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1.
Stem Cells ; 38(2): 231-245, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31648388

RESUMO

Therapeutic applications for mesenchymal stem/stromal cells (MSCs) are growing; however, the successful implementation of these therapies requires the development of appropriate MSC delivery systems. Hydrogels are ideally suited to cultivate MSCs but tuning hydrogel properties to match their specific in vivo applications remains a challenge. Thus, further characterization of how hydrogel-based delivery vehicles broadly influence MSC function and fate will help lead to the next generation of more intelligently designed delivery vehicles. To date, few attempts have been made to comprehensively characterize hydrogel impact on the MSC transcriptome. Herein, we have synthesized cell-degradable hydrogels based on bio-inert poly(ethylene glycol) tethered with specific integrin-binding small molecules and have characterized their resulting effect on the MSC transcriptome when compared with 2D cultured and untethered 3D hydrogel cultured MSCs. The 3D culture systems resulted in alterations in the MSC transcriptome, as is evident by the differential expression of genes related to extracellular matrix production, glycosylation, metabolism, signal transduction, gene epigenetic regulation, and development. For example, genes important for osteogenic differentiation were upregulated in 3D hydrogel cultures, and the expression of these genes could be partially suppressed by tethering an integrin-binding RGD peptide within the hydrogel. Highlighting the utility of tunable hydrogels, when applied to ex vivo human wounds the RGD-tethered hydrogel was able to support wound re-epithelialization, possibly due to its ability to increase PDGF expression and decrease IL-6 expression. These results will aid in future hydrogel design for a broad range of applications.

2.
J Immunol ; 203(5): 1383-1391, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31331973

RESUMO

CD40L plays a major role in immune response and is a major therapeutic target for inflammation. Integrin α5ß1 and CD40 simultaneously bind to CD40L. It is unclear if α5ß1 and CD40 work together in CD40/CD40L signaling or how α5ß1 binds to CD40L. In this article, we describe that the integrin-binding site of human CD40L is predicted to be located in the trimeric interface by docking simulation. Mutations in the predicted integrin-binding site markedly reduced the binding of α5ß1 to CD40L. Several CD40L mutants defective in integrin binding were defective in NF-κB activation and B cell activation and suppressed CD40L signaling induced by wild-type CD40L; however, they still bound to CD40. These findings suggest that integrin α5ß1 binds to monomeric CD40L through the binding site in the trimeric interface of CD40L, and this plays a critical role in CD40/CD40L signaling. Integrin αvß3, a widely distributed vascular integrin, bound to CD40L in a KGD-independent manner, suggesting that αvß3 is a new CD40L receptor. Several missense mutations in CD40L that induce immunodeficiency with hyper-IgM syndrome type 1 (HIGM1) are clustered in the integrin-binding site of the trimeric interface. These HIGM1 CD40L mutants were defective in binding to α5ß1 and αvß3 (but not to CD40), suggesting that the defect in integrin binding may be a causal factor of HIGM1. These findings suggest that α5ß1 and αvß3 bind to the overlapping binding site in the trimeric interface of monomeric CD40L and generate integrin-CD40L-CD40 ternary complex. CD40L mutants defective in integrins have potential as antagonists of CD40/CD40L signaling.

3.
FASEB J ; 33(8): 9131-9141, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31116572

RESUMO

Proper control of cell migration is critically important in many biologic processes, such as wound healing, immune surveillance, and development. Much progress has been made in the initiation of cell migration; however, little is known about termination and sometimes directional reversal. During active cell migration, as in wound healing, development, and immune surveillance, the integrin expression profile undergoes drastic changes. Here, we uncovered the extensive regulatory and even opposing roles of integrins in directional cell migration in electric fields (EFs), a potentially important endogenous guidance mechanism. We established cell lines that stably express specific integrins and determined their responses to applied EFs with a high throughput screen. Expression of specific integrins drove cells to migrate to the cathode or to the anode or to lose migration direction. Cells expressing αMß2, ß1, α2, αIIbß3, and α5 migrated to the cathode, whereas cells expressing ß3, α6, and α9 migrated to the anode. Cells expressing α4, αV, and α6ß4 lost directional electrotaxis. Manipulation of α9 molecules, one of the molecular directional switches, suggested that the intracellular domain is critical for the directional reversal. These data revealed an unreported role for integrins in controlling stop, go, and reversal activity of directional migration of mammalian cells in EFs, which might ensure that cells reach their final destination with well-controlled speed and direction.-Zhu, K., Takada, Y., Nakajima, K., Sun, Y., Jiang, J., Zhang, Y., Zeng, Q., Takada, Y., Zhao, M. Expression of integrins to control migration direction of electrotaxis.

