Regulation of the Matriptase-Prostasin Cell Surface Proteolytic Cascade by Hepatocyte Growth Factor Activator Inhibitor-1 during Epidermal Differentiation

Abstract

Matriptase, a membrane-tethered serine protease, plays essential roles in epidermal differentiation and barrier function, largely mediated via its activation of prostasin, a glycosylphosphatidylinositol-anchored serine protease. Matriptase activity is tightly regulated by its inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) such that free active matriptase is only briefly available to act on its substrates. In the current study we provide evidence for how matriptase activates prostasin under this tight control by HAI-1. When primary human keratinocytes are induced to differentiate in a skin organotypic culture model, both matriptase and prostasin are constitutively activated and then inhibited by HAI-1. These processes also occur in HaCaT human keratinocytes when matriptase activation is induced by exposure of the cells to a pH 6.0 buffer. Using this acid-inducible activation system we demonstrate that prostatin activation is suppressed by matriptase knockdown and by blocking matriptase activation with sodium chloride, suggesting that prostatin activation is dependent on matriptase in this system. Kinetics studies further reveal that the timing of autoactivation of matriptase, prostasin activation, and inhibition of both enzymes by HAI-1 binding are closely correlated. These data suggest that, during epidermal differentiation, the matriptase-prostasin proteolytic cascade is tightly regulated by two mechanisms: 1) prostasin activation temporally coupled to matriptase autoactivation and 2) HAI-1 rapidly inhibiting not only active matriptase but also active prostasin, resulting in an extremely brief window of opportunity for both active matriptase and active prostasin to act on their substrates.Matriptase, a type II transmembrane serine protease, plays essential roles in epidermal differentiation and barrier function (1, 2). Mutations of matriptase are associated with autosomal recessive ichthyosis and hypotrichosis (3). Four mutations have been identified in ST14, the gene that encodes for matriptase (3-5). Targeted deletion of matriptase in mice also causes severe epidermal defects with impairment of desquamation, lipid matrix formation, and profilaggrin processing. These defects lead to compromised epidermal barrier function and postnatal death (2). The severe epidermal defects appear to result from the lack of activation of the glycosylphosphatidylinositolanchored serine protease prostasin by matriptase, because both matriptase-deficient and prostasin-deficient mice share an almost identical pattern of epidermal defects. Furthermore, lack of prostasin activation was observed in matriptase-deficient mouse skin (6, 7), suggesting that much of the function of matriptase in the epidermis is manifested through the activation of prostasin.Hepatocyte growth factor activator inhibitor-1 (HAI-1) 2 is a transmembrane Kunitz-type serine protease inhibitor (8). HAI-1 can inhibit matriptase in a competitive and reversible manner, consistent with the characteristics of Kunitz-type serine protease inhibitors (...

The Membrane-anchored Serine Protease Prostasin (CAP1/PRSS8) Supports Epidermal Development and Postnatal Homeostasis Independent of Its Enzymatic Activity

“…It is now generally recognized that prostasin and the type II transmembrane serine protease, matriptase, form part of a single epithelial proteolytic cascade in the context of placental development, terminal epidermal differentiation, and epithelial tight junction formation (5)(6)(7)(8)(9)(10). The specific mechanistic interrelationship between the two proteases, however, has remained unclear, with different studies placing prostasin either upstream or downstream from matriptase depending on the specific context (5)(6)(7)9).…”

Matriptase Expression and Zymogen Activation in Human Pilosebaceous Unit

“…The total matriptase mAbs M24 and M32 recognize both latent and activated forms of human matriptase. M69 mAb is specific for activated matriptase and does not recognize latent matriptase, and M19 mAb specifically recognizes human HAI-1 (Chen et al, 2010b;Lin et al, 1997;Lin et al, 1999;Tseng et al, 2010). HGF protein was detected using a goat polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) that recognizes the beta subunit of HGF.…”

“…Thus, matriptase might exert an effect on hair follicles via such downstream targets. Thirdly, matriptase can also activate other serine proteases, including the urokinase-type plasminogen activator (uPA) (Chen et al, 2010b;Lee et al, 2000;Netzel-Arnett et al, 2006;Takeuchi et al, 2000). Activation of the uPA/plasmin system by matriptase could impact extracellular matrix (ECM) remodeling processes, which are known to modulate processes important in normal hair biology, such as involution and downward growth (Gao et al, 2010;Jarrousse et al, 2001;Paus et al, 1994;Weinberg et al, 1990).…”

Hepatocyte Growth Factor Activator Inhibitor Type 1 Maintains the Assembly of Keratin into Desmosomes in Keratinocytes by Regulating Protease-Activated Receptor 2–Dependent p38 Signaling

“…The following antibodies were used: anti-matriptase mouse monoclonal antibody (mAb; M24), 32 anti-activated matriptase mouse mAb (M69), 32 antieHAI-1 goat polyclonal antibody (R&D Systems, Minneapolis, MN), anti-prostasin mouse mAb (BD Bioscience, San Jose, CA), antiePAR-2 mouse mAb SAM11 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-p38 and anti-phosphorylated p38 MAPK (Thr180/Tyr182) rabbit mAbs (Cell Signaling Technology Japan, Tokyo, Japan), Alexa Fluor 594econjugated anti-phosphorylated p38 MAPK (Thr180/Tyr182) rabbit mAbs (Cell Signaling Technology), anti-AKT and anti-phosphorylated AKT (Ser473) rabbit mAbs (Cell Signaling Technology), anti-p44/42 MAPK [extracellular signaleregulated kinase (ERK) 1/2] rabbit polyclonal antibody and anti-phosphorylated ERK1/2 (Thr202/Tyr204) rabbit mAb (Cell Signaling Technology), anti-cytokeratin (CK) 5 rabbit mAb (Abcam, Cambridge, UK), anti-CK10 mouse mAb (Dako, Tokyo, Japan), and anti-desmoglein 3 mouse mAb (Life Technologies Japan, Tokyo, Japan). Aprotinin and specific p38 inhibitor, SB203580, were purchased from Sigma (St. Louis, MO).…”