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1.
FASEB J ; 35(9): e21816, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34396583

RESUMO

Proper physiological function of mammalian airways requires the differentiation of basal stem cells into secretory or multiciliated cells, among others. In addition, the self-renewal ability of these basal stem cells is crucial for developing a quick response to toxic agents in order to re-establish the epithelial barrier function of the airways. Although these epithelial missions are vital, little is known about those mechanism controlling airway epithelial regeneration in health and disease. p53 has been recently proposed as the guardian of homeostasis, promoting differentiation programs, and antagonizing a de-differentiation program. Here, we exploit mouse and human tracheal epithelial cell culture models to study the role of MDM2-p53 signaling in self-renewal and differentiation in the airway epithelium. We show that p53 protein regulation by MDM2 is crucial for basal stem cell differentiation and to keep proper cell proliferation. Therefore, we suggest that MDM2/p53 interaction modulation is a potential target to control regeneration of the mammalian airway epithelia without massively affecting the epithelium integrity and differentiation potential.


Assuntos
Diferenciação Celular/fisiologia , Epitélio/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Mucosa Respiratória/metabolismo , Células-Tronco/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proliferação de Células/fisiologia , Células Epiteliais/metabolismo , Feminino , Homeostase/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Traqueia/metabolismo
2.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445563

RESUMO

Choroid plexus (CP) sequesters cadmium and other metals, protecting the brain from these neurotoxins. These metals can induce cellular stress and modulate homeostatic functions of CP, such as solute transport. We previously showed in primary cultured neonatal rat CP epithelial cells (CPECs) that cadmium induced cellular stress and stimulated choline uptake at the apical membrane, which interfaces with cerebrospinal fluid in situ. Here, in CPECs, we characterized the roles of glutathione (GSH) and Zinx supplementation in the adaptive stress response to cadmium. Cadmium increased GSH and decreased the reduced GSH-to-oxidized GSH (GSSG) ratio. Heat shock protein-70 (Hsp70), heme oxygenase (HO-1), and metallothionein (Mt-1) were induced along with the catalytic and modifier subunits of glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis. Inhibition of GCL by l-buthionine sulfoximine (BSO) enhanced stress protein induction and stimulation of choline uptake by cadmium. Zinx alone did not induce Hsp70, HO-1, or GCL subunits, or modulate choline uptake. Zinx supplementation during cadmium exposure attenuated stress protein induction and stimulation of choline uptake; this effect persisted despite inhibition of GSH synthesis. These data indicated up-regulation of GSH synthesis promotes adaptation to cadmium-induced cellular stress in CP, but Zinx may confer cytoprotection independent of GSH.


Assuntos
Cádmio/toxicidade , Colina/metabolismo , Plexo Corióideo/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Glutationa/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Zinco/administração & dosagem , Animais , Animais Recém-Nascidos , Plexo Corióideo/metabolismo , Plexo Corióideo/patologia , Suplementos Nutricionais , Epitélio/metabolismo , Epitélio/patologia , Ratos , Ratos Sprague-Dawley
3.
Development ; 148(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34195802

RESUMO

Tooth formation requires complex signaling interactions both within the oral epithelium and between the epithelium and the underlying mesenchyme. Previous studies of the Wnt/ß-catenin pathway have shown that tooth formation is partly inhibited in loss-of-function mutants, and gain-of-function mutants have perturbed tooth morphology. However, the stage at which Wnt signaling is first important in tooth formation remains unclear. Here, using an Fgf8-promoter-driven, and therefore early, deletion of ß-catenin in mouse molar epithelium, we found that loss of Wnt/ß-catenin signaling completely deletes the molar tooth, demonstrating that this pathway is central to the earliest stages of tooth formation. Early expression of a dominant-active ß-catenin protein also perturbs tooth formation, producing a large domed evagination at early stages and supernumerary teeth later on. The early evaginations are associated with premature mesenchymal condensation marker, and are reduced by inhibition of condensation-associated collagen synthesis. We propose that invagination versus evagination morphogenesis is regulated by the relative timing of epithelial versus mesenchymal cell convergence regulated by canonical Wnt signaling. Together, these studies reveal new aspects of Wnt/ß-catenin signaling in tooth formation and in epithelial morphogenesis more broadly.


