RESUMEN
Enhanced blood vessel (BV) formation is thought to drive tumor growth through elevated nutrient delivery. However, this observation has overlooked potential roles for mural cells in directly affecting tumor growth independent of BV function. Here we provide clinical data correlating high percentages of mural-ß3-integrin-negative tumor BVs with increased tumor sizes but no effect on BV numbers. Mural-ß3-integrin loss also enhances tumor growth in implanted and autochthonous mouse tumor models with no detectable effects on BV numbers or function. At a molecular level, mural-cell ß3-integrin loss enhances signaling via FAK-p-HGFR-p-Akt-p-p65, driving CXCL1, CCL2, and TIMP-1 production. In particular, mural-cell-derived CCL2 stimulates tumor cell MEK1-ERK1/2-ROCK2-dependent signaling and enhances tumor cell survival and tumor growth. Overall, our data indicate that mural cells can control tumor growth via paracrine signals regulated by ß3-integrin, providing a previously unrecognized mechanism of cancer growth control.
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Integrina beta3/metabolismo , Neoplasias/metabolismo , Carga Tumoral/fisiología , Animales , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Femenino , Humanos , Masculino , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/fisiologíaRESUMEN
Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal alloantibodies directed against paternally inherited human platelet alloantigens (HPAs) present on the surface of fetal and neonatal platelets. There are currently no approved therapies for the prevention of FNAIT. We report herein the ability of 2 human HPA-1a-specific therapeutic candidates, one a polyclonal, and the other a monoclonal antibody, to prevent alloimmunization in a novel preclinical mouse model of FNAIT. Both antibody preparations effected the rapid and complete elimination of HPA-1a+ platelets from circulation and prevented the development of HPA-1a alloantibodies. HPA-1a- female mice treated prophylactically with anti-HPA-1a antibody prior to exposure to HPA-1a+ platelets gave birth to HPA-1a+/- pups with significantly improved platelet counts and no bleeding symptoms. These preclinical data establish both the potential and threshold exposure targets for prophylactic treatment with HPA-1a-specific antibodies for the prevention of FNAIT in humans.
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Antígenos de Plaqueta Humana , Trombocitopenia Neonatal Aloinmune , Embarazo , Humanos , Femenino , Ratones , Animales , Trombocitopenia Neonatal Aloinmune/prevención & control , Isoanticuerpos , Integrina beta3 , Atención Prenatal , FetoRESUMEN
INTRODUCTION: This case describes passenger lymphocyte syndrome (PLS) generating human platelet antigen 1a (HPA-1a) alloantibodies against the recipient's platelets after liver transplant. Given the rarity of PLS, especially in liver transplant with HPA-1a alloantibodies, disease course and management options are poorly described. METHODS: The patient had cirrhosis secondary to nonalcoholic steatohepatitis complicated by hepatocellular carcinoma, encephalopathy, and severe ascites. The model for end-stage liver disease (MELD) score was 15 at presentation. The patient developed hepatic artery thrombosis after an orthotopic liver transplant and was relisted for transplant with a MELD score of 40. The patient received a hepatitis C virus antibody positive, hepatitis C virus nucleic amplification test positive donor liver on postoperative day (POD) 7 after first transplant. On POD 7 after the second transplant, the patient developed profound thrombocytopenia refractory to platelet infusion. They were found to have serum antibody to HPA-1a based upon serum platelet alloantibody testing. The donor was later found to be negative for HPA-1a by genetic testing. However, the patient's native platelets were HPA-1a positive. The patient was diagnosed with PLS. RESULTS: The patient's treatment course included 57 units of platelets transfused, emergency splenectomy, rituximab, plasma exchange, intravenous immunoglobulin (IVIG), eltrombopag, romiplostim, and efgartigimod. DISCUSSION: The synergistic effect of efgartigimod with eltrombopag and romiplostim most likely resolved the patient's thrombocytopenia. This case represents a novel use of efgartigimod in the treatment of passenger lymphocyte syndrome following liver transplant.
