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1.
J Cell Sci ; 137(9)2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38712627

RESUMO

Tight junctions (TJs) are specialized regions of contact between cells of epithelial and endothelial tissues that form selective semipermeable paracellular barriers that establish and maintain body compartments with different fluid compositions. As such, the formation of TJs represents a critical step in metazoan evolution, allowing the formation of multicompartmental organisms and true, barrier-forming epithelia and endothelia. In the six decades that have passed since the first observations of TJs by transmission electron microscopy, much progress has been made in understanding the structure, function, molecular composition and regulation of TJs. The goal of this Perspective is to highlight the key concepts that have emerged through this research and the future challenges that lie ahead for the field.


Assuntos
Junções Íntimas , Junções Íntimas/metabolismo , Junções Íntimas/ultraestrutura , Humanos , Animais , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Células Epiteliais/citologia
2.
Int J Mol Sci ; 25(5)2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38473701

RESUMO

This work analyzes the role of the tight junction (TJ) protein ZO-2 on mechanosensation. We found that the lack of ZO-2 reduced apical membrane rigidity measured with atomic force microscopy, inhibited the association of γ-actin and JAM-A to the cell border, and instead facilitated p114RhoGEF and afadin accumulation at the junction, leading to an enhanced mechanical tension at the TJ measured by FRET, with a ZO-1 tension probe, and increased tricellular TJ tension. Simultaneously, adherens junction tension measured with an E-cadherin probe was unaltered. The stability of JAM-A and ZO-2 binding was assessed by a collaborative in silico study. The absence of ZO-2 also impacted the cell response to the substrate, as monolayers plated in 20 kPa hydrogels developed holes not seen in parental cultures and displayed a retarded elongation and formation of cell aggregates. The absence of ZO-2 was sufficient to induce YAP and Snail nuclear accumulation in cells cultured over glass, but when ZO-2 KD cells were plated in nanostructured ridge arrays, they displayed an increased abundance of nuclear Snail and conspicuous internalization of claudin-4. These results indicate that the absence of ZO-2 also impairs the response of cells to substrate stiffness and exacerbates transformation triggered by substrate topography.


Assuntos
Actinas , Junções Íntimas , Actinas/metabolismo , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Fosfoproteínas/metabolismo
3.
Cells ; 11(20)2022 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-36291162

RESUMO

Tight junctions (TJs) regulate the transit of ions and molecules through the paracellular pathway in epithelial cells. Zonula occludens 2 (ZO-2) is a cytoplasmic TJ protein. Here, we studied the ubiquitination of hZO-2 employing mutants of SUMOylation site K730 present in the GuK domain and the putative ubiquitination residues K759 and K992 located at the GuK domain and proline-rich region, respectively. In immunoprecipitation experiments done with MDCK cells transfected with wild-type (WT) hZO-2 or the ubiquitination-site mutants hZO-2-K759R or -K992R, we observed diminished ubiquitination of the mutants, indicating that residues K759 and K992 in hZO-2 are acceptors for ubiquitination. Moreover, using TUBES, we found that residues K759 and K992 of hZO-2 are targets of K48 polyubiquitination, a signal for proteasomal degradation. Accordingly, compared to WT hZO-2, the half-life of hZO-2 mutants K759R and K992R augmented from 19.9 to 37.3 and 23.3 h, respectively. Instead, the ubiquitination of hZO-2 mutant K730R increased, and its half-life diminished to 6.7 h. The lack of these lysine residues in hZO-2 affects TJ sealing as the peak of TER decreased in monolayers of MDCK cells transfected with any of these mutants. These results highlight the importance of ZO-2 ubiquitination and SUMOylation to maintain a healthy and stable pool of ZO-2 molecules at the TJ.


