Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

UMR 201 is a nontransformed rat clonal cell line derived from neonatal calvaria with phenotypic characteristics of preosteoblasts. Retinoic acid strongly induces expression of alkaline phosphatase and its mRNA in these cells. Dexamethasone substantially reduced the retinoic acid-induced expression of alkaline phosphatase. This apparent interaction between dexamethasone and retinoic acid effects raised the possibility that interactions may extend to other osteoblast-related phenotypic characteristics in UMR 201 cells. Treatment with dexamethasone resulted in a decrease in the expression of mRNA for pro-alpha 1(I) collagen, but upon coincubation with 1 microM retinoic acid for 24 h, the decrease in mRNA for pro-alpha 1(I) collagen was abrogated. Dexamethasone (Dex) treatment caused a dose-dependent increase in osteonectin mRNA, half maximally effective between 1 nM and 10 nM Dex. One micromolar of retinoic acid alone led to a small increase in expression of osteonectin mRNA but prevented any further increase when Dex was added to retinoic acid-treated cells. To study transcriptional control, osteonectin genomic fragments were linked to the bacterial reporter gene, chloramphenicol acetyltransferase, and introduced by transfection into UMR 201 cells. Dexamethasone increased the transcriptional activity of an osteonectin-chloramphenicol acetyltransferase construct; 100 nM Dex resulted in a 3-fold increase over control cells which was attenuated when 1 microM retinoic acid was added to the incubation, while retinoic acid alone resulted in a 2-fold increase in transcriptional activity. Finally, it was noted that coincubation with retinoic acid and Dex stimulated the proliferation of UMR 201 cells when compared with either treatment alone. This study shows the potential importance of hormonal interactions in the expression of osteoblast function.
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PMID:Opposing influences of glucocorticoid and retinoic acid on transcriptional control in preosteoblasts. 262 42

The mechanism of mineral formation in bone is seen best where active new bone formation is occurring, e.g., in newly forming subperiosteal bone of the embryo, in the growing bone of young animals, and in healing rickets where the calcification process in osteoid is reactivated. A large body of ultrastructural evidence, using conventional and anhydrous methods for tissue preparation, has shown convincingly that extracellular matrix vesicles are present at or near the mineralization front in all of the above, and that these vesicles are the initial site of apatite mineral deposition. Thus bone resembles growth plate cartilage, predentin, and turkey tendon in having calcification initiated by matrix vesicles. Once the calcification cascade is begun, matrix vesicles are no longer needed to support mineralization and are consumed by the advancing mineralization front in which performed crystals serve as nuclei for the formation of new crystals. The rate of crystal proliferation is promoted by the availability of Ca2+, PO4(3-), and the presence of collagen, and retarded by naturally occurring inhibitors of mineralization such as proteoglycans and several noncollagenous calcium-binding proteins of bone including bone-Gla protein (osteocalcin), phosphoproteins, osteonectin, and alpha-2HS-glycoproteins. New electron microscopic immunocytochemical findings in our laboratory suggest that the origin of alkaline phosphatase-positive bone matrix vesicles is polarized to the mineral-facing side of osteoblasts and may be concentrated near the intercellular junctions of human embryonic osteoblasts.
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PMID:Mechanism of mineral formation in bone. 264 65

Human bone cell cultures were established by maintaining collagenase-treated bone fragments in low Ca++ medium. The resulting cell cultures exhibited a high level of alkaline phosphatase activity and produced a significant increase in intracellular cAMP when exposed to the 1-34 fragment of human parathyroid hormone. With continued culture, the cells formed a thick, extracellular matrix that mineralized when cultures were provided daily with normal levels of calcium, fresh ascorbic acid (50 micrograms/ml) and 10 mM beta-glycerol phosphate. Biosynthetically, these cells produced type I collagen (without any type III collagen), and the bone-specific protein, osteonectin. In addition, the cells produced sulfated macromolecules electrophoretically identical to those positively identified as the bone proteoglycan in parallel cultures of fetal bovine bone cells. This technique provides a useful system for the study of osteoblast metabolism in vitro.
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PMID:Human bone cells in vitro. 299 72

