RESUMEN
Extracellular vesicles (EVs) influence a host of normal and pathophysiological processes in vivo. Compared to soluble mediators, EVs can traffic a wide range of proteins on their surface including extracellular matrix (ECM) binding proteins, and their large size (â¼30-150 nm) limits diffusion. We isolated EVs from the MCF10 series-a model human cell line of breast cancer progression-and demonstrated increasing presence of laminin-binding integrins α3ß1 and α6ß1 on the EVs as the malignant potential of the MCF10 cells increased. Transport of the EVs within a microfluidic device under controlled physiological interstitial flow (0.15-0.75 µm/s) demonstrated that convection was the dominant mechanism of transport. Binding of the EVs to the ECM enhanced the spatial concentration and gradient, which was mitigated by blocking integrins α3ß1 and α6ß1. Our studies demonstrate that convection and ECM binding are the dominant mechanisms controlling EV interstitial transport and should be leveraged in nanotherapeutic design.
Asunto(s)
Vesículas Extracelulares , Laminina , Humanos , Laminina/metabolismo , Convección , Integrina alfa6beta1/metabolismo , Vesículas Extracelulares/metabolismo , Integrina alfa3beta1/metabolismo , Matriz Extracelular/metabolismoRESUMEN
Tissue engineering advancements have made it possible to modify biomaterials to reconstruct a similar three-dimensional structure of the extracellular matrix (ECM) for follicle development and to supply the required biological signals. We postulated that an artificial polysaccharide hydrogel modified with an ECM mimetic peptide may produce efficient irritation signals by binding to specific integrins providing a suitable environment for follicular development and influencing the behavior of human granulosa cells (hGCs). Laminin, an important component of the extracellular matrix, can modulate hGCs and oocyte growth. Specifically, follicles of mice were randomly divided into two-dimensional (2D) and three-dimensional (3D) culture systems established by a hydrogel modified with RGD or laminin mimetic peptides (IKVAV and YIGSR) and RGD (IYR). Our results showed that 3D cultured systems significantly improved follicle survival, growth, and viability. IYR peptides enhanced the oocyte meiosis competence. Additionally, we explored the effect of 3D culture on hGCs, which improved hGCs viability, increased the proportion of S- and G2/M-phase cells, and inhibited cell apoptosis of hGCs. On days 1 and 2, the secretion of progesterone was reduced in 3D-cultured hGCs. Notably, 3D-cultured hGCs exhibited delayed senescence, decreased oxidative stress, and elevated mitochondrial membrane potential. Moreover, the expression levels of cumulus expansion-related genes (COX2, HAS2, and PTX3) and integrin α6ß1 were upregulated in 3D-cultured hGCs. In conclusion, a 3D culture utilizing hydrogels modified with Laminin-mimetic peptides can provide a durable physical environment suitable for follicular development. The laminin-mimetic peptides may regulate the biological activity of hGCs by attaching to the integrin α6ß1.
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Células de la Granulosa , Laminina , Femenino , Humanos , Ratones , Animales , Laminina/farmacología , Integrina alfa6beta1/metabolismo , Células de la Granulosa/metabolismo , Hidrogeles/farmacología , Péptidos/farmacología , Péptidos/metabolismo , Oligopéptidos/farmacología , Oligopéptidos/metabolismo , Estrés OxidativoRESUMEN
Hepatic progenitor cells (HPCs) hold tremendous potential for liver regeneration, but their well-known limitation of proliferation hampers their broader use. There is evidence that laminin is required for the proliferation of HPCs, but the laminin isoform that plays the dominant role and the key intracellular downstream targets that mediate the regulation of HPC proliferation have yet to be determined. Here we showed that p53 expression increased gradually and reached maximal levels around 8 days when laminin α4, α5, ß2, ß1, and γ1 subunit levels also reached a maximum during HPC activation and expansion. Laminin-521 (LN-521) promoted greater proliferation of HPCs than do laminin, matrigel or other laminin isoforms. Inactivation of p53 by PFT-α or Ad-p53V143A inhibited the promotion of proliferation by LN-521. Further complementary MRI and bioluminescence imaging analysis showed that p53 inactivation decreased the proliferation of transplanted HPCs in vivo. p53 was activated by LN-521 through the Integrin α6ß1/FAK-Src-Paxillin/Akt axis. Activated p53 was involved in the nuclear translocation of CDK4 and inactivation of Rb by inducing p27Kip1. Taken together, this study identifies LN-521 as an ideal candidate substrate for HPC culture and uncovers an unexpected positive role for p53 in regulating proliferation of HPCs, which makes it a potential target for HPC-based regenerative medicine.
