RESUMEN
The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a worldwide threat in the past 3 years. Although it has been widely and intensively investigated, the mechanism underlying the coronavirus-host interaction requires further elucidation, which may contribute to the development of new antiviral strategies. Here, we demonstrated that the host cAMP-responsive element-binding protein (CREB1) interacts with the non-structural protein 13 (nsp13) of SARS-CoV-2, a conserved helicase for coronavirus replication, both in cells and in lung tissues subjected to SARS-CoV-2 infection. The ATPase and helicase activity of viral nsp13 were shown to be potentiated by CREB1 association, as well as by Protein kinase A (PKA)-mediated CREB1 activation. SARS-CoV-2 replication is significantly suppressed by PKA Cα, cAMP-activated protein kinase catalytic subunit alpha (PRKACA), and CREB1 knockdown or inhibition. Consistently, the CREB1 inhibitor 666-15 has shown significant antiviral effects against both the WIV04 strain and the Omicron strain of the SARS-CoV-2. Our findings indicate that the PKA-CREB1 signaling axis may serve as a novel therapeutic target against coronavirus infection. IMPORTANCE: In this study, we provide solid evidence that host transcription factor cAMP-responsive element-binding protein (CREB1) interacts directly with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) helicase non-structural protein 13 (nsp13) and potentiate its ATPase and helicase activity. And by live SARS-CoV-2 virus infection, the inhibition of CREB1 dramatically impairs SARS-CoV-2 replication in vivo. Notably, the IC50 of CREB1 inhibitor 666-15 is comparable to that of remdesivir. These results may extend to all highly pathogenic coronaviruses due to the conserved nsp13 sequences in the virus.
Asunto(s)
ARN Polimerasa Dependiente de ARN de Coronavirus , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico , Interacciones Microbiota-Huesped , SARS-CoV-2 , Proteínas no Estructurales Virales , Replicación Viral , Humanos , Adenosina Trifosfatasas/metabolismo , Antivirales/farmacología , ARN Polimerasa Dependiente de ARN de Coronavirus/metabolismo , COVID-19/virología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ADN Helicasas/metabolismo , Concentración 50 Inhibidora , ARN Helicasas/metabolismo , SARS-CoV-2/clasificación , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , SARS-CoV-2/crecimiento & desarrollo , Transducción de Señal/efectos de los fármacos , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos , Femenino , Animales , RatonesRESUMEN
AIMS: The primary focus of this study was to explore the effects of cyclic AMP response element-binding protein H (CREBH) on the development of nonalcoholic fatty liver disease (NAFLD). MATERIALS AND METHODS: CREBH knockout (KO) and wildtype (WT) mice were averagely divided into a methionine and choline-deficient (MCD) or high fat (HF) diet group and respective chow diet (CD) groups. Mice were sacrificed after 4-week treatment for MCD model and 24-week treatment for HF model. KEY FINDINGS: Characteristics of nonalcoholic steatohepatitis-related liver fibrosis in KO-MCD/HF group were verified by hepatic histological analyses. Compared with WT-MCD/HF group, levels of plasma ALT and hepatic hydroxyproline increased in KO-MCD/HF group. Significantly higher levels of MCP-1, αSMA, Desmin, COL-1, TIMP-1, TGF-ß1, TGF-ß2 were found while MMP-9 and FGF21 mRNA levels decreased in KO-MCD/HF group. There was also a distinct difference of mRNA levels of TNFα, CTGF and CCND1 in KO-HF group compared with controls. Protein levels of MCP-1, BAX, αSMA, COL-1, TGF-ß1 and SMAD2/3 significantly increased in KO-MCD/HF group and CCND1 was also upregulated in KO-HF group compared to their counterparts. SIGNIFICANCE: CREBH knockout may primarily regulate the TGF-ß1 signaling pathway via TGF-ß2 and FGF21 resulting in more severe inflammation and fibrosis in NAFLD.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Cirrosis Hepática/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Factor de Crecimiento Transformador beta/metabolismo , Alanina Transaminasa/sangre , Animales , Deficiencia de Colina , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Dieta Alta en Grasa , Factores de Crecimiento de Fibroblastos/biosíntesis , Hidroxiprolina/metabolismo , Lípidos/sangre , Cirrosis Hepática/sangre , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Masculino , Metionina/deficiencia , Ratones , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Transducción de Señal/genéticaRESUMEN
Autophagy, a lysosomal degradative pathway in response to nutrient limitation, plays an important regulatory role in lipid homeostasis upon energy demands. Here, we demonstrated that the endoplasmic reticulum-tethered, stress-sensing transcription factor cAMP-responsive element-binding protein, hepatic-specific (CREBH) functions as a major transcriptional regulator of hepatic autophagy and lysosomal biogenesis in response to nutritional or circadian signals. CREBH deficiency led to decreased hepatic autophagic activities and increased hepatic lipid accumulation upon starvation. Under unfed or during energy-demanding phases of the circadian cycle, CREBH is activated to drive expression of the genes encoding the key enzymes or regulators in autophagosome formation or autophagic process, including microtubule-associated protein 1B-light chain 3, autophagy-related protein (ATG)7, ATG2b, and autophagosome formation Unc-51 like kinase 1, and the genes encoding functions in lysosomal biogenesis and homeostasis. Upon nutrient starvation, CREBH regulates and interacts with peroxisome proliferator-activated receptor α (PPARα) and PPARγ coactivator 1α to synergistically drive expression of the key autophagy genes and transcription factor EB, a master regulator of lysosomal biogenesis. Furthermore, CREBH regulates rhythmic expression of the key autophagy genes in the liver in a circadian-dependent manner. In summary, we identified CREBH as a key transcriptional regulator of hepatic autophagy and lysosomal biogenesis for the purpose of maintaining hepatic lipid homeostasis under nutritional stress or circadian oscillation.-Kim, H., Williams, D., Qiu, Y., Song, Z., Yang, Z., Kimler, V., Goldberg, A., Zhang, R., Yang, Z., Chen, X., Wang, L., Fang, D., Lin, J. D., Zhang, K. Regulation of hepatic autophagy by stress-sensing transcription factor CREBH.
