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)

Numerous studies of experimental hypo- and hypervitaminosis A have long suggested that retinoic acid (RA) is involved in chondrocyte maturation during endochondral ossification and skeletogenesis. However, the specific and direct roles of RA in these complex processes remain unclear. Based on recent studies from our laboratories, we tested the hypothesis that RA induces the expression of genes associated with the terminal mineralization phase of chondrocyte maturation and promotes apatite deposition in the extracellular matrix. Cell populations containing chondrocytes at advanced stages of maturation were isolated from the upper portion of Day 18 chick embryo sterna and grown for 2 weeks in monolayer until confluent. The cells were then treated with low doses (10-100 nM) of RA for up to 6 days in the presence of a phosphate donor (beta-glycerophosphate) but in the absence of ascorbic acid. Within 4 days of treatment, RA dramatically induced expression of the alkaline phosphatase (APase), osteonectin, and osteopontin genes, caused a several-fold increase in APase activity, and provoked massive mineral formation while it left type X collagen gene expression largely unchanged. The mineral had a mean Ca/Pi molar ratio of 1.5; Fourier transform infrared spectra confirmed that it represented hydroxyapatite. Mineralization was completely abolished by treatment with parathyroid hormone; this profound effect confirmed that RA induced cell-mediated mineralization and not nonspecific precipitation. When cultures were treated with both RA and ascorbic acid, there was a slight further increase in APase activity and increased calcium accumulation. The effects of RA were also studied in cultures of immature chondrocytes isolated from the caudal portion of sternum; however, RA only had minimal effects on mineralization and gene expression in these cells. Thus, RA appears to be a rapid, potent, maturation-dependent, ascorbate-independent promoter of terminal maturation and matrix calcification in chondrocytes.
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PMID:Retinoic acid induces rapid mineralization and expression of mineralization-related genes in chondrocytes. 834 89

We have addressed questions raised by the observation in fetal rats of delayed ossification induced by caffeine at maternal doses above 80 mg/kg body weight per day. The effect of caffeine on endochondral bone development and mineralization has been studied in an experimental model system of bone formation which involves implantation of demineralized bone particles (DBP) in subcutaneous pockets of young growing rats. Caffeine's effects on cellular events associated with endochondral ossification were examined directly by quantitating cellular mRNA levels of chondrocyte and osteoblast growth and differentiation markers in DBP implants from caffeine-treated rats harvested at specific stages of development (day 7 through day 15). Oral caffeine administration to rats implanted with DBP resulted in a dose dependent inhibition of the formation of cartilage tissue in the implants. Histologic examination of the implants revealed a decrease in the number of cells which were transformed to chondrocytes compared to control implants. Those cartilaginous areas that did form, however, proceeded through the normal sequelae of calcified cartilage and bone formation. At the 100 mg/kg dose, cellular levels of mRNA for histone, collagen type II, and TGF beta were all reduced by greater than 40% of control implants consistent with the histological findings. Alkaline phosphatase activity in the implants and mRNA levels for proteins reflecting the hypertrophic chondrocyte and bone phenotype, collagen type I and osteocalcin were markedly decreased compared to controls. Lower doses of 50 and 12.5 mg/kg caffeine also resulted in decreased cellular proliferation and transformation to cartilage histologically and reflected by significant inhibition of type II collagen mRNA levels (day 7). The effects of caffeine on gene expression observed in vivo during the period of bone formation (day 11 to day 15) in the DBP model were similar to the inhibited expression of H4, alkaline phosphatase, osteocalcin, and osteopontin found in fetal rat calvarial derived osteoblast cultures following 24 hour exposure of the cultures to 0.4 mM caffeine. Thus the observed delayed mineralization in the fetal skeleton associated with caffeine appears to be related to an inhibition of endochondral bone formation at the early stages of proliferation of undifferentiated mesenchymal cells to cartilage specific cells as well as at later stages of bone formation.
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PMID:Inhibition of induced endochondral bone development in caffeine-treated rats. 836 35

