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1.
PLoS Med ; 12(3): e1001796, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25734483

RESUMO

BACKGROUND: Chronic lymphocytic leukemia (CLL), the most common adulthood leukemia, is characterized by the accumulation of abnormal CD5+ B lymphocytes, which results in a progressive failure of the immune system. Despite intense research efforts, drug resistance remains a major cause of treatment failure in CLL, particularly in patients with dysfunctional TP53. The objective of our work was to identify potential approaches that might overcome CLL drug refractoriness by examining the pro-apoptotic potential of targeting the cell surface receptor CD47 with serum-stable agonist peptides. METHODS AND FINDINGS: In peripheral blood samples collected from 80 patients with CLL with positive and adverse prognostic features, we performed in vitro genetic and molecular analyses that demonstrate that the targeting of CD47 with peptides derived from the C-terminal domain of thrombospondin-1 efficiently kills the malignant CLL B cells, including those from high-risk individuals with a dysfunctional TP53 gene, while sparing the normal T and B lymphocytes from the CLL patients. Further studies reveal that the differential response of normal B lymphocytes, collected from 20 healthy donors, and leukemic B cells to CD47 peptide targeting results from the sustained activation in CLL B cells of phospholipase C gamma-1 (PLCγ1), a protein that is significantly over-expressed in CLL. Once phosphorylated at tyrosine 783, PLCγ1 enables a Ca2+-mediated, caspase-independent programmed cell death (PCD) pathway that is not down-modulated by the lymphocyte microenvironment. Accordingly, down-regulation of PLCγ1 or pharmacological inhibition of PLCγ1 phosphorylation abolishes CD47-mediated killing. Additionally, in a CLL-xenograft model developed in NOD/scid gamma mice, we demonstrate that the injection of CD47 agonist peptides reduces tumor burden without inducing anemia or toxicity in blood, liver, or kidney. The limitations of our study are mainly linked to the affinity of the peptides targeting CD47, which might be improved to reach the standard requirements in drug development, and the lack of a CLL animal model that fully mimics the human disease. CONCLUSIONS: Our work provides substantial progress in (i) the development of serum-stable CD47 agonist peptides that are highly effective at inducing PCD in CLL, (ii) the understanding of the molecular events regulating a novel PCD pathway that overcomes CLL apoptotic avoidance, (iii) the identification of PLCγ1 as an over-expressed protein in CLL B cells, and (iv) the description of a novel peptide-based strategy against CLL.


Assuntos
Apoptose/efeitos dos fármacos , Linfócitos B/metabolismo , Antígeno CD47/metabolismo , Resistencia a Medicamentos Antineoplásicos , Leucemia Linfocítica Crônica de Células B/metabolismo , Peptídeos/farmacologia , Fosfolipase C gama/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Peptídeos/uso terapêutico , Trombospondina 1/uso terapêutico
2.
J Biol Chem ; 288(43): 31080-92, 2013 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-23986439

RESUMO

The structure-function relationships of sugar transporter-receptor hGLUT2 coded by SLC2A2 and their impact on insulin secretion and ß cell differentiation were investigated through the detailed characterization of a panel of mutations along the protein. We studied naturally occurring SLC2A2 variants or mutants: two single-nucleotide polymorphisms and four proposed inactivating mutations associated to Fanconi-Bickel syndrome. We also engineered mutations based on sequence alignment and conserved amino acids in selected domains. The single-nucleotide polymorphisms P68L and T110I did not impact on sugar transport as assayed in Xenopus oocytes. All the Fanconi-Bickel syndrome-associated mutations invalidated glucose transport by hGLUT2 either through absence of protein at the plasma membrane (G20D and S242R) or through loss of transport capacity despite membrane targeting (P417L and W444R), pointing out crucial amino acids for hGLUT2 transport function. In contrast, engineered mutants were located at the plasma membrane and able to transport sugar, albeit with modified kinetic parameters. Notably, these mutations resulted in gain of function. G20S and L368P mutations increased insulin secretion in the absence of glucose. In addition, these mutants increased insulin-positive cell differentiation when expressed in cultured rat embryonic pancreas. F295Y mutation induced ß cell differentiation even in the absence of glucose, suggesting that mutated GLUT2, as a sugar receptor, triggers a signaling pathway independently of glucose transport and metabolism. Our results describe the first gain of function mutations for hGLUT2, revealing the importance of its receptor versus transporter function in pancreatic ß cell development and insulin secretion.