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Anemia , Antígenos de Plaqueta Humana , Benzoatos , Enfermedad Hepática en Estado Terminal , Hidrazinas , Trasplante de Hígado , Pirazoles , Trombocitopenia , Humanos , Isoanticuerpos , Donadores Vivos , Índice de Severidad de la Enfermedad , Trombocitopenia/etiología , Trombocitopenia/terapia , Linfocitos , Integrina beta3RESUMEN
BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a condition during pregnancy, which can lead to thrombocytopenia and a bleeding tendency with intracranial hemorrhage (ICH) being the most concerning complication in the fetus or neonate. An incompatibility between human platelet antigen (HPA)-1a accounts for the majority of FNAIT cases. Binding of HPA-1a-specific alloantibodies to their target on fetal platelets and endothelial cells can induce apoptosis of megakaryocytes, disrupt platelet function, and impair angiogenesis. Currently, there is no screening program to identify pregnancies at risk for severe disease. A better understanding of HPA-1a-specific antibody heterogeneity in FNAIT could aid in identifying pathogenic antibody properties linked to severe disease. STUDY DESIGN AND METHODS: This study aimed to isolate HPA-1a-specific B-cells from an HPA-1a-alloimmunized pregnant woman. Using fluorescently labeled HPA-1a-positive platelets, single B-cells were sorted and cultured for 10 days to stimulate antibody production. Subsequently, supernatants were tested for the presence of antibodies by enzyme-linked immunosorbent assay and their reactivity towards HPA-1a-positive platelets. Amplification and sequencing of variable regions allowed the generation of monoclonal antibodies using a HEK-Freestyle-based expression system. RESULTS: Three platelet-specific B-cells were obtained and cloned of which two were specific for HPA-1a, named D- and M-204, while the third was specific for HLA class I, which was named L-204. DISCUSSION: This study outlined an effective method for the isolation of HPA-1a-specific B-cells and the generation of monoclonal antibodies. Further characterization of these antibodies holds promise for better understanding the pathogenic nature of alloantibodies in FNAIT.
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Antígenos de Plaqueta Humana , Isoanticuerpos , Trombocitopenia Neonatal Aloinmune , Humanos , Antígenos de Plaqueta Humana/inmunología , Embarazo , Femenino , Trombocitopenia Neonatal Aloinmune/inmunología , Isoanticuerpos/inmunología , Integrina beta3/inmunología , Linfocitos B/inmunología , Anticuerpos Monoclonales/inmunología , Plaquetas/inmunología , Plaquetas/metabolismo , Recién NacidoRESUMEN
Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC) compared to other BC subtypes in clinical settings. Currently, there are no effective therapeutic strategies for TNBC treatment. Therefore, there is an urgent need to identify suitable biomarkers or therapeutic targets for TNBC patients. Thrombomodulin (TM) plays a role in cancer progression and metastasis in many different cancers. However, the role of TM in TNBC is not yet fully understood. First, silenced-TM in MDA-MB-231 cells caused an increase in proliferative and metastatic activity. In contrast, overexpression of TM in Hs578T cells caused a reduction in proliferation, invasion, and migration rate. Using RNA-seq analysis, we found that Integrin beta 3 (ITGB3) expression may be a downstream target of TM. Furthermore, we found an increase in ITGB3 levels in TM-KD cells by QPCR and western blot analysis but a decrease in ITGB3 levels in TM-overexpressing cells. We found phospho-smad2/3 levels were increased in TM-KD cells but decreased in TM-overexpressing cells. This implies that TM negatively regulates ITGB3 levels through the activation of the smad2/3 pathway. Silencing ITGB3 in TM-KD cells caused a decrease in proliferation and migration. Finally, we found that higher ITGB3 levels were correlated with poor overall survival and relapse-free survival in patients with TNBC. Our results indicated a novel regulatory relationship between TM and ITGB3 in TNBC.
