HAI-1 is required for the novel role of FGFBP1 in maintenance of cell morphology and F-actin rearrangement in human keratinocytes.

Formation and maintenance of skin barrier function require tightly controlled membrane-associated proteolysis, in which the integral membrane Kunitz-type serine protease inhibitor, HAI-1, functions as the primary inhibitor of the membrane-associated serine proteases, matriptase and prostasin. Previously, HAI-1 loss in HaCaT human keratinocytes resulted in an expected increase in prostasin proteolysis but a paradoxical decrease in matriptase proteolysis. The paradoxical decrease in shed active matriptase is further investigated in this study with an unexpected discovery of novel functions of fibroblast growth factor-binding protein 1 (FGFBP1), which acts as an extracellular ligand that can rapidly elicit F-actin rearrangement and subsequently affect the morphology of human keratinocytes. This novel growth factor-like function is in stark contrast to the canonical activity of this protein through interactions with FGFs for its pathophysiological functions. This discovery began with the observation that HAI-1 KO HaCaT cells lose the characteristic cobblestone morphology of the parental cells and exhibit aberrant F-actin formation along with altered subcellular targeting of matriptase and HAI-2. The alterations in cell morphology and F-actin status caused by targeted HAI-1 deletion can be restored by treatment with conditioned medium from parental HaCaT cells, in which FGFBP1 was identified by tandem mass spectrometry. Recombinant FGFBP1 down to 1 ng/ml was able to revert the changes caused by HAI-1 loss. Our study reveals a novel function of FGFBP1 in the maintenance of keratinocyte morphology, which depends on HAI-1.

Targeted HAI-2 deletion causes excessive proteolysis with prolonged active prostasin and depletion of HAI-1 monomer in intestinal but not epidermal epithelial cells.

Mutations of SPINT2, the gene encoding the integral membrane, Kunitz-type serine inhibitor HAI-2, primarily affect the intestine, while sparing many other HAI-2-expressing tissues, causing sodium loss in patients with syndromic congenital sodium diarrhea. The membrane-bound serine protease prostasin was previously identified as a HAI-2 target protease in intestinal tissues but not in the skin. In both tissues, the highly related inhibitor HAI-1 is, however, the default inhibitor for prostasin and the type 2 transmembrane serine protease matriptase. This cell-type selective functional linkage may contribute to the organ-selective damage associated with SPINT 2 mutations. To this end, the impact of HAI-2 deletion on matriptase and prostasin proteolysis was, here, compared using Caco-2 human colorectal adenocarcinoma cells and HaCaT human keratinocytes. Greatly enhanced prostasin proteolytic activity with a prolonged half-life and significant depletion of HAI-1 monomer were observed with HAI-2 loss in Caco-2 cells but not HaCaT cells. The constitutive, high level prostasin zymogen activation observed in Caco-2 cells, but not in HaCaT cells, also contributes to the excessive prostasin proteolytic activity caused by HAI-2 loss. HAI-2 deletion also caused increased matriptase zymogen activation, likely as an indirect result of increased prostasin proteolysis. This increase in activated matriptase, however, only had a negligible role in depletion of HAI-1 monomer. Our study suggests that the constitutive, high level of prostasin zymogen activation and the cell-type selective functional relationship between HAI-2 and prostasin renders Caco-2 cells more susceptible than HaCaT cells to the loss of HAI-2, causing a severe imbalance favoring prostasin proteolysis.

Matriptase and prostasin proteolytic activities are differentially regulated in normal and wounded skin.

Orchestrated control of multiple overlapping and sequential processes is required for the maintenance of epidermal homeostasis and the response to and recovery from a variety of skin insults. Previous studies indicate that membrane-associated serine protease matriptase and prostasin play essential roles in epidermal development, differentiation, and barrier formation. The control of proteolysis is a highly regulated process, which depends not only on gene expression but also on zymogen activation and the balance between protease and protease inhibitor. Subcellular localization can affect the accessibility of protease inhibitors to proteases and, thus, also represents an integral component of the control of proteolysis. To understand how membrane-associated proteolysis is regulated in human skin, these key aspects of matriptase and prostasin were determined in normal and injured human skin by immunohistochemistry. This staining shows that matriptase is expressed predominantly in the zymogen form at the periphery of basal and spinous keratinocytes, and prostasin appears to be constitutively activated at high levels in polarized organelle-like structures of the granular keratinocytes in the adjacent quiescent skin. The membrane-associated proteolysis appears to be elevated via an increase in matriptase zymogen activation and prostasin protein expression in areas of skin recovering from epidermal insults. There was no noticeable change observed in other regulatory aspects, including the expression and tissue distribution of their cognate inhibitors HAI-1 and HAI-2. This study reveals that the membrane-associated proteolysis may be a critical epidermal mechanism involved in responding to, and recovering from, damage to human skin.

