RESUMEN
Accurate genome replication is essential for all life and a key mechanism of disease prevention, underpinned by the ability of cells to respond to replicative stress (RS) and protect replication forks. These responses rely on the formation of Replication Protein A (RPA)-single stranded (ss) DNA complexes, yet this process remains largely uncharacterized. Here, we establish that actin nucleation-promoting factors (NPFs) associate with replication forks, promote efficient DNA replication and facilitate association of RPA with ssDNA at sites of RS. Accordingly, their loss leads to deprotection of ssDNA at perturbed forks, impaired ATR activation, global replication defects and fork collapse. Supplying an excess of RPA restores RPA foci formation and fork protection, suggesting a chaperoning role for actin nucleators (ANs) (i.e. Arp2/3, DIAPH1) and NPFs (i.e, WASp, N-WASp) in regulating RPA availability upon RS. We also discover that ß-actin interacts with RPA directly in vitro, and in vivo a hyper-depolymerizing ß-actin mutant displays a heightened association with RPA and the same dysfunctional replication phenotypes as loss of ANs/NPFs, which contrasts with the phenotype of a hyper-polymerizing ß-actin mutant. Thus, we identify components of actin polymerization pathways that are essential for preventing ectopic nucleolytic degradation of perturbed forks by modulating RPA activity.
Asunto(s)
Actinas , Replicación del ADN , Actinas/genética , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , ADN de Cadena Simple/genética , Chaperonas Moleculares/genéticaRESUMEN
BACKGROUND: Wiskott-Aldrich syndrome (WAS) is an X-linked primary immune deficiency disorder resulting from Wiskott-Aldrich syndrome protein (WASp) deficiency. Lymphocytes from patients with WAS manifest increased DNA damage and lymphopenia from cell death, yet how WASp influences DNA damage-linked cell survival is unknown. A recently described mechanism promoting cell survival after ionizing radiation (IR)-induced DNA damage involves fragmentation and dispersal of the Golgi apparatus, known as the Golgi-dispersal response (GDR), which uses the Golgi phosphoprotein 3 (GOLPH3)-DNA-dependent protein kinase (DNA-PK)-myosin XVIIIA-F-actin signaling pathway. OBJECTIVE: We sought to define WASp's role in the DNA damage-induced GDR and its disruption as a contributor to the development of radiosensitivity-linked immunodeficiency in patients with WAS. METHODS: In human TH and B-cell culture systems, DNA damage-induced GDR elicited by IR or radiomimetic chemotherapy was monitored in the presence or absence of WASp or GOLPH3 alone or both together. RESULTS: WASp deficiency completely prevents the development of IR-induced GDR in human TH and B cells, despite the high DNA damage load. Loss of WASp impedes nuclear translocation of GOLPH3 and its colocalization with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Surprisingly, however, depletion of GOLPH3 alone or depolymerization of F-actin in WASp-sufficient TH cells still allows development of robust GDR, suggesting that WASp, but not GOLPH3, is essential for GDR and cell survival after IR-induced DNA-damage in human lymphocytes. CONCLUSION: The study identifies WASp as a novel effector of the nucleus-to-Golgi cell-survival pathway triggered by IR-induced DNA damage in cells of the hematolymphoid lineage and proposes an impaired GDR as a new cause for development of a "radiosensitive" form of immune dysregulation in patients with WAS.
Asunto(s)
Linfocitos B/inmunología , Daño del ADN/inmunología , Aparato de Golgi/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Familia de Proteínas del Síndrome de Wiskott-Aldrich/inmunología , Daño del ADN/genética , Proteína Quinasa Activada por ADN/genética , Proteína Quinasa Activada por ADN/inmunología , Aparato de Golgi/genética , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/inmunología , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
BACKGROUND: Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT), and X-linked neutropenia, which are caused by WAS mutations affecting Wiskott-Aldrich syndrome protein (WASp) expression or activity, manifest in immunodeficiency, autoimmunity, genomic instability, and lymphoid and other cancers. WASp supports filamentous actin formation in the cytoplasm and gene transcription in the nucleus. Although the genetic basis for XLT/WAS has been clarified, the relationships between mutant forms of WASp and the diverse features of these disorders remain ill-defined. OBJECTIVE: We sought to define how dysfunctional gene transcription is causally linked to the degree of TH cell deficiency and genomic instability in the XLT/WAS clinical spectrum. METHODS: In human TH1- or TH2-skewing cell culture systems, cotranscriptional R-loops (RNA/DNA duplex and displaced single-stranded DNA) and DNA double-strand breaks (DSBs) were monitored in multiple samples from patients with XLT and WAS and in normal T cells depleted of WASp. RESULTS: WASp deficiency provokes increased R-loops and R-loop-mediated DSBs in TH1 cells relative to TH2 cells. Mechanistically, chromatin occupancy of serine 2-unphosphorylated RNA polymerase II is increased, and that of topoisomerase 1, an R-loop preventing factor, is decreased at R-loop-enriched regions of IFNG and TBX21 (TH1 genes) in TH1 cells. These aberrations accompany increased unspliced (intron-retained) and decreased spliced mRNA of IFNG and TBX21 but not IL13 (TH2 gene). Significantly, increased cellular load of R-loops and DSBs, which are normalized on RNaseH1-mediated suppression of ectopic R-loops, inversely correlates with disease severity scores. CONCLUSION: Transcriptional R-loop imbalance is a novel molecular defect causative in TH1 immunodeficiency and genomic instability in patients with WAS. The study proposes that cellular R-loop load could be used as a potential biomarker for monitoring symptom severity and prognostic outcome in the XLT-WAS clinical spectrum and could be targeted therapeutically.
