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)

Chondrocytes from chicken embryo tibia can be maintained in culture as adherent cells in Coon's modified Ham's F-12 medium supplemented with 10% FCS. In this condition, they dedifferentiate, losing type II collagen expression in favor of type I collagen synthesis. Their differentiation to hypertrophy can be obtained by transferring them to suspension culture. Differentiation is evidenced by the shift from type I to type II and type IX collagen synthesis and the following predominant expression of type X collagen, all markers of specific stages of the differentiation process. To identify the factors required for differentiation, we developed a serum-free culture system where only the addition of triiodothyronine (T3; 10(-11) M), insulin (60 ng/ml), and dexamethasone (10(-9) M) to the F-12 medium was sufficient to obtain hypertrophic chondrocytes. In this hormonal context, chondrocytes display the same changes in the pattern of protein synthesis as described above. For proper and complete cell maturation, T3 and insulin concentrations cannot be modified. Insulin cannot be substituted by insulin-like growth factor-I, but dexamethasone concentration can be decreased to 10(-12) M without chondrogenesis being impaired. In the latter case, the expression of type X collagen and its mRNA are inversely proportional to dexamethasone concentration. When ascorbic acid is added to the hormone-supplemented medium, differentiating chondrocytes organize their matrix leading to a cartilage-like structure with hypertrophic chondrocytes embedded in lacunae. However, this structure does not present detectable calcification, at variance with control cultures maintained in FCS. Accordingly, in the presence of the hormone mixture, the differentiating chondrocytes have low levels of alkaline phosphatase activity. This report indicates that T3 and insulin are primary factors involved in the onset and progression of chondrogenesis, while dexamethasone supports cell viability and modulates some differentiated functions.
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PMID:Thyroid hormone, insulin, and glucocorticoids are sufficient to support chondrocyte differentiation to hypertrophy: a serum-free analysis. 142 44

Chondrogenesis can occur during a bone repair process, which is related to several growth factors. Transforming growth factor beta 1 (TGF-beta 1) downregulates the expression of type II collagen by chondrocytes in vitro, but injection of TGF-beta 1 into the periosteum in vivo increases type II collagen mRNA levels and initiates chondrogenesis. We examined the effect of TGF-beta 1 on collagen gene expression in a bovine periosteum-derived cell culture system to evaluate its direct effect on the periosteum. Cultured cells expressed alkaline phosphatase and collagen pro alpha 1(I) and pro alpha 1(II) mRNAs. A low level of type II collagen synthesis was demonstrated by immunoprecipitation. TGF-beta 1 had no effect on periosteal cell proliferation. Expression of collagen pro alpha 1(I) mRNA did not change with TGF-beta 1 treatment, but alkaline phosphatase mRNA showed a dose-dependent decrease. Expression of collagen pro alpha 1(II) mRNA was stimulated 2.7-fold by TGF-beta 1. TGF-beta 1 also caused a 2.6-fold increase in type II collagen synthesis by immunoprecipitation. These findings indicate that TGF-beta 1 is an enhancer of the expression of the chondrocyte phenotype of the periosteal cells and suggest that TGF-beta 1 is important in initiating and promoting cartilage formation in vivo.
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PMID:Transforming growth factor beta 1 stimulates type II collagen expression in cultured periosteum-derived cells. 154 55

