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1.
Cell ; 156(6): 1340-1340.e1, 2014 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-24630731

RESUMO

Integrin αß transmembrane heterodimers play central roles in metazoan development and physiology by mediating adhesion and by transmitting forces and biochemical signals across the plasma membrane. In this SnapShot, we present a simplified "modular" view of the integrin adhesome, centered on the talin-integrin interaction, and provide examples of how this view can help to unravel the adhesome's remarkable functional diversity and plasticity.


Assuntos
Integrinas/metabolismo , Talina/metabolismo , Sequência de Aminoácidos , Animais , Adesão Celular , Integrinas/química , Dados de Sequência Molecular , Talina/química
2.
Arterioscler Thromb Vasc Biol ; 44(6): 1246-1264, 2024 06.
Artigo em Inglês | MEDLINE | ID: mdl-38660801

RESUMO

BACKGROUND: Heterogeneity in the severity of cerebral cavernous malformations (CCMs) disease, including brain bleedings and thrombosis that cause neurological disabilities in patients, suggests that environmental, genetic, or biological factors act as disease modifiers. Still, the underlying mechanisms are not entirely understood. Here, we report that mild hypoxia accelerates CCM disease by promoting angiogenesis, neuroinflammation, and vascular thrombosis in the brains of CCM mouse models. METHODS: We used genetic studies, RNA sequencing, spatial transcriptome, micro-computed tomography, fluorescence-activated cell sorting, multiplex immunofluorescence, coculture studies, and imaging techniques to reveal that sustained mild hypoxia via the CX3CR1-CX3CL1 (CX3C motif chemokine receptor 1/chemokine [CX3C motif] ligand 1) signaling pathway influences cell-specific neuroinflammatory interactions, contributing to heterogeneity in CCM severity. RESULTS: Histological and expression profiles of CCM neurovascular lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) in male and female mice found that sustained mild hypoxia (12% O2, 7 days) accelerates CCM disease. Our findings indicate that a small reduction in oxygen levels can significantly increase angiogenesis, neuroinflammation, and thrombosis in CCM disease by enhancing the interactions between endothelium, astrocytes, and immune cells. Our study indicates that the interactions between CX3CR1 and CX3CL1 are crucial in the maturation of CCM lesions and propensity to CCM immunothrombosis. In particular, this pathway regulates the recruitment and activation of microglia and other immune cells in CCM lesions, which leads to lesion growth and thrombosis. We found that human CX3CR1 variants are linked to lower lesion burden in familial CCMs, proving it is a genetic modifier in human disease and a potential marker for aggressiveness. Moreover, monoclonal blocking antibody against CX3CL1 or reducing 1 copy of the Cx3cr1 gene significantly reduces hypoxia-induced CCM immunothrombosis. CONCLUSIONS: Our study reveals that interactions between CX3CR1 and CX3CL1 can modify CCM neuropathology when lesions are accelerated by environmental hypoxia. Moreover, a hypoxic environment or hypoxia signaling caused by CCM disease influences the balance between neuroinflammation and neuroprotection mediated by CX3CR1-CX3CL1 signaling. These results establish CX3CR1 as a genetic marker for patient stratification and a potential predictor of CCM aggressiveness.


Assuntos
Receptor 1 de Quimiocina CX3C , Quimiocina CX3CL1 , Modelos Animais de Doenças , Hemangioma Cavernoso do Sistema Nervoso Central , Transdução de Sinais , Animais , Feminino , Humanos , Masculino , Camundongos , Quimiocina CX3CL1/metabolismo , Quimiocina CX3CL1/genética , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Hipóxia/metabolismo , Hipóxia/complicações , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/metabolismo , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Doenças Neuroinflamatórias/genética
3.
Annu Rev Cell Dev Biol ; 27: 321-45, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21663444

RESUMO

Regulation of cell-cell and cell-matrix interaction is essential for the normal physiology of metazoans and is important in many diseases. Integrin adhesion receptors can rapidly increase their affinity (integrin activation) in response to intracellular signaling events in a process termed inside-out signaling. The transmembrane domains of integrins and their interactions with the membrane are important in inside-out signaling. Moreover, integrin activation is tightly regulated by a complex network of signaling pathways. Here, we review recent progress in understanding how the membrane environment can, in cooperation with integrin-binding proteins, regulate integrin activation.


