RESUMO
Scavenger receptor class B, type I (SR-BI) and its adaptor protein PDZK1 mediate responses to HDL cholesterol in endothelium. Whether the receptor-adaptor protein tandem serves functions in other vascular cell types is unknown. The current work determined the roles of SR-BI and PDZK1 in vascular smooth muscle (VSM). To evaluate possible VSM functions of SR-BI and PDZK1 in vivo, neointima formation was assessed 21 days post-ligation in the carotid arteries of wild-type, SR-BI-/- or PDZK1-/- mice. Whereas neointima development was negligible in wild-type and SR-BI-/-, there was marked neointima formation in PDZK1-/- mice. PDZK1 expression was demonstrated in primary mouse VSM cells, and compared to wild-type cells, PDZK1-/- VSM displayed exaggerated proliferation and migration in response to platelet derived growth factor (PDGF). Tandem affinity purification-mass spectrometry revealed that PDZK1 interacts with breakpoint cluster region kinase (Bcr), which contains a C-terminal PDZ binding sequence and is known to enhance responses to PDGF in VSM. PDZK1 interaction with Bcr in VSM was demonstrated by pull-down and by coimmunoprecipitation, and the augmented proliferative response to PDGF in PDZK1-/- VSM was abrogated by Bcr depletion. Furthermore, compared with wild-type Bcr overexpression, the introduction of a Bcr mutant incapable of PDZK1 binding into VSM cells yielded an exaggerated proliferative response to PDGF. Thus, PDZK1 has novel SR-BI-independent function in VSM that affords protection from neointima formation, and this involves PDZK1 suppression of VSM cell proliferation via an inhibitory interaction with Bcr.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Músculo Liso Vascular/enzimologia , Proteínas Proto-Oncogênicas c-bcr/antagonistas & inibidores , Túnica Íntima/crescimento & desenvolvimento , Animais , Movimento Celular , Proliferação de Células , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/citologia , Proteínas Proto-Oncogênicas c-bcr/metabolismoRESUMO
In beating hearts, phosphorylation of myosin regulatory light chain (RLC) at a single site to 0.45 mol of phosphate/mol by cardiac myosin light chain kinase (cMLCK) increases Ca(2+) sensitivity of myofilament contraction necessary for normal cardiac performance. Reduction of RLC phosphorylation in conditional cMLCK knock-out mice caused cardiac dilation and loss of cardiac performance by 1 week, as shown by increased left ventricular internal diameter at end-diastole and decreased fractional shortening. Decreased RLC phosphorylation by conventional or conditional cMLCK gene ablation did not affect troponin-I or myosin-binding protein-C phosphorylation in vivo. The extent of RLC phosphorylation was not changed by prolonged infusion of dobutamine or treatment with a ß-adrenergic antagonist, suggesting that RLC is constitutively phosphorylated to maintain cardiac performance. Biochemical studies with myofilaments showed that RLC phosphorylation up to 90% was a random process. RLC is slowly dephosphorylated in both noncontracting hearts and isolated cardiac myocytes from adult mice. Electrically paced ventricular trabeculae restored RLC phosphorylation, which was increased to 0.91 mol of phosphate/mol of RLC with inhibition of myosin light chain phosphatase (MLCP). The two RLCs in each myosin appear to be readily available for phosphorylation by a soluble cMLCK, but MLCP activity limits the amount of constitutive RLC phosphorylation. MLCP with its regulatory subunit MYPT2 bound tightly to myofilaments was constitutively phosphorylated in beating hearts at a site that inhibits MLCP activity. Thus, the constitutive RLC phosphorylation is limited physiologically by low cMLCK activity in balance with low MLCP activity.
