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PURPOSE: Over 550 loci have been associated with human pulmonary function in genome-wide association studies (GWAS); however, the causal role of most remains uncertain. Single nucleotide polymorphisms in a disintegrin and metalloprotease domain 19 (ADAM19) are consistently related to pulmonary function in GWAS. Thus, we used a mouse model to investigate the causal link between Adam19 and pulmonary function. METHODS: We created an Adam19 knockout (KO) mouse model and validated the gene targeting using RNA-Seq and RT-qPCR. Mouse body composition was assessed using dual-energy X-ray absorptiometry. Mouse lung function was measured using flexiVent. RESULTS: Contrary to prior publications, the KO was not neonatal lethal. KO mice had lower body weight and shorter tibial length than wild-type (WT) mice. Their body composition revealed lower soft weight, fat weight, and bone mineral content. Adam19 KO had decreased baseline respiratory system elastance, minute work of breathing, tissue damping, tissue elastance, and forced expiratory flow at 50% forced vital capacity but higher FEV0.1 and FVC. Adam19 KO had attenuated tissue damping and tissue elastance in response to methacholine following LPS exposure. Adam19 KO also exhibited attenuated neutrophil extravasation into the airway after LPS administration compared to WT. RNA-Seq analysis of KO and WT lungs identified several differentially expressed genes (Cd300lg, Kpna2, and Pttg1) implicated in lung biology and pathogenesis. Gene set enrichment analysis identified negative enrichment for TNF pathways. CONCLUSION: Our murine findings support a causal role of ADAM19, implicated in human GWAS, in regulating pulmonary function.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection involves an initial viral infection phase followed by a host-response phase that includes an eicosanoid and cytokine storm, lung inflammation and respiratory failure. While vaccination and early anti-viral therapies are effective in preventing or limiting the pathogenic host response, this latter phase is poorly understood with no highly effective treatment options. Inhibitors of soluble epoxide hydrolase (sEH) increase levels of anti-inflammatory molecules called epoxyeicosatrienoic acids (EETs). This study aimed to investigate the impact of sEH inhibition on the host response to SARS-CoV-2 infection in a mouse model with human angiotensin-converting enzyme 2 (ACE2) expression. Mice were infected with SARS-CoV-2 and treated with either vehicle or the sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU). At day 5 post-infection, SARS-CoV-2 induced weight loss, clinical signs, a cytokine storm, an eicosanoid storm, and severe lung inflammation with ~50% mortality on days 6-8 post-infection. SARS-CoV-2 infection induced lung expression of phospholipase A2 (PLA2), cyclooxygenase (COX) and lipoxygenase (LOX) pathway genes, while suppressing expression of most cytochrome P450 genes. Treatment with the sEH inhibitor TPPU delayed weight loss but did not alter clinical signs, lung cytokine expression or overall survival of infected mice. Interestingly, TPPU treatment significantly reversed the eicosanoid storm and attenuated viral-induced elevation of 39 fatty acids and oxylipins from COX, LOX and P450 pathways, which suggests the effects at the level of PLA2 activation. The suppression of the eicosanoid storm by TPPU without corresponding changes in lung cytokines, lung inflammation or mortality reveals a surprising dissociation between systemic oxylipin and cytokine signaling pathways during SARS-CoV-2 infection and suggests that the cytokine storm is primarily responsible for morbidity and mortality in this animal model.
