RESUMEN

BACKGROUND: NEK10, a serine/threonine/tyrosine kinase belonging to the NEK (NIMA-related kinases) family, has been associated with diverse cellular processes. However, no specific target pathways have been identified. Our previous work knocking down NEK10 in HeLa cells suggested a functional association with mitochondria, as we observed altered mitochondrial morphology, mitochondrial oxygen consumption, mtDNA integrity, and reactive oxygen species levels. METHODS: To better understand this association, we studied human HAP1 cells fully knockout for NEK10 and confirmed that NEK10 has an important role in mitochondrial homeostasis. We performed the study of mitochondrial respiration, mitochondrial morphology, mitochondrial mass, and mtDNA analysis. Additionally, we showed proteome and phosphoproteome data of crude mitochondrial fraction of Parental and NEK10 KO cells using liquid chromatography-mass spectrometry (LC-MS/MS). RESULTS: In the absence of NEK10 several mitochondrial functions were disturbed. Moreover, proteome and phosphoproteome analyses of mitochondrial fractions showed that NEK10 alters the threonine phosphorylation status of several mitochondrial/endoplasmic reticulum components, including HSP60, NDUFB4, and TOM20. These changes impacted the steady-state levels of a larger group of proteins, preferentially involving respiratory complexes and autophagy pathways. CONCLUSION: We concluded that NEK10 plays a key role in mitochondrial function, possibly by modulating the phosphorylation status of mitochondrial proteins.

RESUMEN

Mayaro virus (MAYV) is an emerging arthropod-borne virus endemic in Latin America and the causative agent of arthritogenic febrile disease. Mayaro fever is poorly understood; thus, we established an in vivo model of infection in susceptible type-I interferon receptor-deficient mice (IFNAR-/-) to characterize the disease. MAYV inoculations in the hind paws of IFNAR-/- mice result in visible paw inflammation, evolve into a disseminated infection and involve the activation of immune responses and inflammation. The histological analysis of inflamed paws indicated edema at the dermis and between muscle fibers and ligaments. Paw edema affected multiple tissues and was associated with MAYV replication, the local production of CXCL1 and the recruitment of granulocytes and mononuclear leukocytes to muscle. We developed a semi-automated X-ray microtomography method to visualize both soft tissue and bone, allowing for the quantification of MAYV-induced paw edema in 3D with a voxel size of 69 µm3. The results confirmed early edema onset and spreading through multiple tissues in inoculated paws. In conclusion, we detailed features of MAYV-induced systemic disease and the manifestation of paw edema in a mouse model extensively used to study infection with alphaviruses. The participation of lymphocytes and neutrophils and expression of CXCL1 are key features in both systemic and local manifestations of MAYV disease.

Asunto(s)

Infecciones por Alphavirus , Alphavirus , Animales , Ratones , Infecciones por Alphavirus/patología , Inflamación , Sincrotrones , Microtomografía por Rayos X

RESUMEN

Although macrophages have long been considered key players in the course of Leishmania infections, other non-professional phagocytes have lately been shown to maintain low levels of the parasite in safe intracellular niches. Recently, it was demonstrated that the adipose tissue is capable of harboring Old World L. (L.) infantum in mice. However, there is no evidence of experimental adipocyte infection with New World Leishmania species so far. In addition, it was not known whether adipocytes would be permissive for formation of the unique, large and communal parasitophorous vacuoles that are typical of L. (L.) amazonensis in macrophages. Here we evaluated the ability of L. (L.) amazonensis and L. (V.) braziliensis promastigotes and amastigotes to infect 3T3-L1 fibroblast-derived adipocytes (3T3-Ad) using light and transmission electron microscopy. Our results indicate that amastigotes and promastigotes of both species were capable of infecting and surviving inside pre- and fully differentiated 3T3-Ad for up to 144 h. Importantly, L. (L.) amazonensis amastigotes resided in large communal parasitophorous vacuoles in pre-adipocytes, which appeared to be compressed between large lipid droplets in mature adipocytes. In parallel, individual L. (V.) braziliensis amastigotes were detected in single vacuoles 144 h post-infection. We conclude that 3T3-Ad may constitute an environment that supports low loads of viable parasites perhaps contributing to parasite maintenance, since amastigotes of both species recovered from these cells differentiated into replicative promastigotes. Our findings shed light on the potential of a new host cell model that can be relevant to the persistence of New World Leishmania species.

