RESUMO
Aberrant activation of GLI transcription factors has been implicated in the pathogenesis of different tumor types including pancreatic ductal adenocarcinoma. However, the mechanistic link with established drivers of this disease remains in part elusive. In this study, using a new genetically engineered mouse model overexpressing constitutively active mouse form of GLI2 and a combination of genome-wide assays, we provide evidence of a novel mechanism underlying the interplay between KRAS, a major driver of pancreatic ductal adenocarcinoma development, and GLI2 to control oncogenic gene expression. These mice, also expressing KrasG12D, show significantly reduced median survival rate and accelerated tumorigenesis compared with the KrasG12D only expressing mice. Analysis of the mechanism using RNA sequencing demonstrate higher levels of GLI2 targets, particularly tumor growth-promoting genes, including Ccnd1, N-Myc, and Bcl2, in KrasG12D mutant cells. Furthermore, chromatin immunoprecipitation sequencing studies showed that in these cells KrasG12D increases the levels of trimethylation of lysine 4 of the histone 3 (H3K4me3) at the promoter of GLI2 targets without affecting significantly the levels of other major active chromatin marks. Importantly, Gli2 knockdown reduces H3K4me3 enrichment and gene expression induced by mutant Kras. In summary, we demonstrate that Gli2 plays a significant role in pancreatic carcinogenesis by acting as a downstream effector of KrasG12D to control gene expression.
Assuntos
Carcinoma Ductal Pancreático , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras) , Proteína Gli2 com Dedos de Zinco , Animais , Humanos , Camundongos , Carcinogênese , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Histonas/metabolismo , Histonas/genética , Camundongos Transgênicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transcrição Gênica , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismoRESUMO
Myeloid neoplasms are clonal hematopoietic stem cell disorders driven by the sequential acquisition of recurrent genetic lesions. Truncating mutations in the chromatin remodeler ASXL1 (ASXL1MT) are associated with a high-risk disease phenotype with increased proliferation, epigenetic therapeutic resistance, and poor survival outcomes. We performed a multi-omics interrogation to define gene expression and chromatin remodeling associated with ASXL1MT in chronic myelomonocytic leukemia (CMML). ASXL1MT are associated with a loss of repressive histone methylation and increase in permissive histone methylation and acetylation in promoter regions. ASXL1MT are further associated with de novo accessibility of distal enhancers binding ETS transcription factors, targeting important leukemogenic driver genes. Chromatin remodeling of promoters and enhancers is strongly associated with gene expression and heterogenous among overexpressed genes. These results provide a comprehensive map of the transcriptome and chromatin landscape of ASXL1MT CMML, forming an important framework for the development of novel therapeutic strategies targeting oncogenic cis interactions.
Assuntos
Leucemia Mielomonocítica Crônica , Epigênese Genética , Expressão Gênica , Humanos , Leucemia Mielomonocítica Crônica/genética , Leucemia Mielomonocítica Crônica/patologia , Mutação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Pancreatitis is a debilitating disease involving inflammation and fibrosis of the exocrine pancreas. Recurrent or chronic forms of pancreatitis are a significant risk factor for pancreatic ductal adenocarcinoma. While genetic factors have been identified for both pathologies, environmental stresses play a large role in their etiology. All cells have adapted mechanisms to handle acute environmental stress that alters energy demands. A common pathway involved in the stress response involves endoplasmic reticulum stress and the unfolded protein response (UPR). While rapidly activated by many external stressors, in the pancreas the UPR plays a fundamental biological role, likely due to the high protein demands in acinar cells. Despite this, increased UPR activity is observed in response to acute injury or following exposure to risk factors associated with pancreatitis and pancreatic cancer. Studies in animal and cell cultures models show the importance of affecting the UPR in the context of both diseases, and inhibitors have been developed for several specific mediators of the UPR. Given the importance of the UPR to normal acinar cell function, efforts to affect the UPR in the context of disease must be able to specifically target pathology vs. physiology. In this review, we highlight the importance of the UPR to normal and pathological conditions of the exocrine pancreas. We discuss recent studies suggesting the UPR may be involved in the initiation and progression of pancreatitis and PDAC, as well as contributing to chemoresistance that occurs in pancreatic cancer. Finally, we discuss the potential of targeting the UPR for treatment.