Calcification occurs in the extracellular matrix of the hypertrophic zone of the growth plate when the extracellular matrix volume is reduced to a minimum and alkaline phosphatase content is maximal. The present study shows that significant quantitative and qualitative changes occur in the composition and structure of macromolecules in the extracellular matrix before and during calcification in the proximal tibial growth plate of the bovine fetus. These were detected in part by using microchemical and microimmuno-chemical analyses of sequential transverse frozen sections at defined sites throughout the growth plate. Concentrations of matrix molecules in the extracellular matrix have not previously been determined biochemically. They were measured per unit matrix volume by using combined immunochemical/chemical-histomorphometric analyses. The concentrations within the extracellular matrix of the C-propeptide of type II collagen, aggregating proteoglycan (aggrecan), and hyaluronic acid all progressively increased in the maturing and hypertrophic zones, being maximal (or near maximal) at the time of initiation of mineralization. These results for proteoglycan are contrary to some earlier reports of a loss of proteoglycan are contrary to some earlier reports of a loss of proteoglycan prior to mineralization which measured the tissue content of proteoglycan rather than that present in the extracellular matrix, the volume of which is progressively reduced as the growth plate matures. The C-propeptide data provides a quantitative confirmation of previous immunohistochemical studies. Total collagen concentration (measured as hydroxyproline) in the extracellular matrix initially increased through the proliferating and maturing zones but then rapidly decreased in the hypertrophic zone.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The extracellular matrix of cartilage in the growth plate before and during calcification: changes in composition and degradation of type II collagen. 157 44

A human malignant fibrous histiocytoma (MFH) cell line, designated as MFH-ino, was established from the maxillary tumor of a 45-year-old woman. Clinically, the original tumor was accompanied by extensive destruction of the surrounding tissues. Cells were obtained from the explant culture of tumor fragments. Both histiocytic and fibroblastic markers were observed in the histochemical and immunocytochemical studies of MFH-ino. The cells were positive for lysozyme, alpha-1-antichymotrypsin, and the collagen types I, III, IV, V, but were negative for alpha-1-antitrypsin, acetate esterase and type II collagen. As biochemical examinations of the culture cells, collagen synthesis was assayed by the measurement of hydroxyproline and the content increase in culture dishes with time after cell inoculation. Collagenase activity secreted in culture medium was also examined with FITC-labeled type I collagen as substrate, and high activity was detected at the late stage of the stationary phase. Further, the MFH-ino cells had high acid phosphatase activity while lacking alkaline phosphatase activity. These findings indicated that MFH-ino cells expressed the various properties of MFH, which will be of importance for understanding the biological behavior, and especially the collagen metabolism, of MFH.
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PMID:Establishment and characterization of a human neoplastic cell line (MFH-ino) derived from malignant fibrous histiocytoma of maxilla. 165 97

The myc oncogene is expressed by proliferating quail embryo chondrocytes (QEC) grown as adherent cells and is repressed in QEC maintained in suspension culture. To investigate the interference of myc expression during chondrocyte differentiation, QEC were infected with a retrovirus carrying the v-myc oncogene (QEC-v-myc). Uninfected or helper virus-infected QEC were used as control. In adherent culture, QEC-v-myc displayed a chondrocytic phenotype and synthesized type II collagen and Ch21 protein, while control chondrocytes synthesized type I and type II collagen with no Ch21 protein detected as long as the attachment to the plastic was kept. In suspension culture, QEC-v-myc readily aggregated and within 1 week the cell aggregates released small single cells; still they secreted only type II collagen and Ch21 protein. In the same conditions control cell aggregates released hypertrophic chondrocytes producing type II and type X collagens and Ch21 protein. In the appropriate culture conditions, QEC-v-myc reconstituted a tissue defined as nonhypertrophic, noncalcifying cartilage by the high cellularity, the low levels of alkaline phosphatase enzymatic activity, and the absence of type X collagen synthesis and of calcium deposition. We conclude that the constitutive expression of the v-myc oncogene keeps chondrocytes in stage I (active proliferation and synthesis of type II collagen) and prevents these cells from reconstituting hypertrophic calcifying cartilage.
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PMID:Constitutive myc expression impairs hypertrophy and calcification in cartilage. 172 87

