RESUMO
During vertebrate gastrulation, canonical Wnt signaling induces the formation of neural plate border (NPB). Wnt is also thought to be required for the subsequent specification of neural crest (NC) lineage at the NPB, but the direct evidence is lacking. We found previously that the disintegrin metalloproteinase ADAM13 is required for Wnt activation and NC induction in Xenopus Here, we report that knockdown of ADAM13 or its close paralog ADAM19 severely downregulates Wnt activity at the NPB, inhibiting NC specification without affecting earlier NPB formation. Surprisingly, ADAM19 functions nonproteolytically in NC specification by interacting with ADAM13 and inhibiting its proteasomal degradation. Ectopic expression of stabilized ADAM13 mutants that function independently of ADAM19 can induce the NC marker/specifier snail2 in the future epidermis via Wnt signaling. These results unveil the essential roles of a novel protease-protease interaction in regulating a distinct wave of Wnt signaling, which directly specifies the NC lineage.
Assuntos
Proteínas ADAM/metabolismo , Padronização Corporal/fisiologia , Crista Neural/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/metabolismo , Crista Neural/embriologia , Placa Neural/metabolismo , Transdução de Sinais , Via de Sinalização Wnt/fisiologia , Xenopus/embriologiaRESUMO
Obesity is associated with increased risk for infections and poor responses to vaccinations, which may be due to compromised B cell function. However, there is limited information about the influence of obesity on B cell function and underlying factors that modulate B cell responses. Therefore, we studied B cell cytokine secretion and/or Ab production across obesity models. In obese humans, B cell IL-6 secretion was lowered and IgM levels were elevated upon ex vivo anti-BCR/TLR9 stimulation. In murine obesity induced by a high fat diet, ex vivo IgM and IgG were elevated with unstimulated B cells. Furthermore, the high fat diet lowered bone marrow B cell frequency accompanied by diminished transcripts of early lymphoid commitment markers. Murine B cell responses were subsequently investigated upon influenza A/Puerto Rico/8/34 infection using a Western diet model in the absence or presence of docosahexaenoic acid (DHA). DHA, an essential fatty acid with immunomodulatory properties, was tested because its plasma levels are lowered in obesity. Relative to controls, mice consuming the Western diet had diminished Ab titers whereas the Western diet plus DHA improved titers. Mechanistically, DHA did not directly target B cells to elevate Ab levels. Instead, DHA increased the concentration of the downstream specialized proresolving lipid mediators (SPMs) 14-hydroxydocosahexaenoic acid, 17-hydroxydocosahexaenoic acid, and protectin DX. All three SPMs were found to be effective in elevating murine Ab levels upon influenza infection. Collectively, the results demonstrate that B cell responses are impaired across human and mouse obesity models and show that essential fatty acid status is a factor influencing humoral immunity, potentially through an SPM-mediated mechanism.
Assuntos
Linfócitos B/imunologia , Ácidos Graxos Essenciais/imunologia , Imunidade Humoral , Interleucina-6/metabolismo , Obesidade/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Dieta Ocidental , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácidos Docosa-Hexaenoicos/sangue , Ácidos Docosa-Hexaenoicos/imunologia , Ácidos Graxos Essenciais/sangue , Humanos , Imunoglobulina M/sangue , Vírus da Influenza A/imunologia , Interleucina-6/imunologia , Ativação Linfocitária , Camundongos , Obesidade/complicações , Infecções por Orthomyxoviridae/complicações , Receptor Toll-Like 9/imunologia , Receptor Toll-Like 9/metabolismoRESUMO
The ADAM (a disintegrin and metalloprotease) protein family uniquely exhibits both catalytic and adhesive properties. In the well-defined process of ectodomain shedding, ADAMs transform latent, cell-bound substrates into soluble, biologically active derivatives to regulate a spectrum of normal and pathological processes. In contrast, the integrin ligand properties of ADAMs are not fully understood. Emerging models posit that ADAM-integrin interactions regulate shedding activity by localizing or sequestering the ADAM sheddase. Interestingly, 8 of the 21 human ADAMs are predicted to be catalytically inactive. Unlike their catalytically active counterparts, integrin recognition of these "dead" enzymes has not been largely reported. The present study delineates the integrin ligand properties of a group of non-catalytic ADAMs. Here we report that human ADAM11, ADAM23, and ADAM29 selectively support integrin α4-dependent cell adhesion. This is the first demonstration that the disintegrin-like domains of multiple catalytically inactive ADAMs are ligands for a select subset of integrin receptors that also recognize catalytically active ADAMs.
