Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A serum-free primary culture system for chicken growth plate chondrocytes has been developed which consistently undergoes mineral deposition. Upon attainment of confluency, the chondrocytes develop locally into multilayer cellular nodules leading to matrix calcification. Mineralization first occurs in matrix vesicles (MV) that are abundant in the extraterritorial matrix between the hypertrophic cells. Studies with 45Ca reveal that significant accumulation of Ca2+ occurs as early as day 12, continuing progressively throughout the culture period. By day 24, the nodules become densely calcified. Fourier transform infrared spectroscopy reveals the mineral to be similar to apatite, with features essentially identical to those of mineral formed by MV in vitro. The presence of ascorbate is critical to the culture system; in its absence, calcification is rarely observed. Ascorbate stimulates MV formation and synthesis of cellular protein, alkaline phosphatase, and especially types II and X collagens. In addition, there is strong evidence that the types II and X collagens are associated with MV. 1) Electron microscopy reveals MV embedded in a type II collagenous network; 2) Western blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of MV using monospecific antibodies to types X and II collagen indicate that both collagens are present in specific MV fractions; 3) sucrose gradient purification of MV does not remove associated collagens; 4) graded salt extraction selectively releases type II collagen from MV; and 5) incubation of radiolabeled types II and X collagens with MV leads to their cosedimentation upon subsequent centrifugation. Taken together, the data suggest that coordinated synthesis of the collagens, alkaline phosphatase, MV formation, and Ca2+ accumulation by the cultures combine to induce mineral deposition in the multilayer nodules.
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PMID:Induction of mineral deposition by primary cultures of chicken growth plate chondrocytes in ascorbate-containing media. Evidence of an association between matrix vesicles and collagen. 259 80

The calcification of cartilage matrix in endochondral bone formation occurs in an extracellular matrix composed of fibrils of type II collagen with which type X collagen is closely associated. Also present within this matrix are the large proteoglycans containing chondroitin sulfate which aggregate with hyaluronic acid. In addition, the matrix contains matrix vesicles containing alkaline phosphatase. There is probably a concentration of calcium as a result of its binding to the many chondroitin sulfate chains. At the time of calcification, these proteoglycans become focally concentrated in sites where mineral is deposited. This would result in an even greater focal concentration of calcium. Release of inorganic phosphate, as a result of the activity of alkaline phosphatase, can lead to the displacement of proteoglycan bound calcium and its precipitation. The C-propeptide of type II collagen becomes concentrated in the mineralizing sites, prior to which it is mainly associated with type II collagen fibrils and is present in dilated cisternae of the enlarged hypertrophic chondrocytes. The synthesis of type II collagen and the C-propeptide, together with alkaline phosphatase, are regulated by the vitamin D metabolites 24,25(OH)2 cholecalciferol and 1,25 (OH)2 cholecalciferol. At the time of calcification, type X collagen remains associated with type II collagen fibrils. It may play a role in preventing the initial calcification of these fibrils focusing mineral formation in focal interfibrillar sites. This process of calcification is clearly very complex, and involves different interacting matrix molecules and is carefully regulated at the cellular level.
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PMID:Cartilage macromolecules and the calcification of cartilage matrix. 267 83

Cell cultures derived from young rat epiphyseal cartilage were grown for approximately 2 wk in BGJb medium supplemented with 10% fetal bovine serum to reach confluence. These cells were identified as chondrocytes as checked by morphology, the presence of alkaline phosphatase, and a positive type II collagen antibody reaction. The cells also responded to different hormonal treatment. Parathyroid hormone (PTH) increased cyclic AMP production by 50% within 15 min of treatment, whereas prostaglandin E2 (PGE2) caused an increase of 160%. Calcitonin (CT) did not affect cAMP production in these cells. DNA synthesis 24 h after hormonal treatment was increased by PTH (2.5-fold) and PGE2 (2-fold), but not by CT. Among the vitamin D metabolites, 24,25(OH)2D3 increased significantly the [3H]thymidine incorporation into DNA, whereas 1,25(OH)2D3 effect was minimal. These results provide evidence for the use of these cell cultures as a model for cartilage in vitro when studying biological and hormonal responsiveness.
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PMID:Rat epiphyseal cells in culture: responsiveness to bone-seeking hormones. 284 Apr 29

