RESUMO
T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with inferior outcome compared with that of B cell ALL. Here, we show that Runt-related transcription factor 2 (RUNX2) was upregulated in high-risk T-ALL with KMT2A rearrangements (KMT2A-R) or an immature immunophenotype. In KMT2A-R cells, we identified RUNX2 as a direct target of the KMT2A chimeras, where it reciprocally bound the KMT2A promoter, establishing a regulatory feed-forward mechanism. Notably, RUNX2 was required for survival of immature and KMT2A-R T-ALL cells in vitro and in vivo. We report direct transcriptional regulation of CXCR4 signaling by RUNX2, thereby promoting chemotaxis, adhesion, and homing to medullary and extramedullary sites. RUNX2 enabled these energy-demanding processes by increasing metabolic activity in T-ALL cells through positive regulation of both glycolysis and oxidative phosphorylation. Concurrently, RUNX2 upregulation increased mitochondrial dynamics and biogenesis in T-ALL cells. Finally, as a proof of concept, we demonstrate that immature and KMT2A-R T-ALL cells were vulnerable to pharmacological targeting of the interaction between RUNX2 and its cofactor CBFß. In conclusion, we show that RUNX2 acts as a dependency factor in high-risk subtypes of human T-ALL through concomitant regulation of tumor metabolism and leukemic cell migration.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Animais , Linhagem Celular Tumoral , Quimiotaxia de Leucócito , Criança , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade beta de Fator de Ligação ao Core/metabolismo , Progressão da Doença , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Rearranjo Gênico , Hematopoese , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Biogênese de Organelas , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CXCR4/metabolismo , Transdução de SinaisRESUMO
While current treatment regimens for acute leukemia can dramatically improve patient survival, there remains room for improvement. Due to its roles in cell differentiation, cell survival, and apoptotic signaling, modulation of the cyclic AMP (cAMP) pathway has provided a meaningful target in hematological malignancies. Several studies have demonstrated that gene expression profiles associated with increased pro-survival cAMP activity or downregulation of various pro-apoptotic factors associated with the cAMP pathway are apparent in acute leukemia patients. Previous work to increase leukemia cell intracellular cAMP focused on the use of cAMP analogs, stimulating cAMP production via transmembrane-associated adenylyl cyclases, or decreasing cAMP degradation by inhibiting phosphodiesterase activity. However, targeting cyclic nucleotide efflux by ATP-binding cassette (ABC) transporters represents an unexplored approach for modulation of intracellular cyclic nucleotide levels. Preliminary studies have shown that inhibition of cAMP efflux can stimulate leukemia cell differentiation, cell growth arrest, and apoptosis, indicating that targeting cAMP efflux may show promise for future therapeutic development. Furthermore, inhibition of cyclic nucleotide transporter activity may also contribute multiple anticancer benefits by reducing extracellular pro-survival signaling in malignant cells. Hence, several opportunities for drug repurposing may exist for targeting cyclic nucleotide transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Descoberta de Drogas , Reposicionamento de Medicamentos/métodos , Leucemia/tratamento farmacológico , Animais , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , HumanosRESUMO
Adhesive properties of leukemia cells shape the degree of organ infiltration and the extent of leukocytosis. CD44 and the integrin VLA-4, a CD49d/CD29 heterodimer, are important factors of progenitor cell adhesion in bone marrow (BM). Here, we report their cooperation in acute myeloid leukemia (AML) by a novel non-classical CD44-mediated way of inside-out VLA-4 activation. In primary AML BM samples from patients and the OCI-AML3 cell line, CD44 engagement by hyaluronan induced inside-out activation of VLA-4 resulting in enhanced leukemia cell adhesion on VCAM-1. This was independent from VLA-4 affinity regulation but based on ligand-induced integrin clustering on the cell surface. CD44-induced VLA-4 activation could be inhibited by the Src family kinase inhibitor PP2 and the multikinase inhibitor midostaurin. In further consequence, the increased adhesion on VCAM-1 allowed AML cells to strongly bind stromal cells. Thereby VLA-4/VCAM-1 interaction promoted activation of Akt, MAPK, NF-kB and mTOR signaling and decreased AML cell apoptosis. Collectively, our investigations provide a mechanistic description of an unusual CD44 function in regulating VLA-4 avidity in AML, supporting AML cell retention in the supportive BM microenvironment.
