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1.
Science ; 371(6531): 839-846, 2021 02 19.
Artículo en Inglés | MEDLINE | ID: mdl-33602855

RESUMEN

Organoid technology holds great promise for regenerative medicine but has not yet been applied to humans. We address this challenge using cholangiocyte organoids in the context of cholangiopathies, which represent a key reason for liver transplantation. Using single-cell RNA sequencing, we show that primary human cholangiocytes display transcriptional diversity that is lost in organoid culture. However, cholangiocyte organoids remain plastic and resume their in vivo signatures when transplanted back in the biliary tree. We then utilize a model of cell engraftment in human livers undergoing ex vivo normothermic perfusion to demonstrate that this property allows extrahepatic organoids to repair human intrahepatic ducts after transplantation. Our results provide proof of principle that cholangiocyte organoids can be used to repair human biliary epithelium.


Asunto(s)
Enfermedades de los Conductos Biliares/terapia , Conductos Biliares Intrahepáticos/fisiología , Conductos Biliares/citología , Tratamiento Basado en Trasplante de Células y Tejidos , Células Epiteliales/citología , Organoides/trasplante , Animales , Bilis , Conductos Biliares/fisiología , Conductos Biliares Intrahepáticos/citología , Conducto Colédoco/citología , Células Epiteliales/fisiología , Vesícula Biliar/citología , Regulación de la Expresión Génica , Humanos , Hígado/fisiología , Trasplante de Hígado , Trasplante de Células Madre Mesenquimatosas , Ratones , Organoides/fisiología , RNA-Seq , Obtención de Tejidos y Órganos , Transcriptoma
2.
Int J Nanomedicine ; 16: 1067-1081, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33603369

RESUMEN

Background: Extracellular vesicles (EVs) are capable of manipulating cellular functions for the maintenance of biological homeostasis and disease progression, such as in glaucoma disease. These nano-particles carry a net negative surface charge under physiological conditions that can contribute to EVs:EVs interaction and their uptake by target cells. Purpose: To investigate the effect of glaucoma drugs on EVs physicochemical characters and the implications for their uptake by trabecular meshwork (TM) cells. Methods: TM or non-pigmented ciliary epithelium (NPCE) cells derived EVs were incubated with commercial anti-glaucoma formulation, Timolol maleate, Brinzolamide or Benzalkonium Cl and their size and zeta potential (ZP) and physical interactions of EVs derived from NPCE cells and TM cells were evaluated. The contribution of EVs interactions to up-take by TM cells was examined using fluorescence-activated cell sorting. Results: EVs size and ZP were affected by the ionic strength of the buffer rather than EVs type. Commercial glaucoma eye drops, including ß-blocker, α-2-agonist and prostaglandin analogs, reduced NPCE EVs ZP, whereas exposure of EVs to carbonic anhydrase inhibitor caused an increase in the ZP. A correlation was found between increased ZP values and increased NPCE EVs uptake by TM cells. We were able to show that Benzalkonium chloride stands behind this ZP effect and not Timolol or Brinzolamide. Conclusion: Altogether, our findings demonstrate that EVs size, surface membrane charge, and ionic strength of the surrounding have an impact on EVs:EVs interactions, which affect the uptake of NPCE EVs by TM cells.


Asunto(s)
Agonistas Adrenérgicos/farmacología , Antagonistas Adrenérgicos beta/farmacología , Células Epiteliales/fisiología , Vesículas Extracelulares/fisiología , Glaucoma/tratamiento farmacológico , Soluciones Oftálmicas/farmacología , Malla Trabecular/fisiología , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Glaucoma/patología , Humanos , Malla Trabecular/efectos de los fármacos
3.
Life Sci ; 271: 119195, 2021 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-33581125

