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1.
Hepatology ; 72(3): 1073-1084, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-31860744

RESUMO

BACKGROUND AND AIMS: Moesin, an ezrin/radixin/moesin family member, is involved in the regulation of cell adhesion, polarity, and migration by cross-linking between the actin cytoskeleton and plasma membrane. The primary effector cell in hepatic fibrosis is the hepatic stellate cell (HSC), which undergoes activation during liver injury leading to increased extracellular matrix production. APPROACH AND RESULTS: Here, we have hypothesized that moesin plays a critical role in linking the HSC cytoskeleton to the fibrogenic cascade during HSC activation. Moesin phosphorylation was up-regulated during HSC activation and fibrogenesis. Using moesin wild-type (WT) and mutant constructs (phosphomimicking T558D and nonphosphorylatable T558A), we found that cellular motility and contraction were increased in moesin WT-infected and T558D-infected cells, paralleled by an increase in smooth muscle α-actin and collagen 1 expression. In contrast, overexpression of nonphosphorylatable moesin and moesin knockout (KO) decreased cellular motility and contraction. Most importantly, moesin KO led to abrogation of liver fibrosis. The mechanism of moesin's effect was a reduction in myocardin-related transcription factor-A and serum-response factor (SRF)-mediated changes in the actin cytoskeleton, which in turn modulated the expression of matrix genes. CONCLUSIONS: Taken together, our findings suggest that the linkage between cytoskeletal dynamics and the correlated MRTF/SRF signaling pathway has a pivotal role in HSC activation and fibrogenesis.


Assuntos
Citoesqueleto de Actina/metabolismo , Células Estreladas do Fígado , Cirrose Hepática , Proteínas dos Microfilamentos/metabolismo , Fator de Resposta Sérica/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Adesão Celular , Membrana Celular/fisiologia , Movimento Celular , Polaridade Celular , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/ultraestrutura , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , Camundongos Knockout , Fragmentos de Peptídeos , Fosforilação , Ratos
3.
Am J Physiol Renal Physiol ; 311(2): F343-51, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27279487

RESUMO

Na(+)/H(+) exchanger (NHE)3, a major Na(+) transporter in the luminal membrane of the proximal tubule, is subject to ANG II regulation in renal Na(+)/fluid absorption and blood pressure control. We have previously shown that inositol 1,4,5-trisphosphate receptor-binding protein released with inositol 1,4,5-trisphosphate (IRBIT) mediates ANG II-induced exocytosis of NHE3 in cultured proximal tubule epithelial cells. In searching for scaffold protein(s) that coordinates with IRBIT in NHE3 trafficking, we found that NHE regulatory factor (NHERF)1, NHE3, and IRBIT proteins were coexpressed in the same macrocomplexes and that loss of ANG II type 1 receptors decreased their expression in the renal brush-border membrane. We found that NHERF1 was required for ANG II-mediated forward trafficking and activation of NHE3 in cultured cells. ANG II induced a concomitant increase of NHERF1 interactions with NHE3 and IRBIT, which were abolished when the NHERF1 PDZ1 domain was removed. Overexpression of a yellow fluorescent protein-NHERF1 construct that lacks PDZ1, but not PDZ2, failed to exaggerate the ANG II-dependent increase of NHE3 expression in the apical membrane. Moreover, exogenous expression of PDZ1 exerted a dominant negative effect on NHE3 activation by ANG II. We further demonstrated that IRBIT was indispensable for the ANG II-provoked increase in NHERF1-NHE3 interactions and that phosphorylation of IRBIT at Ser(68) was necessary for the assembly of the NHEF1-IRBIT-NHE3 complex. Taken together, our findings suggest that NHERF1 mediates ANG II-induced activation of renal NHE3, which requires coordination between IRBIT and the NHERF1 PDZ1 domain in binding and transporting NHE3.


