Caspase-Dependent Inhibition of Mousepox Replication by gzmB

Background

Ectromelia virus is a natural mouse pathogen, causing mousepox. The cytotoxic T (Tc) cell granule serine-protease, granzyme B, is important for its control, but the underlying mechanism is unknown. Using ex vivo virus immune Tc cells, we have previously shown that granzyme B is able to activate several independent pro-apoptotic pathways, including those mediated by Bid/Bak/Bax and caspases-3/-7, in target cells pulsed with Tc cell determinants.

Methods and Findings

Here we analysed the physiological relevance of those pro-apoptotic pathways in ectromelia infection, by incubating ectromelia-immune ex vivo Tc cells from granzyme A deficient (GzmB⁺ Tc cells) or granzyme A and granzyme B deficient (GzmA×B^−/− Tc cell) mice with ectromelia-infected target cells. We found that gzmB-induced apoptosis was totally blocked in ectromelia infected or peptide pulsed cells lacking caspases-3/-7. However ectromelia inhibited only partially apoptosis in cells deficient for Bid/Bak/Bax and not at all when both pathways were operative suggesting that the virus is able to interfere with apoptosis induced by gzmB in case not all pathways are activated. Importantly, inhibition of viral replication in vitro, as seen with wild type cells, was not affected by the lack of Bid/Bak/Bax but was significantly reduced in caspase-3/-7-deficient cells. Both caspase dependent processes were strictly dependent on gzmB, since Tc cells, lacking both gzms, neither induced apoptosis nor reduced viral titers.

Significance

Out findings present the first evidence on the biological importance of the independent gzmB-inducible pro-apoptotic pathways in a physiological relevant virus infection model.     

Mouse Cytotoxic T Cell-derived Granzyme B Activates the Mitochondrial Cell Death Pathway in a Bim-dependent Fashion

Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB⁺Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB⁺Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-X_L, the anti-apoptotic counterparts of Bim. In contrast, gzmB⁺Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB⁺Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB⁺Tc-induced death pathways.     

Mouse granzyme K has pro-inflammatory potential

Granzymes (gzms) are key components of T-killer (Tc) cells believed to mediate pro-apoptotic activities. Recent evidence suggests that gzms also possess non-cytotoxic activities that contribute to host defense. In this study, we show that Tc cells from lymphocytic choriomeningitis virus (LCMV)-infected wild-type (wt) and gzm A/B-deficient mice express similar levels of gzmK protein, with both mouse strains efficiently controlling infection. GzmK, in recombinant form or secreted by ex vivo-derived LCMV-immune gzmAxB(-/-) Tc cells, lacks pro-apoptotic activity. Instead, gzmK induces primary mouse macrophages to process and secrete interleukin-1β, independent of the ATP receptor P2X(7). Together with the finding that IL-1Ra (Anakinra) treatment inhibits virus elimination but not generation of cytotoxic Tc cells in wt mice, the data suggest that Tc cells control LCMV through non-cytotoxic processes that involve gzmK.     

Metabolic and physiologic studies of nonimmune lymphoid cells cytotoxic for fibroblastic cells in vitro

An in vitro reaction between mouse lymphoid cells and target fibroblastic cells in wells of microtest plates, which appears to simulate the in vivo rejection of hemopoietic allografts, has been analyzed for metabolic and physiologic requirements. Protein synthesis was required for only the first few hours of culture. Inhibition of RNA synthesis and alteration of cell surface charge with various agents were without obvious effects. Metabolic slowing at 4 °C or deviation of the pH of the culture medium suppressed the reaction. Thymus cells, which are not cytotoxic in this system, significantly but not completely inhibited the cytotoxicity of lymph node cells. Antiserum directed against target cells specifically protected them from the cytotoxic lymphoid cells in the absence of complement.Precursors of cytotoxic lymphoid cells were radiosensitive, unlike the cytotoxic cells themselves. BALB/c anti-C57BL/6 spleen cell serum and ⁸⁹Sr both are able to prevent rejection of marrow allografts in vitro. Lymphoid cells incubated with this antiserum plus complement lost much of their cytotoxicity but were still effective at high ratios of aggressor to target cells. Lymphoid cells of mice treated with ⁸⁹Sr were effectively cytotoxic but lost practically all of their cytotoxicity afer incubation with the antiserum plus complement. Thus, it appears that this reaction detects two different cytotoxic lymphoid cells, either of which can function in vitro. Both cell types may need to cooperate in vivo during marrow allograft rejections.     

