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)

A new bone cell line was established by transfecting normal adult human osteoblast-like (hOB) cells, derived from a 68-year-old woman, with the plasmid pSV3 neo. The plasmid included coding sequences and promotors for the large and small T antigens of the SV40 virus as well as resistance to the antibiotics neomycin and G418. A single antibiotic-resistant colony was located and cloned. Large tumor antigen production in the clonal cell line was confirmed by indirect immunofluorescence study. Treatment with 1,25-dihydroxy-vitamin D3 increased steady-state concentrations of protein and mRNA for osteocalcin and for alkaline phosphatase. Northern blot analyses also demonstrated the presence of mRNAs for alpha(I)-procollagen, osteopontin 1a, transforming growth factor beta, and interleukin-1 beta. The plasma membrane calcium pump and osteonectin were identified by immunocytochemical analysis. These cells produced a matrix that mineralized when beta-glycerophosphate was added to their cultures. As assessed by functional receptor assays, both estrogen and androgen receptors were present and functional, although at low concentrations. Treatment with parathyroid hormone did not stimulate adenylate cyclase activity. Thus, these cells are a well-differentiated, steroid-responsive clonal cell line that closely approximates the phenotype of the mature osteoblast. They should serve as an excellent model for the study of osteoblast biology.
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PMID:Development and characterization of a rapidly proliferating, well-differentiated cell line derived from normal adult human osteoblast-like cells transfected with SV40 large T antigen. 137 29

Trans-differentiation of hypertrophic chondrocytes into bone-forming cells was observed when femurs from 14-day-old chick embryos were cut through the region of hypertrophic cartilage and the separated pieces were cultured for 2-18 days. Inside many chondrocytic lacunae a new matrix was present which had the staining characteristics of bone matrix including birefringence and the capacity to mineralize. The cells within the lacunae had the characteristics of osteoblasts, such as alkaline phosphatase activity and positive immunocytochemical staining for osteocalcin, osteonectin, osteopontin and type I collagen. Chondrocyte necrosis and empty lacunae were only observed immediately at the cut edge, and in that region no bone-forming cells were present inside the lacunae. Where bone-matrix was present, the lacunae had remained intact, the cells were viable and no evidence of cell migration was observed. This suggested that the bone-forming cells had originated from the hypertrophic chondrocytes. The temporal sequence of events was followed closely. Two days following the cut only a few chondrocytes showed a positive reaction for osteocalcin, osteonectin, osteopontin and the type I collagen. At that time no such reaction product was observed in the chondrocytes of uncut femurs. Many hypertrophic chondrocytes divided, as shown by tritiated thymidine incorporation. The rate of cell division increased between 2-6 days, when several smaller basophilic cells were present inside the lacuna instead of the single hypertrophic chondrocyte. These cells expressed alkaline phosphatase activity, were positive for fibronectin, the above non-collagenous bone proteins and type I collagen. The bone matrix that was observed after 6-18 days was initially confined to the inside of the chondrocytic lacunae, but later spread beyond the lacunar confines. The bone proteins were still associated with the bone-forming cells, but fibronectin was absent when matrix formation was evident. Mineralization of the intra-lacunar osteoid took place after 12-18 days. It is speculated that the trans-differentiation was initiated by disruptions of the normal cell-cell associations.
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PMID:Trans-differentiation of hypertrophic chondrocytes into cells capable of producing a mineralized bone matrix. 142 2

Bone sialoprotein (BSP) is a phosphorylated and sulfated glycoprotein that is a major noncollagenous protein of bone and other mineralizing connective tissues. BSP is characterized by the presence of several polyglutamic acid segments and an RGD motif that mediates cell attachment through a vitronectin-like receptor. Although the precise function of BSP is unknown, the expression of BSP in conjunction with bone formation in vitro indicates a role for this protein in the biomineralization of connective tissues. In this study we used Northern hybridization and in situ hybridization to determine the tissue-specific and developmental expression of BSP during embryogenesis and growth of rat tissues. Analysis of tissues obtained from 13, 17, and 21 day fetuses, and from 4-, 14-, and 100-day-old animals indicates that BSP mRNA expression is restricted to cells actively forming the mineralizing tissues of bone, dentin and cementum. BSP mRNA transcripts were first evident in fully differentiated osteoblasts of 17 day fetal tissues at sites of de novo intramembranous and endochondral bone formation, with maximal expression observed at 21 days of gestation. Thereafter, BSP mRNA levels decreased markedly, and in adult bone hybridization was detected only in the primary spongiosa of long bones. In comparison, mRNAs for osteopontin (OPN), alkaline phosphatase (ALP), and osteocalcin (OC) peaked at 4-14 days postpartum before declining. In the tibiae, Northern hybridization revealed a second peak of mRNA for BSP, ALP, and OPN at 14 days, reflecting an increased osteogenic activity due to the formation of the secondary centers of ossification in the epiphyseal cartilage. In situ hybridization also revealed BSP mRNA in hypertrophic chondrocytes at sites of bone formation, in odontoblasts of the incisor during dentinogenesis, and in cementoblasts during cementogenesis. In view of the restricted distribution and temporal changes in the expression of BSP mRNA that we observed together with the chemical properties of BSP, we believe that this protein has a specific role in mediating the initial stages of connective tissue mineralization.
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PMID:Development expression of bone sialoprotein mRNA in rat mineralized connective tissues. 144 13

