RESUMO
Most epithelial cells contain apical membrane structures associated to bundles of actin filaments, which constitute the brush border. Whereas microtubule participation in the maintenance of the brush border identity has been characterized, their contribution to de novo microvilli organization remained elusive. Hereby, using a cell model of individual enterocyte polarization, we found that nocodazole induced microtubule depolymerization prevented the de novo brush border formation. Microtubule participation in brush border actin organization was confirmed in polarized kidney tubule MDCK cells. We also found that centrosome, but not Golgi derived microtubules, were essential for the initial stages of brush border development. During this process, microtubule plus ends acquired an early asymmetric orientation toward the apical membrane, which clearly differs from their predominant basal orientation in mature epithelia. In addition, overexpression of the microtubule plus ends associated protein CLIP170, which regulate actin nucleation in different cell contexts, facilitated brush border formation. In combination, the present results support the participation of centrosomal microtubule plus ends in the activation of the polarized actin organization associated to brush border formation, unveiling a novel mechanism of microtubule regulation of epithelial polarity.
Assuntos
Colo/fisiologia , Enterócitos/fisiologia , Células Epiteliais/fisiologia , Rim/fisiologia , Microtúbulos/fisiologia , Microvilosidades/fisiologia , Citoesqueleto de Actina/fisiologia , Animais , Polaridade Celular , Centrômero/fisiologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/ultraestrutura , Cães , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Enterócitos/ultraestrutura , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Rim/efeitos dos fármacos , Rim/ultraestrutura , Células Madin Darby de Rim Canino , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Nocodazol/farmacologia , Fatores de Tempo , Moduladores de Tubulina/farmacologiaRESUMO
Intestinal epithelial cell culture is important for biological, functional, and immunological studies. Since enterocytes have a short in vivo life span due to anoikis, we aimed to establish a novel and reproducible method to prolong the survival of mouse and human cells. Cells were isolated following a standard procedure, and cultured on ordered-cow's collagen membranes. A prolonged cell life span was achieved; cells covered the complete surface of bio-membranes and showed a classical enterocyte morphology with high expression of enzymes supporting the possibility of cryopreservation. Apoptosis was dramatically reduced and cultured enterocytes expressed cytokeratin and LGR5 (low frequency). Cells exposed to LPS or flagellin showed the induction of TLR4 and TLR5 expression and a functional phenotype upon exposure to the probiotic Bifidobacterium bifidum or the pathogenic Clostridium difficile. The secretion of the homeostatic (IL-25 and TSLP), inhibitory (IL-10 and TGF-ß), or pro-inflammatory mediators (IL-1ß and TNF) were induced. In conclusion, this novel protocol using cow's collagen-ordered membrane provides a simple and reproducible method to maintain intestinal epithelial cells functional for cell-microorganism interaction studies and stem cell expansion. J. Cell. Physiol. 232: 2489-2496, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Colágeno/metabolismo , Enterócitos/fisiologia , Membranas Artificiais , Cultura Primária de Células/métodos , Animais , Apoptose , Bifidobacterium bifidum/fisiologia , Biomarcadores/metabolismo , Sobrevivência Celular , Células Cultivadas , Clostridioides difficile/fisiologia , Citocinas/metabolismo , Enterócitos/enzimologia , Enterócitos/microbiologia , Enzimas/metabolismo , Feminino , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação , Queratinas/metabolismo , Masculino , Camundongos da Linhagem 129 , Pessoa de Meia-Idade , Fenótipo , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Tempo , Receptores Toll-Like/metabolismoRESUMO
The expression of flagella correlates with different aspects of bacterial pathogenicity, ranging from adherence to host cells to activation of inflammatory responses by the innate immune system. In the present study, we investigated the role of flagella in the adherence of an atypical enteropathogenic Escherichia coli (aEPEC) strain (serotype O51:H40) to human enterocytes. Accordingly, isogenic mutants deficient in flagellin (FliC), the flagellar structural subunit; the flagellar cap protein (FliD); or the MotAB proteins, involved in the control of flagellar motion, were generated and tested for binding to differentiated Caco-2 cells. Binding of the aEPEC strain to enterocytes was significantly impaired in strains with the fliCa nd fliD genes deleted, both of which could not form flagella on the bacterial surface. A nonmotile but flagellated MotAB mutant also showed impaired adhesion to Caco-2 cells. In accordance with these observations, adhesion of a EPEC strain 1711-4 to Caco-2 cells was drastically reduced after the treatment of Caco-2 cells with purified FliD. In addition, incubation of a EPEC bacteria with specific anti-FliD serum impaired binding to Caco-2 cells. Finally, incubation of Caco-2 cells with purified FliD, followed by immunolabeling, showed that the protein was specifically bound to the microvillus tips of differentiated Caco-2 cells. The a EPEC FliD or anti-FliD serum also reduced the adherence of prototype typical enteropathogenic, enterohemorrhagic, and enterotoxigenic E. coli strains to Caco-2 cells. In conclusion, our findings further strengthened the role of flagella in the adherence of a EPEC to human enterocytes and disclosed the relevant structural and functional involvement of FliD in the adhesion process.
