Jehng-Kang Wang

Polarized epithelial cells secrete matriptase as a consequence of zymogen activation and HAI-1-mediated inhibition

Matriptase, a transmembrane serine protease, is broadly expressed by, and crucial for the integrity of, the epithelium. Matriptase is synthesized as a zymogen and undergoes autoactivation to become an active protease that is immediately inhibited by, and forms complexes with, hepatocyte growth factor activator inhibitor (HAI-1). To investigate where matriptase is activated and how it is secreted in vivo, we determined the expression and activation status of matriptase in seminal fluid and urine and the distribution and subcellular localization of the protease in the prostate and kidney. The in vivo studies revealed that while the latent matriptase is localized at the basolateral surface of the ductal epithelial cells of both organs, only matriptase-HAI-1 complexes and not latent matriptase are detected in the body fluids, suggesting that activation, inhibition, and transcytosis of matriptase would have to occur for the secretion of matriptase. These complicated processes involved in the in vivo secretion were also observed in polarized Caco-2 intestinal epithelial cells. The cells target latent matriptase to the basolateral plasma membrane where activation, inhibition, and secretion of matriptase appear to take place. However, a proportion of matriptase-HAI-1 complexes, but not the latent matriptase, appears to undergo transcytosis to the apical plasma membrane for secretion. When epithelial cells lose their polarity, they secrete both latent and activated matriptase. Although most epithelial cells retain very low levels of matriptase-HAI-1 complex by rapidly secreting the complex, gastric chief cells may activate matriptase and store matriptase-HAI-1 complexes in the pepsinogen-secretory granules, suggesting an intracellular activation and regulated secretion in these cells. Taken together, while zymogen activation and closely coupled HAI-1-mediated inhibition are common features for matriptase regulation, the cellular location of matriptase activation and inhibition, and the secretory route for matriptase-HAI-1 complex may vary along with the functional divergence of different epithelial cells.

Regulation of the Matriptase-Prostasin Cell Surface Proteolytic Cascade by Hepatocyte Growth Factor Activator Inhibitor-1 during Epidermal Differentiation

Matriptase, a membrane-tethered serine protease, plays essential roles in epidermal differentiation and barrier function, largely mediated via its activation of prostasin, a glycosylphosphatidylinositol-anchored serine protease. Matriptase activity is tightly regulated by its inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) such that free active matriptase is only briefly available to act on its substrates. In the current study we provide evidence for how matriptase activates prostasin under this tight control by HAI-1. When primary human keratinocytes are induced to differentiate in a skin organotypic culture model, both matriptase and prostasin are constitutively activated and then inhibited by HAI-1. These processes also occur in HaCaT human keratinocytes when matriptase activation is induced by exposure of the cells to a pH 6.0 buffer. Using this acid-inducible activation system we demonstrate that prostatin activation is suppressed by matriptase knockdown and by blocking matriptase activation with sodium chloride, suggesting that prostatin activation is dependent on matriptase in this system. Kinetics studies further reveal that the timing of autoactivation of matriptase, prostasin activation, and inhibition of both enzymes by HAI-1 binding are closely correlated. These data suggest that, during epidermal differentiation, the matriptase-prostasin proteolytic cascade is tightly regulated by two mechanisms: 1) prostasin activation temporally coupled to matriptase autoactivation and 2) HAI-1 rapidly inhibiting not only active matriptase but also active prostasin, resulting in an extremely brief window of opportunity for both active matriptase and active prostasin to act on their substrates.

Purification from human milk of matriptase complexes with secreted serpins: mechanism for inhibition of matriptase other than HAI-1

Matriptase, a type 2 transmembrane serine protease, is predominately expressed by epithelial and carcinoma cells in which hepatocyte growth factor activator inhibitor 1 (HAI-1), a membrane-bound, Kunitz-type serine protease inhibitor, is also expressed. HAI-1 plays dual roles in the regulation of matriptase, as a conventional protease inhibitor and as a factor required for zymogen activation of matriptase. As a consequence, activation of matriptase is immediately followed by HAI-1-mediated inhibition, with the activated matriptase being sequestered into HAI-1 complexes. Matriptase is also expressed by peripheral blood leukocytes, such as monocytes and macrophages; however, in contrast to epithelial cells, monocytes and macrophages were reported not to express HAI-1, suggesting that these leukocytes possess alternate, HAI-1-independent mechanisms regulating the zymogen activation and protease inhibition of matriptase. In the present study, we characterized matriptase complexes of 110 kDa in human milk, which contained no HAI-1 and resisted dissociation in boiling SDS in the absence of reducing agents. These complexes were further purified and dissociated into 80-kDa and 45-kDa fragments by treatment with reducing agents. Proteomic and immunological methods identified the 45-kDa fragment as the noncatalytic domains of matriptase and the 80-kDa fragment as the matriptase serine protease domain covalently linked to one of three different secreted serpin inhibitors: antithrombin III, alpha1-antitrypsin, and alpha2-antiplasmin. Identification of matriptase-serpin inhibitor complexes provides evidence for the first time that the proteolytic activity of matriptase, from those cells that express no or low levels of HAI-1, may be controlled by secreted serpins.

