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)

In vivo, the extracellular matrix modulates the phenotype of the connective tissue cells both through its biochemical composition and the transfer of mechanical information. In this study, the mechanical effect was investigated in collagen gels populated by skin fibroblasts maintained under tension (bound lattices (BL)) compared with free retracting lattices (FL) and monolayer on plastic. The overall proteins and collagen synthesis of human skin fibroblasts, investigated by isotopic labeling, were decreased respectively by a factor of about 20 and 40 in FL compared with monolayers and increased by a factor of 4 and 6 in BL versus FL. As assayed by the degradation of [3H]collagen type I by trypsin-activated medium conditioned by fibroblasts under the three models of culture, collagenase activity was inversely regulated and increased in lattices when compared with monolayer culture. It was four times higher in FL than in BL. The steady-state level of mRNA coding for procollagen types I, III, and VI polypeptides, fibronectin, elastin, beta-actin, and procollagenase was determined by cDNA hybridization. The mRNA coding for beta-actin as well as for the various extracellular matrix macromolecules were increased in BL when compared with FL while the level of procollagenase mRNA was lower. These data demonstrate the existence of a modulation of the function of the fibroblasts performed by mechanical forces. This regulation operates, at least in part, at a pretranslational level.
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PMID:Pretranslational regulation of extracellular matrix macromolecules and collagenase expression in fibroblasts by mechanical forces. 131 27

Collagenase is synthesized and secreted by rat osteoblastic cells in response to PTH. We have previously demonstrated that this effect involves a substantial increase in collagenase mRNA via transcription. Northern blots and nuclear run-on assays were performed to further investigate the induction of collagenase by PTH in the rat osteoblastic cell line UMR 106-01. Detectable amounts of collagenase mRNA were not apparent until 2 h of PTH treatment, showed the greatest abundance at 4 h, and declined to approximately 30% of maximum by 8 h. The changes in the rate of transcription of the collagenase gene in response to PTH paralleled and preceded the changes in the steady state mRNA levels. After an initial lag period of about 1 h, collagenase transcription rates increased from very low levels to a maximal response at 2 h, returning to about 50% of maximum by 10 h. The increased transcriptional rate of the collagenase gene was found to be dependent on the concentration of PTH, with a half-maximal response at approximately 7 x 10(-10) M rat PTH-(1-34) and a maximal effect with a dose of 10(-8) M. The PTH-mediated induction of collagenase transcriptional activity was completely abolished by cycloheximide, while transcription of the beta-actin gene was unaffected by the translation inhibitor. These data suggest that a protein factor(s) is required for PTH-mediated transcriptional induction of collagenase. Since PTH increases intracellular levels of several potential second messengers, agents that mimic these substances were employed to determine which signal transduction pathway is predominant in the PTH-mediated stimulation of collagenase transcription.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Parathyroid hormone induces transcription of collagenase in rat osteoblastic cells by a mechanism using cyclic adenosine 3',5'-monophosphate and requiring protein synthesis. 133 47

Total RNA was extracted from skin biopsies of nine patients suffering from systemic sclerosis (SSc). Steady-state mRNA levels of collagen alpha 1(I) and alpha 1(III), collagenase, fibronectin, and beta-actin were studied using specific cDNA probes and compared to those of 12 sex- and age-matched healthy individuals. There was a more than three-fold elevation of collagen I mRNA levels in SSc skin compared to controls. No difference was found, however, for collagen III, collagenase, and fibronectin mRNA levels in SSc and control biopsies. The selective increase of collagen alpha 1(I) mRNA levels indicates a specific alteration of fibroblast metabolism in scleroderma. Analysis of mRNA levels in skin biopsies might not only offer a direct approach to the understanding of the pathophysiology of SSc, but also facilitate the monitoring of fibrotic activity in SSc patients during therapeutic trials.
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PMID:Steady-state mRNA levels of collagens I, III, fibronectin, and collagenase in skin biopsies of systemic sclerosis patients. 164 27

