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)

Recent studies of intracellular signal transduction mechanisms for the transforming growth factor-beta (TGF-beta) superfamily have focused on Smad proteins, but have paid little attention to mitogen-activated protein (MAP) kinase cascades. Here we demonstrate that growth/differentiation factor-5 (GDF-5), but neither bone morphogenetic protein-2 (BMP-2) nor TGF-beta1, fully promotes the early phase of the chondrogenic response by inducing cellular condensation followed by cartilage nodule formation in a mouse chondrogenic cell line, ATDC5. We investigated which, if any, of the three major types of MAP kinase plays a functional role in the promotion of chondrogenesis induced by GDF-5. GDF-5 induced phosphorylation of p38 MAP kinase and extracellular signal-regulated kinase (ERK) but not that of c-Jun N-terminal kinase (JNK). The phosphorylation of p38 MAP kinase was also induced by BMP-2 and TGF-beta1. An inhibitor of p38 and p38 beta MAP kinase, SB202190, showed complete inhibition of cartilage nodule formation but failed to affect alkaline phosphatase (ALP) activity induced by GDF-5. Expression of the type II collagen gene, a hallmark of chondrogenesis in vertebrates, was also induced by GDF-5 treatment and strongly suppressed by SB202190. On the other hand, although an inhibitor of MAP/ERK kinase, PD98059, inhibited the rapid phosphorylation of ERK by GDF-5, it inhibited neither ALP activity nor cartilage nodule formation induced by GDF-5. These results strongly suggest that the p38 MAP kinase cascade is involved in GDF-5 signaling pathways and that a role of the p38 MAP kinase pathway is necessary over a longer period to promote chondrogenesis in ATDC5 cells.
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PMID:p38 mitogen-activated protein kinase functionally contributes to chondrogenesis induced by growth/differentiation factor-5 in ATDC5 cells. 1041 89

We have developed a method to form reconstituted mineralized articular cartilagenous tissue in vitro from isolated deep zone chondrocytes. The aim of this study was to characterize further these cultures prior to and during mineralization. Histologic examination of the cells up to 8 days in culture showed that the chondrocytes had formed cartilagenous tissue. Similar to the in vivo cartilage, the chondrocytes expressed aggrecan, types II, I, and X collagens, osteopontin, and alkaline phosphatase (ALP). No osteocalcin mRNA expression was detected in either the in vivo cartilage or in vitro-generated tissue. Addition of beta-glycerophosphate (beta-GP) to the medium on day 5 induced mineralization and changes in gene expression. Expression of type X collagen, type II collagen, aggrecan core protein, and ALP were inhibited significantly between 2 h and 24 h after the addition of beta-GP. At 72 h, expression of these genes were still significantly depressed. These changes correlated with a decrease in collagen and proteoglycan synthesis, and ALP activity. Osteopontin expression increased within 8 h but returned to constitutive levels by 72 h. No change in type I collagen expression was detected. The changes in gene expression were not due to a direct effect of beta-GP itself, because similar gene changes occurred in the presence of phosphoethanolamine, another agent which induces mineralization. No changes in gene expression were seen in nonmineralizing cultures. In summary, articular chondrocytes grown on filter culture show expression of similar genes to the chondrocytes in the deep zone of articular cartilage and that changes in expression of specific genes were observed during tissue mineralization, suggesting that it is a suitable model to use to study the mechanism(s) regulating the localized mineralization of articular cartilage.
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PMID:Deep zone articular chondrocytes in vitro express genes that show specific changes with mineralization. 1057 92

