Biosynthesis of tissue inhibitor of metalloproteinases by human fibroblasts in culture. Stimulation by 12-O-tetradecanoylphorbol 13-acetate and interleukin 1 in parallel with collagenase.
Biosynthesis of the glycoprotein tissue inhibitor of metalloproteinases (TIMP) by human fibroblasts in culture has been characterized by functional assays, immunoprecipitation, and immunocytochemistry with a monospecific antiserum. As determined by radiolabeling with [35S]methionine, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the secreted form of TIMP had an Mr of 29,000, whereas the form associated with the cell layer had an Mr of 24,000. Unstimulated human lung fibroblasts (HFL-1) secreted TIMP at the rate of approximately 2 micrograms/10(6) cells/24 h, and normal foreskin fibroblasts (HS 27) and skin fibroblasts from a patient with Hurler's disease (GM 1391) secreted TIMP at 0.3 and 0.2 micrograms/10(6) cells/24 h, respectively. Secretion of TIMP was stimulated up to 10-fold by treating the cells with 20-100 ng/ml of 12-O-tetradecanoylphorbol 13-acetate or 10 units/ml of human interleukin 1. In the stimulated HFL-1 cells, TIMP accounted for 0.03-0.09% of the total [35S]methionine incorporated into protein, and 0.3-0.8% of the [35S]methionine in secreted protein. Although TIMP accounted for a relatively small proportion of total protein synthesis of the fibroblasts, greater than 80% of untreated and greater than 95% of stimulated fibroblasts synthesized TIMP, as determined by indirect immunofluorescence. The treatments of the human fibroblasts that increased TIMP secretion also induced synthesis and secretion of proenzyme forms of collagenase, indicating that degradative enzymes and their controlling inhibitors may be synthesized in parallel under certain conditions.
Murphy
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Reynolds
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Werb
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D000818:Animals; D002460:Cell Line; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D008666:Metalloendopeptidases; D008715:Methionine / Q000378:metabolism; D003012:Microbial Collagenase / Q000378:metabolism*; D010704:Phorbols / Q000494:pharmacology*; D011247:Pregnancy; D011480:Protease Inhibitors / Q000096:biosynthesis*; D012756:Sheep; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D003470:Culture Media; D007375:Interleukin-1; D010704:Phorbols; D011480:Protease Inhibitors; D008715:Methionine; D008666:Metalloendopeptidases; D003012:Microbial Collagenase; D013755:Tetradecanoylphorbol Acetate