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1.
Int J Mol Sci ; 23(13)2022 Jul 03.
Artículo en Inglés | MEDLINE | ID: mdl-35806422

RESUMEN

Adipocyte iron overload is a maladaptation associated with obesity and insulin resistance. The objective of the current study was to determine whether and how adipose tissue macrophages (ATMs) regulate adipocyte iron concentrations and whether this is impacted by obesity. Using bone marrow-derived macrophages (BMDMs) polarized to M0, M1, M2, or metabolically activated (MMe) phenotypes, we showed that MMe BMDMs and ATMs from obese mice have reduced expression of several iron-related proteins. Furthermore, the bioenergetic response to iron in obese ATMs was hampered. ATMs from iron-injected lean mice increased their glycolytic and respiratory capacities, thus maintaining metabolic flexibility, while ATMs from obese mice did not. Using an isotope-based system, we found that iron exchange between BMDMs and adipocytes was regulated by macrophage phenotype. At the end of the co-culture, MMe macrophages transferred and received more iron from adipocytes than M0, M1, and M2 macrophages. This culminated in a decrease in total iron in MMe macrophages and an increase in total iron in adipocytes compared with M2 macrophages. Taken together, in the MMe condition, the redistribution of iron is biased toward macrophage iron deficiency and simultaneous adipocyte iron overload. These data suggest that obesity changes the communication of iron between adipocytes and macrophages and that rectifying this iron communication channel may be a novel therapeutic target to alleviate insulin resistance.


Asunto(s)
Resistencia a la Insulina , Sobrecarga de Hierro , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Animales , Inflamación/metabolismo , Hierro/metabolismo , Sobrecarga de Hierro/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Obesos , Obesidad/metabolismo , Fenotipo
2.
Nat Nanotechnol ; 16(12): 1394-1402, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34764452

RESUMEN

Activating CD8+ T cells by antigen cross-presentation is remarkably effective at eliminating tumours. Although this function is traditionally attributed to dendritic cells, tumour-associated macrophages (TAMs) can also cross-present antigens. TAMs are the most abundant tumour-infiltrating leukocyte. Yet, TAMs have not been leveraged to activate CD8+ T cells because mechanisms that modulate their ability to cross-present antigens are incompletely understood. Here we show that TAMs harbour hyperactive cysteine protease activity in their lysosomes, which impedes antigen cross-presentation, thereby preventing CD8+ T cell activation. We developed a DNA nanodevice (E64-DNA) that targets the lysosomes of TAMs in mice. E64-DNA inhibits the population of cysteine proteases that is present specifically inside the lysosomes of TAMs, improves their ability to cross-present antigens and attenuates tumour growth via CD8+ T cells. When combined with cyclophosphamide, E64-DNA showed sustained tumour regression in a triple-negative-breast-cancer model. Our studies demonstrate that DNA nanodevices can be targeted with organelle-level precision to reprogram macrophages and achieve immunomodulation in vivo.


Asunto(s)
ADN/química , Lisosomas/metabolismo , Nanopartículas/química , Neoplasias/patología , Macrófagos Asociados a Tumores/metabolismo , Animales , Antígenos/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/deficiencia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Terapia Combinada , Reactividad Cruzada/inmunología , Ciclofosfamida , Femenino , Humanos , Inmunidad , Ratones Endogámicos C57BL , Neoplasias/inmunología , Proteómica
3.
STAR Protoc ; 2(4): 100845, 2021 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-34604813

RESUMEN

Monocytes and neutrophils are widely distributed throughout the body and play essential roles in health and disease. Here, we present a detailed protocol to isolate polymorphonuclear neutrophils and monocytes from a single sample of human peripheral blood. We have optimized several aspects of the procedure, including the density gradient, timing of each cell processing step, and the buffer/media conditions to preserve cell viability for subsequent functional assays. This protocol is reproducible and can be scaled as required for downstream applications. For complete details on the use and execution of this protocol, please refer to Cui et al. (2021).