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Tratamiento Farmacológico de COVID-19 , COVID-19 , Síndrome de Liberación de Citoquinas , Eicosanoides , Epóxido Hidrolasas , SARS-CoV-2 , Animales , Ratones , Eicosanoides/metabolismo , COVID-19/inmunología , COVID-19/virología , COVID-19/metabolismo , SARS-CoV-2/efectos de los fármacos , Epóxido Hidrolasas/antagonistas & inhibidores , Epóxido Hidrolasas/metabolismo , Síndrome de Liberación de Citoquinas/tratamiento farmacológico , Piperidinas/farmacología , Piperidinas/uso terapéutico , Citocinas/metabolismo , Humanos , Pulmón/virología , Pulmón/metabolismo , Pulmón/patología , Pulmón/efectos de los fármacos , Enzima Convertidora de Angiotensina 2/metabolismo , Modelos Animales de Enfermedad , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/uso terapéutico , FemeninoRESUMEN
Purpose: Over 550 loci have been associated with human pulmonary function in genome-wide association studies (GWAS); however, the causal role of most remains uncertain. Single nucleotide polymorphisms in a disintegrin and metalloprotease domain 19 (ADAM19) are consistently related to pulmonary function in GWAS. Thus, we used a mouse model to investigate the causal link between Adam19 and pulmonary function. Methods: We created an Adam19 knockout (KO) mouse model and validated the gene targeting using RNA-Seq and RT-qPCR. Contrary to prior publications, the KO was not neonatal lethal. Thus, we phenotyped the Adam19 KO. Results: KO mice had lower body weight and shorter tibial length than wild type (WT). Dual-energy X-ray Absorptiometry indicated lower soft weight, fat weight, and bone mineral content in KO mice. In lung function analyses using flexiVent, compared to WT, Adam19 KO had decreased baseline respiratory system elastance, minute work of breathing, tissue damping, tissue elastance, and forced expiratory flow at 50% forced vital capacity but higher FEV0.1 and FVC. Adam19 KO had attenuated tissue damping and tissue elastance in response to methacholine following LPS exposure. Adam19 KO also exhibited attenuated neutrophil extravasation into the airway after LPS administration compared to WT. RNA-Seq analysis of KO and WT lungs identified several differentially expressed genes (Cd300lg, Kpna2, and Pttg1) implicated in lung biology and pathogenesis. Gene set enrichment analysis identified negative enrichment for TNF pathways. Conclusion: Our murine findings support a causal role of ADAM19, implicated in human GWAS, in regulating pulmonary function.
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Cytochromes P450 can metabolize endogenous fatty acids, such as arachidonic acid, to bioactive lipids such as epoxyeicosatrienoic acids (EETs) that have beneficial effects. EETs protect hearts against ischemic damage, heart failure or fibrosis; however, their effects are limited by hydrolysis to less active dihydroxy oxylipins by soluble epoxide hydrolase (sEH), encoded by the epoxide hydrolase 2 gene (EPHX2, EC 3.3.2.10). Pharmacological inhibition or genetic disruption of sEH/EPHX2 have been widely studied for their impact on cardiovascular diseases. Less well studied is the role of increased EPHX2 expression, which occurs in a substantial human population that carries the EPHX2 K55R polymorphism or after induction by inflammatory stimuli. Herein, we developed a mouse model with cardiomyocyte-selective expression of human EPHX2 (Myh6-EPHX2) that has significantly increased total EPHX2 expression and activity. Myh6-EPHX2 hearts exhibit strong, cardiomyocyte-selective expression of EPHX2. EPHX2 mRNA, protein, and epoxide hydrolysis measurements suggest that Myh6-EPHX2 hearts have 12-fold increase in epoxide hydrolase activity relative to wild type (WT) hearts. This increased activity significantly decreased epoxide:diol ratios in vivo. Isolated, perfused Myh6-EPHX2 hearts were not significantly different from WT hearts in basal parameters of cardiac function; however, compared to WT hearts, Myh6-EPHX2 hearts demonstrated reduced recovery of heart contractile function after ischemia and reperfusion (I/R). This impaired recovery after I/R correlated with reduced activation of PI3K/AKT and GSK3ß signaling pathways in Myh6-EPHX2 hearts compared to WT hearts. In summary, the Myh6-EPHX2 mouse line represents a novel model of cardiomyocyte-selective overexpression of EPHX2 that has detrimental effects on cardiac function.