RESUMEN

AIMS: Ulcerative colitis and Crohn's disease, collectively known as inflammatory bowel disease (IBD), are chronic inflammatory disorders of the intestine for which key elements in disease initiation and perpetuation are defects in epithelial barrier integrity. Achieving mucosal healing is essential to ameliorate disease outcome and so new therapies leading to epithelial homeostasis and repair are under investigation. This study was designed to determine the mechanisms by which IL-22 regulates intestinal epithelial cell function. MAIN METHODS: Human intestinal organoids and resections, as well as mice were used to evaluate the effect of IL-22 on stem cell expansion, proliferation and expression of mucus components. IL-22 effect on barrier function was assessed in polarized T-84 cell monolayers. Butyrate co-treatments and organoid co-cultures with immune cells were performed to monitor the impact of microbial-derived metabolites and inflammatory environments on IL-22 responses. KEY FINDINGS: IL-22 led to epithelial stem cell expansion, proliferation, barrier dysfunction and anti-microbial peptide production in human and mouse models evaluated. IL-22 also altered the mucus layer by inducing an increase in membrane mucus but a decrease in secreted mucus and goblet cell content. IL-22 had the same effect on anti-microbial peptides and membrane mucus in both healthy and IBD human samples. In contrast, this IL-22-associated epithelial phenotype was different when treatments were performed in presence of butyrate and organoids co-cultured with immune cells. SIGNIFICANCE: Our data indicate that IL-22 promotes epithelial regeneration, innate defense and membrane mucus production, strongly supporting the potential clinical utility of IL-22 as a mucosal healing therapy in IBD.


Asunto(s)
Células Epiteliales/fisiología , Homeostasis/fisiología , Interleucinas/fisiología , Interleucinas/uso terapéutico , Mucosa Intestinal/fisiología , Animales , Línea Celular , Técnicas de Cocultivo , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/patología , Células Epiteliales/efectos de los fármacos , Homeostasis/efectos de los fármacos , Humanos , Interleucinas/farmacología , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Organoides/efectos de los fármacos , Organoides/fisiología
4.
J Dairy Sci ; 104(2): 1351-1363, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33309364

RESUMEN

During the thermal processing of milk, Maillard reactions occur between proteins and lactose to generate glycated proteins. In this study, a lactose-glycated caseinate was hydrolyzed by trypsin. The obtained glycated caseinate (GCN) hydrolysate had a lactose content of 10.8 g/kg of protein. We identified its glycation sites and then assessed it for its protective effect against lipopolysaccharide-induced barrier injury using a rat intestinal epithelial cell line (IEC-6 cells) as a cell model and unglycated caseinate (CN) hydrolysate as a reference. Results from our liquid chromatography-mass spectrometry analysis of the GCN hydrolysate verified that lactose glycation occurred at the Lys residues in 3 casein components (αS1-casein, ß-casein, and κ-casein), and this resulted in the formation of 5 peptides with the following amino acid sequences: EMPFPKYPKYPVEPF, HIQKEDVPSE, GSENSEKTTMPL, NQDKTEIPT, and EGIHAQQKEPM. The results from cell experiments showed that the 2 hydrolysates could promote cell growth and decrease lactate dehydrogenase release in the lipopolysaccharide-injured cells; more importantly, they could partially protect the damaged barrier function of the cells by increasing trans-epithelial electrical resistance, decreasing epithelial permeability, and upregulating the expression of the 3 tight junction proteins zonula occludens-1, occludin, and claudin-1. However, compared with CN hydrolysate, GCN hydrolysate showed lower efficacy in protecting against cellular barrier dysfunction. We propose that the different chemical characteristics of the CN hydrolysate and the GCN hydrolysate (i.e., amino acid loss and lactose conjugation) contributed to the lower barrier-protective efficacy of the GCN hydrolysate. During dairy processing, protein glycation of the Maillard type might have a non-negligible, unfavorable effect on dairy proteins, in view of the resulting protein glycation we found and the critical function of proteins for maintaining the integrity of the intestinal barrier.


Asunto(s)
Caseínas/metabolismo , Células Epiteliales/efectos de los fármacos , Lactosa/metabolismo , Lipopolisacáridos/farmacología , Péptidos/farmacología , Animales , Sitios de Unión , Caseínas/química , Línea Celular , Claudina-1/metabolismo , Células Epiteliales/fisiología , Glicosilación , Hidrólisis , Intestinos/citología , Intestinos/efectos de los fármacos , Reacción de Maillard , Péptidos/química , Permeabilidad , Ratas , Tripsina/metabolismo
5.
Methods Mol Biol ; 2179: 275-287, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32939727

RESUMEN

Mesenchymal-to-epithelial transition (MET) describes the ability of loosely associated migratory cells to form a more adherent sheet-like assembly of cells. MET is a conserved motif occurring throughout organogenesis and plays a key role in regeneration and cancer metastasis, and is the first step in producing induced pluripotent stem cells (iPSCs). To resolve fundamental biological questions about MET, its relation to epithelial-to-mesenchymal transition, and to explore MET's role in tissue assembly and remodeling requires live models for MET that are amenable to experimentation. Many cases of clinically important MET are inferred since they occur deep with the body of the embryo or adult. We have developed a tractable model for MET, where cellular transitions can be directly observed under conditions where molecular, mechanical, and cellular contexts can be controlled experimentally. In this chapter, we introduce a 3-dimensional (3D) tissue model to study MET using Xenopus laevis embryonic mesenchymal cell aggregates.


