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1.
Development ; 150(17)2023 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-37539662

RESUMEN

Congenital tufting enteropathy (CTE) is a life-threatening intestinal disorder resulting from loss-of-function mutations in EPCAM and SPINT2. Mice deficient in Spint2, encoding the protease inhibitor HAI-2, develop CTE-like intestinal failure associated with a progressive loss of the EpCAM protein, which is caused by unchecked activity of the serine protease matriptase (ST14). Here, we show that loss of HAI-2 leads to increased proteolytic processing of EpCAM. Elimination of the reported matriptase cleavage site strongly suppressed proteolytic processing of EpCAM in vitro and in vivo. Unexpectedly, expression of cleavage-resistant EpCAM failed to prevent intestinal failure and postnatal lethality in Spint2-deficient mice. In addition, genetic inactivation of intestinal matriptase (St14) counteracted the effect of Spint2 deficiency in mice expressing cleavage-resistant EpCAM, indicating that matriptase does not drive intestinal dysfunction by excessive proteolysis of EpCAM. Interestingly, mice expressing cleavage-resistant EpCAM developed late-onset intestinal defects and exhibited a shortened lifespan even in the presence of HAI-2, suggesting that EpCAM cleavage is indispensable for EpCAM function. Our findings provide new insights into the role of EpCAM and the etiology of the enteropathies driven by Spint2 deficiency.


Asunto(s)
Insuficiencia Intestinal , Animales , Ratones , Molécula de Adhesión Celular Epitelial/genética , Molécula de Adhesión Celular Epitelial/metabolismo , Intestinos , Proteínas Inhibidoras de Proteinasas Secretoras
2.
Proc Natl Acad Sci U S A ; 120(1): e2211927120, 2023 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-36574698

RESUMEN

The limited efficacy of the current antitumor microenvironment strategies is due in part to the poor understanding of the roles and relative contributions of the various tumor stromal cells to tumor development. Here, we describe a versatile in vivo anthrax toxin protein delivery system allowing for the unambiguous genetic evaluation of individual tumor stromal elements in cancer. Our reengineered tumor-selective anthrax toxin exhibits potent antiproliferative activity by disrupting ERK signaling in sensitive cells. Since this activity requires the surface expression of the capillary morphogenesis protein-2 (CMG2) toxin receptor, genetic manipulation of CMG2 expression using our cell-type-specific CMG2 transgenic mice allows us to specifically define the role of individual tumor stromal cell types in tumor development. Here, we established mice with CMG2 only expressed in tumor endothelial cells (ECs) and determined the specific contribution of tumor stromal ECs to the toxin's antitumor activity. Our results demonstrate that disruption of ERK signaling only within tumor ECs is sufficient to halt tumor growth. We discovered that c-Myc is a downstream effector of ERK signaling and that the MEK-ERK-c-Myc central metabolic axis in tumor ECs is essential for tumor progression. As such, disruption of ERK-c-Myc signaling in host-derived tumor ECs by our tumor-selective anthrax toxins explains their high efficacy in solid tumor therapy.


Asunto(s)
Células Endoteliales , Neoplasias , Ratones , Animales , Células Endoteliales/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Transducción de Señal , Antígenos Bacterianos/metabolismo , Neoplasias/genética , Microambiente Tumoral
3.
J Biol Chem ; 300(5): 107284, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38614208

RESUMEN

Receptor-mediated cellular uptake of specific ligands constitutes an important step in the dynamic regulation of individual protein levels in extracellular fluids. With a focus on the inflammatory lung, we here performed a proteomics-based search for novel ligands regulated by the mannose receptor (MR), a macrophage-expressed endocytic receptor. WT and MR-deficient mice were exposed to lipopolysaccharide, after which the protein content in their lung epithelial lining fluid was compared by tandem mass tag-based mass spectrometry. More than 1200 proteins were identified in the epithelial lining fluid using this unbiased approach, but only six showed a statistically different abundance. Among these, an unexpected potential new ligand, thrombospondin-4 (TSP-4), displayed a striking 17-fold increased abundance in the MR-deficient mice. Experiments using exogenous addition of TSP-4 to MR-transfected CHO cells or MR-positive alveolar macrophages confirmed that TSP-4 is a ligand for MR-dependent endocytosis. Similar studies revealed that the molecular interaction with TSP-4 depends on both the lectin activity and the fibronectin type-II domain of MR and that a closely related member of the TSP family, TSP-5, is also efficiently internalized by the receptor. This was unlike the other members of this protein family, including TSPs -1 and -2, which are ligands for a close MR homologue known as urokinase plasminogen activator receptor-associated protein. Our study shows that MR takes part in the regulation of TSP-4, an important inflammatory component in the injured lung, and that two closely related endocytic receptors, expressed on different cell types, undertake the selective endocytosis of distinct members of the TSP family.