Assuntos
Integrina alfa4beta1 , Leucemia Mieloide Aguda , Medula Óssea , Adesão Celular , Humanos , Receptores de Hialuronatos/genética , Microambiente Tumoral , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
Clioquinol, one of the first mass-produced drugs, was considered safe and efficacious for many years. It was used as an antifungal and an antiprotozoal drug until it was linked to an outbreak of subacute myelo-optic neuropathy (SMON), a debilitating disease almost exclusively confined to Japan. Today, new information regarding clioquinol targets and its mechanism of action, as well as genetic variation (SNPs) in efflux transporters in the Japanese population, provide a unique interpretation of the existing phenomena. Further understanding of clioquinol's role in the inhibition of cAMP efflux and promoting apoptosis might offer promise for the treatment of cancer and/or neurodegenerative diseases. Here, we highlight recent developments in the field and discuss possible connections, hypotheses and perspectives in clioquinol-related research.
Assuntos
Anti-Infecciosos/uso terapêutico , Clioquinol/uso terapêutico , Neoplasias/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Anti-Infecciosos/efeitos adversos , Povo Asiático/genética , Clioquinol/efeitos adversos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Mielite/induzido quimicamente , Mielite/genética , Doenças Neurodegenerativas/metabolismo , Neurite Óptica/induzido quimicamente , Neurite Óptica/genética , Polimorfismo de Nucleotídeo Único , SíndromeRESUMO
Förster resonance energy transfer (FRET) continues to be a useful tool to study movement and interaction between proteins within living cells. When FRET as an optical technique is measured with flow cytometry, conformational changes of proteins can be rapidly measured cell-by-cell for the benefit of screening and profiling. We exploit FRET to study the extent of activation of α4ß1 integrin dimers expressed on the surface of leukocytes. The stalk-like transmembrane heterodimers when not active lay bent and upon activation extend outward. Integrin extension is determined by changes in the distance of closest approach between an FRET donor and acceptor, bound at the integrin head and cell membrane, respectively. Time-resolved flow cytometry analysis revealed donor emission increases up to 17%, fluorescence lifetime shifts over 1.0 ns during activation, and FRET efficiencies of 37% and 26% corresponding to the inactive and active integrin state, respectively. Last, a graphical phasor analysis, including population clustering, gating, and formation of an FRET trajectory, added precision to a comparative analysis of populations undergoing FRET, partial donor recovery, and complete donor recovery. This work establishes a quantitative cytometric approach for profiling fluorescence donor decay kinetics during integrin conformational changes on a single-cell level.
