Hepatocyte growth factor activator inhibitor-1 (HAI-1) was initially identified as cognate inhibitor of matriptase, a membrane-bound serine protease. Paradoxically, HAI-1 is also required for matriptase activation, a process that requires sphingosine 1-phosphate (S1P)-mediated translocation of the protease to cell-cell junctions in human mammary epithelial cells. In the present study, we further explored how HAI-1 regulates this protease. First, we observed that after S1P treatment HAI-1 was cotranslocated with matriptase to cell-cell junctions and that the cellular ratio of HAI-1 to matriptase was maintained during this process. However, when this ratio was changed by cell treatment with HAI-1 small interfering RNA or anti-HAI-1 MAb M19, spontaneous activation of matriptase occurred in the absence of S1P-induced translocation; S1P-induced matriptase activation was also enhanced. These results support a role for HAI-1 in protection of cell from uncontrolled matriptase activation. We next expressed matriptase, either alone or with HAI-1 in breast cancer cells that do not endogenously express either protein. A defect in matriptase trafficking to the cell surface occurred if wild-type matriptase was expressed in the absence of HAI-1; this defect appeared to result from matriptase toxicity to cells. Coexpression with matriptase of wild-type HAI-1, but not HAI-1 mutants altered in its Kunitz domain 1, corrected the trafficking defect. In contrast, catalytically defective matriptase mutants were normal in their trafficking in the absence of HAI-1. These results are also consistent with a role for HAI-1 to prevent inappropriate matriptase proteolytic activity during its protein synthesis and trafficking. Taken together, these results support multiple roles for HAI-1 to regulate matriptase, including its proper expression, intracellular trafficking, activation, and inhibition.

.-In live cells, autoactivation of matriptase, a membrane-bound serine protease, can be induced by lysophospholipids, androgens, and the polyanionic compound suramin. These structurally distinct chemicals induce different signaling pathways and cellular events that somehow, in a cell type-specific manner, lead to activation of matriptase immediately followed by inhibition of matriptase by hepatocyte growth factor activator inhibitor 1 (HAI-1).In the current study, we established an analogous matriptase autoactivation system in an in vitro cell-free setting and showed that a burst of matriptase activation and HAI-1-mediated inhibition spontaneously occurred in the insoluble fractions of cell homogenates and that this in vitro activation could be attenuated by a soluble suppressive factor(s) in cytosolic fractions. Immunofluorescence staining and subcellular fractionation studies revealed that matriptase activation occurred in the perinuclear regions. Solubilization of matriptase from cell homogenates by Triton X-100 or sonication of cell homogenates completely inhibited the effect, suggesting that matriptase activation requires proper lipid bilayer microenvironments, potentially allowing appropriate interactions of matriptase zymogens with HAI-1 and other components. Matriptase activation occurred in a narrow pH range (from pH 5.2 to 7.2), with a sharp increase in activation at the transition from pH 5.2 to 5.4, and could be completely suppressed by moderately increased ionic strength. Protease inhibitors only modestly affected activation, whereas 30 nM (5 g/ml) of anti-matriptase LDL receptor domain 3 monoclonal antibodies completely blocked activation. These atypical biochemical features are consistent with a mechanism for autoactivation of matriptase that requires protein-protein interactions but not active proteases.hepatocyte growth factor activator inhibitor 1; protease activation; low-density lipoprotein MATRIPTASE AND HEPATOCYTE growth factor activator inhibitor 1 (HAI-1) are a pair of epithelium-derived, membrane-associated proteins: a proteolytic enzyme and its cognate inhibitor, respectively (22, 49). Matriptase, a member of the type II transmembrane serine protease (8,35,50), contains a transmembrane domain at the amino terminus, followed by a sperm protein, enterokinase, and agrin (SEA) domain, two tandem C1r/s, Uegf, and bone morphogenic protein-1 (CUB) domains, four tandem LDL receptor class A domains, and a trypsin-like serine protease domain (15,22,23,52,54). HAI-1, a type 1 transmembrane protein, contains two Kunitz-type serine protease inhibitor domains and an LDL receptor class A domain (46). Matriptase and HAI-1 are broadly expressed and may have diverging functions in the epithelial cells of most epithelium-containing tissues (15,22,26,36,39,52,54). For example, matriptase was shown to be required for postnatal survival, epidermal barrier function, hair follicle development, and thymic homeostasis in matriptase knockout mice (25). The roles of matriptase in epidermal differentiation apparent...

Lee, Ming-Shyue, Ken-ichi Kiyomiya, Christelle Benaud, Robert B. Dickson, and Chen-Yong Lin. Simultaneous activation and hepatocyte growth factor activator inhibitor 1-mediated inhibition of matriptase induced at activation foci in human mammary epithelial cells.

