation in osteopetrotic (op/op) mice, which carry a mutation of colony-stimulating factor-1 (CSF-1) and demonstrate sparse macrophages and osteoclasts, failure of bone resorption, lack of tooth eruption, and poor root formation. Jaw tissues from 21 mice in different age groups (7 days, 18 days, 26 days, 5 weeks, and 3 months) were prepared for immunocytochemistry and light microscopy. Immunocytochemistry with the neuronal marker protein gene product 9.5 (PGP 9.5), macrophage marker F4/80, double-labeling with F4/80 and PGP 9.5, and histochemical analysis using tartarate-resistant acid phosphatase (TRAPase) were carried out in selected sections. Molar and incisor development were arrested in the op/op mouse, and both types of teeth had bony occlusion of the eruptive pathway and failure of root formation. Third molar development in the normal mouse is delayed until after birth; therefore, it encounters different bone barriers and jaw structures than are present when first and second molars and incisors begin to develop after the second embryonic week. All three molars, however, completed crown formation prior to eruption failure. Partial root formation was seen in several homozygous op/op mice, and, in those cases, there was partial development of the periodontal ligament. Innervation of dental tissues that successfully formed was essentially normal in the mutant mice despite phenotypic deficiencies in macrophages and osteoclasts. The periodontal ligament was innervated with PGP 9.5-immunoreactive Ruffini mechanoreceptive endings in those cases in which the ligament formed, and op/op mice had remarkably normal sensory innervation of molar and incisor pulp despite failure of bone resorption, failure of root development, and arrested eruption. This study shows that op/op mice develop normal innervation in dental tissues and that dental nerve development proceeds independently of bone abnormalities and root failure in this animal.

Topics: Acid Phosphatase; Animals; Antigens, Differentiation; Cell Division; Dental Pulp; Isoenzymes; Macrophage Colony-Stimulating Factor; Macrophages; Mice; Mice, Mutant Strains; Nerve Endings; Nerve Tissue; Neurons, Afferent; Osteoclasts; Osteopetrosis; Periodontium; Tartrate-Resistant Acid Phosphatase; Thiolester Hydrolases; Tooth; Tooth Eruption; Tooth Root; Ubiquitin Thiolesterase

Mature osteoclasts specifically express the purple, band 5 isozyme (Acp 5) of tartrate-resistant acid phosphatase, a binuclear metalloenzyme that can generate reactive oxygen species. The function of Acp 5 was investigated by targeted disruption of the gene in mice. Animals homozygous for the null Acp 5 allele had progressive foreshortening and deformity of the long bones and axial skeleton but apparently normal tooth eruption and skull plate development, indicating a rôle for Acp 5 in endochondral ossification. Histomorphometry and mineralization density analysis of backscattered electron imaging revealed widened and disorganized epiphyseal growth plates with delayed mineralization of cartilage in 6- to 8-week-old mutant mice. The membrane bones of the skull showed increased density at all ages examined, indicating defective osteoclastic bone turnover. Increased mineralization density was observed in the long bones of older animals which showed modelling deformities at their extremities: heterozygotes and homozygous Acp 5 mutant mice had tissue that was more mineralized and occupied a greater proportion of the bone in all regions. Thus the findings reflect a mild osteopetrosis due to an intrinsic defect of osteoclastic modelling activity that was confirmed in the resorption pit assay in vitro. We conclude that this bifunctional metalloprotein of the osteoclast is required for normal mineralization of cartilage in developing bones; it also maintains integrity and turnover of the adult skeleton by a critical contribution to bone matrix resorption.

Topics: Acid Phosphatase; Animals; Bone and Bones; Bone Development; Bone Resorption; Calcification, Physiologic; Gene Deletion; Mice; Mutation; Osteopetrosis; Phenotype; Tartrates

