Abstract
The type II transmembrane serine protease matriptase is a key regulator of epithelial barriers in skin and intestine. In skin, matriptase acts upstream of the glycosylphosphatidylinositol-anchored serine protease, prostasin, to activate the prostasin zymogen and initiate a proteolytic cascade that is required for stratum corneum barrier functionality. Here, we have investigated the relationship between prostasin and matriptase in intestinal epithelial barrier function. We find that similar to skin, matriptase and prostasin are components of a common intestinal epithelial barrier-forming pathway. Depletion of prostasin by siRNA silencing in Caco-2 intestinal epithelium inhibits barrier development similar to loss of matriptase, and the addition of recombinant prostasin to the basal side of polarized Caco-2 epithelium stimulates barrier forming changes similar to the addition of recombinant matriptase. However, in contrast to the proteolytic cascade in skin, prostasin functions upstream of matriptase to activate the endogenous matriptase zymogen. Prostasin is unable to proteolytically activate the matriptase zymogen directly but induces matriptase activation indirectly. Prostasin requires expression of endogenous matriptase to stimulate barrier formation since matriptase depletion by siRNA silencing abrogates prostasin barrier-forming activity. Active recombinant matriptase, however, does not require the expression of endogenous prostasin for barrier-forming activity. Together, these data show that matriptase and not prostasin is the primary effector protease of tight junction assembly in simple columnar epithelia and further highlight a spatial and tissue-specific aspect of cell surface proteolytic cascades.
Highlights
Full-length matriptase is initially synthesized as a 95-kDa zymogen that undergoes spontaneous cleavage at Gly149 in its SEA domain, prior to a second activation inducing cleavage at its canonical activation motif that generates a disulfide-linked active protease, which remains tethered to the plasma membrane [32]
Using an antibody that detects the serine protease domain, the unprocessed full-length matriptase form is not detected in Caco-2 cultures; the predominant form present is the SEA-processed 70-kDa matriptase zymogen (Fig. 1A)
The co-induction of prostasin and matriptase expression during tight junction assembly and the development of transepithelial electrical resistance (TEER) are consistent with a cooperative role for these proteases in intestinal epithelial barrier formation
Summary
Does not require the expression of endogenous prostasin for barrier-forming activity Together, these data show that matriptase and not prostasin is the primary effector protease of tight junction assembly in simple columnar epithelia and further highlight a spatial and tissue-specific aspect of cell surface proteolytic cascades. The onset of expression of these two proteases during embryonic development is coordinated and correlated with acquisition of skin barrier functionality [6] Both matriptase and prostasin are synthesized as zymogens and require cleavage at a conserved activation motif between their pro- and catalytic domains to gain catalytic activity. In contrast to the protease activation cascade that promotes barrier function in stratified epithelia of the epidermis, we found that prostasin functions upstream of matriptase in simple columnar epithelia of intestine, with matriptase being the critical effector of intestinal epithelial barrier formation and tight junction assembly
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