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1.
JCI Insight ; 52019 08 08.
Artículo en Inglés | MEDLINE | ID: mdl-31393853

RESUMEN

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease. A maladaptive epithelium due to chronic injury is a prominent feature and contributor to pathogenic cellular communication in IPF. Recent data highlight the concept of a "reprogrammed" lung epithelium as critical in the development of lung fibrosis. Extracellular vesicles (EVs) are potent mediator of cellular crosstalk, and recent evidence supports their role in lung pathologies such as IPF. Here, we demonstrate that syndecan-1 is overexpressed by the epithelium in the lungs of IPF patients and in murine models after bleomycin injury. Moreover, we find that syndecan-1 is a pro-fibrotic signal that alters alveolar type II (ATII) cell phenotypes by augmenting TGFß and Wnt signaling among other pro-fibrotic pathways. Importantly, we demonstrate that syndecan-1 controls the packaging of several anti-fibrotic microRNAs into EVs that have broad effects over several fibrogenic signaling networks as a mechanism of regulating epithelial plasticity and pulmonary fibrosis. Collectively, our work reveals new insight into how EVs orchestrate cellular signals that promote lung fibrosis and demonstrate the importance of syndecan-1 in coordinating these programs.


Asunto(s)
Células Epiteliales Alveolares/metabolismo , Vesículas Extracelulares/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Sindecano-1/metabolismo , Células Epiteliales Alveolares/patología , Animales , Bleomicina/efectos adversos , Línea Celular , Modelos Animales de Enfermedad , Vesículas Extracelulares/patología , Femenino , Humanos , Fibrosis Pulmonar Idiopática/inducido químicamente , Fibrosis Pulmonar Idiopática/patología , Pulmón/patología , Lesión Pulmonar/inducido químicamente , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , MicroARNs/metabolismo , Transducción de Señal , Sindecano-1/genética , Transcriptoma , Factor de Crecimiento Transformador beta/metabolismo
2.
Lung ; 196(6): 737-743, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30167842

RESUMEN

INTRODUCTION: Influenza infects millions of people each year causing respiratory distress and death in severe cases. On average, 200,000 people annually are hospitalized in the United States for influenza related complications. Tissue inhibitor of metalloproteinase-1 (TIMP-1), a secreted protein that inhibits MMPs, has been found to be involved in lung inflammation. Here, we evaluated the role of TIMP-1 in the host response to influenza-induced lung injury. METHODS: Wild-type (WT) and Timp1-deficient (Timp1-/-) mice that were 8-12 weeks old were administered A/PR/8/34 (PR8), a murine adapted H1N1 influenza virus, and euthanized 6 days after influenza installation. Bronchoalveolar lavage fluid and lungs were harvested from each mouse for ELISA, protein assay, PCR, and histological analysis. Cytospins were executed on bronchoalveolar lavage fluid to identify immune cells based on morphology and cell count. RESULTS: WT mice experienced significantly more weight loss compared to Timp1-/- mice after influenza infection. WT mice demonstrated more immune cell infiltrate and airway inflammation. Interestingly, PR8 levels were identical between the WT and Timp1-/- mice 6 days post-influenza infection. CONCLUSION: The data suggest that Timp1 promotes the immune response in the lungs after influenza infection facilitating an injurious phenotype as a result of influenza infection.


Asunto(s)
Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/metabolismo , Hemorragia/virología , Subtipo H1N1 del Virus de la Influenza A , Infecciones por Orthomyxoviridae/complicaciones , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Lesión Pulmonar Aguda/patología , Lesión Pulmonar Aguda/virología , Animales , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Modelos Animales de Enfermedad , Recuento de Eritrocitos , Eritrocitos , Hemorragia/genética , Recuento de Leucocitos , Macrófagos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos , Infecciones por Orthomyxoviridae/virología , Carga Viral/genética , Pérdida de Peso/genética
3.
Am J Pathol ; 188(4): 1094-1103, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29355516

RESUMEN

Syndecan-1 is a transmembrane proteoglycan expressed prominently by lung epithelium and has pleiotropic functions such as regulating cell migration, proliferation, and survival. Loss of syndecan-1 expression by lung cancer cells is associated with higher-grade cancers and worse clinical prognosis. We evaluated the effects of syndecan-1 in various cell-based and animal models of lung cancer and found that lung tumorigenesis was moderated by syndecan-1. We also demonstrate that syndecan-1 (or lack thereof) alters the miRNA cargo carried within exosomes exported from lung cancer cells. Analysis of the changes in miRNA expression identified a distinct shift toward augmented procancer signaling consistent with the changes found in lung adenocarcinoma. Collectively, our work identifies syndecan-1 as an important factor in lung cancer cells that shapes the tumor microenvironment through alterations in miRNA packaging within exosomes.