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Neoplasias de la Mama Triple Negativas , Humanos , Western Blotting , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Integrina beta3/genética , Trombomodulina/genética , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Invadopodia are integrin-mediated adhesions with abundant PI(3,4)P2 However, the functional role of PI(3,4)P2 in adhesion signaling remains unclear. Here, we find that the PI(3,4)P2 biogenesis regulates the integrin endocytosis at invadopodia. PI(3,4)P2 is locally produced by PIK3CA and SHIP2 and is concentrated at the trailing edge of the invadopodium arc. The PI(3,4)P2-rich compartment locally forms small puncta (membrane buds) in a SNX9-dependent manner, recruits dynein activator Hook1 through AKTIP, and rearranges into micrometer-long tubular invaginations (membrane tubes). The uncurving membrane tube extends rapidly, follows the retrograde movement of dynein along microtubule tracks, and disconnects from the plasma membrane. Activated integrin-beta3 is locally internalized through the pathway of PI(3,4)P2-mediated membrane invagination and is then actively recycled. Blockages of PI3K, SHIP2, and SNX9 suppress integrin-beta3 endocytosis, delay adhesion turnover, and impede transwell invasion of MEF-Src and MDA-MB-231 cells. Thus, the production of PI(3,4)P2 promotes invasive cell migration by stimulating the trafficking of integrin receptor at the invadopodium.
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Movimiento Celular/efectos de los fármacos , Integrinas/metabolismo , Fosfatidilinositoles/metabolismo , Fosfatidilinositoles/farmacología , Adhesión Celular/efectos de los fármacos , Línea Celular , Membrana Celular/metabolismo , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Endocitosis/efectos de los fármacos , Humanos , Integrina beta3/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismo , Podosomas , Transducción de Señal/fisiologíaRESUMEN
Integrin αIIbß3 mediates platelet aggregation by binding the Arginyl-Glycyl-Aspartic acid (RGD) sequence of fibrinogen. RGD binding occurs at a site topographically proximal to the αIIb and ß3 subunits, promoting the conformational activation of the receptor from bent to extended states. While several experimental approaches have characterized RGD binding to αIIbß3 integrin, applying computational methods has been significantly more challenging due to limited sampling and the need for a priori information regarding the interactions between the RGD peptide and integrin. In this study, we employed all-atom simulations using funnel metadynamics (FM) to evaluate the interactions of an RGD peptide with the αIIb and ß3 subunits of integrin. FM incorporates an external history-dependent potential on selected degrees of freedom while applying a funnel-shaped restraint potential to limit RGD exploration of the unbound state. Furthermore, it does not require a priori information about the interactions, enhancing the sampling at a low computational cost. Our FM simulations reveal significant molecular changes in the ß3 subunit of integrin upon RGD binding and provide a free-energy landscape with a low-energy binding mode surrounded by higher-energy prebinding states. The strong agreement between previous experimental and computational data and our results highlights the reliability of FM as a method for studying dynamic interactions of complex systems such as integrin.
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Simulación de Dinámica Molecular , Oligopéptidos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , Unión Proteica , Oligopéptidos/química , Oligopéptidos/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Humanos , Plaquetas/metabolismo , Sitios de Unión , Integrina beta3/metabolismo , Integrina beta3/químicaRESUMEN
HER2-positive breast cancer, representing 15-20% of all breast cancer cases, often develops resistance to the HER2-targeted therapy trastuzumab. Unfortunately, effective treatments for advanced HER2-positive breast cancer remain scarce. This study aims to investigate the roles of ITGß3, and Hedgehog signaling in trastuzumab resistance and explore the potential of combining trastuzumab with cilengitide as a therapeutic strategy. Quantitative gene expression analysis was performed to assess the transcription of EMT (epithelial-mesenchymal transition) markers Slug, Snail, Twist2, and Zeb1 in trastuzumab-resistant HER2-positive breast cancer cells. The effects of ITGß3 and Hedgehog signaling were investigated. Additionally, the combination therapy of trastuzumab and cilengitide was evaluated. Acquired trastuzumab resistance induced the transcription of Slug, Snail, Twist2, and Zeb1, indicating increased EMT. This increased EMT was mediated by ITGB3 and Hedgehog signaling. ITGß3 regulated both the Hedgehog pathway and EMT, with the latter being independent of the Hedgehog pathway. The combination of trastuzumab and cilengitide showed a synergistic effect, reducing both EMT and Hedgehog pathway activity. Targeting ITGß3 with cilengitide, combined with trastuzumab, effectively suppresses the Hedgehog pathway and EMT, offering a potential strategy to overcome trastuzumab resistance and improve outcomes for HER2-positive breast cancer patients.
