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2.
Blood ; 142(12): 1056-1070, 2023 09 21.
Artículo en Inglés | MEDLINE | ID: mdl-37339579

RESUMEN

TP 53-mutant acute myeloid leukemia (AML) remains the ultimate therapeutic challenge. Epichaperomes, formed in malignant cells, consist of heat shock protein 90 (HSP90) and associated proteins that support the maturation, activity, and stability of oncogenic kinases and transcription factors including mutant p53. High-throughput drug screening identified HSP90 inhibitors as top hits in isogenic TP53-wild-type (WT) and -mutant AML cells. We detected epichaperomes in AML cells and stem/progenitor cells with TP53 mutations but not in healthy bone marrow (BM) cells. Hence, we investigated the therapeutic potential of specifically targeting epichaperomes with PU-H71 in TP53-mutant AML based on its preferred binding to HSP90 within epichaperomes. PU-H71 effectively suppressed cell intrinsic stress responses and killed AML cells, primarily by inducing apoptosis; targeted TP53-mutant stem/progenitor cells; and prolonged survival of TP53-mutant AML xenograft and patient-derived xenograft models, but it had minimal effects on healthy human BM CD34+ cells or on murine hematopoiesis. PU-H71 decreased MCL-1 and multiple signal proteins, increased proapoptotic Bcl-2-like protein 11 levels, and synergized with BCL-2 inhibitor venetoclax in TP53-mutant AML. Notably, PU-H71 effectively killed TP53-WT and -mutant cells in isogenic TP53-WT/TP53-R248W Molm13 cell mixtures, whereas MDM2 or BCL-2 inhibition only reduced TP53-WT but favored the outgrowth of TP53-mutant cells. Venetoclax enhanced the killing of both TP53-WT and -mutant cells by PU-H71 in a xenograft model. Our data suggest that epichaperome function is essential for TP53-mutant AML growth and survival and that its inhibition targets mutant AML and stem/progenitor cells, enhances venetoclax activity, and prevents the outgrowth of venetoclax-resistant TP53-mutant AML clones. These concepts warrant clinical evaluation.


Asunto(s)
Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Animales , Ratones , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Apoptosis , Células Madre/metabolismo , Línea Celular Tumoral
3.
Front Immunol ; 13: 1033483, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36389785

RESUMEN

Lung cancer is currently the leading cause of cancer-related deaths worldwide. Significant improvements in lung cancer therapeutics have relied on a better understanding of lung cancer immunity and the development of novel immunotherapies, as best exemplified by the introduction of PD-1/PD-L1-based therapies. However, this improvement is limited to lung cancer patients who respond to anti-PD-1 immunotherapy. Further improvements in immunotherapy may benefit from a better understanding of innate immune response mechanisms in the lung. Toll-like receptors (TLRs) are a key component of the innate immune response and mediate the early recognition of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). TLR signaling modulates the tumor microenvironment from "cold" to "hot" leading to immune sensitization of tumor cells to treatments and improved patient prognosis. In addition, TLR signaling activates the adaptive immune response to improve the response to cancer immunotherapy through the regulation of anti-tumor T cell activity. This review will highlight recent progress in our understanding of the role of TLRs in lung cancer immunity and immunotherapy.


Asunto(s)
Neoplasias Pulmonares , Receptores Toll-Like , Humanos , Neoplasias Pulmonares/terapia , Inmunidad Innata , Inmunidad Adaptativa , Factores Inmunológicos , Moléculas de Patrón Molecular Asociado a Patógenos , Inmunoterapia , Microambiente Tumoral
4.
Front Pharmacol ; 13: 833380, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36105216

