RESUMO
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.
Assuntos
Lectinas Tipo C , Lesão Pulmonar , Receptor de Manose , Lectinas de Ligação a Manose , Proteômica , Receptores de Superfície Celular , Trombospondinas , Animais , Camundongos , Células CHO , Cricetulus , Endocitose , Lectinas Tipo C/metabolismo , Lectinas Tipo C/genética , Ligantes , Lipopolissacarídeos/toxicidade , Pulmão/metabolismo , Pulmão/patologia , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Lectinas de Ligação a Manose/metabolismo , Lectinas de Ligação a Manose/genética , Camundongos Knockout , Proteômica/métodos , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/genética , Trombospondinas/metabolismo , Trombospondinas/genéticaRESUMO
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.
Assuntos
Insuficiência Intestinal , Animais , Camundongos , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Intestinos , Proteínas Secretadas Inibidoras de ProteinasesRESUMO
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.
Assuntos
Células Endoteliais , Neoplasias , Camundongos , Animais , Células Endoteliais/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Transdução de Sinais , Antígenos de Bactérias/metabolismo , Neoplasias/genética , Microambiente TumoralRESUMO
FDA-approved BRAF and MEK small molecule inhibitors have demonstrated some level of efficacy in patients with metastatic melanomas. However, these "targeted" therapeutics have a very low therapeutic index, since these agents affect normal cells, causing undesirable, even fatal, side effects. To address these significant drawbacks, here, we have reengineered the anthrax toxin-based protein delivery system to develop a potent, tumor-selective MEK inactivator. This toxin-based MEK inactivator exhibits potent activity against a wide range of solid tumors, with the highest activity seen when directed toward tumors containing the BRAFV600E mutation. We demonstrate that this reengineered MEK inactivator also exhibits an extremely high therapeutic index (>15), due to its in vitro and in vivo activity being strictly dependent on the expression of multiple tumor-associated factors including tumor-associated proteases matrix metalloproteinase, urokinase plasminogen activator, and anthrax toxin receptor capillary morphogenesis protein-2. Furthermore, we have improved the specificity of this MEK inactivator, restricting its enzymatic activity to only target the ERK pathway, thereby greatly diminishing off-target toxicity. Together, these data suggest that engineered bacterial toxins can be modified to have significant in vitro and in vivo therapeutic effects with high therapeutic index.
RESUMO
Epithelial cell adhesion molecule (EPCAM) is a transmembrane glycoprotein expressed on the surface of most epithelial and epithelium-derived tumor cells and reported to regulate stability of epithelial tight junction proteins, claudins. Despite its widespread expression, loss of EPCAM function has so far only been reported to prominently affect intestinal development, resulting in severe early onset enteropathy associated with impaired growth and decreased survival in both humans and mice. In this study, we show that the critical role of EPCAM is not limited to intestinal tissues and that it shares its essential function with its only known homolog, Trophoblast cell surface antigen 2 (TROP2). EPCAM-deficient mice show significant growth retardation and die within 4 weeks after birth. In addition to changes in small and large intestines, loss of EPCAM results in hyperkeratosis in the skin and forestomach, hair follicle atrophy leading to alopecia, nephron hypoplasia in the kidney, proteinuria, and altered production of digestive enzymes by the pancreas. Expression of TROP2 partially, but not completely, overlaps with EPCAM in a number developing epithelia. Although loss of TROP2 had no gross impact on mouse development and survival, TROP2 deficiency generally compounded developmental defects observed in EPCAM-deficient mice, led to an approximately 60% decrease in embryonic viability, and further shortened postnatal lifespan of born pups. Importantly, TROP2 was able to compensate for the loss of EPCAM in stabilizing claudin-7 expression and cell membrane localization in tissues that co-express both proteins. These findings identify overlapping functions of EPCAM and TROP2 as regulators of epithelial development in both intestinal and extraintestinal tissues.
Assuntos
Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Claudinas , Intestinos , Animais , Claudinas/genética , Claudinas/metabolismo , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Epitélio/metabolismo , CamundongosRESUMO
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.
Assuntos
Antraz , Antígenos de Bactérias , Bacillus anthracis , Toxinas Bacterianas , Animais , Antraz/diagnóstico por imagem , Antraz/metabolismo , Antígenos de Bactérias/química , Antígenos de Bactérias/toxicidade , Bacillus anthracis/metabolismo , Toxinas Bacterianas/toxicidade , Citoplasma/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
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.
