RESUMO
The hourglass model describes the convergence of species within the same phylum to a similar body plan during development; however, the molecular mechanisms underlying this phenomenon in mammals remain poorly described. Here, we compare rabbit and mouse time-resolved differentiation trajectories to revisit this model at single-cell resolution. We modeled gastrulation dynamics using hundreds of embryos sampled between gestation days 6.0 and 8.5 and compared the species using a framework for time-resolved single-cell differentiation-flows analysis. We find convergence toward similar cell-state compositions at E7.5, supported by the quantitatively conserved expression of 76 transcription factors, despite divergence in surrounding trophoblast and hypoblast signaling. However, we observed noticeable changes in specification timing of some lineages and divergence of primordial germ cell programs, which in the rabbit do not activate mesoderm genes. Comparative analysis of temporal differentiation models provides a basis for studying the evolution of gastrulation dynamics across mammals.
Assuntos
Gastrulação , Mesoderma , Animais , Coelhos , Camundongos , Gastrulação/genética , Mesoderma/fisiologia , Diferenciação Celular/fisiologia , Mamíferos/genética , Trofoblastos , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Type 2 diabetes (T2D) is a worldwide epidemic with a medical need for additional targeted therapies. Suppression of hepatic glucose production (HGP) effectively ameliorates diabetes and can be exploited for its treatment. We hypothesized that targeting PGC-1α acetylation in the liver, a chemical modification known to inhibit hepatic gluconeogenesis, could be potentially used for treatment of T2D. Thus, we designed a high-throughput chemical screen platform to quantify PGC-1α acetylation in cells and identified small molecules that increase PGC-1α acetylation, suppress gluconeogenic gene expression, and reduce glucose production in hepatocytes. On the basis of potency and bioavailability, we selected a small molecule, SR-18292, that reduces blood glucose, strongly increases hepatic insulin sensitivity, and improves glucose homeostasis in dietary and genetic mouse models of T2D. These studies have important implications for understanding the regulatory mechanisms of glucose metabolism and treatment of T2D.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Gluconeogênese/efeitos dos fármacos , Hipoglicemiantes/administração & dosagem , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/antagonistas & inibidores , Acetilação , Animais , Glicemia/metabolismo , Células Cultivadas , Glucose/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , Ensaios de Triagem em Larga Escala , Resistência à Insulina , Camundongos , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
PGC-1α is well established as a metazoan transcriptional coactivator of cellular adaptation in response to stress. However, the mechanisms by which PGC-1α activates gene transcription are incompletely understood. Here, we report that PGC-1α serves as a scaffold protein that physically and functionally connects the DNA-binding protein estrogen-related receptor α (ERRα), cap-binding protein 80 (CBP80), and Mediator to overcome promoter-proximal pausing of RNAPII and transcriptionally activate stress-response genes. We show that PGC-1α promotes pausing release in a two-arm mechanism (1) by recruiting the positive transcription elongation factor b (P-TEFb) and (2) by outcompeting the premature transcription termination complex Integrator. Using mice homozygous for five amino acid changes in the CBP80-binding motif (CBM) of PGC-1α that destroy CBM function, we show that efficient differentiation of primary myoblasts to myofibers and timely skeletal muscle regeneration after injury require PGC-1α binding to CBP80. Our findings reveal how PGC-1α activates stress-response gene transcription in a previously unanticipated pre-mRNA quality-control pathway.
Assuntos
Precursores de RNA , Fatores de Transcrição , Animais , Camundongos , Proteínas de Ligação a DNA/genética , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Regiões Promotoras Genéticas , Proteínas de Ligação ao Cap de RNA/genética , RNA Polimerase II/metabolismo , Precursores de RNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The nuclear receptor co-repressor (NCoR) complex mediates transcriptional repression dependent on histone deacetylation by histone deacetylase 3 (HDAC3) as a component of the complex. Unexpectedly, we found that signaling by the receptor activator of nuclear factor κB (RANK) converts the NCoR/HDAC3 co-repressor complex to a co-activator of AP-1 and NF-κB target genes that are required for mouse osteoclast differentiation. Accordingly, the dominant function of NCoR/HDAC3 complexes in response to RANK signaling is to activate, rather than repress, gene expression. Mechanistically, RANK signaling promotes RNA-dependent interaction of the transcriptional co-activator PGC1ß with the NCoR/HDAC3 complex, resulting in the activation of PGC1ß and inhibition of HDAC3 activity for acetylated histone H3. Non-coding RNAs Dancr and Rnu12, which are associated with altered human bone homeostasis, promote NCoR/HDAC3 complex assembly and are necessary for RANKL-induced osteoclast differentiation in vitro. These findings may be prototypic for signal-dependent functions of NCoR in other biological contexts.
