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)

We have shown previously that phosphatidic acid esterified to polyunsaturated fatty acids is mitogenic for primary cultures of mouse mammary epithelial cells embedded within collagen gels. We hypothesized that this mitogenic competence resulted from the ability of this phospholipid to activate multiple signal transduction pathways in mammary epithelium. A closer examination of this hypothesis was undertaken by examining the effect of exogenous phosphatidic acid on phosphoinositide (PI) hydrolysis and its intracellular metabolism to diglyceride, an activator of protein kinase C. For assays of phosphoinositide-specific phospholipase C activation, mammary epithelial cells from virgin Balb/c mice were isolated by collagenase dissociation of mammary glands and cultured on the surface of Type I collagen-coated culture dishes. Phosphatidic acid (PA) stimulated a sustained increase in inositol phosphates and caused inositol phospholipid depletion when added to cells in which inositol phospholipids were prelabeled with 3H-myoinositol. This effect was specific for PA among phospholipids tested. Neither lineoleic acid, that can be released from PA, nor prostaglandin E2 affected PI hydrolysis. When mammary epithelial cells were cultured inside collagen gels in the presence of exogenous PA or phosphatidylcholine (PC) radiolabeled with 3H-glycerol, PA was found to persist intracellularly and be dephosphorylated to diglyceride (an activator of protein kinase C) to a greater extent than PC, a nonmitogenic phospholipid. In contrast to PA, epidermal growth factor (EGF) only slightly stimulated PI hydrolysis, showing that these two different growth-promoting factors do not actively couple to the same signal transduction pathways in mammary epithelial cells. These results show that PA may activate multiple pathways in mammary epithelial cells either directly or via its metabolism to diglyceride.
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PMID:Multifunctional phosphatidic acid signaling in mammary epithelial cells: stimulation of phosphoinositide hydrolysis and conversion to diglyceride. 777 98

Stromelysin-1 and collagenase mRNA levels were assayed in fibrochondrocytes by Northern blot analysis at 0, 2, 4, 8 and 24 h after stimulation with tissue necrosis factor-alpha (TNF-alpha). Peak collagenase mRNA levels occurred 24 h after stimulation and were increased nine-fold over the level at time 0. Stromelysin-1 mRNA levels peaked 8 h after stimulation, with a five-fold increase over the level at time 0. A TNF-alpha dose-related response to both collagenase and stromelysin-1 mRNA accumulation was also demonstrated. Confirmation of the presence of secreted metallo-proteinases in the conditioned media was established by immunoprecipitation of stromelysin-1 and Western blotting of collagenase. Both enzymes were secreted in latent forms. Consistent with stromelysin-1 activity, substrate gels demonstrated a doublet of caseinase activity with molecular masses at 57 kDa and 59 kDa in TNF-alpha stimulated samples. Collagenase assays of conditioned media also demonstrated a significant increase in collagenase activity after stimulation by TNF-alpha. While epidermal growth factor had a minimal effect on stromelysin-1 and collagenase expression, transforming growth factor-beta, and insulin-like growth factor-1 did not induce either enzyme activity.
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PMID:Induction of stromelysin-1 and collagenase synthesis in fibrochondrocytes by tumor necrosis factor-alpha. 792 41

Tumor necrosis factor alpha (TNF alpha) has multiple biological functions including the prolonged activation of the collagenase and c-jun genes, which are regulated via their AP-1 binding sites. We show that incubating human fibroblasts with TNF alpha induces prolonged activation of JNK, the c-Jun kinase, which phosphorylates the transactivation domain of c-Jun. Furthermore, an immune complex kinase assay specifically demonstrates that TNF alpha stimulates the activity of JNK1, the recently described predominant form of JNK. TNF alpha also produces a small and transient increase in extracellular signal-regulated kinase (ERK) activity and no measured increase in Raf-1 kinase activity. On the other hand, epidermal growth factor causes a prolonged activation of Raf-1 kinase and ERK activity and a smaller, more transient activation of JNK, whereas the phorbol ester phorbol 12-myristate 13-acetate causes a small stimulation of Raf-1 kinase and a pronounced stimulation of ERK activity. The activation of JNK by TNF alpha does not correlate with Raf-1 or ERK activity. The kinetics of Raf-1, ERK, and JNK induction by epidermal growth factor, phorbol 12-myristate 13-acetate, or TNF alpha indicate distinct mechanisms of activation in human fibroblasts.
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PMID:Tumor necrosis factor alpha stimulates AP-1 activity through prolonged activation of the c-Jun kinase. 792 60