The biology of cytotoxic cell granule exocytosis pathway: granzymes have evolved to induce cell death and inflammation

The granule exocytosis pathway of cytotoxic lymphocytes (Tc and NK cells) is critical for control of tumor development and viral infections. Granule-associated perforin and granzymes are key components in Tc cell-mediated function(s). On the basis of studies that showed granzymes A, B, C, K and M, to induce apoptosis in vitro, all granzymes were thought to also induce cell death in vivo. This review summarizes our present understanding of the biological processes elicited by purified granzyme A and granzyme as well as the processes induced by the more physiologically relevant cytotoxic cells secreting these proteases. The combined evidence supports the concept that the granule secretion pathway is not mono-specific but rather poly-functional including induction of pro-inflammatory cytokines, besides their widely appreciated apoptotic properties.     

Inducible Dimerization and Inducible Cleavage Reveal a Requirement for Both Processes in Caspase-8 Activation

Caspase-8 is a cysteine protease activated by membrane-bound receptors at the cytosolic face of the cell membrane, initiating the extrinsic pathway of apoptosis. Caspase-8 activation relies on recruitment of inactive monomeric zymogens to activated receptor complexes, where they produce a fully active enzyme composed of two catalytic domains. Although in vitro studies using drug-mediated affinity systems or kosmotropic salts to drive dimerization have indicated that uncleaved caspase-8 can be readily activated by dimerization alone, in vivo results using mouse models have reached the opposite conclusion. Furthermore, in addition to interdomain autoprocessing, caspase-8 can be cleaved by activated executioner caspases, and reports of whether this cleavage event can lead to activation of caspase-8 have been conflicting. Here, we address these questions by carrying out studies of the activation characteristics of caspase-8 mutants bearing prohibitive mutations at the interdomain cleavage sites both in vitro and in cell lines lacking endogenous caspase-8, and we find that elimination of these cleavage sites precludes caspase-8 activation by prodomain-driven dimerization. We then further explore the consequences of interdomain cleavage of caspase-8 by adapting the tobacco etch virus protease to create a system in which both the cleavage and the dimerization of caspase-8 can be independently controlled in living cells. We find that unlike the executioner caspases, which are readily activated by interdomain cleavage alone, neither dimerization nor cleavage of caspase-8 alone is sufficient to activate caspase-8 or induce apoptosis and that only the coordinated dimerization and cleavage of the zymogen produce efficient activation in vitro and apoptosis in cellular systems.     

Viral Cross-Class Serpin Inhibits Vascular Inflammation and T Lymphocyte Fratricide; A Study in Rodent Models In Vivo and Human Cell Lines In Vitro