Asunto(s)
Lectinas Tipo C , Lesión Pulmonar , Receptor de Manosa , Lectinas de Unión a Manosa , Proteómica , Receptores de Superficie Celular , Trombospondinas , Animales , Ratones , Células CHO , Cricetulus , Endocitosis , Lectinas Tipo C/metabolismo , Lectinas Tipo C/genética , Ligandos , Lipopolisacáridos/toxicidad , Pulmón/metabolismo , Pulmón/patología , Lesión Pulmonar/metabolismo , Lesión Pulmonar/patología , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Lectinas de Unión a Manosa/metabolismo , Lectinas de Unión a Manosa/genética , Ratones Noqueados , Proteómica/métodos , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/genética , Trombospondinas/metabolismo , Trombospondinas/genética
4.
Annu Rev Cell Dev Biol ; 27: 213-35, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21721945

RESUMEN

Analysis of vertebrate genome sequences at the turn of the millennium revealed that a vastly larger repertoire of enzymes execute proteolytic cleavage reactions within the pericellular and extracellular environments than was anticipated from biochemical and molecular analysis. Most unexpected was the unveiling of an entire new family of structurally unique multidomain serine proteases that are anchored directly to the plasma membrane. Unlike secreted serine proteases, which function primarily in tissue repair, immunity, and nutrient uptake, these membrane-anchored serine proteases regulate fundamental cellular and developmental processes, including tissue morphogenesis, epithelial barrier function, ion and water transport, cellular iron export, and fertilization. Here the cellular and developmental biology of this fascinating new group of proteases is reviewed. Particularly highlighted is how the study of membrane-anchored serine proteases has expanded our knowledge of the range of physiological processes that require regulated proteolysis at the cell surface.


Asunto(s)
Biología Celular , Membrana Celular/enzimología , Biología Evolutiva , Serina Endopeptidasas/metabolismo , Animales , Membrana Celular/química , Oído Interno/fisiología , Fertilización/fisiología , Humanos , Hierro/metabolismo , Péptidos Natriuréticos/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Transducción de Señal/fisiología , Sodio/metabolismo , Uniones Estrechas/metabolismo
5.
Blood ; 139(9): 1302-1311, 2022 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-34958662

RESUMEN

Fibrinogen plays a pathologic role in multiple diseases. It contributes to thrombosis and modifies inflammatory and immune responses, supported by studies in mice expressing fibrinogen variants with altered function or with a germline fibrinogen deficiency. However, therapeutic strategies to safely and effectively tailor plasma fibrinogen concentration are lacking. Here, we developed a strategy to tune fibrinogen expression by administering lipid nanoparticle (LNP)-encapsulated small interfering RNA (siRNA) targeting the fibrinogen α chain (siFga). Three distinct LNP-siFga reagents reduced both hepatic Fga messenger RNA and fibrinogen levels in platelets and plasma, with plasma levels decreased to 42%, 16%, and 4% of normal within 1 week of administration. Using the most potent siFga, circulating fibrinogen was controllably decreased to 32%, 14%, and 5% of baseline with 0.5, 1.0, and 2.0 mg/kg doses, respectively. Whole blood from mice treated with siFga formed clots with significantly decreased clot strength ex vivo, but siFga treatment did not compromise hemostasis following saphenous vein puncture or tail transection. In an endotoxemia model, siFga suppressed the acute phase response and decreased plasma fibrinogen, D-dimer, and proinflammatory cytokine levels. In a sterile peritonitis model, siFga restored normal macrophage migration in plasminogen-deficient mice. Finally, treatment of mice with siFga decreased the metastatic potential of tumor cells in a manner comparable to that observed in fibrinogen-deficient mice. The results indicate that siFga causes robust and controllable depletion of fibrinogen and provides the proof-of-concept that this strategy can modulate the pleiotropic effects of fibrinogen in relevant disease models.


