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1.
Life Sci ; 231: 116571, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31207308

RESUMO

AIMS: The role of long non-coding RNA's (lncRNA) in the biology of ulcerative colitis (UC) is not well understood. We have previously detected changes in lncRNA's associated with UC. This study aims to characterize one specific lncRNA, CDKN2B-AS1 whose expression was downregulated in UC patients. MAIN METHODS: UC biopsies were used to determine the levels of linear and circular CDKN2B-AS1 relative to healthy controls. In situ hybridization was used to determine the localization of CKDN2B-AS1 in the colon. The intestinal epithelial cell line, Caco-2, was used to study the effects of shRNA mediated loss of CDKN2B-AS1. Transepithelial electrical resistance was used to measure barrier function. An RT-PCR array, immunoblots and immunohistochemistry were used to determine tight junction proteins that CDKN2B-AS1 regulates. KEY FINDINGS: CDKN2B-AS1 is transcribed into not only linear transcripts but also as circular RNA through back-splicing and both forms are decreased in IBD. CDKN2B-AS1 is expressed mainly in colonic epithelial cells. Cells with down-regulated CDKN2B-AS1 exhibited increased proliferation and no alterations in apoptosis. Targeting both the linear and circular transcripts of CDKN2B-AS1 with short hairpin RNAs enhanced barrier function. We subsequently determined that Claudin-2, a "leaky Claudin" known to decrease barrier function, was decreased in CDKN2B-AS1 knockdown cells. SIGNIFICANCE: This study identifies a novel lncRNA with both linear and circular transcripts affecting UC biology.


Assuntos
Doenças Inflamatórias Intestinais/genética , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , Adulto , Apoptose/genética , Células CACO-2 , Proliferação de Células/genética , Claudina-2/genética , Claudina-2/metabolismo , Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , Inibidor de Quinase Dependente de Ciclina p15/biossíntese , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , DNA Circular/genética , Células Epiteliais/metabolismo , Feminino , Humanos , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , RNA/genética , RNA Longo não Codificante/metabolismo
2.
Gastroenterology ; 157(1): 109-118.e5, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31100380

RESUMO

BACKGROUND & AIMS: Confocal laser endomicroscopy (CLE) is a technique that permits real-time detection and quantification of changes in intestinal tissues and cells, including increases in intraepithelial lymphocytes and fluid extravasation through epithelial leaks. Using CLE analysis of patients with irritable bowel syndrome (IBS), we found that more than half have responses to specific food components. Exclusion of the defined food led to long-term symptom relief. We used the results of CLE to detect reactions to food in a larger patient population and analyzed duodenal biopsy samples and fluid from patients to investigate mechanisms of these reactions. METHODS: In a prospective study, 155 patients with IBS received 4 challenges with each of 4 common food components via the endoscope, followed by CLE, at a tertiary medical center. Classical food allergies were excluded by negative results from immunoglobulin E serology analysis and skin tests for common food antigens. Duodenal biopsy samples and fluid were collected 2 weeks before and immediately after CLE and were analyzed by histology, immunohistochemistry, reverse transcription polymerase chain reaction, and immunoblots. Results from patients who had a response to food during CLE (CLE+) were compared with results from patients who did not have a reaction during CLE (CLE-) or healthy individuals (controls). RESULTS: Of the 108 patients who completed the study, 76 were CLE+ (70%), and 46 of these (61%) reacted to wheat. CLE+ patients had a 4-fold increase in prevalence of atopic disorders compared with controls (P = .001). Numbers of intraepithelial lymphocytes were significantly higher in duodenal biopsy samples from CLE+ vs CLE- patients or controls (P = .001). Expression of claudin-2 increased from crypt to villus tip (P < .001) and was up-regulated in CLE+ patients compared with CLE- patients or controls (P = .023). Levels of occludin were lower in duodenal biopsy samples from CLE+ patients vs controls (P = .022) and were lowest in villus tips (P < .001). Levels of messenger RNAs encoding inflammatory cytokines were unchanged in duodenal tissues after CLE challenge, but eosinophil degranulation increased, and levels of eosinophilic cationic protein were higher in duodenal fluid from CLE+ patients than controls (P = .03). CONCLUSIONS: In a CLE analysis of patients with IBS, we found that more than 50% of patients could have nonclassical food allergy, with immediate disruption of the intestinal barrier upon exposure to food antigens. Duodenal tissues from patients with responses to food components during CLE had immediate increases in expression of claudin-2 and decreases in occludin. CLE+ patients also had increased eosinophil degranulation, indicating an atypical food allergy characterized by eosinophil activation.


