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

Co-cultures of human osteosarcoma Takase (OST) cells with various human fibroblasts derived from surgical specimens stimulated production of gelatinase B (92-kDa type-IV collagenase, MMP-9), tissue inhibitors of metalloproteinase (TIMP)-1, and TIMP-2 when compared to cultures of individual cells. The maximum stimulation of gelatinase-B production occurred at a cellular ratio of 1:1. Conditioned media from several fibroblast cultures stimulated OST cells to produce gelatinase B, TIMP-1 and TIMP-2, but not vice versa. Among various recombinant growth factors or cytokines, tumor necrosis factor (TNF)-alpha stimulated gelatinase-B production in cultures of OST cells alone, while recombinant basic fibroblast growth factor (bFGF) stimulated gelatinase-B production in co-cultures of OST cells with skin fibroblasts but not in individual cultures of each cell type. In the co-cultures, gelatinase-B production was inhibited by anti-bFGF monoclonal antibody (MAb), but not by anti TNF-alpha MAb. This co-culture-specific stimulation of gelatinase-B production by bFGF was associated with increased expression of the FGF receptor in the co-culture.
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PMID:Stimulation of gelatinase B and tissue inhibitors of metalloproteinase (TIMP) production in co-culture of human osteosarcoma cells and human fibroblasts: gelatinase B production was stimulated via up-regulation of fibroblast growth factor (FGF) receptor. 860 72

Fibroblast growth factor-2 (FGF-2) is a potent autocrine mitogen for fetal epiphyseal growth plate chondrocytes and exhibits a transient nuclear translocation during G1 of the cell cycle. We have characterized an intracellular binding protein (FGFBP) for FGF-2 that undergoes a juxtanuclear localization coincident with the nuclear translocation of the growth factor. Chondrocytes were isolated from the proliferative zone of the ovine fetal proximal tibial growth plate at 50-130 days gestation by collagenase digestion and were maintained in monolayer at early passage number. Cells were growth restricted by serum starvation for 48 h, and the synchronized culture was restarted into the cell cycle in the presence of 2% FBS. Cells were removed between 4-26 h of incubation, and fractions representing the plasma membrane, cytoplasm, nuclear membrane, and nuclear contents were separated by differential centrifugation. FGFBPs were separated using FGF-2 affinity chromatography. Ligand blot analysis using 125I-labeled FGF-2 showed that a FGFBP of 46-48 kDa (represented by a double band) was present on the nuclear membrane at mid to late G1, and Western blot showed this to be immunologically related to a part of the extracellular domain of the high affinity FGF receptor 1 (FGFR1). Immunocytochemistry with intact cell cultures showed that this protein underwent a juxtanuclear distribution through mid to late G1. Immunoprecipitation was performed to monitor newly synthesized FGFR1 migration throughout the cell cycle. Synchronized cells were cultured in medium containing 35S-labeled methionine/cysteine, and the cellular compartments were separated before immunoprecipitation using an antibody raised against the extracellular domain of FGFR1. Newly synthesized FGFR1-related proteins appeared throughout G1 and migrated multidirectionally within the cell; intact receptor of 125-145 kDa accumulated at the plasma membrane, while both intact receptor and truncated FGFR1 of 46-48 kDa were detected on the nuclear membrane, but not within the nucleus. Cells were incubated with protamine sulfate to prevent the binding of endogenous, cell membrane-associated FGF-2 to high affinity FGFRs and their subsequent internalization. This did not alter the juxtanuclear accumulation of truncated FGFR1 in late G1, suggesting that this was not derived from the plasma membrane. The truncated FGFR1 may mediate the nuclear translocation of FGF-2 during late G1.
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PMID:Perinuclear localization of an intracellular binding protein related to the fibroblast growth factor (FGF) receptor 1 is temporally associated with the nuclear trafficking of FGF-2 in proliferating epiphyseal growth plate chondrocytes. 889 82