RESUMEN

Allergic asthma is a chronic inflammatory respiratory disease associated with eosinophilic infiltration, increased mucus production, airway hyperresponsiveness, and airway remodeling. Epidemiologic data reveal that the prevalence of allergic sensitization and associated diseases has increased in the twentieth century. This has been hypothesized to be partly due to reduced contact with microbial organisms (the hygiene hypothesis) in industrialized society. Airway epithelial cells, once considered a static physical barrier between the body and the external world, are now widely recognized as immunologically active cells that can initiate, maintain, and restrain inflammatory responses, such as those that mediate allergic disease. Airway epithelial cells can sense allergens via expression of myriad Toll-like receptors (TLRs) and other pattern-recognition receptors. We sought to determine whether the innate immune response stimulated by a combination of Pam2CSK4 ("Pam2", TLR2/6 ligand) and a class C oligodeoxynucleotide ODN362 ("ODN", TLR9 ligand), when delivered together by aerosol ("Pam2ODN"), can modulate the allergic immune response to allergens. Treatment with Pam2ODN 7 days before sensitization to House Dust Mite (HDM) extract resulted in a strong reduction in eosinophilic and lymphocytic inflammation. This Pam2ODN immunomodulatory effect was also seen using Ovalbumin (OVA) and A. oryzae (Ao) mouse models. The immunomodulatory effect was observed as much as 30 days before sensitization to HDM, but ineffective just 2 days after sensitization, suggesting that Pam2ODN immunomodulation lowers the allergic responsiveness of the lung, and reduces the likelihood of inappropriate sensitization to aeroallergens. Furthermore, Pam2 and ODN cooperated synergistically suggesting that this treatment is superior to any single agonist in the setting of allergen immunotherapy.

5.
Stem Cells ; 40(3): 260-272, 2022 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-35296897

RESUMEN

Ten-eleven Translocation (TET) dioxygenases mediated DNA methylation oxidation plays an important role in regulating the embryonic stem cells (ESCs) differentiation. Herein, we utilized a CRISPR/Cas9 based genome editing method to generate single, double, and triple Tet-deficient mouse ESCs (mESCs) and differentiated these cells toward cardiac progenitors. By using emerald green fluorescent protein (GFP; emGFP) expression under the control of Nkx2.5 promoter as marker for cardiac progenitor cells, we discovered that Tet1 and Tet2 depletion significantly impaired mESC-to-cardiac progenitor differentiation. Single-cell RNA-seq analysis further revealed that Tet deletion resulted in the accumulation of mesoderm progenitors to hamper cardiac differentiation. Re-expression of the Tet1 catalytic domain (Tet1CD) rescued the differentiation defect in Tet-triple knockout mESCs. Dead Cas9 (dCas9)-Tet1CD mediated loci-specific epigenome editing at the Hand1 loci validated the direct involvement of Tet-mediated epigenetic modifications in transcriptional regulation during cardiac differentiation. Our study establishes that Tet-mediated epigenetic remodeling is essential for maintaining proper transcriptional outputs to safeguard mESC-to-cardiac progenitor differentiation.


Asunto(s)
Células Madre Embrionarias de Ratones , Proteínas Proto-Oncogénicas , Animales , Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/metabolismo , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo
6.
Cancer Res ; 81(8): 1965-1976, 2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33589517

RESUMEN

Inactivation of tumor-infiltrating lymphocytes (TIL) is one of the mechanisms mitigating antitumor immunity during tumor onset and progression. Epigenetic abnormalities are regarded as a major culprit contributing to the dysfunction of TILs within tumor microenvironments. In this study, we used a murine model of melanoma to discover that Tet2 inactivation significantly enhances the antitumor activity of TILs with an efficacy comparable to immune checkpoint inhibition imposed by anti-PD-L1 treatment. Single-cell RNA-sequencing analysis suggested that Tet2-deficient TILs exhibit effector-like features. Transcriptomic and ATAC-sequencing analysis showed that Tet2 ablation reshapes chromatin accessibility and favors binding of transcription factors geared toward CD8+ T-cell activation. Furthermore, the ETS family of transcription factors contributed to augmented CD8+ T-cell function following Tet2 depletion. Overall, our study establishes that Tet2 constitutes one of the epigenetic barriers that account for dysfunction of TILs and that Tet2 inactivation could promote antitumor immunity to suppress tumor growth. SIGNIFICANCE: This study suggests that ablation of TET2+ from TILs could promote their antitumor function by reshaping chromatin accessibility for key transcription factors and enhancing the transcription of genes essential for antitumor activity.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Proteínas de Unión al ADN/deficiencia , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma Experimental/inmunología , Proteínas Proto-Oncogénicas/deficiencia , Traslado Adoptivo/métodos , Animales , Cromatina/metabolismo , Desmetilación del ADN , Proteínas de Unión al ADN/genética , Dioxigenasas , Modelos Animales de Enfermedad , Epigénesis Genética , Eliminación de Gen , Silenciador del Gen , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Quinasas Quinasa Quinasa PAM , Melanoma Experimental/metabolismo , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/inmunología , Perforina/metabolismo , Proteínas Proto-Oncogénicas/genética , Análisis de Secuencia de ARN , Factores de Transcripción/metabolismo , Microambiente Tumoral/inmunología , Factor de Necrosis Tumoral alfa/metabolismo
7.
Nat Commun ; 11(1): 2135, 2020 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-32358509

