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)

We studied the effects of parathyroid hormone (PTH) on two markers of the osteoblast phenotype: alkaline phosphatase (AP) (activity and mRNA) and cyclic adenosine monophosphate (cAMP) accumulation. Osteoblast-like cells derived from fetal rat (ROB) and mouse (MOB) calvariae were isolated by collagenase treatment. Cells were cultured in alpha-Minimal Essential Medium (MEM) with 2% fetal calf serum (FCS) for 4 days. In ROB and MOB bPTH(1-34) induced a fast increase (up to 5 minutes) in cAMP accumulation. When equal amounts of cells were seeded, the cAMP accumulation was higher in MOB than in ROB. No difference in basal AP activity was observed between ROB and MOB. When bpTH (1-34) was added to ROB for the last 24 or 48 hr, AP activity decreased dose dependently. However, MOB treated with bPTH(1-34) for the last 24 or 48 hours showed an increase of AP activity. Basal AP activity was positively correlated with the seeding density of ROB and MOB cultures. Basal AP activity influenced the degree of inhibition (ROB) or stimulation (MOB) after incubation with bPTH(1-34).
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PMID:Parathyroid hormone-induced changes in alkaline phosphatase expression in fetal calvarial osteoblasts: differences between rat and mouse. 838 77

Endosteal bone surface cells were previously shown to be involved in the regulation of bone formation in humans. In this study, we have characterized the cells isolated from the endosteal bone surface in adult rats. Fragments of periosteum-free tibia were obtained from 4-, 6- and 9-month-old rats by collagenase digestion, and the phenotypic characteristics of the osteoblastic cells migrating from the endosteal bone surface were evaluated in culture. Endosteal bone surface cells present a strong alkaline phosphatase (ALP) activity as shown by cytochemistry and measured biochemically. The cells synthesize high levels of osteocalcin as measured by radioimmunoassay. Osteocalcin production was increased after stimulation with 10 nM 1,25 dihydroxyvitamin D (1,25(OH)2 D) and the response to 1,25(OH)2 D was similar at all ages. Endosteal cells from young adult rats (4 months old) but not from older rats (6 and 9 months old) showed increased cAMP production in response to 10 nM parathyroid hormone (PTH), suggesting an age-related decrease in the PTH-responsiveness of the bone surface cells. Immunocytochemistry using specific antibodies showed that preconfluent endosteal bone cells from adult rats expressed collagen and noncollagenous bone proteins in culture in the absence of inducers. The cells synthesized mostly type-I collagen which remained localized intracellularly. Type-III collagen was only expressed at low levels. The bone surface cells also expressed osteocalcin and bone sialoprotein, two markers of differentiated osteoblasts, as well as osteonectin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cells isolated from the endosteal bone surface of adult rats express differentiated osteoblastic characteristics in vitro. 847 7

Bone resorption is a complex multistep process that involves removal of both the organic and mineral constituents of bone matrix by proteolytic enzymes synthesized by osteoblasts and osteoclasts. To further understand the role of matrix metalloproteinases (MMPs) and their specific inhibitors TIMPs (tissue inhibitor of metalloproteinases) in this process, human osteoblasts were obtained by sequential enzymatic digestion from samples of bone from normal donors and patients with various forms of arthritis; first passage cells were used in all experiments and cultured on a type I collagen substratum. Collagenase was detected by an ELISA in supernatants from unstimulated osteoblasts (range 12-730 ng/mL), although the levels did not appear to bear any relationship to the age or clinical status of the patient; treatment with parathyroid hormone (PTH; 2 units/mL) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3, 10 ng/mL] had no added effect, but mononuclear cell conditioned medium (MCM; 5% v/v) and interleukin-1 alpha (IL-1 alpha; 1 ng/mL) both stimulated collagenase synthesis, in the case of MCM by two orders of magnitude. TIMP-1 was detected in unstimulated cultures by an ELISA (range 320-590 ng/mL), the mean level being three-fold greater than for collagenase and was stimulated by 1,25(OH)2D3 and MCM treatment. Degradation studies showed that, over a 120 h culture period, one third of the collagen substratum was degraded by unstimulated cells. PTH and 1,25(OH)2D3 had no effect on this endogenous level of lysis, but addition of MCM and IL-1 alpha resulted in a significant increase in collagen degradation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The synthesis of collagenase, gelatinase-A (72 kDa) and -B (95 kDa), and TIMP-1 and -2 by human osteoblasts from normal and arthritic bone. 854 Nov 38

