RESUMO
Integrins are fundamental for cell adhesion and the formation of focal adhesions (FA). Accordingly, these receptors guide embryonic development, tissue maintenance, and haemostasis but are also involved in cancer invasion and metastasis. A detailed understanding of the molecular interactions that drive integrin activation, FA assembly, and downstream signalling cascades is critical. Here, we reveal a direct association of paxillin, a marker protein of FA sites, with the cytoplasmic tails of the integrin ß1 and ß3 subunits. The binding interface resides in paxillin's LIM3 domain, where based on the NMR structure and functional analyses, a flexible, 7-amino acid loop engages the unstructured part of the integrin cytoplasmic tail. Genetic manipulation of the involved residues in either paxillin or integrin ß3 compromises cell adhesion and motility of murine fibroblasts. This direct interaction between paxillin and the integrin cytoplasmic domain identifies an alternative, kindlin-independent mode of integrin outside-in signalling particularly important for integrin ß3 function.
Assuntos
Paxilina , Ligação Proteica , Paxilina/metabolismo , Animais , Camundongos , Domínios Proteicos , Adesão Celular/fisiologia , Adesões Focais/metabolismo , Humanos , Movimento Celular , Integrina beta3/metabolismo , Integrina beta3/genética , Integrina beta3/química , Fibroblastos/metabolismo , Cadeias beta de Integrinas/metabolismo , Cadeias beta de Integrinas/química , Cadeias beta de Integrinas/genética , Integrina beta1/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos , Neoplasias , Humanos , Integrina beta3/química , Integrina beta3/metabolismo , Aptâmeros de Nucleotídeos/farmacologia , Integrina alfaVbeta3/metabolismo , Movimento Celular , Microambiente TumoralRESUMO
The therapeutic efficacy of 5-fluorouracil (5-FU) is often reduced by the development of drug resistance. We observed significant upregulation of lipocalin 2 (LCN2) expression in a newly established 5-FU-resistant colorectal cancer (CRC) cell line. In this study, we demonstrated that 5-FU-treated CRC cells developed resistance through LCN2 upregulation caused by LCN2 promoter demethylation and that feedback between LCN2 and NF-κB further amplified LCN2 expression. High LCN2 expression was associated with poor prognosis in CRC patients. LCN2 attenuated the cytotoxicity of 5-FU by activating the SRC/AKT/ERK-mediated antiapoptotic program. Mechanistically, the LCN2-integrin ß3 interaction enhanced integrin ß3 stability, thus recruiting SRC to the cytomembrane for autoactivation, leading to downstream AKT/ERK cascade activation. Targeting LCN2 or SRC compromised the growth of CRC cells with LCN2-induced 5-FU resistance. Our findings demonstrate a novel mechanism of acquired resistance to 5-FU, suggesting that LCN2 can be used as a biomarker and/or therapeutic target for advanced CRC.
Assuntos
Neoplasias Colorretais/patologia , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Integrina beta3/metabolismo , Lipocalina-2/genética , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HT29 , Humanos , Integrina beta3/química , Lipocalina-2/metabolismo , Masculino , Camundongos , NF-kappa B/metabolismo , Transplante de Neoplasias , Prognóstico , Regiões Promotoras Genéticas/efeitos dos fármacos , Estabilidade Proteica , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima , Quinases da Família src/metabolismoRESUMO
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.
Assuntos
Adesões Focais/metabolismo , Integrina beta3/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Animais , Linhagem Celular , Cricetinae , Dissulfetos/análise , Adesões Focais/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrina beta3/química , Integrina beta3/genética , Simulação de Dinâmica Molecular , Mutação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/química , Glicoproteína IIb da Membrana de Plaquetas/genéticaRESUMO
A heterodimeric receptor subunit, Integrin αV, often complexed with Integrin ß3 plays a vital role in cell signaling to regulate angiogenesis and promote cancer progression. The paramount ß-turn formed from pentapeptide residues (PPQEE) in the cytoplasmic domain of Integrin αV was previously reported as crucial for cell signaling and its deletion was proved deleterious for protein's cell membrane adhesion and ligand binding properties. This study revealed conformational changes in the Integrin αV subunit upon deletion of PPQEE residues through in silico structural modelling approach followed by analysis of alteration of binding sites. Human Protein Atlas database helped to identify the association of Integrin αV to the unfavourable prognosis of three gastrointestinal cancers: stomach, liver and pancreatic cancers. Molecular modelling and docking techniques were carried out for the necessary complex formations (wild-type and mutant-type). Further comparison was performed for the complexes. The changes in protein's conformation and stability due to PPQEE deletion were observed in both independent subunit and heterodimer. The most noteworthy conformational shift was the disruption of a transmembrane helix into coil, which accounted for protein's impaired cell membrane adhesion, increased solvent accessibility and decreased stability. The deletion also caused a reduction of beta-turn regions, which disrupted ligand binding in the cytoplasmic domain of Integrin αV subunit. This study emphasized on structural basis of how the deletion of PPQEE residues alters stability, ligand binding and signaling activity of Integrin αV subunit highlighting the importance of these residues in maintenance of protein's native structure.
