RESUMEN
Lung cancer is one of the leading causes of death worldwide and non-small cell lung cancer (NSCLC) accounts for approximately 80% of lung cancer. Long noncoding RNAs (lncRNAs) are closely associated with the development and progression of various cancers, including lung cancer. The purpose of this study was to explore the potential role and molecular mechanism of lncRNA plasmacytoma variant translocation 1 (PVT1) in regulating the proliferation, apoptosis, migration, and invasion of NSCLC cells. The expressions of PVT1, integrin ß-8 (ITGB8), and miR-145-5p were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of ITGB8, MEK, p-MEK, ERK, and p-ERK were measured by western blot analysis. Cell proliferation, apoptosis, migration, and invasion were determined by MTT assay, flow cytometry, and transwell assay, respectively. The potential binding sites between miR-145-5p and PVT1 or ITGB8 were predicted by online software and verified by luciferase reporter assay. A xenograft tumor model was established to confirm the effect of PVT1 on NSCLC in vivo. We found out that the expression levels of PVT1 and ITGB8 were upregulated in NSCLC tissues and cells. Knockdown of PVT1 or ITGB8 suppressed cell proliferation, migration, invasion and promoted apoptosis in NSCLC cells, which could be reversed by ITGB8 overexpression in NSCLC cells. Moreover, PVT1 could regulate ITGB8 expression via direct binding to miR-145-5p. Furthermore, PVT1 regulated the MEK/ERK pathway by affecting ITGB8 expression. In addition, knockdown of PVT1 inhibited tumor growth, ITGB8 expression, MEK/ERK signaling pathway, and increased miR-145-5p expression in vivo. In conclusion, the knockdown of PVT1 inhibited proliferation, migration, and invasion but induced apoptosis of NSCLC cells by regulating miR-145-5p/ITGB8 axis and inhibiting MEK/ERK signaling pathway, providing a novel avenue for the treatment of NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , MicroARNs , Plasmacitoma , ARN Largo no Codificante , Apoptosis/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Cadenas beta de Integrinas/fisiología , Integrinas , Neoplasias Pulmonares/genética , MicroARNs/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/fisiologíaRESUMEN
Germinal matrix hemorrhage (GMH), affecting about 1 in 300 births, is a major perinatal disease with lifelong neurological consequences. Yet despite advances in neonatal medicine, there is no effective intervention. GMH is characterized by localized bleeding in the germinal matrix (GM), due to inherent vessel fragility unique to this developing brain region. Studies have shown that reduced TGFß signaling contributes to this vascular immaturity. We have previously shown that a region-specific G-protein-coupled receptor pathway in GM neural progenitor cells regulates integrin ß8, a limiting activator of pro-TGFß. In this study, we use mice to test whether this regional pathway can be harnessed for GMH intervention. We first examined the endogenous dynamics of this pathway and found that it displays specific patterns of activation. We then investigated the functional effects of altering these dynamics by chemogenetics and found that there is a narrow developmental window during which this pathway is amenable to manipulation. Although high-level activity in this time window interferes with vessel growth, moderate enhancement promotes vessel maturation without compromising growth. Furthermore, we found that enhancing the activity of this pathway in a mouse model rescues all GMH phenotypes. Altogether, these results demonstrate that enhancing neurovascular signaling through pharmacological targeting of this pathway may be a viable approach for tissue-specific GMH intervention. They also demonstrate that timing and level are likely two major factors crucial for success. These findings thus provide critical new insights into both brain neurovascular biology and the intervention of GMH.
