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)

Rat bone marrow stromal cells were cultured in the presence of 10 nM dexamethasone and various concentrations of beta-glycerophosphate. At day 12-15, some nodules consisting of polygonal cells were formed in all culture conditions, and these nodules were mineralized 2-3 days later. beta-Glycerophosphate significantly enhanced nodule formation at concentrations of not less than 5 mM. The mineralized nodules formed in the absence of beta-glycerophosphate were examined using phase-contrast microscopy, undemineralized and demineralized tissue histology, histochemistry for alkaline phosphatase, immunohistochemistry for type I, II, and III collagen, energy dispersive X-ray microanalysis, electron diffraction, and Fourier transform infrared spectroscopy (FT-IR). Mineralized nodules had histological characteristic similar to bone. Cells associated with nodules exhibited high alkaline phosphatase activity, and extracellular matrix of the nodules predominantly consisted of type I collagen. X-Ray microanalysis showed the presence of Ca and P in the mineralized area, and electron diffraction pattern showed the mineral to have apatite crystal structure. Moreover FT-IR indicated that the mineral was a mixture of hydroxyapatite and carbonateapatite. From these observations, it is concluded that the mineralized nodules formed in our culture system are truly bone-like.
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PMID:Mineralized nodule formation in rat bone marrow stromal cell culture without beta-glycerophosphate. 186 68

Conditions were defined for promoting cell growth, hypertrophy, and extracellular matrix mineralization of a culture system derived from embryonic chick vertebral chondrocytes. Ascorbic acid supplementation by itself led to the hypertrophic phenotype as assessed by respective 10- and 15-fold increases in alkaline phosphatase enzyme activity and type X synthesis. Maximal extracellular matrix mineralization was obtained, however, when cultures were grown in a nutrient-enriched medium supplemented with both ascorbic acid and 20 mM beta-glycerophosphate. Temporal studies over a 3-wk period showed a 3-4-fold increase in DNA accompanied by a nearly constant DNA to protein ratio. In this period, total collagen increased from 3 to 20% of the cell layer protein; total calcium and phosphorus contents increased 15-20-fold. Proteoglycan synthesis was maximal until day 12 but thereafter showed a fourfold decrease. In contrast, total collagen synthesis showed a greater than 10-fold increase until day 18, a result suggesting that collagen synthesis was replacing proteoglycan synthesis during cellular hypertrophy. Separate analysis of individual collagen types demonstrated a low level of type I collagen synthesis throughout the 21-d time course. Collagen types II and X synthesis increased during the first 2 wk of culture; thereafter, collagen type II synthesis decreased while collagen type X synthesis continued to rise. Type IX synthesis remained at undetectable levels throughout the time course. The levels of collagen types I, II, IX, and X mRNA and the large proteoglycan core protein mRNA paralleled their levels of synthesis, data indicating pretranslational control of synthesis. Ultrastructural examination revealed cellular and extracellular morphology similar to that for a developing hypertrophic phenotype in vivo. Chondrocytes in lacunae were surrounded by a well-formed extracellular matrix of randomly distributed collagen type II fibrils (approximately 20-nm diam) and extensive proteoglycan. Numerous vesicular structures could be detected. Cultures mineralized reproducibly and crystals were located in extracellular matrices, principally associated with collagen fibrils. There was no clear evidence of mineral association with extracellular vesicles. The mineral was composed of calcium and phosphorus on electron probe microanalysis and was identified as a very poorly crystalline hydroxyapatite on electron diffraction. In summary, these data suggest that this culture system consists of chondrocytes which undergo differentiation in vitro as assessed by their elevated levels of alkaline phosphatase and type X collagen and their ultrastructural appearance.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system. 199 93

