Cleavage of hepatocyte growth factor activator inhibitor‐1 by membrane‐type MMP‐1 activates matriptase

Abstract

Co-expression of membrane-type 1 (MT1)-MMP with hepatocyte growth factor activator inhibitor-1 (HAI-1) in HEK293T cells resulted in cleavage of HAI-1 to produce three fragments. Recombinant MT1-MMP was shown to cleave HAI-1 protein in vitro. Hepatocyte growth factor activator inhibitor-1 was initially identified as the cognate inhibitor of matriptase, a transmembrane serine protease that processes urokinase-type plasminogen activator (uPA). Co-expression of HAI-1 with matriptase suppressed matriptase protease activity, and co-expression of MT1-MMP with them resulted in recovery of matriptase activity by stimulating shedding of HAI-1 fragments. Matriptase protein was detected in squamous carcinoma-derived HSC-4 cells, however, matriptase protease activity was undetectable. Transfection of siRNA for HAI-1 enhanced serine protease activity, which was suppressed by cotransfection of matriptase siRNA. Collagen-gel culture or treatment with concanavalin A (ConA) of HSC-4 cells enhanced MT1-MMP activity, which induced shedding of HAI-1 fragments and conversely stimulated uPA activation by these cells. Serine protease activity, including uPA activation of cells treated with ConA, was abrogated by downregulation of either matriptase or MT1-MMP through the transfection of each siRNA. These results suggest that MT1-MMP induced by collagen-gel culture or ConA treatment causes cleavage and shedding of HAI-1 protein, which allows activation of matriptase in HSC-4 cells. HSC-4 cells showed a characteristic invasive growth by forming vacuole-like structures in collagen gel, which was suppressed by transfection of siRNA for either MT1-MMP or matriptase, suggesting that activation of matriptase through the cleavage of HAI-1 is one of the MT1-MMP multifunctions essential for invasive growth of HSC-4 cells. (Cancer Sci 2012; 103: 448-454) H epatocyte growth factor activator inhibitor-1 is a membrane-associated Kunitz-type serine protease inhibitor. (1)(2)(3)(4)(5) It was initially identified as the cognate inhibitor of HGFA, (6) and purified from human milk as a complex with matriptase, a multidomain, transmembrane serine protease of the S1 trypsinlike family.(7-11) Matriptase was detected in a variety of human tumors of epithelial origin or phenotype and has been implicated in the initiation and progression of human carcinomas. (12)(13)(14) Matriptase mediates the degradation of ECM components and activates growth and angiogenic factors, which not only facilitates cellular invasiveness but may also activate oncogenic pathways. These functions are partially attributed to its role in the activation of HGF and uPA.(15-17) Both HGF and uPA have been implicated in cancer invasion and metastasis for their roles in cellular motility, ECM degradation, and tumor vascularization.(1,18) The HAI-1 fragments are often identified in culture supernatant of cells in complex with proteases, suggesting that proteolytic processing of HAI-1 may play roles in regulation of inhibitory activity. (19,20) However, the molecular mechanism of HAI-1 shedd...

Endothelial Sirtuin 1 Deficiency Perpetrates Nephrosclerosis through Downregulation of Matrix Metalloproteinase-14

“…26,27 We confirmed this activity of ConA in vitro by pretreating HUVECs overnight with different concentrations of ConA, which consistently resulted in increased expression of MMP-14 ( Figure 7A). Importantly, ConA effect was independent on the expression of SIRT1, as it occurred in cells treated with SIRT1 inhibitor III.…”

Hepatocyte Growth Factor Activator Inhibitor Type 1 Is a Suppressor of Intestinal Tumorigenesis

“…1B), suggesting that the decreased immunoreactivity may be caused by enhanced shedding of membrane-bound Hai-1/Spint1 from the neoplastic cell surface. Given that HAI-1 shedding is reported to be mediated by metalloprotease activity (29) and in human oral carcinoma cells MT1-MMP/MMP14 is responsible for HAI-1/SPINT1 ectodomain shedding (30), we compared Mmp14 expression in tumor tissue with corresponding normal mucosa in Apc Min/þ intestine and found that the Mmp14 mRNA level was significantly increased in tumor tissue (Fig. 1C).…”

“…In the MT1rev2 cells, MT1-MMP/Mmp14 expression did indeed result in enhanced Hai-1/Spint1 shedding into the culture supernatant generating 58-and 42-kDa secreted forms as previously reported (ref. 30; Fig. 1D).…”

“…We hypothesize that membranous Hai-1/Spint1 may be critical for "normal" matriptase function that is required for epithelial integrity, and loss of Hai-1/Spint1 may cause abnormal subcellular localization or enhanced release of matriptase under adverse cellular conditions. In fact, HAI-1/SPINT1 is required for intracellular matriptase trafficking (15,16,47), and in tumor cells, loss of membrane-associated HAI-1/SPINT1 resulted in decreased membrane-associated matriptase and increased pericellular matriptase activity (27,30) that might activate pericellular molecules involved in tumor progression such as proHGF. In the current study, intestinal tumor cells with decreased membranous Hai-1/Spint1 showed abnormal matriptase subcellular localization.…”

Deregulated hepsin protease activity confers oncogenicity by concomitantly augmenting HGF/MET signalling and disrupting epithelial cohesion

“…28,29 The shorter secreted 58 kDa and 42 kDa HAI-1 forms are cleaved from plasma membrane-associated 66 kDa form and earlier studies have reported an altered or elevated pattern of HAI-1 shedding in context of prostate cancer and experimental systems with elevated protease activity. 28,30,31 MCF10A cells express both the plasma membrane-associated and secreted forms of HAI-1, and we found that Dox-induced hepsin already at early time-points led to the appearance of a 42 kDa HAI-1 form in the conditioned medium (Figures 2i and j). The 42 kDa shed HAI-1 form has been reported earlier and it contains a Kunitz domain required for the inhibition of hepsin and other serine proteases.…”