Asunto(s)
Modelos Animales de Enfermedad , Hemorragias Intracraneales/prevención & control , Neostriado/irrigación sanguínea , Células-Madre Neurales/fisiología , Animales , Vasos Sanguíneos/fisiología , Circulación Cerebrovascular , Clozapina/análogos & derivados , Clozapina/farmacología , Femenino , Cadenas beta de Integrinas/fisiología , Hemorragias Intracraneales/etiología , Ratones , Receptores Acoplados a Proteínas G/fisiología , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Integrin ß6 (ITGB6), an epithelial-specific integrin, is upregulated in oral squamous cell carcinomas (OSCC) and is associated with progression and metastasis of OSCC. Lysophosphatidic acid (LPA), an important bioactive phospholipid present in saliva, has also been related to OSCC cell migration and invasiveness. LPA exerts its biological effects through signal transduction pathways that ultimately regulate gene expression. However, it is unclear whether LPA signaling is involved in ITGB6 upregulation in OSCC. Therefore, the aim of the current study was to investigate the role of LPA in the regulation of ITGB6 expression in OSCC cells, and to delineate the molecular signaling pathways involved. Using SAS and HSC-3 OSCC cell lines, we found that LPA increases ITGB6 mRNA expression without affecting mRNA stability, suggesting that LPA acts by regulating ITGB6 gene transcription. In addition, we show that LPA stimulation increases phosphorylation and binding of the transcription factors SMAD3 and ETS-1 to the ITGB6 promoter resulting in ITGB6 active transcription. Finally, we demonstrate that LPA-induced ITGB6 expression is mediated via the LPA receptors 1 (LPAR1) coupling to Gαi. Our findings provide insights into the molecular mechanism underlying ITGB6 overexpression in OSCC and may have important implications for therapeutic purposes.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Cadenas beta de Integrinas/fisiología , Lisofosfolípidos/fisiología , Neoplasias de la Boca/metabolismo , Línea Celular Tumoral , Humanos , Proteína Proto-Oncogénica c-ets-1/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Proteína smad3/metabolismoRESUMEN
In vitro expansion of undifferentiated porcine primed embryonic stem (ES) cells is facilitated by use of non-cellular niches that mimic three-dimensional (3D) microenvironments enclosing an inner cell mass of porcine blastocysts. Therefore, we investigated the integrin heterodimers on the surface of undifferentiated porcine primed ES cells for the purpose of developing a non-cellular niche to support in vitro maintenance of the self-renewal ability of porcine primed ES cells. Immunocytochemistry and a fluorescence immunoassay were performed to assess integrin α and ß subunit levels, and attachment and antibody inhibition assays were used to evaluate the function of integrin heterodimers. The integrin α3 , α5 , α6 , α9 , αV , and ß1 subunits, but not the α1 , α2 , α4 , α7 , and α8 subunits, were identified on the surface of undifferentiated porcine primed ES cells. Subsequently, significant increase of their adhesion to fibronectin, tenascin C, and vitronectin were observed and functional blocking of integrin heterodimer α5 ß1 , α9 ß1 , or αV ß1 showed significantly inhibited adhesion to fibronectin, tenascin C, or vitronectin. No integrin α6 ß1 heterodimer-mediated adhesion to laminin was detected. These results demonstrate that active α5 ß1 , α9 ß1 , and αV ß1 integrin heterodimers are present on the surface of undifferentiated porcine primed ES cells, together with inactive integrin α3 (presumed) and α6 subunits.
Asunto(s)
Adhesión Celular/fisiología , Células Madre Embrionarias/metabolismo , Integrinas/metabolismo , Animales , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , Células Cultivadas , Células Madre Embrionarias/fisiología , Matriz Extracelular/metabolismo , Células Nutrientes , Fibronectinas/metabolismo , Cadenas alfa de Integrinas/metabolismo , Cadenas alfa de Integrinas/fisiología , Cadenas beta de Integrinas/metabolismo , Cadenas beta de Integrinas/fisiología , Integrinas/fisiología , Laminina/metabolismo , Ratones , Porcinos , Tenascina , VitronectinaRESUMEN
OBJECTIVE: To study the expression of ß-integrin family members in children with T-cell acute lymphoblastic leukemia (T-ALL) and their significance. METHODS: Quantitative real-time PCR analyses were performed to assess the expression levels of ß-integrin family members in bone marrow samples from 22 children with newly-diagnosed T-ALL and 21 controls (16 children with non-malignant hematologic disease and 5 healthy donors with bone marrow transplantation). Jurkat cells were treated with integrin inhibitor arginine-glycine-aspartate (Arg-Gly-Asp, RGD) peptide. The cell viability and apoptosis rate were determined by CCK8 assay and flow cytometry respectively. RESULTS: The mRNA levels of integrins ß2, ß3, and ß5 were significantly lower in children with T-ALL than in controls (P<0.05). In T-ALL patients, high integrin ß3 expression was associated with lower white blood cell counts (<100×109/L), minimal residual disease (MRD) positivity, and day 33 bone marrow negative remission (P<0.05). In T-ALL patients, higher integrin ß5 expression was associated with relapse of T-ALL (P<0.05). Based on survival curve analysis, higher integrin ß3 expression was related to lower event-free survival and overall survival rates. RGD peptide treatment inhibited the proliferation of Jurkat cells and increased their apoptosis rate (P<0.05). CONCLUSIONS: ß-Integrin may play a role in the occurrence and development of T-ALL by affecting cell proliferation and apoptosis. The expression of integrin ß5 is closely related to the risk of relapse of T-ALL. The expression of integrin ß3 is closely related the treatment response and prognosis of T-ALL.
