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1.
Matrix vesicles are extracellular organelles produced by cells that mineralize their matrix. They contain enzymes that are associated with calcification and are regulated by vitamin D metabolites in a cell maturation-dependent manner. Matrix vesicles also contain metalloproteinases that degrade proteoglycans, macromolecules known to inhibit calcificationin vitro, as well as plasminogen activator, a proteinase postulated to play a role in activation of latent TGF-\. In the present study, we examined whether matrix vesicle metalloproteinase and plasminogen activator are regulated by 1,25(OH)2D3 and 24,25 (OH)2D3. Matrix vesicles and plasma membranes were isolated from fourth passage cultures of resting zone chondrocytes that had been incubated with 1010-10-7 M24,25(OH)2D3 or growth zone chondrocytes incubated with 10-11-l0-8 M 1,25(OH)2D3, and their alkaline phosphatase, active and total neutral metalloproteinase, and plasminogen activator activities determined. 24,25(OH)2D3 increased alkaline phosphatase by 35–60%, decreased active and total metalloproteinase by 75%, and increased plasminogen activator by fivefold in matrix vesicles from resting zone chondrocyte cultures. No effect of vitamin D treatment was observed in plasma membranes isolated from these cultures. In contrast, 1,25(OH)2D3 increased alkaline phosphatase by 35–60%, but increased active and total metalloproteinase three- to fivefold and decreased plasminogen activator by as much as 75% in matrix vesicles isolated from growth zone chondrocyte cultures. Vitamin D treatment had no effect on plasma membrane alkaline phosphatase or metalloproteinase, but decreased plasminogen activator activity. The results demonstrate that neutral metalloproteinase and plasminogen activator activity in matrix vesicles are regulated by vitamin D metabolites in a cell maturation-specific manner. In addition, they support the hypothesis that 1,25(OH)2D3 regulation of matrix vesicle function facilitates calcification by increasing alkaline phosphatase and phospholipase A2 specific activities as well as metalloprotemases which degrade proteoglycans.  相似文献   

2.
Summary The present investigation was undertaken to study the role of carbonic anhydrase in 1,25 dihydroxyvitamin D3-induced bone resorption. Calvaria were removed from 5- to 6-day-old mice and cultured for periods up to 96 h in Dulbecco's Modified Eagle Medium (high glucose, 4,500 mg/dl) supplemented with antibiotics and either heat-inactivated horse and fetal calf sera or bovine serum albumin. The experimental cultures contained 1×10−8 M 1,25 dihydroxyvitamin D3 (1,25(OH)2D3). All cultures were incubated at 37°C in 5% CO(in2)/95% air. Bone resorption was assessed by release of stable calcium into the medium. Bone enzymes (acid and alkaline phosphatases and carbonic anhydrase) were determined following homogenization in 0.25 M sucrose. The effects of 1,25(OH)2D3 were studied in the presence and absence of the carbonic anhydrase inhibitor acetazolamide and its analogue (CL 13,850), which lacks inhibitory activity. Acetazolamide inhibited 1,25(OH)2D3-induced calcium release in a dose-dependent fashion from 10−5–10−4 M. When added to the cultures at a concentration of 1×10−4 M, acetazolamide completely blocked the 1,25(OH)2D3-induced calcium release, a phenomenon not seen with an equimolar concentration of CL 13,850. The most significant finding was that 1,25(OH)2D3-induced calcium release was accompanied by a significant increase in the carbonic anhydrase activity of bone at both 48 (treated/control ratio=2.05) and 96 (treated/control ratio=2.59) hours. Bone alkaline phosphatase activity decreased and acid phosphatase activity increased in response to 1,25(OH)2D3. These findings support the concept that carbonic anhydrase is involved in bone resorption inducedin vitro by certain calcemic hormones and related compounds.  相似文献   

3.
Summary The present study was undertaken to test the in vitro action of aluminum on bone phosphatase activities and the possible interaction of this metal with parathyroid hormone (bPTH) or vitamin D3 dihydroxymetabolites [1,25- and 24,25(OH)2D3). Three-day-old rat calvaria were incubated for 24 h with one of the following: bPTH at 5×10−8M, 1,25-or 24,25(OH)2D3 at 2.5×10−9M, Al at concentrations ranging from 3×10−11M to 6×10−6M, or their corresponding solvents. Al effects were also investigated when the medium phosphate or calcium concentrations were modified. In some experiments, Al was added simultaneously with bPTH or one of the vitamin D3 metabolites at the beginning of the 24 h incubation. At the end of all incubations, acid and alkaline phosphatase activities were measured in bone cytoplasmic extract. The results show that: (a) When compared to the value found in half calvaria incubated in a control medium, the bone acid and alkaline phosphatase content is significantly higher in paired halves incubated with Al (3×10−11M to 1.5×10−6M) as well as with bPTH, 1,25-, or 24,25(OH)2D3 and sharply decreased with higher Al concentrations (6×10−6M). (b) The Al effect on phosphatase activities is modified in a free phosphate or a free calcium medium. (c) The presence of Al at 1.5×10−6M or 6×10−6M significantly decreases the bPTH or 1,25(OH)2D3-induced stimulation of bone phosphatase activities. (d) A similar interaction could not be found between Al and 24,25-(OH)2D3.  相似文献   