Asunto(s)
Afibrinogenemia/metabolismo , Fibrina/biosíntesis , Fibrinógeno/biosíntesis , Técnicas de Silenciamiento del Gen , Liposomas/farmacología , ARN Interferente Pequeño , Afibrinogenemia/genética , Animales , Plaquetas/metabolismo , Modelos Animales de Enfermedad , Femenino , Fibrina/genética , Fibrinógeno/genética , Humanos , Masculino , Ratones , Nanopartículas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología
6.
J Biol Chem ; 298(1): 101467, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34871548

RESUMEN

Bacillus anthracis lethal toxin and edema toxin are binary toxins that consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds to either of two receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding and cytoplasmic translocation of LF and EF. However, the distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals has not been fully elucidated. Herein, we describe an assay to image anthrax toxin intoxication in animals, and we use it to visualize TEM-8- and CMG-2-dependent intoxication in mice. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were coadministered to transgenic mice expressing a red fluorescent protein in the absence of Cre and a green fluorescent protein in the presence of Cre, intoxication could be visualized at single-cell resolution by confocal microscopy or flow cytometry. Using this assay, we found that: (a) CMG-2 is critical for intoxication in the liver and heart, (b) TEM-8 is required for intoxication in the kidney and spleen, (c) CMG-2 and TEM-8 are redundant for intoxication of some organs, (d) combined loss of CMG-2 and TEM-8 completely abolishes intoxication, and (e) CMG-2 is the dominant receptor on leukocytes. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for clinical development of reengineered toxin variants for cancer treatment.


Asunto(s)
Carbunco , Antígenos Bacterianos , Bacillus anthracis , Toxinas Bacterianas , Animales , Carbunco/diagnóstico por imagen , Carbunco/metabolismo , Antígenos Bacterianos/química , Antígenos Bacterianos/toxicidad , Bacillus anthracis/metabolismo , Toxinas Bacterianas/toxicidad , Citoplasma/metabolismo , Ratones , Ratones Transgénicos
7.
Pflugers Arch ; 474(6): 613-624, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35312839

RESUMEN

Experimental nephrotic syndrome leads to activation of the epithelial sodium channel (ENaC) by proteolysis and promotes renal sodium retention. The membrane-anchored serine protease prostasin (CAP1/PRSS8) is expressed in the distal nephron and participates in proteolytic ENaC regulation by serving as a scaffold for other serine proteases. However, it is unknown whether prostasin is also involved in ENaC-mediated sodium retention of experimental nephrotic syndrome. In this study, we used genetically modified knock-in mice with Prss8 mutations abolishing its proteolytic activity (Prss8-S238A) or prostasin activation (Prss8-R44Q) to investigate the development of sodium retention in doxorubicin-induced nephrotic syndrome. Healthy Prss8-S238A and Prss8-R44Q mice had normal ENaC activity as reflected by the natriuretic response to the ENaC blocker triamterene. After doxorubicin injection, all genotypes developed similar proteinuria. In all genotypes, urinary prostasin excretion increased while renal expression was not altered. In nephrotic mice of all genotypes, triamterene response was similarly increased, consistent with ENaC activation. As a consequence, urinary sodium excretion dropped in all genotypes and mice similarly gained body weight by + 25 ± 3% in Prss8-wt, + 20 ± 2% in Prss8-S238A and + 28 ± 3% in Prss8-R44Q mice (p = 0.16). In Western blots, expression of fully cleaved α- and γ-ENaC was similarly increased in nephrotic mice of all genotypes. In conclusion, proteolytic ENaC activation and sodium retention in experimental nephrotic syndrome are independent of the activation of prostasin and its enzymatic activity and are consistent with the action of aberrantly filtered serine proteases or proteasuria.


