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

The type X collagen is a short chain collagen associated with calcific cartilage and/or the expression of the hypertrophic chondrocyte phenotype. In articular cartilage, type X collagen is restricted to the basal zone of calcified cartilage adjacent to the subchondral bone. However, during pathological change such as in osteoarthritis, the synthesis of type X collagen becomes more widespread but never extends to the articular surface. Using immunocytochemistry and fluorography of newly synthesised collagens, we report that surface articular chondrocytes (which occupy the uppermost 10-15% of the tissue depth) from normal human cartilage initiate de novo synthesis of both type X collagen and alkaline phosphatase when maintained in suspension culture.
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PMID:Human articular surface chondrocytes initiate alkaline phosphatase and type X collagen synthesis in suspension culture. 148 93

We developed a new 4-dimensional differentiation model of growth-cartilage cells (temporally and spatially regulated). composed of a high-density suspension culture of rabbit costal chondrocytes. In this system, fibroblast growth factor (FGF) or transforming growth factor-beta (TGF beta) stimulated proliferation and matrix synthesis, but reversibly inhibited terminal differentiation-induction of alkaline phosphatase (Alpase) activity, type X collagen synthesis and subsequent calcification. When parathyroid hormone (PTH) was added to the differentiation model, both the induction of Alpase and calcium uptake were reversibly suppressed. In contrast, calcitonin (CT) stimulated Alpase activity and calcium uptake dose-dependently. Bone morphogenetic protein (BMP) strongly stimulated DNA synthesis of chondrocytes in the presence of FGF and induced rapid maturation of chondrocytes at a growing stage. Moreover, BMP stimulated Alpase activity in multilayered chondrocytes at the calcifying stage.
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PMID:Effects of various growth factors on a chondrocyte differentiation model. 149 10

Expression of specific differentiation markers was investigated by histochemistry, immunofluorescence, and biosynthetic studies in osteoblasts outgrown from chips derived from tibia diaphyses of 18-day-old chick embryos. The starting osteoblast population expressed type I collagen and alkaline phosphatase in addition to other bone and cartilage markers as the lipocalin Ch21; the extracellular matrix deposited by these cells was not stainable for cartilage proteoglycans, and mineralization was observed when the culture was maintained in the presence of ascorbic acid, calcium and beta-glycerophosphate. During culture, clones of cells presenting a polygonal chondrocyte morphology and surrounded by an Alcian-positive matrix appeared in the cell population. Type II collagen and type X collagen were synthesized in these areas of chondrogenesis. In addition, chondrocytes isolated from these cultures expressed Ch21 and alkaline phosphatase. Chondrocytes were generated also from homogeneous osteoblast populations derived from a single cloned cell. The coexistence of chondrocytes and osteoblasts was observed during amplification of primary clones as well as in subclones. The data show the existence, within embryonic bone, of cells capable in vitro of both osteogenic and chondrogenic differentiation.
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PMID:Chondrogenic differentiation in chick embryo osteoblast cultures. 151 96

The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) (2.3 x 10(-12) - 1.4 x 10(-6) [M]) on alkaline phosphatase, collagen, and cell proliferation were examined in primary cultured hypertrophic chondrocytes prepared from the distal epiphyseal growth plate of the tibias of 12-day chick embryos. 1,25(OH)2D3 showed time- and dose-dependent inhibitory effects on the alkaline phosphatase and collagen levels. The inhibition of alkaline phosphatase activity became detectable at 2 x 10(-11) [M] and reached 10% of control at 10(-7) [M]. The concentration of 1,25(OH)2D3 giving a 50% inhibition of the enzyme level was approximately 3 x 10(-10) [M]. Of the two extracellular collagen pools, a cell-associated matrix pool showed a more dramatic decrease (to 10% of control) than a culture medium pool (to 50% of control) at increased 1,25(OH)2D3 concentrations. The degree of inhibition was different for each type of chondrocyte-specific collagen (types II, IX, X, and XI). Types II and IX were inhibited in a parallel manner to only 60-80% of control. On the other hand, types X and XI were more greatly reduced up to 10% of control, and their dose-dependent inhibitory curves were similar to that of alkaline phosphatase. On cell proliferation, 1,25(OH)2D3 had a biphasic effect: stimulation at 10(-10)-10(-8) [M] and inhibition at higher levels. The results revealed the significant involvement of 1,25(OH)2D3 in the metabolism of two probable calcification-related products, alkaline phosphatase and type X collagen.
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PMID:Coordinate inhibition of alkaline phosphatase and type X collagen syntheses by 1,25-dihydroxyvitamin D3 in primary cultured hypertrophic chondrocytes. 159 80

