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:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cells obtained from male quail kidneys by digestion with collagenase and hyaluronidase were plated and maintained in a chemically defined, serum-free medium. Culture dishes (35 mm) were inoculated with 1.5 . 10(6) cells which became confluent in 5 days. The cells maintained an epithelial-like morphology over the entire culture period. During a 2 h incubation the cells metabolized 25--30% of the 10 nM 25-hydroxyvitamin D-3 (25-OH-D-3) provided. Seven metabolites were chromatographically separated on Sephadex LH-20. Three have been identified as 1 alpha, 25-dihydroxyvitamin D-3 (1,25(OH)2D-3), 24,25-dihydroxyvitamin D-3 (24,25(OH)2D-3) and 1 alpha, 24,25-trihhydroxyvitamin D-3 (1,24,25(OH)3D-3). The activities of the 25-OH-D-3:1 alpha- and 24-hydroxylases increased eight times faster than the cell number in 5 days. Preincubation of the cells with 10 nM 25-OH-D-3 or 1,25(OH)2D-3 decreased 1,25(OH)2D-3 synthesis, and increased both 24,25(OH)2D-3 and metabolite IV synthesis. The decrease in 25-OH-D-3:1 alpha-hydroxylase activity required a 2 h preincubation with 25-OH-D-3, while stimulation of 25-OH-D-3:24-hydroxylase activity and metabolite IV production required a 6 h preincubation. Incubations of cells for 1 h with parathyroid hormone resulted in a 30-fold increase in cyclic AMP in the medium. A 6 h preincubation with parathyroid hormone decreased 24,25(OH)2D-3) synthesis 50% relative to control cells. These results demonstrate the amenability of this system for studying the regulation of 25-OH-D-3 metabolism, as well as its use for other in vitro studies on renal cell function in a chemically defined culture system.
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PMID:Serum-free culture of Japanese quail kidney cells. Regulation of vitamin D metabolism. 22 48

Transforming growth factor-beta (TGF beta) produced by osteoblasts is present in high levels in bone and influences bone formation, replication of bone cells, and expression of osteoblast protein products. Interactions between bone active hormones and locally released and activated TGF beta were studied by examining the influence of TGF beta preincubation on PTH, calcitonin (CT), and vitamin D receptors in an osteoblastic cell line (UMR 106-06). Preincubation of UMR 106-06 cells with 1 ng/ml TGF beta for 3 days increased specific binding of [125I]PTH-related protein (PTHrP)(1-84) to 140% of that in control cells, but [125I]salmon CT binding decreased to 50% of controls. Binding isotherms indicated that the changes in binding were due to altered receptor numbers since affinities for 125I-labeled PTH and CT remained unchanged. The effect on receptor levels was time dependent, requiring 24 h preincubation with TGF beta for measurable changes, and dose dependent, with maximal effects seen with 1 ng/ml TGF beta. Binding of [3H]1,25(OH)2 vitamin D3 was increased to 130% of control in cytosolic extracts of UMR 106-06 cells pretreated for 3 days with 1 ng/ml TGF beta. Scatchard plots suggested an increase in receptor number without change in affinity. The adenylate cyclase response to PTH increased to 150% of control cells after 3 days of treatment with 1 ng/ml TGF beta; however, the adenylate cyclase response to CT was little changed. Forskolin- and cholera toxin-stimulated adenylate cyclase responses were increased by TGF beta treatment to 130-160% of control, indicating an increase in the stimulatory subunit of the G protein. Increased abundance of both Gs and Gi proteins were indicated by increased cholera toxin- or pertussis toxin-dependent [32P] NAD ribosylation of 47-kilodalton (kDa) and 42-kDa or 40-kDa proteins, respectively, in TGF beta-treated cells. Our data support a complex regulatory effect of TGF beta on UMR 106-06 cells with increases in PTH receptors, vitamin D receptors, and G proteins, whereas there is an apparent down-regulation of CT receptors. TGF beta might induce a more differentiated osteoblast phenotype of these cells, which already express differentiated features such as high alkaline phosphatase activity, PTH and vitamin D receptors, and collagenase production. Since low doses of PTH stimulate bone formation in vivo, TGF beta released or activated at sites of new bone formation might locally modulate PTH activity be allowing increased PTH receptor and postreceptor effectiveness.
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PMID:Transforming growth factor-beta modulates receptor binding of calciotropic hormones and G protein-mediated adenylate cyclase responses in osteoblast-like cells. 132 61