A synthetic peptide corresponding to the N-terminal amino acid residues of stanniocalcin (STC1-20) and including a region that is known to be an active site in teleosts was prepared and tested for its effects on the metabolism of mammalian bone in vitro. STC1-20 (10(-10)-10(-12) M) inhibited increases in the number of tartrate-resistant acid phosphatase-positive, multinucleated cells promoted by an N-terminal fragment of human parathyroid hormone (hPTH1-34) in cultures of murine hemopoietic cells. STC1-20 also slightly decreased the rate of loss of radioactivity from calvariae of fetal rats that had been prelabeled with 45Ca, both with and without stimulation by hPTH1-34. The accumulation of cAMP induced by hPTH1-34 in ROS 17/2.8-5 cells was suppressed by STC1-20 (10(-10)-10(-12) M). Treatment with STC1-20 (10(-11)-10(-13) M) caused increases of the rate of incorporation of [3H]proline into the collagenase-digestible protein of calvariae in newborn mice. From these results, it appears that STC1-20 has diverse effects on the metabolism of mammalian bone, causing a biphasic response. Such effects have not been observed with intact stanniocalcin or with materials from the corpuscles of Stannius and they are also different from the effects of hPTH1-34.
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PMID:Effects of a synthetic N-terminal fragment of stanniocalcin on the metabolism of mammalian bone in vitro. 866 40

The role of matrix metalloproteinases in parathyroid hormone (PTH)-induced bone resorption was assayed using a fetal rat limb bone culture system. Cotreatment of bones with PTH and recombinant inhibitor of metalloproteinases, TIMP-1, in vitro, inhibited the PTH-stimulated 45Ca release from the limb bones without affecting beta-glucuronidase release. TIMP-1 was fully effective when added during only the final 24 h of a 72 h culture with PTH but was ineffective when added for only the first 24 h of the 72 h culture. In contrast, calcitonin (CT) was effective when added for either the first 24 or the final 24 h of the culture. Using in situ hybridization, the mRNA for collagenase was detected in mononuclear cells of cultured bone. Treatment of the bones with PTH resulted in an increase in the number of cells producing collagenase mRNA, some of which had osteoclastic morphology, PTH also caused a dramatic induction of the mRNA for the 92-kD gelatinase B metalloproteinase in both mononuclear and osteoclastic cells. There was no detectable mRNA for the metalloproteinases stromelysin-1, stromelysin-2, or matrilysin in PTH-treated or control cultures. These results suggest that PTH-induced bone resorption is mediated, at least in part, by the induction of collagenase and gelatinase B mRNA in bone cells.
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PMID:Parathyroid hormone-induced resorption in fetal rat limb bones is associated with production of the metalloproteinases collagenase and gelatinase B. 877 Jun 99

Plasminogen activators (PA) are implicated in cell migration and tissue remodeling, two components of the bone resorption processes. Using mice with inactivated tissue PA (tPA), urokinase PA (uPA), or type 1 PA inhibitor (PAI-1) genes, we evaluated whether these processes, or their stimulation by parathyroid hormone (PTH) or 1,25-dihydroxyvitamin (1,25[OH]2D3) are dependent on these genes. Two culture models were used, one involving 19-day fetal calvariae, to evaluate the direct resorptive activity of osteoclasis, and the other involving 45Ca-labeled 17-day fetal metatarsals, in which this activity depends on preliminary (pre)osteoclast migration. PTH similarly increased (about 10-fold) PA activity in calvariae from wild-type tPA+/+ and uPA+/+ or deficient uPA-/- and PAI-/- mice; it affected only tPA, not uPA. In tPA-/- bones, the low PA levels, due to uPA, were not influenced by PTH. Calcitonin did not affect PA responses to PTH. No differences were observed between tPA+/+, tPA-/-, uPA+/+, and uPA-/- calvariae for any parameter related to bone resorption (development of lacunae, release of calcium and lysosomal enzymes, accumulation of collagenase, loss of hydroxyproline), indicating similar responses to PTH or calcitonin. The progressive 45Ca release was largely similar in cultures of tPA+/+, tPA-/-, uPA+/+, uPA-/-, PAI+/+, or PAI-/- metatarsals and it was similarly enhanced by PTH or 1,25(OH)2D3. However, uPA-/- metatarsals released 45Ca at a slower rate at the beginning of the cultures, suggesting an impaired recruitment of the (pre)osteoclasts, which migrate at that time from the periosteum into the calcified cartilage. Thus, it appears that the direct resorptive activity of the osteoclasts does not necessitate the presence of either tPA or uPA, but uPA is likely to facilitate the migration of the (pre)osteoclasts toward the mineralized surfaces. Although considerably enhanced by PTH, tPA does not mediate the actions of PTH (nor of 1,25[OH]2D3) evaluated in these models.
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PMID:Bone resorption and response to calcium-regulating hormones in the absence of tissue or urokinase plasminogen activator or of their type 1 inhibitor. 885 51

