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1.
Gene ; 759: 144999, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32717305

RESUMO

Clostridium perfringens beta2 (CPB2), a key virulence factor, is produced by C. perfringens type C that is the main pathogenic microorganism causing diarrhea in piglets. However, little is known concerning the toxic damage effect of CPB2 on intestinal cells of piglets. In present study, CPB2 toxin obtained by genetic recombination technology was evaluated for its cytotoxicity property using the intestinal porcine epithelial (IPEC-J2) cells, which aims to attempt to understand and explain its mechanism of action in porcine small intestinal epithelial cells. IPEC-J2 cells were treated with different concentrations of CPB2 toxin (5, 10, 20, 30, 40, and 50 µg/mL), and MTT assay results showed that the cell viability of CPB2-treated IPEC-J2 cells decreased in a dose-dependent manner. Lactate dehydrogenase (LDH) assay results revealed that CPB2 significantly increased the LDH release, relative to the control. The expression of tumor necrosis factor α (TNF-α) and interleukin 8 (IL-8) gradually increased, while the expression of interleukin 10 (IL-10) gradually decreased in IPEC-J2 cells with increasing concentration of CPB2 (10-30 µg/mL), as analyzed by quantitative real-time PCR (RT-qPCR). Also, CPB2 increased the content of intracellular reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) of IPEC-J2 cells. Western blot and immunofluorescence results demonstrate that CPB2 decreased the expression of zonula occludens (ZO-1), claudin12 (CLDN12) and occludin (OCLN) in IPEC-J2 cells. In addition, CPB2 increased Bax expression, and inhibited Bcl-2 and Bcl-xL expression, as measured by Western blot. Considering all of these findings, it was concluded that CPB2 toxin shows significant cytotoxicity, cell growth inhibition and increase in cell permeability in IPEC-J2 cells in a concentration-dependent manner, thus leading to abnormal cell apoptosis and functions in porcine small intestinal epithelial cells.


Assuntos
Toxinas Bacterianas/toxicidade , Células Epiteliais/efeitos dos fármacos , Estresse Oxidativo , Animais , Apoptose , Linhagem Celular , Claudinas/genética , Claudinas/metabolismo , Células Epiteliais/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Potencial da Membrana Mitocondrial , Ocludina/genética , Ocludina/metabolismo , Suínos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
2.
Biochim Biophys Acta Biomembr ; 1862(7): 183279, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32224152

RESUMO

The functional and structural concept of tight junctions has developed after discovery of claudin and TAMP proteins. Many of these proteins contribute to epi- and endothelial barrier but some, in contrast, form paracellular channels. Claudins form the backbone of tight junction (TJ) strands whereas other proteins regulate TJ dynamics. The current joined double-row model of TJ strands and channels is crucially based on the linear alignment of claudin-15 in the crystal. Molecular dynamics simulations, protein docking, mutagenesis, cellular TJ reconstitution, and electron microscopy studies largely support stability and functionality of the model. Here, we summarize in silico and in vitro data about TJ strand assembly including comparison of claudin crystal structures and alternative models. Sequence comparisons, experimental and structural data substantiate differentiation of classic and non-classic claudins differing in motifs related to strand assembly. Classic claudins seem to share a similar mechanism of strand formation. Interface variations likely contribute to TJ strand flexibility. Combined in vitro/in silico studies are expected to elucidate mechanistic keys determining TJ regulation.


Assuntos
Claudinas/química , Conformação Proteica , Junções Íntimas/química , Junções Íntimas/genética , Claudinas/genética , Simulação por Computador , Células HEK293 , Humanos , Microscopia Eletrônica , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutagênese , Multimerização Proteica , Junções Íntimas/ultraestrutura
3.
Life Sci ; 249: 117478, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32119960

RESUMO

BACKGROUND: Gastric cancer is a severe disease with a high occurrence rate worldwide. And lncRNAs are demonstrated to be responsible for cancer growth and metastasis. So, it is of great importance to explore the lncRNAs involved mechanism of gastric cancer occurrence and development deeply. METHODS: Transfection was conducted to build over-expression and down-expression models. Moreover, RT-qPCR and western blot were used to detect the transcriptional and translational levels. The biological functions such as proliferation, migration and invasion of AGS cells were evaluated by MTT analysis, colony formation assay, scarification detection and transwell assay, respectively. The potential binding of miR-135b and its downstream and upstream molecules was validated by dual luciferase reporter gene assay or RIP. Also, the in-vivo mice model was further used to demonstrate the role of lncRNA PCAT18 in gastric cancer. RESULTS: PCAT18 down-expression promoted proliferation, migration and invasion of gastric cancer cells. Furtherly, over-expression of miR-135b also promoted these biological characteristics of AGS cells. Importantly, we found that PCAT18 could bind miR-135b which also was bound with CLDN11. We found that miR-135b is negatively correlated with CLDN11; PCAT18 and CLDN11 are positively correlated. Moreover, miR-135b mimics could down-regulate protein level of CLDN11, whereas CLDN11 could reverse this effect. In in-vivo experiment, PCAT18 over-expression restrained tumor growth and metastasis. CONCLUSIONS: Over-expressed lncRNA PCAT18 inhibits proliferation, migration and invasion of gastric cancer cells through regulation of miR-135b/CLDN11.