Assuntos
Miocárdio/metabolismo , Cadeias Leves de Miosina/metabolismo , Animais , Cálcio/metabolismo , Calmodulina/metabolismo , Ventrículos do Coração/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/deficiência , Cadeias Leves de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosforilação , Ratos , Ratos Sprague-DawleyRESUMO
Optimal stress signaling by Hypoxia Inducible Factor 2 (HIF-2) during low oxygen states or hypoxia requires coupled actions of a specific coactivator/lysine acetyltransferase, Creb binding protein (CBP), and a specific deacetylase, Sirtuin 1 (SIRT1). We recently reported that acetylation of HIF-2 by CBP also requires a specific acetyl CoA generator, acetate-dependent acetyl CoA synthetase 2 (ACSS2). In this study, we demonstrate that ACSS2/HIF-2 signaling is active not only during hypoxia, but also during glucose deprivation. Acetate levels increase during stress and coincide with maximal HIF-2α acetylation and CBP/HIF-2α complex formation. Exogenous acetate induces HIF-2α acetylation, CBP/HIF-2α complex formation, and HIF-2 signaling. ACSS2 and HIF-2 are required for maximal colony formation, proliferation, migration, and invasion during stress. Acetate also stimulates flank tumor growth and metastasis in mice in an ACSS2 and HIF-2 dependent manner. Thus, ACSS2/CBP/SIRT1/HIF-2 signaling links nutrient sensing and stress signaling with cancer growth and progression in mammals.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Transdução de Sinais , Microambiente Tumoral , Acetato-CoA Ligase/metabolismo , Acetatos/metabolismo , Animais , Proteína de Ligação a CREB/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Glucose/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Oxigênio/metabolismo , Sirtuína 1/metabolismoRESUMO
It is poorly understood why there is greater cardiovascular disease risk associated with the apolipoprotein E4 (apoE) allele vs. apoE3, and also greater risk with the LRP8/apolipoprotein E receptor 2 (ApoER2) variant ApoER2-R952Q. Little is known about the function of the apoE-ApoER2 tandem outside of the central nervous system. We now report that in endothelial cells apoE3 binding to ApoER2 stimulates endothelial NO synthase (eNOS) and endothelial cell migration, and it also attenuates monocyte-endothelial cell adhesion. However, apoE4 does not stimulate eNOS or endothelial cell migration or dampen cell adhesion, and alternatively it selectively antagonizes apoE3/ApoER2 actions. The contrasting endothelial actions of apoE4 vs. apoE3 require the N-terminal to C-terminal interaction in apoE4 that distinguishes it structurally from apoE3. Reconstitution experiments further reveal that ApoER2-R952Q is a loss-of-function variant of the receptor in endothelium. Carotid artery reendothelialization is decreased in ApoER2(-/-) mice, and whereas adenoviral-driven apoE3 expression in wild-type mice has no effect, apoE4 impairs reendothelialization. Moreover, in a model of neointima formation invoked by carotid artery endothelial denudation, ApoER2(-/-) mice display exaggerated neointima development. Thus, the apoE3/ApoER2 tandem promotes endothelial NO production, endothelial repair, and endothelial anti-inflammatory properties, and it prevents neointima formation. In contrast, apoE4 and ApoER2-R952Q display dominant-negative action and loss of function, respectively. Thus, genetic variants of apoE and ApoER2 impact cardiovascular health by differentially modulating endothelial function.
Assuntos
Apolipoproteínas E/genética , Células Endoteliais/metabolismo , Proteínas Relacionadas a Receptor de LDL/genética , Células 3T3 , Animais , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Artérias Carótidas/metabolismo , Bovinos , Adesão Celular , Movimento Celular , Células Endoteliais/citologia , Humanos , Proteínas Relacionadas a Receptor de LDL/metabolismo , Camundongos , Monócitos/citologia , Proteínas Mutantes/metabolismo , Neointima/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismoRESUMO
The hormone erythropoietin (EPO), which is synthesized in the kidney or liver of adult mammals, controls erythrocyte production and is regulated by the stress-responsive transcription factor hypoxia-inducible factor-2 (HIF-2). We previously reported that the lysine acetyltransferase CREB-binding protein (CBP) is required for HIF-2α acetylation and efficient HIF-2-dependent EPO induction during hypoxia. We now show that these processes require acetate-dependent acetyl CoA synthetase 2 (ACSS2). In human Hep3B hepatoma cells and in EPO-generating organs of hypoxic or acutely anemic mice, acetate levels rise and ACSS2 is required for HIF-2α acetylation, CBP-HIF-2α complex formation, CBP-HIF-2α recruitment to the EPO enhancer and efficient induction of EPO gene expression. In acutely anemic mice, acetate supplementation augments stress erythropoiesis in an ACSS2-dependent manner. Moreover, in acquired and inherited chronic anemia mouse models, acetate supplementation increases EPO expression and the resting hematocrit. Thus, a mammalian stress-responsive acetate switch controls HIF-2 signaling and EPO induction during pathophysiological states marked by tissue hypoxia.
