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1.
Nat Commun ; 11(1): 5063, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033242

RESUMO

Genome-wide chromatin state underlies gene expression potential and cellular function. Epigenetic features and nucleosome positioning contribute to the accessibility of DNA, but widespread regulators of chromatin state are largely unknown. Our study investigates how coordination of ANP32E and H2A.Z contributes to genome-wide chromatin state in mouse fibroblasts. We define H2A.Z as a universal chromatin accessibility factor, and demonstrate that ANP32E antagonizes H2A.Z accumulation to restrict chromatin accessibility genome-wide. In the absence of ANP32E, H2A.Z accumulates at promoters in a hierarchical manner. H2A.Z initially localizes downstream of the transcription start site, and if H2A.Z is already present downstream, additional H2A.Z accumulates upstream. This hierarchical H2A.Z accumulation coincides with improved nucleosome positioning, heightened transcription factor binding, and increased expression of neighboring genes. Thus, ANP32E dramatically influences genome-wide chromatin accessibility through subtle refinement of H2A.Z patterns, providing a means to reprogram chromatin state and to hone gene expression levels.


Assuntos
Cromatina/metabolismo , Genoma , Chaperonas Moleculares/metabolismo , Animais , Diferenciação Celular/genética , DNA Helicases/metabolismo , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/metabolismo
2.
Nat Commun ; 11(1): 5061, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033262

RESUMO

The interplay between the Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC) and transcriptional/epigenetic co-regulators in somatic cell reprogramming is incompletely understood. Here, we demonstrate that the histone H3 lysine 27 trimethylation (H3K27me3) demethylase JMJD3 plays conflicting roles in mouse reprogramming. On one side, JMJD3 induces the pro-senescence factor Ink4a and degrades the pluripotency regulator PHF20 in a reprogramming factor-independent manner. On the other side, JMJD3 is specifically recruited by KLF4 to reduce H3K27me3 at both enhancers and promoters of epithelial and pluripotency genes. JMJD3 also promotes enhancer-promoter looping through the cohesin loading factor NIPBL and ultimately transcriptional elongation. This competition of forces can be shifted towards improved reprogramming by using early passage fibroblasts or boosting JMJD3's catalytic activity with vitamin C. Our work, thus, establishes a multifaceted role for JMJD3, placing it as a key partner of KLF4 and a scaffold that assists chromatin interactions and activates gene transcription.


Assuntos
Reprogramação Celular , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Catálise , Proliferação de Células , Senescência Celular , Desmetilação , Elementos Facilitadores Genéticos/genética , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Histonas/metabolismo , Lisina/metabolismo , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas , Ativação Transcricional/genética
3.
Nat Commun ; 11(1): 4480, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900992

RESUMO

Macroautophagy initiates by formation of isolation membranes, but the source of phospholipids for the membrane biogenesis remains elusive. Here, we show that autophagic membranes incorporate newly synthesized phosphatidylcholine, and that CTP:phosphocholine cytidylyltransferase ß3 (CCTß3), an isoform of the rate-limiting enzyme in the Kennedy pathway, plays an essential role. In starved mouse embryo fibroblasts, CCTß3 is initially recruited to autophagic membranes, but upon prolonged starvation, it concentrates on lipid droplets that are generated from autophagic degradation products. Omegasomes and isolation membranes emanate from around those lipid droplets. Autophagy in prolonged starvation is suppressed by knockdown of CCTß3 and is enhanced by its overexpression. This CCTß3-dependent mechanism is also present in U2OS, an osteosarcoma cell line, and autophagy and cell survival in starvation are decreased by CCTß3 depletion. The results demonstrate that phosphatidylcholine synthesis through CCTß3 activation on lipid droplets is crucial for sustaining autophagy and long-term cell survival.