4.
Sci Rep ; 8(1): 10019, 2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-29968781

RESUMO

The enteric species F human adenovirus types 40 and 41 (HAdV-40 and -41) are the third most common cause of infantile gastroenteritis in the world. Knowledge about HAdV-40 and -41 cellular infection is assumed to be fundamentally different from that of other HAdVs since HAdV-40 and -41 penton bases lack the αV-integrin-interacting RGD motif. This motif is used by other HAdVs mainly for internalization and endosomal escape. We hypothesised that the penton bases of HAdV-40 and -41 interact with integrins independently of the RGD motif. HAdV-41 transduction of a library of rodent cells expressing specific human integrin subunits pointed to the use of laminin-binding α2-, α3- and α6-containing integrins as well as other integrins as candidate co-receptors. Specific laminins prevented internalisation and infection, and recombinant, soluble HAdV-41 penton base proteins prevented infection of human intestinal HT-29 cells. Surface plasmon resonance analysis demonstrated that HAdV-40 and -41 penton base proteins bind to α6-containing integrins with an affinity similar to that of previously characterised penton base:integrin interactions. With these results, we propose that laminin-binding integrins are co-receptors for HAdV-40 and -41.


Assuntos
Adenovírus Humanos/metabolismo , Integrina alfa6/metabolismo , Integrina alfa6beta4/metabolismo , Laminina/metabolismo , Receptores Virais/metabolismo , Ligação Viral , Animais , Células CHO , Linhagem Celular , Cricetulus , Células HT29 , Humanos , Ressonância de Plasmônio de Superfície
5.
Biochem J ; 475(4): 723-732, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-29301984

RESUMO

Leukocyte arrest on the endothelial cell surface during leukocyte extravasation is induced by rapid integrin activation by chemokines. We recently reported that fractalkine induces integrin activation without its receptor CX3CR1 through binding to the allosteric site (site 2) of integrins. Peptides from site 2 bound to fractalkine and suppressed integrin activation by fractalkine. We hypothesized that this is not limited to membrane-bound fractalkine. We studied whether stromal cell-derived factor-1 (SDF1), another chemokine that plays a critical role in leukocyte arrest, activates integrins through binding to site 2. We describe here that (1) SDF1 activated soluble integrin αvß3 in cell-free conditions, suggesting that SDF1 can activate αvß3 without CXCR4; (2) site 2 peptide bound to SDF1, suggesting that SDF1 binds to site 2; (3) SDF1 activated integrins αvß3, α4ß1, and α5ß1 on CHO cells (CXCR4-negative) and site 2 peptide suppressed the activation; (4) A CXCR4 antagonist AMD3100 did not affect the site 2-mediated integrin activation by SDF1; (5) Cell-surface integrins were fully activated in 1 min (much faster than activation of soluble αvß3) and the activation lasted at least for 1 h. We propose that the binding of SDF1 to cell-surface proteoglycan facilitates the allosteric activation process; (6) Mutations in the predicted site 2-binding site in SDF1 suppressed integrin activation. These results suggest that SDF1 (e.g. presented on proteoglycans) can rapidly activate integrins in an allosteric manner by binding to site 2 in the absence of CXCR4. The allosteric integrin activation by SDF1 is a novel target for drug discovery.


Assuntos
Quimiocina CXCL12/química , Integrinas/química , Receptores CXCR4/química , Sítio Alostérico , Animais , Sítios de Ligação , Células CHO , Sistema Livre de Células , Quimiocina CX3CL1/química , Quimiocina CX3CL1/genética , Quimiocina CXCL12/genética , Cricetulus , Humanos , Integrinas/genética , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Receptores CXCR4/genética , Transdução de Sinais/genética
6.
J Biol Chem ; 292(49): 20067-20075, 2017 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-29030430

RESUMO

There is a strong link between integrins and interleukin-1ß (IL-1ß), but the specifics of the role of integrins in IL-1ß signaling are unclear. We describe that IL-1ß specifically bound to integrins αvß3 and α5ß1. The E128K mutation in the IL1R-binding site enhanced integrin binding. We studied whether direct integrin binding is involved in IL-1ß signaling. We compared sequences of IL-1ß and IL-1 receptor antagonist (IL1RN), which is an IL-1ß homologue but has no agonistic activity. Several surface-exposed Lys residues are present in IL-1ß, but not in IL1RN. A disulfide linkage is present in IL1RN, but is not in IL-1ß because of natural C117F mutation. Substitution of the Lys residues to Glu markedly reduced integrin binding of E128K IL-1ß, suggesting that the Lys residues mediate integrin binding. The Lys mutations reduced, but did not completely abrogate, agonistic action of IL-1ß. We studied whether the disulfide linkage plays a role in agonistic action of IL-1ß. Reintroduction of the disulfide linkage by the F117C mutation did not affect agonistic activity of WT IL-1ß, but effectively reduced the remaining agonistic activity of the Lys mutants. Also, deletion of the disulfide linkage in IL1RN by the C116F mutation did not make it agonistic. We propose that the direct binding to IL-1ß to integrins is primarily important for agonistic IL-1ß signaling, and that the disulfide linkage indirectly affects signaling by blocking conformational changes induced by weak integrin binding to the Lys mutants. The integrin-IL-1ß interaction is a potential target for drug discovery.