Osteoclasts are terminally differentiated cells that express tartrate-resistant acid phosphatase (TRAP) at a higher level than other normal cells. Therefore, in an attempt to develop immortalized osteoclasts, we produced two lines of transgenic mice in which expression of the simian virus 40 T antigen oncogene was targeted to osteoclasts using the TRAP gene promoter. Osteoclasts were increased in number in bones from both lines. More than 50% of them appeared morphologically transformed, 2-5% were mitotic, but, unexpectedly, 5% were apoptotic. Osteoclast tumors were observed occasionally in one line of mice (line 4), and sheets of TRAP-positive cells (tumorlets) developed in most mice in both lines. Although cells isolated from these tumorlets formed multinucleated TRAP-positive cells that resorbed bone in vitro, to date we have been unable to develop an immortalized osteoclast cell line from them. Osteoclasts from one line (line 5) had reduced ruffled border formation and a higher level of T-antigen expression than osteoclasts in the other line (line 4), and these features were associated with the presence of osteopetrosis. However, osteoclasts from these osteopetrotic mice and from line 4 mice resorbed bone normally when the mice were treated with interleukin-1. These findings indicate that T antigen can be targeted to osteoclasts in transgenic mice and causes osteoclast transformation, tumors, mitosis, and apoptosis. When T antigen is expressed at high levels, functional impairment of osteoclasts can be detected. Furthermore, these results suggest that T antigen is insufficient on its own to immortalize cells in the osteoclast lineage.

Topics: Acid Phosphatase; Animals; Antigens, Polyomavirus Transforming; Apoptosis; Bone Neoplasms; Cell Transformation, Neoplastic; Female; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Osteoclasts; Osteopetrosis; Simian virus 40

We have reported that osteoblastic cells are required for differentiation of osteoclast progenitors in splenic tissues into multinucleated osteoclasts. In the present study we examined the pathogenesis of the osteoclast deficiency in osteopetrotic (op/op) mice using a coculture system of spleen cells and osteoblastic cells. When spleen cells obtained from op/op or normal (+/?) littermates of op/+ parent mice were cocultured with osteoblastic cells obtained from calvaria of normal ddy strain mice, numerous tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) were formed in the presence of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]. Most of the TRAP-positive MNCs bound [125I]salmon calcitonin. This suggests that there is no abnormality in the osteoclast progenitors present in the splenic tissues of op/op mice. When osteoblastic cells from +/? littermates were cocultured with normal spleen cells from ddy mice, TRAP-positive MNCs were similarly formed in response to 1 alpha,25(OH)2D3. In contrast, in cocultures of op/op osteoblastic cells with normal spleen cells, no TRAP-positive cells appeared, even in the presence of 1 alpha,25(OH)2D3. The op/op mutation was recently reported to exist in the coding region of the macrophage colony-stimulating factor (M-CSF) gene. Adding M-CSF and 1 alpha,25(OH)2D3 to the coculture with op/op osteoblastic cells induced the appearance of TRAP-positive MNCs with calcitonin receptors. These results clearly indicate that osteoclast deficiency in op/op mice is due to a defect in the local microenvironment in bone, in which M-CSF produced by osteoblastic cells plays a critical role in osteoclast development.

Topics: Acid Phosphatase; Animals; Calcitonin; Calcitriol; Cell Differentiation; Cells, Cultured; Female; Macrophage Colony-Stimulating Factor; Male; Mice; Osteoblasts; Osteoclasts; Osteopetrosis; Spleen; Stem Cells; Tartrates

Severe deficiency of osteoclasts, monocytes, and peritoneal macrophages in osteopetrotic (op/op) mutant mice is caused by the absence of functional macrophage colony-stimulating factor (M-CSF). To clarify the role of M-CSF in the osteoclast differentiation, we established a clonal stromal cell line OP6L7 capable of supporting hemopoiesis from newborn op/op mouse calvaria. Although very few macrophages appeared in the cocultures of bone marrow cells and OP6L7 cells, a 50-fold larger number of macrophages was detected in the day 7 cocultures when purified recombinant human M-CSF (rhM-CSF) was exogenously supplied. Tartrate-resistant acid phosphatase (TRACP; a marker enzyme of osteoclasts)-positive cells appeared only when bone marrow cells were cultured in contact with OP6L7 cells and both rhM-CSF and 1 alpha, 25 (OH)2D3 were added. The TRACP-positive cells became multinucleated with increasing time in culture and expressed the c-fms/M-CSF receptor. These results indicate that both contact with stromal cells and M-CSF are requisite for osteoclast differentiation under physiological conditions.