TMPRSS2, a Serine Protease Expressed in the Prostate on the Apical Surface of Luminal Epithelial Cells and Released into Semen in Prostasomes, Is Misregulated in Prostate Cancer Cells

TMPRSS2, a type II transmembrane serine protease, is highly expressed by the epithelium of the human prostate gland. To explore the regulation and function of TMPRSS2 in the prostate, a panel of monoclonal antibodies with high sensitivity and specificity were generated. Immunodetection showed TMPRSS2 on the apical plasma membrane of the prostate luminal cells and demonstrated its release into semen as a component of prostasomes, organelle-like vesicles that may facilitate sperm function and enhance male reproduction. In prostate cancer cells, TMPRSS2 expression was increased and the protein mislocalized over the entire tumor cell membrane. In both LNCaP prostate cancer cells and human semen, TMPRSS2 protein was detected predominantly as inactive zymogen forms as part of an array of multiple noncovalent and disulfide-linked complexes, suggesting that TMPRSS2 activity may be regulated by unconventional mechanisms. Our data suggested that TMPRSS2, an apical surface serine protease, may have a normal role in male reproduction as a component of prostasomes. The aberrant cellular localization, and increased expression of the protease seen in cancer, may contribute to prostate tumorigenesis by providing access of the enzyme to nonphysiological substrates and binding-proteins.

Increased matriptase zymogen activation in inflammatory skin disorders

Matriptase, a type 2 transmembrane serine protease, and its inhibitor hepatocyte growth factor activator inhibitor (HAI)-1 are required for normal epidermal barrier function, and matriptase activity is tightly regulated during this process. We therefore hypothesized that this protease system might be deregulated in skin disease. To test this, we examined the level and activation state of matriptase in examples of 23 human skin disorders. We first examined matriptase and HAI-1 protein distribution in normal epidermis. Matriptase was detected at high levels at cell-cell junctions in the basal layer and spinous layers but was present at minimal levels in the granular layer. HAI-1 was distributed in a similar pattern, except that high-level expression was retained in the granular layer. This pattern of expression was retained in most skin disorders. We next examined the distribution of activated matriptase. Although activated matriptase is not detected in normal epidermis, a dramatic increase is seen in keratinocytes at the site of inflammation in 16 different skin diseases. To gain further evidence that activation is associated with inflammatory stimuli, we challenged HaCaT cells with acidic pH or H(2)O(2) and observed matriptase activation. These findings suggest that inflammation-associated reactive oxygen species and tissue acidity may enhance matriptase activation in some skin diseases.

Kinetic mechanism of the cytosolic malic enzyme from human breast cancer cell line

The kinetic mechanism of the cytosolic NADP(+)-dependent malic enzyme from cultured human breast cancer cell line was studied by steady-state kinetics. In the direction of oxidative decarboxylation, the initial-velocity and product-inhibition studies indicate that the enzyme reaction follows a sequential ordered Bi-Ter kinetic mechanism with NADP+ as the leading substrate followed by L-malate. The products are released in the order of CO2, pyruvate, and NADPH. The enzyme is unstable at high salt concentration and elevated temperature. However, it is stable for at least 20 min under the assay conditions. Tartronate (2-hydroxymalonate) was found to be a noncompetitive inhibitor for the enzyme with respect to L-malate. The kinetic mechanism of the cytosolic tumor malic enzyme is similar to that for the pigeon liver cytosolic malic enzyme but different from those for the mitochondrial enzyme from various sources.

Early hyperbaric oxygen therapy attenuates disease severity in lupus-prone autoimmune (NZB × NZW) F1 mice

The effects of hyperbaric oxygen (HBO(2)) therapy on the immune system are reported including potential changes to the CD4/CD8 ratio and a decreased proliferation of lymphocytes during exposure. The immunosuppressive effect of HBO(2) had been suggested to be applicable for the treatment of certain autoimmune diseases. (NZB x NZW) F1 hybrid mice, the unique lupus-prone mice, have been used for elucidating the pathogenesis of SLE. To investigate the effect of HBO(2) on NZB/W F1 lupus-prone mice, 32 female mice were divided into four groups. Three groups of mice were treated with HBO(2) (2.5 atm abs (ATA) for 90 min daily over 2 weeks) starting at (A) 3 months, (B) 6 months, or (C) 8 months of age, while the remaining group (D) served as control. Animals were followed until 11 months of age. Experimental parameters included life span, proteinuria, peripheral lymphocytes, anti-dsDNA antibody titers, and renal histopathology. HBO(2) treatment resulted in increased survival, decreased proteinuria, alterations in lymphocyte-subset redistribution, reduced anti-dsDNA antibody titers, and amelioration of immune-complex deposition in groups A and B. Our data demonstrated that HBO(2) therapy attenuated disease severity in NZB/W F1 mice. HBO(2) treatment may be of use in the clinical treatment of lupus patients and would benefit from further study.