This study examines the osteoblastic properties of the established human osteosarcoma cell line Saos-2. Saos-2 cells inoculated into diffusion chambers, which were implanted i.p. into nude mice, produced mineralized matrix in 4 of 6 chambers at 8 weeks. In 5 of 6 chambers there was a strong positive alkaline phosphatase reaction. In culture the alkaline phosphatase levels increased with time and cell density, reaching very high levels at confluence: 4-7 mumol/mg protein/min. The cells show a sensitive adenylate cyclase response to parathyroid hormone, 50% effective dose = 2.8 nM, which increases with cell density and is further raised by dexamethasone treatment. They also exhibit typical binding of 1-25-dihydroxyvitamin D3 to 3.2S receptor protein with an apparent Kd of 0.21 nM; the numbers of sites per cell were 3,300 at 50,000 cells/cm2 and 1,800 at 280,000 cells/cm2. The presence of osteonectin was visualized with a monoclonal antibody which revealed a reticular pattern on the cell surface. Osteonectin was also detected in the medium by Western blots, migrating at around Mr 40,000 in nonreduced gels and Mr 44,000 in reduced gels. The Saos-2 cells thus possess several osteoblastic features and could be useful as a permanent line of human osteoblast-like cells and as a source of bone-related molecules.
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PMID:Characterization of a human osteosarcoma cell line (Saos-2) with osteoblastic properties. 304 Feb 34

The non-collagen proteins of bone are a complex set of molecules that arise from local or exogenous sources. Because bone mineral is an excellent adsorbent, many circulatory and/or cell surface proteins bind to bone, where they may have immediate or subsequent effects. These include the alpha 2-HS-glycoprotein from blood and the potent growth factors TGF-beta, PDGF, IGF-1, FGF-a and -b, and IL-1, derived from both bone and non-bone cells. Furthermore, bone cell membrane proteins such as alkaline phosphatase may be cleaved from the cell surface and entrapped in the bone matrix. Bone is enriched in a variety of enzymes and their inhibitors by similar adsorption processes. Even osteocalcin, a bone cell product, is adsorbed to bone via mineral-binding (Gla) groups. The bone sialoproteins (BSP-I or osteopontin and BSP-II) also bind to the mineral via acidic groups. Because of this phenomenon it is difficult to distinguish whether a given protein's presence in bone is advantageous or merely fortuitous. The bone matrix proper consists of type I collagen and other osteoblast products such as osteonectin (a phosphorylated glycoprotein) and small proteoglycans (PG-I and/or PG-II) which are incorporated into bone collagen fibrils. These proteins may have additional roles in tissue morphogenesis and/or differentiation.
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PMID:Non-collagen proteins in bone. 306 9

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

To determine the effects of transforming growth factor-beta (TGF-beta) on the different cell types that exist in bone, cell populations (I-IV), progressively enriched in osteoblastic cells relative to fibroblastic cells, were prepared from fetal rat calvaria using timed collagenase digestions. TGF-beta did not induce anchorage-independent growth of these cells, nor was anchorage-dependent growth stimulated in most populations studied, despite a two- to threefold increase in the synthesis of cellular proteins. In all populations the synthesis of secreted proteins increased 2-3.5-fold. In particular, collagen, fibronectin, and plasminogen activator inhibitor synthesis was stimulated. However, different degrees of stimulation of individual proteins were observed both within and between cell populations. A marked preferential stimulation of plasminogen activator inhibitor was observed in each population, together with a slight preferential stimulation of collagen; the effect on collagen expression being directed primarily at type I collagen. In contrast, the synthesis of SPARC (secreted protein acidic rich in cysteine/osteonectin was stimulated approximately two-fold by TGF-beta, but only in fibroblastic populations. Collectively, these results demonstrate that TGF-beta stimulates matrix production by bone cells and, through differential effects on individual matrix components, may also influence the nature of the matrix formed by different bone cell populations. In the presence of TGF-beta, osteoblastic cells lost their polygonal morphology and alkaline phosphatase activity was decreased, reflecting a suppression of osteoblastic features. The differential effects of TGF-beta on bone cell populations are likely to be important in bone remodeling and fracture repair.
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PMID:Differential effects of transforming growth factor-beta on the synthesis of extracellular matrix proteins by normal fetal rat calvarial bone cell populations. 316 38

