Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.006
Filtrar
1.
Biophys J ; 120(20): 4349-4359, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34509509

RESUMO

Conversion of integrins from low to high affinity states, termed activation, is important in biological processes, including immunity, hemostasis, angiogenesis, and embryonic development. Integrin activation is regulated by large-scale conformational transitions from closed, low affinity states to open, high affinity states. Although it has been suggested that substrate stiffness shifts the conformational equilibrium of integrin and governs its unbinding, here, we address the role of integrin conformational activation in cellular mechanosensing. Comparison of wild-type versus activating mutants of integrin αVß3 show that activating mutants shift cell spreading, focal adhesion kinase activation, traction stress, and force on talin toward high stiffness values at lower stiffness. Although all activated integrin mutants showed equivalent binding affinity for soluble ligands, the ß3 S243E mutant showed the strongest shift in mechanical responses. To understand this behavior, we used coarse-grained computational models derived from molecular level information. The models predicted that wild-type integrin αVß3 displaces under force and that activating mutations shift the required force toward lower values, with S243E showing the strongest effect. Cellular stiffness sensing thus correlates with computed effects of force on integrin conformation. Together, these data identify a role for force-induced integrin conformational deformation in cellular mechanosensing.


Assuntos
Integrinas , Talina , Adesão Celular , Integrina alfaVbeta3/metabolismo , Integrinas/genética , Ligantes , Fenômenos Mecânicos , Ligação Proteica , Talina/metabolismo
2.
Cells ; 10(9)2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34571954

RESUMO

Ischemic vascular diseases are associated with elevated tissue expression of angiomotin (AMOT), a promising molecular target for PET imaging. On that basis, we developed an AMOT-targeting radiotracer, 68Ga-sCD146 and performed the first in vivo evaluation on a myocardial infarction mice model and then, compared AMOT expression and αvß3-integrin expression with 68Ga-sCD146 and 68Ga-RGD2 imaging. After myocardial infarction (MI) induced by permanent ligation of the left anterior descending coronary artery, myocardial perfusion was evaluated by Doppler ultrasound and by 18F-FDG PET imaging. 68Ga-sCD146 and 68Ga-RGD2 PET imaging were performed. In myocardial infarction model, heart-to-muscle ratio of 68Ga-sCD146 imaging showed a significantly higher radiotracer uptake in the infarcted area of MI animals than in sham (* p = 0.04). Interestingly, we also observed significant correlations between 68Ga-sCD146 imaging and delayed residual perfusion assessed by 18F-FDG (* p = 0.04), with lowest tissue fibrosis assessed by histological staining (* p = 0.04) and with functional recovery assessed by ultrasound imaging (** p = 0.01). 68Ga-sCD146 demonstrated an increase in AMOT expression after MI. Altogether, significant correlations of early post-ischemic 68Ga-sCD146 uptake with late heart perfusion, lower tissue fibrosis and better functional recovery, make 68Ga-sCD146 a promising radiotracer for tissue angiogenesis assessment after MI.


Assuntos
Antígeno CD146/metabolismo , Radioisótopos de Gálio/metabolismo , Infarto do Miocárdio/metabolismo , Neovascularização Patológica/metabolismo , Oligopeptídeos/metabolismo , Compostos Radiofarmacêuticos/metabolismo , Animais , Modelos Animais de Doenças , Fibrose/metabolismo , Fibrose/patologia , Fluordesoxiglucose F18/metabolismo , Integrina alfaVbeta3/metabolismo , Masculino , Camundongos , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Neovascularização Patológica/patologia , Tomografia por Emissão de Pósitrons/métodos
3.
Cells ; 10(7)2021 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-34359854

RESUMO

Integrin αvß3, a cell surface receptor, participates in signaling transduction pathways in cancer cell proliferation and metastasis. Several ligands bind to integrin αvß3 to regulate proliferation and metastasis in cancer cells. Crosstalk between the integrin and other signal transduction pathways also plays an important role in modulating cancer proliferation. Carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) activates the downstream integrin FAK to stimulate biological activities including cancer proliferation and metastasis. Blockage of signals related to integrin αvß3 was shown to be a promising target for cancer therapies. 3,3',5,5'-tetraiodothyroacetic acid (tetrac) completely binds to the integrin with the thyroid hormone to suppress cancer proliferation. The (E)-stilbene analog, resveratrol, also binds to integrin αvß3 to inhibit cancer growth. Recently, nanotechnologies have been used in the biomedical field for detection and therapeutic purposes. In the current review, we show and evaluate the potentiation of the nanomaterial carrier RGD peptide, derivatives of PLGA-tetrac (NDAT), and nanoresveratrol targeting integrin αvß3 in cancer therapies.