Asunto(s)

Leishmania , Leishmaniasis Cutánea , Leishmaniasis , Células 3T3-L1 , Adipocitos , Animales , Leishmaniasis/parasitología , Ratones , Ratones Endogámicos BALB C

RESUMEN

Preserving morphological features that are important for cell function and structure is a critical parameter for in vitro experiments with rat cardiomyocytes. Lentiviral vectors are commonly used as gene transfer tool because of its high flexibility, efficiency to deliver expression cassettes and versatility of transducing quiescent cells. The tropism of the recombinant viral particle can be determined depending on the virus envelope, which shows a specific binding to cell surface receptors on the target cell. The combination of promoter arrangement and viral envelope must be optimized to achieve a greater transduction efficiency and a higher transgene expression. In this study we explored the optimization of promoters and heterologous envelopes to transduce primary culture of neonatal rat ventricular myocytes. Our results suggest a robust expression driven by the cytomegalovirus promoter, and high efficiency transduction mediated by VSV-G envelope with no apparent compromising ultrastructural features of genetically modified cells.

Asunto(s)

Lentivirus/genética , Miocitos Cardíacos/citología , Transducción Genética/métodos , Animales , Animales Recién Nacidos , Células Cultivadas , Citomegalovirus/genética , Expresión Génica , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Glicoproteínas de Membrana/genética , Miocitos Cardíacos/metabolismo , Regiones Promotoras Genéticas , Ratas , Sarcómeros/ultraestructura , Transgenes , Proteínas del Envoltorio Viral/genética , Pseudotipado Viral

RESUMEN

The inhibition of immunosuppressive mechanisms may switch the balance between tolerance and surveillance, leading to an increase in antitumor activity. Regulatory T cells play an important role in the control of immunosuppression, exhibiting the unique property of inhibiting T cell proliferation. These cells migrate to tumor sites or may be generated at the tumor site itself from the conversion of lymphocytes exposed to tumor microenvironment signaling. Because of the high similarity between regulatory T cells and other lymphocytes, the available approaches to inhibit this population are nonspecific and may antagonize antitumor response. In this work we explore a new strategy for inhibition of regulatory T cells based on the use of a chimeric aptamer targeting a marker of immune activation harboring a small antisense RNA molecule for transcriptional gene silencing of Fox p 3, which is essential for the control of the immunosuppressive phenotype. The silencing of Fox p 3 inhibits the immunosuppressive phenotype of regulatory T cells and potentiates the effect of the GVAX antitumor vaccine in immunocompetent animals challenged with syngeneic tumors. This novel approach highlights an alternative method to antagonize regulatory T cell function to augment antitumor immune responses.

RESUMEN

Smooth muscle cells (SMCs) are dynamic and transition from a contractile to a synthetic phenotype under different circumstances. Plasma factors (fibrin and transforming growth factors, TGFs) are possible components affecting SMCs differentiation and behavior. Thus, the objective of this work was to investigate how the fibrin matrix and TGFs affect SMCs differentiation and motility behavior. SMCs invaded the fibrin gel and adopted a stellate phenotype while reducing the expression of differentiation markers (Acta2, Myh11, and Smtn). At the ultrastructural level, SMCs did not assemble a basal lamina and showed numerous blebs along the entire cell surface. This transition was not associated with changes in focal adhesion kinase (FAK) content and phosphorylation status but reflected a marked change in FAK distribution in the cytoplasm. After 48 h in culture, SMCs caused an active degradation of the fibrin gel. Additionally, we tested the SMCs response to TGFs in a cell layer wound repair assay. TGFα, but not TGFß1 or TGFß3, had significantly increased motility. In conclusion, prostatic SMCs present a phenotypical transition when cultured on fibrin, adopting a micro-blebbing based motility behavior and increasing migration in response to TGFα.