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Laminina , Proteína p53 Supresora de Tumor , Proliferación Celular/genética , Integrina alfa6beta1/metabolismo , Laminina/genética , Laminina/metabolismo , Células Madre/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
In human induced pluripotent stem cells (hiPSCs), laminin-511/α6ß1 integrin interacts with E-cadherin, an intercellular adhesion molecule, to induce the activation of the phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathway. The interaction between laminin-511/α6ß1 integrin and E-cadherin, an intercellular adhesion molecule, results in protection against apoptosis through the proto-oncogene tyrosine-protein kinase Fyn(Fyn)-RhoA-ROCK signaling pathway and the Ras homolog gene family member A (RhoA)/Rho kinase (ROCK) signaling pathway (the major pathway for cell death). In this article, the impact of laminin-511 on hiPSC on α6ß1 integrin-Fyn-RhoA-ROCK signaling is discussed and explored along with validation experiments. PIK3CA mRNA (mean [standard deviation {SD}]: iMatrix-511, 1.00 [0.61]; collagen+MFGE8, 0.023 [0.02]; **P < 0.01; n = 6) and PIK3R1 mRNA (mean [SD]: iMatrix-511, 1.00 [0.79]; collagen+MFGE8, 0.040 [0.06]; *P < 0.05; n = 6) were upregulated by iMatrix-511 resulting from an increased expression of Integrin α6 mRNA (mean [SD]: iMatrix-511, 1.00 [0.42]; collagen+MFGE8, 0.23 [0.05]; **P < 0.01; n = 6). The iMatrix-511 increased the expression of p120-Catenin mRNA (mean [SD]: iMatrix-511, 1.00 [0.71]; collagen+MFGE8, 0.025 [0.03]; **P < 0.01; n = 6) and RAC1 mRNA (mean [SD]: iMatrix-511, 1.00 [0.28]; collagen+MFGE8, 0.39 [0.15]; **P < 0.01; n = 6) by increasing the expression of E-cadherin mRNA (mean [SD]: iMatrix-511, 1.00 [0.38]; collagen+MFGE8, 0.16 [0.11]; **P < 0.01; n = 6). As a result, iMatrix-511 increased the expression of P190 RhoGAP (GTPase-activating proteins) mRNA, such as ARHGAP1 mRNA (mean [SD]: iMatrix-511, 1.00 [0.57]; collagen+MFGE8, 0.032 [0.03]; **P < 0.01; n = 6), ARHGAP4 mRNA (mean [SD]: iMatrix-511, 1.00 [0.56]; collagen+MFGE8, 0.039 [0.049]; **P < 0.01; n = 6), and ARHGAP5 mRNA (mean [SD]: iMatrix-511, 1.00 [0.39]; collagen+MFGE8, 0.063 [0.043]; **P < 0.01; n = 6). Western blotting showed that phospho-Rac1 remained in the cytoplasm and phospho-Fyn showed nuclear transition in iPSCs cultured on iMatrix-511. Proteome analysis showed that PI3K signaling was enhanced and cytoskeletal actin was activated in iPSCs cultured on iMatrix-511. In conclusion, laminin-511 strongly activated the cell survival by promoting α6ß1 integrin-Fyn-RhoA-ROCK signaling in hiPSCs.
Asunto(s)
Células Madre Pluripotentes Inducidas , Laminina , Humanos , Integrina alfa6beta1/metabolismo , Laminina/metabolismo , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Cadherinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Colágeno/metabolismo , ARN Mensajero , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
CCN1/CYR61 promotes angiogenesis, tumor growth and chemoresistance by binding to its integrin receptor αvß3 in endothelial and breast cancer (BC) cells. CCN1 controls also tissue regeneration by engaging its integrin receptor α6ß1 to induce fibroblast senescence. Here, we explored if the ability of CCN1 to drive an endocrine resistance phenotype in estrogen receptor-positive BC cells relies on interactions with either αvß3 or α6ß1. First, we took advantage of site-specific mutagenesis abolishing the CCN1 receptor-binding sites to αvß3 and α6ß1 to determine the integrin partner responsible for CCN1-driven endocrine resistance. Second, we explored a putative nuclear role of CCN1 in regulating ERα-driven transcriptional responses. Retroviral forced expression of a CCN1 derivative with a single amino acid change (D125A) that abrogates binding to αvß3 partially phenocopied the endocrine resistance phenotype induced upon overexpression of wild-type (WT) CCN1. Forced expression of the CCN1 mutant TM, which abrogates all the T1, H1, and H2 binding sites to α6ß1, failed to bypass the estrogen requirement for anchorage-independent growth or to promote resistance to tamoxifen. Wild-type CCN1 promoted estradiol-independent transcriptional activity of ERα and enhanced ERα agonist response to tamoxifen. The α6ß1-binding-defective TM-CCN1 mutant lost the ERα co-activator-like behavior of WT-CCN1. Co-immunoprecipitation assays revealed a direct interaction between endogenous CCN1 and ERα, and in vitro approaches confirmed the ability of recombinant CCN1 to bind ERα. CCN1 signaling via α6ß1, but not via αvß3, drives an endocrine resistance phenotype that involves a direct binding of CCN1 to ERα to regulate its transcriptional activity in ER+ BC cells.