Assuntos
Carcinoma Ductal Pancreático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neoplasias Pancreáticas/terapia , Pancreatite , Resposta a Proteínas não Dobradas , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/metabolismo , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Estresse do Retículo Endoplasmático/genética , Endorribonucleases , Recidiva Local de Neoplasia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Pancreatite/tratamento farmacológico , Pancreatite/genética , Proteínas Serina-Treonina Quinases , eIF-2 Quinase , Neoplasias PancreáticasRESUMO
Proliferative chronic myelomonocytic leukemia (pCMML), an aggressive CMML subtype, is associated with dismal outcomes. RAS pathway mutations, mainly NRASG12D, define the pCMML phenotype as demonstrated by our exome sequencing, progenitor colony assays and a Vav-Cre-NrasG12D mouse model. Further, these mutations promote CMML transformation to acute myeloid leukemia. Using a multiomics platform and biochemical and molecular studies we show that in pCMML RAS pathway mutations are associated with a unique gene expression profile enriched in mitotic kinases such as polo-like kinase 1 (PLK1). PLK1 transcript levels are shown to be regulated by an unmutated lysine methyl-transferase (KMT2A) resulting in increased promoter monomethylation of lysine 4 of histone 3. Pharmacologic inhibition of PLK1 in RAS mutant patient-derived xenografts, demonstrates the utility of personalized biomarker-driven therapeutics in pCMML.
Assuntos
Proteínas de Ciclo Celular/genética , GTP Fosfo-Hidrolases/genética , Histona-Lisina N-Metiltransferase/genética , Leucemia Mielomonocítica Crônica/genética , Proteínas de Membrana/genética , Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Animais , Proteínas de Ciclo Celular/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Leucêmica da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Estimativa de Kaplan-Meier , Leucemia Mielomonocítica Crônica/metabolismo , Leucemia Mielomonocítica Crônica/terapia , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/genética , Transplante de Células-Tronco/métodos , Transplante Homólogo , Sequenciamento do Exoma/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Quinase 1 Polo-LikeRESUMO
The unfolded protein response (UPR) is activated in pancreatic pathologies and suggested as a target for therapeutic intervention. In this study, we examined activating transcription factor 3 (ATF3), a mediator of the UPR that promotes acinar-to-ductal metaplasia (ADM) in response to pancreatic injury. Since ADM is an initial step in the progression to pancreatic ductal adenocarcinoma (PDAC), we hypothesized that ATF3 is required for initiation and progression of PDAC. We generated mice carrying a germline mutation of Atf3 (Atf3-/-) combined with acinar-specific induction of oncogenic KRAS (Ptf1acreERT/+KrasG12D/+). Atf3-/- mice with (termed APK) and without KRASG12D were exposed to cerulein-induced pancreatitis. In response to recurrent pancreatitis, Atf3-/- mice showed decreased ADM and enhanced regeneration based on morphological and biochemical analysis. Similarly, an absence of ATF3 reduced spontaneous pancreatic intraepithelial neoplasia (PanIN) formation and PDAC in Ptf1acreERT/+KrasG12D/+ mice. In response to injury, KRASG12D bypassed the requirement for ATF3 with a dramatic loss in acinar tissue and PanIN formation observed regardless of ATF3 status. Compared to Ptf1acreERT/+KrasG12D/+ mice, APK mice exhibited a significant decrease in pancreatic and total body weight, did not progress through to PDAC, and showed altered pancreatic fibrosis and immune cell infiltration. These findings suggest a complex, multifaceted role for ATF3 in pancreatic cancer pathology.