Assuntos
Sumoilação , Junções Íntimas , Proteína da Zônula de Oclusão-2/metabolismo , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo , Lisina/metabolismo , Fosfoproteínas/metabolismo , Linhagem Celular , Prolina/metabolismo
4.
Tissue Barriers ; 10(2): 1994351, 2022 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-34689705

RESUMO

ZO-2 is a peripheral tight junction (TJ) protein whose silencing in renal epithelia induces cell hypertrophy. Here, we found that in ZO-2 KD MDCK cells, in compensatory renal hypertrophy triggered in rats by a unilateral nephrectomy and in liver steatosis of obese Zucker (OZ) rats, ZO-2 silencing is accompanied by the diminished activity of LATS, a kinase of the Hippo pathway, and the nuclear concentration of YAP, the final effector of this signaling route. ZO-2 appears to function as a scaffold for the Hippo pathway as it associates to LATS1. ZO-2 silencing in hypertrophic tissue is due to a diminished abundance of ZO-2 mRNA, and the Sp1 transcription factor is critical for ZO-2 transcription in renal cells. Treatment of OZ rats with metformin, an activator of AMPK that blocks JNK activity, augments ZO-2 and claudin-1 expression in the liver, reduces the paracellular permeability of hepatocytes, and serum bile acid content. Our results suggest that ZO-2 silencing is a common feature of hypertrophy, and that ZO-2 is a positive regulator of the Hippo pathway that regulates cell size. Moreover, our observations highlight the importance of AMPK, JNK, and ZO-2 as therapeutic targets for blood-bile barrier dysfunction.


Assuntos
Proteínas Quinases Ativadas por AMP , Fígado Gorduroso , Proteína da Zônula de Oclusão-2/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Via de Sinalização Hippo , Hipertrofia , Ratos , Ratos Zucker , Proteínas de Junções Íntimas
6.
Cells ; 10(10)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34685547

RESUMO

MAGUK protein ZO-2 is present at tight junctions (TJs) and nuclei. In MDCK ZO-2 knockdown (KD) cells, nuclei exhibit an irregular shape with lobules and indentations. This condition correlates with an increase in DNA double strand breaks, however cells are not senescent and instead become resistant to UV-induced senescence. The irregular nuclear shape is also observed in isolated cells and in those without TJs, due to the lack of extracellular calcium. The aberrant nuclear shape of ZO-2 KD cells is not accompanied by a reduced expression of lamins A/C and B and lamin B receptors. Instead, it involves a decrease in constitutive and facultative heterochromatin, and microtubule instability that is restored with docetaxel. ZO-2 KD cells over-express SUN-1 that crosses the inner nuclear membrane and connects the nucleoskeleton of lamin A to nesprins, which traverse the outer nuclear membrane. Nesprins-3 and -4 that indirectly bind on their cytoplasmic face to vimentin and microtubules, respectively, are also over-expressed in ZO-2 KD cells, whereas vimentin is depleted. SUN-1 and lamin B1 co-immunoprecipitate with ZO-2, and SUN-1 associates to ZO-2 in a pull-down assay. Our results suggest that ZO-2 forms a complex with SUN-1 and lamin B1 at the inner nuclear membrane, and that ZO-2 and cell-cell contacts are required for a normal nuclear shape.


Assuntos
Comunicação Celular/imunologia , Epitélio/metabolismo , Proteína da Zônula de Oclusão-2/metabolismo , Humanos , Transfecção
7.
Mol Biol Cell ; 32(15): 1347-1358, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34010016

RESUMO

The presence of tight junction protein zonula occludens 2 (ZO-2) at the nucleus inhibits the transcription of genes regulated by TEAD transcription factor. Here, we analyzed whether the movement of ZO-2 into the nucleus modulates the nuclear concentration of TEAD. In sparse cultures of ZO-2 knockdown Madin-Darby canine kidney cells, nuclear TEAD was diminished, as in parental cells transfected with a ZO-2 construct without nuclear localization signals, indicating that ZO-2 facilitates the entry of TEAD into the nucleus. Inhibition of nPKCδ in parental cells triggers the interaction between ZO-2 and TEAD at the cytoplasm and facilitates TEAD/ZO-2 complex nuclear importation. Using proximity ligation, immunoprecipitation, and pull-down assays, TEAD/ZO-2 interaction was confirmed. Nuclear TEAD is phosphorylated, and its exit in parental cells is enhanced by activation of a ZO-2 nuclear exportation signal by nPKCε, while the nuclear accumulation of ZO-2 triggered by the mutation of ZO-2 nuclear export signals induces no change in TEAD nuclear concentration. In summary, our results indicate that the movements of ZO-2 in and out of the nucleus modulate the intracellular traffic of TEAD through a process regulated by nPKCδ and ε and provide a novel role of ZO-2 as a nuclear translocator of TEAD.