Asunto(s)
Neoplasias de la Mama , Receptor alfa de Estrógeno , Neoplasias de la Mama/genética , Proteína 61 Rica en Cisteína/genética , Proteína 61 Rica en Cisteína/metabolismo , Receptor alfa de Estrógeno/genética , Femenino , Humanos , Integrina alfa6beta1/metabolismo , Integrinas , Tamoxifeno/farmacologíaRESUMEN
The most promising therapy for leukemia is hematopoietic stem cell transplantation. Engraftment of HPSCs mainly depends on some factors such as adhesion molecules, including VLAs. This study tried to delineate the relationship between HPSCs engraftment and expression level of PSGL1 and VLA4, 5, and 6 genes in candidate MM patients for autologous bone marrow transplantation. Firstly, the CD 34+ HPSCs were collected from multiple myeloma (MM) patients after five days of G-CSF therapy through apheresis processes. Then, the patients were categorized into two groups of good and bad prognosis depending on engraftment time (Less or more than 18 days). Followingly, the expression of PSGL1 and VLA4, VLA5, and VLA6 genes were assessed by the qRT-PCR technique in each patient. Finally, the correlation between the genes and engraftment time was investigated to determine the prognostic role of each gene on HPSCs transplantation. Our findings demonstrated that there is a significant correlation between VLA4 (P=< 0.0001) and 5 (P = 0.005) levels and HPSCs engraftment time. As the higher levels of VLA4 and 5, the shorter time HPSCs engraftment occurs. In contrast, there was no significant correlation between VLA6 (P = 0.2) and PSGL1 (P = 0.3) genes levels and engraftment time. So that, the patients with a good prognosis had a higher level of VLA4 and VLA5, but no relation was found between VLA6 and PSGL1. It is concluded that VLA4 and VLA5 expression could be considered a significant prognostic factor for HPSC transplantation.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas/métodos , Integrina alfa4beta1/metabolismo , Integrina alfa5beta1/metabolismo , Integrina alfa6beta1/metabolismo , Glicoproteínas de Membrana/metabolismo , Mieloma Múltiple/genética , Mieloma Múltiple/terapia , Acondicionamiento Pretrasplante/métodos , Trasplante Autólogo/métodos , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/patologíaRESUMEN
RATIONALE: αv integrins, key regulators of transforming growth factor-ß activation and fibrogenesis in in vivo models of pulmonary fibrosis, are expressed on abnormal epithelial cells (αvß6) and fibroblasts (αvß1) in fibrotic lungs. OBJECTIVES: We evaluated multiple αv integrin inhibition strategies to assess which most effectively reduced fibrogenesis in explanted lung tissue from patients with idiopathic pulmonary fibrosis. METHODS: Selective αvß6 and αvß1, dual αvß6/αvß1, and multi-αv integrin inhibitors were characterized for potency, selectivity, and functional activity by ligand binding, cell adhesion, and transforming growth factor-ß cell activation assays. Precision-cut lung slices generated from lung explants from patients with idiopathic pulmonary fibrosis or bleomycin-challenged mouse lungs were treated with integrin inhibitors or standard-of-care drugs (nintedanib or pirfenidone) and analyzed for changes in fibrotic gene expression or TGF-ß signaling. Bleomycin-challenged mice treated with dual αvß6/αvß1 integrin inhibitor, PLN-74809, were assessed for changes in pulmonary collagen deposition and Smad3 phosphorylation. MEASUREMENTS AND MAIN RESULTS: Inhibition of integrins αvß6 and αvß1 was additive in reducing type I collagen gene expression in explanted lung tissue slices from patients with idiopathic pulmonary fibrosis. These data were replicated in fibrotic mouse lung tissue, with no added benefit observed from inhibition of additional αv integrins. Antifibrotic efficacy of dual αvß6/αvß1 integrin inhibitor PLN-74809 was confirmed in vivo, where dose-dependent inhibition of pulmonary Smad3 phosphorylation and collagen deposition was observed. PLN-74809 also, more potently, reduced collagen gene expression in fibrotic human and mouse lung slices than clinically relevant concentrations of nintedanib or pirfenidone. CONCLUSIONS: In the fibrotic lung, dual inhibition of integrins αvß6 and αvß1 offers the optimal approach for blocking fibrogenesis resulting from integrin-mediated activation of transforming growth factor-ß.
