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)

During bone remodeling, activation of resorption is followed by a cycle of formation and this ordered sequence of events has long suggested that local interactions between osteoclasts and osteoblasts are an important regulatory mechanism in bone metabolism. To study this phenomenon, we have prepared bone cells containing primarily osteoclasts by brief digestion of mice calvariae in collagenase, overnight attachment to polystyrene tissue culture flasks in serumless medium supplemented with OB (osteoblast) cell conditioned medium and subsequent growth in low serum. These OC (osteoclast) cells were found to be highly enriched in acid phosphatase activity and expressed cAMP responses to PTH (parathyroid hormone) and prostaglandin E2 but exhibited no PTH-stimulated hyaluronate synthesis in contrast to prostaglandin E2. PTH effects on hyaluronate, however, could be restored upon coculture of OC cells with OB cells (noncontact) or with OB cell conditioned medium, thereby suggesting that OB cells regulate OC cell PTH responsiveness and/or differentiation by soluble cell products secreted into the medium.
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PMID:Paracrine interactions in bone-secreted products of osteoblasts permit osteoclasts to respond to parathyroid hormone. 632 52

The purpose of the present study was to investigate the mechanism of action on bone of Benzo(B)Thiophene-2-Carboxylic Acid (BL-5583). BL-5583, at a dose range of 0.01-100 micrograms/ml, inhibited spontaneous as well as A23187 and PTH-induced bone resorption in tissue culture. This compound also decreased calcium uptake in both osteoclastic and osteoblastic enriched bone cell populations obtained by sequential collagenase digestion of 1-2 day newborn rat calvariae. The decrease occurred after a 5 min. incubation with 45Ca and BL-5583. The effective dose range was 0.01-100 micrograms/ml. No effect on leucine incorporation or lactic acid production by bone cells was observed. BL-5583 also induced a transient decrease in calcium uptake in skin cells isolated from fetal rats by collagenase digestion, suggesting a lack of tissue specificity for this compound. No effect on cyclic AMP in isolated bone cells was observed with the same dose range that produced a calcium effect.
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PMID:Benzo(B)thiophene-2-carboxylic acid: calcium uptake and cyclic AMP production in isolated bone cells. 633 14

An isolated osteoblast-like cell line (MMB-1) was used to study the hormonal regulation of collagen synthesis in bone cells. Collagen synthesis was measured by incorporation of [3H]proline into collagenase-digestible and collagenase-non-digestible proteins after exposure of the cells in culture to varying concentrations of PTH, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], osteoclast-activating factor, and insulin. Collagen synthesis was inhibited by 10(-10) M 1,25-(OH)2D3 and 3 X 10(-10) M PTH after 9-12 h of treatment. Osteoclast-activating factor at 10(-10) M also inhibited collagen synthesis. Insulin at 10(-8) M increased collagen synthesis without stimulating proline incorporation into noncollagen proteins. No effect on collagen synthesis was observed with 24,25-(OH)2D3. Inhibition of collagen synthesis was also observed when cells were treated with either 3 X 10(-5) M 8-bromo-cAMP or 3 X 10(-5) M (Bu)2cAMP. For all agents tested, the onset of the effects was gradual, with differences from controls beginning at 4-8 h, and maximal effects occurring only after 24 h or more of treatment. The collagen synthesized by these cells remained associated primarily with the cell monolayer and was estimated to be greater than 90% type I collagen. No detectable changes in the type or composition of collagen synthesized were found with any of the hormonal treatments. These studies indicate that the synthesis of collagen in bone cells is under multihormonal control, with both cAMP-dependent and cAMP-independent mechanisms involved. The MMB-1 cell line offers a suitable model system for studies of the interactions of hormones in the control of bone turnover.
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PMID:Multiple hormonal mechanisms for the control of collagen synthesis in an osteoblast-like cell line, MMB-1. 633 52

Stimulators of bone resorption, such as PTH, 1,25-dihydroxycholecalciferol [1,25-(OH)2D3], or prostaglandin E2 (PGE2), do not cause calcium release from cultured calvaria of the genetically determined osteopetrotic microphthalmic (mi/ mi) mouse, due to a defect in the function of osteoclasts. To investigate the capacity of cells of mi/mi bone to degrade collagen, calvaria of 1- to 3-day-old normal and mi/mi littermates were labeled in vivo with [3H]proline 16 h before removal, followed by culture in resorption medium. PTH, 1,25-(OH)2D3, and PGE2 stimulated the release of 3H-labeled material into the culture medium from both normal and mi/mi calvaria. The labeled substance released was of collagenous origin, as indicated by its content of hydroxyproline and susceptibility to collagenase. PTH also stimulated the release of 3H-labeled materials from normal calvaria labeled in vivo 112 h before the mice were killed, but had little or no effect on 3H release from the mi/mi bone, indicating that only noncalcified collagen is susceptible to hormone-stimulated degradation in osteopetrotic bone. We conclude that a portion of the hormone-stimulated resorptive mechanism, namely collagenolysis, is functional in bone of mi/mi mice. This result helps to pinpoint the resorptive defect in mi/ mi bone to a failure to dissolve mineral, rather than a more general phenomenon of failure to remove both mineral and matrix.
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PMID:A collagenolytic response to parathormone, 1,25-dihydroxycholecalciferol D3, and prostaglandin E2 in bone of osteopetrotic (mi/mi) mice. 657 20

