Pericellular activation of hepatocyte growth factor by the transmembrane serine proteases matriptase and hepsin, but not by the membrane-associated protease uPA
Résumé
Hepatocyte growth factor (HGF) is a pleiotropic cytokine homologous to the serine protease zymogen plasminogen that requires canonical proteolytic cleavage to gain functional activity. The activating proteases are key components of its regulation, but controversy surrounds their identity. Using quantitative analysis we found no evidence for activation by the urokinase-type plasminogen (uPA), despite reports that this is a principal activator of pro-HGF. This was unaffected by a wide range of experimental conditions, including the use of various molecular forms of both HGF and uPA, and the presence of uPA receptor (uPAR) or heparin. By contrast the catalytic domains of the type-II transmembrane serine proteases (TTSPs) matriptase and hepsin were highly efficient activators (50% activation at 0.1 and 3.4 nM, respectively), at least 4-orders of magnitude more efficient than uPA. Positional scanning-synthetic combinatorial peptide libraries identified consensus sequences for the TTSPs, which in the case of hepsin corresponded to the pro-HGF activation sequence, demonstrating a high specificity for this reaction. Both TTSPs were also found to be efficient activators at the cell surface. Activation of pro-HGF by PC-3 prostate carcinoma cells was abolished by both protease inhibition and matriptase-targeting siRNA, and scattering of MDCK in the presence of pro-HGF was abolished by inhibition of matriptase. Hepsin-transfected 293 cells also activated pro-HGF. These observations demonstrate that, in contrast to the uPA/uPAR system, the TTSPs matriptase and hepsin are direct pericellular activators of pro-HGF, and that together these proteins may form a pathway contributing to their involvement in pathological situations, including cancer.
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