Asunto(s)
Inestabilidad Genómica/genética , Células TH1/patología , Síndrome de Wiskott-Aldrich/genética , Células Cultivadas , Daño del ADN/genética , Humanos , Transcripción Genética , Síndrome de Wiskott-Aldrich/patologíaRESUMEN
In Wiskott-Aldrich syndrome (WAS), immunodeficiency and autoimmunity often comanifest, yet how WAS mutations misregulate chromatin-signaling in Thelper (TH) cells favoring development of auto-inflammation over protective immunity is unclear. Previously, we identified an essential promoter-specific, coactivator role of nuclear-WASp in TH1 gene transcription. Here we identify small ubiquitin-related modifier (SUMO)ylation as a novel posttranslational modification of WASp, impairment of which converts nuclear-WASp from a transcriptional coactivator to a corepressor of nuclear factor (NF)-κB response genes in human (TH)1-differentiating cells. V75M, one of many disease-causing mutations occurring in SUMO*motif (72-ψψψψKDxxxxSY-83) of WASp, compromises WASp-SUMOylation, associates with COMMD1 to attenuate NF-κB signaling, and recruits histone deacetylases-6 (HDAC6) to p300-marked promoters of NF-κB response genes that pattern immunity but not inflammation. Consequently, proteins mediating adaptive immunity (IFNG, STAT1, TLR1) are deficient, whereas those mediating auto-inflammation (GM-CSF, TNFAIP2, IL-1ß) are paradoxically increased in TH1 cells expressing SUMOylation-deficient WASp. Moreover, SUMOylation-deficient WASp favors ectopic development of the TH17-like phenotype (↑IL17A, IL21, IL22, IL23R, RORC, and CSF2) under TH1-skewing conditions, suggesting a role for WASp in modulating TH1/TH17 plasticity. Notably, pan-histone deacetylase inhibitors lift promoter-specific repression imposed by SUMOylation-deficient WASp and restore misregulated gene expression. Our findings uncovering a SUMOylation-based mechanism controlling WASp's dichotomous roles in transcription may have implications for personalized therapy for patients carrying mutations that perturb WASp-SUMOylation.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Mutación , FN-kappa B/inmunología , Células TH1/inmunología , Activación Transcripcional/fisiología , Proteína del Síndrome de Wiskott-Aldrich/genética , Inmunidad Adaptativa/genética , Inmunidad Adaptativa/inmunología , Western Blotting , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Citometría de Flujo , Humanos , Inmunoprecipitación , Espectrometría de Masas , Mutagénesis Sitio-Dirigida , FN-kappa B/genética , Reacción en Cadena de la Polimerasa , Transducción de Señal/genética , Transducción de Señal/inmunología , Sumoilación , TransfecciónRESUMEN
Wiskott-Aldrich syndrome (WAS), an immunodeficiency disorder, and X-linked thrombocytopenia (XLT), a bleeding disorder, both arise from nonsynonymous mutations in WAS, which encodes a hematopoietic-specific WASp. Intriguingly, XLT evolves into WAS in some patients but not in others; yet the biological basis for this cross-phenotype (CP) effect remains unclear. Using human T-helper (TH) cells expressing different disease-causing WAS mutations, we demonstrated that hSWI/SNF-like complexes require nuclear-WASp to execute their chromatin-remodeling activity at promoters of WASp-target, immune function genes during TH1 differentiation. Hot-spot WAS mutations Thr45Met and Arg86Cys, which result in XLT-to-WAS disease progression, impair recruitment of hBRM- but not BRG1-enriched BAF complexes to IFNG and TBX21 promoters. Moreover, promoter enrichment of histone H2A.Z and its catalyzing enzyme EP400 are both impaired. Consequently, activation of Notch signaling, a hBRM-regulated event, and its downstream effector NF-κB are both compromised, along with decreased accessibility of nucleosomal DNA and inefficient transcription-elongation of WASp-target TH1 genes. In contrast, patient mutations Ala236Gly and Arg477Lys that manifest in XLT without progressing to WAS do not disrupt chromatin remodeling or transcriptional reprogramming of TH1 genes. Our study defines an indispensable relationship between nuclear-WASp- and hSWI/SNF-complexes in gene activation and reveals molecular distinctions in TH cells that might contribute to disease severity in the XLT/WAS clinical spectrum.