Assuntos
Osteoclastos , RNA , Humanos , Camundongos , Animais , Proteínas Correpressoras/genética , Osteoclastos/metabolismo , Ligante RANK/genética , Correpressor 1 de Receptor Nuclear/genética , Correpressor 1 de Receptor Nuclear/metabolismo , Expressão GênicaRESUMO
Lipid droplets (LDs) provide a reservoir for triacylglycerol storage and are a central hub for fatty acid trafficking and signaling in cells. Lipolysis promotes mitochondrial biogenesis and oxidative metabolism via a SIRT1/PGC-1α/PPARα-dependent pathway through an unknown mechanism. Herein, we identify that monounsaturated fatty acids (MUFAs) allosterically activate SIRT1 toward select peptide-substrates such as PGC-1α. MUFAs enhance PGC-1α/PPARα signaling and promote oxidative metabolism in cells and animal models in a SIRT1-dependent manner. Moreover, we characterize the LD protein perilipin 5 (PLIN5), which is known to enhance mitochondrial biogenesis and function, to be a fatty-acid-binding protein that preferentially binds LD-derived monounsaturated fatty acids and traffics them to the nucleus following cAMP/PKA-mediated lipolytic stimulation. Thus, these studies identify the first-known endogenous allosteric modulators of SIRT1 and characterize a LD-nuclear signaling axis that underlies the known metabolic benefits of MUFAs and PLIN5.
Assuntos
Ácidos Graxos Monoinsaturados/metabolismo , Gotículas Lipídicas/química , Perilipina-5/metabolismo , Sirtuína 1/metabolismo , Regulação Alostérica , Animais , Transporte Biológico , Linhagem Celular , Células Cultivadas , Dieta , Ácidos Graxos/metabolismo , Lipase/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Azeite de Oliva , Perilipina-5/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transcrição GênicaRESUMO
Hypoxia, which occurs during tumor growth, triggers complex adaptive responses in which peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) plays a critical role in mitochondrial biogenesis and oxidative metabolism. However, how PGC-1α is regulated in response to oxygen availability remains unclear. We demonstrated that lysine demethylase 3A (KDM3A) binds to PGC-1α and demethylates monomethylated lysine (K) 224 of PGC-1α under normoxic conditions. Hypoxic stimulation inhibits KDM3A, which has a high KM of oxygen for its activity, and enhances PGC-1α K224 monomethylation. This modification decreases PGC-1α's activity required for NRF1- and NRF2-dependent transcriptional regulation of TFAM, TFB1M, and TFB2M, resulting in reduced mitochondrial biogenesis. Expression of PGC-1α K224R mutant significantly increases mitochondrial biogenesis, reactive oxygen species (ROS) production, and tumor cell apoptosis under hypoxia and inhibits brain tumor growth in mice. This study revealed that PGC-1α monomethylation, which is dependent on oxygen availability-regulated KDM3A, plays a critical role in the regulation of mitochondrial biogenesis.