Growth plate cartilage from normal and vitamin D-phosphate deficient (-VDP) rats was cultured to study the production of collagenase and tissue inhibitor of metalloproteinases (TIMP) in vitro. All tissues secreted latent collagenase into the medium at a constant rate during the 5 days in culture. Microdissected-VDP growth plates, containing predominatly hypertrophic cells, released up to 8-fold more collagenase into the medium than either intact-VDP or normal growth plates. TIMP was also secreted during the culture, but its rate of production was not as dependent on tissue type as collagenase. The tissue level of collagenase and TIMP before culture was compared with that found in conditioned medium and remnant tissue after culture. During the 5 day culture period microdissected-VDP growth plates, containing predominatly hypertrophic cells, produced 3-times more collagenase/microgram DNA over the starting level than either intact-VDP or normal growth plates. TIMP was never found in tissues after they had been cultured, but was present in all tissues before culture except those containing predominatly hypertrophic cells. The amount of TIMP required to block collagenase was calculated. Growth plates in culture produced enough TIMP to block all collagenase found in the medium and remnant tissue, while extracts of uncultured intact -VDP growth plates, and those divided to contain hypertrophic cells, had an excess of collagenase over TIMP. The results suggest that hypertrophic cells produce far more collagenase than other cells in the growth plate, but all cell types have about the same capacity to synthesize TIMP. As a result, increased collagenase synthesis by hypertrophic cells may surpass increases in TIMP synthesis and lead to collagen removal. This would allow for thinning of the longitudinal septa and expansion of the hypertrophic cells.
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PMID:Production of collagenase and tissue inhibitor of metalloproteinases (TIMP) by rat growth plates in culture. 196 14

In UMR 106 rat osteosarcoma cells, parathormone (1-34hPTH) and calcitonin (sCT) stimulated adenylate cyclase (AC) activity 5.5-and 2.8-fold, respectively. AC in osteoblasts (OB) from collagenase-treated calvaria of 3-day-old rats responded similarly to 1-34hPTH. In contrast, fibroblasts (mouse fibroblastomas) displayed a marginal 1-34hPTH sensitive AC. Osteoclasts (OC) of collagenase-treated rat calvariae, rat monocytes and mouse macrophages did not demonstrate 1-34hPTH inducable AC activity. Physiological concentrations of 24,25-dihydroxyvitamin D-3 attenuated PTH-sensitive AC in OB and UMR 106 cells within 20 min, while 1,25-dihydroxyvitamin D-3 showed no such immediate effect. In contrast, the AC response to Gpp(NH)p was unaffected by 24,25-(OH)2D3, indicating that 24,25-(OH)2D3 interrupts the coupling of the PTH receptor to the GTP binding protein Gs. OB and UMR 106 cells were also subjected to long-term (48 h) incubation with vitamin D-3 metabolites, 1-34hPTH or 20% serum from patients with secondary hyperparathyroidism (sHBT-serum), respectively. PTH-sensitive AC was markedly attenuated by pre-exposure to both 1-34hPTH and 1,25-(OH)2D3, while minimally affected by corresponding 24,25-(OH)2D3 and 20% sHPT-serum treatment. The secretion of alkaline phosphatase (Alphos) from the two cell types was strongly increased by 1-34hPTH, the effect being abolished by the presence of 24,25-(OH)2D3. Iliac crest biopsies of normal individuals exhibited a clear negative correlation between PTH-sensitive AC and corresponding serum 24,25-(OH)2D3 levels. Basal AC activity was, however, negatively correlated to serum 1,25-(OH)2D3 concentrations. In summary, the results show that 24,25-(OH)2D3 reduces PTH-stimulated AC activity in and Alphos secretion from osteoblastic bone cells by rapidly and directly interfering with the plasma membrane. These data reinforce the probable in vivo significance of 24,25-(OH)2D3. Moreover, the negative correlation between basal AC activity and serum 1,25-(OH)2D3 levels indicates a possible role for 1,25-(OH)2D3 in regulating bone cell synthesis of AC components in vivo.
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PMID:1,25-dihydroxyvitamin D-3 and 24,25-dihydroxyvitamin D-3 affect parathormone (PTH) -sensitive adenylate cyclase activity and alkaline phosphatase secretion of osteoblastic cells through different mechanisms of action. 216 95

