Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 7.983
Filtrar
1.
PLoS Genet ; 16(8): e1008644, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776941

RESUMO

Correct regulation of cell contractility is critical for the function of many biological systems. The reproductive system of the hermaphroditic nematode C. elegans contains a contractile tube of myoepithelial cells known as the spermatheca, which stores sperm and is the site of oocyte fertilization. Regulated contraction of the spermatheca pushes the embryo into the uterus. Cell contractility in the spermatheca is dependent on actin and myosin and is regulated, in part, by Ca2+ signaling through the phospholipase PLC-1, which mediates Ca2+ release from the endoplasmic reticulum. Here, we describe a novel role for GSA-1/Gαs, and protein kinase A, composed of the catalytic subunit KIN-1/PKA-C and the regulatory subunit KIN-2/PKA-R, in the regulation of Ca2+ release and contractility in the C. elegans spermatheca. Without GSA-1/Gαs or KIN-1/PKA-C, Ca2+ is not released, and oocytes become trapped in the spermatheca. Conversely, when PKA is activated through either a gain of function allele in GSA-1 (GSA-1(GF)) or by depletion of KIN-2/PKA-R, the transit times and total numbers, although not frequencies, of Ca2+ pulses are increased, and Ca2+ propagates across the spermatheca even in the absence of oocyte entry. In the spermathecal-uterine valve, loss of GSA-1/Gαs or KIN-1/PKA-C results in sustained, high levels of Ca2+ and a loss of coordination between the spermathecal bag and sp-ut valve. Additionally, we show that depleting phosphodiesterase PDE-6 levels alters contractility and Ca2+ dynamics in the spermatheca, and that the GPB-1 and GPB-2 Gß subunits play a central role in regulating spermathecal contractility and Ca2+ signaling. This work identifies a signaling network in which Ca2+ and cAMP pathways work together to coordinate spermathecal contractions for successful ovulations.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Sinalização do Cálcio , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Contração Muscular , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Mutação com Ganho de Função , Células Musculares/metabolismo , Células Musculares/fisiologia , Oócitos/fisiologia
2.
Am J Physiol Gastrointest Liver Physiol ; 319(2): G109-G120, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32508154

RESUMO

Crohn's disease (CD) is a complex and multifactorial illness. There are still considerable gaps in our knowledge regarding its pathophysiology. A transcriptomic approach could shed some light on little-known biological alterations of the disease. We therefore aimed to explore the ileal transcriptome to gain knowledge about CD. We performed whole transcriptome gene expression analysis on ileocecal resections from CD patients and inflammatory bowel disease-free controls, as well as on a CD-independent cohort to replicate selected results. Normalized data were hierarchically clustered, and gene ontology and the molecular network were studied. Cell cultures and molecular methods were used for further evaluations. Genome-wide expression data analysis identified a robust transmembrane immunoglobulin domain-containing 1 (TMIGD1) gene underexpression in CD tissue, which was even more marked in inflamed ileum, and which was replicated in the validation cohort. Immunofluorescence showed TMIGD1 to be located in the apical microvilli of well-differentiated enterocytes but not in intestinal crypt. This apical TMIGD1 was lower in the noninflamed tissue and almost disappeared in the inflamed mucosa of surgical resections. In vitro studies showed hypoxic-dependent TMIGD1 decreased its expression in enterocyte-like cells. The gene enrichment analysis linked TMIGD1 with cell recovery and tissue remodeling in CD settings, involving guanylate cyclase activities. Transcriptomics may be useful for finding new targets that facilitate studies of the CD pathology. This is how TMIGD1 was identified in CD patients, which was related to multiciliate ileal epithelial cell differentiation.NEW & NOTEWORTHY This is a single-center translational research study that aimed to look for key targets involved in Crohn's disease and define molecular pathways through different functional analysis strategies. With this approach, we have identified and described a novel target, the almost unknown TMIGD1 gene, which may be key in the recovery of injured mucosa involving intestinal epithelial cell differentiation.


