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)

The Runx2/Cbfa1 transcription factor is a scaffolding protein that promotes osteoblast differentiation; however, the specific Runx2-functional domains required for induction of the osteogenic lineage remain to be identified. We approached this question using a TERT-immortalized cell line derived from calvaria of Runx2-null mice by reconstituting the osteogenic activity with wild-type and deletion mutants of Runx2. The presence or absence of osteogenic media (beta-glycerol phosphate and ascorbic acid) and/or with BMP2 did not stimulate osteoblastic gene expression in the Runx2-null cells. However, cells infected with wild-type Runx2 adenovirus showed a robust temporal increase in the expression of osteoblast marker genes and were competent to respond to BMP2. Early markers (i.e., collagen type-1, alkaline phosphatase) were induced (four- to eightfold) at Days 4 and 8 of culture. Genes representing mature osteoblasts (e.g., Runx2, osteopontin, bone sialoprotein, osteocalcin) were temporally expressed and induced from 18- to 36-fold at Days 8 and 12. Interestingly, TGFbeta and Vitamin D-mediated transcription of osteoblast genes (except for osteopontin) required the presence of Runx2. Runx2 lacking the C-terminal 96 amino acids (Runx2 Delta432) showed a pattern of gene expression similar to wild-type protein, demonstrating the Groucho interaction and part of the activation domain are dispensable for Runx2 osteogenic activity. Upon further deletion of the Runx2 C-terminus containing the nuclear matrix targeting signal and Smad-interacting domain (Delta391), we find none of the osteoblast markers are expressed. Therefore, the Runx2 391-432 domain is essential for execution of the BMP2 osteogenic signal.
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PMID:Reconstitution of Runx2/Cbfa1-null cells identifies a requirement for BMP2 signaling through a Runx2 functional domain during osteoblast differentiation. 1692 9

Flavonoids, which have been detected in a variety of foods, have been repeatedly reported to affect bone metabolism. However, the effects of flavonoids on osteoblastogenesis remain a matter of some controversy. In this study, the effects of quercetin on the differentiation and proliferation of human adipose tissue-derived stromal cells (hADSC) were determined. Quercetin was found to increase osteogenic differentiation in a dose-dependent manner. Other flavonoids, chrysin and kaempferol, were also shown to increase the osteogenic differentiation of hADSC, but this stimulatory effect was weaker than that associated with quercetin. Quercetin pretreatment administered prior to the induction of differentiation also exerted stimulatory effects on the osteogenic differentiation of hADSC. RT-PCR and real time PCR analysis showed that quercetin treatment induced an increase in the expression of osteopontin, BMP2, alkaline phosphatase and Runx2. Quercetin inhibited the proliferation of hADSC, but did not affect their survival. The pretreatment of quercetin increased ERK phosphorylation during osteogenic differentiation, although it did not increase ERK activity in control culture condition. ICI182780, an specific estrogen receptor antagonist, failed to inhibit the effects of quercetin on osteogenic differentiation. Quercetin-pretreated hADSC showed better bone regenerating ability in skull defect model of nude mice than naive cells. Our findings indicate that quercetin enhances osteogenic differentiation via an independent mechanism from estrogen receptor (ER) activation, and prove useful for in vivo bone engineering, using human mesencymal stem cells (hMSC).
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PMID:Quercetin, a flavonoid, inhibits proliferation and increases osteogenic differentiation in human adipose stromal cells. 1699 34

