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)

In order to explore further the regulatory factors to the potentiality in inducing osteogenesis by fibroblasts, the fibroblasts were isolated, and purified from human skin, and were grown in incubation in the media of EGF, IL-6, TNF-alpha and BMP2 at different concentrations for two weeks, then, the markers for osteogenic features were investigated by biochemistry, histochemistry and electron microscopic observations. It was found that the combined use of TNF-alpha and BMP2 could stimulate fibroblasts to secrete alkaline phosphatase, osteocalcin and collagen, and the morphological changes of the fibroblasts were also very striking. In the extracellular matrix, the collagen fibrils, with or without periodicity, were arranged regularly or randomly oriented, and numerous minute calcium granules were interspersed among them. The fibroblasts were interwoven one on top of another in the form of multilayer structure and on the surface, there were secreting granules and piling up of calcium crystals which coalessed steadily and increased in size in forming bony nodules. It was considered that TNF-alpha and BMP2 were capable of inducing the fibroblasts to form bone.
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PMID:[Regulatory factors of osteogenic phenotypical experession by fibroblasts in vitro]. 1043 77

It has been well established that bone morphogenetic protein-2 (BMP-2) can induce bone formation both in vivo and in vitro, although high concentrations (up to milligrams) of BMP-2 have been required to achieve this effect in vivo. Further, clinical applications are usually limited to a single dose at the time of implantation. In an attempt to prolong the transforming effect of BMP-2 we used a recombinant adenoviral vector carrying the human BMP-2 gene (Adv-BMP2) to transduce marrow-derived mesenchymal stem cells (MSC) of skeletally mature male New Zealand white rabbits. The pluripotential MSC were incubated with Adv-BMP2 overnight followed by culture in growth medium for 1 week. Assays on tissue cultures demonstrated that these Adv-BMP2 transduced MSC produced BMP-2 protein, differentiated into an osteoprogenitor line, and induced bone formation in vitro. These MSC had increased alkaline phosphatase activity, increased expression of type I collagen, osteopontin, and osteocalcin mRNA, and induced matrix mineralization compared with both non-transduced cells and cells transduced with a control adenoviral construct. To analyze the osteogenic potential in vivo, Adv-BMP2-transduced MSC were autologously implanted into the intertransverse process space between L5 and L6 of the donor rabbits. The production of new bone was demonstrated by radiographic examination 4 weeks later in areas implanted with cells transduced with Adv-BMP2, whereas no bone was evident at sites implanted with cells transduced with the control adenoviral construct. Histological examination further confirmed the presence of new bone formation. These accumulated data indicate that it is possible to successfully transduce mesenchymal stem cells with a recombinant adenoviral vector carrying the gene for BMP-2 such that these cells will produce BMP-2, differentiate into an osteoprogenitor line, and induce bone formation both in vitro and in vivo. Moreover, incubation of the Adv-BMP2-transduced cells for an additional 7 days in culture before transplantation enhances the success rate in bone formation (three out of three) as compared with our previous report (one out of five, Calcif Tissue Int 63:357-360, 1998).
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PMID:In vitro and in vivo induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene. 1131 Mar 52

We sought to develop a retroviral vector system that would produce secretion of high levels of bone morphogenetic protein (BMP)-4 by optimizing the expression construct and developing an improved retroviral vector. Replacement of the propeptide domain of BMP4 with that of BMP2 increased the secretion level of mature BMP4 protein in transduced cells. The intact BMP2 pro-peptide sequence was essential, as deletion of a small part of the propeptide sequence of BMP2 from the BMP2/4 hybrid construct diminished BMP4 expression and secretion. Addition of a hemaglutinin tag to the carboxy terminus of BMP4 abolished the bioactivity of secreted BMP4. Transduction of rat marrow stromal cells (and fibroblasts) with an MFG-based retroviral vector pseudotyped with VSV-G envelope containing this BMP2/4 hybrid expression construct led to secretion of very high levels of mature BMP4 in conditioned medium (up to 1 microg/10(6) cells/24 hours). The secreted BMP4 was biologically active, as it induced alkaline phosphatase expression in C2C12 cells. The transduced rat marrow stromal cells expressing mature BMP4 induced de novo ectopic bone formation in syngenic immune-competent rats. We have developed an MFG-based retroviral vector system that causes secretion of high levels of functionally active human BMP4 protein.
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PMID:Development of an MFG-based retroviral vector system for secretion of high levels of functionally active human BMP4. 1148 80