Asunto(s)
Rastreo Celular/métodos , Ectodermo/citología , Imagenología Tridimensional/métodos , Mesodermo/citología , Técnicas de Cultivo de Tejidos/métodos , Animales , Movimiento Celular , Células Epiteliales/citología , Células Epiteliales/fisiología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Xenopus
6.
PLoS One ; 15(12): e0243914, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33326470

RESUMEN

PURPOSE: Seeking to improve the access to regenerative medicine, this study investigated the structural and transcriptional effects of storage temperature on human oral mucosal epithelial cells (OMECs). METHODS: Cells were stored at four different temperatures (4°C, 12°C, 24°C and 37°C) for two weeks. Then, the morphology, cell viability and differential gene expression were examined using light and scanning electron microscopy, trypan blue exclusion test and TaqMan gene expression array cards, respectively. RESULTS: Cells stored at 4°C had the most similar morphology to non-stored controls with the highest viability rate (58%), whereas the 37°C group was most dissimilar with no living cells. The genes involved in stress-induced growth arrest (GADD45B) and cell proliferation inhibition (TGFB2) were upregulated at 12°C and 24°C. Upregulation was also observed in multifunctional genes responsible for morphology, growth, adhesion and motility such as EFEMP1 (12°C) and EPHA4 (4°C-24°C). Among genes used as differentiation markers, PPARA and TP53 (along with its associated gene CDKN1A) were downregulated in all temperature conditions, whereas KRT1 and KRT10 were either unchanged (4°C) or downregulated (24°C and 12°C; and 24°C, respectively), except for upregulation at 12°C for KRT1. CONCLUSIONS: Cells stored at 12°C and 24°C were stressed, although the expression levels of some adhesion-, growth- and apoptosis-related genes were favourable. Collectively, this study suggests that 4°C is the optimal storage temperature for maintenance of structure, viability and function of OMECs after two weeks.


Asunto(s)
Proliferación Celular/fisiología , Células Epiteliales/fisiología , Mucosa Bucal/fisiología , Manejo de Especímenes , Apoptosis/fisiología , Adhesión Celular/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Criopreservación , Humanos , Temperatura
7.
J Toxicol Sci ; 45(11): 701-711, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33132244

RESUMEN

We aimed to investigate the role of programmed cell death protein 1 (PD-1) and T lymphocytes in the proliferation, apoptosis and secretion of cells from patients and mice with Graves' disease (GD). The levels of serum hormones, related antibodies and inflammatory cytokines in GD patients were determined by electrochemiluminescence immunoassay and ELISA. The percentages of CD4 and CD8 T-lymphocytes and PD-1 expression were examined by flow cytometry. A GD mouse model, a thyroid follicular epithelial cell, and a CD4+PD-1+, CD4+PD-1- and CD8+PD-1+, CD8+PD-1- T lymphocyte co-culture system were constructed. The viability, apoptosis-related markers, serum hormones, related antibodies and inflammatory cytokines in thyroid follicular epithelial cells were determined by CCK-8, Western blot, qTR-PCR, electrochemiluminescence immunoassay and ELISA. Elevated free thyroid hormones (FT3, FT4), thyroid hormone antibodies (TRAb, TPOAb and TGAb), inflammatory cytokines, and inhibited TSH were observed in GD patients. The percentage of CD4+ T cells was increased, while that of CD8+ T cells was reduced in GD patients. PD-1 expression level was lifted in both CD4+ and CD8+ cells from GD patients. In mouse thyroid follicular epithelial cells co-cultured with CD4+PD-1+ and CD8+PD-1+ T lymphocytes, the cell viability, TH and TRAb levels and inflammatory cytokines level were the highest, while the TSH level and apoptosis were the lowest. PD-1 positive T lymphocytes were able to promote viability and inhibit apoptosis of thyroid follicular epithelial cells, which further caused a more accelerated development of GD.