Asunto(s)
Autofagia/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Privación de Alimentos/fisiología , Regulación de la Expresión Génica/fisiología , Hígado/metabolismo , Animales , Autofagosomas/metabolismo , Autofagia/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Línea Celular Tumoral , Células Cultivadas , Ritmo Circadiano , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Hígado Graso/etiología , Hígado Graso/genética , Hígado Graso/metabolismo , Hepatocitos/metabolismo , Metabolismo de los Lípidos , Hígado/citología , Lisosomas/metabolismo , Ratones , Ratones Noqueados , PPAR alfa/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Estrés Fisiológico/genética , Estrés Fisiológico/fisiología , Transcripción GenéticaRESUMEN
Renal ischemia reperfusion (IR) is a common cause of acute kidney injury (AKI), and no effective treatment is available to date. In our previous studies, we demonstrated that Tisp40 exacerbates tubular cell apoptosis and tubulointerstitial fibrosis after renal IR injury. However, the role of Tisp40 in renal inflammatory responses and tubular cell proliferation during renal IR injury remains unknown. In this study, Tisp40 knockout (KO) and wild-type (WT) mice were induced with or without renal IR injury. For renal IR, bilateral renal pedicels were exposed and clamped to induce 30â¯min of ischemia. After 48â¯h of reperfusion, the kidneys were collected for analyses. Results showed that Tisp40 deficiency attenuates neutrophil and macrophage infiltration after renal IR. Consistently, the protein levels of TNF-α and MCP-1 were markedly decreased, and the phosphorylation levels of IκBα and P65 were inhibited in Tisp40-deficient mice than in WT mice in renal IR injury. In addition, compared with WT mice, Tisp40 deficiency significantly increased the expression levels of proliferative cellular nuclear antigen and phosphorylated Erk1/2 after renal IR injury. In conclusion, Tisp40 deficiency limits renal inflammatory responses and promotes tubular cell proliferation in ischemic AKI.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Células Epiteliales/metabolismo , Túbulos Renales Proximales/metabolismo , Nefritis Intersticial/genética , Daño por Reperfusión/genética , Animales , Movimiento Celular , Proliferación Celular , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Células Epiteliales/citología , Regulación de la Expresión Génica , Inflamación , Túbulos Renales Proximales/patología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Inhibidor NF-kappaB alfa/genética , Inhibidor NF-kappaB alfa/metabolismo , Nefritis Intersticial/metabolismo , Nefritis Intersticial/patología , Nefritis Intersticial/prevención & control , Neutrófilos/metabolismo , Neutrófilos/patología , Fosforilación , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Daño por Reperfusión/prevención & control , Transducción de Señal , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Cyclic AMP response element-binding protein 1 (CREB1), a member of the CREB family, is known to be involved in follicular growth, ovulation, and ovarian disease. However, the physiological function of CREB1 in mouse granulosa cells (mGCs) remains lagerly unknown. The aim of this study was to determine the role of CREB1 in mGCs by knocking down CREB1 expression. CREB1 knock-down in mGCs at the mRNA and protein levels, was confirmed by quantitative real-time polymerase chain reaction and western blot. Results of enzyme linked immunosorbent assay revealed that CREB1 knockdown significantly decreased the concentrations of estradiol (E2) and progesterone (P4) in mGCs. Furthermore, the CREB1 knockdown in mGCs promoted cell proliferation and apoptosis, and arrested the cell cycle in S-phase. To elucidate the regulatory mechanism underlying the effects of CREB1 knockdown on steroid synthesis, cell cycle, and apoptosis, we measured the protein expression levels of several related genes in mGCs knocked down CREB1. When CREB1 was knocked down, the expression of Cyp1b1 and Cyp19a1, which encode steroidogenic enzymes, was down-regulated; the expression of the cell cycle factors CyclinA1, CyclinB1, and CyclinD2 were significantly decreased. Among apoptosis-related genes, Bcl-2 was down-regulated, whereas Bax and cleaved Caspase3 were upregulated. Moreover, CREB1 knockdown significantly decreased expression level of Has2, Ptgs2, and Igfbp4, which are essential genes for folliculogenesis in mGCs. Taken together, these findings suggested that CREB1 might be a key regulator of mGCs through regulating steroid synthesis, cell proliferation, cell cycle, apoptosis, and other regulators of folliculogenesis.