Assuntos
Neoplasias Encefálicas/enzimologia , Histona Desmetilases com o Domínio Jumonji/metabolismo , Mitocôndrias/enzimologia , Biogênese de Organelas , Oxigênio/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Apoptose , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Metilação , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/patologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Carga Tumoral , Hipóxia Tumoral , Microambiente TumoralRESUMO
Diet-induced obesity can be caused by impaired thermogenesis of beige adipocytes, the brown-like adipocytes in white adipose tissue (WAT). Promoting brown-like features in WAT has been an attractive therapeutic approach for obesity. However, the mechanism underlying beige adipocyte formation is largely unknown. N-α-acetyltransferase 10 protein (Naa10p) catalyzes N-α-acetylation of nascent proteins, and overexpression of human Naa10p is linked to cancer development. Here, we report that both conventional and adipose-specific Naa10p deletions in mice result in increased energy expenditure, thermogenesis, and beige adipocyte differentiation. Mechanistically, Naa10p acetylates the N terminus of Pgc1α, which prevents Pgc1α from interacting with Pparγ to activate key genes, such as Ucp1, involved in beige adipocyte function. Consistently, fat tissues of obese human individuals show higher NAA10 expression. Thus, Naa10p-mediated N-terminal acetylation of Pgc1α downregulates thermogenic gene expression, making inhibition of Naa10p enzymatic activity a potential strategy for treating obesity.
Assuntos
Adipócitos Bege/enzimologia , Tecido Adiposo Bege/enzimologia , Acetiltransferase N-Terminal A/metabolismo , Acetiltransferase N-Terminal E/metabolismo , Obesidade/enzimologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Processamento de Proteína Pós-Traducional , Termogênese , Acetilação , Tecido Adiposo Bege/fisiopatologia , Adiposidade , Adolescente , Adulto , Idoso , Animais , Estudos de Casos e Controles , Dieta Hiperlipídica , Modelos Animais de Doenças , Metabolismo Energético , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Acetiltransferase N-Terminal A/deficiência , Acetiltransferase N-Terminal A/genética , Acetiltransferase N-Terminal E/deficiência , Acetiltransferase N-Terminal E/genética , Células NIH 3T3 , Obesidade/genética , Obesidade/fisiopatologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Fenótipo , Transdução de Sinais , Adulto JovemRESUMO
The nuclear receptor corepressor (NCoR) forms a complex with histone deacetylase 3 (HDAC3) that mediates repressive functions of unliganded nuclear receptors and other transcriptional repressors by deacetylation of histone substrates. Recent studies provide evidence that NCoR/HDAC3 complexes can also exert coactivator functions in brown adipocytes by deacetylating and activating PPARγ coactivator 1α (PGC1α) and that signaling via receptor activator of nuclear factor kappa-B (RANK) promotes the formation of a stable NCoR/HDAC3/PGC1ß complex that coactivates nuclear factor kappa-B (NFκB)- and activator protein 1 (AP-1)-dependent genes required for osteoclast differentiation. Here, we demonstrate that activation of Toll-like receptor (TLR) 4, but not TLR3, the interleukin 4 (IL4) receptor nor the Type I interferon receptor, also promotes assembly of an NCoR/HDAC3/PGC1ß coactivator complex. Receptor-specific utilization of TNF receptor-associated factor 6 (TRAF6) and downstream activation of extracellular signal-regulated kinase 1 (ERK1) and TANK-binding kinase 1 (TBK1) accounts for the common ability of RANK and TLR4 to drive assembly of an NCoR/HDAC3/PGC1ß complex in macrophages. ERK1, the p65 component of NFκB, and the p300 histone acetyltransferase (HAT) are also components of the induced complex and are associated with local histone acetylation and transcriptional activation of TLR4-dependent enhancers and promoters. These observations identify a TLR4/TRAF6-dependent signaling pathway that converts NCoR from a corepressor of nuclear receptors to a coactivator of NFκB and AP-1 that may be relevant to functions of NCoR in other developmental and homeostatic processes.