The effects of Bordetella bronchiseptica dermonecrotic toxin on the structure and function of a clonal osteoblastic cell line, MC3T3-E1, were investigated. The toxin induced a morphological change in the cells from a spindle shape to a spherical form with many blebs. The toxin-treated cells were viable and grew to form confluent cell layers composed of irregularly shaped cells and multinuclear cells. The toxin inhibited elevation of alkaline phosphatase activity in the cells in a dose-dependent manner at concentrations from 10 pg to 10 ng/ml. The accumulation of type I collagen in the cells was also reduced by the toxin. Since high alkaline phosphatase activity and accumulation of collagen are closely linked to differentiation of the cells into osteoblasts, it is considered likely that B. bronchiseptica dermonecrotic toxin impairs the ability of the cells to differentiate.
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PMID:Effects of Bordetella bronchiseptica dermonecrotic toxin on the structure and function of osteoblastic clone MC3T3-e1 cells. 199 14

Normal rat osteoblasts in culture undergo a developmental sequence consisting of a proliferation period in which high levels of the histone and collagen type I genes are expressed, followed by periods of matrix maturation [high levels of alkaline phosphatase (AP)] and mineralization that signal a high level of production of osteopontin (OP) and osteocalcin (OC). Since these parameters are regulated by vitamin D, the effects of both short term and chronic treatment with 1,25-dihydroxyvitamin D3 were examined during osteoblast growth and differentiation. In acute studies, during the proliferation period, histone mRNA (reflecting DNA synthesis) was inhibited (20-60%). Matrix Gla protein (MGP) and OP mRNA were significantly elevated during proliferation (30- and 15-fold), in contrast to OC which is not expressed and was not induced by hormone treatment. OP and MGP remained stimulated throughout the developmental sequence, but to a lesser degree (from 6- to 10-fold). Collagen and AP mRNA were inhibited by hormone at their peak levels of expression, but were stimulated at their lowest basal levels in the mineralization period. OC expression, which was initiated at the onset of mineralization, was stimulated 13- to 15-fold when basal levels were low, then from 6- to 8-fold by hormone throughout its period of expression. In chronic studies a different profile of gene expression was observed. When hormone treatment was initiated during the proliferation period on day 6, type I collagen and AP expression were suppressed, mineralized nodules did not develop, and induced levels of OP and OC gene expression did not occur. When chronic treatment was initiated on day 20 after the development of a mineralized matrix, OC, but not collagen and OP, levels were stimulated by the hormone. This observation is consistent with the requirement of a competent or mineralized bone matrix for expression of OC. In contrast, MGP expression was stimulated in the chronic vitamin D-treated cultures similar to acute treatments. Taken together these studies demonstrate that vitamin D, a physiological mediator of bone formation and remodelling, can both positively and negatively regulate expression of osteoblast phenotypic markers as a function of duration of hormone treatment and basal levels of gene expression, which is a reflection of bone matrix competency and the differentiated state of the osteoblast.
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PMID:Pleiotropic effects of vitamin D on osteoblast gene expression are related to the proliferative and differentiated state of the bone cell phenotype: dependency upon basal levels of gene expression, duration of exposure, and bone matrix competency in normal rat osteoblast cultures. 199 68

We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or 1 microM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25-dihydroxyvitamin D3 to these dexamethasone-treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25-dihydroxyvitamin D3-treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone-treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the osteoblast phenotype in cultures of rat marrow stromal fibroblasts.
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PMID:Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures. 202 91

