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)

Basic fibroblast growth factor (basic FGF) is a mitogen for isolated epiphyseal growth plate chondrocytes. To determine whether basic FGF might function as an autocrine stimulus to longitudinal skeletal growth in utero, we investigated the synthesis and release of basic FGF by isolated growth plate chondrocytes from the ovine fetus, the expression of mRNA for a high affinity basic FGF receptor by these cells, and the contribution of endogenous basic FGF to the DNA synthetic rate of the cells in vitro. Chondrocytes were isolated from the proximal tibial growth plate of the lamb fetuses between 35 and 132 days' gestation using collagenase, and were cultured in monolayer before use between passages 3 and 6. Viability was confirmed over the duration of the experiments by the exclusion of trypan blue, and an absence of lactate dehydrogenase accumulation in conditioned medium. Immunocytochemistry of chondrocyte monolayers showed immunoreactive basic FGF to be present in the cytoplasm of approximately 80% of sub-confluent cells which was accompanied by pronounced nuclear staining in approximately 30% of cells. Serum-free, conditioned culture medium, extracellular matrix and chondrocyte cytoplasm contained 52 +/- 2 pM/micrograms DNA, 66 +/- 2 pM/micrograms DNA and 22 +/- 3 pM/micrograms DNA basic FGF, respectively (mean +/- S.E.M., n = 8 fetuses), for cells obtained from animals of 35-40 days' gestation when assessed by radioimmunoassay. Chondrocyte-conditioned medium increased endothelial cell proliferation in vitro (a specific bio-assay for basic FGF and related peptides); and the mitogenic activity was removed from conditioned medium by incubation with heparin-Sepharose demonstrating that this was due to heparin-binding protein(s). Western blot analysis of conditioned medium using a specific basic FGF antibody revealed a single immunoreactive protein of approximately 18 kDa molecular size. The appearance of radiommunoassayable basic FGF in conditioned medium, extracellular matrix, and chondrocyte cytoplasm observed during culture was blocked by co-incubation with cycloheximide. The levels of immunoreactive basic FGF present in each compartment decreased with gestational age as did basal DNA synthetic rate assessed by the incorporation of [3H] thymidine. Incubation of chondrocytes with transforming growth factor beta, resulted in a significant increase while exposure to insulin-like growth factors or insulin caused a decrease, in the content and release of basic FGF. Basic FGF presence was unaltered when medium was supplemented with varying amounts of glucose (2.7-16.7 mM). In situ hybridization on cell monolayers using a cRNA probe encoding the high affinity flg receptor for FGFs showed an abundant expression of mRNA for the receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Basic fibroblast growth factor is synthesized and released by isolated ovine fetal growth plate chondrocytes: potential role as an autocrine mitogen. 134 Feb 7

Basic fibroblast growth factor (basic FGF) is a potent mitogen for chondrocytes in vitro and is present in developing cartilage in vivo. Studies of intracellular basic FGF localization in other cell types have revealed a transient nuclear presence. We have examined ovine fetal growth plate chondrocytes for the presence of intracellular basic FGF by immunocytochemistry. Chondrocytes were isolated from the proximal tibial growth plate of lamb fetuses between 75 and 80 days' gestation using collagenase, and were cultured in monolayer before use between passages 3 and 6. In non-synchronized cell cultures 58 +/- 6% of cells (mean +/- s.d., n = 3) demonstrated cytoplasmic staining for immunoreactive basic FGF. Of these cells, 18 +/- 3% also exhibited strong nuclear staining. Chondrocytes were growth-restricted and restarted into the cell cycle with 2% (vol/vol) fetal calf serum. The timing of S phase was followed by nuclear labelling of nuclei with [3H] thymidine followed by autoradiography, or by the incorporation of [3H] thymidine into trichloroacetic acid-precipitable DNA in parallel cultures. A cytoplasmic presence of immunoreactive basic FGF did not appear, following immunocytochemistry, until the second half of G1 with 97% of cells immunopositive 2 hr prior to S phase. Nuclear staining for basic FGF appeared 2 hr before peak nuclear labelling index, and 56% of cell nuclei were immunopositive. Following entry into S phase cytoplasmic and nuclear basic FGF immunostaining rapidly disappeared. When these experiments were repeated with or without the presence of anti-basic FGF antibody or heparin, the presence of the antibody significantly reduced peak [3H] thymidine incorporation into DNA during S phase while exposure to heparin increased this. However, the proportion of cells demonstrating cytoplasmic or nuclear staining for immunoreactive basic FGF, and the time of onset of staining, were unaltered. Incubation of cells with suramin blocked subsequent DNA synthesis and no intracellular basic FGF was visualized. Cell-conditioned culture medium, extracellular matrix and cytoplasm from synchronized cultures of chondrocytes were taken at time points throughout the cell cycle and assessed for basic FGF content by radioimmunoassay. Basic FGF was detectable in each compartment and steadily rose throughout the second half of G1 to reach maximum values around the S phase. The accumulation of basic FGF in medium, matrix and cytoplasm was blocked by the presence of cycloheximide.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Cell cycle-dependent localization of immunoreactive basic fibroblast growth factor to cytoplasm and nucleus of isolated ovine fetal growth plate chondrocytes. 145 27