Assuntos
Dente Molar/crescimento & desenvolvimento , Dente Molar/metabolismo , Odontogênese/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Proliferação de Células , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Mesoderma/metabolismo , Camundongos , Dente Molar/citologia , Morfogênese/fisiologia , Odontogênese/genética , beta Catenina/metabolismo
4.
Nutrients ; 13(7)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209455

RESUMO

Glucose-based solutions remain the most used osmotic agents in peritoneal dialysis (PD), but unavoidably they contribute to the loss of peritoneal filtration capacity. Here, we evaluated at a molecular level the effects of XyloCore, a new PD solution with a low glucose content, in mesothelial and endothelial cells. Cell viability, integrity of mesothelial and endothelial cell membrane, activation of mesothelial and endothelial to mesenchymal transition programs, inflammation, and angiogenesis were evaluated by several techniques. Results showed that XyloCore preserves mesothelial and endothelial cell viability and membrane integrity. Moreover XyloCore, unlike glucose-based solutions, does not exert pro-fibrotic, -inflammatory, and -angiogenic effects. Overall, the in vitro evidence suggests that XyloCore could represent a potential biocompatible solution promising better outcomes in clinical practice.


Assuntos
Soluções para Diálise/farmacologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Glucose/farmacologia , Inflamação/patologia , Mesoderma/metabolismo , Neovascularização Fisiológica , Diálise Peritoneal , Biomarcadores/metabolismo , Linhagem Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Impedância Elétrica , Células Epiteliais/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Mesoderma/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Permeabilidade , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
Development ; 148(11)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34061174

RESUMO

During embryonic development, the otic epithelium and surrounding periotic mesenchymal cells originate from distinct lineages and coordinate to form the mammalian cochlea. Epithelial sensory precursors within the cochlear duct first undergo terminal mitosis before differentiating into sensory and non-sensory cells. In parallel, periotic mesenchymal cells differentiate to shape the lateral wall, modiolus and pericochlear spaces. Previously, Wnt activation was shown to promote proliferation and differentiation of both otic epithelial and mesenchymal cells. Here, we fate-mapped Wnt-responsive epithelial and mesenchymal cells in mice and found that Wnt activation resulted in opposing cell fates. In the post-mitotic cochlear epithelium, Wnt activation via ß-catenin stabilization induced clusters of proliferative cells that dedifferentiated and lost epithelial characteristics. In contrast, Wnt-activated periotic mesenchyme formed ectopic pericochlear spaces and cell clusters showing a loss of mesenchymal and gain of epithelial features. Finally, clonal analyses via multi-colored fate-mapping showed that Wnt-activated epithelial cells proliferated and formed clonal colonies, whereas Wnt-activated mesenchymal cells assembled as aggregates of mitotically quiescent cells. Together, we show that Wnt activation drives transition between epithelial and mesenchymal states in a cell type-dependent manner.


Assuntos
Cóclea/embriologia , Epitélio/metabolismo , Células-Tronco Mesenquimais/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Desdiferenciação Celular , Diferenciação Celular , Proliferação de Células , Cóclea/citologia , Cóclea/crescimento & desenvolvimento , Mesoderma/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Wnt , beta Catenina/metabolismo
6.
Development ; 148(12)2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34128985

RESUMO

Epithelial attachment to the basement membrane (BM) is essential for mammary gland development, yet the exact roles of specific BM components remain unclear. Here, we show that Laminin α5 (Lama5) expression specifically in the luminal epithelial cells is necessary for normal mammary gland growth during puberty, and for alveologenesis during pregnancy. Lama5 loss in the keratin 8-expressing cells results in reduced frequency and differentiation of hormone receptor expressing (HR+) luminal cells. Consequently, Wnt4-mediated crosstalk between HR+ luminal cells and basal epithelial cells is compromised during gland remodeling, and results in defective epithelial growth. The effects of Lama5 deletion on gland growth and branching can be rescued by Wnt4 supplementation in the in vitro model of branching morphogenesis. Our results reveal a surprising role for BM-protein expression in the luminal mammary epithelial cells, and highlight the function of Lama5 in mammary gland remodeling and luminal differentiation.