Assuntos
Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Integrinas/metabolismo , Interleucina-1beta/metabolismo , Animais , Células CHO , Cricetulus , Dissulfetos/farmacologia , Humanos , Interleucina-1beta/genética , Células MCF-7 , Mutação , Ligação Proteica , Transdução de Sinais
7.
PLoS One ; 12(9): e0184285, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28873464

RESUMO

We have reported that integrins crosstalk with growth factors through direct binding to growth factors (e.g., fibroblast growth factor-1, insulin-like growth factor 1 (IGF1), neuregulin-1, fractalkine) and subsequent ternary complex formation with cognate receptor [e.g., integrin/IGF1/IGF1 receptor (IGF1R)]. IGF1 and IGF2 are overexpressed in cancer and major therapeutic targets. We previously reported that IGF1 binds to integrins ανß3 and α6ß4, and the R36E/R37E mutant in the C-domain of IGF1 is defective integrin binding and signaling functions of IGF1, and acts as an antagonist of IGF1R. We studied if integrins play a role in the signaling functions of IGF2, another member of the IGF family. Here we describe that IGF2 specifically binds to integrins ανß3 and α6ß4, and induced proliferation of CHO cells (IGF1R+) that express ανß3 or α6ß4 (ß3- or α6ß4-CHO cells). Arg residues to Glu at positions 24, 34, 37 and/or 38 in or close to the C-domain of IGF2 play a critical role in binding to integrins and signaling functions. The R24E/R37E/R38E, R34E/R37E/R38E, and R24E/R34E/R37E/R38E mutants were defective in integrin binding and IGF2 signaling. These mutants suppressed proliferation induced by WT IGF2, suggesting that they are dominant-negative antagonists of IGF1R. These results suggest that IGF2 also requires integrin binding for signaling functions, and the IGF2 mutants that cannot bind to integrins act as antagonists of IGF1R. The present study defines the role of the C-domain in integrin binding and signaling.


Assuntos
Fator de Crescimento Insulin-Like II/química , Fator de Crescimento Insulin-Like II/metabolismo , Integrina alfa6beta4/metabolismo , Integrina alfaVbeta3/metabolismo , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Células CHO , Linhagem Celular Tumoral , Proliferação de Células , Cricetinae , Cricetulus , Humanos , Proteínas Mutantes , Ligação Proteica , Domínios Proteicos , Relação Estrutura-Atividade
8.
Biosci Rep ; 37(2)2017 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-28302677

RESUMO

We recently found that integrin αvß3 binds to fibroblast growth factor (FGF)-αvß31 (FGF1), and that the integrin-binding defective FGF1 mutant (Arg-50 to glutamic acid, R50E) is defective in signalling and antagonistic to FGF1 signalling. R50E suppressed angiogenesis and tumour growth, suggesting that R50E has potential as a therapeutic. However, FGF1 is unstable, and we had to express R50E in cancer cells for xenograft study, since injected R50E may rapidly disappear from circulation. We studied if we can develop antagonist of more stable FGF2. FGF2 is widely involved in important biological processes such as stem cell proliferation and angiogenesis. Previous studies found that FGF2 bound to αvß3 and antagonists to αvß3 suppressed FGF2-induced angiogenesis. However, it is unclear how FGF2 interacts with integrins. Here, we describe that substituting Lys-119/Arg-120 and Lys-125 residues in the predicted integrin-binding interface of FGF2 to glutamic acid (the K119E/R120E and K125E mutations) effectively reduced integrin binding to FGF2. These FGF2 mutants were defective in signalling functions (ERK1/2 activation and DNA synthesis) in NIH3T3 cells. Notably they suppressed, FGF2 signalling induced by WT FGF2 in endothelial cells, suggesting that the FGF2 mutants are antagonists. The FGF2 mutants effectively suppressed tube formation in vitro, sprouting in aorta ring assays ex vivo and angiogenesis in vivo The positions of amino acids critical for integrin binding are different between FGF1 and FGF2, suggesting that they do not interact with integrins in the same manner. The newly developed FGF2 mutants have potential as anti-angiogenic agents and useful tools for studying the role of integrins in FGF2 signalling.