Assuntos
Integrinas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Adesão Celular/fisiologia , Moléculas de Adesão Celular/metabolismo , Proteínas Contráteis/metabolismo , Proteínas do Citoesqueleto/metabolismo , Filaminas , Humanos , Integrinas/química , Integrinas/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Talina/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo
4.
Cell Commun Signal ; 22(1): 23, 2024 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-38195510

RESUMO

Cerebral cavernous malformation (CCM) is a hemorrhagic neurovascular disease with no currently available therapeutics. Prior evidence suggests that different cell types may play a role in CCM pathogenesis. The contribution of each cell type to the dysfunctional cellular crosstalk remains unclear. Herein, RNA-seq was performed on fluorescence-activated cell sorted endothelial cells (ECs), pericytes, and neuroglia from CCM lesions and non-lesional brain tissue controls. Differentially Expressed Gene (DEG), pathway and Ligand-Receptor (LR) analyses were performed to characterize the dysfunctional genes of respective cell types within CCMs. Common DEGs among all three cell types were related to inflammation and endothelial-to-mesenchymal transition (EndMT). DEG and pathway analyses supported a role of lesional ECs in dysregulated angiogenesis and increased permeability. VEGFA was particularly upregulated in pericytes. Further pathway and LR analyses identified vascular endothelial growth factor A/ vascular endothelial growth factor receptor 2 signaling in lesional ECs and pericytes that would result in increased angiogenesis. Moreover, lesional pericytes and neuroglia predominantly showed DEGs and pathways mediating the immune response. Further analyses of cell specific gene alterations in CCM endorsed potential contribution to EndMT, coagulation, and a hypoxic microenvironment. Taken together, these findings motivate mechanistic hypotheses regarding non-endothelial contributions to lesion pathobiology and may lead to novel therapeutic targets. Video Abstract.


Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central , Fator A de Crescimento do Endotélio Vascular , Humanos , Fator A de Crescimento do Endotélio Vascular/genética , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Células Endoteliais , Perfilação da Expressão Gênica , Transcriptoma , Microambiente Tumoral
5.
Blood ; 137(1): 29-38, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-32777822

RESUMO

Integrin-mediated neutrophil adhesion starts by arrest from rolling. Activation of integrins involves conformational changes from an inactive, bent conformation to an extended conformation (E+) with high affinity for ligand binding (H+). The cytoplasmic protein kindlin-3 is necessary for leukocyte adhesion; mutations of kindlin-3 cause leukocyte adhesion deficiency type 3. Kindlin-3 binds the ß2-integrin cytoplasmic tail at a site distinct from talin-1, but the molecular mechanism by which kindlin-3 activates ß2-integrins is unknown. In this study, we measured the spatiotemporal dynamics of kindlin-3 and ß2-integrin conformation changes during neutrophil and HL-60 cell rolling and arrest under flow. Using high-resolution quantitative dynamic footprinting microscopy and kindlin-3-fluorescent protein (FP) fusion proteins, we found that kindlin-3 was recruited to the plasma membrane in response to interleukin-8 (IL-8) before induction of the H+ ß2-integrin conformation. Intravital imaging revealed that EGFP-kindlin-3-reconstituted, kindlin-3-knockout neutrophils arrest in vivo in response to CXCL1. EGFP-kindlin-3 in primary mouse neutrophils was also recruited to the plasma membrane before arrest. Upon arrest, we found small clusters of high-affinity ß2-integrin molecules within large areas of membrane-proximal kindlin-3 FP. Deletion of kindlin-3 or its pleckstrin homology (PH) domain in neutrophil-like HL-60 cells completely abolished H+ ß2-integrin induction. IL-8 also triggered recruitment of the isolated kindlin-3 PH domain to the plasma membrane before arrest. In summary, we showed that the kindlin-3 PH domain is necessary for recruitment to the plasma membrane, where full-length kindlin-3 is indispensable for the induction of high-affinity ß2-integrin.


