Reduced Prostasin (CAP1/PRSS8) Activity Eliminates HAI-1 and HAI-2 Deficiency–Associated Developmental Defects by Preventing Matriptase Activation

Abstract

Loss of either hepatocyte growth factor activator inhibitor (HAI)-1 or -2 is associated with embryonic lethality in mice, which can be rescued by the simultaneous inactivation of the membrane-anchored serine protease, matriptase, thereby demonstrating that a matriptase-dependent proteolytic pathway is a critical developmental target for both protease inhibitors. Here, we performed a genetic epistasis analysis to identify additional components of this pathway by generating mice with combined deficiency in either HAI-1 or HAI-2, along with genes encoding developmentally co-expressed candidate matriptase targets, and screening for the rescue of embryonic development. Hypomorphic mutations in Prss8, encoding the GPI-anchored serine protease, prostasin (CAP1, PRSS8), restored placentation and normal development of HAI-1–deficient embryos and prevented early embryonic lethality, mid-gestation lethality due to placental labyrinth failure, and neural tube defects in HAI-2–deficient embryos. Inactivation of genes encoding c-Met, protease-activated receptor-2 (PAR-2), or the epithelial sodium channel (ENaC) alpha subunit all failed to rescue embryonic lethality, suggesting that deregulated matriptase-prostasin activity causes developmental failure independent of aberrant c-Met and PAR-2 signaling or impaired epithelial sodium transport. Furthermore, phenotypic analysis of PAR-1 and matriptase double-deficient embryos suggests that the protease may not be critical for focal proteolytic activation of PAR-2 during neural tube closure. Paradoxically, although matriptase auto-activates and is a well-established upstream epidermal activator of prostasin, biochemical analysis of matriptase- and prostasin-deficient placental tissues revealed a requirement of prostasin for conversion of the matriptase zymogen to active matriptase, whereas prostasin zymogen activation was matriptase-independent.

Distinct Developmental Functions of Prostasin (CAP1/PRSS8) Zymogen and Activated Prostasin

“…Prostasin, however, is upstream of matriptase in other epithelia (6), and zymogen-locked prostasin supports matriptase auto-activation in cell-based overexpression systems (13). We, therefore, next directly examined the status of matriptase activation in the epidermis of newborn Prss8 Ϫ/Ϫ , Prss8 zym/zym , and Prss8 ϩ/ϩ pups.…”

“…Loss-of-function genetic studies in mice have uncovered critical functions of prostasin in both the formation of epidermal barrier formation and the formation of whiskers and pelage hair (1,2). Prostasin is synthesized as an inactive proform (zymogen) that is converted to a catalytically active protease by a single endoproteolytic cleavage in the conserved activation cleavage site (3)(4)(5)(6). Prostasin zymogen conversion in the epidermis requires the membraneactivated serine protease matriptase (7)(8)(9).…”

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).…”

“…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).…”

Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1

“…It has also been proposed that HAI-1 inhibits the glycophosphatidylinositol membrane-anchored serine protease prostasin (10). Mouse studies also support a tight regulatory relationship between matriptase HAI-1 and prostasin (11). In vitro inhibition of additional membrane-associated serine proteases hepsin (12), TMPRSS13 (13), HAT (14), and HATL5 (15) as well as the soluble proteases trypsin, plasmin, and plasma kallikrein have also been reported (16).…”