Assuntos
Adenosil-Homocisteinase/metabolismo , Angiotensina II/farmacologia , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/agonistas , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Biotinilação , Linhagem Celular , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Knockout , Microvilosidades/metabolismo , Plasmídeos/genética , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Sódio/metabolismo , Trocador 3 de Sódio-Hidrogênio
4.
Dig Dis Sci ; 61(2): 453-63, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26500117

RESUMO

BACKGROUND: The dominant ezrin/radixin/moesin protein in hepatocytes is radixin, which plays an important role in mediating the binding of F-actin to the plasma membrane after a conformational activation by phosphorylation at Thr564. AIM: Here we have investigated the importance of Akt-mediated radixin Thr564 phosphorylation on Mrp-2 distribution and function in WIF-B cells. Mrp-2 is an adenosine triphosphate (ATP)-binding cassette transporter that plays an important role in detoxification and chemoprotection by transporting a wide range of compounds, especially conjugates of lipophilic substances with glutathione, organic anions, and drug metabolites such as glucuronides. METHODS: Akt1 and Akt2 expression were manipulated using dominant active and negative constructs as well as Akt1 and Akt2 siRNA. Cellular distribution of radixin and Mrp-2 was visualized by fluorescence microscopy. A 5-chloromethylfluorescein diacetate, which is a substrate of the Mrp-2 and is actively transported in canalicular lumina, was used to measure Mrp-2 function. RESULTS: Radixin phosphorylation was significantly increased in wild-type and dominant active Akt2 transfected cells. Furthermore, radixin and Mrp-2 were localized at the canalicular membrane, similar to control cells. In contrast, overexpression of dominant negative Akt2, siRNA knockdown of Akt2 and a specific Akt inhibitor prevented radixin phosphorylation and led to alteration of normal radixin and Mrp-2 localization; inhibition of Akt2, but not Akt1 function led to radixin localization to the cytoplasmic space. In addition, dominant negative and Akt2 knockdown led to a dramatically impaired hepatocyte secretory response, while wild-type and dominant active Akt2 transfected cells exhibited increased 5-chloromethylfluorescein diacetate excretion. In contrast to Akt2, Akt1 was not associated with radixin phosphorylation. CONCLUSIONS: These studies, therefore, identify Akt2 as a critical kinase that regulates radixin phosphorylation and leads to Mrp-2 translocation and function.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular , Proteínas do Citoesqueleto/genética , Técnicas de Silenciamento de Genes , Glutationa/análogos & derivados , Glutationa/metabolismo , Hepatócitos/metabolismo , Humanos , Proteínas de Membrana/genética , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética
5.
Am J Physiol Cell Physiol ; 309(4): C264-70, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-26108660

RESUMO

Gastric acid secretion is mediated by the K(+)-dependent proton pump (H(+),K(+)-ATPase), which requires a continuous supply of K(+) at the luminal side of the apical membrane. Several K(+) channels are implicated in gastric acid secretion. However, the identity of the K(+) channel(s) responsible for apical K(+) supply is still elusive. Our previous studies have shown the translocation of KCNJ15 from cytoplasmic vesicles to the apical membrane on stimulation, indicating its involvement in gastric acid secretion. In this study, the stimulation associated trafficking of KCNJ15 was observed in a more native context with a live cell imaging system. KCNJ15 molecules in resting live cells were scattered in cytoplasm but exhibited apical localization after stimulation. Furthermore, knocking down KCNJ15 expression with a short hairpin RNA adenoviral construct abolished histamine-stimulated acid secretion in rabbit primary parietal cells. Moreover, KCNJ15, like H(+),K(+)-ATPase, was detected in all of the parietal cells by immunofluorescence staining, whereas only about half of the parietal cells were positive for KCNQ1 under the same condition. Consistently, the endogenous protein levels of KCNJ15, analyzed by Western blotting, were higher than those of KCNQ1 in the gastric mucosa of human, mouse, and rabbit. These results provide evidence for a major role of KCNJ15 in apical K(+) supply during stimulated acid secretion.