Assuntos
Fator 3 Ativador da Transcrição , Células Acinares , Animais , Ceruletídeo , Humanos , Camundongos , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras) , Neoplasias PancreáticasRESUMO
Non-alcoholic fatty liver (NAFL) and related syndromes affect one-third of the adult population in industrialized and developing countries. Lifestyle and caloric oversupply are the main causes of such array of disorders, but the molecular mechanisms underlying their etiology remain elusive. Nuclear Protein 1 (NUPR1) expression increases upon cell injury in all organs including liver. Recently, we reported NUPR1 actively participates in the activation of the Unfolded Protein Response (UPR). The UPR typically maintains protein homeostasis, but downstream mediators of the pathway regulate metabolic functions including lipid metabolism. As increases in UPR and NUPR1 in obesity and liver disease have been well documented, the goal of this study was to investigate the roles of NUPR1 in this context. To establish whether NUPR1 is involved in these liver conditions we used patient-derived liver biopsies and in vitro and in vivo NUPR1 loss of functions models. First, we analyzed NUPR1 expression in a cohort of morbidly obese patients (MOPs), with simple fatty liver (NAFL) or more severe steatohepatitis (NASH). Next, we explored the metabolic roles of NUPR1 in wild-type (Nupr1+/+ ) or Nupr1 knockout mice (Nupr1-/- ) fed with a high-fat diet (HFD) for 15 weeks. Immunohistochemical and mRNA analysis revealed NUPR1 expression is inversely correlated to hepatic steatosis progression. Mechanistically, we found NUPR1 participates in the activation of PPAR-α signaling via UPR. As PPAR-α signaling is controlled by UPR, collectively, these findings suggest a novel function for NUPR1 in protecting liver from metabolic distress by controlling lipid homeostasis, possibly through the UPR.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Estresse do Retículo Endoplasmático , Metabolismo dos Lipídeos , Fígado/metabolismo , Proteínas de Neoplasias/fisiologia , Animais , Linhagem Celular Tumoral , Dieta Hiperlipídica , Homeostase , Humanos , Camundongos , Resposta a Proteínas não DobradasRESUMO
Nuclear protein 1 (NUPR1) is a stress response protein overexpressed upon cell injury in virtually all organs including the exocrine pancreas. Despite NUPR1's well-established role in the response to cell stress, the molecular and structural machineries triggered by NUPR1 activation remain largely debated. In this study, we uncover a new role for NUPR1, participating in the unfolded protein response (UPR) and the integrated stress response. Biochemical results and ultrastructural morphological observations revealed alterations in the UPR of acinar cells of germline-deleted NUPR1 murine models, consistent with the inability to restore general protein synthesis after stress induction. Bioinformatic analysis of NUPR1-interacting partners showed significant enrichment in translation initiation factors, including eukaryotic initiation factor (eIF) 2α. Co-immunoprecipitation and proximity ligation assays confirmed the interaction between NUPR1 and eIF2α and its phosphorylated form (p-eIF2α). Furthermore, our data suggest loss of NUPR1 in cells results in maintained eIF2α phosphorylation and evaluation of nascent proteins by click chemistry revealed that NUPR1-depleted PANC-1 cells displayed a slower poststress protein synthesis recovery when compared to wild-type. Combined, these data propose a novel role for NUPR1 in the integrated stress response pathway, at least partially through promoting efficient PERK branch activity and resolution through a unique interaction with eIF2α.
Assuntos
Proteínas de Ligação a DNA/genética , Estresse do Retículo Endoplasmático/genética , Fator de Iniciação 2 em Eucariotos/genética , Regulação da Expressão Gênica , Proteínas de Neoplasias/genética , Pâncreas/metabolismo , Resposta a Proteínas não Dobradas/genética , Células Acinares/metabolismo , Células Acinares/ultraestrutura , Animais , Western Blotting , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteínas de Neoplasias/metabolismo , Pâncreas/citologia , Pâncreas/ultraestrutura , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Pancreatic cancer remains among the deadliest forms of cancer with a 5 year survival rate less than 10%. With increasing numbers being observed, there is an urgent need to elucidate the pathogenesis of pancreatic cancer. While both contribute to disease progression, neither genetic nor environmental factors completely explain susceptibility or pathogenesis. Defining the links between genetic and environmental events represents an opportunity to understand the pathogenesis of pancreatic cancer. Epigenetics, the study of mitotically heritable changes in genome function without a change in nucleotide sequence, is an emerging field of research in pancreatic cancer. The main epigenetic mechanisms include DNA methylation, histone modifications and RNA interference, all of which are altered by changes to the environment. Epigenetic mechanisms are being investigated to clarify the underlying pathogenesis of pancreatic cancer including an increasing number of studies examining the role as possible diagnostic and prognostic biomarkers. These mechanisms also provide targets for promising new therapeutic approaches for this devastating malignancy.
