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BACKGROUND: Gestational choriocarcinoma (GC) is a highly malignant trophoblastic tumor that often develops from a complete hydatidiform mole (HM). NLRP7 is the major gene responsible for recurrent HM and is involved in the innate immune response, inflammation and apoptosis. NLRP7 can function in an inflammasome-dependent or -independent pathway. Recently, we have demonstrated that NLRP7 is highly expressed in GC tumor cells and contributes to their tumorigenesis. However, the underlying mechanisms are still unknown. Here, we investigated the mechanism by which NLRP7 controls these processes in malignant (JEG-3) and non-tumor (HTR8/SVneo) trophoblastic cells. Cell survival, dedifferentiation, camouflage, and aggressiveness were compared between normal JEG-3 cells or knockdown for NLRP7, JEG-3 Sh NLRP7. In addition, HTR8/SVneo cells overexpressing NLRP7 were used to determine the impact of NLRP7 overexpression on non-tumor cells. NLRP7 involvement in tumor cell growth and tolerance was further characterized in vivo using the metastatic mouse model of GC. RESULTS: We demonstrate that NLRP7 (i) functions in an inflammasome-dependent and -independent manners in HTR8/SVneo and JEG-3 cells, respectively; (ii) differentially regulates the activity of NF-κB in tumor and non-tumor cells; (iii) increases malignant cell survival, dedifferentiation, and camouflage; and (iv) facilitates tumor cells colonization of the lungs in the preclinical model of GC. CONCLUSIONS: This study demonstrates for the first time the mechanism by which NLRP7, independently of its inflammasome machinery, contributes to GC growth and tumorigenesis. The clinical relevance of NLRP7 in this rare cancer highlights its potential therapeutic promise as a molecular target to treat resistant GC patients.
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Proteínas Adaptadoras Transductoras de Señales , Coriocarcinoma , Animales , Femenino , Humanos , Ratones , Embarazo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Carcinogénesis , Línea Celular Tumoral , Supervivencia Celular , Coriocarcinoma/genética , Coriocarcinoma/metabolismo , Coriocarcinoma/patología , Inflamasomas/metabolismo , Recurrencia Local de NeoplasiaRESUMEN
Receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL) proteins are key regulators of necroptosis, a highly pro-inflammatory mode of cell death, which has been involved in various human diseases. Necroptotic-independent functions of RIPK3 and MLKL also exist, notably in the adipose tissue but remain poorly defined. Using knock-out (KO) cell models, we investigated the role of RIPK3 and MLKL in adipocyte differentiation. Mlkl-KO abolished white adipocyte differentiation via a strong expression of Wnt10b, a ligand of the Wnt/ß-catenin pathway, and a downregulation of genes involved in lipid metabolism. This effect was not recapitulated by the ablation of Ripk3. Conversely, Mlkl and Ripk3 deficiencies did not block beige adipocyte differentiation. These findings indicate that RIPK3 and MLKL have distinct roles in adipogenesis. The absence of MLKL blocks the differentiation of white, but not beige, adipocytes highlighting the therapeutic potential of MLKL inhibition in obesity.
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For many years, clinicians have understood the advantages of listening with two ears compared with one. In addition to improved speech intelligibility in quiet, noisy, and reverberant environments, binaural versus monaural listening improves perceived sound quality and decreases the effort listeners must expend to understand a target voice of interest or to monitor a multitude of potential target voices. For most individuals with bilateral hearing impairment, the body of evidence collected across decades of research has also found that the provision of two compared with one hearing aid yields significant benefit for the user. This article briefly summarizes the major advantages of binaural compared with monaural hearing, followed by a detailed description of the related technological advances in modern hearing aids. Aspects related to the communication and exchange of data between the left and right hearing aids are discussed together with typical algorithmic approaches implemented in modern hearing aids.