Asunto(s)
Síndrome Nefrótico , Serina Endopeptidasas , Sodio , Animales , Doxorrubicina/farmacología , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Ratones , Síndrome Nefrótico/genética , Síndrome Nefrótico/metabolismo , Serina Endopeptidasas/metabolismo , Serina Proteasas/metabolismo , Sodio/metabolismo , Triantereno
8.
Development ; 146(22)2019 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-31628112

RESUMEN

Syndromic congenital tufting enteropathy (CTE) is a life-threatening recessive human genetic disorder that is caused by mutations in SPINT2, encoding the protease inhibitor HAI-2, and is characterized by severe intestinal dysfunction. We recently reported the generation of a Spint2-deficient mouse model of CTE. Here, we show that the CTE-associated early-onset intestinal failure and lethality of Spint2-deficient mice is caused by unchecked activity of the serine protease matriptase. Macroscopic and histological defects observed in the absence of HAI-2, including villous atrophy, luminal bleeding, loss of mucin-producing goblet cells, loss of defined crypt architecture and the resulting acute inflammatory response in the large intestine, were all prevented by intestinal-specific inactivation of the St14 gene encoding matriptase. The CTE-associated loss of the cell junctional proteins EpCAM and claudin 7 was also prevented. As a result, inactivation of intestinal matriptase allowed Spint2-deficient mice to gain weight after birth and dramatically increased their lifespan. These data implicate matriptase as a causative agent in the development of CTE and may provide a new target for the treatment of CTE in individuals carrying SPINT2 mutations.This article has an associated 'The people behind the papers' interview.


Asunto(s)
Diarrea Infantil/genética , Diarrea Infantil/patología , Intestinos/patología , Síndromes de Malabsorción/genética , Síndromes de Malabsorción/patología , Proteínas de la Membrana/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Animales , Claudinas/metabolismo , Cruzamientos Genéticos , Modelos Animales de Enfermedad , Molécula de Adhesión Celular Epitelial/metabolismo , Epitelio/metabolismo , Femenino , Genotipo , Hemorragia , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Fenotipo
9.
Int J Mol Sci ; 23(12)2022 Jun 16.
Artículo en Inglés | MEDLINE | ID: mdl-35743186

RESUMEN

The serine protease prostasin (CAP1/Prss8, channel-activating protease-1) is a confirmed in vitro and in vivo activator of the epithelial sodium channel ENaC. To test whether proteolytic activity or CAP1/Prss8 abundance itself are required for ENaC activation in the kidney, we studied animals either hetero- or homozygous mutant at serine 238 (S238A; Prss8cat/+ and Prss8cat/cat), and renal tubule-specific CAP1/Prss8 knockout (Prss8PaxLC1) mice. When exposed to varying Na+-containing diets, no changes in Na+ and K+ handling and only minor changes in the expression of Na+ and K+ transporting protein were found in both models. Similarly, the α- or γENaC subunit cleavage pattern did not differ from control mice. On standard and low Na+ diet, Prss8cat/+ and Prss8cat/cat mice exhibited standard plasma aldosterone levels and unchanged amiloride-sensitive rectal potential difference indicating adapted ENaC activity. Upon Na+ deprivation, mice lacking the renal CAP1/Prss8 expression (Prss8PaxLC1) exhibit significantly decreased plasma aldosterone and lower K+ levels but compensate by showing significantly higher plasma renin activity. Our data clearly demonstrated that the catalytic activity of CAP1/Prss8 is dispensable for proteolytic ENaC activation. CAP1/Prss8-deficiency uncoupled ENaC activation from its aldosterone dependence, but Na+ homeostasis is maintained through alternative pathways.


Asunto(s)
Aldosterona , Sodio , Animales , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Riñón/metabolismo , Ratones , Oligopéptidos , Serina Endopeptidasas , Sodio/metabolismo
10.
Blood ; 134(3): 291-303, 2019 07 18.
Artículo en Inglés | MEDLINE | ID: mdl-31101623

RESUMEN

Efficient migration of macrophages to sites of inflammation requires cell surface-bound plasmin(ogen). Here, we investigated the mechanisms underlying the deficits of plasmin(ogen)-mediated macrophage migration in 2 models: murine thioglycollate-induced peritonitis and in vitro macrophage migration. As previously reported, macrophage migration into the peritoneal cavity of mice in response to thioglycollate was significantly impaired in the absence of plasminogen. Fibrin(ogen) deposition was noted in the peritoneal cavity in response to thioglycollate, with a significant increase in fibrin(ogen) in the plasminogen-deficient mice. Interestingly, macrophage migration was restored in plasminogen-deficient mice by simultaneous imposition of fibrinogen deficiency. Consistent with this in vivo finding, chemotactic migration of cultured macrophages through a fibrin matrix did not occur in the absence of plasminogen. The macrophage requirement for plasmin-mediated fibrinolysis, both in vivo and in vitro, was negated by deletion of the major myeloid integrin αMß2-binding motif on the γ chain of fibrin(ogen). The study identifies a critical role of fibrinolysis in macrophage migration, presumably through the alleviation of migratory constraints imposed by the interaction of leukocytes with fibrin(ogen) through the integrin αMß2 receptor.