Asunto(s)
Carcinogénesis/metabolismo , Exosomas/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/metabolismo , MicroARNs/genética , Sindecano-1/metabolismo , Células A549 , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Animales , Proliferación Celular , Regulación hacia Abajo/genética , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/patología , Ratones , MicroARNs/metabolismo , Análisis de Supervivencia , Regulación hacia Arriba/genética
4.
Am J Physiol Lung Cell Mol Physiol ; 312(4): L556-L567, 2017 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-28188224

RESUMEN

Pericytes are perivascular PDGF receptor-ß+ (PDGFRß+) stromal cells required for vasculogenesis and maintenance of microvascular homeostasis in many organs. Because of their unique juxtaposition to microvascular endothelium, lung PDGFRß+ cells are well situated to detect proinflammatory molecules released following epithelial injury and promote acute inflammatory responses. Thus we hypothesized that these cells represent an unrecognized immune surveillance or injury-sentinel interstitial cell. To evaluate this hypothesis, we isolated PDGFRß+ cells from murine lung and demonstrated that they have characteristics consistent with a pericyte population (referred to as pericyte-like cells for simplicity hereafter). We showed that pericyte-like cells expressed functional Toll-like receptors and upregulated chemokine expression following exposure to bronchoalveolar lavage fluid (BALF) collected from mice with sterile lung injury. Interestingly, BALF from mice without lung injury also induced chemokine expression in pericyte-like cells, suggesting that pericyte-like cells are primed to sense epithelial injury (permeability changes). Following LPS-induced lung inflammation, increased numbers of pericyte-like cells expressed IL-6, chemokine (C-X-C motif) ligand-1, chemokine (C-C motif) ligand 2/ monocyte chemotactic protein-1, and ICAM-1 in vivo. Sterile lung injury in pericyte-ablated mice was associated with decreased inflammation compared with normal mice. In summary, we found that pericyte-like cells are immune responsive and express diverse chemokines in response to lung injury in vitro and in vivo. Furthermore, pericyte-like cell ablation attenuated inflammation in sterile lung injury, suggesting that these cells play an important functional role in mediating lung inflammatory responses. We propose a model in which pericyte-like cells function as interstitial immune sentinels, detecting proinflammatory molecules released following epithelial barrier damage and participating in recruitment of circulating leukocytes.


Asunto(s)
Sistema Inmunológico/citología , Pulmón/citología , Pericitos/citología , Animales , Líquido del Lavado Bronquioalveolar , Células Cultivadas , Inflamación/patología , Mediadores de Inflamación/metabolismo , Lesión Pulmonar/patología , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Pericitos/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Células del Estroma/metabolismo
5.
JCI Insight ; 1(20): e90301, 2016 12 08.
Artículo en Inglés | MEDLINE | ID: mdl-27942594

RESUMEN

Maladaptive epithelial repair from chronic injury is a common feature in fibrotic diseases, which in turn activates a pathogenic fibroblast response that produces excessive matrix deposition. Dysregulated microRNAs (miRs) can regulate expression of multiple genes and fundamentally alter cellular phenotypes during fibrosis. Although several miRs have been shown to be associated with lung fibrosis, the mechanisms by which miRs modulate epithelial behavior in lung fibrosis are lacking. Here, we identified miR-323a-3p to be downregulated in the epithelium of lungs with bronchiolitis obliterans syndrome (BOS) after lung transplantation, idiopathic pulmonary fibrosis (IPF), and murine bleomycin-induced fibrosis. Antagomirs for miR-323a-3p augment, and mimics suppress, murine lung fibrosis after bleomycin injury, indicating that this miR may govern profibrotic signals. We demonstrate that miR-323a-3p attenuates TGF-α and TGF-ß signaling by directly targeting key adaptors in these important fibrogenic pathways. Moreover, miR-323a-3p lowers caspase-3 expression, thereby limiting programmed cell death from inducers of apoptosis and ER stress. Finally, we find that epithelial expression of miR-323a-3p modulates inhibitory crosstalk with fibroblasts. These studies demonstrate that miR-323a-3p has a central role in lung fibrosis that spans across murine and human disease, and downregulated expression by the lung epithelium releases inhibition of various profibrotic pathways to promote fibroproliferation.