Assuntos
Integrina alfaV/metabolismo , Integrina beta3/metabolismo , Complexos Multiproteicos/metabolismo , Neoplasias/metabolismo , Oligopeptídeos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Simulação por Computador , Deleção de Genes , Humanos , Integrina alfaV/química , Integrina alfaV/genética , Integrina beta3/química , Integrina beta3/genética , Ligantes , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Neoplasias/classificação , Neoplasias/genética , Oligopeptídeos/química , Oligopeptídeos/genética , Conformação Proteica , Transdução de Sinais/genéticaRESUMO
BACKGROUND: Integrins are highly attractive targets in oncology due to their involvement in angiogenesis in a wide spectrum of cancer entities. Among several integrin inhibitors, cilengitide is suggested to be one of the most promising inhibitors. However, little is known about the cellular processes induced during cilengitide chemotherapy in head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: For the current study, 3 HNSCC cell lines, SCC4, SCC15 and SCC25; and 3 primary culture cells, TU53, TU57, and TU63 were used. CD90, cytokeratin, and vimentin were stained immunohistochemically to identify the biological characteristics of these cell lines and primary culture cells and the cytostatic effect of cilengitide was evaluated. Quantitative polymerase chain reaction (qPCR) arrays were applied to evaluate target protein genes ITGAV, ITGB3, and ITGB5 of integrin αvß3 and αvß5 at respective concentrations of 50 and 100 µM cilengitide for 72 h. RESULTS: Cilengitide has significantly inhibited the proliferation of HNSCC cells in a dose-dependent way. At the same concentration, cilengitide suppressed the proliferation of primary culture cells even more strongly than it did that of cell lines, suggesting that primary culture cells retain more of their internal biological characteristics than do cell lines. qPCR assay detected downregulation of ITGAV, ITGB3, and ITGB5 gene expression after exposure to 50 µM of cilengitide. However, after exposure to 100-µM cilengitide, expression of these genes significantly increased both in cell lines and primary culture cells. CONCLUSIONS: RGD-containing small-molecule synthetic peptides might be considered in tumor chemotherapy in the near future. The different reactions of primary culture cells and cell lines demonstrated that individualized chemotherapy plans may be a feasible option. However, research on the role of cilengitide in HNSCC therapy is still in its early stages, and further investigations are required.
Assuntos
Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/patologia , Cadeias beta de Integrinas/química , Integrina beta3/química , Venenos de Serpentes/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Apoptose/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Cadeias beta de Integrinas/genética , Cadeias beta de Integrinas/metabolismo , Integrina beta3/genética , Integrina beta3/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND/AIMS: Neointimal hyperplasia is responsible for stenosis, which requires corrective vascular surgery, and is also a major morphological feature of many cardiovascular diseases. This hyperplasia involves the endothelial-to-mesenchymal transition (EndMT). We investigated whether integrin ß3 can modulate the EndMT, as well as its underlying mechanism. METHODS: Integrin ß3 was overexpressed or knocked down in human umbilical vein endothelial cells (HUVECs). The expression of endothelial markers and mesenchymal markers was determined by real-time reverse transcription PCR (RT-PCR), immunofluorescence staining, and western blot analysis. Notch signaling pathway components were detected by real-time RT-PCR and western blot analysis. Cell mobility was evaluated by wound-healing, Transwell, and spreading assays. Fibroblast-specific protein 1 (FSP-1) promoter activity was determined by luciferase assay. RESULTS: Transforming growth factor (TGF)-ß1 treatment or integrin ß3 overexpression significantly promoted the EndMT by downregulating VE-cadherin and CD31 and upregulating smooth muscle actin α and FSP-1 in HUVECs, and by enhancing cell migration. Knockdown of integrin ß3 reversed these effects. Notch signaling was activated after TGF-ß1 treatment of HUVECs. Knockdown of integrin ß3 suppressed TGF-ß1-induced Notch activation and expression of the Notch downstream target FSP-1. CONCLUSION: Integrin ß3 may promote the EndMT in HUVECs through activation of the Notch signaling pathway.
