RESUMO
The regulation of integrin-mediated adhesion is of vital importance to adaptive and innate immunity. Integrins are versatile proteins and mediate T cell migration and trafficking by binding to extracellular matrix or other cells as well as initiating intracellular signaling cascades promoting survival or activation. The MAPK pathway is known to be downstream from integrins and to regulate survival, differentiation, and motility. However, secondary roles for canonical MAPK pathway members are being discovered. We show that chemical inhibition of RAF by sorafenib or shRNA-mediated knockdown of B-Raf reduces T cell resistance to shear stress to α4ß1 integrin ligands vascular cell adhesion molecule 1 (VCAM-1) and fibronectin, whereas inhibition of MEK/ERK by U0126 had no effect. Microscopy showed that RAF inhibition leads to significant inhibition of T cell spreading on VCAM-1. The association of α4ß1 integrin with the actin cytoskeleton was shown to be dependent on B-Raf activity or expression, whereas α4ß1 integrin affinity for soluble VCAM-1 was not. These effects were shown to be specific for α4ß1 integrin and not other integrins, such as α5ß1 or LFA-1, or a variety of membrane proteins. We demonstrate a novel role for B-Raf in the selective regulation of α4ß1 integrin-mediated adhesion.
Assuntos
Citoesqueleto/metabolismo , Integrina alfa4beta1/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Estresse Fisiológico/fisiologia , Linfócitos T/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Citoesqueleto/genética , Técnicas de Silenciamento de Genes , Humanos , Integrina alfa4beta1/genética , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/metabolismo , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Receptores de Vitronectina/genética , Receptores de Vitronectina/metabolismo , Resistência ao Cisalhamento/efeitos dos fármacos , Resistência ao Cisalhamento/fisiologia , Sorafenibe , Estresse Fisiológico/efeitos dos fármacos , Linfócitos T/citologia , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Activation of the integrin family of cell adhesion receptors on progenitor cells may be a viable approach to enhance the effects of stem cell-based therapies by improving cell retention and engraftment. Here, we describe the synthesis and characterization of the first small molecule agonist identified for the integrin α4ß1 (also known as very late antigen-4 or VLA-4). The agonist, THI0019, was generated via two structural modifications to a previously identified α4ß1 antagonist. THI0019 greatly enhanced the adhesion of cultured cell lines and primary progenitor cells to α4ß1 ligands VCAM-1 and CS1 under both static and flow conditions. Furthermore, THI0019 facilitated the rolling and spreading of cells on VCAM-1 and the migration of cells toward SDF-1α. Molecular modeling predicted that the compound binds at the α/ß subunit interface overlapping the ligand-binding site thus indicating that the compound must be displaced upon ligand binding. In support of this model, an analog of THI0019 modified to contain a photoreactive group was used to demonstrate that when cross-linked to the integrin, the compound behaves as an antagonist instead of an agonist. In addition, THI0019 showed cross-reactivity with the related integrin α4ß7 as well as α5ß1 and αLß2. When cross-linked to αLß2, the photoreactive analog of THI0019 remained an agonist, consistent with it binding at the α/ß subunit interface and not at the ligand-binding site in the inserted ("I") domain of the αL subunit. Co-administering progenitor cells with a compound such as THI0019 may provide a mechanism for enhancing stem cell therapy.
Assuntos
Movimento Celular/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Integrina alfa4beta1/agonistas , Modelos Moleculares , Células-Tronco/metabolismo , Antígeno CD11a/genética , Antígeno CD11a/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Integrina alfa5beta1/genética , Integrina alfa5beta1/metabolismo , Células Jurkat , Células-Tronco/citologia , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Distinct signalling pathways producing diverse cellular outcomes can utilize similar subsets of proteins. For example, proteins from the TCR (T-cell receptor) ESC (early signalling complex) are also involved in interferon-α receptor signalling. Defining the mechanism for how these proteins function within a given pathway is important in understanding the integration and communication of signalling networks with one another. We investigated the contributions of the TCR ESC proteins Lck (lymphocyte-specific kinase), ZAP-70 (ζ-chain-associated protein of 70 kDa), Vav1, SLP-76 [SH2 (Src homology 2)-domain-containing leukocyte protein of 76 kDa] and LAT (linker for activation of T-cells) to integrin outside-in signalling in human T-cells. Lck, ZAP-70, SLP-76, Vav1 and LAT were activated by α4ß1 outside-in signalling, but in a manner different from TCR signalling. TCR stimulation recruits ESC proteins to activate the mitogen-activated protein kinase ERK (extracellular-signal-regulated kinase). α4ß1 outside-in-mediated ERK activation did not require TCR ESC proteins. However, α4ß1 outside-in signalling induced CD25 and co-stimulated CD69 and this was dependent on TCR ESC proteins. TCR and α4ß1 outside-in signalling are integrated through the common use of TCR ESC proteins; however, these proteins display functionally distinct roles in these pathways. These novel insights into the cross-talk between integrin outside-in and TCR signalling pathways are highly relevant to the development of therapeutic strategies to overcome disease associated with T-cell deregulation.