RESUMEN

The eukaryotic translation initiation factor 5A (eIF5A) is the only known protein containing the amino acid residue hypusine, essential for its activity. Hypusine residue is produced by a posttranslational modification involving deoxyhypusine synthetase and deoxyhypusine hydroxylase. Herein, we aimed to describe the role of the alternative human isoform A on mitochondrial processes. Isoform A depletion modulates oxidative metabolism in association with the downregulation of mitochondrial biogenesis-related genes. Through positive feedback, it increases cell respiration leading to highly reactive oxygen species production, which impacts mitochondrial bioenergetics. These metabolic changes compromise mitochondrial morphology, increasing its electron density and fission, observed by transmission electron microscopy. This set of changes leads the cells to apoptosis, evidenced by increased DNA fragmentation and proapoptotic BAK protein content increase. Thus, we show that the alternative eIF5A isoform A is crucial for energy metabolism controlled by mitochondria and cellular survival.

Asunto(s)

Mitocondrias/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Apoptosis/fisiología , Humanos , Lisina/análogos & derivados , Lisina/metabolismo , Microscopía Electrónica de Transmisión , Factores de Iniciación de Péptidos/genética , Isoformas de Proteínas/genética , Proteínas de Unión al ARN/genética , Factor 5A Eucariótico de Iniciación de Traducción

RESUMEN

Prostate cancer is a life-threatening condition worldwide. As the tumor progresses, smooth muscle cells (SMCs) become atrophic/dedifferentiated, within a series of stromal changes named stromal reaction. Here, we tested whether a laminin 111-rich extracellular matrix (Lr-ECM) could affect SMCs phenotype and differentiation status. Using time-lapse microscopy, image analyses, quantitative real-time reverse transcription polymerase chain reaction, immunohistochemistry and immunoblotting, and transmission electron microscopy, we showed that SMCs acquires a migratory behavior with a decreased expression of differentiation markers and relocation of focal adhesion kinase. SMCs set homotypic cell junctions and were active in autophagy/phagocytosis. Analysis of the migratory behavior showed that SMCs polarized and migrated toward each other, recognizing long-distance signals such as matrix tensioning. However, half of the cell population were immotile, irrespective of the nearest neighbor distance, suggesting they do not engage in productive interactions, possibly as a result of back-to-back positioning. In conclusion, the Lr-ECM, mimics the effects of the proliferating and infiltrating tumor epithelium, causing SMCs phenotypical change similar to that observed in the stromal reaction, in addition to a hitherto undescribed, stereotyped pattern of cell motility resulting from cell polarization.

Asunto(s)

Miocitos del Músculo Liso , Próstata , Neoplasias de la Próstata , Animales , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Matriz Extracelular , Laminina/metabolismo , Masculino , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Ratas , Ratas Wistar

RESUMEN

To investigate the role of TYRO3, AXL and TIM1 receptors in the Zika virus (ZIKV) cycle, we determined their mRNA expression in different placental sites of ZIKV infected tissue during pregnancy. Unexpectedly, the ZIKV infection was not related with mRNA upregulation of these receptors or changes in expression of type I and III interferons in different placental sites. Instead, a decrease of TYRO3 mRNA expression was observed in positive sites of ZIKV positive placentas in comparison to negative sites. The basis of this downregulation can help to understand how ZIKV persists in placental tissue during pregnancy.

