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1.
Development ; 143(22): 4236-4248, 2016 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-27729410

RESUMEN

Profiling of RNA from mouse mammary epithelial cells (MECs) isolated on pregnancy day (P)14 and lactation day (L)2 revealed that the majority of differentially expressed microRNA declined precipitously between late pregnancy and lactation. The decline in miR-150, which exhibited the greatest fold-decrease, was verified quantitatively and qualitatively. To test the hypothesis that the decline in miR-150 is crucial for lactation, MEC-specific constitutive miR-150 was achieved by crossing ROSA26-lox-STOP-lox-miR-150 mice with WAP-driven Cre recombinase mice. Both biological and foster pups nursed by bitransgenic dams exhibited a dramatic decrease in survival compared with offspring nursed by littermate control dams. Protein products of predicted miR-150 targets Fasn, Olah, Acaca, and Stat5B were significantly suppressed in MECs of bitransgenic mice with constitutive miR-150 expression as compared with control mice at L2. Lipid profiling revealed a significant reduction in fatty acids synthesized by the de novo pathway in L2 MECs of bitransgenic versus control mice. Collectively, these data support the hypothesis that a synchronized decrease in miRNAs, such as miR-150, at late pregnancy serves to allow translation of targets crucial for lactation.


Asunto(s)
Lactancia/genética , Lipogénesis/genética , Glándulas Mamarias Animales/metabolismo , MicroARNs/genética , Animales , Células Cultivadas , Regulación hacia Abajo/genética , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Lactancia/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/metabolismo , Análisis por Micromatrices , Embarazo/genética , Embarazo/metabolismo
2.
Am J Physiol Regul Integr Comp Physiol ; 317(5): R684-R695, 2019 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-31553623

RESUMEN

Exercise is a potent facilitator of long-term weight loss maintenance (WLM), whereby it decreases appetite and increases energy expenditure beyond the cost of the exercise bout. We have previously shown that exercise may amplify energy expenditure through energetically expensive nutrient deposition. Therefore, we investigated the effect of exercise on hepatic de novo lipogenesis (DNL) during WLM and relapse to obesity. Obese rats were calorically restricted with (EX) or without (SED) treadmill exercise (1 h/day, 6 days/wk, 15 m/min) to induce and maintain weight loss. After 6 wk of WLM, subsets of WLM-SED and WLM-EX rats were allowed ad libitum access to food for 1 day to promote relapse (REL). An energy gap-matched group of sedentary, relapsing rats (REL-GM) were provided a diet matched to the positive energy imbalance of the REL-EX rats. During relapse, exercise increased enrichment of hepatic DN-derived lipids and induced hepatic molecular adaptations favoring DNL compared with the gap-matched controls. In the liver, compared with both REL-SED and REL-GM rats, REL-EX rats had lower hepatic expression of genes required for cholesterol biosynthesis; greater hepatic expression of genes that mediate very low-density lipoprotein synthesis and secretion; and greater mRNA expression of Cyp27a1, which encodes an enzyme involved in the biosynthesis of bile acids. Altogether, these data provide compelling evidence that the liver has an active role in exercise-mediated potentiation of energy expenditure during early relapse.


Asunto(s)
Colesterol/biosíntesis , Metabolismo Energético , Lipogénesis , Hígado/metabolismo , Obesidad/terapia , Condicionamiento Físico Animal , Aumento de Peso , Pérdida de Peso , Animales , Ácidos y Sales Biliares/biosíntesis , Restricción Calórica , Modelos Animales de Enfermedad , Metabolismo Energético/genética , Regulación Enzimológica de la Expresión Génica , Insulina/sangre , Lipogénesis/genética , Masculino , Obesidad/genética , Obesidad/metabolismo , Obesidad/fisiopatología , Recurrencia , Carrera , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
3.
BMC Cancer ; 16(1): 788, 2016 10 11.
Artículo en Inglés | MEDLINE | ID: mdl-27724921

