Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bone morphogenetic proteins (BMPs) were originally identified by their ability to induce ectopic bone formation and have been shown to promote both chondrogenesis and chondrocyte hypertrophy. BMPs have recently been found to activate a membrane serine/threonine kinase signaling mechanism in a variety of cell types, but the downstream effectors of BMP signaling in chondrocyte differentiation remain unidentified. We have previously reported that BMP-2 markedly stimulates type X collagen expression in prehypertrophic chick sternal chondrocytes, and that type X collagen mRNA levels in chondrocytes cultured under serum-free (SF) conditions are elevated 3- to 5-fold within 24 h. To better define the molecular mechanisms of induction of chondrocyte hypertrophy by BMPs, we examined the effect of BMPs on type X collagen production by 15-day chick embryo sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP-2, BMP-4, or BMP-7. Two populations of chondrocytes were used: one representing resting cartilage isolated from the caudal third of the sterna and the second representing prehypertrophic cartilage from the cephalic third of the sterna. BMP-2, BMP-4, and BMP-7 all effectively promoted chondrocyte maturation of cephalic sternal chondrocytes as measured by high levels of alkaline phosphatase, diminished levels of type II collagen, and induction of the hypertrophic chondrocyte-specific marker, type X collagen. To test whether BMP control of type X collagen expression occurs at the transcriptional level, we utilized plasmid constructs containing the chicken collagen X promoter and 5' flanking regions fused to a reporter gene. Constructs were transiently transfected into sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP-2, BMP-4, or BMP-7. A 533 bp region located 2.4-2.9 kb upstream from the type X collagen transcriptional start site was both necessary and sufficient for strong BMP responsiveness in cells destined for hypertrophy, but not in chondrocytes derived from the lower sterna.
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PMID:A BMP responsive transcriptional region in the chicken type X collagen gene. 978 40

Bone morphogenetic proteins (BMPs) induce the differentiation of cells of the osteoblastic lineage and enhance the function of the osteoblast. Growth factors are regulated by binding proteins, but there is no information about binding proteins for BMPs in skeletal cells. Noggin specifically binds BMPs, but its expression by cells of the osteoblastic lineage has not been reported. We tested for the expression of noggin and its induction by BMP-2 in cultures of osteoblast-enriched cells from 22-d-old fetal rat calvariae (Ob cells). BMP-2 caused a time- and dose-dependent increase in noggin mRNA and polypeptide levels, as determined by Northern and Western blot analyses. The effects of BMP-2 on noggin transcripts were dependent on protein, but independent of DNA synthesis. BMP-2 increased the rates of noggin transcription as determined by nuclear run-on assays. BMP-4, BMP-6, and TGF-beta1 increased noggin mRNA in Ob cells, but basic fibroblast growth factor, platelet- derived growth factor BB, and IGF-I did not. Noggin decreased the stimulatory effects of BMPs on DNA and collagen synthesis and alkaline phosphatase activity in Ob cells. In conclusion, BMPs induce noggin transcription in Ob cells, a probable mechanism to limit BMP action in osteoblasts.
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PMID:Bone morphogenetic proteins induce the expression of noggin, which limits their activity in cultured rat osteoblasts. 985 46

Bone tissue has been shown to contain numerous cell-to-cell signalling peptides called growth factors. These growth factors are thought to have important regulating effects for bone remodeling and bone healing, due to their potent effects on bone cell metabolism. In vivo studies over the last half decade have demonstrated that growth factors candidates for future clinical use in orthopedic surgery. In numerous clinical situations enhanced bone formation and bone healing could lead to improved results of surgical procedures. This thesis describes the most important bone growth factors and their actions in vitro and in vivo. In vitro investigations of growth factor effects on osteoblast chemotaxis and metabolism are described as well as in vivo studies with growth factor stimulation of fracture healing and bone healing to prosthetic-like implants. In vitro results: Several growth factors exhibited chemotactic effects towards human osteoblasts. TGF-beta 1 and PDGF-BB had the strongest chemotactic effects, whereas PDGF-AA, IGF-1, and IGF-2 had less but significant chemotactic effects towards human osteoblasts. TGF-beta 1 exhibited the highest chemotactic potency with maximal activity at 100 pg/mL, whereas the other growth factors had maximal effects at 10-100 ng/mL. BMP-2 was found to have chemotactic effects toward human osteoblasts, human bone marrow osteoprogenitor cells, and U2-OS osteosarcoma cells. BMP-4 and BMP-6 were without any chemotactic effects towards these celltypes. Human bone marrow osteoprogenitor cells were the most responsive celltype to BMP-2 stimulation. Growth factor combinations resulted in synergic stimulative effects on different metabolic functions on human osteoblasts. Combinations with TGF-beta 1 and PDGF-BB strongly stimulated proliferation and chemotaxis. Combinations with TGF-beta 1, PDGF-BB and BMP-2 strongly stimulated an osteoblast differentiation parameter (alkaline phosphatase activity). The different growth factor combinations had no effect on collagen synthesis in human osteoblasts. In vivo results: Continuous application of 1 and 10 micrograms natural TGF-beta to a plated tibial osteotomy in rabbits increased mechanical bending strength and callus formation at 6 weeks observation. Diaphyseal cortical bone remodeling was not affected by the local growth factor application. In a dog model with unloaded implants surrounded by a gap, 0.3 microgram rhTGF-beta 1 adsorbed to gritblasted tricalcium phosphate coated implants, was able to enhance mechanical fixation, bone ingrowth and gap bone formation. 3.0 micrograms rhTGF-beta 1 had less but significant stimulative effect. In a weight-loaded model, 0.3 microgram rhTGF-beta 1, adsorbed to gritblasted tricalcium phosphate coated implants, was able to enhance bone ingrowth, without enhancement of mechanical fixation. In the unloaded model, 0.3 microgram rhTGF-beta 1, adsorbed to gritblasted hydroxyapatite coated implants, was able to enhance bone ingrowth, without enhancement of mechanical fixation. 3.0 micrograms rhTGF-beta 1 had no stimulative effects. The establishment of a biological implant fixation concept with growth factor absorbed to ceramic coatings of implants was successful. These data are promising for a possible future clinical usage of growth factors, especially for enhancement of bone healing to cementless prosthetic components.
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PMID:Growth factor stimulation of bone healing. Effects on osteoblasts, osteomies, and implants fixation. 985 74