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In lung, thromboxane A2 (TXA2) activates the TP receptor to induce proinflammatory and bronchoconstrictor effects. Thus, TP receptor antagonists and TXA2 synthase inhibitors have been tested as potential asthma therapeutics in humans. Th9 cells play key roles in asthma and regulate the lung immune response to allergens. Herein, we found that TXA2 reduces Th9 cell differentiation during allergic lung inflammation. Th9 cells were decreased approximately 2-fold and airway hyperresponsiveness was attenuated in lungs of allergic mice treated with TXA2. Naive CD4+ T cell differentiation to Th9 cells and IL-9 production were inhibited dose-dependently by TXA2 in vitro. TP receptor-deficient mice had an approximately 2-fold increase in numbers of Th9 cells in lungs in vivo after OVA exposure compared with wild-type mice. Naive CD4+ T cells from TP-deficient mice exhibited increased Th9 cell differentiation and IL-9 production in vitro compared with CD4+ T cells from wild-type mice. TXA2 also suppressed Th2 and enhanced Treg differentiation both in vitro and in vivo. Thus, in contrast to its acute, proinflammatory effects, TXA2 also has longer-lasting immunosuppressive effects that attenuate the Th9 differentiation that drives asthma progression. These findings may explain the paradoxical failure of anti-thromboxane therapies in the treatment of asthma.
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Asma , Diferenciación Celular , Linfocitos T Reguladores , Células Th2 , Tromboxano A2 , Animales , Ratones , Células Th2/inmunología , Células Th2/patología , Tromboxano A2/metabolismo , Tromboxano A2/inmunología , Linfocitos T Reguladores/inmunología , Asma/inmunología , Asma/patología , Asma/tratamiento farmacológico , Asma/genética , Ratones Noqueados , Interleucina-9/inmunología , Interleucina-9/genética , Interleucina-9/metabolismo , Neumonía/inmunología , Neumonía/patología , Ratones Endogámicos C57BL , Ratones Endogámicos BALB C , Pulmón/inmunología , Pulmón/patología , Ovalbúmina/inmunología , Femenino , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
RATIONALE: Pulmonary hypertension (PH) is a multifactorial disease with a poor prognosis and inadequate treatment options. We found two-fold higher expression of the orphan G-Protein Coupled Receptor 75 (GPR75) in leukocytes and pulmonary arterial smooth muscle cells from idiopathic PH patients and from lungs of C57BL/6 mice exposed to hypoxia. We therefore postulated that GPR75 signaling is critical to the pathogenesis of PH. METHODS: To test this hypothesis, we exposed global (Gpr75-/-) and endothelial cell (EC) GPR75 knockout (EC-Gpr75-/-) mice and wild-type (control) mice to hypoxia (10% oxygen) or normal atmospheric oxygen for 5 weeks. We then recorded echocardiograms and performed right heart catheterizations. RESULTS: Chronic hypoxia increased right ventricular systolic and diastolic pressures in wild-type mice but not Gpr75-/- or EC-Gpr75-/- mice. In situ hybridization and qPCR results revealed that Gpr75 expression was increased in the alveoli, airways and pulmonary arteries of mice exposed to hypoxia. In addition, levels of chemokine (CC motif) ligand 5 (CCL5), a low affinity ligand of GPR75, were increased in the lungs of wild-type, but not Gpr75-/-, mice exposed to hypoxia, and CCL5 enhanced hypoxia-induced contraction of intra-lobar pulmonary arteries in a GPR75-dependent manner. Gpr75 knockout also increased pulmonary cAMP levels and decreased contraction of intra-lobar pulmonary arteries evoked by endothelin-1 or U46619 in cAMP-protein kinase A-dependent manner. CONCLUSION: These results suggest GPR75 has a significant role in the development of hypoxia-induced PH.