We report the effect of UVA irradiation on collagen metabolism of fibroblasts, including both synthesis of the collagen degrading enzyme collagenase and de novo synthesis of type I collagen as the major structural component of the dermis. For this purpose confluent fibroblast monolayers were irradiated under standardized conditions (5, 15, 35, 60 J/cm2 using UVASUN 3000, Mutzhas, Munich, FRG, and UV source Sellas sunlight type 2.001, Sellas, Gevelsberg, FRG). Subsequently, total RNA was isolated and subjected to dot blot and northern blot analysis using oligolabelled cDNA clones for human type I collagen, collagenase and beta-actin. Collagen type I and beta-actin mRNA levels remained unaltered following irradiation, suggesting that the synthetic pathway of collagen metabolism at the pretranslational level is not affected by short-term UVA irradiation. However, collagenase mRNA was found to be dose-dependently induced in fibroblasts after irradiation, thus probably contributing to the actinic damage to the dermis. These in vitro data were confirmed in vivo using in situ hybridization on frozen sections of biopsy material obtained from UVA irradiated patients.
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PMID:UVA irradiation induces collagenase in human dermal fibroblasts in vitro and in vivo. 166 13

Rat livers were perfused and stored for 48 hr in cold University of Wisconsin solution before dissociation by the two-step collagenase method. At that time, glycogen content was significantly reduced, but no obvious changes in albumin, beta-actin and aldolase B mRNAs and in glutathione levels were observed. Enzymatic perfusion yielded 280 +/- 30 x 10(6) viable hepatocytes vs. 520 +/- 40 x 10(6) viable hepatocytes from unstored organs. Cell viability determined by trypan blue exclusion was 74% and 90%, respectively. Hepatocytes from University of Wisconsin-preserved livers had a 29% reduced adenosine triphosphate content, but glutathione levels did not significantly differ from those found in unstored cells. When put into culture, hepatocytes formed typical monolayers of granular epithelial cells and did not exhibit alteration of their fine structure when compared with cells from unstored organs. After 24 and 48 hr, they showed variations in cytochrome P-450 content and ethoxyresorufin O-deethylase activity similar to those observed with unstored cells. By contrast, overall protein synthesis and albumin secretion rate were 40% and 30% lower, respectively. Hepatocytes from University of Wisconsin-preserved organs could be cryopreserved and further cultured as unstored cells. The University of Wisconsin solution was also used to preserve isolated hepatocytes. Viability of freshly isolated hepatocytes was decreased by only 10% after 48 hr of hypothermic liver storage when assayed by intracellular lactate dehydrogenase content. However, after 4 hr of storage, in contrast with hepatocytes preserved in L15 Leibovitz medium, the cells attached poorly to plastic and exhibited morphological alterations.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Primary culture of adult rat hepatocytes after 48-hour preservation of the liver with cold UW solution. 225 48

The effects of recombinant human interleukin-1 alpha (IL-1) on procollagen gene expression were examined in the clonal mouse osteoblastic cell line MC3T3-E1. Cells were grown in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum and 50 micrograms/ml ascorbic acid. Collagen synthesis was assessed as [3H]proline incorporation into collagenase-digestible protein (CDP). Procollagen mRNA levels were determined by Northern blot analysis using a 32P-labeled alpha 1(I) cDNA. Transcription rates were determined by nuclear run-off assay. IL-1 at 1-1000 pg/ml caused a concentration-dependent inhibition of CDP, which was maximally reduced by 75-80%, and a parallel reduction of procollagen alpha 1(I) mRNA levels. The effects of IL-1 were mimicked by the tumor promoter phorbol 12-myristate 13-acetate (PMA) at 1-100 nM, which inhibited CDP and reduced procollagen alpha 1(I) mRNA levels to a similar extent. The effects of IL-1 and PMA were independent of prostaglandin production, since indomethacin did not alter the inhibitory effect of either agent on CDP. Neither IL-1 (up to 10 ng/ml) nor PMA (100 nM) affected adenylate cyclase activity, while forskolin (10 microM), PTH (10 nM) and prostaglandin E2 (1 microM) stimulated adenylate cyclase activity 3- to 5-fold. However, forskolin (10 microM) and (Bu)2cAMP (100 microM) failed to alter CDP or procollagen alpha 1(I) mRNA levels. IL-1 (1 ng/ml) and PMA (100 nM) reduced transcription of the alpha 1(I) procollagen gene by 70% and 80%, respectively, while alpha 2(I) transcription was decreased by 59% and 53%. Neither IL-1 nor PMA affected transcription of the beta-actin or beta-tubulin genes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interleukin-1 alpha and phorbol ester inhibit collagen synthesis in osteoblastic MC3T3-E1 cells by a transcriptional mechanism. 232 98