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/diagnóstico , Linfocitos T/metabolismo , Trombocitopenia/diagnóstico , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/diagnóstico , Núcleo Celular/genética , Células Cultivadas , Diagnóstico Diferencial , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Humanos , Mutación , Regiones Promotoras Genéticas , Células TH1/metabolismo , Trombocitopenia/genética , Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Defects in Wiskott-Aldrich Syndrome protein (WASp) underlie development of WAS, an X-linked immunodeficiency and autoimmunity disorder of childhood. Nucleation-promoting factors (NPFs) of the WASp family generate F-actin in the cytosol via the VCA (verprolin-homology, cofilin-homology, and acidic) domain and support RNA polymerase II-dependent transcription in the nucleus. Whether nuclear-WASp requires the integration of its actin-related protein (ARP)2/3-dependent cytoplasmic function to reprogram gene transcription, however, remains unresolved. Using the model of human TH cell differentiation, we find that WASp has a functional nuclear localizing and nuclear exit sequences, and accordingly, its effects on transcription are controlled mainly at the level of its nuclear entry and exit via the nuclear pore. Human WASp does not use its VCA-dependent, ARP2/3-driven, cytoplasmic effector mechanisms to support histone H3K4 methyltransferase activity in the nucleus of TH1-skewed cells. Accordingly, an isolated deficiency of nuclear-WASp is sufficient to impair the transcriptional reprogramming of TBX21 and IFNG promoters in TH1-skewed cells, whereas an isolated deficiency of cytosolic-WASp does not impair this process. In contrast, nuclear presence of WASp in TH2-skewed cells is small, and its loss does not impair transcriptional reprogramming of GATA3 and IL4 promoters. Our study unveils an ARP2/3:VCA-independent function of nuclear-WASp in TH1 gene activation that is uncoupled from its cytoplasmic role in actin polymerization.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/inmunología , Actinas/inmunología , Núcleo Celular/inmunología , Células TH1/inmunología , Células Th2/inmunología , Transcripción Genética/inmunología , Proteína del Síndrome de Wiskott-Aldrich/inmunología , Complejo 2-3 Proteico Relacionado con la Actina/genética , Actinas/genética , Núcleo Celular/genética , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/inmunología , Células HeLa , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/inmunología , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-4/genética , Interleucina-4/inmunología , Células Jurkat , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/inmunología , Transcripción Genética/genética , Proteína del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
Congenital dyserythropoietic anemia (CDA) type-1 is a rare genetic disorder of ineffective erythropoiesis, which manifests in macrocytic anemia. We report a CDA1 patient who as a newborn presented with macrocytic anemia and persistent pulmonary hypertension of the newborn (PPHN) requiring mechanical ventilation. Post-infancy, the patient developed acral dysmorphism and pectus excavatum the latter rarely found in CDA1. Patient is a compound heterozygote for a known maternal-derived missense-mutation (c.1796A > G/p.Asn589Ser) and a novel paternal-derived deletion-mutation (c.1104_1106del/Phe365del) in CDAN1. This report highlights the importance of recognizing PPHN as a presenting symptom of CDA1 and expands the repertoire of the accompanying mutations and axial skeletal malformations.
Asunto(s)
Anemia Diseritropoyética Congénita , Glicoproteínas/genética , Hipertensión Pulmonar , Mutación Missense , Tórax/anomalías , Sustitución de Aminoácidos , Anemia Diseritropoyética Congénita/complicaciones , Anemia Diseritropoyética Congénita/genética , Anemia Diseritropoyética Congénita/patología , Humanos , Hipertensión Pulmonar/complicaciones , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Recién Nacido , Masculino , Proteínas NuclearesRESUMEN
Accurate genome replication is essential for all life and a key mechanism of disease prevention, underpinned by the ability of cells to respond to replicative stress (RS) and protect replication forks. These responses rely on the formation of Replication Protein A (RPA)-single stranded (ss) DNA complexes, yet this process remains largely uncharacterized. Here we establish that actin nucleation-promoting factors (NPFs) associate with replication forks, promote efficient DNA replication and facilitate association of RPA with ssDNA at sites of RS. Accordingly, their loss leads to deprotection of ssDNA at perturbed forks, impaired ATR activation, global replication defects and fork collapse. Supplying an excess of RPA restores RPA foci formation and fork protection, suggesting a chaperoning role for actin nucleators (ANs) (i.e., Arp2/3, DIAPH1) and NPFs (i.e, WASp, N-WASp) in regulating RPA availability upon RS. We also discover that ß-actin interacts with RPA directly in vitro , and in vivo a hyper-depolymerizing ß-actin mutant displays a heightened association with RPA and the same dysfunctional replication phenotypes as loss of ANs/NPFs, which contrasts with the phenotype of a hyper-polymerizing ß-actin mutant. Thus, we identify components of actin polymerization pathways that are essential for preventing ectopic nucleolytic degradation of perturbed forks by modulating RPA activity.
