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)

To investigate the molecular mechanism underlying the differentiation of osteoblasts and chondroblasts, we established a clonal cell lines, RD-C6, from Runx2-deficient mouse embryos. RD-C6 cells expressed almost undetectable levels of phenotypes related to osteoblast and chondroblast differentiation at basal culture condition, whereas treatment with recombinant human bone morphogenetic protein-2 (rhBMP-2) or transduction of BMP-2 by adenovirus effectively induced this cell line to express mRNA related to the differentiation of osteoblasts and chondroblasts including alkaline phosphatase, osteocalcin, and osterix. Transduction of Runx2 also induced the expression of these mRNA in RD-C6 cells. BMP-2 transduction increased expression levels of mRNA for Msx2 and Dlx5, but Runx2 transduction induced no significant increases in expression levels of these mRNA. Microarray analysis using RD-C6 cells with or without rhBMP-2 treatment demonstrated that BMP-2 upregulated 66 genes including 13 transcription-related molecules such as Id1, Id2, Id4, Hey1, Smad6, Smad7, and Msx2. To confirm bone and cartilage formation ability of RD-C6 cells, we transplanted RD-C6 cells into the peritoneal cavity of athymic mice using diffusion chambers with rhBMP-2. RD-C6 cells generated unmineralized cartilage but not bone. These results indicate that BMP-2 induces Runx2-deficient cells to express markers related to osteoblast and chondroblast differentiation using a Runx2-independent pathway, but it failed to induce these cells to differentiate into bone-forming osteoblasts and mature chondrocytes.
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PMID:BMP-2 promotes differentiation of osteoblasts and chondroblasts in Runx2-deficient cell lines. 1722 53

The sequence of biochemical events involved in mechanical stress-induced signaling in osteoblastic cells remains unclear. Runx2, a transcription factor involved in the control of osteoblast differentiation, has been identified as a target of mechanical stress-induced signaling in osteoblastic cells. In this study, uniaxial sinusoidal stretching (15% strain, 115% peak-to-peak, at 1/12 Hz) stimulated the differentiation of osteoblast-like MC3T3-E1 cells and rat primary osteoblastic cells by activating Runx2. We examined the involvement of diverse mitogen-activated protein kinase (MAPK) pathways in the activation of Runx2 during mechanical stress. Mechanical stress increased alkaline phosphatase activity, a marker of osteoblast differentiation, increased the expression of the osteoblast-specific extracellular matrix (ECM) protein osteocalcin, and induced Runx2 activation, along with increased osterix expression. Furthermore, activation of ERK1/2 and p38 MAPKs increased significantly. U0126, a selective inhibitor of ERK1/2, completely blocked Runx2 activation during periods of mechanical stress, but the p38 MAPK-selective inhibitor SB203580 did not alter nuclear phosphorylation of Runx2. Small interfering RNA (siRNA) targeting Rous sarcoma kinase (RAS), an upstream regulator of both ERK1/2 and p38 MAPKs, inhibited stretch-induced ERK1/2 activation, but not mechanically induced p38 MAPK activity. Furthermore, mechanically induced Runx2 activation was inhibited by Ras depletion, using siRNA. These findings indicate that mechanical stress regulates Runx2 activation and favors osteoblast differentiation through the activation of MAPK signal transduction pathways and Ras/Raf-dependent ERK1/2 activation, independent of p38 MAPK signaling.
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PMID:Mechanical stress-mediated Runx2 activation is dependent on Ras/ERK1/2 MAPK signaling in osteoblasts. 1726 28

