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)

Aging bone shows a progressive decline in mass and strength. Previous studies have suggested that bone marrow stem cells are reduced with aging and that this could be responsible, in part, for age-associated bone deficits. We measured the number of osteoprogenitor cells present in the bone marrow from adult and aged rats as well as their ability to differentiate in vitro and to form bone in vivo. We found that the number of adherent colony-forming cells was significantly lower (65%) in marrow cells isolated from aged compared with adult rats. Furthermore, 88% of the colonies obtained from aged rats were alkaline phosphatase (AP) positive, whereas virtually all the colonies from adult rats were positive. The addition of dexamethasone to the culture medium decreased the proliferation of the adherent cells and reduced the number of colonies obtained from both adult and aged bone marrow, all of which were AP positive. No significant differences were found in the expression of certain major bone cell marker genes as a function of donor age. However, dexamethasone treatment increased expression of osteopontin (OP) by fivefold. Adult stromal cells not treated with dexamethasone and implanted subcutaneously in recipient rats exhibited about 10-fold greater formation of bone compared with cells from aged rats. In contrast, dexamethasone-treated cells exhibited high levels of bone formation, irregardless of donor age or the age of the recipient into which the cells were grafted. These studies are consistent with a deficit of osteoprogenitor cells in the bone marrow site as a contributing, perhaps correctable factor in the decline in bone repair and bone mass with age.
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PMID:Bone progenitor cell deficits and the age-associated decline in bone repair capacity. 773 20

We report the establishment of a human fetal osteoblast cell line derived from biopsies obtained from a spontaneous miscarriage. Primary cultures isolated from fetal tissue were transfected with a gene coding for a temperature-sensitive mutant (tsA58) of SV40 large T antigen along with a gene coding for neomycin (G418) resistance. Individual neomycin resistant colonies were screened for alkaline phosphatase (AP)-specific staining. The clone with the highest AP level, hFOB 1.19, was examined further for other osteoblast phenotypic markers. Incubation of hFOB cells at the permissive temperature (33.5 degrees C) resulted in rapid cell division, whereas little or no cell division occurred at the restrictive temperature (39.5 degrees C). Both AP activity and osteocalcin (OC) secretion increased in a dose-dependent manner following dihydroxyvitamin D3 (1,25-D3) treatment when cultured at either temperature. However, AP and 1,25-D3-induced OC levels were elevated in confluent hFOB cells cultured at 39.5 degrees C compared with 33.5 degrees C. Treatment of hFOB cells with 1-34 parathyroid hormone (PTH) resulted in an increase in cAMP levels. Upon reaching confluence, hFOB cultures went through programmed differentiation and formed mineralized nodules as observed by von Kossa staining. Further, immunostaining of postconfluent, differentiated hFOB cells showed that high levels of osteopontin, osteonectin, bone sialoprotein, and type I collagen were expressed. Therefore, the clonal cell line hFOB 1.19 provides a homogeneous, rapidly proliferating model system to study certain stages of human osteoblast differentiation.
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PMID:Development and characterization of a conditionally immortalized human fetal osteoblastic cell line. 775 97

Advances in the culture of mineralizing growth plate chondrocytes provided an opportunity to study endochondral calcification under controlled conditions. Here we report that these cultures synthesize large amounts of proteins characteristically associated with mineralization: type II and X collagens, sulfated proteoglycans, alkaline phosphatase, and the bone-related proteins, osteonectin and osteopontin. Certain chondrocytes appeared to accumulate large amounts of Ca2+ and Pi during the mineralization process: laser confocal imaging revealed high levels of intracellular Ca2+ in their periphery and X-ray microanalytical mapping revealed the presence of many Ca(2+)- and Pi-rich cell surface structures ranging from filamentous processes 0.14 +/- 0.02 microns by 0.5-2.0 microns, to spherical globules 0.70 +/- 0.27 microns in diameter. Removal of organic matter with alkaline sodium hypochlorite revealed numerous deposits of globular (0.77 +/- 0.19 micron) mineral (calcospherites) in the lacunae around these cells. The size and spatial distribution of these mineral deposits closely corresponded to the Ca(2+)-rich cell surface blebs. The globular mineral progressively transformed into clusters of crystallites. Taken with earlier studies, these findings indicate that cellular uptake of Ca2+ and Pi leads to formation of complexes of amorphous calcium phosphate, membrane lipids, and proteins that are released as cell surface blebs analogous to matrix vesicles. These structures initiate development of crystalline mineral. Thus, the current findings support the concept that the peripheral intracellular accumulation of Ca2+ and Pi is directly involved in endochondral calcification.
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PMID:Morphological and biochemical characterization of mineralizing primary cultures of avian growth plate chondrocytes: evidence for cellular processing of Ca2+ and Pi prior to matrix mineralization. 775 59