The nature and tissue distribution of non-collagenous bone proteins synthesized by adult rat bone marrow cells, induced to differentiate in the presence of dexamethasone (DEX) and beta-glycerophosphate (beta-GP), was studied in vitro to determine the potential role of these proteins in bone formation. Northern hybridization analysis revealed a strong induction of bone sialoprotein (BSP) and osteocalcin in DEX-treated cultures, whereas the constitutive expression of secreted phosphoprotein I (SPP-1), type I collagen, SPARC, and alkaline phosphatase was stimulated 6-, 5-, 3-, and 2.5-told, respectively. Metabolic labeling of proteins showed that the sialoproteins (SPP-1 and BSP) were mostly secreted into the culture medium in the non-mineralizing (-beta-GP) cultures, but were the predominant non-collagenous proteins associated with the hydroxyapatite of the bone nodules in mineralizing cultures (+ beta-GP). Extraction of the tissue matrix with 4 M GuHCl and digestion of the demineralized tissue matrix with bacterial collagenase revealed that some BSP was also associated non-covalently and covalently with the collagenous matrix. SPP-1 was present in two distinct, 44 kDa and 55 kDa, forms in the conditioned medium of all cultures and was preferentially associated with the hydroxyapatite in the mineralizing cultures. In comparison, SPARC was abundant in culture media but could not be detected in de-mineralizing extracts of the mineralized tissue. Radiolabeling with [35SO4] demonstrated that both SPP-1 and BSP synthesized by bone cells are sulfated, and that a 35 kDa protein and some proteoglycan were covalently associated with the collagenous matrix in +DEX cultures. Labeling with [32PO4] was essentially confined to the sialoproteins; the 44 kDa SPP-1 incorporating significantly more [32PO4] than the 55 kDa SPP-1 and the BSP. These studies demonstrate that BSP and osteocalcin are only expressed in differentiated osteoblasts and that most of the major non-collagenous bone proteins associate with the bone mineral. However, some novel proteins together with some of the BSP are associated with the collagenous matrix where they can influence hydroxyapatite formation.
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PMID:Expression of bone matrix proteins associated with mineralized tissue formation by adult rat bone marrow cells in vitro: inductive effects of dexamethasone on the osteoblastic phenotype. 203 18

The effect of leukemia inhibitory factor (LIF) on proliferation and phenotypic expression in murine osteoblast-like (MC3T3E1) cells was examined. LIF inhibited the proliferation of these cells by up to 20% and DNA synthesis was inhibited in a dose-dependent manner with an ED50 of about 0.2 ng/ml. The effect of LIF relative to matched controls increased with decreasing serum concentration, reaching 30% inhibition at 0.2% serum. LIF also reduced the stimulatory effects of platelet-derived growth factor and insulin-like growth factor I on DNA synthesis. The inhibition of the DNA synthesis by saturating concentration of transforming growth factor beta was further enhanced by the addition of LIF, suggesting independent pathways for the action of the two growth inhibitors. In addition, LIF reduced alkaline phosphatase activity and the abundance of type I collagen messenger RNA, but increased the level of osteopontin messenger RNA. These findings suggest that LIF may play a role in regulating the function of osteoblasts.
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PMID:Leukemia inhibitory factor suppresses proliferation, alkaline phosphatase activity, and type I collagen messenger ribonucleic acid level and enhances osteopontin mRNA level in murine osteoblast-like (MC3T3E1) cells. 211 64

Recombinant human interferon-alpha 2C and recombinant human interferon-gamma (5-1000 U/ml) inhibit the proliferation of normal human bone-derived cells and a human osteosarcoma cell line. In the bone-derived cells the inhibitory effect of interferon-gamma was significantly greater than that of interferon-alpha, whereas in the osteosarcoma cell line the inhibitory effects of both interferons were quantitatively similar. Interferon-alpha did not affect the alkaline phosphatase activity of either type of cells. In contrast, interferon-gamma affected the activity of the enzyme in both cell types: in the bone-derived cells the effect of interferon-gamma was stimulatory whereas in the osteosarcoma cells the effect was inhibitory. In both cell types interferon-gamma selectively inhibited the incorporation of radiolabelled proline into type I collagen. In the osteosarcoma cells, the effects of both interferons on collagen synthesis were quantitatively similar. In the bone-derived cells, however, interferon-alpha decreased proline incorporation into collagen and non-collagen proteins to a similar extent and thus did not affect collagen synthesis when expressed as a percentage of total protein synthesis. Two-dimensional polyacrylamide gel electrophoresis of the radiolabelled proteins of the cell layer synthesised by both cell types in the presence of either interferon demonstrated that this treatment enhanced or induced the synthesis of a total of 21 individual proteins (19 in bone cells, 14 in osteosarcoma), ranging in apparent molecular mass over 14-87 kDa. The set of proteins induced was different in all four combinations of cells and interferon. A tentative identification of several of the proteins was possible based upon estimation of molecular mass, preferential induction by interferon-alpha or interferon-gamma and differential induction in normal and transformed bone-derived cells. The results of this study demonstrate that interferons have complex effects upon the proliferative and biosynthetic activities of human bone-derived cells and demonstrate significant differences between the responses of normal cells and transformed bone-derived cell line. Further investigations will be required in order to determine whether or not these differences are unique to the osteosarcoma cell line or are a characteristic of the effects of interferons on bone-derived cells in general.
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PMID:Interferons and bone. A comparison of the effects of interferon-alpha and interferon-gamma in cultures of human bone-derived cells and an osteosarcoma cell line. 212 83

