Differential Subcellular Localization Renders HAI-2 a Matriptase Inhibitor in Breast Cancer Cells but Not in Mammary Epithelial Cells

Abstract

The type 2 transmembrane serine protease matriptase is under tight control primarily by the actions of the integral membrane Kunitz-type serine protease inhibitor HAI-1. Growing evidence indicates that HAI-2 might also be involved in matriptase inhibition in some contexts. Here we showed that matriptase inhibition by HAI-2 depends on the subcellular localizations of HAI-2, and is observed in breast cancer cells but not in mammary epithelial cells. HAI-2 is co-expressed with matriptase in 21 out of 26 human epithelial and carcinoma cells examined. HAI-2 is also a potent matriptase inhibitor in solution, but in spite of this, HAI-2 inhibition of matriptase is not observed in all contexts where HAI-2 is expressed, unlike what is seen for HAI-1. Induction of matriptase zymogen activation in mammary epithelial cells results in the formation of matriptase-HAI-1 complexes, but matriptase-HAI-2 complexes are not observed. In breast cancer cells, however, in addition to the appearance of matriptase-HAI-1 complex, three different matriptase-HAI-2 complexes, are formed following the induction of matriptase activation. Immunofluorescent staining reveals that activated matriptase is focused at the cell-cell junctions upon the induction of matriptase zymogen activation in both mammary epithelial cells and breast cancer cells. HAI-2, in contrast, remains localized in vesicle/granule-like structures during matriptase zymogen activation in human mammary epithelial cells. In breast cancer cells, however, a proportion of the HAI-2 reaches the cell surface where it can gain access to and inhibit active matriptase. Collectively, these data suggest that matriptase inhibition by HAI-2 requires the translocation of HAI-2 to the cell surface, a process which is observed in some breast cancer cells but not in mammary epithelial cells.

HAI‐2 stabilizes, inhibits and regulates SEA‐cleavage‐dependent secretory transport of matriptase

“…43 An unusually large binding interface was observed between the SPD of matriptase and HAI-1, in the order of 1800 Å, 2,47 which corresponds well with the strong binding observed between matriptase and HAI-1, as the complex can withstand separation on an SDS-PAGE gel. 48 Similarly, the matriptase-HAI-2 complex has been reported to withstand separation on an SDS-PAGE gel 49 , thus supporting the presence of at least one unknown ectosite where matriptase and HAI-2 interacts. It is possible, that a HAI-2-matriptase interaction at an undiscovered ectosite is responsible for the stabilization of the membrane-bound form of matriptase; however, it is at present time unclear where this ectosite is located.…”

“…This scheme, that is valid for a large number of protease‐inhibitor pairs, does not seem to apply for matriptase and HAI‐2, as HAI‐2 appears to have multiple roles towards matriptase, of both a stabilizing as well as a regulating nature. The newly synthesized non‐SEA domain cleaved form of matriptase appears to interact with HAI‐2 already in the ER, as HAI‐2 favors binding to the early non‐SEA domain cleaved matriptase, and only in exceptional cases has HAI‐2 been located on the plasma membrane . We suggest that the purpose of this interaction is to ensure the following 3 events: (1) inhibition of the proteolytic activity and/or inhibition of activation/auto‐activation of matriptase within the secretory pathway, to ensure safe passage of other proteins, especially relevant due to the relatively low zymogenicity factor of matriptase; (2) control of the folding, maturation and/or oligomerization of matriptase, possibly reflected by SEA domain cleavage of matriptase, making it transport competent, as mainly SEA domain cleaved matriptase is found on the plasma membrane and in the media; and (3) the previously described stabilization of the membrane bound form of matriptase …”

“…The newly synthesized non-SEA domain cleaved form of matriptase appears to interact with HAI-2 already in the ER, as HAI-2 favors binding to the early non-SEA domain cleaved matriptase, and only in exceptional cases has HAI-2 been located on the plasma membrane. 49 We suggest that the purpose of this interaction is to ensure the following 3 events: (1)…”

Selective Inhibition of Prostasin in Human Enterocytes by the Integral Membrane Kunitz-Type Serine Protease Inhibitor HAI-2

“…The hypothesis that it is excessive proteolysis caused by the loss of protease inhibitor that is responsible for the defect in these patients is supported by the observation that while matriptase, HAI-1, and HAI-2 play dispensable roles in placenta development, the defect in placental development caused by targeted deletion of HAI-1 or HAI-2 can be rescued by simultaneous deletion of matriptase [13,15]. In solution-based experiments, both HAI-1 and HAI-2 are potent inhibitors of matriptase and prostasin [11,20]. At the cellular level, however, the roles of HAIs in the control of matriptase and prostasin are much more complicated and largely depend on whether the proteins are coincided [20].…”

Natural Endogenous Human Matriptase and Prostasin Undergo Zymogen Activation via Independent Mechanisms in an Uncoupled Manner

“…Equal amounts of lysate (non-boiled, non-reducing) diluted with 5X sample loading buffer were fractionated by 10% SDS-PAGE, transferred to Amersham/Protran 0.45 um nitrocellulose (GE Healthcare), and then blocked with 5% non-fat milk in PBS/0.1%Tween20 (Sigma). Blots were probed with 1:2000 dilution of the anti-HAI-2 monoclonal DC16 [29,30] and imaged as described above.…”