Seventeen day chicken embryonic osteoblasts treated over a 30-day period with 1,25(OH)2 D3 showed a 2-10-fold decrease in collagen, osteopontin and osteocalcin protein accumulation, alkaline phosphatase enzyme activity, and mineral deposition. Comparable inhibition in the steady state mRNA levels for alpha 1(I) and alpha 2(I) collagen, osteocalcin, and osteopontin were observed, and the inhibitory action of the hormone was shown to be specific for only the late release populations of cells from sequential enzyme digestions of the chick calvaria. In order to determine whether the continuous hormone treatment blocked osteoblast differentiation, the cells were acutely treated for 24 h with 1,25(OH)2 D3 at culture periods when the cells proliferate (day 5), a culture period when the cells cease further cell division and are increasing in the expression of their differentiated functions (day 17), and a culture period when the cells are encapsulated within a mineralized extracellular matrix (day 30). Inhibition of the expression of collagen, osteocalcin, and osteopontin were observed at days 17 and 30, while no effect could be detected for the 5-day cultures. To further define whether the inhibitory effect was specific for cells expressing their differentiated phenotype, 1,25(OH)2 D3 treatment was initiated at day 17 and continued to day 30 after the cells have established their collagenous matrix. In these experiments further collagenous matrix deposition, mineral deposition, alkaline phosphatase activity, and osteocalcin synthesis were also inhibited after the hormone treatment was initiated. These results, in summary, show that 1,25(OH)2 D3 in primary avian osteoblast cultures derived from 17-day embryonic calvaria inhibits the expression of several genes associated with differentiated osteoblast function and inhibit extracellular matrix mineral deposition.
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PMID:Inhibitory effects of 1,25(OH)2 vitamin D3 on collagen type I, osteopontin, and osteocalcin gene expression in chicken osteoblasts. 776 79

The neonatal rat mandible was used as a model to study bone formation, mineralization, quiescence, and resorption, using immunolocalization and a variety of tissue-processing techniques. Monospecific antibodies for osteopontin (OPN), bone sialoprotein (BSP), alkaline phosphatase (AP) and alpha 2HS-glycoprotein (alpha 2HS-GP) were used on fixed paraffin-embedded tissue, fixed frozen tissue and unfixed frozen tissue. Immunostaining was correlated with mineral content by two procedures, the von Kossa and the morin techniques. Morin fluorescence was used with secondary immunostaining to provide a way of closely correlating bone matrix proteins and matrix mineralization. Co-immunolocalization procedures were used to compare the sites of bone proteins in the matrix. AP was found earliest during osteogenic cell differentiation, appearing in the preosteoblasts, followed by OPN and BSP, which first appeared in osteoblasts. alpha 2HS-GP expression was not observed in cells. The results provide clear evidence for the presence of OPN in osteoid, while BSP and alpha 2HS-GP were confined to the mineralized matrix. Immunostaining of bone proteins is highly technique-dependent: immunolocalization investigations required several methods of approach to ensure adequate demonstration of these proteins in cells and matrix. The results support the contention that osteopontin is multifunctional in bone metabolism, and that alpha 2HS-GP, though produced in the liver, is abundant in bone matrix and may also have a function in bone metabolism.
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PMID:Bone matrix proteins in osteogenesis and remodelling in the neonatal rat mandible as studied by immunolocalization of osteopontin, bone sialoprotein, alpha 2HS-glycoprotein and alkaline phosphatase. 779 28