Assuntos
Antígenos CD/biossíntese , Antígenos de Diferenciação de Linfócitos T/biossíntese , Integrina alfa4beta1/fisiologia , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Lectinas Tipo C/biossíntese , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/fisiologia , Humanos , Células Jurkat , Fatores de TempoRESUMO
PR1 (VLQELNVTV) is a human leukocyte antigen-A2 (HLA-A2)-restricted leukemia-associated peptide from proteinase 3 (P3) and neutrophil elastase (NE) that is recognized by PR1-specific cytotoxic T lymphocytes that contribute to cytogenetic remission of acute myeloid leukemia (AML). We report a novel T-cell receptor (TCR)-like immunoglobulin G2a (IgG2a) antibody (8F4) with high specific binding affinity (dissociation constant [K(D)] = 9.9nM) for a combined epitope of the PR1/HLA-A2 complex. Flow cytometry and confocal microscopy of 8F4-labeled cells showed significantly higher PR1/HLA-A2 expression on AML blasts compared with normal leukocytes (P = .046). 8F4 mediated complement-dependent cytolysis of AML blasts and Lin(-)CD34(+)CD38(-) leukemia stem cells (LSCs) but not normal leukocytes (P < .005). Although PR1 expression was similar on LSCs and hematopoietic stem cells, 8F4 inhibited AML progenitor cell growth, but not normal colony-forming units from healthy donors (P < .05). This study shows that 8F4, a novel TCR-like antibody, binds to a conformational epitope of the PR1/HLA-A2 complex on the cell surface and mediates specific lysis of AML, including LSCs. Therefore, this antibody warrants further study as a novel approach to targeting leukemia-initiating cells in patients with AML.
Assuntos
Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica/imunologia , Epitopos/imunologia , Antígeno HLA-A2/imunologia , Leucemia Mieloide Aguda/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Linhagem Celular , Humanos , Leucemia Mieloide Aguda/patologia , Leucócitos/imunologia , Leucócitos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Receptores de IgG/imunologia , Células-Tronco/imunologiaRESUMO
The activation of leukocyte function-associated antigen-1 (LFA-1) plays a critical role in regulating immune responses. The metal ion-dependent adhesion site on the I-domain of LFA-1 α(L) subunit is the key recognition site for ligand binding. Upon activation, conformation changes in the I-domain can lead LFA-1 from the low affinity state to the high affinity (HA) state. Using the purified HA I-domain locked by disulfide bonds for immunization, we developed an mAb, 2E8, that specifically binds to cells expressing the HA LFA-1. The surface plasmon resonance analysis has shown that 2E8 only binds to the HA I-domain and that the dissociation constant (K(D)) for HA I-domain is 197 nm. The binding of 2E8 to the HA I-domain is metal ion-dependent, and the affinity decreased as Mn(2+) was replaced sequentially by Mg(2+) and Ca(2+). Surface plasmon resonance analysis demonstrates that 2E8 inhibits the interaction of HA I-domain and ICAM-1. Furthermore, we found that 2E8 can detect activated LFA-1 on both JY and Jurkat cells using flow cytometry and parallel plate adhesion assay. In addition, 2E8 inhibits JY cell adhesion to human umbilical vein endothelial cells and homotypic aggregation. 2E8 treatment reduces the proliferation of both human CD4(+) and CD8(+) T cells upon OKT3 stimulation without the impairment of their cytolytic function. Taken together, these data demonstrate that 2E8 is specific for the high affinity form of LFA-1 and that 2E8 inhibits LFA-1/ICAM-1 interactions. As a novel activation-specific monoclonal antibody, 2E8 is a potentially useful reagent for blocking high affinity LFA-1 and modulating T cell activation in research and therapeutics.
