RESUMEN
Maintenance of hepatocyte homeostasis plays an important role in mediating the pathogenesis of many diseases. A growing body of literature has established a critical role played by tumor necrosis factor-α (TNFα) in maintaining hepatocyte homeostasis; however, the transcriptional mechanisms underlying constitutive Tnf expression are unknown. Whole liver fractions and primary hepatocytes from adult control C57BL/6 mice and the murine hepatocyte cell line AML12 were assessed for constitutive Tnf expression. Impacts of glycogen synthase kinase-3 ß (GSK3ß) and nuclear factor κB (NF-κB) inhibition on constitutive Tnf expression were assessed in AML12 cells. Finally, AML12 cell proliferation following GSK3ß and NF-κB inhibition was evaluated. Constitutive Tnf gene expression is present in whole liver, primary hepatocytes, and cultured AML12 hepatocytes. Cytokine-induced Tnf gene expression is regulated by NF-κB activation. Pharmacological inhibition of GSK3ß resulted in a time- and dose-dependent inhibition of Tnf gene expression. GSK3ß inhibition decreased nuclear levels of the NF-κB subunits p65 and p50. We determined that NF-κB transcription factor subunit p65 binds to consensus sequence elements present in the murine TNFα promoter and inhibition of GSK3ß decreases binding and subsequent Tnf expression. Finally, AML12 cell growth was significantly reduced following GSK3ß and NF-κB inhibition. These results demonstrate that GSK3ß and NF-κB are essential for mediating Tnf expression and constitutive hepatocyte cell growth. These findings add to a growing body of literature on TNFα mediated hepatocyte homeostasis and identify novel molecular mechanisms involved in mediating response to various disease states in the liver.NEW & NOTEWORTHY Maintenance of hepatocyte homeostasis plays an important role in controlling the pathogenesis of many diseases. Our findings add to a growing body of literature on tumor necrosis factor-α (TNFα)-mediated hepatocyte homeostasis and identify novel molecular mechanisms involved in regulating this response.
Asunto(s)
FN-kappa B , Factor de Transcripción ReIA , Factor de Necrosis Tumoral alfa , Animales , Ratones , Expresión Génica , Glucógeno Sintasa Quinasa 3 beta , Hepatocitos/metabolismo , Homeostasis , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Transcripción ReIA/metabolismoRESUMEN
BACKGROUND: The developmental immaturity of the innate immune system helps explains the increased risk of infection in the neonatal period. Importantly, innate immune signaling pathways such as p65/NFκB and c-Jun/AP1 are responsible for the prevention of hepatocyte apoptosis in adult animals, yet whether developmental immaturity of these pathways increases the risk of hepatic injury in the neonatal period is unknown. METHODS: Using a murine model of endotoxemia (LPS 5 mg/kg IP x 1) in neonatal (P3) and adult mice, we evaluated histologic evidence of hepatic injury and apoptosis, presence of p65/NFκB and c-Jun/AP1 activation and associated transcriptional regulation of apoptotic genes. RESULTS: We demonstrate that in contrast to adults, endotoxemic neonatal (P3) mice exhibit a significant increase in hepatic apoptosis. This is associated with absent hepatic p65/NFκB signaling and impaired expression of anti-apoptotic target genes. Hepatic c-Jun/AP1 activity was attenuated in endotoxemic P3 mice, with resulting upregulation of pro-apoptotic factors. CONCLUSIONS: These results demonstrate that developmental absence of innate immune p65/NFκB and c-Jun/AP1 signaling, and target gene expression is associated with apoptotic injury in neonatal mice. More work is needed to determine if this contributes to long-term hepatic dysfunction, and whether immunomodulatory approaches can prevent this injury. IMPACT: Various aspects of developmental immaturity of the innate immune system may help explain the increased risk of infection in the neonatal period. In adult models of inflammation and infection, innate immune signaling pathways such as p65/NFκB and c-Jun/AP1 are responsible for a protective, pro-inflammatory transcriptome and regulation of apoptosis. We demonstrate that in contrast to adults, endotoxemic neonatal (P3) mice exhibit a significant increase in hepatic apoptosis associated with absent hepatic p65/NFκB signaling and c-Jun/AP1 activity. We believe that these results may explain in part hepatic dysfunction with neonatal sepsis, and that there may be unrecognized developmental and long-term hepatic implications of early life exposure to systemic inflammatory stress.
