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)

Single cardiac myocytes were isolated from hearts of 9 to 12-week-old rats by means of collagenase (100 U/ml). After assessment of their functional integrity they were processed for immunofluorescence microscopy of the cytoskeletal proteins tubulin, microtubule-associated proteins 1 and 2 (MAP-1 and MAP-2), plectin, vimentin, and vinculin. Antibodies to tubulin decorated a delicate filamentous network that apparently was unrelated to any sarcomeric organization. The distribution of MAP-1 and MAP-2 was strikingly different from that of tubulin, as both antigens were confined to Z-line structures. These structures were also prominently stained by affinity-purified antibodies to plectin and a monoclonal antibody to vimentin. Co-distribution of plectin and vimentin was also observed at the former intercalated disk region of the heart cell. Anti-vinculin antibodies decorated an intricate meshwork consisting of delicate filaments with predominantly irregular orientation and occasional assembly into whorls. These immunolocalization data indicate that the cell shape and cytoskeletal architecture characteristic of cardiac myocytes in tissues is maintained in single isolated cells. Furthermore, intermediate filaments rather than microtubules seem to be instrumental in the preservation of cell morphology.
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PMID:Morphological integrity of single adult cardiac myocytes isolated by collagenase treatment: immunolocalization of tubulin, microtubule-associated proteins 1 and 2, plectin, vimentin, and vinculin. 299 82

Incubation of cultured neonatal rat cardiomyocytes in hypoxic conditions, mimicking the deprivation of O2 which occurs during in situ myocardial ischemia, leads to a progressive change in cardiomyocytes cytoskeletal components. Confocal scanning laser immunofluorescence microscopy (CSLIM) reveals that the typical striated costameric distribution of vinculin gradually disappears to be replaced by circular, vinculin-containing sarcolemmal rosettes. There is little change in distribution of vinculin in the focal adhesions or in the intercalated disks. This cytoskeletal alteration, like that observed in virally transformed fibroblasts and phorbol ester-treated skeletal myoblasts, is inhibited by genistein, a tyrosine kinase inhibitor. Increased exposure to hypoxic conditions also produces an increase in a 92-kDa collagenase which is immunolocalized only to cardiomyocytes. As with the rosette formation, genistein also inhibits the increased expression of the 92-kDa collagenase. We suggest that this cytoskeletal change with attendant release of 92 kDa collagenase may represent a defensive mechanism on the part of the cardiomyocyte to reduce damage by reducing the cellular coupling to the extracellular collagenous matrix, thereby lessening the stresses imposed by contractile forces.
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PMID:Hypoxia-induced alterations in cytoskeleton coincide with collagenase expression in cultured neonatal rat cardiomyocytes. 882 74

Vinculin is a cytoskeletal protein that is believed to be an essential component in the linkage of cytoskeletal actin filaments to the plasma membrane. To investigate the precise function of vinculin in the development of cardiac myofibrils, antisense oligodeoxynucleotides complementary to vinculin mRNA were used to perturb the expression of the protein during myofibril assembly and arrangement in mouse cardiac myocytes. Fetal (day 18-20 post-conception) mouse cardiac myocytes were isolated by collagenase digestion, separated by Percoll density gradient centrifugation, and plated on aligned collagen gels. By 72 h of culture, mouse myocytes displayed an elongated in vivo-like phenotype in parallel with the aligned fibrils of the collagen gels with polarized arrays of myofibrils. Two different antisense oligonucleotides (20-mer) altered the formation of the tissue-like phenotype of myocytes. These antisense oligonucleotides suppressed vinculin protein expression at 43.5+/-26.8% and 48.7+/-20.9% when compared to myocytes that were not treated. Examination of these myocytes by confocal scanning laser and transmission electron microscopy revealed a disruption of the aligned in vivo-like phenotype, assembly of thick and thin filaments, and formulation of Z-bands. Random sequence 20-mer oligonucleotides used as controls had little detectable effect on vinculin protein expression (94.2+/-14.8%), cell shape, normal alignment or assembly of myofibrils. These results indicate that vinculin is a critical cytoskeletal component, that functions in the determination of cell shape and the arrangement and organization of developing myofibrils.
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PMID:Vinculin is an essential component for normal myofibrillar arrangement in fetal mouse cardiac myocytes. 928 37

To identify proteins that are lost during the establishment of the transformed phenotype of a tumor cell, we have prepared a subtracted cDNA library with mRNA from normal human fibroblasts and from their matched SV40 transformed counterparts. More than 40 clones were obtained that showed a dramatic reduction in their relative expression after oncogenic transformation. The proteins encoded by these clones could be grouped into four distinct classes: extracellular matrix proteins (fibronectin, beta ig-h3, collagen VI), enzymes (collagenase, urokinase), cytoskeletal proteins (vinculin, SM22) and regulatory proteins (beta-glycan, integrin-associated protein, myosin kinase, IGFBP-5). Six novel gene products were discovered during these experiments, including a novel serine protease, a zyxin-like protein, an ankyrin-like protein and a GTP-binding protein. Only four of all the transformation-sensitive cDNAs were consistently down-regulated when a variety of cell lines derived from spontaneous mesenchymal tumors was investigated: beta ig-h3, collagen VI, the novel ankyrin-like protein, and IGFBP-5. It is likely that these gene products play an important role in the maintenance of the normal phenotype.
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PMID:Down-regulated proteins of mesenchymal tumor cells. 951 34