Assuntos
Doença de Crohn/genética , Células Epiteliais/fisiologia , Íleo/citologia , Glicoproteínas de Membrana/metabolismo , Transcriptoma , Adulto , Células CACO-2 , Estudos de Casos e Controles , Diferenciação Celular , Doença de Crohn/metabolismo , Regulação da Expressão Gênica , Humanos , Inflamação/metabolismo , Glicoproteínas de Membrana/genética , Consumo de Oxigênio
3.
Proc Natl Acad Sci U S A ; 117(21): 11531-11540, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32414916

RESUMO

A polarized architecture is central to both epithelial structure and function. In many cells, polarity involves mutual antagonism between the Par complex and the Scribble (Scrib) module. While molecular mechanisms underlying Par-mediated apical determination are well-understood, how Scrib module proteins specify the basolateral domain remains unknown. Here, we demonstrate dependent and independent activities of Scrib, Discs-large (Dlg), and Lethal giant larvae (Lgl) using the Drosophila follicle epithelium. Our data support a linear hierarchy for localization, but rule out previously proposed protein-protein interactions as essential for polarization. Cortical recruitment of Scrib does not require palmitoylation or polar phospholipid binding but instead an independent cortically stabilizing activity of Dlg. Scrib and Dlg do not directly antagonize atypical protein kinase C (aPKC), but may instead restrict aPKC localization by enabling the aPKC-inhibiting activity of Lgl. Importantly, while Scrib, Dlg, and Lgl are each required, all three together are not sufficient to antagonize the Par complex. Our data demonstrate previously unappreciated diversity of function within the Scrib module and begin to define the elusive molecular functions of Scrib and Dlg.


Assuntos
Polaridade Celular/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila , Células Epiteliais , Proteínas de Membrana/fisiologia , Animais , Drosophila/citologia , Drosophila/fisiologia , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Epitélio/fisiologia , Feminino , Folículo Ovariano/citologia , Folículo Ovariano/fisiologia , Proteína Quinase C , Proteínas Supressoras de Tumor
4.
Anim Sci J ; 91(1): e13396, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32468659

RESUMO

The objective of this study was to examine the expression profiles of follistatin (FST) and its associated molecules (MSTN, INHA, INHBB, INHBA, ACVR2A, and ACVR2B) in the oviduct of laying hens at 3 hr and 20 hr post-ovulation (p.o., n = 5; 35 weeks old), molting (n = 5; 60 weeks old), and non-laying (n = 4; 35-60 weeks old) hens and also to localize the FST by using immunohistochemistry assay. Expression of FST was significantly higher (p < .05), and MSTN was lower in the uterus of laying hens around 15-20 hr p.o. (during eggshell formation), however, their expressions in the magnum remain unchanged across different physiological stages of hens. FST was mainly expressed in the luminal and glandular epithelium of the uterine tissues, and their expression intensity was highest in laying hens during the eggshell mineralization. There was a relatively increased expression of INHA in the magnum of laying hens around 3 hr p.o. as compared to non-laying and molting hens. At the same time (3 hr p.o.), there was a significant (p < .05) decrease in the expression of the INHBB, ACVR2A, and ACV2B. These results indicate that follistatin may regulate the differentiation of uterine luminal and glandular epithelium during eggshell biomineralization.


Assuntos
Biomineralização/genética , Galinhas/genética , Galinhas/fisiologia , Casca de Ovo/embriologia , Folistatina/genética , Folistatina/metabolismo , Expressão Gênica/genética , Oviductos/metabolismo , Oviposição/genética , Oviposição/fisiologia , Ovulação/genética , Ovulação/fisiologia , Transcriptoma , Animais , Biomineralização/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Casca de Ovo/fisiologia , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Feminino , Oviductos/fisiologia , Útero/citologia , Útero/metabolismo
5.
PLoS One ; 15(5): e0232899, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32392240

RESUMO

Various nanopatterning techniques have been developed to improve cell proliferation and differentiation efficiency. As we previously reported, nanopillars and pores are able to sustain human pluripotent stem cells and differentiate pancreatic cells. From this, the nanoscale patterns would be effective environment for the co-culturing of epithelial and mesenchymal cell types. Interestingly, the nanopatterning selectively reduced the proliferative rate of mesenchymal cells while increasing the expression of adhesion protein in epithelial type cells. Additionally, co-cultured cells on the nanopatterning were not negatively affected in terms of cell function metabolic ability or cell survival. This is in contrast to conventional co-culturing methods such as ultraviolet or chemical treatments. The nanopatterning appears to be an effective environment for mesenchymal co-cultures with typically low proliferative rates cells such as astrocytes, neurons, melanocytes, and fibroblasts without using potentially damaging treatments.