4.
Summary A new organ culture system for the study of bone metabolism has been developed using chicken medullary bone. The presence of viable bone cells in culture was demonstrated by histological and histochemical techniques. Incorporation of3H-proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP) was determined using purified bacterial collagenase. Collagen accounted for approximately 10–15% of the total protein labeled. The addition of 1,25-dihydroxycholecalciferol (1,25 (OH)2D3) resulted in a dose-dependent inhibition of3H-proline incorporation into CDP at doses from 10−10M to 10−7M, with maximal suppression reaching 30% of control. The effect was specific for collagen, since3H-proline incorporation into NCP was unaffected. Hydroxyproline analysis of bone explants and culture medium revealed a 1,25(OH)2D3-induced decrease in the3H-hydroxyproline content of the system (bone + medium), suggesting that the effect of 1,25(OH)2D3 is due to inhibition of collagen synthesis rather than enhanced collagen degradation, impaiored incorporation of collagen into bone matrix, or bone resorption Medullary bone collagen synthesis was not affected by 24,25(OH)2D3, either alone or in combination with 1,25(OH)2D3. Structure-activity studies of vitamin D metabolites showed that 1,25(OH)2D3 and 1,24,25(OH)3D3 were the most potent metabolites tested, followed by 1-alpha(OH)D3. 25(OH)D3 and 24,25(OH)2D3 had no effect at concentrations as high as 10−7M. These results indicate a possible role for vitamin D in the regulation of medullary bone formation during the reproductive cycle of the egg-laying hen, and suggest the potential utility of medullary bone as anin vitro model for the study of bone formation  相似文献   

5.
Summary Calvarial bones from hypophosphatemic (Hyp) mice and normal littermates were cultured in a chemically defined medium to determine: (a) the effect of medium phosphate (Pi) concentration (1, 2, and 3 mM) on collagen synthesis; (b) the effect of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] (10−12M–10−7M) on collagen synthesis; and (c) whether bone responsiveness to 1,25(OH)2D3 was affected by changes in medium Pi concentration. Bone collagen synthesis was evaluated by measuring [3H]hydroxyproline formation. The distribution of labeled hydroxyproline between bone explant and culture medium (total and dialyzable fraction) was studied. These experiments confirm that 1,25(OH)2D3 inhibits specifically bone collagen synthesis in vitro. We did not detect any effect of medium Pi concentration on basal collagen synthesis but were able to demonstrate that lowering medium Pi concentration increased the 1,25(OH)2D3-induced inhibition of collagen synthesis. Bones from both genotypes responded to 1,25(OH)2D3, but modulation of this response by changes in Pi concentration was altered in Hyp bone as, in contrast to normal bone, its response to 1,25(OH)2D3 was unaffected when medium Pi concentration was decreased from 3 to 2 mM. These findings support the hypothesis of an altered response of bone to 1,25(OH)2D3 in the Hyp mouse.  相似文献   

6.
Summary Osteoblastic cells were isolated from periosteum-stripped parietal bones of neonatal rat calvaria, seeded at low density (5,000 cells/35 mm of Falcon dish), and cultured for 6 days in BGJ medium supplemented with 20% of vitamin D-depleted FCS or vitamin D and calcium-depleted FCS, with daily addition of 1,25 dihydroxyvitamin D3 (10−9 M) or 24,25-dihydroxyvitamin D3 (10−9 M). Plating efficiency, clonal growth (number and size distribution of the colonies formed), and the alkaline phosphatase phenotype were evaluated on days 2 and 6 of culture. (1) Culture for 6 days in media not supplemented with 1,25(OH)2D3 led to a significant (P<0.001) loss of the alkaline phosphatase phenotype of the osteoblastic cells; the loss was greater in proliferating cells than in nonproliferating ones and occurred in both 0.12 mM or 1.1 mM ionized calcium concentrations. (2) Daily addition of 1,25(OH)2D3 (10−9 M) but not 24,25(OH)2D3 maintained the basal percentage of Alk Pase positive cell units in nonproliferating cells and significantly reduced the loss of this phenotype in proliferating colonies. (3) This effect did not stem from an action of the hormone on cell growth. 1,25(OH)2D3 was also found to enhance the adhesiveness of the seeded osteoblasts, irrespective of the medium calcium concentration.  相似文献   