Assuntos
Transição Epitelial-Mesenquimal , Integrina beta3/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Actinas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/metabolismo , Fístula Arteriovenosa/metabolismo , Fístula Arteriovenosa/patologia , Caderinas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Movimento Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrina beta3/química , Integrina beta3/genética , Pessoa de Meia-Idade , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100 , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
It has been proposed that integrins adopt a bent, closed conformation with low ligand binding capability at resting state and switch into an extended, open conformation upon activation or interacting with extracellular matrix (ECM) ligand. In this study, we addressed how integrin conformational change at the ß genu affects ligand binding and signaling. We discovered that swapping of the ß3 epidermal growth factor-like (EGF) domain 1 and 2 with that of ß8 greatly promoted ligand binding in ß3 ß8 chimeras. Sequence alignment indicated that ß8 integrin uniquely lacks the interface between the EGF1 and 2. Disrupting this interface of the ß3 integrin increased integrin ligand binding. Furthermore, the interface is critical for integrin affinity regulation but not downstream outside-in signaling.
Assuntos
Cadeias beta de Integrinas/química , Cadeias beta de Integrinas/metabolismo , Integrina beta3/química , Integrina beta3/metabolismo , Domínios Proteicos/fisiologia , Sítios de Ligação , Adesão Celular , Quimera , Complexos de Coordenação/química , Cristalografia , Cisteína/química , Fator de Crescimento Epidérmico/química , Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Ligantes , Microscopia Confocal , Ligação Proteica , Conformação Proteica , Transdução de SinaisRESUMO
BACKGROUND/AIMS: Homeobox D3 (HOXD3) is a member of the homeobox family of genes that is known primarily for its transcriptional regulation of morphogenesis in all multicellular organisms. In this study, we sought to explore the role that HOXD3 plays in the stem-like capacity, or stemness, and drug resistance of breast cancer cells. METHODS: Expression of HOXD3 in clinical breast samples were examined by RT-PCR and immunohistochemistry. HOXD3 expression in breast cancer cell lines were analyzed by RT-PCR and western blot. Ability of drug resistance in breast cancer cells were elevated by MTT cell viability and colony formation assays. We examined stemness using cell fluorescent staining, RT-PCR and western blot for stem cell marker expression. Finally, activity of wnt signaling was analyzed by FOPflash luciferase assays. RT-PCR and western blot were performed for downstream genes of wnt signaling. RESULTS: We demonstrated that HOXD3 is overexpressed in breast cancer tissue as compared to normal breast tissue. HOXD3 overexpression enhances breast cancer cell drug resistance. Furthermore, HOXD3 upregulation in the same cell lines increased sphere formation as well as the expression levels of stem cell biomarkers, suggesting that HOXD3 does indeed increase breast cancer cell stemness. Because we had previously shown that HOXD3 expression is closely associated with integrin ß3 expression in breast cancer patients, we hypothesized that HOXD3 may regulate breast cancer cell stemness and drug resistance through integrin ß 3. Cell viability assays showed that integrin ß 3 knockdown increased cell viability and that HOXD3 could not restore cancer cell stemness or drug resistance. Given integrin ß 3's relationship with Wnt/ß-catenin signaling, we determine whether HOXD3 regulates integrin ß 3 activity through Wnt/ß-catenin signaling. We found that, even though HOXD3 increased the expression of Wnt/ß-catenin downstream genes, it did not restore Wnt/ß-catenin signaling activity, which was inhibited in integrin ß3 knockdown breast cancer cells. CONCLUSION: We demonstrate that HOXD3 plays a critical role in breast cancer stemness and drug resistance via integrin ß3-mediated Wnt/ß-catenin signaling. Our findings open the possibility for improving the current standard of care for breast cancer patients by designing targeted molecular therapies that overcome the barriers of cancer cell stemness and drug resistance.
