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Inhibition of 12-O-Tetradecanoylphorbol-13-acetate Induction of Epidermal Transglutaminase Activity by Protease Inhibitors

In Vitro Pathogenesis Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Cancer Cause and Prevention, National Cancer Institute, Bethesda, Maryland 20205

Pretreatment of primary mouse epidermal cell cultures with chymostation, a protease inhibitor, blocked the increase in transglutaminase (R-glutaminyl-peptide:amine -glutamyltransferase, EC 2.3.2.13) activity resulting from treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or with retinoic acid. This inhibitory effect was dependent upon both the concentration of chymostatin used and preincubation time and was eliminated when chymostatin was inactivated by NaBH₄ reduction. Five other protease inhibitors, antipain, leupeptin, pepstatin, aprotinin, and soybean trypsin inhibitor, also suppressed TPA induction of transglutaminase activity. However, neither chymostatin, nor antipain, nor leupeptin reduced protein synthesis as measured by incorporation of labeled leucine into acid-precipitable products, while pepstatin, aprotinin, and soybean trypsin inhibitor inhibited protein synthesis markedly. L-1-Tosylamide-2-phenylethylchloromethyl ketone, on the other hand, strongly inhibited protein synthesis but did not inhibit the increase of transglutaminase activity after TPA exposure, and elastatinal inhibited neither TPA action nor protein synthesis. Chymostatin did not block phorbol ester binding to epidermal cells or TPA-mediated reduction of epidermal growth factor binding. These results suggest that the apparent induction of intracellular transglutaminase activity is mediated by a chymostatin-sensitive protease, while both phorbol ester binding and the reduction by TPA of epidermal growth factor binding at the cell membrane were independent.

¹ To whom requests for reprints should be addressed, at Room 3A23, Building 37, NIH, Bethesda, Md. 20205.

Received 11/12/82. Accepted 6/ 2/83.