Assuntos
Alérgenos , Claudina-2/metabolismo , Citocinas/metabolismo , Duodeno/patologia , Proteína Catiônica de Eosinófilo/metabolismo , Hipersensibilidade Alimentar/patologia , Linfócitos Intraepiteliais/patologia , Síndrome do Intestino Irritável/patologia , Ocludina/metabolismo , Adolescente , Adulto , Idoso , Animais , Biópsia , Degranulação Celular , Duodeno/metabolismo , Hipersensibilidade a Ovo/metabolismo , Hipersensibilidade a Ovo/patologia , Clara de Ovo , Endoscopia do Sistema Digestório , Eosinófilos/metabolismo , Feminino , Hipersensibilidade Alimentar/metabolismo , Humanos , Imunoglobulina E , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Síndrome do Intestino Irritável/metabolismo , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Leite , Hipersensibilidade a Leite/metabolismo , Hipersensibilidade a Leite/patologia , Permeabilidade , Estudos Prospectivos , RNA Mensageiro/metabolismo , Soja , Junções Íntimas/metabolismo , Junções Íntimas/patologia , Triticum , Hipersensibilidade a Trigo/metabolismo , Hipersensibilidade a Trigo/patologia , Leveduras , Adulto Jovem
3.
Genes Dev ; 33(3-4): 180-193, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30692208

RESUMO

Claudin-2 promotes breast cancer liver metastasis by enabling seeding and early cancer cell survival. We now demonstrate that the PDZ-binding motif of Claudin-2 is necessary for anchorage-independent growth of cancer cells and is required for liver metastasis. Several PDZ domain-containing proteins were identified that interact with the PDZ-binding motif of Claudin-2 in liver metastatic breast cancer cells, including Afadin, Arhgap21, Pdlim2, Pdlim7, Rims2, Scrib, and ZO-1. We specifically examined the role of Afadin as a potential Claudin-2-interacting partner that promotes breast cancer liver metastasis. Afadin associates with Claudin-2, an interaction that requires the PDZ-binding motif of Claudin-2. Loss of Afadin also impairs the ability of breast cancer cells to form colonies in soft agar and metastasize to the lungs or liver. Immunohistochemical analysis of Claudin-2 and/or Afadin expression in 206 metastatic breast cancer tumors revealed that high levels of both Claudin-2 and Afadin in primary tumors were associated with poor disease-specific survival, relapse-free survival, lung-specific relapse, and liver-specific relapse. Our findings indicate that signaling downstream from a Claudin-2/Afadin complex enables the efficient formation of breast cancer metastases. Moreover, combining Claudin-2 and Afadin as prognostic markers better predicts the potential of breast cancer to metastasize to soft tissues.


Assuntos
Neoplasias da Mama/fisiopatologia , Claudina-2/metabolismo , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/secundário , Proteínas dos Microfilamentos/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Claudina-2/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/fisiopatologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/fisiopatologia , Proteínas dos Microfilamentos/genética , Metástase Neoplásica , Domínios PDZ , Prognóstico , Análise de Sobrevida , Células Tumorais Cultivadas
4.
Cell Mol Gastroenterol Hepatol ; 7(2): 255-274, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30686779

RESUMO

BACKGROUND & AIMS: Epithelial regeneration is essential for homeostasis and repair of the mucosal barrier. In the context of infectious and immune-mediated intestinal disease, interleukin (IL) 22 is thought to augment these processes. We sought to define the mechanisms by which IL22 promotes mucosal healing. METHODS: Intestinal stem cell cultures and mice were treated with recombinant IL22. Cell proliferation, death, and differentiation were assessed in vitro and in vivo by morphometric analysis, quantitative reverse transcriptase polymerase chain reaction, and immunohistochemistry. RESULTS: IL22 increased the size and number of proliferating cells within enteroids but decreased the total number of enteroids. Enteroid size increases required IL22-dependent up-regulation of the tight junction cation and water channel claudin-2, indicating that enteroid enlargement reflected paracellular flux-induced swelling. However, claudin-2 did not contribute to IL22-dependent enteroid loss, depletion of Lgr5+ stem cells, or increased epithelial proliferation. IL22 induced stem cell apoptosis but, conversely, enhanced proliferation within and expanded numbers of transit-amplifying cells. These changes were associated with reduced wnt and notch signaling, both in vitro and in vivo, as well as skewing of epithelial differentiation, with increases in Paneth cells and reduced numbers of enteroendocrine cells. CONCLUSIONS: IL22 promotes transit-amplifying cell proliferation but reduces Lgr5+ stem cell survival by inhibiting notch and wnt signaling. IL22 can therefore promote or inhibit mucosal repair, depending on whether effects on transit-amplifying or stem cells predominate. These data may explain why mucosal healing is difficult to achieve in some inflammatory bowel disease patients despite markedly elevated IL22 production.