The vitamin A derivative retinoic acid (RA) is widely thought to be involved in cartilage development, but its precise roles and mechanisms of action in this complex process remain unclear. We have tested the hypothesis that RA is involved in chondrocyte maturation during endochondral ossification and, in particular, is an inducer of maturation-associated traits such as type X collagen and alkaline phosphatase. Immature chondrocytes isolated from the caudal region of Day 19 chick embryo sterna were seeded in secondary monolayer cultures and treated either with a high dose (100 nM) or with physiological doses (10-35 nM) of RA for up to 3 days. We found that after an initial lag of about 24 h, physiological doses of RA indeed induced type X collagen gene expression in the immature cells. This induction was not accompanied by obvious changes in expression of the type II collagen and large aggregating proteoglycan core protein genes. As revealed by immunocytochemistry, 30-35% of the cells in cultures treated with RA for 3 days were engaged in type X collagen production. Interestingly, these cells were relatively similar in size to chondrocytes in which no type X collagen was detected, suggesting that chondrocytes can initiate type X collagen production independent of cell hypertrophy. RA treatment also led to increased alkaline phosphatase activity occurring as early as 24 h after the start of treatment. The data in this study indicate that RA may have a role in endochondral ossification as an inducer/promoter of maturation-associated traits during chondrocyte maturation.
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PMID:Retinoic acid treatment induces type X collagen gene expression in cultured chick chondrocytes. 205 74

Bone morphogenetic protein 2B (BMP-2B) also called BMP-4 is one of a family of cartilage and bone-inductive proteins derived from bone matrix and belongs to the transforming growth factor beta (TGF-beta) superfamily. These bone-inductive proteins isolated from adult bone may be involved in bone repair. However, they may also play a role in cartilage and bone formation during embryonic development. To test whether BMP-2B influences cartilage formation by embryonic cells, recombinant human BMP-2B was applied to cultured limb bud mesoderm plated at three different densities. BMP-2B stimulated cartilage formation as assessed by Alcian blue staining and incorporation of radioactive sulfate into sulfated proteoglycans. Cells cultured at all three densities in the presence of 10 ng/ml BMP-2B formed a nearly continuous sheet of cartilage with abundant extracellular matrix and type II collagen. In addition, when cells were cultured in 0.5% serum in the presence of 10 ng/ml of BMP-2B for 5 days there was an increase in alkaline phosphatase as detected by histochemical and biochemical methods. Transforming growth factor beta isoforms (TGF-beta 1 and TGF-beta 2) inhibited sulfate incorporation into proteoglycans in a dose-dependent manner. This inhibition by TGF beta was overcome by recombinant BMP-2B. This study demonstrates that recombinant BMP-2B stimulates cartilage formation by chick limb bud mesoderm in vitro and is further modulated by TGF-beta isoforms.
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PMID:Stimulation of chondrogenesis in limb bud mesoderm cells by recombinant human bone morphogenetic protein 2B (BMP-2B) and modulation by transforming growth factor beta 1 and beta 2. 207 Aug 31

The present study describes the behavior of mandibular condylar cartilage (MCC) cells as a function of time in primary culture, since it is not yet clear whether these cells maintain their phenotype in culture. MCC cells from New Zealand white rabbits were seeded at high density and cultured in DMEM containing 50 micrograms/mL ascorbic acid and 10% fetal bovine serum. These cells appeared as a heterogeneous population and changed their shape, size, and refractivity as cultures aged. Cartilage-like cells, which always dominated the culture, were infiltrated with a minority of fibroblast-like cells. Cell number increased progressively, and cultures reached confluence at nine days. Antibody activity for cartilage-specific glycosaminoglycan was determined by ELISA assay. This reaction reached a maximum at six days and decreased thereafter. Cultures stained with Alcian blue (pH 1.0) supported these results. Cytoplasmic mRNA analysis indicated that the transcription of type II collagen gene was present at all time points. Type I collagen and alkaline phosphatase mRNA levels showed progressive increases from 12 h to nine days, with significantly higher values in cells cultured for six, nine, and 12 days than in cells collected from earlier time points. These results suggest that in our present culture system, MCC cells undergo phenotypic changes that resemble their maturation processes in vivo.
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PMID:Phenotypic changes of rabbit mandibular condylar cartilage cells in culture. 222 13

