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)

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] stimulates the alkaline phosphatase of rat and human osteoblast-like cells in culture. Here the mechanism of this effect was investigated using the rat osteogenic sarcoma cell line ROS 17/2-8. We found that 50% maximum alkaline phosphatase stimulation is elicited by 1,25(OH)2D3 at 7 X 10(-10) M. The concentration of serum in the culture medium influences inversely the effective 1,25(OH)2D3 concentration. Increased alkaline phosphatase appears after a lag period of cell exposure to 1,25(OH)2D3 which is between 8 and 24 h; during 96 h culture in the presence of 1,25(OH)2D3 the enzyme activity continues to rise. Cycloheximide (0.1-1 micrograms/ml) added in the cultures for 3 days or actinomycin-D (1-30 ng/ml) added for 24 h inhibit the 1,25(OH)2D3 effect on alkaline phosphatase in a dose-dependent fashion; withdrawal of cycloheximide restores the responsiveness of cells to 1,25(OH)2D3 completely, but withdrawal of actinomycin-D restores cell responsiveness only partially. These findings suggest that 1,25(OH)2D3-induced stimulation of alkaline phosphatase in the osteoblast-like cells involves genome activation and de novo protein synthesis.
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PMID:Mechanism of action of 1,25-dihydroxyvitamin D3-induced stimulation of alkaline phosphatase in cultured osteoblast-like cells. 635 97

Osteogenic protein-1 (OP-1) stimulates bone morphogenesis in vivo and modulates osteoblast growth and differentiation in vitro. Treatment of ROS 17/2.8 cells with OP-1 resulted in a time- and concentration-dependent inhibition of [3H]thymidine incorporation. In contrast, OP-1 treatment stimulated phenotypic differentiation in ROS 17/2.8 cells, as indicated by enhanced 1) alkaline phosphatase activity (4-fold); 2) alkaline phosphatase mRNA (5-fold); 3) parathyroid hormone receptor mRNA (2-fold), and 4) parathyroid hormone-stimulated adenosine 3',5'-cyclic monophosphate accumulation (2-fold). OP-1-induced changes in cell growth and gene expression were sensitive to cycloheximide and actinomycin D. Measurement of [3H]thymidine incorporation and alkaline phosphatase activity in situ revealed heterogeneity in the cellular responses to OP-1. Proliferating cells exhibited less alkaline phosphatase activity than nonproliferating cells, whereas cells expressing high levels of alkaline phosphatase incorporated little [3H]thymidine. Our data delineating the responses of mature differentiated osteoblasts to OP-1 suggest that potentiation of osteoblast differentiated function is an important component of bone morphogenesis in vivo.
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PMID:Osteogenic protein-1 enhances phenotypic expression in ROS 17/2.8 cells. 749 44

Recent evidence suggests that the production of nitric oxide (NO) may have important roles in the regulation of osteoblast and osteoclast metabolism. The present study was performed to investigate the effects of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) on the expression of inducible NO-synthase (iNOS) and to measure high-output production of NO by primary rat osteoblasts and osteoblastic cell lines ROS 17/2.8, MC3T3-E1 and MG-63. In addition, we have investigated if NO may mediate some of the effects of these cytokines on osteoblast metabolism. Northern blots and immunocytochemistry revealed time-dependent iNOS messenger RNA and protein expression in primary rat osteoblasts in response to cytokine treatment. Reverse transcription polymerase chain reaction amplified an 807-base pair (bp) product from ROS 17/2.8 cells, which had a size and restriction enzyme-cut pattern identical to that predicted for authentic rat iNOS. Nitrite accumulation in culture medium was induced by IFN-gamma in a time- and dose-dependent manner and inhibited by cotreatment with inhibitors of NOS activity and by dexamethasone. IL-1 beta, TNF-alpha, and bacterial lipopolysaccharide were found to have weak stimulatory effects on nitrite production on their own. However, IL-1 beta and TNF-alpha showed strong synergy with IFN-gamma, but, surprisingly, lipopolysaccharide was found to exert potent inhibitory effects on IFN-gamma-induced nitrite synthesis. Basal production of nitrite and induction of its synthesis was similarly observed with primary rat osteoblasts as well as ROS 17/2.8, MC3T3-E1, and MG-63 cell lines. Cytokine-induced NO production significantly reduced osteoblast activity, as was evidenced by inhibition of DNA synthesis, cell proliferation, alkaline phosphatase activity, and osteocalcin production. The results provide evidence for a basal expression of iNOS activity and show that the iNOS messenger RNA, protein, and enzyme activity are all induced by cytokines across the species. The data further suggest that osteoblast-derived NO may have an important role in mediation of localized bone destruction associated with inflammatory bone diseases such as rheumatoid arthritis.
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PMID:Cytokine-stimulated expression of inducible nitric oxide synthase by mouse, rat, and human osteoblast-like cells and its functional role in osteoblast metabolic activity. 758 94