Assuntos
Anticorpos Monoclonais/farmacologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Metais/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Cátions Bivalentes/imunologia , Cátions Bivalentes/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Dissulfetos/imunologia , Dissulfetos/metabolismo , Células Endoteliais/citologia , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/imunologia , Molécula 1 de Adesão Intercelular/metabolismo , Células Jurkat , Células K562 , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Metais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Muromonab-CD3/imunologia , Muromonab-CD3/metabolismo , Estrutura Terciária de Proteína , Subunidades Proteicas/imunologia , Subunidades Proteicas/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Veias Umbilicais/citologia , Veias Umbilicais/imunologiaRESUMO
A key issue regarding the use of stem cells in cardiovascular regenerative medicine is their retention in target tissues. Here, we have generated and assessed a bispecific antibody heterodimer designed to improve the retention of bone-marrow-derived multipotent stromal cells (BMMSC) in cardiac tissue damaged by myocardial infarction. The heterodimer comprises an anti-human CD90 monoclonal antibody (mAb) (clone 5E10) and an anti-myosin light chain 1 (MLC1) mAb (clone MLM508) covalently cross-linked by a bis-arylhydrazone. We modified the anti-CD90 antibody with a pegylated-4-formylbenzamide moiety to a molar substitution ratio (MSR) of 2.6 and the anti-MLC1 antibody with a 6-hydrazinonicotinamide moiety to a MSR of 0.9. The covalent modifications had no significant deleterious effect on mAb epitope binding. Furthermore, the binding of anti-CD90 antibody to BMMSCs did not prevent their differentiation into adipo-, chondro-, or osteogenic lineages. Modified antibodies were combined under mild conditions (room temperature, pH 6, 1 h) in the presence of a catalyst (aniline) to allow for rapid generation of the covalent bis-arylhydrazone, which was monitored at A(354). We evaluated epitope immunoreactivity for each mAb in the construct. Flow cytometry demonstrated binding of the bispecific construct to BMMSCs that was competed by free anti-CD90 mAb, verifying that modification and cross-linking were not detrimental to the anti-CD90 complementarity-determining region. Similarly, ELISA-based assays demonstrated bispecific antibody binding to plastic-immobilized recombinant MLC1. Excess anti-MLC1 mAb competed for bispecific antibody binding. Finally, the anti-CD90 × anti-MLC1 bispecific antibody construct induced BMMSC adhesion to plastic-immobilized MLC1 that was resistant to shear stress, as measured in parallel-plate flow chamber assays. We used mAbs that bind both human antigens and the respective pig homologues. Thus, the anti-CD90 × anti-MLC1 bispecific antibody may be used in large animal studies of acute myocardial infarction and may provide a starting point for clinical studies.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Terapia de Alvo Molecular/métodos , Células-Tronco Multipotentes/imunologia , Infarto do Miocárdio/tratamento farmacológico , Cadeias Leves de Miosina/imunologia , Células Estromais/imunologia , Antígenos Thy-1/imunologia , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/imunologia , Células da Medula Óssea , Humanos , Infarto do Miocárdio/patologia , Miocárdio , SuínosRESUMO
The development of antagonists to the α4 integrin family of cell adhesion molecules has been an active area of pharmaceutical research to treat inflammatory and autoimmune diseases. Presently being tested in human clinical trials are compounds selective for α4ß1 (VLA-4) as well as several dual antagonists that inhibit both α4ß1 and α4ß7. The value of a dual versus a selective small molecule antagonist as well as the consequences of inhibiting different affinity states of the α4 integrins have been debated in the literature. Here, we characterize TBC3486, a N,N-disubstituted amide, which represents a unique structural class of non-peptidic, small molecule VLA-4 antagonists. Using a variety of adhesion assay formats as well as flow cytometry experiments using mAbs specific for certain activation-dependent integrin epitopes we demonstrate that TBC3486 preferentially targets the high affinity conformation of α4ß1 and behaves as a ligand mimetic. The antagonist is capable of blocking integrin-dependent T-cell co-activation in vitro as well as proves to be efficacious in vivo at low doses in two animal models of allergic inflammation. These data suggest that a small molecule α4 integrin antagonist selective for α4ß1 over α4ß7 and, specifically, selective for the high affinity conformation of α4ß1 may prove to be an effective therapy for multiple inflammatory diseases in humans.