The aim of the study was to determine the binding characteristics of the epidermal growth factor (EGF) receptor in isolated human endometrial glands and stromal cells in culture. Stromal cells and glands were obtained from endometrial tissue by collagenase dispersion followed by sieve filtration. They were plated into 24-well multiwell plates in Ham's F10 medium supplemented with 5% fetal calf serum and used at 70-80% confluence. Scatchard analysis revealed a single class of high-affinity binding sites in both cell types with apparent dissociation constants of 1.17 +/- 0.6 (n = 15) and 1.20 +/- 0.3 (n = 8) nmol 1-1 for stromal cells and glands, respectively. The concentration of receptors was higher in stromal cells than in glands, 719 +/- 377 (n = 16) and 310 +/- 177 (n = 8) fmol mg-1 protein, respectively. Epidermal growth factor labelled with 125I was displaced from the receptor by EGF and transforming growth factor alpha, but not insulin, insulin-like growth factor, fibroblast growth factor, or platelet-derived growth factor. Binding was shown to be dependent on time and temperature. Downregulation of the receptor was demonstrated by preincubating cells with 5 nmol EGF I-1, which reduced receptor concentrations by 75%. 12-O-Tetradecanoylphorbol-13-acetate decreased the affinity of the receptor for EGF changing the dissociation constant from 1.8 to 3.9 nmol l-1. A suitable system for investigating the regulation of this receptor in human endometrium was established.
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PMID:Characterization of epidermal growth factor receptor in human endometrial cells in culture. 793 77

An improved technique for primary short-term culture of prostate carcinoma cells in two phases, with and without serum, for subsequent cytogenetic analysis is reported and compared with four other methods. After mechanical disaggregation and a brief collagenase treatment of tumor specimens, cell clusters were seeded in RPMI 1640 and 15% fetal calf serum (FCS) without any other supplement in the first phase. The culture medium was changed to a serum-free medium supplemented with bovine pituitary extract (BPE) and epidermal growth factor (EGF) when the first outgrowth became apparent. During this second phase, fibroblast growth could be virtually abolished within 48 hr. The epithelial and prostatic origin of the cultured cells was confirmed by immunocytochemical methods in each culture. Metaphase analysis revealed chromosome aberrations in over 80% of cases (both clonal and nonclonal alterations) indicating the presence of neoplastic cells. Clonal numerical chromosome aberrations, found by conventional cytogenetic analysis, were used to provide the reliability of the culture system in interphase nuclei of corresponding uncultured tumor tissue by fluorescence in situ hybridization (FISH). The main points of the described method are: 1) combined mechanical/enzymatic disaggregation, 2) seeding of the disaggregated cell clumps rather than of single cells, 3) initialization of the cultures in RPMI 1640 medium with 18% FCS without any other supplements, and (4) stimulating of selective epithelial proliferation by changing the culture conditions through serum-free medium.
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PMID:Two-phase short-term culture method for cytogenetic investigations from human prostate carcinoma. 797 14

The c-fos proto-oncogene is believed to play a pivotal role in transducing growth factor-mediated signals from the extracellular milieu into the nucleus. c-fos protein dimerizes with c-jun and related proteins and mediates transcription via AP-1 sites. Using c-fos-deficient mice generated through gene knockout techniques, we derived 3T3-type cell lines from primary embryonic fibroblasts. The c-fos-deficient cells grow normally under optimal culture conditions and show only a slight reduction in growth rate in low serum culture compared with control cells. They also express mRNA for most of the Fos and Jun family members at normal levels. The overall levels of AP-1 DNA binding activity are normal and several genes (c-jun, MCP1, metallothionein) known to contain functional AP-1 sites are expressed normally in the c-fos-deficient and control cells. In contrast, mRNA for the metalloproteases stromelysin (MMP-3) and type I collagenase (MMP-1), which are often induced by oncogenes and growth factors and have been implicated in tumor invasiveness, cannot be induced by epidermal growth factor or platelet-derived growth factor in c-fos-deficient cells. Transformation of mutant cells with polyoma middle T oncogene essentially restores wild-type levels of stromelysin expression, while transformation with v-src leads to only a weak induction of the metalloprotease. These results clearly demonstrate that some AP-1-dependent genes require c-fos for full expression while others do not; oncogenes may activate expression of metalloproteases via either fos-dependent or fos-independent mechanisms. These results also imply that c-fos may play an important regulatory role in the invasive behavior of malignant tumors, independent of any role this proto-oncogene might play in cell growth per se.
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PMID:Targeted disruption of the c-fos gene demonstrates c-fos-dependent and -independent pathways for gene expression stimulated by growth factors or oncogenes. 803 3