Asunto(s)
Monocitos , Neutrófilos , Medios de Cultivo , Humanos
4.
Cell ; 184(12): 3163-3177.e21, 2021 06 10.
Artículo en Inglés | MEDLINE | ID: mdl-33964209

RESUMEN

Cancer cell genetic variability and similarity to host cells have stymied development of broad anti-cancer therapeutics. Our innate immune system evolved to clear genetically diverse pathogens and limit host toxicity; however, whether/how innate immunity can produce similar effects in cancer is unknown. Here, we show that human, but not murine, neutrophils release catalytically active neutrophil elastase (ELANE) to kill many cancer cell types while sparing non-cancer cells. ELANE proteolytically liberates the CD95 death domain, which interacts with histone H1 isoforms to selectively eradicate cancer cells. ELANE attenuates primary tumor growth and produces a CD8+T cell-mediated abscopal effect to attack distant metastases. Porcine pancreatic elastase (ELANE homolog) resists tumor-derived protease inhibitors and exhibits markedly improved therapeutic efficacy. Altogether, our studies suggest that ELANE kills genetically diverse cancer cells with minimal toxicity to non-cancer cells, raising the possibility of developing it as a broad anti-cancer therapy.


Asunto(s)
Carcinogénesis/patología , Elastasa de Leucocito/metabolismo , Neoplasias/enzimología , Neoplasias/patología , Regulación Alostérica/efectos de los fármacos , Animales , Linfocitos T CD8-positivos/inmunología , Carcinogénesis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteína Catiónica del Eosinófilo/metabolismo , Histonas/metabolismo , Humanos , Ratones , Neoplasias/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Elastasa Pancreática/metabolismo , Inhibidores de Proteasas/farmacología , Dominios Proteicos , Isoformas de Proteínas/metabolismo , Proteolisis/efectos de los fármacos , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Porcinos , Receptor fas/química , Receptor fas/metabolismo
5.
Front Pharmacol ; 11: 1219, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33013356

RESUMEN

BACKGROUND: CFTR modulators decrease some etiologies of CF airway inflammation; however, data indicate that non-resolving airway infection and inflammation persist in individuals with CF and chronic bacterial infections. Thus, identification of therapies that diminish airway inflammation without allowing unrestrained bacterial growth remains a critical research goal. Novel strategies for combatting deleterious airway inflammation in the CFTR modulator era require better understanding of cellular contributions to chronic CF airway disease, and how inflammatory cells change after initiation of CFTR modulator therapy. Peripheral blood monocytes, which traffic to the CF airway, can develop both pro-inflammatory and inflammation-resolving phenotypes, represent intriguing cellular targets for focused therapies. This therapeutic approach, however, requires a more detailed knowledge of CF monocyte cellular programming and phenotypes. MATERIAL AND METHODS: In order to characterize the inflammatory phenotype of CF monocytes, and how these cells change after initiation of CFTR modulator therapy, we studied adults (n=10) with CF, chronic airway infections, and the CFTR-R117H mutations before and 7 days after initiation of ivacaftor. Transcriptomes of freshly isolated blood monocytes were interrogated by RNA-sequencing (RNA-seq) followed by pathway-based analyses. Plasma concentrations of cytokines and chemokines were evaluated by multiplex ELISA. RESULTS: RNAseq identified approximately 50 monocyte genes for which basal expression was significantly changed in all 10 subjects after 7 days of ivacaftor. Of these, the majority were increased in expression post ivacaftor, including many genes traditionally associated with enhanced inflammation and immune responses. Pathway analyses confirmed that transcriptional programs were overwhelmingly up-regulated in monocytes after 7 days of ivacaftor, including biological modules associated with immunity, cell cycle, oxidative phosphorylation, and the unfolded protein response. Ivacaftor increased plasma concentrations of CXCL2, a neutrophil chemokine secreted by monocytes and macrophages, and CCL2, a monocyte chemokine. CONCLUSIONS: Our results demonstrate that ivacaftor causes acute changes in blood monocyte transcriptional profiles and plasma chemokines, and suggest that increased monocyte inflammatory signals and changes in myeloid cell trafficking may contribute to changes in airway inflammation in people taking CFTR modulators. To our knowledge, this is the first report investigating the transcriptomic response of circulating blood monocytes in CF subjects treated with a CFTR modulator.