Cells of the clonal rat osteogenic sarcoma cell line, UMR 106-01, were used to investigate the regulation of collagen synthesis by PTH in osteoblastic cells. Monolayer cultures of cells were labeled with [3H] proline in order to determine both collagen type and rates of production. Analysis of labeled extracellular polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that UMR 106-01 cells synthesized predominantly type I collagen, accounting for 45.48 +/- 2.09% of the radioactivity incorporated into total protein. After 24-h treatment with bovine PTH (1-34, 10(-8) M), collagen synthesis (i.e. collagenase-digestible protein) was decreased to 29.45 +/- 1.39% of total protein production. This decrease was first observed 12 h after addition of hormone and greatest inhibition was achieved at 24 h. The effect of PTH was dose dependent, with half-maximal inhibition of collagen synthesis occurring at 5 x 10(-10) M after 24-h treatment. In contrast, when steady state levels of mRNA for type I collagen chains were examined by Northern blot analysis, the concentration of PTH that reduced collagen synthesis by 35-45% (10(-8) M), caused a net decrease of approximately 80-96% in the number of procollagen transcripts; a small reduction in beta-actin mRNA levels was also observed. The effect of the hormone on procollagen message level was dose dependent, with significant inhibition observed at 10(-10) M PTH and, as with collagen synthesis, maximal after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Parathyroid hormone inhibits collagen synthesis at both ribonucleic acid and protein levels in rat osteogenic sarcoma cells. 246 7

Recent clinical observations have suggested that retinoids, which are in frequent use in dermatology, can affect the connective tissue metabolism in skin and other tissues. In this study, we examined the effects of several retinoids on the metabolism of collagen by human skin fibroblasts in culture. Incubation of cultured fibroblasts with all-trans-retinoic acid or 13-cis-retinoic acid, in 10(-5) M or higher concentrations, markedly reduced the procollagen production, as measured by synthesis of radioactive hydroxyproline. The effect was selective in that little, if any, inhibition was noted in the incorporation of [3H]leucine into the noncollagenous proteins, when the cells were incubated with the retinoids in 10(-5) M concentration. Similar reduction in procollagen production was noted with retinol and retinal, whereas an aromatic analogue of retinoic acid ethyl ester (RO-10-9359) resulted in a slight increase in procollagen production in these cultures. The reduction in procollagen production by all-trans-retinoic acid was accompanied by a similar reduction in pro alpha 2(I) of type I procollagen specific messenger RNA (mRNA), as detected by dot blot and Northern blot hybridizations. Hybridizations with human fibronectin and beta-actin specific DNA probes indicated that the levels of the corresponding mRNAs were not affected by the retinoids, further suggesting selectivity in the inhibition of procollagen gene expression. Further control experiments indicated that all-trans-retinoic acid, under the culture conditions employed, did not affect the posttranslational hydroxylation of prolyl residues, the mannosylation of newly synthesized procollagen, the specific radioactivity of the intracellular prolyltransfer RNA pool, or DNA replication. All-trans-retinoic acid also elicited a reduction in trypsin-activatable collagenase, but not in the activity of prolyl hydroxylase or an elastaselike neutral protease in the fibroblast cultures. Incubation of three fibroblast lines established from human keloids with all-trans-retinoic acid or 13-cis-retinoic acid also resulted in a marked reduction in procollagen production. The results, therefore, suggest that further development of retinoids might provide a novel means of modulating collagen gene expression in patients with various diseases affecting the connective tissues.
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PMID:Modulation of procollagen gene expression by retinoids. Inhibition of collagen production by retinoic acid accompanied by reduced type I procollagen messenger ribonucleic acid levels in human skin fibroblast cultures. 298 6