Assuntos
Proliferação de Células/genética , Claudinas/genética , MicroRNAs/genética , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , RNA Longo não Codificante/genética , Neoplasias Gástricas/patologia , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos C57BL
4.
J Mol Biol ; 432(7): 2405-2427, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32142789

RESUMO

Tight junctions regulate paracellular permeability size and charge selectively. Models have been proposed for the molecular architecture of tight junction strands and paracellular channels. However, they are not fully consistent with experimental and structural data. Here, we analysed the architecture of claudin-based tight junction strands and channels by cellular reconstitution of strands, structure-guided mutagenesis, in silico protein docking and oligomer modelling. Prototypic channel- (Cldn10b) and barrier-forming (Cldn3) claudins were analysed. Förster resonance energy transfer (FRET) assays indicated multistep claudin polymerisation, starting with cis-oligomerization specific to the claudin subtype, followed by trans-interaction-triggered cis-polymerisation. Alternative protomer interfaces were modelled in silico and tested by cysteine-mediated crosslinking, confocal- and freeze fracture EM-based analysis of strand formation. The analysed claudin mutants included also mutations causing the HELIX syndrome. The results indicated that protomers in Cldn10b and Cldn3 strands form similar antiparallel double rows, as has been suggested for Cldn15. Mutually stabilising -hydrophilic and hydrophobic - cis- and trans-interfaces were identified that contained novel key residues of extracellular segments ECS1 and ECS2. Hydrophobic clustering of the flexible ECS1 ß1ß2 loops together with ECS2-ECS2 trans-interaction is suggested to be the driving force for conjunction of tetrameric building blocks into claudin polymers. Cldn10b and Cldn3 are indicated to share this polymerisation mechanism. However, in the paracellular centre of tetramers, electrostatic repulsion may lead to formation of pores (Cldn10b) and electrostatic attraction to barriers (Cldn3). Combining in vitro data and in silico modelling, this study improves mechanistic understanding of paracellular permeability regulation by elucidating claudin assembly and its pathologic alteration as in HELIX syndrome.


Assuntos
Claudina-3/química , Claudinas/química , Multimerização Proteica , Junções Íntimas/química , Animais , Permeabilidade da Membrana Celular , Claudina-3/genética , Claudina-3/metabolismo , Claudinas/genética , Claudinas/metabolismo , Células HEK293 , Humanos , Canais Iônicos , Camundongos , Mutação , Conformação Proteica , Síndrome , Junções Íntimas/metabolismo
5.
Am J Physiol Renal Physiol ; 318(5): F1138-F1146, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32174144

RESUMO

The proximal renal tubule (PT) is characterized by a highly conductive paracellular pathway, which contributes to a significant amount of solute and water reabsorption by the kidney. Claudins are tight junction proteins that, in part, determine the paracellular permeability of epithelia. In the present study, we determined the expression pattern of the major PT claudins. We found that claudin-2 and claudin-10 are coexpressed throughout the PT, whereas claudin-3 is coexpressed with claudin-2 predominantly in the proximal straight tubule. Additionally, claudin-2 and claudin-3 are expressed separately within mutually exclusive populations of descending thin limbs. We developed a novel double-inducible Madin-Darby canine kidney I cell model to characterize in vitro the functional effect of coexpression of PT claudins. In keeping with previous studies, we found that claudin-2 alone primarily increased cation (Na+ and Ca2+) permeability, whereas claudin-10a alone increased anion (Cl-) permeability. Coexpression of claudin-2 and claudin-10a together led to a weak physical interaction between the isoforms and the formation of a monolayer with high conductance but neutral charge selectivity. Claudin-3 expression had a negligible effect on all measures of cell permeability, whether expressed alone or together with claudin-2. In cells coexpressing a claudin-2 mutant, S68C, together with claudin-10a, inhibition of cation permeability through the claudin-2 pore with a thiol-reactive pore blocker did not block anion permeation through claudin-10a. We conclude that claudin-2 and claudin-10a form independent paracellular cation- and anion-selective channels that function in parallel.