Assuntos
Antígenos CD18/metabolismo , Migração e Rolagem de Leucócitos/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Infiltração de Neutrófilos/fisiologia , Neutrófilos/metabolismo , Animais , Membrana Celular/metabolismo , Células HL-60 , Humanos , Camundongos , Transporte Proteico/fisiologia
6.
FASEB J ; 36(12): e22629, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36349990

RESUMO

ß1 integrins are important in blood vessel formation and function, finely tuning the adhesion of endothelial cells to each other and to the extracellular matrix. The role of integrins in the vascular disease, cerebral cavernous malformation (CCM) has yet to be explored in vivo. Endothelial loss of the gene KRIT1 leads to brain microvascular defects, resulting in debilitating and often fatal consequences. We tested administration of a monoclonal antibody that enforces the active ß1 integrin conformation, (clone 9EG7), on a murine neonatal CCM mouse model, Krit1flox/flox ;Pdgfb-iCreERT2 (Krit1ECKO ), and on KRIT1-silenced human umbilical vein endothelial cells (HUVECs). In addition, endothelial deletion of the master regulator of integrin activation, Talin 1 (Tln1), in Krit1ECKO mice was performed to assess the effect of completely blocking endothelial integrin activation on CCM. Treatment with 9EG7 reduced lesion burden in the Krit1ECKO model and was accompanied by a strong reduction in the phosphorylation of the ROCK substrate, myosin light chain (pMLC), in both retina and brain endothelial cells. Treatment of KRIT1-silenced HUVECs with 9EG7 in vitro stabilized cell-cell junctions. Overnight treatment of HUVECs with 9EG7 resulted in significantly reduced total surface expression of ß1 integrin, which was associated with reduced pMLC levels, supporting our in vivo findings. Genetic blockade of integrin activation by Tln1ECKO enhanced bleeding and did not reduce CCM lesion burden in Krit1ECKO mice. In sum, targeting ß1 integrin with an activated-specific antibody reduces acute murine CCM lesion development, which we found to be associated with suppression of endothelial ROCK activity.


Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central , Animais , Humanos , Camundongos , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Integrina beta1/metabolismo , Anticorpos Monoclonais/metabolismo , Integrinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo
7.
Circ Res ; 129(1): 195-215, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34166073

RESUMO

Cerebral cavernous malformations are acquired vascular anomalies that constitute a common cause of central nervous system hemorrhage and stroke. The past 2 decades have seen a remarkable increase in our understanding of the pathogenesis of this vascular disease. This new knowledge spans genetic causes of sporadic and familial forms of the disease, molecular signaling changes in vascular endothelial cells that underlie the disease, unexpectedly strong environmental effects on disease pathogenesis, and drivers of disease end points such as hemorrhage. These novel insights are the integrated product of human clinical studies, human genetic studies, studies in mouse and zebrafish genetic models, and basic molecular and cellular studies. This review addresses the genetic and molecular underpinnings of cerebral cavernous malformation disease, the mechanisms that lead to lesion hemorrhage, and emerging biomarkers and therapies for clinical treatment of cerebral cavernous malformation disease. It may also serve as an example for how focused basic and clinical investigation and emerging technologies can rapidly unravel a complex disease mechanism.