RESUMEN

A non-immunogenic tumor microenvironment (TME) is a significant barrier to immune checkpoint blockade (ICB) response. The impact of Polybromo-1 (PBRM1) on TME and response to ICB in renal cell carcinoma (RCC) remains to be resolved. Here we show that PBRM1/Pbrm1 deficiency reduces the binding of brahma-related gene 1 (BRG1) to the IFNγ receptor 2 (Ifngr2) promoter, decreasing STAT1 phosphorylation and the subsequent expression of IFNγ target genes. An analysis of 3 independent patient cohorts and of murine pre-clinical models reveals that PBRM1 loss is associated with a less immunogenic TME and upregulated angiogenesis. Pbrm1 deficient Renca subcutaneous tumors in mice are more resistance to ICB, and a retrospective analysis of the IMmotion150 RCC study also suggests that PBRM1 mutation reduces benefit from ICB. Our study sheds light on the influence of PBRM1 mutations on IFNγ-STAT1 signaling and TME, and can inform additional preclinical and clinical studies in RCC.


Asunto(s)
Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/microbiología , Factores de Transcripción/metabolismo , Animales , Complejo Antígeno-Anticuerpo/genética , Complejo Antígeno-Anticuerpo/metabolismo , Carcinoma de Células Renales/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Femenino , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Neoplasias Renales/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Mutación , Fosforilación , Factor de Transcripción STAT1/metabolismo , Análisis de Matrices Tisulares , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transcriptoma/genética
9.
Ann Biomed Eng ; 47(3): 866-877, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30607644

RESUMEN

Despite advances in the development of materials for cardiovascular devices, current strategies generally lack the thromboresistance of the native endothelium both in terms of efficacy and longevity. To harness this innate hemostatic regulation and improve long-term hemocompatibility, biohybrid devices are designed to promote endothelialization. Much of the research effort to date has focused on the use of extracellular matrix (ECM)-mimics and coatings to promote endothelial cell adhesion and migration with less attention given to the effect of the supported ECM binding events on hemostatic regulation. In this study, we developed integrin-targeted hydrogels to investigate the individual and combined effects of integrin binding events supported by many ECM-based coatings (α1ß1, α2ß1, α5ß1, αvß3). Targeted endothelial cell integrin interactions were first confirmed with antibody blocking studies and then correlated with gene expression of hemostatic regulators and a functional assay of platelet attachment and activation. Surfaces that targeted integrins α1ß1 and α2ß1 resulted in an endothelial cell layer that exhibited a thromboresistant phenotype with an associated reduction in platelet attachment and activation. It is anticipated that identification of specific integrins that promote endothelial cell adhesion as well as thromboresistance will enable the design of cardiovascular materials with improved long-term hemocompatibility.


Asunto(s)
Células Endoteliales de la Vena Umbilical Humana/fisiología , Integrinas/fisiología , Plaquetas/fisiología , Adhesión Celular , Células Cultivadas , Hemostáticos , Humanos , Hidrogeles , Activación Plaquetaria
10.
ACS Synth Biol ; 7(3): 814-821, 2018 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-29489336

RESUMEN

Tools capable of modulating gene expression in living organisms are very useful for interrogating the gene regulatory network and controlling biological processes. The catalytically inactive CRISPR/Cas9 (dCas9), when fused with repressive or activating effectors, functions as a versatile platform to reprogram gene transcription at targeted genomic loci. However, without temporal control, the application of these reprogramming tools will likely cause off-target effects and lack strict reversibility. To overcome this limitation, we report herein the development of a chemical or light-inducible transcriptional reprogramming device that combines photoswitchable genetically encoded calcium actuators with dCas9 to control gene expression. By fusing an engineered Ca2+-responsive NFAT fragment with dCas9 and transcriptional coactivators, we harness the power of light to achieve photoinducible transcriptional reprogramming in mammalian cells. This synthetic system (designated CaRROT) can also be used to document calcium-dependent activity in mammals after exposure to ligands or chemicals that would elicit calcium response inside cells.