Assuntos
Claudinas/metabolismo , Túbulos Renais Proximais/metabolismo , Junções Íntimas/metabolismo , Animais , Claudinas/genética , Cães , Condutividade Elétrica , Transporte de Íons , Túbulos Renais Proximais/citologia , Células Madin Darby de Rim Canino , Potenciais da Membrana , Camundongos , Permeabilidade , Transdução de Sinais
6.
Int J Mol Sci ; 21(4)2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-32093310

RESUMO

Dietary NaCl depletion increases Na+ and Cl- absorption in the colon, but the mechanisms are not fully understood. So far, we reported that the expression of claudin-7 (CLDN7), a tight junction (TJ) protein, was upregulated in the mice fed with NaCl-depleted diets, but the regulatory mechanism has not been clarified. Here, we found that angiotensin II (ANGII) increases the mRNA level of CLDN7, which was inhibited by losartan, a type 1 ANGII (AT1) receptor antagonist. Immunofluorescence measurement showed that CLDN7 is colocalized with zonula occludens-1 at the TJ in untreated and ANGII-treated cells. ANGII decreased transepithelial electrical resistance (TER) and increased permeability to C1- without affecting permeability to lucifer yellow, a paracellular flux marker. In contrast, TER was increased by CLDN7 knockdown in the absence and presence of ANGII. ANGII increased the nuclear distribution of phosphorylated p65 subunit of NF-κB, which was inhibited by losartan. The ANGII-induced elevation of CLDN7 expression was blocked by BAY 11-7082 (BAY), an NF-κB inhibitor. Luciferase reporter assay showed that ANGII increases promoter activity of CLDN7, which was inhibited by the treatment with losartan or BAY, and introduction of mutations in κB-binding motifs in the promoter. The binding of p65 on the promoter region of CLDN7 was increased by ANGII, which was inhibited by losartan and BAY in chromatin immunoprecipitation assay. Our data suggest that ANGII acts on AT1 receptor and increases paracellular permeability to Cl- mediated by the elevation of CLDN7 expression in the colon.


Assuntos
Angiotensina II/farmacologia , Claudinas/biossíntese , Colo/metabolismo , Dieta Hipossódica , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Animais , Linhagem Celular , Claudinas/genética , Colo/patologia , Células Epiteliais/patologia , Mucosa Intestinal/patologia , Masculino , Camundongos , Nitrilos/farmacologia , Cloreto de Sódio , Sulfonas/farmacologia , Junções Íntimas/genética , Junções Íntimas/metabolismo , Junções Íntimas/patologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
7.
J Fish Biol ; 96(3): 768-781, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32017083

RESUMO

The role of lamprey epithelium tight junctions (TJs) in the regulation of salt and water balance is poorly understood. This study reported on claudin (Cldn) TJ protein transcripts of pre-metamorphic larval and post-metamorphic juvenile sea lamprey (Petromyzon marinus) and the transcriptional response of genes encoding Cldns to changed environmental ion levels. Transcripts encoding Cldn-3b, -4, -5, -10, -14, -18 and -19 were identified, and mRNA expression profiles revealed the organ-specific presence of cldn-5 and -14, broad expression of cldn-3b, -4, -10, -18 and -19 and spatial differences in the mRNA abundance of cldn-4, -3b and -14 along the ammocoete intestine. Expression profiles were qualitatively similar in ammocoetes and juvenile fishes. Transcript abundance of genes encoding Cldns in osmoregulatory organs (gill, kidney, intestine and skin) was subsequently investigated after exposure of ammocoetes to ion-poor water (IPW) and juveniles to hyperosmotic conditions [60% sea water (SW)]. IPW-acclimated ammocoetes increased mRNA abundance of nearly all cldns in the gill. Simultaneously, cldn-10 abundance increased in the skin, whereas cldn-4, -14 and -18 decreased in the kidney. Ammocoete cldn mRNA abundance in the intestine was altered in a region-specific manner. In contrast, cldn transcript abundance was mostly downregulated in osmoregulatory organs of juvenile fish acclimated to SW - cldn-3b, -10 and -19 in the gill; cldn-3b, -4, -10 and -19 in the skin; cldn-3b in the kidney; and cldn-3b and -14 in the intestine. Data support the idea that Cldn TJ proteins play an important role in the osmoregulatory physiology of pre- and post-metamorphic sea lamprey and that Cldn participation can occur across organs, in an organ-specific manner, as well as differ spatially within organs, which contributes to the regulation of salt and water balance in these fishes.