In the transition from proliferating to hypertrophic cell zones in the growth plate, there is an increased in chondrocyte cell volume and a corresponding decrease in collagen content to allow for cell enlargement. To substantiate our hypothesis that collagenase is responsible for these changes, growth plates from rats treated with bisphosphonate (HEBP) were compared histologically and biochemically with growth plates from normal and vitamin D and phosphate deficient (-VDP) rats. HEBP-treated rats developed an expanded hypertrophic cell zone (HCZ) characterized by the presence of two distinct populations of hypertrophic cells. The proximal hypertrophic cells were only 2-fold enlarged compared to the proliferating cells, whereas 1/6 of the distal hypertrophic cells were enlarged almost 5-fold and appeared morphologically identical with hypertrophic cells from normal and -VDP rats. The HEBP growth plates were divided into cross-sectional thirds and analyzed for active and latent collagenase. The juxta-metaphyseal (lower 1/3) cartilage contained 100% of the fully enlarged hypertrophic cells and appeared identical to those found in normal and -VDP growth plates, along with 81% of the active and 77% of the total collagenase. Collagenase and tissue inhibitor of metalloproteinases (TIMP) were measured in extracts of similarly divided tissues. The presence of true collagenas was confirmed by using [3H]-telopeptide-free collagen. TIMP levels were inversely related to the presence of active collagenase and cellular hypertrophy. Substantial levels of latent collagenase were found in the extracellular fluid at sites of active collagenolysis, but not in the fluid phase surrounding the 2-fold enlarged hypertrophic cells. It is postulated that increased amounts of active collagenase and insufficient levels of TIMP may account for the reduced collagen content seen in the lower HCZ of both -VDP and HEBP rickets. Unlike active collagenase, which remains localized by binding to collagen, latent enzyme is probably restricted in its mobility throughout the extracellular space by diffusion, itself, or the interstices of the extracellular matrix.
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PMID:Association of collagenase and tissue inhibitor of metalloproteinases (TIMP) with hypertrophic cell enlargement in the growth plate. 255 3

Previous studies have suggested that vitamin D metabolites directly influence the differentiation and maturation of chondrocytes in calcifying cartilage. Recently, this laboratory has shown that the response of chondrocyte plasma membrane and matrix vesicle enzymes to 1,25-(OH)2D3 and 24,25-(OH)2D3 is both cell and membrane specific. The current study demonstrates that cell replication and matrix protein synthesis are also modulated by vitamin D. Confluent, third-passage growth zone (GC) and resting zone (RC) costochondral chondrocytes were incubated in medium containing 10(-13)-10(-7) M 1,25-(OH)2D3 or 10(-12)-10(-6) M 24,25-(OH)2D3. The amount of collagenase-digestible protein (CDP) secreted into the media was inversely proportional to the concentration of fetal bovine serum (FBS). At 10% FBS, greater than 80% of the CDP was incorporated into the matrix. 1,25-(OH)2D3 stimulated CDP and percentage collagen synthesis by GC cells but had no effect on the synthesis of noncollagenous protein (NCP). 1,25-(OH)2D3 inhibited CDP and percentage collagen synthesis by RC cells but did not alter NCP synthesis. [3H]thymidine incorporation was inhibited in both cell types, whether confluent or subconfluent cultures were examined. At 10(-6) and 10(-7) M 24,25-(OH)2D3, there was a significant decrease in CDP production and percentage collagen synthesis by RC cells but no effect on NCP. However, at 10(-9) and 10(-10) M hormone there was an increase in NCP production but no effect on CDP, resulting in a decrease in percentage collagen synthesis. CDP and NCP production were unaffected by 24,25-(OH)2D3 in GC cells. High concentrations of hormone inhibited [3H]thymidine incorporation in both cell types.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of vitamin D metabolites on collagen production and cell proliferation of growth zone and resting zone cartilage cells in vitro. 278 22