Active matrix metalloproteinases and degraded collagen are observed in disease states, such as atherosclerosis. To examine whether degraded collagen fragments have distinct effects on vascular smooth muscle cells (SMC), collagenase-digested type I collagen was added to cultured human arterial SMC. After addition of collagen fragments, adherent SMC lose their focal adhesion structures and round up. Analysis of components of the focal adhesion complex demonstrates rapid cleavage of the focal adhesion kinase (pp125(FAK)), paxillin, and talin. Cleavage is suppressed by inhibitors of the proteolytic enzyme, calpain I. In vitro translated pp125(FAK) is a substrate for both calpain I- and II-mediated processing. Mapping of the proteolytic cleavage fragments of pp125(FAK) predicts a dissociation of the focal adhesion targeting (FAT) sequence and second proline-rich domain from the tyrosine kinase domain and integrin-binding sequence. Coimmunoprecipitation studies confirm that the ability of pp125(FAK) to associate with paxillin, vinculin, and p130cas is significantly reduced in SMC treated with degraded collagen fragments. Further, there is a significant reduction in the association of intact pp125(FAK) with the cytoskeletal fraction, while pp125(FAK) cleavage fragments appear in the cytoplasm in SMC treated with degraded collagen fragments. Integrin-blocking studies indicate that integrin-mediated signals are involved in degraded collagen induction of pp125(FAK) cleavage. Thus, collagen fragments induce distinct integrin signals that lead to initiation of calpain-mediated cleavage of pp125(FAK), paxillin, and talin and dissolution of the focal adhesion complex.
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PMID:Degraded collagen fragments promote rapid disassembly of smooth muscle focal adhesions that correlates with cleavage of pp125(FAK), paxillin, and talin. 1054 5

The objective of this study was to establish a technique to isolate porcine mesothelial cells (PMCs) from omental tissue and to compare them to human mesothelial cells (HMCs). The PMCs were dispersed by collagenase digestion and isolated on a Ficoll layer. Their morphologic and ultrastructural features were assessed at confluence by light and electronic microscopy, and they were characterized by immunohistochemistry using specific HMC markers. PMC proliferation was studied in the presence of growth factors platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or transforming growth factors beta1, beta2, or beta3 (TGF). Fibrinolytic PMC activity was detected by zymography for tissue plasminogen activator (tPA) and by reverse zymography for plasminogen activator inhibitor-1 (PAI-1). The recalcification time of cell lysates was used to define PMC procoagulant activity, and gelatinase zymography was used to detect metalloproteinase production. At confluence, PMCs formed typical cobblestone monolayers and exhibited structural features characteristic of HMCs. Weibel Palade bodies were never seen. Specific HMC markers (HBME1, ME1, WT1) cross-reacted with PMCs. As HMCs and PMCs coexpressed cytokeratin and vimentin, and also expressed vinculin and alpha-actin. Addition of PDGF or EGF to the culture medium stimulated PMC proliferation. PMCs constitutively expressed fibrinolytic and procoagulant activity and secreted MMP9 and MMP2. The technique described in this study allows isolation of mesothelial cells from porcine omental tissue. These porcine cells exhibit a mesothelial phenotype and functional properties similar to those of HMCs. Our data warrant an evaluation of mesothelial cells as targets in several therapeutic strategies with porcine models.
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PMID:Phenotypic and functional characteristics of porcine peritoneal mesothelial cells. 1061 73

The properties of two VEGF receptors, Flt-1 and KDR, in the signal transduction of VEGF in human umbilical vein endothelial cells (HUVECs) were investigated by using two newly developed blocking monoclonal antibodies (mAbs) against Flt-1 and KDR. VEGF stimulated the expression of transcription factor Ets-1 as well as matrix metalloproteinase-1 (MMP-1) and Flt-1 in HUVECs. The KDR/Flt-1 heterodimer and the KDR homodimer mediate the expression of Ets-1, MMP-1, and Flt-1. VEGF also stimulated DNA synthesis and migration of HUVECs. DNA synthesis is mediated by the same signaling system as the expression of Ets-1. In contrast, cell migration is regulated by two distinct signaling systems. The Flt-1 homodimer is required for actin reorganization. The KDR/Flt-1 heterodimer and the KDR homodimer are required for the assembly of vinculin in focal adhesion plaque by regulating the phosphorylation of focal adhesion kinase (FAK) and paxillin.
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PMID:Properties of two VEGF receptors, Flt-1 and KDR, in signal transduction. 1086 39