Asunto(s)
Señalización del Calcio/genética , Biología Sintética/métodos , Transcripción Genética , Sistemas CRISPR-Cas/genética , Calcio/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Núcleo Celular/metabolismo , Regulación de la Expresión Génica/efectos de la radiación , Células HEK293 , Células HeLa , Humanos , Luz , Optogenética , Transporte de Proteínas
11.
PLoS Pathog ; 13(7): e1006440, 2017 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-28704539

RESUMEN

Streptococcus gallolyticus subsp. gallolyticus (Sg) has long been known to have a strong association with colorectal cancer (CRC). This knowledge has important clinical implications, and yet little is known about the role of Sg in the development of CRC. Here we demonstrate that Sg promotes human colon cancer cell proliferation in a manner that depends on cell context, bacterial growth phase and direct contact between bacteria and colon cancer cells. In addition, we observed increased level of ß-catenin, c-Myc and PCNA in colon cancer cells following incubation with Sg. Knockdown or inhibition of ß-catenin abolished the effect of Sg. Furthermore, mice administered with Sg had significantly more tumors, higher tumor burden and dysplasia grade, and increased cell proliferation and ß-catenin staining in colonic crypts compared to mice receiving control bacteria. Finally, we showed that Sg is present in the majority of CRC patients and is preferentially associated with tumor compared to normal tissues obtained from CRC patients. These results taken together establish for the first time a tumor-promoting role of Sg that involves specific bacterial and host factors and have important clinical implications.


Asunto(s)
Neoplasias Colorrectales/microbiología , Infecciones Estreptocócicas/microbiología , Streptococcus gallolyticus subspecies gallolyticus/fisiología , Animales , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Femenino , Humanos , Ratones , Transducción de Señal , Infecciones Estreptocócicas/genética , Infecciones Estreptocócicas/metabolismo , Infecciones Estreptocócicas/patología , beta Catenina/genética , beta Catenina/metabolismo
13.
PLoS Pathog ; 12(6): e1005678, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-27304426

RESUMEN

Spores of Bacillus anthracis, the causative agent of anthrax, are known to persist in the host lungs for prolonged periods of time, however the underlying mechanism is poorly understood. In this study, we demonstrated that BclA, a major surface protein of B. anthracis spores, mediated direct binding of complement factor H (CFH) to spores. The surface bound CFH retained its regulatory cofactor activity resulting in C3 degradation and inhibition of downstream complement activation. By comparing results from wild type C57BL/6 mice and complement deficient mice, we further showed that BclA significantly contributed to spore persistence in the mouse lungs and dampened antibody responses to spores in a complement C3-dependent manner. In addition, prior exposure to BclA deletion spores (ΔbclA) provided significant protection against lethal challenges by B. anthracis, whereas the isogenic parent spores did not, indicating that BclA may also impair protective immunity. These results describe for the first time an immune inhibition mechanism of B. anthracis mediated by BclA and CFH that promotes spore persistence in vivo. The findings also suggested an important role of complement in persistent infections and thus have broad implications.


Asunto(s)
Carbunco/inmunología , Factor H de Complemento/inmunología , Tolerancia Inmunológica/inmunología , Glicoproteínas de Membrana/inmunología , Esporas Bacterianas/inmunología , Animales , Carbunco/metabolismo , Bacillus anthracis/inmunología , Bacillus anthracis/metabolismo , Factor H de Complemento/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Esporas Bacterianas/metabolismo
14.
Curr Allergy Asthma Rep ; 12(5): 402-12, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22875242