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Hipertensión Pulmonar , Humanos , Ratones , Animales , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Arteria Pulmonar , Ligandos , Células Cultivadas , Ratones Endogámicos C57BL , Hipoxia/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Oxígeno/metabolismo , Ratones NoqueadosRESUMEN
Receptors for estrogen and progesterone frequently interact, via Cohesin/CTCF loop extrusion, at enhancers distal from regulated genes. Loss-of-function CTCF mutation in >20% of human endometrial tumors indicates its importance in uterine homeostasis. To better understand how CTCF-mediated enhancer-gene interactions impact endometrial development and function, the Ctcf gene was selectively deleted in female reproductive tissues of mice. Prepubertal Ctcfd/d uterine tissue exhibited a marked reduction in the number of uterine glands compared to those without Ctcf deletion (Ctcff/f mice). Post-pubertal Ctcfd/d uteri were hypoplastic with significant reduction in both the amount of the endometrial stroma and number of glands. Transcriptional profiling revealed increased expression of stem cell molecules Lif, EOMES, and Lgr5, and enhanced inflammation pathways following Ctcf deletion. Analysis of the response of the uterus to steroid hormone stimulation showed that CTCF deletion affects a subset of progesterone-responsive genes. This finding indicates (1) Progesterone-mediated signaling remains functional following Ctcf deletion and (2) certain progesterone-regulated genes are sensitive to Ctcf deletion, suggesting they depend on gene-enhancer interactions that require CTCF. The progesterone-responsive genes altered by CTCF ablation included Ihh, Fst, and Errfi1. CTCF-dependent progesterone-responsive uterine genes enhance critical processes including anti-tumorigenesis, which is relevant to the known effectiveness of progesterone in inhibiting progression of early-stage endometrial tumors. Overall, our findings reveal that uterine Ctcf plays a key role in progesterone-dependent expression of uterine genes underlying optimal post-pubertal uterine development.
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Cromatina , Neoplasias Endometriales , Humanos , Femenino , Animales , Ratones , Progesterona , Útero , EndometrioRESUMEN
Genetically engineered mice are widely used to study the impact of altered gene expression in vivo. Within the reproductive tract, the Amhr2-IRES-Cre(Bhr) mouse model is used to ablate genes in ovarian granulosa and uterine stromal cells. There are reports of Amhr2-IRES-Cre(Bhr) inducing recombination in non-target tissues. We hypothesized the inefficiency or off-target Cre action in Amhr2-IRES-Cre(Bhr) mice is due to lack of recombination in every cell that expresses Amhr2. To investigate, we created a new targeted knock-in mouse model, Amhr2-iCre(Fjd), by inserting a codon-optimized improved Cre (iCre) into exon 1 of the Amhr2 gene. Amhr2-iCre(Fjd)/+ males were mated with females that contain a lox-stop-lox cassette in the Sun1 gene so when DNA recombination occurs, SUN1-sfGFP fusion protein is expressed in a peri-nuclear pattern. In adult Amhr2-iCre(Fjd)/+ Sun1LsL/+ mice, Amhr2-iCre(Fjd)-mediated genetic recombination was apparent in uterine epithelial, stromal, and myometrial cells, while Amhr2-IRES-Cre(Bhr)/+ Sun1LsL/+ females demonstrated inter-mouse variability of Amhr2-IRES-Cre(Bhr) activity in uterine cells. Fluorescence was observed in Amhr2-iCre(Fjd)-positive mice at post-natal Day 1, indicating global genetic recombination, while fluorescence of individual Amhr2-IRES-Cre(Bhr)-positive pups varied. To determine the developmental stage that genetic recombination first occurs, Sun1LsL/LsL females were super-ovulated and mated with Amhr2-IRES-Cre(Bhr)/+ or Amhr2(iCre/+)Fjd males, then putative zygotes were collected and cultured. In the four-cell embryo, Amhr2-iCre(Fjd) and Amhr2-IRES-Cre(Bhr) activities were apparent in 100% and 25-100% of cells, respectively. In conclusion, Amhr2-IRES-Cre(Bhr) or Amhr2-iCre(Fjd) driven by the Amhr2 promoter is active in the early embryo and can lead to global genetic modification, rendering this transgenic mouse model ineffective.