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BACKGROUND & AIMS: In non-alcoholic fatty liver disease (NAFLD), hepatocytes can undergo necroptosis: a regulated form of necrotic cell death mediated by the receptor-interacting protein kinase (RIPK) 1. Herein, we assessed the potential for RIPK1 and its downstream effector mixed lineage kinase domain-like protein (MLKL) to act as therapeutic targets and markers of activity in NAFLD. METHODS: C57/BL6J-mice were fed a normal chow diet or a high-fat diet (HFD). The effect of RIPA-56, a highly specific inhibitor of RIPK1, was evaluated in HFD-fed mice and in primary human steatotic hepatocytes. RIPK1 and MLKL concentrations were measured in the serum of patients with NAFLD. RESULTS: When used as either a prophylactic or curative treatment for HFD-fed mice, RIPA-56 caused a downregulation of MLKL and a reduction of liver injury, inflammation and fibrosis, characteristic of non-alcoholic steatohepatitis (NASH), as well as of steatosis. This latter effect was reproduced by treating primary human steatotic hepatocytes with RIPA-56 or necrosulfonamide, a specific inhibitor of human MLKL, and by knockout (KO) of Mlkl in fat-loaded AML-12 mouse hepatocytes. Mlkl-KO led to activation of mitochondrial respiration and an increase in ß-oxidation in steatotic hepatocytes. Along with decreased MLKL activation, Ripk3-KO mice exhibited increased activities of the liver mitochondrial respiratory chain complexes in experimental NASH. In patients with NAFLD, serum concentrations of RIPK1 and MLKL increased in correlation with activity. CONCLUSION: The inhibition of RIPK1 improves NASH features in HFD-fed mice and reverses steatosis via an MLKL-dependent mechanism that, at least partly, involves an increase in mitochondrial respiration. RIPK1 and MLKL are potential serum markers of activity and promising therapeutic targets in NAFLD. LAY SUMMARY: There are currently no pharmacological treatment options for non-alcoholic fatty liver disease (NAFLD), which is now the most frequent liver disease. Necroptosis is a regulated process of cell death that can occur in hepatocytes during NAFLD. Herein, we show that RIPK1, a gatekeeper of the necroptosis pathway that is activated in NAFLD, can be inhibited by RIPA-56 to reduce not only liver injury, inflammation and fibrosis, but also steatosis in experimental models. These results highlight the potential of RIPK1 as a therapeutic target in NAFLD.
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Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/sangre , Acrilamidas/farmacología , Anciano , Animales , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Femenino , Técnicas de Inactivación de Genes , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Necroptosis/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Proteínas Quinasas/sangre , Proteínas Quinasas/deficiencia , Proteínas Quinasas/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Resultado del TratamientoRESUMEN
The auditory system allows the estimation of the distance to sound-emitting objects using multiple spatial cues. In virtual acoustics over headphones, a prerequisite to render auditory distance impression is sound externalization, which denotes the perception of synthesized stimuli outside of the head. Prior studies have found that listeners with mild-to-moderate hearing loss are able to perceive auditory distance and are sensitive to externalization. However, this ability may be degraded by certain factors, such as non-linear amplification in hearing aids or the use of a remote wireless microphone. In this study, 10 normal-hearing and 20 moderate-to-profound hearing-impaired listeners were instructed to estimate the distance of stimuli processed with different methods yielding various perceived auditory distances in the vicinity of the listeners. Two different configurations of non-linear amplification were implemented, and a novel feature aiming to restore a sense of distance in wireless microphone systems was tested. The results showed that the hearing-impaired listeners, even those with a profound hearing loss, were able to discriminate nearby and far sounds that were equalized in level. Their perception of auditory distance was however more contracted than in normal-hearing listeners. Non-linear amplification was found to distort the original spatial cues, but no adverse effect on the ratings of auditory distance was evident. Finally, it was shown that the novel feature was successful in allowing the hearing-impaired participants to perceive externalized sounds with wireless microphone systems.
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Percepción Auditiva/fisiología , Pérdida Auditiva/patología , Personas con Deficiencia Auditiva/estadística & datos numéricos , Acústica , Adulto , Señales (Psicología) , Femenino , Audífonos , Pruebas Auditivas , Humanos , Masculino , Persona de Mediana Edad , Sonido , Percepción del HablaRESUMEN
The nuclear factor κB (NF-κB) signaling pathway ubiquitously controls cell growth and survival in basic conditions as well as rapid resetting of cellular functions following environment changes or pathogenic insults. Moreover, its deregulation is frequently observed during cell transformation, chronic inflammation or autoimmunity. Understanding how it is properly regulated therefore is a prerequisite to managing these adverse situations. Over the last years evidence has accumulated showing that ubiquitination is a key process in NF-κB activation and its resolution. Here, we examine the various functions of ubiquitin in NF-κB signaling and more specifically, how it controls signal transduction at the molecular level and impacts in vivo on NF-κB regulated cellular processes.