Assuntos
Integrina alfaVbeta3/metabolismo , Nanomedicina , Neoplasias/terapia , Animais , Humanos , Terapia de Alvo Molecular , Nanopartículas/química , Transdução de Sinais
4.
Int J Mol Sci ; 22(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34299008

RESUMO

Angiogenesis has a pivotal role in tumor growth and the metastatic process. Molecular imaging was shown to be useful for imaging of tumor-induced angiogenesis. A great variety of radiolabeled peptides have been developed to target αvß3 integrin, a target structure involved in the tumor-induced angiogenic process. The presented study aimed to synthesize deferoxamine (DFO)-based c(RGD) peptide conjugate for radiolabeling with gallium-68 and perform its basic preclinical characterization including testing of its tumor-imaging potential. DFO-c(RGDyK) was labeled with gallium-68 with high radiochemical purity. In vitro characterization including stability, partition coefficient, protein binding determination, tumor cell uptake assays, and ex vivo biodistribution as well as PET/CT imaging was performed. [68Ga]Ga-DFO-c(RGDyK) showed hydrophilic properties, high stability in PBS and human serum, and specific uptake in U-87 MG and M21 tumor cell lines in vitro and in vivo. We have shown here that [68Ga]Ga-DFO-c(RGDyK) can be used for αvß3 integrin targeting, allowing imaging of tumor-induced angiogenesis by positron emission tomography.


Assuntos
Desferroxamina/química , Radioisótopos de Gálio/química , Glioblastoma/diagnóstico por imagem , Integrina alfaVbeta3/metabolismo , Neovascularização Patológica/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Animais , Linhagem Celular Tumoral , Desferroxamina/análogos & derivados , Desferroxamina/síntese química , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Distribuição Tecidual , Tomografia Computadorizada por Raios X/métodos , Transplante Heterólogo
5.
Biochem Biophys Res Commun ; 570: 206-213, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34311201

RESUMO

Implantation is a highly organized process that involves an interaction between a competent blastocyst and a receptive uterus. Despite significant research efforts, the molecular mechanisms governing this complex process remain elusive. Here, we investigated the effect of dicalcin, an S100-like Ca2+-binding protein, on the attachment of choriocarcinoma cells (BeWo cells) onto a monolayer of endometrial carcinoma cells (Ishikawa cells). Extracellularly administered dicalcin bound to both BeWo and Ishikawa cells. Pretreatment of BeWo spheroids with dicalcin reduced the attachment ratio of the spheroids onto the monolayer, whereas that of Ishikawa cells showed no apparent change. We identified the partial amino acid sequence of human dicalcin that exhibited maximum suppression for BeWo spheroid attachment. Transmission electron microscopy analysis revealed that the dicalcin-derived peptide caused a dilation of the intercellular junction between BeWo and ISK cells. Peptide treatment of BeWo spheroids downregulated the expression of integrinαvß3 in BeWo cells, and induced alterations in their phalloidin-staining pattern, as measured by the length of each F-actin fiber and the thickness of the cortical stress fiber. Thus, dicalcin affects reorganization of the intracellular actin meshwork and subsequently the intensity of attachment, functioning as a novel suppressor of implantation.