Assuntos
Proteínas ADAM/metabolismo , Integrina alfa4/metabolismo , Proteínas ADAM/genética , Animais , Células CHO , Adesão Celular/fisiologia , Cricetulus , Humanos , Integrina alfa4/genética , Células Jurkat , Células K562 , LigantesRESUMO
Allosteric regulation of pyruvate carboxylase (PC) activity is pivotal to maintaining metabolic homeostasis. In contrast, dysregulated PC activity contributes to the pathogenesis of numerous diseases, rendering PC a possible target for allosteric therapeutic development. Recent research efforts have focused on demarcating the role of acetyl-CoA, one of the most potent activators of PC, in coordinating catalytic events within the multifunctional enzyme. Herein, we report a kinetic and thermodynamic analysis of acetyl-CoA activation of the Staphylococcus aureus PC (SaPC)-catalyzed carboxylation of pyruvate to identify novel means by which acetyl-CoA synchronizes catalytic events within the PC tetramer. Kinetic and linked-function analysis, or thermodynamic linkage analysis, indicates that the substrates of the biotin carboxylase and carboxyl transferase domain are energetically coupled in the presence of acetyl-CoA. In contrast, both kinetic and energetic coupling between the two domains is lost in the absence of acetyl-CoA, suggesting a functional role for acetyl-CoA in facilitating the long-range transmission of substrate-induced conformational changes within the PC tetramer. Interestingly, thermodynamic activation parameters for the SaPC-catalyzed carboxylation of pyruvate are largely independent of acetyl-CoA. Our results also reveal the possibility that global conformational changes give rise to observed species-specific thermodynamic activation parameters. Taken together, our kinetic and thermodynamic results provide a possible allosteric mechanism by which acetyl-CoA coordinates catalysis within the PC tetramer.
Assuntos
Acetilcoenzima A/metabolismo , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Piruvato Carboxilase/metabolismo , Staphylococcus aureus/enzimologia , Acetilcoenzima A/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Algoritmos , Regulação Alostérica , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Biocatálise , Transferência de Energia , Ativação Enzimática , Estabilidade Enzimática , Cinética , Magnésio/química , Magnésio/metabolismo , Conformação Molecular , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Redobramento de Proteína , Piruvato Carboxilase/química , Piruvato Carboxilase/genética , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
Retinoids, natural and synthetic derivatives of vitamin A, induce cellular changes by activating nuclear retinoic acid receptors (RAR) and retinoid X receptors (RXR). Although the ability of retinoids to govern gene expression is exploited clinically for cancer therapeutics, the full benefit of retinoid-based strategies is unrealized due to detrimental side effects. Delineating the receptors that prompt cellular outcomes is critical to advancing retinoid-based approaches. Here, we identify the receptors that evoke multiple responses in cutaneous T-cell lymphoma (CTCL). The data demonstrate that RARα drives integrin ß7-dependent adhesion and CCR9-mediated chemotaxis in CTCL cells. Of note, concomitant activation of RARα and RXR nuclear receptors yielded synergistic increases in adhesion and migration at concentrations where single agents were ineffective. As the established paradigm of retinoid action in CTCL is apoptosis and growth arrest, the role of RARα/RXR in these events was studied. As with adhesion and migration, RARα/RXR synergism prompted apoptosis and dampened CTCL cell proliferation. Strikingly, RARα/RXR synergism induced responses from CTCL cell lines previously reported to be unresponsive to retinoids. These data provide a novel framework that may further refine a proven CTCL therapy.
