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)

Cells of the craniofacial skeleton are derived from a common mesenchymal progenitor. The regulatory factors that control their differentiation into various cell lineages are unknown. To investigate the biological function of dentin matrix protein 1 (DMP1), an extracellular matrix gene involved in calcified tissue formation, stable transgenic cell lines and adenovirally infected cells overexpressing DMP1 were generated. The findings in this paper demonstrate that overexpression of DMP1 in pluripotent and mesenchyme-derived cells such as C3H10T1/2, MC3T3-E1, and RPC-C2A can induce these cells to differentiate and form functional odontoblast-like cells. Functional differentiation of odontoblasts requires unique sets of genes being turned on and off in a growth- and differentiation-specific manner. The genes studied include transcription factors like core binding factor 1 (Cbfa1), bone morphogenetic protein 2 (BMP2), and BMP4; early markers for extracellular matrix deposition like alkaline phosphatase (ALP), osteopontin, osteonectin, and osteocalcin; and late markers like DMP2 and dentin sialoprotein (DSP) that are expressed by terminally differentiated odontoblasts and are responsible for the formation of tissue-specific dentin matrix. However, this differentiation pathway was limited to mesenchyme-derived cells only. Other cell lines tested by the adenoviral expression system failed to express odontoblast-phenotypic specific genes. An in vitro mineralized nodule formation assay demonstrated that overexpressed cells could differentiate and form a mineralized matrix. Furthermore, we also demonstrate that phosphorylation of Cbfa1 (osteoblast-specific transcription factor) was not required for the expression of odontoblast-specific genes, indicating the involvement of other unidentified odontoblast-specific transcription factors or coactivators. Cell lines that differentiate into odontoblast-like cells are useful tools for studying the mechanism involved in the terminal differentiation process of these postmitotic cells.
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PMID:Differentiation of embryonic mesenchymal cells to odontoblast-like cells by overexpression of dentin matrix protein 1. 1128 60

Full-length cDNA coding for dentin matrix protein 1 (DMP1) has been cloned and sequenced, but the corresponding complete protein has not been isolated. In searching for naturally occurring DMP1, we recently discovered that the extracellular matrix of bone contains fragments originating from DMP1. Shortened forms of DMP1, termed 37K and 57K fragments, were treated with alkaline phosphatase and then digested with trypsin. The resultant peptides were purified by a two-dimensional method: size exclusion followed by reversed-phase high performance liquid chromatography. Purified peptides were sequenced by Edman degradation and mass spectrometry, and the sequences compared with the DMP1 sequence predicted from cDNA. Extensive sequencing of tryptic peptides revealed that the 37K fragments originated from the NH2-terminal region, and the 57K fragments were from the COOH-terminal part of DMP1. Phosphate analysis indicated that the 37K fragments contained 12 phosphates, and the 57K fragments had 41. From 37K fragments, two peptides lacked a COOH-terminal lysine or arginine; instead they ended at Phe173 and Ser180 and were thus COOH termini of 37K fragments. Two peptides were from the NH2 termini of 57K fragments, starting at Asp218 and Asp222. These findings indicated that DMP1 is proteolytically cleaved at four bonds, Phe173-Asp174, Ser180-Asp181, Ser217-Asp218, and Gln221-Asp222, forming eight fragments. The uniformity of cleavages at the NH2-terminal peptide bonds of aspartyl residues suggests that a single proteinase is involved. Based on its reported specificity, we hypothesize that these scissions are catalyzed by PHEX protein. We envision that the proteolytic processing of DMP1 plays a crucial role during osteogenesis and dentinogenesis.
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PMID:Evidence for the proteolytic processing of dentin matrix protein 1. Identification and characterization of processed fragments and cleavage sites. 1281 42

Dental pulp progenitor/stem cells have the capacity to differentiate into odontoblasts and they provide a potential for dentin repair and regeneration by gene therapy. To develop a successful ex vivo gene therapy to induce reparative dentin formation rapidly and effectively after treatment of caries, we developed a three-dimensional pellet culture system of pulp cells electrotransfected with growth/differentiation factor 11 (Gdf11). The viability after electrotransfection was more than 85%, and the efficiency was about 70% as determined by flow cytometry. After 10 days of culture, the total amount of type I and type III collagen was 3-fold higher in the pEGFP-Gdf11-transfected pellet than in the control. Real-time RT-PCR analysis demonstrated that the expression of markers of odontoblast differentiation (alkaline phosphatase, dentin matrix protein 1 [Dmp1], dentin sialophosphoprotein [Dspp], enamelysin, and phosphate-regulating gene with homologies to endopeptidases on X-chromosome [Phex]) was increased in the pEGFP-Gdf11-transfected pellet compared with the control on day 14. On the basis of this in vitro evaluation, an in vivo investigation in the dog was performed. Autogenous transplantation of Gdf11-transfected cells cultured as a pellet on amputated pulp stimulated reparative dentin formation. Thus, Gdf11 gene therapy may be potentially used in endodontic treatment in dentistry.
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PMID:Stimulation of reparative dentin formation by ex vivo gene therapy using dental pulp stem cells electrotransfected with growth/differentiation factor 11 (Gdf11). 1561 Jun 5