Assuntos
Núcleo Celular/metabolismo , Células Epiteliais/metabolismo , Fatores de Transcrição de Domínio TEA/metabolismo , Proteína da Zônula de Oclusão-2/metabolismo , Animais , Linhagem Celular , Cães , Células HEK293 , Humanos , Sinais de Localização Nuclear , Fosforilação , Ligação Proteica , Proteína Quinase C-épsilon/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Ratos , Transdução de Sinais
8.
Cells ; 10(2)2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33578631

RESUMO

Clinical manifestations of coronavirus disease 2019 (COVID-19) in pregnant women are diverse, and little is known of the impact of the disease on placental physiology. Severe acute respiratory syndrome coronavirus (SARS-CoV-2) has been detected in the human placenta, and its binding receptor ACE2 is present in a variety of placental cells, including endothelium. Here, we analyze the impact of COVID-19 in placental endothelium, studying by immunofluorescence the expression of von Willebrand factor (vWf), claudin-5, and vascular endothelial (VE) cadherin in the decidua and chorionic villi of placentas from women with mild and severe COVID-19 in comparison to healthy controls. Our results indicate that: (1) vWf expression increases in the endothelium of decidua and chorionic villi of placentas derived from women with COVID-19, being higher in severe cases; (2) Claudin-5 and VE-cadherin expression decrease in the decidua and chorionic villus of placentas from women with severe COVID-19 but not in those with mild disease. Placental histological analysis reveals thrombosis, infarcts, and vascular wall remodeling, confirming the deleterious effect of COVID-19 on placental vessels. Together, these results suggest that placentas from women with COVID-19 have a condition of leaky endothelium and thrombosis, which is sensitive to disease severity.


Assuntos
COVID-19/complicações , Placenta/irrigação sanguínea , Placenta/patologia , Complicações Cardiovasculares na Gravidez/etiologia , Complicações Infecciosas na Gravidez/etiologia , Trombose/etiologia , Adulto , Antígenos CD/análise , COVID-19/patologia , COVID-19/virologia , Caderinas/análise , Claudina-5/análise , Endotélio/irrigação sanguínea , Endotélio/patologia , Endotélio/virologia , Feminino , Humanos , Recém-Nascido , Microvasos/patologia , Microvasos/virologia , Gravidez , Complicações Cardiovasculares na Gravidez/patologia , Complicações Cardiovasculares na Gravidez/virologia , Complicações Infecciosas na Gravidez/patologia , Complicações Infecciosas na Gravidez/virologia , SARS-CoV-2/isolamento & purificação , Trombose/patologia , Trombose/virologia , Adulto Jovem , Fator de von Willebrand/análise
9.
Int J Oncol ; 57(4): 905-924, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32945372

RESUMO

Tight junctions (TJs) are cell­cell adhesion structures frequently altered by oncogenic transformation. In the present study the role of human papillomavirus (HPV) 16 E7 oncoprotein on the sealing of TJs was investigated and also the expression level of claudins in mouse cervix and in epithelial Madin­Darby Canine Kidney (MDCK) cells. It was found that there was reduced expression of claudins ­1 and ­10 in the cervix of 7­month­old transgenic K14E7 mice treated with 17ß­estradiol (E2), with invasive cancer. In addition, there was also a transient increase in claudin­1 expression in the cervix of 2­month­old K14E7 mice, and claudin­10 accumulated at the border of cells in the upper layer of the cervix in FvB mice treated with E2, and in K14E7 mice treated with or without E2. These changes were accompanied by an augmented paracellular permeability of the cervix in 2­ and 7­month­old FvB mice treated with E2, which became more pronounced in K14E7 mice treated with or without E2. In MDCK cells the stable expression of E7 increased the space between adjacent cells and altered the architecture of the monolayers, induced the development of an acute peak of transepithelial electrical resistance accompanied by a reduced expression of claudins ­1, ­2 and ­10, and an increase in claudin­4. Moreover, E7 enhances the ability of MDCK cells to migrate through a 3D matrix and induces cell stiffening and stress fiber formation. These observations revealed that cell transformation induced by HPV16 E7 oncoprotein was accompanied by changes in the pattern of expression of claudins and the degree of sealing of epithelial TJs.


