Alkaline Phosphatase In Chondrocytes Research Articles

Prevention of Osteoporosis in Mice after Ovariectomy via Allograft of Microencapsulated Ovarian Cells

It is believed that estrogen deficiency is one of the major risk factors associated with osteoporosis. To investigate the effects of the transplantation of microencapsulated ovarian cells in estrogen-deficient mice, ovarian cells from female Kunming (KM) mice (6-weeks old) were separated, cultured, and microencapsulated with alginic acid-polylysine-alginic acid. Female KM mice (8-weeks old) were randomly separated into three groups: intact (normal), ovariectomized (OVX), and treatment (OVX+ implantation). Microencapsulated ovarian cells were found to secrete estrogen at normal levels in vitro. Ninety days after transplantation, serum estradiol levels in the OVX group were significantly lower, and the trabecular bone amount and volume were decreased when compared with the normal group. The expression of alkaline phosphatase in chondrocytes appeared lower, while the expression of matrix metalloproteinase 9 (MMP-9) in the bone matrix was higher. The ratio of MMP-9-positive chondrocytes and osteoblasts to osteoclasts was significantly lower than that of the normal group. The concentrations of hydroxyproline (Hyp), Ca, and P in the left femurs of the OVX group were lower than those of the normal group. However, the aforementioned changes were not seen in the treatment group. In conclusion, microencapsulated ovarian cells survive well after transplantation and secrete estrogen in vivo, and they can prevent in some degree osteoporosis caused by ovariectomy.

Ascorbic acid-induced chondrocyte terminal differentiation: the role of the extracellular matrix and 1,25-dihydroxyvitamin D

Chondrocyte terminal differentiation is associated with cellular hypertrophy increased activity of plasma membrane alkaline phosphatase and the synthesis of collagen type X. The hypertrophic phenotype of cultured chondrocytes can be stimulated by ascorbic acid but the underlying mechanisms for this phenotypic change are unclear. As ascorbic acid is central to many hydroxylation reactions, the possibility was examined that its pro-differentiating effects are mediated by its effects on collagen and vitamin D metabolite formation. In vitro studies indicated that ascorbic acid-induced chondrocyte alkaline phosphatase activity was inhibited by the addition of both collagen and proteoglycan synthesis inhibitors. The addition of arginine-glycine-aspartic acid (RGD)-containing peptides also resulted in lower alkaline phosphatase activity. Chicks supplemented with dietary ascorbic acid had higher concentrations of both collagen and proteoglycans within their growth plates but the chondrocyte maturation rate was unaltered. No evidence was obtained to suggest that ascorbic acid-induced collagen production was mediated by lipid peroxidation. In addition, supplementation with dietary ascorbic acid resulted in higher serum 1,25-dihydroxyvitamin D3 concentrations and increased chondrocyte vitamin D receptor number. Ascorbic acid-treated chondrocytes maintained in vitro also had increased vitamin D receptor numbers but chondrocyte receptor affinity for 1,25-dihydroxyvitamin D3 was unaltered. These results indicate that ascorbic acid promotes both chondrocyte matrix production and 1,25-dihydroxyvitamin D3 synthesis, accompanied by upregulation of the vitamin D receptor. Thus, ascorbic acid may be causing amplification of the vitamin D receptor-dependent genomic response to 1,25-dihydroxyvitamin D, resulting in promotion of terminal differentiation. Strong evidence is provided to support the hypothesis that ascorbic acid-induced chondrocyte terminal differentiation is mediated by interactions between integrins and RGD-containing cartilage matrix proteins.

Effects of deferoximine on chondrocyte alkaline phosphatase activity: proxidant role of deferoximine in thalassemia.