To investigate the role of bone morphogenetic protein (BMP-2) in ossifying rat bone marrow stromal cell cultures, we determined the population of fibroblast-like stromal cells that expressed BMP-2 immunocytochemically (anti-rhBMP-2 monoclonal antibody), and compared that to alkaline phosphatase (AP) and collagen synthesis formed in culture over a 4-week period in control and dexamethasone-supplemented mineralizing media. In control media, the percentage of BMP-2-positive stromal cells (BMP-2(+)) increased from 12 to 25% within the first 4 days of culture. In mineralizing media, the level of BMP-2(+) cells was significantly increased (43-44%). The intensity of immunostaining gradually increased with time. The levels of AP were undetectable at 1 week in both control and mineralizing media, but increased gradually over the next 2 weeks and peaked at 3 weeks. ALP levels were significantly greater in cultures grown in mineralizing medium (P < 0.05 at 3 weeks, P < 0.01 at 4 weeks). Collagen synthesis peaked and was significantly greater at 3 weeks (P < 0.05) in cultures grown in mineralizing medium. The levels of AP and collagen synthesis most closely reflected the changes in the percentage of BMP-2(+) cells from 7 to 28 days. Though these changes may reflect a primary action of BMP-2 on marrow osteoprogenitor-like stromal cells, they do not exclude a mechanism that involves the induction of other members of the BMP family known to stimulate AP and collagen synthesis. We conclude that BMP-2 expression in cultures of fibroblast-like marrow stromal cells is enhanced when those cells are induced to become osteoblasts by exposure to dexamethasone.
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PMID:Expression of BMP-2 by rat bone marrow stromal cells in culture. 986 86

Bone morphogenetic protein (BMP), a member of the transforming growth factor superfamily, is one of the most potent growth factors that stimulate osteoblast differentiation and bone formation. We investigated the effects of recombinant human BMP-2 (rhBMP-2) on osteoblast differentiation and matrix metalloproteinase-1 (MMP-1) production in human bone cells (HBC) isolated from mandibulae of 3 adult patients. rhBMP-2 at concentrations over 50 ng/ml significantly stimulated alkaline phosphatase activity and parathyroid hormone (PTH)-dependent 3', 5'-cyclic adenosine monophosphate accumulation, which are early markers of osteoblast differentiation, in HBCs. rhBMP-2 (500 ng/ml) also enhanced the level of PTH/PTH related-peptide receptor mRNA expression in HBCs. Although neither HBCs untreated nor treated with rhBMP-2 produced measurable amounts of osteocalcin, which is a marker of more mature osteoblasts, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced ostocalcin mRNA expression and its protein synthesis in these cells. rhBMP-2 inhibited 1,25(OH)2D3-induced osteocalcin synthesis in HBCs at both the mRNA and protein level. rhBMP-2 also significantly suppressed MMP-1 production and MMP-1 mRNA expression at concentrations over 500 ng/ml. These results suggest that rhBMP-2 exerts anabolic effects on human osteoblastic cells derived from mandibulae by stimulation of osteoblast differentiation and down-regulation of MMP-1 synthesis.
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PMID:Recombinant human bone morphogenetic protein-2 stimulates osteoblast differentiation and suppresses matrix metalloproteinase-1 production in human bone cells isolated from mandibulae. 987 21