Assuntos
Linfoma Cutâneo de Células T/tratamento farmacológico , Linfoma Cutâneo de Células T/metabolismo , Receptor alfa de Ácido Retinoico/metabolismo , Receptores X de Retinoides/metabolismo , Anticarcinógenos/farmacologia , Antineoplásicos/farmacologia , Apoptose , Benzoatos/farmacologia , Bexaroteno , Adesão Celular , Linhagem Celular , Movimento Celular , Proliferação de Células , Expressão Gênica , Humanos , Cadeias beta de Integrinas/genética , Cadeias beta de Integrinas/metabolismo , RNA Mensageiro/metabolismo , Receptor alfa de Ácido Retinoico/agonistas , Receptores X de Retinoides/agonistas , Tetra-Hidronaftalenos/farmacologia , Tretinoína/farmacologiaRESUMO
Oxidative metabolites of vitamin A, in particular all-trans-retinoic acid (atRA), have emerged as key factors in immunity by specifying the localization of immune cells to the gut. Although it is appreciated that isomers of retinoic acid activate the retinoic acid receptor (RAR) and retinoid X receptor (RXR) family of nuclear receptors to elicit cellular changes, the molecular details of retinoic acid action remain poorly defined in immune processes. Here we employ a battery of agonists and antagonists to delineate the specific nuclear receptors utilized by retinoids to evoke lymphocyte cell adhesion to ADAM (adisintegrin and metalloprotease) protein family members. We report that RAR agonism is sufficient to promote immune cell adhesion in both immortal and primary immune cells. Interestingly, adhesion occurs independent of integrin function, and mutant studies demonstrate that atRA-induced adhesion to ADAM members required a distinct binding interface(s) as compared to integrin recognition. Anti-inflammatory corticosteroids as well as 1,25-(OH)2D3, a vitamin D metabolite that prompts immune cell trafficking to the skin, potently inhibited the observed adhesion. Finally, our data establish that induced adhesion was specifically attributable to the RAR-α receptor isotype. The current study provides novel molecular resolution as to which nuclear receptors transduce retinoid exposure into immune cell adhesion.
Assuntos
Integrinas/metabolismo , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Receptor alfa de Ácido Retinoico/agonistas , Receptor alfa de Ácido Retinoico/metabolismo , Retinoides/farmacologia , Adesão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Linfócitos/metabolismo , Relação Estrutura-AtividadeRESUMO
Pericellular proteolysis by ADAM family metalloproteinases has been widely implicated in cell signaling and development. We recently found that Xenopus ADAM13, an ADAM metalloproteinase, is required for activation of canonical Wnt signaling during cranial neural crest (CNC) induction by regulating a novel crosstalk between Wnt and ephrin B (EfnB) signaling pathways (Wei et al., 2010b). In the present study we show that the metalloproteinase activity of ADAM13 also plays important roles in eye development in Xenopus tropicalis. Knockdown of ADAM13 results in reduced expression of eye field markers pax6 and rx1, as well as that of the pan-neural marker sox2. Activation of canonical Wnt signaling or inhibition of forward EfnB signaling rescues the eye defects caused by loss of ADAM13, suggesting that ADAM13 functions through regulation of the EfnB-Wnt pathway interaction. Downstream of Wnt, the head inducer Cerberus was identified as an effector that mediates ADAM13 function in early eye field formation. Furthermore, ectopic expression of the Wnt target gene snail2 restores cerberus expression and rescues the eye defects caused by ADAM13 knockdown. Together these data suggest an important role of ADAM13-regulated Wnt activity in eye development in Xenopus.