Assuntos
Diferenciação Celular/genética , Epitélio/metabolismo , Laminina/genética , Glândulas Mamárias Animais/metabolismo , Transdução de Sinais , Proteína Wnt4/genética , Animais , Biomarcadores , Células Epiteliais , Feminino , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Laminina/metabolismo , Glândulas Mamárias Animais/embriologia , Camundongos , Modelos Biológicos , Morfogênese/genética , Organogênese/genética , Proteína Wnt4/metabolismo
7.
Molecules ; 26(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34072205

RESUMO

Mitochondria play a key role in energy metabolism within the cell. Potassium channels such as ATP-sensitive, voltage-gated or large-conductance Ca2+-regulated channels have been described in the inner mitochondrial membrane. Several hypotheses have been proposed to describe the important roles of mitochondrial potassium channels in cell survival and death pathways. In the current study, we identified two populations of mitochondrial large-conductance Ca2+-regulated potassium (mitoBKCa) channels in human bronchial epithelial (HBE) cells. The biophysical properties of the channels were characterized using the patch-clamp technique. We observed the activity of the channel with a mean conductance close to 285 pS in symmetric 150/150 mM KCl solution. Channel activity was increased upon application of the potassium channel opener NS11021 in the micromolar concentration range. The channel activity was completely inhibited by 1 µM paxilline and 300 nM iberiotoxin, selective inhibitors of the BKCa channels. Based on calcium and iberiotoxin modulation, we suggest that the C-terminus of the protein is localized to the mitochondrial matrix. Additionally, using RT-PCR, we confirmed the presence of α pore-forming (Slo1) and auxiliary ß3-ß4 subunits of BKCa channel in HBE cells. Western blot analysis of cellular fractions confirmed the mitochondrial localization of α pore-forming and predominately ß3 subunits. Additionally, the regulation of oxygen consumption and membrane potential of human bronchial epithelial mitochondria in the presence of the potassium channel opener NS11021 and inhibitor paxilline were also studied. In summary, for the first time, the electrophysiological and functional properties of the mitoBKCa channel in a bronchial epithelial cell line were described.


Assuntos
Brônquios/metabolismo , Cálcio/metabolismo , Células Epiteliais/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Consumo de Oxigênio , Potássio/metabolismo , Biofísica , Sobrevivência Celular , Eletrofisiologia , Metabolismo Energético , Epitélio/metabolismo , Humanos , Indóis/química , Potencial da Membrana Mitocondrial , Potenciais da Membrana , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Técnicas de Patch-Clamp , Peptídeos/química , Domínios Proteicos
8.
Int J Mol Sci ; 22(9)2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-34067060

RESUMO

Recent findings suggest that epithelial to mesenchymal transition (EMT), a key step during heart development, is involved in cardiac tissue repair following myocardial infarction (MI). MicroRNAs (miRNAs) act as key regulators in EMT processes; however, the mechanisms by which miRNAs target epicardial EMT remain largely unknown. Here, by using an in vitro model of epicardial EMT, we investigated the role of miRNAs as regulators of this process and their potential targets. EMT was induced in murine epicardial-mesothelial cells (EMCs) through TGF ß1 treatment for 48, 72, and 96 h as indicated by the expression of EMT-related genes by qRT-PCR, WB, and immunofluorescence. Further, enhanced expression of stemness genes was also detected. Among several EMT-related miRNAs, miR-200c-3p expression resulted as the most strongly suppressed. Interestingly, we also found a significant upregulation of Follistatin-related protein 1 (FSTL1), a miR-200c predicted target already identified as a potent cardiogenic factor produced by epicardial cells that promotes regeneration following MI. Dual-luciferase reporter assay demonstrated that miR-200c-3p directly targeted the 3'-untranslated region of FSTL1 in EMCs. Consistently, WB analysis showed that knockdown of miR-200c-3p significantly increased FSTL1 expression, whereas overexpression of miR-200c-3p counteracted TGF ß1-mediated FSTL1 upregulation. Importantly, FSTL1 silencing maintained epithelial features in EMCs, despite EMT induction by TGF ß1, and attenuated EMT-associated traits, including migration and stemness. In conclusion, epicardial FSTL1, an important cardiogenic factor in its secreted form, induces EMT, stemness, and migration of EMCs in a miR-200c-3p dependent pathway.