Asunto(s)
Apoptosis/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Estradiol/biosíntesis , Técnicas de Silenciamiento del Gen , Células de la Granulosa/metabolismo , Animales , Proliferación Celular/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Femenino , Técnicas de Silenciamiento del Gen/métodos , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
Evidence exists that chronic antidepressant therapy enhances CREB levels and activity. Nevertheless, the data are not conclusive, as previous analysis of transgenic mouse models has suggested that CREB inactivation in fact contributes to antidepressant-like behavior. The aim of this study was to evaluate the role of CREB in this context by exploiting novel transgenic mouse models, characterized by selective ablation of CREB restricted to noradrenergic (Creb1DBHCre/Crem-/-) or serotonergic (Creb1TPH2CreERT2/Crem-/-) neurons in a CREM-deficient background to avoid possible compensatory effects of CREM. Selective and functional ablation of CREB affected antidepressant-like behavior in a tail suspension test (TST) after antidepressant treatment. Contrary to single Creb1DBHCre mutants, Creb1DBHCre/Crem-/- mice did not respond to acute desipramine administration (20 mg/kg) on the TST. On the other hand, single Creb1TPH2CreERT2 mutants displayed reduced responses to fluoxetine (10 mg/kg) on the TST, while the effects in Creb1TPH2CreERT2/Crem-/- mice differed by gender. Our results provide further evidence for the important role of CREM as a compensatory factor. Additionally, the results indicate that new models based on the functional ablation of CREB in select neuronal populations may represent a valuable tool for investigating the role of CREB in the mechanism of antidepressant therapy.
Asunto(s)
Neuronas Adrenérgicas/metabolismo , Antidepresivos/uso terapéutico , Modulador del Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Depresión/genética , Neuronas Serotoninérgicas/metabolismo , Animales , Modulador del Elemento de Respuesta al AMP Cíclico/deficiencia , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Depresión/tratamiento farmacológico , Depresión/etiología , Desipramina/uso terapéutico , Femenino , Fluoxetina/uso terapéutico , Suspensión Trasera/efectos adversos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Renal ischemia reperfusion (IR) is a major cause of acute kidney injury (AKI) and no effective treatments have been established. Tisp40 is a transcription factor of the CREB/ATF family and involves in cell apoptosis, proliferation and differentiation, but its role in renal IR remains unknown. Here, we investigated the role of Tisp40 in renal IR injury. In vivo, Tisp40 knockout (KO) and wild-type (WT) mice were subjected to thirty minutes of bilateral renal ischemia and 48h reperfusion, the blood and kidneys were harvested for analysis. In vitro, Tisp40 overexpression and vector cells were subjected to hypoxia/reoxygenation (HR), the apoptosis rate and the expressions of related proteins were measured. Following IR, the expressions of Tisp40 protein, serum creatinine (sCr), blood urea nitrogen (BUN) and apoptosis of tubular cells were significantly increased in WT mice. However, Tisp40 deficiency significantly attenuated the increase of sCr, BUN and apoptosis of tubular cells. Following HR, apoptosis of tubular cells was increased in Tisp40 overexpression cells compared with vector cells. Mechanistically, Tisp40 promoted the expressions of C/EBP homologous protein (CHOP), Bax and Cleaved caspase3 and suppressed the expression of Bcl-2 in renal IR injury. In conclusion, Tisp40 aggravates tubular cells apoptosis in renal IR injury.