Assuntos
Claudinas/biossíntese , Papillomavirus Humano 16/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/metabolismo , Junções Íntimas/metabolismo , Neoplasias do Colo do Útero/virologia , Animais , Células Cultivadas , Claudinas/genética , Claudinas/metabolismo , Modelos Animais de Doenças , Cães , Feminino , Papillomavirus Humano 16/isolamento & purificação , Humanos , Camundongos , Camundongos Transgênicos , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia
10.
Toxicology ; 438: 152463, 2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32294493

RESUMO

Methyl parathion (Me-Pa) is an extremely toxic organophosphorus pesticide still used in developing countries. It has been associated with decreased sperm function and fertility and with oxidative and DNA damage. The blood-testis barrier (BTB) is a structure formed by tight junction (TJ) proteins in Sertoli cells and has a critical role in spermatogenesis. We assessed the effect of repeated doses of Me-Pa (3-12 mg/kg/day for 5 days, i.p.) on sperm quality, lipid oxidation, DNA integrity, and BTB permeability in adult male mice and explored oxidation as a mechanism of toxicity. Me-Pa caused dose-dependent effects on sperm quality, lipoperoxidation, and DNA integrity. Testis histology results showed the disruption of spermatogenesis progression and atrophy of seminiferous tubules. The pesticide opened the BTB, as evidenced by the presence of a biotin tracer in the adluminal compartment of the seminiferous tubules. This effect was not observed after 45 days of exposure when a spermatogenic cycle had completed. The coadministration of the antioxidant α-tocopherol (50 mg/kg/day for 5 days, oral) prevented the effects of Me-Pa on sperm quality, DNA and the BTB, indicating the importance of oxidative stress in the damage generated by Me-Pa. As evidenced by immunochemistry, no changes were found in the localization of the TJ proteins of the BTB, although oxidation (carbonylation) of total proteins in testis homogenates was detected. Our results show that Me-Pa disturbs the BTB and that oxidation is involved in the observed toxic effects on sperm cells.


Assuntos
Barreira Hematotesticular/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Inibidores da Colinesterase/toxicidade , Dano ao DNA , Metil Paration/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Praguicidas/toxicidade , Espermatozoides/efeitos dos fármacos , Acetilcolinesterase/metabolismo , Animais , Antioxidantes/farmacologia , Barreira Hematotesticular/metabolismo , Barreira Hematotesticular/patologia , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos ICR , Carbonilação Proteica/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Espermatozoides/metabolismo , Espermatozoides/patologia
11.
Biochim Biophys Acta Biomembr ; 1862(9): 183278, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32240623

RESUMO

The apical junctional complex (AJC) is a cell-cell adhesion system present at the upper portion of the lateral membrane of epithelial cells integrated by the tight junction (TJ) and the adherens junction (AJ). This complex is crucial to initiate and stabilize cell-cell adhesion, to regulate the paracellular transit of ions and molecules and to maintain cell polarity. Moreover, we now consider the AJC as a hub of signal transduction that regulates cell-cell adhesion, gene transcription and cell proliferation and differentiation. The molecular components of the AJC are multiple and diverse and depending on the cellular context some of the proteins in this complex act as tumor suppressors or as promoters of cell transformation, migration and metastasis outgrowth. Here, we describe these new roles played by TJ and AJ proteins and their potential use in cancer diagnostics and as targets for therapeutic intervention.