Asunto(s)
Quimiotaxis de Leucocito , Fibrinolisina/metabolismo , Fibrinólisis , Inflamación/etiología , Inflamación/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Animales , Biomarcadores , Quimiotaxis de Leucocito/inmunología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Fibrinógeno/genética , Fibrinógeno/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunofenotipificación , Inflamación/patología , Recuento de Leucocitos , Ratones , Ratones Noqueados , Plasminógeno/deficiencia , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Células RAW 264.7
11.
Cell Mol Life Sci ; 77(16): 3161-3176, 2020 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-32100084

RESUMEN

As the dominant constituent of the extracellular matrix (ECM), collagens of different types are critical for the structural properties of tissues and make up scaffolds for cellular adhesion and migration. Importantly, collagens also directly modulate the phenotypic state of cells by transmitting signals that influence proliferation, differentiation, polarization, survival, and more, to cells of mesenchymal, epithelial, or endothelial origin. Recently, the potential of collagens to provide immune regulatory signals has also been demonstrated, and it is believed that pathological changes in the ECM shape immune cell phenotype. Collagens are themselves heavily regulated by a multitude of structural modulations or by catabolic pathways. One of these pathways involves a cellular uptake of collagens or soluble collagen-like defense collagens of the innate immune system mediated by endocytic collagen receptors. This cellular uptake is followed by the degradation of collagens in lysosomes. The potential of this pathway to regulate collagens in pathological conditions is evident from the increased extracellular accumulation of both collagens and collagen-like defense collagens following endocytic collagen receptor ablation. Here, we review how endocytic collagen receptors regulate collagen turnover during physiological conditions and in pathological conditions, such as fibrosis and cancer. Furthermore, we highlight the potential of collagens to regulate immune cells and discuss how endocytic collagen receptors can directly regulate immune cell activity in pathological conditions or do it indirectly by altering the extracellular milieu. Finally, we discuss the potential collagen receptors utilized by immune cells to directly detect ECM-related changes in the tissues which they encounter.


Asunto(s)
Colágeno/inmunología , Animales , Endocitosis/inmunología , Matriz Extracelular/inmunología , Fibrosis/inmunología , Humanos , Neoplasias/inmunología
12.
J Biol Chem ; 294(44): 15987-15996, 2019 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-31501243

RESUMEN

Elucidation of gene function by reverse genetics in animal models frequently is complicated by the functional redundancy of homologous genes. This obstacle often is compounded by the tight clustering of homologous genes, which precludes the generation of multigene-deficient animals through standard interbreeding of single-deficient animals. Here, we describe an iterative, multiplexed CRISPR-based approach for simultaneous gene editing in the complex seven-member human airway trypsin-like protease/differentially expressed in a squamous cell carcinoma (HAT/DESC) cluster of membrane-anchored serine proteases. Through four cycles of targeting, we generated a library of 18 unique congenic mouse strains lacking combinations of HAT/DESC proteases, including a mouse strain deficient in all seven proteases. Using this library, we demonstrate that HAT/DESC proteases are dispensable for term development, postnatal health, and fertility and that the recently described function of the HAT-like 4 protease in epidermal barrier formation is unique among all HAT/DESC proteases. The study demonstrates the potential of iterative, multiplexed CRISPR-mediated gene editing for functional analysis of multigene clusters, and it provides a large array of new congenic mouse strains for the study of HAT/DESC proteases in physiological and in pathophysiological processes.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Familia de Multigenes , Serina Endopeptidasas/genética , Animales , Desarrollo Embrionario/genética , Epidermis/crecimiento & desarrollo , Epidermis/metabolismo , Femenino , Fertilidad/genética , Células HEK293 , Humanos , Masculino , Ratones , Serina Endopeptidasas/metabolismo
13.
Biochem Soc Trans ; 48(2): 517-528, 2020 04 29.
Artículo en Inglés | MEDLINE | ID: mdl-32196551