Assuntos
Veias Cerebrais/anormalidades , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/terapia , Mutação , Animais , Veias Cerebrais/metabolismo , Predisposição Genética para Doença , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Humanos , Fenótipo , Transdução de Sinais
8.
J Biol Chem ; 296: 100675, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33865854

RESUMO

Interaction of talin with the cytoplasmic tails of integrin ß triggers integrin activation, leading to an increase of integrin affinity/avidity for extracellular ligands. In talin KO mice, loss of talin interaction with platelet integrin αIIbß3 causes a severe hemostatic defect, and loss of talin interaction with endothelial cell integrin αVß3 affects angiogenesis. In normal cells, talin is autoinhibited and localized in the cytoplasm. Here, we used an optogenetic platform to assess whether recruitment of full-length talin to the plasma membrane was sufficient to induce integrin activation. A dimerization module (Arabidopsis cryptochrome 2 fused to the N terminus of talin; N-terminal of cryptochrome-interacting basic helix-loop-helix domain ended with a CAAX box protein [C: cysteine; A: aliphatic amino acid; X: any C-terminal amino acid]) responsive to 450 nm (blue) light was inserted into Chinese hamster ovary cells and endothelial cells also expressing αIIbß3 or αVß3, respectively. Thus, exposure of the cells to blue light caused a rapid and reversible recruitment of Arabidopsis cryptochrome 2-talin to the N-terminal of cryptochrome-interacting basic helix-loop-helix domain ended with a CAAX box protein [C: cysteine; A: aliphatic amino acid; X: any C-terminal amino acid]-decorated plasma membrane. This resulted in ß3 integrin activation in both cell types, as well as increasing migration of the endothelial cells. However, membrane recruitment of talin was not sufficient for integrin activation, as membrane-associated Ras-related protein 1 (Rap1)-GTP was also required. Moreover, talin mutations that interfered with its direct binding to Rap1 abrogated ß3 integrin activation. Altogether, these results define a role for the plasma membrane recruitment of talin in ß3 integrin activation, and they suggest a nuanced sequence of events thereafter involving Rap1-GTP.


Assuntos
Membrana Celular/metabolismo , Citoplasma/metabolismo , Células Endoteliais/metabolismo , Optogenética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Talina/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Camundongos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Ligação Proteica , Talina/genética , Proteínas rap1 de Ligação ao GTP/genética
9.
Blood ; 136(10): 1180-1190, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32518959

RESUMO

Ras-related protein 1 (Rap1) is a major convergence point of the platelet-signaling pathways that result in talin-1 binding to the integrin ß cytoplasmic domain and consequent integrin activation, platelet aggregation, and effective hemostasis. The nature of the connection between Rap1 and talin-1 in integrin activation is an important remaining gap in our understanding of this process. Previous work identified a low-affinity Rap1-binding site in the talin-1 F0 domain that makes a small contribution to integrin activation in platelets. We recently identified an additional Rap1-binding site in the talin-1 F1 domain that makes a greater contribution than F0 in model systems. Here we generated mice bearing point mutations, which block Rap1 binding without affecting talin-1 expression, in either the talin-1 F1 domain (R118E) alone, which were viable, or in both the F0 and F1 domains (R35E,R118E), which were embryonic lethal. Loss of the Rap1-talin-1 F1 interaction in platelets markedly decreases talin-1-mediated activation of platelet ß1- and ß3-integrins. Integrin activation and platelet aggregation in mice whose platelets express only talin-1(R35E, R118E) are even more impaired, resembling the defect seen in platelets lacking both Rap1a and Rap1b. Although Rap1 is important in thrombopoiesis, platelet secretion, and surface exposure of phosphatidylserine, loss of the Rap1-talin-1 interaction in talin-1(R35E, R118E) platelets had little effect on these processes. These findings show that talin-1 is the principal direct effector of Rap1 GTPases that regulates platelet integrin activation in hemostasis.