When fetal rat calvarial cells are cultured in medium containing vitamin C, osteoid nodules develop after approximately 15 days of culture. Upon addition of an organic phosphate (beta-glycerophosphate, beta GP), these nodules mineralize. We have now used this system to explore the suggestion made by others that a negative feedback may exist between matrix mineralization on the one hand and the synthesis of alkaline phosphatase and bone matrix collagen on the other by analyzing the synthesis of these proteins and the levels of their mRNAs in mineralizing and nonmineralizing cultures. Our results indicate that in the osteoid nodule-bone nodule system, matrix mineralization did not affect the mRNA levels for osteopontin, type I collagen, bone sialoprotein, or osteocalcin. Synthesis of total protein and collagen and the osteocalcin content of culture media were also not different in the mineralizing and nonmineralizing cultures. However, alkaline phosphatase mRNA was increased in early mineralizing cultures and alkaline phosphatase activity in the cell layer was also increased in mineralizing cultures. Thus, the hypothesis that a direct negative feedback exists between mineralization and matrix protein synthesis is not supported by our experiments.
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PMID:beta-Glycerophosphate-induced mineralization of osteoid does not alter expression of extracellular matrix components in fetal rat calvarial cell cultures. 145 88

To understand the mechanisms by which glucocorticoids promote differentiation of fetal rat calvaria derived osteoblasts to produce bone-like mineralized nodules in vitro, a panel of osteoblast growth and differentiation related genes that characterize development of the osteoblast phenotype has been quantitated in glucocorticoid-treated cultures. We compared the mRNA levels of osteoblast expressed genes in control cultures of subcultivated cells where nodule formation is diminished, to cells continuously (35 days) exposed to 10(-7) M dexamethasone, a synthetic glucocorticoid, which promotes nodule formation to levels usually the extent observed in primary cultures. Tritiated thymidine labelling revealed a selective inhibition of internodule cell proliferation and promotion of proliferation and differentiation of cells forming bone nodules. Fibronectin, osteopontin, and c-fos expression were increased in the nodule forming period. Alkaline phosphatase and type I collagen expression were initially inhibited in proliferating cells, then increased after nodule formation to support further growth and mineralization of the nodule. Expression of osteocalcin was 1,000-fold elevated in glucocorticoid-differentiated cultures in relation to nodule formation. Collagenase gene expression was also greater than controls (fivefold) with the highest levels observed in mature cultures (day 35). At this time, a rise in collagen and TGF beta was also observed suggesting turnover of the matrix. Short term (48 h) effects of glucocorticoid on histone H4 (reflecting cell proliferation), alkaline phosphatase, osteopontin, and osteocalcin mRNA levels reveal both up or down regulation as a function of the developmental stage of the osteoblast phenotype. A comparison of transcriptional levels of these genes by nuclear run-on assays to mRNA levels indicates that glucocorticoids exert both transcriptional and post-transcriptional effects. Further, the presence of glucocorticoids enhances the vitamin D3 effect on gene expression. Those genes which are upregulated by 1,25(OH)2D3 are transcribed at an increased rate by dexamethasone, while those genes which are inhibited by vitamin D3 remain inhibited in the presence of dexamethasone and D3. We propose that the glucocorticoids promote changes in gene expression involved in cell-cell and cell-extracellular matrix signaling mechanisms that support the growth and differentiation of cells capable of osteoblast phenotype development and bone tissue-like organization, while inhibiting the growth of cells that cannot progress to the mature osteoblast phenotype in fetal rat calvarial cultures.
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PMID:Glucocorticoids promote development of the osteoblast phenotype by selectively modulating expression of cell growth and differentiation associated genes. 146 73

We have used a model of rapid bone induction and resorption in rats initiated by the removal of bone marrow to define age-associated deficits. Here we report the sequential expression of various genes implicated in the formation and removal of bone following marrow ablation. Significant increases in alkaline phosphatase and procollagen alpha 1(I) mRNA were observed by day 5, and of osteocalcin and osteopontin by day 6. At their peak, these mRNA levels were elevated three- to eight-fold and correlated with histological evidence of bone formation. No change in collagen II mRNA was observed, indicating that there was no cartilage phase. Collagenase activity increased 10-fold at day 9 and coincided with the beginning of bone resorption. Actin mRNA, a reference gene marker, remained at constant levels. Comparison of the response between adult (6 mo.) and old (24 mo.) rats showed the same temporal pattern, but a lower expression of bone-related genes in older rats. Histological examination also showed that the bone volume and osteoblast number at day 6 were significantly lower in old rats. Furthermore, the percentage of mineralized bone was greatly reduced in the aged rat. This model system is currently being used to evaluate the effectiveness of interventions to up-regulate the bone activity in senescent rats.
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PMID:Impaired bone activity in aged rats: alterations at the cellular and molecular levels. 147 22