Pasteurella multocida toxin induces localized osteolysis in the turbinate bones of swine. Osteolysis appears to be due to an increased level of osteoclastic bone resorption, although osteoblast activity may also be impaired. We studied the effects of purified toxin on the osteoblastic phenotype of the ROS 17/2.8 rat osteoblastic osteosarcoma cell line. Treatment of both embryonic bovine lung cells and a nonosteoblastic rat osteosarcoma cell line (ROS 25/1) with nanomolar doses of toxin produced marked cytotoxic actions. In the osteoblastic ROS 17/2.8 cells, this level of toxin reduced expression of an osteoblastic marker (alkaline phosphatase), was associated with matrix mineralization, but had no cytopathologic action. The osteoblastic cell population may be resistant to a direct cytotoxic effect but is nevertheless a target for toxin action.
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PMID:Effects of the Pasteurella multocida toxin on osteoblastic cells in vitro. 760 94

1 alpha,25-Dihydroxyvitamin D3 (D3), T3, and retinoids are necessary for normal skeletal development, and their actions are interdependent due to the heterodimerization capabilities of their receptors. We investigated the hypothesis that these hormones act on osteoblasts directly to produce complex target gene responses resulting from multiple hormone interactions. Physiological interactions among D3, T3, and retinoid signaling were analyzed in serum-free cultures of the osteosarcoma cell lines ROS 25/1, UMR106, and ROS 17/2.8. These cells express distinct stages of the osteoblast phenotype and coexpress appropriate hormone receptors. Regulation of collagen I alpha 1 and alpha 2, alkaline phosphatase, osteopontin, and osteocalcin messenger RNAs was dependent on the dose and duration of hormone stimulation and modified by cell confluence. Retinoids were required for comprehensive expression of phenotypic responses to D3 and T3 in each cell type and hormone interactions were both cell and target gene specific. Differing responses of target genes in each cell line may provide a molecular basis for discrete hormone actions seen at specific stages of osteoblast differentiation or skeletal development.
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PMID:Retinoids modify regulation of endogenous gene expression by vitamin D3 and thyroid hormone in three osteosarcoma cell lines. 766 49

Glucocorticoids and sex-steroids can modulate osteogenesis in vivo and in vitro. Although the effects of glucocorticoids on bone cells in vitro have been described in detail, the role of sex-steroids is not as well defined. We examined whether sex-steroids influence bone metabolism indirectly by regulating glucocorticoid effects on bone. Interactions of the sex-steroid progesterone or its analog RU38486 with the glucocorticoid dexamethasone (dex) were studied in functional assays of osteogenesis. Three osteoblastic models were evaluated: (1) the rat bone marrow stromal cell (RBMC) nodule system; (2) the chick periosteal osteogenesis (CPO) model; and (3) ROS 17/2.8 cells. RU38486, progesterone, and unlabelled dex competitively inhibited 3H-dex uptake by ROS 17/2.8 cells as well as its (3H-dex) binding to cytosol preps. Both RU38486 and progesterone inhibited dex-induced increases in alkaline phosphatase in CPO cultures, in RBMC cultures, and in ROS 17/2.8 cells. Dex-induced decreases in cell proliferation in ROS 17/2.8 cells were reversed by RU38486 but dex-induced increases in proliferation in the CPO model were not affected. In CPO cultures, dex-induced increases in collagen synthesis were inhibited completely by RU38486 and progesterone. Dex-dependent nodule formation in the RBMC was blocked by RU38486. Both RU38486 and dex mediated reduction of calcium uptake in the CPO model but did not affect mineralized tissue area. The data indicate that RU38486 and progesterone competitively inhibit dex-mediated stimulation of osteogenesis in vitro; this inhibition is exerted on early but not late stage differentiation events of osteoprogenitor cells.
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PMID:Probing glucocorticoid-dependent osteogenesis in rat and chick cells in vitro by specific blockade of osteoblastic differentiation with progesterone and RU38486. 766 5