Asunto(s)
Cadenas beta de Integrinas/fisiología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidad , Niño , Preescolar , Femenino , Humanos , Cadenas beta de Integrinas/genética , Células Jurkat , Masculino , Leucemia-Linfoma Linfoblástico de Células T Precursoras/etiología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , ARN Mensajero/análisisRESUMEN
Integrins play key roles in the regulation of tumor cell adhesion, migration, invasion and sensitivity to anticancer drugs. In the present study we investigate the mechanism of resistance of tongue squamous carcinoma cells Cal27 with de novo integrin αvß3 expression to anticancer drugs. Cal27-derived cell clones, obtained by transfection of plasmid containing integrin subunit ß3 cDNA, as compared to control cells demonstrate: expression of integrin αvß3; increased expression of integrin αvß5; increased adhesion to fibronectin and vitronectin; resistance to cisplatin, mitomycin C, doxorubicin and 5-fluorouracil; increased migration and invasion, increased amount of integrin-linked kinase (ILK) and decreased amounts of non-receptor tyrosine kinase (Src) and pSrc(Y418). Knockdown of ILK and integrin ß5 in cells expressing integrin αvß3 ruled out their involvement in drug resistance. Opposite, Src knockdown in Cal27 cells which led to a reduction in pSrc(Y418), as well as treatment with the pSrc(Y418) inhibitors dasatinib and PP2, conferred resistance to all four anticancer drugs, indicating that the loss of pSrc(Y418) is responsible for the observed effect. We identified differential integrin signaling between Cal27 and integrin αvß3-expressing cells. In Cal27 cells integrin αv heterodimers signal through pSrc(Y418) while this is not the case in integrin αvß3-expressing cells. Finally, we show that dasatinib counteracts the effect of cisplatin in two additional head and neck squamous cell carcinoma (HNSCC) cell lines Cal33 and Detroit562. Our results suggest that pSrc(Y418) inhibitors, potential drugs for cancer therapy, may reduce therapeutic efficacy if combined with chemotherapeutics, and might not be recommended for HNSCC treatment.
Asunto(s)
Carcinoma de Células Escamosas/patología , Resistencia a Antineoplásicos/fisiología , Integrina alfaVbeta3/fisiología , Proteínas de Neoplasias/fisiología , Proteínas Proto-Oncogénicas pp60(c-src)/fisiología , Neoplasias de la Lengua/patología , Antineoplásicos/farmacología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Cisplatino/farmacología , Dasatinib/farmacología , Doxorrubicina/farmacología , Sinergismo Farmacológico , Fluorouracilo/farmacología , Regulación Neoplásica de la Expresión Génica , Genes src , Humanos , Integrina alfaVbeta3/biosíntesis , Integrina alfaVbeta3/genética , Cadenas beta de Integrinas/fisiología , Mitomicina/farmacología , Invasividad Neoplásica , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Mutación Puntual , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Interferencia de ARN , Neoplasias de la Lengua/genética , Neoplasias de la Lengua/metabolismoRESUMEN
Hematopoietic cells depend on integrin-mediated adhesion and signaling, which is induced by kindlin-3 and talin-1. To determine whether platelet and polymorphonuclear neutrophil (PMN) functions require specific thresholds of kindlin-3, we generated mouse strains expressing 50%, 10%, or 5% of normal kindlin-3 levels. We report that in contrast to kindlin-3-null mice, which die perinatally of severe bleeding and leukocyte adhesion deficiency, mice expressing as little as 5% of kindlin-3 were viable and protected from spontaneous bleeding and infections. However, platelet adhesion and aggregation were reduced in vitro and bleeding times extended. Similarly, leukocyte adhesion, extravasation, and bacterial clearance were diminished. Quantification of protein copy numbers revealed stoichiometric quantities of kindlin-3 and talin-1 in platelets and neutrophils, indicating that reduction of kindlin-3 in our mouse strains progressively impairs the cooperation with talin-1. Our findings show that very low levels of kindlin-3 enable basal platelet and neutrophil functions, whereas in stress situations such as injury and infection, platelets and neutrophils require a maximum of functional integrins that is achieved with high and stoichiometric quantities of kindlin-3 and talin-1.