Asunto(s)
Apoptosis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Células Epiteliales/metabolismo , Túbulos Renales/patología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Células Epiteliales/patología , Hipoxia/complicaciones , Hipoxia/patología , Túbulos Renales/anomalías , Túbulos Renales/fisiopatología , Ratones Endogámicos C57BL , Ratones Noqueados , Oxígeno , Daño por Reperfusión/fisiopatología , Factor de Transcripción CHOP/metabolismoRESUMEN
By taking advantage of a "floxed" conditional CREB mutant mouse (CREB1loxP/loxP), in which postnatal deletion of the Creb gene in the forebrain is driven by the calcium/calmodulin-dependent protein kinase II-α gene (Camk2a) promoter (BCKO mice), we here show that selective disruption of CREB function in adult forebrain neurons results, in adult mice, in morphological alterations at the hippocampal level, including hippocampal shrinkage, reduced somal volume of neurons, microgliosis and mild reactive astrocytosis, mainly involving the CA1 subfield. The huge increase of microglial cells showing a mild activated profile, and the higher percentage of double-stained GFAP/S100B astrocytes, together with the increased expression of S100b mRNA at hippocampal level, suggest the establishment of a sub-inflammatory environment in the hippocampus of BCKO mice compared with age-matched controls. Collectively, the present data link neuron-specific, adult deletion of CREB to hippocampal structural alterations and to the early appearance of neuropathological features closely resembling those occurring in the aged brain. This information may be valuable for the understanding of the role of CREB in neuroinflammatory pathways.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Eliminación de Gen , Hipocampo/metabolismo , Mediadores de Inflamación/metabolismo , Neuronas/metabolismo , Factores de Edad , Animales , Astrocitos/metabolismo , Astrocitos/patología , Atrofia/genética , Atrofia/metabolismo , Atrofia/patología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Hipocampo/patología , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Microglía/metabolismo , Microglía/patología , Neuronas/patologíaRESUMEN
Altered glucocorticoid sensitivity is believed to contribute to a number of human diseases, including inflammatory and autoimmune conditions as well as disorders characterized by abnormal hypothalamic-pituitary-adrenal axis (HPA) function. LUMAN (or CREB3), originally identified through its interaction with a cell cycle regulator HCFC1, is an endoplasmic reticulum membrane-bound transcription factor that is involved in the unfolded protein response. Here we demonstrate that LUMAN changes the glucocorticoid response by modulating the expression of the glucocorticoid receptor leading to an overall increase in GR activity. Luman-deficient mice exhibited a blunted stress response characterized by low levels of both anxiety and depressive-like behaviour in addition to low circulating corticosterone levels. These mice also have reduced dendritic branching in the CA3 region of the hippocampus, consistent with increased GR responses. These findings are consistent with the notion that elevated GR activities are the primary cause of the observed phenotype in these LUMAN-deficient mice. We thus postulate that LUMAN is a key regulator of GR-mediated signaling and modulates HPA axis reactivity.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Glucocorticoides/farmacología , Estrés Fisiológico , Animales , Animales Recién Nacidos , Conducta Animal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Región CA3 Hipocampal/metabolismo , Corticosterona/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Dendritas/efectos de los fármacos , Dendritas/metabolismo , Ratones Endogámicos C57BL , Receptores de Glucocorticoides/metabolismo , Estrés Fisiológico/efectos de los fármacos , Análisis de SupervivenciaRESUMEN
CREB3L3 is involved in fatty acid oxidation and ketogenesis in a mutual manner with PPARα. To evaluate relative contribution, a combination of knockout and transgenic mice was investigated. On a ketogenic-diet (KD) that highlights capability of hepatic ketogenesis, Creb3l3-/- mice exhibited reduction of expression of genes for fatty oxidation and ketogenesis comparable to Ppara-/- mice. Most of the genes were further suppressed in double knockout mice indicating independent contribution of hepatic CREB3L3. During fasting, dependency of ketogenesis on CREB3L3 is lesser extents than Ppara-/- mice suggesting importance of adipose PPARα for supply of FFA and hyperlipidemia in Creb3l3-/- mice. In conclusion CREB3L3 plays a crucial role in hepatic adaptation to energy starvation via two pathways: direct related gene regulation and an auto-loop activation of PPARα. Furthermore, as KD-fed Creb3l3-/- mice exhibited severe fatty liver, activating inflammation, CREB3L3 could be a therapeutic target for NAFLD.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Ácidos Grasos/química , PPAR alfa/genética , Adenoviridae/genética , Animales , Glucemia/análisis , Carnitina O-Palmitoiltransferasa/química , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Línea Celular Tumoral , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Dieta Cetogénica , Metabolismo Energético , Ácidos Grasos/metabolismo , Hígado Graso/etiología , Hígado Graso/metabolismo , Factores de Crecimiento de Fibroblastos/sangre , Expresión Génica , Peroxidación de Lípido , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , PPAR alfa/deficiencia , Plásmidos/genética , Plásmidos/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Triglicéridos/sangreRESUMEN
OBJECTIVE: Liver-enriched transcription factor cAMP-responsive element-binding protein H (CREBH) regulates plasma triglyceride clearance by inducing lipoprotein lipase cofactors, such as apolipoprotein A-IV (apoA-IV), apoA-V, and apoC-II. CREBH also regulates apoA-I transcription. This study aims to determine whether CREBH has a role in lipoprotein metabolism and development of atherosclerosis. APPROACH AND RESULTS: CREBH-deficient Creb3l3(-/-) mice were bred with Ldlr(-/-) mice creating Ldlr(-/-) Creb3l3(-/-) double knockout mice. Mice were fed on a high-fat and high-sucrose Western diet for 20 weeks. We showed that CREBH deletion in Ldlr(-/-) mice increased very low-density lipoprotein-associated triglyceride and cholesterol levels, consistent with the impairment of lipoprotein lipase-mediated triglyceride clearance in these mice. In contrast, high-density lipoprotein cholesterol levels were decreased in CREBH-deficient mice, which was associated with decreased production of apoA-I from the liver. The results indicate that CREBH directly activated Apoa1 gene transcription. Accompanied by the worsened atherogenic lipid profile, Ldlr(-/-) Creb3l3(-/-) mice developed significantly more atherosclerotic lesions in the aortas than Ldlr(-/-) mice. CONCLUSIONS: We identified CREBH as an important regulator of lipoprotein metabolism and suggest that increasing hepatic CREBH activity may be a novel strategy for prevention and treatment of atherosclerosis.