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Antifibróticos/farmacología , Células Epiteliales/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Integrina alfa6beta1/antagonistas & inhibidores , Pulmón/efectos de los fármacos , Receptores de Vitronectina/antagonistas & inhibidores , Animales , Bleomicina , Línea Celular , Técnicas de Cocultivo , Cadena alfa 1 del Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Integrina alfa6beta1/metabolismo , Pulmón/metabolismo , Pulmón/patología , Ratones Endogámicos C57BL , Fosforilación , Receptores de Vitronectina/metabolismo , Transducción de Señal , Proteína smad3/metabolismoRESUMEN
Recognition of laminin by integrin receptors is central to the epithelial cell adhesion to basement membrane, but the structural background of this molecular interaction remained elusive. Here, we report the structures of the prototypic laminin receptor α6ß1 integrin alone and in complex with three-chain laminin-511 fragment determined via crystallography and cryo-electron microscopy, respectively. The laminin-integrin interface is made up of several binding sites located on all five subunits, with the laminin γ1 chain C-terminal portion providing focal interaction using two carboxylate anchor points to bridge metal-ion dependent adhesion site of integrin ß1 subunit and Asn189 of integrin α6 subunit. Laminin α5 chain also contributes to the affinity and specificity by making electrostatic interactions with large surface on the ß-propeller domain of α6, part of which comprises an alternatively spliced X1 region. The propeller sheet corresponding to this region shows unusually high mobility, suggesting its unique role in ligand capture.
Asunto(s)
Células Epiteliales/metabolismo , Integrina alfa6/metabolismo , Integrina alfa6beta1/metabolismo , Integrina beta1/metabolismo , Laminina/metabolismo , Secuencia de Aminoácidos , Membrana Basal/metabolismo , Sitios de Unión/fisiología , Adhesión Celular/fisiología , Microscopía por Crioelectrón , Cristalografía por Rayos X , Humanos , Conformación Proteica , Dominios Proteicos/fisiología , Electricidad EstáticaRESUMEN
The delivery of glucose and antioxidants is vital to maintain homeostasis and lens transparency. Here, we report a new mechanism whereby mechanically activated connexin (Cx) hemichannels serve as a transport portal for delivering glucose and glutathione (GSH). Integrin α6ß1 in outer cortical lens fiber activated by fluid flow shear stress (FFSS) induced opening of hemichannels. Inhibition of α6 activation prevented hemichannel opening as well as glucose and GSH uptake. The activation of integrin ß1, a heterodimeric partner of α6 in the absence of FFSS, increased Cx50 hemichannel opening. Hemichannel activation by FFSS depended on the interaction of integrin α6 and Cx50 C-terminal domain. Moreover, hemichannels in nuclear fiber were unresponsive owing to Cx50 truncation. Taken together, these results show that mechanically activated α6ß1 integrin in outer cortical lens fibers leads to opening of hemichannels, which transport glucose and GSH into cortical lens fibers. This study unveils a new transport mechanism that maintains metabolic and antioxidative function of the lens.
Asunto(s)
Antioxidantes/metabolismo , Proteínas Aviares/metabolismo , Conexinas/metabolismo , Glutatión/metabolismo , Integrina alfa6beta1/metabolismo , Cristalino/metabolismo , Mecanotransducción Celular , Animales , Proteínas Aviares/genética , Transporte Biológico Activo , Embrión de Pollo , Pollos , Conexinas/genética , Integrina alfa6beta1/genética , Dominios ProteicosRESUMEN
The androgen receptor (AR) is the major driver of prostate cancer growth and survival. However, almost all patients relapse with castration-resistant disease (CRPC) when treated with anti-androgen therapy. In CRPC, AR is often aberrantly activated independent of androgen. Targeting survival pathways downstream of AR could be a viable strategy to overcome CRPC. Surprisingly, little is known about how AR drives prostate cancer survival. Furthermore, CRPC tumors in which Pten is lost are also resistant to eradication by PI3K inhibitors. We sought to identify the mechanism by which AR drives tumor survival in CRPC to identify ways to overcome resistance to PI3K inhibition. We found that integrins α6ß1 and Bnip3 are selectively elevated in CRPC downstream of AR. While integrin α6 promotes survival and is a direct transcriptional target of AR, the ability of AR to induce Bnip3 is dependent on adhesion to laminin and integrin α6ß1-dependent nuclear translocation of HIF1α. Integrins α6ß1 and Bnip3 were found to promote survival of CRPC cells selectively on laminin through the induction of autophagy and mitophagy. Furthermore, blocking Bnip3 or integrin α6ß1 restored sensitivity to PI3K inhibitors in Pten-negative CRPC. We identified an AR driven pathway that cooperates with laminin and hypoxia to drive resistance to PI3K inhibitors. These findings can help explain in part why PI3K inhibitors have failed in clinical trials to overcome AR-dependent CRPC.
