Efficient Purification of TIMP-2 from Culture Medium Conditioned by Human Hepatoma Cell Line, and Its Inhibitory Effects on Metalloproteinases and In Vitro Tumor Invasion

Abstract

Metalloproteinase inhibitors were surveyed with the culture media of 19 kinds of human tumor cell lines, using transin (rat stromelysin) as the target enzyme. This survey showed that most of the cell lines more or less secreted inhibitor activity, and that a human hepatoma cell line, HLE, secreted an extremely high inhibitor activity into the culture medium. Two kinds of metalloproteinase inhibitors were purified from the serum-free conditioned medium of HLE cells. The major inhibitor, which showed a single protein band with a molecular weight (M_r) of 21, 000 (21k) (nonreduced) or 24k (reduced) on SDS-polyacrylamide gel electrophoresis, was identified as TIMP-2 (tissue inhibitor of metallo-proteinases-2) by the analysis of its N-terminal amino acid sequence. The other was immunologically identified as TIMP. Purified TIMP-2 inhibited the activities of transin, matrin (pump-1), M_r 72k gelatinase, and interstitial collagenase with 1 : 1 stoichiometry. When the latent precursor form (M_r 57k) of transin was incubated with p-aminophenylmer-curie acetate as an activating reagent, TIMP-2 inhibited the conversion of the intermediate form (M_r 45k) into the mature enzyme (M_r 42k). This indicated that TIMP-2 regulates not only the activity of the mature enzyme but also the autolytic processing of the proenzyme. TIMP-2 also inhibited in vitro tumor invasion through reconstituted basement membrane (matrigel) in chemotaxis chambers, showing that the metalloproteinase inhibitors as well as the extracellular matrix metalloproteinases are involved in tumor invasion through basement membrane and other extracellular matrices.