Matriptase drives dissemination of ovarian cancer spheroids by a PAR-2/PI3K/Akt/MMP9 signaling axis.

The transmembrane serine protease matriptase is a key regulator of both barrier-disruptive and protective epithelial cell-cell interactions. Elevated matriptase is a consistent feature of epithelial ovarian cancers (OvCa), where multicellular spheroids shed from the primary tumor into the peritoneal cavity are critical drivers of metastasis. Dynamic cell-to-cell adhesive contacts are required for spheroid formation and maintenance. Here, we show that overactive matriptase, reflected in an increased ratio of matriptase to its inhibitor hepatocyte growth factor activator inhibitor 1 (HAI-1), disrupts cell-cell contacts to produce loose prometastatic spheroids that display increased mesothelial cell adhesion and submesothelial invasion. We show that these activities are dependent on the matriptase activation of a protease-activated receptor-2 (PAR-2) signaling pathway involving PI3K/Akt and MMP9-induced disruption of cell-cell adhesion by the release of the soluble E-cadherin ectodomain. These data reveal a novel pathological connection between matriptase activation of PAR-2 and disruption of cell-cell adhesion, and support the clinical investigation of this signaling axis as a therapeutic strategy for aggressive metastatic OvCa.

M2a macrophages facilitate resolution of chemically-induced colitis in TLR4-SNP mice.

IMPORTANCE: Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, impacts millions of individuals worldwide and severely impairs the quality of life for patients. Dysregulation of innate immune signaling pathways reduces barrier function and exacerbates disease progression. Macrophage (Mφ) signaling pathways are potential targets for IBD therapies. While multiple treatments are available for IBD, (i) not all patients respond, (ii) responses may diminish over time, and (iii) treatments often have undesirable side effects. Genetic studies have shown that the inheritance of two co-segregating SNPs expressed in the innate immune receptor, TLR4, is associated with human IBD. Mice expressing homologous SNPs ("TLR4-SNP" mice) exhibited more severe colitis than WT mice in a DSS-induced colonic inflammation/repair model. We identified a critical role for M2a "tissue repair" Mφ in the resolution of colitis. Our findings provide insight into potential development of novel therapies targeting Mφ signaling pathways that aim to alleviate the debilitating symptoms experienced by individuals with IBD.

Intersection of Coagulation and Fibrinolysis by the Glycosylphosphatidylinositol (GPI)-Anchored Serine Protease Testisin.

Hemostasis is a delicate balance between coagulation and fibrinolysis that regulates the formation and removal of fibrin, respectively. Positive and negative feedback loops and crosstalk between coagulation and fibrinolytic serine proteases maintain the hemostatic balance to prevent both excessive bleeding and thrombosis. Here, we identify a novel role for the glycosylphosphatidylinositol (GPI)-anchored serine protease testisin in the regulation of pericellular hemostasis. Using in vitro cell-based fibrin generation assays, we found that the expression of catalytically active testisin on the cell surface accelerates thrombin-dependent fibrin polymerization, and intriguingly, that it subsequently promotes accelerated fibrinolysis. We find that the testisin-dependent fibrin formation is inhibited by rivaroxaban, a specific inhibitor of the central prothrombin-activating serine protease factor Xa (FXa), demonstrating that cell-surface testisin acts upstream of factor X (FX) to promote fibrin formation at the cell surface. Unexpectedly, testisin was also found to accelerate fibrinolysis by stimulating the plasmin-dependent degradation of fibrin and enhancing plasmin-dependent cell invasion through polymerized fibrin. Testisin was not a direct activator of plasminogen, but it is able to induce zymogen cleavage and the activation of pro-urokinase plasminogen activator (pro-uPA), which converts plasminogen to plasmin. These data identify a new proteolytic component that can regulate pericellular hemostatic cascades at the cell surface, which has implications for angiogenesis, cancer biology, and male fertility.

Selective targeting of metastatic ovarian cancer using an engineered anthrax prodrug activated by membrane-anchored serine proteases.

Treatments for advanced and recurrent ovarian cancer remain a challenge due to a lack of potent, selective, and effective therapeutics. Here, we developed the basis for a transformative anticancer strategy based on anthrax toxin that has been engineered to be selectively activated by the catalytic power of zymogen-activating proteases on the surface of malignant tumor cells to induce cell death. Exposure to the engineered toxin is cytotoxic to ovarian tumor cell lines and ovarian tumor spheroids derived from patient ascites. Preclinical studies demonstrate that toxin treatment induces tumor regression in several in vivo ovarian cancer models, including patient-derived xenografts, without adverse side effects, supportive of progression toward clinical evaluation. These data lay the groundwork for developing therapeutics for treating women with late-stage and recurrent ovarian cancers, utilizing a mechanism distinct from current anticancer therapies.