Asunto(s)
Animales Recién Nacidos , Apoptosis , Endotoxemia , Inmunidad Innata , Hígado , Transducción de Señal , Factor de Transcripción AP-1 , Animales , Factor de Transcripción AP-1/metabolismo , Hígado/metabolismo , Hígado/inmunología , Hígado/patología , Ratones , Endotoxemia/inmunología , Endotoxemia/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción ReIA/metabolismo , Lipopolisacáridos , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-jun/metabolismo , Modelos Animales de EnfermedadRESUMEN
BACKGROUND AND AIMS: Parenteral nutrition (PN)-associated cholestasis (PNAC) complicates the care of patients with intestinal failure. In PNAC, phytosterol containing PN synergizes with intestinal injury and IL-1ß derived from activated hepatic macrophages to suppress hepatocyte farnesoid X receptor (FXR) signaling and promote PNAC. We hypothesized that pharmacological activation of FXR would prevent PNAC in a mouse model. APPROACH AND RESULTS: To induce PNAC, male C57BL/6 mice were subjected to intestinal injury (2% dextran sulfate sodium [DSS] for 4 days) followed by central venous catheterization and 14-day infusion of PN with or without the FXR agonist GW4064. Following sacrifice, hepatocellular injury, inflammation, and biliary and sterol transporter expression were determined. GW4064 (30 mg/kg/day) added to PN on days 4-14 prevented hepatic injury and cholestasis; reversed the suppressed mRNA expression of nuclear receptor subfamily 1, group H, member 4 (Nr1h4)/FXR, ATP-binding cassette subfamily B member 11 (Abcb11)/bile salt export pump, ATP-binding cassette subfamily C member 2 (Abcc2), ATP binding cassette subfamily B member 4(Abcb4), and ATP-binding cassette subfamily G members 5/8(Abcg5/8); and normalized serum bile acids. Chromatin immunoprecipitation of liver showed that GW4064 increased FXR binding to the Abcb11 promoter. Furthermore, GW4064 prevented DSS-PN-induced hepatic macrophage accumulation, hepatic expression of genes associated with macrophage recruitment and activation (ll-1b, C-C motif chemokine receptor 2, integrin subunit alpha M, lymphocyte antigen 6 complex locus C), and hepatic macrophage cytokine transcription in response to lipopolysaccharide in vitro. In primary mouse hepatocytes, GW4064 activated transcription of FXR canonical targets, irrespective of IL-1ß exposure. Intestinal inflammation and ileal mRNAs (Nr1h4, Fgf15, and organic solute transporter alpha) were not different among groups, supporting a liver-specific effect of GW4064 in this model. CONCLUSIONS: GW4064 prevents PNAC in mice through restoration of hepatic FXR signaling, resulting in increased expression of canalicular bile and of sterol and phospholipid transporters and suppression of macrophage recruitment and activation. These data support augmenting FXR activity as a therapeutic strategy to alleviate or prevent PNAC.
Asunto(s)
Colestasis/prevención & control , Expresión Génica/efectos de los fármacos , Isoxazoles/farmacología , Nutrición Parenteral/efectos adversos , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/genética , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/genética , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/genética , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/genética , Animales , Ácidos y Sales Biliares/sangre , Colestasis/etiología , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/metabolismo , Interleucina-1beta/farmacología , Enfermedades Intestinales/inducido químicamente , Enfermedades Intestinales/terapia , Isoxazoles/uso terapéutico , Lipoproteínas/genética , Hepatopatías/etiología , Hepatopatías/patología , Hepatopatías/prevención & control , Activación de Macrófagos/efectos de los fármacos , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
ATP-binding cassette, subfamily B member 11 (ABCB11) is an efflux transporter for bile acids on the liver canalicular membrane. The expression of this transporter is reduced in cholestasis; however, the mechanisms contributing to this reduction are unclear. In this study, we sought to determine whether miR-199a-5p contributes to the depletion of ABCB11/Abcb11 in cholestasis in mice. In a microRNA (miRNA) screen of mouse liver after common bile duct ligation (CBDL), we found that miR-199a-5p was significantly upregulated by approximately fourfold. In silico analysis predicted that miR-199a-5p would target the 3'-untranslated region (3'-UTR) of ABCB11/Abcb11 mRNA. The expression of ABCB11-3'-UTR luciferase construct in Huh-7 cells was markedly inhibited by cotransfection of a miRNA-199a-5p mimic, which was reversed by an miRNA-199a-5p mimic inhibitor. We also show treatment of mice after CBDL with the potent nuclear receptor FXR agonist obeticholic acid (OCA) significantly increased Abcb11 mRNA and protein and decreased miR-199a-5p expression. Computational mapping revealed a well-conserved FXR-binding site (FXRE) in the promoter of the gene encoding miR-199a-5, termed miR199a-2. Electromobility shift, chromatin immunoprecipitation, and miR199a-2 promoter-luciferase assays confirmed that this binding site was functional. Finally, CBDL in mice led to depletion of nuclear repressor NcoR1 binding at the miR199a-2 promoter, which facilitates transcription of miR199a-2. In CBDL mice treated with OCA, NcoR1 recruitment to the miR199a-2 FXRE was maintained at levels found in sham-operated mice. In conclusion, we demonstrate that miR-199a-5p is involved in regulating ABCB11/Abcb11 expression, is aberrantly upregulated in obstructive cholestasis, and is downregulated by the FXR agonist OCA.