Asunto(s)
Aorta/metabolismo , Enfermedades de la Aorta/metabolismo , Aterosclerosis/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Receptores de LDL/deficiencia , Animales , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Apolipoproteína A-I/sangre , Apolipoproteína A-I/genética , Apolipoproteína B-100 , Apolipoproteínas B/sangre , Aterosclerosis/genética , Aterosclerosis/patología , Biomarcadores/sangre , Línea Celular Tumoral , HDL-Colesterol/sangre , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Dieta Alta en Grasa , Sacarosa en la Dieta , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Humanos , Hiperlipidemias/genética , Hiperlipidemias/metabolismo , Lipoproteína Lipasa/metabolismo , Lipoproteínas VLDL/sangre , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Placa Aterosclerótica , Receptores de LDL/genética , Índice de Severidad de la Enfermedad , Transcripción Genética , Activación Transcripcional , Triglicéridos/sangreRESUMEN
cAMP responsive element binding protein 3-like 3 (CREB3L3), a transcription factor expressed in the liver and small intestine, governs fasting-response energy homeostasis. Tissue-specific CREB3L3 knockout mice have not been generated till date. To our knowledge, this is the first study using the one-step CRISPR/Cas9 system to generate CREB3L3 floxed mice and subsequently obtain liver- and small intestine-specific Creb3l3 knockout (LKO and IKO, respectively) mice. While LKO mice as well as global KO mice developed hypertriglyceridemia, LKO mice exhibited hypercholesterolemia in contrast to hypocholesterolemia in global KO mice. LKO mice demonstrated up-regulation of hepatic Srebf2 and its corresponding target genes. No phenotypic differences were observed between IKO and floxed mice. Severe liver injury was observed in LKO mice fed a methionine-choline deficient diet, a model for non-alcoholic steatohepatitis. These results provide new evidence regarding the hepatic CREB3L3 role in plasma triglyceride metabolism and hepatic and intestinal CREB3L3 contributions to cholesterol metabolism.
Asunto(s)
Sistemas CRISPR-Cas/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Hepatitis Animal/etiología , Hiperlipidemias/etiología , Hígado/metabolismo , Animales , Colesterol/metabolismo , Deficiencia de Colina , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Femenino , Hepatitis Animal/metabolismo , Hiperlipidemias/metabolismo , Hiperlipidemias/veterinaria , Insulina/sangre , Intestino Delgado/metabolismo , Hígado/patología , Masculino , Metionina/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Triglicéridos/sangre , Triglicéridos/metabolismo , Regulación hacia ArribaRESUMEN
During mammalian lung development, the morphological transition from respiratory tree branching morphogenesis to a predominantly saccular architecture, capable of air-breathing at birth, is dependent on physical forces as well as molecular signaling by a range of transcription factors including the cAMP response element binding protein 1 (Creb1). Creb1(-/-) mutant mice exhibit complete neonatal lethality consistent with a lack of lung maturation beyond the branching phase. To further define its role in the developing mouse lung, we deleted Creb1 separately in the respiratory epithelium and mesenchyme. Surprisingly, we found no evidence of a morphological lung defect nor compromised neonatal survival in either conditional Creb1 mutant. Interestingly however, loss of mesenchymal Creb1 on a genetic background lacking the related Crem protein showed normal lung development but poor neonatal survival. To investigate the underlying requirement for Creb1 for normal lung development, Creb1(-/-) mice were re-examined for defects in both respiratory muscles and glucocorticoid hormone signaling, which are also required for late stage lung maturation. However, these systems appeared normal in Creb1(-/-) mice. Together our results suggest that the requirement of Creb1 for normal mammalian lung morphogenesis is not dependent upon its expression in lung epithelium or mesenchyme, nor its role in musculoskeletal development.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Epitelio/embriología , Pulmón/embriología , Pulmón/metabolismo , Mesodermo/embriología , Morfogénesis , Factor de Transcripción Activador 1/metabolismo , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Diferenciación Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Diafragma/embriología , Diafragma/metabolismo , Epitelio/metabolismo , Feto/metabolismo , Eliminación de Gen , Glucocorticoides/metabolismo , Mesodermo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Transducción de Señal , Análisis de Supervivencia , Regulación hacia ArribaRESUMEN
