title: Production of procollagenase by cultured human keratinocytes.
abstract: Using a collagen film assay utilizing 14C-labeled type I collagen, we demonstrated that cultured human keratinocytes produced a procollagenase after treatment with the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Production of collagenase paralleled alterations in cellular morphology induced by TPA. When procollagenase was immunoprecipitated with antibody to human fibroblast collagenase and analyzed on sodium dodecyl sulfate-polyacrylamide gels, the zymogen was revealed as a 56- and 51-kDa doublet. The keratinocyte-derived collagenase was a neutral metalloprotease, required activation with trypsin for detection in the collagenase assay and produced the characteristic three-quarter and one-quarter length collagen cleavage products when incubated with type I collagen at 25 degrees C. The enzyme was inhibited by serum and cysteine and was largely unaffected by serine, thiol, and carboxyl protease inhibitors. Cycloheximide inhibited the TPA-induced production of collagenase, suggesting that the procollagenase was not stored preformed in the keratinocytes. Keratinocytes treated with a tumor-promoting analogue of TPA also produced collagenase, but cells treated with cytochalasin B, interleukin-1, or two non-tumor promoting phorbol esters did not. Keratinocyte-derived collagenase may play a role in wound healing and morphogenesis.
mesh_terms: D000328:Adult; D000375:Aging; D002478:Cells, Cultured; D017364:Collagenases; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D007231:Infant, Newborn; D007633:Keratins / Q000032:analysis*; D007700:Kinetics; D008297:Male; D003012:Microbial Collagenase / Q000096:biosynthesis* / Q000378:metabolism; D012867:Skin / Q000201:enzymology* / Q000254:growth & development; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D004792:Enzyme Precursors; D007633:Keratins; D017364:Collagenases; C022440:procollagenase; D003012:Microbial Collagenase; D013755:Tetradecanoylphorbol Acetate