Topics: Acid Phosphatase; Animals; Bone Marrow; Bone Marrow Cells; Calcitriol; Cell Communication; Cell Differentiation; Cell Line; Hematopoiesis; Macrophage Colony-Stimulating Factor; Macrophages; Mice; Mice, Mutant Strains; Osteoclasts; Osteopetrosis; Recombinant Proteins

Osteoclast development was studied in cell cultures prepared from calvaria of neonatal osteopetrotic (mi/mi) mice or their normal littermates, using tartrate-resistant acid phosphatase (TRAPase), as an osteoclast marker. In cultures from normal mice, treatment with 10 nM PTH for 4-5 days stimulated the formation of osteoclasts. However in cultures from mi/mi mice, this response was only 7% +/- 5% that of normal mice and they were significantly smaller than osteoclasts of normal mice. Mineralized bone particles elicited osteoclast development in cultures from both normal and mi/mi mice, and osteoclast size was identical for both genotypes. Seventy-eight to 96% of the TRAPase-positive cells bound 125I-CT, as demonstrated by autoradiography. 125I-CT binding characteristics were identical in cultures from both genotypes treated with bone particles, exhibiting a Kd of 3.3-3.6 x 10(-10) M. Addition of PTH stimulated 45Ca release from the added bone particles only in the case of cultures prepared from normal mice, and CT inhibited this response. Cells from normal mice were capable of excavating bone from the surface of smooth cortical bone wafers, but such excavations were rarely seen in the case of calvarial cells from mi/mi mice. Thus, PTH-driven differentiation of osteoclasts is arrested in calvarial cell cultures from mi/mi mice, but mi/mi preosteoclasts retain the ability to express certain osteoclast markers in response to bone derived signals. We hypothesize that the lack of activity of mi/mi osteoclasts is due to the failure of mi/mi preosteoclasts to respond appropriately to resorptive agents, or to cytokines elicited by these agents.

Topics: Acid Phosphatase; Animals; Bone and Bones; Calcitonin; Cell Differentiation; Cells, Cultured; Kinetics; Mice; Osteoclasts; Osteopetrosis; Parathyroid Hormone; Protein Binding; Tartrates

We tested the ability of normal osteoclast progenitors found in neonatal liver and bone marrow to develop into functional osteoclasts when co-cultured with metatarsals from newborn osteopetrotic rabbits; the latter inherit an osteoclast incompetence resistant to cure by bone marrow transplantation. This system, developed by Burger and colleagues, has been shown to produce normal, functional osteoclasts when used with normal metatarsals. Our study tested the competence of the mutant skeletal microenvironment for differentiation of normal osteoclasts. Mutant and normal metatarsals were cultured alone or with normal liver, spleen, or bone marrow for up to 14 days. All normal cultures possessed a marrow cavity and contained numerous osteoclasts with cytochemical characteristics (tartrate-resistant acid phosphatase) of active cells. Mutant metatarsals co-cultured with normal spleen, liver, or bone marrow failed to develop a marrow cavity (evidence in itself of reduced bone resorption) and had osteoclasts reduced in both numbers and cytochemically detectable activity. Similar metatarsal cultures of an osteopetrotic rat mutation (incisors--absent) curable by bone-marrow transplantation exhibited marrow cavity development in mutant metatarsals co-cultured with normal spleen. These data suggest that the skeletal environment of osteopetrotic rabbits contains an inhibitor or lacks a promoter of osteoclast differentiation and function.

Topics: Acid Phosphatase; Animals; Animals, Newborn; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Histocytochemistry; Liver; Osteoclasts; Osteopetrosis; Rabbits; Spleen; Stem Cells