Assuntos
Interleucinas/farmacologia , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Notch/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Claudina-2/metabolismo , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Intestinos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Organoides/metabolismo , Células-Tronco/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
5.
Chem Biol Interact ; 288: 83-90, 2018 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-29680209

RESUMO

6-Formylindolo(3,2-b)carbazole (FICZ), a high-affinity aryl hydrocarbon receptor (AhR) ligand, plays a protective role in inflammatory bowel disease (IBD) through activation of AhR. Interleukin-6 (IL-6) induced intestinal epithelial barrier dysfunction is involved in the pathological process of IBD. In this study, we investigated the protective effects of FICZ on IL-6 induced intestinal epithelial barrier injury. Our data show that AhR activation by FICZ ameliorated colonic inflammation, decreased IL-6 and claudin-2 expression, and maintained intestinal barrier function in a mouse model of dextran sulphate sodium (DSS)-induced colitis. In Caco-2 and T84 intestinal epithelial cells, FICZ also prevented the increase of intestinal epithelial permeability and claudin-2 expression induced by IL-6. Depletion of AhR expression by small interfering (si)RNA reversed FICZ induced decrease of claudin-2. Furthermore, IL-6 induced upregulation of claudin-2 was required for increased caudal-related homeobox 2 (CDX-2) and hepatocyte-nuclear factor (HNF)-1α. However, FICZ repressed the increase of CDX-2 and HNF-1α expression induced by IL-6. These results reveal the protective effects of FICZ on IL-6 induced disruption of intestinal epithelial barrier function through suppressing the expression of claudin-2. In addition, CDX-2 and HNF-1α are involved in the regulation of claudin-2 after IL-6 and FICZ treatment. Therefore compounds related to AhR ligands may be potential pharmaceutical agents to treat IBD.


Assuntos
Carbazóis/farmacologia , Claudina-2/metabolismo , Regulação para Baixo/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Fator de Transcrição CDX2/antagonistas & inibidores , Fator de Transcrição CDX2/genética , Fator de Transcrição CDX2/metabolismo , Células CACO-2 , Carbazóis/química , Claudina-2/genética , Colite/induzido quimicamente , Colite/patologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Interleucina-6/farmacologia , Intestinos/citologia , Intestinos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/genética , Regulação para Cima/efeitos dos fármacos
6.
Kaohsiung J Med Sci ; 34(3): 134-141, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29475460

RESUMO

The intestinal mucosal barrier (IMB) enables the intestine to provide adequate containment of luminal microorganisms and molecules while preserving the ability to absorb nutrients. In this study, we explored the effect of brain-derived neurotrophic factor (BDNF) on IMB function and gut microbiota in mice. BDNF gene knock-out mice (the BDNF+/- group) and wild-type mice (the BDNF+/+ group) were selected. The gut microbiota of these mice was analyzed by denaturing gradient gel electrophoresis (DGGE) assay. The ultrastructure of the ileum and the colonic epithelium obtained from decapitated mice were observed by transmission electron microscopy. The protein expression of epithelial tight junction proteins, zonula occludens-1 (ZO-1) and occludin was detected by immunohistochemistry staining. The protein expression of claudin-1 and claudin-2 was determined by Western blotting. The DGGE band patterns of gut microbiota in the BDNF+/- group were significantly different from that in the BDNF+/+ group, which indicated that the BDNF expression alters the gut microbiota in mice. Compared with the BDNF+/+ group, the BDNF+/- group presented no significant difference in the ultrastructure of ileal epithelium; however, a significant difference was observed in the colonic epithelial barrier, manifested by decreased microvilli, widening intercellular space and bacterial invasion. Compared with the BDNF+/+ group, the expression of ZO-1 and occludin in the BDNF+/- group was significantly decreased. The expression of claudin-1 in the BDNF+/- group was significantly reduced, while the expression of claudin-2 was elevated. These findings indicate that BDNF preserves IMB function and modulates gut microbiota in mice.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Colo/microbiologia , Microbioma Gastrointestinal/fisiologia , Íleo/microbiologia , Mucosa Intestinal/microbiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/deficiência , Claudina-1/genética , Claudina-1/metabolismo , Claudina-2/genética , Claudina-2/metabolismo , Colo/metabolismo , Colo/ultraestrutura , Regulação da Expressão Gênica , Íleo/metabolismo , Íleo/ultraestrutura , Mucosa Intestinal/metabolismo , Mucosa Intestinal/ultraestrutura , Masculino , Camundongos , Camundongos Knockout , Microvilosidades/metabolismo , Microvilosidades/microbiologia , Microvilosidades/ultraestrutura , Ocludina/genética , Ocludina/metabolismo , Junções Íntimas/metabolismo , Junções Íntimas/microbiologia , Junções Íntimas/ultraestrutura , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
7.
PLoS One ; 12(12): e0189221, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29252987

