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)

This study investigated the biochemical structure of proteoglycans synthesized during matrix maturation by mineralizing bone cells in vitro, in the presence and absence of fluoride. Bone cells were obtained from rat femur washes and cultured in alpha MEM media supplemented with fetal calf serum, ascorbic acid, beta-glycerophosphate and dexamethasone. Cells were characterized as osteoblast-like by the expression of alkaline phosphatase activity and the synthesis of collagen type I and osteocalcin. Fluoride, present in the culture media at concentrations of 10(-5) M or 10(-7) M, had negligible effect on cell viability. However, calcium deposition was increased in cell cultures incubated in the presence of fluoride. Proteoglycans were extracted from the extracellular matrix with 4 M guanidinium chloride and purified by anion exchange chromatography. Biochemical analysis identified the presence of the small leucine rich proteoglycan, decorin and biglycan, in addition to degradation products relating to the larger chondroitin sulphate protoeglycan, versican. Fluoride had little effect on the size or amino acid composition of the protein core, but resulted in significant alterations to the GAG chains, including a dramatic reduction in chain length, reduction in sulphation and decrease in the proportion of dermatan sulphate compared to chondroitin sulphate. The influence of fluoride on proteoglycan structure synthesized by mineralizing bone cells provides valuable information, indicating specific roles for dermatan sulphate and chondroitin sulphate proteoglycans. The results suggested that fluoride affected the post-translational assembly of the GAG chains which may be an influential factor in the mineralization process.
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PMID:Structural analysis of proteoglycans synthesized by mineralizing bone cells in vitro in the presence of fluoride. 974 42

The influence of bone morphogenetic protein-2 (BMP-2) and transforming growth factor beta (TGF-beta) on the expression of small proteoglycans, decorin and biglycan was investigated in a clonal rat osteoblastic cell line, ROS-C26 (C26) cells, which is a potential osteoblast precursor cell line and capable of differentiating into mature osteoblasts after treatment with recombinant BMP-2 (rhBMP-2). Following the culture of C26 cells for 3, 6, and 9 days in the presence or absence of rhBMP-2, alkaline phosphatase activity increased in the rhBMP-2 treated cells in direct proportion to their differentiation into more mature osteoblastic cells, whereas decorin mRNA decreased in the cells, when compared to control cells without rhBMP-2 treatment. These results were evident 6 days after treatment. However, rhBMP-2 treatment had no effect on biglycan mRNA expression in the cells. Subsequently, after removal of rhBMP-2 from the culture media, the cells were further cultured for 24 h with graded concentrations of TGF-beta1 (0, 0.1, 1.0, 5.0, and 10 ng/ml). TGF-beta1 decreased decorin mRNA expression in the cells dose dependently, but did not affect their biglycan mRNA expression. Furthermore, either removal of rhBMP-2 from the culture media or addition of TGF-beta1 significantly decreased alkaline phosphatase activity of rhBMP-2-induced cells. These results indicate that osteoblastic differentiation is accompanied by increased alkaline phosphatase activity and decreased expression of decorin mRNA, but continuous expression of biglycan mRNA. Both rhBMP-2 and TGF-beta1 inhibit decorin mRNA expression in osteoblasts at varying stages of differentiation, but their effects on biglycan mRNA expression and alkaline phosphatase are different.
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PMID:Effects of bone morphogenetic protein-2 and transforming growth factor-beta1 on gene expression of decorin and biglycan by cultured osteoblastic cells. 1046 26