OBJECTIVE: Akkermansia muciniphila (A muciniphila) is a mucin-degrading bacterium that resides in the mucus layer whose abundance inversely correlates with body weight and the development of diabetes mellitus in mice and humans. The objective of this study was to explore the regulatory effect of A muciniphila on host lipoprotein metabolism, insulin sensitivity, and hepatic metabolic inflammation. APPROACH AND RESULTS: By establishing a novel mouse model that colonized the A muciniphila in the gastrointestinal tract of the cAMP-responsive binding protein H (CREBH)-deficient mouse and in vivo chylomicron assay, we found that increased colonization of A muciniphila in the gastrointestinal tract of wild-type mice protected mice from an acute fat load-induced hyperlipidemia compared with vehicle-treated mice. A muciniphila administration also significantly ameliorated chronic hypertriglyceridemia, improved insulin sensitivity, and prevented overproduction of postprandial chylomicrons in CREBH-null mice. Mechanistic studies revealed that increased A muciniphila colonization induced expression of low-density lipoprotein receptors and apolipoprotein E in the hepatocytes of CREBH-null mice, which facilitated the uptake of intermediate-density lipoprotein via the mediation of apolipoprotein B100 and apolipoprotein E, leading to the increased clearance of triglyceride-rich lipoprotein remnants, chylomicron remnants, and intermediate-density lipoproteins, from the circulation. Treatment with A muciniphila further improved hepatic endoplasmic reticulum stress and metabolic inflammation in CREBH-null mice. CONCLUSIONS: Increased colonization of the disease-protective gut bacteria A muciniphila protected the host from acute and chronic hyperlipidemia by enhancing the low-density lipoprotein receptor expression and alleviating hepatic endoplasmic reticulum stress and the inflammatory response in CREBH-null mice.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Microbioma Gastrointestinal , Tracto Gastrointestinal/microbiología , Hipertrigliceridemia/prevención & control , Receptores de LDL/metabolismo , Transducción de Señal , Triglicéridos/sangre , Verrucomicrobia/fisiología , Animales , Apolipoproteína B-100/metabolismo , Apolipoproteínas E/metabolismo , Biomarcadores/sangre , Quilomicrones/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo , Estrés del Retículo Endoplásmico , Predisposición Genética a la Enfermedad , Interacciones Huésped-Patógeno , Hipertrigliceridemia/sangre , Hipertrigliceridemia/genética , Hipertrigliceridemia/microbiología , Resistencia a la Insulina , Lipoproteínas IDL/metabolismo , Hígado/metabolismo , Hígado/microbiología , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Factores de TiempoRESUMEN
BACKGROUND & AIMS: Hepatic gluconeogenesis provides fuel during starvation, and is abnormally induced in obese individuals or those with diabetes. Common metabolic disorders associated with active gluconeogenesis and insulin resistance (obesity, metabolic syndrome, diabetes, and nonalcoholic fatty liver disease) have been associated with alterations in iron homeostasis that disrupt insulin sensitivity and promote disease progression. We investigated whether gluconeogenic signals directly control Hepcidin, an important regulator of iron homeostasis, in starving mice (a model of persistently activated gluconeogenesis and insulin resistance). METHODS: We investigated hepatic regulation of Hepcidin expression in C57BL/6Crl, 129S2/SvPas, BALB/c, and Creb3l3-/- null mice. Mice were fed a standard, iron-balanced chow diet or an iron-deficient diet for 9 days before death, or for 7 days before a 24- to 48-hour starvation period; liver and spleen tissues then were collected and analyzed by quantitative reverse-transcription polymerase chain reaction and immunoblot analyses. Serum levels of iron, hemoglobin, Hepcidin, and glucose also were measured. We analyzed human hepatoma (HepG2) cells and mouse primary hepatocytes to study transcriptional control of Hamp (the gene that encodes Hepcidin) in response to gluconeogenic stimuli using small interfering RNA, luciferase promoter, and chromatin immunoprecipitation analyses. RESULTS: Starvation led to increased transcription of the gene that encodes phosphoenolpyruvate carboxykinase 1 (a protein involved in gluconeogenesis) in livers of mice, increased levels of Hepcidin, and degradation of Ferroportin, compared with nonstarved mice. These changes resulted in hypoferremia and iron retention in liver tissue. Livers of starved mice also had increased levels of Ppargc1a mRNA and Creb3l3 mRNA, which encode a transcriptional co-activator involved in energy metabolism and a liver-specific transcription factor, respectively. Glucagon and a cyclic adenosine monophosphate analog increased promoter activity and transcription of Hamp in cultured liver cells; levels of Hamp were reduced after administration of small interfering RNAs against Ppargc1a and Creb3l3. PPARGC1A and CREB3L3 bound the Hamp promoter to activate its transcription in response to a cyclic adenosine monophosphate analog. Creb3l3-/- mice did not up-regulate Hamp or become hypoferremic during starvation. CONCLUSIONS: We identified a link between glucose and iron homeostasis, showing that Hepcidin is a gluconeogenic sensor in mice during starvation. This response is involved in hepatic metabolic adaptation to increased energy demands; it preserves tissue iron for vital activities during food withdrawal, but can cause excessive iron retention and hypoferremia in disorders with persistently activated gluconeogenesis and insulin resistance.