RESUMEN
Perturbation in the replication-stress response (RSR) and DNA-damage response (DDR) causes genomic instability. Genomic instability occurs in Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency disorder, yet the mechanism remains largely uncharacterized. Replication protein A (RPA), a single-strand DNA (ssDNA) binding protein, has key roles in the RSR and DDR. Here we show that human WAS-protein (WASp) modulates RPA functions at perturbed replication forks (RFs). Following genotoxic insult, WASp accumulates at RFs, associates with RPA, and promotes RPA:ssDNA complexation. WASp deficiency in human lymphocytes destabilizes RPA:ssDNA-complexes, impairs accumulation of RPA, ATR, ETAA1, and TOPBP1 at genotoxin-perturbed RFs, decreases CHK1 activation, and provokes global RF dysfunction. las17 (yeast WAS-homolog)-deficient S. cerevisiae also show decreased ScRPA accumulation at perturbed RFs, impaired DNA recombination, and increased frequency of DNA double-strand break (DSB)-induced single-strand annealing (SSA). Consequently, WASp (or Las17)-deficient cells show increased frequency of DSBs upon genotoxic insult. Our study reveals an evolutionarily conserved, essential role of WASp in the DNA stress-resolution pathway, such that WASp deficiency provokes RPA dysfunction-coupled genomic instability.
Asunto(s)
Roturas del ADN de Doble Cadena , Replicación del ADN , ADN de Cadena Simple , Proteína de Replicación A , Proteínas de Saccharomyces cerevisiae , Proteína del Síndrome de Wiskott-Aldrich , Animales , Antígenos de Superficie/metabolismo , Reparación del ADN , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Inestabilidad Genómica , Humanos , Unión Proteica , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Immunodeficiency is associated with cancer risk. Accordingly, hematolymphoid cancers develop in Wiskott-Aldrich syndrome (WAS), an X-linked primary immunodeficiency disorder (PID) resulting from the deficiency of WAS-protein (WASp) expressed predominantly in the hematolymphoid cell lineages. Despite the correlation between WASp deficiency and hematolymphoid cancers, the molecular mechanism underlying the oncogenic role of WASp is incompletely understood. Employing the WASp-sufficient and WASp-deficient cell-pair model of human T and B lymphocytes, we show that WASp deficiency differentially influences hyperactivation versus inhibition of both CDC42:ERK1/2 and NF-κB:AP-1 pro-oncogenic signaling pathways in nonmalignant versus malignant T and B lymphocytes. Furthermore, WASp deficiency induces a cell-type specific up/down-modulation of the DNA-binding activities of NF-κB, AP-1, and multiple other transcription factors with known roles in oncogenesis. We propose that WASp functions as a putative "tumor-suppressor" protein in normal T and B cells, and "oncoprotein" in a subset of established T and B cell malignancies that are not associated with the NPM-ALK fusion.