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Neoplasias de la Mama , Resistencia a Antineoplásicos , Transición Epitelial-Mesenquimal , Integrina beta3 , Receptor ErbB-2 , Trastuzumab , Humanos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Trastuzumab/farmacología , Trastuzumab/uso terapéutico , Integrina beta3/metabolismo , Integrina beta3/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-2/genética , Línea Celular Tumoral , Transducción de Señal/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Hedgehog/metabolismo , Venenos de SerpienteRESUMEN
Metastatic melanoma, a deadly form of skin cancer, often develops resistance to the BRAF inhibitor drug vemurafenib, highlighting the need for understanding the underlying mechanisms of resistance and exploring potential therapeutic strategies targeting integrins and TGF-ß signalling. In this study, the role of integrins and TGF-ß signalling in vemurafenib resistance in melanoma was investigated, and the potential of combining vemurafenib with cilengitide as a therapeutic strategy was investigated. In this study, it was found that the transcription of PAI1 and p21 was induced by acquired vemurafenib resistance, and ITGA5 levels were increased as a result of this resistance. The transcription of ITGA5 was mediated by the TGF-ß pathway in the development of vemurafenib resistance. A synergistic effect on the proliferation of vemurafenib-resistant melanoma cells was observed with the combination therapy of vemurafenib and cilengitide. Additionally, this combination therapy significantly decreased invasion and colony formation in these resistant cells. In conclusion, it is suggested that targeting integrins and TGF-ß signalling, specifically ITGA5, ITGB3, PAI1, and p21, may offer promising approaches to overcoming vemurafenib resistance, thereby improving outcomes for metastatic melanoma patients.
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Resistencia a Antineoplásicos , Melanoma , Venenos de Serpiente , Vemurafenib , Vemurafenib/farmacología , Vemurafenib/uso terapéutico , Humanos , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Melanoma/patología , Melanoma/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Línea Celular Tumoral , Venenos de Serpiente/farmacología , Integrina beta3/metabolismo , Integrina beta3/genética , Factor de Crecimiento Transformador beta/metabolismo , Transducción de Señal/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Integrinas/metabolismo , Integrinas/antagonistas & inhibidores , Integrina alfa5/metabolismo , Integrina alfa5/genética , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Indoles/farmacología , Indoles/uso terapéutico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéuticoRESUMEN
Mutations in the αIIb ß-propeller domain have long been known to disrupt heterodimerization and intracellular trafficking of αIIbß3 complexes leading to diminished surface expression and/or function, resulting in Glanzmann thrombasthenia. Our previous study on three ß-propeller mutations, namely G128S, S287L, and G357S, showed variable defects in protein transport correlated with the patient's clinical phenotypes. Pulse-chase experiments revealed differences in αIIbß3 complex maturation among the three mutations. Hence, the current study aims to correlate conformational changes caused by each one of them. Evolutionary conservation analysis, stability analysis, and molecular dynamics simulations of the three mutant structures were carried out. Stability analysis revealed that, while G128S and G357S mutations destabilized the ß-propeller structure, S287L retained the stability. Wild-type and mutant ß-propeller structures, when subjected to molecular dynamics simulations, confirmed that G128S and G357S were both destabilizing in nature when compared with the wild-type and S287L based on several parameters studied, like RMSD, RMSF, Rg, FEL, PCA, secondary structure, and hydrogen bonds. In our previous study, we demonstrated that mutant S287L αIIbß3 complexes were more stable than the wild-type αIIbß3 complexes, as evidenced in pulse-chase experiments. These findings corroborate variable intracellular fates of mutant αIIbß3 complexes as a result of these ß-propeller mutations.