Assuntos
Acetatos/metabolismo , Eritropoese , Estresse Fisiológico , Acetilação , Animais , Hipóxia Celular , Eritropoetina/genética , Camundongos , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Cancer genome sequencing has identified numerous somatic mutations whose biologic relevance is uncertain. In this study, we used genome-editing tools to create and analyze targeted somatic mutations in murine models of liver cancer. Transcription activator-like effector nucleases (TALEN) were designed against ß-catenin (Ctnnb1) and adenomatous polyposis coli (Apc), two commonly mutated genes in hepatocellular carcinoma (HCC), to generate isogenic HCC cell lines. Both mutant cell lines exhibited evidence of Wnt pathway dysregulation. We asked whether these TALENs could create targeted somatic mutations after hydrodynamic transfection into mouse liver. TALENs targeting ß-catenin promoted endogenous HCC carrying the intended gain-of-function mutations. However, TALENs targeting Apc were not as efficient in inducing in vivo homozygous loss-of-function mutations. We hypothesized that hepatocyte polyploidy might be protective against TALEN-induced loss of heterozygosity, and indeed Apc gene editing was less efficient in tetraploid than in diploid hepatocytes. To increase efficiency, we administered adenoviral Apc TALENs and found that we could achieve a higher mutagenesis rate in vivo. Our results demonstrate that genome-editing tools can enable the in vivo study of cancer genes and faithfully recapitulate the mosaic nature of mutagenesis in mouse cancer models. Cancer Res; 74(18); 5311-21. ©2014 AACR.
Assuntos
Endonucleases/genética , Engenharia Genética/métodos , Neoplasias Experimentais/genética , Fatores de Transcrição/genética , Animais , Genoma , Xenoenxertos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Mutagênese , Ativação Transcricional , TransfecçãoRESUMO
Mammalian skeletal muscle can remodel, repair, and regenerate itself by mobilizing satellite cells, a resident population of myogenic progenitor cells. Muscle injury and subsequent activation of myogenic progenitor cells is associated with oxidative stress. Cytoglobin is a hemoprotein expressed in response to oxidative stress in a variety of tissues, including striated muscle. In this study, we demonstrate that cytoglobin is up-regulated in activated myogenic progenitor cells, where it localizes to the nucleus and contributes to cell viability. siRNA-mediated depletion of cytoglobin from C2C12 myoblasts increased levels of reactive oxygen species and apoptotic cell death both at baseline and in response to stress stimuli. Conversely, overexpression of cytoglobin reduced reactive oxygen species levels, caspase activity, and cell death. Mice in which cytoglobin was knocked out specifically in skeletal muscle were generated to examine the role of cytoglobin in vivo. Myogenic progenitor cells isolated from these mice were severely deficient in their ability to form myotubes as compared with myogenic progenitor cells from wild-type littermates. Consistent with this finding, the capacity for muscle regeneration was severely impaired in mice deficient for skeletal-muscle cytoglobin. Collectively, these data demonstrate that cytoglobin serves an important role in muscle repair and regeneration.
Assuntos
Regulação da Expressão Gênica , Globinas/metabolismo , Músculos/fisiologia , Regeneração/fisiologia , Células Satélites de Músculo Esquelético/citologia , Animais , Apoptose , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Citoglobina , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio , Células Satélites de Músculo Esquelético/patologia , Células-Tronco/citologia , Fatores de TempoRESUMO
Hibernation is a natural adaptation that allows certain mammals to survive physiological extremes that are lethal to humans. Near freezing body temperatures, heart rates of 3-10 beats per minute, absence of food consumption, and depressed metabolism are characteristic of hibernation torpor bouts that are periodically interrupted by brief interbout arousals (IBAs). The molecular basis of torpor induction is unknown, however starved mice overexpressing the metabolic hormone fibroblast growth factor 21 (FGF21) promote fat utilization, reduce body temperature, and readily enter torpor-all hallmarks of mammalian hibernation. In this study we cloned FGF21 from the naturally hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus) and found that levels of FGF21 mRNA in liver and FGF21 protein in serum are elevated during hibernation torpor bouts and significantly elevated during IBAs compared to summer active animals. The effects of artificially elevating circulating FGF21 concentrations 50 to 100-fold via adenoviral-mediated overexpression were examined at three different times of the year. This is the first time that a transgenic approach has been used in a natural hibernator to examine mechanistic aspects of hibernation. Surgically implanted transmitters measured various metrics of the hibernation phenotype over a 7-day period including changes in motor activity, heart rate and core body temperature. In April fed-state animals, FGF21 overexpression decreased blood insulin and free fatty acid concentrations, effects similar to those seen in obese mice. However, elevated FGF21 concentrations did not cause torpor in these fed-state animals nor did they cause torpor or affect metabolic parameters in fasted-state animals in March/April, August or October. We conclude that FGF21 is strongly regulated during torpor and IBA but that its overexpression is not sufficient to cause torpor in naturally hibernating ground squirrels.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Hibernação , Sciuridae/metabolismo , Adipócitos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Temperatura Corporal , Meios de Cultivo Condicionados/farmacologia , DNA Complementar/metabolismo , Frequência Cardíaca , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Estações do Ano , Homologia de Sequência de AminoácidosRESUMO