Assuntos
Técnicas de Cocultura/instrumentação , Células Epiteliais , Células-Tronco Mesenquimais , Nanoestruturas , Animais , Adesão Celular , Proliferação de Células , Sobrevivência Celular , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Camundongos , Propriedades de Superfície
6.
Nat Commun ; 11(1): 2366, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398639

RESUMO

Epithelial bending is a fundamental process that shapes organs during development. Previously known mechanisms involve cells locally changing shape from columnar to wedge-shaped. Here we report a different mechanism that occurs without cell wedging. In mammalian salivary glands and teeth, we show that initial invagination occurs through coordinated vertical cell movement: cells towards the periphery of the placode move vertically upwards while their more central neighbours move downwards. Movement is achieved by active cell-on-cell migration: outer cells migrate with apical, centripetally polarised leading edge protrusions but remain attached to the basal lamina, depressing more central neighbours to "telescope" the epithelium downwards into underlying mesenchyme. Inhibiting protrusion formation by Arp2/3 protein blocks invagination. FGF and Hedgehog morphogen signals are required, with FGF providing a directional cue. These findings show that epithelial bending can be achieved by a morphogenetic mechanism of coordinated cell rearrangement quite distinct from previously recognised invagination processes.


Assuntos
Movimento Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Epitélio/embriologia , Dente Molar/embriologia , Glândulas Salivares/embriologia , Animais , Ectoderma/citologia , Ectoderma/embriologia , Embrião de Mamíferos/citologia , Células Epiteliais/fisiologia , Feminino , Microscopia Intravital , Masculino , Camundongos , Dente Molar/citologia , Glândulas Salivares/citologia , Técnicas de Cultura de Tecidos
7.
Nat Cell Biol ; 22(5): 546-558, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32341550

RESUMO

Macrophages are diverse immune cells that reside in all tissues. Although macrophages have been implicated in mammary-gland function, their diversity has not been fully addressed. By exploiting high-resolution three-dimensional imaging and flow cytometry, we identified a unique population of tissue-resident ductal macrophages that form a contiguous network between the luminal and basal layers of the epithelial tree throughout postnatal development. Ductal macrophages are long lived and constantly survey the epithelium through dendrite movement, revealed via advanced intravital imaging. Although initially originating from embryonic precursors, ductal macrophages derive from circulating monocytes as they expand during puberty. Moreover, they undergo proliferation in pregnancy to maintain complete coverage of the epithelium in lactation, when they are poised to phagocytose milk-producing cells post-lactation and facilitate remodelling. Interestingly, ductal macrophages strongly resemble mammary tumour macrophages and form a network that pervades the tumour. Thus, the mammary epithelium programs specialized resident macrophages in both physiological and tumorigenic contexts.


Assuntos
Células Epiteliais/fisiologia , Epitélio/fisiologia , Animais , Proliferação de Células/fisiologia , Feminino , Lactação/fisiologia , Macrófagos/fisiologia , Glândulas Mamárias Animais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/fisiologia , Fagocitose/fisiologia , Gravidez
8.
Int J Mol Sci ; 21(2)2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-32284519

RESUMO

Maternal stress before or during the sensitive preimplantation phase is associated with reproduction failure. Upon real or perceived threat, glucocorticoids (classic stress hormones) as cortisol are synthesized. The earliest "microenvironment" of the embryo consists of the oviduct epithelium and the oviductal fluid generated via the epithelial barrier. However, to date, the direct effects of cortisol on the oviduct are largely unknown. In the present study, we used a compartmentalized in vitro system to test the hypothesis that a prolonged stimulation with cortisol modifies the physiology of the oviduct epithelium. Porcine oviduct epithelial cells were differentiated at the air-liquid interface and basolaterally stimulated with physiological levels of cortisol representing moderate and severe stress for 21 days. Epithelium structure, transepithelial bioelectric properties, and gene expression were assessed. Furthermore, the distribution and metabolism of cortisol was examined. The polarized oviduct epithelium converted basolateral cortisol to cortisone and thereby reduced the amount of bioactive cortisol reaching the apical compartment. However, extended cortisol stimulation affected its barrier function and the expression of genes involved in hormone signaling and immune response. We conclude that continuing maternal stress with long-term elevated cortisol levels may alter the early embryonic environment by modification of basic oviductal functions.