Asunto(s)
Linfocitos B/patología , Proteínas Oncogénicas/metabolismo , Linfocitos T/patología , Proteínas Supresoras de Tumor/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/deficiencia , Síndrome de Wiskott-Aldrich/patología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Proliferación Celular , Células Cultivadas , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factor de Transcripción AP-1/metabolismo , Síndrome de Wiskott-Aldrich/inmunología , Síndrome de Wiskott-Aldrich/metabolismo , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
The clinical symptomatology in the X-linked Wiskott-Aldrich syndrome (WAS), a combined immunodeficiency and autoimmune disease resulting from WAS protein (WASp) deficiency, reflects the underlying coexistence of an impaired T helper 1 (TH1) immunity alongside intact TH2 immunity. This suggests a role for WASp in patterning T(H) subtype immunity, yet the molecular basis for the TH1-TH2 imbalance in human WAS is unknown. We have discovered a nuclear role for WASp in the transcriptional regulation of the TH1 regulator gene TBX21 at the chromatin level. In primary TH1-differentiating cells, a fraction of WASp is found in the nucleus, where it is recruited to the proximal promoter locus of the TBX21 gene, but not to the core promoter of GATA3 (a TH2 regulator gene) or RORc (a TH17 regulator gene). Genome-wide mapping demonstrates association of WASp in vivo with the gene-regulatory network that orchestrates TH1 cell fate choice in the human TH cell genome. Functionally, nuclear WASp associates with H3K4 trimethyltransferase [RBBP5 (retinoblastoma-binding protein 5)] and H3K9/H3K36 tridemethylase [JMJD2A (Jumonji domain-containing protein 2A)] proteins, and their enzymatic activity in vitro and in vivo is required for achieving transcription-permissive chromatin dynamics at the TBX21 proximal promoter in primary differentiating TH1 cells. During TH1 differentiation, the loss of WASp accompanies decreased enrichment of RBBP5 and, in a subset of WAS patients, also of filamentous actin at the TBX21 proximal promoter locus. Accordingly, human WASp-deficient TH cells, from natural mutation or RNA interference-mediated depletion, demonstrate repressed TBX21 promoter dynamics when driven under TH1-differentiating conditions. These chromatin derangements accompany deficient T-BET messenger RNA and protein expression and impaired TH1 function, defects that are ameliorated by reintroducing WASp. Our findings reveal a previously unappreciated role of WASp in the epigenetic control of T-BET transcription and provide a new mechanism for the pathogenesis of WAS by linking aberrant histone methylation at the TBX21 promoter to dysregulated adaptive immunity.
Asunto(s)
Núcleo Celular/metabolismo , Inmunidad/inmunología , Células TH1/inmunología , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/inmunología , Síndrome de Wiskott-Aldrich/fisiopatología , Actinas/metabolismo , Diferenciación Celular , Células Cultivadas , Reprogramación Celular/genética , Cromatina/metabolismo , ADN/metabolismo , Epigénesis Genética , Sitios Genéticos/genética , Genoma Humano/genética , Histonas/metabolismo , Humanos , Metilación , Proteínas Mutantes/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Transporte de Proteínas , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Células TH1/citología , Transcripción Genética , Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/patología , Proteína del Síndrome de Wiskott-Aldrich/deficienciaRESUMEN
Allergic bronchopulmonary aspergillosis (ABPA) is caused by a dominant Th2 immune response to antigens derived from the opportunistic mold Aspergillus, most commonly Aspergillus fumigatus. It occurs in 4%-15% of patients with cystic fibrosis (CF); however, not all patients with CF infected with A. fumigatus develop ABPA. Therefore, we compared cohorts of A. fumigatus-colonized CF patients with and without ABPA to identify factors mediating tolerance versus sensitization. We found that the costimulatory molecule OX40 ligand (OX40L) was critical in driving Th2 responses to A. fumigatus in peripheral CD4+ T cells isolated from patients with ABPA. In contrast, CD4+ T cells from the non-ABPA cohort did not mount enhanced Th2 responses in vitro and contained a higher frequency of TGF-beta-expressing regulatory T cells. Heightened Th2 reactivity in the ABPA cohort correlated with lower mean serum vitamin D levels. Further, in vitro addition of 1,25 OH-vitamin D3 substantially reduced DC expression of OX40L and increased DC expression of TGF-beta. This in vitro treatment also resulted in increased Treg TGF-beta expression and reduced Th2 responses by CD4+ T cells from patients with ABPA. These data provide rationale for a therapeutic trial of vitamin D to prevent or treat ABPA in patients with CF.