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Receptores de Factores de Crecimiento Transformadores beta , Recombinasas , Femenino , Masculino , Ratones , Animales , Ratones Transgénicos , Integrasas/genética , Integrasas/metabolismo , Proteínas Serina-Treonina QuinasasRESUMEN
Cytochromes P450 metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which have numerous effects. After cardiac ischemia, EET-induced coronary vasodilation increases delivery of oxygen/nutrients to the myocardium, and EET-induced signaling protects cardiomyocytes against postischemic mitochondrial damage. Soluble epoxide hydrolase 2 (EPHX2) diminishes the benefits of EETs through hydrolysis to less active dihydroxyeicosatrienoic acids. EPHX2 inhibition or genetic disruption improves recovery of cardiac function after ischemia. Immunohistochemical staining revealed EPHX2 expression in cardiomyocytes and some endothelial cells but little expression in cardiac smooth muscle cells or fibroblasts. To determine specific roles of EPHX2 in cardiac cell types, we generated mice with cell-specific disruption of Ephx2 in endothelial cells (Ephx2fx/fx/Tek-cre) or cardiomyocytes (Ephx2fx/fx/Myh6-cre) to compare to global Ephx2-deficient mice (global Ephx2-/-) and WT (Ephx2fx/fx) mice in expression, EET hydrolase activity, and heart function studies. Most cardiac EPHX2 expression and activity is in cardiomyocytes with substantially less activity in endothelial cells. Ephx2fx/fx/Tek-cre hearts have similar EPHX2 expression, hydrolase activity, and postischemic cardiac function as control Ephx2fx/fx hearts. However, Ephx2fx/fx/Myh6-cre hearts were similar to global Ephx2-/- hearts with significantly diminished EPHX2 expression, decreased hydrolase activity, and enhanced postischemic cardiac function compared to Ephx2fx/fx hearts. During reperfusion, Ephx2fx/fx/Myh6-cre hearts displayed increased ERK activation compared to Ephx2fx/fx hearts, which could be reversed by EEZE treatment. EPHX2 did not regulate coronary vasodilation in this model. We conclude that EPHX2 is primarily expressed in cardiomyocytes where it regulates EET hydrolysis and postischemic cardiac function, whereas endothelial EPHX2 does not play a significant role in these processes.
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Miocardio , Miocitos Cardíacos , Ratones , Animales , Miocitos Cardíacos/metabolismo , Miocardio/metabolismo , Isquemia/metabolismo , Eicosanoides/metabolismo , Reperfusión , Hidrolasas/metabolismo , Epóxido Hidrolasas/metabolismoRESUMEN
Technical challenges have to date prevented a complete profiling of the levels of myo-inositol phosphates (InsPs) and pyrophosphates (PP-InsPs) in mammalian tissues. Here, we have deployed capillary electrophoresis mass spectrometry to identify and record the levels of InsPs and PP-InsPs in several tissues obtained from wild type mice and a newly created PPIP5K2 knockout strain. We observe that the mouse colon harbours unusually high levels of InsPs and PP-InsPs. Additionally, the PP-InsP profile is considerably more complex than previously reported for animal cells: using chemically synthesized internal stable isotope references and high-resolution mass spectra, we characterize two new PP-InsP isomers as 4/6-PP-InsP5 and 2-PP-InsP5. The latter has not previously been described in nature. The analysis of feces and the commercial mouse diet suggests that the latter is one potential source of noncanonical isomers in the colon. However, we also identify both molecules in the heart, indicating unknown synthesis pathways in mammals. We also demonstrate that the CE-MS method is sensitive enough to measure PP-InsPs from patient samples such as colon biopsies and peripheral blood mononuclear cells (PBMCs). Strikingly, PBMCs also contain 4/6-PP-InsP5 and 2-PP-InsP5. In summary, our study substantially expands PP-InsP biology in mammals.