Assuntos
Afibrinogenemia/metabolismo , Fibrina/biossíntese , Fibrinogênio/biossíntese , Técnicas de Silenciamento de Genes , Lipossomos/farmacologia , RNA Interferente Pequeno , Afibrinogenemia/genética , Animais , Plaquetas/metabolismo , Modelos Animais de Doenças , Feminino , Fibrina/genética , Fibrinogênio/genética , Humanos , Masculino , Camundongos , Nanopartículas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologiaRESUMO
BACKGROUND: Oral squamous cell carcinoma (OSCC) remains a challenging cancer to treat despite all the advances of the last 50 years. Kallikrein 5 (KLK5) is among the serine proteases implicated in OSCC development. However, whether the activity of KLK5 promotes carcinogenesis is still controversial. Moreover, knowledge regarding the role of the KLK5 cognate inhibitor, Lympho-Epithelial Kazal-Type related Inhibitor (LEKTI), in OSCC is scarce. We have, thus, sought to investigate the importance of KLK5 and LEKTI expression in premalignant and malignant lesions of the oral cavity. METHODS: KLK5 and LEKTI protein expression was evaluated in 301 human samples, which were comprised of non-malignant and malignant lesions of the oral cavity. Moreover, a bioinformatic analysis of the overall survival rate from 517 head and neck squamous cell carcinoma (HNSCC) samples was performed. Additionally, to mimic the uncovered KLK5 to serine peptidase inhibitor (SPINK5) imbalance, the KLK5 gene was abrogated in an OSCC cell line using CRISPR-Cas9 technology. The generated cell line was then used for in vivo and in vitro carcinogenesis related experiments. RESULTS: LEKTI was found to be statistically downregulated in OSCCs, with increased KLK5/SPINK5 mRNA ratio being associated with a shorter overall survival (pâ¯=â¯0.091). Indeed, disruption of KLK5 to SPINK5 balance through the generation of KLK5 null OSCC cells led to smaller xenografted tumors and statistically decreased proliferation rates following multiple time points of BrdU treatment in vitro. CONCLUSION: The association of increased enzyme/inhibitor ratio with poor prognosis indicates KLK5 to SPINK5 relative expression as an important prognostic marker in OSCC.
RESUMO
The membrane-anchored matrix metalloprotease MT1-MMP is a potent collagenolytic enzyme with a well-established role in extracellular matrix turnover and cellular invasion into collagen-rich tissues. MT1-MMP is highly expressed in various types of cancer and has been demonstrated to be directly involved in several stages of tumor progression, including primary tumor growth, angiogenesis, invasion and metastasis. Osteosarcoma is the most common type of primary bone cancer. This disease is characterized by invasive tumor growth, leading to extensive bone destruction, and metastasis to the lungs. The tumor cells in human osteosarcoma display a strong expression of MT1-MMP, but the role of MT1-MMP in osteosarcoma progression is currently unknown. In this study, we investigated the role of MT1-MMP during various stages of osteosarcoma development. We utilized an optimized orthotopic murine osteosarcoma model and human osteosarcoma cells in which the MT1-MMP gene was knocked out using CRISPR/Cas9. We observed a strong expression of MT1-MMP in wildtype cells of both primary tumors and lung metastases, but, surprisingly, MT1-MMP deficiency did not affect primary tumor growth, bone degradation or the formation and growth of lung metastases. We therefore propose that, unlike findings reported in other cancers, tumor-expressed MT1-MMP is dispensable for all stages of osteosarcoma progression.
Assuntos
Neoplasias Ósseas/genética , Osso e Ossos/patologia , Proliferação de Células/genética , Neoplasias Pulmonares/genética , Metaloproteinase 14 da Matriz/genética , Osteossarcoma/genética , Animais , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Osso e Ossos/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Progressão da Doença , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Metaloproteinase 14 da Matriz/metabolismo , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Osteossarcoma/metabolismo , Osteossarcoma/secundárioRESUMO
Anthrax lethal toxin (LT), produced by Bacillus anthracis, comprises a receptor-binding moiety, protective antigen and the lethal factor (LF) protease1,2. Although LF is known to cleave mitogen-activated protein kinase kinases (MEKs/MKKs) and some variants of the NLRP1 inflammasome sensor, targeting of these pathways does not explain the lethality of anthrax toxin1,2. Here we report that the regulatory subunits of phosphoinositide-3 kinase (PI3K)-p85α (PIK3R1) and p85ß (PIK3R2)3,4-are substrates of LF. Cleavage of these proteins in a proline-rich region between their N-terminal Src homology and Bcr homology domains disrupts homodimer formation and impacts PI3K signalling. Mice carrying a mutated p85α that cannot be cleaved by LF show a greater resistance to anthrax toxin challenge. The LF(W271A) mutant cleaves p85α with lower efficiency and is non-toxic to mice but can regain lethality when combined with PI3K pathway inhibitors. We provide evidence that LF targets two signalling pathways that are essential for growth and metabolism and that the disabling of both pathways is likely necessary for lethal anthrax infection.