Assuntos
Neoplasias da Mama/patologia , Proteínas de Homeodomínio/metabolismo , Antineoplásicos/uso terapêutico , Antineoplásicos/toxicidade , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/uso terapêutico , Cisplatino/toxicidade , Doxorrubicina/uso terapêutico , Doxorrubicina/toxicidade , Resistencia a Medicamentos Antineoplásicos , Feminino , Proteínas de Homeodomínio/genética , Humanos , Imuno-Histoquímica , Integrina beta3/química , Integrina beta3/genética , Integrina beta3/metabolismo , Células-Tronco Neoplásicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição , Via de Sinalização WntRESUMO
Acute myeloid leukemia (AML) is an aggressive bone marrow malignancy where the immature leukemia cells communicate with neighboring cells through constitutive cytokine release and through their cell surface adhesion molecules. The primary AML cells express various integrins. These heterodimeric molecules containing an α and a ß chain are cell surface molecules that bind extracellular matrix molecules, cell surface molecules and soluble mediators. The ß3 integrin (ITGB3) chain can form heterodimers only with the two α chains αIIb and αV. These integrins are among the most promiscuous and bind to a large number of ligands, including extracellular matrix molecules, cell surface molecules and soluble mediators. Recent studies suggest that the two ß3 integrins are important for leukemogenesis and chemosensitivity in human AML. Firstly, αIIb and ß3 are both important for adhesion of AML cells to vitronectin and fibronectin. Secondly, ß3 is important for the development of murine AML and also for the homing and maintenance of the proliferation for xenografted primary human AML cells, and for maintaining a stem cell transcriptional program. These last effects seem to be mediated through Syk kinase. The ß3 expression seems to be regulated by HomeboxA9 (HoxA9) and HoxA10, and the increased ß3 expression then activates spleen tyrosine kinase (Syk) and thereby contributes to cytokine hypersensitivity and activation of ß2 integrins. Finally, high integrin αV/ß3 expression is associated with an adverse prognosis in AML and decreased sensitivity to the kinase inhibitor sorafenib; this integrin can also be essential for osteopontin-induced sorafenib resistance in AML. In the present article, we review the experimental and clinical evidence for a role of ß3 integrins for leukemogenesis and chemosensitivity in AML.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Integrina beta3/genética , Integrina beta3/metabolismo , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina beta3/química , Integrinas/química , Integrinas/genética , Integrinas/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Ligantes , Família Multigênica , Prognóstico , Ligação Proteica , Transdução de SinaisRESUMO
Kindlin-2 (K2), a 4.1R-ezrin-radixin-moesin (FERM) domain adaptor protein, mediates numerous cellular responses, including integrin activation. The C-terminal 15-amino acid sequence of K2 is remarkably conserved across species but is absent in canonical FERM proteins, including talin. In CHO cells expressing integrin αIIbß3, co-expression of K2 with talin head domain resulted in robust integrin activation, but this co-activation was lost after deletion of as few as seven amino acids from the K2 C terminus. This dependence on the C terminus was also observed in activation of endogenous αIIbß3 in human erythroleukemia (HEL) cells and ß1 integrin activation in macrophage-like RAW264.1 cells. Kindlin-1 (K1) exhibited a similar dependence on its C terminus for integrin activation. Expression of the K2 C terminus as an extension of membrane-anchored P-selectin glycoprotein ligand-1 (PSGL-1) inhibited integrin-dependent cell spreading. Deletion of the K2 C terminus did not affect its binding to the integrin ß3 cytoplasmic tail, but combined biochemical and NMR analyses indicated that it can insert into the F2 subdomain. We suggest that this insertion determines the topology of the K2 FERM domain, and its deletion may affect the positioning of the membrane-binding functions of the F2 subdomain and the integrin-binding properties of its F3 subdomain. Free C-terminal peptide can still bind to K2 and displace the endogenous K2 C terminus but may not restore the conformation needed for integrin co-activation. Our findings indicate that the extreme C terminus of K2 is essential for integrin co-activation and highlight the importance of an atypical architecture of the K2 FERM domain in regulating integrin activation.