RESUMO
Acinar cell exocytosis requires spatiotemporal Ca2+ signals regulated through endoplasmic reticulum (ER) stores, Ca2+ATPases, and store-operated Ca2+ entry (SOCE). The secretory pathway Ca2+ATPase 2 (SPCA2) interacts with Orai1, which is involved in SOCE and store independent Ca2+ entry (SICE). However, in the pancreas, only a C-terminally truncated form of SPCA2 (termed SPAC2C) exists. The goal of this study was to determine if SPCA2C effects Ca2+ homeostasis in a similar fashion to the full-length SPCA2. Using epitope-tagged SPCA2C (SPCA2CFLAG) expressed in HEK293A cells and Fura2 imaging, cytosolic [Ca2+] was examined during SICE, SOCE and secretagogue-stimulated signaling. Exogenous SPCA2C expression increased resting cytosolic [Ca2+], Ca2+ release in response to carbachol, ER Ca2+ stores, and store-mediated and independent Ca2+ influx. Co-IP detected Orai1-SPCA2C interaction, which was altered by co-expression of STIM1. Importantly, SPCA2C's effects on store-mediated Ca2+ entry were independent of Orai1. These findings indicate SPCA2C influences Ca2+ homeostasis through multiple mechanisms, some of which are independent of Orai1, suggesting novel and possibly cell-specific Ca2+ regulation.
Assuntos
Sinalização do Cálcio/fisiologia , ATPases Transportadoras de Cálcio/fisiologia , Cálcio/metabolismo , Pâncreas/metabolismo , Canais de Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Homeostase , Humanos , Proteína ORAI2/genética , Proteína ORAI2/metabolismo , Especificidade de Órgãos/genética , Isoformas de Proteínas/fisiologia , Via Secretória/fisiologiaRESUMO
The enzyme enhancer of zeste homologue 2 (EZH2) plays a catalytic role in histone methylation (H3K27me3), one of the epigenetic modifications that is dysregulated in cancer. The development of a positron emission tomography (PET) imaging agent targeting EZH2 has the potential to provide a method of stratifying patients for epigenetic therapies. In this study, we designed and synthesized a series of fluoroethyl analogs based upon the structure of EZH2 inhibitors UNC1999 and EPZ6438. Among the candidate compounds, 20b exhibited a high binding affinity to EZH2 (IC50 = 6 nM) with selectivity versus EZH1 (IC50 = 200 nM) by SAM competition assay, and furthermore, EZH2 inhibition was demonstrated in the pancreatic cancer cell line PANC-1 (IC50 = 9.8 nM). [18F]20b was synthesized successfully and showed 5-fold higher uptake in PANC-1 cells than in MCF-7 cells. MicroPET imaging in a PANC-1 cell xenograft mouse model indicates that [18F]20b has specific binding to EZH2, which was identified by ex vivo Western blot analysis of the tumor tissue.
RESUMO
Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in North America, accounting for >30,000 deaths annually. Although somatic activating mutations in KRAS appear in 97% of PDAC patients, additional factors are required to initiate PDAC. Because mutations in genes encoding chromatin remodelling proteins have been implicated in KRAS-mediated PDAC, we investigated whether loss of chromatin remodeler É-thalassemia, mental-retardation, X-linked (ATRX) affects oncogenic KRAS's ability to promote PDAC. ATRX affects DNA replication, repair, and gene expression and is implicated in other cancers including glioblastomas and pancreatic neuroendocrine tumors. The hypothesis was that deletion of Atrx in pancreatic acinar cells will increase susceptibility to injury and oncogenic KRAS. Methods: Mice allowing conditional loss of Atrx within pancreatic acinar cells were examined after induction of recurrent cerulein-induced pancreatitis or oncogenic KRAS (KRASG12D ). Histologic, biochemical, and molecular analysis examined pancreatic pathologies up to 2 months after induction of Atrx deletion. Results: Mice lacking Atrx showed more progressive damage, inflammation, and acinar-to-duct cell metaplasia in response to injury relative to wild-type mice. In combination with KRASG12D, Atrx-deficient acinar cells showed increased fibrosis, inflammation, progression to acinar-to-duct cell metaplasia, and pre-cancerous lesions relative to mice expressing only KRASG12D. This sensitivity appears only in female mice, mimicking a significant prevalence of ATRX mutations in human female PDAC patients. Conclusions: Our results indicate the absence of ATRX increases sensitivity to injury and oncogenic KRAS only in female mice. This is an instance of a sex-specific mutation that enhances oncogenic KRAS's ability to promote pancreatic intraepithelial lesion formation.