Assuntos
Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Integrina alfaVbeta3/metabolismo , Mutação de Sentido Incorreto , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Sítios de Ligação/genética , Células Cultivadas , Fator 2 de Crescimento de Fibroblastos/química , Humanos , Integrina alfaVbeta3/química , Células K562 , Cinética , Camundongos , Modelos Moleculares , Células NIH 3T3 , Neovascularização Fisiológica/genética , Ligação Proteica , Domínios Proteicos , Ratos , Transdução de Sinais/genética
9.
Cytokine Growth Factor Rev ; 34: 67-72, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28190785

RESUMO

It has been generally accepted that integrin cell adhesion receptors are involved in growth factor signaling (integrin-growth factor crosstalk), since antagonists to integrins often suppress growth factor signaling. Partly because integrins have been originally identified as cell adhesion receptors to extracellular matrix (ECM) proteins, current models of the crosstalk between IGF1 and integrins propose that ECM ligands (e.g., vitronectin) bind to integrins and IGF1 binds to IGF receptor type 1 (IGF1R), and two separate signals merge inside the cells. Our research proves otherwise. We discovered that IGF1 interacts directly with integrins, and induces integrin-IGF-IGF1R complex formation on the cell surface. IGF1 signaling can be detected in the absence of ECM (anchorage-independent conditions). Integrin antagonists block both ECM-integrin interaction and IGF-integrin interaction, and do not distinguish the two. This is one possible reason why integrin-IGF1 interaction has not been detected. With these new discoveries, we believe that the direct IGF-integrin interaction should be incorporated into models of IGF1 signaling. The integrin-binding defective mutant of IGF1 is defective in inducing IGF signaling, although the mutant still binds to IGF1R. Notably, the IGF1 mutant is dominant-negative and suppresses cell proliferation induced by wt IGF1, and suppresses tumorigenesis in vivo, and thus the IGF1 mutant has potential as a therapeutic.


Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Integrinas/metabolismo , Receptor Cross-Talk , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Animais , Adesão Celular , Proliferação de Células , Transformação Celular Neoplásica , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/uso terapêutico , Camundongos , Ligação Proteica , Transporte Proteico , Receptores de Somatomedina
10.
Biochem J ; 474(4): 589-596, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-27993971

RESUMO

Tetraspanins play important roles in normal (e.g. cell adhesion, motility, activation, and proliferation) and pathological conditions (e.g. metastasis and viral infection). Tetraspanins interact with integrins and regulate integrin functions, but the specifics of tetraspanin-integrin interactions are unclear. Using co-immunoprecipitation with integrins as a sole method to detect interaction between integrins and full-length tetraspanins, it has been proposed that the variable region (helices D and E) of the extracellular-2 (EC2) domain of tetraspanins laterally associates with a non-ligand-binding site of integrins. We describe that, using adhesion assays, the EC2 domain of CD81, CD9, and CD151 bound to integrin αvß3, and this binding was suppressed by cRGDfV, a specific inhibitor of αvß3, and antibody 7E3, which is mapped to the ligand-binding site of ß3. We also present evidence that the specificity loop of ß3 directly bound to the EC2 domains. This suggests that the EC2 domains specifically bind to the classical ligand-binding site of αvß3. αvß3 was a more effective receptor for the EC2 domains than the previously known tetraspanin receptors α3ß1, α4ß1, and α6ß1. Docking simulation predicted that the helices A and B of CD81 EC2 bind to the RGD-binding site of αvß3. Substituting Lys residues at positions 116 and 144/148 of CD81 EC2 in the predicted integrin-binding interface reduced the binding of CD81 EC2 to αvß3, consistent with the docking model. These findings suggest that, in contrast with previous models, the ligand-binding site of integrin αvß3, a new tetraspanin receptor, binds to the constant region (helices A and B) of the EC2 domain.


Assuntos
Integrina alfaVbeta3/química , Tetraspanina 24/química , Tetraspanina 28/química , Tetraspanina 29/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Sítios de Ligação , Células CHO , Clonagem Molecular , Cricetulus , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/imunologia , Cinética , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tetraspanina 24/genética , Tetraspanina 24/imunologia , Tetraspanina 28/genética , Tetraspanina 28/imunologia , Tetraspanina 29/genética , Tetraspanina 29/imunologia
11.
Adv Exp Med Biol ; 925: 103-115, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27864802

RESUMO

Secreted phospholipase A2 type IIA (sPLA2-IIA) is a well-established pro-inflammatory protein and has been a major target for drug discovery. However, the mechanism of its signaling action has not been fully understood. We previously found that sPLA2-IIA binds to integrins αvß3 and α4ß1 in human and that this interaction plays a role in sPLA2-IIA's signaling action. Our recent studies found that sPLA2-IIA activates integrins in an allosteric manner through direct binding to a newly identified binding site of integrins (site 2), which is distinct from the classical RGD-binding site (site 1). The sPLA2-IIA-induced integrin activation may be related to the signaling action of sPLA2-IIA. Since sPLA2-IIA is present in normal human tears in addition to rheumatoid synovial fluid at high concentrations the sPLA2-IIA-mediated integrin activation on leukocytes may be involved in immune responses in normal and pathological conditions.