We recently showed that osteogenic protein-1(OP-1), a bone morphogenetic protein member of TGF-beta superfamily, induces endochondral bone formation in vivo, and stimulates growth and differentiation of osteoblasts in rat calvarial-derived cell cultures. In the present study, we examined the effect of OP-1 on cell growth and expression of markers that are characteristic of osteoblast phenotype using the clonal rat osteosarcoma cells (ROS 17/2.8). A comparison of OP-1 and TGF-beta 1 effects on cell growth showed that, both OP-1 and TGF-beta 1 inhibited DNA synthesis up to 90 percent and 60 percent of the controls at concentrations of 10 ng/ml and 1 ng/ml, respectively, in serum-free medium. In the presence of 5% serum, TGF-beta 1 did not have any significant inhibitory effects while 40 ng OP-1/ml inhibited the DNA synthesis up to 80% of the controls. Examination of collagen synthesis showed that 40 ng OP-1/ml increased the expression of type I collagen mRNA, and thus increased collagen synthesis (4-fold), as examined by collagenase-digestible protein. Evaluation of markers that are characteristic of the osteoblast phenotype demonstrated that OP-1 stimulated cAMP production in response to PTH (10-fold at 200 ng/ml), alkaline phosphatase specific activity (ALPase) (4-fold at 80 ng/ml), and osteocalcin (OC) synthesis (4.5-fold at 40 ng/ml). Northern blot analysis revealed that OP-1 increased mRNA expression for both ALPase and OC in a dose-dependent manner. These data collectively demonstrate that OP-1 suppresses cell proliferation and stimulates the expression of markers characteristic of osteoblast phenotype in rat clonal osteoblastic osteosarcoma cells (ROS 17/2.8).
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PMID:Osteogenic protein-1 (BMP-7) inhibits cell proliferation and stimulates the expression of markers characteristic of osteoblast phenotype in rat osteosarcoma (17/2.8) cells. 773 48

Transforming growth factor beta (TGF-beta), a potent regulator of bone formation, has bifunctional effects on osteoblast replication and biochemical activity that appear differentiation dependent. We now show that cell surface binding sites for TGF-beta vary markedly among fibroblasts, bone-derived cells, and highly differentiated osteosarcoma cultures from fetal rats. Expression of betaglycan and type II receptors decline relative to type I receptor expression in parallel with an increase in osteoblast-like activity, predicting that the ratio among various TGF-beta binding sites could influence how its signals are perceived. Bone morphogenetic protein 2 (BMP-2), which induces osteoblast function, does not alter TGF-beta binding or biochemical activity in fibroblasts and has only small effects in less differentiated bone cells. In contrast, BMP-2 rapidly reduces TGF-beta binding to betaglycan and type II receptors in osteoblast-enriched primary cell cultures and increases its relative binding to type I receptors in these cells and in ROS 17/2.8 cultures. Pretreatment with BMP-2 diminishes TGF-beta-induced DNA synthesis in osteoblast-enriched cultures but synergistically enhances its stimulatory effects on either collagen synthesis or alkaline phosphatase activity, depending on the present state of bone cell differentiation. Therefore, BMP-2 shifts the TGF-beta binding profile on bone cells in ways that are consistent with progressive expression of osteoblast phenotype, and these changes distinguish the biochemical effects mediated by each receptor. Our observations indicate specific stepwise actions by TGF-beta family members during osteoblast differentiation, developing in part from changes imprinted by BMP-2 on TGF-beta receptor stoichiometry.
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PMID:Independent changes in type I and type II receptors for transforming growth factor beta induced by bone morphogenetic protein 2 parallel expression of the osteoblast phenotype. 776 Aug 23

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

Streptolysin-O (SLO), a cholesterol-binding agent, was used for studies on the release of glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase (AP) from ROS cells. Treatment of cells with SLO resulted in a time- and concentration-dependent release of AP into the extracellular medium. This release was potentiated by Ca2+ and bovine serum, but not by GPI-specific phospholipase D (GPI-PLD) purified from bovine serum. The released AP distributed to the detergent phase after Triton X-114 phase separation. This result suggested that the released AP contained an intact GPI anchor, and thus both proteolysis and anchor degradation by anchor-specific hydrolases, including GPI-PLD, as the potential mechanisms for SLO-mediated AP release were ruled out. The released AP sedimented at 100,000 g. A substantial amount of lipids was detected in the 100,000 g pellet. Cholesterol and sphingomyelin were enriched in SLO-released material, compared with intact cells. These results were consistent with vesiculation as the mechanism for SLO induction of AP release. Two other cholesterol-binding agents, saponin and digitonin, were also able to release AP, possibly by a similar vesiculation mechanism, whereas others, including nystatin, filipin and beta-escin, failed to elicit any AP release. Eight GPI-anchored proteins were identified in ROS cells, and all were substantially enriched in the vesicles released by SLO. Taken together, these results do not provide any support for the hypothesis that the clustering of GPI-anchored proteins in the plasma membrane is responsible for their resistance to GPI-PLD cleavage.
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PMID:Streptolysin-O induces release of glycosylphosphatidylinositol-anchored alkaline phosphatase from ROS cells by vesiculation independently of phospholipase action. 783 71


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