Endochondral bone formation occurs by a series of developmentally regulated cellular events from initial formation of cartilage tissue to stages of calcified cartilage, resorption, and replacement by bone tissue. Several studies have raised the question of the possibility that the hypertrophic chondrocytes associated with the calcifying cartilage matrix can acquire properties similar to osteoblasts. We have addressed this possibility by measuring synthesis within hypertrophic chondrocytes in vitro of two bone-related proteins, osteopontin and osteocalcin. Chondrocytes derived from chick embryo ventral vertebral tissue were cultured under conditions that promoted extracellular matrix mineralization and differentiation towards the hypertrophic phenotype as indicated by the induction of Type X collagen, alkaline phosphatase, and diminished expression of Type II collagen and the core protein of large proteoglycan. In these cultures, osteopontin synthesis was detected in early cultures in the absence of a calcified matrix; in contrast, an absence of the bone-specific protein osteocalcin was observed. However, with onset of development of the hypertrophic phenotype an induction of protein expression for osteocalcin was observed with a significant (twofold) increase in osteopontin. Maximal levels of osteocalcin synthesis occurred with the peak of alkaline phosphatase activity and Type X collagen mRNA levels. The levels of osteocalcin synthesis were induced fiftyfold from the earliest level of detection but this level was only one one-hundredth of that observed for mature chick osteoblast cultures. Osteocalcin and osteopontin were characterized by several criteria (electrophoresis, immunoblotting, chromatographic characteristics, and response to 1,25(OH)2D3) which confirmed their molecular properties as being identical to osteoblast synthesized proteins. The coordinate change in the cellular phenotype to the hypertrophic chondrocyte was shown to be concurrent with ultrastructural maturation of the cells and the accumulation of osteocalcin and osteopontin in the extracellular matrix associated with hydroxyapatite at sites of mineralization. Since the ultrastructural features of the cells in vitro and the extracellular matrix surrounding the lacunae have features of the hypertrophic chondrocyte and associated matrix in vivo, the induction of the bone-specific protein osteocalcin suggests that at least a population of these cells may develop osteoblastic phenotypic markers in association with mineralizing matrix. The detection of osteocalcin and the high level of synthesis of osteopontin may represent an advanced stage of chondrocyte hypertrophy or the possibility of a trans-differentiation of the chondrocytes to an osteoblastic-like cell.
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PMID:Induction of bone-related proteins, osteocalcin and osteopontin, and their matrix ultrastructural localization with development of chondrocyte hypertrophy in vitro. 836 37

The purpose of this study was to determine the response of bone cells to physical stress. Intermittent compressive force (ICF) was applied to 13 kPa to subconfluent ROS 17/2.8 cells at 18 cycles/min. After 48 h of this application, the cells were labelled with [35S]-methionine or [32PO4]. Application of ICF over this time did not alter the synthesis of type I collagen, fibronectin or bone SPARC (osteonectin) compared to that of control cells. However, the activity of alkaline phosphatase was increased 1.5-fold, and the synthesis of a 32PO4-labelled, 75-kDa phosphoprotein, recognized as osteopontin by immunoprecipitation with specific antibodies, was increased 1.4-fold. Also, an increase in osteopontin mRNA starting within 12h of ICF application was observed. The selective increase in osteopontin expression associated with ICF may be important in the remodelling of bone tissues during growth and development and in response to functional forces.
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PMID:Influence of an intermittent compressive force on matrix protein expression by ROS 17/2.8 cells, with selective stimulation of osteopontin. 838 21

The responses of the immortalized rat preosteoblast UMR-201-10B to ascorbic acid (AA), 1,25(OH)2D3 (calcitriol), and retinoic acid (RA) were examined. UMR-201-10B cells have an undetectable basal alkaline phosphatase (ALP) activity that is induced after 24 h of treatment with 10(-6) M RA (4.64 +/- 0.06 mumol/h/mg of protein). The addition of 10(-8) M calcitriol resulted in a slight induction of ALP activity after 72 h (0.43 +/- 0.07 mumol/h/mg of protein). When calcitriol was added to RA, however, over the same period ALP activity was enhanced significantly compared with treatment with RA alone (RA and calcitriol, 12.29 +/- 0.86 mumol/h/mg of protein). Treatment with AA (50 micrograms/ml) alone had no effect on ALP activity but increased RA-induced ALP activity to 6.78 +/- 0.28 mumol/h/mg of protein at 24 h. In contrast, AA inhibited calcitriol-induced ALP activity after 7 days of combined treatment with calcitriol (calcitriol, 7.73 +/- 0.16 mumol/h/mg of protein; AA and calcitriol, 1.44 +/- 0.06 mumol/h/mg of protein). Individually, RA and calcitriol induced mRNA expression for ALP, matrix-gla protein (MGP), and osteopontin (OP). The steady state level of pro-alpha 1(I) collagen mRNA also was increased significantly by treatment with RA and AA individually. The combination of RA and calcitriol had a synergistic effect on ALP, OP, and especially MGP mRNA expression but significantly reduced the expression of pro-alpha 1(I) collagen mRNA. AA enhanced the effect of RA on the expression of pro-alpha 1(I) collagen, MGP, and ALP mRNAs as well as the effect of calcitriol on OP and MGP. The addition of AA to RA resulted in a decrease in the steady state level of OP, whereas its cotreatment with calcitriol caused a decrease in pro-alpha 1(I) collagen and ALP mRNA. In conclusion, these studies identify RA, calcitriol, and AA as regulators of differentiated osteoblast function.
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PMID:Effects of ascorbic acid, calcitriol, and retinoic acid on the differentiation of preosteoblasts. 841 Apr 63