Apoptosis is a process important for the development and homeostasis of self-renewing tissues, including bone. However, little is known about the function of Bcl-2, a key player of apoptosis, in the regulation of osteoblast activity. Ex vivo cultures of osteoblasts from Col2.3Bcl-2 mice, in which human Bcl-2 was targeted to bone by the 2.3 kb fragment of the type I collagen promoter, were used to study the effect of Bcl-2 in osteoblasts. During 35 days of culture, hBcl-2 expression increased without any effect on endogenous mouse Bcl-2 and Bax expression. Adhesion of transgenic (TG) osteoblasts was twofold more than that of wild-type (WT) cells, with significantly higher expression of integrins alpha(1), alpha(2), and alpha(5) but similar levels of alpha(v) and beta(1) relative to WT cells. Proliferation of osteoblasts was not affected. Overexpression of hBcl-2 promoted the differentiation of osteoblasts, as shown by increased message levels of alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin in the TG compared to WT cells throughout the culture period. The two transcription factors essential for osteoblast differentiation, core binding factor alpha 1 (Cbfa-1) and osterix, had significantly higher expression in TG than WT cells during the early culture period. ss-Catenin, a central player in the canonical Wnt pathway, also had higher expression in TG than WT cultures. Mineralization was significantly decreased in TG cultures, with less osteoblast apoptosis, compared to WT. Thus, Bcl-2 seems to have multiple roles in modulating osteoblast activities.
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PMID:Bone-targeted overexpression of Bcl-2 increases osteoblast adhesion and differentiation and inhibits mineralization in vitro. 1730 93

Mechanical stress is considered to be an important factor in the progression of thoracic ossification of the ligament flavum (TOLF). To elucidate the mechanism underlying mechanical stress-induced TOLF, we investigated the effect of stretching on cultured flavum ligament cells derived from TOLF and non-TOLF patients. We found that the mRNA expression of alkaline phosphatase (ALP), osteocalcin, Runx2, and osterix, but not that of Dlx5 and Msx2, was significantly increased by stretching in TOLF cells. In addition, the effect seems to be finely tuned by stretching-triggered activation of distinct mitogen-activated protein kinase cascades. Specifically, a p38 specific inhibitor, SB203580, significantly inhibited stretching-induced osterix expression as well as ALP activity, whereas a specific inhibitor of ERK1/2, U0126, prevented stretching-induced Runx2 expression. We showed that overexpression of osterix resulted in a significant increase of ALP activity in TOLF cells, and osterix-specific RNAi completely abrogated the stretching-induced ALP activity, indicating that osterix plays a key role in stretching-stimulated osteogenic effect in TOLF cells. These results suggest that mechanical stress plays important roles in the progression of TOLF through induction of osteogenic differentiation of TOLF cells, and our findings support that osterix functions as a molecular link between mechanostressing and osteogenic differentiation.
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PMID:Osterix is a key target for mechanical signals in human thoracic ligament flavum cells. 1731 Dec 98

Osteoblasts, derived from pluripotent mesenchymal precursor cells, acquire their differentiated phenotypes under the control of a series of regulatory factors, the best known of which is BMP-2. Our recent preliminary studies suggest that expression of deltaEF1, a member of the zinc finger-homeodomain transcription factor family, is significantly down-regulated as human mesenchymal stem cells (MSCs) are subjected to osteoblastic differentiation in the presence of BMP-2. Here we demonstrate that overexpression of deltaEF1 in murine pre-myoblast C2C12 cells resulted in a decrease in the mRNA levels of early osteoblast marker genes induced by BMP-2 including osterix and collagen type I. This inhibitory effect was further confirmed by decreased alkaline phosphatase (ALP) activities. Neither of the zinc finger clusters of deltaEF1 is necessary for its repressive effect on BMP-2-induced osteoblastic differentiation of C2C12 cells. Immunoprecipitation results indicated that deltaEF1 did not physically associate with Smads proteins, suggesting that the inhibitory effect of deltaEF1 may be Smad-independent. deltaEF1 overexpression in C2C12 cells resulted in down-regulation of activating protein-1 (AP-1) activities promoted by BMP-2. Moreover, deltaEF1 exhibited transrepression on murine osteocalcin gene which effect is partially mediated through diminishing of AP-1 signaling. These results suggest that deltaEF1 acts as a potent inhibitor of BMP-2-induced osteogenesis in vitro, in part, by differentially regulating the AP-1 signaling pathway.
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PMID:deltaEF1 represses BMP-2-induced differentiation of C2C12 myoblasts into the osteoblast lineage. 1747 58