Bone morphogenetic proteins (BMPs) are factors that promote osteoblastic cell differentiation and osteogenesis. It is unknown whether BMPs may act on human osteoblastic cells by increasing immature cell growth and/or differentiation. We investigated the short- and long-term effects of recombinant human (rh)BMP-2 on cell growth and osteoblast phenotype in a new model of human neonatal pre-osteoblastic calvaria cells (HNC). In short-term culture, rhBMP-2 (20-100 ng/ml) inhibited DNA synthesis and increased alkaline phosphatase (ALP) activity without affecting osteocalcin (OC) production. When cultured for 3 weeks in the presence of ascorbic acid and inorganic phosphate to induce cell differentiation, HNC cells initially proliferated, type 1 collagen mRNA and protein levels rose, and then decreased, whereas OC mRNA and protein levels, and calcium accumulation into the extracellular matrix increased at 2 to 3 weeks. A transient treatment with rhBMP-2 (50 ng/ml) for 1 to 7 days which affected immature HNC cells, decreased cell growth, increased ALP activity and mRNA, and induced cells to express ALP, osteopontin, and OC at 7 days, as shown by immunocytochemistry. At 2 to 3 weeks, matrix mineralization was markedly increased despite cessation of treatment, and although OC and Col 1 mRNA and protein levels were not changed. A continuous treatment with rhBMP-2 for 3 weeks which affected immature and mature cells reduced cell growth, increased ALP activity and mRNA at 1 week and increased OC mRNA and protein levels and calcium content in the matrix at 3 weeks, indicating complete osteoblast differentiation. These results indicate that the differentiating effects of BMP-2 on human neonatal calvaria are dependent on duration of exposure. Although long-term exposure led to complete differentiation of OC-synthesizing osteoblasts, the primary effect of rhBMP-2 was to promote osteoblast marker expression in immature cells, which was sufficient to induce optimal matrix mineralization independently of cell growth and type 1 collagen expression.
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PMID:Effects of bone morphogenetic protein-2 on human neonatal calvaria cell differentiation. 1002 69

BMP-3b (also called GDF-10) is a novel BMP-3-related protein recently discovered in rat femur tissue. Gene expression of BMP-3b in osteoblastic cells and its regulation by prolonged culture, BMP-2 and transforming growth factor beta1 (TGF-beta1) were examined. The BMP-3b gene was highly expressed in rat osteoblasts obtained from calvarial bones but not in the osteoblastic cell lines (MC3T3-E1 and U2-OS). BMP-3b mRNA increased during osteoblastic differentiation in prolonged culture and was associated with increased alkaline phosphatase (ALPase) activity. When BMP-2, an enhancer of ALPase activity, was added to the primary osteoblast culture, BMP-3b mRNA increased 6.9-fold after 24 h. In contrast, TGF-beta1 treatment, which suppresses ALPase activity, rapidly and completely inhibited gene expression of BMP-3b. The regulation of BMP-3 mRNA differed from that of BMP-3b, even though both proteins share 81% identity. These findings indicate that BMP-3b gene expression is regulated by osteoblastic differentiation and BMP-3b functions in highly differentiated osteoblasts.
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PMID:Bone morphogenetic protein-3b (BMP-3b) gene expression is correlated with differentiation in rat calvarial osteoblasts. 1007

The bone morphogenetic protein (BMP)-2 is a potent osteoinductive signal, inducing bone formation in vivo and osteoblast differentiation from non-osseous cells in vitro. The runt domain-related protein Cbfa1/PEBP2alphaA/AML-3 is a critical component of bone formation in vivo and transcriptional regulator of osteoblast differentiation. To investigate the relationship between the extracellular BMP-2 signal, Cbfa1, and osteogenesis, we examined expression of Cbfa1 and osteoblastic genes during the BMP-2 induced osteogenic transdifferentiation of the myoblastic cell line C2C12. BMP-2 treatment completely blocked myotube formation and transiently induced expression of Cbfa1 and the bone-related homeodomain protein Msx-2 concomitant with loss of the myoblast phenotype. While induction of collagen type I and alkaline phosphatase (AP) expression coincided with Cbfa1 expression, Cbfa1 mRNA was strikingly downregulated at the onset of expression of osteopontin (OPN) and osteocalcin (OCN) genes, reflecting the mature osteoblast phenotype. TGF-beta1 treatment effectively suppressed myogenesis and induced Cbfa1 expression but was insufficient to support osteoblast differentiation reflected by the absence of ALP, OPN, and OCN. We addressed whether induction of Cbfa1 in response to BMP-2 results in the transcriptional activation of the OC promoter which contains three enhancer Cbfa1 elements. Transfection studies show BMP-2 suppresses OC promoter activity in C2C12, but not in osteoblastic ROS 17/2.8 cells. Maximal suppression of OC promoter activity in response to BMP-2 requires sequences in the proximal promoter (up to nt -365) and may occur independent of the three Cbfa sites. Taken together, our results demonstrate a dissociation of Cbfa1 expression from development of the osteoblast phenotype. Our findings suggest that Cbfal may function transiently to divert a committed myoblast to a potentially osteogenic cell. However, other factors induced by BMP-2 appear to be necessary for complete expression of the osteoblast phenotype.
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PMID:Transient upregulation of CBFA1 in response to bone morphogenetic protein-2 and transforming growth factor beta1 in C2C12 myogenic cells coincides with suppression of the myogenic phenotype but is not sufficient for osteoblast differentiation. 1008 30