In this study, the effects of incubating two clonal rat osteoblastic cell lines at different stages of differentiation, ROB-C26 (C26) and ROB-C20 (C20), with transforming growth factor-beta1 (TGF-beta1) on the gene expression of decorin, biglycan, and alkaline phosphatase were examined. C26 cells are a potential osteoblast precursor cell line that is also capable of differentiating into muscle cells and adipocytes and is differentiated into osteoblasts after treatment with bone morphogenetic protein-2. C20 cells are a more differentiated osteoblastic cell line. Our Northern blot studies demonstrated that after treatment with TGF-beta1 (0, 0.1, 1.0, 5.0, and 10 ng/ml), a dose- and time-dependent decrease in decorin mRNA expression was found in C26 cells. In contrast, the effect of decorin mRNA with TGF-beta1 was not determined in C20 cells, since decorin mRNA expression was extremely low in this cell line even in the absence or presence of TGF-beta1. Although TGF-beta1 treatment resulted in no appreciable effect on biglycan mRNA expression in both cell lines in a dose- and time-dependent manner, it decreased significantly the expression of alkaline phosphatase in both cell lines at the gene and protein level. Reverse transcriptase-polymerase chain reaction analysis revealed the gene expression of decorin, and TGF-beta type I and type II receptors in both cell lines. These results indicate that osteoblasts progenitor cells express both decorin and biglycan mRNAs. In contrast, more differentiated and mature osteoblastic cells express preferentially biglycan mRNA. TGF-beta1 exerts different effects on the expression of decorin and biglycan mRNAs, and is a potent inhibitor of the gene expression of alkaline phosphatase during osteoblast differentiation.
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PMID:Effects of transforming growth factor-beta1 on the gene expression of decorin, biglycan, and alkaline phosphatase in osteoblast precursor cells and more differentiated osteoblast cells. 1057 18

To explore how the loss of Phex function contributes to the pathogenesis of osteomalacia, we examined the abnormalities of mineralization, Phex, and bone matrix protein expression occurring in Hyp mice in vivo and in ex vivo bone marrow cell cultures. The results in vivo show that mineralization was decreased significantly in Hyp mouse bone. Phex protein was identifiable in osteoblasts and osteocytes in wild-type mice, but not in Hyp mice. In Hyp mice, osteocalcin, bone sialoprotein, and vitronectin expression were down-regulated, whereas biglycan and fibrillin-1 expression were up-regulated in osteocytes and bone matrix relative to those in their wild-type counterparts. Parallel studies ex vivo demonstrated that cells derived from 18-day Hyp mouse bone marrow cell cultures had a 3'-Phex deletion, no Phex protein expression, decreased alkaline phosphatase activity, collagen deposition, and calcium accumulation, and reduced osteocalcin, bone sialoprotein, and vitronectin at both the protein and messenger RNA levels. Furthermore conditioned medium from Hyp mouse bone marrow cultures could induce analogous defects in bone marrow cell cultures of wild-type cells. These novel findings indicate that there is an intrinsic osteogenic cell differentiation defect in addition to the known hypomineralization of bone in Hyp mice, which may be inducible by an autocrine/paracrine secreted factor. These results suggest that alterations in the Phex gene may control bone matrix mineralization indirectly by regulating the synthesis and deposition of bone matrix proteins.
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PMID:Osteomalacia in hyp mice is associated with abnormal phex expression and with altered bone matrix protein expression and deposition. 1115 66

We report the establishment in vitro of three-dimensional (3D) cultures of human osteoblasts (hOB) derived from normal adults and supported uniquely by the extracellular matrix (ECM) they deposit. Osteoblasts were cultured in 3D cultures in vitro for up to 120 days. The 3D cultures, examined at 25, 31, and 48 days, expressed protein markers of osteoblastic cells, namely osteonectin, collagen type I, fibronectin, osteopontin, bone sialoprotein, biglycan, and decorin. Sequentially, alkaline phosphatase (AP) and then Ca incorporation, mineralization of matrix (monitored by histochemistry and transmission electron microscopy), and finally osteocalcin expression, were detected in the 3D cultures. Ultrastructurally, morphology progressed from early to mature osteoblast and to osteocyte-like. Cells were embedded in a matrix with organized collagen type I fibers containing, increasingly with time of culture, needle-shaped crystals, often associated with matrix vesicles, characteristic of those in bone. During the culture (up to 120 days) there was an outgrowth of proliferating osteogenic cells from the 3D structure. Subcutaneous implantation in nude mice for 20 days of osteoblasts cultured in 3D culture for different lengths of time in vitro, showed progression of mineralization from the inner region of the implant outward, with peripheral cells being embedded in nonmineralized, collagen-rich matrix. The 3D implants were invaded by vessels derived from the host.
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PMID:Three-dimensional cultures of normal human osteoblasts: proliferation and differentiation potential in vitro and upon ectopic implantation in nude mice. 1199 10