RESUMO

BACKGROUND: Claudin-2, ZO-1, and occludin are major components of tight junctions (TJs) in the proximal tubule. However, their roles in maintaining paracellular permeability as leaky epithelia have yet to be defined. METHODS: To investigate the contributory role of TJ proteins in the leaky proximal tubule, we xamined the effect of inhibiting claudin-2, occludin, and ZO-1 expression on transepithelial electrical resistance (TER) and paracellular permeability using the immortalized human proximal tubule epithelial cell line HK-2. For this, small-interfering RNAs (siRNAs) against claudin-2, occludin and ZO-1 were transfected into HK-2 cells. TER and transepithelial flux rates of dextrans (4 and 70 kDa) were determined after 24 h. RESULTS: Transfection of siRNAs (25 nM) knocked down TJ protein expression. Control HK-2 monolayers achieved a steady-state TER of 6-8 Ω·cm2 when grown in 12-well Transwell filters, which are compatible with leaky epithelia. Knockdown of claudin-2 decreased in TER and increased occludin expression. Transfection with siRNA against either occludin or ZO-1 increased TER and decreased claudin-2 expression. TER was decreased by co-inhibition of claudin-2 and ZO-1 but increased by co-inhibition of claudin-2 and occludin. TER was suppressed when claudin-2, occludin, and ZO-1 were all inhibited. Dextran flux rate was increased by claudin-2, occludin, or ZO-1 siRNA transfection. Increased dextran flux was enhanced by co-transfection of claudin-2, ZO-1, and occludin siRNA. CONCLUSIONS: The depletion of claudin-2, occludin and ZO-1 in HK-2 cells had differential effects on TER and macromolecule flux. We demonstrated that integration of claudin-2, occludin and ZO-1 is necessary for maintaining the function of the proximal tubular epithelium.


Assuntos
Claudina-2/metabolismo , Células Epiteliais/metabolismo , Ocludina/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular , Dextranos/metabolismo , Impedância Elétrica , Técnicas de Silenciamento de Genes , Humanos , RNA Interferente Pequeno/metabolismo , Transfecção
8.
Exp Parasitol ; 183: 69-75, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29097064

RESUMO

Free-living amoebae of the genus Acanthamoeba are protozoa ubiquitously found in nature. Some species of the genus are potentially pathogenic for humans provoking keratitis in healthy individuals, often in contact lens wearers and opportunistic infections such as pneumonitis, fatal granulomatous encephalitis and skin infections, particularly in immunocompromised individuals. The pathogenic mechanisms of these amoebae are poorly understood, however it had been suggested that contact dependent mechanisms are important during invasion, regardless of the epithelia type, since amoebae penetrate epithelia separating tight junction (TJ). This study was undertaken to determine whether Acanthamoeba sp. (T4) damages the barrier function of the TJ in MDCK epithelial monolayers. Actin cytoskeleton staining and electron microscopy analyses were performed; paracellular permeability and TJ sealing were evaluated by apicobasolateral diffusion of ruthenium red and transepithelial resistance (TER) measurements; immunofluorescence and Western blot assays were performed to locate and estimate expression of TJ protein claudins 2 (Cldn2) and 4 (Cldn4). The results show that Acanthamoeba sp. crosses the MDCK monolayer without altering the actin cytoskeleton or the morphology of the cells. When trophozoites or conditioned medium interact with the monolayer, paracellular diffusion of ruthenium red increases. After 6 h, the amoebae, but not their conditioned medium, increase the TER, and Cldn2 is removed from the TJ, and its overall content in the cells diminishes, while Cldn4 is targeted to the TJ without changing its expression level. In conclusion Acanthamoeba (T4) crosses MDCK monolayer without damaging the cells, increasing permeability and TER through Cldn2 degradation, and redirecting Cldn4 to TJ. These results strongly suggest that contact-dependent mechanisms are relevant during amoebae invasion.


Assuntos
Acanthamoeba/fisiologia , Células Madin Darby de Rim Canino/parasitologia , Junções Íntimas/parasitologia , Acanthamoeba/patogenicidade , Acanthamoeba/ultraestrutura , Animais , Western Blotting , Claudina-2/metabolismo , Claudina-4/metabolismo , Meios de Cultivo Condicionados , Cães , Impedância Elétrica , Imunofluorescência , Indicadores e Reagentes/metabolismo , Células Madin Darby de Rim Canino/ultraestrutura , Microscopia Eletrônica de Transmissão , Permeabilidade , Rutênio Vermelho/metabolismo , Junções Íntimas/química , Junções Íntimas/metabolismo , Trofozoítos/fisiologia , Trofozoítos/ultraestrutura
9.
Artigo em Inglês | MEDLINE | ID: mdl-28861400

RESUMO

During intestinal invasion, Entamoeba histolytica opens tight junctions (TJs) reflected by transepithelial electrical resistance (TEER) dropping. To explore the molecular mechanisms underlying this, we studied in vitro and in vivo the damage produced by the recombinant E. histolytica cysteine protease (rEhCP112) on TJ functions and proteins. rEhCP112 reduced TEER in Caco-2 cells in a dose- and time-dependent manner; and EhCP112-overexpressing trophozoites provoked major epithelial injury compared to control trophozoites. rEhCP112 penetrated through the intercellular space, and consequently the ion flux increased and the TJs fence function was disturbed. However, macromolecular flux was not altered. Functional in vitro assays revealed specific association of rEhCP112 with claudin-1 and claudin-2, that are both involved in regulating ion flux and fence function. Of note, rEhCP112 did not interact with occludin that is responsible for regulating macromolecular flux. Moreover, rEhCP112 degraded and delocalized claudin-1, thus affecting interepithelial adhesion. Concomitantly, expression of the leaky claudin-2 at TJ, first increased and then it was degraded. In vivo, rEhCP112 increased intestinal epithelial permeability in the mouse colon, likely due to apical erosion and claudin-1 and claudin-2 degradation. In conclusion, we provide evidence that EhCP112 causes epithelial dysfunction by specifically altering claudins at TJ. Thus, EhCP112 could be a potential target for therapeutic approaches against amoebiasis.