Asunto(s)
Plaquetas/fisiología , Proteínas del Citoesqueleto/fisiología , Leucocitos/inmunología , Animales , Tiempo de Sangría , Plaquetas/química , Adhesión Celular , Proteínas del Citoesqueleto/sangre , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/genética , Gastritis/sangre , Gastritis/inmunología , Gastritis/microbiología , Infecciones por Helicobacter/sangre , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/inmunología , Helicobacter pylori/aislamiento & purificación , Trastornos Hemorrágicos/genética , Cadenas beta de Integrinas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neutrófilos/química , Neutrófilos/inmunología , Fagocitosis/genética , Adhesividad Plaquetaria/genética , Agregación Plaquetaria/genética , Talina/sangre , Talina/genéticaRESUMEN
BACKGROUND: Mechanical strain plays a great role in growth and differentiation of osteoblast. A previous study indicated that integrin-ß (ß1, ß5) mediated osteoblast proliferation promoted by mechanical tensile strain. However, the involvement of integrin-ß in osteoblastic differentiation and extracellular matrix (ECM) formation induced by mechanical tensile strain, remains unclear. RESULTS: After transfection with integrin-ß1 siRNA or integrin-ß5 siRNA, mouse MC3T3-E1 preosteoblasts were cultured in cell culture dishes and stimulated with mechanical tensile strain of 2500 microstrain (µÎµ) at 0.5 Hz applied once a day for 1 h over 3 or 5 consecutive days. The cyclic tensile strain promoted osteoblastic differentiation of MC3T3-E1 cells. Transfection with integrin-ß1 siRNA attenuated the osteoblastic diffenentiation induced by the tensile strain. By contrast, transfection with integrin-ß5 siRNA had little effect on the osteoblastic differentiation induced by the strain. At the same time, the result of ECM formation promoted by the strain, was similar to the osteoblastic differentiation. CONCLUSION: Integrin-ß1 mediates osteoblast differentiation and osteoblastic ECM formation promoted by cyclic tensile strain, and integrin-ß5 is not involved in the osteoblasts response to the tensile strain.
Asunto(s)
Diferenciación Celular/fisiología , Matriz Extracelular/fisiología , Cadenas beta de Integrinas/fisiología , Integrina beta1/fisiología , Osteoblastos/fisiología , Resistencia a la Tracción/fisiología , Animales , Western Blotting , Línea Celular , Proliferación Celular/fisiología , Ratones , ARN Interferente Pequeño , Reacción en Cadena en Tiempo Real de la Polimerasa , Estrés Mecánico , TransfecciónRESUMEN
Integrins (ITGs) are mediators of cell-cell and cell-matrix interactions, which are also associated with embryo implantation processes by controlling the interaction of blastocyst with endometrium. During early pregnancy, ITGbeta8 (ITGB8) has been shown to interact with latent transforming growth factor (TGF) beta 1 (TGFB1) at the fetomaternal interface. However, the precise role of ITGB8 in the uterus and its association with embryo implantation has not been elucidated. Therefore, we attempted to ascertain the role of ITGB8 during the window of embryo implantation process by inhibiting its function or protein expression. Uterine plasma membrane-anchored ITGB8 was augmented at peri-implantation and postimplantation stages. A similar pattern of mRNA expression was also found during the embryo implantation period. An immunolocalization study revealed the presence of ITGB8 on luminal epithelial cells along with mild expression on the stromal cells throughout the implantation period studied; however, an intense fluorescence was noted only during the peri- and postimplantation stages. Bioneutralization and mRNA silencing of the uterine Itgb8 at preimplantation stage reduced the rate/frequency of embryo implantation and subsequent pregnancy, suggesting its indispensable role during the embryo implantation period. ITGB8 can also regulate the liberation of active TGFB1 from its latent complex, which, in turn, acts on SMAD2/3 phosphorylation (activation) in the uterus during embryo implantation. This indicates involvement of ITGB8 in the embryo implantation process through regulation of activation of TGFB1.
Asunto(s)
Implantación del Embrión/fisiología , Cadenas beta de Integrinas/fisiología , Factor de Crecimiento Transformador beta1/fisiología , Animales , Blastocisto/fisiología , Membrana Celular/metabolismo , ADN Complementario/biosíntesis , ADN Complementario/genética , Embrión de Mamíferos/fisiología , Femenino , Técnicas de Silenciamiento del Gen , Cadenas beta de Integrinas/genética , Ratones , Oligonucleótidos Antisentido/farmacología , Embarazo , Proteína Smad2/genética , Proteína Smad2/fisiología , Proteína smad3/genética , Proteína smad3/fisiología , Útero/fisiologíaRESUMEN
BACKGROUND: Mechanical strain plays a great role in growth and differentiation of osteoblast. A previous study indicated that integrin-ß (ß1, ß5) mediated osteoblast proliferation promoted by mechanical tensile strain. However, the involvement of integrin-ß; in osteoblastic differentiation and extracellular matrix (ECM) formation induced by mechanical tensile strain, remains unclear. RESULTS: After transfection with integrin-ß1 siRNA or integrin-ß5 siRNA, mouse MC3T3-E1 preosteoblasts were cultured in cell culture dishes and stimulated with mechanical tensile strain of 2500 microstrain (µÎµ) at 0.5 Hz applied once a day for 1 h over 3 or 5 consecutive days. The cyclic tensile strain promoted osteoblastic differentiation of MC3T3-E1 cells. Transfection with integrin-ß1 siRNA attenuated the osteoblastic diffenentiation induced by the tensile strain. By contrast, transfection with integrin-ß5 siRNA had little effect on the osteoblastic differentiation induced by thestrain. At thesametime, theresultofECM formation promoted by the strain, was similar to the osteoblastic differentiation. CONCLUSION: Integrin-ß1 mediates osteoblast differentiation and osteoblastic ECM formation promoted by cyclic tensile strain, and integrin-ß5 is not involved in the osteoblasts response to the tensile strain.
