HGF (hepatocyte growth factor) 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 uPA (urokinase plasminogen activator), 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 uPAR (uPA receptor) or heparin. In contrast the catalytic domains of the TTSPs (type-II transmembrane serine proteases) matriptase and hepsin were highly efficient activators (50% activation at 0.1 and 3.4 nM respectively), at least four orders of magnitude more efficient than uPA. PS-SCL (positional-scanning synthetic combinatorial peptide libraries) were used to identify 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 PC3 prostate carcinoma cells was abolished by both protease inhibition and matriptase-targeting siRNA (small interfering RNA), and scattering of MDCK (Madin-Darby canine kidney) cells in the presence of pro-HGF was abolished by inhibition of matriptase. Hepsin-transfected HEK (human embryonic kidney)-293 cells also activated pro-HGF. These observations demonstrate that, in contrast with 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.
SummaryThe analysis of the not well understood composition of the stalk, a key ribosomal structure, in eukaryotes having multiple 12 kDa P1/P2 acidic protein components has been approached using these proteins tagged with a histidine tail at the C-terminus. Tagged
Pericellular proteolytic activity affects many aspects of cellular behaviour, via mechanisms involving processing of the extracellular matrix, growth factors and receptors. The serine proteases have exquisitely sensitive regulatory mechanisms in this setting, involving both receptor-bound and transmembrane proteases. Receptor-bound proteases are exemplified by the uPA (urokinase plasminogen activator)/uPAR (uPAR receptor) plasminogen activation system. The mechanisms initiating the activity of this proteolytic system on the cell surface, a critical regulatory point, are poorly understood. We have found that the expression of the TTSP (type II transmembrane serine protease) matriptase is highly regulated in leucocytes, and correlates with the presence of active uPA on their surface. Using siRNA (small interfering RNA), we have demonstrated that matriptase specifically activates uPAR-associated pro-uPA. The uPA/uPAR system has been implicated in the activation of the plasminogen-related growth factor HGF (hepatocyte growth factor). However, we find no evidence for this, but instead that HGF can be activated by both matriptase and the related TTSP hepsin in purified systems. Hepsin is of particular interest, as the proteolytic cleavage sequence of HGF is an 'ideal substrate' for hepsin and membrane-associated hepsin activates HGF with high efficiency. Both of these TTSPs can be activated autocatalytically at the cell surface, an unusual mechanism among the serine proteases. Therefore these TTSPs have the capacity to be true upstream initiators of proteolytic activity with subsequent downstream effects on cell behaviour.
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