Bone-induced multinucleated cells have been suggested as surrogates for the study of osteoclastic lineage and function. This study evaluates this proposal by comparing acid phosphatase localization in tibial osteoclasts (in situ) with that of cell populations elicited by subcutaneous implantation of devitalized trabecular bone chips from two week old rats and suture into normal and osteopetrotic (ia) rats, emphasizing tartrate-resistant acid phosphatase, an osteoclastic marker. The ia rat mutation of osteopetrosis is characterized by defective osteoclasts which typically express enhanced TRAP activity when compared to normal; ia macrophage populations do not share the same osteoclastic defect and demonstrate normal amounts of acid phosphatase reactivity. The majority of the acid phosphatase activity expressed by implant-elicited mononuclear cells was tartrate sensitive. An increase in the percentage of tartrate-sensitive, but not TRAP-positive, mononuclear cells was observed during the 14-day implantation period, suggesting the mononuclear cells did not undergo osteoclastic differentiation. Both normal and ia osteoclasts contained high concentrations of TRAP reaction product (++) while bone- and suture-induced multinucleated cells examined at 14 days post-implantation were negative (0) or mildly (+) TRAP reactive. We conclude that devitalized bone matrix implanted at this ectopic site is capable of the formation of TRAP-positive (+) multinucleated cells, but when compared on the basis of strength of TRAP activity, the bone-induced multinucleated cells do not resemble active osteoclasts, but are similar to suture-elicited macrophage polykaryons. Therefore, we suggest caution in the use of bone-induced multinucleated cells as surrogates for the study of osteoclasts and normal bone resorption. Instead, these cells may represent a population of cells involved in pathological bone loss due to inflammation.

Topics: Acid Phosphatase; Animals; Bone and Bones; Bone Transplantation; Histocytochemistry; Insect Proteins; Macrophages; Osteoclasts; Osteopetrosis; Proteins; Rats; Rats, Mutant Strains; Silk; Sutures; Tartrates; Tibia

Cell surface structures of isolated osteopetrotic (mi/mi) and normal murine (+/+ and +/mi) and human osteoclasts were examined in a microscope combining light and scanning electron microscopy (LM/SEM). Tartrate-resistant acid phosphatase (TrAP) was used as an histochemical osteoclast marker. In osteopetrotic bone, as in normal murine bone, TrAP activity was exclusively seen in osteoclasts and preosteoclasts and was therefore judged a suitable marker for identification of isolated osteoclasts. A method was developed for preparation of LM/SEM specimens from osteoclast-enriched cell suspensions. In the LM/SEM isolated osteoclasts were easily recognized in the LM mode by TrAP contents. In specimens prepared from murine cells, but not human cells, LM identification of osteoclasts by TrAP was essential. This was in particular true for small, mononuclear, mi/mi osteoclasts. All osteoclasts examined had a villous appearance and were well spread over the glass substrate. There were no differences in cell surface morphology and in adherence to glass between osteopetrotic and normal osteoclasts.

Topics: Acid Phosphatase; Animals; Fetus; Histocytochemistry; Humans; Mice; Mice, Mutant Strains; Microphthalmos; Microscopy, Electron, Scanning; Osteoclasts; Osteopetrosis

In order to investigate skeletal abnormalities in a case of idiopathic osteopetrosis, a bone biopsy was taken from the anterior iliac crest and prepared for ultrastructural and histochemical study. There was a drastic reduction in osteoclastic bone resorption. The ruffle border and sealing zone, which are the osteoclast cell surface markers of bone resorption, were absent. The cells were highly vacuolated, and the vacuoles contained large amounts of a residual organic material which reacted strongly with acid phosphatase. Acid phosphatase activity was never found outside the cell, and in particular, not at the bone-cell interface. This suggests that the defect in bone resorption is caused by cell membrane abnormalities and the lack of ruffle border formation, rather than the inability of the lysosomal enzymes to digest the bone matrix.

Topics: Acid Phosphatase; Adult; Female; Humans; Lysosomes; Microscopy, Electron; Osteoclasts; Osteopetrosis

The staining intensity and inhibitor sensitivity of acid phosphatase activity was determined histochemically in various tissues of normal and ia rat pups by the use of freeze-dried whole body sections. Activity was determined using alpha-naphthylphosphate as substrate and hexazonium pararosaniline as coupler. Sections from ia rats (6 and 24 days old) showed markedly higher enzyme activity in bone than sections from normal littermates. However, there were no differences between ia and normal pups in acid phosphatase activity in soft tissues and developing teeth. Preincubation of sections with 1-100 mM sodium dithionite (an iron-binding agent) caused a dose-related inhibition of enzyme activity in bone of ia and normal pups, but only slight inhibition of activity in soft tissues. Partial restoration of the dithionite-inhibited activity in bone was achieved by subsequent preincubation in 1 mM FeCl2. Addition of 100 mM sodium tartrate to the staining solution of non-preincubated sections caused almost complete inhibition of activity in soft tissues and the developing teeth but no inhibition of the activity in bone that was sensitive to sodium dithionite. These data indicate a) that sodium dithionite can be used as a specific histochemical inhibitor of the tartrate-resistant acid phosphatase and b) that the source of increased acid phosphatase activity in bone from ia rats is mostly from the tartrate-resistant acid phosphatase.