Assuntos
Oncogenes , Pâncreas/lesões , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína Nuclear Ligada ao X/deficiência , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Análise Mutacional de DNA , Feminino , Deleção de Genes , Masculino , Camundongos , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Proteína Nuclear Ligada ao X/metabolismo , Neoplasias PancreáticasRESUMO
International experts gathered at the Mayo Clinic (Rochester MN, USA) on February 27th-28th, 2017 for a meeting entitled 'Basic and Translational Facets of the Epigenetics of GI Diseases'. This workshop summarized recent advances on the role of epigenetics in the pathobiology of gastrointestinal (GI) diseases. Highlights of the meeting included recent advances on the involvement of different epigenetic mechanisms in malignant and nonmalignant GI disorders and the epigenetic heterogeneity exhibited in these diseases. The translational value of epigenetic drugs, as well as the current and future use of epigenetic changes (i.e., DNA methylation patterns) as biomarkers for early detection tools or disease stratification were also important topics of discussion.
Assuntos
Epigênese Genética , Gastroenteropatias/genética , Metilação de DNA , Heterogeneidade Genética , Marcadores Genéticos , Humanos , Pesquisa Translacional BiomédicaRESUMO
Pancreatitis is a debilitating disease of the exocrine pancreas that, under chronic conditions, is a major susceptibility factor for pancreatic ductal adenocarcinoma (PDAC). Although down-regulation of genes that promote the mature acinar cell fate is required to reduce injury associated with pancreatitis, the factors that promote this repression are unknown. Activating transcription factor 3 (ATF3) is a key mediator of the unfolded protein response, a pathway rapidly activated during pancreatic insult. Using chromatin immunoprecipitation followed by next-generation sequencing, we show that ATF3 is bound to the transcriptional regulatory regions of >30% of differentially expressed genes during the initiation of pancreatitis. Of importance, ATF3-dependent regulation of these genes was observed only upon induction of pancreatitis, with pathways involved in inflammation, acinar cell differentiation, and cell junctions being specifically targeted. Characterizing expression of transcription factors that affect acinar cell differentiation suggested that acinar cells lacking ATF3 maintain a mature cell phenotype during pancreatitis, a finding supported by maintenance of junctional proteins and polarity markers. As a result, Atf3-/- pancreatic tissue displayed increased tissue damage and inflammatory cell infiltration at early time points during injury but, at later time points, showed reduced acinar-to-duct cell metaplasia. Thus our results reveal a critical role for ATF3 as a key regulator of the acinar cell transcriptional response during injury and may provide a link between chronic pancreatitis and PDAC.