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Integrina alfa2 , Integrina beta3 , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , Trombastenia , Humanos , Integrina beta3/genética , Simulación de Dinámica Molecular , Mutación , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Estructura Secundaria de Proteína , Trombastenia/genética , Trombastenia/metabolismo , Integrina alfa2/genética , Integrina alfa2/metabolismoRESUMEN
Mechanical ventilation (MV) has become a clinical first-line treatment option for patients with respiratory failure. However, it was unclear whether MV further aggravates the process of sepsis-associated pulmonary fibrosis and eventually leads to sepsis and mechanical ventilation-associated pulmonary fibrosis (S-MVPF). This study aimed to explore the mechanism of S-MVPF concerning integrin ß3 activation in glycometabolic reprogramming of lung fibroblasts. We found that MV exacerbated sepsis-associated pulmonary fibrosis induced by lipopolysaccharide, which was accompanied by proliferation of lung fibroblasts, increased deposition of collagen in lung tissue, and increased procollagen type I carboxy-terminal propeptide in the bronchoalveolar lavage fluid. A large number of integrin ß3- and pyruvate kinase M2-positive fibroblasts were detected in lung tissue after stimulation with lipopolysaccharide and MV, with an increase in lactate dehydrogenase A expression and lactate levels. S-MVPF was primarily attenuated in integrin ß3-knockout mice, which also resulted in a decrease in the levels of pyruvate kinase M2, lactate dehydrogenase A, and lactate. In conclusion, MV aggravated sepsis-associated pulmonary fibrosis, with glycometabolic reprogramming mediated by integrin ß3 activation. Thus, integrin ß3-mediated glycometabolic reprogramming might be a potential therapeutic target for S-MVPF.
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Fibrosis Pulmonar , Sepsis , Ratones , Animales , Fibrosis Pulmonar/etiología , Fibrosis Pulmonar/metabolismo , Integrina beta3/metabolismo , Respiración Artificial , Lipopolisacáridos , Lactato Deshidrogenasa 5 , Piruvato Quinasa , Sepsis/complicacionesRESUMEN
In excitatory hippocampal pyramidal neurons, integrin ß3 is critical for synaptic maturation and plasticity in vitro. Itgb3 is a potential autism susceptibility gene that regulates dendritic morphology in the cerebral cortex in a cell-specific manner. However, it is unknown what role Itgb3 could have in regulating hippocampal pyramidal dendritic morphology in vivo, a key feature that is aberrant in many forms of autism and intellectual disability. We found that Itgb3 mRNA is expressed in the stratum pyramidale of CA3. We examined the apical dendritic morphology of CA3 hippocampal pyramidal neurons in conditional Itgb3 knockouts and controls, utilizing the Thy1-GFP-M line. We fully reconstructed the apical dendrite of each neuron and determined each neuron's precise location along the dorsoventral, proximodistal, and radial axes of the stratum pyramidale. We found a very strong effect for Itgb3 expression on CA3 apical dendritic morphology: neurons from conditional Itgb3 knockouts had longer and thinner apical dendrites than controls, particularly in higher branch orders. We also assessed potential relationships between pairs of topographic or morphological variables, finding that most variable pairs were free from any linear relationships to each other. We also found that some neurons from controls, but not conditional Itgb3 knockouts, had a graded pattern of overall diameter along the dorsoventral and proximodistal axes of the stratum pyramidale of CA3. Taken together, Itgb3 is essential for constructing normal dendritic morphology in pyramidal neurons throughout CA3.
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Dendritas , Integrina beta3 , Integrina beta3/genética , Dendritas/fisiología , Hipocampo/fisiología , Células Piramidales/fisiología , NeuronasRESUMEN
Due to its key roles in malignant tumor progression and reprograming of the tumor microenvironment, integrin ß3 has attracted great attention as a new target for tumor therapy. However, the structure-function relationship of integrins ß3 remains incompletely understood, leading to the shortage of specific and effective targeting probes. This work uses a purified extracellular domain of integrin ß3 and integrin ß3-positive cells to screen aptamers, specifically targeting integrin ß3 in the native conformation on live cells through the SELEX approach. Following meticulous truncation and characterization of the initial aptamer candidates, the optimized aptamer S10yh2 was produced, exhibiting a low equilibrium dissociation constant (Kd) in the nanomolar range. S10yh2 displays specific recognition of cancer cells with varying levels of integrin ß3 expression and demonstrates favorable stability in serum. Subsequent analysis of docking sites revealed that S10yh2 binds to the seven amino acid residues located in the core region of integrin ß3. The S10yh2 aptamer can downregulate the level of integrin heterodimer αvß3 on integrin ß3 overexpressed cancer cells and partially inhibit cell migration behavior. In summary, S10yh2 is a promising probe with a small size, simple synthesis, good stability, high binding affinity, and selectivity. It therefore holds great potential for investigating the structure-function relationship of integrins.