Assuntos
Claudinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Íons/farmacologia , Petromyzon/genética , Água/química , Aclimatação/genética , Animais , Epitélio/efeitos dos fármacos , Brânquias/efeitos dos fármacos , Osmorregulação/genética , RNA Mensageiro/genética , Água do Mar , Equilíbrio Hidroeletrolítico/genética
8.
Biochim Biophys Acta Biomembr ; 1862(5): 183205, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32001212

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterized by an abnormal healing response to injury of the alveolar epithelium. Tight junctions provide a physical barrier at the apical intercellular space between epithelial cells and regulate paracellular flux. While tight junction alterations are known to contribute to barrier dysfunction in a number of disease states, the role of tight junction proteins in IPF is poorly defined. To determine a potential role for tight junction protein alterations in IPF, we performed immunohistochemical staining for tight junction proteins ZO-1, occludin, claudin-2, claudin-3, claudin-4, claudin-5, and claudin-18. Staining intensity and localization were compared between IPF and control lung tissues. IPF was associated with type II pneumocyte hyperplasia and altered tight junction protein expression. While there was no difference in the expression of ZO-1, claudin-3, or claudin-5, between IPF and normal control, there was an overall increase in claudin-2 expression in bronchiolar and alveolar epithelium and a decrease in claudin-4 expression in type II pneumocytes. There was also increased occludin and decreased claudin-18 expression in pneumocytes overlying fibroblastic foci. These findings suggest that epithelial barrier alterations may be important to the pathogenesis of IPF.


Assuntos
Fibrose Pulmonar Idiopática/metabolismo , Junções Íntimas/metabolismo , Idoso , Células Epiteliais Alveolares/metabolismo , Claudina-4/genética , Claudina-4/metabolismo , Claudinas/genética , Claudinas/metabolismo , Feminino , Humanos , Fibrose Pulmonar Idiopática/fisiopatologia , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Ocludina/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Proteínas de Junções Íntimas/análise , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/fisiologia , Proteína da Zônula de Oclusão-1/metabolismo
9.
J Exp Clin Cancer Res ; 39(1): 42, 2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32093760

RESUMO

BACKGROUND: We have previously described CLDN6 as a tumor suppressor gene in breast cancer. Here, a new finding is that CLDN6 was upregulated under hypoxia, a commonly recognized factor that promotes tumor metastasis. In this study, we aim to explain this confusing finding and delineate the role of CLDN6 in the breast cancer metastasis induced by hypoxia. METHODS: RNAi and ChIP assays were used to confirm that CLDN6 is transcriptional regulated by HIF-1α. mRNA seq and KEGG analysis were performed to define the downstream pathways of CLDN6. The roles of the CLDN6/SENP1/HIF-1α signaling on tumor metastasis were evaluated by function experiments and clinical samples. Finally, the possible transcription factor of SENP1 was suspected and then validated by ChIP assay. RESULTS: We demonstrated a previously unrecognized negative feedback loop exists between CLDN6 and HIF-1α. CLDN6 was transcriptionally up-regulated by HIF-1α under hypoxia. On the other hand, in cytoplasm CLDN6 combines and retains ß-catenin, a transcription factor of SENP1, causing ß-catenin degradation and preventing its nuclear translocation. This process reduced SENP1 expression and prevented the deSUMOylation of HIF-1α, ultimately leading to HIF-1α degradation and breast cancer metastasis suppression. CONCLUSIONS: Our data provide a molecular mechanistic insight indicating that CLDN6 loss may lead to elevated HIF-1α-driven breast cancer metastasis in a SUMOylation-dependent manner.


Assuntos
Neoplasias da Mama/patologia , Claudinas/genética , Claudinas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Retroalimentação Fisiológica , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Proteólise , Transdução de Sinais/efeitos dos fármacos , Sumoilação
10.
J Exp Clin Cancer Res ; 39(1): 5, 2020 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900207