6.
Endocrinology ; 161(10)2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-32770234

RESUMEN

The Silencing Mediator of Retinoid and Thyroid Hormone Receptors (SMRT) is a nuclear corepressor, regulating the transcriptional activity of many transcription factors critical for metabolic processes. While the importance of the role of SMRT in the adipocyte has been well-established, our comprehensive understanding of its in vivo function in the context of homeostatic maintenance is limited due to contradictory phenotypes yielded by prior generalized knockout mouse models. Multiple such models agree that SMRT deficiency leads to increased adiposity, although the effects of SMRT loss on glucose tolerance and insulin sensitivity have been variable. We therefore generated an adipocyte-specific SMRT knockout (adSMRT-/-) mouse to more clearly define the metabolic contributions of SMRT. In doing so, we found that SMRT deletion in the adipocyte does not cause obesity-even when mice are challenged with a high-fat diet. This suggests that adiposity phenotypes of previously described models were due to effects of SMRT loss beyond the adipocyte. However, an adipocyte-specific SMRT deficiency still led to dramatic effects on systemic glucose tolerance and adipocyte insulin sensitivity, impairing both. This metabolically deleterious outcome was coupled with a surprising immune phenotype, wherein most genes differentially expressed in the adipose tissue of adSMRT-/- mice were upregulated in pro-inflammatory pathways. Flow cytometry and conditioned media experiments demonstrated that secreted factors from knockout adipose tissue strongly informed resident macrophages to develop a pro-inflammatory, MMe (metabolically activated) phenotype. Together, these studies suggest a novel role for SMRT as an integrator of metabolic and inflammatory signals to maintain physiological homeostasis.


Asunto(s)
Tejido Adiposo/metabolismo , Diferenciación Celular/genética , Metabolismo Energético/genética , Macrófagos/fisiología , Co-Represor 2 de Receptor Nuclear/fisiología , Adipocitos/fisiología , Tejido Adiposo/citología , Animales , Homeostasis/genética , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Co-Represor 2 de Receptor Nuclear/genética , Obesidad/genética , Obesidad/metabolismo , Obesidad/patología , Especificidad de Órganos/genética , Fenotipo
7.
ERJ Open Res ; 6(2)2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32337217

RESUMEN

This study demonstrates that initiation of the CFTR modulator ivacaftor in people with cystic fibrosis and susceptible CFTR mutations causes an acute reduction in blood monocyte sensitivity to the key proinflammatory cytokine IFN-γ http://bit.ly/2TeI6LG.

8.
J Exp Med ; 216(6): 1345-1358, 2019 06 03.
Artículo en Inglés | MEDLINE | ID: mdl-31053611

RESUMEN

Obesity is associated with increased incidence and severity of triple-negative breast cancer (TNBC); however, mechanisms underlying this relationship are incompletely understood. Here, we show that obesity reprograms mammary adipose tissue macrophages to a pro-inflammatory metabolically activated phenotype (MMe) that alters the niche to support tumor formation. Unlike pro-inflammatory M1 macrophages that antagonize tumorigenesis, MMe macrophages are pro-tumorigenic and represent the dominant macrophage phenotype in mammary adipose tissue of obese humans and mice. MMe macrophages release IL-6 in an NADPH oxidase 2 (NOX2)-dependent manner, which signals through glycoprotein 130 (GP130) on TNBC cells to promote stem-like properties including tumor formation. Deleting Nox2 in myeloid cells or depleting GP130 in TNBC cells attenuates obesity-augmented TNBC stemness. Moreover, weight loss reverses the effects of obesity on MMe macrophage inflammation and TNBC tumor formation. Our studies implicate MMe macrophage accumulation in mammary adipose tissue as a mechanism for promoting TNBC stemness and tumorigenesis during obesity.


Asunto(s)
Tejido Adiposo/patología , Macrófagos/metabolismo , Obesidad/patología , Neoplasias de la Mama Triple Negativas/patología , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Receptor gp130 de Citocinas/metabolismo , Dieta , Femenino , Humanos , Interleucina-6/metabolismo , Glándulas Mamarias Animales/patología , Glándulas Mamarias Humanas/patología , Ratones Endogámicos C57BL , Células Madre Neoplásicas/patología , Fenotipo , Pérdida de Peso
9.
Cell Rep ; 23(10): 3021-3030, 2018 06 05.
Artículo en Inglés | MEDLINE | ID: mdl-29874587