RATIONALE: Signal initiation by the high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), which is important to actions of HDL on endothelium and other processes, requires cholesterol efflux and the C-terminal transmembrane domain. The C-terminal transmembrane domain uniquely interacts with plasma membrane (PM) cholesterol. OBJECTIVE: The molecular basis and functional significance of SR-BI interaction with PM cholesterol are unknown. We tested the hypotheses that the interaction is required for SR-BI signaling, and that it enables SR-BI to serve as a PM cholesterol sensor. METHODS AND RESULTS: In studies performed in COS-M6 cells, mutation of a highly conserved C-terminal transmembrane domain glutamine to alanine (SR-BI-Q445A) decreased PM cholesterol interaction with the receptor by 71% without altering HDL binding or cholesterol uptake or efflux, and it yielded a receptor incapable of HDL-induced signaling. Signaling prompted by cholesterol efflux to methyl-ß-cyclodextrin also was prevented, indicating that PM cholesterol interaction with the receptor enables it to serve as a PM cholesterol sensor. Using SR-BI-Q445A, we further demonstrated that PM cholesterol sensing by SR-BI does not influence SR-BI-mediated reverse cholesterol transport to the liver in mice. However, the PM cholesterol sensing does underlie apolipoprotein B intracellular trafficking in response to postprandial micelles or methyl-ß-cyclodextrin in cultured enterocytes, and it is required for HDL activation of endothelial NO synthase and migration in cultured endothelial cells and HDL-induced angiogenesis in vivo. CONCLUSIONS: Through interaction with PM cholesterol, SR-BI serves as a PM cholesterol sensor, and the resulting intracellular signaling governs processes in both enterocytes and endothelial cells.
Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Células Endoteliais/metabolismo , Enterócitos/metabolismo , Receptores Depuradores Classe B/metabolismo , Transdução de Sinais , Alanina , Animais , Apolipoproteínas B/metabolismo , Células CACO-2 , Bovinos , Membrana Celular/efeitos dos fármacos , HDL-Colesterol/metabolismo , Células Endoteliais/efeitos dos fármacos , Enterócitos/efeitos dos fármacos , Glutamina , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/metabolismo , Masculino , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Neovascularização Fisiológica , Óxido Nítrico Sintase Tipo III/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Depuradores Classe B/química , Receptores Depuradores Classe B/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , beta-Ciclodextrinas/farmacologiaRESUMO
The epicardium encapsulates the heart and functions as a source of multipotent progenitor cells and paracrine factors essential for cardiac development and repair. Injury of the adult heart results in reactivation of a developmental gene program in the epicardium, but the transcriptional basis of epicardial gene expression has not been delineated. We established a mouse embryonic heart organ culture and gene expression system that facilitated the identification of epicardial enhancers activated during heart development and injury. Epicardial activation of these enhancers depends on a combinatorial transcriptional code centered on CCAAT/enhancer binding protein (C/EBP) transcription factors. Disruption of C/EBP signaling in the adult epicardium reduced injury-induced neutrophil infiltration and improved cardiac function. These findings reveal a transcriptional basis for epicardial activation and heart injury, providing a platform for enhancing cardiac regeneration.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica , Coração/fisiopatologia , Infarto do Miocárdio/genética , Traumatismo por Reperfusão Miocárdica/genética , Pericárdio/embriologia , Pericárdio/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Animais , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/genética , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Elementos Facilitadores Genéticos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Masculino , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Contração Miocárdica , Infarto do Miocárdio/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Infiltração de Neutrófilos , Análise de Sequência com Séries de Oligonucleotídeos , Técnicas de Cultura de Órgãos , Pericárdio/citologia , Transdução de Sinais , Uroplaquina III/genética , Uroplaquina III/metabolismo , Remodelação Ventricular , Proteínas WT1/genética , Proteínas WT1/metabolismoRESUMO
Fibroblast growth factor 19 (FGF19) is a postprandial enterokine induced by the nuclear bile acid receptor, FXR, in ileum. FGF19 inhibits bile acid synthesis in liver through transcriptional repression of cholesterol 7α-hydroxylase (CYP7A1) via a mechanism involving the nuclear receptor SHP. Here, in a series of loss-of-function studies, we show that the nuclear receptors HNF4α and LRH-1 have dual roles in regulating Cyp7a1 in vivo. First, they cooperate in maintaining basal Cyp7a1 expression. Second, they enable SHP binding to the Cyp7a1 promoter and facilitate FGF19-mediated repression of bile acid synthesis. HNF4α and LRH-1 promote active transcription histone marks on the Cyp7a1 promoter that are reversed by FGF19 in a SHP-dependent manner. These findings demonstrate that both HNF4α and LRH-1 are important regulators of Cyp7a1 transcription in vivo.