Assuntos
Fosfolipases A2 do Grupo II/química , Integrina alfa4beta1/química , Integrina alfaVbeta3/química , Transdução de Sinais/imunologia , Regulação Alostérica , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Sítios de Ligação , Regulação da Expressão Gênica , Fosfolipases A2 do Grupo II/genética , Fosfolipases A2 do Grupo II/imunologia , Humanos , Integrina alfa4beta1/genética , Integrina alfa4beta1/imunologia , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/imunologia , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Líquido Sinovial/química , Líquido Sinovial/imunologia , Lágrimas/química , Lágrimas/imunologia
12.
J Biol Chem ; 291(40): 20993-21007, 2016 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-27484800

RESUMO

We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5ß1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-ß1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-ß1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5ß1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking.


Assuntos
Anticorpos Monoclonais Murinos/química , Epitopos/química , Fibronectinas/química , Integrina alfa5beta1/química , Modelos Moleculares , Oligopeptídeos/química , Regulação Alostérica/imunologia , Anticorpos Monoclonais Murinos/imunologia , Epitopos/genética , Epitopos/imunologia , Fibronectinas/genética , Fibronectinas/imunologia , Humanos , Integrina alfa5beta1/genética , Integrina alfa5beta1/imunologia , Células Jurkat , Oligopeptídeos/genética , Oligopeptídeos/imunologia
13.
Mol Cancer Ther ; 15(2): 232-40, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26719578

RESUMO

We have previously reported the use of one-bead-one-compound (OBOC) combinatorial technology to develop a disulfide cyclic, Arg-Gly-Asp-containing octapeptide LXW7 (cGRGDdvc), that targets αvß3 integrin with high affinity and specificity. αvß3 integrin is known to be overexpressed in many cancers and in tumor vasculature, and it has been established as a cancer therapeutic target. To further optimize LXW7, we have performed systematic structure-activity relationship studies. On the basis of the results, two highly focused OBOC peptide libraries were designed, synthesized, and screened against αvß3 integrin-transfected K562 cells. One of the best ligands, LXW64, was found to have 6.6-fold higher binding affinity than LXW7, and showed preferential binding to cells expressing αvß3 integrin. In addition to binding strongly to U-87MG glioblastoma cells in vitro, LXW64 also targets U-87MG xenografts implanted in nude mice, indicating that it is an excellent vehicle for the delivery of cytotoxic payload to tumors and tumor blood vessels that overexpress αvß3 integrin. Mol Cancer Ther; 15(2); 232-40. ©2015 AACR.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Glioblastoma/metabolismo , Integrina alfaVbeta3/metabolismo , Peptídeos Cíclicos/síntese química , Animais , Linhagem Celular Tumoral , Técnicas de Química Combinatória , Humanos , Células K562 , Camundongos , Camundongos Nus , Estrutura Molecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Relação Estrutura-Atividade
14.
PLoS One ; 10(9): e0137486, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26334633

RESUMO

Epithelial-to-mesenchymal transition (EMT) plays a critical role in cancer metastasis, and is regulated by growth factors such as transforming growth factor ß (TGF-ß) and fibroblast growth factors (FGF) secreted from the stromal and tumor cells. However, the role of growth factors in EMT has not been fully established. Several integrins are upregulated by TGF-ß1 during EMT. Integrins are involved in growth factor signaling through integrin-growth factor receptor crosstalk. We previously reported that FGF1 directly binds to integrin αvß3 and the interaction was required for FGF1 functions such as cell proliferation and migration. We studied the role of αvß3 induced by TGF-ß on TGF-ß-induced EMT. Here, we describe that FGF1 augmented EMT induced by TGF-ß1 in MCF10A and MCF12A mammary epithelial cells. TGF-ß1 markedly amplified integrin αvß3 and FGFR1 (but not FGFR2). We studied if the enhancing effect of FGF1 on TGF-ß1-induced EMT requires enhanced levels of both integrin αvß3 expression and FGFR1. Knockdown of ß3 suppressed the enhancement by FGF1 of TGF-ß1-induced EMT in MCF10A cells. Antagonists to FGFR suppressed the enhancing effect of FGF1 on EMT. Integrin-binding defective FGF1 mutant did not augment TGF-ß1-induced EMT in MCF10A cells. These findings suggest that enhanced integrin αvß3 expression in addition to enhanced FGFR1 expression is critical for FGF1 to augment TGF-ß1-induced EMT in mammary epithelial cells.