Assuntos
Ácido Gástrico/metabolismo , Mucosa Gástrica/metabolismo , Histamina/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Adolescente , Animais , Feminino , Mucosa Gástrica/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Camundongos , Células Parietais Gástricas/metabolismo , Coelhos
6.
Am J Physiol Gastrointest Liver Physiol ; 308(4): G313-24, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25501552

RESUMO

The most prominent ezrin-radixin-moesin protein in hepatocytes is radixin, which is localized primarily at the canalicular microvilli and appears to be important in regulation of cell polarity and in localizing the multidrug resistance-associated protein 2 (Mrp-2) function. Our aim was to investigate how hypoxia affects radixin distribution and Mrp-2 function. We created wild-type and mutant constructs (in adenoviral vectors), which were expressed in WIF-B cells. The cellular distribution of Mrp-2 and radixin was visualized by fluorescence microscopy, and a 5-chloromethylfluorescein diacetate (CMFDA) assay was used to measure Mrp-2 function. Under usual conditions, cells infected with wild-type radixin, nonphosphorylatable radixin-T564A, and radixin-T564D (active phospho-mimicking mutant) were found to be heavily expressed in canalicular membrane compartment vacuoles, typically colocalizing with Mrp-2. In contrast, after hypoxia for 24 h, both endogenous and overexpressed wild-type radixin and the radixin-T564A mutant were found to be translocated to the cytoplasmic space. However, distribution of the radixin-T564D mutant, which mimics constant phosphorylation, was remarkably different, being associated with canalicular membranes even in hypoxic conditions. This dominant-active construct also prevented dissociation of radixin from the plasma membrane. Hypoxia also led to Mrp-2 mislocalization and caused Mrp-2 to be dissociated from radixin; the radixin phospho-mimicking mutant (T564D) abrogated this effect of hypoxia. Finally, hypoxia diminished the secretory response (measured using the CMFDA assay) in WIF-B cells, and the dominant-active construct (radixin-T567D) rescued this phenotype. Taken collectively, these findings suggest that radixin regulates Mrp-2 localization and function in hepatocytes and is important in hypoxic liver injury.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Hepatócitos/metabolismo , Proteínas de Membrana/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sobrevivência Celular , Citoplasma/metabolismo , Proteínas do Citoesqueleto/genética , Genótipo , Hepatócitos/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Cinética , Proteínas de Membrana/genética , Mutação , Fenótipo , Fosforilação , Transporte Proteico , Interferência de RNA , Ratos , Transfecção
7.
Am J Physiol Cell Physiol ; 307(8): C727-37, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25163515

RESUMO

Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) is a multifunctional scaffolding protein that interacts with receptors and ion transporters in its PDZ domains and with the ezrin-radixin-moesin (ERM) family of proteins in its COOH terminus. The role of NHERF1 in hepatocyte function remains largely unknown. We examine the distribution and physiological significance of NHERF1 and multidrug resistance-associated protein 2 (Mrp-2) in hepatocytes. A WT radixin binding site mutant (F355R) and NHERF1 PDZ1 and PDZ2 domain adenoviral mutant constructs were tagged with yellow fluorescent protein and expressed in polarized hepatocytes to study localization and function of NHERF1. Cellular distribution of NHERF1 and radixin was visualized by fluorescence microscopy. A 5-chloromethylfluorescein diacetate (CMFDA) assay was used to characterize Mrp-2 function. Similar to Mrp-2, WT NHERF1 and the NHERF1 PDZ2 deletion mutant were localized to the canalicular membrane. In contrast, the radixin binding site mutant (F355R) and the NHERF1 PDZ1 deletion mutant, which interacts poorly with Mrp-2, were rarely associated with the canalicular membrane. Knockdown of NHERF1 led to dramatically impaired CMFDA secretory response. Use of CMFDA showed that the NHERF1 PDZ1 and F355R mutants were devoid of a secretory response, while WT NHERF1-infected cells exhibited increased secretion of glutathione-methylfluorescein. The data indicate that NHERF1 interacts with Mrp-2 via the PDZ1 domain of NHERF1 and, furthermore, that NHERF1 is essential for maintaining the localization and function of Mrp-2.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Hepatócitos/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla , Mutação de Sentido Incorreto , Fosfoproteínas/genética , Ligação Proteica , Transporte Proteico , Ratos , Trocadores de Sódio-Hidrogênio/genética , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATP
8.
Gastroenterology ; 140(3): 868-78, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20888820