Assuntos
Células Acinares/metabolismo , Fator 3 Ativador da Transcrição/metabolismo , Pancreatite/metabolismo , Pancreatite/patologia , Células Acinares/citologia , Fator 3 Ativador da Transcrição/genética , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Diferenciação Celular/fisiologia , Ceruletídeo , Regulação para Baixo , Masculino , Camundongos , Camundongos Knockout , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Pancreatite/induzido quimicamente , Fenótipo , Neoplasias PancreáticasRESUMO
Organic anion-transporting polypeptides (OATPs) are membrane proteins that mediate cellular uptake of structurally diverse endogenous and exogenous compounds, including bile salts, thyroid and sex hormones, pharmacological agents, and toxins. Roles of OATPs in human liver are well established. Our recent report suggested the presence of the hepatic transporter OATP1B3 in human ß cells. The aim of this study was to better characterize cellular localization and interindividual variation in OATP1B3 expression in human adult islets as a function of age, sex, and pancreatic disease, and to assess the expression of other OATPs. High transcript levels of OATP1B3, OATP2B1, OATP1A2, but not OATP1B1 were observed in isolated human adult islets. While OATP1B3 protein expression was variable, the carrier co-localized more frequently with glucagon-positive α cells than insulin-positive ß cells in islets of normal pancreatic tissues from ten subjects using dual immunostaining. Moreover, OATP1B3 co-staining with endocrine cells was two- to three-fold higher in older (≥60 years) than younger (<60 years) subjects. In comparison, in a subset of three individuals, OATP2B1 was primarily found in ß cells, suggesting a distinct expression pattern for OATP1B3 and OATP2B1 in islets. Abundant OATP1B3 staining was also observed in islet as well as ductal cells of diseased tissues of patients with pancreatitis or pancreatic adenocarcinoma. Considering the abundance of key OATP carriers in ß and α cells, potential implications of OATP transport in islet cell function may be suggested. Future studies are needed to gain insights into their specific endocrine roles as well as pharmacological relevance.
Assuntos
Ilhotas Pancreáticas/metabolismo , Transportadores de Ânions Orgânicos/genética , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/genética , Adulto , Humanos , Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/citologia , Transportadores de Ânions Orgânicos/análise , Transportadores de Ânions Orgânicos/metabolismo , RNA Mensageiro/genética , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/análise , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismoRESUMO
Nonalcoholic fatty liver disease (NAFLD) alters drug response. We previously reported that NAFLD is associated with reduced in vivo CYP3A drug-metabolism activity and hepatic CYP3A4 expression in humans as well as mouse and human hepatoma models of the disease. Here, we investigated the role of the lipid- and glucose-modulating hormone fibroblast growth factor 21 (FGF21) in the molecular mechanism regulating CYP3A4 expression in NAFLD. In human subjects, mouse and cellular NAFLD models with lower CYP3A4 expression, circulating FGF21, or hepatic FGF21 mRNA levels were elevated. Administration of recombinant FGF21 or transient hepatic overexpression of FGF21 resulted in reduced liver CYP3A4 luciferase reporter activity in mice and decreased CYP3A4 mRNA expression and activity in cultured Huh7 hepatoma cells. Blocking canonical FGF21 signaling by pharmacological inhibition of MEK1 kinase in Huh7 cells caused de-repression of CYP3A4 mRNA expression with FGF21 treatment. Mice with high-fat diet-induced simple hepatic steatosis and lipid-loaded Huh7 cells had reduced nuclear localization of the pregnane X receptor (PXR), a key transcriptional regulator of CYP3A4 Furthermore, decreased nuclear PXR was observed in mouse liver and Huh7 cells after FGF21 treatment or FGF21 overexpression. Decreased PXR binding to the CYP3A4 proximal promoter was found in FGF21-treated Huh7 cells. An FGF21-PXR signaling pathway may be involved in decreased hepatic CYP3A4 metabolic activity in NAFLD.