We studied the effects of parathyroid hormone (PTH) on PTH parathyroid hormone related peptide (PTHrP) receptor mRNA level, PTHrP binding and PTH-stimulated cyclic adenosine monophosphate (cAMP) accumulation in osteoblasts, derived from fetal rat calvariae (ROB). Cells isolated during 10-70 minutes of collagenase treatment were seeded at a density of 25,000 cells/cm2 and cultured for 4 days. These cells show a fast increase in cAMP production after stimulation for 5 minutes with 20 nM bovine parathyroid hormone(1-34) (bPTH(1-34)). When ROB are incubated with bPTH(1-34) (0.04-40nM) for 24 h, a dose-dependent decrease of the PTH/PTHrP receptor mRNA level, PTHrP binding, and PTH-stimulated cAMP accumulation can be observed. Pretreatment of ROB with a high concentration of bPTH(1-34) (40 nM) leads within 15 minutes to a decrease in PTH-stimulated cAMP accumulation. However, it takes > or = 3 h before a significant decrease in PTH/PTHrP receptor mRNA level can be observed. Also a significant decrease in PTHrP binding is observed after only 4 h of incubation with bPTH(1-34). Compared with bPTH(1-34), pretreatment of ROB with bPTH(3-34) (40 and 100 nM) for 24 h causes smaller decreases in PTH-stimulated cAMP accumulation, PTHrP binding, and in the PTH/PTHrP receptor mRNA level. We investigated the possible involvement of the protein kinase A signaling pathway in the regulation of the PTH/PTHrP receptor mRNA expression. Both forskolin and (Bu)2cAMP decreased PTHrP binding and PTH/PTHrP mRNA levels. These observations suggest that chronic activation of the PKA signaling pathway may down-regulate PTH/PTHrP receptor expression and thus hormone responsiveness in "normal" osteoblasts. In short, we found that the decrease of the PTH-stimulated cAMP accumulation after long-term pretreatment with bPTH(1-34) is correlated with both PTH/PTHrP receptor mRNA level and PTHrP binding. These data also suggest that the initial desensitization (< 30 minutes) of PTH-stimulated cAMP responsiveness by pretreatment with a high concentration of bPTH(1-34) (40 nM) is not dependent on the number of available PTH/PTHrP receptors. The protein kinase A signaling pathway is involved in the regulation of the PTH/PTHrP receptor, but, regarding the effect of bPTH(3-34), other signaling systems are also involved.
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PMID:Down-regulation of the receptor for parathyroid hormone (PTH) and PTH-related peptide by PTH in primary fetal rat osteoblasts. 886 95

Interstitial collagenase plays an important role in both the normal and pathological remodeling of collagenous extracellular matrices, including skeletal tissues. The enzyme is a member of the family of matrix metalloproteinases. Only one rodent interstitial collagenase has been found but there are two human enzymes, human collagenase-1 and -3, the latter being the homologue of the rat enzyme. In developing rat and mouse bone, collagenase is expressed by hypertrophic chondrocytes, osteoblasts, and osteocytes, a situation that is replicated in a fracture callus. Cultured osteoblasts derived from neonatal rat calvariae show greater amounts of collagenase transcripts late in differentiation. These levels can be regulated by parathyroid hormone (PTH), retinoic acid, and insulin-like growth factors, as well as the degree of matrix mineralization. Much of the work on collagenase in bone has been derived from studies on the rat osteosarcoma cell line, UMR 106-01. All bone-resorbing agents stimulate these cells to produce collagenase mRNA and protein, with PTH being the most potent stimulator. Determination of secreted levels of collagenase has been difficult because UMR cells, normal rat osteoblasts, and rat fibroblasts possess a scavenger receptor that removes the enzyme from the extracellular space, internalizes and degrades it, thus imposing another level of control. PTH can also regulate the abundance of the receptor as well as the expression and synthesis of the enzyme. Regulation of the collagenase gene by PTH appears to involve the cAMP pathway as well as a primary response gene, possibly Fos, which then contributes to induction of the collagenase gene. The rat collagenase gene contains an activator protein-1 sequence that is necessary for basal expression, but other promoter regions may also participate in PTH regulation. Thus, there are many levels of regulation of collagenase in bone perhaps constraining what would otherwise be a rampant enzyme.
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PMID:The regulation and regulatory role of collagenase in bone. 888 5