RESUMO

BACKGROUND & AIMS: Rabs are monomeric guanosine triphosphatases that regulate membrane trafficking and acid secretion in gastric parietal cells. Using a proteomics approach, we identified a new Rab, Rab27b, in tubulovesicle membranes and determined its role in parietal cell activation. METHODS: We used mass spectrometry (MS) to identify Rab27b in purified tubulovesicular membrane fractions and used immunoblot and immunofluorescence analyses to study its expression. Wild-type, constitutively active (Rab27bQ78L), and dominant negative (Rab27bN133I) forms of Rab27b were tagged with yellow fluorescent protein (YFP) and expressed in parietal cells using adenoviral constructs to study localization and function. Localization was visualized by fluorescence microscopy in resting and stimulated cells. Acid secretion in primary cell cultures was measured by aminopyrine accumulation. RESULTS: A tandem MS peptide mass fingerprint was matched to 7 peptides of Rab27b. Rab27b localized to tubulovesicle membranes, based on immunoblot and immunocytochemical analyses. Endogenous Rab27b, YFP/wild-type Rab27b, Rab27bQ78L, and Rab27bN133I all distributed throughout the cytoplasm of resting parietal cells. After stimulation, wild-type Rab27b and YFP-Rab27bQ78L translocated to the apical membrane, but YFPR-ab27bN133I did not. Expression of wild-type YFP-Rab27b or YFP-Rab27bQ78L did not affect acid secretion, whereas expression of Rab27bN133I almost completely inhibited acid secretion. CONCLUSIONS: Rab27b is associated with tubulovesicle membranes in the parietal cell and Rab27b may play a role in stimulation-associated membrane recruitment and gastric acid secretion.


Assuntos
Ácido Gástrico/metabolismo , Membranas Intracelulares/metabolismo , Células Parietais Gástricas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Aminopirina/metabolismo , Animais , Western Blotting , Células Cultivadas , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Humanos , Imuno-Histoquímica , Microscopia de Fluorescência , Mutação , Mapeamento de Peptídeos , Transporte Proteico , Proteômica/métodos , Coelhos , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Massas em Tandem , Transfecção , Proteínas rab de Ligação ao GTP/genética
9.
Am J Physiol Cell Physiol ; 300(3): C416-24, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21160029

RESUMO

Radixin, the dominant ezrin-radixin-moesin (ERM) protein in hepatocytes, has two important binding domains: an NH(2)-terminal region that binds to plasma membrane and a COOH-terminal region that binds to F-actin after a conformational activation by phosphorylation at Thr564. The present studies were undertaken to investigate the cellular changes in expression of radixin in WIF-B cells and to assess radixin distribution and its influence on cell polarity. We used a recombinant adenoviral expression system encoding radixin wild-type and Thr564 mutants fused to cyan fluorescent protein (CFP), as well as conventional immunostaining procedures. Functional analyses were characterized quantitatively. Similar to endogenous radixin, adenovirus-infected radixin-CFP-wild type and nonphosphorylatable radixin-CFP-T564A were found to be expressed heavily in the compartment of canalicular membrane vacuoles, typically colocalizing with multidrug resistance-associated protein 2 (Mrp-2). Expression of radixin-CFP-T564D, which mimics constant phosphorylation, was quite different, being rarely associated with canalicular membranes. The WIF-B cells were devoid of a secretory response, T567D radixin became predominantly redistributed to the basolateral membrane, usually in the form of dense, long spikes and fingerlike projections, and the altered cell polarity involved changes in apical membrane markers. Differences in polar distribution of radixin suggest a role for the linker protein in promoting formation and plasticity of membrane surface projections and also suggest that radixin might be an organizer and regulator of Mrp-2 and cell polarity in hepatocytes.