A growing body of data suggests that the bone marrow stroma contains a population of pluripotent cells capable of differentiating into adipocytes, osteoblasts, and lymphohematopoietic supporting cells. In this work, the murine stromal cell lines BMS2 and +/+ 2.4 have been examined as preadipocytes and adipocytes for evidence of osteoblastic gene expression. Adipocyte differentiation has been quantitated using fluorescence activated cell sorting. Within 7-10 days of adipocyte induction by treatment with glucocorticoids, indomethacin, and methylisobutylxanthine, between 40% to 50% of the cells contain lipid vacuoles and exhibit a characteristic adipocyte morphology. Based on immunocytochemistry, both the adipocytes and preadipocytes express a number of osteoblastic markers; these include alkaline phosphatase, osteopontin, collagen (I, III), bone sialoprotein II, and fibronectin. Based on biochemical assays, the level of alkaline phosphatase expression is not significantly different between preadipocyte and adipocyte cells. However, unlike rat cell lines, dexamethasone exposure causes a dose-dependent decrease in enzyme activity. The steady-state mRNA levels of the osteoblast associated genes varies during the process of adiopogenesis. The relative level of collagen I and collagen III mRNA is lower in adipocyte-induced cells when compared to the uninduced controls. Osteocalcin mRNA is detected in preadipocytes but absent in adipocytes. These data indicate that osteoblastic gene expression is detected in cells capable of undergoing adipocyte differentiation, consistent with the hypothesis that these cell lineages are interrelated.
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PMID:Osteoblastic gene expression during adipogenesis in hematopoietic supporting murine bone marrow stromal cells. 842 12

Following injury to bone marrow there is a phase of osteogenesis in which bone trabeculae replace the initial blood clot and fill the marrow cavity. The newly formed bone is subsequently fully resorbed by osteoclasts and normal bone marrow is restored. In this study we correlated the morphologic events with the pattern of gene expression that defines this sequence. Following marrow ablation, the trabecular bone volume in the affected section of the marrow cavity increased from control to 27% at day 6, declined to 18% at day 8, and eventually returned to control levels at day 14. Osteoblast number increased up to day 6 and declined substantially by day 8, but the number of osteoclasts peaked between days 8 and 10. Histologic analysis of alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) activity correlated with the observed cellular changes. Northern blot analysis of the levels of AP, osteocalcin (OC), and osteopontin (OP) mRNA shows a specific pattern of regulated gene expression, with AP mRNA maximal at day 6, OC mRNA very low until days 6-8, and OP mRNA expressed at very high levels throughout. In addition, procollagen alpha 1(I) and alpha 1(III) mRNAs show a regulated pattern of expression, with procollagen alpha 1(I) maximally expressed between days 4 and 10 and procollagen alpha 1(III) expressed at lower levels between days 4 and 6. The mRNA encoding insulin-like growth factor I (IGF-I) was found to be highly expressed between days 5 and 12; however, transforming growth factor beta 1 (TGF-beta 1) and TGF-beta 3 mRNA were only weakly expressed between days 4 and 10. These data demonstrate a temporal pattern of gene expression consistent with the observed morphologic profile, identify changes in growth factor mRNA that may be related to this repair process, and suggest that this is a suitable model for studying in vivo a synchronized sequence of bone formation and resorption at a well-defined anatomic site.
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PMID:Pattern of gene expression following rat tibial marrow ablation. 845 91