Assuntos
Integrina alfa2/metabolismo , Integrina beta3/metabolismo , Leucemia Eritroblástica Aguda/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Substituição de Aminoácidos , Animais , Células CHO , Linhagem Celular Tumoral , Cricetulus , Deleção de Genes , Humanos , Integrina alfa2/química , Integrina alfa2/genética , Integrina beta3/química , Integrina beta3/genética , Leucemia Eritroblástica Aguda/patologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Macrófagos/citologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Mutação , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Células RAW 264.7 , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Talina/química , Talina/genética , Talina/metabolismoRESUMO
Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrinαIIbß3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbß3 activation, but it activates αIIbß3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of αIIbß3 is tightly regulated by the topology of ß3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the embedding of integrin ß3 TMD, resulting in integrin activation, whereas we observed here that EGCG decreases the embedding, thus opposing talin-induced integrin activation. In the absence of talin, EGCG decreases the TMD embedding, which can also disrupt the integrin α-ß TMD interaction, leading to integrin activation. EGCG exhibited similar paradoxical behavior in EGFR signaling. EGCG alters the topology of EGFR TMD and activates the receptor in the absence of EGF, but inhibits EGF-induced EGFR activation. Thus, this widely ingested polyphenol exhibits pleiotropic effects on transmembrane signaling by modifying the topology of TMDs.
Assuntos
Antioxidantes/metabolismo , Catequina/análogos & derivados , Receptores ErbB/metabolismo , Integrina beta3/metabolismo , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Transdução de Sinais , Substituição de Aminoácidos , Animais , Antioxidantes/química , Antioxidantes/uso terapêutico , Células CHO , Catequina/química , Catequina/metabolismo , Catequina/uso terapêutico , Cricetulus , Suplementos Nutricionais , Dimerização , Receptores ErbB/agonistas , Receptores ErbB/química , Receptores ErbB/genética , Humanos , Integrina alfa2/química , Integrina alfa2/genética , Integrina alfa2/metabolismo , Integrina beta3/química , Integrina beta3/genética , Ligantes , Bicamadas Lipídicas/química , Mutação , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/agonistas , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Talina/antagonistas & inibidores , Talina/química , Talina/metabolismoRESUMO
Integrins are transmembrane receptors composed of α and ß subunits. Although most integrins contain ß1, canonical activation mechanisms are based on studies of the platelet integrin, αIIbß3. Its inactive conformation is characterized by the association of the αIIb transmembrane and cytosolic domain (TM/CT) with a tilted ß3 TM/CT that leads to activation when disrupted. We show significant structural differences between ß1 and ß3 TM/CT in bicelles. Moreover, the 'snorkeling' lysine at the TM/CT interface of ß subunits, previously proposed to regulate αIIbß3 activation by ion pairing with nearby lipids, plays opposite roles in ß1 and ß3 integrin function and in neither case is responsible for TM tilt. A range of affinities from almost no interaction to the relatively high avidity that characterizes αIIbß3 is seen between various α subunits and ß1 TM/CTs. The αIIbß3-based canonical model for the roles of the TM/CT in integrin activation and function clearly does not extend to all mammalian integrins.
Assuntos
Células Epiteliais/fisiologia , Integrina alfa1/metabolismo , Integrina beta1/metabolismo , Integrina beta3/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Multimerização Proteica , Substituição de Aminoácidos , Adesão Celular , Células Cultivadas , Células Epiteliais/química , Humanos , Integrina alfa1/química , Integrina beta1/química , Integrina beta1/genética , Integrina beta3/química , Integrina beta3/genética , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/química , Ligação ProteicaRESUMO
Previous studies in Chinese hamster ovary cells showed that truncational mutations of ß3 at sites of F754 and Y759 mimicking calpain cleavage regulate integrin signaling. The roles of the sequence from F754 to C-terminus and the conservative N756ITY759 motif in platelet function have yet to be elaborated. Mice expressing ß3 with F754 and Y759 truncations, or NITY deletion (ß3-ΔTNITYRGT, ß3-ΔRGT, or ß3-ΔNITY) were established through transplanting the homozygous ß3-deficient mouse bone marrow cells infected by the GFP tagged MSCV MigR1 retroviral vector encoding different ß3 mutants into lethally radiated wild-type mice. The platelets were harvested for soluble fibrinogen binding and platelet spreading on immobilized fibrinogen. Platelet adhesion on fibrinogen- and collagen-coated surface under flow was also tested to assess the ability of the platelets to resist hydrodynamic drag forces. Data showed a drastic inhibition of the ß3-ΔTNITYRGT platelets to bind soluble fibrinogen and spread on immobilized fibrinogen in contrast to a partially impaired fibrinogen binding and an almost unaffected spreading exhibited in the ß3-ΔNITY platelets. Behaviors of the ß3-ΔRGT platelets were consistent with the previous observations in the ß3-ΔRGT knock-in platelets. The adhesion impairment of platelets with the ß3 mutants under flow was in different orders of magnitude shown as: ß3-ΔTNITYRGT>ß3-ΔRGT>ß3-ΔNITY to fibrinogen-coated surface, and ß3-ΔTNITYRGT>ß3-ΔNITY>ß3-ΔRGT to collagen-coated surface. To evaluate the interaction of the ß3 mutants with signaling molecules, GST pull-down and immunofluorescent assays were performed. Results showed that ß3-ΔRGT interacted with kindlin but not c-Src, ß3-ΔNITY interacted with c-Src but not kindlin, while ß3-ΔTNITYRGT did not interact with both proteins. This study provided evidence in platelets at both static and flow conditions that the calpain cleavage-related sequences of integrin ß3, i.e. T755NITYRGT762, R760GT762, and N756ITY759 participate in bidirectional, outside-in, and inside-out signaling, respectively and the association of c-Src or kindlin with ß3 integrin may regulate these processes.
