RESUMEN
CD95 ligand (CD95L) is expressed by immune cells and triggers apoptotic death. Metalloprotease-cleaved CD95L (cl-CD95L) is released into the bloodstream but does not trigger apoptotic signaling. Hence, the pathophysiological role of cl-CD95L remains unclear. We observed that skin-derived endothelial cells from systemic lupus erythematosus (SLE) patients expressed CD95L and that after cleavage, cl-CD95L promoted T helper 17 (Th17) lymphocyte transmigration across the endothelial barrier at the expense of T regulatory cells. T cell migration relied on a direct interaction between the CD95 domain called calcium-inducing domain (CID) and the Src homology 3 domain of phospholipase Cγ1. Th17 cells stimulated with cl-CD95L produced sphingosine-1-phosphate (S1P), which promoted endothelial transmigration by activating the S1P receptor 3. We generated a cell-penetrating CID peptide that prevented Th17 cell transmigration and alleviated clinical symptoms in lupus mice. Therefore, neutralizing the CD95 non-apoptotic signaling pathway could be an attractive therapeutic approach for SLE treatment.
Asunto(s)
Señalización del Calcio , Inflamación/inmunología , Lupus Eritematoso Sistémico/inmunología , Fosfolipasa C gamma/metabolismo , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Receptor fas/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Lisofosfolípidos/metabolismo , Ratones , Ratones Endogámicos MRL lpr , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/genética , Fosfolipasa C gamma/genética , Dominios y Motivos de Interacción de Proteínas/genética , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Transcriptoma , Migración Transendotelial y Transepitelial , Receptor fas/genéticaRESUMEN
The anti-CD20 mAb, rituximab, is routinely used to treat B cell malignancies. However, a majority of patients relapse. An improvement in the complete response was obtained by combining rituximab with chemotherapy, at the cost of increased toxicity. We reported that rituximab induced the colocalization of both the Orai1 Ca(2+) release-activated Ca(2+) channel (CRAC) and the endoplasmic reticulum Ca(2+) sensor stromal interaction molecule 1 with CD20 and CD95 into a cluster, eliciting a polarized store-operated Ca(2+) entry (SOCE). We observed that blocking this Ca(2+) entry with downregulation of Orai1, pharmacological inhibitors, or reducing calcemia with hypocalcemic drugs sensitized human B lymphoma cell lines and primary human lymphoma cells to rituximab-induced apoptosis in vitro, and improved the antitumoral effect of rituximab in xenografted mice. This revealed that Ca(2+) entry exerted a negative feedback loop on rituximab-induced apoptosis, suggesting that associating CRAC channel inhibitors or hypocalcemic agents with rituximab may improve the treatment of patients with B cell malignancies. The calcium-dependent proteins involved in this process appear to vary according to the B lymphoma cell type, suggesting that CRAC-channel targeting is likely to be more efficient than calcium-dependent protein targeting.
Asunto(s)
Apoptosis/efectos de los fármacos , Canales de Calcio/metabolismo , Calcio/metabolismo , Linfoma no Hodgkin/tratamiento farmacológico , Rituximab/farmacología , Receptor fas/metabolismo , Animales , Antígenos CD20/inmunología , Antígenos CD20/metabolismo , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Western Blotting , Canales de Calcio/genética , Línea Celular Tumoral , Difosfonatos/farmacología , Retículo Endoplásmico/metabolismo , Femenino , Células HEK293 , Humanos , Imidazoles/farmacología , Linfoma no Hodgkin/genética , Linfoma no Hodgkin/patología , Proteínas de la Membrana/metabolismo , Ratones Noqueados , Microscopía Confocal , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , Interferencia de ARN , Rituximab/administración & dosificación , Molécula de Interacción Estromal 1 , Ensayos Antitumor por Modelo de Xenoinjerto , Ácido ZoledrónicoRESUMEN
The death receptor CD95 plays a pivotal role in immune surveillance and immune tolerance. Binding of CD95L to CD95 leads to recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates caspase-8 and caspase-10. Efficient formation of the CD95/FADD/caspase complex, known as the death-inducing signaling complex (DISC), culminates in the induction of apoptosis. We show that cells exposed to CD95L undergo a reorganization of the plasma membrane in which the Ca(2+) release-activated Ca(2+) channel Orai1 and the endoplasmic reticulum-resident activator stromal interaction molecule 1 colocalize with CD95 into a micrometer-sized cluster in which the channel elicits a polarized entry of calcium. Orai1 knockdown and expression of a dominant negative construct (Orai1E106A) reveal that on CD95 engagement, the Orai1-driven localized Ca(2+) influx is fundamental to recruiting the Ca(2+)-dependent protein kinase C (PKC) ß2 to the DISC. PKCß2 in turn transiently holds the complex in an inactive status, preventing caspase activation and transmission of the apoptotic signal. This study identifies a biological role of Ca(2+) and the Orai1 channel that drives a transient negative feedback loop, introducing a lag phase in the early steps of the CD95 signal. We suggest that these localized events provide a time of decision to prevent accidental cell death.