RESUMEN

The eosinophil is a multifunctional granulocyte best known for providing host defense against parasites. Paradoxically, eosinophils are also implicated in the pathogenesis of allergic inflammation, asthma, and hypereosinophilic syndromes. Emerging evidence also supports the potential for harnessing the cytotoxic power of eosinophils and redirecting it to kill solid tumors. Central to eosinophil physiology is interleukin-5 (IL-5) and its receptor (IL-5R) which is composed of a ligand-specific alpha chain (IL-5Rα) and the common beta chain (ßc). Eosinophil activation can lead to their degranulation, resulting in rapid release of an arsenal of tissue-destructive proinflammatory mediators and cytotoxic proteins that can be both beneficial and detrimental to the host. This review discusses eosinophil immunobiology and therapeutic strategies for targeting of IL-5 and IL-5R, as well as the potential for harnessing eosinophil cytotoxicity as a tumoricide.


Asunto(s)
Citotoxicidad Inmunológica , Eosinófilos/inmunología , Síndrome Hipereosinofílico/tratamiento farmacológico , Hipersensibilidad/tratamiento farmacológico , Interleucina-5/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Receptores de Interleucina-5/antagonistas & inhibidores , Anticuerpos Monoclonales Humanizados/uso terapéutico , Asma/tratamiento farmacológico , Síndrome de Churg-Strauss/tratamiento farmacológico , Ensayos Clínicos como Asunto , Dermatitis Atópica/tratamiento farmacológico , Humanos , Síndrome Hipereosinofílico/inmunología , Hipersensibilidad/inmunología , Inflamación/tratamiento farmacológico , Pólipos Nasales/tratamiento farmacológico , Neoplasias/inmunología , Oligonucleótidos Fosforotioatos/uso terapéutico
15.
J Biol Chem ; 286(46): 40091-103, 2011 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-21965659

RESUMEN

Eosinophils are multifunctional leukocytes implicated in the pathogenesis of numerous inflammatory diseases including allergic asthma and hypereosinophilic syndrome. Eosinophil physiology is critically dependent on IL-5 and the IL-5 receptor (IL-5R), composed of a ligand binding α chain (IL-5Rα), and a common ß chain, ßc. Previously, we demonstrated that the ßc cytoplasmic tail is ubiquitinated and degraded by proteasomes following IL-5 stimulation. However, a complete understanding of the role of ßc ubiquitination in IL-5R biology is currently lacking. By using a well established, stably transduced HEK293 cell model system, we show here that in the absence of ubiquitination, ßc subcellular localization, IL-5-induced endocytosis, turnover, and IL-5R signaling were significantly impaired. Whereas ubiquitinated IL-5Rs internalized into trafficking endosomes for their degradation, ubiquitination-deficient IL-5Rs accumulated on the cell surface and displayed blunted signaling even after IL-5 stimulation. Importantly, we identified a cluster of three membrane-proximal ßc lysine residues (Lys(457), Lys(461), and Lys(467)) whose presence was required for both JAK1/2 binding to ßc and receptor ubiquitination. These findings establish that JAK kinase binding to ßc requires the presence of three critical ßc lysine residues, and this binding event is essential for receptor ubiquitination, endocytosis, and signaling.


Asunto(s)
Subunidad beta Común de los Receptores de Citocinas/metabolismo , Endocitosis/fisiología , Janus Quinasa 1/metabolismo , Janus Quinasa 2/metabolismo , Lisina/metabolismo , Transducción de Señal/fisiología , Ubiquitinación/fisiología , Subunidad beta Común de los Receptores de Citocinas/genética , Endosomas/genética , Endosomas/metabolismo , Células HEK293 , Humanos , Janus Quinasa 1/genética , Janus Quinasa 2/genética , Lisina/genética , Unión Proteica/fisiología , Transporte de Proteínas/fisiología
16.
Cell Microbiol ; 13(10): 1479-96, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21722286