Assuntos
Proteínas S100/metabolismo , Trofoblastos/citologia , Trofoblastos/metabolismo , Actinas/metabolismo , Animais , Adesão Celular , Linhagem Celular , Humanos , Integrina alfaVbeta3/metabolismo , Junções Intercelulares/metabolismo , Junções Intercelulares/ultraestrutura , Camundongos , Esferoides Celulares/patologia
6.
PLoS One ; 16(6): e0253347, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34161337

RESUMO

The unprecedented global COVID-19 pandemic has prompted a desperate international effort to accelerate the development of anti-viral candidates. For unknown reasons, COVID-19 infections are associated with adverse cardiovascular complications, implicating that vascular endothelial cells are essential in viral propagation. The etiological pathogen, SARS-CoV-2, has a higher reproductive number and infection rate than its predecessors, indicating it possesses novel characteristics that infers enhanced transmissibility. A unique K403R spike protein substitution encodes an Arg-Gly-Asp (RGD) motif, introducing a potential role for RGD-binding host integrins. Integrin αVß3 is widely expressed across the host, particularly in the endothelium, which acts as the final barrier before microbial entry into the bloodstream. This mutagenesis creates an additional binding site, which may be sufficient to increase SARS-CoV-2 pathogenicity. Here, we investigate how SARS-CoV-2 passes from the epithelium to endothelium, the effects of αVß3 antagonist, Cilengitide, on viral adhesion, vasculature permeability and leakage, and also report on a simulated interaction between the viral and host protein in-silico.


Assuntos
Endotélio Vascular/virologia , Integrina alfaVbeta3/metabolismo , SARS-CoV-2/patogenicidade , Venenos de Serpentes/farmacologia , Antígenos CD/metabolismo , Sítios de Ligação , COVID-19/metabolismo , COVID-19/fisiopatologia , Células CACO-2 , Caderinas/metabolismo , Simulação por Computador , Endotélio Vascular/citologia , Endotélio Vascular/fisiopatologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Integrina alfaVbeta3/química , Modelos Moleculares , Mutação , Permeabilidade , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus
7.
Biochim Biophys Acta Mol Basis Dis ; 1867(10): 166186, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34166766

RESUMO

The soluble urokinase plasminogen activator receptor (suPAR) has been implicated in the pathogenesis of kidney diseases including primary and recurrent focal and segmental glomerulosclerosis (FSGS), diabetic nephropathy, and acute kidney injuries (AKI). Elevated serum suPAR concentration is a negative prognostic indicator in multiple critical clinical conditions. This study has examined the initial transduction steps used by suPAR in cultured mouse podocytes. We now report that the receptor for advanced glycation end-products (RAGE) co-immunoprecipitates with αV and ß3 integrin subunits, which have been previously shown to initiate suPAR signal transduction at the podocyte cell surface. siRNA knock-down of RAGE attenuated Src phosphorylation evoked by either suPAR or by glycated albumin (AGE-BSA), a prototypical RAGE agonist. suPAR effects on Src phosphorylation were also blocked by the structurally dissimilar RAGE antagonists FPS-ZM1 and azeliragon, as well as by cilengitide, an inhibitor of outside-in signaling through αV-integrins. FPS-ZM1 also blocked Src phosphorylation evoked by AGE-BSA. FPS-ZM1 blocked increases in cell surface TRPC6 abundance, cytosolic reactive oxygen species (ROS) and activation of the small GTPase Rac1 evoked by either suPAR or AGE-BSA. In addition, FPS-ZM1 inhibited Src phosphorylation evoked by serum collected from a patient with recurrent FSGS during a relapse. The magnitude of this inhibition was indistinguishable from the effect produced by a neutralizing antibody against suPAR. These data suggest that orally bioavailable small molecule RAGE antagonists could represent a useful therapeutic strategy for a wide range of clinical conditions associated with elevated serum suPAR, including primary FSGS and AKI.


Assuntos
Integrina alfaVbeta3/metabolismo , Podócitos/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Humanos , Nefropatias/metabolismo , Camundongos , Espécies Reativas de Oxigênio/metabolismo
8.
Cells ; 10(5)2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068767

RESUMO

Asparagine endopeptidase (AEP), also called legumain, is currently the only known cysteine protease that specifically cleaves peptide bonds in asparaginyl residue in the mammalian genome. Since 2003, AEP has been reported to be widely expressed in a variety of carcinomas and is considered a potential therapeutic target. In the following years, researchers intensively investigated the substrates of AEP and the mechanism of AEP in partial tumors. With the identification of substrate proteins such as P53, integrin αvß3, MMP-2, and MMP-9, the biochemical mechanism of AEP in carcinomas is also more precise. This review will clarify the probable mechanisms of AEP in the progression of breast carcinoma, glioblastoma, gastric carcinoma, and epithelial ovarian carcinoma. This review will also discuss the feasibility of targeted therapy with AEP inhibitor (AEPI) in these carcinomas.