RESUMEN

BACKGROUND: Claudin-4 is a transmembrane protein expressed at high levels in the majority of epithelial ovarian tumors, irrespective of subtype, and has been associated with tumor cells that are both chemoresistant and highly mobile. The objective of this study was to determine the functional role that claudin-4 plays in apoptosis resistance and migration as well as the therapeutic utility of targeting claudin-4 activity with a small mimic peptide. METHODS: We examined claudin-4 activity in human ovarian tumor cell lines (SKOV3, OVCAR3, PEO4) using in vitro caspase and scratch assays as well as an in vivo mouse model of ovarian cancer. Claudin-4 activity was disrupted by treating cells with a small peptide that mimics the DFYNP sequence in the second extracellular loop of claudin-4. Claudin-4 expression was also altered using shRNA-mediated gene silencing. RESULTS: Both the disruption of claudin-4 activity and the loss of claudin-4 expression significantly increased tumor cell caspase-3 activation (4 to 10-fold, respectively) in response to the apoptotic inducer staurosporine and reduced tumor cell migration by 50 %. The mimic peptide had no effect on cells that lacked claudin-4 expression. Female athymic nude mice bearing ZsGreen-PEO4 ovarian tumors showed a significant decrease in ovarian tumor burden, due to increased apoptosis, after treatment with intraperitoneal injections of 4 mg/kg mimic peptide every 48 h for three weeks, compared to control peptide treated mice. CONCLUSION: Claudin-4 functionally contributes to both ovarian tumor cell apoptosis resistance and migration and targeting extracellular loop interactions of claudin-4 may have therapeutic implications for reducing ovarian tumor burden.


Asunto(s)
Apoptosis/genética , Movimiento Celular/genética , Claudina-4/genética , Neoplasias Ováricas/genética , Animales , Caspasa 3/metabolismo , Línea Celular Tumoral , Supervivencia Celular/genética , Claudina-4/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Inmunohistoquímica , Ratones , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Interferencia de ARN , ARN Interferente Pequeño/genética , Carga Tumoral
4.
BMC Cell Biol ; 14: 19, 2013 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-23521713

RESUMEN

BACKGROUND: Claudins are key integral proteins of the tight junction. Although they play an essential role in controlling paracellular diffusion in epithelia, increasing evidence supports a role for these proteins in non-barrier forming activities. To elucidate a potential function for claudins outside of their traditional role in tight junctions, subcellular localization of claudin-4 was determined in normal mammary epithelial cells as well as breast and ovarian cancer cell lines and the effects of a claudin mimic peptide on cell motility were determined. RESULTS: Immunofluorescence revealed that claudin-4 was localized along cellular projections. Using a fluorescent peptide that mimics a conserved sequence in the second extracellular loop of a set of claudin subtypes, that includes claudin-4, exposure of this loop to the extracellular environment was confirmed in non-polarized cells. This peptide inhibited cell motility when normal mammary epithelial cells as well as breast and ovarian tumor cells were subjected to a wound healing assay. Knockdown of claudin-4 also inhibited cell motility and the mimic peptide had no effect on motility in the claudin-4 knockdown cells. This effect on motility was seen when cells were grown on collagen, but not when cells were grown on non-physiological cell adhesive or fibronectin. CONCLUSION: The second extracellular loop of claudins is able to interact with the extracellular environment to promote normal and tumor cell motility when it is not associated with tight junction structures.


Asunto(s)
Claudina-4/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Claudina-4/antagonistas & inhibidores , Claudina-4/genética , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Células MCF-7 , Péptidos/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo
5.
iScience ; 26(1): 105750, 2023 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-36590177

RESUMEN

Establishing metabolic programming begins during fetal and postnatal development, and early-life lipid exposures play a critical role during neonatal adipogenesis. We define how neonatal consumption of a low omega-6 to -3 fatty acid ratio (n6/n3 FA ratio) establishes FA oxidation in adipocyte precursor cells (APCs) before they become adipocytes. In vivo, APCs isolated from mouse pups exposed to the low n6/n3 FA ratio had superior FA oxidation capacity, elevated beige adipocyte mRNAs Ppargc1α, Ucp2, and Runx1, and increased nuclear receptor NR2F2 protein. In vitro, APC treatment with NR2F2 ligand-induced beige adipocyte mRNAs and increased mitochondrial potential but not mass. Single-cell RNA-sequencing analysis revealed low n6/n3 FA ratio yielded more mitochondrial-high APCs and linked APC NR2F2 levels with beige adipocyte signatures and FA oxidation. Establishing beige adipogenesis is of clinical relevance, because fat depots with energetically active, smaller, and more numerous adipocytes improve metabolism and delay metabolic dysfunction.