Cells from fetal or neonatal skeleton can synthesize bone-like tissue in vitro. In contrast, formation of bone-like tissue in vitro by cells derived from adult animals has rarely been reported and has not been achieved using cells from bone marrow. We have explored development of bone-like tissue in vitro by bone marrow stromal cells. Marrow stromal cells obtained from 40-43-day-old Wistar rats were grown in primary culture for 7 days and then subcultured for 20-30 days. Cells were cultured in either alpha-minimal essential medium containing 15% fetal bovine serum, antibiotics, and 50 micrograms/ml ascorbic acid, or the above medium supplemented with either 10 mM Na-beta-glycerophosphate, 10(-8) M dexamethasone, or a combination of both. Cultures were examined using phase-contrast microscopy, undemineralized and demineralized tissue histology, histochemistry (for alkaline phosphatase activity), immunohistochemistry (for collagen type, osteonectin, and bone Gla-protein), scanning and transmission electron microscopy, energy dispersive X-ray microanalysis, and X-ray diffraction. Collagenous, mineralized nodules exhibiting morphological and ultrastructural characteristics similar to bone were formed in the cultures, but only in the presence of both beta-glycerophosphate and dexamethasone. Cells associated with the nodules exhibited alkaline phosphatase activity. The matrix of the nodules was composed predominantly of type-I collagen and both osteonectin and Gla-protein were present. X-ray microanalysis showed the presence of Ca and P, and X-ray diffraction indicated the mineral to be hydroxyapatite. The nodules were also examined for bone morphogenetic protein-like activity. Paired diffusion chambers containing partly demineralized nodules and fetal muscle were implanted intraperitonealy in rats. Induction of cartilage in relation to muscle was observed histologically after 40 days in the chambers. This finding provided further support for the bone-like nature of the nodules. The observations show that bone-like tissue can be synthesized in vitro by cells cultured from young-adult bone marrow, provided that the medium contains both beta-glycerophosphate and, particularly, dexamethasone.
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PMID:Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. 319 89

TGF beta 1 from porcine platelets increased alkaline phosphatase (AP) activity in the rat osteoblastic cell line ROS 17/2.8 about three-fold. This effect was dose-dependent with an ED50 of about approximately 0.2 ng/ml and was larger during logarithmic growth than at confluence. TGF beta 1 inhibited cell growth by about 30% with similar dose dependence. Thirty min exposure to TGF beta 1 was sufficient to increase AP activity 3 days later by about two-fold but did not affect cell growth, suggesting dissociation between effects on proliferation and differentiation. The rise in AP activity started 6 h after TGF beta 1 addition and was blocked by cycloheximide and actinomycin D. TGF beta 1 also increased AP mRNA by two- to three-fold and this effect was not blocked by cycloheximide. The half-life of AP mRNA, estimated following the addition of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole was about ten h in both control and TGF beta 1-treated cells. The mRNAs for type I procollagen and osteonectin were also increased by TGF beta 1 but fibronectin mRNA was decreased. TGF beta 2 effects on AP and cell growth were similar to those of TGF beta 1, except for lack of activity following transient exposure. At saturating concentrations, TGF beta 2 (2 ng/ml) or dexamethasone (10(-7) M), which has similar effects on these cells, did not further augment the effects of TGF beta 1 (at 2 ng/ml). Above findings suggest that TGF beta promotes osteoblastic differentiation in rat osteosarcoma cells at least in part by acting at the pretranslational level.
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PMID:Type beta transforming growth factor (TGF beta) regulation of alkaline phosphatase expression and other phenotype-related mRNAs in osteoblastic rat osteosarcoma cells. 348 Feb 88

We isolated cells from both calvaria and the outer cortices of long bones from 3- to 5-mo bovine fetuses. The cells were identified as functional osteoblasts by indirect immunofluorescence using antibodies against three bone-specific, noncollagenous matrix proteins (osteonectin, the bone proteoglycan, and the bone sialoprotein) and against type 1 collagen. In separate experiments, confluent cultures of the cells were radiolabeled and shown to synthesize and secrete osteonectin, the bone proteoglycan and the bone sialoprotein by immunoprecipitation and fluorography of SDS polyacrylamide gels. Analysis of the radiolabeled collagens synthesized by the cultures showed that they produced predominantly (approximately 94%) type I collagen, with small amounts of types III and V collagens. In agreement with previous investigators who have employed the rodent bone cell system, we confirmed in bovine bone cells that (a) there was a typical cyclic AMP response to parathyroid hormone, (b) freshly isolated cells possessed high levels of alkaline phosphatase, which diminished during culture but returned to normal levels in mineralizing cultures, and (c) cells grown in the presence of ascorbic acid and beta-glycerophosphate rapidly produced and mineralized an extracellular matrix containing largely type I collagen. These results show that antibodies directed against bone-specific, noncollagenous proteins can be used to clearly identify bone cells in vitro.
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PMID:Fetal bovine bone cells synthesize bone-specific matrix proteins. 608 72


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