Assuntos
Microambiente Celular , Desenvolvimento Embrionário , Células Epiteliais/fisiologia , Hidrocortisona/metabolismo , Suínos/fisiologia , Animais , Diferenciação Celular , Epitélio/fisiologia , Tubas Uterinas/embriologia , Tubas Uterinas/fisiologia , Feminino , Glucocorticoides/metabolismo , Gravidez , Estresse Fisiológico , Suínos/embriologia
9.
Arch Med Res ; 51(3): 233-244, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32139108

RESUMO

OBJECTIVE: To evaluate the anti-cancer effect of unmethylated cytosine-phosphorothioate-guanine (CpG)-containing oligodeoxynucleotides (ODNs) on human bladder cancer UM-UC-3 cells, our study was carried out. METHODS: The viability of cells (UM-UC-3, T24 and SV-HUC-1) with CpG ODN treatments was examined by cell counting kit-8 (CCK-8) assay. Apoptosis and cell cycle phase were determined by flow cytometry analysis. Pre-apoptosis factors of caspase-3, p53, B-cell lymphoma 2 associated X protein (Bax) and anti-apoptosis factor of B-cell lymphoma 2 (Bcl-2) were detected by western blot. RESULTS: Experimental results showed that the viability of human bladder cancer cells (UM-UC-3 and T24) with CpG ODN treatment was decreased and the viability of human normal urothelial cells (SV-HUC-1) with CpG ODN treatment was increased with time-dependance manner. Moreover, CpG ODN increased the apoptosis rate of UM-UC-3 cells and arrested more cells in G0G1 phase. Furthermore, the expression of caspase-3, p53 and Bax were increased and the expression of Bcl-2 was decreased with CpG ODN treatment on UM-UC-3 cells. CONCLUSION: CpG ODN promoted the proliferation of normal urinary transitional epithelial cells (SV-HUC-1) and inhibited the cell viability of human bladder cancer cells (UM-UC-3 and T24) in vitro. CpG ODN induced the apoptosis of human bladder cancer (UM-UC-3) cells in a cascade progress via enhancing the expression of caspase-3, p53 and Bax, and inhibiting the expression of Bcl-2 with significant time-dependancy. CpG ODN inhibited cell cycle distribution of human bladder cancer (UM-UC-3) cells with more cells were arrested in G0G1 phase. This study suggested that the CpG ODN is the potential candidate on human bladder cancer.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citosina/farmacologia , Células Epiteliais/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Guanina/farmacologia , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Neoplasias da Bexiga Urinária/patologia , Proteína X Associada a bcl-2/metabolismo
10.
Proc Natl Acad Sci U S A ; 117(11): 5655-5663, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123100

RESUMO

Epithelial tissues mechanically deform the surrounding extracellular matrix during embryonic development, wound repair, and tumor invasion. Ex vivo measurements of such multicellular tractions within three-dimensional (3D) biomaterials could elucidate collective dissemination during disease progression and enable preclinical testing of targeted antimigration therapies. However, past 3D traction measurements have been low throughput due to the challenges of imaging and analyzing information-rich 3D material deformations. Here, we demonstrate a method to profile multicellular clusters in a 96-well-plate format based on spatially heterogeneous contractile, protrusive, and circumferential tractions. As a case study, we profile multicellular clusters across varying states of the epithelial-mesenchymal transition, revealing a successive loss of protrusive and circumferential tractions, as well as the formation of localized contractile tractions with elongated cluster morphologies. These cluster phenotypes were biochemically perturbed by using drugs, biasing toward traction signatures of different epithelial or mesenchymal states. This higher-throughput analysis is promising to systematically interrogate and perturb aberrant mechanobiology, which could be utilized with human-patient samples to guide personalized therapies.