Acetaldehyde, the first metabolite of ethanol, mediates many of the biological effects of ethanol. We have previously shown that acetaldehyde, but not ethanol, stimulates collagen production in cultured human fibroblasts (Holt, K., Bennett, M., and Chojkier, M. (1984) Hepatology 4, 843-848). Here, we examined the effects of acetaldehyde on collagen gene expression. Confluent human fetal fibroblasts were incubated for up to 4 h in the presence of ascorbate (0.2 mM) alone or with the addition of either ethanol (12 mM) or acetaldehyde (200 microM). Acetaldehyde induced the production of collagen (up to 2.5-fold) and had a small inhibitory effect on procollagen secretion (-20%). The steady-state levels of mRNAs were measured by hybridizing total cellular RNA to specific cDNA probes at high stringency. Acetaldehyde increased the steady-state level of collagen alpha 1(I) and collagen alpha 2(I) mRNAs about 3-fold and had small effects on beta-actin mRNA (+50%) and collagenase mRNA (-50%). Northern blots revealed that the RNAs were intact and that acetaldehyde preferentially increased the abundance of the longer of the two collagen alpha 1(I) transcripts. Acetaldehyde increased both collagen alpha 1(I) and collagen alpha 1(III) transcriptional activity by 2.5-fold and had small effects on beta-actin and collagenase gene transcription. The increase in both collagen production and collagen mRNA levels induced by acetaldehyde was blocked by methylene blue, a scavenger of reducing equivalents. These data indicate that reducing equivalents, which enhance the formation and stability of acetaldehyde-protein adducts, may be required for acetaldehyde-stimulated collagen production. Thus, this study suggests that acetaldehyde increases collagen production by increasing collagen gene transcription in cultured human fibroblasts.
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PMID:Acetaldehyde increases collagen gene transcription in cultured human fibroblasts. 369 68

Vascular endothelial cell (EC) wound healing was characterized on an EC-synthesized extracellular matrix (ECM) previously treated with enzymes and antibodies specific for ECM components. Using a computer-assisted video-microscope recording system capable of automatic EC recognition, we learned whether components of the EC-synthesized matrix influenced post-injury migration and wound healing in vitro. Localization of actin and its encoded mRNA using isoform-specific antibodies and labeled cDNA probes allowed for a direct correlation of living-cell behavior with cytoskeletal form and distribution. Results of these studies indicate that the computer-assisted EC tracking system allows for an automatic and reproducible analysis of EC behavior following injury in vitro. EC migrate fastest immediately following injury and then achieve a new, slower migration rate that is maintained until EC from one edge of 200- to 300-microns-wide wound zone contact EC from the other wound face. Treatment of EC-synthesized matrices with antibodies against fibronectin and laminin has no effect on EC migration following injury (-0.25 microns/min) or on cytoskeletal array. Similarly, digestion of these matrices with heparinase and hyaluronidase has no effect on wound healing rates. Slowly spreading EC cytoplasm, which borders the intact and antibody-treated EC matrices, is rich in actin but lacks myosin II. Two different preparations of collagenase (bacterial and mammalian) each potentiate EC wound healing in vitro. Bacterial collagenase treatment of the EC-synthesized matrices potentiates EC migration fivefold (1 micron/min) while treatment of EC-matrices with mammalian cell collagenase stimulates EC migration following injury some twofold (0.4 micron/min) over control values. Whereas EC on control matrices migrate in unison as a tissue-like sheet, EC on the collagenase-treated EC matrices migrate as individuals. Concomitant with the increased rates of migration following injury on the collagenase-treated EC-matrices is a two- to fourfold increase in the steady-state levels of beta-actin mRNA. This increase in actin mRNA abundance is observable by its preferential localization (seen by in situ hybridization) in the lamellae bordering the wound edge in association with beta-actin, which is exclusively localized there. Because beta-actin and its encoded mRNA are positioned together in association with the plasma membrane in regions of moving cytoplasm, it seems likely that beta-actin filament assembly is required for motility following endothelial injury.
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PMID:Molecular mechanisms regulating the vascular endothelial cell motile response to injury. 752 70


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