Asunto(s)
Apoptosis/fisiología , Canales de Calcio/metabolismo , Calcio/metabolismo , Complejos Multiproteicos/metabolismo , Proteína Quinasa C/metabolismo , Receptor fas/metabolismo , Western Blotting , Caspasa 10/metabolismo , Caspasa 8/metabolismo , Línea Celular , Proteína Ligando Fas/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Citometría de Flujo , Humanos , Inmunoprecipitación , Microscopía Confocal , Proteína ORAI1 , Proteína Quinasa C beta , Estadísticas no ParamétricasRESUMEN
Death receptors play a crucial role in immune surveillance and cellular homeostasis, two processes circumvented by tumor cells. CD95 (also termed Fas or APO1) is a transmembrane receptor, which belongs to the tumor necrosis factor receptor superfamily, and induces a potent apoptotic signal. Initial steps of the CD95 signal take place through protein/protein interactions that bring zymogens such as caspase-8 and caspase-10 closer. Aggregation of these procaspases leads to their autoprocessing, to the release of activated caspases in the cytosol, which causes a caspase cascade, and to the transmission of the apoptotic signal. In parallel, CD95 engagement drives an increase in the intracellular calcium concentration (Ca(2+))i whose origin and functions remain controversial. Although Ca(2+) ions play a central role in apoptosis/necrosis induction, recent studies have highlighted a protective role of Ca(2+) in death receptor signaling. In the light of these findings, we discuss the role of Ca(2+) ions as modulators of CD95 signaling.
Asunto(s)
Calcio/fisiología , Transducción de Señal/fisiología , Receptor fas/fisiología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Humanos , Receptores de Muerte Celular/efectos de los fármacosRESUMEN
GA101/obinutuzumab is a novel type II anti-CD20 monoclonal antibody (mAb), which is more effective than rituximab (RTX) in preclinical and clinical studies when used in combination with chemotherapy. Ca2+ signaling was shown to play a role in RTX-induced cell death. This report concerns the effect of GA101 on Ca2+ signaling and its involvement in the direct cell death induced by GA101. We reveal that GA101 triggered an intracellular Ca2+ increase by mobilizing intracellular Ca2+ stores and activating Orai1-dependent Ca2+ influx in non-Hodgkin lymphoma cell lines and primary B-Cell Chronic Lymphocytic Leukemia (B-CLL) cells. According to the cell type, Ca2+ was mobilized from two distinct intracellular compartments. In Raji, BL2, and B-CLL cells, GA101 induced a Ca2+ release from lysosomes, leading to the subsequent lysosomal membrane permeabilization and cell death. Inhibition of this calcium signaling reduced GA101-induced cell death in these cells. In SU-DHL-4 cells, GA101 mobilized Ca2+ from the endoplasmic reticulum (ER). Inhibition of ER replenishment, by blocking Orai1-dependent Ca2+ influx, led to an ER stress and unfolded protein response (UPR) which sensitized these cells to GA101-induced cell death. These results revealed the central role of Ca2+ signaling in GA101's action mechanism, which may contribute to designing new rational drug combinations improving its clinical efficacy.