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Inflamação/tratamento farmacológico , Integrina alfa4beta1/antagonistas & inibidores , Tiofenos/farmacologia , Ureia/análogos & derivados , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Humanos , Hipersensibilidade/tratamento farmacológico , Integrina alfa4beta1/química , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Conformação Proteica/efeitos dos fármacos , Eosinofilia Pulmonar/tratamento farmacológico , Linfócitos T/efeitos dos fármacos , Tiofenos/uso terapêutico , Ureia/farmacologia , Ureia/uso terapêuticoRESUMO
Staphylococcus aureus (SA) is an opportunistic pathogen that affects a variety of organ systems and is responsible for many diseases worldwide. SA express an MHC class II analog protein (Map), which may potentiate SA survival by modulating host immunity. We tested this hypothesis in mice by generating Map-deficient SA (Map(-)SA) and comparing disease outcome to wild-type Map(+)SA-infected mice. Map(-)SA-infected mice presented with significantly reduced levels of arthritis, osteomyelitis, and abscess formation compared with control animals. Furthermore, Map(-)SA-infected nude mice developed arthritis and osteomyelitis to a severity similar to Map(+)SA-infected controls, suggesting that T cells can affect disease outcome following SA infection and Map may attenuate cellular immunity against SA. The capacity of Map to alter T cell function was tested more specifically in vitro and in vivo using native and recombinant forms of Map. T cells or mice treated with recombinant Map had reduced T cell proliferative responses and a significantly reduced delayed-type hypersensitivity response to challenge antigen, respectively. These data suggest a role for Map as an immunomodulatory protein that may play a role in persistent SA infections by affecting protective cellular immunity.
Assuntos
Proteínas de Bactérias/imunologia , Staphylococcus aureus/imunologia , Linfócitos T/imunologia , Adjuvantes Imunológicos/farmacologia , Transferência Adotiva , Animais , Apoptose , Artrite Infecciosa/etiologia , Artrite Infecciosa/imunologia , Artrite Infecciosa/patologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Hipersensibilidade Tardia , Imunidade Celular , Técnicas In Vitro , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Nus , Osteomielite/etiologia , Osteomielite/imunologia , Osteomielite/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidadeRESUMO
Considerable advances in understanding the mechanisms associated with anoikis resistance of normal and malignant epithelial cells have been made. However, little is still known about the pathways involved in anoikis resistance of non-epithelial cells such as fibroblasts and sarcomas. Our results show that Src activity contributes to anoikis resistance of human osteosarcoma SAOS-2 cells. Src was found to be upregulated in anoikis resistant SAOS cells, and pharmacological inhibition of its activity resulted in the restoration of anoikis sensitivity. A normal pattern of dephosphorylation of FAK was observed upon cell detachment of both anoikis sensitive and resistant SAOS-2 cells, suggesting that FAK activity during anoikis resistance is not essential. The activity of Akt was found to be upregulated in anoikis resistant SAOSar cells and the pharmacological inhibition of PI3-K activity restored sensitivity to anoikis resistant cells, reconfirming the critical role of PI3-K/Akt pathway in cell survival. Furthermore, pharmacological inhibition of Src resulted in a decrease of Akt phosphorylation at Ser473. Altogether, these studies indicated a survival pathway mediated by the Src-dependent activation of the PI3-K/Akt pathway in a manner independent of FAK activity.