Sequential collagenase digestion of mice calvariae provides populations of bone cells that express either osteoclasts (OC) or osteoblastic (OB) activities after growth for 6 days in similar culture conditions consisting of minimal essential medium supplemented with 10% fetal calf serum (FCS). The OC characteristics (acid phosphatase activity and hyaluronate synthesis, and their stimulation by PTH) were recovered in the cell populations released early from calvariae, but these also contained OB cells and numerous spindle-shaped alkaline phosphatase positive cells that resembled fibroblasts. We have attempted to select for growth of OC cells in these early populations by exploiting differences in growth requirements of OC, OB, and fibroblastic cells. We find that after growth for 6 days in low serum (2% FCS), OC cell populations demonstrated a threefold increase in OC activity/cell, and cell yield was reduced to one-third of that obtained in 10% FCS. Spindle-shaped cells were absent in 2% FCS and OB marker activities (alkaline phosphatase and citrate decarboxylation) were reduced threefold. In contrast to OC cells, high serum (10% FCS) favored the growth and phenotypic expression of OB cells (late populations). Cell yield and OB marker activities/cell were twofold higher in OB cells grown in 10% FCS vs 2% FCS, whereas growth but not phenotypic expression was retained at 5% FCS. These data suggest that differential serum dependence of OC and OB cells may provide a basis for further enrichment for each cell type following sequential digestion.
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PMID:Differential serum dependence of cultured osteoclastic and osteoblastic bone cells. 665 53

Cell aggregates of bovine parathyroid tissue were prepared by limited collagenase digestion and placed in culture in Weymouth's MB752/1 (calcium = 3.3 mg/100 ml) containing 5% fetal bovine serum and supplemented with insulin alone, or insulin, hydrocortisone, transferrin and epidermal growth factor. Only insulin was required for the maintenance of PTH secretion over a 9-day period. The cell aggregates spread to form monolayer in 3-5 days. The majority of the cells in monolayer were polygonal with well-defined borders. Nuclei were round and the cytoplasm was free of vacuoles. Cell cultures responded to secretory stimulation by low calcium or by isoproterenol with increases in the secretion of PTH and SP-1. At low calcium, about 18% of both the cellular PTH and SP-1 was secreted per hour, and up to 50% of the cell content of these proteins was released per hour upon stimulation by isoproterenol and low calcium combined. The responses to calcium and isoproterenol decreased as a function of time in culture, and calcium responses often disappeared completely by 10 days of culture. When cells were cultured in medium containing a higher (5 mg%) than standard concentration of calcium between days 3-6 of culture, the degree of secretory inhibition attainable with high calcium was greater than that of cells cultured in the standard medium. When secreted hormonal peptides were separated by SDS-gel electrophoresis prior to RIA, it was found that the secretion of intact hormone was sensitive to calcium. For every molecule of PTH secreted into the medium, 1.5-2 mole-equivalents of carboxyl fragments were also released. Calcium control of fragment release was not as stringent as that of PTH release.
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PMID:Primary monolayer cell culture of bovine parathyroids: effects of calcium, isoproterenol and growth factors. 686 97

Suppression of PTH secretion by extracellular calcium is mediated by a plasma membrane calcium receptor (CaR). However, primary cultures of bovine parathyroid cells are known to quickly lose their responsiveness to extracellular calcium. The present study was designed to determine if the loss of calcium responsiveness is due to changes in CaR expression. In primary monolayer cultures of parathyroid cells, calcium-mediated suppression of PTH was still evident after 24 hours in culture but was completely absent after 6 days. This was preceded by a 75% drop in CaR mRNA content within 24 hours. CaR mRNA levels remained low for the 6-day culture. Earlier time points, examined in parathyroid cell suspensions, showed a 70% drop in CaR mRNA by 4 hours after collagenase-dispersion of the glands and an 85% drop after 24 hours. The decreased expression of CaR mRNA was not influenced by altering medium serum, calcium, or 1,25-dihydroxyvitamin D3. Our results indicate that the loss of responsiveness of cultured parathyroid cells to calcium is due to decreased CaR mRNA and, presumably, CaR protein expression.
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PMID:Loss of calcium responsiveness in cultured bovine parathyroid cells is associated with decreased calcium receptor expression. 762 22