Poxviruses express highly active inhibitors, including serine proteinase inhibitors (serpins), designed to target host immune defense pathways. Recent work has demonstrated clinical efficacy for a secreted, myxomaviral serpin, Serp-1, which targets the thrombotic and thrombolytic proteases, suggesting that other viral serpins may have therapeutic application. Serp-2 and CrmA are intracellular cross-class poxviral serpins, with entirely distinct functions from the Serp-1 protein. Serp-2 and CrmA block the serine protease granzyme B (GzmB) and cysteine proteases, caspases 1 and 8, in apoptotic pathways, but have not been examined for extracellular anti-inflammatory activity. We examined the ability of these cross-class serpins to inhibit plaque growth after arterial damage or transplant and to reduce leukocyte apoptosis. We observed that purified Serp-2, but not CrmA, given as a systemic infusion after angioplasty, transplant, or cuff-compression injury markedly reduced plaque growth in mouse and rat models in vivo. Plaque growth was inhibited both locally at sites of surgical trauma, angioplasty or transplant, and systemically at non-injured sites in ApoE-deficient hyperlipidemic mice. With analysis in vitro of human cells in culture, Serp-2 selectively inhibited T cell caspase activity and blocked cytotoxic T cell (CTL) mediated killing of T lymphocytes (termed fratricide). Conversely, both Serp-2 and CrmA inhibited monocyte apoptosis. Serp-2 inhibitory activity was significantly compromised either in vitro with GzmB antibody or in vivo in ApoE/GzmB double knockout mice. Conclusions The viral cross-class serpin, Serp-2, that targets both apoptotic and inflammatory pathways, reduces vascular inflammation in a GzmB-dependent fashion in vivo, and inhibits human T cell apoptosis in vitro. These findings indicate that therapies targeting Granzyme B and/or T cell apoptosis may be used to inhibit T lymphocyte apoptosis and inflammation in response to arterial injury.     

LYG-202, a new flavonoid with a piperazine substitution, shows antitumor effects in vivo and in vitro

LYG-202 is a newly synthesized flavonoid with a piperazine substitution. We investigated the antitumor effect of LYG-202 in vivo and in vitro. We show that, LYG-202 significantly decreases tumor growth in mice inoculated with S180 sarcoma cells, compared with the control group. Meanwhile, the viabilities of various kinds of tumor cells were inhibited by LYG-202 with IC₅₀ values in the range of 4.80 to 27.70 μM. Then apoptosis induced by LYG-202 in HepG2 cells was characterized by DAPI staining and Annexin V/PI double staining and degradation of PARP was observed. Activation of the caspase cascade for both the extrinsic and intrinsic pathways was demonstrated, including caspase-8, -9, and -3. The results also showed that the expression of Bcl-2 protein decreased whereas that of Bax protein increased, leading to an increase of the Bax/Bcl-2 ratio. Our results demonstrated that LYG-202 exhibited strong antitumor effect in vivo and in vitro, involving with apoptosis induction.     

Modulation of the recognition and lysis of EL4 tumor target cells by cytotoxic T lymphocytes

When the EL4 targets were harvested from the peritoneal cavity (in vivo), they had less than half as much cell-surface sialic acid as EL4 cells harvested from tissue culture (in vitro), apparently due to the presence of a neuraminidase activity in the peritoneal cavity. Both the recognition and the lysis of either EL4 in vivo or EL4 in vitro target cells by allogeneically primed cytotoxic T lymphocytes were enhanced upon removal of cell-surface sialic acid by neuraminidase treatment. However, even after neuraminidase treatment, there still remained a difference in the lytic profile when using EL4 targets that were harvested in vivo versus in vitro. Both conjugate formation between the target and the T cells and anti-H-2D^b adsorption by the target cells were unaffected by the culture conditions of the target line. However, antibody-induced capping and exocytosis of vesicles differed between the differently cultured target cells, suggesting that there was a membrane organizational difference between them that was detected by the cytotoxic T cells. These data are consistent with the idea that cell surface sialic acid as well as the membrane organization can influence T-cell recognition and lysis of target cells.     

Granzyme A Is Expressed in Mouse Lungs during Mycobacterium tuberculosis Infection but Does Not Contribute to Protection In Vivo

Granzyme A, a serine protease expressed in the granules of cytotoxic T and Natural Killer cells, is involved in the generation of pro-inflammatory cytokines by macrophages. Granzyme A has been described to induce in macrophages in vitro the activation of pro-inflammatory pathways that impair intracellular mycobacterial replication. In the present study, we explored the physiological relevance of Granzyme A in the control of pulmonary Mycobacterium tuberculosis infection in vivo. Our results show that, even though Granzyme A is expressed by cytotoxic cells from mouse lungs during pulmonary infection, its deficiency in knockout mice does not have an effect in the control of M. tuberculosis infection. In addition our findings indicate that absence of Granzyme A does not affect the protection conferred by the live-attenuated M. tuberculosis vaccine MTBVAC. Altogether, our findings are in apparent contradiction with previously published in vitro results and suggest that Granzyme A does not have a crucial role in vivo in the protective response to tuberculosis.     