Assuntos
Olho/metabolismo , Via de Sinalização Wnt , Proteínas de Xenopus/genética , Xenopus/genética , Animais , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Efrina-B1/genética , Olho/embriologia , Proteínas do Olho/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Morfogênese/genética , Morfogênese/fisiologia , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Proteínas Wnt/genética , Xenopus/embriologia , Proteínas de Xenopus/fisiologiaRESUMO
BACKGROUND: Members of the disintegrin metalloproteinase (ADAM) family play important roles in cellular and developmental processes through their functions as proteases and/or binding partners for other proteins. The amphibian Xenopus has long been used as a model for early vertebrate development, but genome-wide analyses for large gene families were not possible until the recent completion of the X. tropicalis genome sequence and the availability of large scale expression sequence tag (EST) databases. In this study we carried out a systematic analysis of the X. tropicalis genome and uncovered several interesting features of ADAM genes in this species. RESULTS: Based on the X. tropicalis genome sequence and EST databases, we identified Xenopus orthologues of mammalian ADAMs and obtained full-length cDNA clones for these genes. The deduced protein sequences, synteny and exon-intron boundaries are conserved between most human and X. tropicalis orthologues. The alternative splicing patterns of certain Xenopus ADAM genes, such as adams 22 and 28, are similar to those of their mammalian orthologues. However, we were unable to identify an orthologue for ADAM7 or 8. The Xenopus orthologue of ADAM15, an active metalloproteinase in mammals, does not contain the conserved zinc-binding motif and is hence considered proteolytically inactive. We also found evidence for gain of ADAM genes in Xenopus as compared to other species. There is a homologue of ADAM10 in Xenopus that is missing in most mammals. Furthermore, a single scaffold of X. tropicalis genome contains four genes encoding ADAM28 homologues, suggesting genome duplication in this region. CONCLUSIONS: Our genome-wide analysis of ADAM genes in X. tropicalis revealed both conservation and evolutionary divergence of these genes in this amphibian species. On the one hand, all ADAMs implicated in normal development and health in other species are conserved in X. tropicalis. On the other hand, some ADAM genes and ADAM protease activities are absent, while other novel ADAM proteins in this species are predicted by this study. The conservation and unique divergence of ADAM genes in Xenopus probably reflect the particular selective pressures these amphibian species faced during evolution.
Assuntos
Proteínas ADAM/genética , Evolução Molecular , Família Multigênica , Xenopus/genética , Sequência de Aminoácidos , Animais , Biologia Computacional , Sequência Conservada , Etiquetas de Sequências Expressas , Humanos , Mamíferos/genética , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , SinteniaRESUMO
A disintegrin and a metalloprotease (ADAM) family members have been implicated in many biological processes. Although it is recognized that recombinant ADAM disintegrin domains can interact with integrins, little is known about ADAM-integrin interactions in cellular context. Here, we tested whether ADAMs can selectively regulate integrin-mediated cell migration. ADAMs were expressed in Chinese hamster ovary cells that express defined integrins (alpha4beta1, alpha5beta1, or both), and cell migration on full-length fibronectin or on its alpha4beta1 or alpha5beta1 binding fragments was studied. We found that ADAMs inhibit integrin-mediated cell migration in patterns dictated by the integrin binding profiles of their isolated disintegrin domains. ADAM12 inhibited cell migration mediated by the alpha4beta1 but not the alpha5beta1 integrin. ADAM17 had the reciprocal effect; it inhibited alpha5beta1- but not alpha4beta1-mediated cell migration. ADAM19 and ADAM33 inhibited migration mediated by both alpha4beta1 and alpha5beta1 integrins. A point mutation in the ADAM12 disintegrin loop partially reduced the inhibitory effect of ADAM12 on cell migration on the alpha4beta1 binding fragment of fibronectin, whereas mutations that block metalloprotease activity had no effect. Our results indicate that distinct ADAMs can modulate cell migration mediated by specific integrins in a pattern dictated, at least in part, by their disintegrin domains.
Assuntos
Proteínas ADAM/fisiologia , Movimento Celular/fisiologia , Proteínas ADAM/genética , Proteína ADAM12 , Proteína ADAM17 , Animais , Células CHO , Adesão Celular/fisiologia , Cricetinae , Cricetulus , Fibronectinas/metabolismo , Integrina alfa4beta1/metabolismo , Integrina alfa5beta1/metabolismo , Mutação PuntualRESUMO
The ADAM (a disintegrin and metalloprotease) family of proteins possess both proteolytic and adhesive domains. We have established previously that the disintegrin domain of ADAM28, an ADAM expressed by human lymphocytes, is recognized by the integrin alpha4beta1. The present study characterizes the integrin binding properties of the disintegrin-like domains of human ADAM7, ADAM28 and ADAM33 with the integrins alpha4beta1, alpha4beta7 and alpha9beta1. Cell-adhesion assays demonstrated that, similar to ADAM28, the ADAM7 disintegrin domain supported alpha4beta1-dependent Jurkat cell adhesion, whereas the ADAM33 disintegrin domain did not. The lymphocyte integrin alpha4beta7 was also found to recognize both disintegrin domains of ADAM7 and ADAM28, but not of ADAM33. This is the first demonstration that mammalian disintegrins are capable of interacting with alpha4beta7. All three disintegrin domains supported alpha9beta1-dependent cell adhesion. Recognition by both alpha4beta1 and alpha4beta7 of ADAM7 and ADAM28 was activation-dependent, requiring either the presence of Mn2+ or an activating monoclonal antibody for cell attachment. Charge-to-alanine mutagenesis experiments revealed that the same residues within an individual ADAM disintegrin domain function in recognizing multiple integrins. However, the residues within a specific region of each ADAM disintegrin-like domain required for integrin binding were distinct. These results establish that ADAM7 and ADAM28 are recognized by the leucocyte integrins alpha4beta1, alpha4beta7 and alpha9beta1. ADAM33 exclusively supported only alpha9beta1-dependent adhesion.