Assuntos
Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fator 1 de Crescimento de Fibroblastos/farmacologia , Integrina alfaVbeta3/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Humanos , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos
15.
J Biol Chem ; 290(19): 12403-14, 2015 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-25814665

RESUMO

Human rotavirus is the leading cause of severe gastroenteritis in infants and children under the age of 5 years in both developed and developing countries. Human lactadherin, a milk fat globule membrane glycoprotein, inhibits human rotavirus infection in vitro, whereas bovine lactadherin is not active. Moreover, it protects breastfed infants against symptomatic rotavirus infections. To explore the potential antiviral activity of lactadherin sourced by equines, we undertook a proteomic analysis of milk fat globule membrane proteins from donkey milk and elucidated its amino acid sequence. Alignment of the human, bovine, and donkey lactadherin sequences revealed the presence of an Asp-Gly-Glu (DGE) α2ß1 integrin-binding motif in the N-terminal domain of donkey sequence only. Because integrin α2ß1 plays a critical role during early steps of rotavirus host cell adhesion, we tested a minilibrary of donkey lactadherin-derived peptides containing DGE sequence for anti-rotavirus activity. A 20-amino acid peptide containing both DGE and RGD motifs (named pDGE-RGD) showed the greatest activity, and its mechanism of antiviral action was characterized; pDGE-RGD binds to integrin α2ß1 by means of the DGE motif and inhibits rotavirus attachment to the cell surface. These findings suggest the potential anti-rotavirus activity of equine lactadherin and support the feasibility of developing an anti-rotavirus peptide that acts by hindering virus-receptor binding.


Assuntos
Antígenos de Superfície/química , Glicolipídeos/química , Glicoproteínas/química , Glicoproteínas de Membrana/química , Proteínas do Leite/química , Peptídeos/química , Infecções por Rotavirus/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Bovinos , Membrana Celular/metabolismo , Sobrevivência Celular , Equidae , Cavalos , Humanos , Concentração Inibidora 50 , Integrinas/química , Leite , Dados de Sequência Molecular , Proteômica , Rotavirus/metabolismo , Infecções por Rotavirus/tratamento farmacológico , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
16.
J Biol Chem ; 290(1): 259-71, 2015 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-25398877

RESUMO

Integrins are activated by signaling from inside the cell (inside-out signaling) through global conformational changes of integrins. We recently discovered that fractalkine activates integrins in the absence of CX3CR1 through the direct binding of fractalkine to a ligand-binding site in the integrin headpiece (site 2) that is distinct from the classical RGD-binding site (site 1). We propose that fractalkine binding to the newly identified site 2 induces activation of site 1 though conformational changes (in an allosteric mechanism). We reasoned that site 2-mediated activation of integrins is not limited to fractalkine. Human secreted phospholipase A2 type IIA (sPLA2-IIA), a proinflammatory protein, binds to integrins αvß3 and α4ß1 (site 1), and this interaction initiates a signaling pathway that leads to cell proliferation and inflammation. Human sPLA2-IIA does not bind to M-type receptor very well. Here we describe that sPLA2-IIA directly activated purified soluble integrin αvß3 and transmembrane αvß3 on the cell surface. This activation did not require catalytic activity or M-type receptor. Docking simulation predicted that sPLA2-IIA binds to site 2 in the closed-headpiece of αvß3. A peptide from site 2 of integrin ß1 specifically bound to sPLA2-IIA and suppressed sPLA2-IIA-induced integrin activation. This suggests that sPLA2-IIA activates αvß3 through binding to site 2. sPLA2-IIA also activated integrins α4ß1 and α5ß1 in a site 2-mediated manner. We recently identified small compounds that bind to sPLA2-IIA and suppress integrin-sPLA2-IIA interaction (e.g. compound 21 (Cmpd21)). Cmpd21 effectively suppressed sPLA2-IIA-induced integrin activation. These results define a novel mechanism of proinflammatory action of sPLA2-IIA through integrin activation.


Assuntos
Fosfolipases A2 do Grupo II/química , Integrina alfa4beta1/química , Integrina alfaVbeta3/química , Receptores de Vitronectina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cricetulus , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Fosfolipases A2 do Grupo II/antagonistas & inibidores , Fosfolipases A2 do Grupo II/genética , Fosfolipases A2 do Grupo II/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Células K562 , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Ligação Proteica , Receptores de Vitronectina/genética , Receptores de Vitronectina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Transdução de Sinais
17.
PLoS One ; 9(5): e96372, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24789099