The transcription factor osterix (Osx) is a key regulator of osteoblast differentiation and induces bone formation in embryonic but not adult stem cells. We investigated the effect of up-regulating Osx on an intermediate stem cell type, first trimester fetal mesenchymal stem cells (MSCs), which are more expandable than adult MSCs. Human fetal (hf ) MSCs were transduced with a lentiviral vector encoding human Osx. In undifferentiating MSCs cultures, forced expression of Osx stimulated osteopontin and alkaline phosphatase expression. However, Osx did not up-regulate osteocalcin, a late marker of osteoblast differentiation or result in extracellular calcium crystals, indicating that Osx does not directly mediate terminal differentiation in primary hfMSCs. To understand the downstream effects of Osx expression in primary hfMSCs, we next investigated the regulatory relationship between Osx, and the transcription factors Dlx5, Runx2, and Msx2. Osx induced Dlx5 but did not affect Runx2 and Msx2, whereas stealth ribonucleic acid interference of Osx inhibited Dlx5 without affecting expression of Runx2 and Msx2. In conclusion, Osx regulates osteogenic gene expression in hfMSCs but is insufficient to induce terminal osteogenic differentiation.
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PMID:Osterix induces osteogenic gene expression but not differentiation in primary human fetal mesenchymal stem cells. 1751 20

There are increasing reports regarding differentiation of bone marrow stromal cells (BMSC) from human and various species of animals including pigs. The phenotype and function of BMSC along a mesenchymal lineage differentiation are well characterized by specific transcription factors and marker genes. However, it is not fully clear whether multilineage differentiation (osteogenesis, chondrogenesis, and adipogenesis) of BMSC is associated with a specific gene expression pattern. In the present study, we investigated the gene expression pattern of representative transcription factors and marker genes along those three mesenchymal lineages during a particular lineage differentiation of porcine BMSC by means of real-time PCR measurement. In an osteogenic medium, the mRNA levels of cbfa1, osterix, alkaline phosphatase, type 1 collagen, osteonectin, bone sialoprotein, and osteocalcin were induced stepwise. Meanwhile, sox9, specific to chondrogenic differentiation, was inhibited but not PPARgamma2 specific to adipogenic differentiation. In an adipogenic medium, adipogenic differentiation was confirmed by upregulation of PPARgamma2 and aP2 and downregulation of osteogenic genes and sox9. Chondrogenic differentiation was induced in cell pellet culture by expression of sox9, type 2 collagen, and aggrecan. Cbfa1 and PPARgamma2 were inhibited in chondrogenic medium. These results indicate that the differentiation potential of BMSC to a particular mesenchymal lineage relies upon specific gene expression pattern, namely upregulation of genes specific for this lineage and suppression of other lineage differentiation.
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PMID:Multilineage differentiation of porcine bone marrow stromal cells associated with specific gene expression pattern. 1767 6

Glycation reactions resulting in the generation and accumulation of advanced glycation endproducts (AGEs) are potential mechanisms by which bone protein may be altered in vivo. AGEs accumulate in the bone increasingly with age come into close contact with osteoblasts or osteoclasts. The direct effect of AGEs on bone cells has not been thoroughly investigated. This study aimed to examine whether glycated bovine serum albumin (AGE - BSA) as an AGE modulate the mRNA expression of various genes in primary human osteoblast cultures. The following parameters were included: RAGE (receptor for AGEs), alkaline phosphatase, osteocalcin, osterix and RANKL (receptor activator of nuclear factor-kappaB ligand). Primary human osteoblast cultures were obtained from bone specimens of six patients with osteoarthrosis. Human osteoblasts were treated in AGE - BSA or control-BSA (non-glycated BSA) containing medium (5 mg/ml each) over a time course of seven days. After RT-PCR the mRNA expression was measured by real-time PCR. Related to control - BSA exposure, the mRNA expression of RAGE, RANKL and osterix increased during AGE - BSA treament. For alkaline phosphatase and osteocalcin a tendency of down-regulation was found. In summary, the study presents evidence that advanced glycation end products accumulated in bone alter osteoblasts by activation the AGE - RAGE pathway (RAGE mRNA up-regulation), inducing enhanced osteoclastogenesis (RANKL mRNA up-regulation) and impaired matrix mineralization (down-regulation of alkaline phosphatase and osteocalcin mRNA). Thus, AGEs may play a functional role in the development of bone diseases (e.g. osteoporosis).
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PMID:Advanced glycation endproducts influence the mRNA expression of RAGE, RANKL and various osteoblastic genes in human osteoblasts. 1792 11