The effect of interleukin-1 alpha (IL-1 alpha) and murine epidermal growth factor (EGF) on incorporation of endogenously produced collagenase in the extracellular matrix of soft connective tissue was studied in an in vitro model system using periosteal explants obtained from rabbit calvariae. Immunohistochemical analysis indicated the highest level of collagenase in explants cultured for 72 hours with IL-1 alpha in combination with EGF. Most enzyme appeared to be associated with the extracellular matrix, but labeling was also found in numerous fibroblast-like cells. Explants cultured in the presence of IL-1 alpha alone contained less enzyme and in periostea treated without cytokines, or with EGF alone, only a faint label, if any, was seen. Freshly isolated, non-cultured periostea contained no detectable enzyme. Extraction of collagenase from periostea revealed that: (1) non-cultured periosteum did not contain detectable levels of enzyme. (2) The amount of total activatable enzyme synergistically increased (10-fold) under the influence of IL-1 alpha and EGF, whereas IL-1 alpha alone showed a 4-fold enhancement compared to control or EGF-incubated explants. (3) The latent fraction of the enzyme was synergistically increased (up to 100-fold or more) in periostea cultured in the presence of IL-1 alpha + EGF (21.17 mU/explant versus 0.05 mU/explant in controls). (4) Active collagenase, on the other hand, appeared to be present in a relatively high concentration in explants cultured without cytokines (2.45 mU/explant versus 0.36 mU/explant in IL-1 alpha + EGF-treated explants).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Immunolocalisation of collagenase in rabbit periosteal tissue explants and extraction of the enzyme. The effect of the cytokines IL-1 alpha and EGF. 805 29

Previous investigations have shown that culture of freshly isolated hepatocytes under conventional conditions, i.e., on dried rat tail collagen in the presence of growth factors, facilitates cell growth but also causes an extensive down-regulation of most liver-specific functions. This dedifferentiation process can be prevented if the cells are cultured on a reconstituted basement membrane gel matrix derived from the Englebreth-Holm-Swarm mouse sarcoma tumor (EHS gel). To gain insight into the mechanisms regulating this response to extracellular matrix, we are analyzing the activities of two families of transcription factors, C/EBP and AP-1, which control the transcription of hepatic and growth-responsive genes, respectively. We demonstrate that isolation of hepatocytes from the normal quiescent rat liver by collagenase perfusion activates the immediate-early growth response program, as indicated by increased expression of c-jun, junB, c-fos, and c-myc mRNAs. Adhesion of these activated cells to dried rat tail collagen augments the elevated levels of these mRNAs for the initial 1 to 2 h postplating; junB and c-myc mRNA levels then drop steeply, with junB returning to normal quiescence and the c-myc level remaining slightly elevated during the 3-day culture period. Levels of c-jun mRNA and AP-1 DNA binding activity, however, remain elevated from the outset, while C/EBP alpha mRNA expression is down-regulated, resulting in a decrease in the steady-state levels of the 42- and 30-kDa C/EBP alpha polypeptides and C/EBP alpha DNA binding activity. In contrast, C/EBP beta mRNA production remains at near-normal hepatic levels for 5 to 8 days of culture, although its DNA binding activity decreases severalfold during this time. Adhesion of hepatocytes to the EHS gel for the same period of time dramatically alters this program: it arrests growth and inhibits AP-1 DNA binding activity and the expression of c-jun, junB, and c-myc mRNAs, but, in addition, it restores C/EBP alpha mRNA and protein as well as C/EBP alpha and C/EBP beta DNA binding activities to the abundant levels present in freshly isolated hepatocytes. These changes are not due merely to growth inhibition, because suppression of hepatocyte proliferation on collagen by epidermal growth factor starvation or addition of transforming growth factor beta does not inhibit AP-1 activity or restore C/EBP alpha DNA binding activity to normal hepatic levels. These data suggest that expression of the normal hepatic phenotype requires that hepatocytes exist in a G0 state of growth arrest, facilitated here by adhesion of cells to the EHS gel, in order to express high levels of hepatic transcription factors such as C/EBP alpha.
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PMID:Cell-extracellular matrix interactions can regulate the switch between growth and differentiation in rat hepatocytes: reciprocal expression of C/EBP alpha and immediate-early growth response transcription factors. 806 19