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Integrina alfa6beta1/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Proteínas Proto-Oncogénicas/metabolismo , Receptores Androgénicos/genética , Animales , Humanos , Masculino , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptores Androgénicos/metabolismo , Análisis de SupervivenciaRESUMEN
The endothelial cell basement membrane (BM) is a barrier to migrating leukocytes and a rich source of signaling molecules that can influence extravasating cells. Using mice lacking the major endothelial BM components, laminin 411 or 511, in murine experimental autoimmune encephalomyelitis (EAE), we show here that loss of endothelial laminin 511 results in enhanced disease severity due to increased T cell infiltration and altered polarization and pathogenicity of infiltrating T cells. In vitro adhesion and migration assays reveal higher binding to laminin 511 than laminin 411 but faster migration across laminin 411. In vivo and in vitro analyses suggest that integrin α6ß1- and αvß1-mediated binding to laminin 511-high sites not only holds T cells at such sites but also limits their differentiation to pathogenic Th17 cells. This highlights the importance of the interface between the endothelial monolayer and the underlying BM for modulation of immune cell phenotype.
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Membrana Basal/metabolismo , Encéfalo/patología , Células Endoteliales/metabolismo , Linfocitos T/inmunología , Animales , Membrana Basal/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Células Endoteliales/efectos de los fármacos , Integrina alfa6beta1/metabolismo , Laminina/farmacología , Ratones , Receptores de Vitronectina/metabolismo , Médula Espinal/patología , Linfocitos T/efectos de los fármacos , Células Th17/efectos de los fármacos , Células Th17/metabolismoRESUMEN
BACKGROUND: Atherosclerosis occurs preferentially at the blood vessels encountering blood flow turbulence. The matricellular protein CCN1 is induced in endothelial cells by disturbed flow, and is expressed in advanced atherosclerotic lesions in patients and in the Apoe-/- mouse model. The role of CCN1 in atherosclerosis remains undefined. METHODS: To assess the function of CCN1 in vivo, knock-in mice carrying the integrin α6ß1-binding-defective mutant allele Ccn1-dm on the Apoe-/- background were tested in an atherosclerosis model generated by carotid artery ligation. Additionally, CCN1-regulated functional phenotypes of human umbilical vein endothelial cells, or primary mouse aortic endothelial cells isolated from wild-type and Ccn1 dm/dm mice, were investigated in the in vitro shear stress experiments under unidirectional laminar shear stress (12 dyn/cm2) versus oscillatory shear stress (±5 dyn/cm2) conditions. RESULTS: We found that Ccn1 expression was upregulated in the arterial endothelium 3 days after ligation before any detectable structural changes, and intensified with the progression of atherosclerotic lesions. Compared with Apoe-/- controls, Ccn1 dm/dm/ Apoe-/- mice were remarkably resistant to ligation-induced plaque formation (n=6). These mice exhibited lower oxidative stress, expression of endothelin-1 and monocyte chemoattractant protein-1, and monocyte homing. CCN1/α6ß1 critically mediated flow-induced activation of the pleiotropic transcription factor nuclear factor-κB and therefore the induction of atheroprone gene expression in the mouse arterial endothelium after ligation (n=6), or in cultured human umbilical vein endothelial cells or primary mouse aortic endothelial cells exposed to oscillatory shear stress (n=3 in triplicate). Interestingly, the activation of nuclear factor-κB by CCN1/α6ß1 signaling prompted more production of CCN1 and α6ß1. Blocking CCN1-α6ß1 binding by the Ccn1-dm mutation or by T1 peptide (derived from an α6ß1-binding sequence of CCN1) disrupted the positive-feedback regulation between CCN1/α6ß1 and nuclear factor-κB, and prevented flow-induced atheroprone phenotypic alterations in endothelial cells or atherosclerosis in mice. CONCLUSIONS: These data demonstrate a causative role of CCN1 in atherosclerosis via modulating endothelial phenotypes. CCN1 binds to its receptor integrin α6ß1 to activate nuclear factor-κB, thereby instigating a vicious circle to persistently promote atherogenesis. T1, a peptide antagonist selectively targeting CCN1-α6ß1, can be further optimized for developing T1-mimetics to treat atherosclerosis.