Assuntos
Aderência Bacteriana/fisiologia , Proteínas de Bactérias/metabolismo , Enterócitos/microbiologia , Escherichia coli Enteropatogênica/fisiologia , Microvilosidades/fisiologia , Animais , Anticorpos , Proteínas de Bactérias/genética , Células CACO-2 , Enterócitos/fisiologia , Escherichia coli Enteropatogênica/genética , Humanos , Imuno-Histoquímica , Camundongos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Mutação , Coelhos , Proteínas RecombinantesRESUMO
The aim of this work was to study the effect of sodium deoxycholate (NaDOC) and ursodeoxycholic acid (UDCA) on Ca(2+) uptake by enterocytes and the underlying mechanisms. Rats were divided into four groups: a) controls, b) treated with NaDOC, c) treated with UDCA d) treated with NaDOC and UDCA. Ca(2+) uptake was studied in enterocytes with different degrees of maturation. Apoptosis, autophagy and NO content and iNOS protein expression were evaluated. NaDOC decreased and UDCA increased Ca(2+) uptake only in mature enterocytes. The enhancement of protein expression of Fas, FasL, caspase-8 and caspase-3 activity by NaDOC indicates triggering of the apoptotic extrinsic pathway, which was blocked by UDCA. NO content and iNOS protein expression were enhanced by NaDOC, and avoided by UDCA. The increment of acidic vesicular organelles and LC3 II produced by NaDOC was also prevented by UDCA. In conclusion, the inhibitory effects of NaDOC on intestinal Ca(2+) absorption occur by decreasing the Ca(2+) uptake by mature enterocytes. NaDOC triggers apoptosis and autophagy, in part as a result of nitrosative stress. In contrast, UDCA increases the Ca(2+) uptake by mature enterocytes, and in combination with NaDOC acts as an antiapoptotic and antiautophagic agent normalizing the transcellular Ca(2+) pathway.
Assuntos
Cálcio/metabolismo , Ácido Desoxicólico/administração & dosagem , Enterócitos/citologia , Enterócitos/fisiologia , Absorção Intestinal/fisiologia , Ácido Ursodesoxicólico/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Enterócitos/efeitos dos fármacos , Masculino , Ratos , Ratos WistarRESUMO
Adherence to the intestinal epithelia is a key feature in enteroaggregative Escherichia coli (EAEC) infection. The aggregative adherence fimbriae (AAFs) are involved in EAEC interaction with receptors at the surface of intestinal cells. We and others have demonstrated that fibronectin is a receptor for AAF/II fimbriae. Considering that the major cellular receptor of fibronectin is integrin α5ß1, in this study we evaluated the participation of this receptor in the fibronectin-mediated adherence of EAEC strain 042 to intestinal cells. We found that EAEC strain 042 has the ability to bind directly and indirectly to integrin α5ß1; direct binding was not mediated by AAF/II fimbriae and indirect binding was mediated by AAF/II and fibronectin. Coimmunoprecipitation assays confirmed the formation of the complex AafA/fibronectin/integrin α5ß1. To evaluate EAEC adherence to intestinal cells, T84 cells were incubated with fibronectin and an antibody that blocks the interaction region of integrin α5ß1 to fibronectin, the RGD site. Under these conditions, we found the number of adherent bacteria to epithelial cells significantly reduced. Additionally, fibronectin-mediated adherence of EAEC strain 042 was abolished in HEp-2 cells transfected with integrin α5 shRNA. Altogether, our data support the involvement of integrin α5ß1 in the fibronectin-mediated EAEC binding to intestinal cells.