RESUMEN

Cleavage of proteins in the extracellular milieu, including hormones, growth factors and their receptors, ion channels, and various cell adhesion and extracellular matrix molecules, plays a key role in the regulation of cell behavior. Among more than 500 proteolytic enzymes encoded by mammalian genomes, membrane-anchored serine proteases (MASPs), which are expressed on the surface of epithelial cells of all major organs, are excellently suited to mediate signal transduction across the epithelia and are increasingly being recognized as important regulators of epithelial development, function, and disease [ 1-3]. In this minireview, we summarize current knowledge of the in vivo roles of MASPs in acquisition and maintenance of some of the defining functions of epithelial tissues, such as barrier formation, ion transport, and sensory perception.


Asunto(s)
Membrana Celular/enzimología , Células Epiteliales/enzimología , Serina Proteasas/metabolismo , Transducción de Señal , Animales , Adhesión Celular , Matriz Extracelular/metabolismo , Humanos , Iones , Proteínas de la Membrana/metabolismo , Ratones , Serina Endopeptidasas/metabolismo
14.
Traffic ; 18(6): 378-391, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28371047

RESUMEN

It has recently been shown that hepatocyte growth factor activator inhibitor-2 (HAI-2) is able to suppress carcinogenesis induced by overexpression of matriptase, as well as cause regression of individual established tumors in a mouse model system. However, the role of HAI-2 is poorly understood. In this study, we describe 3 mutations in the binding loop of the HAI-2 Kunitz domain 1 (K42N, C47F and R48L) that cause a delay in the SEA domain cleavage of matriptase, leading to accumulation of non-SEA domain cleaved matriptase in the endoplasmic reticulum (ER). We suggest that, like other known SEA domains, the matriptase SEA domain auto-cleaves and reflects that correct oligomerization, maturation, and/or folding has been obtained. Our results suggest that the HAI-2 Kunitz domain 1 mutants influence the flux of matriptase to the plasma membrane by affecting the oligomerization, maturation and/or folding of matriptase, and as a result the SEA domain cleavage of matriptase. Two of the HAI-2 Kunitz domain 1 mutants investigated (C47F, R48L and C47F/R48L) also displayed a reduced ability to proteolytically silence matriptase. Hence, HAI-2 separately stabilizes matriptase, regulates the secretory transport, possibly via maturation/oligomerization and inhibits the proteolytic activity of matriptase in the ER, and possible throughout the secretory pathway.


Asunto(s)
Retículo Endoplásmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Serina Endopeptidasas/metabolismo , Transporte Biológico/fisiología , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Glicoproteínas de Membrana/genética , Dominios Proteicos , Proteolisis
15.
Development ; 143(15): 2818-28, 2016 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-27385010

RESUMEN

The membrane-anchored serine proteases prostasin (PRSS8) and matriptase (ST14) initiate a cell surface proteolytic pathway essential for epithelial function. Mice expressing only catalytically inactive prostasin are viable, unlike prostasin null mice, indicating that at least some prostasin functions are non-proteolytic. Here we used knock-in mice expressing catalytically inactive prostasin (Prss8(Ki/Ki)) to show that the physiological and pathological functions of prostasin vary in their dependence on its catalytic activity. Whereas prostasin null mice exhibited partial embryonic and complete perinatal lethality, Prss8(Ki/Ki) mice displayed normal prenatal and postnatal survival. Unexpectedly, catalytically inactive prostasin caused embryonic lethality in mice lacking its cognate inhibitors HAI-1 (SPINT1) or HAI-2 (SPINT2). Proteolytically inactive prostasin, unlike the wild-type protease, was unable to activate matriptase during placentation. Surprisingly, all essential functions of prostasin in embryonic and postnatal development were compensated for by loss of HAI-1, indicating that prostasin is only required for mouse development and overall viability in the presence of this inhibitor. This study expands our knowledge of non-proteolytic functions of membrane-anchored serine proteases and provides unexpected new data on the mechanistic interactions between matriptase and prostasin in the context of epithelial development.