Asunto(s)

Receptor Celular 1 del Virus de la Hepatitis A/metabolismo , Placenta/enzimología , Complicaciones Infecciosas del Embarazo/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Infección por el Virus Zika/metabolismo , Estudios de Casos y Controles , Femenino , Interacciones Huésped-Patógeno , Humanos , Interferón Tipo I/metabolismo , Interferones/metabolismo , Placenta/inmunología , Placenta/virología , Embarazo , Complicaciones Infecciosas del Embarazo/inmunología , Complicaciones Infecciosas del Embarazo/virología , Virus Zika/fisiología , Infección por el Virus Zika/inmunología , Interferón lambda , Tirosina Quinasa del Receptor Axl

RESUMEN

BACKGROUND: A pathophysiological link exists between dysregulation of MEF2C transcription factors and heart failure (HF), but the underlying mechanisms remain elusive. Alternative splicing of MEF2C exons α, ß and γ provides transcript diversity with gene activation or repression functionalities. METHODS: Neonatal and adult rat ventricular myocytes were used to overexpress MEF2C splicing variants γ+ (repressor) or γ-, or the inactive MEF2Cγ+23/24 (K23T/R24L). Phenotypic alterations in cardiomyocytes were determined by confocal and electron microscopy, flow cytometry and DNA microarray. We used transgenic mice with cardiac-specific overexpression of MEF2Cγ+ or MEF2Cγ- to explore the impact of MEF2C variants in cardiac phenotype. Samples of non-infarcted areas of the left ventricle from patients and mouse model of myocardial infarction were used to detect the expression of MEF2Cγ+ in failing hearts. FINDINGS: We demonstrate a previously unrealized upregulation of the transrepressor MEF2Cγ+ isoform in human and mouse failing hearts. We show that adenovirus-mediated overexpression of MEF2Cγ+ downregulates multiple MEF2-target genes, and drives incomplete cell-cycle reentry, partial dedifferentiation and apoptosis in the neonatal and adult rat. None of these changes was observed in cardiomyocytes overexpressing MEF2Cγ-. Transgenic mice overexpressing MEF2Cγ+, but not the MEF2Cγ-, developed dilated cardiomyopathy, correlated to cell-cycle reentry and apoptosis of cardiomyocytes. INTERPRETATION: Our results provide a mechanistic link between MEF2Cγ+ and deleterious abnormalities in cardiomyocytes, supporting the notion that splicing dysregulation in MEF2C towards the selection of the MEF2Cγ+ variant contributes to the pathogenesis of HF by promoting cardiomyocyte dropout. FUNDING: São Paulo Research Foundation (FAPESP); Brazilian National Research Council (CNPq).

Asunto(s)

Ciclo Celular/genética , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Variación Genética , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Empalme Alternativo , Animales , Apoptosis/genética , Modelos Animales de Enfermedad , Estudios de Asociación Genética , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/terapia , Humanos , Factores de Transcripción MEF2/genética , Ratones , Ratones Transgénicos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/ultraestructura , Ratas

RESUMEN

There is no consensus on the effects of omega-3 (ω-3) fatty acids (FA) on cutaneous repair. To solve this problem, we used 2 different approaches: (1) FAT-1 transgenic mice, capable of producing endogenous ω-3 FA; (2) wild-type (WT) mice orally supplemented with DHA-enriched fish oil. FAT-1 mice had higher systemic (serum) and local (skin tissue) ω-3 FA levels, mainly docosahexaenoic acid (DHA), in comparison with WT mice. FAT-1 mice had increased myeloperoxidase (MPO) activity and content of CXCL-1 and CXCL-2, and reduced IL-10 in the skin wound tissue three days after the wound induction. Inflammation was maintained by an elevated TNF-α concentration and presence of inflammatory cells and edema. Neutrophils and macrophages, isolated from FAT-1 mice, also produced increased TNF-α and reduced IL-10 levels. In these mice, the wound closure was delayed, with a wound area 6-fold bigger in relation with WT group, on the last day of analysis (14 days post-wounding). This was associated with poor orientation of collagen fibers and structural aspects in repaired tissue. Similarly, DHA group had a delay during late inflammatory phase. This group had increased TNF-α content and CD45+F4/80+ cells at the third day after skin wounding and increased concentrations of important metabolites derived from ω-3, like 18-HEPE, and reduced concentrations of those from ω-6 FA. In conclusion, elevated DHA content, achieved in both FAT-1 and DHA groups, slowed inflammation resolution and impaired the quality of healed skin tissue.

