Showing papers on "Tartrate-resistant acid phosphatase published in 1991"

Essential role of macrophage colony-stimulating factor in the osteoclast differentiation supported by stromal cells.

Abstract: 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.

Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phosphatase activity of protein phosphatases.

Abstract: The phosphatase activities of type 2A, type 1 and type 2C protein phosphatase preparations were measured against p-nitrophenyl phosphate (pNPP), a commonly used substrate for alkaline phosphatases. Of the three types of phosphatase examined, the type 2A phosphatase exhibited an especially high pNPP phosphatase activity (119 +/- 8 mumol/min per mg of protein; n = 4). This activity was strongly inhibited by pico- to nano-molar concentrations of okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases that has been shown to have no effect on alkaline phosphatases. The dose-inhibition relationship was markedly shifted to the right and became steeper by increasing the concentration of the enzyme, as predicted by the kinetic theory for tightly binding inhibitors. The enzyme concentration estimated by titration with okadaic acid agreed well with that calculated from the protein content and the molecular mass for type 2A phosphatase. These results strongly support the idea that the pNPP phosphatase activity is intrinsic to type 2A protein phosphatase and is not due to contamination by alkaline phosphatases. pNPP was also dephosphorylated, but at much lower rates, by type 1 phosphatase (6.4 +/- 8 nmol/min per mg of protein; n = 4) and type 2C phosphatase (1.2 +/- 3 nmol/min per mg of protein; n = 4). The pNPP phosphatase activity of the type 1 phosphatase preparation shows a susceptibility to okadaic acid similar to that of its protein phosphatase activity, whereas it was interestingly very resistant to inhibitor 2, an endogenous inhibitory factor of type 1 protein phosphatase. The pNPP phosphatase activity of type 2C phosphatase preparation was not affected by up to 10 microM-okadaic acid.

Differentiation of HL-60 cells into cells with the osteoclast phenotype.

Abstract: The osteoclast is the unique multinucleated cell that is responsible for bone degradation in both physiological and pathological circumstances. However, knowledge of the lineage of this inaccessible cell, the nature of its precursors, and the regulation of its formation and activation is limited and controversial. Here we show that the human promyelocytic cell line HL-60 has the potential, under the appropriate culture conditions, to differentiate into cells that have morphological and functional characteristics of osteoclasts, including multinucleation, presence of tartrate-resistant acid phosphatase activity, cross-reactivity with monoclonal antibodies that preferentially recognize osteoclasts, and capacity to resorb bone and respond to calcitonin. The multinucleated cells also contain high affinity receptors to calcitonin, in contrast to wild-type undifferentiated HL-60 cells. These data suggest that osteoclasts share a common precursor with hematopoietic cells. These undifferentiated and differentiated HL-60 cells should provide a unique model for study of the cell biology of human osteoclast differentiation, allowing molecular biological and biochemical studies heretofore not possible.

Tartrate-resistant acid phosphatase from human osteoclastomas is translated as a single polypeptide.

Abstract: Tartrate-resistant acid phosphatases have been isolated from a number of sources. These enzymes consist of one subunit (Mr 30,000-40,000) or two dissimilar subunits (Mr 15,000-20,000). Previously we isolated the enzyme from human osteoclastomas, as a two-subunit protein. By Northern blotting and hybridization with radiolabelled oligonucleotides corresponding to the N-terminal sequences of the two subunits, we demonstrate here that the enzyme is transcribed as one mRNA which is translated in vitro to produce a single polypeptide of approx. Mr 33,000. Transcription as a single mRNA species is also the case in other tissues. These results suggest that the osteoclastoma enzyme undergoes post-translational modification in the form of cleavage of a single peptide bond to give a disulphide-bonded two-subunit protein.

Ultrastructural localization of tartrate-resistant acid phosphatase (purple acid phosphatase) activity in chicken cartilage and bone.