Assuntos
Integrina beta1/metabolismo , Integrina beta3/metabolismo , Mutação Puntual , Talina/fisiologia , Trombopoese , Proteínas rap de Ligação ao GTP/fisiologia , Proteínas rap1 de Ligação ao GTP/fisiologia , Animais , Feminino , Integrina beta1/genética , Integrina beta3/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ativação Plaquetária , Agregação Plaquetária , Domínios Proteicos , Transdução de Sinais
10.
Nat Rev Mol Cell Biol ; 11(4): 288-300, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20308986

RESUMO

Cell-directed changes in the ligand-binding affinity ('activation') of integrins regulate cell adhesion and migration, extracellular matrix assembly and mechanotransduction, thereby contributing to embryonic development and diseases such as atherothrombosis and cancer. Integrin activation comprises triggering events, intermediate signalling events and, finally, the interaction of integrins with cytoplasmic regulators, which changes an integrin's affinity for its ligands. The first two events involve diverse interacting signalling pathways, whereas the final steps are immediately proximal to integrins, thus enabling integrin-focused therapeutic strategies. Recent progress provides insight into the structure of integrin transmembrane domains, and reveals how the final steps of integrin activation are mediated by integrin-binding proteins such as talins and kindlins.


Assuntos
Citoplasma/metabolismo , Integrinas/metabolismo , Transdução de Sinais , Talina/metabolismo , Animais , Humanos
11.
J Immunol ; 205(10): 2883-2892, 2020 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-33077644

RESUMO

CD98, which is required for the rapid proliferation of both normal and cancer cells, and MET, the hepatocyte growth factor receptor, are potential targets for therapeutic antitumor Abs. In this study, we report that the antiproliferative activity of a prototype anti-CD98 Ab, UM7F8, is due to Ab-induced membrane-associated ring CH (MARCH) E3 ubiquitin ligase-mediated ubiquitination and downregulation of cell surface CD98. MARCH1-mediated ubiquitination of CD98 is required for UM7F8's capacity to reduce CD98 surface expression and its capacity to inhibit the proliferation of murine T cells. Similarly, CD98 ubiquitination is required for UM7F8's capacity to block the colony-forming ability of murine leukemia-initiating cells. To test the potential generality of the paradigm that MARCH E3 ligases can mediate the antiproliferative response to antitumor Abs, we examined the potential effects of MARCH proteins on responses to emibetuzumab, an anti-MET Ab currently in clinical trials for various cancers. We report that MET surface expression is reduced by MARCH1, 4, or 8-mediated ubiquitination and that emibetuzumab-induced MET ubiquitination contributes to its capacity to downregulate MET and inhibit human tumor cell proliferation. Thus, MARCH E3 ligases can act as cofactors for antitumor Abs that target cell surface proteins, suggesting that the MARCH protein repertoire of cells is a determinant of their response to such Abs.


Assuntos
Antígenos de Neoplasias/metabolismo , Antineoplásicos Imunológicos/farmacologia , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Neoplasias/tratamento farmacológico , Ubiquitina-Proteína Ligases/metabolismo , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antineoplásicos Imunológicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Cadeia Pesada da Proteína-1 Reguladora de Fusão/antagonistas & inibidores , Cadeia Pesada da Proteína-1 Reguladora de Fusão/genética , Cadeia Pesada da Proteína-1 Reguladora de Fusão/imunologia , Técnicas de Inativação de Genes , Células HeLa , Humanos , Células Jurkat , Camundongos , Camundongos Knockout , Neoplasias/imunologia , Neoplasias/patologia , Proteólise , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/imunologia
12.
Nat Immunol ; 10(4): 412-9, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19270713

RESUMO

The proliferation of antigen-specific lymphocytes and resulting clonal expansion are essential for adaptive immunity. We report here that B cell-specific deletion of the heavy chain of CD98 (CD98hc) resulted in lower antibody responses due to total suppression of B cell proliferation and subsequent plasma cell formation. Deletion of CD98hc did not impair early B cell activation but did inhibit later activation of the mitogen-activated protein kinase Erk1/2 and downregulation of the cell cycle inhibitor p27. Reconstitution of CD98hc-deficient B cells with CD98hc mutants showed that the integrin-binding domain of CD98hc was required for B cell proliferation but that the amino acid-transport function of CD98hc was dispensable for this. Thus, CD98hc supports integrin-dependent rapid proliferation of B cells. We propose that the advantage of adaptive immunity favored the appearance of CD98hc in vertebrates.