Increasing evidence suggests that morphogenesis of the distinct developmental structures derived from the same organ-committed epithelium is controlled by differential mechanisms. As was recently shown in mice with mutations in the downless (dL) gene, induction of primary or tylotrich hair follicles is strikingly dependent of signaling through the Tnf receptor homologue, Edar. Here, we show that dorsal skin of murine embryos with constitutive deletion of the BMP2/4 antagonist noggin, after transplantation into SCID mice, is characterized by the lack of induction of secondary hair follicles, and by the arrest of primary hair follicle development prior to hair shaft formation. The loss of noggin activity was associated with failure to express genes that specify hair follicle cell fates in the epidermis (Lef-1, beta-catenin, Shh) and dermal papilla (p75 kDa neurotrophin receptor, alkaline phosphatase). This suggests that regulation of BMP2/4 signaling by noggin is essential for the induction of secondary hair follicles, as well as for advanced stages of development in primary hair follicles.
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PMID:Modulation of BMP signaling by noggin is required for induction of the secondary (nontylotrich) hair follicles. 1185 69

Bone morphogenetic protein (BMP) signaling regulates body axis determination, apoptosis, and differentiation of various types of cells including neuron, gut, and bone cells. However, the molecules involved in such BMP regulation of biological events have not been fully understood. Here, we examined the involvement of Cas-interacting zinc finger protein (CIZ) in the modulation of BMP2-induced osteoblastic cell differentiation. CIZ overexpression in osteoblastic MC3T3E1 cells suppressed BMP2-enhanced expression of alkaline phosphatase, osteocalcin, and type I collagen genes. Upstream analyses revealed that CIZ overexpression also suppressed BMP2-induced enhancement of the mRNA expression of Cbfa1, which is a critical transcription factor for osteoblastic differentiation. BMP-induced Smad1 and Smad5 activation of GCCG-mediated transcription was blocked in the presence of CIZ overexpression. CIZ overexpression alone in the absence of BMP2 moderately enhanced basal levels of Cbfa1 mRNA expression. CIZ overexpression also enhanced 1.8-kb Cbfa1 promoter activity in the absence of BMP2, whereas it suppressed the promoter activity in the presence of BMP2. Finally, CIZ overexpression suppressed the formation of mineralized nodules in osteoblastic cell cultures. These data indicate that CIZ is a novel type inhibitor of BMP/Smad signaling.
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PMID:Negative regulation of bone morphogenetic protein/Smad signaling by Cas-interacting zinc finger protein in osteoblasts. 1202 67

Wnt glycoproteins are important regulators of cellular differentiation and embryonic development. Some Wnt proteins induce stabilization of beta-catenin which cooperatively regulates gene expression with LEF/Tcf transcription factors. Here we demonstrate a direct role for beta-catenin signaling in osteoblast differentiation and in BMP2-mediated signal transduction. Similar to treatment with BMP-2 protein, ectopic expression of stabilized beta-catenin in C3H10T1/2 cells or activation of endogenous beta-catenin signaling with LiCl induces expression of alkaline phosphatase mRNA and protein, a defined marker of early osteoblast differentiation. Unlike BMP2 protein, stabilized beta-catenin does not induce osteocalcin gene expression, a marker of late osteoblast differentiation. BMP2-induced differentiation also leads to activation of endogenous beta-catenin signaling thus implicating beta-catenin in early steps of BMP2-mediated osteoblast differentiation. Effects of beta-catenin and BMP2 on C3H10T1/2 differentiation are not completely overlapping, implying that some aspects of BMP2-induced differentiation may be mediated by beta-catenin signaling and that beta-catenin can also participate in non-BMP2-dependent differentiation processes.
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PMID:Activated beta-catenin induces osteoblast differentiation of C3H10T1/2 cells and participates in BMP2 mediated signal transduction. 1253 44

Daidzein, a natural isoflavonoid found in Leguminosae, has received increasing attention because of its possible role in the prevention of osteoporosis. In the present investigation, primary osteoblastic cells isolated from newborn Wistar rats were used to investigate the effect of this isoflavonoid on osteoblasts. Daidzein (2-50 microM) increased the viability (P<0.05) of osteoblasts by about 1.4-fold. In addition, daidzein (2-100 microM) increased the alkaline phosphatase activity and osteocalcin synthesis (P<0.05) of osteoblasts by about 1.4- and 2.0-fold, respectively. Alkaline phosphatase and osteocalcin are phenotypic markers for early-stage differentiated osteoblasts and terminally differentiated osteoblasts, respectively. Our results indicated that daidzein stimulated osteoblast differentiation at various stages (from osteoprogenitors to terminally differentiated osteoblasts). We also investigated the effect of daidzein on bone morphogenetic protein (BMP) production in osteoblasts that display the mature osteoblast phenotype. The results indicated that BMP2 synthesis was elevated significantly in response to daidzein (the mRNA increased 5.0-fold, and the protein increased 7.0-fold), suggesting that some of the effects of daidzein on the cell may be mediated by the increased production of BMPs by the osteoblasts. In conclusion, daidzein has a direct stimulatory effect on bone formation in cultured osteoblastic cells in vitro, which may be mediated by increased production of BMPs in osteoblasts.
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PMID:Daidzein enhances osteoblast growth that may be mediated by increased bone morphogenetic protein (BMP) production. 1262 84