The organotelluride catalyst LAB027 prevents colon cancer growth in the mice

Organotellurides are newly described redox-catalyst molecules with original pro-oxidative properties. We have investigated the in vitro and in vivo antitumoral effects of the organotelluride catalyst LAB027 in a mouse model of colon cancer and determined its profile of toxicity in vivo. LAB027 induced an overproduction of H₂O₂ by both human HT29 and murine CT26 colon cancer cell lines in vitro. This oxidative stress was associated with a decrease in proliferation and survival rates of the two cell lines. LAB027 triggered a caspase-independent, ROS-mediated cell death by necrosis associated with mitochondrial damages and autophagy. LAB027 also synergized with the cytotoxic drug oxaliplatin to augment its cytostatic and cytotoxic effects on colon cancer cell lines but not on normal fibroblasts. The opposite effects of LAB027 on tumor and on non-transformed cells were linked to differences in the modulation of reduced glutathione metabolism between the two types of cells. In mice grafted with CT26 tumor cells, LAB027 alone decreased tumor growth compared with untreated mice, and synergized with oxaliplatin to further decrease tumor development compared with mice treated with oxaliplatin alone. LAB027 an organotelluride catalyst compound synergized with oxaliplatin to prevent both in vitro and in vivo colon cancer cell proliferation while decreasing the in vivo toxicity of oxaliplatin. No in vivo adverse effect of LAB027 was observed in this model.     

Apoptotic pathways are selectively activated by granzyme A and/or granzyme B in CTL-mediated target cell lysis

Purified cytolytic T lymphocyte (CTL) proteases granzyme (gzm)A and gzmB with sublytic dose of perforin (perf) initiate distinct proapoptotic pathways. Their physiological relevance in CTL-mediated target cell apoptosis is elusive. Using ex vivo virus-immune CD8(+) T cells from mice deficient in perf, gzmA and/or gzmB, and the Fas-resistant EL4.F15 tumor target cell, we show that (a) CTL from gzmA(-/-) or gzmB(-/-) mice similarly induced early proapoptotic features, such as phosphatidyl serine (PS) exposure on plasma membrane, Delta Psi(m) loss, and reactive oxygen radical generation, though with distinct kinetics; (b) CTL from gzmA(-/-) but not from gzmB(-/-) mice activate caspase 3 and 9; (c) PS exposure induced by CTL from gzmA(-/-) or gzmB(-/-) mice is prevented, respectively, by caspase inhibitors or by reactive oxygen scavengers without interfering with target cell death; and (d) all gzm-induced apoptotic features analyzed depend critically on perf. Thus, perf is the principal regulator in CTL-mediated and gzm-facilitated intracellular processes. The ability of gzmA and gzmB to induce multiple independent cell death pathways may be the hosts response to circumvent evasion strategies of pathogens and tumors.     

Possible mechanism for target cell lysis by cytotoxic T-cells

The mechanism of the lysis of target cells by cytotoxic T-cells (Tc) is still obscure; there is no evidence for transfer of material from the Tc and prior to lysis, despite intimate contact, the plasma membranes of both types of cell appear to remain intact. The effects on the target cell lysosomes of brief contact between anti-viral Tc and targets bearing both the appropriate histocompatibility and viral antigens, have been examined cytochemically. Both the distribution of acid phosphatase activity and the percentage bound lysosomal naphthylamidase activity indicated that, in virus-infected target cells exposed to Tc, the lysosomal membranes became totally labilized. Thus the contact between Tc and targets appears to cause sufficient perturbation of the target plasma membrane as to cause the intracellular release of some agent that activates 'suicide capsule' lysosomes.     

A Novel Entamoeba histolytica Cysteine Proteinase,EhCP4, Is Key for Invasive Amebiasis and a Therapeutic Target

Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization, and biochemical properties of EhCP4 could be exploited as a potential target for drug design.     

Identification of Serpinb6b as a Species-specific Mouse Granzyme A Inhibitor Suggests Functional Divergence between Human and Mouse Granzyme A

The granzyme family serine proteases are key effector molecules expressed by cytotoxic lymphocytes. The physiological role of granzyme (Gzm) A is controversial, with significant debate over its ability to induce death in target cells. Here, we investigate the natural inhibitors of GzmA. We employed substrate phage display and positional proteomics to compare substrate specificities of mouse (m) and human (h) GzmA at the peptide and proteome-wide levels and we used the resulting substrate specificity profiles to search for potential inhibitors from the intracellular serpin family. We identified Serpinb6b as a potent inhibitor of mGzmA. Serpinb6b interacts with mGzmA, but not hGzmA, with an association constant of 1.9 ± 0.8 × 10⁵ m⁻¹ s⁻¹ and a stoichiometry of inhibition of 1.8. Mouse GzmA is over five times more cytotoxic than hGzmA when delivered into P815 target cells with streptolysin O, whereas transfection of target cells with a Serpinb6b cDNA increases the EC₅₀ value of mGzmA 13-fold, without affecting hGzmA cytotoxicity. Unexpectedly, we also found that Serpinb6b employs an exosite to specifically inhibit dimeric but not monomeric mGzmA. The identification of an intracellular inhibitor specific for mGzmA only indicates that a lineage-specific increase in GzmA cytotoxic potential has driven cognate inhibitor evolution.     

Ginsenoside F2 induces apoptosis in humor gastric carcinoma cells through reactive oxygen species-mitochondria pathway and modulation of ASK-1/JNK signaling cascade in vitro and in vivo

Ginsenoside F₂ (F₂) is a potential bioactive metabolite of major ginsenosides. The potential anti-cancer effect of F₂ in gastric cancer cells has not been appraised. This study investigated the effects of F₂ on the production of reactive oxygen species (ROS). We also investigated the in vitro and in vivo effects of F₂ on the downstream signaling pathways leading to apoptosis in human gastric cancer cells. The in vitro data revealed that F₂ induces ROS accumulation followed by a decrease in mitochondrial transmembrane potential (MTP), and the release of cytochrome c (cyto c), which induced the caspase-dependent apoptosis. Further assay indicated that modulation of ASK-1/JNK pathway contributes to apoptosis. In vivo, F₂ exhibits the obvious anti-cancer effect compared with cisplatin with no obvious toxicity. Jointly, these results suggest that F₂ induces apoptosis by causing an accumulation of ROS and activating the ASK-1/JNK signaling pathway. This provides further support for the use of F₂ as a novel anticancer therapeutic candidate.     

Docosahexaenoic Acid Induces Apoptosis in MCF-7 Cells In Vitro and In Vivo via Reactive Oxygen Species Formation and Caspase 8 Activation

Background

The present study sought to further investigate the in vitro and in vivo anticancer effects of a representative omega-3 fatty acid, docosahexaenoic acid (DHA), with a focus on assessing the induction of oxidative stress and apoptosis as an important mechanism for its anticancer actions.

Methodology/Principal Findings

In vitro studies showed that DHA strongly reduces the viability and DNA synthesis of MCF-7 human breast cancer cells in culture, and also promotes cell death via apoptosis. Mechanistically, accumulation of reactive oxygen species and activation of caspase 8 contribute critically to the induction of apoptotic cell death. Co-presence of antioxidants or selective inhibition or knockdown of caspase 8 each effectively abrogates the cytotoxic effect of DHA. Using athymic nude mice as an in vivo model, we found that feeding animals the 5% fish oil-supplemented diet for 6 weeks significantly reduces the growth of MCF-7 human breast cancer cells in vivo through inhibition of cancer cell proliferation as well as promotion of cell death. Using 3-nitrotyrosine as a parameter, we confirmed that the fish oil-supplemented diet significantly increases oxidative stress in tumor cells in vivo. Analysis of fatty acid content in plasma and tissues showed that feeding animals a 5% fish oil diet increases the levels of DHA and eicosapentaenoic acid in both normal and tumorous mammary tissues by 329% and 300%, respectively.

Conclusions/Significance

DHA can strongly induce apoptosis in human MCF-7 breast cancer cells both in vitro and in vivo. The induction of apoptosis in these cells is selectively mediated via caspase 8 activation. These observations call for further studies to assess the effectiveness of fish oil as a dietary supplement in the prevention and treatment of human breast cancer.     

The ClpXP Protease Is Responsible for the Degradation of the Epsilon Antidote to the Zeta Toxin of the Streptococcal pSM19035 Plasmid

Most bacterial genomes contain different types of toxin-antitoxin (TA) systems. The ω-ϵ-ζ proteinaceous type II TA cassette from the streptococcal pSM19035 plasmid is a member of the ϵ/ζ family, which is commonly found in multiresistance plasmids and chromosomes of various human pathogens. Regulation of type II TA systems relies on the proteolysis of antitoxin proteins. Under normal conditions, the Epsilon antidote neutralizes the Zeta toxin through the formation of a tight complex. In this study, we show, using both in vivo and in vitro analyses, that the ClpXP protease is responsible for Epsilon antitoxin degradation. Using in vivo studies, we examined the stability of the plasmids with active or inactive ω-ϵ-ζ TA cassettes in B. subtilis mutants that were defective for different proteases. Using in vitro assays, the degradation of purified His₆-Epsilon by the His₆-Lon_Bs, ClpP_Bs, and ClpX_Bs proteases from B. subtilis was analyzed. Additionally, we showed that purified Zeta toxin protects the Epsilon protein from rapid ClpXP-catalyzed degradation.     

Matriptase Activation, an Early Cellular Response to Acidosis

Extracellular acidosis often rapidly causes intracellular acidification, alters ion channel activities, and activates G protein-coupled receptors. In this report, we demonstrated a novel cellular response to acidosis: induction of the zymogen activation of matriptase. Acid-induced matriptase activation is ubiquitous among epithelial and carcinoma cells and is characterized by rapid onset, fast kinetics, and the magnitude of activation seen. Trace amounts of activated matriptase can be detected 1 min after cells are exposed to pH 6.0 buffer, and the vast majority of latent matriptase within the cells is converted to activated matriptase within 20 min. Matriptase activation may be a direct response to proton exposure because acid-induced matriptase activation also occurs in an in vitro, cell-free setting in which intracellular signaling molecules and ion channel activities are largely absent. Acid-induced matriptase activation takes place both on the cell surface and inside the cells, likely due to the parallel intracellular acidification that activates intracellular matriptase. Following matriptase activation, the active enzyme is immediately inhibited by binding to hepatocyte growth factor activator inhibitor 1, resulting in stable matriptase-hepatocyte growth factor activator inhibitor 1 complexes that are rapidly secreted. As an early response to acidosis, matriptase activation can also be induced by perturbation of intracellular pH homeostasis by 5-(N-methyl-N-isobutyl)-amiloride and 5-(N-ethyl-N-isopropyl)-amiloride, both of which inhibit Na⁺/H⁺ exchangers, and diisothiocyanostilbene-2,2′-disulfonic acid, which can inhibit other acid-base ion channels. This study uncovers a novel mechanism regulating proteolysis in epithelial and carcinoma cells, and also demonstrates that a likely function of matriptase is as an early response to acidosis.