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Enfermedades de las Arterias Carótidas/metabolismo , Arteria Carótida Común/metabolismo , Proteína 61 Rica en Cisteína/metabolismo , Células Endoteliales/metabolismo , Mecanotransducción Celular , Placa Aterosclerótica , Animales , Enfermedades de las Arterias Carótidas/diagnóstico , Enfermedades de las Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/fisiopatología , Arteria Carótida Común/patología , Arteria Carótida Común/fisiopatología , Células Cultivadas , Proteína 61 Rica en Cisteína/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/patología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Integrina alfa6beta1/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Mutación , FN-kappa B/metabolismo , Fenotipo , Flujo Sanguíneo Regional , Estrés MecánicoRESUMEN
In recent years, osteosarcoma survival rates have failed to improve significantly with conventional treatment modalities because of the development of chemotherapeutic resistance. The human breast cancer resistance protein/ATP binding cassette subfamily G member 2 (BCRP/ABCG2), a member of the ATP-binding cassette family, uses ATP hydrolysis to expel xenobiotics and chemotherapeutics from cells. CCN family member 2 (CCN2) is a secreted protein that modulates the biological function of cancer cells, enhanced ABCG2 protein expression and activation in this study via the α6ß1 integrin receptor and increased osteosarcoma cell viability. CCN2 treatment downregulated miR-519d expression, which promoted ABCG2 expression. In a mouse xenograft model, knockdown of CCN2 expression increased the therapeutic effect of doxorubicin, which was reversed by ABCG2 overexpression. Our data show that CCN2 increases ABCG2 expression and promotes drug resistance through the α6ß1 integrin receptor, whereas CCN2 downregulates miR-519d. CCN2 inhibition may represent a new therapeutic concept in osteosarcoma.
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Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/genética , Osteosarcoma/genética , Animales , Secuencia de Bases , Línea Celular Tumoral , Supervivencia Celular/genética , Humanos , Integrina alfa6beta1/metabolismo , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Modelos Biológicos , Transducción de SeñalRESUMEN
Integrins, the major receptors for cell-extracellular matrix (ECM) interactions, regulate multiple cell biological processes including adhesion, migration, proliferation and growth factor-dependent signaling. The principal laminin (LM) binding integrins α3ß1, α6ß1 and α6ß4 are usually co-expressed in cells and bind to multiple laminins with different affinities making it difficult to define their specific function. In this study, we generated kidney epithelial collecting duct (CD) cells that lack both the α3 and α6 integrin subunits. This deletion impaired cell adhesion and migration to LM-332 and LM-511 more than deleting α3 or α6 alone. Cell adhesion mediated by both α3ß1 and α6 integrins was PI3K independent, but required K63-linked polyubiquitination of Akt by the ubiquitin-modifying enzyme TRAF6. Moreover, we provide evidence that glial-derived neurotrophic factor (GDNF) and fibroblast growth factor 10 (FGF10)- mediated cell signaling, spreading and proliferation were severely compromised in double integrin α3/α6- but not single α3- or α6-null CD cells. Interestingly, these growth factor-dependent cell functions required both PI3K- and TRAF6-dependent Akt activation. These data suggest that expression of the integrin α3 or α6 subunit is sufficient to mediate GDNF- and FGF10-dependent spreading, proliferation and signaling on LM-511. Thus, our study shows that α3 and α6 containing integrins promote distinct functions and signaling by CD cells on laminin substrata.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Integrina alfa3/metabolismo , Integrina alfa6/metabolismo , Laminina/metabolismo , Transducción de Señal , Animales , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/química , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Matriz Extracelular/química , Matriz Extracelular/efectos de los fármacos , Factor 10 de Crecimiento de Fibroblastos/farmacología , Eliminación de Gen , Regulación de la Expresión Génica , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Humanos , Integrina alfa3/genética , Integrina alfa3beta1/genética , Integrina alfa3beta1/metabolismo , Integrina alfa6/genética , Integrina alfa6beta1/genética , Integrina alfa6beta1/metabolismo , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/metabolismo , Laminina/química , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Cultivo Primario de Células , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , KalininaRESUMEN
Autoimmune pancreatitis (AIP), a major manifestation of immunoglobulin G4-related disease (IgG4-RD), is an immune-mediated disorder, but the target autoantigens are still unknown. We previously reported that IgG in patients with AIP induces pancreatic injuries in mice by binding the extracellular matrix (ECM). In the current study, we identified an autoantibody against laminin 511-E8, a truncated laminin 511, one of the ECM proteins, in patients with AIP. Anti-laminin 511-E8 IgG was present in 26 of 51 AIP patients (51.0%), but only in 2 of 122 controls (1.6%), by enzyme-linked immunosorbent assay. Because truncated forms of other laminin family members in other organs have been reported, we confirmed that truncated forms of laminin 511 also exist in human and mouse pancreas. Histologic studies with patient pancreatic tissues showed colocalization of patient IgG and laminin 511. Immunization of mice with human laminin 511-E8 induced antibodies and pancreatic injury, fulfilling the pathologic criteria for human AIP. Four of 25 AIP patients without laminin 511-E8 antibodies had antibodies against integrin α6ß1, a laminin 511 ligand. AIP patients with laminin 511-E8 antibodies exhibited distinctive clinical features, as the frequencies of malignancies or allergic diseases were significantly lower in patients with laminin 511-E8 antibodies than in those without. The discovery of these autoantibodies should aid in the understanding of AIP pathophysiology and possibly improve the diagnosis of AIP.