Assuntos
Proteínas de Bactérias/farmacologia , Claudina-1/efeitos dos fármacos , Claudina-2/efeitos dos fármacos , Cisteína Endopeptidases/farmacologia , Entamoeba histolytica/metabolismo , Células Epiteliais/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Claudina-1/metabolismo , Claudina-2/metabolismo , Claudina-4/efeitos dos fármacos , Colo/efeitos dos fármacos , Colo/parasitologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Modelos Animais de Doenças , Cães , Entamoeba histolytica/genética , Entamoeba histolytica/patogenicidade , Entamebíase/patologia , Regulação da Expressão Gênica , Humanos , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ocludina/efeitos dos fármacos , Permeabilidade , Proteínas Recombinantes/farmacologia , Junções Íntimas/metabolismo , Trofozoítos/genética , Trofozoítos/metabolismo , Proteína da Zônula de Oclusão-1/efeitos dos fármacos
10.
Life Sci ; 188: 149-157, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28882647

RESUMO

AIMS: Type 2 diabetes mellitus (T2DM) is one of the most prevalent diseases worldwide. Diabetic nephropathy (DN) is a complication of diabetes and the mechanisms underlying onset and progression of this disease are not fully understood. It has been shown that hyperglycemia is an independent factor to predict the development of DN in individuals with T2DM, however, a link between high plasma glucose levels and renal tubular injuries in DN remains unknown. In this study, we investigated the effect of high levels of glucose (i.e. 180 or 360mg/dL) for up to 24h (acute) or over 72h (chronic) upon tight junction (TJ)-mediated epithelial barrier integrity of the kidney tubular cell line, MDCK. METHODS/KEY FINDINGS: High levels of glucose (180 and 360mg/dL) induced a decrease in transepithelial electrical resistance associated with an increase in TJ cation selectivity at 24h or in TJ permeability to a paracellular marker, Lucifer Yellow, at 72h-exposure when compared to control group (exposed to 100mg/dL glucose). Immunofluorescence analyses showed that glucose treatment induced a significant decrease in the tight junctional content of claudins-1 and -3 as well as a significant increase in claudin-2 (particularly at 24h-exposure) and a time-dependent change in occludin/ZO-1 junctional content. The analyses of total cell content of these junctional proteins by Western blot did not reveal significant changes, except in claudin-2 expression. SIGNIFICANCE: Our data suggest that high levels of glucose induce time-dependence changes in TJ structure in MDCK monolayers, suggesting a possible link between hyperglycemia-induced tubular epithelial barrier disruption and diabetic nephropathy.


Assuntos
Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Glucose/efeitos adversos , Túbulos Renais/citologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/fisiologia , Animais , Células Cultivadas , Claudina-1/metabolismo , Claudina-2/metabolismo , Claudina-3/metabolismo , Cães , Células Madin Darby de Rim Canino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Ocludina/metabolismo , Junções Íntimas/metabolismo
11.
Br J Nutr ; 118(5): 321-332, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28901890

RESUMO

We investigated the effects of dietary l-arginine level and feeding duration on the intestinal damage of broilers induced by Clostridium perfringens (CP) in vivo, and the antimicrobial effect of its metabolite nitric oxide (NO) in vitro. The in vivo experiment was designed as a factorial arrangement of three dietary treatments×two challenge statuses. Broilers were fed a basal diet (CON) or a high-arginine diet (ARG) containing 1·87 % l-arginine, or CON for the first 8 d and ARG from days 9 to 28 (CON/ARG). Birds were co-infected with or without Eimeria and CP (EM/CP). EM/CP challenge led to intestinal injury, as evidenced by lower plasma d-xylose concentration (P<0·01), higher paracellular permeability in the ileum (P<0·05) and higher numbers of Escherichia coli (P<0·05) and CP (P<0·001) in caecal digesta; however, this situation could be alleviated by l-arginine supplementation (P<0·05). The intestinal claudin-1 and occludin mRNA expression levels were decreased (P<0·05) following EM/CP challenge; this was reversed by l-arginine supplementation (P<0·05). Moreover, EM/CP challenge up-regulated (P<0·05) claudin-2, interferon-γ (IFN-γ), toll-like receptor 2 and nucleotide-binding oligomerisation domain 1 (NOD1) mRNA expression, and l-arginine supplementation elevated (P<0·05) IFN-γ, IL-10 and NOD1 mRNA expression. In vitro study showed that NO had bacteriostatic activity against CP (P<0·001). In conclusion, l-arginine supplementation could inhibit CP overgrowth and alleviate intestinal mucosal injury by modulating innate immune responses, enhancing barrier function and producing NO.