Asunto(s)
Fibrosis Pulmonar Idiopática/genética , MicroARNs/genética , Mucosa Respiratoria/fisiopatología , Animales , Bleomicina , Bronquiolitis Obliterante/genética , Bronquiolitis Obliterante/patología , Células Cultivadas , Fibroblastos/citología , Humanos , Fibrosis Pulmonar Idiopática/patología , Pulmón , Trasplante de Pulmón , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Factores de Crecimiento Transformadores/metabolismo
6.
J Clin Invest ; 126(4): 1245-7, 2016 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-26999598

RESUMEN

Influenza infection can cause acute respiratory distress syndrome (ARDS), leading to poor disease outcome with high mortality. One of the driving features in the pathogenesis of ARDS is the accumulation of fluid in the alveoli, which causes severe pulmonary edema and impaired oxygen uptake. In this issue of the JCI, Peteranderl and colleagues define a paracrine communication between macrophages and type II alveolar epithelial cells during influenza infection where IFNα induces macrophage secretion of TRAIL that causes endocytosis of Na,K-ATPase by the alveolar epithelium. This reduction of Na,K-ATPase expression decreases alveolar fluid clearance, which in turn leads to pulmonary edema. Inhibition of the TRAIL signaling pathway has been shown to improve lung injury after influenza infection, and future studies will be needed to determine if blocking this pathway is a viable option in the treatment of ARDS.


Asunto(s)
Virus de la Influenza A/inmunología , Interferón Tipo I/inmunología , Macrófagos Alveolares/inmunología , Infecciones por Orthomyxoviridae/inmunología , Comunicación Paracrina/inmunología , Edema Pulmonar/inmunología , Mucosa Respiratoria/inmunología , Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Animales , Humanos
7.
Am J Respir Crit Care Med ; 194(3): 333-44, 2016 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-26959387

RESUMEN

RATIONALE: Syndecan-1 is a cell surface heparan sulfate proteoglycan primarily expressed in the lung epithelium. Because the influenza virus is tropic to the airway epithelium, we investigated the role of syndecan-1 in influenza infection. OBJECTIVES: To determine the mechanism by which syndecan-1 regulates the lung mucosal response to influenza infection. METHODS: Wild-type (WT) and Sdc1(-/-) mice were infected with a H1N1 virus (PR8) as an experimental model of influenza infection. Human and murine airway epithelial cell cultures were also infected with PR8 to study the mechanism by which syndecan-1 regulates the inflammatory response. MEASUREMENT AND MAIN RESULTS: We found worsened outcomes and lung injury in Sdc1(-/-) mice compared with WT mice after influenza infection. Our data demonstrated that syndecan-1 suppresses bronchial epithelial apoptosis during influenza infection to limit widespread lung inflammation. Furthermore, we determined that syndecan-1 attenuated apoptosis by crosstalking with c-Met to potentiate its cytoprotective signals in airway epithelial cells during influenza infection. CONCLUSIONS: Our work shows that cell-associated syndecan-1 has an important role in regulating lung injury. Our findings demonstrate a novel mechanism in which cell membrane-associated syndecan-1 regulates the innate immune response to influenza infection by facilitating cytoprotective signals through c-Met signaling to limit bronchial epithelial apoptosis, thereby attenuating lung injury and inflammation.