Asunto(s)
Animales , Ratones , Osteoblastos/fisiología , Resistencia a la Tracción/fisiología , Diferenciación Celular/fisiología , Integrina beta1/fisiología , Cadenas beta de Integrinas/fisiología , Matriz Extracelular/fisiología , Estrés Mecánico , Transfección , Línea Celular , Western Blotting , ARN Interferente Pequeño , Proliferación Celular/fisiología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The recognition and attachment of virus to its host cell surface is a critical step for viral infection. Recent research revealed that beta-integrin was involved in White spot syndrome virus (WSSV) infection. In this study, the interaction of beta-integrin with structure proteins of WSSV and motifs involved in WSSV infection was examined. The results showed that envelope proteins VP26, VP31, VP37, VP90 and nucleocapsid protein VP136 interacted with LvInt. RGD-, YGL- and LDV-related peptide functioned as motifs of WSSV proteins binding with beta-integrin. The beta-integrin ligand of RGDT had better blocking effect compared with that of YGL- and LDV-related peptides. In vivo assay indicated that RGD-, LDV- and YGL-related peptides could partially block WSSV infection. These data collectively indicate that multiple proteins were involved in recognition of beta-integrin. Identification of proteins in WSSV that are associated with beta-integrin will assist development of new agents for effective control of the white spot syndrome.
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Interacciones Huésped-Patógeno , Penaeidae/virología , Acoplamiento Viral , Virus del Síndrome de la Mancha Blanca 1/fisiología , Secuencias de Aminoácidos , Animales , Cadenas beta de Integrinas/inmunología , Cadenas beta de Integrinas/metabolismo , Cadenas beta de Integrinas/fisiología , Estructura Terciaria de Proteína , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/fisiología , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/metabolismo , Proteínas Estructurales Virales/fisiologíaRESUMEN
The medical management of idiopathic inflammatory bowel disease (IBD) has historically been based upon the use of broad-spectrum anti-inflammatory drugs such as corticosteroids and thiopurines. Recently, the identification of novel mechanisms central to the pathophysiology of IBD has provided more specific targets, including inhibition of leukocyte trafficking to the gut. In this article, we discuss the molecular biology of intestinal leukocyte trafficking and review the emerging therapies that target this process, including vedolizumab, natalizumab, etrolizumab, PF-547659, alicaforsen, efalizumab, and emerging members of this class.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Linfocitos T/efectos de los fármacos , Quimiocina CXCL10/antagonistas & inhibidores , Humanos , Enfermedades Inflamatorias del Intestino/inmunología , Integrina alfa4/fisiología , Cadenas beta de Integrinas/fisiología , Integrinas/antagonistas & inhibidores , Leucoencefalopatía Multifocal Progresiva/inducido químicamente , Receptores de Lisoesfingolípidos/agonistas , Receptores de Lisoesfingolípidos/fisiología , Transducción de Señal , Linfocitos T/fisiologíaRESUMEN
OBJECTIVE: To study the effect of ß8 expression on transforming growth factor ß1(TGF-ß1) activation in astrocytes with oxygen glucose deprivation (OGD). METHODS: Astrocytes were cultured and then subjected to OGD to generate hypoxia-ischemia (HI) model in vitro. Immunocytochemistry was used to detect the expression and distribution of ß8 in nomoxia cultured cells. ß8 protein expression was quantified by Western blot at 12 hours, 1 day and 2 days after OGD. Astrocytes and luciferase reporter cells (TMLC) were co-cultured. ß8 RNA interference system was established to specifically inhibit ß8 expression in cultured astrocytes. TGF-ß1 activation was then detected in the co-culture system. RESULTS: ß8 was mainly located in the cytoplasm and neurites of astrocytes. OGD resulted in increase of ß8 protein expression at 12 hours after reoxygenation in astrocytes, which was peaked at 1 day after reoxygenation. TGF-ß1 activation was in accordance with ß8 expression in astrocyte-TMLC co-culture system after reoxygenation. After the inhibition of ß8, TGF-ß1 activation was significantly reduced in all time points. CONCLUSIONS: The highly expressed ß8 plays important roles in the regulation of TGF-ß1 activation in neonatal rats with hypoxic-ischemic brain damage.