Assuntos
Polaridade Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Hepatócitos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Animais , Linhagem Celular Tumoral , Extensões da Superfície Celular/metabolismo , Proteínas do Citoesqueleto/genética , Vetores Genéticos/genética , Hepatócitos/citologia , Humanos , Hibridomas , Proteínas de Membrana/genética , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Mutantes/genética , Fosforilação/genética , Ratos , Transfecção , Vacúolos/metabolismo
10.
Am J Physiol Cell Physiol ; 301(2): C507-21, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21525430

RESUMO

Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b(-/-) and Rab27(ash/ash)/Rab27b(-/-) mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release.


Assuntos
Células Epiteliais/metabolismo , Exocitose , Aparelho Lacrimal/metabolismo , Vesículas Secretórias/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Análise de Variância , Animais , Carbacol/farmacologia , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Exocitose/efeitos dos fármacos , Aparelho Lacrimal/efeitos dos fármacos , Aparelho Lacrimal/ultraestrutura , Masculino , Fusão de Membrana , Camundongos , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Vídeo , Miosinas/metabolismo , Transporte Proteico , Coelhos , Proteínas Recombinantes de Fusão/metabolismo , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/ultraestrutura , Fatores de Tempo , Transfecção , Proteínas rab de Ligação ao GTP/deficiência , Proteínas rab de Ligação ao GTP/genética , Proteínas rab3 de Ligação ao GTP/metabolismo
11.
Curr Pharm Des ; 23(34): 5104-5114, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28847302

RESUMO

The genus Digitalis L. containing species, commonly known as the "foxglove", is the main source of cardenolides, which have various pharmacological properties effective against certain pathological conditions including myocardial infarction, arterial hypertension, cardiac dysfunction, angina, and hypertrophy. Togehter with a prime effect of controlling the heart rhythm, many workers demonstrated that lanatoside C and some other cardiac glycosides are effective in several cancer treatments such as prostate and breast cancers. Due to digoxigenin derivatives of cardenolides, which are mainly used for medicinal purposes, such as digoxigenin, D. lanata as a main source is of great interest for commercial scale production of cardenolides in Europe. Phytochemical studies on cardenolides, naturally occurring plant secondary metabolites, have mainly focused on the species of the genus Digitalis L., as the members of this family have a high level and diverse content of cardenolides. During the last few decades, plant tissue culture techniques have been optimised for many plant species including Digitalis, however, the production capacity of cardenolides somehow failed to reach a commercially desired extent. In this review paper, the genus Digitalis is evaluated in terms of its main botanical and physiological features, traditional uses, molecular genetics and metabolomics, cellular mechanism of action, medicinal uses, clinical pharmacology, drug interactions, therapy in the management of cardiovascular disorders, potential utility of therapy in extracardiac conditions, and toxicity.


Assuntos
Cardenolídeos/uso terapêutico , Cardiotônicos/uso terapêutico , Doenças Cardiovasculares/tratamento farmacológico , Digitalis , Extratos Vegetais/uso terapêutico , Animais , Cardenolídeos/química , Cardenolídeos/isolamento & purificação , Cardiotônicos/química , Cardiotônicos/isolamento & purificação , Doenças Cardiovasculares/fisiopatologia , Digitalis/química , Humanos , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação
12.
J Clin Invest ; 127(4): 1321-1337, 2017 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-28287407