Assuntos
Integrina alfa4beta1/metabolismo , Integrinas/metabolismo , Linfócitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Proteínas ADAM , Sequência de Aminoácidos , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Células Jurkat/química , Células Jurkat/metabolismo , Células K562/química , Células K562/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Receptores de Retorno de Linfócitos/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Colorectal cancer (CRC) is highly metastatic, significantly so to liver, a characteristic that embodies one of the most challenging aspects of treatment. The integrin family of cell-cell and cell-matrix adhesion receptors plays a central role in migration and invasion, functions that underlie metastatic potential. In the present work we sought to determine the impact of ceramide, which plays a key modulatory role in cancer suppression, on integrin cell surface expression and function in CRC cells in order to reveal possible ceramide-centric effects on tumor cell motility. Human CRC cells LoVo, HT-29, and HCT-116 were employed, which represent lines established from primary and metastatic sites. A cell-permeable, short-chain analog, C6-ceramide, was used as ceramide mimic. Exposure of cells to C6-ceramide (24 h) promoted a dose-dependent (2.5-10 µM) decrease in the expression of cell surface ß1 and ß4 integrin subunits in all cell lines; at 10 µM C6-ceramide, the decreases ranged from 30 to 50% of the control. Expression of cell surface αVß6 integrin, which is associated with advanced invasion in CRC, was also suppressed by C6-ceramide. Decreases in integrin expression translated to diminished cellular adhesion, 50% of the control at 5 µM C6-ceramide, and markedly reduced cellular migration, approximately 30-40% of the control in all cell lines. Physicochemical examination revealed potent efficacy of nano-formulated C6-ceramide, but inferior activity of dihydro-C6-ceramide and L-C6-ceramide, compared to the unsaturated counterpart and the natural d-enantiomer, respectively. These studies demonstrate novel actions of ceramides that may have application in suppression of tumor metastasis, in addition to their known tumor suppressor effects.
Assuntos
Antineoplásicos/farmacologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Ceramidas/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Integrinas/metabolismo , Antígenos de Neoplasias/metabolismo , Antineoplásicos/química , Sobrevivência Celular/efeitos dos fármacos , Ceramidas/química , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Regulação para Baixo , Composição de Medicamentos , Células HCT116 , Células HT29 , Humanos , Integrina beta1/metabolismo , Integrina beta4/metabolismo , Estrutura Molecular , Metástase Neoplásica , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
ADAMs (a disintegrin and metalloprotease) are a family of cell surface proteins related to the Class III snake venom metalloproteases (SVMP). ADAMs are members of the Metazincin family which includes the matrix matalloproteases and the ADAMTS proteins. Unlike their snake venom relatives, ADAMs are expressed as transmembrane cell surface proteins. The domain structure of ADAMs suggests that these proteins posses both proteolytic and adhesive functions. Several members of the ADAM protein family have been shown to be involved in ectodomain shedding of many important cell surface proteins resulting in the release of biologically active soluble factors. The carboxyl-terminal domains, especially the disintegrin-like domain of ADAMs, have been demonstrated to support cell adhesion. The disintegrin-like domains of many ADAMs are capable of acting as integrin ligands. Integrins known to interact with ADAM disintegrin-like domains include alpha4beta1, alpha4beta7, alpha5beta1, alpha6beta1, alpha9beta1, alphavbeta3, and alphavbeta5. This integrin mediated interaction of the disintegrin-like domains with the cell surface suggests that ADAMs may function as cellular counter receptors. In this review we discuss the individual functions ascribed to members of the ADAM family especially those related to integrin interactions and the potential for integrin mediated regulation of ectodomain shedding.