RESUMEN
Intracellular calcium signals regulate cell function and cell survival by controlling many processes. CD95 engagement results in distinct intracellular calcium signals that control the cell fate, apoptosis, or survival, depending on the ligand (membrane or soluble). Intracellular calcium determination is an exquisite readout to explore the molecular mechanisms elicited by CD95 engagement. The most widely applied methods for studying calcium signaling pathways use fluorescent indicators and imaging methods with fluorescence microscopy. This technical approach, however, requires many precautions that we discuss in this chapter.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Receptor fas/metabolismo , Línea Celular , Proteína Ligando Fas/metabolismo , Humanos , Microscopía Confocal , Mitocondrias/metabolismo , Imagen Molecular/métodos , Unión ProteicaRESUMEN
Selective Serotonin Reuptake Inhibitor antidepressants, such as fluoxetine (Prozac), have been shown to induce cell death in cancer cells, paving the way for their potential use as cancer therapy. These compounds are able to increase cytosolic calcium concentration ([Ca2+]cyt), but the involved mechanisms and their physiological consequences are still not well understood. Here, we show that fluoxetine induces an increase in [Ca2+]cyt by emptying the endoplasmic reticulum (ER) through the translocon, an ER Ca2+ leakage structure. Our data also show that fluoxetine inhibits oxygen consumption and lowers mitochondrial ATP. This latter is essential for Ca2+ reuptake into the ER, and we postulated therefore that the fluoxetine-induced decrease in mitochondrial ATP production results in the emptying of the ER, leading to capacitative calcium entry. Furthermore, Ca2+ quickly accumulated in the mitochondria, leading to mitochondrial Ca2+ overload and cell death. We found that fluoxetine could induce an early necrosis in human peripheral blood lymphocytes and Jurkat cells, and could also induce late apoptosis, especially in the tumor cell line. These results shed light on fluoxetine-induced cell death and its potential use in cancer treatment.
Asunto(s)
Antidepresivos de Segunda Generación/farmacología , Calcio/metabolismo , Metabolismo Energético/efectos de los fármacos , Fluoxetina/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Necrosis/inducido químicamente , Necrosis/metabolismo , Adenosina Trifosfato/metabolismo , Apoptosis/efectos de los fármacos , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular , Respiración de la Célula/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Imagen Molecular , Consumo de Oxígeno , Fosfoinositido Fosfolipasa C/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Although the human malignant astrocytoma cell line U87-MG has been used in numerous studies, few findings are available on the properties of its membrane ion conductances. Characterization of the ion channels expressed in these cells will make it possible to study membrane ion conductance changes when a receptor is activated by its ligand. This will help to elucidate the functional properties of these receptors and their signal-transduction pathways in pathophysiological events. This work studied the voltage-dependent ionic conductances of U87-MG cells using the Whole-Cell Recording patch-clamp technique. Six types of voltage-dependent ionic currents were identified: (i) a TEA-, 4-AP- and CTX-sensitive Ca2+-dependent K+ current, (ii) a transient K+ current inhibited by 4-AP, (iii) an inwardly rectifying K+ current blocked by Ba2+ and 4-AP, (iv) a DIDS- and SITS-sensitive Cl- current, (v) a TTX-sensitive Na+ conductance and (vi) a L-type Ca2+ conductance activated by BayK-8644 and inhibited by Ni and the L-type Ca2+ channel inhibitor, nifedipine. In addition, electrical depolarizations elicited inward currents due to voltage-independent, Ca2+-dependent K+ influx against the electrochemical gradient, probably via an ouabain-sensitive Na+-K+ pump.