RESUMO

BACKGROUND: LncRNA LINC00662 is closely related to the occurrence and development of cancer. This study aims to explore the effect of LINC00662 on colon cancer tumor growth and metastasis and its molecular mechanism. METHODS: CCK8, colony formation, transwell, scratch wound, TUNEL, flow cytometry, RT-PCR, western blotting and immunohistochemistry assays were used to detect the proliferation, apoptosis, invasion and migration of colon cancer cell and mRNA and protein expressions. Luciferase reporter and RNA pull down assays were used to detect the combination of LINC00662 and miR-340-5p or IL22 and the combination of miR-340-5p and CLDN8/IL22. Co-immunoprecipitation were used to detect the co-expression of CLDN8 and IL22 in colon cell lines. The targets of LINC00662 were predicated by Starbase v2.0. The target genes of miR-340-5p were predicated by miRDB and TargetScan. GO and KEGG enrichment analysis were performed by DAVID website. RESULTS: LINC00662 was up-regulation in colon cancer tissues and cell lines. Univariate Cox regression analysis showed that the LINC00662 expression level was related to the poor prognosis. LINC00662-WT and miR-340-5p mimics co-transfection depressed luciferase activity and IL22/CLDN8-WT and miR-340-5p inhibitors co-transfection memorably motivated luciferase activity. LINC00662 overexpression promoted cell proliferation, invasion and migration, and inhibited cell apoptosis in colon cancer. In vivo xenograft studies in nude mice manifested that LINC00662 overexpression prominently accelerate tumor growth. There was an opposite reaction in the biological functions of colon cells and tumor growth between LINC00662 overexpression and LINC00662 inhibition in vitro and in vivo. The functions of miR-340-5p mimics regulating the biological functions of colon cells and tumor growth were consistent with those of LINC00662 inhibition. CLDN8 and IL22, as target genes of miR-340-5p, reversed the functions of LINC00662 affecting the biological functions of colon cells and the protein levels of Bax, Bcl-2, XIAP, VEGF, MMP-2, E-cadherin and N-cadherin. Co-immunoprecipitation experiments indicated that CLDN8 directly interact with IL22 in colon cell lines. LINC00662 regulated CLDN8 and IL22 expressions and the activation of ERK signaling pathway via targeting miR-340-5p. CONCLUSION: LINC00662 overexpression promoted the occurrence and development of colon cancer by competitively binding with miR-340-5p to regulate CLDN8/IL22 co-expression and activating ERK signaling pathway.


Assuntos
Claudinas/genética , Neoplasias do Colo/patologia , Interleucinas/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Regiões 3' não Traduzidas , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Claudinas/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Interleucinas/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Prognóstico , Regulação para Cima
11.
Biochim Biophys Acta Mol Cell Res ; 1867(4): 118642, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31923533

RESUMO

Claudin-2 (CLDN2), a tight junctional protein, is involved in the chemoresistance in spheroid culture models of human lung adenocarcinoma A549 cells. However, there is no chemical which can improve the sensitivity to anticancer drugs. So far, we reported that DFYSP, a short peptide which mimics the second extracellular loop (ECL2) of CLDN2, decreases CLDN2 expression in A549 cells, but the concentration is relatively high. Here, we found that the effects of VPDSM and DSMKF are stronger than that of DFYSP. Both VPDSM and DSMKF decreased the protein levels of CLDN2 without affecting the mRNA levels of CLDN2. The peptide-induced decrease in CLDN2 expression was suppressed by monodansylcadaverine (MDC), a clathrin-dependent endocytosis (CDE) inhibitor, and chloroquine, a lysosome inhibitor. CLDN2 was colocalized with ZO-1, an adapter protein, in tight junctions (TJs) under control conditions, whereas it disappeared from the TJs in the peptide-treated cells. Quartz crystal microbalance assay showed that both peptides can bind to recombinant CLDN2 protein. Both peptides increased permeability to paracellular transport marker lucifer yellow. In three-dimensional spheroid culture models, both peptides enhanced the sensitivity to doxorubicin, a cytotoxic anticancer drug, which was inhibited by MDC. We suggest that VPDSM and DSMKF enhance the chemosensitivity to anticancer drugs in aggregated adenocarcinoma cells mediated by the CDE pathway and lysosomal degradation of CLDN2 in lung adenocarcinoma cells. VPDSM and DSMKF, which mimic the ECL2 of CLDN2, may become novel adjuvant therapeutic drugs for lung adenocarcinoma.


Assuntos
Claudinas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Oligopeptídeos/farmacologia , Células A549 , Antibióticos Antineoplásicos/farmacologia , Claudinas/genética , Doxorrubicina/farmacologia , Humanos , Oligopeptídeos/química , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Junções Íntimas/metabolismo
12.
Int J Mol Sci ; 21(1)2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31935940