Collagenase 3 degrades collagen fibrils and is necessary for bone resorption. Cortisol increases collagenase 3 mRNA in osteoblasts by stabilizing collagenase 3 transcripts. To understand mechanisms involved, we used RNA electrophoretic mobility shift assay and RNA turnover studies. Cortisol increased the binding of Ob cell cytosolic extracts to AU-rich sequences in the collagenase 3 3'-untranslated region (UTR). No cortisol-dependent protein complexes were formed with the coding region or the 5'-UTR. Functional assays, using transient transfections of CMV-driven c-fos collagenase 3'-UTR chimeric constructs, demonstrated that the 3'-UTR of collagenase 3 stabilizes c-fos mRNA in transcriptionally arrested Ob cells, cortisol prolongs the transcript half-life, and mutations of AU-rich sequences destabilize c-fos transcripts precluding the cortisol effect. Purification of osteoblast cytosolic extracts by ultracentrifugation, ion exchange, and RNA affinity chromatography, and polyacrylamide gel electrophoresis followed by mass spectroscopy identified specific proteins. RNA gel mobility supershift assays demonstrated that vinculin and far upstream element (FUSE)-binding protein 2 interacted with collagenase 3 3'-UTR sequences, and RNA interference demonstrated these proteins altered collagenase mRNA stability. In conclusion, AU-rich sequences of the 3'-UTR of collagenase 3 and vinculin and FUSE-binding protein 2 regulate collagenase mRNA stability in osteoblasts.
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PMID:AU-rich elements in the collagenase 3 mRNA mediate stabilization of the transcript by cortisol in osteoblasts. 1464 43

Clinically significant elevations in the expression of manganese superoxide dismutase (Sod2) are associated with an increased frequency of tumor invasion and metastasis in certain cancers. The aim of this study was to examine whether increases in Sod2 activity modulate the migratory potential of tumor cells, contributing to their enhanced metastatic behavior. Overexpression of Sod2 in HT-1080 fibrosarcoma cells significantly enhanced their migration 2-fold in a wound healing assay and their invasive potential 3-fold in a transwell invasion assay. Severity of invasion was directly correlated to Sod2 expression levels and this invasive phenotype was similarly observed in 253J bladder tumor cells, in which Sod expression resulted in a 3-fold increase in invasion compared with controls. Further, migration and invasion of the Sod2-expressing cells was inhibited following overexpression of catalase, indicating that the promigratory/invasive phenotype of Sod2-expressing cells is H(2)O(2) dependent. Sod2 overexpression was associated with a loss of vinculin-positive focal adhesions that were recovered in cells coexpressing catalase. Tail vein injections of Sod2-GFP-expressing HT-1080 cells in NCR nude mice led to the development of pulmonary metastatic nodules displaying high Sod2-GFP expression. Isolated tumors were shown to retain high Sod2 activity in culture and elevated levels of the matrix degrading protein matrix metalloproteinase-1, and a promigratory phenotype was observed in a population of cells growing out from the tumor nodule. These findings suggest that the association between increased Sod2 activity and poor prognosis in cancer can be attributed to alterations in their migratory and invasive capacity.
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PMID:Manganese superoxide dismutase enhances the invasive and migratory activity of tumor cells. 1797 67

Quinolones and glucocorticoids are frequently used drugs that may cause tendinopathy as a rare adverse effect. We exposed human tenocyte cultures to the steroid dexamethasone alone or in combination with either ciprofloxacin or levofloxacin at concentrations of 3mg/L and 10mg/L. At concentrations corresponding to peak levels in plasma and tissues during therapy (ca. 3-10mg/L), ciprofloxacin caused a significant decrease in collagen type I and the beta(1)-integrin receptor. In contrast, no corresponding effect was induced by 3mg/L levofloxacin. With both quinolones at 3mg/L and 10mg/L, the amount of matrix metalloproteinase-1 (MMP-1) and MMP-13 was increased. In addition, 3mg/L ciprofloxacin and 10mg/L levofloxacin activated caspase-3. Apoptotic changes were confirmed by electron microscopy. Incubation of human tenocytes with dexamethasone decreased the main matrix protein collagen type I, the transmembrane beta(1)-integrin receptor and the cytoskeleton protein vinculin, but only at the high concentrations tested (0.1 microM or 10 microM). Concentrations of 0.1 microM and 10 microM dexamethasone increased the amount of MMPs and activated caspase-3 as an indicator of apoptosis. Combined exposure to quinolones and dexamethasone led to more pronounced effects in tenocyte cultures at most of the analysed endpoints. The clinical observations of an increased risk of quinolone-induced tendinopathy by glucocorticoids are supported by these in vitro data.
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PMID:Synergistic effects of dexamethasone and quinolones on human-derived tendon cells. 2003 66


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