Asunto(s)
Serina Endopeptidasas/metabolismo , Serina Proteasas/metabolismo , Animales , Femenino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Placentación , Embarazo , Proteínas Inhibidoras de Proteinasas Secretoras , Proteolisis , Serina Endopeptidasas/genética , Serina Proteasas/genética
16.
Proc Natl Acad Sci U S A ; 113(28): E4079-87, 2016 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-27357689

RESUMEN

Engineered tumor-targeted anthrax lethal toxin proteins have been shown to strongly suppress growth of solid tumors in mice. These toxins work through the native toxin receptors tumor endothelium marker-8 and capillary morphogenesis protein-2 (CMG2), which, in other contexts, have been described as markers of tumor endothelium. We found that neither receptor is required for tumor growth. We further demonstrate that tumor cells, which are resistant to the toxin when grown in vitro, become highly sensitive when implanted in mice. Using a range of tissue-specific loss-of-function and gain-of-function genetic models, we determined that this in vivo toxin sensitivity requires CMG2 expression on host-derived tumor endothelial cells. Notably, engineered toxins were shown to suppress the proliferation of isolated tumor endothelial cells. Finally, we demonstrate that administering an immunosuppressive regimen allows animals to receive multiple toxin dosages and thereby produces a strong and durable antitumor effect. The ability to give repeated doses of toxins, coupled with the specific targeting of tumor endothelial cells, suggests that our strategy should be efficacious for a wide range of solid tumors.


Asunto(s)
Antígenos Bacterianos/uso terapéutico , Toxinas Bacterianas/uso terapéutico , Biomarcadores de Tumor/metabolismo , Células Endoteliales/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Receptores de Péptidos/metabolismo , Animales , Antígenos Bacterianos/farmacología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica , Toxinas Bacterianas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclofosfamida/farmacología , Ciclofosfamida/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales , Linfocitos/efectos de los fármacos , Ratones , Proteínas de Microfilamentos , Terapia Molecular Dirigida , Neoplasias/genética , Pentostatina/farmacología , Pentostatina/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/genética , Receptores de Superficie Celular
17.
Blood ; 127(25): 3260-9, 2016 06 23.
Artículo en Inglés | MEDLINE | ID: mdl-27114461

RESUMEN

The coagulation cascade is designed to sense tissue injury by physical separation of the membrane-anchored cofactor tissue factor (TF) from inactive precursors of coagulation proteases circulating in plasma. Once TF on epithelial and other extravascular cells is exposed to plasma, sequential activation of coagulation proteases coordinates hemostasis and contributes to host defense and tissue repair. Membrane-anchored serine proteases (MASPs) play critical roles in the development and homeostasis of epithelial barrier tissues; how MASPs are activated in mature epithelia is unknown. We here report that proteases of the extrinsic pathway of blood coagulation transactivate the MASP matriptase, thus connecting coagulation initiation to epithelial proteolysis and signaling. Exposure of TF-expressing cells to factors (F) VIIa and Xa triggered the conversion of latent pro-matriptase to an active protease, which in turn cleaved the pericellular substrates protease-activated receptor-2 (PAR2) and pro-urokinase. An activation pathway-selective PAR2 mutant resistant to direct cleavage by TF:FVIIa and FXa was activated by these proteases when cells co-expressed pro-matriptase, and matriptase transactivation was necessary for efficient cleavage and activation of wild-type PAR2 by physiological concentrations of TF:FVIIa and FXa. The coagulation initiation complex induced rapid and prolonged enhancement of the barrier function of epithelial monolayers that was dependent on matriptase transactivation and PAR2 signaling. These observations suggest that the coagulation cascade engages matriptase to help coordinate epithelial defense and repair programs after injury or infection, and that matriptase may contribute to TF-driven pathogenesis in cancer and inflammation.


Asunto(s)
Coagulación Sanguínea/fisiología , Células Epiteliales/metabolismo , Proteolisis , Serina Endopeptidasas/metabolismo , Tromboplastina/fisiología , Línea Celular Tumoral , Activación Enzimática , Factor VIIa/metabolismo , Factor Xa/metabolismo , Células HeLa , Humanos , Células MCF-7 , Proteínas Mutantes/metabolismo , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Transducción de Señal
18.
Blood ; 127(9): 1085-96, 2016 Mar 03.
Artículo en Inglés | MEDLINE | ID: mdl-26647393