Assuntos
Diferenciação Celular/fisiologia , Transportador de Glucose Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Substituição de Aminoácidos , Animais , Transporte Biológico Ativo/genética , Linhagem Celular Tumoral , Glucose/genética , Glucose/metabolismo , Transportador de Glucose Tipo 2/genética , Humanos , Insulina/genética , Secreção de Insulina , Células Secretoras de Insulina/citologia , Camundongos , Ratos , Transdução de Sinais , Xenopus laevis
3.
Gastroenterology ; 143(1): 122-32.e15, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22446194

RESUMO

BACKGROUND & AIMS: Cell adhesion is one function regulated by cellular prion protein (PrP(c)), a ubiquitous, glycosylphosphatidylinositol-anchored glycoprotein. PrP(c) is located in cell-cell junctions and interacts with desmosome proteins in the intestinal epithelium. We investigated its role in intestinal barrier function. METHODS: We analyzed permeability and structure of cell-cell junctions in intestine tissues from PrP(c) knockout (PrP(c-/-)) and wild-type mice. PrP(c) expression was knocked down in cultured human Caco-2/TC7 enterocytes using small hairpin RNAs. We analyzed colon samples from 24 patients with inflammatory bowel disease (IBD). RESULTS: Intestine tissues from PrP(c-/-) mice had greater paracellular permeability than from wild-type mice (105.9 ± 13.4 vs 59.6 ± 10.1 mg/mL fluorescein isothiocyanate-dextran flux; P < .05) and impaired intercellular junctions. PrP(c-/-) mice did not develop spontaneous disease but were more sensitive than wild-type mice to induction of colitis with dextran sulfate (32% mortality vs 4%, respectively; P = .0033). Such barrier defects were observed also in Caco-2/TC7 enterocytes following PrP(c) knockdown; the cells had increased paracellular permeability (1.5-fold over 48 hours; P < .001) and reduced transepithelial electrical resistance (281.1 ± 4.9 vs 370.6 ± 5.7 Ω.cm(2); P < .001). Monolayer shape and cell-cell junctions were altered in cultures of PrP(c) knockdown cells; levels of E-cadherin, desmoplakin, plakoglobin, claudin-4, occludin, zonula occludens 1, and tricellulin were decreased at cell contacts. Cell shape and junctions were restored on PrP(c) re-expression. Levels of PrP(c) were decreased at cell-cell junctions in colonic epithelia from patients with Crohn's disease or ulcerative colitis. CONCLUSIONS: PrP(c) regulates intestinal epithelial cell-cell junctions and barrier function. Its localization is altered in colonic epithelia from patients with IBD, supporting the concept that disrupted barrier function contributes to this disorder.


Assuntos
Doenças Inflamatórias Intestinais/metabolismo , Junções Intercelulares/metabolismo , Mucosa Intestinal/metabolismo , Proteínas PrPC/metabolismo , Animais , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , Colo/metabolismo , Enterócitos/metabolismo , Humanos , Camundongos , Camundongos Knockout
4.
Biol Cell ; 103(11): 499-517, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21787361

RESUMO

BACKGROUND INFORMATION: Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)-rich lipoprotein] assembly and secretion. The accumulation of circulating intestine-derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose-gradient centrifugation from differentiated Caco-2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD-associated TAG mobilization. RESULTS: Among the 105 proteins identified in the CLD fraction by LC-MS/MS (liquid chromatography coupled with tandem MS), 27 were directly involved in lipid metabolism pathways potentially relevant to enterocyte-specific functions. The transient feature of CLDs was consistent with the presence of proteins necessary for fatty acid activation (acyl-CoA synthetases) and for TAG hydrolysis. In differentiated Caco-2/TC7 enterocytes, we identified for the first time LPCAT2 (lysophosphatidylcholine acyltransferase 2), involved in PC (phosphatidylcholine) synthesis, and 3BHS1 (3-ß-hydroxysteroid dehydrogenase 1), involved in steroid metabolism, and confirmed their partial CLD localization by immunofluorescence. In enterocytes, LPCAT2 may provide an economical source of PC, necessary for membrane synthesis and lipoprotein assembly, from the lysoPC present in the intestinal lumen. We also identified proteins involved in lipoprotein metabolism, such as ApoA-IV (apolipoprotein A-IV), which is specifically expressed by enterocytes and has been proposed to play many functions in vivo, including the formation of lipoproteins and the control of their size. The association of ApoA-IV with CLD was confirmed by confocal and immunoelectron microscopy and validated in vivo in the jejunum of mice fed with a high-fat diet. CONCLUSIONS: We report for the first time the protein endowment of Caco-2/TC7 enterocyte CLDs. Our results suggest that their formation and mobilization may participate in the control of enterocyte TRL secretion in a cell-specific manner.