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Little is known about the perception of artificial spatial hearing by hearing-impaired subjects. The purpose of this study was to investigate how listeners with hearing disorders perceived the effect of a spatialization feature designed for wireless microphone systems. Forty listeners took part in the experiments. They were arranged in four groups: normal-hearing, moderate, severe, and profound hearing loss. Their performance in terms of speech understanding and speaker localization was assessed with diotic and binaural stimuli. The results of the speech intelligibility experiment revealed that the subjects presenting a moderate or severe hearing impairment better understood speech with the spatialization feature. Thus, it was demonstrated that the conventional diotic binaural summation operated by current wireless systems can be transformed to reproduce the spatial cues required to localize the speaker, without any loss of intelligibility. The speaker localization experiment showed that a majority of the hearing-impaired listeners had similar performance with natural and artificial spatial hearing, contrary to the normal-hearing listeners. This suggests that certain subjects with hearing impairment preserve their localization abilities with approximated generic head-related transfer functions in the frontal horizontal plane.
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Audífonos , Pérdida Auditiva Sensorineural , Percepción del Habla , Adulto , Anciano , Audición , Pruebas Auditivas , Humanos , Persona de Mediana Edad , Ruido , Localización de Sonidos , Adulto JovenAsunto(s)
Enfermedades de los Pequeños Vasos Cerebrales/metabolismo , Enfermedades de los Pequeños Vasos Cerebrales/patología , Enfermedades de los Pequeños Vasos Cerebrales/fisiopatología , Animales , Humanos , Incontinencia Pigmentaria/metabolismo , Incontinencia Pigmentaria/patología , Incontinencia Pigmentaria/fisiopatología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Receptor-interacting protein kinase 3 (RIPK3) mediates necroptosis, a form of programmed cell death that promotes inflammation in various pathological conditions, suggesting that it might be a privileged pharmacological target. However, its function in glucose homeostasis and obesity has been unknown. Here we show that RIPK3 is over expressed in the white adipose tissue (WAT) of obese mice fed with a choline-deficient high-fat diet. Genetic inactivation of Ripk3 promotes increased Caspase-8-dependent adipocyte apoptosis and WAT inflammation, associated with impaired insulin signalling in WAT as the basis for glucose intolerance. Similarly to mice, in visceral WAT of obese humans, RIPK3 is overexpressed and correlates with the body mass index and metabolic serum markers. Together, these findings provide evidence that RIPK3 in WAT maintains tissue homeostasis and suppresses inflammation and adipocyte apoptosis, suggesting that systemic targeting of necroptosis might be associated with the risk of promoting insulin resistance in obese patients.
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Tejido Adiposo Blanco/enzimología , Deficiencia de Colina/genética , Intolerancia a la Glucosa/genética , Grasa Intraabdominal/enzimología , Necrosis/enzimología , Obesidad/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Adipocitos/enzimología , Adipocitos/patología , Tejido Adiposo Blanco/patología , Animales , Apoptosis/genética , Índice de Masa Corporal , Caspasa 8/genética , Caspasa 8/metabolismo , Colina/metabolismo , Deficiencia de Colina/enzimología , Deficiencia de Colina/etiología , Deficiencia de Colina/patología , Dieta Alta en Grasa , Regulación de la Expresión Génica , Intolerancia a la Glucosa/enzimología , Intolerancia a la Glucosa/etiología , Intolerancia a la Glucosa/patología , Homeostasis , Humanos , Inflamación , Insulina/metabolismo , Resistencia a la Insulina , Grasa Intraabdominal/patología , Masculino , Ratones , Necrosis/genética , Necrosis/patología , Obesidad/enzimología , Obesidad/etiología , Obesidad/patología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismoRESUMEN
The transcription factor NF-κB plays a key role in numerous physiological processes such as inflammation, immunity, cell proliferation or control of cell death. Its activation is tightly controlled by a kinase complex, IκB kinase (IKK), composed of three core proteins: IKK1/IKKα, IKK2/IKKß and NEMO/IKKγ. The first two are structurally related kinases whereas the third one is a regulatory subunit exhibiting affinity for upstream activators modified by polyubiquitin chains. Over the years, several inherited diseases caused by mutations of each of the three subunits of IKK have been identified in humans together with diseases caused by mutations of several of its substrates. They are associated with very specific and complex phenotypes involving a broad range of abnormalities such as impaired innate and acquired immune response, perturbed skin development and defects of the central nervous system. Here, we summarize the diverse clinical, cellular and molecular manifestations of IKK-related genetic diseases and show that studying patient-related mutations affecting the IKK subunits and some of their substrates offers the opportunity to understand the various functions of NF-κB in humans, complementing studies performed with mouse models. This analysis also provides glimpses about putative functions of IKK subunits that may be NF-κB-independent.