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Astrocitos/metabolismo , Hipoxia-Isquemia Encefálica/metabolismo , Cadenas beta de Integrinas/fisiología , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Femenino , Glucosa/metabolismo , Masculino , Oxígeno/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
We identified a family in which pitted hypomineralized amelogenesis imperfecta (AI) with premature enamel failure segregated in an autosomal recessive fashion. Whole-exome sequencing revealed a missense mutation (c.586C>A, p.P196T) in the I-domain of integrin-ß6 (ITGB6), which is consistently predicted to be pathogenic by all available programmes and is the only variant that segregates with the disease phenotype. Furthermore, a recent study revealed that mice lacking a functional allele of Itgb6 display a hypomaturation AI phenotype. Phenotypic characterization of affected human teeth in this study showed areas of abnormal prismatic organization, areas of low mineral density and severe abnormal surface pitting in the tooth's coronal portion. We suggest that the pathogenesis of this form of AI may be due to ineffective ligand binding of ITGB6 resulting in either compromised cell-matrix interaction or compromised ITGB6 activation of transforming growth factor-ß (TGF-ß) impacting indirectly on ameloblast-ameloblast interactions and proteolytic processing of extracellular matrix proteins via MMP20. This study adds to the list of genes mutated in AI and further highlights the importance of cell-matrix interactions during enamel formation.
Asunto(s)
Amelogénesis Imperfecta/genética , Amelogénesis Imperfecta/patología , Genes Recesivos , Cadenas beta de Integrinas/fisiología , Mutación Missense/genética , Amelogénesis Imperfecta/metabolismo , Secuencia de Aminoácidos , Animales , ADN/genética , Esmalte Dental/metabolismo , Esmalte Dental/patología , Exoma/genética , Humanos , Técnicas para Inmunoenzimas , Ratones , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Linaje , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Homología de Secuencia de Aminoácido , Microtomografía por Rayos XRESUMEN
BACKGROUND: L-selectin (CD62L) and ß(7) integrins are important for trafficking of naive T cells under steady-state conditions. The objectives of this study were to dissect the requirements for T cell-associated CD62L and ß(7) integrins during initiation, progression, and regulation of chronic colitis. METHODS: Using the T-cell transfer model, we compared colitogenic potential between T cells lacking one or both of these molecules with wild-type T cells. To assess trafficking of cells to the secondary lymphoid tissue and the gut, we performed co-homing experiments. RESULTS: Adoptive transfer of wild-type, CD62L(-/-) or ß(7)(-/-) single-deficient T cells induced moderate to severe disease with slightly different kinetics. However, transfer of CD62L(-/-) ß(7)(-/-) double-deficient (DKO) T cells produced significantly attenuated gut inflammation, which correlated with fewer T cells and reduced levels of proinflammatory cytokines in the colon lamina propria. Our subsequent experiments established that lack of colitogenic potential of these cells was due to inability of DKO T cells to home to the secondary lymphoid tissue. Furthermore, homing of in vitro-generated effector DKO T cells to the inflamed intestine was significantly impaired. Lastly, DKO regulatory T cells were ineffective at suppressing colitis induced by wild-type T cells. CONCLUSIONS: We established that T cells can use either CD62L(-/-) or ß(7)(-/-) integrins to induce chronic colitis, but lack of both abrogates their colitogenic potential. Effector T cells critically rely on ß(7) integrin during their recruitment to the inflamed intestinal mucosa. Finally, regulation of intestinal inflammation by regulatory T cells requires one or both of these adhesion molecules.