Assuntos
Plaquetas/metabolismo , Citoplasma/metabolismo , Integrina beta3/química , Integrina beta3/genética , Mutação/genética , Ativação Plaquetária/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Feminino , Fibrinogênio/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Hemorreologia , Proteínas Imobilizadas/metabolismo , Integrina beta3/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Adesividade Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Ligação Proteica , Retroviridae/metabolismo , Transdução de Sinais , Solubilidade , TransfecçãoRESUMO
PURPOSE: In HLA-matched allogeneic hematopoietic stem cell transplantation (alloSCT), donor T cells recognizing minor histocompatibility antigens (MiHAs) can mediate desired antitumor immunity as well as undesired side effects. MiHAs with hematopoiesis-restricted expression are relevant targets to augment antitumor immunity after alloSCT without side effects. To identify therapeutic MiHAs, we analyzed the in vivo immune response in a patient with strong antitumor immunity after alloSCT. EXPERIMENTAL DESIGN: T-cell clones recognizing patient, but not donor, hematopoietic cells were selected for MiHA discovery by whole genome association scanning. RNA-sequence data from the GEUVADIS project were analyzed to investigate alternative transcripts, and expression patterns were determined by microarray analysis and qPCR. T-cell reactivity was measured by cytokine release and cytotoxicity. RESULTS: T-cell clones were isolated for two HLA-B*15:01-restricted MiHA. LB-GLE1-1V is encoded by a nonsynonymous SNP in exon 6 of GLE1 For the other MiHAs, an associating SNP in intron 3 of ITGB2 was found, but no SNP disparity was present in the normal gene transcript between patient and donor. RNA-sequence analysis identified an alternative ITGB2 transcript containing part of intron 3. qPCR demonstrated that this transcript is restricted to hematopoietic cells and SNP-positive individuals. In silico translation revealed LB-ITGB2-1 as HLA-B*15:01-binding peptide, which was validated as hematopoietic MiHA by T-cell experiments. CONCLUSIONS: Whole genome and transcriptome analysis identified LB-ITGB2-1 as MiHAs encoded by an alternative transcript. Our data support the therapeutic relevance of LB-ITGB2-1 and illustrate the value of RNA-sequence analysis for discovery of immune targets encoded by alternative transcripts. Clin Cancer Res; 22(16); 4185-96. ©2016 AACR.