Treatment of the U-2 OS human osteosarcoma cell line with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) dramatically decreased the rate of DNA synthesis. This decrease in proliferation as well as the change in morphology of the TPA-treated cells can be blocked by the protein kinase C inhibitor GF 109203X. The U-2 OS cells are known to express the c-sis oncogene [platelet-derived growth factor (PDGF) B-chain], PDGF-A, and receptors for PDGF, thus providing a potential autocrine loop of growth stimulation. TPA was found to induce the expression of both the PDGF-A and the PDGF-B chains. However, the levels of the PDGF receptor beta subunits and of the PDGF-BB inducable tyrosine phosphorylation of the PDGF receptor were markedly reduced. The TPA treatment of the U-2 OS cells also induced changes typical for maturing bone cells, such as increased expression levels of alkaline phosphatase and osteopontin. The expression levels of type I collagen and bone sialoprotein were reduced. The results show a TPA-dependent down-regulation of the PDGF receptor beta subunits that correlates with an increased expression of osteoblast phenotypic markers.
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PMID:Phenotypic modification of human osteosarcoma cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. 779 13

Osteoblasts derived from Day 21 fetal rat calvaria grown on films of collagen type I exhibit an earlier and enhanced expression of the differentiated phenotype, compared to cells cultured on plastic. The temporal expression of genes characterizing three distinct periods of growth and differentiation are dramatically modified. During the initial proliferation period, expression of genes normally expressed at high levels on plastic (fibronectin, beta 1 integrin, and actin) was decreased from 50 to 70% in cells grown on collagen. Genes normally expressed at maximal levels in the postproliferative period (osteonectin, osteocalcin, and osteopontin) were up-regulated severalfold very early. Alkaline phosphatase enzyme activity was elevated 2- to 3-fold during the proliferation period, while mRNA levels remained low, suggesting post-transcriptional modifications. The most dramatic consequence of culture of cells on collagen is the accelerated and uniform mineralization of the matrix in contrast to the focal mineralization confined to bone nodules in cultures on plastic. Type I collagen supports maintenance of osteoblast phenotypic properties of passaged cells in the absence of glucocorticoid supplementation required for differentiation of osteoblasts subcultivated on plastic. Treatment of proliferating rat osteoblasts on plastic with 1,25(OH)2D3 blocks osteoblast differentiation and matrix mineralization. Although differentiation-related genes (alkaline phosphatase and osteocalcin) were up-regulated by vitamin D, culture on the collagen matrix could not overcome the inhibition of mineralization. Taken together, these studies define the critical role of type I collagen in mediating the signaling cascade for expression of a mature osteoblast phenotype and mineralization of the extracellular matrix in a physiological manner.
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PMID:The influence of type I collagen on the development and maintenance of the osteoblast phenotype in primary and passaged rat calvarial osteoblasts: modification of expression of genes supporting cell growth, adhesion, and extracellular matrix mineralization. 781 31

We investigated the sequence of expression of osteoblast gene markers during bone formation in vivo by in situ hybridization. Cylindrical lesions were induced in the femora of sheep with titanium analytic bone implants that allow removal of serial core samples to study bone formation. At 2 weeks (2W), granulation tissue made up of spindle-shaped cells had partially replaced the blood clot. Islands of osseous tissue, first noted in the periphery of the ingrowing tissue at 3W, became the predominant tissue by 6W. The surfaces of newly forming bone at 3W were apposed by cuboidal cells, which in some areas were several layers thick. By 6W, most of the cells lining bone trabeculae had assumed a flattened morphology. The temporal and spatial distribution of osteoblast gene markers was examined by in situ hybridization with nonradioactive digoxigenin probes for alpha 1(I) procollagen, alkaline phosphatase (ALP), osteopontin (OP), and bone Gla protein (BGP). The spindle-shaped cells in the granulation tissue expressed mRNA for alpha 1(I) procollagen, ALP, and OP but not BGP, suggesting that they may be osteoblast precursor cells. alpha 1(I) procollagen mRNA was strongly expressed by all cells on the surface of bone, with a peak intensity at 3W and then reducing sharply by 6W. Initially, only pockets of cuboidal cells on bone surfaces expressed ALP mRNA, with a peak intensity at 5W. Similarly, only a proportion of cuboidal cells expressed OP mRNA early in bone formation, but the number of cells expressing OP mRNA increased with time. Clumps of cuboidal cells expressed BGP mRNA only when bone was present, and the degree of expression increased with the amount of bone formed. This model allows the study of temporal and spatial sequence of gene expression in cells participating in osteogenesis. The temporal sequence is similar to that shown in vitro in other models of mineralization. The geographic localization of cells expressing mRNA for alpha 1(I) procollagen, ALP, OP, and BGP implies subspecialization of osteoblasts in bone formation.
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PMID:In situ hybridization to show sequential expression of osteoblast gene markers during bone formation in vivo. 781 34