Assuntos
Movimento Celular , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal , Tecidos Suporte/química , Fenômenos Biomecânicos , Linhagem Celular , Colágeno/química , Fibroínas/química , Humanos , Hidrogéis/química , Fenótipo , Medicina de Precisão/métodos , Cultura Primária de Células/métodos , Esferoides Celulares/fisiologia
11.
J Mycol Med ; 30(2): 100939, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32111506

RESUMO

Nosocomial infections by fungi are important causes of morbidity and mortality, and the adhesion capacity of yeast on abiotic and biotic surfaces has been considered an important step in this process. Als3 proteins are widely studied for their ability to allow Candida albicans to bind to various surfaces. The objective of the present study was to verify, with more details, the action of F2768-0318 in relation to its antifungal activity as well as its ability to act on C. albicans virulence factors related to adhesion and biofilm formation in vitro and in vivo by inhibiting the Als3 protein. F2768-0318 was assessed in tests of biofilm formation and adhesion on abiotic surfaces (polystyrene plates) and adherence on biotic surfaces, including human endocervical (HeLa) cells, human umbilical vein endothelial cells (HUVECs), and fresh buccal epithelial cells (BEC). Our results showed F2768-0318 was useful in reducing the adhesion and biofilm formation of C. albicans on abiotic surfaces, indicating the possibility of treating hospital materials and preventing biofilm formation on these types of equipment. Further studies are still needed, including optimization of the molecule to allow this molecule to be effective on other types of surfaces, such as human cells.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Animais , Antifúngicos/uso terapêutico , Candida albicans/crescimento & desenvolvimento , Candida albicans/fisiologia , Candidemia/tratamento farmacológico , Candidemia/microbiologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Células Epiteliais/fisiologia , Feminino , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Testes de Toxicidade
12.
J Biomed Sci ; 27(1): 32, 2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-32035490

RESUMO

BACKGROUND: Fallopian tube epithelial cells (FTEC) were thought to be the origin of high-grade serous ovarian carcinoma (HGSOC). Knowledge of the stemness or initiating characteristics of FTEC is insufficient. Previously, we have characterized the stemness cell marker of FTEC, this study aims to further characterize the clonogenicity and spheroid features of FTEC. METHODS: We successfully derived FTECs from the epithelial layer of the human fallopian tubes. We examined the morphology, proliferation rate, doubling time, and clonal growth of them. At passage 3, the sphere formations on gelatin-coated culture, suspension culture, and matrigel culture were observed, and the expression of LGR5, SSEA3, SSEA4, and other stemness markers was examined. Furthermore, tissue-reconstituted organoids from coculture of FTEC, fallopian stromal cells (FTMSC) and endothelial cells (HUVEC) were examined. RESULTS: FTEC exhibited cuboidal cell morphology and maintained at a constant proliferation rate for up to nine passages (P9). FTEC could proliferate from a single cell with a clonogenic efficiency of 4%. Flow cytometry revealed expressions of normal stem cell markers (SSEA3, SSEA4, and LGR5) and cancer stem cell markers (CD24, CD44, CD117, ROR1, and CD133). FTEC formed spheres and colonies when cultured on low attach dish. In the presence of Matrigel, the stemness and colony formation activity were much enhanced. In co-culturing with FTMSC and HUVEC, FTEC could form organoids that could be blocked by Wnt inhibitor DKK1. Expressions of LGR5 and FOXJ1 expression were also decreased by adding DKK1. CONCLUSION: We demonstrated abundantly presence of stem cells in human FTECs which are efficient in forming colonies, spheres and organoids, relying on Wnt signaling. We also reported for the first time the generation of organoid from reconstitutied cell lineages in the tissue. This may provide a new model for studying the regneration and malignant transformation of the tubal epithelium.


Assuntos
Autorrenovação Celular/fisiologia , Células Epiteliais/fisiologia , Tubas Uterinas/fisiologia , Expressão Gênica , Organoides/fisiologia , Proteínas Wnt/genética , Feminino , Marcadores Genéticos , Humanos , Proteínas Wnt/metabolismo
13.
Dev Cell ; 52(3): 321-334.e6, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32049039

RESUMO

Epithelial fusion is a key process of morphogenesis by which tissue connectivity is established between adjacent epithelial sheets. A striking and poorly understood feature of this process is "zippering," whereby a fusion point moves directionally along an organ rudiment. Here, we uncover the molecular mechanism underlying zippering during mouse spinal neural tube closure. Fusion is initiated via local activation of integrin ß1 and focal anchorage of surface ectoderm cells to a shared point of fibronectin-rich basement membrane, where the neural folds first contact each other. Surface ectoderm cells undergo proximal junction shortening, establishing a transitory semi-rosette-like structure at the zippering point that promotes juxtaposition of cells across the midline enabling fusion propagation. Tissue-specific ablation of integrin ß1 abolishes the semi-rosette formation, preventing zippering and causing spina bifida. We propose integrin-mediated anchorage as an evolutionarily conserved mechanism of general relevance for zippering closure of epithelial gaps whose disturbance can produce clinically important birth defects.