RESUMO

The abnormal deposition of calcium within renal parenchyma, termed nephrocalcinosis, frequently occurs as a result of impaired renal calcium handling. It is closely associated with renal stone formation (nephrolithiasis) as elevated urinary calcium levels (hypercalciuria) are a key common pathological feature underlying these clinical presentations. Although monogenic causes of nephrocalcinosis and nephrolithiasis are rare, they account for a significant disease burden with many patients developing chronic or end-stage renal disease. Identifying underlying genetic mutations in hereditary cases of nephrocalcinosis has provided valuable insights into renal tubulopathies that include hypercalciuria within their varied phenotypes. Genotypes affecting other enzyme pathways, including vitamin D metabolism and hepatic glyoxylate metabolism, are also associated with nephrocalcinosis. As the availability of genetic testing becomes widespread, we cannot be imprecise in our approach to nephrocalcinosis. Monogenic causes of nephrocalcinosis account for a broad range of phenotypes. In cases such as Dent disease, supportive therapies are limited, and early renal replacement therapies are necessitated. In cases such as renal tubular acidosis, a good renal prognosis can be expected providing effective treatment is implemented. It is imperative we adopt a precision-medicine approach to ensure patients and their families receive prompt diagnosis, effective, tailored treatment and accurate prognostic information.


Assuntos
Heterogeneidade Genética , Mutação , Nefrocalcinose/genética , Fenótipo , Adenilil Ciclases/genética , Animais , Claudinas/genética , Humanos , Proteínas de Membrana Transportadoras/genética , Nefrocalcinose/patologia , Receptores de Detecção de Cálcio/genética , Vitamina D3 24-Hidroxilase/genética
13.
Cell Prolif ; 53(1): e12729, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31746095

RESUMO

OBJECTIVES: The successional dental lamina is the distinctive structure on the lingual side of the vertebrate tooth germ. The aim of this study was to investigate the relationship among Sox2, Claudin10 and laminin5 and the role of Sox2 in successional dental lamina proliferation during vertebrate tooth development. MATERIALS AND METHODS: To understand the successional dental lamina, two types of successional tooth formation, that in geckos (with multiple rounds of tooth generation) and that in mice (with only one round of tooth generation), were analysed. RESULTS: Unique coexpression patterns of Sox2 and Claudin10 expression were compared in the successional dental lamina from the cap stage to the late bell stage in the mouse tooth germ and in juvenile gecko teeth to support continuous tooth replacement. Furthermore, Laminin5 expression was shown in the cap stage and decreased after the bell stage. Upon comparing the epithelial cell cycles and cell proliferation in successional dental lamina regions between mouse and gecko molars using BrdU and IdU staining and pulse-chase methods, distinctive patterns of continuous expression were revealed. Moreover, Sox2 overexpression with a lentiviral system resulted in hyperplastic dental epithelium in mouse molars. CONCLUSIONS: Our findings indicate that the regulation of Sox2 in dental lamina proliferation is fundamental to the successional dental lamina in both species.


Assuntos
Proliferação de Células , Células Epiteliais/metabolismo , Dente Molar/embriologia , Fatores de Transcrição SOXB1/metabolismo , Germe de Dente/embriologia , Animais , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Claudinas/biossíntese , Claudinas/genética , Células Epiteliais/citologia , Lagartos/embriologia , Camundongos , Camundongos Endogâmicos ICR , Dente Molar/citologia , Proteínas de Répteis/genética , Proteínas de Répteis/metabolismo , Fatores de Transcrição SOXB1/genética , Germe de Dente/citologia
14.
Int J Mol Sci ; 20(21)2019 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-31694170

RESUMO

In higher organisms, epithelia separate compartments in order to guarantee their proper function. Such structures are able to seal but also to allow substances to pass. Within the paracellular pathway, a supramolecular structure, the tight junction transport is largely controlled by the temporospatial regulation of its major protein family called claudins. Besides the fact that the expression of claudins has been identified in different forms of human diseases like cancer, clearly defined mutations in the corresponding claudin genes have been shown to cause distinct human disorders. Such disorders comprise the skin and its adjacent structures, liver, kidney, the inner ear, and the eye. From the phenotype analysis, it has also become clear that different claudins can cause a complex phenotype when expressed in different organs. To gain deeper insights into the physiology and pathophysiology of claudin-associated disorders, several mouse models have been generated. In order to model human disorders in detail, they have been designed either as full knockouts, knock-downs or knock-ins by a variety of techniques. Here, we review human disorders caused by CLDN mutations and their corresponding mouse models that have been generated thus far and assess their usefulness as a model for the corresponding human disorder.