Establishing regulatory mechanisms that mediate proliferation of osteoblasts while restricting expression of genes associated with mature bone cell phenotypic properties to post-proliferative cells is fundamental to understanding skeletal development. To gain insight into relationships between growth control and the developmental expression of genes during osteoblast differentiation, we have examined expression of three classes of genes during the cell cycle of normal diploid rat calvarial-derived osteoblasts and rat osteosarcoma cells (ROS 17/2.8): cell cycle and growth-related genes (e.g., histone), genes that encode major structural proteins (e.g., actin and vimentin), and genes related to the biosynthesis, organization, and mineralization of the bone extracellular matrix (e.g., alkaline phosphatase, collagen I, osteocalcin, and osteopontin). In normal diploid osteoblasts as well as in osteosarcoma cells we found that histone genes, required for cell progression, are selectively expressed during S phase. All other genes studied were constitutively expressed both at the transcriptional and posttranscriptional levels. Alkaline phosphatase, an integral membrane protein in both osteoblasts and osteosarcoma cells, exhibited only minimal changes in activity during the osteoblast and osteosarcoma cell cycles. Our findings clearly indicate that despite the loss of normal proliferation-differentiation interrelationships in osteosarcoma cells, cell cycle regulation or constitutive expression of growth and phenotypic genes is maintained.
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PMID:Expression of cell growth and bone phenotypic genes during the cell cycle of normal diploid osteoblasts and osteosarcoma cells. 782 87

Ipriflavone (IP), an isoflavone derivative, has been shown to interfere with bone remodeling by inhibiting bone resorption and perhaps stimulating bone formation. In this study, we have analyzed the effect of IP and its metabolites on the differentiation and function of human osteoblastic cells. Bone marrow stromal osteoprogenitor cells (BMC) and trabecular bone osteoblasts (HOB) were isolated from human donors. The former can be induced to differentiate by treatment with dexamethasone, whereas the latter represent a more differentiated osteoblast. Incubation of BMC with metabolite III (10(-5) M) for 1 week induced modest but significant changes of alkaline phosphatase activity. Though both IP and metabolite III stimulated the expression of bone sialoprotein mRNA, a protein involved in cell attachment to the matrix, only metabolite III increased the steady-state level of decorin mRNA, a collagen fibrillogenesis-regulating proteoglycan. Metabolites III and V, but not the other isoflavones, increased the expression of type I collagen mRNA in HOB, whereas no detectable changes were observed in BMC cells with any of the experimental compounds. In HOB, an increased abundance of osteopontin and bone sialoprotein mRNA were also obtained after 1-week treatment with IP or metabolite V. No appreciable effects of IP or its metabolites were seen on osteocalcin expression and synthesis by either cell type. Finally, IP consistently increased the amount of 45Ca incorporated into the cell layer by BMC, and stimulated mineralization of both BMC and HOB, assessed by von Kossa staining. Thus, IP and its metabolites regulate the differentiation and biosynthetic properties of human bone-forming cells by enhancing the expression of some important matrix proteins and facilitating the mineralization process.
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PMID:Stimulation of human osteoblast differentiation and function by ipriflavone and its metabolites. 786 17


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