Gastrin-releasing polypeptide (GRP) has been implicated in the development of the human fetal lung. To determine whether GRP has a wider role in fetal development, its actions on DNA synthesis and cell replication by isolated epiphyseal growth plate chondrocytes obtained from ovine fetuses between 35 days gestation and near term (145 days) were examined. Chondrocytes were isolated using collagenase from the proximal tibia and cultured in monolayer. Synthesis of DNA was assessed from the incorporation of [3H]thymidine into previously growth-restricted cells after incubation in medium supplemented with GRP1-27 (40-1280 nM). Increase in cell number was assessed after incubation with test medium for 1 week. GRP caused a dose-dependent increase in both cell number and DNA synthetic rate compared to control incubations. Cell number was increased by 50% in the presence of a maximally effective 160 nM GRP and DNA synthesis by up to 800% utilizing chondrocytes obtained from animals of 75-80 days gestation. The mean (+/- SEM) half-maximal concentration of GRP for the stimulation of DNA synthesis was 97 +/- 12 nM (5 separate fetuses). Concentrations of GRP in excess of 160 nM caused a sharp reduction in both cell replication and DNA synthesis. To determine where within the cell cycle GRP exerted its mitogenic action, synchronized chondrocytes were transiently exposed to fetal bovine serum and cultured with GRP for increasing periods of time before pulse labeling with [3H]thymidine during S phase. GRP was as effective in stimulating DNA synthesis when present for the initial 4 h of G1 as when present for the entire G1 period. Since isolated fetal growth plate chondrocytes release insulin-like growth factor II (IGF II) and basic fibroblast growth factor (basic FGF) the possible mediation of GRP action by the release of these peptides or synergistic interactions were examined. Specific antibodies shown to negate the mitogenic actions of exogenous IGFs or basic FGF on chondrocytes did not alter GRP-stimulated DNA synthesis. The release of radioimmunoassayable IGF II by chondrocytes was not altered in the presence of GRP. Coincubation of GRP with submaximal concentrations of IGF I or basic FGF showed additive effects on DNA synthesis. When the actions of galanin were examined it was found to inhibit basal DNA synthesis by chondrocytes at a concentration of 167 nM. However, 66 nM or greater galanin was able to render 160 nM GRP inactive as a mitogen. These results suggest that GRP may potentially influence skeletal development in the ovine fetus and may interact with locally released peptide growth factors or other neuropeptides.
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PMID:Mitogenic action of gastrin-releasing polypeptide on isolated epiphyseal growth plate chondrocytes from the ovine fetus. 157 94

Using human heart fibroblasts (HHF), we studied the effect of basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) on the gene expression of type I collagen, collagenase and tissue inhibitor of metalloproteinases (TIMP). Initially, treatment of HHF with bFGF alone (10 ng/ml) resulted in elevated secretion of collagenase into the culture medium. Subsequent treatment of HHF with TGF-beta in combination with bFGF suppressed collagenase secretion. Northern blot analysis reinforced this observation by revealing an enhancement of the steady-state mRNA level of collagenase in response to bFGF. In order to examine if the collagenase gene was affected by bFGF at the transcriptional level, transfection experiments were carried out with a plasmid containing collagenase promoter linked to chloramphenicol acetyltransferase gene (CAT). Basic FGF stimulated CAT activity by four-fold, indicating increased promoter activity whereas the combination of TGF-beta and bFGF resulted in decreased CAT activity. TGF-beta was shown to increase type I collagen and TIMP mRNA levels by 2.5- and 2.1-fold, respectively. These results suggest that TGF-beta and bFGF may play a pivotal role in regulating collagen metabolism in HHF.
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PMID:Effect of growth factors on collagen metabolism in cultured human heart fibroblasts. 166 Aug 2