Assuntos
Citometria de Fluxo/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Integrinas/análise , Integrinas/química , Linhagem Celular Tumoral , Humanos , Integrinas/metabolismo , Conformação Proteica , Processamento de Sinais Assistido por ComputadorRESUMO
Kinase inhibitors have dramatically increased patient survival in a multitude of cancers, including hematological malignancies. However, kinase inhibitors have not yet been integrated into current clinical trials for patients with T-cell-lineage acute lymphoblastic leukemia (T-ALL). In this study, we used a high-throughput flow cytometry (HTFC) approach to test a collection of small-molecule inhibitors, including 26 FDA-approved tyrosine kinase inhibitors in a panel of T-ALL cell lines and patient-derived xenografts. Because hypoxia is known to cause resistance to chemotherapy, we developed a synthetic niche that mimics the low oxygen levels found in leukemic bone marrow to evaluate the effects of hypoxia on the tested inhibitors. Drug sensitivity screening was performed using the Agilent BioCel automated liquid handling system integrated with the HyperCyt HT flow cytometry platform, and the uptake of propidium iodide was used as an indication of cell viability. The HTFC dose-response testing identified several compounds that were efficacious in both normal and hypoxic conditions. This study shows that some clinically approved kinase inhibitors target T-ALL in the hypoxic niche of the bone marrow.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Reposicionamento de Medicamentos , Citometria de Fluxo , Ensaios de Triagem em Larga Escala , Bibliotecas de Moléculas Pequenas , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Descoberta de Drogas/métodos , Reposicionamento de Medicamentos/métodos , Citometria de Fluxo/métodos , Humanos , Camundongos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, which antagonizes B cell receptor (BCR) signals, demonstrates remarkable clinical activity in chronic lymphocytic leukemia (CLL). The lymphocytosis experienced by most patients under ibrutinib has previously been attributed to inhibition of BTK-dependent integrin and chemokine cues operating to retain the tumor cells in nodal compartments. Here, we show that the VLA-4 integrin, as expressed by CD49d-positive CLL, can be inside-out activated upon BCR triggering, thus reinforcing the adhesive capacities of CLL cells. In vitro and in vivo ibrutinib treatment, although reducing the constitutive VLA-4 activation and cell adhesion, can be overcome by exogenous BCR triggering in a BTK-independent manner involving PI3K. Clinically, in three independent ibrutinib-treated CLL cohorts, CD49d expression identifies cases with reduced lymphocytosis and inferior nodal response and behaves as independent predictor of shorter progression-free survival, suggesting the retention of CD49d-expressing CLL cells in tissue sites via activated VLA-4. Evaluation of CD49d expression should be incorporated in the characterization of CLL undergoing therapy with BCR inhibitors.
Assuntos
Integrina alfa4beta1/metabolismo , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/metabolismo , Adesão Celular/efeitos dos fármacos , Humanos , Imunoglobulina M/metabolismo , Estimativa de Kaplan-Meier , Linfonodos/efeitos dos fármacos , Linfonodos/metabolismo , Linfonodos/patologia , Linfocitose/metabolismo , Linfocitose/patologia , Análise Multivariada , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Piperidinas , Intervalo Livre de Progressão , Modelos de Riscos Proporcionais , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptores de Antígenos de Linfócitos B/metabolismoRESUMO
Assays to identify small molecule inhibitors of cell transporters have long been used to develop potential therapies for reversing drug resistance in cancer cells. In flow cytometry, these approaches rely on the use of fluorescent substrates of transporters. Compounds which prevent the loss of cell fluorescence have typically been pursued as inhibitors of specific transporters, but further drug development has been largely unsuccessful. One possible reason for this low success rate could be a substantial overlap in substrate specificities and functions between transporters of different families. Additionally, the fluorescent substrates are often synthetic dyes that exhibit promiscuity among transporters as well. Here, we describe an assay in which a fluorescent analog of a natural metabolite, 3',5'-cyclic adenosine monophosphate (F-cAMP), is actively effluxed by malignant leukemia cells. The F-cAMP is loaded into the cell cytoplasm using a procedure based on the osmotic lysis of pinocytic vesicles. The flow cytometric analysis of the fluorescence retained in F-cAMP-loaded cells incubated with various compounds can subsequently identify inhibitors of cyclic AMP efflux (ICE).