Asunto(s)
Antígeno B7-H1/inmunología , Antígeno B7-H1/fisiología , Proliferación Celular , Citocinas/metabolismo , Células Epiteliales/inmunología , Células Epiteliales/fisiología , Enfermedad de Graves/genética , Enfermedad de Graves/inmunología , Mediadores de Inflamación/metabolismo , Linfocitos/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/fisiología , Glándula Tiroides/citología , Adulto , Animales , Apoptosis , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Enfermedad de Graves/patología , Humanos , Masculino , Ratones Endogámicos BALB C , Persona de Mediana Edad
8.
Science ; 370(6514)2020 10 16.
Artículo en Inglés | MEDLINE | ID: mdl-33060329

RESUMEN

Biological systems tailor their properties and behavior to their size throughout development and in numerous aspects of physiology. However, such size scaling remains poorly understood as it applies to cell mechanics and mechanosensing. By examining how the Drosophila pupal dorsal thorax epithelium responds to morphogenetic forces, we found that the number of apical stress fibers (aSFs) anchored to adherens junctions scales with cell apical area to limit larger cell elongation under mechanical stress. aSFs cluster Hippo pathway components, thereby scaling Hippo signaling and proliferation with area. This scaling is promoted by tricellular junctions mediating an increase in aSF nucleation rate and lifetime in larger cells. Development, homeostasis, and repair entail epithelial cell size changes driven by mechanical forces; our work highlights how, in turn, mechanosensitivity scales with cell size.


Asunto(s)
Epitelio/fisiología , Mecanotransducción Celular , Fibras de Estrés/fisiología , Estrés Mecánico , Animales , Cadherinas/metabolismo , Tamaño de la Célula , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Células Epiteliales/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Miosina Tipo II/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transactivadores/metabolismo
9.
Nat Commun ; 11(1): 5053, 2020 10 07.
Artículo en Inglés | MEDLINE | ID: mdl-33028821

RESUMEN

The epithelial-to-mesenchymal transition (EMT) and the unjamming transition (UJT) each comprises a gateway to cellular migration, plasticity and remodeling, but the extent to which these core programs are distinct, overlapping, or identical has remained undefined. Here, we triggered partial EMT (pEMT) or UJT in differentiated primary human bronchial epithelial cells. After triggering UJT, cell-cell junctions, apico-basal polarity, and barrier function remain intact, cells elongate and align into cooperative migratory packs, and mesenchymal markers of EMT remain unapparent. After triggering pEMT these and other metrics of UJT versus pEMT diverge. A computational model attributes effects of pEMT mainly to diminished junctional tension but attributes those of UJT mainly to augmented cellular propulsion. Through the actions of UJT and pEMT working independently, sequentially, or interactively, those tissues that are subject to development, injury, or disease become endowed with rich mechanisms for cellular migration, plasticity, self-repair, and regeneration.


Asunto(s)
Movimiento Celular/fisiología , Células Epiteliales/fisiología , Transición Epitelial-Mesenquimal/fisiología , Regeneración , Mucosa Respiratoria/fisiología , Bronquios/citología , Bronquios/fisiología , Plasticidad de la Célula/fisiología , Células Cultivadas , Humanos , Cultivo Primario de Células , Mucosa Respiratoria/citología
10.
Arch Virol ; 165(12): 2817-2828, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-32990841

RESUMEN

Enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) are the major pathogens responsible for hand, foot and mouth disease (HFMD), but the mechanism by which these viruses cause disease remains unclear. In this study, we used transcriptome sequencing technology to investigate changes in the transcriptome profiles after infection with EV-A71 and CV-A16 in human bronchial epithelial (16HBE) cells. Using systematic bioinformatics analysis, we then searched for useful clues regarding the pathogenesis of HFMD. As a result, a total of 111 common differentially expressed genes were present in both EV-A71- and CV-A16-infected cells. A trend analysis of these 111 genes showed that 91 of them displayed the same trend in EV-A71 and CV-A16 infection, including 49 upregulated genes and 42 downregulated genes. These 91 genes were further used to conduct GO, pathway, and coexpression network analysis. It was discovered that enriched GO terms (such as histone acetylation and positive regulation of phosphorylation) and pathways (such as glycosylphosphatidylinositol (GPI)-anchor biosynthesis and DNA replication) might be closely associated with the pathogenic mechanism of these two viruses, and key genes (such as TBCK and GPC) might be involved in the progression of HFMD. Finally, we randomly selected 10 differentially expressed genes for qRT-PCR to validate the transcriptome sequencing data. The experimental qRT-PCR results were roughly in agreement with the results of transcriptome sequencing. Collectively, our results provide clues to the mechanism of pathogenesis of HFMD induced by EV-A71 and CV-A16.