Assuntos
Neoplasias da Mama/enzimologia , Cisteína Endopeptidases/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/enzimologia , Animais , Neoplasias da Mama/patologia , Carcinoma Epitelial do Ovário/enzimologia , Carcinoma Epitelial do Ovário/patologia , Domínio Catalítico , Progressão da Doença , Matriz Extracelular/metabolismo , Feminino , Humanos , Integrina alfaVbeta3/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Metástase Neoplásica , Neoplasias Ovarianas/patologia , Peptídeos/química , Fosfatidilinositol 3-Quinases/metabolismo , Fatores de Risco , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/patologia , Proteína Supressora de Tumor p53/metabolismo
9.
ACS Appl Mater Interfaces ; 13(18): 21018-21029, 2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-33938725

RESUMO

The native extracellular matrix communicates and interacts with cells by dynamically displaying signals to control their behavior. Mimicking this dynamic environment in vitro is essential in order to unravel how cell-matrix interactions guide cell fate. Here, we present a synthetic platform for the temporal display of cell-adhesive signals using coiled-coil peptides. By designing an integrin-engaging coiled-coil pair to have a toehold (unpaired domain), we were able to use a peptide strand displacement reaction to remove the cell cue from the surface. This allowed us to test how the user-defined display of RGDS ligands at variable duration and periodicity of ligand exposure influence cell spreading degree and kinetics. Transient display of αVß3-selective ligands instructed fibroblast cells to reversibly spread and contract in response to changes in ligand exposure over multiple cycles, exhibiting a universal kinetic response. Also, cells that were triggered to spread and contract repeatedly exhibited greater enrichment of integrins in focal adhesions versus cells cultured on persistent RGDS-displaying surfaces. This dynamic platform will allow us to uncover the molecular code by which cells sense and respond to changes in their environment and will provide insights into ways to program cellular behavior.


Assuntos
Matriz Extracelular/metabolismo , Oligopeptídeos/metabolismo , Transdução de Sinais , Adesão Celular , Dimerização , Fibroblastos/citologia , Humanos , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Ligantes
10.
Molecules ; 26(6)2021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-33810198

RESUMO

Multimeric ligands consisting of multiple pharmacophores connected to a single backbone have been widely investigated for diagnostic and therapeutic applications. In this review, we summarize recent developments regarding multimeric radioligands targeting integrin αvß3 receptors on cancer cells for molecular imaging and diagnostic applications using positron emission tomography (PET). Integrin αvß3 receptors are glycoproteins expressed on the cell surface, which have a significant role in tumor angiogenesis. They act as receptors for several extracellular matrix proteins exposing the tripeptide sequence arginine-glycine-aspartic (RGD). Cyclic RDG peptidic ligands c(RGD) have been developed for integrin αvß3 tumor-targeting positron emission tomography (PET) diagnosis. Several c(RGD) pharmacophores, connected with the linker and conjugated to a chelator or precursor for radiolabeling with different PET radionuclides (18F, 64Cu, and 68Ga), have resulted in multimeric ligands superior to c(RGD) monomers. The binding avidity, pharmacodynamic, and PET imaging properties of these multimeric c(RGD) radioligands, in relation to their structural characteristics are analyzed and discussed. Furthermore, specific examples from preclinical studies and clinical investigations are included.


Assuntos
Integrina alfaVbeta3/metabolismo , Peptídeos Cíclicos , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Humanos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/uso terapêutico , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/uso terapêutico
11.
Commun Biol ; 4(1): 490, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33883697