Assuntos
Histonas , Fator 6 Associado a Receptor de TNF , Ativação Transcricional , Proteínas Correpressoras/genética , Histonas/genética , Histonas/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Fator de Transcrição AP-1/metabolismo , Receptor 4 Toll-Like/metabolismo , Transdução de Sinais , NF-kappa B/genética , NF-kappa B/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Current models of SIRT1 enzymatic regulation primarily consider the effects of fluctuating levels of its co-substrate NAD+, which binds to the stably folded catalytic domain. By contrast, the roles of the sizeable disordered N- and C-terminal regions of SIRT1 are largely unexplored. Here we identify an insulin-responsive sensor in the SIRT1 N-terminal region (NTR), comprising an acidic cluster (AC) and a 3-helix bundle (3HB), controlling deacetylase activity. The allosteric assistor DBC1 removes a distal N-terminal shield from the 3-helix bundle, permitting PACS-2 to engage the acidic cluster and the transiently exposed helix 3 of the 3-helix bundle, disrupting its structure and inhibiting catalysis. The SIRT1 activator (STAC) SRT1720 binds and stabilizes the 3-helix bundle, protecting SIRT1 from inhibition by PACS-2. Identification of the SIRT1 insulin-responsive sensor and its engagement by the DBC1 and PACS-2 regulatory hub provides important insight into the roles of disordered regions in enzyme regulation and the mode by which STACs promote metabolic fitness.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Hepatócitos/enzimologia , Insulina/metabolismo , Sirtuína 1/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Regulação Alostérica , Animais , Sítios de Ligação , Dieta Hiperlipídica , Modelos Animais de Doenças , Regulação da Expressão Gênica , Células HCT116 , Hepatócitos/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Resistência à Insulina , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/enzimologia , Obesidade/genética , Obesidade/prevenção & controle , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Sirtuína 1/genética , Proteínas de Transporte Vesicular/deficiência , Proteínas de Transporte Vesicular/genéticaRESUMO
Although peroxisome proliferator-activated receptor-γ (PPARγ) coactivator 1α (PGC-1α) is a well-established transcriptional coactivator for the metabolic adaptation of mammalian cells to diverse physiological stresses, the molecular mechanism by which it functions is incompletely understood. Here we used in vitro binding assays, X-ray crystallography, and immunoprecipitations of mouse myoblast cell lysates to define a previously unknown cap-binding protein 80 (CBP80)-binding motif (CBM) in the C terminus of PGC-1α. We show that the CBM, which consists of a nine-amino-acid α helix, is critical for the association of PGC-1α with CBP80 at the 5' cap of target transcripts. Results from RNA sequencing demonstrate that the PGC-1α CBM promotes RNA synthesis from promyogenic genes. Our findings reveal a new conduit between DNA-associated and RNA-associated proteins that functions in a cap-binding protein surveillance mechanism, without which efficient differentiation of myoblasts to myotubes fails to occur.
Assuntos
Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/química , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Ativação Transcricional , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Diferenciação Celular , Humanos , Células MCF-7 , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Capuzes de RNA/metabolismo , Proteínas de Ligação a RNA , Transcrição GênicaRESUMO
A delicate balance in the levels of proteins that regulate the p53 tumor suppressor pathway exists such that subtle changes alter p53 tumor suppressor activity and cancer risk. Many single-nucleotide polymorphisms (SNPs) in the p53 pathway alter p53 transcriptional activity and are associated with cancer risk. In addition, some SNPs influence the gain-of-function (GOF) activities of mutant p53 through unknown mechanisms. In this issue of Genes & Development, Basu and colleagues (pp. 230-243) provide direct evidence that the presence of an R72 polymorphism enhances the GOF invasive and metastatic properties of mutant p53 by regulating interactions with PGC-1α, an important regulator of mitochondrial biogenesis and oxidative phosphorylation. The study culminates with evidence that R72 is associated with worse outcomes in human breast cancer.
Assuntos
Neoplasias da Mama , Proteína Supressora de Tumor p53/genética , Humanos , Fosforilação Oxidativa , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Mutant forms of p53 protein often possess protumorigenic functions, conferring increased survival and migration to tumor cells via their "gain-of-function" activity. Whether and how a common polymorphism in TP53 at amino acid 72 (Pro72Arg; referred to here as P72 and R72) impacts this gain of function has not been determined. We show that mutant p53 enhances migration and metastasis of tumors through the ability to bind and regulate PGC-1α and that this regulation is markedly impacted by the codon 72 polymorphism. Tumor cells with the R72 variant of mutant p53 show increased PGC-1α function along with greatly increased mitochondrial function and metastatic capability. Breast cancers containing mutant p53 and the R72 variant show poorer prognosis compared with P72. The combined results reveal PGC-1α as a novel "gain-of-function" partner of mutant p53 and indicate that the codon 72 polymorphism influences the impact of mutant p53 on metabolism and metastasis.