Assuntos
Anoikis/fisiologia , Osteossarcoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Western Blotting , Humanos , PTEN Fosfo-Hidrolase/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Chemotherapy-induced cell death can involve the induction of apoptosis. Thus, aberrant function of the pathways involved might result in chemoresistance. Since cell adhesion to the extracellular matrix acts as a survival factor that homeostatically maintains normal tissue architecture, it was tested whether acquisition of resistance to deadhesion-induced apoptosis (anoikis) in human osteosarcoma would result in resistance to chemotherapy. METHODS: Osteosarcoma cell lines (SAOS-2 and TE-85) obtained from ATCC and were maintained in complete Eagle's MEM medium. Suspension culture was established by placing cells in tissue culture wells coated with poly-HEMA. Cell cytotoxicity was determined using a live/dead cytotoxicity assay. Cell cycle/apoptosis analyses were performed using propidium iodide (PI) staining with subsequent FACS analysis. Apoptosis was also assayed by Annexin-FITC/PI staining. RESULTS: Etoposide, adriamycin, vinblastine, cisplatin and paclitaxel were able to induce apoptosis in human osteosarcoma cells SAOS-2 regardless of their anoikis resistance phenotype or the culture conditions (adhered vs. suspended). Moreover, suspended anoikis resistant TE-85 cells (TE-85ar) retained their sensitivity to chemotherapy as well. CONCLUSION: Acquisition of anoikis resistance in human osteosarcoma cells does not result in a generalized resistance to all apoptotic stimuli, including chemotherapy. Moreover, our results suggest that the pathways regulating anoikis resistance and chemotherapy resistance might involve the action of different mediators.
Assuntos
Anoikis , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Apoptose , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Separação Celular , Sobrevivência Celular , Cisplatino/farmacologia , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Matriz Extracelular/metabolismo , Citometria de Fluxo , Humanos , Hipóxia , Metástase Neoplásica , Paclitaxel/farmacologia , Propídio/farmacologia , Vimblastina/farmacologiaRESUMO
CD98 is a cell surface protein previously characterized as a cell activation marker, an amino acid transporter, and has recently been implicated in integrin-related functions. Integrins are cell surface proteins, important for homotypic cell aggregation, cell adhesion, and coactivation of T lymphocytes. We have previously shown that the anti-CD98 mAb 80A10, when coimmobilized with anti-CD3 mAb OKT3, is able to mediate human T cell coactivation that is inhibited by anti-beta1 integrin specific mAb 18D3. These results indicated a functional association of CD98 and beta1 integrin signaling but left open the question of a physical association. We now show the induction of homotypic aggregation through CD98 among human T cells and this aggregation was inhibited by anti-beta1 integrin mAb. Therefore, CD98-dependent lymphocyte proliferation and adhesion may involve integrins. Competitive binding assays and fluorescence colocalization analysis suggested that CD98 and beta1 integrin were physically associated. Differential extraction techniques and immunoprecipitations provided the first evidence that the alpha4beta1 integrin and CD98 are specifically associated on human T lymphocytes.
Assuntos
Proteína-1 Reguladora de Fusão/metabolismo , Integrina beta1/metabolismo , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Adesão Celular , Agregação Celular , Linhagem Celular , Humanos , Ativação Linfocitária , Transdução de Sinais , Linfócitos T/citologiaRESUMO
BACKGROUND: Rho family GTPase regulation of the actin cytoskeleton governs a variety of cell responses. In this report, we have analyzed the role of the GTPase Rho in maintenance of the T lymphocyte actin cytoskeleton. RESULTS: Inactivation of the GTPase Rho in the human T lymphocytic cell line HPB-ALL does not inhibit constitutively high adhesion to the integrin beta1 substrate fibronectin. It did however result in the aberrant extension of finger-like dendritic processes on the substrates VCAM-1, Fn, and mAb specific to beta1 integrins. Time-lapse video microscopy demonstrated that C3 induced extensions were primarily the result of an altered pseudopod elongation rather than retraction. Once the stellate pseudopodia extended, none retracted, and cells became completely immobile. Filipodial structures were absent and the dendritic-like processes in C3 treated cells were rich in filamentous actin. Immunolocalization of RhoA in untreated HPB-ALL cells spreading on fibronectin demonstrated a diffuse staining pattern within the pseudopodia. In C3 treated cells, clusters of RhoA were pronounced and localized within the altered extensions. CONCLUSIONS: GTPase Rho is actively involved in the regulation of T lymphocyte morphology and motility.