Assuntos
Processamento Alternativo , Perfilação da Expressão Gênica , Integrina beta3/genética , Antígenos de Histocompatibilidade Menor/genética , Sequenciamento Completo do Genoma , Sequência de Aminoácidos , Sequência de Bases , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Antígeno HLA-B15/genética , Antígeno HLA-B15/imunologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Integrina beta3/química , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/imunologia , Peptídeos/genética , Peptídeos/imunologia , Linfócitos T , Transplante HomólogoRESUMO
In the present study, we tested the hypothesis that aldosterone regulates osteopontin (OPN)-related signaling pathways to promote nuclear factor κB (NF-κB) activation in primary human umbilical vein endothelial cells (HUVECs) and that kaempferol, a flavonoid compound, blocks those changes. Aldosterone induced productions of reactive oxygen species (ROS), OPN, interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) and expression of nicotinamide adenine dinucleotide phosphate-oxidase 4 (Nox4), NF-κB, OPN, alphavbeta3 (αvß3) integrin, and inhibitor of NF-κB alpha phosphorylation (P-IκBα) in HUVEC. HUVECs were pretreated with kaempferol (0, 1, 3, or 10 µM) for 1 h and exposed to aldosterone (10(-6) M) for 24 h. Kaempferol reduced ROS, OPN, NF-κB, IL-6, and TNF-α levels; Nox4, αvß3 integrin; and P-IκBα expressions. The effect of aldosterone was also abrogated by spironolactone (10(-6) M). In addition, vitamin C (20 mmol/L) reduced ROS production. Vitamin C and LM609 (10 µg/mL) treatment decreased expressions of OPN, αvß3 integrin, and NF-κB (P < 0.05 or P < 0.01). The present results suggest that kaempferol may modulate OPN-αvß3 integrin pathway to inhibit NF-κB activation in HUVECs.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antioxidantes/farmacologia , Regulação para Baixo/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Quempferóis/farmacologia , Espécies Reativas de Oxigênio/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Aldosterona/química , Aldosterona/farmacologia , Anticorpos Monoclonais/farmacologia , Ácido Ascórbico/farmacologia , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Integrina alfaV/química , Integrina alfaV/metabolismo , Integrina alfaVbeta3/antagonistas & inibidores , Integrina beta3/química , Integrina beta3/metabolismo , Antagonistas de Receptores de Mineralocorticoides/farmacologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Osteopontina/antagonistas & inibidores , Osteopontina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Espironolactona/farmacologiaRESUMO
OBJECTIVES: Previous studies have shown that germ-like cells can be induced from human umbilical cord mesenchymal stem cell (hUC-MSCs) in vitro. However, induction efficiency was low and a stable system had not been built. CD61, also called integrin-ß3, plays a significant role in cell differentiation, in that CD61-positive-cell-derived pluripotent stem cells easily differentiate into primordial germ-like cells (PGC). Here, we have explored whether overexpression of CD61 would promote hUC-MSC differentiation into PGC and male germ-like cells. MATERIALS AND METHODS: hUC-MSCs were cultured and transduced using pCD61-CAGG-TRIP-pur (oCD61) and pTRIP-CAGG plasmid (Control), and hUC-MSCs overexpressed CD61 were induced by bone morphogenetic protein 4 (BMP4, 12.5 ng/ml), to differentiate into PGC and male germ cells. Quantitative real-time PCR (RT-qPCR), western blotting and immunofluorescence staining were used to examine PGC- and germ cell-specific markers. RESULTS: High expression levels of PGC-specific markers were detected in oCD61 hUC-MSCs compared to controls. After BMP4 induction, expression levels of male germ cell markers such as Acrosin (ACR), Prm1 and meiotic markers including Stra8, Scp3 in oCD61 were significantly higher than those of the Control group. CONCLUSIONS: Under induction of BMP4, CD61-overexpressing hUC-MSCs, which had turned into PGC-like cells, could be further differentiated into male germ-like cells. Thus, a simple and efficient approach to study male germ cell development by using hUC-MSCs has been established.
Assuntos
Diferenciação Celular , Células Germinativas/citologia , Integrina beta3/metabolismo , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , Biomarcadores/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Clonagem Molecular , Códon/genética , Humanos , Integrina beta3/química , Integrina beta3/genética , Masculino , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Análise de Sequência de Proteína , Homologia de Sequência do Ácido NucleicoRESUMO
UNLABELLED: Essentials Protein disulfide isomerases may have an essential role in thrombus formation. A platelet-binding sensor (PDI-sAb) was developed to detect thiol reductase activity under flow. Primary human platelet adhesion to collagen at 200 s(-1) was correlated with the PDI-sAb signal. Detected thiol reductase activity was localized in the core of growing thrombi at the site of injury in mice. SUMMARY: Background Protein disulfide isomerases (PDIs) may regulate thrombus formation in vivo, although the sources and targets of PDIs are not fully understood. Methods and results Using click chemistry to link anti-CD61 and a C-terminal azido disulfide-linked peptide construct with a quenched reporter, we developed a fluorogenic platelet-targeting antibody (PDI-sAb) for thiol reductase activity detection in whole blood under flow conditions. PDI-sAb was highly responsive to various exogenous reducing agents (dithiothreitol, glutathione and recombinant PDI) and detected thiol reductase activity on P-selectin/phosphatidylserine-positive platelets activated with convulxin/PAR1 agonist peptide, a signal partially blocked by PDI inhibitors and antibody. In a microfluidic thrombosis model using 4 µg mL(-1) corn trypsin inhibitor-treated human blood perfused over collagen (wall shear rate = 100 s(-1) ), the PDI-sAb signal increased mostly over the first 200 s, whereas platelets continually accumulated for over 500 s, indicating that primary adhesion to collagen, but not secondary aggregation, was correlated with the PDI-sAb signal. Rutin and the PDI blocking antibody RL90 reduced platelet adhesion and the PDI-sAb signal only when thrombin production was inhibited with PPACK, suggesting limited effects of platelet thiol isomerase activity on platelet aggregation on collagen in the presence of thrombin. With anti-mouse CD41 PDI-sAb used in an arteriolar laser injury model, thiol reductase activity was localized in the core of growing thrombi where platelets displayed P-selectin and were in close proximity to disrupted endothelium. Conclusion PDI-sAb is a sensitive and real-time reporter of platelet- and vascular-derived disulfide reduction that targets clots as they form under flow to reveal spatial gradients.