7.
Growth plate chondrocytes are affected by 1,25(OH)2D3 and androgens, which may critically interact to regulate proliferation and differentiation during the male pubertal growth spurt. We investigated possible interactions of 1,25(OH)2D3 and the non-aromatizable androgen dihydrotestosterone (DHT) in primary chondrocyte cultures from young male rats. DHT and 1,25(OH)2D3 independently stimulated DNA synthesis and cell proliferation in a dose-dependent manner with maximally effective doses of [10-8 M] and [10-12 M], respectively. Both DHT and 1,25(OH)2D3 stimulated the expression and release of IGF-I, and the proliferative effects of each hormone were prevented by an IGF-I antibody. DHT and 1,25(OH)2D3 increased messenger RNAs (mRNAs) of their cognate receptors and of IGF-I receptor mRNA (IGF-I-R). 1,25(OH)2D3 also stimulated mRNA of the androgen receptor (AR), whereas DHT did not affect mRNA of the vitamin-D receptor (VDR). Coincubation with both steroid hormones did not stimulate receptor mRNAs more than either hormone alone. The proliferative effects of DHT and 1,25(OH)2D3 were completely inhibited by simultaneous incubation with both hormones, despite potentiation of IGF-I synthesis. In contrast, both hormones synergistically stimulated cell differentiation as judged by alkaline phosphatase activity, collagen X mRNA, and matrix calcification in long-term experiments. We conclude that DHT and 1,25(OH)2D3 interact with respect to chondrocyte proliferation and cell differentiation. The proliferative effects of both hormones are mediated by local IGF-I synthesis. Simultaneous coincubation with both hormones blunts the proliferative effect exerted by either hormone alone, in favor of a more marked stimulation of cell differentiation.  相似文献   

8.
Summary The in vitro effects of vitamin D3 metabolites, parathyroid extract (PTE), purified parathyroid hormone (bPTH), vitamin A, and heparin on acid and alkaline phosphatases in rat or mouse calvaria in culture were investigated. Results show that: (a) when compared to values found in half calvaria incubated for 24 h in control medium, the bone acid and alkaline phosphatase content is significantly higher in paired halves incubated with PTE (1 USP/ml), bPTH (4×10−8M), heparin (5 USP/ml), vitamin A (23 USP/ml), 25-(OH)D3 (2.5×10−11 to 2.5×10−8M), 24,25-(OH)2D3, and 1,25(OH)2D3 (2.5×10−12 to 2.5×10−7M); (b) the presence of 24,25-(OH)2D3 at low concentrations in the incubation medium decreases significantly the PTE, bPTH, vitamin A, or heparin induced stimulation of the phosphatase activities. This interaction is also observed when measuringβ glucuronidase and glucose-6-phosphatase activities and45Ca release from previously labeled mouse calvaria; (c) a similar activity could not be found with 1,25-(OH)2D3 suggesting that 24,25-(OH)2D3 may have a specific role in bone metabolism.  相似文献   

9.
Summary The active vitamin D metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] causes marked suppression of both pre-proparathyroid hormone messenger RNA (pre-proPTH mRNA) and parathyroid hormone (PTH) secretion. These effects are dose dependent and reversible when tested in anin vitro primary tissue culture cell system using normal bovine parathyroid cells. In the current studies, the precursors of 1,25(OH)2D3 and the related metabolite 24,25-dihydroxyvitamin D3 [24,25(OH)2D3], were used in the same culture system to test for possible regulatory effects. The results were compared with identically prepared cells exposed to 1,25(OH)2D3. In short-term studies (30–120 minutes), none of the vitamin D-related compounds produced any effect on PTH secretion. In long-term studies (24–48 hours, using primary tissue culture in the presence of test agents), neither vitamin D3 nor 25(OH)D3 affected PTH secretion or pre-proPTH mRNA over the concentration range 10−11–10−7M. On the other hand, 24,25(OH)2D3 produced significant suppression of both pre-proPTH mRNA (77% of control,P<.01) and PTH secretion (75% of control,P<.005) at 10−7 M. By comparison, 10−11 M 1,25(OH)2D3 produced levels of suppression (25–30%) of both pre-proPTH mRNA and PTH secretion comparable to 10−7 M 24,25(OH)2D3, while even greater suppression (40–50%) occurred at 10−9-10−7 M 1,25(OH)2D3. From these studies, we conclude that vitamin D3 and 25(OH)D3 do not have significant effects on PTH synthesis and secretion over the range of doses tested. Compared with 1,25(OH)2D3, 24,25(OH)2D3 exhibits mild suppression at pharmacologic concentrations. The effect of 24,25(OH)2D3 prabably occurs through weak interaction of 24,25(OH)2D3 with the 1,25(OH)2D3 receptor.  相似文献   

10.
Summary Binding of [3H] 1,25 (OH)2D3 and effects of 1,25 (OH)2D3 on cell ultrastructure were evaluated in vascular smooth muscle cells (VSMC) primary cultures (aortic media). Specific reversible binding of [3H] 1,25 (OH)2D3 by a 3.5 S macromolecule with DNA binding, KD 6.2×10−10M and Nmax 16 fmol/mg protein was demonstrated. Incubation of VSMC with 10−8 M 1,25 (OH)2D3, but not 25 (OH)D3, in the presence of 10% FCS for up to three weeks caused rapid reversible appearance in the cytoplasm of membrane-bounded electron-dense lysosomal particles which on electronspectroscopic imaging contained Ca and Pi. VSMC are targets for vitamin D.  相似文献   

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