Matriptase regulates proliferation and early, but not terminal, differentiation of human keratinocytes

Genetic defects in matriptase are linked to two congenital ichthyosis, autosomal recessive ichthyosis with hypotrichosis (ARIH, OMIM 610765) and, ichthyosis, follicular atrophoderma, hypotrichosis, and hypohidrosis (IFAH, OMIM602400). Mouse models with matriptase deficiency indicate an involvement of matriptase in suprabasal keratinocytes in the maintenance of the epidermal barrier. In contrast to what has been reported for mouse skin, we show that in human skin, matriptase is primarily expressed in the basal and spinous keratinocytes, but not in the more differentiated keratinocytes of the granular layer. In addition, matriptase zymogen activation was predominantly detected in the basal cells. Furthermore, using skin organotypic cultures as a model system to monitor the course of human epidermal differentiation, we found elevated matriptase zymogen activation during early stages of epidermal differentiation, coupled with a loss of matriptase expression in the late stages of this process. We also show here that matriptase deficiency in HaCaT cells modestly reduces cell proliferation and temporally affects calcium-induced expression of differentiation markers. These collective data suggests that, unlike mouse matriptase, human matriptase may be involved in regulation of keratinocyte growth and early differentiation, rather than terminal differentiation, providing mechanistic insights for the pathology of the two congenital ichthyoses, ARIH and IFAH.

Imbalanced matriptase pericellular proteolysis contributes to the pathogenesis of malignant B-Cell lymphomas

Membrane-associated serine protease matriptase is widely expressed by epithelial/carcinoma cells in which its proteolytic activity is tightly controlled by the Kunitz-type protease inhibitor, hepatocyte growth factor activator inhibitor (HAI-1). We demonstrate that, although matriptase is not expressed in lymphoid hyperplasia, roughly half of the non-Hodgkin B-cell lymphomas analyzed express significant amounts of matriptase. Furthermore, a significant proportion of these tumors express matriptase in the absence of HAI-1. Aggressive Burkitt lymphoma was more likely than indolent follicular lymphoma to express matriptase alone (86% versus 36%). In the absence of significant HAI-1 expression, the lymphoma cells activate and shed active matriptase when the cells are stimulated with mildly acidic buffer or the hypoxia-mimicking agent, CoCl2. The shed active matriptase can initiate pericellular proteolytic cascades by activating urokinase-type plasminogen activator on the cell surface of monocytes, and it can activate prohepatocyte growth factor. In addition, matriptase knockdown suppressed proliferation and colony-forming ability of neoplastic B cells in culture and growth as tumor xenografts in mice. Furthermore, exogenous expression of HAI-1 significantly suppressed proliferation of neoplastic B cells. These studies suggest that dysregulated pericellular proteolysis as a result of unregulated matriptase expression with limited HAI-1 may contribute to the pathological characteristics of several human B-cell lymphomas through modulation of the tumor microenvironment and enhanced tumor growth.

Antithrombin Regulates Matriptase Activity Involved in Plasmin Generation, Syndecan Shedding, and HGF Activation in Keratinocytes

Matriptase, a membrane-associated serine protease, plays an essential role in epidermal barrier function through activation of the glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin. The matriptase-prostasin proteolytic cascade is tightly regulated by hepatocyte growth factor activator inhibitor (HAI)-1 such that matriptase autoactivation and prostasin activation occur simultaneously and are followed immediately by the inhibition of both enzymes by HAI-1. However, the mechanisms whereby matriptase acts on extracellular substrates remain elusive. Here we report that some active matriptase can escape HAI-1 inhibition by being rapidly shed from the cell surface. In the pericellular environment, shed active matriptase is able to activate hepatocyte growth factor (HGF), accelerate plasminogen activation, and shed syndecan 1. The amount of active matriptase shed is inversely correlated with the amount of antithrombin (AT) bound to the surface of the keratinocytes. Binding of AT to the surface of keratinocytes is dependent on a functional heparin binding site, Lys-125, and that the N-glycosylation site Asn-135 be unglycosylated. This suggests that β-AT, and not α-AT, is responsible for regulation of pericellular matriptase activity in keratinocytes. Keratinocytes appear to rely on AT to regulate the level of pericellular active matriptase much more than breast and prostate epithelial cells in which AT regulation of matriptase activity occurs at much lower levels than keratinocytes. These results suggest that keratinocytes employ two distinct serine protease inhibitors to control the activation and processing of two different sets of matriptase substrates leading to different biological events: 1) HAI-1 for prostasin activation/inhibition, and 2) AT for the pericellular proteolysis involved in HGF activation, accelerating plasminogen activation, and shedding of syndecans.