RESUMO

Epithelial-mesenchymal transition (EMT) plays a pivotal role for tumor progression. Recent studies have revealed the existence of distinct intermediate states in EMT (partial EMT); however, the mechanisms underlying partial EMT are not fully understood. Here, we demonstrate that αvß3 integrin induces partial EMT, which is characterized by acquiring mesenchymal phenotypes while retaining epithelial markers. We found αvß3 integrin to be associated with poor survival in patients with lung adenocarcinoma. Moreover, αvß3 integrin-induced partial EMT promoted migration, invasion, tumorigenesis, stemness, and metastasis of lung cancer cells in a TGF-ß-independent fashion. Additionally, TGF-ß1 promoted EMT progression synergistically with αvß3 integrin, while a TGF-ß signaling inhibitor showed no effect on αvß3 integrin-induced partial EMT. Meanwhile, the microRNA-200 family abolished the αvß3 integrin-induced partial EMT by suppressing αvß3 integrin cell surface expression. These findings indicate that αvß3 integrin is a key inducer of partial EMT, and highlight a new mechanism for cancer progression.


Assuntos
Transição Epitelial-Mesenquimal/genética , Integrina alfaVbeta3/genética , Fator de Crescimento Transformador beta1/genética , Animais , Feminino , Integrina alfaVbeta3/metabolismo , Camundongos , Camundongos Nus , Fator de Crescimento Transformador beta1/metabolismo
12.
BMC Mol Cell Biol ; 22(1): 21, 2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33827416

RESUMO

BACKGROUND: Schwann cells (SCs) play a crucial role in the repair of peripheral nerves. This is due to their ability to proliferate, migrate, and provide trophic support to axon regrowth. During peripheral nerve injury, SCs de-differentiate and reprogram to gain the ability to repair nerves. Cysteine-rich 61 (Cyr61/CCN1) is a member of the CCN family of matrix cell proteins and have been reported to be abundant in the secretome of repair mediating SCs. In this study we investigate the function of Cyr61 in SCs. RESULTS: We observed Cyr61 was expressed both in vivo and in vitro. The promoting effect of Cyr61 on SC proliferation and migration was through autocrine and paracrine mechanisms. SCs expressed αvß3 integrin and the effect of Cyr61 on SC proliferation and migration could be blocked via αvß3 integrin. Cyr61 could influence c-Jun protein expression in cultured SCs. CONCLUSIONS: In this study, we found that Cyr61 promotes SC proliferation and migration via αvß3 integrin and regulates c-Jun expression. Our study contributes to the understanding of cellular and molecular mechanisms underlying SC's function during nerve injury, and thus, may facilitate the regeneration of peripheral nerves after injury.


Assuntos
Proliferação de Células/efeitos dos fármacos , Proteína Rica em Cisteína 61/farmacologia , Integrina alfaVbeta3/metabolismo , Células de Schwann/efeitos dos fármacos , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley
13.
J Immunol Res ; 2021: 9958239, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33880384

RESUMO

Methods: Superparamagnetic iron oxide nanoclusters (SPIOCs) were located within the core, which resulted in high photothermal conversion and outstanding generation of reactive oxygen species (ROS). The shell consisted of a human serum albumin- (HSA-) paclitaxel (PTX) layer, which extended the blood circulation time and ensured the effectiveness of the chemotherapy. Arg-Gly-Asp peptides (RGD) were linked to the naked cysteine moieties in HSA to promote the specific targeting of human glioma U87 cells by α v ß 3 integrins. Continuous near-infrared light irradiation triggered and promoted the synergistic chemo/CDT therapy through the photothermal effect. Results: Our SPIOCs@HSA-RGD nanoplatform showed well biocompatibility and could target glioma specifically. Photothermal conversion and ROS burst were detected after continuous 808 nm light irradiation, and a significant antitumor effect was achieved. Conclusion: Experimental in vitro and in vivo evaluations showed that our photothermal-mediated chemo/CDT therapy could efficiently inhibit tumor growth and is therefore promising for cancer therapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quimiorradioterapia/métodos , Glioma/terapia , Integrina alfaVbeta3/uso terapêutico , Oligopeptídeos/uso terapêutico , Paclitaxel/uso terapêutico , Nanomedicina Teranóstica/métodos , Animais , Processos de Crescimento Celular , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Raios Infravermelhos , Integrina alfaVbeta3/metabolismo , Nanopartículas Magnéticas de Óxido de Ferro/administração & dosagem , Nanopartículas Magnéticas de Óxido de Ferro/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oligopeptídeos/química , Paclitaxel/química , Ratos , Espécies Reativas de Oxigênio/metabolismo , Albumina Sérica Humana/química , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Chem Commun (Camb) ; 57(27): 3407-3410, 2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33687395