RESUMEN

Extravascular fibrin deposition accompanies many human diseases and causes chronic inflammation and organ damage, unless removed in a timely manner. Here, we used intravital microscopy to investigate how fibrin is removed from extravascular space. Fibrin placed into the dermis of mice underwent cellular endocytosis and lysosomal targeting, revealing a novel intracellular pathway for extravascular fibrin degradation. A C-C chemokine receptor type 2 (CCR2)-positive macrophage subpopulation constituted the majority of fibrin-uptaking cells. Consequently, cellular fibrin uptake was diminished by elimination of CCR2-expressing cells. The CCR2-positive macrophage subtype was different from collagen-internalizing M2-like macrophages. Cellular fibrin uptake was strictly dependent on plasminogen and plasminogen activator. Surprisingly, however, fibrin endocytosis was unimpeded by the absence of the fibrin(ogen) receptors, αMß2 and ICAM-1, the myeloid cell integrin-binding site on fibrin or the endocytic collagen receptor, the mannose receptor. The study identifies a novel fibrin endocytic pathway engaged in extravascular fibrin clearance and shows that interstitial fibrin and collagen are cleared by different subsets of macrophages employing distinct molecular pathways.


Asunto(s)
Endocitosis , Fibrina/metabolismo , Macrófagos/metabolismo , Receptores CCR2/metabolismo , Animales , Bioensayo , Receptor 1 de Quimiocinas CX3C , Proliferación Celular , Fibrinolisina/metabolismo , Ratones , Células Mieloides/metabolismo , Plasminógeno/metabolismo , Activadores Plasminogénicos/metabolismo , Proteolisis , Receptores de Quimiocina/metabolismo , Receptores de Péptidos/metabolismo
19.
BMC Biol ; 15(1): 46, 2017 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-28571576

RESUMEN

BACKGROUND: Matriptase is a membrane serine protease essential for epithelial development, homeostasis, and regeneration, as well as a central orchestrator of pathogenic pericellular signaling in the context of inflammatory and proliferative diseases. Matriptase is an unusual protease in that its zymogen displays measurable enzymatic activity. RESULTS: Here, we used gain and loss of function genetics to investigate the possible biological functions of zymogen matriptase. Unexpectedly, transgenic mice mis-expressing a zymogen-locked version of matriptase in the epidermis displayed pathologies previously reported for transgenic mice mis-expressing wildtype epidermal matriptase. Equally surprising, mice engineered to express only zymogen-locked endogenous matriptase, unlike matriptase null mice, were viable, developed epithelial barrier function, and regenerated the injured epithelium. Compatible with these observations, wildtype and zymogen-locked matriptase were equipotent activators of PAR-2 inflammatory signaling. CONCLUSION: The study demonstrates that the matriptase zymogen is biologically active and is capable of executing developmental and homeostatic functions of the protease.


Asunto(s)
Precursores Enzimáticos/metabolismo , Epitelio/crecimiento & desarrollo , Homeostasis/genética , Regeneración/genética , Serina Endopeptidasas/metabolismo , Animales , Precursores Enzimáticos/genética , Epitelio/metabolismo , Femenino , Mutación con Ganancia de Función , Expresión Génica , Mutación con Pérdida de Función , Masculino , Ratones Transgénicos , Serina Endopeptidasas/genética
20.
J Biol Chem ; 291(6): 2577-82, 2016 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-26719335

RESUMEN

The membrane-anchored serine prostasin (CAP1/PRSS8) is essential for barrier acquisition of the interfollicular epidermis and for normal hair follicle development. Consequently, prostasin null mice die shortly after birth. Prostasin is found in two forms in the epidermis: a one-chain zymogen and a two-chain proteolytically active form, generated by matriptase-dependent activation site cleavage. Here we used gene editing to generate mice expressing only activation site cleavage-resistant (zymogen-locked) endogenous prostasin. Interestingly, these mutant mice displayed normal interfollicular epidermal development and postnatal survival, but had defects in whisker and pelage hair formation. These findings identify two distinct in vivo functions of epidermal prostasin: a function in the interfollicular epidermis, not requiring activation site cleavage, that can be mediated by the zymogen-locked version of prostasin and a proteolysis-dependent function of activated prostasin in hair follicles, dependent on zymogen conversion by matriptase.


Asunto(s)
Precursores Enzimáticos/metabolismo , Folículo Piloso/enzimología , Serina Endopeptidasas/metabolismo , Animales , Activación Enzimática , Precursores Enzimáticos/genética , Ratones , Serina Endopeptidasas/genética
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