Assuntos
Colesterol 7-alfa-Hidroxilase/biossíntese , Regulação da Expressão Gênica , Fator 4 Nuclear de Hepatócito/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Animais , Ácidos e Sais Biliares/metabolismo , Sítios de Ligação , Fatores de Crescimento de Fibroblastos/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Fígado/metabolismo , Camundongos , Regiões Promotoras Genéticas , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transcrição GênicaRESUMO
Progesterone (P(4)) and estradiol-17ß (E(2)) play critical and opposing roles in regulating myometrial quiescence and contractility during pregnancy and labor. Although these contrasting hormonal effects are likely mediated via differential regulation of inflammatory and contractile genes, the underlying mechanisms remain incompletely understood. Recently we discovered that targets of the microRNA (miR)-200 family, transcription factors zinc finger E-box binding homeobox (ZEB)-1 and ZEB2, serve as P(4)/progesterone receptor-mediated regulators of uterine quiescence during pregnancy. In the present study, we found that levels of the clustered miRNAs, miR-199a-3p and miR-214, were significantly decreased in laboring myometrium of pregnant mice and humans and in an inflammatory mouse model of preterm labor, whereas the miR-199a-3p/miR-214 target, cyclooxygenase-2, a critical enzyme in synthesis of proinflammatory prostaglandins, was coordinately increased. Overexpression of miR-199a-3p and miR-214 in cultured human myometrial cells inhibited cyclooxygenase-2 protein and blocked TNF-α-induced myometrial cell contractility, suggesting their physiological relevance. Notably, E(2) treatment of ovariectomized mice suppressed, whereas P(4) enhanced uterine miR-199a-3p/214 expression. Intriguingly, these opposing hormonal effects were mediated by ZEB1, which is induced by P(4), inhibited by E(2) and activates miR199a/214 transcription. Together, these findings identify miR-199a-3p/miR-214 as important regulators of myometrial contractility and provide new insight into strategies to prevent preterm birth.
Assuntos
Estrogênios/farmacologia , Trabalho de Parto/efeitos dos fármacos , Trabalho de Parto/genética , MicroRNAs/metabolismo , Progesterona/farmacologia , Contração Uterina/efeitos dos fármacos , Contração Uterina/genética , Animais , Sequência de Bases , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos ICR , MicroRNAs/genética , Dados de Sequência Molecular , Família Multigênica/genética , Miométrio/citologia , Miométrio/efeitos dos fármacos , Miométrio/metabolismo , Gravidez , Fatores de Transcrição/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
In response to severe injury, adult skeletal muscle exhibits a remarkable regenerative capacity due to a resident muscle stem/progenitor cell population. While a number of factors are expressed in the muscle progenitor cell (MPC) population, the molecular networks that govern this cell population remain an area of active investigation. In this study, utilizing knockdown techniques and overexpression of Foxk1 in the myogenic lineage, we observed dysregulation of Foxo and Mef2 downstream targets. Utilizing an array of technologies, we establish that Foxk1 represses the transcriptional activity of Foxo4 and Mef2 and physically interacts with Foxo4 and Mef2, thus promoting MPC proliferation and antagonizing the myogenic lineage differentiation program, respectively. Correspondingly, knockdown of Foxk1 in C2C12 myoblasts results in cell cycle arrest, and Foxk1 overexpression in C2C12CAR myoblasts retards muscle differentiation. Collectively, we have established that Foxk1 promotes MPC proliferation by repressing Foxo4 transcriptional activity and inhibits myogenic differentiation by repressing Mef2 activity. These studies enhance our understanding of the transcriptional networks that regulate the MPC population and muscle regeneration.