RESUMEN

Type 2 diabetes (T2D) is associated with increased risk for atherosclerosis; however, the mechanisms underlying this relationship are poorly understood. Macrophages, which are activated in T2D and causatively linked to atherogenesis, are an attractive mechanistic link. Here, we use proteomics to show that diet-induced obesity and insulin resistance (obesity/IR) modulate a pro-atherogenic "macrophage-sterol-responsive-network" (MSRN), which, in turn, predisposes macrophages to cholesterol accumulation. We identify IFNγ as the mediator of obesity/IR-induced MSRN dysregulation and increased macrophage cholesterol accumulation and show that obesity/IR primes T cells to increase IFNγ production. Accordingly, myeloid cell-specific deletion of the IFNγ receptor (Ifngr1-/-) restores MSRN proteins, attenuates macrophage cholesterol accumulation and atherogenesis, and uncouples the strong relationship between hyperinsulinemia and aortic root lesion size in hypercholesterolemic Ldlr-/- mice with obesity/IR, but does not affect these parameters in Ldlr-/- mice without obesity/IR. Collectively, our findings identify an IFNγ-macrophage pathway as a mechanistic link between obesity/IR and accelerated atherogenesis.


Asunto(s)
Aterosclerosis/metabolismo , Aterosclerosis/patología , Resistencia a la Insulina , Interferón gamma/metabolismo , Macrófagos/metabolismo , Obesidad/metabolismo , Obesidad/patología , Animales , Aterosclerosis/genética , Colesterol/metabolismo , Células Espumosas/metabolismo , Regulación de la Expresión Génica , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/metabolismo , Receptores de Interferón/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/metabolismo , Receptor de Interferón gamma
10.
Oncotarget ; 9(4): 4282-4300, 2018 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-29435103

RESUMEN

Major roadblocks to developing effective progesterone receptor (PR)-targeted therapies in breast cancer include the lack of highly-specific PR modulators, a poor understanding of the pro- or anti-tumorigenic networks for PR isoforms and ligands, and an incomplete understanding of the cross talk between PR and estrogen receptor (ER) signaling. Through genomic analyses of xenografts treated with various clinically-relevant ER and PR-targeting drugs, we describe how the activation or inhibition of PR differentially reprograms estrogen signaling, resulting in the segregation of transcriptomes into separate PR agonist and antagonist-mediated groups. These findings address an ongoing controversy regarding the clinical utility of PR agonists and antagonists, alone or in combination with tamoxifen, for breast cancer management. Additionally, the two PR isoforms PRA and PRB, bind distinct but overlapping genomic sites and interact with different sets of co-regulators to differentially modulate estrogen signaling to be either pro- or anti-tumorigenic. Of the two isoforms, PRA inhibited gene expression and ER chromatin binding significantly more than PRB. Differential gene expression was observed in PRA and PRB-rich patient tumors and PRA-rich gene signatures had poorer survival outcomes. In support of antiprogestin responsiveness of PRA-rich tumors, gene signatures associated with PR antagonists, but not PR agonists, predicted better survival outcomes. The better patient survival associated with PR antagonists versus PR agonists treatments was further reflected in the higher in vivo anti-tumor activity of therapies that combine tamoxifen with PR antagonists and modulators. This study suggests that distinguishing common effects observed due to concomitant interaction of another receptor with its ligand (agonist or antagonist), from unique isoform and ligand-specific effects will inform the development of biomarkers for patient selection and translation of PR-targeted therapies to the clinic.

11.
Cell Rep ; 20(13): 3149-3161, 2017 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-28954231

RESUMEN

During obesity, adipose tissue macrophages (ATMs) adopt a metabolically activated (MMe) phenotype. However, the functions of MMe macrophages are poorly understood. Here, we combine proteomic and functional methods to demonstrate that, in addition to potentiating inflammation, MMe macrophages promote dead adipocyte clearance through lysosomal exocytosis. We identify NADPH oxidase 2 (NOX2) as a driver of the inflammatory and adipocyte-clearing properties of MMe macrophages and show that, compared to wild-type, Nox2-/- mice exhibit a time-dependent metabolic phenotype during diet-induced obesity. After 8 weeks of high-fat feeding, Nox2-/- mice exhibit attenuated ATM inflammation and mildly improved glucose tolerance. After 16 weeks of high-fat feeding, Nox2-/- mice develop severe insulin resistance, hepatosteatosis, and visceral lipoatrophy characterized by dead adipocyte accumulation and defective ATM lysosomal exocytosis, a phenotype reproduced in myeloid cell-specific Nox2-/- mice. Collectively, our findings suggest that MMe macrophages perform detrimental and beneficial functions whose contribution to metabolic phenotypes during obesity is determined by disease progression.