Assuntos
Transição Epitelial-Mesenquimal , Epitélio/metabolismo , Proteínas Relacionadas à Folistatina/metabolismo , MicroRNAs/metabolismo , Pericárdio/patologia , Animais , Biomarcadores/metabolismo , Transição Epitelial-Mesenquimal/genética , Feminino , Mesoderma/patologia , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator de Crescimento Transformador beta1/farmacologia
9.
Eur J Med Res ; 26(1): 52, 2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108042

RESUMO

BACKGROUND: We are laryngologists. We observe natural phonatory and swallowing functions in clinical examinations with a trans-nasal laryngeal fiberscope (TNLF). Before each observation, we use epinephrine to enlarge and smooth the common nasal meatus (bottom of nostril) and then insert a wet swab inside the nose, as in taking a swab culture in the nasopharynx. During the current COVID-19 pandemic situation, this careful technique prevents any complications, including nasal bleeding, painfulness, and induced sneezing. Here, we introduce our routine to observe esophageal movement in swallowing in a natural (sitting) position without anesthesia. CASE PRESENTATION: The case was a 70-year-old female who complained that something was stuck in her esophagus; there was a strange sensation below the larynx and pharynx. After enlarging and smoothing the common nasal meatus, we inserted the TNLF (slim type ⌀2.9 mm fiberscope, VNL8-J10, PENTAX Medical, Tokyo, Japan.) in the normal way. We then observed the phonatory and swallowing movements of the vocal folds. As usual, to not interfere with natural movements, we used no anesthesia. We found no pathological condition in the pyriform sinus. We asked the patient to swallow the fiberscope. During the swallow, we pushed the TNLF and inserted the tip a bit deeper, which made the fiberscope easily enter the esophagus, like in the insertion of a nasogastric tube. We then asked the patient to swallow a sip of water or saliva to clear and enlarge the lumen of the esophagus. This made it possible to observe the esophagus easily without any air supply. With tone enhancement scan, the esophagus was found to be completely normal except for glycogenic acanthosis. CONCLUSIONS: The advantage of this examination is that it is easily able to perform without anesthesia and with the patient in sitting position. It is quick and minimally invasive, enabling observation the physiologically natural swallowing. It is also possible to observe without anesthesia down to the level of the esophagogastric junction using with a thin type flexible bronchoscope. In the future, gastric fiberscopes might be thinner, even with narrow band imaging (NBI) function. Before that time, physicians should remember to just insert along the bottom of the nose.


Assuntos
COVID-19/prevenção & controle , Esôfago/metabolismo , Glicogênio/metabolismo , Intubação Intratraqueal/métodos , Idoso , Anestesia , COVID-19/epidemiologia , COVID-19/virologia , Epitélio/metabolismo , Feminino , Humanos , Intubação Intratraqueal/instrumentação , Membrana Mucosa/metabolismo , Cavidade Nasal , Pandemias , Reprodutibilidade dos Testes , SARS-CoV-2/fisiologia , Espirro
10.
Molecules ; 26(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067773

RESUMO

BACKGROUND: DNA-RNA compounds have shown promising protection against cell oxidative stress. This study aimed to assess the cytotoxicity, protective, or preventive effect of different experimental formulations on oral epithelia's oxidative stress in vitro. METHODS: Reconstituted human oral epithelia (RHOE) were grown air-lifted in a continuous-flow bioreactor. Mouthwashes and gels containing DNA-RNA compounds and other bioactive molecules were tested on a model of oxidative stress generated by hydrogen peroxide treatment. Epithelia viability was evaluated using a biochemical MTT-based assay and confocal microscopy; structural and ultrastructural morphology was evaluated by light microscopy and TEM. RESULTS: DNA-RNA showed non-cytotoxic activity and effectively protected against oxidative stress, but did not help in its prevention. Gel formulations did not express adequate activity compared to the mouthwashes. Excipients played a fundamental role in enhancing or even decreasing the bioactive molecules' effect. CONCLUSION: A mouthwash formulation with hydrolyzed DNA-RNA effectively protected against oxidative stress without additional enhancement by other bioactive molecules. Active compounds, such as hyaluronic acid, ß-Glucan, allantoin, bisabolol, ruscogenin, and essential oils, showed a protective effect against oxidative stress, which was not synergistic with the one of DNA-RNA. Incorporation of surfactant agents showed a reduced, yet significant, cytotoxic effect.