Assuntos
Claudinas/genética , Mutação , Sequência de Aminoácidos , Animais , Claudinas/química , Modelos Animais de Doenças , Oftalmopatias/genética , Humanos , Nefropatias/genética , Hepatopatias/genética , Camundongos , Neoplasias/genética , Dermatopatias/genética
15.
Proc Natl Acad Sci U S A ; 116(49): 24600-24609, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31740618

RESUMO

Cell adhesion is essential for proper tissue architecture and function in multicellular organisms. Cell adhesion molecules not only maintain tissue integrity but also possess signaling properties that contribute to diverse cellular events such as cell growth, survival, differentiation, polarity, and migration; however, the underlying molecular basis remains poorly defined. Here we identify that the cell adhesion signal initiated by the tight-junction protein claudin-6 (CLDN6) regulates nuclear receptor activity. We show that CLDN6 recruits and activates Src-family kinases (SFKs) in second extracellular domain-dependent and Y196/200-dependent manners, and SFKs in turn phosphorylate CLDN6 at Y196/200. We demonstrate that the CLDN6/SFK/PI3K/AKT axis targets the AKT phosphorylation sites in the retinoic acid receptor γ (RARγ) and the estrogen receptor α (ERα) and stimulates their activities. Interestingly, these phosphorylation motifs are conserved in 14 of 48 members of human nuclear receptors. We propose that a similar link between diverse cell adhesion and nuclear receptor signalings coordinates a wide variety of physiological and pathological processes.


Assuntos
Adesão Celular/fisiologia , Claudinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Linhagem Celular , Claudinas/genética , Receptor alfa de Estrogênio/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Domínios Proteicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Transdução de Sinais , Tirosina/genética , Quinases da Família src/metabolismo
16.
Int J Mol Sci ; 20(21)2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31671507

RESUMO

Claudins are key components of the tight junction, sealing the paracellular cleft or composing size-, charge- and water-selective paracellular channels. Claudin-10 occurs in two major isoforms, claudin-10a and claudin-10b, which constitute paracellular anion or cation channels, respectively. For several years after the discovery of claudin-10, its functional relevance in men has remained elusive. Within the past two years, several studies appeared, describing patients with different pathogenic variants of the CLDN10 gene. Patients presented with dysfunction of kidney, exocrine glands and skin. This review summarizes and compares the recently published studies reporting on a novel autosomal-recessive disorder based on claudin-10 mutations.


Assuntos
Claudinas/genética , Claudinas/metabolismo , Nefropatias/genética , Mutação , Predisposição Genética para Doença , Humanos , Hipo-Hidrose/genética , Ictiose/genética , Nefropatias/metabolismo , Doenças do Aparelho Lacrimal/genética , Domínios Proteicos , Xerostomia/genética
17.
Int J Biol Sci ; 15(10): 2224-2239, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31592143

RESUMO

Microvesicles are the body's most powerful intercellular communication system and cancer-initiating cell microvesicles (CIC-TEX) reprogram Non-CIC towards fortified malignancy. Claudin7, a CIC-biomarker in gastrointestinal tumors, is recovered in TEX. Recent evidence suggesting individual cells delivering distinct microvesicles became of particular interest for claudin7, which is part of tight junctions (TJ) and glycolipid-enriched membrane domains (GEM), GEM-located claudin7 is palmitoylated. This offered the unique possibility of exploring the contribution of a CIC marker and its origin from distinct membrane domains on CIC-TEX biogenesis and activities. Proteome and miRNA analysis of wild-type, claudin7-knockdown and a rescue with claudin7 harboring a mutated palmitoylation site (mP) of a rat pancreatic and a human colon cancer line uncovered significant, only partly overlapping contributions of palmitoylated and non-palmitoylated claudin7 to TEX composition. Palmitoylated claudin7 facilitates GEM-integrated plasma membrane and associated signaling molecule recruitment; non-palmitoylated claudin7 supports recruitment of trafficking components, proteins engaged in fatty acid metabolism and TJ proteins into TEX. Claudin7mP also assists TEX recovery of selected miRNA. Thus, distinctly located claudin7 affects CIC-TEX composition and TJ-derived cld7 might play a unique role in equipping CIC-TEX with transporters and lipid metabolism-regulating molecules, awareness of distinct TEX populations being crucial facing therapeutic translation.


Assuntos
Membrana Celular/metabolismo , Claudinas/metabolismo , Animais , Linhagem Celular Tumoral , Claudinas/genética , Exossomos/metabolismo , Humanos , Imunoprecipitação , Mutação/genética , Ratos , Transdução de Sinais
18.
Int J Mol Sci ; 20(19)2019 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-31581662

RESUMO

: Kidney stones affect 10% of the population. Yet, there is relatively little known about how they form or how to prevent and treat them. The claudin family of tight junction proteins has been linked to the formation of kidney stones. The flavonoid quercetin has been shown to prevent kidney stone formation and to modify claudin expression in different models. Here we investigate the effect of quercetin on claudin expression and localization in MDCK II cells, a cation-selective cell line, derived from the proximal tubule. For this study, we focused our analyses on claudin family members that confer different tight junction properties: barrier-sealing (Cldn1, -3, and -7), cation-selective (Cldn2) or anion-selective (Cldn4). Our data revealed that quercetin's effects on the expression and localization of different claudins over time corresponded with changes in transepithelial resistance, which was measured continuously throughout the treatment. In addition, these effects appear to be independent of PI3K/AKT signaling, one of the pathways that is known to act downstream of quercetin. In conclusion, our data suggest that quercetin's effects on claudins result in a tighter epithelial barrier, which may reduce the reabsorption of sodium, calcium and water, thereby preventing the formation of a kidney stone.