Assuntos
Aderência Bacteriana/fisiologia , Enterócitos/fisiologia , Escherichia coli/fisiologia , Fibronectinas/metabolismo , Integrina alfa5beta1/metabolismo , Linhagem Celular , Enterócitos/citologia , Escherichia coli/citologia , HumanosRESUMO
Iron deficiency is the most common nutritional deficiency in the world. Special molecules have evolved for iron acquisition, transport and storage in soluble, nontoxic forms. Studies about the effects of iron on health are focused on iron metabolism or nutrition to prevent or treat iron deficiency and anemia. These studies are focused in two main aspects: (1) basic studies to elucidate iron metabolism and (2) nutritional studies to evaluate the efficacy of iron supplementation to prevent or treat iron deficiency and anemia. This paper reviews the advantages and disadvantages of the experimental models commonly used as well as the methods that are more used in studies related to iron. In vitro studies have used different parts of the gut. In vivo studies are done in humans and animals such as mice, rats, pigs and monkeys. Iron metabolism is a complex process that includes interactions at the systemic level. In vitro studies, despite physiological differences to humans, are useful to increase knowledge related to this essential micronutrient. Isotopic techniques are the most recommended in studies related to iron, but their high cost and required logistic, making them difficult to use. The depletion-repletion of hemoglobin is a method commonly used in animal studies. Three depletion-repletion techniques are mostly used: hemoglobin regeneration efficiency, relative biological values (RBV) and metabolic balance, which are official methods of the association of official analytical chemists. These techniques are well-validated to be used as studies related to iron and their results can be extrapolated to humans. Knowledge about the main advantages and disadvantages of the in vitro and animal models, and methods used in these studies, could increase confidence of researchers in the experimental results with less costs.
Assuntos
Enterócitos/fisiologia , Ferro/metabolismo , Fenômenos Fisiológicos da Nutrição Animal , Animais , Transporte Biológico Ativo/fisiologia , Feminino , GravidezRESUMO
The increase in cardiovascular morbidity and mortality associated to insulin resistance (IR) states (obesity, metabolic syndrome, type 2 diabetes) represents a major public health problem. In IR, dyslipidemia typically include hypertriglyceridemia, low high density lipoprotein cholesterol, increased small and dense low density lipoprotein particles, and post-prandial hyperlipidemia, which play a direct or indirect role in the mechanisms of atherosclerosis. Dyslipidemia is mainly due to accumulation of circulating triglyceride-rich lipoproteins from the liver and bowel. The bowel has traditionally been seen as a passive organ, but current evidence confirms that it is an active organ subject to regulation by free fatty acids, insulin, incretins, and inflammation. Two new concepts have emerged: intestinal IR and overproduction of chylomicrons in hyperinsulinemic/IR states. A better understanding of intestinal IR may make the enterocyte a therapeutic target.