In this study, an organ culture system is defined which demonstrates complete loss of cartilage matrix from embryonic chick tibiae. Efficient loss of the cartilage matrix occurs within 30 days of serum-free culture only when the intact tibiae containing bone, marrow, and cartilage tissue are cultured. During organ culture nonhypertrophic chondrocytes become hypertrophic and stain positively for type X collagen and alkaline phosphatase. The cartilage loses Safranin O staining, and finally all cartilage matrix disappears leaving the bony collar and marrow cells. If the tibial cartilage is separated from the bony collar and cultured alone in serum-free medium, the nonhypertrophic chondrocytes also hypertrophy; the matrix loses Safranin O staining; however, some components of the matrix including type X collagen still remain after 30 days. In the presence of serum, the chondrocytes will hypertrophy but cartilage degradation is not evident. The results of this study support the conclusions that 1) hypertrophy is inherently programmed in the chondrocyte and 2) while Safranin O staining of cartilage cultured alone is diminished in serum-free organ culture, the degradation of cartilage is complete only when bone and marrow are also present.
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PMID:The influence of bone and marrow on cartilage hypertrophy and degradation during 30-day serum-free culture of the embryonic chick tibia. 160 Feb 46

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

In bone forming cartilage in vivo, cells undergo terminal differentiation, whereas most of the cells in normal articular cartilage do not. Chondrocyte hypertrophy can be induced also in vitro by diffusible signals. We have identified growth factors or hormones acting individually on 17-d chick embryo sternal chondrocytes cultured in agarose gels under strictly serum-free conditions. Insulin-like growth factor I or insulin triggered the first steps of chondrocyte maturation, i.e., cell proliferation and increased matrix deposition while the chondrocytic phenotype was maintained. However, cells did not progress to the hypertrophic stage. Proliferation and stimulated collagen production was preceded by a lag period, indicating that synthesis of other components was required before cells became responsive to insulin-like growth factor I or insulin. Very small amounts of FBS exerted effects similar to those of insulin-like growth factor I or insulin. However, FBS could act directly and elicited hypertrophy when constituting greater than 1% of the culture media. Basic FGF has been claimed to be the most potent chondrocyte mitogen, but had negligible effects under serum-free conditions. The same is true for PDGF, a major serum-mitogen. Under the direction of thyroxine, cells did not proliferate but became typical hypertrophic chondrocytes, extensively synthesizing collagen X and alkaline phosphatase.
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PMID:Induction of proliferation or hypertrophy of chondrocytes in serum-free culture: the role of insulin-like growth factor-I, insulin, or thyroxine. 173 18

Bones grow in length because of the activities of cartilage cells in the epiphyseal growth plate. We have examined selected events that occur in the growth cartilage by the use of cultured epiphyseal cells; we have also evaluated the influence of ascorbate on these activities. Our studies indicate that 1) ascorbate induces the expression of a unique collagen isoform, type X collagen; 2) ascorbate stimulates alkaline phosphatase activity of maturing chondrocytes; and 3) ascorbate regulates the energy status of the maturing chondrocyte. We have found that in the presence of ascorbate there is a change in oxidative activity. Thus, lactate formation is inhibited, there is an increase in the adenylate energy charge ratio, and there is an elevation in the activity of isocitrate dehydrogenase. The results of these studies point to multiple effects of vitamin C on chondrocyte maturation involving changes in protein synthesis and energy metabolism.
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PMID:Ascorbic acid regulates multiple metabolic activities of cartilage cells. 196 72

Conditions were defined for promoting cell growth, hypertrophy, and extracellular matrix mineralization of a culture system derived from embryonic chick vertebral chondrocytes. Ascorbic acid supplementation by itself led to the hypertrophic phenotype as assessed by respective 10- and 15-fold increases in alkaline phosphatase enzyme activity and type X synthesis. Maximal extracellular matrix mineralization was obtained, however, when cultures were grown in a nutrient-enriched medium supplemented with both ascorbic acid and 20 mM beta-glycerophosphate. Temporal studies over a 3-wk period showed a 3-4-fold increase in DNA accompanied by a nearly constant DNA to protein ratio. In this period, total collagen increased from 3 to 20% of the cell layer protein; total calcium and phosphorus contents increased 15-20-fold. Proteoglycan synthesis was maximal until day 12 but thereafter showed a fourfold decrease. In contrast, total collagen synthesis showed a greater than 10-fold increase until day 18, a result suggesting that collagen synthesis was replacing proteoglycan synthesis during cellular hypertrophy. Separate analysis of individual collagen types demonstrated a low level of type I collagen synthesis throughout the 21-d time course. Collagen types II and X synthesis increased during the first 2 wk of culture; thereafter, collagen type II synthesis decreased while collagen type X synthesis continued to rise. Type IX synthesis remained at undetectable levels throughout the time course. The levels of collagen types I, II, IX, and X mRNA and the large proteoglycan core protein mRNA paralleled their levels of synthesis, data indicating pretranslational control of synthesis. Ultrastructural examination revealed cellular and extracellular morphology similar to that for a developing hypertrophic phenotype in vivo. Chondrocytes in lacunae were surrounded by a well-formed extracellular matrix of randomly distributed collagen type II fibrils (approximately 20-nm diam) and extensive proteoglycan. Numerous vesicular structures could be detected. Cultures mineralized reproducibly and crystals were located in extracellular matrices, principally associated with collagen fibrils. There was no clear evidence of mineral association with extracellular vesicles. The mineral was composed of calcium and phosphorus on electron probe microanalysis and was identified as a very poorly crystalline hydroxyapatite on electron diffraction. In summary, these data suggest that this culture system consists of chondrocytes which undergo differentiation in vitro as assessed by their elevated levels of alkaline phosphatase and type X collagen and their ultrastructural appearance.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system. 199 93


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