Asunto(s)
Aspergilosis Broncopulmonar Alérgica/inmunología , Aspergillus fumigatus/inmunología , Linfocitos T CD4-Positivos/inmunología , Colecalciferol/farmacología , Fibrosis Quística/inmunología , Células Th2/inmunología , Adulto , Aspergillus/inmunología , Femenino , Humanos , Masculino , Receptores OX40/metabolismo , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
Cigarette smoke, a major risk factor in emphysema, causes cell death by incompletely understood mechanisms. Death-inducing signaling complex (DISC) formation is an initial event in Fas-mediated apoptosis. We demonstrate that cigarette smoke extract (CSE) induces DISC formation in human lung fibroblasts (MRC-5) and promotes DISC trafficking from the Golgi complex to membrane lipid rafts. We demonstrate a novel role of protein kinase C (PKC) in the regulation of DISC formation and trafficking. The PKC isoforms, PKCalpha, zeta, epsilon, and eta, were activated by CSE exposure. Overexpression of wild-type PKCalpha inhibited, while PKCzeta promoted, CSE-induced cell death. Dominant-negative (dn)PKCzeta protected against CSE-induced cell death by suppressing DISC formation and caspase-3 activation, while dnPKCalpha enhanced cell death by promoting these events. DISC formation was augmented by wortmannin, an inhibitor of PI3K. CSE-induced Akt phosphorylation was reduced by dnPKCalpha, but it was increased by dnPKCzeta. Expression of PKCalpha in vivo inhibited DISC formation, caspase-3/8 activation, lung injury, and cell death after prolonged cigarette smoke exposure, whereas expression of PKCzeta promoted caspase-3 activation. In conclusion, CSE-induced DISC formation is differentially regulated by PKCalpha and PKCzeta via the PI3K/Akt pathway. These results suggest that modulation of PKC may have therapeutic potential in the prevention of smoke-related lung injury.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Nicotiana/química , Proteína Quinasa C-alfa/metabolismo , Proteína Quinasa C/metabolismo , Contaminación por Humo de Tabaco , Animales , Caspasas/metabolismo , Línea Celular , Activación Enzimática/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfoserina/metabolismo , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Direct T cell allorecognition underlies the development of a vigorous immune response in the clinical setting of acute graft rejection. The Notch pathway is an important regulator of Th immune responses, yet the molecular underpinnings of directional Notch signaling, otherwise critical for binary cell fate decisions, are unknown during autologous or allogeneic Th:DC interactions. Using the development of immune synapses (IS) in the allogeneic, human physiological Th:DC interaction, we demonstrate that Th-Notch1 receptor and DC-Notch ligands (Delta-like1, Jagged1) cluster in their apposed central-supramolecular-activation-clusters (cSMAC), whereas DC-Notch1 receptor and Th-Notch ligands cluster in their apposed peripheral-SMAC (pSMAC). Numb, a negative regulator of Notch, is excluded from the IS-microdomains where Notch1 receptor accumulates. This antiparallel arrangement across the partnering halves of the IS supports reciprocal Notch signal propagation in the DC-to-Th direction via the cSMAC and Th-to-DC direction via the pSMAC. As a result, processed Notch1 receptor (Notch-intracellular-domain, NICD1) and its ligands, as well as their downstream targets, HES-1 and phosphorylated-STAT3, accumulate in the nuclei of both cell-types. There is also enhancement of GLUT1 expression in both cell-types, as well as increased production of Th-IFN-gamma. Significantly, neutralizing Notch1R Ab inhibits NICD1 and HES-1 nuclear translocation, and production of IFN-gamma. In contrast, the IS formed during Ag-nonspecific, autologous Th:DC interaction is immature, resulting in failure of Notch1 receptor segregation and subsequent nuclear translocation of NICD1. Our results provide the first evidence for the asymmetric recruitment of Notch components in the Th:DC immunological synapse, which regulates the bidirectional Notch signal propagation.
Asunto(s)
Células Dendríticas/inmunología , Activación de Linfocitos/inmunología , Receptores Notch/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Presentación de Antígeno , Comunicación Celular/inmunología , Células Dendríticas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Humanos , Imagenología Tridimensional , Receptores Notch/metabolismo , Linfocitos T/metabolismo , Trasplante HomólogoRESUMEN
Statins, best known for their lipid-lowering actions, also possess immunomodulatory properties. Recent studies have shown a Th2-biasing effect of statins, although the underlying mechanism has not been identified. In this study, we investigated whether simvastatin can exercise a Th2-promoting effect through modulation of function of dendritic cells (DCs) without direct interaction with CD4+ T cells. Exposure of DCs to simvastatin induced the differentiation of a distinct subset of DCs characterized by a high expression of B220. These simvastatin-conditioned DCs up-regulated GATA-3 expression and down-regulated T-bet expression in cocultured CD4+ T cells in the absence of additional simvastatin added to the coculture. The Th2-biased transcription factor profile induced by simvastatin-treated DCs also was accompanied by increased Th2 (IL-4, IL-5, and IL-13) and decreased Th1 (IFN-gamma) cytokine secretion from the T cells. The Th2-promoting effect of simvastatin was found to depend on the chitinase family member Ym1, known to be a lectin. Anti-Ym1 antibody abolished the Th2-promoting effect of simvastatin-treated DCs. Also, simvastatin was unable to augment Ym1 expression in DCs developed from STAT6-/- or IL-4R alpha-/- mice. Thus, modulation of Ym1 production by DCs identifies a previously undescribed mechanism of Th2 polarization by statin.