The purpose of this study was to develop a primary culture system using serum-free medium for rat placental trophoblast cells and to investigate the factors that control rat placental lactogen-II (rPL-II) secretion in vitro. The placentae of day 13 pregnant rats were dissociated in Medium 199 containing 0.1% collagenase and 0.002% DNAase. Dissociated cells were fractionated into five segments by centrifugation through a 40% Percoll density gradient and incubated on rat tail collagen bed in medium SFM-101 for up to 7 days. Fraction B at the Percoll gradient density of 1.05 g ml-1 was enriched with rPL-II-producing cells and the time course of rPL-II secretion was characterized by a rapid increase in the first 2 days, remaining at high values (mean: 14-16 ng micrograms-1 DNA) for the following 2-3 days and decreasing thereafter. The rPL-II-producing cells from faction B identified by immunocytochemical examination accounted for approximately 69% of total cultured cells and consisted of a few giant cells and polygonal cells. Growth factors (bovine insulin, 0.1-20 micrograms ml-1; recombinant human insulin-like growth factor (IGF)-I, IGF-II, 0.1-1.0 micrograms ml-1; murine epidermal growth factor (EGF), 0.001-10 micrograms ml-1), rat pituitary hormones (rat growth hormone, rat prolactin, 0.1-10 micrograms ml-1) and hypothalamic hormones (human growth hormone-releasing hormone (GHRH), corticotrophin-releasing hormone (CRH), LHRH, 0.1-10 micrograms ml-1) were individually added to the culture medium to investigate the putative factors that directly control rPL-II secretion by the trophoblast cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Stimulation of rat placental lactogen-II (rPL-II) secretion by cultured trophoblasts by insulin: development of a rat placental cell culture system and effects of peptide hormones on rPL-II secretion in vitro. 810 35

In this study the localization and regulation of steady-state follistatin messenger ribonucleic acid (mRNA) levels in testicular cell cultures were examined with a solution-hybridization assay using a specific 32P-labelled cytosolic RNA antisense probe for follistatin and a 35S-labelled cytosolic RNA antisense probe for cyclophilin as internal standard. Testes from immature rats were dispersed with collagenase and fractionated in Sertoli and Leydig cell-enriched cultures. Follistatin mRNA was mainly localized to the Sertoli cell-enriched fraction and the expression of follistatin mRNA could be stimulated in vitro with fetal calf serum, epidermal growth factor or phorbol-12-myristate-13-acetate (an activator of protein kinase C), whereas follicle-stimulating hormone and forskolin (an activator of protein kinase A) had no effect. Neither prostaglandin E2, the synthetic glucocorticoid RU 28362 or all-trans-retinoic acid, which all regulate follistatin mRNA levels in non-testicular cell types, nor extracellular adenosine triphosphate (a purinergic receptor agonist) or testosterone had any obvious influence on follistatin mRNA levels in Sertoli cell-enriched cultures. From this study it is concluded that Sertoli cells are likely to be the source of follistatin expression in the rat testis, that follistatin mRNA levels in Sertoli cell-enriched cultures are subjected to regulation by epidermal growth factor and the protein kinase C-dependent pathway but are not regulated by extracellular adenosine triphosphate, follicle-stimulating hormone, all-trans-retinoic acid, prostaglandin E2, forskolin, testosterone or the glucocorticoid RU 28362 and that the regulation of follistatin mRNA is sex- and tissue-specific.
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PMID:Expression of follistatin messenger ribonucleic acid in Sertoli cell-enriched cultures: regulation by epidermal growth factor and protein kinase C-dependent pathway but not by follicle-stimulating hormone and protein kinase A-dependent pathway. 810 86


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