Assuntos
Linfócitos T/enzimologia , Proteínas rho de Ligação ao GTP/metabolismo , ADP Ribose Transferases/metabolismo , ADP Ribose Transferases/farmacologia , Actinas/metabolismo , Adenosina Difosfato Ribose/metabolismo , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas/farmacologia , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/metabolismo , Fibronectinas/farmacologia , Humanos , Microscopia de Vídeo/métodos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Células Tumorais Cultivadas , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are acidic proteins found in the extracellular matrices of bones and teeth. Recent data from gene knockouts, along with those of gene mutations, indicate that these two phosphoproteins are critical for bone and tooth development and/or maintenance. However, the precise functions of the two proteins have not been elucidated. In order to gain insights into their functions in tooth formation, we performed systematic, comparative investigations on the immunolocalization of DMP1 and dentin sialoprotein (DSP, a cleaved fragment of DSPP), using the rat first molar at different developmental stages as a model. Immunohistochemistry (IHC) was performed with specific, monoclonal antibodies against the COOH-terminal fragments of DMP1 and against DSP. In 1-day- and 1-week-old rats, weak immunoreactions for DMP1 were observed in dentinal tubules while stronger reactions for DSP were seen in the tubules and predentin. In rats older than 2 weeks, immunoreactions for DMP1 were found in dentinal tubules, predentin and odontoblasts. In 5-week- and 8-week-old rats, strong immunoreactions for DMP1 were widely distributed in odontoblasts and predentin. The distribution pattern of DSP was strikingly similar to that of DMP1 after 2 weeks and the localization of each was distinctly different from that of bone sialoprotein (BSP). The unique colocalization of DMP1 and DSPP in tooth development suggests that the two proteins play complementary and/or synergistic roles in formation and maintenance of healthy teeth.
Assuntos
Proteínas da Matriz Extracelular/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Dente Molar/crescimento & desenvolvimento , Sialoglicoproteínas/biossíntese , Animais , Anticorpos Monoclonais/química , Osso e Ossos/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Sialoproteína de Ligação à Integrina , Odontoblastos/metabolismo , Fosfoproteínas/química , Precursores de Proteínas , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Sialoglicoproteínas/metabolismo , Fatores de TempoRESUMO
T lymphocytes are the primary cells responsible for maintaining the immune system. There are many intricate mechanisms involved in the regulation of T cells and the integrin family of adhesive surface proteins plays a pivotal role in the control of T lymphocyte activation and functions. Integrins are heterodimeric transmembrane proteins that are not merely adhesion molecules but also function in T cell coactivation by providing a scaffold for signaling and cytoskeletal proteins that are adept at transmitting signals from the inside of the cell to the outside ("inside-out signaling") or from the outside of the cell to the inside ("outside-in signaling"). The signaling property of integrins allows for rapid responses to changes in the microenviroment of the lymphocyte. Therefore, whether the T cell needs to adhere or detach, integrins can quickly accommodate either state of the cell. Once cells are guided to sites of infection, inflammation, or antigen presentation, integrins can also participate in the initiation, maintenance, or termination of the response. This review will focus on the aspects of integrin-mediated T cell coactivation, affinity and avidity control of integrins, signaling molecules involved with integrins, association of integrins in lipid microdomains, and negative regulation of integrins.
Assuntos
Integrinas/imunologia , Linfócitos T/imunologia , Proteínas de Bactérias/imunologia , Adesão Celular/imunologia , Humanos , Ativação Linfocitária/imunologia , Microdomínios da Membrana/imunologia , Transdução de Sinais/imunologiaRESUMO
T-lymphocytes are responsible for cell-mediated immunity, and recognize antigens on target cells (e.g., tumor cells, virus-infected cells) and antigen presenting cells (e.g., macrophages, dendritic cells). While mechanical forces applied to a cell surface can produce alterations in the cytoskeletal structure, leading to global structural rearrangements and changes in the intracellular biochemistry and gene expression, it remains unknown if local mechanical forces acting at the lymphocyte-antigen interaction site play any role in lymphocyte activation following antigen recognition. In this study we investigate the effect of such forces induced by optical tweezers on the lymphocyte's morphological response. We brought optically trapped polystyrene beads, coated with a specific antibody against a clonotypic epitope of the T-cell receptors (TCRs), in contact with individual lymphocytes and applied mechanical forces at the TCR-antibody interaction site. Although bead size was a factor, simple bead contact tended to induce formation of pseudopodia that appeared randomly over the cell's surface, while application of tangential forces at the interaction site redirected pseudopodia formation toward that site and promoted endocytosis activity. We propose that local forces play a key role in the initial lymphocyte adhesion to antigen-bearing cells, and may be implicated in antigen-specific motility, transendothelial migration, and tissue homing to sites of inflammation.