Single-cell suspensions obtained from sequential enzymatic digestions of fetal rat calvaria were grown in long-term culture in the presence of ascorbic acid, Na beta-glycerophosphate, and dexamethasone to determine the capacity of these populations to form mineralized bone. In cultures of osteoblastlike cells grown in the presence of ascorbic acid and beta-glycerophosphate or ascorbic acid alone, three-dimensional nodules (approximately 75 micron thick) covered by polygonal cells resembling osteoblasts could be detected 3 days after confluency. The nodules became macroscopic (up to 3 mm in diameter) after a further 3-4 days. Only in the presence of organic phosphate did they mineralize. Nodules did not develop without ascorbic acid in the medium. Dexamethasone caused a significant increase in the number of nodules. Histologically, nodules resembled woven bone and the cells covering the nodules stained strongly for alkaline phosphatase. Immunolabeling with specific antibodies demonstrated intense staining for type I collagen that was mineral-associated, a weaker staining for type III collagen and osteonectin, and undetectable staining for type II collagen. Nodules did not develop from population I and the number of nodules formed by populations II-V bore a linear relationship to the number of cells plated (r = .99). The results indicate that enzymatically released calvaria cells can form mineralized bone nodules in vitro in the presence of ascorbic acid and organic phosphate.
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PMID:Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations. 308 92

Transforming growth factor beta (TGF-beta) has been shown to induce chondrogenesis by embryonic rat mesenchymal cells (Seyedin et al., J. Biol. Chem., 261: 5693, 1986). Here we report the effects of bovine TGF-beta on the phenotypic expression of differentiated primary rat osteoblastic and chondroblastic cells. Culture of rat calvarial osteoblasts with TGF-beta resulted in a dose and time-dependent decrease in alkaline phosphatase activity. Levels of alkaline phosphatase were reduced to less than 10% of control values by 0.4 nM TGF-beta. The decrease became apparent after 24 hours and reached a maximum by 72 hours. Similarly, treatment of chondroblasts with 0.4 nM TGF-beta resulted in decreased production of cartilage-specific macromolecules: type II collagen and cartilage proteoglycan. Both cell types exhibited dramatic changes in cell shape after treatment with TGF-beta. Modulation of these differentiated markers by TGF-beta could be mimicked, in part, by addition of fibronectin. Addition of dihydrocytochalasin B blocked the inhibition of phenotypic expression by TGF-beta. These results indicate that TGF-beta inhibits phenotypic expression by osteoblasts and chondroblasts in vitro and suggest that this activity of TGF-beta may be mediated through interactions between the extracellular matrix and cytoskeletal elements.
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PMID:Transforming growth factor-beta modulates the expression of osteoblast and chondroblast phenotypes in vitro. 316 57

Chondroprogenitor cells of newborn murine mandibular condyles were cultured on top of collagen sponges for up to 18 days. After 24 h in culture, new chondroblasts developed which subsequently matured showing signs of hypertrophy, while the extracellular matrix revealed positive reactivity for type II collagen, cartilage proteoglycans and mineralization. Light and electron microscopy examinations showed signs of new osteoid formation, a feature that was preceded by positive immunohistochemical reaction for type I collagen, fibronectin and bone specific sialoprotein. A close temporal and spatial association was noted between the development of mature, mineralized cartilage and new osteoid. The differentiation of new cartilage and bone cells was linked to an increased activity of DNA synthesis and cellular proliferation. The de novo bone formation was accompanied by increasing rates of alkaline phosphatase activity and uptake of [45Ca] features that were found to be tightly correlated to each other. The collagen substrata appeared also to facilitate the migration of cells, their replication and their subsequent differentiation to their respective cellular lineage. Hence, collagen sponges in vitro appear to serve as a promising substrata for culture systems involved with the growth and differentiation of mineralizing tissues such as cartilage and bone.
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PMID:Acceleration of cartilage and bone differentiation on collagenous substrata. 331 77

Mandibular condyles of fetal mice 19 to 20 days in utero comprising clean cartilage and its perichondrium were cultured for up to 14 days, and their capacity to develop osteoid and to mineralize in vitro was examined. After 3 days in culture the cartilage of the mandibular condyle appeared to have lost its inherent structural characteristics, including its various cell layers: chondroprogenitor, chondroblastic, and hypertrophic cells. At that time interval no chondroblasts could be seen; instead, most of the cartilage consisted of hypertrophic chondrocytes. By that time, the surrounding perichondrium, which contains pluripotential mesenchymal stem cells, revealed the first signs of extracellular matrix enclosing type I collagen, bone alkaline phosphatase, osteonection, fibronectin, and bone sialoprotein as demonstrated by immunofluorescent techniques. Electron microscopic examinations of the newly formed matrix revealed foci of mineralization within and along collagen fibers as is normally observed during bone development. The composition of the latter mineral deposits resembled calcium pyrophosphate crystals. Following 14 days in culture larger portions of the condyle revealed signs of osseous matrix, yet the tissue reacted positively for type II collagen. Hence, the condylar cartilage, a genuine representative of secondary-type cartilage, elaborated in vitro a unique type of bone that would be most appropriately defined as chondroid bone. Biochemical assays indicated that the de novo formation of chondroid bone was correlated with changes in alkaline phosphatase activity and 45Ca incorporation. The findings of the present study imply that mesenchymal stem cells that ordinarily differentiate into cartilage possess the capacity to differentiate into osteogenic cells and form chondroid bone.
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PMID:Chondroid bone arises from mesenchymal stem cells in organ culture of mandibular condyles. 359 22