RESUMO

Moesin is a member of the ezrin-radixin-moesin (ERM) family of proteins that are important for organizing membrane domains and receptor signaling and regulating the migration of effector T cells. Whether moesin plays any role during the generation of TGF-ß-induced Tregs (iTregs) is unknown. Here, we have discovered that moesin is translationally regulated by TGF-ß and is also required for optimal TGF-ß signaling that promotes efficient development of iTregs. Loss of moesin impaired the development and function of both peripherally derived iTregs and in vitro-induced Tregs. Mechanistically, we identified an interaction between moesin and TGF-ß receptor II (TßRII) that allows moesin to control the surface abundance and stability of TßRI and TßRII. We also found that moesin is required for iTreg conversion in the tumor microenvironment, and the deletion of moesin from recipient mice supported the rapid expansion of adoptively transferred CD8+ T cells against melanoma. Our study establishes moesin as an important regulator of the surface abundance and stability of TßRII and identifies moesin's role in facilitating the efficient generation of iTregs. It also provides an advancement to our understanding about the role of the ERM proteins in regulating signal transduction pathways and suggests that modulation of moesin is a potential therapeutic target for Treg-related immune disorders.


Assuntos
Melanoma Experimental/imunologia , Proteínas dos Microfilamentos/fisiologia , Neoplasias Cutâneas/imunologia , Linfócitos T Reguladores/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Transferência Adotiva , Animais , Diferenciação Celular , Membrana Celular/metabolismo , Células Cultivadas , Feminino , Células HEK293 , Humanos , Masculino , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transplante de Neoplasias , Ligação Proteica , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade Proteica , Transporte Proteico , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Ativação Transcricional , Evasão Tumoral , Regulação para Cima
13.
PLoS One ; 7(2): e31789, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22363735

RESUMO

This study uses YFP-tagged Rab27b expression in rabbit lacrimal gland acinar cells, which are polarized secretory epithelial cells, to characterize early stages of secretory vesicle trafficking. Here we demonstrate the utility of YFP-Rab27b to delineate new perspectives on the mechanisms of early vesicle biogenesis in lacrimal gland acinar cells, where information is significantly limited. Protocols were developed to deplete the mature YFP-Rab27b-enriched secretory vesicle pool in the subapical region of the cell, and confocal fluorescence microscopy was used to track vesicle replenishment. This analysis revealed a basally-localized organelle, which we termed the "nascent vesicle site," from which nascent vesicles appeared to emerge. Subapical vesicular YFP-Rab27b was co-localized with p150(Glued), a component of the dynactin cofactor of cytoplasmic dynein. Treatment with the microtubule-targeted agent, nocodazole, did not affect release of mature secretory vesicles, although during vesicle repletion it significantly altered nascent YFP-Rab27b-enriched secretory vesicle localization. Instead of moving to the subapical region, these vesicles were trapped at the nascent vesicle site which was adjacent to, if not a sub-compartment of, the trans-Golgi network. Finally, YFP-Rab27b-enriched secretory vesicles which reached the subapical cytoplasm appeared to acquire the actin-based motor protein, Myosin 5C. Our findings show that Rab27b enrichment occurs early in secretory vesicle formation, that secretory vesicles bud from a visually discernable nascent vesicle site, and that transport from the nascent vesicle site to the subapical region requires intact microtubules.


Assuntos
Células Acinares/metabolismo , Imageamento Tridimensional/métodos , Aparelho Lacrimal/citologia , Vesículas Secretórias/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Células Acinares/citologia , Animais , Transporte Biológico , Compartimento Celular , Feminino , Complexo de Golgi/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Miosinas/metabolismo , Ligação Proteica , Coelhos , Proteínas Recombinantes de Fusão/metabolismo
14.
Traffic ; 6(8): 654-66, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15998321