Assuntos
Autofagia/fisiologia , Colina-Fosfato Citidililtransferase/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Animais , Autofagossomos/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Colina-Fosfato Citidililtransferase/antagonistas & inibidores , Colina-Fosfato Citidililtransferase/genética , Meios de Cultura , Ativação Enzimática , Técnicas de Silenciamento de Genes , Humanos , Gotículas Lipídicas/metabolismo , Camundongos , Modelos Biológicos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Fosfatidilcolinas/metabolismo
5.
Int J Nanomedicine ; 15: 4943-4956, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764927

RESUMO

Background: Hydroxyapatite (HA) [Ca5(PO4)3(OH)] is a naturally occurring calcium phosphate which makes up 60-70% of the dry weight of human bones. Nano-scale HA particles are increasingly being used as carriers for controlled and targeted delivery of bioactive agents like drugs, proteins, and nucleic acids due to their high porosity, negative charge, and biodegradability. Purpose: Although much effort has been devoted to understanding the delivery kinetics and effects of the payloads in such carriers, a thorough understanding of the influence of the carriers themselves is lacking. Methods: HA particles (300 µg/mL) were administered to primary human dermal fibroblasts (HDFs). The uptake and intracellular localization of the particles were determined by flow cytometry, confocal imaging, and transmission electron microscopy (TEM). Immunological assays and PCR were performed to determine the levels of pro-inflammatory cytokines and collagens in cell lysates and media supernatant. Results: The current study explores the effects of poly-dispersed HA particles on primary HDFs as a model system. The majority of the particles were determined to range between 150 and 200 nm in diameter. Upon exposure to HA suspensions, primary HDFs internalized the particles by endocytosis within 6 hours of exposure, showing maximum uptake at 72 hours following which the particles were exocytosed by 168 hours. This correlated to reduced secretion of various pro-inflammatory and pro-collagenic cytokines. Biochemical analysis further revealed a reduction in Type I collagen expression and secretion. Conclusion: HA particles have an immune-modulatory effect on dermal fibroblasts and reduce collagen production, which may impact the integrity of the extracellular matrix (ECM). This study demonstrates the need to consider the secondary effects of particulate carriers like HA, beyond basic cytotoxicity, in the specific tissue environment where the intended function is to be realized.


Assuntos
Colágeno/metabolismo , Durapatita/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Pele/citologia , Colágeno Tipo I/metabolismo , Citocinas/metabolismo , Durapatita/química , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Humanos
6.
Int J Nanomedicine ; 15: 5097-5111, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764939

RESUMO

Introduction: In this in-vitro study, we designed a 3D printed composite of zinc oxide (ZnO) nanoparticles (NPs) with photocatalytic activities encapsulated within hydrogel (alginate) constructs, for antibacterial purposes applicable towards wound healing. We primarily sought to confirm the mechanical properties and cell compatibility of these ZnO NP infused scaffolds. Methods: The antibacterial property of the ZnO NPs was confirmed by hydroxyl radical generation using ultraviolet (U.V.) photocatalysis. Titanium dioxide (TiO2), a well-known antibacterial compound, was used as a positive control (1% w/v) for the ZnO NP-based alginate constructs and their antibacterial efficacies compared. Among the ZnO group, 3D printed gels containing 0.5% and 1% w/v of ZnO were analyzed and compared with manually casted samples via SEM, swelling evaluation, and rheological analysis. Envisioning an in-vivo application for the 3D printed ZnO NP-based alginates, we studied their antibacterial properties by bacterial broth testing, cytocompatibility via live/dead assay, and moisture retention capabilities utilizing a humidity sensor. Results: 3D printed constructs revealed significantly greater pore sizes and enhanced structural stability compared to manually casted samples. For all samples, the addition of ZnO or TiO2 resulted in significantly stiffer gels in comparison with the alginate control. Bacterial resistance testing on Staphylococcus epidermidis indicated the addition of ZnO NPs to the gels decreased bacterial growth when compared to the alginate only gels. Cell viability of STO-fibroblasts was not adversely affected by the addition of ZnO NPs to the alginate gels. Furthermore, the addition of increasing doses of ZnO NPs to the alginate demonstrated increased humidity retention in gels. Discussion: The customization of 3D printed alginates containing antibacterial ZnO NPs leads to an alternative that allows accessible mobility of molecular exchange required for improving chronic wound healing. This scaffold can provide a cost-effective and durable antibacterial treatment option.