Assuntos
Formação de Anticorpos , Linfócitos B/imunologia , Proliferação de Células , Cadeia Pesada da Proteína-1 Reguladora de Fusão/imunologia , Animais , Linfócitos B/citologia , Transporte Biológico Ativo , Diferenciação Celular/fisiologia , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Integrinas/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Plasmócitos/citologia , Plasmócitos/imunologia , Ligação Proteica
13.
Blood ; 133(3): 193-204, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30442679

RESUMO

Cerebral cavernous malformations (CCMs) are common brain vascular dysplasias that are prone to acute and chronic hemorrhage with significant clinical sequelae. The pathogenesis of recurrent bleeding in CCM is incompletely understood. Here, we show that central nervous system hemorrhage in CCMs is associated with locally elevated expression of the anticoagulant endothelial receptors thrombomodulin (TM) and endothelial protein C receptor (EPCR). TM levels are increased in human CCM lesions, as well as in the plasma of patients with CCMs. In mice, endothelial-specific genetic inactivation of Krit1 (Krit1 ECKO ) or Pdcd10 (Pdcd10 ECKO ), which cause CCM formation, results in increased levels of vascular TM and EPCR, as well as in enhanced generation of activated protein C (APC) on endothelial cells. Increased TM expression is due to upregulation of transcription factors KLF2 and KLF4 consequent to the loss of KRIT1 or PDCD10. Increased TM expression contributes to CCM hemorrhage, because genetic inactivation of 1 or 2 copies of the Thbd gene decreases brain hemorrhage in Pdcd10 ECKO mice. Moreover, administration of blocking antibodies against TM and EPCR significantly reduced CCM hemorrhage in Pdcd10 ECKO mice. Thus, a local increase in the endothelial cofactors that generate anticoagulant APC can contribute to bleeding in CCMs, and plasma soluble TM may represent a biomarker for hemorrhagic risk in CCMs.


Assuntos
Anticoagulantes/metabolismo , Proteínas Reguladoras de Apoptose/fisiologia , Hemorragia Cerebral/diagnóstico , Endotélio Vascular/patologia , Hemangioma Cavernoso do Sistema Nervoso Central/complicações , Proteína KRIT1/fisiologia , Proteínas de Membrana/fisiologia , Proteína C/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Trombomodulina/sangue , Adulto , Animais , Coagulação Sanguínea , Estudos de Casos e Controles , Hemorragia Cerebral/sangue , Hemorragia Cerebral/etiologia , Receptor de Proteína C Endotelial/metabolismo , Endotélio Vascular/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/fisiopatologia , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Knockout , Transdução de Sinais , Adulto Jovem
14.
J Immunol ; 200(12): 4012-4023, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29703862

RESUMO

Maintenance of the regulatory T (Treg) cell pool is essential for peripheral tolerance and prevention of autoimmunity. Integrins, heterodimeric transmembrane proteins consisting of α and ß subunits that mediate cell-to-cell and cell-to-extracellular matrix interactions, play an important role in facilitating Treg cell contact-mediated suppression. In this article, we show that integrin activation plays an essential, previously unappreciated role in maintaining murine Treg cell function. Treg cell-specific loss of talin, a ß integrin-binding protein, or expression of talin(L325R), a mutant that selectively abrogates integrin activation, resulted in lethal systemic autoimmunity. This dysfunction could be attributed, in part, to a global dysregulation of the Treg cell transcriptome. Activation of integrin α4ß1 led to increased suppressive capacity of the Treg cell pool, suggesting that modulating integrin activation on Treg cells may be a useful therapeutic strategy for autoimmune and inflammatory disorders. Taken together, these results reveal a critical role for integrin-mediated signals in controlling peripheral tolerance by virtue of maintaining Treg cell function.