The isolation of human adrenal capillary endothelial (HACE) cells without resort to fluorescence activated cell sorting is described, together with their properties in culture. HACE cells were isolated by plating collagenase digests at high dilution in the presence of endothelial cell growth supplement, followed by clonal selection of endothelial colonies. HACE cells exhibit a typical endothelial 'cobblestone' morphology at confluence and formed 'tubes' when seeded onto 'Matrigel'. They are positive for human MHC1, and the endothelial markers ENDOCAM (CD31) and weakly CD34, they also take up dil-acetyl low density lipoprotein but are negative for Factor VIII. Their growth is strongly stimulated by FGF and inhibited by TGF-beta I. Like their much studied bovine counterparts they are robust in culture, retaining the properties described up to senescence. HACE cells provide a readily available alternative to human umbilical vein endothelial cells in that they are easily isolated pure and in quantity. They should be particularly useful in studies where human capillary, as opposed to large vessel endothelium, is required.
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PMID:Isolation and properties in culture of human adrenal capillary endothelial cells. 199 80

Endothelial cells play a fundamental role in the pathogenesis of chronic inflammatory arthritis in humans such as rheumatoid arthritis (RA), as well as experimental animal models such as streptococcal cell wall (SCW) arthritis in Lewis (LEW/N) rats. This review summarizes data in support of this concept. The earliest apparent abnormalities in synovial tissues of patients with RA and Lewis rats with SCW arthritis appear to reflect microvascular endothelial cell activation or injury. At the molecular level, the abnormalities include enhanced expression by endothelial cells of activation markers such as class II major histocompatibility complex antigens, phosphotyrosine, leukocyte adhesion molecules, oncoproteins such as c-Fos and c-Myc, and metalloproteinases such as collagenase and transin/stromelysin. The development of severe, chronic, destructive arthritis is dependent upon thymic-derived lymphocytes and is accompanied by tumorlike proliferation of cells in the synovial connective tissue stroma (blood vessels and fibroblastlike cells), which results in resorptive destruction of bone and cartilage. Multiple criteria support the analogy to a neoplastic process. Paracrine and autocrine factors such as interleukin-1 (IL-1), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), and heparin-binding fibroblast growth factors (HBGF, FGF) appear to play important roles in the generation of these lesions. Finally, in addition to the autocrine and paracrine regulatory factors, neuroendocrine factors, particularly the hypothalamic-pituitary-adrenal axis, appear to be involved in the counterregulation of the inflammatory process. The counterregulatory effects are mediated, in part, by inhibition of endothelial cell activation by corticosteroids.
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PMID:Endothelial cells and the pathogenesis of rheumatoid arthritis in humans and streptococcal cell wall arthritis in Lewis rats. 205 44

The labeling pattern of mouse embryonic eye frozen sections incubated with radioiodinated brain acidic and basic fibroblasts growth factors (aFGF and bFGF) was investigated by autoradiography. Both growth factors bind to basement membranes in a dose-dependent way, with a higher affinity for bFGF. Similar data were obtained with eye-derived growth factors (EDGF), the retinal forms of FGF. There was a heterogeneity in the affinity of the various basement membranes toward these growth factors. The inner limiting membrane of the retina and the posterior part of the lens capsule have a higher binding capacity than the posterior part of the Bruch's membrane. The specificity of the growth factor-basement membrane interaction was demonstrated by the following experiments: (i) an excess of unlabeled growth factor displaced the labeling; (ii) unrelated proteins with different isoelectric points--gelatin, serum albumin, histones--did not modify the labeling; and (iii) iodinated EGF or PDGF did not label basement membrane. In order to get a better understanding of the nature of this binding, we performed the incubation of the frozen sections with iodinated FGFs preincubated with various compounds: (i) heparin which is known to have a strong affinity for aFGF and bFGF partially decreases the labeling, and (ii) chondroitin sulfate B and dextran sulfate at high concentrations were also partially effective. In addition, enzymatic treatment of the sections reveals that only heparitinase, not collagenase or chondroitinase ABC, completely prevents the labeling without destroying the overall structure of the basement membrane. An antibody against the proteic part of EHS mouse proteoheparan sulfate does not affect the signal. Esterification of the acidic groups cancelled the binding. These results demonstrate that FGFs bind specifically to basement membranes, probably on the polysaccharidic part of the proteoheparan sulfate, and suggest that this type of interaction may be a general feature of the mechanism of action of these growth factors.
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PMID:Specific fixation of bovine brain and retinal acidic and basic fibroblast growth factors to mouse embryonic eye basement membranes. 244 16

