Abstract
Hepatocyte growth factor (HGF) activator is a serine protease that is produced and secreted by the liver and circulates in the blood as an inactive zymogen. In response to tissue injury, the HGF activator zymogen is converted to the active form by limited proteolysis. The activated HGF activator converts an inactive single chain precursor of HGF to a biologically active heterodimer in injured tissue. The activated HGF may be involved in the regeneration of the injured tissue. In this study, we purified an inhibitor of HGF activator from the conditioned medium of a human MKN45 stomach carcinoma cell line and molecularly cloned its cDNA. The sequence of the cDNA revealed that the inhibitor has two well defined Kunitz domains, suggesting that the inhibitor is a member of the Kunitz family of serine protease inhibitors. The sequence also showed that the primary translation product of the inhibitor has a hydrophobic sequence at the COOH-terminal region. Inhibitory activity toward HGF activator was detected in the membrane fraction as well as in the conditioned medium of MKN45 cells. These results suggest that the inhibitor may be produced as a membrane-associated form and secreted by the producing cells as a proteolytically truncated form.
Highlights
Hepatocyte growth factor (HGF),1 known as scatter factor, is a mesenchymally derived heparin-binding glycoprotein that is secreted as an inactive single chain precursor from producing cells, and normally it remains in this form [1], probably associated with the extracellular matrix in the producing tissues [2]
The zymogen is activated by limited proteolysis by thrombin, and the NH2-terminal half region of the zymogen is removed by plasma kallikrein in vitro [11]
We found an inhibitory activity toward HGF activator in serum-free conditioned media of various human cell lines
Summary
Cell Lines and Cell Culture—The following cell lines were obtained from the indicated sources: human melanoma cell lines C32 and A375, human lung carcinoma cell line A549, human lung fibroblast cell line HLF, human hepatoma cell lines Hep G2 and PLC/PRF/5, and human colon carcinoma cell line CCL229 from American Type Culture Collection (Rockville, MD); human stomach carcinoma cell lines HSC-3 and MKN45 and human lung carcinoma cell lines PC-9 and PC-3 from IBL (Gunma, Japan); human lung carcinoma cell line HLC-1 and human stomach carcinoma cell line IG-1 from Department of Physiology, Keio This is an Open Access article under the CC BY license.
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