Asunto(s)
Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/biosíntesis , Colestasis/metabolismo , Regulación de la Expresión Génica , MicroARNs/metabolismo , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/genética , Animales , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/farmacología , Colestasis/tratamiento farmacológico , Colestasis/genética , Colestasis/patología , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Noqueados , MicroARNs/genética , Co-Represor 1 de Receptor Nuclear/genética , Co-Represor 1 de Receptor Nuclear/metabolismoRESUMEN
BACKGROUND: Biliary atresia (BA) is an inflammatory pediatric cholangiopathy with only surgical means for treatment. Many contributors to bile acid synthesis and transport have previously been reported to be downregulated in patients with BA; yet, the driving factors of the abnormal bile acid synthesis and transport in regard to BA have not been previously studied. MATERIALS AND METHODS: Wild type or Ig-α-/- mice were injected with salt solution (control) or rotavirus on day of life 0, and analyses were performed on day of life 14. The mRNA levels of bile acid transporters/nuclear receptors and liver microRNAs (miRNAs) were compared between groups. A mouse hepatocyte cell line was used to examine the effects of innate cytokines on miRNA levels and bile acid transporter/nuclear receptor expression and miRNAs on bile acid transporter/nuclear receptor expression. RESULTS: BA mice had significantly increased mRNA expression of innate cytokines and miRNAs known to bind bile acid transporters/nuclear receptors (miRNAs -22-5p, -34a-5p, and -222-3p) and decreased mRNA expression of bile acid transporters and nuclear receptors. In vitro, TNF-α and IL-1ß decreased BSEP and CYP7A1 while increasing miRNA-34a-5p and miRNA 222-3p. LXR, SHP, CYP7A1, NTCP, and MRP2 were decreased by miRNA-34a-5p, whereas miRNA-222-3p decreased NTCP and MRP4. TNF-α and IL-1ß increased expression of miRNAs 34a-5p and 222-3p and these miRNAs then decrease expression of multiple bile acid transporters and nuclear receptors. CONCLUSIONS: Loss of bile acid transporters increases hepatotoxicity via bile acid retention. Therapeutic agents that increase bile acid transport or nuclear receptor functioning should be investigated in BA.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Atresia Biliar/inmunología , Colestasis/inmunología , Inflamación/genética , MicroARNs/metabolismo , Animales , Conductos Biliares/inmunología , Conductos Biliares/patología , Atresia Biliar/patología , Antígenos CD79/genética , Antígenos CD79/inmunología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Colestasis/patología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/inmunología , Hepatocitos/metabolismo , Humanos , Inflamación/complicaciones , Inflamación/inmunología , Hígado/citología , Hígado/inmunología , Hígado/patología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
The control of transcription is regulated through the well-coordinated spatial and temporal interactions between distal genomic regulatory elements required for specialized cell-type and developmental gene expression programs. With recent findings CFTR has served as a model to understand the principles that govern genome-wide and topological organization of distal intra-chromosomal contacts as it relates to transcriptional control. This is due to the extensive characterization of the DNase hypersensitivity sites, modification of chromatin, transcription factor binding sites and the arrangement of these sites in CFTR consistent with the restrictive expression in epithelial cell types. Here, we identified CHD6 from a screen among several chromatin-remodeling proteins as a putative epigenetic modulator of CFTR expression. Moreover, our findings of CTCF interactions with CHD6 are consistent with the role described previously for CTCF in CFTR regulation. Our results now reveal that the CHD6 protein lies within the infrastructure of multiple transcriptional complexes, such as the FACT, PBAF, PAF1C, Mediator, SMC/Cohesion and MLL complexes. This model underlies the fundamental role CHD6 facilitates by tethering cis-acting regulatory elements of CFTR in proximity to these multi-subunit transcriptional protein complexes. Finally, we indicate that CHD6 structurally coordinates a three-dimensional stricture between intragenic elements of CFTR bound by several cell-type specific transcription factors, such as CDX2, SOX18, HNF4α and HNF1α. Therefore, our results reveal new insights into the epigenetic regulation of CFTR expression, whereas the manipulation of CFTR gene topology could be considered for treating specific indications of cystic fibrosis and/or pancreatitis.
Asunto(s)
Cromatina/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , ADN Helicasas/metabolismo , Sitios Genéticos , Proteínas del Tejido Nervioso/metabolismo , Elementos Reguladores de la Transcripción , Epigénesis Genética , Humanos , Conformación de Ácido Nucleico , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/metabolismoRESUMEN
Elevated serum concentrations of the vasoactive protein endothelin-1 (ET-1) occur in the setting of systemic inflammatory response syndrome and contribute to distal organ hypoperfusion and pulmonary hypertension. Thus, understanding the cellular source and transcriptional regulation of systemic inflammatory stress-induced ET-1 expression may reveal therapeutic targets. Using a murine model of LPS-induced septic shock, we demonstrate that the hepatic macrophage is the primary source of elevated circulating ET-1, rather than the endothelium as previously proposed. Using pharmacologic inhibitors, ET-1 promoter luciferase assays, and by silencing and overexpressing NF-κB inhibitory protein IκB expression, we demonstrate that LPS-induced ET-1 expression occurs via an NF-κB-dependent pathway. Finally, the specific role of the cRel/p65 inhibitory protein IκBß was evaluated. Although cytoplasmic IκBß inhibits activity of cRel-containing NF-κB dimers, nuclear IκBß stabilizes NF-κB/DNA binding and enhances gene expression. Using targeted pharmacologic therapies to specifically prevent IκBß/NF-κB signaling, as well as mice genetically modified to overexpress IκBß, we show that nuclear IκBß is both necessary and sufficient to drive LPS-induced ET-1 expression. Together, these results mechanistically link the innate immune response mediated by IκBß/NF-κB to ET-1 expression and potentially reveal therapeutic targets for patients with Gram-negative septic shock.
Asunto(s)
Endotelina-1/inmunología , Endotoxemia/inmunología , Regulación de la Expresión Génica/inmunología , Proteínas I-kappa B/inmunología , Hígado/inmunología , Macrófagos/inmunología , FN-kappa B/inmunología , Transducción de Señal/inmunología , Animales , Línea Celular , Endotelina-1/genética , Endotoxemia/genética , Endotoxemia/patología , Proteínas I-kappa B/genética , Hígado/patología , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , FN-kappa B/genética , Transducción de Señal/genéticaRESUMEN
In this study we identified the mechanisms underlying the inhibitory effects of NF-κB on the expression of genes encoding multiple liver transport proteins. Well-conserved NF-κB binding sites were found in the promoters of farnesoid X receptor (FXR)-target genes. An electromobility shift assay (EMSA) demonstrated the specific interaction between the NF-κB p65 protein and a (32)P-labeled BSEP NF-κB response element (NF-κBE). Chromatin immunoprecipitation (ChIP) analysis confirmed binding of NF-κB p65 to the BSEP locus but not the FXRE in vitro. NF-κB p65 overexpression in Huh-7 cells markedly repressed FXR/RXR transactivation of the BSEP, ABCG5/G8, MRP2, and FXR promoters, which was totally reversed by expression of the IκBα super-repressor. NF-κB interacted directly with FXR on coimmunoprecipitation, suggesting another level for the inhibitory effects of NF-κB on FXR-target genes. In vivo ChIP analysis with liver nuclei obtained from mice after 3 days of common bile duct ligation (BDL) or 6 h post-lipopolysaccharide (LPS) injection showed a markedly increased recruitment of NF-κB p65 to the Bsep promoter compared with controls. There was also increased recruitment of the corepressor silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and histone deacetylase (HDAC)3 and HDAC2 to the NF-κB sites. We also found that NF-κB p65 was recruited to NF-κB binding sites in the promoters of organic solute transporter, OSTα and OSTß, and unexpectedly activated rather than repressed gene expression. In mouse liver after BDL NF-κB recruitment to Ostα and Ostß promoters was associated with increased binding of the potent coactivator cAMP response element binding protein (CREB)-binding protein (CBP)/p300 to the NF-κBE and depletion of CBP/p300 at the FXR element. Overall, these studies demonstrate a novel role for NF-κB in adaptation to obstructive and LPS-induced cholestasis acting through recruitment to specific NF-κB binding sites in the promoters of FXR-target genes and possibly through direct interaction with FXR.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Hígado/metabolismo , Elementos de Respuesta , Factor de Transcripción ReIA/metabolismo , Activación Transcripcional , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Línea Celular Tumoral , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Co-Represor 2 de Receptor Nuclear/genética , Co-Represor 2 de Receptor Nuclear/metabolismo , Unión ProteicaRESUMEN
BACKGROUND: Small ubiquitin-like modifiers (SUMO) are covalently conjugated to other proteins including nuclear receptors leading to modification of various cellular processes. RESULTS: Ligand-dependent SUMOylation of farnesoid X receptor (FXR) negatively regulates the expression of its target genes. CONCLUSION: SUMO modification attenuates the capacity of FXR to function as a transcriptional activator. SIGNIFICANCE: Defining post-translation modification of FXR bySUMOis important to understanding how this nuclear receptor functions in health and disease. The farnesoid X receptor (FXR) belongs to a family of ligand-activated transcription factors that regulate many aspects of metabolism including bile acid homeostasis. Here we show that FXR is covalently modified by the small ubiquitin-like modifier (Sumo1), an important regulator of cell signaling and transcription. Well conserved consensus sites at lysine 122 and 275 in the AF-1 and ligand binding domains, respectively, of FXR were subject to SUMOylation in vitro and in vivo. Chromatin immunoprecipitation (ChIP) analysis showed that Sumo1 was recruited to the bile salt export pump (BSEP), the small heterodimer partner (SHP), and the OSTα-OSTß organic solute transporter loci in a ligand-dependent fashion. Sequential chromatin immunoprecipitation (ChIP-ReChIP) verified the concurrent binding of FXR and Sumo1 to the BSEP and SHP promoters. Overexpression of Sumo1 markedly decreased binding and/or recruitment of FXR to the BSEP and SHP promoters on ChIP-ReChIP. SUMOylation did not have an apparent effect on nuclear localization of FXR. Expression of Sumo1 markedly inhibited the ligand-dependent, transactivation of BSEP and SHP promoters by FXR/retinoid X receptor α (RXRα) in HepG2 cells. In contrast, mutations that abolished SUMOylation of FXR or siRNA knockdown of Sumo1 expression augmented the transactivation of BSEP and SHP promoters by FXR. Pathways for SUMOylation were significantly altered during obstructive cholestasis with differential Sumo1 recruitment to the promoters of FXR target genes. In conclusion, FXR is subject to SUMOylation that regulates its capacity to transactivate its target genes in normal liver and during obstructive cholestasis.
Asunto(s)
Regulación de la Expresión Génica , Receptores Citoplasmáticos y Nucleares/metabolismo , Sumoilación , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/genética , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Colestasis/metabolismo , Cromatina/metabolismo , Secuencia de Consenso , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Unión Proteica , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , ARN Interferente Pequeño/genética , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/genética , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Transcripción GenéticaRESUMEN
Researchers have been motivated to develop photovoltaic systems that can efficiently convert artificial light into power with the growing use of indoor electrical devices for the Internet of Things. Understanding the impact of molecular design strategies involving morphological optimization through the terminal group of the non-fullerene acceptors (NFAs) is crucial. This is critically important to enhancing the photovoltaic efficiency of organic photovoltaic devices under diverse irradiation conditions. Halogenation of terminal groups proves to be a standout approach for adjusting energy levels, refining light-harvesting capabilities, crystallinity, and bolstering the intermolecular stacking in NFAs. Herein, we have designed two simple NFAs, DICTF-4F and DICTF-4Cl, to explore the dihalogenation (F and Cl) effect on the terminal group on the optical and electrochemical properties. After combining with the BODIPY-thiophene-backboned donor polymer P(BdP-HT), the organic solar cells (OSCs) using an optimized active layer with P(BdP-HT):DICTF-4F and P(BdP-HT):DICTF-4Cl attained a power conversion efficiency (PCE) of about 8.03 and 14.16%, respectively, under 1 sun illumination. Moreover, the OSC-based P(BdP-HT):DICTF-4Cl active layer showed a PCE approaching 24% under 1000 lx indoor conditions.
RESUMEN
Base amino acid lysine residues play an important role in regulation of nuclear receptors [e.g., farnesyl X receptor (FXR)], leading to enhanced or suppressed biologic activity. To understand the molecular mechanisms and the subsequent effects in modulating FXR functions in diverse biologic processes, we individually replaced eight highly conserved lysine residues of human FXR (hFXR) with arginine. The effects of each mutated FXR on target gene activation, subcellular localization, protein-protein association, and protein-DNA interaction were investigated. Results demonstrated that K122R, K210R, K339R, and K460R mutants of hFXR significantly impaired target gene [organic solute transporter α/ß and bile salt export pump (BSEP)] promoter reporter activity in a ligand-dependent fashion. None of the four mutants affected the nuclear localization of FXR. Protein interaction studies show that K210R slightly but significantly decreased FXR/retinoid X receptor (RXR) binding affinity but enhanced the interaction of FXR with lysine methyltransferase Set7/9 by â¼21%. K460R decreased the FXR interaction with Set7/9 by â¼45% but had no significant effects on interaction with RXR. Electrophoretic mobility shift assays demonstrated that hFXR-K210R and -K339R reduced the protein-DNA (IR1 element at hBSEP promoter) binding affinity by â¼80 and â¼90%, respectively. Computational-based protein modeling studies were consistent with these results and provided further insights into the potential underlying mechanisms responsible for these results. In conclusion, four highly conserved lysine residues of hFXR, K122, K210, K339, and K460, have been identified that play a critical role in FXR target gene regulation and molecular interaction (protein-protein and protein-DNA).
Asunto(s)
Lisina/genética , Lisina/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Activación Transcripcional/genética , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Arginina/genética , Arginina/metabolismo , Células CACO-2 , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ligandos , Metiltransferasas/genética , Metiltransferasas/metabolismo , Datos de Secuencia Molecular , Mutación/efectos de los fármacos , Mutación/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Dominios y Motivos de Interacción de Proteínas/genética , Receptores X Retinoide/genética , Receptores X Retinoide/metabolismo , Activación Transcripcional/efectos de los fármacosRESUMEN
Establishing an S-scheme heterojunction is a promising method for increasing the photocatalytic activity of synthetic materials. In this study, nitrogen-doped g-C3N5/TiO2 S-scheme photocatalysts have been synthesized and examined for photocatalytic hydrogen production using thermal decomposition methods. Nitrogen-doped g-C3N5/TiO2 composites performed better than pure nitrogen-doped g-C3N5 and TiO2 alone. Using experiments and density functional theory (DFT) calculations, nitrogen (N) doping was identified as being introduced by replacing the carbon (C) atoms in the matrix of g-C3N5. In addition to its narrow band gap, N-doped g-C3N5 showed efficient carrier separation and charge transfer, resulting in the enhanced absorption of visible light and photocatalytic activity. DFT, XPS, optical property characteristics, and PL spectra confirmed these findings, which were attributed to the successful nitrogen doping, and the composite was proven to be a potential candidate for photocatalytic hydrogen generation under light irradiation. The quantity of H2 produced from the nitrogen-doped g-C3N5/TiO2 composite for 3 hours (3515.1 µmol g-1) was about three times that of N-doped g-C3N5. The H2 production percentage of the nitrogen-doped g-C3N5/TiO2 catalyst with Pt as the cocatalyst was improved by nearly ten times as compared to N-doped g-C3N5/TiO2 without a cocatalyst. Herein, we report the successful preparation of the N-doped g-C3N5/TiO2 S-scheme heterojunction and highlight a simple and efficient catalyst for energy storage requirements and environmental monitoring.
RESUMEN
Herein, simple acceptor-donor-acceptor (A-D-A)-type small molecules denoted as DICTF and DRCTF with modification in terminal units were synthesized and used as electron acceptors. With the tuning of the electron-withdrawing units in electron acceptors, their photovoltaic properties were investigated when combined with low-band-gap BODIPY-thiophene-backboned donor material, named P(BdP-HT). The P(BdP-HT):DICTF-based organic solar cells (OSCs) displayed excellent efficiency of around 11.94%, which is superior to the P(BdP-HT):DRCTF counterpart (8.78%). Although the open-circuit voltage (VOC) of the P(BdP-HT):DRCTF-based OSC is greater than that for the P(BdP-HT):DICTF counterpart, the rise in the short-circuit current density (JSC) may be attributed to the fact that the P(BdP-HT):DICTF blend displayed impressive panchromatic absorption compared to P(BdP-HT):DRCTF. The improved fill factor (FF) is responsible for the balanced transport of charges in the P(BdP-HT):DICTF-based device. Moreover, the P(BdP-HT):DRCTF- and P(BdP-HT):DICTF-based OSCs showed 17.68 and 21.84%, respectively, under indoor illumination (1000 lx). To the best of our observation, this might be the first report on BODIPY-based donors with power conversion efficiency (PCE) of 21.84% under indoor illumination conditions.
RESUMEN
The farnesoid X receptor (FXR) is a ligand (bile acid)-dependent nuclear receptor that regulates target genes involved in every aspect of bile acid homeostasis. Upon binding of ligand, FXR recruits an array of coactivators and associated proteins, some of which have intrinsic enzymatic activity that modify histones or even components of the transcriptional complex. In this study, we show chromatin occupancy by the Set7/9 methyltransferase at the FXR response element (FXRE) and direct methylation of FXR in vivo and in vitro at lysine 206. siRNA depletion of Set7/9 in the Huh-7 liver cell line decreased endogenous mRNAs of the FXR target genes, the short heterodimer partner (SHP) and bile salt export pump (BSEP). Mutation of the methylation site at K206 of FXR to an arginine prevented methylation by Set7/9. A pan-methyllysine antibody recognized the wild-type FXR but not the K206R mutant form. An electromobility shift assay showed that methylation by Set7/9 enhanced binding of FXR/retinoic X receptor-α to the FXRE. Interaction between hinge domain of FXR (containing K206) and Set7/9 was confirmed by coimmunoprecipitation, GST pull down, and mammalian two-hybrid experiments. Set7/9 overexpression in Huh-7 cells significantly enhanced transactivation of the SHP and BSEP promoters in a ligand-dependent fashion by wild-type FXR but not the K206R mutant FXR. A Set7/9 mutant deficient in methyltransferase activity was also not effective in increasing transactivation of the BSEP promoter. These studies demonstrate that posttranslational methylation of FXR by Set7/9 contributes to the transcriptional activation of FXR-target genes.
Asunto(s)
Hepatocitos/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Riñón/metabolismo , Activación Transcripcional/fisiología , Animales , Línea Celular , Regulación de la Expresión Génica/fisiología , Haplorrinos , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Metilación , Regiones Promotoras Genéticas/genética , Unión Proteica , Receptores Citoplasmáticos y NuclearesRESUMEN
BACKGROUND: We have recently reported a mouse model of PN-associated cholestasis (PNAC) in which combining intestinal inflammation and PN infusion results in cholestasis, hepatic macrophage activation, and transcriptional suppression of canalicular bile acid, bilirubin and sterol transporters Abcb11, Abcc2 and Abcg5/8. The aim of this study was to examine the role of TNFα in promoting PNAC in mice. METHODS: First, recombinant TNFα was administered to mice as well as in hepatocyte cell culture. Second, Tnfr1/2KO or wild-type (WT) mice were exposed to dextran sulfate sodium (DSS) for 4 days followed by soy-oil lipid emulsion-based PN infusion through a central venous catheter for 14 days (DSS-PN). Finally, WT/DSS-PN mice were also infused with infliximab at 10 mg/kg on days 3 and 10 of PN. PNAC was defined by increased serum aspartate aminotransferase, alanine aminotransferase, total bile acids, and bilirubin. RESULTS: Intraperitoneal injection of TNFα into WT mice or TNFα treatment of Huh7 hepatocarcinoma cells and primary mouse hepatocytes suppressed messenger RNA (mRNA) transcription of bile (Abcb11, Abcc2]) and sterol transporters (Abcg5/8) and their regulators Nr1h3 and Nr1h4. DSS-PN mice with PNAC had increased hepatic TNFα mRNA expression and significant reduction of mRNA expression of Abcb11, Abcc2, Abcg5/8, Nr1h3, and Nr1h4. In contrast, PNAC development was prevented and mRNA expression normalized in both Tnfr1/2KO /DSS-PN mice and DSS-PN mice treated with infliximab. CONCLUSIONS: TNFα is a key mediator in the pathogenesis of PNAC through suppression of hepatocyte Abcb11, Abcc2, and Abcg5/8. Pharmacologic targeting of TNFα as a therapeutic strategy for PNAC thus deserves further investigation.
Asunto(s)
Colestasis , Factor de Necrosis Tumoral alfa , Animales , Ácidos y Sales Biliares , Bilirrubina , Colestasis/etiología , Infliximab , Ratones , Nutrición Parenteral , ARN Mensajero , Receptores Tipo I de Factores de Necrosis Tumoral/genética , EsterolesRESUMEN
UNLABELLED: The organic anion (99m)Tc-N-[2-[(3-bromo-2,4,6-trimethylphenyl)-amino]-2-oxoethyl]-N-(carboxymethyl)-glycine ((99m)Tc-mebrofenin) and its analogs are widely used for hepatobiliary imaging. Identification of the mechanisms directing bile canalicular transport of these agents will provide insights into the basis of their hepatic handling for assessing perturbations. METHODS: We performed studies in animals, including healthy Fischer 344 rats or rats treated with carbon tetrachloride or intrasplenic cell transplantation and healthy Wistar rats or HsdAMC:TR-Abcc2 mutant rats in Wistar background. Onset of hepatic inflammation was verified by analysis of carbon uptake in Kupffer cells. Hepatic clearance of (99m)Tc-mebrofenin was studied with dynamic imaging, and fractional retention of peak hepatic mebrofenin activity after 60 min was determined. Changes in the expression of bile canalicular transporters were analyzed by real-time polymerase chain reaction and Western blots. RESULTS: Carbon tetrachloride and cell transplantation produced hepatic inflammation with activation of Kupffer cells, resulting in a rapid decline in the expression of the bile canalicular transporters Abcb4, Abcb11, and Abcc2. Among these transporters, decreased expression of Abcc2 was most prominent, and this decline persisted for 4 wk. Next, we examined (99m)Tc-mebrofenin excretion in HsdAMC:TR-Abcc2 mutant rats (in which Abcc2 expression is naturally inactivated), compared with their healthy counterparts. In healthy HsdRccHan:WIST rats, only 23% +/- 3% of the peak (99m)Tc-mebrofenin activity was retained after 60 min. By contrast, in HsdAMC:TR-Abcc2 mutant rats, 73% +/- 5% of the peak (99m)Tc-mebrofenin activity was retained (P < 0.001). Moreover, the administration of cyclosporin A markedly inhibited (99m)Tc-mebrofenin excretion in healthy rats, with no further effect on already impaired (99m)Tc-mebrofenin excretion in HsdAMC:TR-Abcc2 mutant rats. Hepatic excretion of (99m)Tc-mebrofenin was largely dependent on Abcc2. This molecular basis of (99m)Tc-mebrofenin excretion will advance studies of pathophysiologic mechanisms in hepatic Abcc2 pathways.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Iminoácidos/farmacocinética , Cirrosis Hepática Experimental/diagnóstico por imagen , Cirrosis Hepática Experimental/metabolismo , Hígado/diagnóstico por imagen , Hígado/metabolismo , Compuestos de Organotecnecio/farmacocinética , Compuestos de Anilina , Animales , Tetracloruro de Carbono , Glicina , Cirrosis Hepática Experimental/inducido químicamente , Tasa de Depuración Metabólica , Cintigrafía , Ratas , Ratas Endogámicas F344 , Ratas Wistar , Distribución TisularRESUMEN
UNLABELLED: Prior loss-of-function analyses revealed that ATPase class I type 8B member 1 [familial intrahepatic cholestasis 1 (FIC1)] posttranslationally activated the farnesoid X receptor (FXR). Mechanisms underlying this regulation were examined by gain-of-function studies in UPS cells, which lack endogenous FIC1 expression. FXR function was assayed in response to wild-type and mutated FIC1 expression constructs with a human bile salt export pump (BSEP) promoter and a variety of cellular localization techniques. FIC1 overexpression led to enhanced phosphorylation and nuclear localization of FXR that was associated with FXR-dependent activation of the BSEP promoter. The FIC1 effect was lost after mutation of the FXR response element in the BSEP promoter. Despite similar levels of FIC1 protein expression, Byler disease FIC1 mutants did not activate BSEP, whereas benign recurrent intrahepatic cholestasis mutants partially activated BSEP. The FIC1 effect was dependent on the presence of the FXR ligand, chenodeoxycholic acid. The effect of FIC1 on FXR phosphorylation and nuclear localization and its effects on BSEP promoter activity could be blocked with protein kinase C zeta (PKC zeta) inhibitors (pseudosubstrate or small interfering RNA silencing). Recombinant PKC zeta directly phosphorylated immunoprecipitated FXR. The mutation of threonine 442 of FXR to alanine yielded a dominant negative protein, whereas the phosphomimetic conversion to glutamate resulted in FXR with enhanced activity and nuclear localization. Inhibition of PKC zeta in Caco-2 cells resulted in activation of the human apical sodium-dependent bile acid transporter promoter. CONCLUSION: These results demonstrate that FIC1 signals to FXR via PKC zeta. FIC1-related liver disease is likely related to downstream effects of FXR on bile acid homeostasis. Benign recurrent intrahepatic cholestasis emanates from a partially functional FIC1 protein. Phosphorylation of FXR is an important mechanism for regulating its activity.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína Quinasa C/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Ácidos y Sales Biliares/metabolismo , Células CACO-2 , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Proteínas Fluorescentes Verdes , Haplorrinos , Homeostasis , Humanos , Riñón/citología , Riñón/embriología , Fosforilación , Plásmidos , Proteína Quinasa C/antagonistas & inhibidores , TransfecciónRESUMEN