Assuntos
Antraz/enzimologia , Antígenos de Bactérias/metabolismo , Antígenos de Bactérias/toxicidade , Bacillus anthracis/enzimologia , Bacillus anthracis/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Peptídeo Hidrolases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Motivos de Aminoácidos , Animais , Antraz/genética , Antraz/microbiologia , Classe Ia de Fosfatidilinositol 3-Quinase/química , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeo Hidrolases/genética , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genéticaRESUMO
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.
Assuntos
Membrana Celular/enzimologia , Células Epiteliais/enzimologia , Serina Proteases/metabolismo , Transdução de Sinais , Animais , Adesão Celular , Matriz Extracelular/metabolismo , Humanos , Íons , Proteínas de Membrana/metabolismo , Camundongos , Serina Endopeptidases/metabolismoRESUMO
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.
Assuntos
Colágeno/imunologia , Animais , Endocitose/imunologia , Matriz Extracelular/imunologia , Fibrose/imunologia , Humanos , Neoplasias/imunologiaRESUMO
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.
Assuntos
Diarreia Infantil/genética , Diarreia Infantil/patologia , Intestinos/patologia , Síndromes de Malabsorção/genética , Síndromes de Malabsorção/patologia , Proteínas de Membrana/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Animais , Claudinas/metabolismo , Cruzamentos Genéticos , Modelos Animais de Doenças , Molécula de Adesão da Célula Epitelial/metabolismo , Epitélio/metabolismo , Feminino , Genótipo , Hemorragia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , FenótipoRESUMO
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.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Família Multigênica , Serina Endopeptidases/genética , Animais , Desenvolvimento Embrionário/genética , Epiderme/crescimento & desenvolvimento , Epiderme/metabolismo , Feminino , Fertilidade/genética , Células HEK293 , Humanos , Masculino , Camundongos , Serina Endopeptidases/metabolismoRESUMO
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.
Assuntos
Quimiotaxia de Leucócito , Fibrinolisina/metabolismo , Fibrinólise , Inflamação/etiologia , Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Animais , Biomarcadores , Quimiotaxia de Leucócito/imunologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Fibrinogênio/genética , Fibrinogênio/metabolismo , Imunofluorescência , Humanos , Imunofenotipagem , Inflamação/patologia , Contagem de Leucócitos , Camundongos , Camundongos Knockout , Plasminogênio/deficiência , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Células RAW 264.7RESUMO
Increased activity of matrix metalloproteinases (MMPs) is associated with worse prognosis in different cancer types. The wild-type protective antigen (PA-WT) of the binary anthrax lethal toxin was modified to form a pore in cell membranes only when cleaved by MMPs (to form PA-L1). Anthrax lethal factor (LF) is then able to translocate through these pores. Here, we used a 111In-radiolabeled form of LF with the PA/LF system for noninvasive in vivo imaging of MMP activity in tumor tissue by SPECT. Methods: MMP-mediated activation of PA-L1 was correlated to anthrax receptor expression and MMP activity in a panel of cancer cells (HT1080, MDA-MB-231, B8484, and MCF7). Uptake of 111In-radiolabeled PA-L1, 111In-PA-WTK563C, or 111In-LFE687A (a catalytically inactive LF mutant) in tumor and normal tissues was measured using SPECT/CT imaging in vivo. Results: Activation of PA-L1 in vitro correlated with anthrax receptor expression and MMP activity (HT1080 > MDA-MB-231 > B8484 > MCF7). PA-L1-mediated delivery of 111In-LFE687A was demonstrated and was corroborated using confocal microscopy with fluorescently labeled LFE687A Uptake was blocked by the broad-spectrum MMP inhibitor GM6001. In vivo imaging showed selective accumulation of 111In-PA-L1 in MDA-MB-231 tumor xenografts (5.7 ± 0.9 percentage injected dose [%ID]/g) at 3 h after intravenous administration. 111In-LFE687A was selectively delivered to MMP-positive MDA-MB-231 tumor tissue by MMP-activatable PA-L1 (5.98 ± 0.62 %ID/g) but not by furin-cleavable PA-WT (1.05 ± 0.21 %ID/g) or a noncleavable PA variant control, PA-U7 (2.74 ± 0.24 %ID/g). Conclusion: Taken together, our results indicate that radiolabeled forms of mutated anthrax lethal toxin hold promise for noninvasive imaging of MMP activity in tumor tissue.
Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Radioisótopos de Índio/química , Neoplasias/diagnóstico por imagem , Tomografia Computadorizada de Emissão de Fóton Único , Animais , Transporte Biológico , Linhagem Celular Tumoral , Humanos , Cinética , Células MCF-7 , Metaloproteinase 14 da Matriz/química , Metaloproteinase 2 da Matriz/química , Metaloproteinases da Matriz/metabolismo , Camundongos , Mutação , Transplante de NeoplasiasRESUMO
In recent years, anthrax toxin has been reengineered to act as a highly specific antiangiogenic cancer therapeutic, shown to kill tumors in animal models. This has been achieved by modifying protective antigen (PA) so that its activation and toxicity require the presence of two proteases, matrix metalloproteinase (MMP) and urokinase plasminogen activator (uPA), which are upregulated in tumor microenvironments. These therapeutics consist of intercomplementing PA variants, which are individually nontoxic, but form functional toxins upon complementary oligomerization. Here, we have created a dual-protease requiring PA targeting system which utilizes bismaleimide cross-linked PA (CLPA) rather than the intercomplementing PA variants. Three different CLPA agents were tested and, as expected, found to exclusively form octamers. Two of the CLPA agents have in vitro toxicities equal to those of previous intercomplementing agents, while the third CLPA agent had compromised in vitro cleavage and was significantly less cytotoxic. We hypothesize this difference was due to steric hindrance caused by cross-linking two PA monomers in close proximity to the PA cleavage site. Overall, this work advances the development and use of the PA and LF tumor-targeting system as a practical cancer therapeutic, as it provides a way to reduce the drug components of the anthrax toxin drug delivery system from three to two, which may lower the cost and simplify testing in clinical trials. HIGHLIGHT: Previously, anthrax toxin has been reengineered to act as a highly specific antiangiogenic cancer therapeutic. Here, we present a version, which utilizes bismaleimide cross-linked protective antigen (PA) rather than intercomplementing PA variants. This advances the development of anthrax toxin as a practical cancer therapeutic as it reduces the components of the drug delivery system to two, which may lower the cost and simplify testing in clinical trials.
Assuntos
Antígenos de Bactérias/farmacologia , Antineoplásicos/farmacologia , Toxinas Bacterianas/farmacologia , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antineoplásicos/química , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células HT29 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Células RAW 264.7RESUMO
Collectins such as mannose-binding lectin (MBL) and surfactant protein D (SP-D) become temporarily deposited in extravascular compartments after tissue injury and perform immune-stimulatory or inflammation-limiting functions. However, their turnover mechanisms, necessary to prevent excessive tissue damage, are virtually unknown. In this study, we show that fibroblasts in injured tissues undertake the clearance of collectins by using the endocytic collagen receptor uPARAP. In cellular assays, several types of collectins were endocytosed in a highly specific uPARAP-dependent process, not shared by the closely related receptor MR/CD206. When introduced into dermis or bleomycin-injured lungs of mice, collectins MBL and SP-D were endocytosed and routed for lysosomal degradation by uPARAP-positive fibroblasts. Fibroblast-specific expression of uPARAP governed endogenous SP-D levels and overall survival after lung injury. In lung tissue from idiopathic pulmonary fibrosis patients, a strong up-regulation of uPARAP was observed in fibroblasts adjacent to regions with SP-D secretion. This study demonstrates a novel immune-regulatory function of fibroblasts and identifies uPARAP as an endocytic receptor in immunity.