RESUMO

The chemokine domain of fractalkine (FKN-CD) binds to the classical RGD-binding site of αvß3 and that the resulting ternary complex formation (integrin-FKN-CX3CR1) is critical for CX3CR1 signaling and FKN-induced integrin activation. However, only certain cell types express CX3CR1. Here we studied if FKN-CD can activate integrins in the absence of CX3CR1. We describe that WT FKN-CD activated recombinant soluble αvß3 in cell-free conditions, but the integrin-binding defective mutant of FKN-CD (K36E/R37E) did not. This suggests that FKN-CD can activate αvß3 in the absence of CX3CR1 through the direct binding of FKN-CD to αvß3. WT FKN-CD activated αvß3 on CX3CR1-negative cells (K562 and CHO) but K36E/R37E did not, suggesting that FKN-CD can activate integrin at the cellular levels in a manner similar to that in cell-free conditions. We hypothesized that FKN-CD enhances ligand binding to the classical RGD-binding site (site 1) through binding to a second binding site (site 2) that is distinct from site 1 in αvß3. To identify the possible second FKN-CD binding site we performed docking simulation of αvß3-FKN-CD interaction using αvß3 with a closed inactive conformation as a target. The simulation predicted a potential FKN-CD-binding site in inactive αvß3 (site 2), which is located at a crevice between αv and ß3 on the opposite side of site 1 in the αvß3 headpiece. We studied if FKN-CD really binds to site 2 using a peptide that is predicted to interact with FKN-CD in site 2. Notably the peptide specifically bound to FKN-CD and effectively suppressed integrin activation by FKN-CD. This suggests that FKN-CD actually binds to site 2, and this leads to integrin activation. We obtained very similar results in α4ß1 and α5ß1. The FKN binding to site 2 and resulting integrin activation may be a novel mechanism of integrin activation and of FKN signaling.


Assuntos
Quimiocina CX3CL1/metabolismo , Integrina alfaVbeta3/metabolismo , Oligopeptídeos/metabolismo , Receptores de Quimiocinas/metabolismo , Proteínas ADAM/química , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Western Blotting , Células CHO , Receptor 1 de Quimiocina CX3C , Quimiocina CX3CL1/química , Quimiocina CX3CL1/genética , Cricetinae , Cricetulus , Humanos , Integrina alfa4beta1/química , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Integrina alfaVbeta3/química , Integrina alfaVbeta3/genética , Células K562 , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Mutação , Oligopeptídeos/química , Oligopeptídeos/genética , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Quimiocinas/genética , Receptores de Vitronectina/química , Receptores de Vitronectina/genética , Receptores de Vitronectina/metabolismo , Células U937
18.
PLoS One ; 9(4): e93738, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24695572

RESUMO

The prototypic acute phase reactant C-reactive protein (CRP) is not only a marker but also a potential contributor to inflammatory diseases. CRP exists as the circulating native, pentameric CRP (pCRP) and the monomeric isoform (mCRP), formed as a result of a dissociation process of pCRP. mCRP is highly pro-inflammatory, but pCRP is not. The mechanism of pro-inflammatory action of mCRP is unclear. We studied the role of integrins in pro-inflammatory action of mCRP. Docking simulation of interaction between mCRP and integrin αvß3 predicted that mCRP binds to αvß3 well. We found that mCRP actually bound to integrins αvß3 and α4ß1 well. Antagonists to αvß3 or α4ß1 effectively suppressed the interaction, suggesting that the interaction is specific. Using an integrin ß1 mutant (ß1-3-1) that has a small fragment from the ligand binding site of ß3, we showed that mCRP bound to the classical RGD-binding site in αvß3. We studied the role of integrins in CRP signaling in monocytic U937 cells. Integrins αvß3 and α4ß1 specifically mediated binding of mCRP to U937 cells. mCRP induced AKT phosphorylation, but not ERK1/2 phosphorylation, in U937 cells. Notably, mCRP induced robust chemotaxis in U937 cells, and antagonists to integrins αvß3 and α4ß1 and an inhibitor to phosphatidylinositide 3-kinase, but not an MEK inhibitor, effectively suppressed mCRP-induced chemotaxis in U937 cells. These results suggest that the integrin and AKT/phosphatidylinositide 3-kinase pathways play a role in pro-inflammatory action of mCRP in U937 cells. In contrast, pCRP is predicted to have a limited access to αvß3 due to steric hindrance in the simulation. Consistent with the prediction, pCRP was much less effective in integrin binding, chemotaxis, or AKT phosphorylation. These findings suggest that the ability of CRP isoforms to bind to the integrins is related to their pro-inflammatory action.