Assuntos
Linfócitos/citologia , Linfócitos/fisiologia , Mecanotransdução Celular/fisiologia , Fluidez de Membrana/fisiologia , Micromanipulação/métodos , Pseudópodes/fisiologia , Pseudópodes/ultraestrutura , Células Cultivadas , Homeostase/fisiologia , Humanos , Micromanipulação/instrumentação , Óptica e Fotônica/instrumentação , Estimulação Física/instrumentação , Estimulação Física/métodos , Estresse MecânicoRESUMO
CLL cell trafficking between blood and tissue compartments is an integral part of the disease process. Idelalisib, a phosphoinositide 3-kinase delta (PI3Kδ) inhibitor causes rapid lymph node shrinkage, along with an increase in lymphocytosis, prior to inducing objective responses in CLL patients. This characteristic activity presumably is due to CLL cell redistribution from tissues into the blood, but the underlying mechanisms are not fully understood. We therefore analyzed idelalisib effects on CLL cell adhesion to endothelial and bone marrow stromal cells (EC, BMSC). We found that idelalisib inhibited CLL cell adhesion to EC and BMSC under static and shear flow conditions. TNFα-induced VCAM-1 (CD106) expression in supporting layers increased CLL cell adhesion and accentuated the inhibitory effect of idelalisib. Co-culture with EC and BMSC also protected CLL from undergoing apoptosis, and this EC- and BMSC-mediated protection was antagonized by idelalisib. Furthermore, we demonstrate that CLL cell adhesion to EC and VLA-4 (CD49d) resulted in the phosphorylation of Akt, which was sensitive to inhibition by idelalisib. These findings demonstrate that idelalisib interferes with integrin-mediated CLL cell adhesion to EC and BMSC, providing a novel mechanism to explain idelalisib-induced redistribution of CLL cells from tissues into the blood.
Assuntos
Células da Medula Óssea/patologia , Células Endoteliais/patologia , Inibidores Enzimáticos/farmacologia , Integrinas/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Inibidores de Fosfoinositídeo-3 Quinase , Purinas/farmacologia , Quinazolinonas/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Integrina alfa4beta1/metabolismo , Camundongos , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/patologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Disseminated prostate cancer cells must survive in circulation for metastasis to occur. Mechanisms by which these cells survive are not well understood. By immunohistochemistry of human tissues, we found that levels of ß1 integrins and integrin-induced autophosphorylation of FAK (pFAK-Y397) are increased in prostate cancer cells in primary prostate cancer and lymph node metastases, suggesting that ß1 integrin activation occurs in metastatic progression of prostate cancer. A conformation-sensitive antibody, 9EG7, was used to examine ß1 integrin activation. We found that ß1 integrins are constitutively activated in highly metastatic PC3 and PC3-mm2 cells, with less activation in low metastatic LNCaP and C4-2B4 cells. Increased ß1 integrin activation as well as the anoikis resistance in prostate cancer cells correlated with metastatic potential in vivo. Knockdown of ß1 integrin abrogated anoikis resistance in PC3-mm2 cells. In agreement with ß1 integrin activation, PC3-mm2 cells strongly adhered to type I collagen and fibronectin, a process inhibited by the ß1 integrin-neutralizing antibody mAb 33B6. mAb 33B6 also inhibited the phosphorylation of ß1 integrin downstream effectors, focal adhesion kinase (FAK) and AKT, leading to a 3-fold increase in PC3-mm2 apoptosis. Systemic delivery of mAb 33B6 suppressed spontaneous metastasis of PC3-mm2 from the prostate to distant lymph nodes following intraprostatic injection and suppressed metastasis of PC3-mm2 to multiple organs following intracardiac injection. Thus, constitutively activated ß1 integrins play a role in survival of PC3-mm2 cells in circulation and represent a potential target for metastasis prevention.