Assuntos
Mucosa Bucal/metabolismo , Antissépticos Bucais/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Reatores Biológicos/microbiologia , DNA/farmacologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Géis/farmacologia , Engenharia Genética/métodos , Humanos , Mucosa Bucal/efeitos dos fármacos , Antissépticos Bucais/metabolismo , RNA/farmacologia
11.
Environ Toxicol Pharmacol ; 86: 103674, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34029728

RESUMO

Copper (Cu) is an essential element for organism's metabolism, being controversially listed as a priority pollutant. Importantly, the toxicity of Cu has been linked to several cell death pathways. Thus, this study aimed to assess if macroautophagic pathways are triggered by Cu in zebrafish gill, the main target of waterborne pollutants. The electron microscopy findings indicated that Cu induced profound impacts on zebrafish gill structure and functions, being this tissue a biomarker sensitive enough to indicate early toxic effects. The findings also support a clear impairment of autophagy, througth the absence of phagossomes and the significant down-regulation mRNA transcript levels of microtubule-associated protein light chain 3 (LC3). The reduction of LC3 levels was often associated to an increase of apoptotic activation, indicating that the inhibition of macroautophagy triggers apoptosis in zebrafish gills. This study highlighted that the autophagic down-regulation might be affected through the activation of other cell death signaling pathway.


Assuntos
Autofagia/efeitos dos fármacos , Cobre/toxicidade , Epitélio/efeitos dos fármacos , Brânquias/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Epitélio/anatomia & histologia , Epitélio/metabolismo , Epitélio/ultraestrutura , Brânquias/anatomia & histologia , Brânquias/metabolismo , Brânquias/ultraestrutura , Proteínas Associadas aos Microtúbulos/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
12.
Nat Commun ; 12(1): 2886, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001900

RESUMO

The brush border is comprised of microvilli surface protrusions on the apical surface of epithelia. This specialized structure greatly increases absorptive surface area and plays crucial roles in human health. However, transcriptional regulatory networks controlling brush border genes are not fully understood. Here, we identify that hepatocyte nuclear factor 4 (HNF4) transcription factor is a conserved and important regulator of brush border gene program in multiple organs, such as intestine, kidney and yolk sac. Compromised brush border gene signatures and impaired transport were observed in these tissues upon HNF4 loss. By ChIP-seq, we find HNF4 binds and activates brush border genes in the intestine and kidney. H3K4me3 HiChIP-seq identifies that HNF4 loss results in impaired chromatin looping between enhancers and promoters at gene loci of brush border genes, and instead enhanced chromatin looping at gene loci of stress fiber genes in the intestine. This study provides comprehensive transcriptional regulatory mechanisms and a functional demonstration of a critical role for HNF4 in brush border gene regulation across multiple murine epithelial tissues.


Assuntos
Regulação da Expressão Gênica , Fator 4 Nuclear de Hepatócito/genética , Mucosa Intestinal/metabolismo , Rim/metabolismo , Microvilosidades/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Saco Vitelino/metabolismo , Animais , Epitélio/metabolismo , Perfilação da Expressão Gênica/métodos , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Intestinos/ultraestrutura , Rim/ultraestrutura , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Receptores Citoplasmáticos e Nucleares/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Nat Cell Biol ; 23(5): 511-525, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33972733

RESUMO

Epithelial cells rapidly adapt their behaviour in response to increasing tissue demands. However, the processes that finely control these cell decisions remain largely unknown. The postnatal period covering the transition between early tissue expansion and the establishment of adult homeostasis provides a convenient model with which to explore this question. Here, we demonstrate that the onset of homeostasis in the epithelium of the mouse oesophagus is guided by the progressive build-up of mechanical strain at the organ level. Single-cell RNA sequencing and whole-organ stretching experiments revealed that the mechanical stress experienced by the growing oesophagus triggers the emergence of a bright Krüppel-like factor 4 (KLF4) committed basal population, which balances cell proliferation and marks the transition towards homeostasis in a yes-associated protein (YAP)-dependent manner. Our results point to a simple mechanism whereby mechanical changes experienced at the whole-tissue level are integrated with those sensed at the cellular level to control epithelial cell fate.


Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Epiteliais/metabolismo , Homeostase/fisiologia , Animais , Epitélio/metabolismo , Mucosa Esofágica/metabolismo , Humanos , Camundongos , Células-Tronco/metabolismo
14.
Nat Commun ; 12(1): 2861, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001891

RESUMO

Hair cells detect sound, head position or water movements when their mechanosensory hair bundle is deflected. Each hair bundle has an asymmetric architecture that restricts stimulus detection to a single axis. Coordinated hair cell orientations within sensory epithelia further tune stimulus detection at the organ level. Here, we identify GPR156, an orphan GPCR of unknown function, as a critical regulator of hair cell orientation. We demonstrate that the transcription factor EMX2 polarizes GPR156 distribution, enabling it to signal through Gαi and trigger a 180° reversal in hair cell orientation. GPR156-Gαi mediated reversal is essential to establish hair cells with mirror-image orientations in mouse otolith organs in the vestibular system and in zebrafish lateral line. Remarkably, GPR156-Gαi also instructs hair cell reversal in the auditory epithelium, despite a lack of mirror-image organization. Overall, our work demonstrates that conserved GPR156-Gαi signaling is integral to the framework that builds directional responses into mechanosensory epithelia.


Assuntos
Epitélio/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Células Ciliadas Auditivas/metabolismo , Proteínas de Homeodomínio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Transcrição/metabolismo , Animais , Polaridade Celular/genética , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Células Ciliadas Auditivas/citologia , Proteínas de Homeodomínio/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal/métodos , Receptores Acoplados a Proteínas G/genética , Fatores de Transcrição/genética , Peixe-Zebra/metabolismo
15.
Nat Commun ; 12(1): 2587, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972537

RESUMO

Host cells use several anti-bacterial pathways to defend against pathogens. Here, using a uropathogenic Escherichia coli (UPEC) infection model, we demonstrate that bacterial infection upregulates RhoB, which subsequently promotes intracellular bacteria clearance by inducing LC3 lipidation and autophagosome formation. RhoB binds with Beclin 1 through its residues at 118 to 140 and the Beclin 1 CCD domain, with RhoB Arg133 being the key binding residue. Binding of RhoB to Beclin 1 enhances the Hsp90-Beclin 1 interaction, preventing Beclin 1 degradation. RhoB also directly interacts with Hsp90, maintaining RhoB levels. UPEC infections increase RhoB, Beclin 1 and LC3 levels in bladder epithelium in vivo, whereas Beclin 1 and LC3 levels as well as UPEC clearance are substantially reduced in RhoB+/- and RhoB-/- mice upon infection. We conclude that when stimulated by UPEC infections, host cells promote UPEC clearance through the RhoB-Beclin 1-HSP90 complex, indicating RhoB may be a useful target when developing UPEC treatment strategies.


Assuntos
Autofagossomos/metabolismo , Proteína Beclina-1/metabolismo , Infecções por Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Infecções Urinárias/metabolismo , Escherichia coli Uropatogênica/crescimento & desenvolvimento , Proteína rhoB de Ligação ao GTP/metabolismo , Animais , Autofagossomos/genética , Autofagossomos/ultraestrutura , Proteína Beclina-1/genética , Linhagem Celular , Epitélio/metabolismo , Epitélio/microbiologia , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Feminino , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP90/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica , Estabilidade Proteica , RNA Interferente Pequeno , Proteínas Recombinantes , Bexiga Urinária/metabolismo , Bexiga Urinária/microbiologia , Infecções Urinárias/genética , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/patogenicidade , Proteína rhoB de Ligação ao GTP/genética
16.
Dev Cell ; 56(8): 1083-1099.e5, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33831351

RESUMO

Paracellular permeability is regulated to allow solute transport or cell migration across epithelial or endothelial barriers. However, how cell-cell junction dynamics controls paracellular permeability is poorly understood. Here, we describe patency, a developmentally regulated process in Drosophila oogenesis, during which cell vertices in the follicular epithelium open transiently to allow paracellular transport of yolk proteins for uptake by the oocyte. We show that the sequential removal of E-cadherin, N-cadherin, NCAM/Fasciclin 2, and Sidekick from vertices precedes their basal-to-apical opening, while the subsequent assembly of tricellular occluding junctions marks the termination of patency and seals the paracellular barrier. E-cadherin-based adhesion is required to limit paracellular channel size, whereas stabilized adherens junctions, prolonged NCAM/Fasciclin 2 expression, blocked endocytosis, or increased actomyosin contractility prevent patency. Our findings reveal a key role of cell vertices as gateways controlling paracellular transport and demonstrate that dynamic regulation of adhesion and actomyosin contractility at vertices governs epithelial barrier properties.