Assuntos
Desintegrinas/farmacologia , Integrinas/efeitos dos fármacos , Metaloproteases/farmacologia , Animais , Humanos , Ligantes , Modelos Moleculares , Venenos de Serpentes/química , Venenos de Serpentes/farmacologiaRESUMO
Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of malignancies characterized by accumulation of malignant T-cells within the skin. Retinoids, metabolic derivatives, and synthetic analogs of vitamin A embody an effective CTCL therapy with over three decades of clinical use. The established mechanism of action is induction of growth arrest and apoptosis. However, the natural role of retinoids in T-cell biology is imprinting gut-homing properties by inducing integrin α4ß7 expression. How the natural role of retinoids relates to therapeutic effectiveness in CTCL has not been addressed and merits investigation. Here we provide evidence that retinoids, including Bexarotene, selectively induce CTCL lineages to increase integrin ß7 expression and function prior to growth arrest and apoptosis. Interestingly, augmented CTCL cell adhesion obtained with retinoid exposure was potently attenuated by 1,25-dihydroxyvitamin D3, a metabolic vitamin derivative involved in prompting immune cell skin homing. The integrin-dependent adhesion changes in CTCL cells occurred through synergistic activation of RAR and RXR nuclear receptors. These data explore the early cellular changes induced by retinoids that may be pivotal to sensitizing CTCL cells to growth arrest and apoptosis.
Assuntos
Cadeias beta de Integrinas/metabolismo , Linfoma Cutâneo de Células T/metabolismo , Retinoides/farmacologia , Neoplasias Cutâneas/metabolismo , Apoptose/efeitos dos fármacos , Calcitriol/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Cadeias beta de Integrinas/genética , Linfoma Cutâneo de Células T/patologia , Neoplasias Cutâneas/patologiaRESUMO
Fish oil-enriched long chain n-3 polyunsaturated fatty acids disrupt the molecular organization of T-cell proteins in the immunological synapse. The impact of fish oil derived n-3 fatty acids on antigen-presenting cells, particularly at the animal level, is unknown. We previously demonstrated B-cells isolated from mice fed with fish oil-suppressed naïve CD4(+) T-cell activation. Therefore, here we determined the mechanistic effects of fish oil on murine B-cell major histocompatibility complex (MHC) class II molecular distribution using a combination of total internal reflection fluorescence, Förster resonance energy transfer and confocal imaging. Fish oil had no impact on presynaptic B-cell MHC II clustering. Upon conjugation with transgenic T-cells, fish-oil suppressed MHC II accumulation at the immunological synapse. As a consequence, T-cell protein kinase C theta (PKCθ) recruitment to the synapse was also diminished. The effects were independent of changes in B-T cell adhesion, as measured with microscopy, flow cytometry and static cell adhesion assays with select immune ligands. Given that fish oil can reorganize the membrane by lowering membrane cholesterol levels, we then compared the results with fish oil to cholesterol depletion using methyl-B-cyclodextrin (MßCD). MßCD treatment of B-cells suppressed MHC II and T-cell PKCθ recruitment to the immunological synapse, similar to fish oil. Overall, the results reveal commonality in the mechanism by which fish oil manipulates protein lateral organization of B-cells compared to T-cells. Furthermore, the data establish MHC class II lateral organization on the B-cell side of the immunological synapse as a novel molecular target of fish oil.