Assuntos
Diferenciação Celular , Citosol/metabolismo , Enterócitos/citologia , Enterócitos/metabolismo , Lipídeos/isolamento & purificação , Proteoma/metabolismo , Animais , Células CACO-2 , Células Cultivadas , Células HeLa , Humanos , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos
5.
Cell Death Differ ; 25(5): 983-1001, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29323266

RESUMO

Mitochondrial metabolism is a tightly regulated process that plays a central role throughout the lifespan of hematopoietic cells. Herein, we analyze the consequences of the mitochondrial oxidative phosphorylation (OXPHOS)/metabolism disorder associated with the cell-specific hematopoietic ablation of apoptosis-inducing factor (AIF). AIF-null (AIF-/Y ) mice developed pancytopenia that was associated with hypocellular bone marrow (BM) and thymus atrophy. Although myeloid cells were relatively spared, the B-cell and erythroid lineages were altered with increased frequencies of precursor B cells, pro-erythroblasts I, and basophilic erythroblasts II. T-cell populations were dramatically reduced with a thymopoiesis blockade at a double negative (DN) immature state, with DN1 accumulation and delayed DN2/DN3 and DN3/DN4 transitions. In BM cells, the OXPHOS/metabolism dysfunction provoked by the loss of AIF was counterbalanced by the augmentation of the mitochondrial biogenesis and a shift towards anaerobic glycolysis. Nevertheless, in a caspase-independent process, the resulting excess of reactive oxygen species compromised the viability of the hematopoietic stem cells (HSC) and progenitors. This led to the progressive exhaustion of the HSC pool, a reduced capacity of the BM progenitors to differentiate into colonies in methylcellulose assays, and the absence of cell-autonomous HSC repopulating potential in vivo. In contrast to BM cells, AIF-/Y thymocytes compensated for the OXPHOS breakdown by enhancing fatty acid ß-oxidation. By over-expressing CPT1, ACADL and PDK4, three key enzymes facilitating fatty acid ß-oxidation (e.g., palmitic acid assimilation), the AIF-/Y thymocytes retrieved the ATP levels of the AIF +/Y cells. As a consequence, it was possible to significantly reestablish AIF-/Y thymopoiesis in vivo by feeding the animals with a high-fat diet complemented with an antioxidant. Overall, our data reveal that the mitochondrial signals regulated by AIF are critical to hematopoietic decision-making. Emerging as a link between mitochondrial metabolism and hematopoietic cell fate, AIF-mediated OXPHOS regulation represents a target for the development of new immunomodulatory therapeutics.


Assuntos
Fator de Indução de Apoptose/deficiência , Linfócitos B/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Fosforilação Oxidativa , Timócitos/metabolismo , Animais , Linfócitos B/citologia , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Timócitos/citologia
6.
Biochem J ; 395(2): 393-403, 2006 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-16393142

RESUMO

Enterocytes are responsible for the absorption of dietary lipids, which involves TRL [TG (triacylglycerol)-rich lipoprotein] assembly and secretion. In the present study, we analysed the effect on TRL secretion of Caco-2 enterocyte adaptation to a differential glucose supply. We showed that TG secretion in cells adapted to a low glucose supply for 2 weeks after confluence was double that of control cells maintained in high-glucose-containing medium, whereas the level of TG synthesis remained similar in both conditions. This increased secretion resulted mainly from an enlargement of the mean size of the secreted TRL. The increased TG availability for TRL assembly and secretion was not due to an increase in the MTP (microsomal TG transfer protein) activity that is required for lipid droplet biogenesis in the ER (endoplasmic reticulum) lumen, or to the channelling of absorbed fatty acids towards the monoacylglycerol pathway for TG synthesis. Interestingly, by electron microscopy and subcellular fractionation studies, we observed, in the low glucose condition, an increase in the TG content available for lipoprotein assembly in the ER lumen, with the cytosolic/microsomal TG levels being verapamil-sensitive. Overall, we demonstrate that Caco-2 enterocytes modulate TRL secretion through TG partitioning between the cytosol and the ER lumen according to the glucose supply. Our model will help in identifying the proteins involved in the control of the balance between TRL assembly and cytosolic lipid storage. This mechanism may be a way for enterocytes to regulate TRL secretion after a meal, and thus impact on our understanding of post-prandial hypertriglyceridaemia.