Assuntos
Claudinas/genética , Claudinas/metabolismo , Expressão Gênica , Quercetina/metabolismo , Junções Íntimas/metabolismo , Animais , Biomarcadores , Membrana Celular/metabolismo , Cães , Células Madin Darby de Rim Canino , Fosfatidilinositol 3-Quinases/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
19.
Int J Mol Sci ; 20(18)2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31500278

RESUMO

Extracellular vesicles (EVs) are nanometer-sized membranous vesicles used for primitive cell-to-cell communication. We previously reported that colon cancer-derived EVs contain abundant miR-92a-3p and have a pro-angiogenic function. We previously identified Dickkopf-3 (Dkk-3) as a direct target of miR-92a-3p; however, the pro-angiogenic function of miR-92a-3p cannot only be attributed to downregulation of Dkk-3. Therefore, the complete molecular mechanism by which miR-92a-3p exerts pro-angiogenic effects is still unclear. Here, we comprehensively analyzed the gene sets affected by ectopic expression of miR-92a-3p in endothelial cells to elucidate processes underlying EV-induced angiogenesis. We found that the ectopic expression of miR-92a-3p upregulated cell cycle- and mitosis-related gene expression and downregulated adhesion-related gene expression in endothelial cells. We also identified a novel target gene of miR-92a-3p, claudin-11. Claudin-11 belongs to the claudin gene family, which encodes essential components expressed at tight junctions (TJs). Disruption of TJs with a concomitant loss of claudin expression is a significant event in the process of epithelial-to-mesenchymal transition. Our findings have unveiled a new EV-mediated mechanism for tumor angiogenesis through the induction of partial endothelial-to-mesenchymal transition in endothelial cells.


Assuntos
Claudinas/genética , Neoplasias do Colo/irrigação sanguínea , Vesículas Extracelulares/genética , MicroRNAs/genética , Neovascularização Patológica/genética , Linhagem Celular Tumoral , Claudinas/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Células Endoteliais/química , Células Endoteliais/citologia , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Neovascularização Patológica/metabolismo , Mapas de Interação de Proteínas , Junções Íntimas/genética , Junções Íntimas/metabolismo
20.
Int J Mol Sci ; 20(18)2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31527509

RESUMO

Tight junctions are cellular junctions that play a major role in the epithelial barrier function. In the inner ear, claudins, occludin, tricellulin, and angulins form the bicellular or tricellular binding of membrane proteins. In these, one type of claudin gene, CLDN14, was reported to be responsible for human hereditary hearing loss, DFNB29. Until now, nine pathogenic variants have been reported, and most phenotypic features remain unclear. In the present study, genetic screening for 68 previously reported deafness causative genes was carried out to identify CLDN14 variants in a large series of Japanese hearing loss patients, and to clarify the prevalence and clinical characteristics of DFNB29 in the Japanese population. One patient had a homozygous novel variant (c.241C>T: p.Arg81Cys) (0.04%: 1/2549). The patient showed progressive bilateral hearing loss, with post-lingual onset. Pure-tone audiograms indicated a high-frequency hearing loss type, and the deterioration gradually spread to other frequencies. The patient showed normal vestibular function. Cochlear implantation improved the patient's sound field threshold levels, but not speech discrimination scores. This report indicated that claudin-14 is essential for maintaining the inner ear environment and suggested the possible phenotypic expansion of DFNB29. This is the first report of a patient with a tight junction variant receiving a cochlear implantation.


Assuntos
Claudinas/genética , Surdez/diagnóstico , Surdez/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Fenótipo , Adolescente , Adulto , Idoso , Alelos , Substituição de Aminoácidos , Criança , Pré-Escolar , Claudinas/metabolismo , Surdez/metabolismo , Surdez/terapia , Feminino , Estudos de Associação Genética/métodos , Genótipo , Humanos , Lactente , Recém-Nascido , Japão , Masculino , Pessoa de Meia-Idade , Linhagem , Junções Íntimas/genética , Junções Íntimas/metabolismo , Adulto Jovem
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