Abstract: Tartrate-resistant acid adenosine triphosphatase activity at pH 6.5, using a lead-salt method, was localized at light and electron microscopic levels in cartilage and bone matrices, osteoclasts, and chondroclasts. Cartilage matrix staining occurred after vascular invasion of the growth plate. In osteoclasts, activity was present in lysosomes, extracellular ruffled border channels, and the underlying cartilage and bone matrices. Staining artifacts occurred at lower pH levels (pH 5.4, 5.0). Adenosine diphosphate, p-nitrophenylphosphate, thiamine pyrophosphate, and alpha-naphthylphosphate also acted as substrates; but no activity was observed when adenosine monophosphate, adenylate-(beta, gamma-methylene) diphosphate, and beta-glycerophosphate were used. The activity was inhibited by NaF, dithionite, and a high concentration of p-chloromercuribenzoic acid, and activated by simultaneous addition of FeCl2 and ascorbic acid, as has been shown in biochemical studies. These histochemical results support the view that the adenosine triphosphate hydrolyzing activity at pH 6.5 is due to tartrate-resistant acid phosphatase (TRAP). There were some differences in ultrastructural localization between TRAP and tartrate-sensitive acid phosphatase (TSAP) activities in osteoclasts: TSAP activity was more intense in lysosomes and Golgi complexes and TRAP was stronger in the cartilage and bone matrices. It is suggested, therefore, that most of TRAP is in an inactive form in cells and is activated when secreted.

Histochemistry of tartrate-resistant acid phosphatase and carbonic anhydrase isoenzyme II in osteoclast-like giant cells in bone tumours

Abstract: Using routinely processed, paraffin-embedded tissue specimens, osteoclast-like giant cells in giant cell tumour of bone (GCT), chondroblastoma, osteoblastoma and osteoblastic osteosarcoma were examined histochemically for osteoclast-specific enzymes tartrateresistant acid phosphatase (TRAP) and carbonic anhydrase isoenzyme II (CA-II). Osteoclast-like giant cells and some mononuclear cells possessed TRAP activity. These were further classified with respect to CA-II immunoreactivity, i.e. cells with CA-II were seen in GCT and chondroblastoma, while those in osteoblastoma and osteoblastic osteosarcoma were negative for CA-II. All the cellular components in malignant fibrous histiocytoma and various extraosseous inflammatory lesions including malignant giant cells and macrophage polykaryons were negative for both TRAP and CA-II. These results indicate that osteoclast-like giant cells in GCT, chondroblastoma, osteoblastoma and osteoblastic osteosarcoma are all osteoclasts and generated by fusion of mononuclear cells with the same histochemical characteristics as osteoclast-like giant cells. The difference in CA-II immunoreactivity suggests the functional or maturational difference between osteoclast-like giant cells in GCT and chondroblastoma and those in osteoblastoma and osteosarcoma.

Identification of osteoclasts and their mononuclear precursors. A comparative histological and histochemical study in hamster periodontitis.

Abstract: Quantification of osteoclast resorption, a good index of periodontitis destruction, is primarily based on osteoclast identification. As their identification is sometimes dubious, we compared osteoclastic counts in hamster specimens processed for either routine histology or tartrate-resistant acid phosphatase (TRAP) staining. No difference was found between the two approaches concerning the number of osteoclasts. However the mean bone-osteoclast interface was higher in the TRAP-stained specimens (+30%, p less than 0.02). As osteoclast precursors are also TRAP+ cells, they were quantified too. Compared with controls, there was a dramatic increase (p less than 0.0001) in periodontitis-affected animals. Precursors were strongly correlated to active osteoclasts (r = 0.97). Our data suggest that precursors are recruited only when the disease is active in a given site.

Clinical usefulness of serum tartrate-resistant acid phosphatase in Paget's disease of bone: correlation with other biochemical markers of bone remodelling.

Abstract: Tartrate resistant acid phosphatase (TRAP) has been proposed as a new biochemical marker for bone resorption. We have compared this new marker, TRAP, with the classical biochemical markers of bone remodelling, serum alkaline phosphatase (sAP), serum osteocalcin (sBGP), and with the urinary hydroxyproline/creatinine ratio (uOHProl/creatinine), a routine marker of bone resorption. Serum TRAP was significantly higher in pagetic patients (n=43) than in control subjects (n=12) (13.02±4.7 vs 5.48 ±1.31 IU/L,P<0.001) and a significantly positive linear correlation was found between the sTRAP and uOHProl/creatinine ratio (y=0.005x–0.0069,r=0.82,P<0.001), between sTRAP and sAP (y=19.3x–85.0,r=0.71,P<0.001) and also between sTRAP and sBGP (y=0.02x+2.23,r=0.52,P<0.01). Serum TRAP levels were higher than the upper limit of normality in all our pagetic patients except for two, whose uOHProl/creatinine levels were in the normal range. We conclude that (1) sTRAP could be a parameter as sensitive as uOHProl/creatinine in the diagnosis of Paget's disease; (2) sTRAP and uOHProl/creatinine are both good markers of bone resorption; (3) the correlation found between sTRAP and formation markers (sAP and sBGP) makes sdTRAP a marker of disease activity in Paget's disease of bone; (4) the assay of sTRAP is easier, faster, and of lower cost than the urinary hydroxyproline determination. We suggest that sTRAP determination could be used as a routine marker of bone resorption in Paget's disease of bone, as is the case with uOHProl determination.