Assuntos
Adaptação Fisiológica , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Glucose/farmacologia , Lipoproteínas/química , Lipoproteínas/metabolismo , Triglicerídeos/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Apolipoproteínas B/metabolismo , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Retículo Endoplasmático/ultraestrutura , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Enterócitos/ultraestrutura , Glicogênio/metabolismo , Humanos , Lipoproteínas/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Verapamil/farmacologia
7.
Mol Biol Cell ; 15(1): 132-41, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14565984

RESUMO

Enterocytes are highly polarized cells that transfer nutrients across the intestinal epithelium from the apical to the basolateral pole. Apolipoprotein B (apoB) is a secretory protein that plays a key role in the transepithelial transport of dietary fatty acids as triacylglycerol. The evaluation of the control of apoB traffic by lipids is therefore of particular interest. To get a dynamic insight into this process, we used the enterocytic Caco-2 cells cultured on microporous filters, a system in which the apical and basal compartments can be delimited. Combining biochemical and morphological approaches, our results showed that, besides their role in protection from degradation, lipids control the intracellular traffic of apoB in enterocytes. A supply of fatty acids and cholesterol is sufficient for the export of apoB from the endoplasmic reticulum and its post-Golgi traffic up to the apical brush-border domain, where it remains until an apical supply of complex lipid micelles signals its chase down to the basolateral secretory domain. This downward traffic of apoB involves a microtubule-dependent process. Our results demonstrate an enterocyte-specific bidirectional process for the lipid-dependent traffic of a secretory protein.


Assuntos
Apolipoproteínas B/metabolismo , Colesterol/biossíntese , Enterócitos/metabolismo , Ácidos Graxos/biossíntese , Complexo de Golgi/metabolismo , Animais , Transporte Biológico/fisiologia , Células CACO-2 , Diferenciação Celular , Membrana Celular/metabolismo , Polaridade Celular , Retículo Endoplasmático/metabolismo , Humanos , Microscopia Confocal , Microscopia Eletrônica
8.
Front Physiol ; 8: 338, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28596739

RESUMO

Periodontitis is based on a complex inflammatory over-response combined with possible genetic predisposition factors. The RANKL/RANK/OPG signaling pathway is implicated in bone resorption through its key function in osteoclast differentiation and activation, as well as in the inflammatory response. This central element of osteo-immunology has been suggested to be perturbed in several diseases, including periodontitis, as it is a predisposing factor for this disease. The aim of the present study was to validate this hypothesis using a transgenic mouse line, which over-expresses RANK (RTg) and develops a periodontitis-like phenotype at 5 months of age. RTg mice exhibited severe alveolar bone loss, an increased number of TRAP positive cells, and disorganization of periodontal ligaments. This phenotype was more pronounced in females. We also observed dental root resorption lacunas. Hyperplasia of the gingival epithelium, including Malassez epithelial rests, was visible as early as 25 days, preceding any other symptoms. These results demonstrate that perturbations of the RANKL/RANK/OPG system constitute a core element of periodontitis, and more globally, osteo-immune diseases.

9.
Mol Metab ; 6(1): 61-72, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28123938

RESUMO

OBJECTIVE: Intestinal glucose absorption is orchestrated by specialized glucose transporters such as SGLT1 and GLUT2. However, the role of GLUT2 in the regulation of glucose absorption remains to be fully elucidated. METHODS: We wanted to evaluate the role of GLUT2 on glucose absorption and glucose homeostasis after intestinal-specific deletion of GLUT2 in mice (GLUT2ΔIEC mice). RESULTS: As anticipated, intestinal GLUT2 deletion provoked glucose malabsorption as visualized by the delay in the distribution of oral sugar in tissues. Consequences of intestinal GLUT2 deletion in GLUT2ΔIEC mice were limiting body weight gain despite normal food intake, improving glucose tolerance, and increasing ketone body production. These features were reminiscent of calorie restriction. Other adaptations to intestinal GLUT2 deletion were reduced microvillus length and altered gut microbiota composition, which was associated with improved inflammatory status. Moreover, a reduced density of glucagon-like peptide-1 (GLP-1) positive cells was compensated by increased GLP-1 content per L-cell, suggesting a preserved enteroendocrine function in GLUT2ΔIEC mice. CONCLUSIONS: Intestinal GLUT2 modulates glucose absorption and constitutes a control step for the distribution of dietary sugar to tissues. Consequently, metabolic and gut homeostasis are improved in the absence of functional GLUT2 in the intestine, thus mimicking calorie restriction.