Assuntos
Diferenciação Celular , Fatores de Transcrição Forkhead/metabolismo , Desenvolvimento Muscular , Fatores de Regulação Miogênica/metabolismo , Animais , Ciclo Celular , Proteínas de Ciclo Celular , Proliferação de Células , DNA/metabolismo , Fatores de Transcrição MEF2 , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Ligação Proteica , Regeneração , Proteínas Repressoras/metabolismo , Transcrição GênicaRESUMO
Hypoxia-inducible factors (HIFs) are oxygen-sensitive transcription factors. HIF-1α plays a prominent role in hypoxic gene induction. HIF-2α target genes are more restricted but include erythropoietin (Epo), one of the most highly hypoxia-inducible genes in mammals. We previously reported that HIF-2α is acetylated during hypoxia but is rapidly deacetylated by the stress-responsive deacetylase Sirtuin 1. We now demonstrate that the lysine acetyltransferases cAMP-response element-binding protein-binding protein (CBP) and p300 are required for efficient Epo induction during hypoxia. However, despite close structural similarity, the roles of CBP and p300 differ in HIF signaling. CBP acetylates HIF-2α, is a major coactivator for HIF-2-mediated Epo induction, and is required for Sirt1 augmentation of HIF-2 signaling during hypoxia in Hep3B cells. In comparison, p300 is a major contributor for HIF-1 signaling as indicated by induction of Pgk1. Whereas CBP can bind with HIF-2α independent of the HIF-2α C-terminal activation domain via enzyme/substrate interactions, p300 only complexes with HIF-2α through the C-terminal activation domain. Maximal CBP/HIF-2 signaling requires intact CBP acetyltransferase activity in both Hep3B cells as well as in mice.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteína de Ligação a CREB/metabolismo , Fragmentos de Peptídeos/metabolismo , Sialoglicoproteínas/metabolismo , Transdução de Sinais/fisiologia , Sirtuína 1/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteína de Ligação a CREB/genética , Linhagem Celular , Eritropoetina/biossíntese , Eritropoetina/genética , Humanos , Camundongos , Fragmentos de Peptídeos/genética , Fosfoglicerato Quinase/genética , Fosfoglicerato Quinase/metabolismo , Estrutura Terciária de Proteína , Sialoglicoproteínas/genética , Sirtuína 1/genética , Fatores de Transcrição de p300-CBP/genéticaRESUMO
During pregnancy, uterine quiescence is maintained by increased progesterone receptor (PR) activity, but labor is facilitated by a series of events that impair PR function. Previously, we discovered that miR-200 family members serve as progesterone (P(4))-modulated activators of contraction-associated genes in the pregnant uterus. In this study, we identified a unique role for miR-200a to enhance the local metabolism of P(4) in myometrium and, thus, decrease PR function during the progression toward labor. miR-200a exerts this action by direct repression of STAT5b, a transcriptional repressor of the P(4)-metabolizing enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD). We observed that miR-200a expression increased and STAT5b expression coordinately decreased in myometrium of mice as they progressed to labor and in laboring myometrium from pregnant women. These changes were associated with a dramatic increase in expression and activity of 20α-HSD in laboring myometrium from mouse and human. Notably, overexpression of miR-200a in cultured human myometrial cells (hTERT-HM) suppressed STAT5b and increased 20α-HSD mRNA levels. In uterine tissues of ovariectomized mice injected with P(4), miR-200 expression was significantly decreased, STAT5b expression was up-regulated, and 20α-HSD mRNA was decreased, but in 15 d postcoitum pregnant mice injected with the PR antagonist RU486, preterm labor was associated with increased miR-200a, decreased STAT5b, and enhanced 20α-HSD expression. Taken together, these findings implicate miR-200a as an important regulator of increased local P(4) metabolism in the pregnant uterus near term and provide insight into the importance of miR-200s in the decline in PR function leading to labor.