Bone morphogenetic protein (BMP), a member of the transforming growth factor superfamily, is one of the most potent growth factors that stimulate osteoblast differentiation and bone formation. We investigated the effects of recombinant human BMP-2 (rhBMP-2) on osteoblast differentiation and matrix metalloproteinase-1 (MMP-1) production in human bone cells (HBC) isolated from mandibulae of 3 adult patients. rhBMP-2 at concentrations over 50 ng/ml significantly stimulated alkaline phosphatase activity and parathyroid hormone (PTH)-dependent 3', 5'-cyclic adenosine monophosphate accumulation, which are early markers of osteoblast differentiation, in HBCs. rhBMP-2 (500 ng/ml) also enhanced the level of PTH/PTH related-peptide receptor mRNA expression in HBCs. Although neither HBCs untreated nor treated with rhBMP-2 produced measurable amounts of osteocalcin, which is a marker of more mature osteoblasts, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced ostocalcin mRNA expression and its protein synthesis in these cells. rhBMP-2 inhibited 1,25(OH)2D3-induced osteocalcin synthesis in HBCs at both the mRNA and protein level. rhBMP-2 also significantly suppressed MMP-1 production and MMP-1 mRNA expression at concentrations over 500 ng/ml. These results suggest that rhBMP-2 exerts anabolic effects on human osteoblastic cells derived from mandibulae by stimulation of osteoblast differentiation and down-regulation of MMP-1 synthesis.
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PMID:Recombinant human bone morphogenetic protein-2 stimulates osteoblast differentiation and suppresses matrix metalloproteinase-1 production in human bone cells isolated from mandibulae. 987 21

It is well documented that glucocorticoid excess causes bone loss, but the mechanisms of these effects remain poorly defined. To understand further the mechanisms of glucocorticoid-induced osteoporosis, we investigated the effects of glucocorticoids on bone formation and bone resorption by examining the proliferation, functional activities, and cytokine secretion of cultured human bone marrow stromal cells (hBMSC). Treatment with dexamethasone for 24 h at the concentration of 10(-8) M significantly suppressed [(3)H]thymidine incorporation and further inhibition was observed with longer treatment (8 days) or higher concentration (10(-7) M). Alkaline phosphatase activity of hBMSC was markedly stimulated with addition of dexamethasone (10(-8) M), to 191 +/- 22% (after 4 days) and 317 +/- 46% (after 7 days) of control. Dexamethasone (10(-8) M) treatment for 48 h decreased the incorporation of [(3)H]proline into collagenase-digestible protein (CDP; 43.7+/-7.9% of control) and non-collagen protein (65.2+/-8.4% of control), with a greater effect on CDP. Northern blot analysis indicated that alpha1(I)-collagen mRNA level was decreased by dexamethasone to 27.6 +/- 9.0% of the control value after 1 day of exposure, and to 55.2 +/- 6.2% after 7 days. Dexamethasone markedly suppressed basal production of interleukin (IL)-6 and IL-11 and that stimulated by parathyroid hormone (PTH), IL-1alpha, or tumour necrosis factor-alpha in a dose-dependent manner. These results suggest that the glucocorticoid-induced bone loss is derived at least in part via inhibition of bone formation, which includes the suppression of osteoblast proliferation and collagen synthesis. As both basal and PTH-stimulated production of IL-6 and IL-11 are decreased by dexamethasone, the increased bone resorption observed in glucocorticoid-induced osteopenia does not appear to be mediated by IL-6 or IL-11.
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PMID:Effects of dexamethasone on proliferation, activity, and cytokine secretion of normal human bone marrow stromal cells: possible mechanisms of glucocorticoid-induced bone loss. 1046 28


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