In the accompanying study, we report an in vitro culture system from bovine bone cells that can be applied to investigate bone cell growth and differentiation. In this system, bovine bone cells placed in mineralization medium formed multilayers (days 2-3), began deposition of mineral (days 5-6), and eventually acquired a mineralized matrix sheet (days 14-20) through the stages of mineralizing nodules and trabecular-like structure. In the current study we used this system to investigate the relative expression of bone matrix genes that may play an important role in bone development and metabolism. alpha 1(I)-collagen, alkaline phosphatase, osteonectin, biglycan (PgI), decorin (PgII), osteopontin, and bone sialoprotein mRNA gene expression were measured on days 0, 2, 6, 10, and 20 (date when the cells were placed in mineralization medium as day 0). Total RNA was purified and analyzed by northern blot using radiolabeled cDNA encoding these genes. To comprehend the relationship between gene expression and mineralization, total calcium content in the cultures was also measured. During the culture period we observed several very different gene expression profiles. The expression of both alpha 1(I)-collagen and biglycan increased 3- to 4-fold by day 6 and then returned to basal levels by day 20. The osteonectin gene was highly expressed throughout the culture, with no significant increase in induction found during any time of culture. A significant induction of alkaline phosphatase (13.8-fold) gene expression was observed by day 6. Osteopontin showed a similar profile to that of alkaline phosphatase but had a much greater level of relative expression (26-fold) compared to day 0. Interestingly, downregulation during mineral accumulation seemed a common occurrence among many of the genes measured. In contrast, the bone sialoprotein gene showed a significant and distinct expression pattern, increasing rapidly after the onset of mineralization on day 6 and ultimately reaching 140-fold that of day 0. Decorin (Pg II) showed an increasing pattern, with the final relative level of induction 5-fold on day 20. These data suggest that the development of the mature osteoblastic phenotype, complete with the ability to produce a thick mineralized matrix, requires the differential regulation of a series of genes and their gene products over the culture period.
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PMID:Bone matrix mRNA expression in differentiating fetal bovine osteoblasts. 164 43

The cartilagenous tissue of mandibular condyles of newborn mice contains progenitor cells as well as young and mature chondrogenic cells. During in vitro cultivation of the tissue, progenitor cells undergo osteogenic differentiation and form new bone (Silbermann, M., D. Lewinson, H. Gonen, M. A. Lizarbe, and K. von der Mark. 1983. Anat. Rec. 206:373-383). We have studied the expression of genes that typify osteogenic differentiation in mandibular condyles during in vitro cultivation. RNAs of the genes for collagen type I, osteonectin, alkaline phosphatase, and bone gla protein were sequentially expressed in progenitor cells and hypertrophic chondrocytes during culture. Osteopontin expression peaked in both the early and the late phase of the differentiation process. The data indicate a distinct sequence of expression of osteoblast-specific genes during osteogenic differentiation and new bone formation in mandibular condyles.
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PMID:Gene expression during osteogenic differentiation in mandibular condyles in vitro. 169 Nov 90

The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periods--proliferation, extracellular matrix maturation, and mineralization--and 2) two restriction points to which the cells can progress but cannot pass without further signals--the first when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle- and cell growth-regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid-induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.
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PMID:Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. 169 81

The role of 5'-methylthioadenosine (MTA), formed during the process of polyamine biosynthesis, on differentiation of osteoprogenitor cells was assessed by its effects on alkaline phosphatase (ALP) activity, bone nodule formation and osteopontin contents of cultured rat calvaria (RC) cells. These three markers were stimulated by exogenous MTA and were depressed by 5'-difluoromethylthioadenosine (DFMTA), a synthetic inhibitor of MTA phosphorylase, which cleaves MTA to adenine and 5-methylthioribose-1-phosphate. 5-Methylthioribose and 2-keto-4-methylthiobutyrate, metabolites of 5-methylthioribose-1-phosphate, had no effects on ALP activity and bone nodule formation in the presence or absence of DFMTA. On the other hand, adenine enhanced ALP activity, bone nodule formation and osteopontin contents in mineralized nodules and also partially reversed DFMTA-induced inhibition of these three markers. MTA, its metabolites and DFMTA did not affect the growth of RC cells under these culture conditions. These results suggest that adenine formed from MTA is important in the differentiation of RC cells.
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PMID:The role of 5'-methylthioadenosine on rat calvaria cell differentiation. 173 84


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