Asunto(s)
Gluconeogénesis , Hepatocitos/metabolismo , Hepcidinas/sangre , Hierro/sangre , Hígado/metabolismo , Transducción de Señal , Inanición/sangre , Animales , Sitios de Unión , Glucemia/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Modelos Animales de Enfermedad , Hemoglobinas/metabolismo , Células Hep G2 , Homeostasis , Humanos , Resistencia a la Insulina , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Regiones Promotoras Genéticas , Interferencia de ARN , Bazo/metabolismo , Inanición/genética , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Transfección , Regulación hacia ArribaRESUMEN
Bile acids concentration in liver is tightly regulated to prevent cell damage. Previous studies have demonstrated that deregulation of bile acid homeostasis can lead to cholestatic liver disease. Recently, we have shown that ER-bound transcription factor Crebh is a downstream effector of hepatic Cb1r signaling pathway. In this study, we have investigated the effect of alcohol exposure on hepatic bile acid homeostasis and elucidated the mediatory roles of Cb1r and Crebh in this process. We found that alcohol exposure or Cb1r-agonist 2-AG treatment increases hepatic bile acid synthesis and serum ALT, AST levels in vivo alongwith significant increase in Crebh gene expression and activation. Alcohol exposure activated Cb1r, Crebh, and perturbed bile acid homeostasis. Overexpression of Crebh increased the expression of key bile acid synthesis enzyme genes via direct binding of Crebh to their promoters, whereas Cb1r knockout and Crebh-knockdown mice were protected against alcohol-induced perturbation of bile acid homeostasis. Interestingly, insulin treatment protected against Cb1r-mediated Crebh-induced disruption of bile acid homeostasis. Furthermore, Crebh expression and activation was found to be markedly increased in insulin resistance conditions and Crebh knockdown in diabetic mice model (db/db) significantly reversed alcohol-induced disruption of bile acid homeostasis. Overall, our study demonstrates a novel regulatory mechanism of hepatic bile acid metabolism by alcohol via Cb1r-mediated activation of Crebh, and suggests that targeting Crebh can be of therapeutic potential in ameliorating alcohol-induced perturbation of bile acid homeostasis.