6.
Heliyon ; 8(10): e10862, 2022 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-36237976

RESUMEN

Claudin-4, a protein with the structure of classic claudins most often found in cell-cell junctions, is frequently overexpressed in epithelial cancers where its localization has not been studied. In this study we aimed to find out where this membrane protein is localized in an ovarian tumor model, OVCAR3 cells, that express high levels of the protein. Immunohistochemical studies showed claudin-4 staining in a perinuclear region, at most plasma membranes and in cytoplasmic puncta. Native claudin-4 did not overlap with phosphorylated claudin-4, which was partially located in focal adhesions. Using claudin-4 BioID technology we confirmed that large amounts of claudin-4 are localized to the Golgi compartment, including in dispersed Golgi in cells where claudin-4 is partially knocked down and in dividing cells. Claudin-4 appears to be present in the vicinity of several types of cell-cell junctions, but there is no evidence that it forms tight junctions in these tumor cells. Both claudin-4, the Golgi marker GM130, and the plasma membrane receptor Notch2 were found in dispersed Golgi in dividing cells. This definition of the cellular architecture of claudin-4 should provide a framework for better understanding of the function of claudin-4 in tumor cells and its molecular interactions.

7.
Mol Cancer Ther ; 21(4): 647-657, 2022 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-35373300

RESUMEN

High-grade serous ovarian cancer is the deadliest gynecologic malignancy due to progression to resistant disease. Claudin-4 is classically defined as a tight junction protein and is often associated with epithelial cancers. Claudin-4 is aberrantly expressed in nearly 70% of all ovarian cancer tumors and conveys a worse overall prognosis. Elevated claudin-4 expression correlates to increased DNA repair activity and resistance to DNA damaging agents. PARP inhibitors are emerging as an effective therapeutic option for patients with ovarian cancer and function by promoting DNA damage. The study examines the relationship between claudin-4 expression and the response to PARP inhibitors using both genetic and pharmacologic inhibition of claudin-4 in in vitro and ex vivo models of ovarian cancer to examine DNA repair markers and functional activity. Genetic inhibition of claudin-4 results in the downregulation of several DNA damage repair effectors, including 53BP1 and XRCC1. Claudin-4 knockdown did not change homology-directed repair but inhibited nonhomologous end-joining and reduced 53BP1 foci formation. In 15 primary ovarian cancer tumors, higher claudin-4 expression significantly correlated to a dampened PARP inhibitor-mediated antiproliferation response. Further, claudin-4 inhibition in high claudin-4 tumors sensitized tumor sections to PARP inhibition. These data highlight that claudin-4 expression in ovarian cancer tumors could serve as both a marker of PARP inhibitor response and a therapeutic target to improve PARP inhibitor response.


Asunto(s)
Neoplasias Ováricas , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Claudina-4/genética , Daño del ADN , Reparación del ADN , Femenino , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética
8.
BMC Cell Biol ; 10: 85, 2009 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-20003227

RESUMEN

BACKGROUND: Occludin is a tetraspanin protein normally localized to tight junctions. The protein interacts with a variety of pathogens including viruses and bacteria, an interaction that sometimes leads to its extrajunctional localization. RESULTS: Here we report that treatment of mammary epithelial monolayers with a circularized peptide containing a four amino acid sequence found in the second extracellular loop of occludin, LHYH, leads to the appearance of extrajunctional occludin and activation of the extrinsic apoptotic pathway. At early times after peptide treatment endogenous occludin and the LYHY peptide were co-localized in extrajunctional patches, which were also shown to contain components of the death inducing signaling complex (DISC), caspases 8 and 3, the death receptor FAS and the adaptor molecule FADD. After this treatment occludin could be immunoprecipitated with FADD, confirming its interaction with the DISC. Extrusion after LYHY treatment was accomplished with no loss of epithelial resistance. CONCLUSION: These observations provide strong evidence that, following disruption, occludin forms a complex with the extrinsic death receptor leading to extrusion of apoptotic cells from the epithelial monolayer. They suggest that occludin has a protective as well as a barrier forming role in epithelia; pathogenic agents which utilize this protein as an entry point into the cell might set off an apoptotic reaction allowing extrusion of the infected cell before the pathogen can gain entry to the interstitial space.


