title: Collagenase is a major gene product of induced rabbit synovial fibroblasts.
abstract: We have investigated the effects of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), on rabbit synovial fibroblasts, and found that this agent induced a major switch in gene expression in these cells that was marked by the specific induction of the neutral proteinase, collagenase, and was always accompanied by alterations in cell morphology. Procollagenase synthesis and secretion was first observed 6-12 h after the addition of TPA. The rate of collagenase production (1-5 U, or approximately 0.2-1 micrograms secreted procollagenase protein per 10(5) cells per 24 h) depended on the TPA concentration (1-400 ng/ml) and time of exposure (1-72 h). Procollagenase was the most prominent protein visible by direct silver staining or by autoradiography after SDS PAGE of [35S]methionine-labeled proteins. The two procollagenase bands of Mr 53,000 and 57,000, which migrated as a family of spots on two-dimensional gels and were immunoprecipitated by antibodies to purified rabbit collagenase, accounted for 23% of the newly synthesized, secreted protein in TPA-treated cells. Cell-free translation of mRNA from TPA-treated cells in rabbit reticulocyte lysate produced a single band of immunoprecipitable preprocollagenase (Mr 55,000) as a major product (5% of total) that migrated as a single spot on two-dimensional gels. Secreted procollagenase, preprocollagenase , and active collagenase (purified to homogeneity; specific activity 1.2 X 10(4) U/mg protein) had related peptide maps. Two other major secreted proteins, a neutral metalloproteinase of Mr 51,000 and a polypeptide of Mr 47,000, were also induced by TPA. In contrast to the induction of these four polypeptides, TPA decreased synthesis and secretion of a number of proteins, including collagen and fibronectin. Thus, collagenase is a convenient marker for major alterations in the pattern of protein synthesis and secretion by rabbit synovial fibroblasts treated with TPA.
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D017364:Collagenases; D004792:Enzyme Precursors / Q000096:biosynthesis; D005347:Fibroblasts / Q000201:enzymology; D005786:Gene Expression Regulation / Q000187:drug effects; D007700:Kinetics; D003012:Microbial Collagenase / Q000096:biosynthesis* / Q000235:genetics; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011817:Rabbits; D013583:Synovial Membrane / Q000201:enzymology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D004792:Enzyme Precursors; D017364:Collagenases; C022440:procollagenase; D003012:Microbial Collagenase; D013755:Tetradecanoylphorbol Acetate