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , AMP Cíclico/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Citometria de Fluxo/métodos , Corantes Fluorescentes/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , AMP Cíclico/análogos & derivados , Corantes Fluorescentes/química , Humanos , Leucemia/metabolismoRESUMO
Apoptotic evasion is a hallmark of cancer. We propose that some cancers may evade cell death by regulating 3'-5'-cyclic adenosine monophosphate (cAMP), which is associated with pro-apoptotic signaling. We hypothesize that leukemic cells possess mechanisms that efflux cAMP from the cytoplasm, thus protecting them from apoptosis. Accordingly, cAMP efflux inhibition should result in: cAMP accumulation, activation of cAMP-dependent downstream signaling, viability loss, and apoptosis. We developed a novel assay to assess cAMP efflux and performed screens to identify inhibitors. In an acute myeloid leukemia (AML) model, several identified compounds reduced cAMP efflux, appropriately modulated pathways that are responsive to cAMP elevation (cAMP-responsive element-binding protein phosphorylation, and deactivation of Very Late Antigen-4 integrin), and induced mitochondrial depolarization and caspase activation. Blocking adenylyl cyclase activity was sufficient to reduce effects of the most potent compounds. These compounds also decreased cAMP efflux and viability of B-lineage acute lymphoblastic leukemia (B-ALL) cell lines and primary patient samples, but not of normal primary peripheral blood mononuclear cells. Our data suggest that cAMP efflux is a functional feature that could be therapeutically targeted in leukemia. Furthermore, because some of the identified drugs are currently used for treating other illnesses, this work creates an opportunity for repurposing.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Adenilil Ciclases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Descoberta de Drogas/métodos , Reposicionamento de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Integrina alfa4beta1/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Fosforilação , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Células Tumorais Cultivadas , Células U937RESUMO
Acute myeloid leukemia in adults is a highly heterogeneous disease. Gene expression profiling performed using unsupervised algorithms can be used to distinguish specific groups of patients within a large patient cohort. The identified gene expression signatures can offer insights into underlying physiological mechanisms of disease pathogenesis. Here, the analysis of several related gene expression clusters associated with poor outcome, worst overall survival and highest rates of resistant disease and obtained from the patients at the time of diagnosis or from previously untreated individuals is presented. Surprisingly, these gene clusters appear to be enriched for genes corresponding to proteins involved in transport across membranes (transporters, carriers and channels). Several ideas describing the possible relationship of membrane transport activity and leukemic cell biology, including the "Warburg effect," the specific role of chloride ion transport, direct "import" of metabolic energy through uptake of creatine phosphate, and modification of the bone marrow niche microenvironment are discussed.
RESUMO
Homing to distinct lymphoid organs enables chronic lymphocytic leukemia (CLL) cells to receive pro-survival and proliferative signals. Cytogenetic aberrations can significantly affect CLL cell compartmentalization. Trisomy 12 (tri12) defines a CLL subgroup with specific clinical features and increased levels of the negative prognostic marker CD49d, the α4-subunit of the integrin VLA-4, which is a key regulator of CLL cell homing to bone marrow (BM). Chemokine-induced inside-out VLA-4 activation, particularly via the CXCL12-CXCR4 axis, increases the arrest of various cell types on VCAM-1 presenting endothelium. Here, we demonstrate that high CD49d expression in tri12 CLL is accompanied by decreased CXCR4 expression. Dissecting functional consequences of these alterations, we observed that tri12 CLL cell homing to murine BM is not affected by CXCR4-CXCL12 blockage using AMD3100 or olaptesed pegol/NOX-A12. In line, CCL21-CCR7 rather than CXCL12-CXCR4 interactions triggered VLA-4-mediated arrests of tri12 CLL cells to VCAM-1 under blood flow conditions. Concordantly, in real-time kinetic analyses we found CCL21 but not CXCL12 being capable to induce inside-out VLA-4 conformational changes in this CLL subgroup. Our results provide novel insights into the peculiar clinico-biological behaviour of tri12 CLL and emphasize its specific chemokine and integrin utilization during pathophysiologically and therapeutically relevant interactions with the microenvironment.
Assuntos
Quimiocina CCL21/genética , Quimiocina CCL21/metabolismo , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Animais , Humanos , Camundongos , Transdução de SinaisRESUMO
Lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18, αLß2-integrin) and its ligands are essential for adhesion between T-cells and antigen-presenting cells, formation of the immunological synapse, and other immune cell interactions. LFA-1 function is regulated through conformational changes that include the modulation of ligand binding affinity and molecular extension. However, the relationship between molecular conformation and function is unclear. Here fluorescence resonance energy transfer (FRET) with new LFA-1-specific fluorescent probes showed that triggering of the pathway used for T-cell activation induced rapid unquenching of the FRET signal consistent with extension of the molecule. Analysis of the FRET quenching at rest revealed an unexpected result that can be interpreted as a previously unknown LFA-1 conformation.