Several homeodomain (HD) proteins are critical for skeletal patterning and respond directly to BMP2 as an early step in bone formation. RUNX2, the earliest transcription factor proven essential for commitment to osteoblastogenesis, is also expressed in response to BMP2. However, there is a gap in our knowledge of the regulatory cascade from BMP2 signaling to the onset of osteogenesis. Here we show that BMP2 induces DLX3, a homeodomain protein that activates Runx2 gene transcription. Small interfering RNA knockdown studies in osteoblasts validate that DLX3 is a potent regulator of Runx2. Furthermore in Runx2 null cells, DLX3 forced expression suffices to induce transcription of Runx2, osteocalcin, and alkaline phosphatase genes, thus defining DLX3 as an osteogenic regulator independent of RUNX2. Our studies further show regulation of the Runx2 gene by several homeodomain proteins: MSX2 and CDP/cut repress whereas DLX3 and DLX5 activate endogenous Runx2 expression and promoter activity in non-osseous cells and osteoblasts. These HD proteins exhibit distinct temporal expression profiles during osteoblast differentiation as well as selective association with Runx2 chromatin that is related to Runx2 transcriptional activity and recruitment of RNA polymerase II. Runx2 promoter mutagenesis shows that multiple HD elements control expression of Runx2 in relation to the stages of osteoblast maturation. Our studies establish mechanisms for commitment to the osteogenic lineage directly through BMP2 induction of HD proteins DLX3 and DLX5 that activate Runx2, thus delineating a transcriptional regulatory pathway mediating osteoblast differentiation. We propose that the three homeodomain proteins MSX2, DLX3, and DLX5 provide a key series of molecular switches that regulate expression of Runx2 throughout bone formation.
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PMID:BMP2 commitment to the osteogenic lineage involves activation of Runx2 by DLX3 and a homeodomain transcriptional network. 1706 Mar 21

HOXA10 is necessary for embryonic patterning of skeletal elements, but its function in bone formation beyond this early developmental stage is unknown. Here we show that HOXA10 contributes to osteogenic lineage determination through activation of Runx2 and directly regulates osteoblastic phenotypic genes. In response to bone morphogenic protein BMP2, Hoxa10 is rapidly induced and functions to activate the Runx2 transcription factor essential for bone formation. A functional element with the Hox core motif was characterized for the bone-related Runx2 P1 promoter. HOXA10 also activates other osteogenic genes, including the alkaline phosphatase, osteocalcin, and bone sialoprotein genes, and temporally associates with these target gene promoters during stages of osteoblast differentiation prior to the recruitment of RUNX2. Exogenous expression and small interfering RNA knockdown studies establish that HOXA10 mediates chromatin hyperacetylation and trimethyl histone K4 (H3K4) methylation of these genes, correlating to active transcription. HOXA10 therefore contributes to early expression of osteogenic genes through chromatin remodeling. Importantly, HOXA10 can induce osteoblast genes in Runx2 null cells, providing evidence for a direct role in mediating osteoblast differentiation independent of RUNX2. We propose that HOXA10 activates RUNX2 in mesenchymal cells, contributing to the onset of osteogenesis, and that HOXA10 subsequently supports bone formation by direct regulation of osteoblast phenotypic genes.
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PMID:HOXA10 controls osteoblastogenesis by directly activating bone regulatory and phenotypic genes. 1732 44

The aim of the present report was to test a system for controlled release of recombinant human bone morphogenic protein (rhBMP2) incorporated into polylactic acid (PLA) implants. Incorporation of rhBMP2 into the polymer was accomplished by mixing rhBMP2 solution with granular powder of amorphous poly-DL-lactic acid, subsequent lyophilization, and high pressure CO(2) treatment at 100 bar. Porous cylindrical implants of 8 mm diameter and 3 mm thickness were fabricated with 100, 200, 400, and 800 microg BMP2/g polymer and submitted to in vitro testing. Polymer degradation was assessed during immersion of PLA implants into PBS for 176 days by measuring the inherent viscosity at days 0, 99, and 131. BMP2 release was evaluated by immersion of both the lyophilized powder and the implants into cell culture medium for up to 27 days. BMP2 release was assessed using a custom made ELISA. The biological activity of the released growth factor was determined by measuring the induction of alkaline phosphatase (AP) in C2C12 cells. There was a significant retardation in the release of BMP2 from the implants compared to the granular powder. Detectable amounts of BMP2 were found for all concentrations of BMP2 until the end of the observation period. Significant induction of AP was detected by BMP released from the implants after 3, 6, and 9 days. The present in-vitro study has shown that incorporation of rhBMP2 into PLA implants with subsequent slow release of biologically active growth factor is possible.
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PMID:In vitro characterization of a slow release system of polylactic acid and rhBMP2. 1747 90