Collagenase-1 is invariantly expressed by migrating basal keratinocytes in all forms of human skin wounds, and its expression is induced by contact with native type I collagen. However, net differences in enzyme production between acute and chronic wounds may be modulated by soluble factors present within the tissue environment. Basic fibroblast growth factor (bFGF, FGF-2) and keratinocyte growth factor (KGF, FGF-9), which are produced during wound healing, inhibited collagenase-1 expression by keratinocytes in a dose-dependent manner. However, KGF was >100-fold more effective than bFGF at inhibiting collagenase-1 expression, suggesting that this differential signaling is transduced via an FGF receptor that binds these ligands with different affinities. Reverse transcriptase-polymerase chain reaction analysis of human keratinocyte mRNA for fibroblast growth factor receptors (FGFRs) revealed expression of only FGFR-2 IIIb, the KGF-specific receptor, which also binds bFGF with low affinity, and FGFR-3 IIIb, which does not bind bFGF or KGF. FGFRs that bind bFGF with high affinity were not detected. Our results suggest that bFGF and KGF inhibit collagenase-1 expression through the KGF cell-surface receptor (FGFR-2 IIIb). Because bFGF induces collagenase-1 in most cell types, cell-specific expression of FGFR family members may dictate the regulation of matrix metalloproteinases in a tissue-specific manner.
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PMID:Cell type-specific inhibition of keratinocyte collagenase-1 expression by basic fibroblast growth factor and keratinocyte growth factor. A common receptor pathway. 921 49

A coordinated interaction between fibroblast growth factors (FGFs) and matrix metalloproteinases (MMPs) is implicated in migration of microvascular endothelial cells (ECs), an early stage of angiogenesis. Specifically, we investigated microvascular ECs migration in vitro, which can be initiated by the overexpression of a secretory form of the angiogenic fibroblast growth factor-1 (FGF-1) and mediated through the enzymatic activity of matrix metalloproteinase-1 (MMP-1). MMP-1 is a member of the MMP family with a propensity for degradation of interstitial type I collagen. We stably overexpressed a chimeric FGF-1 construct composed of the FGF-4 signal-peptide gene, linked in-frame to the FGF-1 coding frame gene (sp-FGF-1), in cultured postcapillary venular ECs. The presence of the biologically active form of FGF-1 was readily detected in the conditioned medium of ECs transfected with sp-FGF-1 construct as demonstrated by DNA synthesis assay. The sp-FGF-1-, but not the plasmid vector alone-transfected ECs, exhibited an altered morphology as demonstrated by their conversion from a classic cobblestone form to a fibroblastlike shape that featured prominent neuritelike extensions. Addition of the anti-FGF receptor 1 antibody (FGFR1 Ab) reverted the transformed phenotype of sp-FGF-1 transfectants. This suggests that the resulting phenotypic transformation in sp-FGF-1 transfectants requires an uninterrupted interaction between the FGF-1 ligand and its receptor. We studied migration of cells through matrices of either highly pure collagen I or reconstituted basement membrane (matrigel) and found that sp-FGF-1-transfected cells migrated two times and six times faster than the vector control transfectants in the respective matrices. We further demonstrated that the enhanced migration rate of sp-FGF-1-transfected EC coincided with the induction of their MMP-1 mRNA level and increased enzymatic activity. The enhanced migratory activity of sp-FGF-1 could be blocked with a selective inhibitor of MMP-1. These results suggest that the multipotent FGF-1 plays a key role in the early stages of angiogenesis, by mediating MMP-1 proteolytic activity.
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PMID:Overexpression of a secretory form of FGF-1 promotes MMP-1-mediated endothelial cell migration. 1086 46

From collagenase digests of human thyroid, endothelial cells were separated from follicular cells by their greater adherence to gelatin-coated plates. Endothelial cells were further purified using fluorescence-activated cell sorting, selecting for cells expressing factor VIII-related antigen. Isolated cells were negative for thyroglobulin and calcitonin when examined by immunostaining. The receptor for the angiopoietins, Tie-2, was expressed by the cells, and expression was increased by agents that elevate cAMP. Nitric oxide synthase (NOS) 3, the endothelial form of NOS, was expressed by the cells and similarly regulated. Cells responded strongly to the mitogen fibroblast growth factor (FGF)-2 in growth assays but only weakly to vascular endothelial growth factor (VEGF). VEGF was, however, able to stimulate nitric oxide release from the cells consistent with their endothelial origin. The FGF receptor (FGFR1) was full length (120 kDa) and immunolocalized to the cytosol and nucleus. Thyrotropin (TSH) did not regulate FGFR1, but its expression was increased by VEGF. Thrombospondin, a product of follicular cells, was a growth inhibitor, but neither TSH nor 3,5,3'-triiodothyronine had direct mitogenic effects. Thyroid follicular cell conditioned medium contained plasminogen activator activity and stimulated the growth of the endothelial cells, but when treated with plasminogen to produce the endothelial-specific inhibitor, angiostatin, growth was inhibited. Human thyroid endothelial cell cultures will be invaluable in determining the cross talk between endothelial and follicular cells during goitrogenesis.
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PMID:Isolation and characterization of human thyroid endothelial cells. 1962 78