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Endotoxemia elicits a multiorgan inflammatory response that results in cardiac dysfunction and often leads to death. Inflammation-induced metabolism of endogenous N-3 and N-6 polyunsaturated fatty acids generates numerous lipid mediators, such as epoxy fatty acids (EpFAs), which protect the heart. However, EpFAs are hydrolyzed by soluble epoxide hydrolase (sEH), which attenuates their cardioprotective actions. Global genetic disruption of sEH preserves EpFA levels and attenuates cardiac dysfunction in mice following acute lipopolysaccharide (LPS)-induced inflammatory injury. In leukocytes, EpFAs modulate the innate immune system through the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. However, the mechanisms by which both EpFAs and sEH inhibition exert their protective effects in the cardiomyocyte are still elusive. This study investigated whether cardiomyocyte-specific sEH disruption attenuates inflammation and cardiac dysfunction in acute LPS inflammatory injury via modulation of the NLRP3 inflammasome. We use tamoxifen-inducible CreER recombinase technology to target sEH genetic disruption to the cardiomyocyte. Primary cardiomyocyte studies provide mechanistic insight into inflammasome signaling. For the first time, we demonstrate that cardiomyocyte-specific sEH disruption preserves cardiac function and attenuates inflammatory responses by limiting local cardiac inflammation and activation of the systemic immune response. Mechanistically, inhibition of cardiomyocyte-specific sEH activity or exogenous EpFA treatment do not prevent upregulation of NLRP3 inflammasome machinery in neonatal rat cardiomyocytes. Rather, they limit downstream activation of the pathway leading to release of fewer chemoattractant factors and recruitment of immune cells to the heart. These data emphasize that cardiomyocyte sEH is vital for mediating detrimental systemic inflammation.NEW & NOTEWORTHY The cardioprotective effects of genetic disruption and pharmacological inhibition of sEH have been demonstrated in a variety of cardiac disease models, including acute LPS inflammatory injury. For the first time, it has been demonstrated that sEH genetic disruption limited to the cardiomyocyte profoundly preserves cardiac function and limits local and systemic inflammation following acute LPS exposure. Hence, cardiomyocytes serve a critical role in the innate immune response that can be modulated to protect the heart.
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Cardiopatías , Miocitos Cardíacos , Animales , Factores Quimiotácticos/uso terapéutico , Epóxido Hidrolasas/genética , Ácidos Grasos/metabolismo , Ácidos Grasos Insaturados/uso terapéutico , Inflamasomas , Inflamación/tratamiento farmacológico , Lipopolisacáridos/farmacología , Ratones , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Ratas , Recombinasas/uso terapéutico , Tamoxifeno/uso terapéuticoRESUMEN
The Notch signaling pathway is required for reproductive success. This pathway activates its transcriptional effector, recombination signal binding protein for immunoglobulin kappa J (Rbpj), to induce transcription of its target genes. This signaling pathway is required for successful decidualization, implantation, and uterine repair following parturition. To identify the compartmental specific roles of the Notch signaling pathway in the establishment of pregnancy, we generated epithelial and decidual stromal cell specific knockouts of Rbpj utilizing lactoferrin iCre and Prl8A2 iCre, respectively. Both conditional knockout mouse models were fertile. The Rbpj epithelial knockout mice displayed 27% resorption sites at E15.5, but this did not significantly impact the number of live born pups compared with controls. In addition, the Rbpj epithelial knockout mice displayed increased estrogen signaling in their stromal compartment. Given that both mouse models exhibited fertility comparable to control animals, the epithelial and stromal specific nature of the iCre recombinases utilized, and previously published Rbpj total uterine knockout mouse models, we conclude that Notch effector Rbpj signaling is required at the initiation of pregnancy to support decidualization in stromal cells, but that Rbpj is not required in the epithelial compartment nor is it required for post-implantation pregnancy success.
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Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas , Receptores Notch , Animales , Proteínas Portadoras/metabolismo , Estrógenos , Femenino , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Lactoferrina/metabolismo , Ratones , Ratones Noqueados , Embarazo , Receptores Notch/genética , Receptores Notch/metabolismo , Recombinasas/genética , Recombinasas/metabolismo , Recombinación Genética , Transducción de Señal/fisiología , Células del Estroma/metabolismoRESUMEN
Nuclear receptor Pregnane X Receptor (PXR; NR1I2) has transcriptional regulation functions for energy homeostasis in the liver. Mouse PXR has a conserved phosphorylation motif at serine 347 (serine 350 in humans) within the ligand-binding domain. PXR phosphorylated at this motif is expressed in mouse livers in response to fasting. Mice with a PXR∗Ser347Ala knockin mutation (PXR KI) were generated to block phosphorylation, and utilized to investigate the role of Ser347 phosphorylation in vivo. PXR KI mice had decreased body weight at 8-weeks of age and had much greater weight loss after fasting compared with PXR WT mice. The cDNA microarray analysis of hepatic mRNAs showed that cell death or apoptotic signaling was induced in fasting PXR KI mice. Moreover, increasing hepatic lipids, triglycerides and the development of hypertriglyceridemia were observed in fasting PXR KI mice. These findings are indicative that blocking phosphorylation prevents mice from maintaining hepatic energy homeostasis. Thus, phosphorylated PXR may be an essential factor to prevent the liver from developing damage caused by fasting.