BMP6 is a member of TGF-beta superfamily, represent more effective osteogenic activity. Two recombinant plasmids were constructed to expression rhBMP6 in mammalian cells, one contained the cDNA encoding the signal peptide, propeptide and mature peptide of human BMP6, wich was named pcDNA-BMP6, the other contained the recombinant DNA encoding the signal peptide, propeptide of human BMP2 and the mature peptide of BMP6, which was named pcDNA-BMP2/6. Transient expression in Cos7 cells demonstrated that the pcDNA-BMP2/6 produced more rhBMP6 than pcDNA-BMP6. For stable expression, the CHO-dhfr- cells were transfected with pcDNA-BMP2/6 and pSV2-dhfr, then screened by G418 and treated with MTX for targeting gene amplification. The partially purified rhBMP6 by heparin affinity chromatography was shown to possess bone induction activity tested by the induction of alkaline phosphatase activity in C2C12 cells.
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PMID:[Expression of recombinant human BMP6 in CHO cells by fused to the signal peptide and propeptide of another homologue protein]. 1757 85

Dental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenic potential.Because bone morphogenetic proteins (BMPs) are well-established agents to induce bone and dentin formation,in this study STRO-1-selected rat dental pulp-derived stem cells were transfected with the adenoviral mediated human BMP-2 gene. Subsequently, the cells were evaluated for their odontogenic differentiation ability in medium not containing dexamethasone or other stimuli. Cultures were investigated using light microscopy and scanning electron microscopy (SEM) and evaluated for cell proliferation, alkaline phosphatase(ALP) activity, and calcium content. Real-time polymerase chain reaction (PCR) was performed for gene expression of Alp, osteocalcin, collagen type I, bone sialoprotein, dentin sialophosphoprotein, and dentin matrix acidic phosphoprotein 1. Finally, an oligo-microarray was used to profile the expression of odontogenesis-related genes. Results of ALP activity, calcium content, and real-time PCR showed that only BMP2-transfected cells had the ability to differentiate into the odontoblast phenotype and to produce a calcified extracellular matrix. SEM and oligo-microarray confirmed these results. In contrast, the non-transfected cells represented a less differentiated cell phenotype. Based on our results, we concluded that the adenovirus can transfect STRO-1 selected cells with high efficacy. After BMP2 gene transfection, these cells had the ability to differentiate into odontoblast phenotype, even without the addition of odontogenic supplements to the medium.
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PMID:STRO-1 selected rat dental pulp stem cells transfected with adenoviral-mediated human bone morphogenetic protein 2 gene show enhanced odontogenic differentiation. 1782 31