RESUMO

Syntaxins are differentially localized in polarized cells and play an important role in vesicle trafficking and membrane fusion. These soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are believed to be involved in tubulovesicle trafficking and membrane fusion during the secretory cycle of the gastric parietal cell. We examined the cellular localization and distribution of syntaxin-1 and syntaxin-3 in rabbit parietal cells. Fractionation of gastric epithelial cell membranes showed that syntaxin-1 was more abundant in a fraction enriched in apical plasma membranes, whereas syntaxin-3 was found predominantly in the H,K-ATPase-rich tubulovesicle fraction. We also examined the cellular localization of syntaxins in cultured parietal cells. Parietal cells were infected with CFP-syntaxin-1 and CFP-syntaxin-3 adenoviral constructs. Fluorescence microscopy of live and fixed cells demonstrated that syntaxin-1 was primarily on the apical membrane vacuoles of infected cells, but there was also the expression of syntaxin-1 in a subadjacent cytoplasmic compartment. In resting, non-secreting parietal cells, syntaxin-3 was distributed throughout the cytoplasmic compartment; after stimulation, syntaxin-3 translocated to the apical membrane vacuoles, there co-localizing with H,K-ATPase, syntaxin-1 and F-actin. The differential location of these syntaxin isoforms in gastric parietal cells suggests that these proteins may be critical for maintaining membrane compartment identity and that they may play important, but somewhat different, roles in the membrane recruitment processes associated with secretory activation.


Assuntos
Antígenos de Superfície/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Parietais Gástricas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Actinas/metabolismo , Adenoviridae/genética , Animais , Antígenos de Superfície/genética , Fracionamento Celular , Células Cultivadas , Proteínas de Fluorescência Verde/genética , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Histamina/farmacologia , Proteínas de Membrana/genética , Microscopia Confocal , Proteínas do Tecido Nervoso/genética , Células Parietais Gástricas/efeitos dos fármacos , Células Parietais Gástricas/ultraestrutura , Regiões Promotoras Genéticas , Proteínas Qa-SNARE , Coelhos , Proteínas SNARE , Sintaxina 1 , Transfecção
15.
J Biol Chem ; 277(51): 50030-5, 2002 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-12386166

RESUMO

The soluble N-ethylmaleimide-sensitive factor attachment protein of 25 kDa (SNAP-25) plays an important role in vesicle trafficking. Together with vesicle-associated membrane protein-2 (VAMP-2) and syntaxin, SNAP-25 forms a ternary complex implicated in docking and fusion of secretory vesicles with the plasma membrane during exocytosis. These so-called SNARE proteins are believed to regulate tubulovesicle trafficking and fusion during the secretory cycle of the gastric parietal cell. Here we examined the cellular localization and functional importance of SNAP-25 in parietal cell cultures. Adenoviral constructs were used to express SNAP-25 tagged with cyan fluorescent protein, VAMP-2 tagged with yellow fluorescent protein, and SNAP-25 in which the C-terminal 25 amino acids were deleted (SNAP-25 Delta181-206). Membrane fractionation experiments and fluorescent imaging showed that SNAP-25 is localized to the apical plasma membrane. The expression of the mutant SNAP-25 Delta181-226 inhibited the acid secretory response of parietal cells. Also, SNAP Delta181-226 bound poorly in vitro with recombinant syntaxin-1 compared with wild type SNAP-25, indicating that pairing between syntaxin-1 and SNAP-25 is required for parietal cell activation. Dual expression of SNAP-25 tagged with cyan fluorescent protein and VAMP-2 tagged with yellow fluorescent protein revealed a dynamic change in distribution associated with acid secretion. In resting cells, SNAP-25 is at the apical plasma membrane and VAMP-2 is associated with cytoplasmic H,K-ATPase-rich tubulovesicles. After stimulation, the two proteins co-localize on the apical plasma membrane. These data demonstrate the functional significance of SNAP-25 as a SNARE protein in the parietal cell and show the dynamic stimulation-associated redistribution of VAMP-2 from H,K-ATPase-rich tubulovesicles to co-localize with SNAP-25 on the apical plasma membrane.