Assuntos
Drosophila melanogaster/metabolismo , Epitélio/metabolismo , Oogênese , Folículo Ovariano/metabolismo , Actomiosina/metabolismo , Junções Aderentes/metabolismo , Animais , Transporte Biológico , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Permeabilidade da Membrana Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Endocitose , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Junções Íntimas/metabolismo , Vitelogênese
17.
Cell Rep ; 35(5): 109055, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33905739

RESUMO

Coronavirus disease 2019 (COVID-19) is the latest respiratory pandemic caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Although infection initiates in the proximal airways, severe and sometimes fatal symptoms of the disease are caused by infection of the alveolar type 2 (AT2) cells of the distal lung and associated inflammation. In this study, we develop primary human lung epithelial infection models to understand initial responses of proximal and distal lung epithelium to SARS-CoV-2 infection. Differentiated air-liquid interface (ALI) cultures of proximal airway epithelium and alveosphere cultures of distal lung AT2 cells are readily infected by SARS-CoV-2, leading to an epithelial cell-autonomous proinflammatory response with increased expression of interferon signaling genes. Studies to validate the efficacy of selected candidate COVID-19 drugs confirm that remdesivir strongly suppresses viral infection/replication. We provide a relevant platform for study of COVID-19 pathobiology and for rapid drug screening against SARS-CoV-2 and emergent respiratory pathogens.


Assuntos
Células Epiteliais Alveolares/virologia , COVID-19/tratamento farmacológico , COVID-19/patologia , Pulmão/virologia , SARS-CoV-2/efeitos dos fármacos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Adulto , Idoso , Alanina/análogos & derivados , Alanina/farmacologia , Células Epiteliais Alveolares/metabolismo , COVID-19/metabolismo , COVID-19/virologia , Pré-Escolar , Descoberta de Drogas/métodos , Células Epiteliais/virologia , Epitélio/metabolismo , Epitélio/virologia , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Cultura Primária de Células , Mucosa Respiratória/virologia , SARS-CoV-2/fisiologia , Replicação Viral/efeitos dos fármacos
18.
PLoS Negl Trop Dis ; 15(4): e0009319, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33861752

RESUMO

Trichomonas vaginalis is a common protozoan parasite, which causes trichomoniasis associated with severe adverse reproductive outcomes. However, the underlying pathogenesis has not been fully understood. As the first line of defense against invading pathogens, the vaginal epithelial cells are highly responsive to environmental stimuli and contribute to the formation of the optimal luminal fluid microenvironment. The cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel widely distributed at the apical membrane of epithelial cells, plays a crucial role in mediating the secretion of Cl- and HCO3-. In this study, we investigated the effect of T. vaginalis on vaginal epithelial ion transport elicited by prostaglandin E2 (PGE2), a major prostaglandin in the semen. Luminal administration of PGE2 triggered a remarkable and sustained increase of short-circuit current (ISC) in rat vaginal epithelium, which was mainly due to Cl- and HCO3- secretion mediated by the cAMP-activated CFTR. However, T. vaginalis infection significantly abrogated the ISC response evoked by PGE2, indicating impaired transepithelial anion transport via CFTR. Using a primary cell culture system of rat vaginal epithelium and a human vaginal epithelial cell line, we demonstrated that the expression of CFTR was significantly down-regulated after T. vaginalis infection. In addition, defective Cl- transport function of CFTR was observed in T. vaginalis-infected cells by measuring intracellular Cl- signals. Conclusively, T. vaginalis restrained exogenous PGE2-induced anion secretion through down-regulation of CFTR in vaginal epithelium. These results provide novel insights into the intervention of reproductive complications associated with T. vaginalis infection such as infertility and disequilibrium in vaginal fluid microenvironment.


Assuntos
Ânions/metabolismo , Cloretos/metabolismo , Vaginite por Trichomonas/tratamento farmacológico , Vagina/patologia , Animais , Ânions/farmacologia , Transporte Biológico , Linhagem Celular , Células Cultivadas , Antiportadores de Cloreto-Bicarbonato/fisiologia , AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dinoprostona/metabolismo , Células Epiteliais/efeitos dos fármacos , Epitélio/metabolismo , Epitélio/parasitologia , Epitélio/patologia , Feminino , Humanos , Ratos , Ratos Sprague-Dawley , Vaginite por Trichomonas/parasitologia , Trichomonas vaginalis/metabolismo , Vagina/metabolismo , Vagina/parasitologia
19.
PLoS Pathog ; 17(4): e1009041, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33914843