A 4 base-pair deletion mutation in the Distal-less 3 (DLX3) gene is etiologic for Tricho-Dento-Osseous syndrome (TDO). A cardinal feature of TDO is an increased thickness and density of bone. We tested the effects of the DLX3 gene mutation responsible for TDO on the osteoblastic differentiation of preosteoblastic MC3T3E1 cells and multipontent mesenchymal C2C12 cells. Differential expression analysis of C2C12 cells transfected with wild type DLX3 or mutant DLX3 was performed and desmin gene expression, an early myoblastic differentiation marker in mesenchymal cells, was evaluated by RT-PCR, western blot analysis, and desmin promoter transcriptional activity. Transfection of wild type DLX3 into MC3T3E1 and C2C12 cells increased alkaline phosphatase-2 activity, mineral deposition, and promoter activities of the osteocalcin and type 1 collagen genes compared to empty vector transfected cells. Transfection of mutant DLX3 into these cells further enhanced alkaline phosphatase activity, mineral deposition, and osteocalcin promoter activities, but did not further enhance type 1 collagen promoter activity. Transfection of mutant DLX3 into C2C12 cells markedly down regulated desmin gene expression, and protein expression of desmin and MyoD, while increasing protein expression of osterix and Runx2. These results demonstrate that the DLX3 deletion mutation associated with TDO enhances mesenchymal cell differentiation to an osteoblastic lineage rather than a myoblastic lineage by changing the fate of mesenchymal cells. This DLX3 mutation also accelerates the differentiation of osteoprogenitor cells to osteoblasts at later stages of osteogenesis.
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PMID:A 4 bp deletion mutation in DLX3 enhances osteoblastic differentiation and bone formation in vitro. 1795 Jun 83

For bone regeneration constructs using human jaw periosteal cells (JPC) the extent of osteoinductive ability of different three-dimensional scaffolds is not yet established. We analyzed open-cell polylactic acid (OPLA) scaffolds for their suitability as bone engineering constructs using human JPC. Cell adhesion and spreading was visualized on the surface of scaffolds by scanning electron microscopy. JPC proliferation within OPLA scaffolds was compared with proliferation within collagen and calcium phosphate scaffolds. We found a significant increase of proliferation rates in OPLA scaffolds versus Coll/CaP scaffolds at three time points. Live-measurements of oxygen consumption within the cell-seeded scaffolds indicate that the in vitro culturing time should not exceed 12-15 days. OPLA scaffolds, which were turned out to be the most beneficial for JPC growth, were chosen for osteogenic differentiation experiments with or without BMP-2. Gene expression analyses demonstrated induction of several osteogenic genes (alkaline phosphatase, osterix, Runx-2 and insulin-like growth factor) within the 3D-scaffolds after 12 days of in vitro culturing. Element analysis by EDX spectrometry of arising nodules during osteogenesis demonstrated that JPC growing within OPLA scaffolds are able to form CaP particles. We conclude that OPLA scaffolds provide a promising environment for bone substitutes using human JPC.
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PMID:Analysis of OPLA scaffolds for bone engineering constructs using human jaw periosteal cells. 1815 13


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