Asunto(s)
Antígenos/sangre , Enfermedades Autoinmunes/sangre , Laminina/sangre , Pancreatitis/sangre , Anciano , Animales , Autoanticuerpos/sangre , Enfermedades Autoinmunes/inmunología , Femenino , Humanos , Inmunización , Inmunoglobulina G/sangre , Integrina alfa6beta1/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Pancreatitis/inmunologíaRESUMEN
Group A Streptococcus (GAS) is a human-specific pathogen responsible for a wide range of diseases, ranging from superficial to life-threatening invasive infections, including endometritis, and autoimmune sequelae. GAS strains express a vast repertoire of virulence factors that varies depending on the strain genotype, and many adhesin proteins that enable GAS to adhere to host cells are restricted to some genotypes. GAS emm28 is the third most prevalent genotype in invasive infections in France and is associated with gyneco-obstetrical infections. emm28 strains harbor R28, a cell wall-anchored surface protein that has previously been reported to promote adhesion to cervical epithelial cells. Here, using cellular and biochemical approaches, we sought to determine whether R28 supports adhesion also to other cells and to characterize its cognate receptor. We show that through its N-terminal domain, R28Nt, R28 promotes bacterial adhesion to both endometrial-epithelial and endometrial-stromal cells. R28Nt was further subdivided into two domains, and we found that both are involved in cell binding. R28Nt and both subdomains interacted directly with the laminin-binding α3ß1, α6ß1, and α6ß4 integrins; interestingly, these bindings events did not require divalent cations. R28 is the first GAS adhesin reported to bind directly to integrins that are expressed in most epithelial cells. Finally, R28Nt also promoted binding to keratinocytes and pulmonary epithelial cells, suggesting that it may be involved in supporting the prevalence in invasive infections of the emm28 genotype.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Proteínas Bacterianas/metabolismo , Adhesión Celular/fisiología , Integrina alfa3beta1/metabolismo , Integrina alfa6beta1/metabolismo , Integrina alfa6beta4/metabolismo , Adhesinas Bacterianas/química , Adhesión Bacteriana/fisiología , Proteínas Bacterianas/química , Línea Celular Tumoral , Endometrio/metabolismo , Células Epiteliales/metabolismo , Femenino , Humanos , Queratinocitos/metabolismo , Unión Proteica , Dominios Proteicos , Streptococcus pyogenes/química , Células del Estroma/metabolismoRESUMEN
The laminin family of glycoproteins are major constituents of the basal lamina of blood vessels, and play a fundamental role in promoting endothelial differentiation and blood-brain barrier (BBB) stability. Chronic mild hypoxia (CMH), in which mice are exposed to 8% O2 for two weeks, induces a strong vascular remodeling response in the central nervous system (CNS) that includes endothelial proliferation, angiogenesis, arteriogenesis as well as increased expression of tight junction proteins, suggestive of enhanced vascular integrity. As previous studies highlight an important role for laminin in promoting vascular differentiation and BBB stability, the goal of this study was to determine if CMH influences the expression of the laminins and their cell surface receptors in cerebral blood vessels. Our studies revealed that over a 14â¯day period of CMH, blood vessels in the brain showed strong upregulation of the specific laminin subunits α1 and α4, corresponding to increased expression of laminins 111 and 411 respectively, with no discernible changes in the expression levels of the α2 or α5 laminin subunits. This was accompanied by marked endothelial upregulation of the laminin receptor α6ß1 integrin but no alterations in the other laminin receptors α1ß1 integrin or dystroglycan. In light of the instructive role for laminins in promoting vascular differentiation and stability, these data suggest that upregulation of the laminin-α6ß1 integrin axis is part of the molecular response triggered by mild hypoxia that leads to enhanced BBB stability.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Hipoxia/metabolismo , Integrina alfa6beta1/metabolismo , Laminina/metabolismo , Animales , Barrera Hematoencefálica/patología , Trastornos Cerebrovasculares/metabolismo , Trastornos Cerebrovasculares/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Hipoxia/patología , Ratones , Remodelación Vascular/fisiologíaRESUMEN
Fibroblast-derived melanogenic paracrine mediators are known to play a role in melanogenesis. To investigate the effect of CCN1 (also called CYR61 or cysteine-rich 61) on melanogenesis, normal human epidermal melanocytes were treated with recombinant CCN1 protein. Our findings show that CCN1 activates melanogenesis through promoting melanosome maturation and up-regulation of MITF, TRP-1, and tyrosinase via the integrin α6ß1, p38 MAPK, and ERK signaling pathways. Furthermore, we found that UVB irradiation stimulates the secretion of CCN1 from normal human dermal fibroblasts, and CCN1 knockdown in fibroblasts attenuates melanogenesis in melanocyte-fibroblast co-culture system. Moreover, using immunohistochemistry and immunofluorescence staining, we discovered that CCN1 is overexpressed in the dermis of both solar lentigines and Riehl's melanosis lesions. These findings suggest that CCN1 is a fibroblast-derived melanogenic paracrine mediator that is secreted under UVB irradiation, and it may play an important role in the development of hyperpigmentation diseases such as Riehl's melanosis.