Assuntos
Embrião de Mamíferos/fisiologia , Células Epiteliais/fisiologia , Adesões Focais , Integrina beta1/fisiologia , Crista Neural/embriologia , Tubo Neural/embriologia , Neurulação , Actomiosina/metabolismo , Animais , Fusão Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese , Crista Neural/metabolismo , Crista Neural/fisiologia , Tubo Neural/metabolismo , Tubo Neural/fisiologia
14.
Infect Immun ; 88(5)2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32094249

RESUMO

Staphylococcus aureus is a noted human and animal pathogen. Despite decades of research on this important bacterium, there are still many unanswered questions regarding the pathogenic mechanisms it uses to infect the mammalian host. This can be attributed to it possessing a plethora of virulence factors and complex virulence factor and metabolic regulation. PurR, the purine biosynthesis regulator, was recently also shown to regulate virulence factors in S. aureus, and mutations in purR result in derepression of fibronectin binding proteins (FnBPs) and extracellular toxins, required for a so-called hypervirulent phenotype. Here, we show that hypervirulent strains containing purR mutations can be attenuated with the addition of purine biosynthesis mutations, implicating the necessity for de novo purine biosynthesis in this phenotype and indicating that S. aureus in the mammalian host experiences purine limitation. Using cell culture, we showed that while purR mutants are not altered in epithelial cell binding, compared to that of wild-type (WT) S. aureus, purR mutants have enhanced invasion of these nonprofessional phagocytes, consistent with the requirement of FnBPs for invasion of these cells. This correlates with purR mutants having increased transcription of fnb genes, resulting in higher levels of surface-exposed FnBPs to promote invasion. These data provide important contributions to our understanding of how the pathogenesis of S. aureus is affected by sensing of purine levels during infection of the mammalian host.


Assuntos
Mutação/genética , Purinas/biossíntese , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/genética , Fatores de Virulência/genética , Células A549 , Animais , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Linhagem Celular , Citoplasma/genética , Células Epiteliais/fisiologia , Feminino , Fibronectinas/genética , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fagócitos/fisiologia , Células RAW 264.7 , Infecções Estafilocócicas/microbiologia , Transcrição Genética/genética
15.
Nat Commun ; 11(1): 472, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980653

RESUMO

The cadherin-catenin complex at adherens junctions (AJs) is essential for the formation of cell-cell adhesion and epithelium integrity; however, studying the dynamic regulation of AJs at high spatio-temporal resolution remains challenging. Here we present an optochemical tool which allows reconstitution of AJs by chemical dimerization of the force bearing structures and their precise light-induced dissociation. For the dimerization, we reconstitute acto-myosin connection of a tailless E-cadherin by two ways: direct recruitment of α-catenin, and linking its cytosolic tail to the transmembrane domain. Our approach enables a specific ON-OFF switch for mechanical coupling between cells that can be controlled spatially on subcellular or tissue scale via photocleavage. The combination with cell migration analysis and traction force microscopy shows a wide-range of applicability and confirms the mechanical contribution of the reconstituted AJs. Remarkably, in vivo our tool is able to control structural and functional integrity of the epidermal layer in developing Xenopus embryos.


Assuntos
Junções Aderentes/fisiologia , Junções Aderentes/efeitos da radiação , Actomiosina/química , Animais , Antígenos CD/química , Fenômenos Biomecânicos , Caderinas/química , Linhagem Celular , Movimento Celular/fisiologia , Células Epiteliais/fisiologia , Células Epiteliais/efeitos da radiação , Células Epiteliais/ultraestrutura , Humanos , Luz , Microscopia de Força Atômica , Fenômenos Ópticos , Processos Fotoquímicos , Xenopus laevis/embriologia , alfa Catenina/química
16.
FASEB J ; 34(1): 386-398, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914653