Assuntos
Genes p53 , Mutação , Neoplasias/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Movimento Celular , Feminino , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Masculino , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/genética , Neoplasias/patologia , Fosforilação Oxidativa , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/antagonistas & inibidoresRESUMO
The purpose of this review is to explore and discuss the impacts of augmented training volume, intensity, and duration on the phosphorylation/activation of key signaling protein - AMPK, CaMKII and PGC-1α - involved in the initiation of mitochondrial biogenesis. Specifically, we explore the impacts of augmented exercise protocols on AMP/ADP and Ca2+ signaling and changes in post exercise PGC - 1α gene expression. Although AMP/ADP concentrations appear to increase with increasing intensity and during extended durations of higher intensity exercise AMPK activation results are varied with some results supporting and intensity/duration effect and others not. Similarly, CaMKII activation and signaling results following exercise of different intensities and durations are inconsistent. The PGC-1α literature is equally inconsistent with only some studies demonstrating an effect of intensity on post exercise mRNA expression. We present a novel meta-analysis that suggests that the inconsistency in the PGC-1α literature may be due to sample size and statistical power limitations owing to the effect of intensity on PGC-1α expression being small. There is little data available regarding the impact of exercise duration on PGC-1α expression. We highlight the need for future well designed, adequately statistically powered, studies to clarify our understanding of the effects of volume, intensity, and duration on the induction of mitochondrial biogenesis by exercise.
Assuntos
Proteínas Quinases Ativadas por AMP , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Exercício Físico/fisiologia , Músculo Esquelético/metabolismo , Fosforilação , RNA Mensageiro/genética , HumanosRESUMO
The function of spargel/dPGC-1 in Drosophila oogenesis has been unequivocally established. Here, we sought to assess whether Spargel protein or RNA is essential for developmentally competent eggs. The trans-heterozygotic combination of two spargel mutant alleles allowed us to decrease Spargel expression to very low levels. Using this model, we now demonstrated the requirement for Spargel in eggshell patterning and embryonic development, which led us to establish that spargel is a maternal effect gene. Further examination of Spargel's potential mechanism of action in eggshell biogenesis revealed that low levels of Spargel in the adult ovary cause diminished Cyclin E activity, resulting in reduced chorion gene amplification levels, leading to eggshell biogenesis defects. Thus, another novel role for spargel/dPGC-1 is exposed whereby, through Cyclin E activity, this conserved transcriptional coactivator regulates the chorion gene amplification process.
RESUMO
The tricarboxylic acid (TCA) cycle plays a crucial role in mitochondrial ATP production in the healthy heart. However, in heart failure, the TCA cycle becomes dysregulated. Understanding the mechanism by which TCA cycle genes are transcribed in the healthy heart is an important prerequisite to understanding how these genes become dysregulated in the failing heart. PGC-1α is a transcriptional coactivator that broadly induces genes involved in mitochondrial ATP production. PGC-1α potentiates its effects through coactivation of coupled transcription factors, such as ERR, Nrf1, Gabpa, and YY1. We hypothesized that PGC-1α plays an essential role in transcription of TCA cycle genes. Thus, by utilizing localization peaks of PGC-1α to TCA cycle gene promoters, it would allow the identification of coupled transcription factors. PGC-1α potentiated the transcription of 13 out of 14 TCA cycle genes, partly through ERR, Nrf1, Gabpa, and YY1. ChIP-sequencing showed PGC-1α localization peaks in TCA cycle gene promoters. Transcription factors with binding elements that were found proximal to PGC-1α peak localization were generally essential for transcription of the gene. These transcription factor binding elements were well conserved between mice and humans. Among the four transcription factors, ERR and Gabpa played a major role in potentiating transcription when compared to Nrf1 and YY1. These transcription factor-dependent PGC-1α recruitment was verified with Idh3a, Idh3g, and Sdha promoters with DNA binding assay. Taken together, this study clarifies the mechanism by which TCA cycle genes are transcribed, which could be useful to understand how those genes are dysregulated in pathological conditions.