Assuntos
Proteína C-Reativa/metabolismo , Quimiotaxia/fisiologia , Inflamação/metabolismo , Integrina alfa4beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Animais , Proteína C-Reativa/farmacologia , Células CHO , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Quimiotaxia/efeitos dos fármacos , Cricetulus , Humanos , Monócitos/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ligação Proteica , Células U937
19.
J Biol Chem ; 288(27): 19593-603, 2013 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-23696648

RESUMO

Insulin-like growth factor-1 (IGF1) is a major therapeutic target for cancer. We recently reported that IGF1 directly binds to integrins (αvß3 and α6ß4) and induces ternary complex formation (integrin-IGF1-IGF1 receptor (IGF1R)) and that the integrin binding-defective mutant of IGF1 (R36E/R37E) is defective in signaling and ternary complex formation. These findings predict that R36E/R37E competes with WT IGF1 for binding to IGF1R and inhibits IGF signaling. Here, we described that excess R36E/R37E suppressed cell viability increased by WT IGF1 in vitro in non-transformed cells. We studied the effect of R36E/R37E on viability and tumorigenesis in cancer cell lines. We did not detect an effect of WT IGF1 or R36E/R37E in cancer cells under anchorage-dependent conditions. However, under anchorage-independent conditions, WT IGF1 enhanced cell viability and induced signals, whereas R36E/R37E did not. Notably, excess R36E/R37E suppressed cell viability and signaling induced by WT IGF1 under anchorage-independent conditions. Using cancer cells stably expressing WT IGF1 or R36E/R37E, we determined that R36E/R37E suppressed tumorigenesis in vivo, whereas WT IGF1 markedly enhanced it. R36E/R37E suppressed the binding of WT IGF1 to the cell surface and the subsequent ternary complex formation induced by WT IGF1. R36E/R37E suppressed activation of IGF1R by insulin. WT IGF1, but not R36E/R37E, induced ternary complex formation with the IGF1R/insulin receptor hybrid. These findings suggest that 1) IGF1 induces signals under anchorage-independent conditions and that 2) R36E/R37E acts as a dominant-negative inhibitor of IGF1R (IGF1 decoy). Our results are consistent with a model in which ternary complex formation is critical for IGF signaling.


Assuntos
Substituição de Aminoácidos , Transformação Celular Neoplásica/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Mutação de Sentido Incorreto , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Integrinas , Camundongos , Modelos Biológicos , Células NIH 3T3 , Ligação Proteica , Estrutura Quaternária de Proteína , Receptor IGF Tipo 1/genética , Transdução de Sinais/genética
20.
PLoS One ; 8(2): e57927, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23469107

RESUMO

Fibroblast growth factor-1 (FGF1) and FGF2 play a critical role in angiogenesis, a formation of new blood vessels from existing blood vessels. Integrins are critically involved in FGF signaling through crosstalk. We previously reported that FGF1 directly binds to integrin αvß3 and induces FGF receptor-1 (FGFR1)-FGF1-integrin αvß3 ternary complex. We previously generated an integrin binding defective FGF1 mutant (Arg-50 to Glu, R50E). R50E is defective in inducing ternary complex formation, cell proliferation, and cell migration, and suppresses FGF signaling induced by WT FGF1 (a dominant-negative effect) in vitro. These findings suggest that FGFR and αvß3 crosstalk through direct integrin binding to FGF, and that R50E acts as an antagonist to FGFR. We studied if R50E suppresses tumorigenesis and angiogenesis. Here we describe that R50E suppressed tumor growth in vivo while WT FGF1 enhanced it using cancer cells that stably express WT FGF1 or R50E. Since R50E did not affect proliferation of cancer cells in vitro, we hypothesized that R50E suppressed tumorigenesis indirectly through suppressing angiogenesis. We thus studied the effect of R50E on angiogenesis in several angiogenesis models. We found that excess R50E suppressed FGF1-induced migration and tube formation of endothelial cells, FGF1-induced angiogenesis in matrigel plug assays, and the outgrowth of cells in aorta ring assays. Excess R50E suppressed FGF1-induced angiogenesis in chick embryo chorioallantoic membrane (CAM) assays. Interestingly, excess R50E suppressed FGF2-induced angiogenesis in CAM assays as well, suggesting that R50E may uniquely suppress signaling from other members of the FGF family. Taken together, our results suggest that R50E suppresses angiogenesis induced by FGF1 or FGF2, and thereby indirectly suppresses tumorigenesis, in addition to its possible direct effect on tumor cell proliferation in vivo. We propose that R50E has potential as an anti-cancer and anti-angiogenesis therapeutic agent ("FGF1 decoy").


Assuntos
Neoplasias do Colo/patologia , Fator 1 de Crescimento de Fibroblastos/genética , Mutação , Neovascularização Patológica/genética , Animais , Aorta/metabolismo , Aorta/fisiologia , Linhagem Celular Tumoral , Movimento Celular , Embrião de Galinha , Membrana Corioalantoide/metabolismo , Neoplasias do Colo/genética , Células Endoteliais/citologia , Fator 1 de Crescimento de Fibroblastos/metabolismo , Humanos , Integrinas/metabolismo , Camundongos , Ratos , Transdução de Sinais/genética
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