Assuntos
Plaquetas/enzimologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Compostos de Sulfidrila/química , Animais , Anticorpos/química , Velocidade do Fluxo Sanguíneo , Transtornos Plaquetários/metabolismo , Fibrina/química , Hemodinâmica , Humanos , Integrina beta3/química , Microscopia Intravital , Camundongos , Microfluídica , Peptídeos/química , Adesividade Plaquetária , Trombina/química , Trombose/metabolismoRESUMO
Endometrial cancer is the fourth most common female cancer and the most common gynecological malignancy. Although it comprises only ~10% of all endometrial cancers, the serous histological subtype accounts for ~40% of deaths due to its aggressive behavior and propensity to metastasize. Histopathological studies suggest that elevated expression of activin/inhibin ßB subunit is associated with reduced survival in non-endometrioid endometrial cancers (type II, mostly serous). However, little is known about the specific roles and mechanisms of activin B (ßB dimer) in serous endometrial cancer growth and progression. In the present study, we examined the biological functions of activin B in type II endometrial cancer cell lines, HEC-1B and KLE. Our results demonstrate that treatment with activin B increases cell migration, invasion and adhesion to vitronectin, but does not affect cell viability. Moreover, we show that activin B treatment increases integrin ß3 mRNA and protein levels via SMAD2/3-SMAD4 signaling. Importantly, siRNA knockdown studies revealed that integrin ß3 is required for basal and activin B-induced cell migration, invasion and adhesion. Our results suggest that activin B-SMAD2/3-integrin ß3 signaling could contribute to poor patient survival by promoting the invasion and/or metastasis of type II endometrial cancers.
Assuntos
Adesão Celular , Movimento Celular , Neoplasias do Endométrio/patologia , Regulação Neoplásica da Expressão Gênica , Subunidades beta de Inibinas/farmacologia , Integrina beta3/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Apoptose , Western Blotting , Proliferação de Células , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas , Integrina beta3/química , Integrina beta3/genética , Invasividade Neoplásica , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteína Smad2/antagonistas & inibidores , Proteína Smad2/genética , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/genética , Células Tumorais CultivadasRESUMO
BACKGROUND: Increases in ligand binding to integrins (activation) play critical roles in platelet and leukocyte function. Integrin activation requires talin and kindlin binding to integrin ß cytoplasmic tails. Research has focused on the conserved GFFKR motif in integrin αII b tails, integrin ß cytoplasmic tails and the binding partners of ß tails. However, the roles of αII b tail distal of GFFKR motif are unexplored. OBJECTIVE: To investigate the role of αII b tail distal of GFFKR in talin-mediated inside-out integrin signaling. METHODS: We used model cell systems to examine the role of αII b tail distal of GFFKR in bidirectional αII b ß3 signaling and αII b ß3 -talin interactions. RESULTS: Deletion of amino acid residues after the GFFKR motif in αII b tail moderately decreased ß3 (D723R)-induced activation, abolished talin-induced αII b ß3 activation in model cells, and inhibited agonist-induced αII b ß3 activation in megakaryocytic cells. Furthermore, residues in αII b tail distal of GFFKR did not affect outside-in αII b ß3 signaling or αII b ß3 -talin interaction. Addition of non-homologous or non-specific amino acids to the GFFKR motif restored αII b ß3 activation in model cells and in megakaryocytic cells. Molecular modeling indicates that ß3 -bound talin sterically clashes with the αII b tail in the αII b ß3 complexes, potentially disfavoring the α-ß interactions that keep αII b ß3 inactive. CONCLUSION: The αII b tail sequences distal of GFFKR participate in talin-mediated inside-out αII b ß3 activation through its steric clashes with ß3 -bound talin.