No abstract

The extracellular protease urokinase is known to be crucially involved in morphogenesis, tissue repair and tumor invasion by mediating matrix degradation and cell migration. Hepatocyte growth factor/scatter factor (HGF/SF) is a secretory product of stromal fibroblasts, sharing structural motifs with enzymes of the blood clotting cascade, including a zymogen cleavage site. HGF/SF promotes motility, invasion and growth of epithelial and endothelial cells. Here we show that HGF/SF is secreted as a single‐chain biologically inactive precursor (pro‐HGF/SF), mostly found in a matrix‐associated form. Maturation of the precursor into the active alpha beta heterodimer takes place in the extracellular environment and results from a serum‐dependent proteolytic cleavage. In vitro, pro‐HGF/SF was cleaved at a single site by nanomolar concentrations of pure urokinase, generating the active mature HGF/SF heterodimer. This cleavage was prevented by specific urokinase inhibitors, such as plasminogen activator inhibitor type‐1 and protease nexin‐1, and by antibodies directed against the urokinase catalytic domain. Addition of these inhibitors to HGF/SF responsive cells prevented activation of the HGF/SF precursor. These data show that urokinase acts as a pro‐HGF/SF convertase, and suggest that some of the growth and invasive cellular responses mediated by this enzyme may involve activation of HGF/SF.

Scatter factor (SF), a secretory protein of fibroblasts, dissociates and increases the motility of epithelial cells and may be involved in cell migration processes during embryogenesis and tumor progression. Hepatocyte growth factor (HGF), a protein isolated from serum of patients with liver failure, is a potent mitogen for hepatocytes and is thought to play a role in liver regeneration. Here we present structural and functional evidence that human SF and human HGF (and also the human lung fibroblast-derived mitogen) are identical proteins encoded by a single gene, since (a) no major difference could be found by protein sequencing, by cDNA analysis, and by immunological comparison and (ii) SF in fact acts as a hepatocyte growth factor-i.e., stimulates DNA synthesis of primary hepatocytes-whereas HGF exhibits scatter factor activity-i.e., dissociates and induces invasiveness of various epithelial cells. The human SF/HGF gene was localized to chromosome bands 7q11.2-21. These results have important consequences for further studies on the involvement of SF/HGF as a modulator of cellular growth and motility in embryonal, malignant, and regenerative processes.

Human hepatocyte growth factor (hHGF) has been purified 209,000-fold with 18% yield from plasma of a patient with fulminant hepatic failure. The purification involves heat treatment of.plasma, ammonium sulfate precipitation, and chromatography on Affi-Gel Blue, heparin-Sepharose, and hydroxylapatite. Purified hHGF shows several bands with molecular weights between 76,000 and 92,000. Each band shows growthstimulating activity on cultured hepatocytes which is proportional to the intensity of the band. After reduction of the sample with 2-mercaptoethanol, SDS-PAGE yields two chains with molecular weights of 31,500-34,500 and 54,000-65,000. The effect of hHGF on DNA synthesis by hepatocytes is halfmaximal at 3.5 ng/ml. hHGF stimulates proliferation of cultured hepatocytes more effectively than human epidermal growth factor (hEGF) or insulin, and the effect of hHGF is additive or synergistic with the maximal effects of hEGF and insulin. These results suggest that hHGF is a new growth factor which is different from hEGF.

Human hepatocyte growth factor (hHGF) has recently been expressed as a recombinant polypeptide from Chinese hampster ovary cell transfectants. Using a primary rat hepatocyte bioassay, we have tested the biological activity of recombinant hHGF and compared it with native hHGF. Dose-response curves were almost identical, with half-maximal stimulation of DNA synthesis at 1-2 ng/ml (equivalent to -10 pM