Asunto(s)
Apoptosis/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Lesión Pulmonar/prevención & control , Infecciones por Orthomyxoviridae/inmunología , Proteínas Proto-Oncogénicas c-met/inmunología , Transducción de Señal/inmunología , Sindecano-1/farmacología , Animales , Modelos Animales de Enfermedad , Células Epiteliales/inmunología , Humanos , Inmunidad Innata/inmunología , Pulmón/inmunología , Lesión Pulmonar/inmunología , Ratones , Proteínas Proto-Oncogénicas c-met/genética , Sindecano-1/inmunología
8.
J Vis Exp ; (97)2015 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-25867050

RESUMEN

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 °C and 55-60% humidity for 10-11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 °C, the eggs are chilled for at least 4 hr at 4 °C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 °C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.


Asunto(s)
Embrión de Pollo/virología , Subtipo H1N1 del Virus de la Influenza A/fisiología , Animales , Pollos , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Replicación Viral
9.
Am J Respir Cell Mol Biol ; 48(3): 390-6, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23258229

RESUMEN

Matrix metalloproteinase-7 (MMP7) expression is quickly up-regulated after injury, and functions to regulate wound repair and various mucosal immune processes. We evaluated the global transcriptional response of airway epithelial cells from wild-type and Mmp7-null mice cultured at an air-liquid interface. The analysis of differentially expressed genes between genotypes after injury revealed an enrichment of functional categories associated with inflammation, cilia, and differentiation. Because these analyses suggested that MMP7 regulated ciliated cell formation, we evaluated the recovery of the airway epithelium in wild-type and Mmp7-null mice in vivo after naphthalene injury, which revealed augmented ciliated cell formation in the absence of MMP7. Moreover, in vitro studies evaluating cell differentiation in air-liquid interface cultures also showed faster ciliated cell production under Mmp7-null conditions compared with wild-type conditions. These studies identified a new role for MMP7 in attenuating ciliated cell differentiation during wound repair.


Asunto(s)
Células Epiteliales/patología , Metaloproteinasa 7 de la Matriz/metabolismo , Mucosa Respiratoria/lesiones , Mucosa Respiratoria/inervación , Mucosa Respiratoria/fisiopatología , Cicatrización de Heridas/genética , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Células Epiteliales/enzimología , Expresión Génica , Genotipo , Lesión Pulmonar/enzimología , Lesión Pulmonar/genética , Lesión Pulmonar/fisiopatología , Masculino , Metaloproteinasa 7 de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Mucosa Respiratoria/enzimología , Mucosa Respiratoria/patología , Transcripción Genética , Transcriptoma , Regulación hacia Arriba , Cicatrización de Heridas/fisiología
10.
J Biol Chem ; 287(42): 34927-34935, 2012 Oct 12.
Artículo en Inglés | MEDLINE | ID: mdl-22936802

RESUMEN

Syndecan-1 is a cell surface proteoglycan that can organize co-receptors into a multimeric complex to transduce intracellular signals. The syndecan-1 core protein has multiple domains that confer distinct cell- and tissue-specific functions. Indeed, the extracellular, transmembrane, and cytoplasmic domains have all been found to regulate specific cellular processes. Our previous work demonstrated that syndecan-1 controls lung epithelial migration and adhesion. Here, we identified the necessary domains of the syndecan-1 core protein that modulate its function in lung epithelial repair. We found that the syndecan-1 transmembrane domain has a regulatory function in controlling focal adhesion disassembly, which in turn controls cell migration speed. In contrast, the extracellular domain facilitates cell adhesion through affinity modulation of α(2)ß(1) integrin. These findings highlight the fact that syndecan-1 is a multidimensional cell surface receptor that has several regulatory domains to control various biological processes. In particular, the lung epithelium requires the syndecan-1 transmembrane domain to govern cell migration and is independent from its ability to control cell adhesion via the extracellular domain.


Asunto(s)
Movimiento Celular/fisiología , Células Epiteliales/metabolismo , Mucosa Respiratoria/metabolismo , Sindecano-1/metabolismo , Animales , Adhesión Celular/fisiología , Línea Celular Transformada , Células Epiteliales/citología , Adhesiones Focales/genética , Adhesiones Focales/metabolismo , Humanos , Integrina alfa2beta1/genética , Integrina alfa2beta1/metabolismo , Ratones , Estructura Terciaria de Proteína , Mucosa Respiratoria/citología , Sindecano-1/genética
11.
Exp Dermatol ; 20(11): 905-10, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21899598