Asunto(s)

Ácidos Docosahexaenoicos/fisiología , Cicatrización de Heridas , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Suplementos Dietéticos , Ácido Graso Desaturasas/genética , Inflamación , Macrófagos/fisiología , Masculino , Ratones Transgénicos , Neutrófilos/fisiología , Piel/metabolismo

RESUMEN

Wound healing is an essential process for organism survival. Some fatty acids have been described as modulators of wound healing. However, the role of omega-3 fatty acids is unclear. In the present work, we investigate the effects of oral administration of eicosapentaenoic acid (EPA)-rich oil on wound healing in mice. After 4 weeks of EPA-rich oil supplementation (2 g/kg of body weight), mice had increased serum concentrations of EPA (20:5ω-3) (6-fold) and docosahexaenoic acid (DHA; 22:6ω-3) (33%) in relation to control mice. Omega-3 fatty acids were also incorporated into skin in the EPA fed mice. The wound healing process was delayed at the 3rd and 7th days after wounding in mice that received EPA-rich oil when compared to control mice but there was no effect on the total time required for wound closure. Collagen reorganization, that impacts the quality of the wound tissue, was impaired after EPA-rich oil supplementation. These effects were associated with an increase of M2 macrophages (twice in relation to control animals) and interleukin-10 (IL-10) concentrations in tissue in the initial stages of wound healing. In the absence of IL-10 (IL-10-/- mice), wound closure and organization of collagen were normalized even when EPA was fed, supporting that the deleterious effects of EPA-rich oil supplementation were due to the excessive production of IL-10. In conclusion, oral administration of EPA-rich oil impairs the quality of wound healing without affecting the wound closure time likely due to an elevation of the anti-inflammatory cytokine IL-10.

Asunto(s)

Colágeno/metabolismo , Ácido Eicosapentaenoico/análisis , Ácido Eicosapentaenoico/farmacología , Interleucina-10/biosíntesis , Aceites/química , Piel/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Administración Oral , Animales , Ácido Eicosapentaenoico/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL

RESUMEN

Appropriate remodeling of the female lower reproductive tract and pelvic floor is essential during normal mammalian pregnancy, labor, and postpartum recovery. During mouse pregnancy, in addition to reproductive tract modifications, the pubic symphysis (PS) is remodeled into a soft interpubic ligament (IpL) to provide safe delivery of the offspring and fast postpartum recovery. Although temporal changes in the phenotypes of myeloid cells, such as mononuclear phagocytes, are crucial to remodeling the lower reproductive tract organs in preparation for a safe delivery, little is known about the involvement of recruited monocytes or macrophages in mouse PS remodeling. We used combined light microscopy, electron microscopy, and qPCR analysis to investigate the profile of recruited monocytes and macrophage polarization markers in C57Bl6 mouse interpubic tissues during pregnancy (D12, D18, and D19) and early days postpartum (1 dpp and 3 dpp) to better identify their presence in proper remodeling of the mouse PS. Our morphological data show that the number of recruited monocytes is increased in interpubic tissues and that recruited monocytes differentiate into proinflammatory or anti-inflammatory macrophage phenotypes from D18 to 3 dpp, which may contribute to dynamic changes in the gene expression of specific inflammatory mediators involved in interpubic tissue remodeling at these time points. Therefore, our morphological and quantitative gene expression data suggest that both differentiated macrophages from recruited monocytes and polarized macrophages may collaborate for IpL relaxation at labor and the appropriate repair of the PS after the first pregnancy.