Rat frontonasal and mandibular mesenchyme was isolated from day-12 1/2 (stage-22) rat embryos and cultured at high density for up to 12 days. The stage chosen was based on the observation that mandibular mesenchyme at this stage became independent of its epithelium with respect to the production of both cartilage and bone. Frontonasal cultures developed aggregates of anastomosing columns of cells within 2 days. These grew as the cells enlarged, laying down an Alcian-blue-positive matrix by day 3 of culture. Significant mineral was detected by von Kossa staining by day 5 at which time the aggregates covered a large portion of the culture, eventually covering the entire micromass by day 10-12. Mandibular cultures developed centrally located nodular aggregates by 3 days of culture. These nodules increased in number, spreading outwards as the cells enlarged, laying down an Alcian-blue-positive matrix by day 4 and mineral by days 6-7. At this time the nodules began to elongate and coalesce, but never covered the entire culture over the 12-day period. Antibody staining revealed that in both cultures the cells were initially positive for type I collagen. Subsequently, the aggregates began expressing type II collagen, followed by type X, which coincided with the onset of mineralization. At this time some cells were negative for these cartilage markers, but positive for osteoblast markers, bone sialoprotein II, osteocalcin and type I collagen. In addition osteonectin and alkaline phosphatase were demonstrable in all of the aggregate cells late in the culture period. This provided clear evidence that chondroblast and osteoblast differentiation was proceeding within these cultures. The culture of rat facial mesenchyme should prove very useful, not only for the analysis of bone and cartilage induction and lineage relationships, but also in furthering our knowledge of craniofacial differentiation, growth and pattern formation by extending our analysis to a mammalian system.
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PMID:Formation of chondrous and osseous tissues in micromass cultures of rat frontonasal and mandibular ectomesenchyme. 227 11

We have investigated the regulation of chondroblast/chondrocyte differentiation using a unique clonal cell population, designated RCJ 3.1C5 (C5), which differentiates into discrete three-dimensional cartilage nodules when grown in the presence of 15% fetal calf serum. Histologically, the nodules resembled hyaline cartilage; they contained large rounded chondrocytes surrounded by a refractile matrix which stained intensely with Alcian blue, exhibited metachromasia after Toluidine blue staining, and stained with an antibody against type II collagen. The cartilage nodules that formed did not mineralize, despite the presence of organic phosphate in the culture medium. The synthetic glucocorticoid dexamethasone (DEX) increased the number of cartilage nodules formed in a dose-dependent manner (ED50, approximately 10(-9) M), with a maximal stimulatory dose of 10(-8) M. DEX had no effect on the population doubling time and saturation density. The effects of DEX on the number of cartilage nodules were similar whether it was added from the beginning of the culture period (starting during exponential growth) or at confluence. In contrast, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited cartilage nodule formation in a dose-dependent manner (IC50, approximately 5 x 10(-10) M), with maximum inhibition at 10(-7) M. In addition, 1,25-(OH)2D3 decreased cell proliferation and saturation density. Equimolar doses of the vitamin D3 metabolites 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 had no effect. C5 cells treated with 1,25-(OH)2D3 in the absence of DEX during the exponential growth phase exhibited a reduced capacity to form cartilage nodules upon subsequent exposure to DEX. At confluence, before cartilage nodules had formed, C5 cells responded to PTH and prostaglandin-E2 with increases in intracellular cAMP of about 10- and 95-fold respectively. After cartilage nodules were present, prostaglandin-E2 responsiveness decreased to about 25-fold, whereas there was no significant change in PTH responsiveness. DEX decreased the population alkaline phosphatase levels at all times measured, whereas 1,25-(OH)2D3 had a biphasic effect: an increase at 5 days in culture, followed by a decrease at later times in culture. These data indicate that the clonal cell line RCJ 3.1C5 is a useful model system in which to investigate cartilage differentiation.
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PMID:Effects of dexamethasone and vitamin D3 on cartilage differentiation in a clonal chondrogenic cell population. 255 36


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