RESUMEN
Anterior gradient 2 (AGR2) is a dimeric protein disulfide isomerase family member involved in the regulation of protein quality control in the endoplasmic reticulum (ER). Mouse AGR2 deletion increases intestinal inflammation and promotes the development of inflammatory bowel disease (IBD). Although these biological effects are well established, the underlying molecular mechanisms of AGR2 function toward inflammation remain poorly defined. Here, using a protein-protein interaction screen to identify cellular regulators of AGR2 dimerization, we unveiled specific enhancers, including TMED2, and inhibitors of AGR2 dimerization, that control AGR2 functions. We demonstrate that modulation of AGR2 dimer formation, whether enhancing or inhibiting the process, yields pro-inflammatory phenotypes, through either autophagy-dependent processes or secretion of AGR2, respectively. We also demonstrate that in IBD and specifically in Crohn's disease, the levels of AGR2 dimerization modulators are selectively deregulated, and this correlates with severity of disease. Our study demonstrates that AGR2 dimers act as sensors of ER homeostasis which are disrupted upon ER stress and promote the secretion of AGR2 monomers. The latter might represent systemic alarm signals for pro-inflammatory responses.
Asunto(s)
Estrés del Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Mucoproteínas/metabolismo , Proteínas Oncogénicas/metabolismo , Multimerización de Proteína , Proteostasis , Animales , Retículo Endoplásmico/genética , Células HEK293 , Humanos , Masculino , Ratones , Mucoproteínas/genética , Proteínas Oncogénicas/genéticaRESUMEN
TMED2, a member of the transmembrane emp24 domain (TMED) family, is required for transport of cargo proteins between the ER and Golgi. TMED2 is also important for normal morphogenesis of mouse embryos and their associated placenta, and in fact Tmed2 homozygous mutant embryos arrest at mid-gestation due to a failure of placental labyrinth layer formation. Differentiation of the placental labyrinth layer depends on chorioallantoic attachment (contact between the chorion and allantois), and branching morphogenesis (mingling of cells from these two tissues). Since Tmed2 mRNA was found in both the chorion and allantois, and 50% of Tmed2 homozygous mutant embryos failed to undergo chorioallantoic attachment, the tissue-specific requirement of Tmed2 during placental labyrinth layer formation remained a mystery. Herein, we report differential localization of TMED2 protein in the chorion and allantois, abnormal ER retention of Fibronectin in Tmed2 homozygous mutant allantoises and cell-autonomous requirement for Tmed2 in the chorion for chorioallantoic attachment and fusion. Using an ex vivo model of explanted chorions and allantoises, we showed that chorioallantoic attachment failed to occur in 50% of samples when homozygous mutant chorions were recombined with wild type allantoises. Furthermore, though expression of genes associated with trophoblast differentiation was maintained in Tmed2 mutant chorions with chorioallantoic attachment, expression of these genes was attenuated. In addition, Tmed2 homozygous mutant allantoises could undergo branching morphogenesis, however the region of mixing between mutant and wild type cells was reduced, and expression of genes associated with trophoblast differentiation was also attenuated. Our data also suggest that Fibronectin is a cargo protein of TMED2 and indicates that Tmed2 is required cell-autonomously and non-autonomously in the chorion and the allantois for placental labyrinth layer formation.