Topics: Acid Phosphatase; Animals; Bone and Bones; Brain; Dithionite; Ferrous Compounds; Ganglia, Spinal; Muscles; Osteopetrosis; Rats; Rats, Mutant Strains; Tartrates; Tooth Abnormalities

This study of two cases documents increased bone mass in a 30-yr old man and osteopetrosis in a 38-yr-old woman and examines the patients in terms of radiologic features, bone photon absorptiometry and bone biopsy analyses which include tetracycline kinetics. Both patients had elevated bone mass based on quantitative bone histology of iliac crest biopsies normal, or low PTH, and normal calcitonin values. However, they differed markedly from each other in other respects: One patient possessed significantly increased rates of bone formation and mineralization, had elevated serum alkaline phosphatase, increased total hydroxyproline over a 24 hour urine collection but displayed a normal amount of bone surface involved in resorption. The other subject had normal bone formation parameters, but had elevated serum acid phosphatase and a significantly elevated resorbing (crenated) surface. Although most theories ascribe and cause of osteopetrosis to decreased resorption, our study shows that increased bone formation, even when accompanied by normal resorption, can lead to the abnormal accumulation of bone mass. In this respect, the resorptive response of this patient was abnormal; the normal coupled relationship between bone formation and bone resorption, which ensures proper control of endosteal bone volume, had been lost.

Topics: Acid Phosphatase; Adult; Alkaline Phosphatase; Biopsy; Bone and Bones; Bone Resorption; Calcium; Female; Growth Hormone; Humans; Male; Osteogenesis; Osteopetrosis; Parathyroid Hormone; Radiography

Morphokinetic measurements after tetracycline labeling were performed on bone specimens of the iliac crest in a 30-year-old man and a 38-year-old women. On radiographs, the osteopetrosis (=marble bone disease Albers-Schönberg) was characterized by an almost homogenous osteosclerosis in the man and by variable zones of high and normal density in the woman. Histologically, the man revealed increased rates of bone formation and mineralisation as signs of hyperactivity of the osteoblasts accompanied, biochemically, by an elevation of serum alkaline phosphatase and a high urinary output of hydroxyproline. In the woman increased resorbing bone seams, and elevation of serum acid phosphatase and a slightly lowered urinary output of hydroxyproline were apparent as signs of an osteoclastic deficiency. The cause of osteopetrosis has been generally ascribed to decreased bone resorption. This study shows that the accumulation of bone mass in osteopetrosis may be due also to increased bone formation and that by pathomechanism, probably, two forms of osteopetrosis in the adult may be differentiated.

Topics: Acid Phosphatase; Adult; Alkaline Phosphatase; Female; Humans; Hydroxyproline; Male; Osteopetrosis; Osteosclerosis; Radiography

Topics: Acid Phosphatase; Animals; Kupffer Cells; Lymph Nodes; Macrophages; Osteopetrosis; Phagocytes; Phagocytosis; Rats; Spleen

Macrophages from osteopetrotic mice caused less bone resorption than normal sib macrophages in culture. The bone-resorbing activity lay in the culture supernatant, and reduced activity in cultures of macrophages from osteopetrotic mice correlated with reduced glass spreading and reduced latex phagocytosis. We suggest that macrophages from osteopetrotic mice show defective phagocytic recognition of glass, latex, and bone and that this defective recognition may reflect the cause of the failure of resorption which results in osteopetrosis.