Assuntos
Arginina/administração & dosagem , Clostridium perfringens/efeitos dos fármacos , Dieta/veterinária , Imunidade Inata , Mucosa Intestinal/efeitos dos fármacos , Intestinos/microbiologia , Ração Animal/análise , Animais , Galinhas , Claudina-1/genética , Claudina-1/metabolismo , Claudina-2/genética , Claudina-2/metabolismo , Suplementos Nutricionais , Eimeria/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD1/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Regulação para Cima , Xilose/sangue
12.
J Immunol ; 199(8): 2976-2984, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28893958

RESUMO

Commensal interactions between the enteric microbiota and distal intestine play important roles in regulating human health. Short-chain fatty acids (SCFAs), such as butyrate, produced through anaerobic microbial metabolism represent a major energy source for the host colonic epithelium and enhance epithelial barrier function through unclear mechanisms. Separate studies revealed that the epithelial anti-inflammatory IL-10 receptor α subunit (IL-10RA) is also important for barrier formation. Based on these findings, we examined if SCFAs promote epithelial barrier through IL-10RA-dependent mechanisms. Using human intestinal epithelial cells (IECs), we discovered that SCFAs, particularly butyrate, enhanced IEC barrier formation, induced IL-10RA mRNA, IL-10RA protein, and transactivation through activated Stat3 and HDAC inhibition. Loss and gain of IL-10RA expression directly correlates with IEC barrier formation and butyrate represses permeability-promoting claudin-2 tight-junction protein expression through an IL-10RA-dependent mechanism. Our findings provide a novel mechanism by which microbial-derived butyrate promotes barrier through IL-10RA-dependent repression of claudin-2.


Assuntos
Bactérias Anaeróbias/fisiologia , Butiratos/metabolismo , Colo/patologia , Microbioma Gastrointestinal/imunologia , Mucosa Intestinal/fisiologia , Receptores de Interleucina-10/metabolismo , Junções Íntimas/metabolismo , Butiratos/imunologia , Linhagem Celular , Células Cultivadas , Claudina-2/metabolismo , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Humanos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Receptores de Interleucina-10/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Simbiose , Ativação Transcricional , Migração Transendotelial e Transepitelial , Regulação para Cima
13.
Ann N Y Acad Sci ; 1405(1): 116-130, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28804910

RESUMO

T cell protein tyrosine phosphatase (TCPTP) dephosphorylates a number of substrates, including JAK-STAT (signal transducer and activator of transcription) signaling proteins, which are activated by interferon (IFN)-γ, a major proinflammatory cytokine involved in conditions such as inflammatory bowel disease. A critical function of the intestinal epithelium is formation of a selective barrier to luminal contents. The structural units of the epithelium that regulate barrier function are the tight junctions (TJs), and the protein composition of the TJ determines the tightness of the barrier. Claudin-2 is a TJ protein that increases permeability to cations and reduces transepithelial electrical resistance (TER). We previously showed that transient knockdown (KD) of TCPTP permits increased expression of claudin-2 by IFN-γ. Here, we demonstrate that the decreased TER in TCPTP-deficient epithelial cells is alleviated by STAT1 KD. Moreover, increased claudin-2 in TCPTP-deficient cells requires enhanced STAT1 activation and STAT1 binding to the CLDN2 promoter. We also show that mutation of this STAT-binding site prevents elevated CLDN2 promoter activity in TCPTP-deficient epithelial cells. In summary, we demonstrate that TCPTP protects the intestinal epithelial barrier by restricting STAT-induced claudin-2 expression. This is a potential mechanism by which loss-of-function mutations in the gene encoding TCPTP may contribute to barrier defects in chronic intestinal inflammatory disease.


Assuntos
Claudina-2/metabolismo , Mucosa Intestinal/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Fator de Transcrição STAT1/metabolismo , Junções Íntimas/metabolismo , Linhagem Celular , Claudina-2/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Mucosa Intestinal/citologia , Regiões Promotoras Genéticas , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Fator de Transcrição STAT1/genética
14.
Biomed Pharmacother ; 94: 468-473, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28779708

RESUMO

Our research group previously isolated and identified a strain of pathogenic Escherichia coli from clinical samples called E. coli O124 K72. The present study was aimed at determining the potential effects of E. coli O124 K72 on intestinal barrier functions and structural proteins integrity in guinea pig. Guinea pigs were grouped into three groups; control (CG); E. coli O124 K72 (E. coli); and probiotics Lactobacillus rhamnosus (LGG). Initially, we create intestinal dysbiosis by giving all animals Levofloxacin for 10days, but the control group (CG) received the same volume of saline. Then, the animals received either E. coli O124 K72 (E. coli) or Lactobacillus rhamnosus (LGG) according to their assigned group. E. coli O124 K72 treatment significantly affected colon morphology and distorted intestinal barrier function by up-regulating Claudin2 and down-regulating Occludin. In addition, E. coli upregulated the mRNA expression of MUC1, MUC2, MUC13 and MUC15. Furthermore, suspected tumor was found in the E. coli treated animals. Our results suggested that E. coli O124 K72 strain has adverse effects on intestinal barrier functions and is capable of altering integrity of structural proteins in guinea pig model while at same time it may have a role in colon carcinogenesis.