Asunto(s)
Células Dendríticas/inmunología , Hipolipemiantes/farmacología , Lectinas/metabolismo , Simvastatina/farmacología , Células Th2 , beta-N-Acetilhexosaminidasas/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Forma de la Célula , Células Cultivadas , Técnicas de Cocultivo , Células Dendríticas/citología , Factor de Transcripción GATA3/metabolismo , Humanos , Interferón gamma/metabolismo , Lectinas/genética , Antígenos Comunes de Leucocito/metabolismo , Subgrupos Linfocitarios , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Fenotipo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Factor de Transcripción STAT6/genética , Factor de Transcripción STAT6/metabolismo , Células TH1/efectos de los fármacos , Células TH1/metabolismo , Células Th2/efectos de los fármacos , Células Th2/metabolismo , beta-N-Acetilhexosaminidasas/genéticaRESUMEN
Effector functions mediated by NK cells involve cytotoxicity and transcription-dependent production and release of cytokines and chemokines. Although the JAK/STAT pathway mediates lymphokine-induced transcriptional regulation in NK cells, very little is known about transcriptional regulation induced during cell-cell contact. We demonstrate that the Wiskott-Aldrich syndrome protein (WASp) is an important component for integration of signals leading to nuclear translocation of NFAT2 and NF-kappaB (RelA) during cell-cell contact and NKp46-dependent signaling. This WASp function is independent of its known role in F-actin polymerization and cytoskeletal rearrangement. Absence of WASp results in decreased accumulation of calcineurin, WASp-interacting protein, and molecules upstream of calcium mobilization, i.e., activated ZAP70 and phospholipase C-gamma1, in the disorganized NK cell immune synapse. Production of GM-CSF, but not IFN-gamma, is decreased, while natural cytotoxicity of Wiskott-Aldrich syndrome-NK cells is maintained. Our results indicate that WASp independently regulates its dual functions, i.e., actin cytoskeletal remodeling and transcription in NK cells.
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Actinas/metabolismo , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , FN-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Proteínas/fisiología , Factores de Transcripción/metabolismo , Síndrome de Wiskott-Aldrich/inmunología , Síndrome de Wiskott-Aldrich/metabolismo , Transporte Activo de Núcleo Celular/genética , Comunicación Celular/genética , Comunicación Celular/inmunología , Línea Celular Transformada , Células Clonales , Citotoxicidad Inmunológica/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Humanos , Interferón gamma/biosíntesis , Células Asesinas Naturales/enzimología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Activación de Linfocitos/genética , Glicoproteínas de Membrana/fisiología , Microdominios de Membrana/enzimología , Microdominios de Membrana/inmunología , Microdominios de Membrana/metabolismo , Factores de Transcripción NFATC , Receptor 1 Gatillante de la Citotoxidad Natural , Fosfolipasa C gamma , Proteínas/genética , Receptores Inmunológicos/fisiología , Transducción de Señal/genética , Transducción de Señal/inmunología , Factor de Transcripción ReIA , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismo , Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-AldrichRESUMEN
BACKGROUND: Overall survival after recurrence of osteosarcoma (OS) is < 30%. The authors reported their experience treating recurrent OS at the time of first recurrence (R1). METHODS: Patients with high-grade OS who achieved complete disease remission (CR) after primary surgery and chemotherapy, and patients who were treated at R1 at Memorial Sloan-Kettering Cancer Center (New York, NY) after 1990 were analyzed by retrospective chart review. RESULTS: For 43 eligible patients, the median time to R1 from initial diagnosis was 21.7 months (range, 4.6-135.7 mos). The lungs were the most common sites of disease recurrence (n = 33 of 43). With a median follow-up of 15.2 months (range, 0.7-158.3 mos) after R1, 15 of 43 (35%) patients were alive. Four of 43 patients were treated with surgery alone (3 patients were alive and 1 had died of progressive disease at the time of last follow-up). Due to unresectable disease, eight patients received only chemotherapy, none of whom survived. For patients with disease recurrence treated with chemotherapy and surgery (n = 31), 22 patients achieved a second CR (CR2). Nine patients were alive and in disease remission (29%) at the time of last follow-up. Twenty-three patients received ifosfamide as part of their retrieval regimen. Of the 18 who achieved a CR2, 8 experienced disease recurrence, 7 remain alive in CR2, and 3 died due to toxicity. Eight patients did not receive ifosfamide. Of these, 4 achieved a CR2 but 3 subsequently experienced disease recurrence. CONCLUSIONS: At R1, 22 of 31 patients achieved a CR2 with aggressive surgery and chemotherapy. The majority of these patients subsequently developed a disease recurrence. Patients appeared to benefit from the addition of ifosfamide to their retrieval regimens. In the end, the role of chemotherapy in recurrent OS continues to remain undefined.
