Participation of Cbfa1 in regulation of chondrocyte maturation.
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OBJECTIVE: Cbfa1 is a transcription factor, which is classified into the runt family. The mice lacking this gene display complete loss of bone formation, indicating that Cbfa1 is an essential factor for osteoblast differentiation. The Cbfa1-deficient mice also show an abnormality in cartilage development. Although cartilage anlagens are well formed in these mice, endochondral ossification is blocked, and most of chondrocytes fail to differentiate into their maturation form as characterized by the absence of type X collagen and low levels of alkaline phosphatase activity. It is suggested that Cbfa1 may participate in chondrocyte differentiation. In this study, we have investigated the role of Cbfa1 in chondrocytes during their cytodifferentiation in vitro. DESIGN: To investigate the role of Cbfa1 in regulation of chondrocyte differentiation, we over-expressed Cbfa1 or its dominant negative form in cultured chick chondrocytes using a retrovirus (RCAS)system and examined changes in chondrocyte behaviour induced by the introduced genes. RESULTS: Mature chondrocytes isolated form the cephalic portion of sterna seemed to express Cbfa1 more prominently than immature chondrocytes isolated from the one-third caudal portion of sterna. Over-expression of Cbfa1 in immature chondrocytes strongly stimulated alkaline phosphatase activity and matrix calcification. In contrast, expression of a dominant negative form of Cbfa1, which lacks the C-terminal PST domain, severely inhibited alkaline phosphatase activity and matrix calcification in mature chondrocytes. CONCLUSION: Taken together with the observation that Cbfa1 transcripts dominantly localized in hypertrophic chondrocytes as well as in osteoblasts, it is suggested that Cbfa1 plays an important role in the progression of chondrocyte maturation. (+info)
Excretion of inhibitors of calcification in urine Part II. Findings in patients with chronic renal failure.
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By means of a semiquantitative method incorporating the rachitic rat cartilage technique, the total urinary inhibitory activity with respect to calcification was compared in 11 control subjects and 20 patients with renal failure. The patients had significantly lower mean values of inhibiting units per day than did the control subjects. Both groups showed a significant positive correlation between the number of inhibiting units per day and urine volume. When urine volume was taken into account in the comparison, the numbers of inhibiting units for patients continued to be lower than the numbers for controls. These findings are consistent with the hypothesis that the increase of inhibitory activity observed in uremic serum is secondary to a decrease in excretion of the responsible factor (or factors) in the urine, and that the factor (or factors) in serum responsible for the inhibition are identical to those in the urine. (+info)
Phase-independent inhibition by retinoic acid of mineralization correlated with loss of tetranectin expression in a human osteoblastic cell line.
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We have recently reported that retinoic acid inhibits dexamethasone-induced alkaline phosphatase activity and mineralization in human osteoblastic cell line SV-HFO. In this study, we show that this inhibitory effect on alkaline phosphatase activity depends on the stage of cell differentiation; however, expression of tetranectin, which is a recently reported bone matrix protein, was completely inhibited by treatment with retinoic acid, irrespective of the stage of cell differentiation. Similarly, mineral deposit formation in SV-HFO cells was phase-independently inhibited by retinoic acid. To our knowledge, this is the first report that retinoic acid downregulates the tetranectin expression in human osteoblastic cells independent of the stage of cell differentiation, and is correlated with inhibition of mineralization. (+info)
Overexpression of Phex in osteoblasts fails to rescue the Hyp mouse phenotype.
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Inactivating mutations of Phex, a phosphate-regulating endopeptidase, cause hypophosphatemia and impaired mineralization in X-linked hypophosphatemia (XLH) and its mouse homologue, Hyp. Because Phex is predominantly expressed in bone and cultured osteoblasts from Hyp mice display an apparent intrinsic mineralization defect, it is thought that reduced expression of Phex in mature osteoblasts is the primary cause of XLH. To test this hypothesis, we studied both targeted expression of Phex to osteoblasts in vivo under the control of the mouse osteocalcin (OG2) promoter and retroviral mediated overexpression of Phex in Hyp-derived osteoblasts (TMOb-Hyp) in vitro. Targeted overexpression of Phex to osteoblasts of OG2 Phex transgenic Hyp mice normalized Phex endopeptidase activity in bone but failed to correct the hypophosphatemia, rickets, or osteomalacia. OG2 Phex transgenic Hyp mice did exhibit a small, but significant, increase in bone mineral density and dry ashed weight, suggesting a partial mineralization effect from restoration of Phex function in mature osteoblasts. Similarly, retroviral mediated overexpression of Phex in TMOb-Hyp osteoblasts restored Phex mRNA levels, protein expression, and endopeptidase activity but failed to correct their intrinsic mineralization defect. In addition, we failed to detect the Phex substrate FGF-23 in osteoblasts. Taken together, these in vivo and in vitro data indicate that expression of Phex in osteoblasts is not sufficient to rescue the Hyp phenotype and that other sites of Phex expression and/or additional factors are likely to be important in the pathogenesis of XLH. (+info)
Self-assembly and mineralization of peptide-amphiphile nanofibers.