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Hígado Graso , Hipertrigliceridemia , Receptor X de Pregnano/metabolismo , Receptores de Esteroides , Animales , Ayuno/metabolismo , Hígado Graso/metabolismo , Hipertrigliceridemia/genética , Hipertrigliceridemia/metabolismo , Hígado/metabolismo , Ratones , Fosforilación , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Serina/metabolismoRESUMEN
An estimated 75% of unsuccessful pregnancies are due to implantation failure. Investigating the causes of implantation failure is difficult as decidualization and embryo implantation is a dynamic process. Here, we describe a new decidua-specific iCre recombinase mouse strain. Utilizing CRISPR/Cas9-based genome editing, a mouse strain was developed that expresses iCre recombinase under the control of the endogenous prolactin family 8, subfamily a, member 2 (Prl8a2) promoter. iCre recombinase activity was examined by crossing with mTmG/+ or Sun1-GFP reporter alleles. iCre activity initiated reporter expression at gestational day 5.5 in the primary decidual zone and continued into mid-gestation (gestational day 9.5), with expression highly concentrated in the anti-mesometrial region. No reporter expression was observed in the ovary, oviduct, pituitary, or skeletal muscle, supporting the tissue specificity of the Prl8a2iCre in the primary decidual zone. This novel iCre line will be a valuable tool for in vivo genetic manipulation and lineage tracing to investigate functions of genetic networks and cellular dynamics associated with decidualization and infertility.
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Integrasas , Prolactina , Animales , Decidua/metabolismo , Femenino , Integrasas/genética , Integrasas/metabolismo , Ratones , Ratones Transgénicos , Embarazo , Prolactina/genética , Recombinación GenéticaRESUMEN
Nongenomic effects of estrogen receptor α (ERα) signaling have been described for decades. Several distinct animal models have been generated previously to analyze the nongenomic ERα signaling (eg, membrane-only ER, and ERαC451A). However, the mechanisms and physiological processes resulting solely from nongenomic signaling are still poorly understood. Herein, we describe a novel mouse model for analyzing nongenomic ERα actions named H2NES knock-in (KI). H2NES ERα possesses a nuclear export signal (NES) in the hinge region of ERα protein resulting in exclusive cytoplasmic localization that involves only the nongenomic action but not nuclear genomic actions. We generated H2NESKI mice by homologous recombination method and have characterized the phenotypes. H2NESKI homozygote mice possess almost identical phenotypes with ERα null mice except for the vascular activity on reendothelialization. We conclude that ERα-mediated nongenomic estrogenic signaling alone is insufficient to control most estrogen-mediated endocrine physiological responses; however, there could be some physiological responses that are nongenomic action dominant. H2NESKI mice have been deposited in the repository at Jax (stock no. 032176). These mice should be useful for analyzing nongenomic estrogenic responses and could expand analysis along with other ERα mutant mice lacking membrane-bound ERα. We expect the H2NESKI mouse model to aid our understanding of ERα-mediated nongenomic physiological responses and serve as an in vivo model for evaluating the nongenomic action of various estrogenic agents.
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PGC1α is a transcriptional coactivator in peripheral tissues, but its function in the brain remains poorly understood. Various brain-specific Pgc1α isoforms have been reported in mice and humans, including two fusion transcripts (FTs) with non-coding repetitive sequences, but their function is unknown. The FTs initiate at a simple sequence repeat locus â¼570 Kb upstream from the reference promoter; one also includes a portion of a short interspersed nuclear element (SINE). Using publicly available genomics data, here we show that the SINE FT is the predominant form of Pgc1α in neurons. Furthermore, mutation of the SINE in mice leads to altered behavioural phenotypes and significant up-regulation of genes in the female, but not male, cerebellum. Surprisingly, these genes are largely involved in neurotransmission, having poor association with the classical mitochondrial or antioxidant programs. These data expand our knowledge on the role of Pgc1α in neuronal physiology and suggest that different isoforms may have distinct functions. They also highlight the need for further studies before modulating levels of Pgc1α in the brain for therapeutic purposes.