Asunto(s)
Alantoides/metabolismo , Corion/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Diferenciación Celular/fisiología , Retículo Endoplásmico/metabolismo , Femenino , Fibronectinas/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Morfogénesis/fisiología , Placenta/metabolismo , Embarazo , Ratas , TrofoblastosRESUMEN
Background: Suboptimal folate intake, a risk factor for birth defects, is common even in areas with folate fortification. A polymorphism in methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), R653Q (MTHFD1 c.1958 G > A), has also been associated with increased birth defect risk, likely through reduced purine synthesis. Objective: We aimed to determine if the interaction of MTHFD1 synthetase deficiency and low folate intake increases developmental abnormalities in a mouse model for MTHFD1 R653Q. Methods: Female Mthfd1S+/+ and Mthfd1S+/- mice were fed control or low-folate diets (2 and 0.3 mg folic acid/kg diet, respectively) before mating and during pregnancy. Embryos and placentas were examined for anomalies at embryonic day 10.5. Maternal 1-carbon metabolites were measured in plasma and liver. Results: Delays and defects doubled in litters of Mthfd1S+/- females fed low-folate diets compared to wild-type females fed either diet, or Mthfd1S+/- females fed control diets [P values (defects): diet 0.003, maternal genotype 0.012, diet × maternal genotype 0.014]. These adverse outcomes were associated with placental dysmorphology. Intrauterine growth restriction was increased by embryonic Mthfd1S+/- genotype, folate deficiency, and interaction of maternal Mthfd1S+/- genotype with folate deficiency (P values: embryonic genotype 0.045, diet 0.0081, diet × maternal genotype 0.0019). Despite a 50% increase in methylenetetrahydrofolate reductase expression in low-folate maternal liver (P diet = 0.0007), methyltetrahydrofolate concentration decreased 70% (P diet <0.0001) and homocysteine concentration doubled in plasma (P diet = 0.0001); S-adenosylmethionine decreased 40% and S-adenosylhomocysteine increased 20% in low-folate maternal liver (P diet = 0.002 and 0.0002, respectively). Conclusions: MTHFD1 synthetase-deficient mice are more sensitive to low folate intake than wild-type mice during pregnancy. Reduced purine synthesis due to synthetase deficiency and altered methylation potential due to low folate may increase pregnancy complications. Further studies and individualized intake recommendations may be required for women homozygous for the MTHFD1 R653Q variant.
Asunto(s)
Anomalías Congénitas/etiología , Deficiencia de Ácido Fólico/complicaciones , Ácido Fólico/administración & dosificación , Formiato-Tetrahidrofolato Ligasa/deficiencia , Genotipo , Meteniltetrahidrofolato Ciclohidrolasa/deficiencia , Metilenotetrahidrofolato Deshidrogenasa (NADP)/deficiencia , Enzimas Multifuncionales/deficiencia , Polimorfismo Genético , Complicaciones del Embarazo/etiología , Animales , Metilación de ADN , Dieta , Modelos Animales de Enfermedad , Femenino , Desarrollo Fetal , Retardo del Crecimiento Fetal/etiología , Ácido Fólico/sangre , Deficiencia de Ácido Fólico/sangre , Deficiencia de Ácido Fólico/genética , Deficiencia de Ácido Fólico/metabolismo , Formiato-Tetrahidrofolato Ligasa/genética , Formiato-Tetrahidrofolato Ligasa/metabolismo , Ligasas , Hígado/metabolismo , Meteniltetrahidrofolato Ciclohidrolasa/genética , Meteniltetrahidrofolato Ciclohidrolasa/metabolismo , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Metilenotetrahidrofolato Reductasa (NADPH2)/metabolismo , Ratones , Enzimas Multifuncionales/genética , Enzimas Multifuncionales/metabolismo , Placenta , Embarazo , Complicaciones del Embarazo/sangre , Complicaciones del Embarazo/genética , Complicaciones del Embarazo/metabolismo , Preñez , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Tetrahidrofolatos/sangreRESUMEN
The transmembrane emp24 domain/p24 (TMED) family are essential components of the vesicular transport machinery. Members of the TMED family serve as cargo receptors implicated in selection and packaging of endoplasmic reticulum (ER) luminal proteins into coatomer (COP) II coated vesicles for anterograde transport to the Golgi. Deletion or mutations of Tmed genes in yeast and Drosophila results in ER-stress and activation of the unfolded protein response (UPR). The UPR leads to expression of genes and proteins important for expanding the folding capacity of the ER, degrading misfolded proteins, and reducing the load of new proteins entering the ER. The UPR is activated in non-alcoholic fatty liver disease (NAFLD) in human and mouse and may contribute to the development and the progression of NAFLD. Tmed2, the sole member of the vertebrate Tmed ß subfamily, exhibits tissue and temporal specific patterns of expression in embryos and developing placenta but is ubiquitously expressed in all adult organs. We previously identified a single point mutation, the 99J mutation, in the signal sequence of Tmed2 in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Histological and molecular analysis of livers from heterozygous mice carrying the 99J mutation, Tmed299J/+, revealed a requirement for TMED2 in liver health. We show that Tmed299J/+ mice had decreased levels of TMED2 and TMED10, dilated endoplasmic reticulum membrane, and increased phosphorylation of eIF2α, indicating ER-stress and activation of the UPR. Increased expression of Srebp1a and 2 at the newborn stage and increased incidence of NAFLD were also found in Tmed299J/+ mice. Our data establishes Tmed299J/+ mice as a novel mouse model for NAFLD and supports a role for TMED2 in liver health.