Assuntos
Integrinas/imunologia , Tolerância Periférica/imunologia , Linfócitos T Reguladores/imunologia , Animais , Autoimunidade/imunologia , Inflamação/imunologia , Camundongos , Talina/imunologia , Transcriptoma/imunologia
15.
J Immunol ; 198(9): 3410-3415, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28348273

RESUMO

Rap1-interacting adaptor molecule (RIAM) is a Rap1 effector that mediates the recruitment of talin to integrins, thereby supporting their activation. In this study, we investigated the role of RIAM in an adoptive transfer model for type I diabetes and report that RIAM expression in T cells is necessary for diabetes development. Loss of RIAM did not prevent lymphocyte recruitment to draining lymph nodes 24 h after transfer, but it was required for Ag-driven proliferation and cytotoxic killing. RIAM is recruited to immune synapses along with talin and LFA-1, and loss of RIAM profoundly suppresses Ag-dependent conjugate formation in primary naive and effector T cells. These data identify the requirement of RIAM for formation of immunological synapses and in resulting T cell functions in autoimmunity. Moreover, because RIAM-null mice are healthy, fertile, and display no bleeding abnormalities, our results identify RIAM and its regulators as potential targets for therapies of T cell-mediated autoimmunity.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Apresentadoras de Antígenos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Proteínas de Membrana/metabolismo , Linfócitos T/imunologia , Talina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Transferência Adotiva , Animais , Proliferação de Células/genética , Células Cultivadas , Citotoxicidade Imunológica/genética , Humanos , Sinapses Imunológicas/imunologia , Proteínas de Membrana/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/transplante
16.
J Immunol ; 198(12): 4639-4651, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28515282

RESUMO

Talin, a cytoskeletal protein essential in mediating integrin activation, has been previously shown to be involved in the regulation of T cell proliferation and function. In this study, we describe a role for talin in maintaining the homeostasis and survival of the regulatory T (Treg) cell pool. T cell-specific deletion of talin in Tln1fl/flCd4Cre mice resulted in spontaneous lymphocyte activation, primarily due to numerical and functional deficiencies of Treg cells in the periphery. Peripheral talin-deficient Treg cells were unable to maintain high expression of IL-2Rα, resulting in impaired IL-2 signaling and ultimately leading to increased apoptosis through downregulation of prosurvival proteins Bcl-2 and Mcl-1. The requirement for talin in maintaining high IL-2Rα expression by Treg cells was due, in part, to integrin LFA-1-mediated interactions between Treg cells and dendritic cells. Collectively, our data suggest a critical role for talin in Treg cell-mediated maintenance of immune homeostasis.


Assuntos
Homeostase , Ativação Linfocitária , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Talina/metabolismo , Animais , Apoptose , Células Dendríticas/imunologia , Genes bcl-2 , Interleucina-2/imunologia , Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Linfócitos T Reguladores/fisiologia , Talina/deficiência , Talina/imunologia
17.
J Biol Chem ; 292(24): 9858-9864, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28487468

RESUMO

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/metabolismo
18.
Blood ; 128(4): 479-87, 2016 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-27207789

RESUMO

Integrin adhesion receptors mediate the adhesion of blood cells, such as leukocytes, to other cells, such as endothelial cells. Integrins also are critical for anchorage of hematopoietic precursors to the extracellular matrix. Blood cells can dynamically regulate the affinities of integrins for their ligands ("activation"), an event central to their functions. Here we review recent progress in understanding the mechanisms of integrin activation with a focus on the functions of blood cells. We discuss how talin binding to the integrin ß cytoplasmic domain, in conjunction with the plasma membrane, induces long-range allosteric rearrangements that lead to integrin activation. Second, we review our understanding of how signaling events, particularly those involving Rap1 small guanosine triphosphate (GTP)hydrolases, can regulate the talin-integrin interaction and resulting activation. Third, we review recent findings that highlight the role of the Rap1-GTP-interacting adapter molecule (RIAM), encoded by the APBB1IP gene, in leukocyte integrin activation and consequently in leukocyte trafficking.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular/fisiologia , Integrinas/metabolismo , Leucócitos/metabolismo , Proteínas de Membrana/metabolismo , Talina/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Humanos , Leucócitos/citologia , Domínios Proteicos , Complexo Shelterina
19.
Arterioscler Thromb Vasc Biol ; 37(7): 1323-1331, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28495929