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Aptámeros de Nucleótidos , Neoplasias , Humanos , Integrina beta3/química , Integrina beta3/metabolismo , Aptámeros de Nucleótidos/farmacología , Integrina alfaVbeta3/metabolismo , Movimiento Celular , Microambiente TumoralRESUMEN
BACKGROUND: In response to brain injury or inflammation, astrocytes undergo hypertrophy, proliferate, and migrate to the damaged zone. These changes, collectively known as "astrogliosis", initially protect the brain; however, astrogliosis can also cause neuronal dysfunction. Additionally, these astrocytes undergo intracellular changes involving alterations in the expression and localization of many proteins, including αvß3 integrin. Our previous reports indicate that Thy-1, a neuronal glycoprotein, binds to this integrin inducing Connexin43 (Cx43) hemichannel (HC) opening, ATP release, and astrocyte migration. Despite such insight, important links and molecular events leading to astrogliosis remain to be defined. METHODS: Using bioinformatics approaches, we analyzed different Gene Expression Omnibus datasets to identify changes occurring in reactive astrocytes as compared to astrocytes from the normal mouse brain. In silico analysis was validated by both qRT-PCR and immunoblotting using reactive astrocyte cultures from the normal rat brain treated with TNF and from the brain of a hSOD1G93A transgenic mouse model. We evaluated the phosphorylation of Cx43 serine residue 373 (S373) by AKT and ATP release as a functional assay for HC opening. In vivo experiments were also performed with an AKT inhibitor (AKTi). RESULTS: The bioinformatics analysis revealed that genes of the PI3K/AKT signaling pathway were among the most significantly altered in reactive astrocytes. mRNA and protein levels of PI3K, AKT, as well as Cx43, were elevated in reactive astrocytes from normal rats and from hSOD1G93A transgenic mice, as compared to controls. In vitro, reactive astrocytes stimulated with Thy-1 responded by activating AKT, which phosphorylated S373Cx43. Increased pS373Cx43 augmented the release of ATP to the extracellular medium and AKTi inhibited these Thy-1-induced responses. Furthermore, in an in vivo model of inflammation (brain damage), AKTi decreased the levels of astrocyte reactivity markers and S373Cx43 phosphorylation. CONCLUSIONS: Here, we identify changes in the PI3K/AKT molecular signaling network and show how they participate in astrogliosis by regulating the HC protein Cx43. Moreover, because HC opening and ATP release are important in astrocyte reactivity, the phosphorylation of Cx43 by AKT and the associated increase in ATP release identify a potential therapeutic window of opportunity to limit the adverse effects of astrogliosis.
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Lesiones Encefálicas , Conexina 43 , Animales , Ratones , Ratas , Adenosina Trifosfato/farmacología , Adenosina Trifosfato/metabolismo , Astrocitos/metabolismo , Lesiones Encefálicas/metabolismo , Conexina 43/metabolismo , Gliosis/metabolismo , Inflamación/metabolismo , Integrina beta3/genética , Integrina beta3/metabolismo , Integrina beta3/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Regulación hacia Arriba , Antígenos Thy-1/metabolismo , Integrina alfa5/metabolismoRESUMEN
The αIIbß3 integrin receptor coordinates platelet adhesion, activation, and mechanosensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in â¼1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbß3 is predetermined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts transfected with recombinant integrin. From coimmunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in baby hamster kidney fibroblast cells expressing a mutant integrin with an ablated C490-C545 disulfide bond. The disulfide mutant integrin has functional outside-in signaling but extended residency time in focal adhesions due to a reduced rate of clathrin-mediated integrin internalization and recycling, which is associated with enhanced affinity of the αIIb subunit for clathrin adaptor protein 2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the αIIbß3 integrin receptor is produced in various covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin α subunits, and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that the alternate integrin form and function are also conserved.