Asunto(s)
Hígado/patología , Enfermedad del Hígado Graso no Alcohólico/genética , Mutación Puntual , Proteínas de Transporte Vesicular/genética , Animales , Estrés del Retículo Endoplásmico , Células Hep G2 , Heterocigoto , Humanos , Hígado/metabolismo , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/análisis , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/análisis , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Respuesta de Proteína Desplegada , Regulación hacia Arriba , Proteínas de Transporte Vesicular/análisisRESUMEN
BACKGROUND: Moderately high folic acid intake in pregnant women has led to concerns about deleterious effects on the mother and fetus. Common polymorphisms in folate genes, such as methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (MTHFD1) R653Q, may modulate the effects of elevated folic acid intake. OBJECTIVES: We investigated the effects of moderate folic acid supplementation on reproductive outcomes and assessed the potential interaction of the supplemented diet with MTHFD1-synthetase (Mthfd1S) deficiency in mice, which is a model for the R653Q variant. DESIGN: Female Mthfd1S+/+ and Mthfd1S+/- mice were fed a folic acid-supplemented diet (FASD) (5-fold higher than recommended) or control diets before mating and during pregnancy. Embryos and placentas were assessed for developmental defects at embryonic day 10.5 (E10.5). Maternal folate and choline metabolites and gene expression in folate-related pathways were examined. RESULTS: The combination of FASD and maternal MTHFD1-synthetase deficiency led to a greater incidence of defects in E10.5 embryos (diet × maternal genotype, P = 0.0016; diet × embryonic genotype, P = 0.054). The methylenetetrahydrofolate reductase (MTHFR) protein and methylation potential [ratio of S-adenosylmethionine (major methyl donor):S-adenosylhomocysteine) were reduced in maternal liver. Although 5-methyltetrahydrofolate (methylTHF) was higher in maternal circulation, the methylation potential was lower in embryos. The presence of developmental delays and defects in Mthfd1S+/- embryos was associated with placental defects (P = 0.003). The labyrinth layer failed to form properly in the majority of abnormal placentas, which compromised the integration of the maternal and fetal circulation and presumably the transfer of methylTHF and other nutrients. CONCLUSIONS: Moderately higher folate intake and MTHFD1-synthetase deficiency in pregnant mice result in a lower methylation potential in maternal liver and embryos and a greater incidence of defects in embryos. Although maternal circulating methylTHF was higher, it may not have reached the embryos because of abnormal placental development; abnormal placentas were observed predominantly in abnormally developed embryos. These findings have implications for women with high folate intakes, particularly if they are polymorphic for MTHFD1 R653Q.