The osteocyte is the most abundant cell type in bone and is embedded in mineralized bone matrix. Osteocytes are still poorly characterized because of their location and the lack of primary osteocyte isolation methods. Data on the cell biology of osteocytes is especially limited. We have isolated primary osteocytes from rat cortical bone by applying repeated enzymatic digestion and decalcification. The isolated osteocytes expressed typical osteocytic morphology with cell-cell contacts via long protrusions after a 1-day culture. These cells were negative or faintly positive for alkaline phosphatase but expressed high levels of osteocalcin, PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome), and DMP1 (dentin matrix protein 1). These cells also revealed patchy membrane staining for connexin43. For studying the function of gap junctions in isolated osteocytes, we microinjected rhodamine-labeled dextran (MW: 10,000) and Lucifer yellow (MW: 457) and found that Lucifer yellow was rapidly transmitted to several surrounding cells, whereas dextran remained in the injected cells. Heptanol and 18alpha-glycyrrhetinic acid inhibited the transfer of Lucifer yellow. This clearly showed the existence of functional gap junctions in cultured osteocytes. Enveloped viruses, such as vesicular stomatitis virus and influenza A virus, were used for studying cell polarity. We were unable to demonstrate plasma membrane polarization with enveloped viruses in isolated primary osteocytes in culture. Our results suggest that osteocytes do not possess apical and basolateral plasma membrane domains as do osteoblasts, which are their precursors.
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PMID:Isolated primary osteocytes express functional gap junctions in vitro. 1617 87

We investigated gene expression patterns and functional classifications regarding the clusters of human dental pulp stem cells (hDPSCs) and human mesenchymal stem cells (hMSCs)--which possess a multipotent ability--because little is known about the precise moleculobiological clues by which these cells activate their differentiating ability or functionality to eventually form dentin and bone, respectively. We first verified the expressions of the alkaline phosphatase (ALP) gene, dentin matrix protein 1 (DMP-1), and dentinsialophosphoprotein (DSPP) by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and consequently discovered the high expressions of these genes. Total RNA was also followed by hybridization with a human microarray system consisting of 12,814 genes. Analyses of gene expression patterns indicated several genes which encode extracellular matrix components, cell adhesion molecules, growth factors, and transcription regulators. Functional and clustering analyses of differences in gene expression levels revealed cell signaling, cell communication, or cell metabolism. In the future, information on the gene expression patterns of hDPSCs and hMSCs might be useful in determining the detailed functional roles of the relevant genes and applicable to stem cell therapies, and these cells could also be used as multipotent cell sources for gene technology and tissue engineering technology.
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PMID:Cluster analysis and gene expression profiles: a cDNA microarray system-based comparison between human dental pulp stem cells (hDPSCs) and human mesenchymal stem cells (hMSCs) for tissue engineering cell therapy. 1656 96

Investigations of the odontoblast phenotype are hindered by obstacles such as the limited number of odontoblasts within the dental pulp and the difficulty in purification of these cells. Therefore, it is necessary to develop a cell culture system in which the local environment is inductive and can promote dental pulp stem cells (DPSCs) to differentiate into odontoblast lineage. In this study, we investigated the effect of conditioned medium from developing tooth germ cells (TGCs) on the differentiation and dentinogenesis of DPSCs both in vitro and in vivo. DPSCs were enzymatically isolated from the lower incisors of 4-week-old Sprague-Dawley rats and co-cultured with TGC conditioned medium (TGC-CM). The cell phenotype of induced DPSCs presents many features of odontoblasts, as assessed by the morphologic appearance, cell cycle modification, increased alkaline phosphatase level, synthesis of dentin sialoprotein, type I collagen and several other noncollagenous proteins, expression of the dentin sialophosphoprotein and dentin matrix protein 1 genes, and the formation of mineralized nodules in vitro. The induced DPSC pellets in vivo generated a regular-shaped dentin-pulp complex containing distinct dentinal tubules and predentin, while untreated pellets spontaneously differentiated into bone-like tissues. To our knowledge, this is the first study to mimic the dentinogenic microenvironment from TGCs in vitro, and our data suggest that TGC-CM creates the most odontogenic microenvironment, a feature essential and effective for the regular dentinogenesis mediated by DPSCs.
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PMID:Differentiation of dental pulp stem cells into regular-shaped dentin-pulp complex induced by tooth germ cell conditioned medium. 1751 25