Asunto(s)
Tejido Adiposo/metabolismo , Dieta Alta en Grasa/efectos adversos , Macrófagos/metabolismo , Obesidad/etiología , Animales , Humanos , Ratones
13.
Cell Metab ; 20(4): 614-25, 2014 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-25242226

RESUMEN

Adipose tissue macrophage (ATM)-driven inflammation plays a key role in insulin resistance; however, factors activating ATMs are poorly understood. Using a proteomics approach, we show that markers of classical activation are absent on ATMs from obese humans but are readily detectable on airway macrophages of patients with cystic fibrosis, a disease associated with chronic bacterial infection. Moreover, treating macrophages with glucose, insulin, and palmitate-conditions characteristic of the metabolic syndrome-produces a "metabolically activated" phenotype distinct from classical activation. Markers of metabolic activation are expressed by proinflammatory ATMs in obese humans/mice and are positively correlated with adiposity. Metabolic activation is driven by independent proinflammatory and anti-inflammatory pathways, which regulate balance between cytokine production and lipid metabolism. We identify PPARγ and p62/SQSTM1 as two key proteins that promote lipid metabolism and limit inflammation in metabolically activated macrophages. Collectively, our data provide important mechanistic insights into pathways that drive the metabolic-disease-specific phenotype of macrophages.


Asunto(s)
Tejido Adiposo/metabolismo , Macrófagos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Antígenos de Superficie/metabolismo , Autofagia/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Glucosa/farmacología , Humanos , Inflamación/metabolismo , Insulina/farmacología , Metabolismo de los Lípidos/fisiología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Monocitos/citología , PPAR gamma/metabolismo , Palmitatos/farmacología , Fenotipo
14.
Sleep Med ; 15(5): 541-9, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24726570

RESUMEN

BACKGROUND: Intrinsic variance of the urine proteome limits the discriminative power of proteomic analysis and complicates potential biomarker detection in the context of paediatric sleep disorders. METHODS AND RESULTS: Using a rigorous workflow for proteomic analysis of urine, we demonstrate that gender and diurnal effects constitute two important sources of variability in healthy children. In the context of disease, complex pathophysiological perturbations magnify these proteomic differences and therefore require contextualised biomarker analysis. Indeed, by performing biomarker discovery in a gender- and diurnal-dependent manner, we identified ∼30-fold more candidate biomarkers of paediatric obstructive sleep apnoea (OSA), a highly prevalent condition in children characterised by repetitive episodes of intermittent hypoxia and hypercapnia, and sleep fragmentation in the context of recurrent upper airway obstructive events during sleep. Remarkably, biomarkers were highly specific for gender and sampling time as poor overlap (∼3%) was observed in the proteins identified in boys and girls across morning and bedtime samples. CONCLUSIONS: As no clinical basis to explain gender-specific effects in OSA or healthy children is apparent, we propose that implementation of contextualised biomarker strategies will be applicable to a broad range of human diseases, and may be specifically applicable to paediatric OSA.


Asunto(s)
Apnea Obstructiva del Sueño/orina , Biomarcadores/orina , Estudios de Casos y Controles , Niño , Preescolar , Cromatografía Liquida , Ritmo Circadiano , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Péptidos/orina , Polisomnografía , Proteinuria/orina , Proteómica , Factores Sexuales , Apnea Obstructiva del Sueño/diagnóstico , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem
15.
Am J Respir Crit Care Med ; 189(5): 593-601, 2014 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-24471484

RESUMEN

RATIONALE: An increased cancer aggressiveness and mortality have been recently reported among patients with obstructive sleep apnea (OSA). Intermittent hypoxia (IH), a hallmark of OSA, enhances melanoma growth and metastasis in mice. OBJECTIVES: To assess whether OSA-related adverse cancer outcomes occur via IH-induced changes in host immune responses, namely tumor-associated macrophages (TAMs). MEASUREMENTS AND MAIN RESULTS: Lung epithelial TC1 cell tumors were 84% greater in mice subjected to IH for 28 days compared with room air (RA). In addition, TAMs in IH-exposed tumors exhibited reductions in M1 polarity with a shift toward M2 protumoral phenotype. Although TAMs from tumors harvested from RA-exposed mice increased TC1 migration and extravasation, TAMs from IH-exposed mice markedly enhanced such effects and also promoted proliferative rates and invasiveness of TC1 cells. Proliferative rates of melanoma (B16F10) and TC1 cells exposed to IH either in single culture or in coculture with macrophages (RAW 264.7) increased only when RAW 264.7 macrophages were concurrently present. CONCLUSIONS: Our findings support the notion that IH-induced alterations in TAMs participate in the adverse cancer outcomes reported in OSA.