Assuntos
Junções Aderentes/genética , Junções Intercelulares/genética , Neoplasias/genética , Junções Íntimas/genética , Transcrição Gênica , Junções Aderentes/metabolismo , Adesão Celular/genética , Diferenciação Celular/genética , Movimento Celular/genética , Polaridade Celular/genética , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Regulação da Expressão Gênica/genética , Humanos , Transdução de Sinais/genética , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/genética
12.
Cells ; 8(10)2019 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-31569528

RESUMO

The cytotrophoblast of human placenta transitions into an outer multinucleated syncytiotrophoblast (STB) layer that covers chorionic villi which are in contact with maternal blood in the intervillous space. During pregnancy, the Zika virus (ZIKV) poses a serious prenatal threat. STB cells are resistant to ZIKV infections, yet placental cells within the mesenchyme of chorionic villi are targets of ZIKV infection. We seek to determine whether ZIKV can open the paracellular pathway of STB cells. This route is regulated by tight junctions (TJs) which are present in the uppermost portion of the lateral membranes of STB cells. We analyzed the paracellular permeability and expression of E-cadherin, occludin, JAMs -B and -C, claudins -1, -3, -4, -5 and -7, and ZO-1, and ZO-2 in the STB of placentae from ZIKV-infected and non-infected women. In ZIKV-infected placentae, the pattern of expression of TJ proteins was preserved, but the amount of claudin-4 diminished. Placentae from ZIKV-infected women were permeable to ruthenium red, and had chorionic villi with a higher mean diameter and Hofbauer hyperplasia. Finally, ZIKV added to the basolateral surface of a trophoblast cell line reduced the transepithelial electrical resistance. These results suggest that ZIKV can open the paracellular pathway of STB cells.


Assuntos
Biomarcadores/metabolismo , Complicações Infecciosas na Gravidez/virologia , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo , Trofoblastos/metabolismo , Infecção por Zika virus/metabolismo , Adulto , Linhagem Celular , Feminino , Humanos , Recém-Nascido , Permeabilidade , Gravidez , Trofoblastos/patologia
13.
Int J Mol Sci ; 20(17)2019 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-31450555

RESUMO

ZO-2 is a cytoplasmic protein of tight junctions (TJs). Here, we describe ZO-2 involvement in the formation of the apical junctional complex during early development and in TJ biogenesis in epithelial cultured cells. ZO-2 acts as a scaffold for the polymerization of claudins at TJs and plays a unique role in the blood-testis barrier, as well as at TJs of the human liver and the inner ear. ZO-2 movement between the cytoplasm and nucleus is regulated by nuclear localization and exportation signals and post-translation modifications, while ZO-2 arrival at the cell border is triggered by activation of calcium sensing receptors and corresponding downstream signaling. Depending on its location, ZO-2 associates with junctional proteins and the actomyosin cytoskeleton or a variety of nuclear proteins, playing a role as a transcriptional repressor that leads to inhibition of cell proliferation and transformation. ZO-2 regulates cell architecture through modulation of Rho proteins and its absence induces hypertrophy due to inactivation of the Hippo pathway and activation of mTOR and S6K. The interaction of ZO-2 with viral oncoproteins and kinases and its silencing in diverse carcinomas reinforce the view of ZO-2 as a tumor regulator protein.


Assuntos
Regulação da Expressão Gênica , Transdução de Sinais , Proteína da Zônula de Oclusão-2/genética , Proteína da Zônula de Oclusão-2/metabolismo , Actomiosina/metabolismo , Animais , Apoptose/genética , Proliferação de Células , Forma Celular , Tamanho Celular , Desenvolvimento Embrionário/genética , Humanos , Proteínas Nucleares/metabolismo , Especificidade de Órgãos/genética , Ligação Proteica , Transporte Proteico , Junções Íntimas/metabolismo , Transcrição Gênica , Proteína da Zônula de Oclusão-2/química
14.
Mol Biol Cell ; 30(18): 2377-2398, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31318316