RESUMO

Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus with latent and lytic cycles. EBV replicates in the stratified epithelium but the nasopharynx is also composed of pseudostratified epithelium with distinct cell types. Latent infection is associated with nasopharyngeal carcinoma (NPC). Here, we show with nasopharyngeal conditionally reprogrammed cells cultured at the air-liquid interface that pseudostratified epithelial cells are susceptible to EBV infection. Donors varied in susceptibility to de novo EBV infection, but susceptible cultures also displayed differences with respect to pathogenesis. The cultures from one donor yielded lytic infection but cells from two other donors were positive for EBV-encoded EBERs and negative for other lytic infection markers. All cultures stained positive for the pseudostratified markers CK7, MUC5AC, α-tubulin in cilia, and the EBV epithelial cell receptor Ephrin receptor A2. To define EBV transcriptional programs by cell type and to elucidate latent/lytic infection-differential changes, we performed single cell RNA-sequencing on one EBV-infected culture that resulted in alignment with many EBV transcripts. EBV transcripts represented a small portion of the total transcriptome (~0.17%). All cell types in the pseudostratified epithelium had detectable EBV transcripts with suprabasal cells showing the highest number of reads aligning to many EBV genes. Several restriction factors (IRF1, MX1, STAT1, C18orf25) known to limit lytic infection were expressed at lower levels in the lytic subcluster. A third of the differentially-expressed genes in NPC tumors compared to an uninfected pseudostratified ALI culture overlapped with the differentially-expressed genes in the latent subcluster. A third of these commonly perturbed genes were specific to EBV infection and changed in the same direction. Collectively, these findings suggest that the pseudostratified epithelium could harbor EBV infection and that the pseudostratified infection model mirrors many of the transcriptional changes imposed by EBV infection in NPC.


Assuntos
Células Epiteliais/virologia , Infecções por Vírus Epstein-Barr/virologia , Interações Hospedeiro-Patógeno/imunologia , Neoplasias Nasofaríngeas/virologia , Carcinoma/metabolismo , Carcinoma/virologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Epitélio/virologia , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidade , Humanos , Carcinoma Nasofaríngeo/virologia , RNA Viral/genética
20.
Int J Mol Sci ; 22(5)2021 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-33802611

RESUMO

The objective of this work has been to characterize the estrogenic activity of bisphenol-A (BPA) and the adverse effects on the endocannabinoid system (ECS) in modulating germ cell progression. Male offspring exposed to BPA during the foetal-perinatal period at doses below the no-observed-adverse-effect-level were used to investigate the exposure effects in adulthood. Results showed that BPA accumulates specifically in epididymal fat rather than in abdominal fat and targets testicular expression of 3ß-hydroxysteroid dehydrogenase and cytochrome P450 aromatase, thus promoting sustained increase of estrogens and a decrease of testosterone. The exposure to BPA affects the expression levels of some ECS components, namely type-1 (CB1) and type-2 cannabinoid (CB2) receptor and monoacylglycerol-lipase (MAGL). Furthermore, it affects the temporal progression of germ cells reported to be responsive to ECS and promotes epithelial germ cell exfoliation. In particular, it increases the germ cell content (i.e., spermatogonia while reducing spermatocytes and spermatids), accelerates progression of spermatocytes and spermatids, promotes epithelial detachment of round and condensed spermatids and interferes with expression of cell-cell junction genes (i.e., zonula occcludens protein-1, vimentin and ß-catenin). Altogether, our study provides evidence that early exposure to BPA produces in adulthood sustained and site-specific BPA accumulation in epididymal fat, becoming a risk factor for the reproductive endocrine pathways associated to ECS.


Assuntos
Tecido Adiposo/efeitos dos fármacos , Compostos Benzidrílicos/efeitos adversos , Compostos Benzidrílicos/metabolismo , Endocanabinoides/metabolismo , Epididimo/efeitos dos fármacos , Estrogênios/metabolismo , Células Germinativas/efeitos dos fármacos , Fenóis/efeitos adversos , Fenóis/metabolismo , Tecido Adiposo/metabolismo , Animais , Sistema Endócrino/efeitos dos fármacos , Sistema Endócrino/metabolismo , Epididimo/metabolismo , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Células Germinativas/metabolismo , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/metabolismo , Masculino , Camundongos , Fatores de Risco , Testosterona/metabolismo
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