Assuntos
Adesão Celular , Transferência Ressonante de Energia de Fluorescência , Ativação Linfocitária , Antígeno-1 Associado à Função Linfocitária/química , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Humanos , Ligantes , Estrutura Molecular , Conformação Proteica , Linfócitos T/citologiaRESUMO
Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases, and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended toward toxicity. We report here the characterization of a Cdc42-selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analog was identified via structure-activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition toward Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as noncompetitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42-selective allosteric inhibitor and a related analog, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.
Assuntos
Inibidores Enzimáticos/farmacologia , Sondas Moleculares/farmacologia , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Células 3T3 , Regulação Alostérica , Animais , Antivirais/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Integrina alfa4beta1/antagonistas & inibidores , Integrina alfa4beta1/fisiologia , Camundongos , Oligopeptídeos/metabolismo , Compostos de Fenilureia/metabolismo , Ligação Proteica , Pseudópodes/efeitos dos fármacos , Vírus Sin Nombre/fisiologia , Relação Estrutura-Atividade , Internalização do Vírus/efeitos dos fármacos , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Multiple hematological side effects have been reported to result from treatment with psychoactive phenothiazines. These reported toxicities include leucopenia, granulocytopenia, thrombocytopenia, agranulocytosis, and bone marrow aplasia. The physiological mechanism causing these potentially life-threatening blood dyscrasias is unknown. Recently, we discovered that phenothiazines exhibit antagonistic properties towards the VLA-4 integrin, an adhesion molecule that is responsible for homing and retention of hematological stem/progenitor cells (HSPCs) in the bone marrow. After administration of thioridazine we detected rapid mobilization of HSPCs into the peripheral blood. We propose that in patients receiving phenothiazines over a prolonged time period, continuous mobilization of stem cells out of the stem cell niche, results in a disorder of hematopoiesis. Furthermore, we also postulate that such cytopenias are caused by a loss of the niche environment, which is known to be essential for stem cell maintenance.
Assuntos
Antipsicóticos/efeitos adversos , Hematopoese/efeitos dos fármacos , Integrina alfa4beta1/antagonistas & inibidores , Modelos Biológicos , Fenotiazinas/efeitos adversos , Antipsicóticos/farmacologia , Medula Óssea/efeitos dos fármacos , Hematopoese/fisiologia , Humanos , Paraproteinemias/etiologia , Fenotiazinas/farmacologiaRESUMO
BACKGROUND: Integrin activation in response to inside-out signaling serves as the basis for rapid leukocyte arrest on endothelium, migration, and mobilization of immune cells. Integrin-dependent adhesion is controlled by the conformational state of the molecule, which is regulated by seven-transmembrane Guanine nucleotide binding Protein-Coupled Receptors (GPCRs). α4ß1-integrin (CD49d/CD29, Very Late Antigen-4, VLA-4) is expressed on leukocytes, hematopoietic progenitors, stem cells, hematopoietic cancer cells, and others. VLA-4 conformation is rapidly up-regulated by inside-out signaling through Gαi-coupled GPCRs and down-regulated by Gαs-coupled GPCRs. However, other signaling pathways, which include nitric oxide-dependent signaling, have been implicated in the regulation of cell adhesion. The goal of the current report was to study the effect of nitric oxide/cGMP signaling pathway on VLA-4 conformational regulation. RESULTS: Using fluorescent ligand binding to evaluate the integrin activation state on live cells in real-time, we show that several small molecules, which specifically modulate nitric oxide/cGMP signaling pathway, as well as a cell permeable cGMP analog, can rapidly down-modulate binding of a VLA-4 specific ligand on cells pre-activated through three Gαi-coupled receptors: wild type CXCR4, CXCR2 (IL-8RB), and a non-desensitizing mutant of formyl peptide receptor (FPR ΔST). Upon signaling, we detected rapid changes in the ligand dissociation rate. The dissociation rate after inside-out integrin de-activation was similar to the rate for resting cells. In a VLA-4/VCAM-1-specific myeloid cell adhesion system, inhibition of the VLA-4 affinity change by nitric oxide had a statistically significant effect on real-time cell aggregation. CONCLUSIONS: We conclude that nitric oxide/cGMP signaling pathway can rapidly down-modulate the affinity state of the VLA-4 binding pocket, especially under the condition of sustained Gαi-coupled GPCR signaling, generated by a non-desensitizing receptor mutant. This suggests a fundamental role of this pathway in de-activation of integrin-dependent cell adhesion.