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Predisposición Genética a la Enfermedad/genética , Quinasa I-kappa B/genética , Mutación , FN-kappa B/genética , Animales , Humanos , Quinasa I-kappa B/metabolismo , Inflamación/genética , Inflamación/metabolismo , Modelos Genéticos , FN-kappa B/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/metabolismoRESUMEN
CYLD is a tumour suppressor gene mutated in familial cylindromatosis, a genetic disorder leading to the development of skin appendage tumours. It encodes a deubiquitinating enzyme that removes Lys63- or linear-linked ubiquitin chains. CYLD was shown to regulate cell proliferation, cell survival and inflammatory responses, through various signalling pathways. Here we show that CYLD localizes at centrosomes and basal bodies via interaction with the centrosomal protein CAP350 and demonstrate that CYLD must be both at the centrosome and catalytically active to promote ciliogenesis independently of NF-κB. In transgenic mice engineered to mimic the smallest truncation found in cylindromatosis patients, CYLD interaction with CAP350 is lost disrupting CYLD centrosome localization, which results in cilia formation defects due to impairment of basal body migration and docking. These results point to an undiscovered regulation of ciliogenesis by Lys63 ubiquitination and provide new perspectives regarding CYLD function that should be considered in the context of cylindromatosis.
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Cuerpos Basales/fisiología , Comunicación Celular/fisiología , Centrosoma/fisiología , Cilios/fisiología , Cisteína Endopeptidasas/fisiología , Células Epiteliales/fisiología , Animales , Células Cultivadas , Cisteína Endopeptidasas/genética , Proteínas del Citoesqueleto/fisiología , Enzima Desubiquitinante CYLD , Células Epiteliales/citología , Femenino , Humanos , Riñón/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microtúbulos/fisiología , FN-kappa B/fisiología , Proteínas Nucleares/fisiología , Retina/citología , Transducción de Señal/fisiologíaRESUMEN
A molecular feature of Sézary syndrome (SS) is the abnormal expression of T-plastin by malignant T cells. Herein, we investigated the molecular mechanisms involved in T-plastin synthesis and the functions of this actin-binding protein, with a special interest in chemoresistance and migration. We confirm the specific expression of T-plastin in peripheral blood lymphocytes (PBLs) from SS patients and its total absence in PBLs from patients with mycosis fungoides, inflammatory cutaneous or hematologic diseases, and from healthy volunteers. Only 3 of 4 SS patients did constitutively express T-plastin. To assess whether T-plastin expression was inducible, T-plastin-negative PBLs were stimulated by phorbol 12-myristate 13-acetate and ionomycin. Our results demonstrate that T-plastin synthesis was induced in negative PBLs from SS patients, other studied patients, and healthy volunteers. Both constitutive and calcium-induced T-plastin expression was down-regulated by calcineurin inhibitors and involved nuclear factor of activated T cells transcription pathway. Constitutive T-plastin expression in SS was associated with resistance to etoposide-induced apoptosis and cell migration toward chemokines (TARC/CCL17, IP-10). In conclusion, T-plastin is a marker restricted to malignant lymphocytes from SS patients and plays a role for cell survival and migration. This opens new strategies for the treatment of SS advanced stages.
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Linfoma de Células T/fisiopatología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Síndrome de Sézary/fisiopatología , Neoplasias Cutáneas/fisiopatología , Anciano , Anciano de 80 o más Años , Apoptosis/fisiología , Biomarcadores/metabolismo , Calcineurina/metabolismo , Calcio/metabolismo , Línea Celular Tumoral , Movimiento Celular/fisiología , Supervivencia Celular/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Células Jurkat , Linfoma de Células T/genética , Linfoma de Células T/patología , Masculino , Persona de Mediana Edad , Factores de Transcripción NFATC/metabolismo , Síndrome de Sézary/genética , Síndrome de Sézary/patología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Activación Transcripcional/fisiologíaRESUMEN
CYLD deubiquitinase has been originally defined as a tumor suppressor based exclusively on genetic findings. Indeed, inactivation of CYLD in humans results in familial cylindromatosis and multiple trichoepithelioma, two pathologies characterized by the development of tumors originating specifically from the skin appendages. A set of recent publications has revealed that recurrent inactivation of CYLD occurs through diverse mechanisms in several forms of cancer, unequivocally confirming its tumor suppressor function. This property is associated with the critical role played by CYLD in negatively regulating several signaling pathway, among them the NF-κB signaling pathway.
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Transformación Celular Neoplásica , Neoplasias/enzimología , Transducción de Señal/fisiología , Proteínas Supresoras de Tumor/fisiología , Transformación Celular Neoplásica/genética , Enzima Desubiquitinante CYLD , Regulación de la Expresión Génica/fisiología , Genes Supresores de Tumor , Humanos , Modelos Biológicos , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patología , Síndromes Neoplásicos Hereditarios/enzimología , Síndromes Neoplásicos Hereditarios/genética , Procesamiento Proteico-Postraduccional/fisiología , Transducción de Señal/genética , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/genética , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Ubiquitinación/fisiologíaRESUMEN
The IKK kinase complex is the core element of the NF-κB cascade. It is essentially made of two kinases (IKKα and IKKß) and a regulatory subunit, NEMO/IKKγ. Additional components may exist, transiently or permanently, but their characterization is still uncertain. In this review, we will focus on the NEMO molecule, and describe the results which have been obtained, and the hypotheses which have been proposed, to explain how NEMO controls the activation of the IKK complex. NEMO is one of the very few non-redundant components of the NF-κB cascade, and the localization of the gene that encodes it on the X chromosome suggests it is likely to be the target of mutations leading to pathologies: this is indeed the case, and we will also present the current status of our knowledge regarding NEMO-associated pathologies.
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Quinasa I-kappa B/fisiología , Displasia Ectodérmica/genética , Humanos , Quinasa I-kappa B/química , Quinasa I-kappa B/genética , Incontinencia Pigmentaria/etiología , Incontinencia Pigmentaria/genética , Procesamiento Proteico-PostraduccionalRESUMEN
NF-kappaB Essential MOdulator (NEMO) has been shown to play a critical role in NF-kappaB activation, as the regulatory subunit of IkappaB kinase. Upon cell stimulation, NEMO can be modified through phosphorylation, sumoylation or ubiquitination. In the latter case, not much is known regarding the exact function of this posttranslational modification. One of the E3 ligase responsible for K63-linked NEMO polyubiquitination is TRAF6, which participates in several signaling pathways controlling immunity, osteoclastogenesis, skin development and brain functions. We previously observed a potentially important interaction between NEMO and TRAF6. In this study, we defined in more detail the domains required for this interaction, uncovering a new binding site for TRAF6 located at the amino-terminus of NEMO and recognized by the coiled-coil domain of TRAF6. This site appears to work in concert with the previously identified NEMO ubiquitin-binding domain which binds polyubiquitinated chains, suggesting a dual mode of TRAF6 recognition. We also showed that E57K mutation of NEMO found in a mild form of the genetic disease incontinentia pigmenti, resulted in impaired TRAF6 binding and IL-1beta signaling. In contrast, activation of NF-kappaB by TNF-alpha was not affected. These data demonstrate that NEMO/TRAF6 interaction has physiological relevance and might represent a new target for therapeutic purposes.
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Quinasa I-kappa B/genética , Incontinencia Pigmentaria/genética , Mutación , Factor 6 Asociado a Receptor de TNF/genética , Sustitución de Aminoácidos , Animales , Sitios de Unión/genética , Western Blotting , Línea Celular , Células Cultivadas , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Quinasa I-kappa B/química , Quinasa I-kappa B/metabolismo , Inmunoprecipitación , Incontinencia Pigmentaria/metabolismo , Incontinencia Pigmentaria/patología , Interleucina-1beta/farmacología , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Noqueados , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Factor 6 Asociado a Receptor de TNF/metabolismoRESUMEN
Ubiquitination has emerged over the years as the most sophisticated way to modify proteins to affect their fate and function. In particular, it has been reported to be instrumental in regulating several steps of the NF-kappaB signalling pathway which controls inflammation, immunity, adhesion and cell survival. Integrating ubiquitination into NF-kappaB activation requires the regulatory subunit of IKK, NEMO, which not only displays affinity for polyubiquitin chains, but is also posttranslationally modified by a complex set of reactions involving ubiquitin. Here, we examine how studies of the NEMO/ubiquitin relationship have provided novel insights into the IKK activation process and have uncovered molecular mechanisms that should represent in the future attractive targets for specifically modulating NF-kappaB function.
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Quinasa I-kappa B/metabolismo , FN-kappa B/metabolismo , Ubiquitina/metabolismo , Ubiquitinación , Secuencia de Aminoácidos , Animales , Activación Enzimática , Humanos , Datos de Secuencia Molecular , Transducción de SeñalRESUMEN
The key role of NEMO, the regulatory subunit of IKK, in the NF-kB activation process is firmly established. A series of recent publications suggests that this protein also participates in the genotoxic response, after modification by sumoylation in the nucleus, and coordinates the activation of both NF-kB and IRFs during viral infection, through its interaction with TANK adaptor and TBK1/IKKepsilon kinases.
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Quinasa I-kappa B/fisiología , Núcleo Celular/fisiología , Citoplasma/fisiología , Humanos , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiología , Subunidades de Proteína/fisiología , Virosis/fisiopatologíaRESUMEN
PURPOSE: A key molecular feature of cutaneous T-cell lymphomas (CTCL) is the constitutive activation of the nuclear factor-kappaB (NF-kappaB) transcription factor. We investigated in vitro the effects on CTCL survival and chemoresistance of a specific inhibition of IkappaB kinase subunit 2 (IKK2). EXPERIMENTAL DESIGN: Selective IKK2 inhibition was carried out by transfection of SeAx and MyLa CTCL lines with an inactive form of IKK2 and by exposing these lines and tumor cells from 10 patients with Sézary syndrome (SS) to AS602868, a new IKK2 inhibitor. The constitutive nuclear translocation of NF-kappaB was analyzed by electrophoretic mobility shift assay and confocal microscopy. Apoptosis was determined by Annexin V/propidium iodide-positive staining and mitochondrial transmembrane potential alterations as well as poly(ADP-ribose)polymerase cleavage. The expression of Bcl-2 family oncoproteins and survivin was studied by immunoblotting. RESULTS: Specific IKK2 inhibition resulting from transfection or from incubation with AS602868 allowed a down-regulation of NF-kappaB transcriptional activity. As shown by electrophoretic mobility shift assay and apoptosis assays, AS602868 down-regulated the nuclear translocation of NF-kappaB and induced a potent apoptotic response in CTCL lines and in tumor cells from patients with SS while preserving the viability of both peripheral blood lymphocytes from healthy donors and of nonmalignant T cells from SS patients. Moreover, CTCL death induction by conventional antineoplastic agents etoposide and vincristine was potentiated by AS602868. Finally, AS602868-induced apoptosis of CTCL cells was associated with an up-regulation of Bax dimers and a decrease of survivin. CONCLUSION: These results indicate that IKK2 inhibition represents a promising strategy for the treatment of advanced stages of CTCL.
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Antineoplásicos/uso terapéutico , Quinasa I-kappa B/antagonistas & inhibidores , Linfoma Cutáneo de Células T/enzimología , FN-kappa B/metabolismo , Subunidades de Proteína/antagonistas & inhibidores , Neoplasias Cutáneas/enzimología , Anciano , Apoptosis/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Humanos , Linfoma Cutáneo de Células T/patología , Persona de Mediana Edad , FN-kappa B/genética , Pirimidinas/uso terapéutico , Pirimidinas/toxicidad , Valores de Referencia , Síndrome de Sézary/enzimología , Neoplasias Cutáneas/patología , Células Tumorales CultivadasRESUMEN
The regulatory subunit NEMO is involved in the mechanism of activation of IkappaB kinase (IKK), the kinase complex that controls the NF-kappaB signaling pathway. During this process, NEMO is modified post-translationally through K63-linked polyubiquitination. We report the molecular characterization of a new missense mutation of NEMO (A323P) which causes a severe form of incontinentia pigmenti (OMIM#308300), an inherited disease characterized predominantly by skin inflammation. The A323P mutation was found to impair TNF-, IL-1-, LPS- and PMA/ionomycin-induced NF-kappaB activation, as well as to disrupt TRAF6-dependent NEMO polyubiquitination, due to a defective NEMO/TRAF6 interaction. Mutagenesis identified the affected ubiquitination sites as three lysine residues located in the vicinity of A323. Unexpectedly, these lysines were ubiquitinated together with two previously identified lysines not connected to TRAF6. Mutation of all these ubiquitination sites severely impaired NF-kappaB activation induced by stimulation with IL-1, LPS, Nod2/RICK or serum/LPA. In contrast, mutation at all of these sites had only a limited effect on stimulation by TNF. These findings indicate that post-translational modification of NEMO through K63-linked polyubiquitination is a key event in IKK activation and that perturbation of this step may cause human pathophysiology.