Assuntos
Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Transportador de Glucose Tipo 2/metabolismo , Glucose/metabolismo , Animais , Glicemia/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/fisiologia , Homeostase , Absorção Intestinal , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Knockout , Transportador 1 de Glucose-Sódio/metabolismo , Distribuição Tecidual
10.
J Histochem Cytochem ; 50(10): 1417-20, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12364574

RESUMO

Thin layer-based technology in cervical cancer screening now allows both Papanicolaou staining and HPV testing on the same sample. Here, we show that in situ hybridization with catalyzed reporter deposition is a powerful HPV detection method when applied on thin-layer cervical smears, allowing distinction between two staining patterns suggestive of two different physical states of HPV DNA, where diffuse signals are suggestive of episomes and punctate signals are suggestive of viral DNA integration.


Assuntos
DNA Viral/análise , Papillomaviridae/genética , Neoplasias do Colo do Útero/diagnóstico , Feminino , Humanos , Hibridização In Situ/métodos , Teste de Papanicolaou , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Esfregaço Vaginal
11.
J Bone Miner Res ; 29(6): 1446-55, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24967458

RESUMO

Microarray analysis of odontoblastic cells treated with sodium fluoride has identified the asporin gene as a fluoride target. Asporin is a member of the small leucine-rich repeat proteoglycan/protein (SLRP) family that is believed to be important in the mineralization process. In this study, asporin expression and distribution were investigated by systematic analysis of dentin and enamel, with and without fluoride treatment. Specific attention was focused on a major difference between the two mineralized tissues: the presence of a collagenous scaffold in dentin, and its absence in enamel. Normal and fluorotic, continually growing incisors from Wistar rats treated with 2.5 to 7.5 mM sodium fluoride (NaF) were studied by immunochemistry, in situ hybridization, Western blotting, and RT-qPCR. Asporin was continuously expressed in odontoblasts throughout dentin formation as expected. Asporin was also found, for the first time, in dental epithelial cells, particularly in maturation-stage ameloblasts. NaF decreased asporin expression in odontoblasts and enhanced it in ameloblasts, both in vivo and in vitro. The inverse response in the two cell types suggests that the effector, fluoride, is a trigger that elicits a cell-type-specific reaction. Confocal and ultrastructural immunohistochemistry evidenced an association between asporin and type 1 collagen in the pericellular nonmineralized compartments of both bone and dentin. In addition, transmission electron microscopy revealed asporin in the microenvironment of all cells observed. Thus, asporin is produced by collagen-matrix-forming and non-collagen-matrix-forming cells but may have different effects on the mineralization process. A model is proposed that predicts impaired mineral formation associated with the deficiency and excess of asporin.


Assuntos
Calcificação Fisiológica/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Fluoreto de Sódio/farmacologia , Ameloblastos/efeitos dos fármacos , Ameloblastos/metabolismo , Animais , Linhagem Celular , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Proteínas da Matriz Extracelular/genética , Fluorose Dentária/genética , Fluorose Dentária/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Incisivo/efeitos dos fármacos , Incisivo/metabolismo , Incisivo/ultraestrutura , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Odontoblastos/efeitos dos fármacos , Odontoblastos/metabolismo , Odontoblastos/ultraestrutura , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar
12.
Mol Biol Cell ; 25(1): 118-32, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24173715

RESUMO

Enterocytes, the intestinal absorptive cells, have to deal with massive alimentary lipids upon food consumption. They orchestrate complex lipid-trafficking events that lead to the secretion of triglyceride-rich lipoproteins and/or the intracellular transient storage of lipids as lipid droplets (LDs). LDs originate from the endoplasmic reticulum (ER) membrane and are mainly composed of a triglyceride (TG) and cholesterol-ester core surrounded by a phospholipid and cholesterol monolayer and specific coat proteins. The pivotal role of LDs in cellular lipid homeostasis is clearly established, but processes regulating LD dynamics in enterocytes are poorly understood. Here we show that delivery of alimentary lipid micelles to polarized human enterocytes induces an immediate autophagic response, accompanied by phosphatidylinositol-3-phosphate appearance at the ER membrane. We observe a specific and rapid capture of newly synthesized LD at the ER membrane by nascent autophagosomal structures. By combining pharmacological and genetic approaches, we demonstrate that autophagy is a key player in TG targeting to lysosomes. Our results highlight the yet-unraveled role of autophagy in the regulation of TG distribution, trafficking, and turnover in human enterocytes.