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Linfocitos T CD4-Positivos/metabolismo , Colitis/metabolismo , Tracto Gastrointestinal/metabolismo , Cadenas beta de Integrinas/fisiología , Selectina L/fisiología , Animales , Linfocitos T CD4-Positivos/patología , Proliferación Celular , Enfermedad Crónica , Citocinas/metabolismo , Femenino , Citometría de Flujo , Tracto Gastrointestinal/patología , Proteínas de Homeodominio/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The full sequence of white shrimp Litopenaeus vannamei integrin ß (LV-B) is 2879bp which encodes 787 amino acids (aa) of the open reading frame (ORF). The mature protein (764 aa) contains (1) an extracellular domain (ED) of 692 aa, (2) a transmembrane domain (TD) of 23 aa, and (3) a cytoplasmic domain (CD) of 49 aa. The cloned LV-B grouped together with crayfish Pacifastacus leniusculus integrin ß (PL-B1), but was far away from vertebrate integrin ß1, ß3, ß5, ß6, ß7, and ß8, and another L. vannamei integrin ß (LV). A Southern blot analysis indicated that the cloned LV-B was a single copy of genomic DNA. LV-B mRNA was expressed in all tissues, and was highly expressed in haemocytes. LV-B was downregulated in shrimp 24 and 96h after having received white spot syndrome virus (WSSV). LV-B expression by haemocytes of shrimp was higher in the postmoult (A and B) stage, and lower in the premoult (D2/D3) stage. LV-B expression was significantly higher by shrimp reared in 2.5 and 5 salinities. Shrimp injected with integrin ß dsRNA showed gene silencing of integrin ß after 36h. LV-B-silenced shrimp showed decreased hyaline cells (HCs), granular cells (GCs, including semi-granular cells), the total haemocyte count (THC), respiratory bursts (RBs), and lysozyme activity, but showed increased RB/HC, superoxide dismutase (SOD) activity/HC, and the phenoloxidase (PO) activity/GC. LV-B-silenced shrimp showed upregulated expressions of lipopolysaccharide- and ß-glucan-binding protein (LGBP), peroxinectin (PX), prophenoloxidase I (proPO I), proPO II, proPO-activating enzyme (ppA), α2-macroglobulin (α2-M), cytMnSOD, mtMnSOD, and heat shock protein 70 (HSP70). It was concluded that integrin ß plays important roles in proPO activation, phagocytosis, and the antioxidant system for immunomodulation in shrimp.
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Proteínas de Artrópodos/fisiología , Inmunomodulación , Cadenas beta de Integrinas/fisiología , Penaeidae/fisiología , Interferencia de ARN , ARN Bicatenario/genética , Secuencia de Aminoácidos , Animales , Antioxidantes/metabolismo , Proteínas de Artrópodos/química , Secuencia de Bases , Secuencia Conservada , Dosificación de Gen , Expresión Génica , Hemocitos/inmunología , Hemocitos/metabolismo , Hemocitos/virología , Interacciones Huésped-Patógeno , Cadenas beta de Integrinas/química , Datos de Secuencia Molecular , Muda , Especificidad de Órganos , Penaeidae/virología , Filogenia , Estallido Respiratorio , Salinidad , Virus del Síndrome de la Mancha Blanca 1/inmunologíaRESUMEN
OBJECTIVES: There is a clear need for better therapeutics and diagnostics for pancreatic cancer. We aimed to discover plasma membrane-associated proteins overexpressed in pancreatic cancer using quantitative proteomics and apply RNA interference (RNAi) to uncover proteins associated with cancer cell survival. METHODS: Cell surface glycoproteins from 5 pancreatic cancer cell lines were isolated, and differential analyses were performed using mass spectrometry and the "normoid" cell line Hs766T as the comparator. For validation, immunohistochemistry was performed on tissues from 10 independent patients and 2 normal donors. Correlation of protein and mRNA expression level was determined, and functional activity characterized using RNAi. RESULTS: Integrin ß6, CD46, tissue factor, and a novel protein, chromosome 14 open reading frame 1, were identified as overexpressed on pancreatic cancer cell lines. Immunohistochemistry demonstrated the 4 targets were overexpressed in 20% to 70% of primary pancreatic tumor specimens. Small interfering RNA knockdown resulted in a reduction of cellular proliferation by inhibiting DNA synthesis, blocking S-phase progression or induction of apoptosis. CONCLUSIONS: By combining a mass spectrometry identification platform and an RNAi validation platform, we have identified a panel of cell surface glycoproteins that not only are overexpressed, but also play a functional role in pancreatic tumor cell survival.