RESUMO
Echinoderms represent a broad phylum with many tractable features to test evolutionary changes and constraints. Here, we present a single-cell RNA-sequencing analysis of early development in the sea star Patiria miniata, to complement the recent analysis of two sea urchin species. We identified 20 cell states across six developmental stages from 8â hpf to mid-gastrula stage, using the analysis of 25,703 cells. The clusters were assigned cell states based on known marker gene expression and by in situ RNA hybridization. We found that early (morula, 8-14â hpf) and late (blastula-to-mid-gastrula) cell states are transcriptionally distinct. Cells surrounding the blastopore undergo rapid cell state changes that include endomesoderm diversification. Of particular import to understanding germ cell specification is that we never see Nodal pathway members within Nanos/Vasa-positive cells in the region known to give rise to the primordial germ cells (PGCs). The results from this work contrast the results of PGC specification in the sea urchin, and the dataset presented here enables deeper comparative studies in tractable developmental models for testing a variety of developmental mechanisms.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Estrelas-do-Mar , Animais , Estrelas-do-Mar/genética , Ouriços-do-Mar/genética , Células Germinativas/metabolismo , RNA/genéticaRESUMO
Acetaminophen (APAP) is one of the most clinically relevant medications associated with acute liver damage. A prolific deal of research validated the hepatoprotective effect of empagliflozin (EMPA); however, its effect on APAP-induced hepatotoxicity has still not been investigated. In this study, the prospective hepatoprotective impact of EMPA against APAP-induced hepatotoxicity was investigated. Twenty-eight Balb-C mice were assigned to four groups: control, APAP, EMPA10/APAP, and EMPA25/APAP. At the end of the experiment, serum hepatotoxicity biomarkers, MDA level, and GSH content were estimated. Hepatic mitofusin-2 (MFN2), optic atrophy 1 (OPA1), dynamin-related protein 1 (Drp1), and mitochondrial fission 1 protein (FIS1) were immunoassayed. PGC-1α, cGAS, and STING mRNA expression were assessed by real-time PCR. Histopathological changes and immunohistochemistry of INF-ß, p-NF-κB, and iNOS were evaluated. APAP treatment caused significant hepatic functional impairment and increased hepatic MDA levels, as well as a concomitant decrease in GSH content. Marked elevation in Drp1 and FIS1 levels, INF-ß, p-NF-κB, and iNOS immunoreactivity, and reduction in MFN2 and OPA1 levels in the APAP-injected group, PGC-1α downregulation, and high expression of cGAS and STING were also documented. EMPA effectively ameliorated APAP-generated structural and functional changes in the liver, restored redox homeostasis and mitochondrial dynamics balance, and enhanced mitochondrial biogenesis, remarkably diminished hepatic expression of cGAS and STING, and elicited a reduction in hepatic inflammation. Moreover, the computational modeling data support the interaction of APAP with antioxidant system-related proteins as well as the interactions of EMPA against Drp1, cGAS, IKKA, and iNOS proteins. Our findings demonstrated for the first time that EMPA has an ameliorative impact against APAP-induced hepatotoxicity in mice via modulation of mitochondrial dynamics, biogenesis, and cGAS/STING-dependent inflammation. Thus, this study concluded that EMPA could be a promising therapeutic modality for acute liver toxicity.
Assuntos
Acetaminofen , Compostos Benzidrílicos , Doença Hepática Induzida por Substâncias e Drogas , Dinaminas , GTP Fosfo-Hidrolases , Glucosídeos , Proteínas de Membrana , Dinâmica Mitocondrial , Nucleotidiltransferases , Animais , Masculino , Camundongos , Acetaminofen/toxicidade , Acetaminofen/efeitos adversos , Compostos Benzidrílicos/farmacologia , Compostos Benzidrílicos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Dinaminas/metabolismo , Dinaminas/genética , Glucosídeos/farmacologia , GTP Fosfo-Hidrolases/metabolismo , Fígado/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos Endogâmicos BALB C , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , NF-kappa B/metabolismo , Nucleotidiltransferases/metabolismo , Biogênese de Organelas , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Miscarriage is a common complication during early pregnancy and affects approximately 10%-15% of all pregnant women. Several studies have reported that the abnormal expression of mitochondrial oxidative stress-related genes might be involved in the occurrence and progression of miscarriage. The present study attempted to uncover the role of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in miscarriage chorionic villous tissue. The hypothesis that PGC-1α is crucial for glycolysis and oxidative phosphorylation during early pregnancy was tested. The results showed that the mRNA and protein levels of PGC-1α were significantly increased in the miscarriage chorionic villous tissues compared with the artificial selective abortion group, and that the expression was regulated by mTOR in knockdown and overexpression of mTOR in HTR8 cell lines. PGC-1α also promoted mitochondrion oxidative phosphorylation but inhibited glycolysis process. In addition, PGC-1α could drive ROS production, reduce mitochondrial membrane potential and block NADPH synthesis, resulting in cell cycle arrest and cell apoptosis, eventually leading to miscarriage. These results suggested that the aberrant expression of PGC-1α is involved in the etiology of early miscarriage, providing new perspectives regarding the mechanisms of miscarriage and a potential therapeutic target for miscarriage.