Asunto(s)

Macrófagos/fisiología , Monocitos/fisiología , Periodo Posparto/fisiología , Sínfisis Pubiana/fisiología , Animales , Femenino , Ligamentos/citología , Ratones , Embarazo

RESUMEN

AIMS: A disintegrin and metalloprotease 17 (ADAM17) modulates signaling events by releasing surface protein ectodomains such as TNFa and the EGFR-ligands. We have previously characterized cytoplasmic thioredoxin-1 (Trx-1) as a partner of ADAM17 cytoplasmic domain. Still, the mechanism of ADAM17 regulation by Trx-1 is unknown, and it has become of paramount importance to assess the degree of influence that Trx-1 has on metalloproteinase ADAM17. RESULTS: Combining discovery and targeted proteomic approaches, we uncovered that Trx-1 negatively regulates ADAM17 by direct and indirect effect. We performed cell-based assays with synthetic peptides and site-directed mutagenesis, and we demonstrated that the interaction interface of Trx-1 and ADAM17 is important for the negative regulation of ADAM17 activity. However, both Trx-1K72A and catalytic site mutant Trx-1C32/35S rescued ADAM17 activity, although the interaction with Trx-1C32/35S was unaffected, suggesting an indirect effect of Trx-1. We confirmed that the Trx-1C32/35S mutant showed diminished reductive capacity, explaining this indirect effect on increasing ADAM17 activity through oxidant levels. Interestingly, Trx-1K72A mutant showed similar oxidant levels to Trx-1C32/35S, even though its catalytic site was preserved. We further demonstrated that the general reactive oxygen species inhibitor, Nacetylcysteine (NAC), maintained the regulation of ADAM17 dependent of Trx-1 reductase activity levels; whereas the electron transport chain modulator, rotenone, abolished Trx-1 effect on ADAM17 activity. INNOVATION: We show for the first time that the mechanism of ADAM17 regulation, Trx-1 dependent, can be by direct interaction and indirect effect, bringing new insights into the cross-talk between isomerases and mammalian metalloproteinases. CONCLUSION: This unexpected Trx-1K72A behavior was due to more dimer formation and, consequently, the reduction of its Trx-1 reductase activity, evaluated through dimer verification, by gel filtration and mass spectrometry analysis. Antioxid. Redox Signal. 29, 717-734.

Asunto(s)

Proteína ADAM17/metabolismo , Tiorredoxinas/metabolismo , Sitios de Unión , Células HEK293 , Humanos , Modelos Moleculares , Oxidación-Reducción , Tiorredoxinas/análisis , Tiorredoxinas/genética , Células Tumorales Cultivadas

RESUMEN

Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here we show that αB-crystallin interacts with and confers protection to FAK against calpain-mediated proteolysis in cardiomyocytes. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the ß4-ß8 groove of αB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation. αB-crystallin silencing results in calpain-dependent FAK depletion and in the increased apoptosis of cardiomyocytes in response to mechanical stress. FAK overexpression protects cardiomyocytes depleted of αB-crystallin against the stretch-induced apoptosis. Consistently, load-induced apoptosis is blunted in the hearts from cardiac-specific FAK transgenic mice transiently depleted of αB-crystallin by RNA interference. These studies define a role for αB-crystallin in controlling FAK function and cardiomyocyte survival through the prevention of calpain-mediated degradation of FAK.

Asunto(s)

Calpaína/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Regulación Enzimológica de la Expresión Génica , Miocitos Cardíacos/citología , Cadena B de alfa-Cristalina/química , Animales , Aorta/metabolismo , Apoptosis , Supervivencia Celular , Transferencia Resonante de Energía de Fluorescencia , Silenciador del Gen , Homeostasis , Masculino , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Modelos Moleculares , Miocardio/metabolismo , Fosforilación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Estrés Mecánico , Familia-src Quinasas/metabolismo