RESUMEN

A balanced proteolytic activity in the epidermis is vital to maintain epidermal homoeostasis and barrier function. Distinct protease-inhibitor systems are operating in different epidermal layers. In the uppermost layer, the stratum corneum, kallikrein-like proteases and their inhibitors are responsible for desquamation of the cornified keratinocytes, thus regulating the integrity of the epidermal barrier. Following discovery and characterisation of the human multidomain inhibitor LEKTI (lympho-epithelial Kazal-type-related inhibitor, encoded by hspink5), several new members of the Kazal-type inhibitor family have been identified. Here we describe expression and regulation of murine SPINK12, a potential orthologue of human LEKTI2. Its expression was analysed by RT-PCR and immunohistochemistry revealing organ-specific pattern with high level of expression in the epidermis and several epithelia including the stomach, kidney and uterus. In addition, mSPINK12 expression in the epidermis of skin at footpads, where stratification is markedly pronounced, was several folds higher than in the abdominal epidermis. mSPINK12 mRNA levels were not affected by any cytokines tested while treatment of primary murine keratinocytes with the combination of calcium and sorbitol resulted in a strong increase in its mRNA. It appears that mspink12 is especially expressed in the epidermal areas with thick skin and that its regulation generally responds to differentiation signals. mrSPINK12 shows an inhibitory activity against murine keratinocyte-derived trypsin-like proteolytic activity, thus, the protein does appear orthologous to human LEKTI2 and may play an role in the regulation of epithelial cell functions.


Asunto(s)
Proteínas Inhibidoras de Proteinasas Secretoras/genética , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Piel/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , ADN Complementario/genética , Femenino , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidores de Serinpeptidasas Tipo Kazal , Especificidad de la Especie
12.
BMC Biochem ; 12: 38, 2011 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-21787393

RESUMEN

BACKGROUND: Angiogenesis is the process of forming new blood vessels from existing ones and requires degradation of the vascular basement membrane and remodeling of extracellular matrix (ECM) in order to allow endothelial cells to migrate and invade into the surrounding tissue. Matrix metalloproteinases (MMPs) are considered to play a central role in the remodeling of basement membranes and ECM. However, MMPs contribute to vascular remodeling not only by degrading ECM components. Specific MMPs enhance angiogenesis via several ways; they help pericytes to detach from vessels undergoing angiogenesis, release ECM-bound angiogenic growth factors, expose cryptic pro-angiogenic integrin binding sites in the ECM, generate promigratory ECM component fragments, and cleave endothelial cell-cell adhesions. MMPs can also negatively influence the angiogenic process through generating endogenous angiogenesis inhibitors by proteolytic cleavage. Angiostatin, a proteolytic fragment of plasminogen, is one of the most potent antagonists of angiogenesis that inhibits migration and proliferation of endothelial cells. Reports have shown that metalloelastase, pancreas elastase, plasmin reductase, and plasmin convert plasminogen to angiostatin. RESULTS: We report here that MMP-19 processes human plasminogen in a characteristic cleavage pattern to generate three angiostatin-like fragments with a molecular weight of 35, 38, and 42 kDa. These fragments released by MMP-19 significantly inhibited the proliferation of HMEC cells by 27% (p = 0.01) and reduced formation of capillary-like structures by 45% (p = 0.05) compared with control cells. As it is known that angiostatin blocks hepatocyte growth factor (HGF)-induced pro-angiogenic signaling in endothelial cells due to structural similarities to HGF, we have analyzed if the plasminogen fragments generated by MMP-19 interfere with this pathway. As it involves the activation of c-met, the receptor of HGF, we could show that MMP-19-dependent processing of plasminogen decreases the phosphorylation of c-met. CONCLUSION: Altogether, MMP-19 exhibits an anti-angiogenic effect on endothelial cells via generation of angiostatin-like fragments.