Assuntos
GMP Cíclico/metabolismo , Integrina alfa4beta1/metabolismo , Monócitos/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais , Adesão Celular/imunologia , Comunicação Celular/imunologia , Movimento Celular/imunologia , Regulação para Baixo/imunologia , Endotélio Vascular/imunologia , Corantes Fluorescentes , Humanos , Integrina alfa4beta1/genética , Integrina alfa4beta1/imunologia , Monócitos/imunologia , Monócitos/patologia , Ligação Proteica/imunologia , Transdução de Sinais/imunologia , Células U937RESUMO
Integrins are cell adhesion receptors that mediate cell-to-cell, or cell-to-extracellular matrix adhesion. They represent an attractive target for treatment of multiple diseases. Two classes of small molecule integrin inhibitors have been developed. Competitive antagonists bind directly to the integrin ligand binding pocket and thus disrupt the ligand-receptor interaction. Allosteric antagonists have been developed primarily for α(L)ß(2)- integrin (LFA-1, lymphocyte function-associated antigen-1). Here we present the results of screening the Prestwick Chemical Library using a recently developed assay for the detection of α(4)ß(1)-integrin allosteric antagonists. Secondary assays confirmed that the compounds identified: 1) do not behave like competitive (direct) antagonists; 2) decrease ligand binding affinity for VLA-4 â¼2 orders of magnitude; 3) exhibit antagonistic properties at low temperature. In a cell based adhesion assay in vitro, the compounds rapidly disrupted cellular aggregates. In accord with reports that VLA-4 antagonists in vivo induce mobilization of hematopoietic progenitors into the peripheral blood, we found that administration of one of the compounds significantly increased the number of colony-forming units in mice. This effect was comparable to AMD3100, a well known progenitor mobilizing agent. Because all the identified compounds are structurally related, previously used, or currently marketed drugs, this result opens a range of therapeutic possibilities for VLA-4-related pathologies.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Integrina alfa4beta1/antagonistas & inibidores , Regulação Alostérica/efeitos dos fármacos , Animais , Mobilização de Células-Tronco Hematopoéticas/métodos , Humanos , Camundongos , Células U937RESUMO
Integrins are heterodimeric adhesion receptors that regulate immune cell adhesion. Integrin-dependent adhesion is controlled by multiple conformational states that include states with different affinity to the ligand, states with various degrees of molecule unbending, and others. Affinity change and molecule unbending play major roles in the regulation of cell adhesion. The relationship between different conformational states of the integrin is unclear. Here we have used conformationally sensitive antibodies and a small LDV-containing ligand to study the role of the inside-out signaling through formyl peptide receptor and CXCR4 in the regulation of alpha(4)beta(1) integrin conformation. We found that in the absence of ligand, activation by formyl peptide or SDF-1 did not result in a significant exposure of HUTS-21 epitope. Occupancy of the ligand binding pocket without cell activation was sufficient to induce epitope exposure. EC(50) for HUTS-21 binding in the presence of LDV was identical to a previously reported ligand equilibrium dissociation constant at rest and after activation. Furthermore, the rate of HUTS-21 binding was also related to the VLA-4 activation state even at saturating ligand concentration. We propose that the unbending of the integrin molecule after guanine nucleotide-binding protein-coupled receptor-induced signaling accounts for the enhanced rate of HUTS-21 binding. Taken together, current results support the existence of multiple conformational states independently regulated by both inside-out signaling and ligand binding. Our data suggest that VLA-4 integrin hybrid domain movement does not depend on the affinity state of the ligand binding pocket.