RESUMO

To date, there is no direct evidence of telomerase activity in adult lung epithelial cells, but typical culture conditions only support cell proliferation for 30-40 population doublings (PD), a point at which telomeres remain relatively long. Here we report that in in vitro low stress culture conditions consisting of a fibroblast feeder layer, rho-associated coiled coil protein kinase inhibitor (ROCKi), and low oxygen (2%), normal human bronchial epithelial basal progenitor cells (HBECs) divide for over 200 PD without engaging a telomere maintenance mechanism (almost four times the "Hayflick limit"). HBECs exhibit critically short telomeres at 200 PD and the population of cells start to undergo replicative senescence. Subcloning these late passage cells to clonal density, to mimic lung injury in vivo, selects for rare subsets of HBECs that activate low levels of telomerase activity to maintain short telomeres. CRISPR/Cas9 knockout of human telomerase reverse transcriptase or treatment with the telomerase-mediated telomere targeting agent 6-thio-2'deoxyguanosine abrogates colony growth in these late passage cultures (>200 PD) but not in early passage cultures (<200 PD). To our knowledge, this is the first study to report such long-term growth of HBECs without a telomere maintenance mechanism. This report also provides direct evidence of telomerase activation in HBECs near senescence when telomeres are critically short. This novel cell culture system provides an experimental model to understand how telomerase is regulated in normal adult tissues.


Assuntos
Brônquios/citologia , Técnicas de Cultura de Células/métodos , Proliferação de Células , Senescência Celular , Células Epiteliais/citologia , Fibroblastos/citologia , Telômero/fisiologia , Adulto , Brônquios/fisiologia , Divisão Celular , Células Cultivadas , Células Epiteliais/fisiologia , Fibroblastos/fisiologia , Humanos , Telomerase/metabolismo , Encurtamento do Telômero
17.
Gut ; 69(3): 578-590, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31792136

RESUMO

OBJECTIVE: The functional role of interleukin-22 (IL22) in chronic inflammation is controversial, and mechanistic insights into how it regulates target tissue are lacking. In this study, we evaluated the functional role of IL22 in chronic colitis and probed mechanisms of IL22-mediated regulation of colonic epithelial cells. DESIGN: To investigate the functional role of IL22 in chronic colitis and how it regulates colonic epithelial cells, we employed a three-dimentional mini-gut epithelial organoid system, in vivo disease models and transcriptomic datasets in human IBD. RESULTS: As well as inducing transcriptional modules implicated in antimicrobial responses, IL22 also coordinated an endoplasmic reticulum (ER) stress response transcriptional programme in colonic epithelial cells. In the colon of patients with active colonic Crohn's disease (CD), there was enrichment of IL22-responsive transcriptional modules and ER stress response modules. Strikingly, in an IL22-dependent model of chronic colitis, targeting IL22 alleviated colonic epithelial ER stress and attenuated colitis. Pharmacological modulation of the ER stress response similarly impacted the severity of colitis. In patients with colonic CD, antibody blockade of IL12p40, which simultaneously blocks IL12 and IL23, the key upstream regulator of IL22 production, alleviated the colonic epithelial ER stress response. CONCLUSIONS: Our data challenge perceptions of IL22 as a predominantly beneficial cytokine in IBD and provide novel insights into the molecular mechanisms of IL22-mediated pathogenicity in chronic colitis. Targeting IL22-regulated pathways and alleviating colonic epithelial ER stress may represent promising therapeutic strategies in patients with colitis. TRIAL REGISTRATION NUMBER: NCT02749630.


Assuntos
Colite/genética , Doença de Crohn/fisiopatologia , Estresse do Retículo Endoplasmático/genética , Células Epiteliais/fisiologia , Interleucinas/farmacologia , Transcrição Genética , Animais , Antibacterianos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Doença Crônica , Colite/sangue , Colite/tratamento farmacológico , Colite/patologia , Colo/patologia , Doença de Crohn/patologia , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fármacos Gastrointestinais/farmacologia , Fármacos Gastrointestinais/uso terapêutico , Humanos , Interleucina-17/farmacologia , Interleucina-23/antagonistas & inibidores , Interleucinas/sangue , Interleucinas/genética , Mucosa Intestinal/patologia , Camundongos , Organoides , Gravidade do Paciente , Fenilbutiratos/farmacologia , Proteínas Recombinantes/farmacologia , Transcrição Genética/efeitos dos fármacos , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas , Ustekinumab/farmacologia , Ustekinumab/uso terapêutico
18.
Biochim Biophys Acta Biomembr ; 1862(2): 183145, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31809710

RESUMO

Epithelial cells are polarized cells and typically display distinct plasma membrane domains: basal plasma membrane domains face the underlying tissue, lateral domains contact adjacent cells and apical domains face the exterior lumen. Each membrane domain is endowed with a specific macromolecular composition that constitutes the functional identity of that domain. Defects in apical-basal plasma membrane polarity altogether or more subtle defects in the composition of either apical or basal plasma membrane domain can give rise to severe diseases. Lipids are the main component of cellular membranes and mechanisms that control their polarized distribution in epithelial cells are emerging. In particular sphingolipids and phosphatidylinositol lipids have taken center stage in the organization of the apical and basolateral plasma membrane domain. This short review article discusses mechanisms that contribute to the polarized distribution of lipids in epithelial cells.