Activins and bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta family of growth and differentiation factors that induce signaling in target cells by assembling type II and type I receptors at the cell surface. Ligand residues involved in type II binding are located predominantly in the C-terminal region that forms an extended beta-sheet, whereas residues involved in type I binding are located in the alpha-helical and preceding loop central portion of the molecule. To test whether the central residues are sufficient to determine specificity toward type I receptors, activin A/BMP chimeras were constructed in which the central residues (45-79) of activin A were replaced with corresponding residues of BMP2 and BMP7. The chimeras were assessed for activin type II receptor (Act RII) binding, activin-like bioactivity, and BMP-like activity as well as antagonistic properties toward activin A and myostatin. ActA/BMP7 chimera retained Act RII binding affinity comparable with wild type activin A, whereas ActA/BMP2 chimera showed a slightly reduced affinity toward Act RII. Both the chimeras were devoid of significant activin bioactivity in 293T cells in the A3 Lux reporter assay up to concentrations 10-fold higher than the minimal effective activin A concentration (approximately 4 nM). In contrast, these chimeras showed BMP-like activity in a BRE-Luc assay in HepG2 cells as well as induced osteoblast-like phenotype in C2C12 cells expressing alkaline phosphatase. Furthermore, both the chimeras activated Smad1 but not Smad2 in C2C12 cells. Also, both the chimeras antagonized ligands that signal via activin type II receptor, such as activin A and myostatin. These data indicate that activin residues in the central region determine its specificity toward type I receptors. ActA/BMP chimeras can be useful in the study of receptor specificities and modulation of transforming growth factor-beta members, activins, and BMPs.
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PMID:Activin A/bone morphogenetic protein (BMP) chimeras exhibit BMP-like activity and antagonize activin and myostatin. 1805 65

The development is expected of scaffold biomaterials that feature a shape-maintaining property in addition to high porosity and large pores that cells can easily invade. To develop a new biodegradable scaffold biomaterial reinforced with a frame, synthesized carbonate apatite (CO3Ap) was mixed with neutralized collagen gel, and the CO3Ap-collagen mixtures were lyophilized into sponges in a porous hydroxyapatite (HAp) frame ring. X-ray diffraction and Fourier transform infrared spectroscopy (FT-IR) analyses together with chemical analysis indicated that the synthesized CO3Ap had a crystalline nature and a chemical composition similar to that of bone. Scanning electron microscope (SEM) observation showed that the CO3Ap-collagen sponge had a sui pore size for cell invasion. In proliferation and differentiation experiments with osteoblasts, alkaline phosphatase and osteopontin activity were clearly detected. When these sponge-frame complexes with bone morphogenic protein (rh-BMP2) were implanted beneath the periosteum cranii of rats, significant new bone was created at the surface of the periosteum cranii after 4 weeks of implantation. These reinforced CO3Ap-collagen sponges with rh-BMP2 are expected to be used as hard tissue scaffold biomaterials for the therapeutic purpose of the rapid cure of bone defects.
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PMID:Acceleration of bone formation with BMP2 in frame-reinforced carbonate apatite-collagen sponge scaffolds. 1807 50

Since efficient transfer of foreign genes into primary articular chondrocytes (CC) is difficult, a VSV.G pseudotyped retroviral vector (Bullet) was developed for marker and growth factor gene transfer. Transduction efficiency was analysed by FACS. BMP2 production was determined by specific hBMP2-ELISA. BMP2 effect on cells regarding proteoglycan production was measured by alcian blue staining and dye quantification. Alkaline phosphatase activity was determined by enzymatic reaction with p-nitrophenyl phosphate at OD 405nm and proliferation rate was analysed by MTT-assay. ATDC5 cells (98.3+/-0.6%SD) were transduced to express the reporter gene eGFP. After 52 weeks 94.7+/-0.6%SD of cells were positive. Retroviral transduction efficiency for nlslacZ exceeded 92.3+/-6.1%SD in rabbit CC and expression remained high after 15 weeks (75.7+/-14.2%SD). ATDC5 cells and CC expressed the growth factor gene hBMP2 after retroviral transduction at different time-points. BMP2 led to an increase in proteoglycan and alkaline phosphatase production. Initially, the proliferation rate detected by MTT-assay increased in both the cell types; afterwards the proliferation rate was similar to controls. The described retroviral vector system achieved high initial transduction rates in ATDC5 cells and CC. Gene transfer was very stable over the time period analysed, rendering it a useful tool for future in vitro and in vivo studies on cartilage remodelling.
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PMID:Efficient and stable gene transfer of growth factors into chondrogenic cells and primary articular chondrocytes using a VSV.G pseudotyped retroviral vector. 1807 87


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