The minimal structural requirements for the interaction of heparin with acidic fibroblast growth factor (aFGF) were investigated. Oligosaccharides (tetra- to decasaccharides) obtained by nitrous acid depolymerisation of standard heparin were separated by affinity chromatography on Sepharose-immobilised aFGF. The shortest fragment retained by the affinity column at 0.2 M NaCl and eluted at 1 M NaCl was a "regular" hexasaccharide, a trimer of the most abundant disaccharide sequence in heparin. More complex octa- and decasaccharides were also retained by the column. The oligosaccharides eluted by 1 M NaCl from the affinity column ("high-affinity" oligosaccharides) and those washed from the column at 0.2 M NaCl ("low-affinity" oligosaccharides) were compared for their capacity to protect aFGF from proteolysis and to potentiate its mitogenic activity. At a low ionic strength, all oligosaccharides tested, except the "regular" disaccharide, protected aFGF against trypsin and collagenase digestion. At higher ionic strength (greater than 0.2 M NaCl), only high-affinity oligosaccharides showed a protective effect. The high-affinity oligosaccharides (hexa- to decasaccharides) potentiated the mitogenic activity of aFGF, as measured by [3H]thymidine incorporation into DNA of human fibroblasts. The effect of the oligosaccharides on human endothelial cell proliferation was more complex: inhibition of proliferation was observed in the presence of serum and low concentrations of aFGF (1-5 ng/ml) and potentiation in the presence of higher concentrations of aFGF. The potentiating effect increased as a function of molecular size of the heparin fragments and, for a given size, as a function of the anionic charge of the oligosaccharide. Our results suggest that inhibition of cell proliferation by heparin may result from interference with an autocrine basic FGF-like activity.
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PMID:Heparin-derived oligosaccharides: affinity for acidic fibroblast growth factor and effect on its growth-promoting activity for human endothelial cells. 255 Apr 75

The regeneration of connective tissue attachment is a major goal of clinical periodontics. Recent investigations on biochemically mediated periodontal regeneration have attempted to define the various biological response modifiers which may provide a mechanism for periodontal regeneration. Fibronectin and endothelial cell growth factor have been shown to selectively enhance periodontal ligament (PDL) cell adhesion, migration, and proliferation. In addition, dentin preconditioned with tetracycline HCl (TTC) or citric acid (CA) supports PDL cell adhesion, presumably by exposing collagen fibers. We have now extended these studies to include basic fibroblast growth factor (b-FGF) as a potential meditor of periodontal regeneration. Using AFSCM (assays for specific cell migration), b-FGF in concentrations as low as 10 ng per dentin block significantly stimulated PDL cell chemotaxis, while the antibody against b-FGF inhibited both the chemotactic and proliferative characteristics of the mitogen. We also found that 5 ng and above of b-FGF per dentin block significantly stimulated human endothelial cell migration and proliferation. Using 125I-b-FGF, we demonstrated that the factor binds to native dentin. This binding was increased when the dentin blocks were preconditioned by TTC or CA and reduced when the dentin was subsequently treated with collagenase. 125I-b-FGF also bound with moderate affinity to a type I collagen affinity column whereas the binding to a hydroxylapatite affinity column was negligible. The combination of FN and b-FGF was a marginally more potent chemo-attractant than b-FGF alone for PDL cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Repopulation of dentin surfaces by periodontal ligament cells and endothelial cells. Effect of basic fibroblast growth factor. 255 Jun 5

Studies on the direct effects of hormones and growth factors on bone alkaline phosphatase have been limited to parathyroid hormone (PTH) and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] and have not been compared to other parameters of bone formation. Insulin, PTH, 1,25(OH)2D3, epidermal and fibroblast growth factors (EGF, FGF) were examined for their effects on alkaline phosphatase activity and type I, [alpha 1 (I)]2 alpha 2, collagen synthesis in cultures of 21-day fetal rat calvariae. After 24 hr and 96 hr of treatment, insulin increased whereas PTH, 1,25(OH)2D3, EGF and FGF inhibited calvarial alkaline phosphatase activity and the incorporation of 3H-proline into collagenase-digestible protein and type I collagen. The agents tested did not affect the release of alkaline phosphatase into the culture medium. Although type I collagen was the only collagen detected, a small amount of another collagen might have been also synthesized. The hormonal effects on alkaline phosphatase activity and type I collagen synthesis were of greater magnitude after 96 hr than after 24 hr of continuous exposure to the agents tested and the two parameters correlated well (r = 0.88 after 96 hr and r = 0.97 after 24 hr of treatment. These studies indicate that insulin increases bone alkaline phosphatase activity and type I collagen synthesis in calvariae whereas PTH, 1,25(OH)2D3, EGF and FGF have an inhibitory effect. The results suggest that these agents affect osteoblastic function.
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PMID:Effect of hormones and growth factors on alkaline phosphatase activity and collagen synthesis in cultured rat calvariae. 621 95


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