Assuntos
Linfócitos B/imunologia , Óleos de Peixe/farmacologia , Antígenos de Histocompatibilidade Classe II/imunologia , Sinapses Imunológicas/fisiologia , Animais , Colesterol/deficiência , Antígenos de Histocompatibilidade Classe II/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/metabolismo , Isoenzimas/metabolismo , Masculino , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase C/metabolismo , Proteína Quinase C-theta , beta-Ciclodextrinas/farmacologiaRESUMO
Retinoids are essential in the proper establishment and maintenance of immunity. Although retinoids are implicated in immune related processes, their role in immune cell adhesion has not been well established. In this study, the effect of 9-cis-retinoic acid (9-cis-RA) on human hematopoietic cell adhesion was investigated. 9-cis-RA treatment specifically induced cell adhesion of the human immune cell lines HuT-78, NB4, RPMI 8866 and U937. Due to the prominent role of integrin receptors in mediating immune cell adhesion, we sought to evaluate if cell adhesion was integrin-dependent. By employing a variety of integrin antagonist including function-blocking antibodies and EDTA, we establish that 9-cis-RA prompts immune cell adhesion through established integrin receptors in addition to a novel integrin-independent process. The novel integrin-independent adhesion required the presence of retinoid and was attenuated by treatment with synthetic corticosteroids. Finally, we demonstrate that 9-cis-RA treatment of primary murine B-cells induces ex vivo adhesion that persists in the absence of integrin function. Our study is the first to demonstrate that 9-cis-RA influences immune cell adhesion through at least two functionally distinct mechanisms.
Assuntos
Antineoplásicos/farmacologia , Linfócitos B/citologia , Adesão Celular/efeitos dos fármacos , Integrinas/metabolismo , Tretinoína/farmacologia , Corticosteroides/metabolismo , Alitretinoína , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Proteínas Recombinantes de FusãoRESUMO
The cranial neural crest (CNC) consists of multipotent embryonic cells that contribute to craniofacial structures and other cells and tissues of the vertebrate head. During embryogenesis, CNC is induced at the neural plate boundary through the interplay of several major signaling pathways. Here, we report that the metalloproteinase activity of ADAM13 is required for early induction of CNC in Xenopus. In both cultured cells and X. tropicalis embryos, membrane-bound Ephrins (Efns) B1 and B2 were identified as substrates for ADAM13. ADAM13 upregulates canonical Wnt signaling and early expression of the transcription factor snail2, whereas EfnB1 inhibits the canonical Wnt pathway and snail2 expression. We propose that by cleaving class B Efns, ADAM13 promotes canonical Wnt signaling and early CNC induction.
Assuntos
Proteínas ADAM/metabolismo , Efrinas/metabolismo , Proteínas de Membrana/metabolismo , Crista Neural/embriologia , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Proteínas ADAM/genética , Animais , Padronização Corporal/fisiologia , Linhagem Celular , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/fisiologia , Indução Embrionária , Efrinas/genética , Humanos , Proteínas de Membrana/genética , Crista Neural/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Wnt/genética , Xenopus/anatomia & histologia , Xenopus/metabolismo , Proteínas de Xenopus/genéticaRESUMO
Cell adhesion is an integral aspect of immunity facilitating extravasation of immune cells during homing and activation. All -trans-Retinoic acid ( t-RA) regulates leukocyte differentiation, proliferation, and transmigration. However, the role of t-RA in immune cell adhesion is poorly defined. In this study, we evaluated the impact of t-RA and its metabolism on B and T cell adhesion. Specifically, we address the impact of t-RA on the adhesive properties of the human mature B and T cell lines RPMI 8866, Daudi and Jurkats. The effect of t-RA exposure on cell adhesion to vascular cell adhesion molecule-1 (VCAM-1), a well-established integrin counter receptor involved in immunity, and to nonconventional ADAM integrin ligands was assessed. We show for the first time that t-RA potently induces B cell adhesion in an integrin-independent manner to both VCAM-1 and select ADAM disintegrin domains. Using retinoid extraction and reverse-phase HPLC analysis, we identify the retinoid that is functionally responsible for this augmented adhesion. We also provide evidence that this novel t-RA adhesive response is not prototypical of lymphocytes since both Daudi and Jurkats do not alter their adhesive properties upon t-RA treatment. Further, the t-RA metabolic profiles between these lineages is distinct with 9- cis-retinoic acid being exclusively detected in Jurkat media. This study is the first to demonstrate that t-RA directly induces B cell adhesion in an integrin-independent manner and is not contingent upon t-RA metabolism.