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Proliferación Celular , Glicoproteínas de Membrana/fisiología , Proteómica/métodos , Interferencia de ARN , Línea Celular Tumoral , Supervivencia Celular , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Cadenas beta de Integrinas/genética , Cadenas beta de Integrinas/metabolismo , Cadenas beta de Integrinas/fisiología , Proteína Cofactora de Membrana/genética , Proteína Cofactora de Membrana/metabolismo , Proteína Cofactora de Membrana/fisiología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tromboplastina/genética , Tromboplastina/metabolismo , Tromboplastina/fisiologíaRESUMEN
Integrin αvß6 is an epithelial-specific receptor that binds and activates latent transforming growth factor-ß1 (TGF-ß1). TGF-ß1 has been implicated as an endogenous inducer of hair follicle (HF) regression during hair cycling. We hypothesized that αvß6 integrin-mediated TGF-ß1 signaling regulates hair regeneration and HF involution. In wild-type (WT) mice, the expression of integrin αvß6 was strongly upregulated in the outer root sheath (ORS) during early hair regeneration, and was specifically enhanced in the HF bulge region. Expression gradually decreased in late anagen and remained restricted to the bulge region in the catagen and telogen stage HFs. The first spontaneous hair cycle was not altered in ß6 integrin knockout (ß6(-/-)) mice. However, after depilation, ß6(-/-) mice exhibited retarded HF regression compared with WT controls. ß6(-/-) follicles contained significantly higher numbers of proliferating Ki67-positive keratinocytes than WT follicles at an identical cycle stage. The ß6(-/-) follicles also demonstrated significantly reduced levels of TGF-ß1 expression and Smad2 phosphorylation during early anagen and anagen-catagen transition. Our study indicates that αvß6 integrin has an important inhibitory role in keratinocyte proliferation in both HFs and interfollicular epidermis. Thus, downregulated TGF-ß1 signaling in ß6(-/-) mice may affect bulge niche stem cell behavior.
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Proliferación Celular , Folículo Piloso/fisiología , Cadenas beta de Integrinas/fisiología , Queratinocitos/fisiología , Animales , Femenino , Folículo Piloso/citología , Folículo Piloso/metabolismo , Remoción del Cabello , Cadenas beta de Integrinas/genética , Queratinocitos/citología , Queratinocitos/metabolismo , Antígeno Ki-67/análisis , Ratones , Ratones Noqueados , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta1/biosíntesis , Regulación hacia ArribaRESUMEN
Neurogenesis in the post-natal brain occurs in two primary locations: the subgranular layer of the hippocampal dentate gyrus and the subventricular zone (SVZ) of the lateral ventricles. Following differentiation, neuroblasts within the SVZ migrate several millimeters to the olfactory bulbs (OBs) via a distinct anatomic route, or rostral migratory stream (RMS). The genes that govern neuroblast directional migration, and particularly those encoding cell adhesion and signaling factors, remain largely uncharacterized. Here, we report that the extracellular matrix adhesion receptor, ß8 integrin, is essential for proper neuroblast chain formation and directional navigation in the RMS. Primary neuroblasts isolated from the mouse brain express robust levels of ß8 integrin protein, and selective ablation of ß8 integrin gene expression in neuroblasts leads to aberrant chain migration and size-reduced OBs. These integrin-dependent defects can be recapitulated ex vivo using isolated neurospheres or SVZ explants. Collectively, these data identify essential cell-intrinsic functions for ß8 integrin in regulating neuroblast polarity and directional navigation in the mouse forebrain.
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Movimiento Celular/fisiología , Cadenas beta de Integrinas/genética , Cadenas beta de Integrinas/fisiología , Células-Madre Neurales/fisiología , Neurogénesis/genética , Neurogénesis/fisiología , Neuronas/fisiología , Animales , Western Blotting , Movimiento Celular/genética , Polaridad Celular/genética , Polaridad Celular/fisiología , Células Cultivadas , Giro Dentado/citología , Giro Dentado/fisiología , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Inmunoprecipitación , Ventrículos Laterales/citología , Ventrículos Laterales/fisiología , Ratones , Ratones Noqueados , Bulbo Olfatorio/citología , Bulbo Olfatorio/crecimiento & desarrolloRESUMEN
CD98 H chain (4F2 Ag, Slc3a2) was discovered as a lymphocyte-activation Ag. Deletion of CD98 H chain in B cells leads to complete failure of B cell proliferation, plasma cell formation, and Ab secretion. In this study, we examined the role of T cell CD98 in cell-mediated immunity and autoimmune disease pathogenesis by specifically deleting it in murine T cells. Deletion of T cell CD98 prevented experimental autoimmune diabetes associated with dramatically reduced T cell clonal expansion. Nevertheless, initial T cell homing to pancreatic islets was unimpaired. In sharp contrast to B cells, CD98-null T cells showed only modestly impaired Ag-driven proliferation and nearly normal homeostatic proliferation. Furthermore, these cells were activated by Ag, leading to cytokine production (CD4) and efficient cytolytic killing of targets (CD8). The integrin-binding domain of CD98 was necessary and sufficient for full clonal expansion, pointing to a role for adhesive signaling in T cell proliferation and autoimmune disease. When we expanded CD98-null T cells in vitro, they adoptively transferred diabetes, establishing that impaired clonal expansion was responsible for protection from disease. Thus, the integrin-binding domain of CD98 is required for Ag-driven T cell clonal expansion in the pathogenesis of an autoimmune disease and may represent a useful therapeutic target.