Transport via the Transcytotic Pathway Makes Prostasin Available as a Substrate for Matriptase

Abstract

The matriptase-prostasin proteolytic cascade is essential for epidermal tight junction formation and terminal epidermal differentiation. This proteolytic pathway may also be operative in a variety of other epithelia, as both matriptase and prostasin are involved in tight junction formation in epithelial monolayers. However, in polarized epithelial cells matriptase is mainly located on the basolateral plasma membrane whereas prostasin is mainly located on the apical plasma membrane. To determine how matriptase and prostasin interact, we mapped the subcellular itinerary of matriptase and prostasin in polarized colonic epithelial cells. We show that zymogen matriptase is activated on the basolateral plasma membrane where it is able to cleave relevant substrates. After activation, matriptase forms a complex with the cognate matriptase inhibitor, hepatocyte growth factor activator inhibitor (HAI)-1 and is efficiently endocytosed. The majority of prostasin is located on the apical plasma membrane albeit a minor fraction of prostasin is present on the basolateral plasma membrane. Basolateral prostasin is endocytosed and transcytosed to the apical plasma membrane where a long retention time causes an accumulation of prostasin. Furthermore, we show that prostasin on the basolateral membrane is activated before it is transcytosed. This study shows that matriptase and prostasin co-localize for a brief period of time at the basolateral plasma membrane after which prostasin is transported to the apical membrane as an active protease. This study suggests a possible explanation for how matriptase or other basolateral serine proteases activate prostasin on its way to its apical destination.The trypsin-like membrane serine protease matriptase is essential for maintenance of multiple types of epithelia. Conditional ablation of the St14 gene coding for matriptase in intestine, kidney, and lung of adult mice results in weight loss, severe decline in health and death within 2 weeks, caused by organ dysfunction associated with increased permeability and loss of tight junctions (1). Knock down of matriptase by siRNA in a cell model of the intestinal epithelium caused a leaky barrier, impaired ability to develop transepithelial electrical resistance (TEER) 2 and enhanced paracellular permeability through regulation of tight junction proteins (2). Together these data suggest a key role for matriptase in epithelial barrier function and tight junction assembly.Prostasin (also known as CAP1 and PRSS8) is a GPI-anchored trypsin-like serine protease. Prostasin is co-expressed with matriptase in most epithelial tissues including the epidermis, kidney, and colon (3). Prostasin proteolytic activity has also been suggested to promote the development of functional tight junctions, TEER and paracellular permeability (4 -6). Unlike matriptase, which undergoes efficient autoactivation, the prostasin zymogen is not able to auto-activate, and formation of active prostasin requires activation site cleavage by other trypsin-like serine proteases. Strong...

Activity and inhibition of prostasin and matriptase on apical and basolateral surfaces of human airway epithelial cells

“…The finding in the present study that most of the tryptic activity on the apical/lumenal surface of airway epithelial cells is prostasin-like is consistent with recent findings in colonic epithelial cells, suggesting that matriptase is activated on the basolateral surface (16). However, the present studies, which differ not only in the type of cell studied but in culture conditions (air-liquid interface), suggest that matriptase was present on the apical surface although largely inactive given lack of inhibition by scF v anti-matriptase, which is capable of inactivating membrane-bound matriptase in its active form (14,30).…”

“…Whole cell lysates contain prostasin originating from both polarized surfaces and from membrane-bound intracellular organelles, like endoplasmic reticulum and transport vesicles. This finding is consistent with prostasin being activated on the cell surface or being transported promptly to the surface after activation and is also consistent with the recent suggestion that prostasin can be activated by basolateral matriptase and conveyed to the apical surface in transcytotic vesicles (16). These observations regarding surface localization of mature prostasin, in combination with comparisons of surface and whole cell lysate prostasin-like activity, suggest that a substantial pool of activated prostasin lies on each of the membrane surfaces.…”

“…In hypertension, retention of Na ϩ increases blood volume and pressure. Recent evidence suggests that expression of matriptase and prostasin is polarized and that the enzymes are transported vectorially across epithelium (16). Although tryptic activity has been detected on the surface of frog oocytes and cultured epithelial cells (18), little is known concerning relative surface expression of active matriptase and prostasin.…”

Essential Role of Endocytosis of the Type II Transmembrane Serine Protease TMPRSS6 in Regulating Its Functionality

“…Reports have also shown that membrane type 1 matrix metalloproteinase, an integral type I transmembrane metalloproteinase involved in cleaving extracellular matrix proteins, undergoes dynamin-dependent endocytosis using both clathrin-mediated and clathrin-independent pathways (36). Finally, very recent data reveal how another member of the type II transmembrane serine protease family, matriptase, accumulates in intracellular structures (37), bringing support to the notion that these proteins undergo dynamic trafficking.…”

Breaking the epithelial polarity barrier in cancer: the strange case of LKB1/PAR-4

“…It still remains a mystery how cytosolic hepsin interacts with basement membrane but it is interesting to note that vesicular trafficking plays an important role in the activation cascades of other TTSPs. A TTSP family member, matriptase has been reported to activate a downstream TTSP prostasin on the basolateral membrane of polarized colonic epithelial cells from which activated prostasin is intracellularly transcytosed to apical membrane of the cells [111]. It is tempting to speculate that loss of cues for directional secretion in depolarized cancer cells perturbs cell polarity-coupled proteolytic cascades.…”