Assuntos
Proteínas ADAM/metabolismo , Linfócitos B/imunologia , Adesão Celular , Glicoproteínas de Membrana/metabolismo , Tretinoína/farmacologia , Proteínas ADAM/química , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Integrinas/metabolismo , Células Jurkat , Glicoproteínas de Membrana/química , Estrutura Terciária de Proteína , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Tretinoína/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
ADAMs (a disintegrin and metalloprotease) are a family of proteins that possess functional adhesive and proteolytic domains. ADAM 28 (MDC-L) is expressed by human lymphocytes and contains a disintegrin-like domain that serves as a ligand for the leukocyte integrin, alpha4beta1. To elucidate which residues comprise the alpha4beta1 binding site in the ADAM 28 disintegrin domain, a charge-to-alanine mutagenesis strategy was utilized. Each alanine substitution mutant was evaluated and compared to the native sequence for its ability to support cell adhesion of the T-lymphoma cell line, Jurkat. This approach identified ADAM 28 residues Lys(437), Lys(442), Lys(455), Lys(459), Lys(460), Lys(469), and Glu(476) as being essential for alpha4beta1-dependent cell adhesion. The epitope for a function-blocking monoclonal antibody, Dis 1-1, was localized to the N-terminal end of the ADAM 28 disintegrin domain using these same charge-to-alanine mutants. Three distinct molecular models based upon the known structures of snake venom disintegrins suggested that residues contributing to alpha4beta1 recognition are aligned on one face of the domain. This study demonstrates that residues located outside of the disintegrin loop participate in integrin recognition of mammalian disintegrins.
Assuntos
Desintegrinas/metabolismo , Integrina alfa4beta1/metabolismo , Metaloendopeptidases/metabolismo , Proteínas ADAM , Alanina/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Adesão Celular , Desintegrinas/química , Mapeamento de Epitopos , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Células Jurkat , Linfócitos/enzimologia , Linfoma de Células T/metabolismo , Metaloendopeptidases/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas Recombinantes , Homologia de Sequência de AminoácidosRESUMO
The interaction of lymphocytes with other cells is critical for normal immune surveillance and response. MDC-L (ADAM 28), a member of the ADAM (a disintegrin and metalloprotease) protein family, is expressed on the surface of human lymphocytes. ADAMs possess a disintegrin-like domain similar in sequence to small non-enzymatic snake venom peptides that act as integrin antagonists. We report here that the disintegrin domain of MDC-L is recognized by the leukocyte integrin alpha(4)beta(1). Recombinant Fc fusion proteins possessing the disintegrin domain of MDC-L supported adhesion of the T-lymphoma cell line, Jurkat, in a concentration- and divalent cation-dependent manner. Adhesion of Jurkat cells to the disintegrin domain of MDC-L was inhibited by an anti-MDC-L monoclonal antibody (mAb), Dis1-1. The epitope for mAb Dis1-1 was localized within 59 residues of the disintegrin domain. Recombinant expression of this 59-residue fragment of the disintegrin domain also supported cell adhesion. Adhesion of Jurkat cells to the MDC-L disintegrin domain was specifically inhibited by anti-alpha(4) and anti-beta(1) function-blocking mAbs. Furthermore, adhesion of various cell lines to MDC-L correlated with expression of the integrin alpha(4)-subunit. Transfected K562 cells expressing alpha(4)beta(1) adhered to the disintegrin domain in contrast to non-transfected K562 cells. We further investigated the binding of recombinant MDC-L disintegrin domain (rDis-Fc) in solution. The rDis-Fc was found to bind to Jurkat cells in solution in a concentration-dependent and saturable manner. Both adhesion and solution binding of rDis-Fc was inhibited by the alpha(4)beta(1) ligand mimetic CS-1 peptide. Additionally, recognition of the MDC-L disintegrin domain required "activation" of lymphocyte beta(1) integrins. The interaction of MDC-L with alpha(4)beta(1) may potentially regulate metalloprotease function by targeting or sequestering the active protease on the cell surface. These results suggest a potential role for the lymphocyte ADAM, MDC-L, in the interaction of lymphocytes with alpha(4)beta(1)-expressing leukocytes.