Assuntos
Polaridade Celular , Células Epiteliais/metabolismo , Fosfatidilinositóis/metabolismo , Animais , Endossomos/metabolismo , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Humanos , Fosfatidilinositóis/química
19.
Am J Physiol Lung Cell Mol Physiol ; 318(1): L149-L164, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31693390

RESUMO

Disturbances in mitochondrial structure and function in lung epithelial cells have been implicated in the pathogenesis of various lung diseases, including chronic obstructive pulmonary disease (COPD). Such disturbances affect not only cellular energy metabolism but also alter a range of indispensable cellular homeostatic functions in which mitochondria are known to be involved. These range from cellular differentiation, cell death pathways, and cellular remodeling to physical barrier function and innate immunity, all of which are known to be impacted by exposure to cigarette smoke and have been linked to COPD pathogenesis. Next to their well-established role as the first physical frontline against external insults, lung epithelial cells are immunologically active. Malfunctioning epithelial cells with defective mitochondria are unable to maintain homeostasis and respond adequately to further stress or injury, which may ultimately shape the phenotype of lung diseases. In this review, we provide a comprehensive overview of the impact of cigarette smoke on the development of mitochondrial dysfunction in the lung epithelium and highlight the consequences for cell function, innate immune responses, epithelial remodeling, and epithelial barrier function in COPD. We also discuss the applicability and potential therapeutic value of recently proposed strategies for the restoration of mitochondrial function in the treatment of COPD.


Assuntos
Células Epiteliais/fisiologia , Pulmão/fisiopatologia , Mitocôndrias/fisiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Animais , Células Epiteliais/efeitos dos fármacos , Humanos , Pulmão/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/fisiopatologia , Fumar/efeitos adversos , Tabaco/efeitos adversos
20.
Cell Prolif ; 53(2): e12737, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31821660

RESUMO

OBJECTIVE: Embryo implantation needs a reciprocal interaction between competent embryo and receptive endometrium. Adenosine triphosphate (ATP) produced by stressed or injured cells acts as an important signalling molecule. This study aims to investigate whether adenosine triphosphate (ATP) plays an important role in the dialogue of human blastocyst-endometrium. MATERIALS AND METHODS: The concentration of lactate was analysed in culture medium from human embryos collected from in vitro fertilization patients. Extracellular ATP was measured by ATP Bioluminescent Assay Kit. Ishikawa cells and T-HESCs were treated with ATP, ATP receptor antagonist, ATP hydrolysis enzyme or inhibitors of ATP metabolic enzymes. The levels of gene expression were evaluated by real-time PCR and immunoassay. RESULTS: We showed that injured human endometrial epithelial cells could rapidly release ATP into the extracellular environment as an important signalling molecule. In addition, blastocyst-derived lactate induces the release of non-lytic ATP from human endometrial receptive epithelial cells via connexins. Extracellular ATP stimulates the secretion of IL8 from epithelial cells to promote the process of in vitro decidualization. Extracellular ATP could also directly promote the decidualization of human endometrial stromal cells via P2Y-purinoceptors. More importantly, the supernatants of injured epithelial cells clearly induce the decidualization of stromal cells in time-dependent manner. CONCLUSION: Our results suggest that ATP should play an important role in human blastocyst-endometrium dialogue for the initiation of decidualization.


Assuntos
Trifosfato de Adenosina/metabolismo , Blastocisto/metabolismo , Blastocisto/fisiologia , Endométrio/metabolismo , Endométrio/fisiologia , Linhagem Celular , Técnicas de Cocultura/métodos , Implantação do Embrião/fisiologia , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Feminino , Expressão Gênica/fisiologia , Humanos , Transdução de Sinais/fisiologia , Células Estromais/metabolismo , Células Estromais/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA