RESUMO
Melatonin is widely present in Nature. It has pleiotropic activities, in part mediated by interactions with high-affinity G-protein-coupled melatonin type 1 and 2 (MT1 and MT2) receptors or under extreme conditions, e.g., ischemia/reperfusion. In pharmacological concentrations, it is given to counteract the massive damage caused by MT1- and MT2-independent mechanisms. The aryl hydrocarbon receptor (AhR) is a perfect candidate for mediating the latter effects because melatonin has structural similarity to its natural ligands, including tryptophan metabolites and indolic compounds. Using a cell-based Human AhR Reporter Assay System, we demonstrated that melatonin and its indolic and kynuric metabolites act as agonists on the AhR with EC50's between 10-4 and 10-6 M. This was further validated via the stimulation of the transcriptional activation of the CYP1A1 promoter. Furthermore, melatonin and its metabolites stimulated AhR translocation from the cytoplasm to the nucleus in human keratinocytes, as demonstrated by ImageStream II cytometry and Western blot (WB) analyses of cytoplasmic and nuclear fractions of human keratinocytes. These functional analyses are supported by in silico analyses. We also investigated the peroxisome proliferator-activated receptor (PPAR)γ as a potential target for melatonin and metabolites bioregulation. The binding studies using a TR-TFRET kit to assay the interaction of the ligand with the ligand-binding domain (LBD) of the PPARγ showed agonistic activities of melatonin, 6-hydroxymelatonin and N-acetyl-N-formyl-5-methoxykynuramine with EC50's in the 10-4 M range showing significantly lower affinities that those of rosiglitazone, e.g., a 10-8 M range. These interactions were substantiated by stimulation of the luciferase activity of the construct containing PPARE by melatonin and its metabolites at 10-4 M. As confirmed by the functional assays, binding mode predictions using a homology model of the AhR and a crystal structure of the PPARγ suggest that melatonin and its metabolites, including 6-hydroxymelatonin, 5-methoxytryptamine and N-acetyl-N-formyl-5-methoxykynuramine, are excellent candidates to act on the AhR and PPARγ with docking scores comparable to their corresponding natural ligands. Melatonin and its metabolites were modeled into the same ligand-binding pockets (LBDs) as their natural ligands. Thus, functional assays supported by molecular modeling have shown that melatonin and its indolic and kynuric metabolites can act as agonists on the AhR and they can interact with the PPARγ at high concentrations. This provides a mechanistic explanation for previously reported cytoprotective actions of melatonin and its metabolites that require high local concentrations of the ligands to reduce cellular damage under elevated oxidative stress conditions. It also identifies these compounds as therapeutic agents to be used at pharmacological doses in the prevention or therapy of skin diseases.
Assuntos
Melatonina , Receptores de Hidrocarboneto Arílico , Humanos , Queratinócitos/metabolismo , Ligantes , Melatonina/metabolismo , PPAR gama/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismoRESUMO
We recently identified upregulation of a novel aryl hydrocarbon receptor (AhR) target gene, stanniocalcin 2 (STC2), by an endogenous AhR agonist, cinnabarinic acid (CA). STC2 is a disulfide-linked homodimeric secreted glycoprotein that plays a role in various physiologic processes, including cell metabolism, inflammation, endoplasmic reticulum (ER) and oxidative stress, calcium regulation, cell proliferation, and apoptosis. Our previous studies have confirmed that CA-induced AhR-dependent STC2 expression was able to confer cytoprotection both in vitro and in vivo in response to injury induced by variety of ER/oxidative insults. Here, we used mouse models of chronic and acute ethanol feeding and demonstrated that upregulation of STC2 by CA was critical for cytoprotection. In STC2 knockout mice (STC2-/-), CA failed to protect against both acute as well as chronic-plus-binge ethanol-induced liver injury, whereas re-expression of STC2 in the liver using in vivo gene delivery restored cytoprotection against injury based on measures of apoptosis and serum levels of liver enzymes, underlining STC2's indispensable function in cell survival. In conclusion, the identification of STC2 as an AhR target gene receptive to CA-mediated endogenous AhR signaling and STC2's role in providing cytoprotection against liver injury represents a key finding with potentially significant therapeutic implications. SIGNIFICANCE STATEMENT: We recently identified stanniocalcin 2 (STC2) as a novel aryl hydrocarbon receptor (AhR) target gene regulated by endogenous AhR agonist and tryptophan metabolite, cinnabarinic acid (CA). Here, we showed that CA-induced STC2 expression conferred cytoprotection against apoptosis, steatosis, and liver injury in chronic as well as acute models of ethanol feeding. Therefore, this study will prove instrumental in developing CA as a promising lead compound for future drug development against hepatic diseases.
Assuntos
Doença Hepática Crônica Induzida por Substâncias e Drogas , Receptores de Hidrocarboneto Arílico , Animais , Citoproteção , Etanol/toxicidade , Glicoproteínas , Camundongos , Oxazinas , Receptores de Hidrocarboneto Arílico/genéticaRESUMO
Viral-mediated in vivo gene delivery methods currently dominate among therapeutic strategies within the clinical and experimental settings, albeit with well documented limitations arising from immunologic constraints. In this study, we demonstrate the utility of nonviral hepatotropic in vivo gene delivery of unpackaged expression constructs, including one encoding fibroblast growth factor 21 (FGF21). FGF21 is an important hepatokine whose expression positively correlates with therapeutic outcomes across various animal models of obesity. Our data demonstrate that FGF21 expression can be restored into the livers of immunocompetent FGF21 knockout mice for at least 2 weeks after a single injection with an FGF21 expression plasmid. In wild-type C57BL6/J mice, in vivo transfection with an FGF21-expressing plasmid induced weight loss, decreased adiposity, and activated thermogenesis in white fat within 2 weeks. Furthermore, in vivo FGF21 gene delivery protected C57BL6/J mice against diet-induced obesity by decreasing adiposity and increasing uncoupling protein 1-dependent thermogenesis in brown fat and by boosting respiratory capacity in subcutaneous and perigonadal white fat. Together, the data illustrate a facile and effective methodology for delivering prolonged protein expression specifically to the liver. We contend that this method will find utility in basic science research as a practical means to enhance in vivo studies characterizing liver protein function. We further believe our data provide a rationale for further exploring the potential clinical utility of nonviral gene therapy in mouse models of disease. SIGNIFICANCE STATEMENT: This study presents a valuable method for nonviral gene delivery in mice that improves upon existing techniques. The data provide a rationale for further exploring the potential clinical utility of nonviral gene therapy in mouse models of disease and will likely enhance in vivo studies characterizing liver protein function.
Assuntos
Fatores de Crescimento de Fibroblastos , Tecido Adiposo Marrom , Animais , Camundongos , Processamento de Proteína Pós-TraducionalRESUMO
In the wake of the Deepwater Horizon (DWH) oil spill, a number of government agencies, academic institutions, consultants, and nonprofit organizations conducted lab- and field-based research to understand the toxic effects of the oil. Lab testing was performed with a variety of fish, birds, turtles, and vertebrate cell lines (as well as invertebrates); field biologists conducted observations on fish, birds, turtles, and marine mammals; and epidemiologists carried out observational studies in humans. Eight years after the spill, scientists and resource managers held a workshop to summarize the similarities and differences in the effects of DWH oil on vertebrate taxa and to identify remaining gaps in our understanding of oil toxicity in wildlife and humans, building upon the cross-taxonomic synthesis initiated during the Natural Resource Damage Assessment. Across the studies, consistency was found in the types of toxic response observed in the different organisms. Impairment of stress responses and adrenal gland function, cardiotoxicity, immune system dysfunction, disruption of blood cells and their function, effects on locomotion, and oxidative damage were observed across taxa. This consistency suggests conservation in the mechanisms of action and disease pathogenesis. From a toxicological perspective, a logical progression of impacts was noted: from molecular and cellular effects that manifest as organ dysfunction, to systemic effects that compromise fitness, growth, reproductive potential, and survival. From a clinical perspective, adverse health effects from DWH oil spill exposure formed a suite of signs/symptomatic responses that at the highest doses/concentrations resulted in multi-organ system failure.
Assuntos
Exposição Ambiental/efeitos adversos , Poluição por Petróleo/efeitos adversos , Poluentes Químicos da Água/toxicidade , Animais , Aves , Monitoramento Ambiental/métodos , Peixes , Humanos , Insuficiência de Múltiplos Órgãos/etiologia , Petróleo/toxicidade , Tartarugas , VertebradosRESUMO
The aryl hydrocarbon receptor (AHR) has been studied for over 40 years, yet our understanding of this ligand-activated transcription factor remains incomplete. Each year, novel findings continually force us to rethink the role of the AHR in mammalian biology. The AHR has historically been studied within the context of potent activation via AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), with a focus on how the AHR mediates TCDD toxicity. Research has subsequently revealed that the AHR is actively involved in distinct physiological processes ranging from the development of the liver and reproductive organs, to immune system function and wound healing. More recently, the AHR was implicated in the regulation of energy metabolism and is currently being investigated as a potential therapeutic target for obesity. In this review, we re-trace the steps through which the early toxicological studies of TCDD led to the conceptual framework for the AHR as a potential therapeutic target in metabolic disease. We additionally discuss the key discoveries that have been made concerning the role of the AHR in energy metabolism, as well as the current and future directions of the field.
Assuntos
Metabolismo Energético , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Dioxinas/efeitos adversos , Modelos Animais de Doenças , Suscetibilidade a Doenças , Desenvolvimento de Medicamentos , Metabolismo Energético/genética , Regulação da Expressão Gênica , Humanos , Ligantes , Camundongos Transgênicos , Terapia de Alvo Molecular , Obesidade/tratamento farmacológico , Obesidade/etiologia , Obesidade/metabolismo , Dibenzodioxinas Policloradas/efeitos adversos , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/genética , Síndrome de Emaciação/etiologia , Síndrome de Emaciação/metabolismoRESUMO
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor highly expressed in hepatocytes. Researchers have employed global and liver-specific conditional Ahr knockout mouse models to characterize the physiological roles of the AHR; however, the gestational timing of AHR loss in these models can complicate efforts to distinguish the direct and indirect effects of post-gestational AHR deficiency. Utilizing a novel tamoxifen-inducible AHR knockout mouse model, we analyzed the effects of hepatocyte-targeted AHR loss in adult mice. The data demonstrate that AHR deficiency significantly reduces weight gain and adiposity, and increases multilocular lipid droplet formation within perigonadal white adipose tissue (gWAT). Protein and mRNA expression of fibroblast growth factor 21 (FGF21), an important hepatokine that activates thermogenesis in brown adipose tissue (BAT) and gWAT, significantly increases upon AHR loss and correlates with a significant increase of BAT and gWAT respiratory capacity. Confirming the role of FGF21 in mediating these effects, this phenotype is reversed in mice concomitantly lacking AHR and FGF21 expression. Chromatin immunoprecipitation analyses suggest that the AHR may constitutively suppress Fgf21 transcription through binding to a newly identified xenobiotic response element within the Fgf21 promoter. The data demonstrate an important AHR-FGF21 regulatory axis that influences adipose biology and may represent a "druggable" therapeutic target for obesity and its related metabolic disorders.
Assuntos
Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Respiração Celular , Fatores de Crescimento de Fibroblastos/metabolismo , Gônadas/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Termogênese , Tecido Adiposo Branco/efeitos dos fármacos , Adiposidade/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Dieta Hiperlipídica , Ingestão de Líquidos , Metabolismo Energético/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Feminino , Fatores de Crescimento de Fibroblastos/genética , Gônadas/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Gotículas Lipídicas/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Knockout , Fenótipo , Condicionamento Físico Animal , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Tamoxifeno/farmacologia , Termogênese/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates a plethora of target genes. Historically, the AhR has been studied as a regulator of xenobiotic metabolizing enzyme genes, notably cytochrome P4501A1 encoded by CYP1A1, in response to the exogenous prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AhR activity depends on its binding to the xenobiotic response element (XRE) in partnership with the AhR nuclear translocator (Arnt). Recent studies identified stanniocalcin 2 (Stc2) as a novel AhR target gene responsive to the endogenous AhR agonist cinnabarinic acid (CA). CA-dependent AhR-XRE-mediated Stc2 upregulation is responsible for cytoprotection against ectoplasmic reticulum/oxidative stress-induced apoptosis both in vitro and in vivo. Significantly, CA but not TCDD induces expression of Stc2 in hepatocytes. In contrast to TCDD, CA is unable to induce the CYP1A1 gene, thus revealing an AhR agonist-specific mutually exclusive dichotomous transcriptional response. Studies reported here provide a mechanistic explanation for this differential response by identifying an interaction between the AhR and the metastasis-associated protein 2 (MTA2). Moreover, the AhR-MTA2 interaction is CA-dependent and results in MTA2 recruitment to the Stc2 promoter, concomitant with agonist-specific epigenetic modifications targeting histone H4 lysine acetylation. The results demonstrate that histone H4 acetylation is absolutely dependent on CA-induced AhR and MTA2 recruitment to the Stc2 regulatory region and induced Stc2 gene expression, which in turn confers cytoprotection to liver cells exposed to chemical insults.
Assuntos
Epigênese Genética , Glicoproteínas/genética , Oxazinas/farmacologia , Receptores de Hidrocarboneto Arílico/fisiologia , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Animais , Citoproteção , Feminino , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos BALB C , Dibenzodioxinas Policloradas/farmacologia , Regiões Promotoras Genéticas , Receptores de Hidrocarboneto Arílico/agonistas , Elementos de Resposta/fisiologiaRESUMO
The aryl hydrocarbon receptor (AhR), a regulator of xenobiotic toxicity, is a member of the eukaryotic Per-Arnt-Sim domain protein family of transcription factors. Recent evidence identified a novel AhR DNA recognition sequence called the nonconsensus xenobiotic response element (NC-XRE). AhR binding to the NC-XRE in response to activation by the canonical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin resulted in concomitant recruitment of carbamoyl phosphate synthase 1 (CPS1) to the NC-XRE. Studies presented here demonstrate that CPS1 is a bona fide nuclear protein involved in homocitrullination (hcit), including a key lysine residue on histone H1 (H1K34hcit). H1K34hcit represents a hitherto unknown epigenetic mark implicated in enhanced gene expression of the peptidylarginine deiminase 2 gene, itself a chromatin-modifying protein. Collectively, our data suggest that AhR activation promotes CPS1 recruitment to DNA enhancer sites in the genome, resulting in a specific enzyme-independent post-translational modification of the linker histone H1 protein (H1K34hcit), pivotal in altering local chromatin structure and transcriptional activation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Citrulina/análogos & derivados , Epigênese Genética , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Cromatina/metabolismo , Cromatina/ultraestrutura , Citrulina/metabolismo , Feminino , Hidrolases/genética , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dibenzodioxinas Policloradas/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Desiminases de Arginina em Proteínas , Elementos de Resposta , Ativação TranscricionalRESUMO
The aryl hydrocarbon receptor (AhR) is a cytosolic ligand-activated transcription factor historically known for its role in xenobiotic metabolism. Although AhR activity has previously been shown to play a cytoprotective role against intrinsic apoptotic stimuli, the underlying mechanism by which AhR confers cytoprotection against apoptosis is largely unknown. Here, we demonstrate that activation of AhR by the tryptophan catabolite cinnabarinic acid (CA) directly upregulates expression of stanniocalcin 2 (Stc2) to elicit cytoprotection against apoptosis induced by endoplasmic reticulum stress and oxidative stress. Chromatin immunoprecipitation studies demonstrated that CA treatment induces direct AhR binding to a region of the Stc2 promoter containing multiple xenobiotic response elements. Using isolated primary hepatocytes from AhR wild-type (AhR floxed) and liver-specific AhR conditional knockout mice, we showed that pretreatment with CA conferred cytoprotection against hydrogen peroxide (H(2)O(2))-, thapsigargin-, and ethanol-induced apoptosis in an AhR-dependent manner. Furthermore, suppressing Stc2 expression using RNA interference confirmed that the cytoprotective properties of CA against H(2)O(2), thapsigargin, and ethanol injury were absolutely dependent on Stc2. Immunochemistry revealed the presence of Stc2 in the endoplasmic reticulum and on the cell surface, consistent with Stc2 secretion and autocrine and/or paracrine signaling. Finally, in vivo data using a mouse model of acute alcohol hepatotoxicity demonstrated that CA provided cytoprotection against ethanol-induced apoptosis, hepatic microvesicular steatosis, and liver injury. Collectively, our data uncovered a novel mechanism for AhR-mediated cytoprotection in the liver that is dependent on CA-induced Stc2 activity.
Assuntos
Estresse do Retículo Endoplasmático , Glicoproteínas/biossíntese , Fígado/citologia , Oxazinas/farmacologia , Estresse Oxidativo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Apoptose/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Citoproteção , Retículo Endoplasmático/metabolismo , Etanol/farmacologia , Glicoproteínas/genética , Peróxido de Hidrogênio/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos Knockout , Oxazinas/metabolismo , Regiões Promotoras Genéticas , Receptores de Hidrocarboneto Arílico/genética , Tapsigargina/farmacologia , Regulação para CimaRESUMO
Chronic hepatic disease damages the liver, and the resulting wound-healing process leads to liver fibrosis and the subsequent development of cirrhosis. The leading cause of hepatic fibrosis and cirrhosis is infection with hepatitis C virus (HCV), and of the patients with HCV-induced cirrhosis, 2% to 5% develop hepatocellular carcinoma (HCC), with a survival rate of 7%. HCC is one of the leading causes of cancer-related death worldwide, and the poor survival rate is largely due to late-stage diagnosis, which makes successful intervention difficult, if not impossible. The lack of sensitive and specific diagnostic tools and the urgent need for early-stage diagnosis prompted us to discover new candidate biomarkers for HCV and HCC. We used aptamer-based fractionation technology to reduce serum complexity, differentially labeled samples (six HCV and six HCC) with fluorescent dyes, and resolved proteins in pairwise two-dimensional difference gel electrophoresis. DeCyder software was used to identify differentially expressed proteins and spots picked, and MALDI-MS/MS was used to determine that ApoA1 was down-regulated by 22% (p < 0.004) in HCC relative to HCV. Differential expression quantified via two-dimensional difference gel electrophoresis was confirmed by means of (18)O/(16)O stable isotope differential labeling with LC-MS/MS zoom scans. Technically independent confirmation was demonstrated by triple quadrupole LC-MS/MS selected reaction monitoring (SRM) assays with three peptides specific to human ApoA1 (DLATVYVDVLK, WQEEMELYR, and VSFLSALEEYTK) using (18)O/(16)O-labeled samples and further verified with AQUA peptides as internal standards for quantification. In 50 patient samples (24 HCV and 26 HCC), all three SRM assays yielded highly similar differential expression of ApoA1 in HCC and HCV patients. These results validated the SRM assays, which were independently confirmed by Western blotting. Thus, ApoA1 is a candidate member of an SRM biomarker panel for early diagnosis, prognosis, and monitoring of HCC. Future multiplexing of SRM assays for other candidate biomarkers is envisioned to develop a biomarker panel for subsequent verification and validation studies.
Assuntos
Apolipoproteína A-I/genética , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Hepatite C Crônica/genética , Cirrose Hepática/genética , Neoplasias Hepáticas/genética , Adulto , Sequência de Aminoácidos , Apolipoproteína A-I/sangue , Aptâmeros de Peptídeos/química , Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/etiologia , Diagnóstico Precoce , Eletroforese em Gel Bidimensional , Corantes Fluorescentes/química , Hepacivirus/isolamento & purificação , Hepatite C Crônica/sangue , Hepatite C Crônica/complicações , Hepatite C Crônica/diagnóstico , Humanos , Marcação por Isótopo , Cirrose Hepática/sangue , Cirrose Hepática/diagnóstico , Cirrose Hepática/etiologia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/etiologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Isótopos de Oxigênio , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Previous studies in hepatocyte-derived cell lines and the whole liver established that the aryl hydrocarbon receptor (AhR) can disrupt G1-phase cell cycle progression following exposure to persistent AhR agonists, such as TCDD (dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin). Growth arrest was attributed to inhibition of G1-phase cyclin-dependent kinase 2 (CDK2) activity. The present study examined the effect of TCDD exposure on liver regeneration following 70% partial hepatectomy in mice lacking the Cip/Kip inhibitors p21(Cip1) or p27(Kip1) responsible for regulating CDK2 activity. Assessment of the regenerative process in wild-type, p21(Cip1) knockout, and p27(Kip1) knockout mice confirmed that TCDD-induced inhibition of liver regeneration is entirely dependent on p21(Cip1) expression. Compared with wild-type mice, the absence of p21(Cip1) expression completely abrogated the TCDD inhibition, and accelerated hepatocyte progression through G1 phase during the regenerative process. Analysis of the transcriptional response determined that increased p21(Cip1) expression during liver regeneration involved an AhR-dependent mechanism. Chromatin immunoprecipitation studies revealed that p21(Cip1) induction required AhR binding to the newly characterized nonconsensus xenobiotic response element, in conjunction with the tumor suppressor protein Kruppel-like factor 6 functioning as an AhR binding partner. The evidence also suggests that AhR functionality following partial hepatectomy is dependent on a p21(Cip1)-regulated signaling process, intimately linking AhR biology to the G1-phase cell cycle program.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regeneração Hepática , Receptores de Hidrocarboneto Arílico/metabolismo , Elementos de Resposta , Animais , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Hepatectomia , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dibenzodioxinas Policloradas/toxicidade , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Hidrocarboneto Arílico/agonistasRESUMO
The aryl hydrocarbon receptor (AhR) is a ligand-mediated basic helix-loop-helix transcription factor of the Per/Arnt/Sim family that regulates adaptive and toxic responses to a variety of chemical pollutants, including polycyclic aromatic hydrocarbons and halogenated aromatic hydrocarbons, most notably 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Ligand activation leads to AhR nuclear translocation and binding to a xenobiotic response element (XRE) in association with the Arnt to regulate gene expression. Several recent genome-wide transcriptional studies identified numerous AhR target genes that lack the canonical XRE recognition site in the promoter regions. Characterization of one such target gene, the plasminogen activator inhibitor 1, identified a novel nonconsensus XRE (NC-XRE) that confers TCDD responsiveness independently of the Arnt protein. Studies reported here show that the NC-XRE is a recognition site for the AhR and a new binding partner, the Kruppel-like factor (KLF) family member KLF6. In vivo chromatin immunoprecipitations and in vitro DNA binding studies demonstrate that the AhR and KLF6 proteins form an obligatory heterodimer necessary for NC-XRE binding. Mutational analyses show that the protein-protein interactions involve the AhR C terminus and KLF6 N terminus, respectively. Moreover, NC-XRE binding depends on the 5' basic region in KLF6 rather than the previously characterized zinc finger DNA binding domain. Collectively, the results unmask a novel AhR signaling mechanism distinct from the canonical XRE-driven process that will enrich our future understanding of AhR biology.
Assuntos
DNA/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Western Blotting , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Poluentes Ambientais , Feminino , Humanos , Fator 6 Semelhante a Kruppel , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dibenzodioxinas Policloradas/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Elementos de Resposta/genética , Transcrição Gênica/efeitos dos fármacos , Xenobióticos/farmacologia , Dedos de ZincoRESUMO
Proper hepatocyte function is vital for survival; thus, unrepaired destruction of the parenchymal tissue leading to liver decompensation is devastating. Therefore, understanding the homeostatic process regulating liver regeneration is clinically important, and evidence that the aryl hydrocarbon receptor (AhR) can promote cell survival after intrinsic apoptotic stimuli is integral to the regenerative process. The current study uses primary hepatocytes to identify survival mechanisms consistent with normal AhR biology. Taking advantage of the Cre-lox system to manipulate AhR status, we designed a comprehensive microarray analysis to identify immediate and direct changes in the transcriptome concomitant with the loss of the AhR. As a result, we identified a unique data set with minimal overlap, compared with previous array studies, culminating in the identification of Stanniocalcin 2 (Stc2) as a novel receptor target gene previously reported to have a cytoprotective role in endoplasmic reticulum stress. The Stc2 promoter contains multiple putative xenobiotic response elements clustered in a 250-bp region that was shown to recruit the AhR by chromatin immunoprecipitation. Of interest, Stc2 gene expression is refractory to classic exogenous AhR agonists, but responds to cellular stress in an AhR-dependent mechanism consistent with a process promoting cell survival.
Assuntos
Glicoproteínas/metabolismo , Hepatócitos/metabolismo , Regeneração Hepática/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/genética , Citoproteção/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Feminino , Expressão Gênica , Glicoproteínas/genética , Hepatócitos/citologia , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Fígado/citologia , Fígado/metabolismo , Fígado/fisiologia , Camundongos , Regiões Promotoras Genéticas , Receptores de Hidrocarboneto Arílico/genética , Elementos de Resposta , TranscriptomaRESUMO
The aryl hydrocarbon receptor (AhR) is a mediator of xenobiotic toxicity, best recognized for conveying the deleterious effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. The AhR functions as a ligand-activated transcription factor that binds to a canonical xenobiotic response element (XRE) in association with the heterodimerization partner, the AhR nuclear translocator (Arnt) protein. However, within the repertoire of AhR target genes identified in recent years, many lack a clearly defined XRE highlighting the growing realization that AhR-mediated gene expression seems to involve additional mechanisms distinct from the well characterized process involving the XRE. The present study characterized a novel nonconsensus XRE (NC-XRE) in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene that recruits a novel protein-DNA complex responsible for TCDD-inducible expression. DNA binding studies and reporter assays identified key residues in the NC-XRE necessary for protein-DNA binding and function, respectively. Functional studies with AhR expression constructs confirm that TCDD-inducibility is AhR-dependent and requires direct AhR-DNA binding to the NC-XRE. Chromatin immunoprecipitation and RNA interference studies reveal that the Arnt protein is not a component of the NC-XRE-bound AhR complex, suggesting that in contrast to the XRE, AhR-dependent gene expression mediated through the NC-XRE may involve a new DNA binding partner.
Assuntos
Expressão Gênica/fisiologia , Dibenzodioxinas Policloradas/farmacologia , Receptores de Hidrocarboneto Arílico/fisiologia , Animais , Sequência de Bases , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Camundongos , Camundongos Endogâmicos C57BL , Inibidor 1 de Ativador de Plasminogênio/genética , Reação em Cadeia da Polimerase , Regiões Promotoras GenéticasRESUMO
Fas receptor is a member of the tumor necrosis factor-alpha family of death receptors that mediate physiologic apoptotic signaling. To investigate the molecular mechanisms regulating calcium mobilization during Fas-mediated apoptosis, we have analyzed the sequential steps leading to altered calcium homeostasis and cell death in response to activation of the Fas receptor. We show that Fas-mediated apoptosis requires endoplasmic reticulum-mediated calcium release in a mechanism dependent on phospholipase C-gamma1 (PLC-gamma1) activation and Ca2+ release from inositol 1,4,5-trisphosphate receptor (IP3R) channels. The kinetics of Ca2+ release were biphasic, demonstrating a rapid elevation caused by PLC-gamma1 activation and a delayed and sustained increase caused by cytochrome c binding to IP3R. Blocking either phase of Ca2+ mobilization was cytoprotective, highlighting PLC-gamma1 and IP3R as possible therapeutic targets for disorders associated with Fas signaling.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Fosfolipase C gama/metabolismo , Receptor fas/fisiologia , Apoptose , Linhagem Celular , Citocromos c/metabolismo , Citocromos c/fisiologia , Proteína Ligante Fas/metabolismo , Proteína Ligante Fas/fisiologia , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Modelos Biológicos , Transdução de Sinais , Receptor fas/metabolismoRESUMO
The cycling and fate of polycyclic aromatic hydrocarbons (PAHs) is not well understood in estuarine systems. It is critical now more than ever given the increased ecosystem pressures on these critical coastal habitats. A budget of PAHs and cycling has been created for Galveston Bay (Texas) in the northwestern Gulf of Mexico, an estuary surrounded by 30-50% of the US capacity of oil refineries and chemical industry. We estimate that approximately 3 to 4 mt per year of pyrogenic PAHs are introduced to Galveston Bay via gaseous exchange from the atmosphere (ca. 2 mt/year) in addition to numerous spills of petrogenic PAHs from oil and gas operations (ca. 1.0 to 1.9 mt/year). PAHs are cycled through and stored in the biota, and ca. 20 to 30% of the total (0.8 to 1.5 mt per year) are estimated to be buried in the sediments. Oysters concentrate PAHs to levels above their surroundings (water and sediments) and contain substantially greater concentrations than other fish catch (shrimp, blue crabs and fin fish). Smaller organisms (infaunal invertebrates, phytoplankton and zooplankton) might also retain a significant fraction of the total, but direct evidence for this is lacking. The amount of PAHs delivered to humans in seafood, based on reported landings, is trivially small compared to the total inputs, sediment accumulation and other possible fates (metabolic remineralization, export in tides, etc.), which remain poorly known. The generally higher concentrations in biota from Galveston Bay compared to other coastal habitats can be attributed to both intermittent spills of gas and oil and the bay's close proximity to high production of pyrogenic PAHs within the urban industrial complex of the city of Houston as well as periodic flood events that transport PAHs from land surfaces to the Bay.