RESUMO

A new convergent chemoenzymatic synthesis strategy, integrating enzymatic synthesis of heparan sulfate, sortase A ligation, copper(i)-catalyzed alkyne-azide cycloaddition, and solid phase peptide synthesis, has been established to efficiently synthesize a mimetic of heparan sulfate proteoglycan syndecan-1 glyco-polypeptide at a milligram scale. The mimic was able to bind with αvß3 integrin faster and exhibit stronger inhibition of breast cancer cell migration compared to the glycan or the polypeptide alone. This novel approach could serve as a general approach for heparan sulfate proteoglycan mimetic synthesis.


Assuntos
Heparitina Sulfato/farmacologia , Proteoglicanas/farmacologia , Sindecana-1/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Heparitina Sulfato/química , Humanos , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/metabolismo , Conformação Molecular , Proteoglicanas/química , Sindecana-1/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
15.
Commun Biol ; 4(1): 380, 2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33782527

RESUMO

The LIM domain-dependent localization of the adapter protein paxillin to ß3 integrin-positive focal adhesions (FAs) is not mechanistically understood. Here, by combining molecular biology, photoactivation and FA-isolation experiments, we demonstrate specific contributions of each LIM domain of paxillin and reveal multiple paxillin interactions in adhesion-complexes. Mutation of ß3 integrin at a putative paxillin binding site (ß3VE/YA) leads to rapidly inward-sliding FAs, correlating with actin retrograde flow and enhanced paxillin dissociation kinetics. Induced mechanical coupling of paxillin to ß3VE/YA integrin arrests the FA-sliding, thereby disclosing an essential structural function of paxillin for the maturation of ß3 integrin/talin clusters. Moreover, bimolecular fluorescence complementation unveils the spatial orientation of the paxillin LIM-array, juxtaposing the positive LIM4 to the plasma membrane and the ß3 integrin-tail, while in vitro binding assays point to LIM1 and/or LIM2 interaction with talin-head domain. These data provide structural insights into the molecular organization of ß3 integrin-FAs.


Assuntos
Fibroblastos/metabolismo , Adesões Focais/metabolismo , Integrina alfaVbeta3/metabolismo , Paxilina/metabolismo , Animais , Sítios de Ligação , Recuperação de Fluorescência Após Fotodegradação , Adesões Focais/genética , Integrina alfaVbeta3/genética , Cinética , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Células NIH 3T3 , Paxilina/genética , Fenótipo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Relação Estrutura-Atividade
16.
DNA Cell Biol ; 40(4): 629-637, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33646053

RESUMO

Our study aimed at validating the effect of WISP1 on osteoarthritis (OA) and the pathway involved in the WISP1-induced protection against OA. The expression of WISP1 was measured by immunohistochemical analyses. We found that WISP1 expression was shown to be upregulated within human OA cartilage compared with controls. WISP1 expression was related to knee OA severity. rhWISP1 inhibited OA chondrocyte senescence and apoptosis in vitro, which was reversed by the αvß3 antibody and PI3K/Akt inhibitor LY294002. WISP1 overexpression induced by knee injection of LiCI could also prevent the senescence and apoptosis of rat chondrocytes. Safranin-O staining and Mankin score revealed that WISP1 overexpression can protect rat chondrocytes from degeneration. Nearly opposite results were obtained in the treatment of ICG-001 and siRNA-WISP1 in vivo. These data strongly suggest that WISP1 can protect against the senescence and apoptosis of chondrocytes via modulating the αvß3 receptor and PI3K/Akt signaling pathway within OA. Therefore, the development of specific activators of WISP1 may present the value of an underlying OA treatment.