Asunto(s)
Enterovirus Humano A/patogenicidad , Células Epiteliales/virología , Perfilación de la Expresión Génica , Enfermedad de Boca, Mano y Pie/patología , Línea Celular , Replicación del ADN , Células Epiteliales/fisiología , Regulación de la Expresión Génica , Enfermedad de Boca, Mano y Pie/virología , Humanos
11.
Nat Cell Biol ; 22(9): 1091-1102, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32868900

RESUMEN

Organs and cells must adapt to shear stress induced by biological fluids, but how fluid flow contributes to the execution of specific cell programs is poorly understood. Here we show that shear stress favours mitochondrial biogenesis and metabolic reprogramming to ensure energy production and cellular adaptation in kidney epithelial cells. Shear stress stimulates lipophagy, contributing to the production of fatty acids that provide mitochondrial substrates to generate ATP through ß-oxidation. This flow-induced process is dependent on the primary cilia located on the apical side of epithelial cells. The interplay between fluid flow and lipid metabolism was confirmed in vivo using a unilateral ureteral obstruction mouse model. Finally, primary cilium-dependent lipophagy and mitochondrial biogenesis are required to support energy-consuming cellular processes such as glucose reabsorption, gluconeogenesis and cytoskeletal remodelling. Our findings demonstrate how primary cilia and autophagy are involved in the translation of mechanical forces into metabolic adaptation.


Asunto(s)
Autofagia/fisiología , Cilios/metabolismo , Cilios/fisiología , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Riñón/metabolismo , Riñón/fisiología , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Gluconeogénesis/fisiología , Glucosa/metabolismo , Metabolismo de los Lípidos/fisiología , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Estrés Mecánico
12.
PLoS Genet ; 16(8): e1008644, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32776941

RESUMEN

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.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Señalización del Calcio , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Contracción Muscular , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Mutación con Ganancia de Función , Células Musculares/metabolismo , Células Musculares/fisiología , Oocitos/fisiología
13.
Anim Sci J ; 91(1): e13422, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32648312

RESUMEN

The aim of this study was to identify factors that regulate ruminal epithelial insulin-like growth factor-binding protein (IGFBP) expression and determine its role in rumen epithelial cell proliferation. Primary bovine rumen epithelial cells (BREC) were incubated with short-chain fatty acids (SCFAs) at pH 7.4 or 5.6, lactate, lipopolysaccharide (LPS), insulin-like growth factor-I (IGF-I), -II (IGF-II), or recombinant bovine IGFBP2 (rbIGFBP2). The mRNA expression levels of IGFBP in BREC were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation rate of BREC was analyzed using a WST-1 assay. IGFBP2 gene expression tended to be lower with SCFA treatment (p < .1), and IGFBP6 gene expression was significantly lower with SCFA treatment (p < .05). IGFBP3 and IGFBP6 gene expression tended to be higher with d-Lactate treatment (p < .1). IGFBP3 gene expression was significantly higher (p < .05) with LPS treatment. BREC treated with IGF-I grew more rapidly than vehicle control-treated cells (p < .01); however, recombinant bovine rbIGFBP2 inhibited IGF-I-induced proliferation. IGF-II and/or rbIGFBP2 did not affect BREC proliferation. Taken together, SCFA treatment decreased IGFBP2 and IGFBP6 expression in rumen epithelial cells, and lower expression of these IGFBP might promote rumen epithelial cell proliferation by facilitating IGF-I.