Assuntos
Trabalho de Parto/genética , MicroRNAs/genética , Trabalho de Parto Prematuro/genética , Receptores de Progesterona/genética , 20-alfa-Hidroxiesteroide Desidrogenase/genética , 20-alfa-Hidroxiesteroide Desidrogenase/metabolismo , Animais , Células Cultivadas , Feminino , Expressão Gênica/efeitos dos fármacos , Antagonistas de Hormônios/farmacologia , Humanos , Immunoblotting , Trabalho de Parto/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mifepristona/farmacologia , Miométrio/citologia , Miométrio/metabolismo , Trabalho de Parto Prematuro/metabolismo , Ovariectomia , Gravidez , Progesterona/metabolismo , Progesterona/farmacologia , Receptores de Progesterona/antagonistas & inibidores , Receptores de Progesterona/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
The leading cause of death in diabetic patients is cardiovascular disease; diabetic cardiomyopathy is typified by alterations in cardiac morphology and function, independent of hypertension or coronary disease. However, the molecular mechanism that links diabetes to cardiomyopathy is incompletely understood. Insulin resistance is a hallmark feature of diabetes, and the FoxO family of transcription factors, which regulate cell size, viability, and metabolism, are established targets of insulin and growth factor signaling. Here, we set out to evaluate a possible role of FoxO proteins in diabetic cardiomyopathy. We found that FoxO proteins were persistently activated in cardiac tissue in mice with diabetes induced either genetically or by high-fat diet (HFD). FoxO activity was critically linked with development of cardiomyopathy: cardiomyocyte-specific deletion of FoxO1 rescued HFD-induced declines in cardiac function and preserved cardiomyocyte insulin responsiveness. FoxO1-depleted cells displayed a shift in their metabolic substrate usage, from free fatty acids to glucose, associated with decreased accumulation of lipids in the heart. Furthermore, we found that FoxO1-dependent downregulation of IRS1 resulted in blunted Akt signaling and insulin resistance. Together, these data suggest that activation of FoxO1 is an important mediator of diabetic cardiomyopathy and is a promising therapeutic target for the disease.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Animais , Peso Corporal , Tamanho Celular , Sobrevivência Celular , Cardiomiopatias Diabéticas/metabolismo , Ácidos Graxos/metabolismo , Proteína Forkhead Box O1 , Glucose/metabolismo , Insuficiência Cardíaca/metabolismo , Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Somatomedinas/metabolismo , Frações SubcelularesRESUMO
The endocrine hormone fibroblast growth factor 21 (FGF21) is a powerful modulator of glucose and lipid metabolism and a promising drug for type 2 diabetes. Here we identify FGF21 as a potent regulator of skeletal homeostasis. Both genetic and pharmacologic FGF21 gain of function lead to a striking decrease in bone mass. In contrast, FGF21 loss of function leads to a reciprocal high-bone-mass phenotype. Mechanistically, FGF21 inhibits osteoblastogenesis and stimulates adipogenesis from bone marrow mesenchymal stem cells by potentiating the activity of peroxisome proliferator-activated receptor γ (PPAR-γ). Consequently, FGF21 deletion prevents the deleterious bone loss side effect of the PPAR-γ agonist rosiglitazone. Therefore, FGF21 is a critical rheostat for bone turnover and a key integrator of bone and energy metabolism. These results reveal that skeletal fragility may be an undesirable consequence of chronic FGF21 administration.