Assuntos
Escherichia coli/fisiologia , Intestinos/microbiologia , Intestinos/patologia , Proteínas de Junções Íntimas/metabolismo , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Claudina-2/metabolismo , Eletroforese em Gel de Gradiente Desnaturante , Modelos Animais de Doenças , Disbiose/tratamento farmacológico , Disbiose/metabolismo , Disbiose/microbiologia , Disbiose/patologia , Microbioma Gastrointestinal , Cobaias , Intestinos/efeitos dos fármacos , Mucina-2/metabolismo , Ocludina/metabolismo
15.
Cancer Res ; 77(18): 4809-4822, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28754672

RESUMO

Dissociation from epithelial sheets and invasion through the surrounding stroma are critical early events during epithelial cancer metastasis. Here we find that a lymphocyte lineage-restricted transcription factor, Spi-B, is frequently expressed in human lung cancer tissues. The Spi-B-expressing cancer cells coexpressed vimentin but repressed E-cadherin and exhibited invasive behavior. Increased Spi-B expression was associated with tumor grade, lymphatic metastasis, and short overall survival. Mechanistically, Spi-B disrupted intercellular junctions and enhanced invasiveness by reconfiguring the chromatin structure of the tight junction gene claudin-2 (CLDN2) and repressing its transcription. These data suggest that Spi-B participates in mesenchymal invasion, linking epithelial cancer metastasis with a lymphatic transcriptional program. Cancer Res; 77(18); 4809-22. ©2017 AACR.


Assuntos
Carcinoma Pulmonar de Lewis/secundário , Claudina-2/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/patologia , Fatores de Transcrição/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Animais , Apoptose , Biomarcadores Tumorais , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/secundário , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/secundário , Proliferação de Células , Claudina-2/genética , Proteínas de Ligação a DNA/genética , Humanos , Junções Intercelulares , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , Estadiamento de Neoplasias , Prognóstico , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/secundário , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Cell Host Microbe ; 21(6): 671-681.e4, 2017 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-28618266

RESUMO

Diarrhea is a host response to enteric pathogens, but its impact on pathogenesis remains poorly defined. By infecting mice with the attaching and effacing bacteria Citrobacter rodentium, we defined the mechanisms and contributions of diarrhea and intestinal barrier loss to host defense. Increased permeability occurred within 2 days of infection and coincided with IL-22-dependent upregulation of the epithelial tight junction protein claudin-2. Permeability increases were limited to small molecules, as expected for the paracellular water and Na+ channel formed by claudin-2. Relative to wild-type, claudin-2-deficient mice experienced severe disease, including increased mucosal colonization by C. rodentium, prolonged pathogen shedding, exaggerated cytokine responses, and greater tissue injury. Conversely, transgenic claudin-2 overexpression reduced disease severity. Chemically induced osmotic diarrhea reduced colitis severity and C. rodentium burden in claudin-2-deficient, but not transgenic, mice, demonstrating that claudin-2-mediated protection is the result of enhanced water efflux. Thus, IL-22-induced claudin-2 upregulation drives diarrhea and pathogen clearance.


Assuntos
Claudina-2/metabolismo , Diarreia/metabolismo , Infecções por Enterobacteriaceae/imunologia , Epitélio/metabolismo , Interleucinas/metabolismo , Mucosa Intestinal/metabolismo , Regulação para Cima , Animais , Permeabilidade da Membrana Celular , Citrobacter rodentium/imunologia , Citrobacter rodentium/patogenicidade , Colite/microbiologia , Citocinas/metabolismo , Diarreia/imunologia , Diarreia/microbiologia , Diarreia/patologia , Modelos Animais de Doenças , Infecções por Enterobacteriaceae/microbiologia , Epitélio/imunologia , Epitélio/microbiologia , Epitélio/patologia , Imunidade Inata/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Intestinos/microbiologia , Intestinos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Sódio/metabolismo , Junções Íntimas/metabolismo , Água/metabolismo
17.
Ann N Y Acad Sci ; 1397(1): 100-109, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28636801

RESUMO

Physiological studies in leaky epithelia, like kidney proximal tubules and the small intestine, have documented water transport via both transcellular and paracellular pathways. The discovery of aquaporin water channels provided a molecular basis for transcellular water movement. In contrast, the contribution, or even existence, of a specific paracellular water pathway has been disputed for a long time, until the cation channel-forming tight junction protein claudin-2 was shown to also permit the paracellular passage of water through its pore. In proximal kidney tubules, claudin-2-based water transport contributes 23-30% of the total water transport. Other paracellular ion channels (claudin-10a, -10b, and -17) proved to be impermeable to water, although their pore size would be sufficient for water molecules to pass. Studies of barrier-forming claudins, like claudin-1 and claudin-3, which tighten the paracellular pathway against ions and larger solutes, indicate that changes in the expression of these sealing claudins do not influence transepithelial water permeability. The present genetic, molecular, computational, and physiological studies are just now beginning to probe the mechanisms and regulation of paracellular permeation.