Phenotypic expression of chondrocytes can be modulated in vitro by changing the culture technique and by agents such vitamins and growth factors. We studied the effects of ascorbic acid, retinoic acid (0.5 and 10 microM), and dihydrocytochalasin B (3, 10, 20 microM DHCB), separately or in combination (ascorbic acid + retinoic acid or ascorbic acid + DHCB), on the induction of maturation of fetal bovine epiphyseal chondrocytes grown for up to 4 weeks at high density in medium containing 10% fetal calf serum and the various agents. In the absence of any agent or with retinoic acid or DHCB alone, the metabolic activity of the cells remained very low after day 6, with no induction of type I or X collagen synthesis nor increase in alkaline phosphatase activity. Chondrocytes treated with fresh ascorbic acid showed active protein synthesis associated with expression of types I and X after 6 and 13 days, respectively. This maturation was not accompanied by obvious hypertrophy of the cells or high alkaline phosphatase activity. Addition of retinoic acid to the ascorbic acid-treated cultures decreased the level of type II collagen synthesis and delayed the induction of types I and X collagen, which were present only after 30 days. A striking increase in alkaline phosphatase activity (15-20-fold) was observed in the presence of both ascorbic acid and the highest dose of retinoic acid (10 microM). DHCB was also a potent inhibitor of the maturation induced by treatment with ascorbic acid, as the chondrocytes maintained their rounded shape and synthesized type II collagen without induction of type I or X collagen. The pattern of protein secretion was compared under all culture conditions by two-dimensional gel electrophoresis. The different regulations of chondrocyte differentiation by ascorbic acid, retinoic acid, and DHCB were confirmed by the important qualitative and quantitative changes in the pattern of secreted proteins observed by two-dimensional gel electrophoresis along the study.
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PMID:Regulation of growth, protein synthesis, and maturation of fetal bovine epiphyseal chondrocytes grown in high-density culture in the presence of ascorbic acid, retinoic acid, and dihydrocytochalasin B. 1058 Oct 3

Multipotential mesenchymal stem cells capable of chondro-osseous induction contribute to the endochondral callus of healing fractured bone. Microvascular pericytes serving the role of multipotential mesenchymal stem cells are considered osteoprogenitors because they express type I collagen, alkaline phosphatase enzyme activity, osteocalcin immunoreactivity, and bone sialoprotein mRNA. Previous electron microscopic studies indicate that this cell type has a contribution to the fracture callus. Limited data suggest that pericytes may also assume a chondrogenic phenotype. We undertook in vitro studies to understand how the chondro-osseous phenotype of the pericyte might be regulated. Using Northern analysis and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we found that cultured pericytes produce aggrecan and type II collagen mRNA indicating their chondrogenic potential. Aggrecan message is elevated by BMP-2 as analyzed by both Northern hybridization and RT-PCR. This finding suggests a regulatory role for this morphogen on this phenotype in pericytes. RT-PCR amplified versican product was also associated with pericyte cultures but was not affected by BMP-2. Our data strongly support a chondrogenic role for the pericyte and that the phenotype is regulated at least in part by BMP.
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PMID:Microvascular pericytes express aggrecan message which is regulated by BMP-2. 1069 96

Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can be expressed in specific in vitro and in vivo microenvironments.
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PMID:Transient chondrogenic phase in the intramembranous pathway during normal skeletal development. 1075 May 67