Assuntos
Dislipidemias/etiologia , Enterócitos/fisiologia , Resistência à Insulina , Animais , Humanos , Mucosa Intestinal/metabolismo , Lipoproteínas/metabolismo , Triglicerídeos/metabolismoRESUMO
Haematophagy, the utilization of blood as food, has evolved independently among insects such as mosquitoes, bedbugs, fleas, and others. Accordingly, several distinct biological adaptations have occurred in order to facilitate the finding, ingestion and digestion of blood from vertebrate sources. Although blood meals are essential for survival and reproduction of these insects, mechanical and chemical stresses are caused by the ingestion of a sizable meal (frequently twice or more times the weight of the insect) containing large amounts of cytotoxic molecules such as haem. Here we present data showing that the stresses caused by a blood meal induce cell death in the midgut epithelium of Culex quinquefasciatus mosquitoes. The process involves apoptosis, ejection of dead cells to the midgut lumen and differentiation of basal regenerative cells to replace the lost digestive cells. The basal cell differentiation in blood-fed mosquito midguts represents an additional mechanism by which insects cope with the stresses caused by blood meals. C. quinquefasciatus adult females are unable to replace lost cells following a third or fourth blood meal, which may have a significant impact on mosquito longevity, reproduction and vectorial capacity.
Assuntos
Apoptose/fisiologia , Culex/citologia , Culex/fisiologia , Trato Gastrointestinal/citologia , Regeneração/fisiologia , Animais , Sangue/metabolismo , Enterócitos/citologia , Enterócitos/fisiologia , Comportamento Alimentar , Feminino , Mucosa Intestinal/ultraestruturaRESUMO
Iron (Fe) deficiency is one of principal problems of public health associated with nutrition, specially in the developing countries where the content of hem iron (Fe-Hem) of diets is very low and the Fe non hem (Fe-No Hem) has a bioavailability that usually is low, due to the fact that it is influenced by several inhibitors found in diets rich on vegetables and poor on meats. The aim of this article is to discuss aspects related to the interactions of the Fe and other compounds that modify the bioavailability of this metal, specially in the form of Fe-No Hem, in addition, some key concepts to understand the role of the liver in the regulation of the absorption of this micromineral are discussed.
La deficiencia de Fe continua siendo uno de lo principales problemas de salud pública asociados a la nutrición, especialmente en los países en vías de desarrollo donde las dietas tienen bajo contenido de Fe-Hem y alto aporte de Fe-No Hem. Este último tiene una biodisponibilidad que usualmente es baja, debido a que está afectada por los inhibidores presentes en las dietas ricas en productos de origen vegetal y pobres en carnes. El objetivo de esta revisión es discutir los aspectos relacionados con las interacciones del Fe dietario y otros compuestos que modifican la biodisponibilidad del metal, especialmente en la forma de Fe-No Hem, además, se abordan algunos conceptos claves para entender el papel del hígado en la regulación de la absorción de este micromineral.
Assuntos
Humanos , Enterócitos/fisiologia , Alimentos , Heme/farmacocinética , Ferro/farmacocinética , Ácido Ascórbico/farmacologia , Disponibilidade Biológica , Compostos Ferrosos/farmacocinética , Fígado/metabolismoRESUMO
Hepatobiliary transport systems mediate hepatic uptake and biliary excretion of bile acids, bilirubin and other biliary constituents. Hereditary or acquired defects of these transporters may cause or maintain cholestasis and jaundice under various clinical conditions including progressive familial intrahepatic cholestasis (PFIC) 1-3 or its milder forms, benign recurrent intrahepatic cholestasis (BRIC) 1 and 2 , Dubin-Johnson syndrome, drug and inflammation-induced cholestasis and intrahepatic cholestasis of pregnancy. Moreover, induction of alternative efflux pumps for bile acids/bilirubin and phase I/II detoxifying enzymes may counteract hepatic accumulation of potentially toxic biliary constituents in cholestasis by providing alternative escape routes. Transcriptional and post-transcriptional regulation of hepatobiliary transporters in health and disease is mediated by multiple factors such as bile acids, proinflammatory cytokines, drugs and hormones. Ligand-activated nuclear receptors (NR) and hepatocyte-enriched transcription factors play a critical role in transcriptional transporter regulation. Many hepatobiliary transporter alterations in cholestatic liver disease can now be explained by ligand binding of accumulating cholephiles to NRs. Moreover, NR-mediated actions may be targeted by pharmacological ligands. Understanding the transcriptional mechanisms leading to transporter changes therefore not only represents a key for understanding the pathophysiology of the cholestatic liver disease, but also represents a prerequisite for designing novel therapeutic strategies.