A serum-free medium for postembryonic chicken epiphyseal growth plate chondrocytes has been developed from 104 MCDB medium. To enable these fastidious cells to survive, grow, and express normal phenotype, a substantial increase over MCDB 104 in the level of many of the amino acids was required, as well as a change in the buffer system and the addition of SerXtend, a defined, serum-free product containing various growth factors, including fibroblast growth factor. Also required was the provision of cell attachment factors, either by coating culture surfaces with type II collagen, or better, by allowing the freshly released cells to recover for several hours in a medium supplemented with 10% fetal bovine serum before plating. This new serum-free medium, which we call USC-HC1, supports growth and replication, the retention of normal polygonal morphology, the expression of significant levels of cellular alkaline phosphatase activity, the production of sulfated proteoglycans, type II collagen, and the formation of alkaline phosphatase-rich matrix vesicles by the chondrocytes. The major advantage of USC-HC1, however, is that it will provide for the first time an opportunity to examine the effects of various defined growth and hormonal factors on the phenotypic expression and differentiation of growth plate chondrocytes, in the absence of the variable (stimulatory and inhibitory) factors present in fetal bovine serum.
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PMID:Development of a new serum-free medium, USC-HC1, for growth and normal phenotype in postembryonic chicken growth plate chondrocytes. 377 40

Matrix vesicles (MV) can be readily isolated from culture media of chicken growth plate hypertrophic chondrocytes grown in primary culture. The chondrocytes maintain normal morphology and synthesize type II collagen throughout the culture period. The culture-derived MV are morphologically indistinguishable from MV seen in situ and are rich in alkaline phosphatase. Formation of alkaline phosphatase-rich MV is strongly influenced by the stage of culture: large numbers are released shortly after cell seeding; marked decline is seen during cell spreading and rapid cell division; notable resurgence in alkaline phosphatase-rich MV production occurs as the cells attain confluency. Increasing the initial chondrocyte seeding density proportionately increases MV production. Cells derived from the hypertrophic region are much more capable of forming alkaline phosphatase-rich MV than those from the proliferating zone, indicating that MV formation is dependent on cellular differentiation. MV released by the cultured chondrocytes were compared in protein and phospholipid composition and in their ability to accumulate mineral ions, with plasma membrane fractions and collagenase-released MV obtained from the same tissue. Electrophoretic patterns of proteins, and the phospholipid profiles, suggest that significant modification of the plasma membrane occurs during MV formation. The vesicles are capable of accumulating large amounts of mineral ions from a metastable synthetic cartilage lymph when supplied with alkaline phosphatase substrates. This culture system thus appears to be a useful model for isolating native MV and characterizing factors required for vesicle formation and mineralization.
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PMID:Isolation and characterization of calcium-accumulating matrix vesicles from chondrocytes of chicken epiphyseal growth plate cartilage in primary culture. 390

Lysosomal storage diseases such as GM1-gangliosidosis are associated with skeletal abnormalities. Radiological and histological studies, both in human and corresponding animal models, indicate retarded bone formation. Since cartilage maturation leads to bone formation, we developed an in vitro system to study and compare the biological features of cartilage from dogs affected with GM1-gangliosidosis with age-matched controls. Costochondral chondrocytes were grown in monolayer and in agarose culture. Both affected and control cells dedifferentiated in monolayer; however, in agarose culture they re-expressed the chondrocytic phenotype. Cells from affected dogs were enlarged and contained numerous large vacuoles when compared with control cells. This morphology was similar to that seen in vivo. In addition, the affected cells appeared to have a reduction in mitosis and alcian blue staining proteoglycans. Cultures from affected animals contained fewer cells positive for alkaline phosphatase activity. Both affected and control cells expressed collagen types I and II and were positive for the lectin Ricinus communis agglutinin-I. However, the staining of the control culture for type II collagen was more prominent than in the affected cells. These findings suggest that culture of chondrocytes in agarose may be a useful method for studying the biology of cartilage which leads to skeletal abnormalities in lysosomal storage diseases.
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PMID:An in vitro model for abnormal skeletal development in the lysosomal storage diseases. 775 83


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