Asunto(s)
Proliferación Celular/genética , Células Epiteliales/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Expresión Génica/genética , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rumen/citología , Animales , Bovinos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ácidos Grasos Volátiles/farmacología , Expresión Génica/efectos de los fármacos , Concentración de Iones de Hidrógeno , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología
14.
Am J Physiol Lung Cell Mol Physiol ; 319(2): L369-L379, 2020 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-32579851

RESUMEN

Proper development of the respiratory bronchiole and alveolar epithelium proceeds through coordinated cross talk between the interface of epithelium and neighboring mesenchyme. Signals that facilitate and coordinate the cross talk as the bronchial forming canalicular stage transitions to construction of air-exchanging capillary-alveoli niche in the alveolar stage are poorly understood. Expressed within this decisive region, levels of aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1) inversely correlate with the maturation of the lung. The present study addresses the role of AIMP1 in lung development through the generation and characterization of Aimp1-/- mutant mice. Mating of Aimp1+/- produced offspring in expected Mendelian ratios throughout embryonic development. However, newborn Aimp1-/- pups exhibited neonatal lethality with mild cyanosis. Imaging both structure and ultrastructure of Aimp1-/- lungs showed disorganized bronchial epithelium, decreased type I but not type II cell differentiation, increased distal vessels, and disruption of E-cadherin deposition in cell-cell junctions. Supporting the in vivo findings of disrupted epithelial cell-cell junctions, in vitro biochemical experiments show that a portion of AIMP1 binds to phosphoinositides, the lipid anchor of proteins that have a fundamental role in both cellular membrane and actin cytoskeleton organization; a dramatic disruption in F-actin cytoskeleton was observed in Aimp1-/- mouse embryonic fibroblasts. Such observed structural defects may lead to disrupted cell-cell boundaries. Together, these results suggest a requirement of AIMP1 in epithelial cell differentiation in proper lung development.


Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Diferenciación Celular/fisiología , Citocinas/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Pulmón/metabolismo , Pulmón/fisiología , Actinas/metabolismo , Animales , Membrana Celular/metabolismo , Membrana Celular/fisiología , Femenino , Uniones Intercelulares/metabolismo , Uniones Intercelulares/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL
15.
Am J Physiol Gastrointest Liver Physiol ; 319(2): G109-G120, 2020 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-32508154

RESUMEN

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.


Asunto(s)
Enfermedad de Crohn/genética , Células Epiteliales/fisiología , Íleon/citología , Glicoproteínas de Membrana/metabolismo , Transcriptoma , Adulto , Células CACO-2 , Estudios de Casos y Controles , Diferenciación Celular , Enfermedad de Crohn/metabolismo , Regulación de la Expresión Génica , Humanos , Inflamación/metabolismo , Glicoproteínas de Membrana/genética , Consumo de Oxígeno
16.
J Nutr ; 150(8): 2077-2088, 2020 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-32542361

RESUMEN

BACKGROUND: The intestinal epithelial cells, food molecules, and gut microbiota are continuously exposed to intestinal peristaltic shear force. Shear force may impact the crosstalk of human milk oligosaccharides (hMOs) with commensal bacteria and intestinal epithelial cells. OBJECTIVES: We investigated how hMOs combined with intestinal peristaltic shear force impact intestinal epithelial cells and crosstalk with a commensal bacterium. METHODS: We applied the Ibidi system to mimic intestinal peristaltic shear force. Caco-2 cells were exposed to a shear force (5 dynes/cm2) for 3 d, and then stimulated with the hMOs, 2'-fucosyllactose (2'-FL), 3-FL, and lacto-N-triose II (LNT2). In separate experiments, Lactobacillus plantarumWCFS1 adhesion to Caco-2 cells was studied with the same hMOs and shear force. Effects were tested on gene expression of glycocalyx-related molecules (glypican 1 [GPC1], hyaluronan synthase 1 [HAS1], HAS2, HAS3, exostosin glycosyltransferase 1 [EXT1], EXT2), defensin ß-1 (DEFB1), and tight junction (tight junction protein 1 [TJP1], claudin 3 [CLDN3]) in Caco-2 cells. Protein expression of tight junctions was also quantified. RESULTS: Shear force dramatically decreased gene expression of the main enzymes for making glycosaminoglycan side chains (HAS3 by 43.3% and EXT1 by 68.7%) (P <0.01), but did not affect GPC1 which is the gene responsible for the synthesis of glypican 1 which is a major protein backbone of glycocalyx. Expression of DEFB1, TJP1, and CLDN3 genes was decreased 60.0-94.9% by shear force (P <0.001). The presence of L. plantarumWCFS1 increased GPC1, HAS2, HAS3, and ZO-1 expression by 1.78- to 3.34-fold (P <0.05). Under shear force, all hMOs significantly stimulated DEFB1 and ZO-1, whereas only 3-FL and LNT2 enhanced L. plantarumWCFS1 adhesion by 1.85- to 1.90-fold (P <0.01). CONCLUSIONS: 3-FL and LNT2 support the crosstalk between the commensal bacterium L. plantarumWCFS1 and Caco-2 intestinal epithelial cells, and shear force can increase the modulating effects of hMOs.