Testisin/Prss21 deficiency causes increased vascular permeability and a hemorrhagic phenotype during luteal angiogenesis.

Testisin (encoded by PRSS21) is a membrane anchored serine protease, which is tethered to the cell surface via a glycosylphosphatidylinositol (GPI)-anchor. While testisin is found in abundance in spermatozoa, it is also expressed in microvascular endothelial cells where its function is unknown. Here we identify testisin as a novel regulator of physiological hormone-induced angiogenesis and microvascular endothelial permeability. Using a murine model of rapid physiological angiogenesis during corpus luteal development in the ovary, we found that mice genetically deficient in testisin (Prss21-/-) show a substantially increased incidence of hemorrhages which are significantly more severe than in littermate control Prss21+/+ mice. This phenotype was associated with increased vascular leakiness, demonstrated by a greater accumulation of extravasated Evans blue dye in Prss21-/- ovaries. Live cell imaging of in vitro cultured microvascular endothelial cells depleted of testisin by siRNA knockdown revealed that loss of testisin markedly impaired reorganization and tubule-like formation on Matrigel basement membranes. Moreover testisin siRNA knockdown increased the paracellular permeability to FITC-albumin across endothelial cell monolayers, which was associated with decreased expression of the adherens junction protein VE-cadherin and increased levels of phospho(Tyr658)-VE-cadherin, without affecting the levels of the tight junction proteins occludin and claudin-5, or ZO-1. Decreased expression of VE-cadherin in the neovasculature of Prss21-/- ovaries was also observed without marked differences in endothelial cell content, vascular claudin-5 expression or pericyte recruitment. Together, these data identify testisin as a novel regulator of VE-cadherin adhesions during angiogenesis and indicate a potential new target for regulating neovascular integrity and associated pathologies.

Correction to: PRSS21/testisin inhibits ovarian tumor metastasis and antagonizes proangiogenic angiopoietins ANG2 and ANGPTL4.

The affiliation of Erik W. Martin is corrected in this paper.

Fibrinolysis and Inflammation in Venous Thrombus Resolution.

Clinical observations and accumulating laboratory evidence support a complex interplay between coagulation, inflammation, innate immunity and fibrinolysis in venous thromboembolism (VTE). VTE, which includes deep vein thrombosis (DVT) and pulmonary embolism (PE), and the subsequent complications of post-thrombotic syndrome (PTS), are significant causes of morbidity and mortality in patients. Clinical risk factors for VTE include cancer, major trauma, surgery, sepsis, inflammatory bowel disease, paralysis, prolonged periods of immobility, and aging. Abnormalities in venous blood flow or stasis initiates the activation of endothelial cells, and in concert with platelets, neutrophils and monocytes, propagates VTE in an intact vein. In addition, inflammatory cells play crucial roles in thrombus recanalization and restoration of blood flow via fibrinolysis and vascular remodeling. Faster resolution of the thrombus is key for improved disease prognosis. While in the clinical setting, anticoagulation therapy is successful in preventing propagation of venous thrombi, current therapies are not designed to inhibit inflammation, which can lead to the development of PTS. Animal models of DVT have provided many insights into the molecular and cellular mechanisms involved in the formation, propagation, and resolution of venous thrombi as well as the roles of key components of the fibrinolytic system in these processes. Here, we review the recent advances in our understanding of fibrinolysis and inflammation in the resolution of VTE.

PRSS21/testisin inhibits ovarian tumor metastasis and antagonizes proangiogenic angiopoietins ANG2 and ANGPTL4.

Ovarian cancer is the leading cause of death among all the gynecological cancers in the USA. Ovarian cancer employs a unique mode of metastasis, as exfoliated tumor cells disseminate within the peritoneal cavity, colonizing in several sites as well as accumulating ascites. Tumor recurrence and widespread metastasis are significant factors contributing to poor prognosis. PRSS21 is a metastasis-associated ovarian cancer gene that encodes the glycosyl-phosphatidylinositol-linked serine protease, testisin. Testisin expression is increased in multiple ovarian tumor types, with relatively little expression in normal tissues, but is differentially decreased in metastatic ovarian serous carcinomas compared to primary tumors. Here we explored the function of testisin in late-stage ovarian cancer progression using a murine xenograft model of ovarian intraperitoneal tumor metastasis. Increased tumor testisin expression inhibited intra-peritoneal tumor seeding and colonization, ascites accumulation, and metastatic tumor burden that was dependent on catalytically active testisin. The known testisin substrate, protease-activated receptor-2 (PAR-2), is a target of testisin activity. Gene profiling and mechanistic studies demonstrate that testisin activity suppresses the synthesis and secretion of pro-angiogenic angiopoietins, ANG2 and ANGPTL4, which normally promote vascular leak and edema. These observations support a model wherein testisin activates PAR-2 to antagonize proangiogenic angiopoietins that modulate vascular permeability and ascites accumulation associated with ovarian tumor metastasis. KEY MESSAGES: Testisin inhibits metastatic ovarian tumor burden and ascites production. Testisin activity antagonizes ANG2 and ANGPTL4 synthesis and secretion. PAR-2 is a proteolytic target of testisin on the surface of ovarian cancer cells.

Membrane-Anchored Serine Proteases and Protease-Activated Receptor-2-Mediated Signaling: Co-Conspirators in Cancer Progression.

Pericellular proteolysis provides a significant advantage to developing tumors through the ability to remodel the extracellular matrix, promote cell invasion and migration, and facilitate angiogenesis. Recent advances demonstrate that pericellular proteases can also communicate directly to cells by activation of a unique group of transmembrane G-protein-coupled receptors (GPCR) known as protease-activated receptors (PAR). In this review, we discuss the specific roles of one of four mammalian PARs, namely PAR-2, which is overexpressed in advanced stage tumors and is activated by trypsin-like serine proteases that are highly expressed or otherwise dysregulated in many cancers. We highlight recent insights into the ability of different protease agonists to bias PAR-2 signaling and the newly emerging evidence for an interplay between PAR-2 and membrane-anchored serine proteases, which may co-conspire to promote tumor progression and metastasis. Interfering with these pathways might provide unique opportunities for the development of new mechanism-based strategies for the treatment of advanced and metastatic cancers.

Inflammatory cytokines down-regulate the barrier-protective prostasin-matriptase proteolytic cascade early in experimental colitis.

Compromised gastrointestinal barrier function is strongly associated with the progressive and destructive pathologies of the two main forms of irritable bowel disease (IBD), ulcerative colitis (UC), and Crohn's disease (CD). Matriptase is a membrane-anchored serine protease encoded by suppression of tumorigenicity-14 (ST14) gene, which is critical for epithelial barrier development and homeostasis. Matriptase barrier-protective activity is linked with the glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin, which is a co-factor for matriptase zymogen activation. Here we show that mRNA and protein expression of both matriptase and prostasin are rapidly down-regulated in the initiating inflammatory phases of dextran sulfate sodium (DSS)-induced experimental colitis in mice, and, significantly, the loss of these proteases precedes the appearance of clinical symptoms, suggesting their loss may contribute to disease susceptibility. We used heterozygous St14 hypomorphic mice expressing a promoter-linked ß-gal reporter to show that inflammatory colitis suppresses the activity of the St14 gene promoter. Studies in colonic T84 cell monolayers revealed that barrier disruption by the colitis-associated Th2-type cytokines, IL-4 and IL-13, down-regulates matriptase as well as prostasin through phosphorylation of the transcriptional regulator STAT6 and that inhibition of STAT6 with suberoylanilide hydroxamic acid (SAHA) restores protease expression and reverses cytokine-induced barrier dysfunction. Both matriptase and prostasin are significantly down-regulated in colonic tissues from human subjects with active ulcerative colitis or Crohn's disease, implicating the loss of this barrier-protective protease pathway in the pathogenesis of irritable bowel disease.

Membrane-anchored proteases in endothelial cell biology.

PURPOSE OF REVIEW: The endothelial cell plasma membrane is a metabolically active, dynamic, and fluid microenvironment where pericellular proteolysis plays a critical role. Membrane-anchored proteases may be expressed by endothelial cells as well as mural cells and leukocytes with distribution both inside and outside of the vascular system. Here, we will review the recent advances in our understanding of the direct and indirect roles of membrane-anchored proteases in vascular biology and the possible conservation of their extravascular functions in endothelial cell biology. RECENT FINDINGS: Membrane-anchored proteases belonging to the serine or metalloprotease families contain amino-terminal or carboxy-terminal domains, which serve to tether their extracellular protease domains directly at the plasma membrane. This architecture enables protease function and substrate repertoire to be regulated through dynamic localization in distinct areas of the cell membrane. These proteases are proving to be key components of the cell machinery for regulating vascular permeability, generation of vasoactive peptides, receptor tyrosine kinase transactivation, extracellular matrix proteolysis, and angiogenesis. SUMMARY: A complex picture of the interdependence between membrane-anchored protease localization and function is emerging that may provide a mechanism for precise coordination of extracellular signals and intracellular responses through communication with the cytoskeleton and with cellular signaling molecules.

Targeting the membrane-anchored serine protease testisin with a novel engineered anthrax toxin prodrug to kill tumor cells and reduce tumor burden.

The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent. PrAg's native activation sequence was mutated to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PrAg-PCIS. PrAg-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PrAg-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PrAg-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data indicates PrAg can be engineered to target tumor cell-expressed membrane-anchored serine proteases to function as a potent tumoricidal agent.

Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin.

Protease-activated receptors (PARs) are a family of seven-transmembrane, G-protein-coupled receptors that are activated by multiple serine proteases through specific N-terminal proteolytic cleavage and the unmasking of a tethered ligand. The majority of PAR-activating proteases described to date are soluble proteases that are active during injury, coagulation, and inflammation. Less investigation, however, has focused on the potential for membrane-anchored serine proteases to regulate PAR activation. Testisin is a unique trypsin-like serine protease that is tethered to the extracellular membrane of cells through a glycophosphatidylinositol (GPI) anchor. Here, we show that the N-terminal domain of PAR-2 is a substrate for testisin and that proteolytic cleavage of PAR-2 by recombinant testisin activates downstream signaling pathways, including intracellular Ca(2+) mobilization and ERK1/2 phosphorylation. When testisin and PAR-2 are co-expressed in HeLa cells, GPI-anchored testisin specifically releases the PAR-2 tethered ligand. Conversely, knockdown of endogenous testisin in NCI/ADR-Res ovarian tumor cells reduces PAR-2 N-terminal proteolytic cleavage. The cleavage of PAR-2 by testisin induces activation of the intracellular serum-response element and NFκB signaling pathways and the induction of IL-8 and IL-6 cytokine gene expression. Furthermore, the activation of PAR-2 by testisin results in the loss and internalization of PAR-2 from the cell surface. This study reveals a new biological substrate for testisin and is the first demonstration of the activation of a PAR by a serine protease GPI-linked to the cell surface.

CANDIDATE GENES FOR LIMITING CHOLESTATIC INTESTINAL INJURY IDENTIFIED BY GENE EXPRESSION PROFILING.

The lack of bile flow from the liver into the intestine can have devastating complications including hepatic failure, sepsis and even death. This pathologic condition known as cholestasis can result from etiologies as diverse as total parenteral nutrition (TPN), hepatitis and pancreatic cancer. The intestinal injury associated with cholestasis has been shown to result in decreased intestinal resistance, increased bacterial translocation and increased endotoxemia. Anecdotal clinical evidence suggests a genetic predisposition to exaggerated injury. Recent animal research on two different strains of inbred mice demonstrating different rates of bacterial translocation with different mortality rates supports this premise. In this study, a microarray analysis of intestinal tissue following common bile duct ligation (CBDL) performed under general anesthesia on these same two strains of inbred mice was done with the goal of identifying the potential molecular mechanistic pathways responsible. Over 500 genes were increased more than 2.0 fold following CBDL. The most promising candidate genes included MUPs, Serpina1a and LCN-2. RT-PCR validated the microarray results for these candidate genes. In an in vitro experiment using differentiated intestinal epithelial cells, inhibition of MUP-1 by siRNA resulted in increased intestinal epithelial cell permeability. Diverse novel mechanisms involving the growth hormone pathway, the acute phase response and the innate immune response are thus potential avenues for limiting cholestatic intestinal injury. Changes in gene expression were at times found to be not only due to the CBDL but also due to the murine strain. Should further studies in cholestatic patients demonstrate inter-individual variability similar to what we have shown in mice, then a "personalized medicine" approach to cholestatic patients may become possible.

The transcription factor C/EBP-ß mediates constitutive and LPS-inducible transcription of murine SerpinB2.

SerpinB2 or plasminogen activator inhibitor type 2 (PAI-2) is highly induced in macrophages in response to inflammatory stimuli and is linked to the modulation of innate immunity, macrophage survival, and inhibition of plasminogen activators. Lipopolysaccharide (LPS), a potent bacterial endotoxin, can induce SerpinB2 expression via the toll-like receptor 4 (TLR4) by ∼1000-fold over a period of 24 hrs in murine macrophages. To map the LPS-regulated SerpinB2 promoter regions, we transfected reporter constructs driven by the ∼5 kb 5'-flanking region of the murine SerpinB2 gene and several deletion mutants into murine macrophages. In addition, we compared the DNA sequence of the murine 5' flanking sequence with the sequence of the human gene for homologous functional regulatory elements and identified several regulatory cis-acting elements in the human SERPINB2 promoter conserved in the mouse. Mutation analyses revealed that a CCAAT enhancer binding (C/EBP) element, a cyclic AMP response element (CRE) and two activator protein 1 (AP-1) response elements in the murine SerpinB2 proximal promoter are essential for optimal LPS-inducibility. Electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrated that LPS induces the formation of C/EBP-ß containing complexes with the SerpinB2 promoter. Importantly, both constitutive and LPS-induced SerpinB2 expression was severely abrogated in C/EBP-ß-null mouse embryonic fibroblasts (MEFs) and primary C/EBP-ß-deficient peritoneal macrophages. Together, these data provide new insight into C/EBP-ß-dependent regulation of inflammation-associated SerpinB2 expression.

Prostasin is required for matriptase activation in intestinal epithelial cells to regulate closure of the paracellular pathway.

The type II transmembrane serine protease matriptase is a key regulator of epithelial barriers in skin and intestine. In skin, matriptase acts upstream of the glycosylphosphatidylinositol-anchored serine protease, prostasin, to activate the prostasin zymogen and initiate a proteolytic cascade that is required for stratum corneum barrier functionality. Here, we have investigated the relationship between prostasin and matriptase in intestinal epithelial barrier function. We find that similar to skin, matriptase and prostasin are components of a common intestinal epithelial barrier-forming pathway. Depletion of prostasin by siRNA silencing in Caco-2 intestinal epithelium inhibits barrier development similar to loss of matriptase, and the addition of recombinant prostasin to the basal side of polarized Caco-2 epithelium stimulates barrier forming changes similar to the addition of recombinant matriptase. However, in contrast to the proteolytic cascade in skin, prostasin functions upstream of matriptase to activate the endogenous matriptase zymogen. Prostasin is unable to proteolytically activate the matriptase zymogen directly but induces matriptase activation indirectly. Prostasin requires expression of endogenous matriptase to stimulate barrier formation since matriptase depletion by siRNA silencing abrogates prostasin barrier-forming activity. Active recombinant matriptase, however, does not require the expression of endogenous prostasin for barrier-forming activity. Together, these data show that matriptase and not prostasin is the primary effector protease of tight junction assembly in simple columnar epithelia and further highlight a spatial and tissue-specific aspect of cell surface proteolytic cascades.

Matriptase protects against experimental colitis and promotes intestinal barrier recovery.

BACKGROUND: Matriptase is a membrane-anchored serine protease encoded by suppression of tumorigenicity-14 (ST14) that is required for epithelial barrier homeostasis. However, its functional role in inflammatory bowel disease (IBD) is unexplored. METHODS: Matriptase expression in control, Crohn's disease, and ulcerative colitis tissue specimens was studied by quantitative polymerase chain reaction (qPCR) and immunostaining. Matriptase function was investigated by subjecting St14 hypomorphic and control littermates to dextran sodium sulfate (DSS)-induced colitis and by siRNA silencing in cultured monolayers. Mice were analyzed for clinical, histological, molecular, and cellular effects. RESULTS: Matriptase protein and ST14 mRNA levels are significantly downregulated in inflamed colonic tissues from Crohn's disease and ulcerative colitis patients. Matriptase-deficient St14 hypomorphic mice administered DSS for 7 days followed by water without DSS for 3 days develop a severe colitis, with only 30% of the St14 hypomorphic mice surviving to day 14, compared with 100% of control littermates. Persistent colitis in surviving St14 hypomorphic mice was associated with sustained cytokine production, an inability to recover barrier integrity, and enhanced claudin-2 expression. Cytokines implicated in barrier disruption during IBD suppress matriptase expression in T84 epithelial monolayers and restoration of matriptase improves barrier integrity in the cytokine-perturbed monolayers. CONCLUSIONS: These data demonstrate a critical role for matriptase in restoring barrier function to injured intestinal mucosa during colitis, which is suppressed by excessive activation of the immune system. Strategies to enhance matriptase-mediated barrier recovery could be important for intervening in the cycle of inflammation associated with IBD.

The cutting edge: membrane-anchored serine protease activities in the pericellular microenvironment.

The serine proteases of the trypsin-like (S1) family play critical roles in many key biological processes including digestion, blood coagulation, and immunity. Members of this family contain N- or C-terminal domains that serve to tether the serine protease catalytic domain directly to the plasma membrane. These membrane-anchored serine proteases are proving to be key components of the cell machinery for activation of precursor molecules in the pericellular microenvironment, playing vital functions in the maintenance of homoeostasis. Substrates activated by membrane-anchored serine proteases include peptide hormones, growth and differentiation factors, receptors, enzymes, adhesion molecules and viral coat proteins. In addition, new insights into our understanding of the physiological functions of these proteases and their involvement in human pathology have come from animal models and patient studies. The present review discusses emerging evidence for the diversity of this fascinating group of membrane serine proteases as potent modifiers of the pericellular microenvironment through proteolytic processing of diverse substrates. We also discuss the functional consequences of the activities of these proteases on mammalian physiology and disease.

Membrane-anchored serine protease matriptase regulates epithelial barrier formation and permeability in the intestine.

The intestinal epithelium serves as a major protective barrier between the mammalian host and the external environment. Here we show that the transmembrane serine protease matriptase plays a pivotol role in the formation and integrity of the intestinal epithelial barrier. St14 hypomorphic mice, which have a 100-fold reduction in intestinal matriptase mRNA levels, display a 35% reduction in intestinal transepithelial electrical resistance (TEER). Matriptase is expressed during intestinal epithelial differentiation and colocalizes with E-cadherin to apical junctional complexes (AJC) in differentiated polarized Caco-2 monolayers. Inhibition of matriptase activity using a specific peptide inhibitor or by knockdown of matriptase by siRNA disrupts the development of TEER in barrier-forming Caco-2 monolayers and increases paracellular permeability to macromolecular FITC-dextran. Loss of matriptase was associated with enhanced expression and incorporation of the permeability-associated, "leaky" tight junction protein claudin-2 at intercellular junctions. Knockdown of claudin-2 enhanced the development of TEER in matriptase-silenced Caco-2 monolayers, suggesting that the reduced barrier integrity was caused, at least in part, by an inability to regulate claudin-2 expression and incorporation into junctions. We find that matriptase enhances the rate of claudin-2 protein turnover, and that this is mediated indirectly through an atypical PKCzeta-dependent signaling pathway. These results support a key role for matriptase in regulating intestinal epithelial barrier competence, and suggest an intriguing link between pericellular serine protease activity and tight junction assembly in polarized epithelia.

Identification of human zonulin, a physiological modulator of tight junctions, as prehaptoglobin-2.

Increased intestinal permeability (IP) has emerged recently as a common underlying mechanism in the pathogenesis of allergic, inflammatory, and autoimmune diseases. The characterization of zonulin, the only physiological mediator known to regulate IP reversibly, has remained elusive. Through proteomic analysis of human sera, we have now identified human zonulin as the precursor for haptoglobin-2 (pre-HP2). Although mature HP is known to scavenge free hemoglobin (Hb) to inhibit its oxidative activity, no function has ever been ascribed to its uncleaved precursor form. We found that the single-chain zonulin contains an EGF-like motif that leads to transactivation of EGF receptor (EGFR) via proteinase-activated receptor 2 (PAR(2)) activation. Activation of these 2 receptors was coupled to increased IP. The siRNA-induced silencing of PAR(2) or the use of PAR(2)(-/-) mice prevented loss of barrier integrity. Proteolytic cleavage of zonulin into its alpha(2)- and beta-subunits neutralized its ability to both activate EGFR and increase IP. Quantitative gene expression revealed that zonulin is overexpressed in the intestinal mucosa of subjects with celiac disease. To our knowledge, this is the initial example of a molecule that exerts a biological activity in its precursor form that is distinct from the function of its mature form. Our results therefore characterize zonulin as a previously undescribed ligand that engages a key signalosome involved in the pathogenesis of human immune-mediated diseases that can be targeted for therapeutic interventions.