Asunto(s)
Proteína 61 Rica en Cisteína/metabolismo , Fibroblastos/metabolismo , Lentigo/patología , Sistema de Señalización de MAP Quinasas , Melaninas/biosíntesis , Melanocitos/metabolismo , Adulto , Células Cultivadas , Proteína 61 Rica en Cisteína/genética , Dermis/patología , Dermis/efectos de la radiación , Epidermis/metabolismo , Femenino , Fibroblastos/efectos de la radiación , Técnicas de Silenciamiento del Gen , Humanos , Integrina alfa6beta1/metabolismo , Lentigo/etiología , Masculino , Persona de Mediana Edad , Rayos Ultravioleta , Regulación hacia ArribaRESUMEN
Extracellular vesicles (EVs) are small membrane vesicles secreted from cells and have great potential as drug delivery carriers. Surface proteins on EV membranes might play roles in pharmacokinetics. One method which can be used to study the role of surface membrane of EV is to modify the inner space of EV. In the present study, we constructed a plasmid DNA expressing a fusion protein of Gag protein derived from Moloney murine leukemia virus (Gag) and Gaussia luciferase (gLuc) (Gag-gLuc) to modify the inner space of EVs. EVs were collected from B16BL6 melanoma cells, transfected with the plasmid, and isolated by a differential ultracentrifugation method. Gag-gLuc EVs were negatively charged globular vesicles with a diameter of approximately 100 nm. gLuc labeling of the Gag-gLuc EVs was stable in serum. gLuc activity of Gag-gLuc EVs was minimally decreased by proteinase K (ProK) treatment, indicating that gLuc was modified in the inner space of EV. Then, to evaluate the effect of the surface proteins of EVs on their pharmacokinetics, Gag-gLuc EVs treated with ProK were intravenously administered to mice. Volume of distribution (Vd) was significantly smaller for treated EVs than untreated EVs. Moreover, integrin α6ß1, an integrin known to be involved in lung targeting, was degraded after ProK treatment. The ProK treatment significantly reduced the lung distribution of EVs after intravenous injection. These results indicate that the surface proteins of EVs such as integrin α6ß1 play some roles in pharmacokinetics in terms of reducing Vd and their distribution to the lung.
Asunto(s)
Portadores de Fármacos/farmacocinética , Vesículas Extracelulares/metabolismo , Integrina alfa6beta1/metabolismo , Proteínas de la Membrana/farmacocinética , Animales , Línea Celular Tumoral , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/metabolismo , Vesículas Extracelulares/genética , Productos del Gen gag/genética , Vectores Genéticos/genética , Inyecciones Intravenosas , Integrina alfa6beta1/administración & dosificación , Luciferasas/genética , Pulmón/metabolismo , Macrófagos , Masculino , Proteínas de la Membrana/administración & dosificación , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Virus de la Leucemia Murina de Moloney/genética , Proteínas Recombinantes de Fusión/genética , Distribución Tisular , TransfecciónRESUMEN
Extracellular matrix protein-integrin interaction on neurons plays an important role in the development of neuroplasticity in the brain. However, the role of fibronectin-integrin signaling in epilepsy is elusive. Here, we examined the functional role of fibronectin-integrin signaling by utilizing a combination approach involving atomic force microscopy (AFM), immunocytochemistry, and pharmacology in epileptic mouse dentate gyrus granule cells (DGGCs). There was marked increase in the fibronectin receptor α5ß1-integrin staining intensity in DGGCs in epileptic mice. In the AFM study, the unbinding force and binding probability between the fibronectin-coated AFM probe and the membrane integrins were significantly reduced; while the cell stiffness was strikingly increased in epileptic DGGCs. Pretreatment with α5ß1-integrin monoclonal antibody partially reversed this membrane dysfunction. In patch-clamp recordings, fibronectin significantly inhibited GABA current, while RGD, which is known to disrupt fibronectin-integrin-dependent cell adhesive events, strikingly enhanced GABA tonic currents in DGGCs in hippocampal slices. The α5ß1-integrin antibody significantly reduced 4-aminopyridine-induced epileptiform discharges in brain slices. In systemic behavioral studies, susceptibility to hippocampus kindling epileptogenesis was significantly attenuated in mice treated with RGD or ß1-integrin antibody. These pilot studies provide new insights on the functional role of integrin receptor signaling in epileptogenesis and may help identify novel targets for the prevention and treatment of epilepsy.