Asunto(s)
Células Epiteliales/efectos de los fármacos , Mucosa Intestinal/citología , Lactobacillus plantarum/efectos de los fármacos , Leche Humana/química , Oligosacáridos/farmacología , Células CACO-2 , Células Epiteliales/fisiología , Humanos , Lactobacillus plantarum/fisiología , Peristaltismo
17.
Nat Commun ; 11(1): 2366, 2020 05 12.
Artículo en Inglés | MEDLINE | ID: mdl-32398639

RESUMEN

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.


Asunto(s)
Movimiento Celular/fisiología , Desarrollo Embrionario/fisiología , Epitelio/embriología , Diente Molar/embriología , Glándulas Salivales/embriología , Animales , Ectodermo/citología , Ectodermo/embriología , Embrión de Mamíferos/citología , Células Epiteliales/fisiología , Femenino , Microscopía Intravital , Masculino , Ratones , Diente Molar/citología , Glándulas Salivales/citología , Técnicas de Cultivo de Tejidos
18.
Mol Cells ; 43(5): 491-499, 2020 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-32451369

RESUMEN

Hippo signaling acts as a tumor suppressor pathway by inhibiting the proliferation of adult stem cells and progenitor cells in various organs. Liver-specific deletion of Hippo pathway components in mice induces liver cancer development through activation of the transcriptional coactivators, YAP and TAZ, which exhibit nuclear enrichment and are activated in numerous types of cancer. The upstream-most regulators of Warts, the Drosophila ortholog of mammalian LATS1/2, are Kibra, Expanded, and Merlin. However, the roles of the corresponding mammalian orthologs, WWC1, FRMD6 and NF2, in the regulation of LATS1/2 activity and liver tumorigenesis in vivo are not fully understood. Here, we show that deletion of both Wwc1 and Nf2 in the liver accelerates intrahepatic cholangiocarcinoma (iCCA) development through activation of YAP/TAZ. Additionally, biliary epithelial cell-specific deletion of both Lats1 and Lats2 using a Sox9-CreERT2 system resulted in iCCA development through hyperactivation of YAP/TAZ. These findings suggest that WWC1 and NF2 cooperate to promote suppression of cholangiocarcinoma development by inhibiting the oncogenic activity of YAP/TAZ via LATS1/2.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Colangiocarcinoma/genética , Células Epiteliales/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Hígado/fisiología , Neurofibromina 2/genética , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Carcinogénesis/genética , Proteínas de Ciclo Celular/genética , Proliferación Celular/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patología , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Transgénicos , Neurofibromina 2/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Análisis de Matrices Tisulares , Factores de Transcripción/genética
19.
PLoS One ; 15(5): e0232899, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32392240

RESUMEN

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.


Asunto(s)
Técnicas de Cocultivo/instrumentación , Células Epiteliales , Células Madre Mesenquimatosas , Nanoestructuras , Animales , Adhesión Celular , Proliferación Celular , Supervivencia Celular , Células Epiteliales/citología , Células Epiteliales/fisiología , Fibroblastos/citología , Fibroblastos/fisiología , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Ratones , Propiedades de Superficie
20.
Proc Natl Acad Sci U S A ; 117(21): 11531-11540, 2020 05 26.
Artículo en Inglés | MEDLINE | ID: mdl-32414916

RESUMEN

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.


Asunto(s)
Polaridad Celular/fisiología , Proteínas de Drosophila/fisiología , Drosophila , Células Epiteliales , Proteínas de la Membrana/fisiología , Animales , Drosophila/citología , Drosophila/fisiología , Células Epiteliales/citología , Células Epiteliales/fisiología , Epitelio/fisiología , Femenino , Folículo Ovárico/citología , Folículo Ovárico/fisiología , Proteína Quinasa C , Proteínas Supresoras de Tumor
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