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Neoplasias Óseas/terapia , Recurrencia Local de Neoplasia/terapia , Osteosarcoma/terapia , Adolescente , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Óseas/mortalidad , Niño , Preescolar , Femenino , Humanos , Masculino , Recurrencia Local de Neoplasia/mortalidad , Procedimientos Ortopédicos , Osteosarcoma/mortalidad , Estudios Retrospectivos , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
Inhibitory NK receptors with ligand specificity for MHC class I recruit Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) phosphatase and prevent autocytotoxicity. Activation of SHP-1 depends upon Src kinase-mediated tyrosine phosphorylation of the cytoplasmic domain of the inhibitory receptor. In this study we demonstrate, by quantitative temporal analysis, that talin, Lck, and SHP-1 are recruited to the synapse within 1 min in both cytolytic and noncytolytic conjugates. Polarization of talin and Lck rapidly disappears in the noncytolytic interactions but persists in cytolytic interactions, where protein kinase C-theta;, Src homology 2 domain-containing leukocyte protein of 76 kDa, and lysosomes are recruited within 5 min. At 1 min SHP-1 clusters in the periphery of the cytolytic synapse, whereas it clusters in the center of the noncytolytic synapse. Lck has multifocal distribution in both synapses consistent with the shared requirement for early tyrosine phosphorylation. Our studies indicate that the spatial location of SHP-1 in the synapse distinguishes noncytolytic from cytolytic interactions within the first minute.
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Comunicación Celular/inmunología , Citotoxicidad Inmunológica , Células Asesinas Naturales/enzimología , Células Asesinas Naturales/inmunología , Proteínas Tirosina Fosfatasas/metabolismo , Dominios Homologos src/inmunología , Proteínas Adaptadoras Transductoras de Señales , Línea Celular Transformada , Células Clonales , Pruebas Inmunológicas de Citotoxicidad , Humanos , Péptidos y Proteínas de Señalización Intracelular , Isoenzimas/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Lisosomas/enzimología , Lisosomas/inmunología , Fosfoproteínas/metabolismo , Proteína Quinasa C/metabolismo , Proteína Quinasa C-theta , Proteína Fosfatasa 1 , Transporte de Proteínas/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Transducción de Señal/inmunología , Talina/metabolismoRESUMEN
Recent applications of imaging approaches and other methods of cell biology have provided high-resolution visualization of the location of fluorescent proteins in living and fixed cells during cell-cell interactions between lymphocytes, antigen presenting cells and target cells. We review the composition and dynamics of molecular and cytoskeletal events occurring during natural killer cell interactions with susceptible and nonsusceptible target cells. The natural killer cell immune synapse and the concomitant changes in cytoskeletal components and cytoplasmic organelles are described. The findings are compared with the observations made in T helper cells and cytotoxic T cells. It is concluded that the cytolytic immune synapses display spatial-temporal dynamics that are accelerated as compared with T helper cells. In addition, the cytolytic conjugates have unique characteristics relating to their effector function. Furthermore, the natural killer cell immune synapses in cytolytic and noncytolytic interactions are distinctly different and display patterns consistent with characteristic signaling pathways identified in biochemical studies of disrupted cells. The precise relationship between different stages of the natural killer cell immune synapse formation and progression in signal transduction pathways is yet to be established.
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Uniones Intercelulares/inmunología , Células Asesinas Naturales/inmunología , Actinas/inmunología , Animales , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Citoesqueleto/inmunología , Citotoxicidad Inmunológica , Humanos , Células Asesinas Naturales/ultraestructura , Ratones , Microscopía Electrónica , Centro Organizador de los Microtúbulos/inmunología , Modelos Inmunológicos , Receptores Inmunológicos/metabolismo , Transducción de Señal/inmunologíaRESUMEN
Interaction of NK cells with target cells leads to formation of an immunological synapse (IS) at the contact site. NK cells form two distinctly different IS, the inhibitory NK cell IS (NKIS) and the cytolytic NKIS. Cognate ligand binding is sufficient to induce clustering of inhibitory killer cell Ig-like receptors (KIR) and phosphorylation of both the receptor and the phosphatase Src homology domain 2-containing protein tyrosine phosphatase 1 (SHP-1). Recruitment and activation of SHP-1 by a signaling competent inhibitory receptor are essential early events for NK cell inhibition. We have in the present study used three-dimensional immunofluorescence microscopy to analyze distribution of inhibitory KIR, SHP-1, LFA-1, and lipid rafts within the NKIS during cytolytic and noncytolytic interactions. NK clones retrovirally transduced with the inhibitory KIR2DL3 gene fused to GFP demonstrate colocalization of KIR2DL3 with SHP-1 in the center of early inhibitory NKIS. Ligand binding translocates the receptor to the center of the IS where activation signals are accumulating and provides a docking site for SHP-1. SHP-1 and rafts cluster in the center of early inhibitory NKIS and late cytolytic NKIS, and whereas rafts continue to increase in size in cytolytic conjugates, they are rapidly dissolved in inhibitory conjugates. Furthermore, rafts are essential only for cytolytic, not for inhibitory, outcome. These results indicate that the outcome of NK cell-target cell interactions is dictated by early quantitative differences in cumulative activating and inhibitory signals.