Osteocalcin, a bone-specific protein and marker of the mature osteoblast, is expressed only in nonproliferating osteoblasts in a mineralizing extracellular matrix, while type I collagen is expressed in proliferating cells. The nuclear proteins encoded by the c-fos and c-jun protooncogenes are expressed during the proliferation period of osteoblast phenotype development. We present evidence that AP-1 (HeLa cell-activating protein 1) sites residing within two promoter elements of the osteocalcin gene bind the Fos-Jun protein complex: the osteocalcin box (OC box; nucleotides -99 to -76), which contains a CCAAT motif as a central element and influences tissue-specific basal levels of osteocalcin gene transcription, and the vitamin D-responsive element (VDRE; nucleotides -462 to -440), which mediates enhancement of osteocalcin gene transcription. Gel electrophoretic mobility-shift analysis demonstrated high AP-1 binding activity in proliferating osteoblasts and dramatic changes in this activity after the down-regulation of proliferation and the initiation of extracellular-matrix mineralization in primary cultures of normal diploid osteoblasts. Methylation interference analysis established at single nucleotide resolution that purified recombinant Fos and Jun proteins bind in a sequence-specific manner to the AP-1 sites within the VDRE and OC box. Similarly, an AP-1 motif within a putative VDRE of the alkaline phosphatase gene, which is also expressed after the completion of proliferation, binds the Fos-Jun complex. These results support a model in which coordinate occupancy of the AP-1 sites in the VDRE and OC box in proliferating osteoblasts may suppress both basal level and vitamin D-enhanced osteocalcin gene transcription as well as transcription of other genes associated with osteoblast differentiation--a phenomenon we describe as phenotype suppression. This model is further supported by binding of the Fos-Jun complex at an AP-1 site in the type alpha I collagen promoter that is contiguous with, but not overlapping, the VDRE. Such a sequence organization in the collagen VDRE motif is compatible with vitamin D modulation of collagen but not with osteocalcin and alkaline phosphatase expression in proliferating osteoblasts.
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PMID:Coordinate occupancy of AP-1 sites in the vitamin D-responsive and CCAAT box elements by Fos-Jun in the osteocalcin gene: model for phenotype suppression of transcription. 212 10

A new apatite-collagen complex was prepared in calcium beta-glycerophosphate solutions at pH 8.50. For this preparation, reconstituted type I collagen was cross-linked with phosvitin in the presence of alkaline phosphatase by use of a cross-linking agent of dimethyl suberimidate. After two weeks of immersion in daily-renewed solution of calcium beta-glycerophosphate, the complex contained apatite approximately two times the modified collagen in weight. When viewed in a scanning electron microscope, needle-like crystals precipitated densely on the collagen fibrils. However, in some portion of the complex, dot-like precipitate was observed as well. X-ray diffraction and IR analyses of the complex suggested that the apatite precipitated on the collagen fibrils was very similar to bone mineral in two aspects, crystallinity and carbonate content.
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PMID:[Apatite-collagen complex. Preparation of a new apatite-collagen complex]. 213 50


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