Pericellular activation of hepatocyte growth factor by the transmembrane serine proteases matriptase and hepsin, but not by the membrane-associated protease uPA.

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.

Proteomic identification of caldesmon as a physiological substrate of proprotein convertase 6 in human uterine decidual cells essential for pregnancy establishment.

Proprotein convertase 5/6 (PC6), a member of the proprotein convertase (PC) family, is a critical regulator in the uterus for embryo implantation. In particular, PC6 is essential for the differentiation of uterine stromal fibroblasts into decidual cells (decidualization). Knockdown of PC6 in the mouse uterus leads to complete failure of decidualization and implantation. It has been envisaged that PC6 functions by proteolytically activating a number of important growth factors and other precursor proteins. However, to date, the precise mechanism of PC6 action in the uterus, particularly during decidualization, is unknown. In this study, we aimed to investigate the mechanisms of PC6 action in decidualization by identifying its physiological substrates using a proteomic approach. Primary human endometrial stromal cells were decidualized and treated with or without recombinant human PC6 (rhPC6). The proteins cleaved by rhPC6 were identified by 2-dimensional fluorescent differential gel electrophoresis. The candidate proteins were validated as PC6 substrates by a number of approaches, including determining their coexpression with PC6 in vivo in decidual cells in the human uterus. A total of 18 protein spots were significantly altered by rhPC6 treatment, 8 of which were assigned clear identities by mass spectrometry. One of these was confirmed to be caldesmon, a key protein involved in organizing the actin microfilaments and regulating cytoskeletal structure. This study demonstrates a novel approach to identify PC-regulated proteins of physiological relevance, and provides important insight into the mechanism by which PC6 regulates decidualization.

Initiation of plasminogen activation on the surface of monocytes expressing the type II transmembrane serine protease matriptase.

uPA (urokinase-type plasminogen activator) activates plasminogen with high efficiency when bound to its cellular receptor uPAR, but only after a prolonged lag phase during which generated plasmin activates pro-uPA. How the activity of this proteolytic system might be rapidly initiated is unknown. We have now found that 2 monocytic cell lines display distinct patterns of plasminogen activation. U937 cells, but not THP-1 cells, displayed the expected lag phase, suggesting a constitutive initiation mechanism on the latter. This was shown to be due to the plasmin-independent activation of uPAR-bound pro-uPA by a cell surface-associated protease and to correlate with the expression of matriptase, a type II transmembrane serine protease that was highly expressed in THP-1 cells but undetectable in U937 cells. Kinetic analysis demonstrated that matriptase is a relatively poor activator of pro-uPA in solution, approximately 100-fold less efficient than plasmin (k(cat)/K(m) 1.16 x 10(5) M(-1)s(-1) cf 1.21 x 10(7) M(-1)s(-1)). However, down-regulation of matriptase expression in THP-1 cells by siRNA reduced the activation of cell-associated pro-uPA and the subsequent rapid initiation of plasminogen activation by 76% to 93%. Matriptase was also found to be expressed by peripheral blood monocytes and may therefore be a specific mechanism for the rapid initiation and regulation of plasminogen activation by these cells.