Assuntos
Enterócitos/metabolismo , Membranas Intracelulares/metabolismo , Metabolismo dos Lipídeos , Fagossomos/fisiologia , Animais , Apolipoproteína A-I/metabolismo , Autofagia , Transporte Biológico , Células CACO-2 , Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Cinética , Lipoproteínas HDL/metabolismo , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia de Fluorescência , Fosfatos de Fosfatidilinositol/metabolismo
13.
J Clin Invest ; 122(6): 2239-51, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22565313

RESUMO

Intestinal barrier function requires intricate cooperation between intestinal epithelial cells and immune cells. Enteropathogens are able to invade the intestinal lymphoid tissue known as Peyer's patches (PPs) and disrupt the integrity of the intestinal barrier. However, the underlying molecular mechanisms of this process are poorly understood. In mice infected with Yersinia pseudotuberculosis, we found that PP barrier dysfunction is dependent on the Yersinia virulence plasmid and the expression of TLR-2 by hematopoietic cells, but not by intestinal epithelial cells. Upon TLR-2 stimulation, Y. pseudotuberculosis-infected monocytes activated caspase-1 and produced IL-1ß. In turn, IL-1ß increased NF-κB and myosin light chain kinase activation in intestinal epithelial cells, thus disrupting the intestinal barrier by opening the tight junctions. Therefore, Y. pseudotuberculosis subverts intestinal barrier function by altering the interplay between immune and epithelial cells during infection.


Assuntos
Células-Tronco Hematopoéticas/imunologia , Mucosa Intestinal/imunologia , Nódulos Linfáticos Agregados/imunologia , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/imunologia , Infecções por Yersinia pseudotuberculosis/imunologia , Yersinia pseudotuberculosis/imunologia , Animais , Células CACO-2 , Caspase 1/genética , Caspase 1/imunologia , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Células-Tronco Hematopoéticas/microbiologia , Células-Tronco Hematopoéticas/patologia , Humanos , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Monócitos/imunologia , Monócitos/microbiologia , Monócitos/patologia , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Nódulos Linfáticos Agregados/microbiologia , Nódulos Linfáticos Agregados/patologia , Transdução de Sinais/genética , Receptor 2 Toll-Like/genética , Yersinia pseudotuberculosis/genética , Infecções por Yersinia pseudotuberculosis/genética , Infecções por Yersinia pseudotuberculosis/patologia
14.
Autophagy ; 6(6): 754-63, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20574167

RESUMO

Adipose tissue lipoatrophy caused by caveolin gene deletion in mice is not linked to defective adipocyte differentiation. We show that adipose tissue development cannot be rescued by endothelial specific caveolin-1 re-expression, indicating primordial role of caveolin in mature adipocytes. Partial or total caveolin deficiency in adipocytes induced broad protein expression defects, including but not limited to previously described downregulation of insulin receptor. Global alterations in protein turnover, and accelerated degradation of long-lived proteins were found in caveolin-deficient adipocytes. Lipidation of endogenous LC3 autophagy marker and distribution of GFP-LC3 into aggregates demonstrated activated autophagy in the absence of caveolin-1 in adipocytes. Furthermore, electron microscopy revealed autophagic vacuoles in caveolin-1 deficient but not control adipocytes. Surprisingly, significant levels of lipidated LC3-II were found around lipid droplets of normal adipocytes, maintained in nutrient-rich conditions or isolated from fed mice, which do not display autophagy. Altogether, these data indicate that caveolin deficiency induce autophagy in adipocytes, a feature that is not a physiological response to fasting in normal fat cells. This likely resulted from defective insulin and lipolytic responses that converge in chronic nutrient shortage in adipocytes lacking caveolin-1. This is the first report of a pathological situation with autophagy as an adaptative response to adipocyte failure.


Assuntos
Adipócitos/citologia , Autofagia , Caveolina 1/deficiência , Diferenciação Celular , Metabolismo dos Lipídeos , Adipócitos/ultraestrutura , Animais , Caveolina 1/metabolismo , Células Cultivadas , Embrião de Mamíferos/citologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Inativação Gênica , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Animais , Modelos Biológicos , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Células Estromais/metabolismo , Fatores de Tempo
15.
PLoS One ; 4(1): e4278, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19169357