Members of the transforming growth factor-beta (TGF-beta) superfamily, the bone morphogenetic and osteogenic proteins (BMPs/OPs) but not the TGF-beta proteins themselves, induce endochondral bone formation in vivo, when implanted in extraskeletal heterotopic sites of rodents. Here we show that recombinant human TGF-beta2 (hTGF-beta2) induces endochondral bone formation 30 days after implantation in heterotopic intramuscular sites of the baboon (Papio ursinus) at doses of 1, 5 and 25 microg per 100 mg of guanidinium-inactivated collagenous bone matrix as carrier. On day 90 there was generation of large radiopaque and corticalized intramuscular ossicles. Five and 25 microg hTGF-beta2 induced large ossicles in the rectus abdominis of the primate as evaluated by key parameters of bone formation, including generated tissue area, mineralized bone and osteoid volumes, and tissue alkaline phosphatase activity. On day 30 and 90 after healing, hTGF-beta2 also induced bone formation when implanted in the rectus abdominis in conjunction with a sintered porous hydroxyapatite as carrier. mRNA expression in tissues from heterotopic specimens showed OP-1 (BMP-7) and BMP-3 transcripts in low abundance and with a linear dose-dependent increase both in collagenous matrix and hydroxyapatite samples. Type IV collagen mRNA expression, a marker of angiogenesis, was stronger in collagenous than hydroxyapatite samples. Growth and differentiation factor-10 (GDF-10) mRNA transcripts were expressed in ossicles with a distinctly chondrogenic phase, but its expression was greater in ossicles generated in porous hydroxyapatites, in which bone formation is not via a chondrogenic phase, but is rather intramembranous, without expression of type II collagen mRNA. In the same animals, however, 10 and 100 microg of the recombinant morphogen delivered by identical carriers (collagenous matrix and sintered hydroxyapatite) failed to heal calvarial defects. Thus in the primate, TGF-betas themselves are inducers of endochondral bone formation, although the present data strongly indicate that the bone inductive activity of hTGF-beta2 is site and tissue specific, since a single application of hTGF-beta2, or hTGF-beta1 in previously published experiments, did not induce bone in calvarial defects, but did induce endochondral bone differentiation in heterotopic sites.
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PMID:Induction of endochondral bone formation by recombinant human transforming growth factor-beta2 in the baboon (Papio ursinus). 1151 29

Core-binding factor A1 (Cbfa1), also called Pebp2 alpha A/AML3, is a transcription factor that belongs to the runt-domain gene family. Cbfa1-deficient mice are completely incapable of both endochondral and intramembranous bone formation, indicating that Cbfa1 is indispensable for osteogenesis. Maturation of chondrocytes in these mice is also disorganized, suggesting that Cbfa1 may also play a role in chondrogenesis. The aim of this study was to examine the expression and regulation of Pebp2 alpha A/AML3/Cbfa1 expression in the chondrocyte-like cell line, TC6. Northern blot analysis indicated that Cbfa1 mRNA was constitutively expressed as a 6.3 kb message in TC6 cells and the level of Cbfa1 expression was enhanced by treatment with bone morphogenetic protein-2 (BMP2) in a time- and dose-dependent manner. This effect was blocked by an RNA polymerase inhibitor, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole, but not by a protein synthesis inhibitor, cycloheximide. Western blot analysis of the cell lysates using polyclonal antibody raised against Cbfa1 indicated that BMP2 treatment increased the Cbfa1 protein level in TC6 cells. In TC6 cells, BMP2 treatment enhanced expression of alkaline phosphatase and type I collagen mRNAs but suppressed that of type II collagen mRNA. In addition to TC6 cells, Cbfa1 mRNA was also expressed in primary cultures of chondrocytes and BMP2 treatment enhanced Cbfa1 mRNA expression in these cells similarly to its effect on TC6 cells. These data indicate that the Pebp2 alpha A/AML3/Cbfa1 gene is expressed in a chondrocyte-like cell line, TC6, and its expression is enhanced by treatment with BMP.
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PMID:An osteogenesis-related transcription factor, core-binding factor A1, is constitutively expressed in the chondrocytic cell line TC6, and its expression is upregulated by bone morphogenetic protein-2. 1082 41

We have succeeded in transplanting a human osteogenic sarcoma of the mandible into athymic mice. The transplanted tumor showed marked chondrogenesis and mineralization. Recently, a cell line (USAC) with phenotypes of chondrocyte has been established from the transplanted tumor. USAC cells were stellate or spindle-shaped in sparse culture, but polygonal or spherical at sub-confluency to confluency. In long-term culture, the cells were condensed and calcified nodules were formed. Production of types I, II and X collagen were detected by immunohistochemical staining and Western blot analysis. Type I collagen was strongly expressed in the stellate or spindle-shaped cells. Although type II collagen was usually present in all cells during culture, it was strongly stained in polygonal cells at confluency. Type X collagen was seen in large polygonal cells around calcified nodules. Marked [35S]-sulfate uptake and metachromasia were seen at the confluent stage and in the nodule. The cells around the nodules were positive for alkaline phosphatase, and the center of the nodules was stained with alizarin red. The potentiality of cartilage formation was confirmed by in vivo experiments using a diffusion chamber in athymic mice. These observations indicate that USAC cells maintain characteristics of chondrocyte progenitor cells and thus may serve as a useful model to study the sequential events of chondrogenesis and the process of morbid endochondral calcification. This experiment also demonstrated that transplantation of tumor tissue into athymic mice is a convenient strategy for establishment of a cell line.
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PMID:Establishment of a cell line with phenotypes of chondrocyte from a human osteogenic sarcoma of the mandible. 1094 48