RESUMEN

The bacterial virulence factors Shiga toxins (Stxs) are expressed by Shigella dysenteriae serotype 1 and certain Escherichia coli strains. Stxs are protein synthesis inhibitors and induce apoptosis in many cell types. Stxs induce apoptosis via prolonged endoplasmic reticulum stress signalling to activate both extrinsic and intrinsic pathways in human myeloid cells. Studies have shown that autophagy, a lysosome-dependent catabolic process, may be associated with activation of pro-survival or death processes. It is currently unknown if autophagy contributes to apoptosis or protects cells from Stxs. To study cellular responses to Stxs, we intoxicated toxin-sensitive cells (THP-1 and HK-2 cells), and toxin-resistant cells (primary human monocyte-derived macrophages) and examined toxin intracellular trafficking and autophagosome formation. Stxs translocated to different cell compartments in toxin-resistant versus toxin-sensitive cells. Confocal microscopy revealed autophagosome formation in both toxin-resistant and toxin-sensitive cells. Proteolytic cleavage of Atg5 and Beclin-1 plays pivotal roles in switching non-cytotoxic autophagy to cell death signalling. We detected cleaved forms of Atg5 and Beclin-1 in Stx-treated toxin-sensitive cells, while cleaved caspases, calpains, Atg5 and Beclin-1 were not detected in toxin-resistant primary human monocytes and macrophages. These findings suggest that toxin sensitivity correlates with caspase and calpain activation, leading to Atg5 and Beclin-1 cleavage.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia , Escherichia coli/patogenicidad , Interacciones Huésped-Patógeno , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Toxinas Shiga/toxicidad , Shigella dysenteriae/patogenicidad , Proteína 5 Relacionada con la Autofagia , Beclina-1 , Calpaína/metabolismo , Caspasas/metabolismo , Células Cultivadas , Humanos , Toxina Shiga , Transducción de Señal
17.
Curr Allergy Asthma Rep ; 10(5): 320-5, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20623372

RESUMEN

Pulmonary alveolar proteinosis (PAP) is a rare disease of the lung characterized by the accumulation of surfactant-derived lipoproteins within pulmonary alveolar macrophages and alveoli, resulting in respiratory insufficiency and increased infections. The disease is caused by a disruption in surfactant catabolism by alveolar macrophages due to loss of functional granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling. The underlying molecular mechanisms causing deficiencies in GM-CSF signaling are as follows: 1) high levels of neutralizing GM-CSF autoantibodies observed in autoimmune PAP; 2) mutations in CSF2RA, the gene encoding the alpha chain of the GM-CSF receptor, observed in hereditary PAP; and 3) reduced numbers and function of alveolar macrophages as a result of other clinical diseases seen in secondary PAP. Recent studies investigating the biology of GM-CSF have revealed that not only does this cytokine have an indispensable role in lung physiology, but it is also a critical regulator of innate immunity and lung host defense.


Asunto(s)
Proteinosis Alveolar Pulmonar/inmunología , Proteinosis Alveolar Pulmonar/fisiopatología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos , Autoinmunidad/inmunología , Recuento de Células , Humanos , Lipoproteínas/metabolismo , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/patología , Mutación , Proteinosis Alveolar Pulmonar/terapia , Alveolos Pulmonares/fisiopatología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Transducción de Señal
18.
J Exp Med ; 205(12): 2711-6, 2008 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-18955567

RESUMEN

Pulmonary alveolar proteinosis (PAP) is a rare lung disorder in which surfactant-derived lipoproteins accumulate excessively within pulmonary alveoli, causing severe respiratory distress. The importance of granulocyte/macrophage colony-stimulating factor (GM-CSF) in the pathogenesis of PAP has been confirmed in humans and mice, wherein GM-CSF signaling is required for pulmonary alveolar macrophage catabolism of surfactant. PAP is caused by disruption of GM-CSF signaling in these cells, and is usually caused by neutralizing autoantibodies to GM-CSF or is secondary to other underlying diseases. Rarely, genetic defects in surfactant proteins or the common beta chain for the GM-CSF receptor (GM-CSFR) are causal. Using a combination of cellular, molecular, and genomic approaches, we provide the first evidence that PAP can result from a genetic deficiency of the GM-CSFR alpha chain, encoded in the X-chromosome pseudoautosomal region 1.


