Date Published: February 13, 2018
Publisher: Public Library of Science
Author(s): Shiao-Pieng Lee, Chen-Yu Kao, Shun-Cheng Chang, Yi-Lin Chiu, Yen-Ju Chen, Ming-Hsing G. Chen, Chun-Chia Chang, Yu-Wen Lin, Chien-Ping Chiang, Jehng-Kang Wang, Chen-Yong Lin, Michael D. Johnson, Mohammad Saleem.
The membrane-bound serine proteases prostasin and matriptase and the Kunitz-type protease inhibitors HAI-1 and HAI-2 are all expressed in human skin and may form a tightly regulated proteolysis network, contributing to skin pathophysiology. Evidence from other systems, however, suggests that the relationship between matriptase and prostasin and between the proteases and the inhibitors can be context-dependent. In this study the in vivo zymogen activation and protease inhibition status of matriptase and prostasin were investigated in the human skin. Immunohistochemistry detected high levels of activated prostasin in the granular layer, but only low levels of activated matriptase restricted to the basal layer. Immunoblot analysis of foreskin lysates confirmed this in vivo zymogen activation status and further revealed that HAI-1 but not HAI-2 is the prominent inhibitor for prostasin and matriptase in skin. The zymogen activation status and location of the proteases does not support a close functional relation between matriptase and prostasin in the human skin. The limited role for HAI-2 in the inhibition of matriptase and prostasin is the result of its primarily intracellular localization in basal and spinous layer keratinocytes, which probably prevents the Kunitz inhibitor from interacting with active prostasin or matriptase. In contrast, the cell surface expression of HAI-1 in all viable epidermal layers renders it an effective regulator for matriptase and prostasin. Collectively, our study suggests the importance of tissue distribution and subcellular localization in the functional relationship between proteases and protease inhibitors.
The somewhat contradictory descriptions of the functional relationship between matriptase and prostasin present in the literature provide an interesting example of the diversity and divergence of life in apparently similar systems. Matriptase is a type 2 transmembrane serine protease [1–3] and prostasin is a glycosylphosphatidylinositol (GPI)-anchored or transmembrane serine protease [4, 5]. Matriptase and prostasin can function in concert as a tightly coupled proteolytic cascade [6, 7]. Both proteases are broadly co-expressed in many epithelial tissues in the mouse , synthesized and processed through the secretory pathway and anchored on the cell membrane. Matriptase and prostasin resemble one another in that they both possess trypsin-like proteolytic activity, undergoing zymogen activation via cleavage at an Arg residue within an activation motif, and being under the tight control of the hepatocyte growth factor activator inhibitors (HAIs) [9–13]. Almost identical epidermal defects have been observed in the skin of matriptase knockout and prostasin knockout mice [14, 15], and evidence for a functional link between these proteins is further supported by their co-expression in the uppermost viable epidermal layer in mouse skin . The biochemical characteristics of the regulation of proteolytic activity also suggest a functional partnership. Matriptase and prostasin are synthesized as zymogen forms, a shared mechanism among many serine proteases, by which the potential hazards of unfettered proteolytic activity can be moderated by activating the zymogen only at the time and place where the proteolytic activity is needed. Most serine protease zymogens are activated by the action of other proteases that have already been activated, whereas few undergo zymogen activation via autoactivation, an alternative mechanism by which the first active protease in a cascade can be generated in the absence of other active proteases. Matriptase is such a serine protease and acquires proteolytic activity via autoactivation . A functional relationship in which matriptase acts as the upstream activator of the downstream substrate prostasin was initially suggested by the lack of prostasin zymogen activation observed in the skin of matriptase knockout mice . Assessment of the activation state of prostasin in this study depended, however, on being able to discriminate between the zymogen and active forms of prostasin by western blot, based on size (a difference of only 12 amino acids, or less than 5%), raising the possibility that some level of prostasin activation remains. Nevertheless, in HaCaT human keratinocytes, prostasin zymogen activation is induced simultaneously when matriptase zymogen activation is induced, and matriptase is required for the induction of prostasin zymogen activation . Concomitant induction of prostasin and matriptase activation can also be observed in some other epithelial cells , indicating that matriptase and prostasin can function as a tightly coupled proteolytic cascade, at least, in vitro in cultured cells.
The tissue distribution, the in vivo zymogen activation status, the in vivo and in vitro subcellular localization, and the level of protease-protease inhibitor complexes demonstrated in the current study provide important information that informs an assessment of the putative functional relationship between matriptase and prostasin and between the two proteases and HAIs in human skin. A close functional linkage between the two serine proteases and among these proteases and protease inhibitors have been suggested by an array of previous studies using a variety of model systems. The proposed functional relationships include one involving a reciprocal activator-substrate interaction between matriptase and prostasin and another involving potent inhibition of matriptase and prostasin by HAI-1 and HAI-2. Our previous studies, however, suggested that though they may be correct in some contexts these proposed functional linkages can not be generalized and are somewhat tissue-selective or context-dependent [11, 17, 18]. In the current study, the context-dependent nature of any functional relationship is further supported by the distinct pattern of zymogen activation observed in human skin samples, in which prostasin is activated at high levels primarily in the granular layer whereas matriptase zymogen activation occurs at much lower levels predominantly in the cells of the basal layer. Similarly, although HAI-2 has been identified as a relevant matriptase inhibitor in breast cancer cells, and prostasin inhibitor in human enterocytes and colon carcinoma cells, HAI-2 does not appear to be an important protease inhibitor for matriptase or prostasin in human skin, in which the tissue distribution and subcellular localization of HAI-2 suggests that HAI-2 has limited access to active matriptase or prostasin. Although mouse HAI-2 may also play no role in the control of matriptase and prostasin in mouse epidermis, the mechanism responsible for the lack of a functional interactions appears to be different. HAI-2 is apparently not expressed in mouse skin , a piece of evidence that further underlines the genuine physiology differences between human skin and mouse skin.