Assuntos
Fibroblastos/imunologia , Lesão Pulmonar/imunologia , Lectina de Ligação a Manose/imunologia , Lectinas de Ligação a Manose/imunologia , Glicoproteínas de Membrana/imunologia , Fibrose Pulmonar/imunologia , Proteína D Associada a Surfactante Pulmonar/imunologia , Receptores de Superfície Celular/imunologia , Animais , Bleomicina/administração & dosagem , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Endocitose , Fibroblastos/patologia , Expressão Gênica , Humanos , Imunidade Inata , Interleucina-6/genética , Interleucina-6/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Pulmão/imunologia , Pulmão/patologia , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/genética , Lesão Pulmonar/mortalidade , Lisossomos/imunologia , Lisossomos/metabolismo , Receptor de Manose , Lectina de Ligação a Manose/genética , Lectinas de Ligação a Manose/genética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteólise , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/genética , Fibrose Pulmonar/mortalidade , Proteína D Associada a Surfactante Pulmonar/genética , Receptores de Superfície Celular/genética , Análise de SobrevidaRESUMO
Prostasin (CAP1/PRSS8) is a glycosylphosphatidylinositol (GPI)-anchored serine protease that is essential for epithelial development and overall survival in mice. Prostasin is regulated primarily by the transmembrane serine protease inhibitor, hepatocyte growth factor activator inhibitor (HAI)-2, and loss of HAI-2 function leads to early embryonic lethality in mice due to an unregulated prostasin activity. We have recently reported that critical in vivo functions of prostasin can be performed by proteolytically-inactive or zymogen-locked variants of the protease. Here we show that the zymogen form of prostasin does not bind to HAI-2 and, as a result, loss of HAI-2 does not affect prenatal development and survival of mice expressing only zymogen-locked variant of prostasin (Prss8 R44Q). Indeed, HAI-2-deficient mice homozygous for R44Q mutation (Spint2-/-;Prss8R44Q/R44Q) are born in the expected numbers and do not exhibit any obvious developmental abnormality at birth. However, postnatal growth in these mice is severely impaired and they all die within 4 to 7 days after birth due to a critical failure in the development of small and large intestines, characterized by a widespread villous atrophy, tufted villi, near-complete loss of mucin-producing goblet cells, loss of colonic crypt structure, and bleeding into the intestinal lumen. Intestines of Spint2-/-;Prss8R44Q/R44Q mice showed altered expression of epithelial junctional proteins, including reduced levels of EpCAM, E-cadherin, occludin, claudin-1 and -7, as well as an increased level of claudin-4, indicating that the loss of HAI-2 compromises intestinal epithelial barrier function. Our data indicate that the loss of HAI-2 in Prss8R44Q/R44Q mice leads to development of progressive intestinal failure that at both histological and molecular level bears a striking resemblance to human congenital tufting enteropathy, and may provide important clues for understanding and treating this debilitating human disease.
Assuntos
Diarreia Infantil/patologia , Síndromes de Malabsorção/patologia , Proteínas de Membrana/genética , Serina Endopeptidases/metabolismo , Animais , Claudina-1/metabolismo , Diarreia Infantil/metabolismo , Diarreia Infantil/veterinária , Regulação para Baixo , Desenvolvimento Embrionário , Precursores Enzimáticos/metabolismo , Molécula de Adesão da Célula Epitelial/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/crescimento & desenvolvimento , Intestinos/patologia , Síndromes de Malabsorção/metabolismo , Síndromes de Malabsorção/veterinária , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Ocludina/metabolismo , Ligação ProteicaRESUMO
Anthrax lethal toxin is a typical A-B type protein toxin secreted by Bacillus anthracis. Lethal factor (LF) is the catalytic A-subunit, a metalloprotease having MEKs as targets. LF relies on the cell-binding B-subunit, protective antigen (PA), to gain entry into the cytosol of target cells. PA binds to cell surface toxin receptors and is activated by furin protease to form an LF-binding-competent oligomer-PA pre-pore, which converts to a functional protein-conductive pore in the acidic endocytic vesicles, allowing translocation of LF into the cytosol. During PA pre-pore-to-pore conversion, the intermolecular salt bridge interactions between Lys397 and Asp426 on adjacent PA protomers play a critical role in positioning neighboring luminal Phe427 residues to form the Phe-clamp, an essential element of the PA functional pore. This essential intermolecular interaction affords the opportunity to create pairs of PA variants that depend on intermolecular complementation to form a functional pore. We have previously generated PA variants with furin-cleavage site replaced by substrate sequences of tumor-associated proteases, such as urokinase or MMPs. Here we show that PA-U2-K397Q, a urokinase-activated PA variant with Lys397 residue replaced by glutamine, and PA-L1-D426K, a MMP-activated PA variant with Asp426 changed to lysine, do not form functional pores both in vitro or in vivo unless they are used together. Further, the mixture of PA-U2-K397Q and PA-L1-D426K displayed potent anti-tumor activity in the presence of LF. Thus, PA-U2-K397Q and PA-L1-D426K form a novel intermolecular complementation system with toxin activation relying on the presence of two distinct tumor-associated proteases, i.e., urokinase and MMPs.