Assuntos
Citocromo P-450 CYP3A/genética , Regulação para Baixo , Fatores de Crescimento de Fibroblastos/metabolismo , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/genética , Receptores de Esteroides/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Citocromo P-450 CYP3A/metabolismo , Modelos Animais de Doenças , Feminino , Fatores de Crescimento de Fibroblastos/administração & dosagem , Fatores de Crescimento de Fibroblastos/sangue , Fatores de Crescimento de Fibroblastos/genética , Glucuronidase/metabolismo , Humanos , Proteínas Klotho , Fígado , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/sangue , Receptor de Pregnano X , Regiões Promotoras Genéticas/genética , Ligação Proteica , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Frações Subcelulares/metabolismo , Transcrição GênicaRESUMO
Proper regulation of cytosolic Ca(2+) is critical for pancreatic acinar cell function. Disruptions in normal Ca(2+) concentrations affect numerous cellular functions and are associated with pancreatitis. Membrane pumps and channels regulate cytosolic Ca(2+) homeostasis by promoting rapid Ca(2+) movement. Determining how expression of Ca(2+) modulators is regulated and the cellular alterations that occur upon changes in expression can provide insight into initiating events of pancreatitis. The goal of this study was to delineate the gene structure and regulation of a novel pancreas-specific isoform for Secretory Pathway Ca(2+) ATPase 2 (termed SPCA2C), which is encoded from the Atp2c2 gene. Using Next Generation Sequencing of RNA (RNA-seq), chromatin immunoprecipitation for epigenetic modifications and promoter-reporter assays, a novel transcriptional start site was identified that promotes expression of a transcript containing the last four exons of the Atp2c2 gene (Atp2c2c). This region was enriched for epigenetic marks and pancreatic transcription factors that promote gene activation. Promoter activity for regions upstream of the ATG codon in Atp2c2's 24th exon was observed in vitro but not in in vivo. Translation from this ATG encodes a protein aligned with the carboxy terminal of SPCA2. Functional analysis in HEK 293A cells indicates a unique role for SPCA2C in increasing cytosolic Ca(2+) . RNA analysis indicates that the decreased Atp2c2c expression observed early in experimental pancreatitis reflects a global molecular response of acinar cells to reduce cytosolic Ca(2+) levels. Combined, these results suggest SPCA2C affects Ca(2+) homeostasis in pancreatic acinar cells in a unique fashion relative to other Ca(2+) ATPases. J. Cell. Physiol. 231: 2768-2778, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Células Acinares/metabolismo , ATPases Transportadoras de Cálcio/genética , Pâncreas/patologia , Sítio de Iniciação de Transcrição , Transcrição Gênica , Células Acinares/patologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/metabolismo , Ceruletídeo , Epigênese Genética , Éxons/genética , Feminino , Genoma , Células HEK293 , Histonas/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pancreatite/genética , Pancreatite/patologia , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
UNLABELLED: Osteoarthritis (OA) is a highly prevalent, disabling joint disease with no existing therapies to slow or halt its progression. Cartilage degeneration hallmarks OA pathogenesis, and pannexin 3 (Panx3), a member of a novel family of channel proteins, is upregulated during this process. The function of Panx3 remains poorly understood, but we consistently observed a strong increase in Panx3 immunostaining in OA lesions in both mice and humans. Here, we developed and characterized the first global and conditional Panx3 knockout mice to investigate the role of Panx3 in OA. Interestingly, global Panx3 deletion produced no overt phenotype and had no obvious effect on early skeletal development. Mice lacking Panx3 specifically in the cartilage and global Panx3 knockout mice were markedly resistant to the development of OA following destabilization of medial meniscus surgery. These data indicate a specific catabolic role of Panx3 in articular cartilage and identify Panx3 as a potential therapeutic target for OA. Lastly, while Panx1 has been linked to over a dozen human pathologies, this is the first in vivo evidence for a role of Panx3 in disease. KEY MESSAGE: Panx3 is localized to cartilage lesions in mice and humans. Global Panx3 deletion does not result in any developmental abnormalities. Mice lacking Panx3 are resistant to the development of osteoarthritis. Panx3 is a novel therapeutic target for the treatment of osteoarthritis.
Assuntos
Cartilagem Articular/patologia , Conexinas/genética , Deleção de Genes , Osteoartrite/genética , Osteoartrite/patologia , Animais , Cartilagem Articular/metabolismo , Linhagem Celular , Colágeno Tipo II/análise , Colágeno Tipo II/metabolismo , Conexinas/análise , Conexinas/metabolismo , Feminino , Humanos , Masculino , Metaloproteinase 13 da Matriz/análise , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Osteoartrite/etiologia , Osteoartrite/metabolismoRESUMO
Acinar cells of the pancreas produce the majority of enzymes required for digestion and make up >90% of the cells within the pancreas. Due to a common developmental origin and the plastic nature of the acinar cell phenotype, these cells have been identified as a possible source of ß cells as a therapeutic option for Type I diabetes. However, recent evidence indicates that acinar cells are the main source of pancreatic intraepithelial neoplasias (PanINs), the predecessor of pancreatic ductal adenocarcinoma (PDAC). The conversion of acinar cells to either ß cells or precursors to PDAC is dependent on reprogramming of the cells to a more primitive, progenitor-like phenotype, which involves changes in transcription factor expression and activity, and changes in their epigenetic program. This review will focus on the mechanisms that promote acinar cell reprogramming, as well as the factors that may affect these mechanisms.