The homozygous form of beta-thalassemia, the most common single gene disorder, is treated by red cell transfusion therapy. Following transfusion, the chelator, deferoximine, is administered to patients to remove excess iron. However, when this drug is given to young children, metaphyseal dysplasia and abnormalities of linear growth are frequently observed. To explore the notion that deferoximine interferes with endochondral growth by chelating zinc, we examined the effect of the drug on chondrocytes maintained in long-term culture. We found that deferoximine caused a dose-dependent inhibition of a wide range of functions including cell proliferation, protein synthesis (and possibly under-hydroxylation of type X collagen), and mineral deposition. Directly relevant to the mineralization process was the observation that the drug dramatically lowered the activity of alkaline phosphatase, a zinc-requiring enzyme. To test the hypothesis that enzyme inhibition was due to chelation of zinc by deferoximine, the cell culture medium was supplemented with excess zinc. However, this treatment did not overcome the deferoximine-dependent change in enzyme activity. We next examined the possibility that deferoximine, in the presence of ascorbate, could form a free radical system that would serve to inactivate the enzyme. Using alkaline phosphatase extracted from chick cartilage, we noted that the activity of the phosphatase was markedly reduced in the presence of deferoximine and ascorbate. These effects were consistant with the notion that deferoximine and ascorbate can act as a prooxidant couple. This conclusion was confirmed when we measured the oxidative activities of the system using nitrobule tetrazolium and cytochrome c. Indeed, we noted that deferoximine markedly activates the autocatalytic oxidation of ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)

Fetuin and alpha‐2HS glycoprotein induce alkaline phosphatase in epiphyseal growth plate chondrocytes

A previously described chondrocyte alkaline phosphatase induction factor (CAP-IF) for chicken epiphyseal growth plate chondrocytes has been purified to SDS-PAGE homogeneity from fetal bovine serum by ammonium sulfate precipitation and by dye-ligand affinity (Affi-Gel Blue and Reactive Green-19 agarose) and hydroxyapatite column chromatographies. As determined by immunoprecipitation of [35S]methionine-labeled cellular proteins after 3 day treatment, this highly purified CAP-IF increases the level of AP and certain other membrane proteins 2- to 3-fold over control values. The pure protein of apparent 64.5 kDa molecular weight has been identified as fetuin by N-terminal amino acid sequencing. This was confirmed by the finding that high alkaline phosphatase (AP)-inducing activity is present in fetuin prepared by the Spiro method. However, fetuins prepared by the Pedersen or Deutsch procedures are inactive. At least half of the CAP-IF activity of fetuin was irreversibly destroyed by treatment with EDTA and addition of Zn2+ did not reactivate the EDTA-treated fetuin. Ascorbate synergistically enhanced the effect of fetuin on chondrocyte AP activity by over 8-fold during 3 day exposure. Because of the very high homology between fetuin and the A-chain of alpha 2-HS glycoprotein, we also tested and found that alpha 2HS glycoproteins from human serum and bovine bone are both strong AP inducers. Our findings suggest that the AP-inducing activity resides in a labile, cystatin/Zn(2+)-binding domain common to these related serum glycoproteins. These proteins appear to play a role in enhancing AP expression in normal growth plate cartilage differentiation.

The purification and molecular characterization of alkaline phosphatases from chondrocytes and matrix vesicles of bovine fetal epiphyseal cartilage

Intact matrix vesicles and chondrocytes were obtained from a crude coilagenase digest of bovine fetal epiphyseal cartilage. Alkaline phosphatase activity from the chondrocytes was purified 1800-fold to homogeneity. This was accomplished by solubilizing the chondrocytes in sodium deoxycholate, followed by subsequent gel filtration on Sepharose 6B and DE-52 cellulose chromatography. A 30-fold purification of the matrix vesicle alkaline phosphatase was obtained by solubilization of vesicles with sodium deoxycholate, extraction with n-butanol and gel filtration on Sepharose 6B. The purified chondrocyte alkaline phosphatase possessed a specific activity of 900 μmoles p-nitrophenyl phosphate hydrolyzed/minute/mg purified protein at pH 10.5, while the matrix vesicle alkaline phosphatase has a specific activity of 300 μmoles p-nitrophenyi phosphate hydrolyzed/minute/mg purified enzyme. The native molecular weight of the chondrocytic alkaline phosphatase and the matrix vesicle alkaline phosphatase were determined to be 141,000 and 310,00, respectively, by sucrose density gradient centrifugation. A functional subunit of the matrix vesicle alkaline phosphatase, identical to the isolated chondrocyte alkaline phosphatase, could be derived from the intact matrix vesicle enzyme by chromatography on DE-52 cellulose in the presence of 1 mM β-mercaptoethanol SDS polyacrylamide gel electrophoresis demonstrated that the chondrocytic alkaline phosphatase had one protein band of approximately 18,000 molecular weight and the matrix vesicle alkaline phosphatase had two bands, one of 18,000 and the other of 45,000 molecular weight.