Asunto(s)
Apoptosis , Movimiento Celular , Polaridad Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Caspasas/metabolismo , Línea Celular , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Ocludina , Unión Proteica , Receptores de Muerte Celular/metabolismo , Uniones Estrechas/metabolismo
9.
Mol Cancer Res ; 17(3): 741-750, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30606772

RESUMEN

A significant factor contributing to poor survival rates for patients with ovarian cancer is the insensitivity of tumors to standard-of-care chemotherapy. In this study, we investigated the effect of claudin-4 expression on ovarian tumor cell apoptotic response to cisplatin and paclitaxel. We manipulated claudin-4 gene expression by silencing expression [short hairpin RNA (shRNA)] in cells with endogenously expressed claudin-4 or overexpressing claudin-4 in cells that natively do not express claudin-4. In addition, we inhibited claudin-4 activity with a claudin mimic peptide (CMP). We monitored apoptotic response by caspase-3 and Annexin V binding. We examined proliferation rate by counting the cell number over time as well as measuring the number of mitotic cells. Proximity ligation assays, immunoprecipitation (IP), and immunofluorescence were performed to examine interactions of claudin-4. Western blot analysis of tubulin in cell fractions was used to determine the changes in tubulin polymerization with changes in claudin-4 expression. Results show that claudin-4 expression reduced epithelial ovarian cancer (EOC) cell apoptotic response to paclitaxel. EOCs without claudin-4 proliferated more slowly with enhanced mitotic arrest compared with the cells expressing claudin-4. Furthermore, our results indicate that claudin-4 interacts with tubulin, having a profound effect on the structure and polymerization of the microtubule network. In conclusion, we demonstrate that claudin-4 reduces the ovarian tumor cell response to microtubule-targeting paclitaxel and disrupting claudin-4 with CMP can restore apoptotic response. IMPLICATIONS: These results suggest that claudin-4 expression may provide a biomarker for paclitaxel response and can be a target for new therapeutic strategies to improve response.


Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Claudina-4/metabolismo , Paclitaxel/uso terapéutico , Antineoplásicos Fitogénicos/farmacología , Apoptosis , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Paclitaxel/farmacología
10.
Diabetes ; 67(4): 651-661, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29138256

RESUMEN

Adipose tissue expansion progresses rapidly during postnatal life, influenced by both prenatal maternal factors and postnatal developmental cues. The ratio of omega-6 (n-6) relative to n-3 polyunsaturated fatty acids (PUFAs) is believed to regulate perinatal adipogenesis, but the cellular mechanisms and long-term effects are not well understood. We lowered the fetal and postnatal n-6/n-3 PUFA ratio exposure in wild-type offspring under standard maternal dietary fat amounts to test the effects of low n-6/n-3 ratios on offspring adipogenesis and adipogenic potential. Relative to wild-type pups receiving high perinatal n-6/n-3 ratios, subcutaneous adipose tissue in 14-day-old wild-type pups receiving low n-6/n-3 ratios had more adipocytes that were smaller in size; decreased Pparγ2, Fabp4, and Plin1; several lipid metabolism mRNAs; coincident hypermethylation of the PPARγ2 proximal promoter; and elevated circulating adiponectin. As adults, offspring that received low perinatal n-6/n-3 ratios were diet-induced obesity (DIO) resistant and had a lower positive energy balance and energy intake, greater lipid fuel preference and non-resting energy expenditure, one-half the body fat, and better glucose clearance. Together, the findings support a model in which low early-life n-6/n-3 ratios remodel adipose morphology to increase circulating adiponectin, resulting in a persistent adult phenotype with improved metabolic flexibility that prevents DIO.


Asunto(s)
Adipogénesis , Glucemia/metabolismo , Ácidos Grasos Omega-3/sangre , Ácidos Grasos Omega-6/sangre , Metabolismo de los Lípidos , Obesidad/epidemiología , Efectos Tardíos de la Exposición Prenatal/epidemiología , Adipocitos/citología , Adiponectina/metabolismo , Animales , Animales Recién Nacidos , Proliferación Celular , Tamaño de la Célula , Metilación de ADN , Dieta Alta en Grasa , Grasas de la Dieta , Ingestión de Energía , Metabolismo Energético , Proteínas de Unión a Ácidos Grasos/metabolismo , Femenino , Ratones , Obesidad/sangre , PPAR gamma/metabolismo , Perilipina-1/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal/sangre , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Factores de Riesgo
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