To investigate the pathogenesis of the degenerative changes of the ligamentum flavum occurring in lumbar spine stenosis, yellow ligament cells from patients with lumbar spine stenosis were cultured for the first time and subjected to biochemical, histochemical and immunohistochemical study. Stenotic ligamentum flavum (SLF) cells were seen to express high levels of alkaline phosphatase (ALP) activity and to produce a matrix rich in type I and III collagen, fibronectin and osteonectin. The matrix mineralized only following beta-glycerophosphate (betaGP) and ascorbic acid supplementation. Stimulation with human parathyroid hormone (PTH) increased intracellular cAMP concentration. These findings indicate that there was significant evidence of osteoblast-like activity in these cells. SLF cells also stained for S100 protein, type II and type X collagen, and co-localized type II collagen and ALP labelling, reflecting the presence of hypertrophic chondrocyte-like cells. Cultures from control patients showed neither osteoblastic nor chondrocytic features: they expressed type I and type III collagen and fibronectin, but did not stain for osteonectin, nor were bone-like calcifications observed in presence or absence of betaGP. Normal ligamentum flavum (NLF) cells did not synthesized S100 protein or type II or type X collagen, and showed a weaker response to PTH stimulation. Our data demonstrated the presence of hypertrophic chondrocytes with an osteoblast-like activity in the ligamentum flavum of patients with spinal stenosis suggesting that they could have a role in the pathophysiology of the heterotopic ossification of ligamentum flavum (OLF) in lumbar spine stenosis.
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PMID:Characterization of cultured human ligamentum flavum cells in lumbar spine stenosis. 1134 4

During endochondral bone formation and fracture healing, cells committed to chondrogenesis undergo a temporally restricted program of differentiation that is characterized by sequential changes in their phenotype and gene expression. This results in the manufacture, remodeling, and mineralization of a cartilage template on which bone is laid down. Articular chondrocytes undergo a similar but restricted differentiation program that does not proceed to mineralization, except in pathologic conditions such as osteoarthritis. The pathogenesis of disorders of cartilage development and metabolism, including osteochondrodysplasia, fracture non-union, and osteoarthritis remain poorly defined. We used the CFK2 model to examine the potential roles of phosphate and calcium ions in the regulatory pathways that mediate chondrogenesis and cartilage maturation. Differentiation was monitored over a 4-week period using a combination of morphological, biochemical, and molecular markers that have been characterized in vivo and in vitro. CFK2 cells expressed the type III sodium-dependent phosphate transporters Glvr-1 and Ram-1, as well as a calcium-sensing mechanism. Regulated expression and activity of Glvr-1 by extracellular phosphate and parathyroid hormone-related protein was restricted to an early stage of CFK2 differentiation, as evidenced by expression of type II collagen, proteoglycan, and Ihh. On the other hand, regulated expression and activity of a calcium-sensing receptor by extracellular calcium was most evident after 2 weeks of differentiation, concomitant with an increase in type X collagen expression, alkaline phosphatase activity and parathyroid hormone/parathyroid hormone-related protein receptor expression. On the basis of these temporally restricted changes in the sensing and transport of phosphate and calcium, we predict that extracellular phosphate plays a role in the commitment of chondrogenic cells to differentiation, whereas extracellular calcium plays a role at a later stage in their differentiation program.
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PMID:Alterations in the sensing and transport of phosphate and calcium by differentiating chondrocytes. 1140 53


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