Asunto(s)
Hipoxia/inmunología , Neoplasias Pulmonares/patología , Macrófagos/patología , Melanoma Experimental/patología , Apnea Obstructiva del Sueño/fisiopatología , Animales , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Citometría de Flujo , Hipoxia/etiología , Neoplasias Pulmonares/inmunología , Masculino , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Invasividad Neoplásica , Fenotipo , Apnea Obstructiva del Sueño/inmunología , Linfocitos T Citotóxicos/patología
16.
J Biol Chem ; 288(41): 29901-10, 2013 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-23990460

RESUMEN

The MLL CXXC domain binds nonmethylated CpG-containing DNA and is essential for the oncogenic properties of MLL fusion proteins. To determine potential functional promiscuity of similar DNA binding domains, we replaced the MLL CXXC domain in the context of the leukemogenic MLL-AF9 fusion with CXXC domains from DNMT1, CGBP (CFP1), and MBD1, or with a methyl-CpG-binding domain (MBD) from MBD1. MLL(DNMT1 CXXC)-AF9 shows robust in vitro colony forming activity and in vivo leukemogenesis, similar to MLL-AF9. However, colony forming ability and leukemogenicity are abrogated in MLL-AF9 containing either the CGBP or MBD1 CXXC domains or the MBD1 MBD domain. Direct comparison of in vitro DNA binding affinity of the isolated CXXC or MBD domains demonstrated that MLL, DNMT1, and CGBP CXXC domains could each bind to unmethylated DNA but with differing affinity. In contrast, the isolated MBD1 CXXC and MBD1 MBD domains were unable to bind to the same DNA. However, all substituted domains still allowed targeting of the MLL fusions to the functionally important Hoxa9 locus in primary bone marrow progenitor cells. In addition to DNA binding activity, it was critical that specific CpG residues in the Hoxa9 locus were protected from methylation for leukemia development. This ultimately prevented histone 3 lysine 9 trimethylation (H3K9me3) of the locus and enabled Hoxa9 expression. These were properties shared by MLL and DNMT1 CXXC domains but not by CGBP CXXC or the other swapped fusions tested. We demonstrate that similar CXXC domains can be mechanistically distinguished by specificity of CpG nucleotides preferentially protected from DNA methylation.


Asunto(s)
Islas de CpG , ADN/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Línea Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , ADN/genética , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Leucemia Experimental/genética , Leucemia Experimental/metabolismo , Leucemia Experimental/patología , Lisina/metabolismo , Metilación , Ratones , Datos de Secuencia Molecular , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Unión Proteica , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
17.
Am J Physiol Heart Circ Physiol ; 300(6): H2155-60, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21460195

RESUMEN

The cellular mechanism underlying the Frank-Starling law of the heart is myofilament length-dependent activation. The mechanism(s) whereby sarcomeres detect changes in length and translate this into increased sensitivity to activating calcium has been elusive. Small-angle X-ray diffraction studies have revealed that the intact myofilament lattice undergoes numerous structural changes upon an increase in sarcomere length (SL): lattice spacing and the I(1,1)/I(1,0) intensity ratio decreases, whereas the M3 meridional reflection intensity (I(M3)) increases, concomitant with increases in diastolic and systolic force. Using a short (∼10 ms) X-ray exposure just before electrical stimulation, we were able to obtain detailed structural information regarding the effects of external osmotic compression (with mannitol) and obtain SL on thin intact electrically stimulated isolated rat right ventricular trabeculae. We show that over the same incremental increases in SL, the relative changes in systolic force track more closely to the relative changes in myosin head orientation (as reported by I(M3)) than to the relative changes in lattice spacing. We conclude that myosin head orientation before activation determines myocardial sarcomere activation levels and that this may be the dominant mechanism for length-dependent activation.