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Ácidos y Sales Biliares/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Etanol/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hígado/enzimología , Receptor Cannabinoide CB1/metabolismo , Animales , Ácidos y Sales Biliares/biosíntesis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Endocannabinoides/farmacología , Eliminación de Gen , Células Hep G2 , Homeostasis/efectos de los fármacos , Humanos , Insulina/deficiencia , Insulina/farmacología , Resistencia a la Insulina , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Receptor Cannabinoide CB1/deficiencia , Receptor Cannabinoide CB1/genética , Activación Transcripcional/efectos de los fármacosRESUMEN
Cyclic AMP Response Element-Binding Protein 1 (Creb1) is a transcription factor that mediates cyclic adenosine 3', 5'-monophosphate (cAMP) signalling in many tissues. Creb1(-/-) mice die at birth due to respiratory failure and previous genome-wide microarray analysis of E17.5 Creb1(-/-) fetal mouse lung identified important Creb1-regulated gene targets during lung development. The lipogenic enzymes stearoyl-CoA desaturase 1 (Scd1) and fatty acid synthase (Fasn) showed highly reduced gene expression in Creb1(-/-) lungs. We therefore hypothesized that Creb1 plays a crucial role in the transcriptional regulation of genes involved in pulmonary lipid biosynthetic pathways during lung development. In this study we confirmed that Scd1 and Fasn mRNA levels were down regulated in the E17.5 Creb1(-/-) mouse lung while the lipogenic-associated transcription factors SrebpF1, C/ebpα and Pparγ were increased. In vivo studies using germline (Creb1(-/-) ) and lung epithelial-specific (Creb1(EpiΔ/Δ) ) Creb1 knockout mice showed strongly reduced Scd1, but not Fasn gene expression and protein levels in lung epithelial cells. In vitro studies using mouse MLE-15 epithelial cells showed that forskolin-mediated activation of Creb1 increased both Scd1 gene expression and protein synthesis. Additionally, MLE15 cells transfected with a dominant-negative ACreb vector blocked forskolin-mediated stimulation of Scd1 gene expression. Lipid profiling in MLE15 cells showed that dominant-negative ACreb suppressed forskolin-induced desaturation of ether linked lipids to produce plasmalogens, as well as levels of phosphatidylethanolamine, ceramide and lysophosphatidylcholine. Taken together these results demonstrate that Creb1 is essential for the induction and maintenance of Scd1 in developing fetal mouse lung epithelial cells.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Ácido Graso Sintasas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Pulmón/metabolismo , Alveolos Pulmonares/embriología , Estearoil-CoA Desaturasa/metabolismo , Animales , Línea Celular , Colforsina/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Células Epiteliales/metabolismo , Metabolismo de los Lípidos/genética , Pulmón/embriología , Ratones , Ratones Transgénicos , Regulación hacia ArribaRESUMEN
The gastrointestinal (GI) epithelium is constantly renewing, depending upon the intestinal stem cells (ISC) regulated by a spectrum of transcription factors (TFs), including Myb. We noted previously in mice with a p300 mutation (plt6) within the Myb-interaction-domain phenocopied Myb hypomorphic mutant mice with regard to thrombopoiesis, and here, changes in GI homeostasis. p300 is a transcriptional coactivator for many TFs, most prominently cyclic-AMP response element-binding protein (CREB), and also Myb. Studies have highlighted the importance of CREB in proliferation and radiosensitivity, but not in the GI. This prompted us to directly investigate the p300-Myb-CREB axis in the GI. Here, the role of CREB has been defined by generating GI-specific inducible creb knockout (KO) mice. KO mice show efficient and specific deletion of CREB, with no evident compensation by CREM and ATF1. Despite complete KO, only modest effects on proliferation, radiosensitivity and differentiation in the GI under homeostatic or stress conditions were evident, even though CREB target gene pcna (proliferating cell nuclear antigen) was downregulated. creb and p300 mutant lines show increased goblet cells, whereas a reduction in enteroendocrine cells was apparent only in the p300 line, further resembling the Myb hypomorphs. When propagated in vitro, crebKO ISC were defective in organoid formation, suggesting that the GI stroma compensates for CREB loss in vivo, unlike in MybKO studies. Thus, it appears that p300 regulates GI differentiation primarily through Myb, rather than CREB. Finally, active pCREB is elevated in colorectal cancer (CRC) cells and adenomas, and is required for the expression of drug transporter, MRP2, associated with resistance to Oxaliplatin as well as several chromatin cohesion protein that are relevant to CRC therapy. These data raise the prospect that CREB may have a role in GI malignancy as it does in other cancer types, but unlike Myb, is not critical for GI homeostasis.
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Colon/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Intestino Delgado/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Secuencia de Aminoácidos , Animales , Transformación Celular Neoplásica , Células Cultivadas , Colon/patología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Intestino Delgado/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Mutación , Organoides/citología , Organoides/efectos de los fármacos , Organoides/metabolismo , Compuestos Organoplatinos/farmacología , Oxaliplatino , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-myb/genética , Tolerancia a Radiación , Alineación de Secuencia , Irradiación Corporal Total , Factores de Transcripción p300-CBP/genéticaRESUMEN
Although the pyrazolone derivative sulpyrine is widely used as an antipyretic analgesic drug, side effects, including fatal shock, have been reported. However, the molecular mechanism underlying such a severe side effect is largely unclear. Here, we report that the transcription factor CREBH that is highly expressed in the liver plays an important role in fatal shock induced by sulpyrine in mice. CREBH-deficient mice were resistant to experimental fatal sulpyrine shock. We found that sulpyrine-induced expression of cytochrome P450 2B (CYP2B) family genes, which are involved in sulpyrine metabolism, in the liver was severely impaired in CREBH-deficient mice. Moreover, introduction of CYP2B in CREBH-deficient liver restored susceptibility to sulpyrine. Furthermore, ectopic expression of CREBH up-regulated CYP2B10 promoter activity, and in vivo knockdown of CREBH in wild-type mice conferred a significant resistance to fatal sulpyrine shock. These data demonstrate that CREBH is a positive regulator of CYP2B in response to sulpyrine administration, which possibly results in fatal shock.