Ultrathin sections from the dermis of five normal subjects and from 10 patients suffering from pseudoxanthoma elasticum (PXE) were analyzed by immunoelectron microscopy with the aim of identifying and localizing proteins associated with the mineral precipitates within the altered elastic fibers. Serial sections were processed by indirect immunogold cytochemistry using primary antibodies against human fibronectin, vitronectin, bone sialoprotein, alkaline phosphatase, osteonectin, and osteopontin. In the latter two cases, antibodies against synthetic peptides were also used. The results indicate that normal elastic fibers contained osteopontin, and that this protein was associated with the apparently normal elastin as well as with the needle-shaped mineral precipitates in the elastic fibers of patients. On the contrary, significant amounts of vitronectin, alkaline phosphatase and, less, of bone sialoprotein were associated with the polymorphous mineral precipitates inside the elastic fibers. Large amounts of osteonectin and fibronectin, together with vitronectin, were localized on the microfilament aggregates, which were often associated with altered elastic fibers in PXE dermis and were never visualized in the dermis of control subjects. The results seem to indicate once more that PXE is a complex disorder of the fibroblast synthetic control. Elastic fiber mineralization might be considered a secondary event, which could depend on the abnormal synthesis and accumulation within the elastic fibers of proteins that are normally involved in mineralization processes.
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PMID:Matrix proteins with high affinity for calcium ions are associated with mineralization within the elastic fibers of pseudoxanthoma elasticum dermis. 857 19

Recognition of discrete commitment and differentiation stages requires characterization of changes in proliferative capacity together with the temporal acquisition or loss of expression of molecular and morphological traits. Both cell lines and primary cultures have been useful for analysis of transitional steps in the chondroblast (CB) and osteoblast (OB) lineages. One striking feature is that OBs and CBs share expression of some molecules, including newer markers such as epsilon BP (galectin-3), while also having unique markers. The fact that hypertrophic chondrocytes appear able to downregulate cartilage markers and upregulate OB markers also points to an interesting lineage relationship that needs to be explored further. Recently, we have focused on the osteoprogenitors that divide and differentiate into mature OBs forming bone nodules in fetal rat calvaria cell cultures. We use cellular, immunocytochemical, and molecular approaches, including PCR on small numbers of cells, to discriminate stages. Nodule formation is characterized by loss of proliferative capacity and sequential increased marker expression, that is, alkaline phosphatase (AP), followed by bone sialoprotein (BSP), and osteocalcin. Upregulation of collagen type I and biphasic expression of osteopontin, with two peaks corresponding to proliferation and differentiation stages, also occurs. A variety of other molecules are also upregulated in the mature OB, including epsilon BP and CD44s. By replica plating and PCR, we have begun to study the expression of the messenger RNAs (mRNAs) for potential regulatory molecules (e.g., PTHrP) and their receptors (e.g., PTHR, FGFR-1, and PDGFR alpha) and have found all to be modulated during the progression from committed osteoprogenitor to mature OB.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Osteoblast and chondroblast differentiation. 857 3

Mineralization was induced by glucocorticoid treatment in a human osteoblastic cell line derived from normal bone in vitro, designated SV-HFO, immortalized with simian virus 40 (CHIBA, H. et al. (1993). Jpn. J. Cancer Res., 84: 290-297). Mineralization was revealed by electron microscopy, von Kossa staining and electron spectroscopic analysis, which indicated that the Ca/P ratio was approximately 1.70, corresponding to the value of hydroxyapatite. The effect was dose- (10(-8)-10(-6) M) and time-dependent (days 7-28), was greatest at day 28, and was preceded by expression of alkaline phosphatase (ALP) and osteopontin (OPN). The ALP activity induced was highest at day 7, whereas OPN reached its highest level at day 28. When the induction of ALP activity was inhibited by 10(-4) M levamisole, mineralization of SV-HFO cells by glucocorticoid treatment was markedly reduced, suggesting that elevated ALP activity in the early phase is important in the mineralization of human osteoblastic cells. Glucocorticoid treatment did not alter cell proliferation. These results indicated that glucocorticoids play crucial roles in the formation of mineralized matrix in human osteoblasts by inducing differentiation of SV-HFO cells without modulating their proliferative activity.
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PMID:Glucocorticoids induce mineralization coupled with bone protein expression without influence on growth of a human osteoblastic cell line. 858 88


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