Assuntos
Baías/química , Monitoramento Ambiental/estatística & dados numéricos , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Poluentes Químicos da Água/metabolismo , Animais , Organismos Aquáticos/química , Organismos Aquáticos/metabolismo , Atmosfera/química , Braquiúros/química , Braquiúros/metabolismo , Peixes/metabolismo , Sedimentos Geológicos/química , Golfo do México , Ostreidae/química , Ostreidae/metabolismo , Poluição por Petróleo/estatística & dados numéricos , Texas , Poluentes Químicos da Água/análiseRESUMO
BACKGROUND: The association between proximity to oil refineries and cancer rate is largely unknown. We sought to compare the rate of cancer (bladder, breast, colon, lung, lymphoma, and prostate) according to proximity to an oil refinery in Texas. METHODS: A total of 6 302 265 persons aged 20 years or older resided within 30 miles of an oil refinery from 2010 to 2014. We used multilevel zero-inflated Poisson regression models to examine the association between proximity to an oil refinery and cancer rate. RESULTS: We observed that proximity to an oil refinery was associated with a statistically significantly increased risk of incident cancer diagnosis across all cancer types. For example, persons residing within 0-10 (risk ratio [RR] = 1.13, 95% confidence interval [CI] = 1.07 to 1.19) and 11-20 (RR = 1.05, 95% CI = 1.00 to 1.11) miles were statistically significantly more likely to be diagnosed with lymphoma than individuals who lived within 21-30 miles of an oil refinery. We also observed differences in stage of cancer at diagnosis according to proximity to an oil refinery. Moreover, persons residing within 0-10 miles were more likely to be diagnosed with distant metastasis and/or systemic disease than people residing 21-30 miles from an oil refinery. The greatest risk of distant disease was observed in patients diagnosed with bladder cancer living within 0-10 vs 21-30 miles (RR = 1.30, 95% CI = 1.02 to 1.65), respectively. CONCLUSIONS: Proximity to an oil refinery was associated with an increased risk of multiple cancer types. We also observed statistically significantly increased risk of regional and distant/metastatic disease according to proximity to an oil refinery.
RESUMO
This paper is intended to complement our extended documentation and analysis of the activities of the Gulf Coast Health Alliance: Health Risks related to the Macondo Spill project Community Outreach and Dissemination Core entitled, "Building and maintaining a citizen science network with fishermen and fishing communities after the Deepwater Horizon oil disaster using a Community-Based Participatory Research (CBPR) approach." We discuss nuances of CBPR practice, including trust-building, clarification of stakeholder expectations, balancing timelines and agendas, cultural fluency, and the importance of regional history-political-economic context, regulatory practices, and cultural life-ways-in creating social dynamics that overarch and underpin the entire process. We examine the unique role of knowledge-making hybrid structures like the project's Fishermen's citizen science network and compare/contrast this structure with other models of participatory science or deliberation. Finally, we reiterate the importance of environmental health literacy efforts, summarize project outcomes, and offer thoughts on the future roles of collaborative efforts among communities and institutional science in environmental public health.
Assuntos
Ciência do Cidadão , Desastres , Pesqueiros , Poluição por Petróleo/efeitos adversos , Poluentes Químicos da Água/efeitos adversos , Animais , Pesquisa Participativa Baseada na Comunidade , Saúde Ambiental , Monitoramento Ambiental , Golfo do México , Alimentos MarinhosRESUMO
This article provides a description of the rationale and processes adopted by the Gulf Coast Health Alliance: Health Risks related to the Macondo Spill consortium to evaluate and communicate the risk of exposure to polycyclic aromatic hydrocarbons (PAHs) in seafood over several years following the Deepwater Horizon disaster and subsequent oil spill. We examined gaps in knowledge associated with PAH toxicity following exposure to petrogenic (oil-derived) PAHs by studying the metabolic fate of PAHs and their potential toxicity using sophisticated analytical methods. Using the data generated, we developed a risk communication strategy designed to meet the needs of the stakeholder communities including a consumption guideline calculator, a web-based tool to reconcile seafood consumption with risk of adverse health effects.
Assuntos
Desastres , Saúde Ambiental , Monitoramento Ambiental/métodos , Poluição por Petróleo/efeitos adversos , Poluentes Químicos da Água/efeitos adversos , Inocuidade dos Alimentos , Golfo do México , Hidrocarbonetos Policíclicos Aromáticos/isolamento & purificação , Medição de Risco , Alimentos Marinhos/toxicidade , Estados UnidosRESUMO
A community-based participatory research was utilized to address the coastal community's concern regarding Deepwater Horizon oil contamination of seafood. Therefore, we analyzed polycyclic aromatic hydrocarbons (PAHs), major toxic constituents of crude oil, in the seafood collected from gulf coast (Louisiana, Alabama and Mississippi) during December 2011-February 2014. PAHs were extracted from edible part of shrimp, oysters, and crabs by the QuEChERS/dsPE procedure and analyzed by gas chromatography-mass spectrometry. The total PAHs data were further analyzed using the General Linear Mixed Model procedure of the SAS (Version 9.3, SAS Institute, Inc., Cary, NC) statistical software. Brown shrimp showed statistically significant differences in PAHs levels with respect to time and locations while white shrimp showed differences at various time points. PAHs levels in oyster and crab samples were not statistically different at the Type I error of 0.05. Overall, the PAHs levels are far below FDA levels of concern for human consumption.