RESUMO

BACKGROUND: The intestine is responsible for absorbing dietary lipids and delivering them to the organism as triglyceride-rich lipoproteins (TRL). It is important to determine how this process is regulated in enterocytes, the absorptive cells of the intestine, as prolonged postprandial hypertriglyceridemia is a known risk factor for atherosclerosis. During the postprandial period, dietary lipids, mostly triglycerides (TG) hydrolyzed by pancreatic enzymes, are combined with bile products and reach the apical membrane of enterocytes as postprandial micelles (PPM). Our aim was to determine whether these micelles induce, in enterocytes, specific early cell signaling events that could control the processes leading to TRL secretion. METHODOLOGY/PRINCIPAL FINDINGS: The effects of supplying PPM to the apex of Caco-2/TC7 enterocytes were analyzed. Micelles devoid of TG hydrolysis products, like those present in the intestinal lumen in the interprandial period, were used as controls. The apical delivery of PPM specifically induced a number of cellular events that are not induced by interprandial micelles. These early events included the trafficking of apolipoprotein B, a structural component of TRL, from apical towards secretory domains, and the rapid, dose-dependent activation of ERK and p38MAPK. PPM supply induced the scavenger receptor SR-BI/CLA-1 to cluster at the apical brush border membrane and to move from non-raft to raft domains. Competition, inhibition or knockdown of SR-BI/CLA-1 impaired the PPM-dependent apoB trafficking and ERK activation. CONCLUSIONS/SIGNIFICANCE: These results are the first evidence that enterocytes specifically sense postprandial dietary lipid-containing micelles. SR-BI/CLA-1 is involved in this process and could be a target for further study with a view to modifying intestinal TRL secretion early in the control pathway.


Assuntos
Enterócitos/metabolismo , Lipídeos/química , Receptores Depuradores Classe B/metabolismo , Apolipoproteínas B/metabolismo , Bile/metabolismo , Células CACO-2 , Gorduras na Dieta/metabolismo , Humanos , Hidrólise , Metabolismo dos Lipídeos , Micelas , Modelos Biológicos , Fatores de Risco , Transdução de Sinais , Triglicerídeos/metabolismo
16.
J Lipid Res ; 48(10): 2151-61, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17652309

RESUMO

We investigated in vivo catabolism of apolipoprotein A-II (apo A-II), a major determinant of plasma HDL levels. Like apoA-I, murine apoA-II (mapoA-II) and human apoA-II (hapoA-II) were reabsorbed in the first segment of kidney proximal tubules of control and hapoA-II-transgenic mice, respectively. ApoA-II colocalized in brush border membranes with cubilin and megalin (the apoA-I receptor and coreceptor, respectively), with mapoA-I in intracellular vesicles of tubular epithelial cells, and was targeted to lysosomes, suggestive of degradation. By use of three transgenic lines with plasma hapoA-II concentrations ranging from normal to three times higher, we established an association between plasma concentration and renal catabolism of hapoA-II. HapoA-II was rapidly internalized in yolk sac epithelial cells expressing high levels of cubilin and megalin, colocalized with cubilin and megalin on the cell surface, and effectively competed with apoA-I for uptake, which was inhibitable by anti-cubilin antibodies. Kidney cortical cells that only express megalin internalized LDL but not apoA-II, apoA-I, or HDL, suggesting that megalin is not an apoA-II receptor. We show that apoA-II is efficiently reabsorbed in kidney proximal tubules in relation to its plasma concentration.


Assuntos
Apolipoproteína A-II/sangue , Apolipoproteína A-II/metabolismo , Rim/metabolismo , Animais , Apolipoproteínas/metabolismo , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Feminino , Humanos , Túbulos Renais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Metabolismo , Camundongos , Camundongos Transgênicos , Ratos , Receptores de Superfície Celular/metabolismo , Saco Vitelino/metabolismo
17.
J Cell Physiol ; 202(3): 767-76, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15389567

RESUMO

Intestinal triglyceride-rich lipoproteins (TRL) are synthesized from dietary lipids. This study was designed to evaluate the effects of lipid micelles, mimicking post-digestive duodenal micelles, on the fate of apolipoprotein B (apoB)48-containing lipoproteins by Caco-2 cells. Such micelles, consisting of oleic acid (OA), taurocholate, 2-monooleoylglycerol (2-MO), cholesterol (Chol), and L-alpha-lysophospatidylcholine, were the most efficient inducers of OA uptake and esterification. The efficiency of TG and apoB48 secretion increased specifically as a function of cell differentiation. PAGE analysis of secreted lipoproteins separated by sequential ultracentrifugation after [35S] labeling revealed differences in the secretion of apoB100- and apoB48-containing lipoproteins. In absence of micelles, apoB48 was secreted mostly in "HDL-like" particles, as observed in enterocytes in vivo. Micelle application increased 2.7-fold the secretion of apoB, resulting in 53 times more apoB48 being recovered as TG-enriched lipoproteins at d < 1.006 g/ml. Electron microscopy revealed the presence of lipid droplets in the secretory pathway and the accumulation of newly synthesized TG in cytoplasmic lipid droplets, as in enterocytes in vivo. We showed that these droplets could be used for secretion. However, apoB48 preferentially bound to newly synthesized TG in the presence of micelles, accounting in part for the functional advantage of apoB editing in the intestine. While Caco-2 cells express both apoB isoforms, our results show that the apical supply of complex lipid micelles favors the physiological route of apoB48-containing TG-enriched lipoproteins.