Asunto(s)
Aminohidrolasas/deficiencia , Aminohidrolasas/genética , Ácido Fólico/farmacología , Formiato-Tetrahidrofolato Ligasa/deficiencia , Formiato-Tetrahidrofolato Ligasa/genética , Metilenotetrahidrofolato Deshidrogenasa (NADP)/deficiencia , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Complejos Multienzimáticos/deficiencia , Complejos Multienzimáticos/genética , Placenta/anomalías , Placenta/enzimología , Polimorfismo de Nucleótido Simple , Aminohidrolasas/metabolismo , Animales , Colina/farmacología , Suplementos Dietéticos , Embrión de Mamíferos/enzimología , Desarrollo Embrionario/efectos de los fármacos , Femenino , Formiato-Tetrahidrofolato Ligasa/metabolismo , Modelos Logísticos , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/metabolismo , Ratones , Ratones Transgénicos , Complejos Multienzimáticos/metabolismo , Embarazo , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismoRESUMEN
INTRODUCTION: Chorioallantoic fusion is essential for development of the labyrinth layer of the mouse placenta. However, events that occur after chorioallantoic attachment remain poorly described, partly due to difficulties of conducting ex vivo analysis of the placenta. Herein, we report conditions for ex vivo culture of the developing murine placenta. METHODS: Mesometrial halves of decidua containing pre-attachment chorions were cultured alone or with explants of allantoides from stage-matched controls and analyzed by confocal and immunofluorescence microscopy. Expression and levels of marker genes associated with specific placental cell types were measured by in situ hybridization and qRT-PCR, respectively. RESULTS: After 24 h (hr) of co-culture, a mosaic pattern of eGFP+ and eGFP- cells were found when explants of pre-attachment chorions from eGFP+ embryos were co-cultured with stage-matched allantoides from eGFP- embryos or vice versa. In addition, proliferation increased in the allantoic region and folds formed on the chorionic plate. PECAM positive cells derived from the allantois were found in the chorionic region. Levels of the SynT-II marker, Gcm1, significantly increased at 24 h, although expression of Gcm1, was only found in explants co-cultured with an allantois at 12 h and 24 h. In addition, though levels of Tpbpα was not altered by co-culture with an allantois, Tpbpα was only detected in explants co-cultured with an allantois for 24 h. DISCUSSION: Our data show that chorioallantoic fusion and events associated with initiation of labyrinth layer formation can be modeled ex vivo, and reveal a previously unsuspected requirement of chorioallantoic fusion for Tpbpα expression.
Asunto(s)
Alantoides/metabolismo , Corion/metabolismo , Neuropéptidos/metabolismo , Placenta/metabolismo , Proteínas Gestacionales/metabolismo , Alantoides/citología , Animales , Corion/citología , Técnicas de Cocultivo , Proteínas de Unión al ADN , Femenino , Ratones , Neuropéptidos/genética , Placenta/citología , Placentación/fisiología , Embarazo , Proteínas Gestacionales/genética , Factores de TranscripciónRESUMEN
Extracellular vesicles (EVs) enable the exit of regulatory, mutant and oncogenic macromolecules (proteins, RNA and DNA) from their parental tumor cells and uptake of this material by unrelated cellular populations. Among the resulting biological effects of interest is the notion that cancer-derived EVs may mediate horizontal transformation of normal cells through transfer of mutant genes, including mutant ras. Here, we report that H-ras-mediated transformation of intestinal epithelial cells (IEC-18) results in the emission of exosome-like EVs containing genomic DNA, HRAS oncoprotein and transcript. However, EV-mediated horizontal transformation of non-transformed cells (epithelial, astrocytic, fibroblastic and endothelial) is transient, limited or absent due to barrier mechanisms that curtail the uptake, retention and function of oncogenic H-ras in recipient cells. Thus, epithelial cells and astrocytes are resistant to EV uptake, unless they undergo malignant transformation. In contrast, primary and immortalized fibroblasts are susceptible to the EV uptake, retention of H-ras DNA and phenotypic transformation, but these effects are transient and fail to produce a permanent tumorigenic conversion of these cells in vitro and in vivo, even after several months of observation. Increased exposure to EVs isolated from H-ras-transformed cancer cells, but not to those from their indolent counterparts, triggers demise of recipient fibroblasts. Uptake of H-ras-containing EVs stimulates but fails to transform primary endothelial cells. Thus, we suggest that intercellular transfer of oncogenes exerts regulatory rather than transforming influence on recipient cells, while cancer cells may often act as preferential EV recipients.