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We have used the pH-induced self-assembly of a peptide-amphiphile to make a nanostructured fibrous scaffold reminiscent of extracellular matrix. The design of this peptide-amphiphile allows the nanofibers to be reversibly cross-linked to enhance or decrease their structural integrity. After cross-linking, the fibers are able to direct mineralization of hydroxyapatite to form a composite material in which the crystallographic c axes of hydroxyapatite are aligned with the long axes of the fibers. This alignment is the same as that observed between collagen fibrils and hydroxyapatite crystals in bone. (+info)
Interaction of bone proteoglycans and proteoglycan components with hydroxyapatite.
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The small leucine-rich proteoglycans (SLRPs) of bone interact with hydroxyapatite (HAP) and are proposed to play an important role in the regulation of the mineralisation process. The present study has examined the interaction of bone SLRPs, purified, liberated bone glycosaminoglycan (GAG) chains and core proteins, as well as commercial chondroitin 4-sulphate (C4S) with HAP. Isotherm data (0.02 M sodium acetate) revealed that the intact proteoglycans (PGs) and bone GAGs showed greater binding onto HAP with higher adsorption maxima than the constituent core proteins and commercial C4S. Adsorption was dependent on pH and ionic strength, increasing with decreasing pH and in the presence of calcium whilst decreasing in the presence of phosphate, suggesting that electrostatic effects are important. The data indicates that PG/GAG chemistry and conformation in solution are significant determinants in the adsorption process and provides important information concerning interfacial adsorption phenomena between the organic-inorganic phases of mineralised systems. (+info)
Isolation and some characterization of an acidic polysaccharide with anti-calcification activity from coccoliths of a marine alga, Pleurochrysis carterae.
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Coccolith is a calcified scale with species-specific fine structure produced by marine unicellular coccolithophorid algae, and consists of calcium carbonate crystals and organic matrices. EDTA-soluble organic materials extracted from coccoliths of Pleurochrysis carterae showed anti-calcification activity. They were separated by anion-exchange HPLC, and two fractions, fractions A and B, were obtained. Fraction B, which was more active than fraction A, was further separated into six consecutive fractions, B1-B6, by second anion-exchange HPLC. 1H NMR spectral analyses of these fractions suggested that a novel acidic polysaccharide, designated CMAP, existed throughout B1-B6 and that the latter four fractions mainly contained another acidic polysaccharide, PS-2, characterized previously. Since PS-2 did not show anti-calcification activity, CMAP was found to be the active principle. (+info)
Effect of polyphenols on calcium content and alkaline phosphatase activity in rat femoral tissues in vitro.
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The effect of various polyphenols on calcium content and alkaline phosphatase activity in the femoral-diaphyseal and -metaphyseal tissues of young rats in vitro was investigated. Bone tissues were cultured for 24 h in serum-free Dulbecco's modified Eagle's medium containing either vehicle or various polyphenols (10(-7) - 10(-4) M). The presence of genistein (10(-6) - 10(-4) M) caused a significant increase in calcium content and alkaline phosphatase activity in the femoral-diaphyseal and -metaphyseal tissues. Resveratrol (10(-4) m) decreased metaphyseal calcium content significantly, and it (10(-6) - 10(-4) M) had a significant inhibitory effect on diaphyseal enzyme activity. Epigallocatechin gallate (EGCg; 10(-4) M) significantly inhibited alkaline phosphatase activity in the diaphyseal and metaphyseal tissues. EGCg (10(-7) - 10(-4) M) had no effect on bone calcium content. Meanwhile, glycitein, quercetin, or catechin in the range of 10(-7) to 10(-4) ml did not have an effect on calcium content and alkaline phosphatase activity in the femoral-diaphyseal and -metaphyseal tissues. The present study suggests that a phytoestrogen genistein has a unique anabolic effect on bone calcification in vitro. (+info)