Asunto(s)
Citoesqueleto de Actina/diagnóstico por imagen , Corazón/fisiología , Cadenas Pesadas de Miosina/química , Miosinas/química , Volumen Sistólico/fisiología , Citoesqueleto de Actina/fisiología , Animales , Estimulación Eléctrica , Masculino , Modelos Animales , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Miosinas/metabolismo , Radiografía , Ratas , Ratas Endogámicas , Sarcómeros/diagnóstico por imagen , Sarcómeros/fisiología , Difracción de Rayos X
18.
Biophys J ; 99(9): 2978-86, 2010 Nov 03.
Artículo en Inglés | MEDLINE | ID: mdl-21044595

RESUMEN

Length-dependent activation (LDA) is a prominent feature of cardiac muscle characterized by decreases in the Ca(2+) levels required to generate force (i.e., increases in Ca(2+) sensitivity) when muscle is stretched. Previous studies have concluded that LDA originates from the increased ability of (strong) cross-bridges to attach when muscle is lengthened, which in turn enhances Ca(2+) binding to the troponin C (TnC) subunit of the troponin complex. However, our results demonstrate that inhibition of strong cross-bridge attachment with blebbistatin had no effect on the length-dependent modulation of Ca(2+) sensitivity (i.e., EC(50)) or Ca(2+) cooperativity, suggesting that LDA originates upstream of cross-bridge attachment. To test whether LDA arises from length dependence of thin-filament activation, we replaced native cTnC with a mutant cTnC (DM-TnC) that is incapable of binding Ca(2+). Although progressive replacement of native cTnC with DM-TnC caused an expected monotonic decrease in the maximal force (F(max)), DM-TnC incorporation induced much larger increases in EC(50) and decreases in Ca(2+) cooperativity at short lengths than at long lengths. These findings support the conclusion that LDA arises primarily from the influence of length on the modulation of the Ca(2+) cooperativity arising from interaction between adjacent troponin-tropomyosin complexes on the thin filament.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Señalización del Calcio/fisiología , Contracción Miocárdica/fisiología , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Fenómenos Biofísicos , Cartilla de ADN/genética , Humanos , Técnicas In Vitro , Masculino , Microscopía Confocal , Modelos Cardiovasculares , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Miocardio/metabolismo , Ratas , Ratas Sprague-Dawley , Tropomiosina/metabolismo , Troponina C/genética , Troponina C/metabolismo
19.
Circ Res ; 101(2): 195-204, 2007 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-17556659

RESUMEN

It is becoming clear that upregulated protein kinase C (PKC) signaling plays a role in reduced ventricular myofilament contractility observed in congestive heart failure. However, data are scant regarding which PKC isozymes are involved. There is evidence that PKC-alpha may be of particular importance. Here, we examined PKC-alpha quantity, activity, and signaling to myofilaments in chronically remodeled myocytes obtained from rats in either early heart failure or end-stage congestive heart failure. Immunoblotting revealed that PKC-alpha expression and activation was unaltered in early heart failure but increased in end-stage congestive heart failure. Left ventricular myocytes were isolated by mechanical homogenization, Triton-skinned, and attached to micropipettes that projected from a force transducer and motor. Myofilament function was characterized by an active force-[Ca(2+)] relation to obtain Ca(2+)-saturated maximal force (F(max)) and myofilament Ca(2+) sensitivity (indexed by EC(50)) before and after incubation with PKC-alpha, protein phosphatase type 1 (PP1), or PP2a. PKC-alpha treatment induced a 30% decline in F(max) and 55% increase in the EC(50) in control cells but had no impact on myofilament function in failing cells. PP1-mediated dephosphorylation increased F(max) (15%) and decreased EC(50) ( approximately 20%) in failing myofilaments but had no effect in control cells. PP2a-dependent dephosphorylation had no effect on myofilament function in either group. Lastly, PP1 dephosphorylation restored myofilament function in control cells hyperphosphorylated with PKC-alpha. Collectively, our results suggest that in end-stage congestive heart failure, the myofilament proteins exist in a hyperphosphorylated state attributable, in part, to increased activity and signaling of PKC-alpha.


Asunto(s)
Citoesqueleto de Actina/enzimología , Insuficiencia Cardíaca/enzimología , Contracción Muscular , Miocitos Cardíacos/enzimología , Proteína Quinasa C-alfa/metabolismo , Transducción de Señal , Citoesqueleto de Actina/patología , Animales , Calcio/metabolismo , Calcio/farmacología , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Insuficiencia Cardíaca/patología , Ventrículos Cardíacos/enzimología , Ventrículos Cardíacos/patología , Contracción Muscular/efectos de los fármacos , Miocitos Cardíacos/patología , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación/efectos de los fármacos , Proteína Quinasa C-alfa/farmacología , Proteína Fosfatasa 2 , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/efectos de los fármacos , Transducción de Señal/efectos de los fármacos
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