Regulation and expression of type V (tartrate-resistant) acid phosphatase in human mononuclear phagocytes.

Abstract: Human type V (tartrate-resistant) acid phosphatase belongs to a unique group of iron-binding proteins that includes uteroferrin and other purple phosphatases. The enzyme is normally restricted to osteoclasts and certain phagocytic cells but its role is unknown. We show that phosphatase mRNA is abundant in cells of monohistiocytic phenotype and that enzyme expression in cultured human monocyte-derived macrophages is depressed by gamma-interferon and bacterial lipopolysaccharide, agents that promote functional differentiation in these cells. In contrast, phorbol ester, which stimulates intracellular calcium-mediated events, greatly enhances type V phosphatase expression and mRNA abundance. Lymphokine and phorbol ester-modulated expression of type V acid phosphatase expression thus represents a model system for investigating proliferative responses that are specific to cells of the mononuclear macrophage system.

What's new in osteoclast ontogeny?

Abstract: The osteoclast (OC) is a multinuclear bone-resorbing cell which shares several characteristics with cells of the mononuclear phagocyte system. Unlike terminally differentiated macrophages, OCs possess specialized characteristics such as tartrate resistant acid phosphatase activity and the presence of calcitonin receptors. It appears that myeloid progenitor cells, probably granulocyte-macrophage colony-forming units, generate OC precursors which then differentiate and fuse into OCs under the regulation of osteotropic hormones, cytokines and other local factors. Parathyroid hormone and 1,25 dihydroxy Vitamin D3 induce both the formation and fusion of OC precursors, while calcitonin inhibits fusion. Osteoblasts also produce factor(s) which regulate OC precursor differentiation and therefore bone resorption; the nature of these factor(s), however, is unknown. In addition, the OC surface interacts specifically with a range of cellular and extracellular matrix-associated ligands which influence OC differentiation. The precise regulation of OC formation, however, is complex and awaits further investigation.

Osteoclast origin of giant cells in giant cell tumors of bone: ultrastructural and cytochemical study of six cases.

Abstract: To clarify the histogenesis of giant cells appearing in giant cell tumors of bone (GCTB), an ultrastructural and cytochemical study of six cases was performed with both acid phosphatase (ACPase) and tartrate-resistant acid phosphatase (TRACPase) as marker enzymes. TRACPase is considered a specific marker for osteoclasts. ACPase was demonstrated in the macrophagelike stromal cells, the multinucleated giant cells, and the infiltrating macrophages. The enzyme reaction was localized in lysosomal dense bodies and Golgi areas. Intense TRACPase activity was demonstrated in the multinucleated giant cells, whereas a weak reaction was found in the macrophagelike stromal cells. The multinucleated giant cells and macrophagelike stromal cells resembled osteoclasts with regard to the subcellular localization of TRACPase. The present results suggest that the giant cells in GCTB are indeed derived from osteoclasts.

Quantification of tartrate resistant acid phosphatase activity using a computerized image analysis system.

Abstract: Tartrate resistant acid phosphatase (TRAP) has been accepted as a marker for identification of osteoclasts. A method is reported here for quantitating TRAP using an image analysis system. The amount of the enzyme specific to osteoclasts can be used to differentiate osteoclasts from other cells capable of TRAP expression. TRAP expression characteristic of the osteoclast was compared with that of multi-nucleated giant cells (MNGC)s recruited to the site of subcutaneously implanted mineralized bone matrix. Two weeks post-implantation, the pellets were removed and processed for the demonstration of TRAP along with rat proximal tibiae. A large amount of TRAP was consistently expressed by the in situ osteoclasts. The MNGCs associated with the mineralized bone implants expressed little if any TRAP reaction product. Using this system, the amount of TRAP reaction product or any other enzyme reaction product expressed can be objectively and reproducibly quantitated.

Hydrochlorothiazide inhibits parathormone-stimulated increase in plasma tartrate-resistant acid phosphatase in mice.