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Adhesiones Focales/metabolismo , Integrina beta3/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Animales , Línea Celular , Cricetinae , Disulfuros/análisis , Adhesiones Focales/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Integrina beta3/química , Integrina beta3/genética , Simulación de Dinámica Molecular , Mutación , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Glicoproteína IIb de Membrana Plaquetaria/química , Glicoproteína IIb de Membrana Plaquetaria/genéticaRESUMEN
BACKGROUND: The tumour microenvironment consists of a complex and dynamic milieu of cancer cells, including tumour-associated stromal cells (leukocytes, fibroblasts, vascular cells, etc.) and their extracellular products. During invasion and metastasis, cancer cells actively remodel the tumour microenvironment and alterations of microenvironment, particularly cancer-associated fibroblasts (CAFs), can promote tumour progression. However, the underlying mechanisms of the CAF formation and their metastasis-promoting functions remain unclear. METHODS: Primary liver fibroblasts and CAFs were isolated and characterized. CAFs in clinical samples were identified by immunohistochemical staining and the clinical significance of CAFs was also analysed in two independent cohorts. A transwell coculture system was used to confirm the role of HCC cells in CAF recruitment and activation. qRT-PCR, western blotting and ELISA were used to screen paracrine cytokines. The role and mechanism of Egfl7 in CAFs were explored via an in vitro coculture system and an in vivo mouse orthotopic transplantation model. RESULTS: We showed that CAFs in hepatocellular carcinoma (HCC) are characterized by the expression of α-SMA and that HCC cells can recruit liver fibroblasts (LFs) and activate them to promote their transformation into CAFs. High α-SMA expression, indicating high CAF infiltration, was correlated with malignant characteristics. It was also an independent risk factor for HCC survival and could predict a poor prognosis in HCC patients. Then, we demonstrated that EGF-like domain multiple 7 (Egfl7) was preferentially secreted by HCC cells, and exhibited high potential to recruit and activate LFs into the CAF phenotype. The ability of Egfl7 to modulate LFs relies upon increased phosphorylation of FAK and AKT via the receptor ανß3 integrin. Strikingly, CAFs activated by paracrine Egfl7 could further remodel the tumour microenvironment by depositing fibrils and collagen and in turn facilitate HCC cell proliferation, invasion and metastasis. CONCLUSION: Our data highlighted a novel role of Egfl7 in remodelling the tumour microenvironment: it recruits LFs and activates them to promote their transformation into CAFs via the ανß3 integrin signaling pathway, which further promotes HCC progression and contributes to poor clinical outcomes in HCC patients. Video Abstract.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Ratones , Fibroblastos , Integrina beta3 , Péptidos y Proteínas de Señalización Intercelular , Microambiente TumoralRESUMEN
Optical imaging (OI) is gaining increasing attention in medicine as a non-invasive diagnostic imaging technology and as a useful tool for image-guided surgery. OI exploits the light emitted in the near-infrared region by fluorescent molecules able to penetrate living tissues. Cyanines are an important class of fluorescent molecules and by their conjugation to peptides it is possible to achieve optical imaging of tumours by selective targeting. We report here the improvements obtained in the synthesis of DA364, a small fluorescent probe (1.5 kDa) prepared by conjugation of pentamethine cyanine Cy5.5 to an RGD peptidomimetic, which can target tumour cells overexpressing integrin αvß3 receptors.