Assuntos
Aborto Espontâneo , Gravidez , Humanos , Feminino , Aborto Espontâneo/genética , Transdução de Sinais/genética , Apoptose , Estresse Oxidativo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismoRESUMO
Gut microbiota of the gastrointestinal tract provide health benefits to the human host via bacterial metabolites. Bacterial butyrate has beneficial effects on intestinal homeostasis and is the preferred energy source of intestinal epithelial cells, capable of inducing differentiation. It was previously observed that changes in the expression of specific proteins as well as protein glycosylation occur with differentiation. In this study, specific mucin O-glycans were identified that mark butyrate-induced epithelial differentiation of the intestinal cell line CaCo-2 (Cancer Coli-2), by applying porous graphitized carbon nano-liquid chromatography with electrospray ionization tandem mass spectrometry. Moreover, a quantitative proteomic approach was used to decipher changes in the cell proteome. It was found that the fully differentiated butyrate-stimulated cells are characterized by a higher expression of sialylated O-glycan structures, whereas fucosylation is downregulated with differentiation. By performing an integrative approach, we generated hypotheses about the origin of the observed O-glycome changes. These insights pave the way for future endeavors to study the dynamic O-glycosylation patterns in the gut, either produced via cellular biosynthesis or through the action of bacterial glycosidases as well as the functional role of these patterns in homeostasis and dysbiosis at the gut-microbiota interface.
Assuntos
Neoplasias Colorretais , Proteômica , Humanos , Células CACO-2 , Proteômica/métodos , Glicômica/métodos , Butiratos/farmacologia , Diferenciação Celular , Polissacarídeos/metabolismoRESUMO
Bronchopulmonary dysplasia (BPD) is characterized by impaired lung alveolar and vascular growth. We investigated the hypothesis that neonatal exposure to hyperoxia leads to persistent BPD phenotype due to decreased expression of liver kinase B1 (LKB1), a key regulator of mitochondrial function. We exposed mouse pups from postnatal day 1- day 10 (P1-P10) to 21% or 75% oxygen. Half of the pups in each group received metformin or saline intraperitoneally from P1-P10. Pups were euthanized at P4 or P10 or recovered in 21% O2 until euthanasia at P21. Lung histology/morphometry, immunofluorescence and immunoblots were done for changes in lung structure and expression of LKB1 and downstream targets, AMPK, PGC-1α, electron transport chain complexes (ETC) and Notch ligands, Jagged 1 and delta like 4 (Dll4). LKB1 signaling and in vitro angiogenesis were assessed in human pulmonary artery endothelial cells (PAEC) exposed to 21% or 95% O2 for 36h. Levels of LKB1, phosphorylated-AMPK (p-AMPK), PGC-1α, and ETC complexes were decreased in lungs at P10 and P21 in hyperoxia. Metformin increased LKB1, p-AMPK, PGC-1α, and ETC complexes at P10 and P21 in hyperoxia pups. Radial alveolar count was decreased and mean linear intercept increased in hyperoxia pups at P10 and P21; these were improved by metformin. Lung capillary density was decreased in hyperoxia at P10 and P21 and was increased by metformin. In vitro angiogenesis was decreased in HPAEC by 95% O2 and was improved by metformin. Decreased LKB1 signaling may contribute to decreased alveolar and vascular growth in a mouse model of BPD.