RESUMO

Zonula occludens-2 (ZO-2) is a tight junction (TJ) cytoplasmic protein, whose localization varies according to cell density and Ca2+ in the media. In cells cultured in low calcium (LC), ZO-2 displays a diffuse cytoplasmic distribution, but activation of the Ca2+ sensing receptor (CaSR) with Gd3+ triggers the appearance of ZO-2 at the cell borders. CaSR downstream signaling involves activation of protein kinase C, which phosphorylates and activates with no lysine kinase-4 that phosphorylates ZO-2 inducing its concentration at TJs. In LC, ZO-2 is protected from degradation by association to 14-3-3 proteins. When monolayers are transferred to normal calcium, the complexes ZO-2/14-3-3ζ and ZO-2/14-3-3σ move to the cell borders and dissociate. The 14-3-3 proteins are then degraded in proteosomes, whereas ZO-2 integrates to TJs. From the plasma membrane residual ZO-2 is endocyted and degradaded in lysosomes. The unique region 2 of ZO-2, and S261 located within a nuclear localization signal, are critical for the interaction with 14-3-3 ζ and σ and for the efficient nuclear importation of ZO-2. These results explain the molecular mechanism through which extracellular Ca2+ triggers the appearance of ZO-2 at TJs in epithelial cells and reveal the novel interaction between ZO-2 and 14-3-3 proteins, which is critical for ZO-2 protection and intracellular traffic.


Assuntos
Proteínas 14-3-3/metabolismo , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-2/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Proliferação de Células , Cães , Células Epiteliais/metabolismo , Células Madin Darby de Rim Canino , Fosfoproteínas/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais , Proteína da Zônula de Oclusão-1/metabolismo
15.
Toxicol Appl Pharmacol ; 360: 257-272, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30291936

RESUMO

Methamidophos (MET) is an organophosphate (OP) pesticide widely used in agriculture in developing countries. MET causes adverse effects in male reproductive function in humans and experimental animals, but the underlying mechanisms remain largely unknown. We explored the effect of MET on mice testes (5 mg/kg/day/4 days), finding that this pesticide opens the blood-testis barrier and perturbs spermatogenesis, generating the appearance of immature germ cells in the epididymis. In the seminiferous tubules, MET treatment changed the level of expression or modified the stage-specific localization of tight junction (TJ) proteins ZO-1, ZO-2, occludin, and claudin-3. In contrast, claudin-11 was barely altered. MET also modified the shape of claudin-11, and ZO-2 at the cell border, from a zigzag to a more linear pattern. In addition, MET diminished the expression of ZO-2 in spermatids present in seminiferous tubules, induced the phosphorylation of ZO-2 and occludin in testes and reduced the interaction between these proteins assessed by co-immunoprecipitation. MET formed covalent bonds with ZO-2 in serine, tyrosine and lysine residues. The covalent modifications formed on ZO-2 at putative phosphorylation sites might interfere with ZO-2 interaction with regulatory molecules and other TJ proteins. MET bonds formed at ZO-2 ubiquitination sites likely interfere with ZO-2 degradation and TJ sealing, based on results obtained in cultured epithelial cells transfected with ZO-2 mutated at a MET target lysine residue. Our results shed light on MET male reproductive toxicity and are important to improve regulations regarding the use of OP pesticides and to protect the health of agricultural workers.


Assuntos
Barreira Hematotesticular/efeitos dos fármacos , Inseticidas/farmacologia , Organofosfatos/farmacologia , Compostos Organotiofosforados/farmacologia , Proteína da Zônula de Oclusão-2/metabolismo , Animais , Barreira Hematotesticular/metabolismo , Claudinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Ocludina/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Túbulos Seminíferos/efeitos dos fármacos , Túbulos Seminíferos/metabolismo , Espermatogênese/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
16.
Virus Res ; 258: 39-49, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30278191

RESUMO

Dengue is the most relevant mosquito-borne viral disease in the world. It has been estimated that 390 million infections of dengue occur each year. Dengue virus (DENV) infection can be asymptomatic or can produce a self-limited febrile illness called dengue fever (DF) or a severe form of the infection called severe dengue. In some viruses, the entry and egress from cells, occur in a specific domain of polarized endothelial and epithelial cells. In this study, we investigated whether the entry and release of DENV was polarized in epithelial cells, and evaluated the effect of DENV infection on cellular junctions of epithelial cells. We used MDCK epithelial cells, which serve as an excellent model to study a functional barrier due to the presence of an apical junctional complex (AJC), and showed that entry and release of DENV from the cells, is bipolar. Additionally, we performed paracellular flux, diffusion of membrane lipid, immunofluorescence and immunoblotting assays to evaluate the integrity of the AJC during DENV infection. We observed that at later stages of infection, DENV altered the barrier function causing a decrease in the transepithelial electrical resistance and the degradation and delocalization of TJ and AJ proteins. The present study contributes to understand how DENV traverse epithelia in order to cause a productive infection, and provides insights into the mechanism of DENV pathogenesis.