Chondrogenic differentiation of mesenchymal cells is generally thought to be initiated by the inductive action of specific growth factors and depends on intimate cell-cell interactions. In this study, we have used multipotential murine C3H10T1/2 cells to analyze the effect and mechanism of action of bone morphogenetic protein 2 (BMP-2) on chondrogenesis. C3H10T1/2 cells have been previously shown to undergo multiple differentiation pathways. While chondrogenesis, osteogenesis, myogenesis and adipogenesis have been observed, chondrocytes appear significantly less frequently than the other cell types, and the appearance of chondrocytes exclusive of the other cell types has not been observed. We report here that the appearance of chondrocytes in C3H10T1/2 cells is markedly enhanced as a result of culture under conditions favorable for chondrogenesis, i.e. plating as high-density micromass and treatment with BMP-2. Such cultures contain chondrocyte-like cells, elaborate an Alcian blue stained cartilage-like matrix, express link protein and type II collagen, both cartilage matrix markers, and show increased [35S]sulfate incorporation. The appearance of Alcian blue positive material and increased sulfate incorporation are dependent on the dose of BMP-2, culture time, and cell plating density of the micromass cultures. Differentiation of cells within the micromass was specific to the chondrogenic lineage, as alkaline phosphatase staining revealed only faint staining in the micromass at the highest BMP-2 concentration. The importance of enhanced cell-cell interaction in the chondroinductive effects of BMP-2 on high-density C3H10T1/2 cultures was further implicated by the additional promotion of chondrogenesis in the presence of the polycationic compound, poly-L-lysine, which has been previously reported to enhance cellular interactions and chondrogenesis in embryonic limb mesenchymal cells. Taken together, these findings suggest that chondrogenesis in C3H10T1/2 cells is inducible by BMP-2 and requires cell-cell interaction.
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PMID:Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: I. Stimulation by bone morphogenetic protein-2 in high-density micromass cultures. 1023 4

Studies in several cell types indicate that the actions of integrin receptors for extracellular matrix and receptors for growth factors are synergistic in regulating cellular differentiation and function. We studied the roles of the alpha1beta1 and alpha2beta1 integrin collagen receptors in regulating the differentiation of 2T3 osteoblastic cells in response to bone morphogenetic protein (BMP)-2. The immortalized 2T3 cell line was established from the calvaria of mice transgenic for a BMP-2 promoter driving SV40 T-antigen. These cells require exogenous BMP-2, as well as ascorbic acid and beta-glycerolphosphate, for expression of a mature osteoblast phenotype and formation of a mineralized matrix. To determine how integrin receptors for collagen-I affect BMP-2 signaling, function-perturbing anti-rat alpha1 and/or alpha2 integrin subunit, or anti-type I collagen (Col-I), antibodies were added to human recombinant (hr)BMP-2-treated 2T3 cultures at confluence (C0) or at 4 or 8 days postconfluence (C4, C8). After 4 days of exposure to the antibodies, cultures were assayed for alkaline phosphatase (ALP) mRNA levels and enzyme activity and for cAMP production in response to parathyroid hormone. Addition of anti-collagen-I or both anti-integrin-alpha1 and -alpha2 antibodies to C0 cultures blocked expression of these early osteoblast markers by more than 90%, and also blocked mineralization (0.5-1.8% control) of these cells. In all cases, adding anti-alpha1 or anti-alpha2 antibodies separately produced partial effects, while their combined effect approached that of anti-collagen-I. When antibodies were added to more differentiated 2T3 cells, the inhibitory effects decreased. 2T3 cells carrying constitutively active BMP receptor (caBMPR-IB) showed elevated ALP activity without hrBMP-2; this constitutive activity was also suppressed by alpha1 and alpha2 integrin antibodies and by anti-Col-I antibody. Together, our data suggest that a signal(s) from collagen integrin receptors regulates the response to BMP downstream of BMPR-IB and upstream of the regulation of ALP mRNA and other early markers of osteoblast differentiation.
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PMID:Collagen integrin receptors regulate early osteoblast differentiation induced by BMP-2. 1040 7


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