Asunto(s)
Integrina alfaVbeta3 , Integrina beta3 , Línea Celular Tumoral , Oligopéptidos/químicaRESUMEN
Binding of the intracellular adapter proteins talin and its cofactor, kindlin, to the integrin receptors induces integrin activation and clustering. These processes are essential for cell adhesion, migration, and organ development. Although the talin head, the integrin-binding segment in talin, possesses a typical FERM-domain sequence, a truncated form has been crystallized in an unexpected, elongated form. This form, however, lacks a C-terminal fragment and possesses reduced ß3-integrin binding. Here, we present a crystal structure of a full-length talin head in complex with the ß3-integrin tail. The structure reveals a compact FERM-like conformation and a tightly associated N-P-L-Y motif of ß3-integrin. A critical C-terminal poly-lysine motif mediates FERM interdomain contacts and assures the tight association with the ß3-integrin cytoplasmic segment. Removal of the poly-lysine motif or disrupting the FERM-folded configuration of the talin head significantly impairs integrin activation and clustering. Therefore, structural characterization of the FERM-folded active talin head provides fundamental understanding of the regulatory mechanism of integrin function.
Asunto(s)
Integrina beta3/metabolismo , Talina/química , Talina/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Humanos , Integrina beta3/química , Leucina/metabolismo , Ratones , Microscopía Electrónica de Transmisión , Modelos Moleculares , Mutagénesis , Polilisina/química , Dominios Proteicos , Pliegue de Proteína , Talina/genéticaRESUMEN
Tumor tissues often exhibit unique integrin receptor presentation during development, such as high exposures of αvß3 and αIIbß3 integrins. These features are not present in normal tissues. The induction of selective thrombosis and infarction in the tumor-feeding vessels, as well as specific antagonism of αvß3 integrin on the surface of tumor endothelial cells, is a potential novel antitumor strategy. The Echistatin-Annexin V (EAV) fusion protein is a novel Annexin V (ANV) derivative that possesses a high degree of αvß3 and αIIbß3 integrin receptor recognition and binding characteristics while retaining the specific binding ability of the natural ANV molecule for phosphatidylserine (PS). We systematically investigated the biological effects of this novel molecule with superimposed functions on mouse melanoma. We found that EAV inhibited the viability and migration of B16F10 murine melanoma cells in a dose-dependent manner, exhibited good tumor suppressive effects in a xenograft mouse melanoma model, strongly induced tumor tissue necrosis in mice, and targeted the inhibition of angiogenesis in mouse melanoma tumor tissue. EAV exhibited stronger biological effects than natural ANV molecules in inhibiting melanoma in mice. The unique biological effects of EAV are based on its high ß3-type integrin receptor-specific recognition and binding ability, as well as its highly selective binding to PS molecules. Based on these findings, we propose that EAV-mediated tumor suppression is a novel and promising antitumor strategy that targets both PS- and integrin ß3-positive tumor neovascularization and the tumor cells themselves, thus providing a possible mechanism for the treatment of melanoma.
Asunto(s)
Integrina beta3 , Melanoma , Humanos , Ratones , Animales , Integrina beta3/metabolismo , Anexina A5/metabolismo , Células Endoteliales/metabolismo , Melanoma/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Integrina alfaVbeta3/metabolismoRESUMEN
The Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) is known to dephosphorylate PtdIns(3,4,5)P3 into PtdIns(3,4)P2 and to interact with several signaling proteins though its docking functions. It has been shown to negatively regulate platelet adhesion and spreading on a fibrinogen surface and to positively regulate thrombus growth. In the present study, we have investigated its role during the early phase of platelet activation. Using confocal-based morphometric analysis, we found that SHIP1 is involved in the regulation of cytoskeletal organization and internal contractile activity in thrombin-activated platelets. The absence of SHIP1 has no significant impact on thrombin-induced Akt or Erk1/2 activation, but it selectively affects the RhoA/Rho-kinase pathway and myosin IIA relocalization to the cytoskeleton. SHIP1 interacts with the spectrin-based membrane skeleton, and its absence induces a loss of sustained association of integrins to this network together with a decrease in αIIbß3 integrin clustering following thrombin stimulation. This αIIbß3 integrin dynamics requires the contractile cytoskeleton under the control of SHIP1. RhoA activation, internal platelet contraction, and membrane skeleton integrin association were insensitive to the inhibition of PtdIns(3,4,5)P3 synthesis or SHIP1 phosphatase activity, indicating a role for the docking properties of SHIP1 in these processes. Altogether, our data reveal a lipid-independent function for SHIP1 in the regulation of the contractile cytoskeleton and integrin dynamics in platelets.