Assuntos
Células Acinares/citologia , Reprogramação Celular , Epigênese Genética , Pâncreas/citologia , Células Acinares/metabolismo , Animais , Diferenciação Celular , Diabetes Mellitus Tipo 1/terapia , Humanos , Células Secretoras de Insulina/citologia , Camundongos , Pâncreas/embriologia , Pancreatopatias , Ductos Pancreáticos/citologia , Neoplasias Pancreáticas/etiologiaRESUMO
Thyroid hormone (TH) acts through specific receptors (TRs), which are conditional transcription factors, to induce fibroblast growth factor 21 (FGF21), a peptide hormone that is usually induced by fasting and that influences lipid and carbohydrate metabolism via local hepatic and systemic endocrine effects. While TH and FGF21 display overlapping actions when administered, including reductions in serum lipids, according to the current models these hormones act independently in vivo. In this study, we examined mechanisms of regulation of FGF21 expression by TH and tested the possibility that FGF21 is required for induction of hepatic TH-responsive genes. We confirm that active TH (triiodothyronine (T3)) and the TRß-selective thyromimetic GC1 increase FGF21 transcript and peptide levels in mouse liver and that this effect requires TRß. T3 also induces FGF21 in cultured hepatocytes and this effect involves direct actions of TRß1, which binds a TRE within intron 2 of FGF21. Gene expression profiles of WT and Fgf21-knockout mice are very similar, indicating that FGF21 is dispensable for the majority of hepatic T3 gene responses. A small subset of genes displays diminished T3 response in the absence of FGF21. However, most of these are not obviously directly involved in T3-dependent hepatic metabolic processes. Consistent with these results, T3-dependent effects on serum cholesterol are maintained in the Fgf21(-/-) background and we observe no effect of the Fgf21-knockout background on serum triglycerides and glucose. Our findings indicate that T3 regulates the genes involved in classical hepatic metabolic responses independently of FGF21.
Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Fígado/metabolismo , Receptores beta dos Hormônios Tireóideos/fisiologia , Animais , Fatores de Crescimento de Fibroblastos/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise em Microsséries , Elementos de Resposta , Tri-Iodotironina/farmacologiaRESUMO
Fibroblast growth factor 21 (FGF21) is a key regulator of metabolism under conditions of stress such as starvation, obesity, and hypothermia. Rapid induction of FGF21 is also observed in experimental models of pancreatitis, and FGF21 reduces tissue damage observed in these models, suggesting a nonmetabolic function. Pancreatitis is a debilitating disease with significant morbidity that greatly increases the risk of pancreatic ductal adenocarcinoma. The goals of this study were to examine the regulation and function of FGF21 in acinar cell injury, specifically in a mouse model of pancreatic injury (Mist1(-/-)). Mist1(-/-) mice exhibit acinar cell disorganization, decreased acinar cell communication and exocytosis, and increased sensitivity to cerulein-induced pancreatitis (CIP). Examination of Fgf21 expression in Mist1(-/-) mice by qRT-PCR, Northern blot, and Western blot analyses showed a marked decrease in pancreatic Fgf21 expression before and after induction of CIP compared with C57Bl/6 mice. To determine whether the loss of FGF21 accounted for the Mist1(-/-) phenotypes, we generated Mist1(-/-) mice overexpressing human FGF21 from the ApoE promoter (Mist1(-/-)ApoE-FGF21). Reexpression of FGF21 partially mitigated pancreatic damage in Mist1(-/-) tissue based on reduced intrapancreatic enzyme activation, reduced expression of genes involved in fibrosis, and restored cell-cell junctions. Interestingly, alteration of Fgf21 expression in Mist1(-/-) tissue was not simply due to a loss of direct transcriptional regulation by MIST1. Chromatin immunopreciptation indicated that the loss of Fgf21 in the Mist1(-/-) pancreas is due, in part, to epigenetic silencing. Thus, our studies identify a new role for FGF21 in reducing acinar cell injury and uncover a novel mechanism for regulating Fgf21 gene expression.