Assuntos
Etilmaleimida/farmacologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/fisiologia , Células Parietais Gástricas/metabolismo , Proteínas de Transporte Vesicular , Adenoviridae/genética , Animais , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Eletroforese em Gel de Poliacrilamida , Deleção de Genes , Proteínas de Fluorescência Verde , Cobaias , ATPase Trocadora de Hidrogênio-Potássio/química , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Mutação , Proteínas do Tecido Nervoso/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas R-SNARE , Coelhos , Ratos , Proteínas SNARE , Proteína 25 Associada a Sinaptossoma , Sintaxina 1 , Fatores de Tempo
16.
Gastroenterology ; 123(1): 281-90, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12105856

RESUMO

BACKGROUND & AIMS: Acid secretion by parietal cells involves secretagogue-dependent recycling of the H+-K+-ATPase. Proteins called soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) have been implicated as participants in membrane trafficking, docking, and fusing processes. Here we studied the intracellular distribution and functional importance of one SNARE protein, vesicle associated membrane protein-2 (VAMP-2), in gastric parietal cells. METHODS: Using an adenoviral recombinant expression system encoding VAMP-2 (synaptobrevin-2) fused to the green fluorescent protein (GFP), we expressed the GFP-VAMP-2 protein in primary cultures of rabbit parietal cells, which enables us to visualize the dynamics of GFP-VAMP-2 in a variety of functional states by fluorescence microscopy. To ascertain the function of VAMP-2 in parietal cell activation, streptolysin-O permeabilized gastric glands were treated with tetanus toxin, a potent and preferential protease for VAMP-2, and acid secretion was measured. RESULTS: In resting parietal cells GFP was detected throughout the cytoplasm in a pattern of distribution that was very similar to that of H+-K+-ATPase. After stimulation, we observed that the GFP-VAMP-2 translocated to the apical plasma membrane along with the H+-K+-ATPase. A relatively high degree of co-localization was detected between GFP-VAMP-2 and H+-K+-ATPase. Tetanus toxin inhibited cAMP/ATP-stimulated acid secretion by about 45% in permeabilized gastric glands with a concomitant reduction in the level of immunoreactive VAMP-2. CONCLUSIONS: Adenovirus-based GFP reporter fusion proteins can be used to efficiently study the functional dynamics of SNAREs. VAMP-2 is associated with tubulovesicle membranes in the parietal cell and plays a role in stimulation-associated membrane recruitment and acid secretion.


Assuntos
Membranas Intracelulares/metabolismo , Proteínas de Membrana/fisiologia , Células Parietais Gástricas/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Ácido Gástrico/metabolismo , Proteínas de Fluorescência Verde , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Histamina/farmacologia , Indicadores e Reagentes , Proteínas Luminescentes , Proteínas de Membrana/química , Proteínas de Membrana/efeitos dos fármacos , Proteínas R-SNARE , Coelhos , Toxina Tetânica/farmacologia , Distribuição Tecidual
17.
Nutr Cancer ; 46(2): 125-30, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14690787

RESUMO

Selenoproteins such as glutathione peroxidases (GPx), thioredoxin reductases (TrxR), and selenoprotein P (SePP) contain molecular selenium in form of selenocysteines within their active center. They are involved in the defense of reactive oxygen species, which otherwise may cause DNA damage and alterations of protein function. Selenium intake has been linked to colon carcinogenesis in epidemiological and interventional studies. In a double-blinded, placebo-controlled trial, we demonstrate that carriers of colon adenomas present with low basal serum levels of selenium and plasma glutathione peroxidase (pGPx) activity before treatment, but both parameters can be normalized by interventional selenium supplementation. GPx activity in colon mucosa was enhanced in the verum group, albeit this had only borderline significance. No change of activity was observed for mucosal TrxR activity on selenium supplementation. In summary, our results confirm the existence of low selenium levels in patients prone to colon adenomas and show that by selenium supplementation this can be normalized. If prospective trials confirm that selenium supplementation reduces colon cancer incidence rates, it may be concluded that selenium supplementation should be recommended for patients at risk.


Assuntos
Adenoma/enzimologia , Colo/enzimologia , Neoplasias do Colo/enzimologia , Glutationa Peroxidase/sangue , Selênio/administração & dosagem , Selênio/sangue , Neoplasias do Colo/prevenção & controle , Suplementos Nutricionais , Método Duplo-Cego , Humanos , Mucosa Intestinal/enzimologia , Placebos , Selenito de Sódio/administração & dosagem , Tiorredoxina Dissulfeto Redutase/metabolismo
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