Bone regeneration requires interactions between a number of factors including bone morphogenetic proteins (BMPs), growth factors, and transcriptional regulators such as Runx2/Cbfal (Runx2). Because each component may provide a unique contribution to the overall osteogenic response, we hypothesized that bone formation may be enhanced by using combinations of complimentary factors. As an initial test of this concept, interactions between BMP2 and Runx2 were examined using adenovirus-based expression vectors (AdCMV-Runx2, AdCMV-BMP2) in the pluripotent C3H10T1/2 cell line. Cells transduced with AdCMV-Runx2 strongly expressed osteoblast markers, such as alkaline phosphatase and osteocalcin, but formed only a weakly mineralized extracellular matrix in vitro, whereas cells transduced with AdCMV-BMP2 exhibited higher levels of mineralization, but only expressed low levels of Runx2 and osteocalcin mRNA. Significantly, when cells were transduced with optimal titers of both viruses, osteoblast differentiation was stimulated to levels that were 10-fold greater than those seen with either AdCMV-Runx2 or AdCMV-BMP2 alone. To measure in vivo osteogenic activity, virally transduced cells were subcutaneously implanted into immunodeficient mice. Cells transduced with control virus produced only fibrous tissue while those with AdCMV-Runx2 produced limited amounts of both cartilage and bone. In contrast, cells transduced with either AdCMV-BMP2 alone or AdCMV-BMP2 plus AdCMV-Cbfal generated large ossicles containing cartilage, bone, and a marrow cavity. However, ossification in the AdCMV-BMP2 plus AdCMV-Cbfal group was more extensive in that both mineral content and fractional bone area were greater than that seen in the AdCMV-BMP2 group. Thus, the increased osteoblast differentiation observed with combined adenovirus treatment in vitro is also manifested by increased bone formation in vivo. These results suggest that Runx2 and BMP2 have distinct, but complementary, roles in osteogenesis and that their combined actions may be necessary for optimal bone formation.
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PMID:In vitro and in vivo synergistic interactions between the Runx2/Cbfa1 transcription factor and bone morphogenetic protein-2 in stimulating osteoblast differentiation. 1267 31

Previous reports have suggested that bone morphogenetic protein (BMP) gene therapy could be applied for in vivo bone regeneration. However, these studies were conducted either using immunodeficient animals because of immunogenicity of adenovirus vectors, or using ex vivo gene transfer technique, which is much more difficult to handle. Adeno-associated virus (AAV) is a replication-defective virus without any association with immunogenicity and human disease. This study was conducted to investigate whether orthotopic new bone formation could be induced by in vivo gene therapy using AAV-based BMP2 vectors. To test the feasibility of this approach, we constructed an AAV vector carrying human BMP2 gene. Mouse myoblast cells (C2C12) transduced with this vector could produce and secrete biologically active BMP2 protein and induce osteogenic activity, which was confirmed by ELISA and alkaline phosphatase activity assay. For in vivo study, AAV-BMP2 vectors were directly injected into the hindlimb muscle of immunocompetent Sprague-Dawley rats. Significant new bone under X-ray films could be detected as early as 3 weeks postinjection. The ossification tissue was further examined by histological and immunohistochemical analysis. This study is, to our knowledge, the first to establish the feasibility of AAV-based BMP2 gene therapy for endochondral ossification in immunocompetent animals.
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PMID:Gene therapy for new bone formation using adeno-associated viral bone morphogenetic protein-2 vectors. 1288 31

Chondrocyte differentiation is a fundamental process during endochondral ossification. Several factors regulate maturation via the activity of downstream signaling pathways that target specific transcription factors and regulate chondrocyte-specific genes. In this study, we investigated the mechanisms involved in the regulation of chick lower sternal chondrocyte maturation upon stimulation by retinoic acid (RA) and the bone morphogenetic protein BMP2. RA-induced Runx2 in lower sternal chondrocyte cultures and over-expression of wild-type (WT) Runx2 enhanced colX and alkaline phosphatase activity, while over-expression of dominant negative Runx2 was inhibitory. Furthermore, WT Runx2 enhanced the effects of both BMP2 and RA on colX expression, while the effects of both growth factors were completely blocked in cultures over-expressing dominant negative Runx2. Similarly, WT Runx2 enhanced the induction of colX by Smad1. Smad1 and Runx2 were found to act cooperatively at the chicken type X collagen promoter and elimination of either the putative Smad binding site or Runx2 binding site eliminated responsiveness to BMP2, RA, or either of the transcription factors. Altogether the results show cross talk between the BMP-associated Smads and Runx2 during chondrocyte differentiation and dependence upon both signals for induction of the type X collagen promoter. Factors or signals that alter either of these transcription factors regulate the rate of chondrocyte differentiation.
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PMID:Runx2/Cbfa1 stimulation by retinoic acid is potentiated by BMP2 signaling through interaction with Smad1 on the collagen X promoter in chondrocytes. 1463


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