We recently showed that osteogenic protein-1(OP-1), a bone morphogenetic protein member of TGF-beta superfamily, induces endochondral bone formation in vivo, and stimulates growth and differentiation of osteoblasts in rat calvarial-derived cell cultures. In the present study, we examined the effect of OP-1 on cell growth and expression of markers that are characteristic of osteoblast phenotype using the clonal rat osteosarcoma cells (ROS 17/2.8). A comparison of OP-1 and TGF-beta 1 effects on cell growth showed that, both OP-1 and TGF-beta 1 inhibited DNA synthesis up to 90 percent and 60 percent of the controls at concentrations of 10 ng/ml and 1 ng/ml, respectively, in serum-free medium. In the presence of 5% serum, TGF-beta 1 did not have any significant inhibitory effects while 40 ng OP-1/ml inhibited the DNA synthesis up to 80% of the controls. Examination of collagen synthesis showed that 40 ng OP-1/ml increased the expression of type I collagen mRNA, and thus increased collagen synthesis (4-fold), as examined by collagenase-digestible protein. Evaluation of markers that are characteristic of the osteoblast phenotype demonstrated that OP-1 stimulated cAMP production in response to PTH (10-fold at 200 ng/ml), alkaline phosphatase specific activity (ALPase) (4-fold at 80 ng/ml), and osteocalcin (OC) synthesis (4.5-fold at 40 ng/ml). Northern blot analysis revealed that OP-1 increased mRNA expression for both ALPase and OC in a dose-dependent manner. These data collectively demonstrate that OP-1 suppresses cell proliferation and stimulates the expression of markers characteristic of osteoblast phenotype in rat clonal osteoblastic osteosarcoma cells (ROS 17/2.8).
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PMID:Osteogenic protein-1 (BMP-7) inhibits cell proliferation and stimulates the expression of markers characteristic of osteoblast phenotype in rat osteosarcoma (17/2.8) cells. 773 48

To determine how progestins increase bone formation in vivo, the effects of the synthetic progestin norethindrone (NET), on aspects of bone formation in vitro were determined. NET at picomolar concentrations in vitro stimulated the proliferation of human TE85 osteosarcoma cells as assessed by the increase in [3H]thymidine incorporation into DNA and in cell number and also stimulated the release of osteocalcin in both the presence and absence of 10 nM 1,25-(OH)2D3. NET increased cellular alkaline phosphatase specific activity (an index of osteoblastic differentiation), but at much higher concentrations, that is, nanomolar. These findings suggest that low concentrations of NET act directly on human TE85 osteosarcoma cells to stimulate their proliferation, differentiation, and cell activity. Furthermore, mitogenic doses of NET stimulated bone collagen synthesis both in a chicken calvarial organ culture assay (assessed by the incorporation and hydroxylation of [3H]proline) and in a human TE85 osteosarcoma cell culture assay (determined by the incorporation of [3H]proline into collagenase-digestible proteins). In contrast, NET at 10(-6)-10(-12) M had no apparent effect on the rate of basal or PTH-stimulated release of 45Ca from prelabeled mouse calvariae in vitro. In summary, this study has demonstrated for the first time that picomolar NET acted directly on human TE85 osteosarcoma cells to increase (1) cell proliferation and differentiation, (2) osteoblastic activity (i.e., osteocalcin synthesis), and (3) bone collagen synthesis in vitro. The same doses of NET in vitro did not reduce the bone resorption rate under our assay conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Picomolar norethindrone in vitro stimulates the cell proliferation and activity of a human osteosarcoma cell line and increases bone collagen synthesis without an effect on bone resorption. 805 99

PTH administration in vivo increases osteoblast number and activity, resulting in increased bone formation, and also increases osteoclast-mediated bone resorption. Studies in vitro, however, have shown that the actions of PTH on osteoblast-like cells are inhibitory and catabolic, as shown by decreases in growth rate and collagen synthesis and increases in collagenase production. The present studies were designed to investigate possible mechanisms for these observations by examining the effects of PTH on the response of osteoblast-like cells to the osteoblast growth factor, epidermal growth factor (EGF). Confluent cultures of UMR 106-01 cells were treated with rat PTH-(1-34) for periods up to 72 h, and EGF receptors were measured with [125I]EGF. PTH, in a dose- and time-dependent manner, increased the number of EGF receptors 2-fold. The half-maximal effect of PTH occurred at a concentration of 1 nM, the same PTH concentration that resulted in half-maximal increases in cAMP generation. The increase in EGF binding was associated with an enhanced biological effect, as shown by augmentation of EGF-stimulated diglyceride production. The effect of PTH could be reproduced by the addition of 8-bromo-cAMP, but not by the phorbol ester phorbol myristate acetate. In the presence of cyclohexamide, the effect of PTH on EGF binding was abolished, suggesting that new protein synthesis was required to increase the number of EGF receptors. Northern blots of total RNA, using a cDNA probe encoding the extracellular domain of the rat EGF receptor, revealed that PTH treatment resulted in a 2- to 3-fold increase in the level of EGF receptor mRNA. These data suggest that the proliferative effects of PTH on the osteoblast may be mediated indirectly by a PTH-induced increase in the number of EGF receptors.
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PMID:Parathyroid hormone increases the expression of receptors for epidermal growth factor in UMR 106-01 cells. 813 37


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