Assuntos
Proteínas de Sinalização Intercelular CCN/metabolismo , Condrócitos/metabolismo , Osteoartrite do Joelho/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Adulto , Envelhecimento/fisiologia , Animais , Apoptose/fisiologia , Proteínas de Sinalização Intercelular CCN/fisiologia , Cartilagem Articular/metabolismo , Feminino , Humanos , Integrina alfaVbeta3/metabolismo , Interleucina-1beta/metabolismo , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Osteoartrite/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
17.
Cell Prolif ; 54(4): e13012, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33656760

RESUMO

OBJECTIVES: Vitronectin (VTN) has been widely used for the maintenance and expansion of human pluripotent stem cells (hPSCs) as feeder-free conditions. However, the effect of VTN on hPSC differentiation remains unclear. Here, we investigated the role of VTN in early haematopoietic development of hPSCs. MATERIALS AND METHODS: A chemically defined monolayer system was applied to study the role of different matrix or basement membrane proteins in haematopoietic development of hPSCs. The role of integrin signalling in VTN-mediated haematopoietic differentiation was investigated by integrin antagonists. Finally, small interfering RNA was used to knock down integrin gene expression in differentiated cells. RESULTS: We found that the haematopoietic differentiation of hPSCs on VTN was far more efficient than that on Matrigel that is also often used for hPSC culture. VTN promoted the fate determination of endothelial-haematopoietic lineage during mesoderm development to generate haemogenic endothelium (HE). Moreover, we demonstrated that the signals through αvß3 and αvß5 integrins were required for VTN-promoted haematopoietic differentiation. Blocking αvß3 and αvß5 integrins by the integrin antagonists impaired the development of HE, but not endothelial-to-haematopoietic transition (EHT). Finally, both αvß3 and αvß5 were confirmed acting synergistically for early haematopoietic differentiation by knockdown the expression of αv, ß3 or ß5. CONCLUSION: The established VTN-based monolayer system of haematopoietic differentiation of hPSCs presents a valuable platform for further investigating niche signals involved in human haematopoietic development.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Integrina alfaVbeta3/metabolismo , Receptores de Vitronectina/metabolismo , Vitronectina/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/genética , Mesoderma/citologia , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de Vitronectina/antagonistas & inibidores , Receptores de Vitronectina/genética , Transdução de Sinais/efeitos dos fármacos , Venenos de Serpentes/farmacologia
18.
Environ Pollut ; 279: 116933, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33773180

RESUMO

Di(2-ethylhexyl) phthalate (DEHP), a manufactured chemical, is suitable for large-scale production and has extensive applications. Although restricted for use, DEHP is still ubiquitous in the environment and shows potential to disrupt the structure or function of the thyroid system. However, its toxic mechanism is complex and not clearly understood. In this study, a battery of methods was employed to investigate DEHP-induced thyroid-disrupting effects and their mechanism of action, focusing on a newly discovered membrane receptor-mediated mechanism. The results showed that DEHP promoted rat pituitary tumor (GH3) cell proliferation and c-fos gene expression at environment level concentrations (2 and 5 µmol/L) in a manner similar to that of the natural thyroid hormone 3,3',5-triiodo-L-thyronine (T3). The macromolecule DEHP-BSA cannot pass through the cell membrane to interact with nuclear receptors but upregulated the c-fos gene expression when administered at concentrations comparable to DEHP concentrations; molecular docking demonstrated that DEHP has affinity for the membrane receptor integrin αvß3; DEHP at 2 µmol/L upregulated the ß3 gene expression in GH3 cells; after the addition of integrin αvß3-inhibiting RGD peptide, DEHP-induced c-fos gene upregulation decreased. All of these findings support the supposition that DEHP-induced thyroid-disrupting effects might be mediated by the membrane receptor integrin αvß3. Moreover, DEHP activated the downstream extracellular regulated protein kinase (ERK1/2) pathway, upregulating the gene expression of raf-1, MEK-1 and MAPK1 and increasing the protein levels of p-ERK; interestingly, ERK1/2 activation and c-fos upregulation induced by DEHP were attenuated by PD98059 (an ERK1/2 inhibitor). Taken together, the data suggest that the membrane receptor integrin αvß3 and the downstream ERK1/2 pathway might be involved in DEHP-induced thyroid-disrupting effects. This study provides new insight into the thyroid-disrupting effect and the underlying mechanism and will advance the effort to construct adverse outcome pathways of DEHP and other thyroid hormone disrupting chemicals.