Topics: Acid Phosphatase; Animals; Bone Resorption; Calcium; Cells, Cultured; Female; Macrophages; Male; Mice; Mice, Neurologic Mutants; Osteopetrosis; Phagocytosis

Chicks infected as 12-day-old embryos with an end-point purified derivative of avian myeloblastosis virus developed a rapidly progressive osteopetrosis that manifested within 1 week of hatching. A detailed comparison of osteopetrotic chicks and normal hatchmates revealed the following. (i) Osteopetrotic chicks exhibited a stunting syndrome, growing at a mean rate that was 26% of the control rats. (ii) At autopsy, the mass of the lymphoid organs was reduced, whereas the mass of the heart, pancreas, kidneys, lungs, brain, liver, and bones of osteopetrotic chicks was increased. Edema was likely responsible for most of the increase in organ weight. (iii) Infected chicks exhibited a normochromic, normocytic anemia that was virus dose dependent and was not required for the development of osteopetrosis. (iv) Bone collagen content was normal. (v) Osteopetrotic bone was initially hypomineralized, but later became more fully mineralized. (vi) The concentrations of alpha, beta, and gamma globulins in the plasma were elevated in osteopetrotic chicks, whereas albumin concentration was decreased. (vii) The level of plasma alkaline phosphatase was elevated in osteopetrotic chicks, yet the level of acid phosphatase was unchanged. (viii) Body and bone temperatures were unchanged.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Avian Leukosis Virus; Avian Myeloblastosis Virus; Blood Proteins; Body Temperature; Bone and Bones; Chick Embryo; Chickens; Collagen; Organ Specificity; Osteopetrosis; Osteoporosis; Water

Topics: Acid Phosphatase; Alkaline Phosphatase; Child; Genes, Dominant; Humans; Male; Osteopetrosis; Radiography

Topics: Acid Phosphatase; Animals; Glucuronidase; Osteoclasts; Osteopetrosis; Palate; Rats; Succinate Dehydrogenase

Topics: Acid Phosphatase; Animals; Bone and Bones; Glucuronidase; Histocytochemistry; Mandible; Maxilla; Mutation; Osteoclasts; Osteopetrosis; Rats

Topics: Acid Phosphatase; Animals; Biopsy; Bone Resorption; Calcium; Calcium Radioisotopes; Culture Techniques; Frontal Bone; Hypercalcemia; Osteoclasts; Osteopetrosis; Parathyroid Hormone; Parietal Bone; Rats; Tibia; Tritium

Topics: Acid Phosphatase; Alkaline Phosphatase; Bone and Bones; Child; Female; Genes, Dominant; Humans; Osteopetrosis; Radiography

Topics: Acid Phosphatase; Age Factors; Animals; Bone and Bones; Bone Development; Bone Matrix; Bone Resorption; Calcium; Collagen; Cytoplasm; Injections, Intraperitoneal; Lysosomes; Osteoclasts; Osteopetrosis; Proline; Rats; Remission, Spontaneous; Time Factors; Tritium

Topics: Acid Phosphatase; Adult; Alkaline Phosphatase; Bone Diseases; Child; Child, Preschool; Female; Humans; Infant; Male; Middle Aged; Osteopetrosis

Topics: Acid Phosphatase; Adenine Nucleotides; Adenosine Triphosphate; Adult; Alanine Transaminase; Citrates; Citric Acid Cycle; Creatine Kinase; Electrophoresis; Fructose-Bisphosphate Aldolase; Gels; Glucosephosphate Dehydrogenase; Humans; Ketoglutaric Acids; L-Lactate Dehydrogenase; Lactates; Male; Middle Aged; Osteopetrosis; Pyruvates; Starch

Topics: Acid Phosphatase; Adolescent; Adult; Child; Child, Preschool; Female; Fructose-Bisphosphate Aldolase; Genetic Diseases, Inborn; Humans; Infant; L-Lactate Dehydrogenase; Male; Metabolism, Inborn Errors; Middle Aged; Osteopetrosis

Topics: Acid Phosphatase; Electrophoresis; Gaucher Disease; Glycerophosphates; Humans; Hyperparathyroidism; Male; Osteopetrosis; Phosphates; Prostatic Neoplasms

Topics: Acid Phosphatase; Bone Diseases; Bone Neoplasms; Clinical Enzyme Tests; Diagnosis, Differential; Gaucher Disease; Humans; Hyperparathyroidism; Male; Osteitis Deformans; Osteitis Fibrosa Cystica; Osteogenesis Imperfecta; Osteopetrosis; Osteosclerosis; Prostatic Neoplasms