Assuntos
Anticorpos Monoclonais/farmacologia , Integrina beta1/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Matriz Extracelular , Humanos , Imuno-Histoquímica , Integrina beta1/imunologia , Metástase Linfática , Masculino , Camundongos , Camundongos SCID , Metástase Neoplásica , Fosforilação , TransfecçãoRESUMO
The alpha(4)beta(1) integrin VLA-4 (very-late activation antigen-4) and the lineage-specific CD4 and CD8 receptors have been proposed as putative co-stimulatory receptors on T cells. To assess the relative contribution of signaling through the TCR, CD28 and these accessory molecules, we activated human T cells using soluble antibodies recognizing all four of these T-cell receptor classes (CD3, CD28, CD4/CD8, and VLA-4), and we assessed the degree of activation using higher-order flow cytometry detecting intracellular Erk1/2 phosphorylation and production of IL-2 and IFN-gamma. We found that: (1) co-stimulation via CD4/CD8, in addition to CD28, is required for optimal T-cell activation; (2) VLA-4 binding consistently potentiates CD4(+) and CD8(+) T-cell activation; (3) augmentation of T-cell activation through VLA-4 binding is most pronounced following engagement of CD4/CD8. These results confirm that multiple signals, including VLA-4 engagement, are necessary for maximal T-cell activation beyond that induced via the TCR and CD28.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/imunologia , Integrina alfa4beta1/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Citocinas/biossíntese , Ativação Enzimática , Humanos , Ativação Linfocitária , Sistema de Sinalização das MAP Quinases , Fosforilação , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Chronic stress is associated with hormonal changes that are known to affect multiple systems, including the immune and endocrine systems, but the effects of stress on cancer growth and progression are not fully understood. Here, we demonstrate that human ovarian cancer cells exposed to either norepinephrine or epinephrine exhibit lower levels of anoikis, the process by which cells enter apoptosis when separated from ECM and neighboring cells. In an orthotopic mouse model of human ovarian cancer, restraint stress and the associated increases in norepinephrine and epinephrine protected the tumor cells from anoikis and promoted their growth by activating focal adhesion kinase (FAK). These effects involved phosphorylation of FAKY397, which was itself associated with actin-dependent Src interaction with membrane-associated FAK. Importantly, in human ovarian cancer patients, behavioral states related to greater adrenergic activity were associated with higher levels of pFAKY397, which was in turn linked to substantially accelerated mortality. These data suggest that FAK modulation by stress hormones, especially norepinephrine and epinephrine, can contribute to tumor progression in patients with ovarian cancer and may point to potential new therapeutic targets for cancer management.
Assuntos
Adrenérgicos/metabolismo , Anoikis , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Neoplasias Ovarianas/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Epinefrina/metabolismo , Feminino , Humanos , Camundongos , Camundongos Nus , Norepinefrina/metabolismo , FosforilaçãoRESUMO
Lipid rafts are small laterally mobile microdomains that are highly enriched in lymphocyte signaling molecules. GM1 gangliosides are a common lipid raft component and have been shown to be important in many T-cell functions. The aggregation of specific GM1 lipid rafts can control many T-cell activation events, including their novel association with T-cell integrins. We found that clustering GM1 lipid rafts can regulate beta1 integrin function. This was apparent through increased resistance to shear flow-dependent detachment of T cells adherent to the alpha4beta1 and alpha5beta1 integrin ligand fibronectin (FN). Adhesion strengthening as a result of clustering GM1 enriched lipid rafts correlated with increased cellular rigidity and morphology through the localization of cortical F-actin, the resistance to shear-induced cell stretching, and an increase in the surface area and symmetry of the contact area between the cell surface and adhesive substrate. Furthermore, clustering GM1 lipid rafts could initiate integrin 'inside-out' signaling mechanisms. This was seen through increased integrin-cytoskeleton associations and enhanced soluble binding of FN and VCAM-1, suggesting the induction of high-affinity integrin conformations. The activation of these adhesion-strengthening characteristics appears to be specific for the aggregation of GM1 lipid rafts as the aggregation of the heterogeneous raft-associated molecule CD59 failed to activate these functions. These findings indicate a novel mechanism to signal to beta1 integrins and to activate adhesion-strengthening processes.