Hypophosphatemic transgenic (tg) mice overexpressing FGF23 in osteoblasts display disorganized growth plates and reduced bone mineral density characteristic of rickets/osteomalacia. These FGF23 tg mice were used as an in vivo model to examine the relation between osteoclast polarization, secretion of proteolytic enzymes and resorptive activity. Tg mice had increased mRNA expression levels of the osteoblast differentiation marker Runx2 and mineralization-promoting proteins alkaline phosphatase and bone sialoprotein in the long bones compared to wild type (wt) mice. In contrast, expression of alpha1(I) collagen, osteocalcin, dentin matrix protein 1 and osteopontin was unchanged, indicating selective activation of osteoblasts promoting mineralization. The number of osteoclasts was unchanged in tg compared to wt mice, as determined by histomorphometry, serum levels of TRAP 5b activity as well as mRNA expression levels of TRAP and cathepsin K. However, tg mice displayed elevated serum concentrations of C-terminal telopeptide of collagen I (CTX) indicative of increased bone matrix degradation. The majority of osteoclasts in FGF23 tg mice lacked ultrastructural morphological signs of proper polarization. However, they secreted both cathepsin K and MMP-9 at levels comparable to osteoclasts with ruffled borders. Mineralization of bone matrix thus appears essential for inducing osteoclast polarization but not for secretion of osteoclast proteases. Finally, release of CTX by freshly isolated osteoclasts was increased on demineralized compared to mineralized bovine bone slices, indicating that the mineral component limits collagen degradation. We conclude that ruffled borders are implicated in acidification and subsequent demineralization of the bone matrix, however not required for matrix degradation. The data collectively provide evidence that osteoclasts, despite absence of ruffled borders, effectively participate in the degradation of hypomineralized bone matrix in rachitic FGF23 tg mice.
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PMID:Osteoclast polarization is not required for degradation of bone matrix in rachitic FGF23 transgenic mice. 1834 51

Poly(vinyl alcohol) (PVA) has been widely used in the field of biomedical applications because of its hydrophilic properties for desired functions. Nonetheless, the role of PVA in tooth germ (TG) cell differentiation and mineralization has seldom been explored. To test the capacity of PVA in regulating TG cell differentiation and mineralization, TG cells obtained from 4-day-old Wistar rats were cultured on the PVA substrate. It was found that PVA was able to promote TG cell exhibiting high levels of alkaline phosphatase (ALP) activity, mineralization, and mRNA expression of osteocalcin (OCN), osteopontin (OPN), dentin matrix protein 1 (DMP1) and enamelin. Even when the additives routinely administrated in the differentiation medium such as dexamethasone, beta-glycerophosphate and ascorbic acid were removed from the culture system, PVA itself still stimulated TG cells with the differentiation and mineralization ability. By showing the direct suppression of extracellular signaling-regulated kinase1/2 (ERK1/2) of TG cells treated with U0126, known to suppress the activation of ERK1/2, and significant synergistic effects between PVA and U0126, we demonstrated the suppression of ERK1/2 pathway is one of the effects of PVA-promoted TG cell differentiation and mineralization. Taken together, this study demonstrated a novel role of PVA in promoting the differentiation and mineralization of TG cells through ERK1/2 acting as a negative regulator.
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PMID:Induction of differentiation and mineralization in rat tooth germ cells on PVA through inhibition of ERK1/2. 1900 Jun 37

The purpose of this study was to evaluate the behaviors of rat tooth germ (TG) cells cultured on poly(vinyl alcohol) (PVA). It was found that TG cells suspended and aggregated to form three-dimensional spheroids on PVA. Compared with traditional monolayered cells on tissue culture polystyrene, TG cell spheroids on PVA obviously increased the alkaline phosphatase activity, the degree of mineralization, and upregulated both osteopontin and dentin matrix protein 1 genes, regardless of the seeding density. Surprisingly, PVA appears to activate the alkaline phosphatase activity and mineralization effects on TG cell spheroids in the absence of a differentiation medium. Furthermore, the present study indicates that integrins may play an important role in the mineralization on TG cell spheroids by adding Arg-Gly-Asp (RGD) peptides. Therefore, the information presented here should help to clarify the role of PVA in the regulation of the mineralization, differentiation and integrin-mediation of TG cells.
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PMID:The behavior of rat tooth germ cells on poly(vinyl alcohol). 1913 19

Wnt11 signals through both canonical (beta-catenin) and non-canonical pathways and is up-regulated during osteoblast differentiation and fracture healing. In these studies, we evaluated the role of Wnt11 during osteoblastogenesis. Wnt11 overexpression in MC3T3E1 pre-osteoblasts increases beta-catenin accumulation and promotes bone morphogenetic protein (BMP)-induced expression of alkaline phosphatase and mineralization. Wnt11 dramatically increases expression of the osteoblast-associated genes Dmp1 (dentin matrix protein 1), Phex (phosphate-regulating endopeptidase homolog), and Bsp (bone sialoprotein). Wnt11 also increases expression of Rspo2 (R-spondin 2), a secreted factor known to enhance Wnt signaling. Overexpression of Rspo2 is sufficient for increasing Dmp1, Phex, and Bsp expression and promotes bone morphogenetic protein-induced mineralization. Knockdown of Rspo2 abrogates Wnt11-mediated osteoblast maturation. Antagonism of T-cell factor (Tcf)/beta-catenin signaling with dominant negative Tcf blocks Wnt11-mediated expression of Dmp1, Phex, and Rspo2 and decreases mineralization. However, dominant negative Tcf fails to block the osteogenic effects of Rspo2 overexpression. These studies show that Wnt11 signals through beta-catenin, activating Rspo2 expression, which is then required for Wnt11-mediated osteoblast maturation.
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PMID:Wnt11 promotes osteoblast maturation and mineralization through R-spondin 2. 1921 27


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