The localized expression of a number of extracellular matrix genes was evaluated over time in a novel rat rotator cuff injury model. The supraspinatus tendons of rats were severed at the bony insertion and repaired surgically. The healing response was evaluated at 1, 2, 4, and 8 weeks post-injury using histologic and in situ hybridization techniques. Expression patterns of collagens (I, II, III, IX, X, XII), proteoglycans (decorin, aggrecan, versican, biglycan, fibromodulin), and other extracellular matrix proteins (elastin, osteocalcin, alkaline phosphatase) were evaluated at the healing tendon to bone insertion site. Histologic results indicate a poor healing response to the injury, with only partial recreation of the insertion site by 8 weeks. In situ hybridization results indicate a specific pattern of genes expressed in each zone of the insertion site (i.e., tendon, fibrocartilage, mineralized cartilage, bone). Overall, expression of collagen types I and XII, aggrecan, and biglycan was increased, while expression of collagen type X and decorin was decreased. Expression of collagen type I, collagen type XII, and biglycan decreased over time, but remained above normal at 8 weeks. Results indicate that the rat supraspinatus tendon is ineffective in recreating the original insertion site, even at 8 weeks post-injury, in the absence of biological or biomechanical enhancements.
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PMID:The localized expression of extracellular matrix components in healing tendon insertion sites: an in situ hybridization study. 1203 18

Marrow stroma cells (MSC) play a major role in osteogenesis. The potential of the MSC to differentiate to bone-forming cells relies upon molecular regulation. This study analyzed MBA-15 cells for the expression of genes and proteins that are key regulators of osteoblast differentiation. These cells express Cbfa1 and c-fos transcription factors (TF) of osteoprogenitor proliferating cells. RT-PCR and immunohistochemistry were used to demonstrate the message and protein expression of extracellular matrix proteins that are a prerequisite for matrix formation and mineralization, including alkaline phosphatase (ALP), osteocalcin, osteopontin, biglycan, and bone sialoprotein (BSP). The activity of ALP was correlated at various cell densities with co-expression of osteocalcin or osteopontin. Adhering cells must attach to the appropriate matrix to enable survival and differentiation. Using attachment assays, we demonstrated that MBA-15 cells adhered to collagenous matrix and the effect on survival measured by changes in intracellular calcium (Ca) levels. The cells' adhesion to matrix is mediated via cell surface molecules. We quantified the expression of cells surface molecules that are important players in mediating cell-matrix interaction. Flow cytometry analysis (FACS) was used to determine the expression of CD-31 (36%), and lower levels were identified for CD-62E and CD11b. In summary, the present study demonstrates the expression of molecular markers that are distinctive for the osteoblastic phenotype in MBA-15 marrow stroma cells and have crucial role in cell-matrix interaction, in establishing the cellular osteogenic phenotype and their survival.
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PMID:Characterization of adhesion and differentiation markers of osteogenic marrow stromal cells. 1538 28