Matriptase and prostasin are expressed in human skin in an inverse trend over the course of differentiation and are targeted to different regions of the plasma membrane.

Matriptase and prostasin, acting as a tightly coupled proteolytic cascade, were reported to be required for epidermal barrier formation in mouse skin. Here we show that, in human skin, matriptase and prostasin are expressed with an inverse pattern over the course of differentiation. Matriptase was detected primarily in epidermal basal keratinocytes and the basaloid cells in the outer root sheath of hair follicles and the sebaceous gland, where prostasin was not detected. In contrast, prostasin was detected primarily in differentiated cells in the epidermal granular layer, the inner root sheath of hair follicles, and the sebaceous gland, where matriptase expression is negligible. While co-expressed in the middle stage of differentiation, prostasin was detected as polarized patches, and matriptase at intercellular junctions. Targeting to different subcellular localizations is also observed in HaCaT human keratinocytes, in which matriptase was detected primarily at intercellular junctions, and prostasin primarily on membrane protrusion. Furthermore, upon induction of zymogen activation, free active prostasin remains cell-associated and free active matriptase is rapidly shed into the extracellular milieu. Our data suggest that matriptase and prostasin likely function as independent entities in human skin rather than as a tightly coupled proteolytic cascade as observed in mouse skin.

Matriptase Complexes and Prostasin Complexes with HAI-1 and HAI-2 in Human Milk: Significant Proteolysis in Lactation.

Significant proteolysis may occur during milk synthesis and secretion, as evidenced by the presence of protease-protease inhibitor complex containing the activated form of the type 2 transmembrane serine protease matriptase and the transmembrane Kunitz-type serine protease inhibitor HAI-1. In order to identify other proteolysis events that may occur during lactation, human milk was analyzed for species containing HAI-1 and HAI-2 which is closely related to HAI-1. In addition to the previously demonstrated matriptase-HAI-1 complex, HAI-1 was also detected in complex with prostasin, a glycosylphosphatidylinositol (GPI)-anchored serine protease. HAI-2 was also detected in complexes, the majority of which appear to be part of higher-order complexes, which do not bind to ionic exchange columns or immunoaffinity columns, suggesting that HAI-2 and its target proteases may be incorporated into special protein structures during lactation. The small proportion HAI-2 species that could be purified contain matriptase or prostasin. Human mammary epithelial cells are the likely cellular sources for these HAI-1 and HAI-2 complexes with matriptase and prostasin given that these protease-inhibitor complexes with the exception of prostasin-HAI-2 complex were detected in milk-derived mammary epithelial cells. The presence of these protease-inhibitor complexes in human milk provides in vivo evidence that the proteolytic activity of matriptase and prostasin are significantly elevated at least during lactation, and possibly contribute to the process of lactation, and that they are under tight control by HAI-1 and HAI-2.

Increased matriptase zymogen activation by UV irradiation protects keratinocyte from cell death.

BACKGROUND: Overexposure to ultraviolet (UV) derived from solar light causes skin damage by causing DNA lesions and the generation of reactive oxygen species (ROS) in keratinocytes and other epidermal cells. The type 2 transmembrane serine protease matriptase has characteristics that allow keratinocytes to respond to/recover from, environmental insults to the skin. This response may depend on its roles in epidermal proliferation and early differentiation, and its rapid activation in response to changes in the cellular chemical milieu, including increased oxidative stress. OBJECTIVE: We investigate the regulation of matriptase activation and its role in the response of the skin to exposure to different parts of the UV spectrum including UVA UVB, and UVR. METHODS: The activation state and distribution of matriptase in ex vivo UV exposed human skin specimens and sun damaged skin samples were analyzed by immunohistochemistry. HaCaT immortalized human keratinocytes were also used to investigate the mechanism of matriptase zymogen activation induced by UV irradiation. Levels of cytosolic ROS were determined by H2DCF assay. Activated matriptase, PARP and caspase 3 cleavage was analyzed by Western blotting, and the apoptotic ratio was measured by Hoechst 33258 staining. RESULTS: UVB exposure rapidly increased matriptase zymogen activation in the basal keratinocytes of skin samples. Activated matriptase was also detected at much higher levels in both the basal and spinous layer keratinocytes in sun damaged skin with actinic elastosis. UVB and solar light-induced matriptase zymogen activation likely results from UV-induced ROS generation, given that UVR, UVA, and UVB irradiation induced HaCaT human keratinocytes to activate matriptase in a dose- and time-dependent manner, and that this was suppressed by the ROS scavenger N-tert-butyl-α-phenylnitrone and reducing agent dithiothreitol. Matriptase deficient HaCaT keratinocytes were more susceptible to UV-induced apoptosis than control cells, suggesting a protective role for matriptase in UV exposed keratinocytes. CONCLUSION: UV irradiation/ROS induced matriptase proteolysis may have short term protective effects and contribute to the recovery from acute epidermal damage and/or pathology of skin with chronic sun damage.