RESUMO

OBJECTIVE: Coagulation initiation by tissue factor (TF) is regulated by cellular inhibitors, cell surface availability of procoagulant phosphatidylserine, and thiol-disulfide exchange. How these mechanisms contribute to keeping TF in a noncoagulant state and to generating prothrombotic TF remain incompletely understood. APPROACH AND RESULTS: Here, we study the activation of TF in primary macrophages by a combination of pharmacological, genetic, and biochemical approaches. We demonstrate that primed macrophages effectively control TF cell surface activity by receptor internalization. After cell injury, ATP signals through the purinergic receptor P2rx7 induce release of TF+ microvesicles. TF cell surface availability for release onto microvesicles is regulated by the GTPase arf6 associated with integrin α4ß1. Furthermore, microvesicles proteome analysis identifies activation of Gαi2 as a participating factor in the release of microvesicles with prothrombotic activity in flowing blood. ATP not only prevents TF and phosphatidylserine internalization but also induces TF conversion to a conformation with high affinity for its ligand, coagulation factor VII. Although inhibition of dynamin-dependent internalization also exposes outer membrane procoagulant phosphatidylserine, the resulting TF+ microvesicles distinctly lack protein disulfide isomerase and high affinity TF and fail to produce fibrin strands typical for microvesicles generated by thrombo-inflammatory P2rx7 activation. CONCLUSIONS: These data show that procoagulant phospholipid exposure is not sufficient and that TF affinity maturation is required to generate prothrombotic microvesicles from a variety of cell types. These findings are significant for understanding TF-initiated thrombosis and should be considered in designing functional microvesicles-based diagnostic approaches.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Coagulação Sanguínea , Integrina alfa4/metabolismo , Integrina alfa4beta1/metabolismo , Macrófagos/metabolismo , Tromboplastina/metabolismo , Trombose/metabolismo , Fator 6 de Ribosilação do ADP , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Micropartículas Derivadas de Células/metabolismo , Fator VIIa/metabolismo , Técnicas de Introdução de Genes , Genótipo , Humanos , Integrina alfa4/genética , Integrina alfa4beta1/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Fosfolipídeos/metabolismo , Transporte Proteico , Receptores Purinérgicos P2X7/metabolismo , Transdução de Sinais , Trombose/sangue , Trombose/genética , Transfecção
20.
J Biol Chem ; 291(34): 17536-46, 2016 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-27365391

RESUMO

In many families of cell surface receptors, a single transmembrane (TM) α-helix separates ecto- and cytosolic domains. A defined coupling of ecto- and TM domains must be essential to allosteric receptor regulation but remains little understood. Here, we characterize the linker structure, dynamics, and resulting ecto-TM domain coupling of integrin αIIb in model constructs and relate it to other integrin α subunits by mutagenesis. Cellular integrin activation assays subsequently validate the findings in intact receptors. Our results indicate a flexible yet carefully tuned ecto-TM coupling that modulates the signaling threshold of integrin receptors. Interestingly, a proline at the N-terminal TM helix border, termed NBP, is critical to linker flexibility in integrins. NBP is further predicted in 21% of human single-pass TM proteins and validated in cytokine receptors by the TM domain structure of the cytokine receptor common subunit ß and its P441A-substituted variant. Thus, NBP is a conserved uncoupling motif of the ecto-TM domain transition and the degree of ecto-TM domain coupling represents an important parameter in the allosteric regulation of diverse cell surface receptors.


Assuntos
Subunidade beta Comum dos Receptores de Citocinas/química , Cadeias beta de Integrinas/química , Regulação Alostérica/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Subunidade beta Comum dos Receptores de Citocinas/genética , Subunidade beta Comum dos Receptores de Citocinas/metabolismo , Humanos , Cadeias beta de Integrinas/genética , Cadeias beta de Integrinas/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína
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