. Matriptase activation and shedding with HAI-1 is induced by steroid sex hormones in human prostate cancer cells, but not in breast cancer cells. Am J Physiol Cell Physiol 291: C40 -C49, 2006. First published February 8, 2006 doi:10.1152/ajpcell.00351.2005.-Matriptase and its cognate inhibitor, hepatocyte growth factor activator inhibitor-1 (HAI-1), have been implicated in carcinoma onset and malignant progression. However, the pathological mechanisms of matriptase activation are not defined. Steroid sex hormones play crucial roles in prostate and breast cancer. Therefore, we investigated the questions of whether and how steroid sex hormones regulate matriptase activation in these cancer cells. Treatment of cells with 17␤-estradiol had no effect on activation of matriptase in hormonestarved breast cancer cells, in part due to their high constitutive level of activated matriptase. In striking contrast, very low levels of activated matriptase were detected in hormone-starved lymph node prostatic adenocarcinoma (LNCaP) cells. Robust activation of matriptase was observed as early as 6 h after exposure of these cells to 5␣-dihydrotestosterone (DHT). Activation of matriptase was closely followed by shedding of the activated matriptase with Ͼ90% of total activated matriptase present in the culture media 24 h after DHT treatment. Activated matriptase was shed in a complex with HAI-1 and may result from simultaneously proteolytic cleavages of both membrane-bound proteins. Latent matriptase and free HAI-1 were also shed into culture media. As a result of shedding, the cellular levels of matriptase and HAI-1 were significantly reduced 24 h after exposure to DHT. DHT-induced matriptase activation and shedding were significantly inhibited by the androgen antagonist bicalutamide, by the RNA transcription inhibitor actinomycin D, and by the protein synthesis inhibitor cycloheximide. These results suggest that in LNCaP cells, androgen induces matriptase activation via the androgen receptor, and requires transcription and protein synthesis.androgen; hepatocyte growth factor activator inhibitor-1 MATRIPTASE and its inhibitor, hepatocyte growth factor activator inhibitor-1 (HAI-1), represent a cognate pair, a cell surface proteolytic enzyme and its inhibitor (29). Both are broadly coexpressed by epithelial elements in most epithelium-containing tissues (18, 45). Matriptase, a type 2 transmembrane serine protease (40, 54), contains a COOH-terminal serine protease domain and several noncatalytic domains, including a sperm protein-enterokinase-agrin module, two tandem C1r/s-UegfBone morphogenic protein-1 domains, and four tandem lowdensity lipoprotein (LDL) receptor class A domains (19,30,57,59). Matriptase was shown to play an important role in epidermal barrier function, terminal epidermal differentiation, hair follicle development and thymic homeostasis in matriptase knockout mice (32). Some of these functions may result from its role in the maturation of pro-filaggrin (34), a marker for the terminal differentiation of keratinoc...

These results suggest that ADM exerts cytostatic effects on neoplastic and normal undifferentiated cells through the inhibition of DNA synthesis by DNA intercalation, and cytotoxic effects on neoplastic cells through the accumulation of reactive oxygen species resulting from low scavenger enzyme activities. The cytotoxic effects on normal differentiated cells may be related to the high levels of production of reactive oxygen species due to high mitochondrial NADH-cytochrome c reductase activity.

No abstract

In enamel fluorosis model rats treated with sodium fluoride, secretory ameloblasts of incisor tooth germs exhibited disruption of intracellular trafficking. We examined whether heterotrimeric G proteins participated in the disruption of vesicular trafficking of the secretory ameloblast exposed to fluoride, using immunoblotting and pertussis toxin (IAP)-induced adenosyl diphosphate (ADP)-ribosylation for membrane fractions of the cell. Immunoblotting of crude membranes, post supernatants of the ameloblast, with anti-G(alpha i3/alpha o) and anti-G(alpha s) antibodies showed that Gi3 or Go proteins existed in the secretory ameloblast, but Gs protein did not. Immunoblotting of the subcellular membrane fractions indicated that the Gi3 or Go proteins were located in the Golgi membrane, but were not in the rough endoplasmic reticulum (rER) membrane. Autoradiograph of IAP-induced ADP-ribosylation, however, showed the existence of IAP-sensitive G proteins both in rER and Golgi membranes. Fluoride treatment decreased the G proteins bound to both membranes. These findings indicate that different G proteins, both of which are IAP-sensitive, are present in the rER and Golgi apparatus, and suggest that these G proteins participate in the disturbance of intracellular transport of the secretory ameloblast exposed to fluoride.

Fluoride, which is an environmental toxicant, is a potent inducer of mottled enamel in humans and rats. To define the influence of fluoride on the secretory pathway in enamel fluorosis, mottled enamel was induced in the incisor tooth germs of rats by subcutaneous injections of sodium fluoride for 4 days, and then morphological and cytochemical changes of the secretory ameloblast were examined in the tooth germs with HRP-labeled lectin (Con A, GS-I, SBA and PNA) and En3 antibody labeling amelogenins. The accumulation of small vesicles on the route of the secretory pathway between the rER and the Golgi apparatus, disorder of Golgi stacks, and formation of abnormal large granules in distal cytoplasm were seen in the secretory ameloblast. Lectin staining patterns of the secretory ameloblast indicated the disturbance of the vesicular transport between the rER and the Golgi apparatus, and disorganization of the Golgi stack. Immunolabeling of the cell showed disruption of the sorting and fusion process on the secretory pathway. These results suggest that the fluoride disturbs the intracellular transport in the synthesis-secretory pathway of the ameloblast, and that this effect of fluoride on the synthesis-secretory pathway participates in the formation of enamel fluorosis.