Assuntos
Vírus da Dengue/fisiologia , Células Epiteliais/citologia , Células Epiteliais/virologia , Internalização do Vírus , Animais , Dengue/virologia , Cães , Células Madin Darby de Rim Canino
17.
J Am Soc Nephrol ; 29(7): 1838-1848, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29848507

RESUMO

Background Hypercalciuria can result from activation of the basolateral calcium-sensing receptor (CaSR), which in the thick ascending limb of Henle's loop controls Ca2+ excretion and NaCl reabsorption in response to extracellular Ca2+ However, the function of CaSR in the regulation of NaCl reabsorption in the distal convoluted tubule (DCT) is unknown. We hypothesized that CaSR in this location is involved in activating the thiazide-sensitive NaCl cotransporter (NCC) to prevent NaCl loss.Methods We used a combination of in vitro and in vivo models to examine the effects of CaSR on NCC activity. Because the KLHL3-WNK4-SPAK pathway is involved in regulating NaCl reabsorption in the DCT, we assessed the involvement of this pathway as well.Results Thiazide-sensitive 22Na+ uptake assays in Xenopus laevis oocytes revealed that NCC activity increased in a WNK4-dependent manner upon activation of CaSR with Gd3+ In HEK293 cells, treatment with the calcimimetic R-568 stimulated SPAK phosphorylation only in the presence of WNK4. The WNK4 inhibitor WNK463 also prevented this effect. Furthermore, CaSR activation in HEK293 cells led to phosphorylation of KLHL3 and WNK4 and increased WNK4 abundance and activity. Finally, acute oral administration of R-568 in mice led to the phosphorylation of NCC.Conclusions Activation of CaSR can increase NCC activity via the WNK4-SPAK pathway. It is possible that activation of CaSR by Ca2+ in the apical membrane of the DCT increases NaCl reabsorption by NCC, with the consequent, well known decrease of Ca2+ reabsorption, further promoting hypercalciuria.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sódio/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ativação Enzimática/genética , Células HEK293 , Humanos , Imidazóis/farmacologia , Masculino , Camundongos , Proteínas dos Microfilamentos , Oócitos , Fenetilaminas/farmacologia , Fosforilação/efeitos dos fármacos , Propilaminas/farmacologia , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Pirrolidinas/farmacologia , Receptores de Detecção de Cálcio/genética , Transdução de Sinais , Membro 1 da Família 12 de Carreador de Soluto/antagonistas & inibidores , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Transfecção , Proteínas de Xenopus/metabolismo , Xenopus laevis
18.
Int J Parasitol ; 48(8): 621-639, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29571981