Asunto(s)
Astrocitoma/metabolismo , Activación del Canal Iónico , Canales Iónicos/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Cloruros/metabolismo , Conductividad Eléctrica , Humanos , Potenciales de la Membrana , Técnicas de Placa-Clamp , Potasio/metabolismo , Sodio/metabolismoRESUMEN
Prolactin (PRL) is involved in numerous biological processes in peripheral tissues and the brain. Although numerous studies have been conducted to elucidate the signal transduction pathways associated with the PRL receptor, very few have examined the role of ion conductances in PRL actions. We used the patch-clamp technique in "whole cell" configuration and microspectrofluorimetry to investigate the effects of PRL on membrane ion conductances in the U87-MG human malignant astrocytoma cell line, which naturally expresses the PRL receptor. We found that a physiological concentration (4 nM) of PRL exerted a biphasic action on membrane conductances. First, PRL activated a Ca(2+)-dependent K(+) current that was sensitive to CTX and TEA. This current depended on PRL-induced Ca(2+) mobilization, through a JAK2-dependent pathway from a thapsigargin- and 2-APB-sensitive Ca(2+) pool. Second, PRL also activated an inwardly directed current, mainly due to the stimulation of calcium influx via nickel- and 2-APB-sensitive calcium channels. Both phases resulted in membrane hyperpolarizations, mainly through the activation of Ca(2+)-dependent K(+) channels. As shown by combined experiments (electrophysiology and microspectrofluorimetry), the PRL-induced Ca(2+) influx increased with cell membrane hyperpolarization and conversely decreased with cell membrane depolarization. Thus PRL-induced membrane hyperpolarizations facilitated Ca(2+) influx through voltage-independent Ca(2+) channels. Finally, PRL also activated a DIDS-sensitive Cl(-) current, which may participate in the PRL-induced hyperpolarization. These PRL-induced conductance activations are probably related to the PRL proliferative effect we have already described in U87-MG cells.
Asunto(s)
Astrocitoma/fisiopatología , Neoplasias Encefálicas/fisiopatología , Canales Iónicos/efectos de los fármacos , Prolactina/farmacología , Proteínas Proto-Oncogénicas , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Línea Celular Tumoral , Canales de Cloruro/efectos de los fármacos , Canales de Cloruro/fisiología , Electrofisiología , Humanos , Janus Quinasa 2 , Potenciales de la Membrana/efectos de los fármacos , Níquel/farmacología , Técnicas de Placa-Clamp , Canales de Potasio Calcio-Activados/efectos de los fármacos , Canales de Potasio Calcio-Activados/fisiología , Proteínas Tirosina Quinasas/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Espectrometría de FluorescenciaRESUMEN
Prolactin (PRL) has several physiological effects on peripheral tissues and the brain. This hormone acts via its membrane receptor (PRL-R) to induce cell differentiation or proliferation. Using reverse transcription-polymerase chain reaction (RT-PCR) combined with Southern blot analysis, we detected PRL-R transcripts in a human glioma cell line (U87-MG) and in primary cultured human glioblastoma cells. These transcripts were deleted or not in their extracellular domains. We examined the effects of PRL on intracellular free Ca2+ concentration ([Ca2+](i)) in these cells in order to improve our understanding of the PRL transduction mechanism, which is still poorly documented. [Ca2+](i) was measured by microspectrofluorimetry using indo-1 as the Ca2+ fluorescent probe. Spatiotemporal aspects of PRL-induced Ca2+ signals were investigated using high-speed fluo-3 confocal imaging. We found that physiological concentrations (0.4-4 nM) of PRL-stimulated Ca2+ entry and intracellular Ca2+ mobilization via a tyrosine kinase-dependent mechanism. The two types of Ca2+ responses observed were distinguishable by their kinetics: one showing a slow (type I) and the other a fast (type II) increase in [Ca2+](i). The amplitude of PRL-induced Ca2+ increases may be sufficient to provoke several physiological responses, such as stimulating proliferation. Furthermore, PRL induced a dose-dependent increase in [3H]thymidine incorporation levels and in cellular growth and survival, detected by the MTT method. These data indicate that PRL induced mitogenesis of human glioma cells.