The effect of Ca2+ ion concentration on the 25 hydroxylation of tritiated cholecalciferol (3HD3) was investigated using homogenates of ovine liver from vitamin D replete sheep. A significant decrease in the production of 25 hydroxycholecalciferol (25OHD3) was observed when the concentration of Ca2+ in the homogenate was raised above 0.68 mmol/l by the addition of calcium gluconate. Similarly, a final concentration of 37 mumol EGTA/1 (equivalent to a Ca2+ concentration of 26.5 nmol/l) was associated with a 50% reduction of 25OHD3 production. That is, a broad bell-shaped relationship was observed between the production of 25OHD3 and the Ca2+ concentration in the homogenate. These changes in the rate of production of 25OHD3 were reproduced with hepatocytes from vitamin D replete rats, prepared by collagenase perfusion, using the drugs dantrolene sodium (DaNa) to reduce (ED50 = 57 mmol/l) and veratridine to increase (ED50 = 550 mmol/l) the intracellular Ca2+ concentration. Hepatocytes from vitamin D replete rats also showed a reduction in 25 hydroxylation of D3 (ED50 = 6 ng/ml) in response to the addition of 1-25 dihydroxycholecalciferol (1-25 (OH)2D3). The calmodulin antagonists; W7, compound 48/80, trifluoperazine (TFP) and calmidazolium (R24571) were all found to effect a dose response inhibition of the 25 hydroxylation of cholecalciferol by homogenates of ovine liver. R24571 had a similar inhibitory effect (ED50 = 70 mumol/l) upon the 25 hydroxylase enzyme of rat hepatocytes. It is concluded that the 25 hydroxylation of cholecalciferol in liver of vitamin D replete rats and sheep is calcium sensitive and is reduced in the presence of increased concentrations of 1,25(OH)2D3. Calmodulin may also be involved in the regulation of hepatocyte 25-hydroxylase activity by Ca2+.
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PMID:The involvement of intracellular calcium ion concentration and calmodulin in the 25-hydroxylation of cholecalciferol in ovine and rat liver. 282 May 80

The presence of a specific receptor for 1,25-dihydroxy-vitamin D3 was investigated in myoblasts released from chick embryo skeletal muscle by trypsin and collagenase treatment. Density gradient analysis of the cytosol obtained from these muscle cell preparations showed that 1,25-dihydroxy-vitamin D3 binds specifically to a 3.7 S macromolecule. Scatchard analysis yielded an equilibrium dissociation constant of 2.46 x 10(-10) M and a Nmax of 74 fmol/mg of cytosol protein. The data is in agreement with previous evidence which indicates that the action of the vitamin D metabolite on muscle Ca uptake is mediated by de novo protein and RNA synthesis, and supports the concept that muscle is a target organ for 1,25-dihydroxy-vitamin D3.
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PMID:Presence of a 1,25-dihydroxy-vitamin D3 receptor in chick skeletal muscle myoblasts. 298 76

In the transition from proliferation to hypertrophic cell zones in the growth plate, there is an increase in chondrocyte volume and a corresponding decrease in collagen content to accommodate the enlarging cells. It is postulated that collagenase accounts for this collagen loss. To test this hypothesis, tibial growth plates were obtained from normal rats, rachitic rats deficient in vitamin D and phosphate, and rats after 48 and 72 h of healing from rickets. Collagenase was quantitated by a pellet assay based on the release of solubilized collagen from the endogenous insoluble collagen in the tissue homogenates. A fourfold greater collagen release and a concomitant sixfold greater hypertrophic cell volume were measured in rachitic growth plates compared with normal age-matched controls. During healing of rickets, collagenase activity and hypertrophic cell volume returned almost to control levels. Rachitic growth plates were dissected into the juxtaepiphyseal 1/3 and the juxtametaphyseal 2/3. The latter portion contained greater than 95% of the hypertrophic cells and 86% of the collagenase. The collagen-degrading activity was extracted from this region and was shown to be a true collagenase by its production of typical A fragments of tropocollagen produced by collagenase action. The enzyme was activated by aminophenylmercuric acetate and trypsin and was inhibited by EDTA, 1,10-phenanthroline, and a tissue inhibitor of metalloproteinases from human articular cartilage. Inhibitors of aspartic, cysteine, and serine proteases had no effect. Micropuncture fluids aspirated from rachitic cartilage contained latent collagenase activity, indicating an extracellular localization. Negative tests for hemoglobin in the rachitic cartilage samples indicated that there was no contamination by capillaries and that this was not a source of collagenase. It is concluded that extracellular collagenase accounts for the loss of cartilage matrix in the hypertrophic zone, and that this process may be distinct from that of capillary invasion.
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PMID:Localization of collagenase in the growth plate of rachitic rats. 299 64

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 of 3H-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 of 3H-proline incorporation into CDP at doses from 10(-10)M to 10(-7)M, with maximal suppression reaching 30% of control. The effect was specific for collagen, since 3H-proline incorporation into NCP was unaffected. Hydroxyproline analysis of bone explants and culture medium revealed a 1,25(OH)2D3-induced decrease in the 3H-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, impaired 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(-7)M. 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 an in vitro model for the study of bone formation.
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PMID:Avian medullary bone in organ culture: effects of vitamin D metabolites on collagen synthesis. 301 64


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