RESUMO

The adhesion of Giardia duodenalis trophozoites to intestinal epithelial cells allows the onset and maintenance of giardiasis. During these interactions, epithelial cells can be committed to apoptosis by enzymes secreted by the parasites, including cysteine proteases that are increasingly identified as virulence factors in parasitic protozoa. In this work, a monoclonal antibody (mAb1G3) raised against G. duodenalis surface components was found to react with a 25 kDa protein expressed in the cell surface and flagella of G. duodenalis trophozoites. When trophozoites expressing this protein were cultured with IEC-6 intestinal epithelial cell monolayers, a dynamic release of this protein was observed with mAbIG3. Proteomic analysis identified the protein as a mature cathepsin B-like (gCatB) enzyme, whose proteolytic activity, detected in zymograms, was eliminated by CatB inhibitor E-64. This protein was named giardipain-1 due to its functional papain-like features and was purified by affinity chromatography using mAbIG3. Upon exposure to the purified, mature and secreted forms of giardipain-1, IEC-6 epithelial cell monolayers displayed membrane blebbing and phosphatidylserine exposure on the outer cell surface, indicating an apoptotic process. In Madin Darby Canine Kidney (MDCK) cell monolayers, giardipain-1 leads to the appearance of pore-like regions and of gaps along cell-cell junctions, to decreased transepithelial electrical resistance (TER), caspase-3 activation and poly-ADP-ribose polymerase (PARP) fragmentation. At early times during exposure, giardipain-1 co-localized at cell-cell junctions, associated with occludin and induced the delocalization and degradation of tight junction proteins occludin and claudin-1. The damage caused to epithelial monolayers by giardipain-1 was blocked by pre-incubation with the CatB B Inhibitor E-64. Furthermore, silencing the giardipain-1 gene in trophozoites lowered the proteolytic activity of giardipain-1 and reduced the damage in IEC-6 monolayers. The damage observed appears to be specific to giardipain activity since almost no damage was observed when IEC-6 monolayers were incubated with papain, a non-related cysteine protease. Hence this study suggests that giardipain-1 triggers, in epithelial cells, degradation of cell-cell junctional components and apoptotic damage, supporting the notion of giardiapain-1 as a virulence factor of Giardia.


Assuntos
Células Epiteliais/efeitos dos fármacos , Giardia lamblia/enzimologia , Peptídeo Hidrolases/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Apoptose , Domínio Catalítico , Células Epiteliais/fisiologia , Regulação Enzimológica da Expressão Gênica , Giardia lamblia/genética , Giardia lamblia/metabolismo , Humanos , Modelos Moleculares , Peptídeo Hidrolases/genética , Conformação Proteica , Ratos
19.
Tissue Barriers ; 6(1): e1414015, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29420165

RESUMO

Tight junctions (TJs) are sites of cell-cell adhesion, constituted by a cytoplasmic plaque of molecules linked to integral proteins that form a network of strands around epithelial and endothelial cells at the uppermost portion of the lateral membrane. TJs maintain plasma membrane polarity and form channels and barriers that regulate the transit of ions and molecules through the paracellular pathway. This structure that regulates traffic between the external milieu and the organism is affected in numerous pathological conditions and constitutes an important target for therapeutic intervention. Here, we describe how a wide array of G protein-coupled receptors that are activated by diverse stimuli including light, ions, hormones, peptides, lipids, nucleotides and proteases, signal through heterotrimeric G proteins, arrestins and kinases to regulate TJs present in the blood-brain barrier, the blood-retinal barrier, renal tubular cells, keratinocytes, lung and colon, and the slit diaphragm of the glomerulus.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Junções Íntimas/metabolismo , Humanos
20.
Sci Rep ; 7(1): 13351, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-29042633

RESUMO

The Catsper1 gene, whose expression is restricted to male germ cells, has great importance in reproductive biology because of its function in sperm motility and fertilization. We previously reported that the promoter of this gene has transcriptional activity in either direction in a heterologous system. In the present study, we found that the Catsper1 promoter has in vitro transcriptional activity in either orientation in GC-1 spg mouse spermatogonial cells. The results also showed that this promoter regulates the expression of a new divergent Catsper1 gene named Catsper1au (Catsper1 antisense upstream transcript). Catsper1au is expressed in adult male mouse testis and liver tissues but not in female mouse liver or ovary tissues. In the testis, Catsper1au is expressed in embryos at 11.5 days post-coitum and from newborns to adults. This gene is also expressed in 1- to 3-week postnatal hearts and in 1-week to adult stage livers. The analysis of the 1402 bp whole genome sequence revealed that Catsper1au is an intronless and polyadenylated lncRNA, located in the nuclei of Sertoli and spermatogenic cells from adult testis. These data indicate that Catsper1au is divergently expressed from the Catsper1 promoter and could regulate gene expression during spermatogenesis.


Assuntos
Canais de Cálcio/genética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Animais , Perfilação da Expressão Gênica , Ordem dos Genes , Genes Reporter , Vetores Genéticos/genética , Células Germinativas , Masculino , Camundongos , Espermatozoides/metabolismo , Transcriptoma
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