Abstract: The inhibitory effect of hydrochlorothiazide (HTZ) on parathormone-induced bone changes in mice was studied with the aid of the analysis of plasma calcium and tartrate-resistant acid phosphatase. We have found that HTZ alone had no effect on plasma tartrate-resistant acid phosphatase (Tr-ACP), phosphate, and creatinine concentration. Parathormone (PTH) administration increased plasma Tr-ACP from 15.00±1.50 to 20.62±2.35 U/liter in intac mice. The increase of plasma Tr-ACP in HTZ-treated mice after PTH administration was not significant. The plasma calcium was affected in a way similar to Tr-ACP. HTZ reduced the sensitivity of bone to resorbing effects of PTH, using Tr-ACP as a useful biomarker of bone resorption.

Plasma acid and alkaline phosphatase in patients with breast cancer.

Abstract: Acid and alkaline phosphatase were determined in 107 breast cancer patients to study their potential value in case of bone metastases. The patients were divided into 4 groups: A, patients without metastases (n = 34); B, metastatic patients without bone lesions (n = 37); C, patients with metastases in and outside of bones (n = 24), D, patients with bone-only metastases (n = 12). Tartrate resistant acid phosphatase (TR-ACP), and bone alkaline phosphatase (bone-ALP) were significantly higher in patients with metastases than in patients without. However, no difference in TR-ACP was observed between subgroups of metastatic patients.

Ultrastructure, tartrate-resistant acid phosphatase activity and calcitonin responsiveness of osteoclasts at sites of demineralized bone matrix implant-induced osteogenesis.

Abstract: The morphology, ultrastructure, tartrate-resistance acid phosphatase reactivity, and calcitonin responsiveness of osteoclasts induced at sites of demineralized bone matrix (DBM) implant-induced osteogenesis in rats were determined. Osteoclasts at these ectopic sites had a morphologic and ultrastructural appearance similar to osteoclasts normally found in skeletal tissues. When observed by scanning electron microscopy, resorption surfaces on the implants had well-defined resorption pits (Howship's lacunae), indicative of active bone resorption. The osteoclasts stained intensely for tartrate-resistance acid phosphatase, an enzyme that is specific for osteoclasts. In response to human calcitonin, hypocalcemia occurred and osteoclasts lost their ruffled borders, indicating that these cells are responsive to exogenous hormonal stimulation. The osteoclasts induced by subcutaneous implantation of DBM had morphologic and functional characteristics similar to osteoclasts normally found in skeletal tissues.

An Enzyme Histochemical Investigation on Bone Acid Phosphatases: The use of Fluoride to Distinguish the Isoenzymes

Abstract: The histochemical distribution of acid phosphatase activity in chicken tibial metaphyses was investigated with the azo-dye method, using naphthol AS-BI phosphate as a substrate, and the lead-salt method, usingβ-glycerophosphate, p-nitrophenylphosphate or adenosine triphosphate as substrates Tartrate-resistant activity was found in cartilage and bone matrices and in osteoclasts when naphthol AS-BI phosphate, p-nitrophenylphosphate or adenosine triphosphate were used Fluoride-resistant activity was observed in the cytoplasm of osteoclasts with naphthol AS-BI phosphate or p-nitrophenylphosphate; this activity was also insensitive to tartrate The tartrate-resistant acid adenosine triphosphatase activity, which is due to purple acid phosphatse (type V acid phosphatase isoenzyme), was significantly weaker in the cytoplasm of osteoclasts than the tartrate-resistant acid phosphatase (TRAP) activity with naphthol AS-BI phosphate or p-nitrophenylphosphate as substrates Furthermore, the purple acid phosphatase activity was strongly inhibited by fluoride

Enzyme histochemical study of multinucleated giant cells responding to synthetic hydroxyapatite in rat jaw bone and subcutaneous tissue

Abstract: ラット顎骨および皮下組織内に埋入された合成水酸化アパタイト (HA) 表面に出現する多核巨細胞について酸性フォスファターゼ (ACPase), 酒石酸耐性酸性フォスファターゼ (TRAP) を用い酵素組織化学的に検索した。顎骨内埋入HA表面に出現した多核巨細胞はHAとの界面に波状縁様構造を有し, また酵素組織化学的にACPase, TRAP活性を示した。これらの特徴は既存の骨梁表面にみられた破骨細胞に類似していた。また, 多核巨細胞周囲にはTAPR陽性の単核細胞が観察された。一方, 皮下組織内では多核巨細胞は扁平な細胞質によってHAを包囲するように接し, TRAP活性は陰性であった。また, ACPase活性も少数の多核巨細胞に認められたのみであった。従って, HA表面の多核巨細胞は, 顎骨内と皮下組織内で酵素組織化学的に異なる性状を示したことから, HAが埋入される組織環境の影響を受けることが示唆された。