Asunto(s)
Cromosomas Humanos X/genética , Proteinosis Alveolar Pulmonar/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Animales , Antígeno CD11b/metabolismo , Preescolar , Exones , Femenino , Genotipo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Masculino , Ratones , Monocitos/citología , Monocitos/metabolismo , Surfactantes Pulmonares/metabolismo , Transducción de Señal/fisiología , Síndrome de Turner
19.
J Leukoc Biol ; 84(2): 499-509, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18511572

RESUMEN

Eosinophils are critically dependent on IL-5 for their activation, differentiation, survival, and augmentation of cytotoxic activity. We previously showed that the cytoplasmic domain of the hematopoietic receptor, betac, which is shared by IL-5, IL-3, and GM-CSF, is directly ubiquitinated and degraded by the proteasomes in a JAK2-dependent manner. However, studies describing the spatial distribution, endocytic regulation, and trafficking of betac-sharing receptors in human eosinophils are currently lacking. Using deconvolution microscopy and biochemical methods, we clearly demonstrate that IL-5Rs reside in and are internalized by clathrin- and lipid raft-dependent endocytic pathways. Microscopy analyses in TF1 cells and human eosinophils revealed significant colocalization of betac, IL-5Ralpha, and Cy3-labeled IL-5 with transferrin- (clathrin) and cholera toxin-B- (lipid raft) positive vesicles. Moreover, whereas internalized IL-5Rs were detected in both clathrin- and lipid raft-positive vesicles, biochemical data revealed that tyrosine phosphorylated, ubiquitinated, and proteasome-degraded IL-5Rs partitioned to the soluble, nonraft fractions (clathrin-containing). Lastly, we show that optimal IL-5-induced signaling requires entry of activated IL-5Rs into the intracellular compartment, as coimmunoprecipitation of key signaling molecules with the IL-5R was completely blocked when either endocytic pathway was inhibited. These data provide the first evidence that IL-5Rs segregate and traffic into two distinct plasma membrane compartments, and they further establish that IL-5R endocytosis regulates signaling both positively and negatively.


Asunto(s)
Endocitosis/fisiología , Eosinófilos/fisiología , Receptores de Interleucina-5/fisiología , Transferrina/fisiología , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Toxina del Cólera/farmacología , Clatrina/fisiología , Endocitosis/efectos de los fármacos , Citometría de Flujo , Humanos , Interleucina-5/farmacología , Leucemia Eritroblástica Aguda , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transferrina/farmacología
20.
J Leukoc Biol ; 81(4): 1137-48, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17227823

RESUMEN

IL-5, IL-3, and GM-CSF are related hematopoietic cytokines, which regulate the function of myeloid cells and are mediators of the allergic inflammatory response. These cytokines signal through heteromeric receptors containing a specific alpha chain and a shared signaling chain, betac. Previous studies demonstrated that the ubiquitin (Ub) proteasome degradation pathway was involved in signal termination of the betac-sharing receptors. In this study, the upstream molecular events leading to proteasome degradation of the IL-5 receptor (IL-5R) were examined. By using biochemical and flow cytometric methods, we show that JAK kinase activity is required for betac ubiquitination and proteasome degradation but only partially required for IL-5R internalization. Furthermore, we demonstrate the direct ubiquitination of the betac cytoplasmic domain and identify lysine residues 566 and 603 as sites of betac ubiquitination. Lastly, we show that ubiquitination of the betac cytoplasmic domain begins at the plasma membrane, increases after receptor internalization, and is degraded by the proteasome after IL-5R internalization. We propose an updated working model of IL-5R down-regulation, whereby IL-5 ligation of its receptor activates JAK2/1 kinases, resulting in betac tyrosine phosphorylation, ubiquitination, and IL-5R internalization. Once inside the cell, proteasomes degrade the betac cytoplasmic domain, and the truncated receptor complex is terminally degraded in the lysosomes. These data establish a critical role for JAK kinases and the Ub/proteasome degradation pathway in IL-5R down-regulation.


Asunto(s)
Regulación hacia Abajo , Quinasas Janus/metabolismo , Receptores de Interleucina-5/metabolismo , Transducción de Señal , Ubiquitina/metabolismo , Familia-src Quinasas/metabolismo , Línea Celular , Células Cultivadas , Citoplasma/metabolismo , Endocitosis , Inhibidores Enzimáticos/farmacología , Eosinófilos/enzimología , Eosinófilos/metabolismo , Eosinófilos/fisiología , Humanos , Quinasas Janus/antagonistas & inhibidores , Quinasas Janus/fisiología , Lisina/química , Modelos Biológicos , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/fisiología , Tirfostinos/farmacología
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