Assuntos
Apolipoproteínas B/metabolismo , Enterócitos/metabolismo , Metabolismo dos Lipídeos , Lipoproteínas/metabolismo , Micelas , Triglicerídeos/metabolismo , Apolipoproteína B-48 , Células CACO-2 , Humanos , Lipídeos/química , Lipoproteínas/química , Fatores de Tempo
18.
J Cell Sci ; 115(Pt 8): 1663-74, 2002 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-11950885

RESUMO

Two human Fzo-homologs, mitofusins Mfn1 and Mfn2, are shown by RT-PCR and western blot to be ubiquitous mitochondrial proteins. Protease digestion experiments reveal that Mfn2 is an outer membrane protein with N-terminal and C-terminal domains exposed towards the cytosol. The transmembrane and C-terminal domains of Mfn2 (Mfn2-TMCT) are targeted to mitochondria and deletion of these domains leads to the cytosolic localization of truncated Mfn2 (Mfn2-NT). Mfn2 is targeted to the endoplasmic reticulum or to mitochondria when the C-terminal domain is replaced by short stretches of neutral/hydrophobic (Mfn2-IYFFT) or polar/basic (Mfn2-RRD) amino acids. The coiled-coil domains of Mfn2, upstream and downstream of the transmembrane domain, are also important for mitochondrial targeting: Mfn2-mutants deleted of any of its coiled-coil domains are only partially targeted to mitochondria and significant protein amounts remain cytosolic. We show that these coiled-coil domains interact with each other: mistargeted Mfn2-NT or Mfn2-IYFFT localize to mitochondria if co-expressed with Mfn2-TMCT. This relocalization is abolished when the coiled-coil domain is deleted in any of the co-transfected molecules. We also found that Mfn2 can cluster active mitochondria in the perinuclear region independently of the cytoskeleton, bring mitochondrial membranes into close contact and modify mitochondrial structure, without disturbing the integrity of the inner and outer membrane.


Assuntos
Proteínas de Drosophila , GTP Fosfo-Hidrolases/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Estrutura Terciária de Proteína , Actinas/metabolismo , Animais , Fracionamento Celular , Citoesqueleto/metabolismo , Células HeLa , Humanos , Imuno-Histoquímica , Proteínas de Membrana/química , Proteínas de Membrana/classificação , Proteínas de Membrana Transportadoras , Microtúbulos/metabolismo , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial , Proteínas Mitocondriais/química , Proteínas Mitocondriais/classificação , Proteínas Mitocondriais/genética , Filogenia
19.
J Biol Chem ; 279(2): 1499-505, 2004 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-14576159

RESUMO

The physiological function of PrPc, the cellular isoform of prion protein, still remains unclear, although it has been established, in vitro or by using nerve cells, that it can homodimerize, bind copper, or interact with other proteins. Expression of PrPc was demonstrated as necessary for prion infection propagation. Considering the importance of the intestinal barrier in the process of oral prion infectivity, we have analyzed the expression of PrPc in enterocytes, which represent the major cell population of the intestinal epithelium. Our study, conducted both on normal human intestinal tissues and on the enterocytic cell line Caco-2/TC7, shows for the first time that PrPc is present in enterocytes. Interestingly, we found that this glycosylphosphatidylinositol-anchored glycoprotein was localized in cholesterol-dependent raft domains of the upper lateral membranes of enterocytes, beneath tight junctions, in cell-cell junctional domains. We observed that PrPc, E-cadherin, and Src co-localized in adherens junctions and that PrPc was co-immunoprecipitated with Src kinase but not with E-cadherin. Alteration of cell polarity after cholesterol depletion or loosening of the cell-cell junctions after EGTA treatment rapidly impaired membrane targeting of PrPc. Overall, our results point out the signaling of cell-cell contacts as a putative role for PrPc in epithelial cells.


Assuntos
Enterócitos/metabolismo , Proteínas PrPC/biossíntese , Proteínas PrPC/química , Junções Aderentes/metabolismo , Células CACO-2 , Caderinas/metabolismo , Linhagem Celular , Colesterol/metabolismo , Cobre/química , Dimerização , Células Epiteliais/metabolismo , Glicoproteínas/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Microscopia Confocal , Microscopia Imunoeletrônica , Testes de Precipitina , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Quinases da Família src/metabolismo
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