Browsing School, Graduate by Subject "HAI-1"
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Matriptase in Skin: Function and RegulationEpidermal differentiation is a carefully orchestrated process that leads to the formation of the critical protective barrier provided by the skin. The process of generating a functional epidermal layer requires progressive remodeling of cell morphology and tissue structure, and involves significant pericellular proteolysis that must be regulated in a precisely controlled manner. In particular, the matriptase-driven protease network plays a critical role in epidermal barrier construction as well as in the regenerative processes required for wound healing. In this dissertation, I have identified and characterized novel molecular mechanisms governing the regulation of matriptase, and cellular mechanisms by which matriptase activates its molecular targets and contributes to keratinocyte differentiation and formation of epidermal barrier. First, I identified plasminogen as a keratinocyte-selective extracellular stimulus for matriptase activation. The discovery of plasminogen as an initiating signal of the protease cascade reaffirms the theory that the matriptase-uPA-plasmin cascade is not unidirectional in the activation of its components, but it is reciprocal. In addition to HAI-1, I also revealed keratinocytes employ antithrombin as a significant endogenous protease inhibitor. The enhanced role of antithrombin in matriptase inhibition in keratinocytes reveals the regulatory adaptation in stratified epithelial cells due to the changes in tissue structure, compared to the polarized epithelial cells. With the dual inhibitory mechanisms, I further revealed that matriptase acts on its molecular targets in two different ways: a rapid activation of prostasin by cell-associated active matriptase under extremely tight control of HAI-1, and the action on several other substrates, including uPA, HGF, and syndecan-1, by secreted active matriptase that is controlled by antithrombin. Last but not least, I also demonstrated that the physiological role of matriptase in human skin likely lies in the basal and spinous keratinocytes that are involved in proliferation and early differentiation. The role of matriptase in the early stages of keratinocyte life history was further supported by the increased matriptase zymogen activation in the keratinocytes of the bulge area. By charactering the prominent regulators, downstream effectors, and the expression and activation states of matriptase in human skin, a clearer picture is emerging regarding the role of matriptase in skin biology.
Molecular Mechanisms Governing MatriptaseMatriptase, a type II transmembrane serine protease, and its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) is broadly expressed by epithelial and carcinoma cells. The critical interaction between matriptase and HAI-1 are essential for placenta development, epithelial integrity, and epidermal functions. Deregulation of matriptase has been implicated in cancer development and progression. It is thought that the proteolytic activity of matriptase is how this protease participates in these physiological and pathological functions. Since searching for the physiological substrates of matriptase has not been completely successful due to the rapid inhibition by HAI-1, to elucidate the molecular regulatory mechanisms of matriptase becomes the best alternative option to understand its function. Therefore, in this dissertation, I elucidated and revealed the biochemical and cellular details of matriptase autoactivation as well as the alternative inhibitory mechanism. Although matriptase autoactivation can be induced by a variety of structurally unrelated stimuli in a loose cell-type specific manner, they all share some common features including tight coupling with HAI-1 inhibition on the cell surface, which indicates the existence of core autoactivation machinery. Based on this concept, cell-free and intact-cell autoactivation activation systems have been set up to study the molecular mechanisms governing matriptase autoactivation. Among all the chemical and physical factors that could alter the autoactivation, acids seem to be the most likely factor capable of conducting activation as a rapid onset, fast kinetics, in the magnitude of activation ever seen. This could further imply the role of matriptase under acidic pathological conditions, such as in a tumor microenvironment. In the last part of the dissertation, three blood-borne serpin type serine protease inhibitors from human breast milk have been characterized as the alternative mechanisms for matriptase inhibition. This suggests an alternative inhibitory mechanism that may be used physiologically in tissues with low or no HAI-1, such as in hematopoietic cells. Altogether, the dissertation provides insights into the regulatory mechanisms of matriptase. Those insights can be applied to the functional study of matriptase, especially to facilitate the search of its substrates, in the future.