Asunto(s)
Angiostatinas/metabolismo , Células Endoteliales/efectos de los fármacos , Metaloproteinasas de la Matriz Secretadas/metabolismo , Fragmentos de Péptidos/farmacología , Plasminógeno/metabolismo , Inhibidores de la Angiogénesis/metabolismo , Inhibidores de la Angiogénesis/farmacología , Capilares/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Metaloproteinasas de la Matriz Secretadas/genética , Peso Molecular , Fragmentos de Péptidos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato
13.
J Neuropathol Exp Neurol ; 69(3): 215-23, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20142769

RESUMEN

Glial tumors exhibit a high morbidity and mortality because of their invasive nature. Matrix metalloproteinase 19 (MMP19) is a secreted protease that together with epilysin (MMP28) forms a structural subgroup of MMPs. We analyzed their expression by quantitative reverse transcription polymerase chain reaction, Western blot, and immunohistochemistry in tumor and normal control brain tissues and in glioblastoma (GB) cells and performed MMP19 silencing functional assays. Matrix metalloproteinase 28 was transcribed to the same extent in normal brain samples and gliomas but was undetectable in GB cell lines. In contrast, MMP19 was detected by immunohistochemistry in normal brain samples only in endothelial cells but was found at high levels in astrocytomas of different World Health Organization grades in situ and in GB cells in vitro. Matrix metalloproteinase 19 was upregulated in GB cells after exposure to proinflammatory cytokines. In Transwell invasion assays, MMP19-silenced cells migrated more slowly through laminin-, basal lamina-, and brevican-coated membranes than controls. Matrix metalloproteinase 19-silenced GB cells also migrated into brain tissue slices compared with control cells. Brevican, a brain-specific proteoglycan and major component of brain extracellular matrix, was degraded by recombinant human MMP19. Taken together, these results indicate that MMP19 is highly expressed in proliferating astrocytoma/glioma cells, and that its expression may facilitate their invasion through brain extracellular matrix components.


Asunto(s)
Astrocitoma/enzimología , Astrocitoma/fisiopatología , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/fisiopatología , Metaloproteinasas de la Matriz Secretadas/metabolismo , Invasividad Neoplásica/fisiopatología , Astrocitoma/patología , Membrana Basal/metabolismo , Membrana Basal/ultraestructura , Western Blotting , Neoplasias Encefálicas/patología , Brevicano , Línea Celular Tumoral , Movimiento Celular/fisiología , Proteoglicanos Tipo Condroitín Sulfato/efectos de los fármacos , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Citocinas/metabolismo , Citocinas/farmacología , Células Endoteliales/enzimología , Matriz Extracelular , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Lectinas Tipo C/efectos de los fármacos , Lectinas Tipo C/metabolismo , Metaloproteinasas de la Matriz Secretadas/genética , Metaloproteinasas de la Matriz Secretadas/farmacología , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Interferencia de ARN , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología
14.
PLoS One ; 3(6): e2343, 2008 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-18523579

RESUMEN

Matrix metalloproteinase-19 (MMP19) affects cell proliferation, adhesion, and migration in vitro but its physiological role in vivo is poorly understood. To determine the function of MMP19, we generated mice deficient for MMP19 by disrupting the catalytic domain of mmp19 gene. Although MMP19-deficient mice do not show overt developmental and morphological abnormalities they display a distinct physiological phenotype. In a model of contact hypersensitivity (CHS) MMP19-deficient mice showed impaired T cell-mediated immune reaction that was characterized by limited influx of inflammatory cells, low proliferation of keratinocytes, and reduced number of activated CD8(+) T cells in draining lymph nodes. In the inflamed tissue, the low number of CD8(+) T cells in MMP19-deficient mice correlated with low amounts of proinflammatory cytokines, especially lymphotactin and interferon-inducible T cell alpha chemoattractant (I-TAC). Further analyses showed that T cell populations in the blood of immature, unsensitized mice were diminished and that this alteration originated from an altered maturation of thymocytes. In the thymus, thymocytes exhibited low proliferation rates and the number of CD4(+)CD8(+) double-positive cells was remarkably augmented. Based on the phenotype of MMP19-deficient mice we propose that MMP19 is an important factor in cutaneous immune responses and influences the development of T cells.


Asunto(s)
Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Metaloproteinasas de la Matriz Secretadas/fisiología , Piel/inmunología , Animales , Secuencia de Bases , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Citocinas/biosíntesis , Cartilla de ADN , Citometría de Flujo , Inmunohistoquímica , Mediadores de Inflamación/metabolismo , Activación de Linfocitos , Metaloproteinasas de la Matriz Secretadas/genética , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa
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