Assuntos
Alginatos/química , Alginatos/farmacologia , Hidrogéis/química , Nanopartículas/química , Cicatrização/efeitos dos fármacos , Óxido de Zinco/química , Sobrevivência Celular/efeitos dos fármacos , Fibroblastos/citologia
7.
Mol Cell ; 79(4): 660-676.e8, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32755593

RESUMO

Specific combinations of two transcription factors (Hnf4α plus Foxa1, Foxa2, or Foxa3) can induce direct conversion of mouse fibroblasts into hepatocyte-like cells. However, the molecular mechanisms underlying hepatic reprogramming are largely unknown. Here, we show that the Foxa protein family members and Hnf4α sequentially and cooperatively bind to chromatin to activate liver-specific gene expression. Although all Foxa proteins bind to and open regions of closed chromatin as pioneer factors, Foxa3 has the unique potential of transferring from the distal to proximal regions of the transcription start site of target genes, binding RNA polymerase II, and co-traversing target genes. These distinctive characteristics of Foxa3 are essential for inducing the hepatic fate in fibroblasts. Similar functional coupling of transcription factors to RNA polymerase II may occur in other contexts whereby transcriptional activation can induce cell differentiation.


Assuntos
Fator 3-gama Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Fígado/citologia , Fígado/fisiologia , Ativação Transcricional , Animais , Sítios de Ligação , Células Cultivadas , Reprogramação Celular/fisiologia , Cromatina/metabolismo , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Fibroblastos/citologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Fator 3-gama Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/genética , Camundongos Endogâmicos C57BL , Domínios Proteicos , Sítio de Iniciação de Transcrição
8.
Nat Commun ; 11(1): 3953, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769974

RESUMO

Many important cell types in adult vertebrates have a mesenchymal origin, including fibroblasts and vascular mural cells. Although their biological importance is undisputed, the level of mesenchymal cell heterogeneity within and between organs, while appreciated, has not been analyzed in detail. Here, we compare single-cell transcriptional profiles of fibroblasts and vascular mural cells across four murine muscular organs: heart, skeletal muscle, intestine and bladder. We reveal gene expression signatures that demarcate fibroblasts from mural cells and provide molecular signatures for cell subtype identification. We observe striking inter- and intra-organ heterogeneity amongst the fibroblasts, primarily reflecting differences in the expression of extracellular matrix components. Fibroblast subtypes localize to discrete anatomical positions offering novel predictions about physiological function(s) and regulatory signaling circuits. Our data shed new light on the diversity of poorly defined classes of cells and provide a foundation for improved understanding of their roles in physiological and pathological processes.


Assuntos
Diferenciação Celular , Fibroblastos/fisiologia , Células-Tronco Mesenquimais/fisiologia , Miócitos de Músculo Liso/fisiologia , Pericitos/fisiologia , Animais , Separação Celular , Vasos Coronários/citologia , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Citometria de Fluxo , Intestinos/irrigação sanguínea , Intestinos/citologia , Masculino , Camundongos , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/citologia , Músculo Liso Vascular/citologia , Miocárdio/citologia , Miócitos de Músculo Liso/citologia , Pericitos/citologia , RNA-Seq , Análise de Célula Única , Bexiga Urinária/irrigação sanguínea , Bexiga Urinária/citologia
9.
Phys Rev Lett ; 125(6): 068101, 2020 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-32845697

RESUMO

Shape, dynamics, and viscoelastic properties of eukaryotic cells are primarily governed by a thin, reversibly cross-linked actomyosin cortex located directly beneath the plasma membrane. We obtain time-dependent rheological responses of fibroblasts and MDCK II cells from deformation-relaxation curves using an atomic force microscope to access the dependence of cortex fluidity on prestress. We introduce a viscoelastic model that treats the cell as a composite shell and assumes that relaxation of the cortex follows a power law giving access to cortical prestress, area-compressibility modulus, and the power law exponent (fluidity). Cortex fluidity is modulated by interfering with myosin activity. We find that the power law exponent of the cell cortex decreases with increasing intrinsic prestress and area-compressibility modulus, in accordance with previous finding for isolated actin networks subject to external stress. Extrapolation to zero tension returns the theoretically predicted power law exponent for transiently cross-linked polymer networks. In contrast to the widely used Hertzian mechanics, our model provides viscoelastic parameters independent of indenter geometry and compression velocity.


Assuntos
Actinas/química , Fibroblastos/química , Fibroblastos/citologia , Modelos Biológicos , Actinas/fisiologia , Animais , Fenômenos Biomecânicos , Linhagem Celular , Membrana Celular/química , Membrana Celular/fisiologia , Força Compressiva , Cães , Elasticidade , Microscopia de Força Atômica , Miosinas/química , Miosinas/fisiologia , Reologia/métodos , Viscosidade
10.
PLoS One ; 15(8): e0237040, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764823

RESUMO

As type-I-allergies show an increasing prevalence in the general populace, orthodontic patients may also be affected by histamine release during treatment. Human periodontal ligament fibroblasts (PDLF) are regulators of orthodontic tooth movement. However, the impact of histamine on PDLF in this regard is unknown. Therefore PDLF were incubated without or with an orthodontic compressive force of 2g/cm2 with and without additional histamine. To assess the role of histamine-1-receptor (H1R) H1R-antagonist cetirizine was used. Expression of histamine receptors and important mediators of orthodontic tooth movement were investigated. PDLF expressed histamine receptors H1R, H2R and H4R, but not H3R. Histamine increased the expression of H1R, H2R and H4R as well as of interleukin-6, cyclooxygenase-2, and prostaglandin-E2 secretion even without pressure application and induced receptor activator of NF-kB ligand (RANKL) protein expression with unchanged osteoprotegerin secretion. These effects were not observed in presence of H1R antagonist cetirizine. By expressing histamine receptors, PDLF seem to be able to respond to fluctuating histamine levels in the periodontal tissue. Increased histamine concentration was associated with enhanced expression of proinflammatory mediators and RANKL, suggesting an inductive effect of histamine on PDLF-mediated osteoclastogenesis and orthodontic tooth movement. Since cetirizine inhibited these effects, they seem to be mainly mediated via histamine receptor H1R.


Assuntos
Histamina/farmacologia , Ligamento Periodontal/efeitos dos fármacos , Ligamento Periodontal/fisiologia , Técnicas de Movimentação Dentária , Células Cultivadas , Cetirizina/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Expressão Gênica/efeitos dos fármacos , Histamina/fisiologia , Antagonistas dos Receptores Histamínicos H1/farmacologia , Humanos , Mediadores da Inflamação/metabolismo , Modelos Biológicos , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ligamento Periodontal/citologia , Ligante RANK/genética , Ligante RANK/metabolismo , Receptores Histamínicos H1/fisiologia , Estresse Mecânico
11.
PLoS One ; 15(7): e0235965, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701960

RESUMO

Secondary lymphedema (SL)is a frequent and devastating complication of modern oncological therapy and filarial infections. A lack of a reliable preclinical model to investigate the underlying mechanism of clinical stage progression has limited the development of new therapeutic strategies. Current first line treatment has shown to be merely symptomatic and relies on lifetime use of compression garments and decongestive physiotherapy. In this study, we present the development of a secondary lymphedema model in 35 rats using pre- and intraoperative fluorescence-guided mapping of the lymphatics and microsurgical induction. In contrast to the few models reported so far, we decided to avoid the use of radiation for lymphedema induction. It turned out, that the model is nearly free of complications and capable of generating a statistically significant limb volume increase by water displacement measurements, sustained for at least 48 days. A translational, accurate lymphatic dysfunction was visualized by a novel VIS-NIR X-ray ICG-Clearance-Capacity imaging technology. For the first-time SL stage progression was validated by characteristic histological alterations, such as subdermal mast cell infiltration, adipose tissue deposition, and fibrosis by increased skin collagen content. Immunofluorescence confocal microscopy analysis suggested that stage progression is related to the presence of a characteristic α SMA+/HSP-47+/vimentin+ fibroblast subpopulation phenotype. These findings demonstrate that the in-vivo model is a reliable and clinically relevant SL model for the development of further secondary lymphedema therapeutic strategies and the analysis of the veiled molecular mechanisms of lymphatic dysfunction.


Assuntos
Corantes Fluorescentes/química , Linfedema/patologia , Microcirurgia/efeitos adversos , Actinas/metabolismo , Animais , Colágeno/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Verde de Indocianina/química , Linfedema/etiologia , Microscopia de Fluorescência , Ratos , Ratos Endogâmicos Lew , Pele/patologia , Vimentina/metabolismo
12.
Nature ; 583(7815): 296-302, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32612232

RESUMO

The mammalian immune system implements a remarkably effective set of mechanisms for fighting pathogens1. Its main components are haematopoietic immune cells, including myeloid cells that control innate immunity, and lymphoid cells that constitute adaptive immunity2. However, immune functions are not unique to haematopoietic cells, and many other cell types display basic mechanisms of pathogen defence3-5. To advance our understanding of immunology outside the haematopoietic system, here we systematically investigate the regulation of immune genes in the three major types of structural cells: epithelium, endothelium and fibroblasts. We characterize these cell types across twelve organs in mice, using cellular phenotyping, transcriptome sequencing, chromatin accessibility profiling and epigenome mapping. This comprehensive dataset revealed complex immune gene activity and regulation in structural cells. The observed patterns were highly organ-specific and seem to modulate the extensive interactions between structural cells and haematopoietic immune cells. Moreover, we identified an epigenetically encoded immune potential in structural cells under tissue homeostasis, which was triggered in response to systemic viral infection. This study highlights the prevalence and organ-specific complexity of immune gene activity in non-haematopoietic structural cells, and it provides a high-resolution, multi-omics atlas of the epigenetic and transcriptional networks that regulate structural cells in the mouse.


Assuntos
Endotélio/imunologia , Células Epiteliais/imunologia , Fibroblastos/imunologia , Regulação da Expressão Gênica/imunologia , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Especificidade de Órgãos/imunologia , Imunidade Adaptativa , Animais , Cromatina/genética , Cromatina/metabolismo , Endotélio/citologia , Epigênese Genética/imunologia , Epigenoma/genética , Células Epiteliais/citologia , Feminino , Fibroblastos/citologia , Regulação da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/imunologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Sistema Imunitário/virologia , Imunidade Inata , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Masculino , Camundongos , Especificidade de Órgãos/genética , Transcrição Genética/imunologia , Transcriptoma/genética
13.
Int J Nanomedicine ; 15: 4275-4288, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606677

RESUMO

Purpose: Selenium nanoparticles (Se NPs) are promising antibacterial agents to tackle the growing problem of antimicrobial resistance. The aim of this study was to fabricate Se NPs with a net positive charge to enhance their antibacterial efficacy. Methods: Se NPs were coated with a positively charged protein - recombinant spider silk protein eADF4(κ16) - to give them a net positive surface charge. Their cytotoxicity and antibacterial activity were investigated, with negatively charged polyvinyl alcohol coated Se NPs as a control. Besides, these eADF4(κ16)-coated Se NPs were immobilized on the spider silk films, and the antibacterial activity of these films was investigated. Results: Compared to the negatively charged polyvinyl alcohol coated Se NPs, the positively charged eADF4(κ16)-coated Se NPs demonstrated a much higher bactericidal efficacy against the Gram-negative bacteria E. coli, with a minimum bactericidal concentration (MBC) approximately 50 times lower than that of negatively charged Se NPs. Cytotoxicity testing showed that the eADF4(κ16)-coated Se NPs are safe to both Balb/3T3 mouse embryo fibroblasts and HaCaT human skin keratinocytes up to 31 µg/mL, which is much higher than the MBC of these particles against E. coli (8 ± 1 µg/mL). In addition, antibacterial coatings were created by immobilising the eADF4(κ16)-coated Se NPs on positively charged spider silk films and these were shown to retain good bactericidal efficacy and overcome the issue of low particle stability in culture broth. It was found that these Se NPs needed to be released from the film surface in order to exert their antibacterial effects and this release can be regulated by the surface charge of the film, such as the change of the spider silk protein used. Conclusion: Overall, eADF4(κ16)-coated Se NPs are promising new antibacterial agents against life-threatening bacteria.


Assuntos
Antibacterianos/farmacologia , Nanopartículas/química , Proteínas Recombinantes/farmacologia , Selênio/farmacologia , Seda/farmacologia , Células 3T3 , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Contagem de Colônia Microbiana , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Nanopartículas/ultraestrutura , Tamanho da Partícula
14.
Proc Natl Acad Sci U S A ; 117(28): 16500-16508, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601199

RESUMO

Despite the implementation of multiple HER2-targeted therapies, patients with advanced HER2+ breast cancer ultimately develop drug resistance. Stromal fibroblasts represent an abundant cell type in the tumor microenvironment and have been linked to poor outcomes and drug resistance. Here, we show that fibroblasts counteract the cytotoxic effects of HER2 kinase-targeted therapy in a subset of HER2+ breast cancer cell lines and allow cancer cells to proliferate in the presence of the HER2 kinase inhibitor lapatinib. Fibroblasts from primary breast tumors, normal breast tissue, and lung tissue have similar protective effects on tumor cells via paracrine factors. This fibroblast-mediated reduction in drug sensitivity involves increased expression of antiapoptotic proteins and sustained activation of the PI3K/AKT/MTOR pathway, despite inhibition of the HER2 and the RAS-ERK pathways in tumor cells. HER2 therapy sensitivity is restored in the fibroblast cocultures by combination treatment with inhibitors of MTOR or the antiapoptotic proteins BCL-XL and MCL-1. Expression of activated AKT in tumor cells recapitulates the effects of fibroblasts resulting in sustained MTOR signaling and poor lapatinib response. Lapatinib sensitivity was not altered by fibroblasts in tumor cells that exhibited sustained MTOR signaling due to a strong gain-of-function PI3KCA mutation. These findings indicate that in addition to tumor cell-intrinsic mechanisms that cause constitutive PI3K/AKT/MTOR pathway activation, secreted factors from fibroblasts can maintain this pathway in the context of HER2 inhibition. Our integrated proteomic-phenotypic approach presents a strategy for the discovery of protective mechanisms in fibroblast-rich tumors and the design of rational combination therapies to restore drug sensitivity.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Fibroblastos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/fisiopatologia , Linhagem Celular Tumoral , Feminino , Fibroblastos/citologia , Fibroblastos/enzimologia , Humanos , Lapatinib/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética
15.
PLoS One ; 15(7): e0235655, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628740

RESUMO

Biallelic variants in RNU4ATAC, a non-coding gene transcribed into the minor spliceosome component U4atac snRNA, are responsible for three rare recessive developmental diseases, namely Taybi-Linder/MOPD1, Roifman and Lowry-Wood syndromes. Next-generation sequencing of clinically heterogeneous cohorts (children with either a suspected genetic disorder or a congenital microcephaly) recently identified mutations in this gene, illustrating how profoundly these technologies are modifying genetic testing and assessment. As RNU4ATAC has a single non-coding exon, the bioinformatic prediction algorithms assessing the effect of sequence variants on splicing or protein function are irrelevant, which makes variant interpretation challenging to molecular diagnostic laboratories. In order to facilitate and improve clinical diagnostic assessment and genetic counseling, we present i) an update of the previously reported RNU4ATAC mutations and an analysis of the genetic variations affecting this gene using the Genome Aggregation Database (gnomAD) resource; ii) the pathogenicity prediction performances of scores computed based on an RNA structure prediction tool and of those produced by the Combined Annotation Dependent Depletion tool for the 285 RNU4ATAC variants identified in patients or in large-scale sequencing projects; iii) a method, based on a cellular assay, that allows to measure the effect of RNU4ATAC variants on splicing efficiency of a minor (U12-type) reporter intron. Lastly, the concordance of bioinformatic predictions and cellular assay results was investigated.


Assuntos
RNA Nuclear Pequeno/metabolismo , Spliceossomos/metabolismo , Criança , Bases de Dados Genéticas , Nanismo/genética , Nanismo/patologia , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/patologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Variação Genética , Humanos , Microcefalia/genética , Microcefalia/patologia , Conformação de Ácido Nucleico , Osteocondrodisplasias/genética , Osteocondrodisplasias/patologia , Processamento de RNA , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética
16.
Nat Cell Biol ; 22(7): 842-855, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32514071

RESUMO

Senescent cells affect many physiological and pathophysiological processes. While select genetic and epigenetic elements for senescence induction have been identified, the dynamics, epigenetic mechanisms and regulatory networks defining senescence competence, induction and maintenance remain poorly understood, precluding the deliberate therapeutic targeting of senescence for health benefits. Here, we examined the possibility that the epigenetic state of enhancers determines senescent cell fate. We explored this by generating time-resolved transcriptomes and epigenome profiles during oncogenic RAS-induced senescence and validating central findings in different cell biology and disease models of senescence. Through integrative analysis and functional validation, we reveal links between enhancer chromatin, transcription factor recruitment and senescence competence. We demonstrate that activator protein 1 (AP-1) 'pioneers' the senescence enhancer landscape and defines the organizational principles of the transcription factor network that drives the transcriptional programme of senescent cells. Together, our findings enabled us to manipulate the senescence phenotype with potential therapeutic implications.


Assuntos
Senescência Celular , Cromatina/metabolismo , Epigênese Genética , Fibroblastos/citologia , Regulação da Expressão Gênica , Histonas/metabolismo , Fator de Transcrição AP-1/metabolismo , Transcriptoma , Animais , Cromatina/genética , Feminino , Fibroblastos/metabolismo , Histonas/genética , Humanos , Camundongos Endogâmicos C57BL , Fator de Transcrição AP-1/genética
17.
J Vis Exp ; (159)2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32510487

RESUMO

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were >90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.


Assuntos
Separação Celular/métodos , Fibroblastos/citologia , Neurônios Motores/citologia , Células de Schwann/citologia , Células Receptoras Sensoriais/citologia , Animais , Regeneração Nervosa , Fenótipo , Ratos
18.
PLoS One ; 15(6): e0233880, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32497112

RESUMO

The efficiency of somatic cell nuclear transfer (SCNT) is low due to the strong resistance of somatic donor cells to epigenetic reprogramming. Many epigenetic drugs targeting DNA methylation and histone acetylation have been used in attempts to improve the in vitro and in vivo development of SCNT embryos. H3K9me3 has been shown to be an important reprogramming barrier for generating induced pluripotent stem cells (iPSCs) and SCNT embryos in mice and humans. In this study, we examined the effects of selective siRNA and chemical inhibition of H3K9me3 in somatic donor cells on the in vitro development of bovine SCNT embryos. Chaetocin, an inhibitor of SUV39H1/H2, was supplemented during the culture of donor cells. In addition, the siRNA knockdown of SUV39H1/H2 was performed in the donor cells. The effects of chaetocin and siSUV39H1/H2 on H3K9me3 and H3K9ac were quantified using flow cytometry. Furthermore, we assessed chaetocin treatment and SUV39H1/H2 knockdown on the blastocyst formation rate. Both chaetocin and siSUV39H1/H2 significantly reduced and elevated the relative intensity level of H3K9me3 and H3K9ac in treated fibroblast cells, respectively. siSUV39H1/H2 transfection, but not chaetocin treatment, improved the in vitro development of SCNT embryos. Moreover, siSUV39H1/H2 altered the expression profile of the selected genes in the derived blastocysts, similar to those derived from in vitro fertilization (IVF). In conclusion, our results demonstrated H3K9me3 as an epigenetic barrier in the reprogramming process mediated by SCNT in bovine species, a finding which supports the role of H3K9me3 as a reprogramming barrier in mammalian species. Our findings provide a promising approach for improving the efficiency of mammalian cloning for agricultural and biomedical purposes.


Assuntos
Bovinos/embriologia , Desenvolvimento Embrionário , Histona-Lisina N-Metiltransferase/genética , Técnicas de Transferência Nuclear , Proteínas Repressoras/genética , Animais , Bovinos/genética , Bovinos/metabolismo , Células Cultivadas , Desenvolvimento Embrionário/efeitos dos fármacos , Epigênese Genética , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histonas/genética , Histonas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Repressoras/antagonistas & inibidores
19.
PLoS One ; 15(6): e0234180, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32511278

RESUMO

The autophagy-endolysosomal pathway is an evolutionally conserved degradation system that is tightly linked to a wide variety of physiological processes. Dysfunction of this system is associated with many pathological conditions such as cancer, inflammation and neurodegenerative diseases. Therefore, monitoring the cellular autophagy-endolysosomal activity is crucial for studies on the pathogenesis as well as therapeutics of such disorders. To this end, we here sought to create a novel means exploiting Keima, an acid-stable fluorescent protein possessing pH-dependent fluorescence excitation spectra, for precisely monitoring the autophagy-endolysosomal system. First, we generated three lines of transgenic (tg) mouse expressing monomeric Keima-fused MAP1LC3B (mKeima-LC3B). Then, these tg mice were subjected to starvation by food-restriction, and also challenged to neurodegeneration by genetically crossing with a mouse model of amyotrophic lateral sclerosis; i.e., SOD1H46R transgenic mouse. Unexpectedly, despite that a lipidated-form of endogenous LC3 (LC3-II) was significantly increased, those of mKeima-LC3B (mKeima-LC3B-II) were not changed under both stressed conditions. It was also noted that mKeima-LC3B-positive aggregates were progressively accumulated in the spinal cord of SOD1H46R;mKeima-LC3B double-tg mice, suggestive of acid-resistance and aggregate-prone natures of long-term overexpressed mKeima-LC3B in vivo. Next, we characterized mouse embryonic fibroblasts (MEFs) derived from mKeima-LC3B-tg mice. In contrast with in vivo, levels of mKeima-LC3B-I were decreased under starved conditions. Furthermore, when starved MEFs were treated with chloroquine (CQ), the abundance of mKeima-LC3B-II was significantly increased. Remarkably, when cultured medium was repeatedly changed between DMEM (nutrient-rich) and EBSS (starvation), acidic/neutral signal ratios of mKeima-LC3B-positive compartments were rapidly and reversibly shifted, which were suppressed by the CQ treatment, indicating that intraluminal pH of mKeima-LC3B-positive vesicles was changeable upon nutritional conditions of culture media. Taken together, although mKeima-LC3B-tg mice may not be an appropriate tool to monitor the autophagy-endolysosomal system in vivo, mKeima-LC3B must be one of the most sensitive reporter molecules for monitoring this system under in vitro cultured conditions.


Assuntos
Autofagia/fisiologia , Endossomos/metabolismo , Proteínas Luminescentes/genética , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Animais , Células Cultivadas , Meios de Cultura/farmacologia , Endossomos/genética , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/metabolismo , Lisossomos/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Inanição , Superóxido Dismutase-1/genética , Imagem com Lapso de Tempo
20.
Arch Biochem Biophys ; 689: 108437, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32526201

RESUMO

Skin aging is influenced by several genetic, physiological, and environmental factors. In particular, ultraviolet (UV) exposure is an important factor involved in inducing skin photoaging. Autophagy controlling homeostatic balance between the synthesis, degradation, and recycling of cellular organelles and proteins plays important regulatory roles in several biological processes, including aging. The opioid neuropeptide α-neoendorphin (named NEP) is an endogenous decapeptide (N-YGGFLRKYPK-C) that activates the kappa opioid receptor and exhibits certain anti-aging and anti-wrinkling effects on skin cells; however, its action mechanism has not yet been elucidated. Therefore, the aim of this study was to determine the effects of NEP on anti-skin aging and autophagy activation in human dermal fibroblast cells. Western blot results showed that NEP down-regulates the production of phospho-mammalian target of rapamycin (p-mTOR), whereas increases the expression of key autophagy-related molecules such as Beclin-1, Atg5-Atg12, and LC3-II. The immunocytochemical analysis performed with anti-LC3-II antibody also showed that the autophagic indicators, autophagosomes are formed by NEP. These results suggest that NEP can activate cellular autophagy through mTOR-Beclin-1-mediated signaling pathway. It was also revealed by CM-H2DCF-DA assay and Western blottings that NEP can reduce the production of ultraviolet B (UVB)-induced reactive oxygen species (ROS) like with N-acetylcysteine (NAC), resulting in decreasing the expression levels of skin aging-related proteins, such as phospho-ERK (p-ERK), phospho-p38 (p-p38), and phospho-JNK (p-JNK). Furthermore, NEP could increase the type I procollagen production, while decreasing MMP-1, MMP-2, and MMP-9 activities. Taken together, the results demonstrate that NEP can reduce UVB-induced photoaging by activating autophagy.


Assuntos
Autofagia , Endorfinas/metabolismo , Precursores de Proteínas/metabolismo , Envelhecimento da Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Linhagem Celular , Derme/citologia , Derme/metabolismo , Derme/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Pró-Colágeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo
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