Assuntos
Aquaporinas/metabolismo , Claudina-2/metabolismo , Claudinas/metabolismo , Junções Íntimas/metabolismo , Água/metabolismo , Animais , Transporte Biológico , Humanos , Túbulos Renais Proximais/metabolismo , Permeabilidade
18.
Ann N Y Acad Sci ; 1397(1): 209-218, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28605031

RESUMO

Claudins define paracellular permeability to small molecules by forming ion-selective pores within the tight junction. We recently demonstrated that claudin-2 channels are gated and open and close on a submillisecond timescale. To determine if and how the ensemble behavior of this unique class of entirely extracellular gated ion channels could define global epithelial barrier function, we have developed an in silico model of local claudin-2 behavior. This model considers the complex anastomosing ultrastructure of tight junction strands and can be scaled to show that local behavior defines global epithelial barrier function of epithelial monolayers expressing different levels of claudin-2. This is the first mathematical model to describe global epithelial barrier function in terms of the dynamic behavior of single tight junction channels and establishes a framework to consider gating kinetics as a means to regulate barrier function.


Assuntos
Claudina-2/fisiologia , Células Epiteliais/fisiologia , Canais Iônicos/fisiologia , Junções Íntimas/fisiologia , Algoritmos , Animais , Claudina-2/genética , Claudina-2/metabolismo , Simulação por Computador , Cães , Células Epiteliais/metabolismo , Ativação do Canal Iônico , Canais Iônicos/metabolismo , Cinética , Células Madin Darby de Rim Canino , Camundongos , Modelos Biológicos , Técnicas de Patch-Clamp , Junções Íntimas/metabolismo
19.
Biochim Biophys Acta Mol Cell Res ; 1864(10): 1714-1733, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28554775

RESUMO

Silencing Zonula occludens 2 (ZO-2), a tight junctions (TJ) scaffold protein, in epithelial cells (MDCK ZO-2 KD) triggers: 1) Decreased cell to substratum attachment, accompanied by reduced expression of claudin-7 and integrin ß1, and increased vinculin recruitment to focal adhesions and stress fibers formation; 2) Lowered cell-cell aggregation and appearance of wider intercellular spaces; 3) Increased RhoA/ROCK activity, mediated by GEF-HI recruitment to cell borders by cingulin; 4) Increased Cdc42 activity, mitotic spindle disorientation and the appearance of cysts with multiple lumens; 5) Increased Rac and cofilin activity, multiple lamellipodia formation and random cell migration but increased wound closure; 6) Diminished cingulin phosphorylation and disappearance of planar network of microtubules at the TJ region; and 7) Increased transepithelial electrical resistance at steady state, coupled to an increased expression of ZO-1 and claudin-4 and a decreased expression of claudin-2 and paracingulin. Hence, ZO-2 is a crucial regulator of Rho proteins activity and the development of epithelial cytoarchitecture and barrier function.


Assuntos
Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-2/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genética , Animais , Claudina-2/metabolismo , Claudina-4/genética , Claudina-4/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Cães , Células Epiteliais/metabolismo , Humanos , Células Madin Darby de Rim Canino , Fosforilação , Junções Íntimas/genética , Transfecção
20.
PLoS One ; 12(3): e0172914, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28328972

RESUMO

Many of the symptoms of Gulf War Illness (GWI) that include neurological abnormalities, neuroinflammation, chronic fatigue and gastrointestinal disturbances have been traced to Gulf War chemical exposure. Though the association and subsequent evidences are strong, the mechanisms that connect exposure to intestinal and neurological abnormalities remain unclear. Using an established rodent model of Gulf War Illness, we show that chemical exposure caused significant dysbiosis in the gut that included increased abundance of phylum Firmicutes and Tenericutes, and decreased abundance of Bacteroidetes. Several gram negative bacterial genera were enriched in the GWI-model that included Allobaculum sp. Altered microbiome caused significant decrease in tight junction protein Occludin with a concomitant increase in Claudin-2, a signature of a leaky gut. Resultant leaching of gut caused portal endotoxemia that led to upregulation of toll like receptor 4 (TLR4) activation in the small intestine and the brain. TLR4 knock out mice and mice that had gut decontamination showed significant decrease in tyrosine nitration and inflammatory mediators IL1ß and MCP-1 in both the small intestine and frontal cortex. These events signified that gut dysbiosis with simultaneous leaky gut and systemic endotoxemia-induced TLR4 activation contributes to GW chemical-induced neuroinflammation and gastrointestinal disturbances.


Assuntos
Lobo Frontal/metabolismo , Microbioma Gastrointestinal/fisiologia , Inflamação/metabolismo , Enteropatias/metabolismo , Enteropatias/microbiologia , Síndrome do Golfo Pérsico/microbiologia , Receptor 4 Toll-Like/metabolismo , Animais , Claudina-2/metabolismo , Modelos Animais de Doenças , Disbiose/metabolismo , Endotoxemia/metabolismo , Guerra do Golfo , Inflamação/microbiologia , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Síndrome do Golfo Pérsico/metabolismo
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