We investigated porcine dental follicle cells at the early crown-formation stage and examined the behavior of cells grown in a collagen type I (Col-I) matrix. Clone-porcine dental follicle cells (DFC-I) and controls, viz., dental follicle itself, nonclone-dental follicle cells, periodontal ligament cells (PDLC), and bone marrow stromal cells, were obtained from 6-month-old pigs. DFC-I showed a different gene expression pattern from controls by reverse-transcription polymerase chain reaction analysis. In addition, Col-I treatment enhanced DFC-I proliferation and increased their alkaline phosphatase activity compared with nontreated DFC-I. The expression of periostin, biglycan, and osteocalcin (OCN) in cells growing on collagen was upregulated, similar to the pattern seen in PDLC. DFC-I with and without Col-I treatment were combined with beta-tricalcium phosphate particles and implanted into immunodeficient mice. Significant differences were found in the gene expression patterns of bone sialoprotein, OCN, and periostin in both treated and non-treated implants at 2 and/or 4 weeks. The results showed that Col-I induced the mineralization pathway in these cells. Hard tissue formation was observed in both implant types at 8 weeks. Our results suggest that Col-I facilitates the differentiation of DFC-I along the mineralization process.
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PMID:Collagen type I matrix affects molecular and cellular behavior of purified porcine dental follicle cells. 1799 85

A prime pathogenic agent associated with periodontitis is lipopolysaccharide (LPS) derived from Porphyromonas gingivalis. This study investigated the effects of P. gingivalis LPS on osteoblasts, which are responsible for alveolar bone repair. Bone cells were obtained from explants of rat alveolar bone chips and cultured with 0-200 ng ml(-1) of P. gingivalis LPS. Porphyromonas gingivalis LPS significantly increased cell proliferation and inhibited osteoblast differentiation, as judged by reduced alkaline phosphatase activity. Analysis of biglycan mRNA and protein levels indicated that P. gingivalis LPS significantly delayed the normally high expression of biglycan during the early stages of culture, which are associated with cell proliferation and early differentiation of progenitor cells. In the presence of P. gingivalis LPS, decorin expression by the alveolar bone cells was reduced during periods of culture relating to collagen fibrillogenesis and mineral deposition. Analysis of glycosaminoglycan chains conjugated to these proteoglycans suggested that in the presence of P. gingivalis LPS, dermatan sulfate persisted within the matrix. This study suggests that P. gingivalis LPS influences the expression and processing of decorin and biglycan in the matrix, altering alveolar bone cell activity and osteoblast phenotype development. The consequences of this altered expression in relation to hindering bone repair as part of the cycle of events during periodontal disease are discussed.
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PMID:Lipopolysaccharide alters decorin and biglycan synthesis in rat alveolar bone osteoblasts: consequences for bone repair during periodontal disease. 1847 Dec 38

Small leucine-rich proteoglycans, such as biglycan, and their side chain sulfated glycosaminoglycans (GAGs), have been suggested to be involved in bone formation and mineralization processes. The present study was designed to investigate whether chondroitin sulfate (CS), one of the GAG, and its oversulfated structures coupled with bone morphogenetic protein-4 (BMP-4) alter the differentiation and subsequent mineralization of MC3T3-E1 osteoblastic cells. CS-E, one of the oversulfated CS structure, enhanced cell growth, alkaline phosphatase (ALP) activity, collagen deposition, and mineralization whereas heparin enhanced only ALP activity and mineralization. As well as CS-E, CS-H, and CPS also enhanced the mineralization of the cells. CS-E enhanced the mineralization of the cells by interacting with protein in the conditioned medium. CS-E induced mineralization was significantly inhibited by an antibody against BMP-4. The addition of exogenous BMP-4 further increased the capacity of CS-E to enhance mineralization. Fluorescence correlation spectroscopy method using fluoresceinamine-labeled GAG revealed that the oversulfated GAGs have a high affinity for BMP-4. The disaccharide analysis of the cells indicated that MC3T3-E1 cells are capable of producing oversulfated structures of CS by themselves. The lack of CS from the cells after chondroitinase treatment resulted in the inhibition of mineralization. These results in the present study indicate that oversulfated CS, which possesses 4,6-disulfates in N-acetyl-galactosamine, binds to BMP-4 and promotes osteoblast differentiation and subsequent mineralization.
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PMID:Oversulfated chondroitin sulfate-E binds to BMP-4 and enhances osteoblast differentiation. 1872 Mar 84


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