Adenosine triphosphate-binding cassette subfamily C member 2 (ABCC2/Abcc2) is critically important to biliary excretion of many endobiotic and xenobiotic compounds, and is a major driving force for bile acid-independent bile flow. Abcc2 expression is reduced at the messenger RNA (mRNA) and protein levels in various forms of experimental cholestasis. In a microRNA (miRNA) screen of mouse liver after biliary obstruction, we found that miRNA let7a-5p was significantly up-regulated approximately 4-fold. Similarly, ABCC2 mRNA was depleted and miRNA let7a-5p was elevated over 4-fold in livers of children with biliary atresia compared with normal livers. In silico analysis predicted that let7a-5p would target the 3' untranslated region (3' UTR) of ABCC2/Abcc2 RNA. The objective of this study was to determine whether let7a-5p contributes to the depletion of ABCC2/Abcc2 in cholestasis. To demonstrate the functional importance of miRNA let7a-5p in regulating the expression of ABCC2, co-transfection of a let7a-5p mimic and an ABCC2-3' UTR luciferase construct into Huh-7 cells led to a marked inhibition of luciferase activity by about 60%-70% compared with controls, which was reversed by a let7a-5p mimic inhibitor. Expression of this mimic led to a significant decrease in endogenous ABCC2 mRNA and protein levels in a Huh-7 liver cell line, which could be blocked by expression of a let7a-5p mimic inhibitor. Injection of a lentivirus let7a-5p inhibitor into normal mouse liver or into mouse liver after common bile duct ligation led to a significant increase in endogenous Abcc2 mRNA and protein levels and a depletion of let7a-5p mRNA levels compared with untreated, saline-injected livers or livers treated with an inactive lentivirus control. Conclusion: These studies demonstrate that miR-let7a-5p is involved in regulating ABCC2/Abcc2 expression, and is aberrantly up-regulated in obstructive cholestasis.
RESUMEN
The DYT1 gene encodes for torsinA, a protein with widespread tissue distribution, involved in early onset dystonia (EOD). Numerous studies have focused on torsinA function but no information is available on its transcriptional regulation. We cloned mouse and human 5'-upstream DYT1 DNA fragments, exhibiting high transcriptional activity, as well as tissue specificity. We identified a proximal minimal DYT1 promoter within -141 bp for mouse and -191 bp for human with respect to the ATG codon. Primer extension analysis indicated multiple transcription start sites. In silico analysis of approximately 500 bp 5'-upstream DYT1 fragment demonstrated lack of a classical TATA or CAAT box and the presence of a highly conserved direct repeat of two Ets binding cores within -86 bp to -77 bp and -78 bp to -69 bp of the mouse and human DYT1 gene, respectively. A single or a two base nucleotide alteration within the downstream Ets core resulted in approximately 90% (mouse) or 45-60% (human) drop in activity. Interestingly, a 3-bp distance increase between the two Ets cores dramatically decreased transcriptional activity which was partially restored when the distance was increased up to 10 bp. Ets-like dominant negatives confirmed the Ets factors as DYT1 transcriptional activators.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Chaperonas Moleculares/biosíntesis , Chaperonas Moleculares/genética , Proteínas Proto-Oncogénicas c-ets/fisiología , Transactivadores/fisiología , Animales , Secuencia de Bases/fisiología , Sitios de Unión/fisiología , Línea Celular , Distonía Muscular Deformante/genética , Distonía Muscular Deformante/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Ratas , Síndrome de Tourette/genética , Síndrome de Tourette/metabolismoRESUMEN
In infants intolerant of enteral feeding because of intestinal disease, parenteral nutrition may be associated with cholestasis, which can progress to end-stage liver disease. Here we show the function of hepatic macrophages and phytosterols in parenteral nutrition-associated cholestasis (PNAC) pathogenesis using a mouse model that recapitulates the human pathophysiology and combines intestinal injury with parenteral nutrition. We combine genetic, molecular, and pharmacological approaches to identify an essential function of hepatic macrophages and IL-1ß in PNAC. Pharmacological antagonism of IL-1 signaling or genetic deficiency in CCR2, caspase-1 and caspase-11, or IL-1 receptor (which binds both IL-1α and IL-1ß) prevents PNAC in mice. IL-1ß increases hepatocyte NF-κB signaling, which interferes with farnesoid X receptor and liver X receptor bonding to respective promoters of canalicular bile and sterol transporter genes (Abcc2, Abcb11, and Abcg5/8), resulting in transcriptional suppression and subsequent cholestasis. Thus, hepatic macrophages, IL-1ß, or NF-κB may be targets for restoring bile and sterol transport to treat PNAC.