Assuntos
Reabsorção Óssea/patologia , Fatores de Crescimento de Fibroblastos/metabolismo , PPAR gama/metabolismo , Adipogenia/efeitos dos fármacos , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Reabsorção Óssea/metabolismo , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Resistência a Medicamentos/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/administração & dosagem , Fatores de Crescimento de Fibroblastos/farmacologia , Humanos , Camundongos , Camundongos Knockout , Tamanho do Órgão/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteogênese/efeitos dos fármacos , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Rosiglitazona , Tiazolidinedionas/farmacologiaRESUMO
Loss-of-function mutations in 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) 2 in humans and mice result in loss of both the white and brown adipose tissues from birth. AGPAT2 generates precursors for the synthesis of glycerophospholipids and triacylglycerols. Loss of adipose tissue, or lipodystrophy, results in hyperinsulinemia, diabetes mellitus, and severe hepatic steatosis. Here, we analyzed biochemical properties of human AGPAT2 and its close homolog, AGPAT1, and we studied their role in liver by transducing their expression via recombinant adenoviruses in Agpat2(-/-) mice. The in vitro substrate specificities of AGPAT1 and AGPAT2 are quite similar for lysophosphatidic acid and acyl-CoA. Protein homology modeling of both the AGPATs with glycerol-3-phosphate acyltransferase 1 (GPAT1) revealed that they have similar tertiary protein structure, which is consistent with their similar substrate specificities. When co-expressed, both isoforms co-localize to the endoplasmic reticulum. Despite such similarities, restoring AGPAT activity in liver by overexpression of either AGPAT1 or AGPAT2 in Agpat2(-/-) mice failed to ameliorate the hepatic steatosis. From these studies, we suggest that the role of AGPAT1 or AGPAT2 in liver lipogenesis is minimal and that accumulation of liver fat is primarily a consequence of insulin resistance and loss of adipose tissue in Agpat2(-/-) mice.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Tecido Adiposo/enzimologia , Retículo Endoplasmático/enzimologia , Fígado Gorduroso/enzimologia , Lipodistrofia/enzimologia , Fígado/enzimologia , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Tecido Adiposo/patologia , Animais , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Glicerofosfolipídeos/biossíntese , Glicerofosfolipídeos/genética , Células HEK293 , Humanos , Resistência à Insulina/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Lipodistrofia/genética , Lipodistrofia/patologia , Fígado/patologia , Camundongos , Camundongos Knockout , Transdução Genética , Triglicerídeos/biossíntese , Triglicerídeos/genéticaRESUMO
Regulation of hepatic carbohydrate homeostasis is crucial for maintaining energy balance in the face of fluctuating nutrient availability. Here, we show that the hormone fibroblast growth factor 15/19 (FGF15/19), which is released postprandially from the small intestine, inhibits hepatic gluconeogenesis, like insulin. However, unlike insulin, which peaks in serum 15 min after feeding, FGF15/19 expression peaks approximately 45 min later, when bile acid concentrations increase in the small intestine. FGF15/19 blocks the expression of genes involved in gluconeogenesis through a mechanism involving the dephosphorylation and inactivation of the transcription factor cAMP regulatory element-binding protein (CREB). This in turn blunts expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and other genes involved in hepatic metabolism. Overexpression of PGC-1α blocks the inhibitory effect of FGF15/19 on gluconeogenic gene expression. These results demonstrate that FGF15/19 works subsequent to insulin as a postprandial regulator of hepatic carbohydrate homeostasis.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/farmacologia , Glucose/metabolismo , Fígado/metabolismo , Transativadores/genética , Animais , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ácidos Graxos/metabolismo , Fatores de Crescimento de Fibroblastos/fisiologia , Expressão Gênica , Perfilação da Expressão Gênica , Genes Reporter , Gluconeogênese , Fígado/efeitos dos fármacos , Luciferases/biossíntese , Luciferases/genética , Masculino , Redes e Vias Metabólicas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Transdução de Sinais , Transativadores/metabolismo , Fatores de TranscriçãoRESUMO
G protein-coupled receptor (GPCR) pathways control glucose and fatty acid metabolism and the onset of obesity and diabetes. Regulators of G protein signaling (RGS) are GTPase-activating proteins (GAPs) for G(i) and G(q) α-subunits that control the intensity and duration of GPCR signaling. Herein we determined the role of Rgs16 in GPCR regulation of liver metabolism. Rgs16 is expressed during the last few hours of the daily fast in periportal hepatocytes, the oxygen-rich zone of the liver where lipolysis and gluconeogenesis predominate. Rgs16 knock-out mice had elevated expression of fatty acid oxidation genes in liver, higher rates of fatty acid oxidation in liver extracts, and higher plasma ß-ketone levels compared with wild type mice. By contrast, transgenic mice that overexpressed RGS16 protein specifically in liver exhibited reciprocal phenotypes as well as low blood glucose levels compared with wild type littermates and fatty liver after overnight fasting. The transcription factor carbohydrate response element-binding protein (ChREBP), which induces fatty acid synthesis genes in response to high carbohydrate feeding, was unexpectedly required during fasting for maximal Rgs16 transcription in liver and in cultured primary hepatocytes during gluconeogenesis. Thus, RGS16 provides a signaling mechanism for glucose production to inhibit GPCR-stimulated fatty acid oxidation in hepatocytes.