title: The proteinase inhibitors leupeptin, pepstatin A, and TLCK cause reduced collagen production in freshly isolated embryonic chick fibroblasts in suspension culture.
abstract: The production of [14C]proline-labeled collagen by embryonic chick tendon cells in suspension culture is reduced when the cells are incubated in the presence of lysosomotropic agents NH4Cl or chloroquine. Since these agents have multiple effects on fibroblasts, including inhibition of collagen secretion, specific proteinase inhibitors were tested for their effect on collagen production. Here the proteinase inhibitors N-p-tosyl-L-lysine chloromethylketone (TLCK) and leupeptin, specific for certain cysteine and serine proteinases, and pepstatin A, specific for aspartic proteinases, were tested for their effects on both the production and secretion of collagen. When treated with the proteinase inhibitor TLCK, the percentage of protein synthesis devoted to collagen decreased from control levels of 19.0 +/- 1.4% to 10.5 +/- 2.4% with 10 microM TLCK. Collagen synthesis was further reduced to only 1.2% of total protein synthesis with 100 microM TLCK. The incorporation of [14C]proline into collagenase-digestible peptides was only slightly decreased in the samples treated separately with 50 micrograms/ml leupeptin or 60 micrograms/ml pepstatin A. However, the production of collagen was reduced to 10.9 +/- 1.4% of total protein synthesis in samples treated with leupeptin and pepstatin A together. The basal intracellular degradation of newly synthesized, [14C]proline-labeled collagen was not significantly altered by any of the reagents tested, and secretion of the collagen which was produced was not impaired except in samples treated with 100 microM TLCK. The data presented are consistent with the hypothesis that a proteolytic mechanism utilizing some combination of cysteine, serine, and aspartic proteinases is necessary for continued collagen synthesis in freshly isolated embryonic chick tendon fibroblasts, and suggests that a heretofore unknown regulatory system may be operative in controlling the synthesis of collagen in fibroblasts.
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D000818:Animals; D002642:Chick Embryo; D003094:Collagen / Q000096:biosynthesis*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D007976:Leupeptins / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology*; D010436:Pepstatins / Q000494:pharmacology*; D013710:Tendons / Q000378:metabolism; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology*
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D007976:Leupeptins; D009842:Oligopeptides; D010436:Pepstatins; C014195:Streptomyces pepsin inhibitor; D014107:Tosyllysine Chloromethyl Ketone; D003094:Collagen; C032854:leupeptin; C031375:pepstatin