Assuntos
Integrina alfaVbeta3 , Glândula Tireoide , Animais , Integrina alfaVbeta3/metabolismo , Sistema de Sinalização das MAP Quinases , Simulação de Acoplamento Molecular , Ratos , Glândula Tireoide/metabolismo , Hormônios Tireóideos
19.
Theranostics ; 11(7): 3301-3316, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33537088

RESUMO

Background: A tumor microenvironment is a complicated multicellular system comprised of tumor cells, immune cells and blood vessels. Blood vessels are the barriers for drug tissue penetration. Effectively treating a cancer requires drug delivery systems to overcome biological barriers present in tumor microenvironments (TMEs). Methods: We designed a drug delivery system made of bacterial (Escherichia coli) double layer membrane-derived nanovesicles (DMVs) with the expression of RGD peptides and endogenous targeting ligands of bacteria. The physical and biological characteristics of DMVs were assessed by cryogenic transmission electron microscopy, western blotting, flow cytometry and confocal microscopy. Doxorubicin (DOX) was loaded in DMVs via a pH gradient driven drug loading method. Therapeutical effects of DOX-loaded DMVs were studied in a melanoma xenograft mouse model. Results: In vitro and in vivo experiments showed that DMVs can target neutrophils and monocytes that mediated the transport of DMVs across blood vessel barriers and they can also directly target tumor vasculature and tumor cells, resulting in enhanced delivery of therapeutics to TMEs. Furthermore, we developed a remote drug loading approach to efficiently encapsulate DOX inside DMVs, and the drug loading was 12% (w/w). In the B16-F10 melanoma mouse model, we showed that DOX-RGD-DMVs significantly inhibited the tumor growth compared to several controls. Conclusion: Our studies reveal that DMVs are a powerful tool to simultaneously target multiple cells in TMEs, thus increasing drug delivery for improved cancer therapies.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Melanoma Experimental/tratamento farmacológico , Oligopeptídeos/genética , Proteínas Recombinantes de Fusão/genética , Neoplasias Cutâneas/tratamento farmacológico , Animais , Antibióticos Antineoplásicos/metabolismo , Membrana Celular/química , Doxorrubicina/metabolismo , Composição de Medicamentos/métodos , Escherichia coli/química , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Masculino , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/patologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neutrófilos/patologia , Oligopeptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Resultado do Tratamento , Microambiente Tumoral
20.
J Mater Chem B ; 9(9): 2240-2248, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33596297

RESUMO

Stimuli-responsive and active targeted drug release is highly significant and challenging for precise and effective cancer therapy. Herein, a reactive oxygen species (ROS)-responsive drug delivery system iRGD-BDOX@CPNs with active targeting for chemo-/photodynamic (PDT) synergistic therapy has been reported. This nanocarrier iRGD-BDOX@CPNs is constructed by the self-assembly of conjugated polymer poly(fluorene-co-vinylene) (PFV), prodrug BDOX (doxorubicin modified with a phenylboronic acid ester group) and an amphiphilic polymer (DSPE-PEG) modified with internalized RGD (DSPE-PEG-iRGD). The hydrophobic inner cores formed by PFV main chains tightly enclose BDOX. Due to PFV generating many ROS by light triggering, the BDOX prodrug can be in situ activated, resulting in the highly efficient drug release. In addition, the remarkable fluorescence recovery could be used for real-time monitoring of drug delivery and guiding antitumor therapy. Contributing to the specific recognition between iRGD and integrin αvß3 receptors over-expressed on the surface of tumor cells, the active targeting and uptake of iRGD-BDOX@CPNs in tumor cells are greatly enhanced. The prominent anti-cancer effect of iRGD-BDOX@CPNs is realized by targeted drug delivery and synergistic therapeutic effects of PDT/chemotherapy. This study illustrates that the development of ROS-responsive and targeted drug delivery nanocarriers iRGD-BDOX@CPNs provides a new insight for controllable drug release and tumor precision therapy.


Assuntos
Portadores de Fármacos/química , Nanopartículas/química , Fotoquimioterapia , Polímeros/química , Espécies Reativas de Oxigênio/metabolismo , Ácidos Borônicos/química , Linhagem Celular Tumoral , Terapia Combinada , Doxorrubicina/química , Doxorrubicina/farmacologia , Humanos , Integrina alfaVbeta3/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...