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Conducta Animal , Cerebelo/metabolismo , Expresión Génica , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Isoformas de Proteínas/genética , Transducción de Señal/genética , Regulación hacia Arriba/genética , Animales , Prueba de Laberinto Elevado , Femenino , Locomoción/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/genética , Mutación , Neuronas/metabolismo , Prueba de Campo Abierto , Regiones Promotoras Genéticas/genética , Elementos de Nucleótido Esparcido Corto/genéticaRESUMEN
Epoxyeicosatrienoic acids (EETs) have potent antiinflammatory properties. Hydrolysis of EETs by soluble epoxide hydrolase/ epoxide hydrolase 2 (sEH/EPHX2) to less active diols attenuates their antiinflammatory effects. Macrophage activation is critical to many inflammatory responses; however, the role of EETs and sEH in regulating macrophage function remains unknown. Lung bacterial clearance of Streptococcus pneumoniae was impaired in Ephx2-deficient (Ephx2-/-) mice and in mice treated with an sEH inhibitor. The EET receptor antagonist EEZE restored lung clearance of S. pneumoniae in Ephx2-/- mice. Ephx2-/- mice had normal lung Il1b, Il6, and Tnfa expression levels and macrophage recruitment to the lungs during S. pneumoniae infection; however, Ephx2 disruption attenuated proinflammatory cytokine induction, Tlr2 and Pgylrp1 receptor upregulation, and Ras-related C3 botulinum toxin substrates 1 and 2 (Rac1/2) and cell division control protein 42 homolog (Cdc42) activation in PGN-stimulated macrophages. Consistent with these observations, Ephx2-/- macrophages displayed reduced phagocytosis of S. pneumoniae in vivo and in vitro. Heterologous overexpression of TLR2 and peptidoglycan recognition protein 1 (PGLYRP1) in Ephx2-/- macrophages restored macrophage activation and phagocytosis. Human macrophage function was similarly regulated by EETs. Together, these results demonstrate that EETs reduced macrophage activation and phagocytosis of S. pneumoniae through the downregulation of TLR2 and PGLYRP1 expression. Defining the role of EETs and sEH in macrophage function may lead to the development of new therapeutic approaches for bacterial diseases.
Asunto(s)
Eicosanoides/fisiología , Epóxido Hidrolasas/fisiología , Pulmón/inmunología , Macrófagos/inmunología , Fagocitosis/fisiología , Streptococcus pneumoniae/inmunología , Animales , Proteínas Portadoras/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Moléculas de Patrón Molecular Asociado a Patógenos/farmacología , Receptor Toll-Like 2/fisiologíaRESUMEN
Recent advances in high throughput RNA sequencing have revealed that, in addition to messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs) play an important role in the regulation of many cell functions and of organ development. While a number of lncRNAs have been identified in pancreatic islets, their function remains largely undetermined. Here, we identify a novel long ncRNA regulated by the transcription factor GLIS3, which we refer to as GLIS3 regulated 1 (G3R1). This lncRNA was identified for its significant loss of expression in GLIS3 knockout mouse pancreatic islets. G3R1 appears to be specifically expressed in mouse pancreatic ß-cells and in a ß-cell line (ßTC-6). ChIP-seq analysis indicated that GLIS3 and other islet-enriched transcription factors bind near the G3R1 gene, suggesting they directly regulate G3R1 transcription. Similarly, an apparent human homolog of G3R1 displays a similar expression pattern, with additional expression seen in human brain. In order to determine the function of G3R1 in mouse pancreatic ß-cells, we utilized CRISPR to develop a knockout mouse where ~80% of G3R1 sequence is deleted. Phenotypic analysis of these mice did not reveal any impairment in ß-cell function or glucose regulation, indicating the complexity underlying the study of lncRNA function.