RESUMO
The Zfp296 gene encodes a zinc finger-type protein. Its expression is high in mouse embryonic stem cells (ESCs) but rapidly decreases following differentiation. Zfp296-knockout (KO) ESCs grew as flat colonies, which were reverted to rounded colonies by exogenous expression of Zfp296. KO ESCs could not form teratomas when transplanted into mice but could efficiently contribute to germline-competent chimeric mice following blastocyst injection. Transcriptome analysis revealed that Zfp296 deficiency up- and down-regulates a distinct group of genes, among which Dppa3, Otx2, and Pou3f1 were markedly downregulated. Chromatin immunoprecipitation sequencing demonstrated that ZFP296 binding is predominantly seen in the vicinity of the transcription start sites (TSSs) of a number of genes, and ZFP296 was suggested to negatively regulate transcription. Consistently, chromatin accessibility assay clearly showed that ZFP296 binding reduces the accessibility of the TSS regions of target genes. Zfp296-KO ESCs showed increased histone H3K9 di- and trimethylation. Co-immunoprecipitation analyses revealed interaction of ZFP296 with G9a and GLP. These results show that ZFP296 plays essential roles in maintaining the global epigenetic state of ESCs through multiple mechanisms including activation of Dppa3, attenuation of chromatin accessibility, and repression of H3K9 methylation, but that Zfp296-KO ESCs retain a unique state of pluripotency while lacking the teratoma-forming ability.
Assuntos
Cromatina , Teratoma , Animais , Camundongos , Células-Tronco Embrionárias , Histonas , Células-Tronco Embrionárias Murinas , Bioensaio , Proteínas Cromossômicas não Histona , Fator 6 de Transcrição de OctâmeroRESUMO
Accurate species identification is of primary importance in ecology and evolutionary biology. For a long time, the unionid mussels Beringiana and Sinanodonta have puzzled researchers trying to unravel their diversity because of their poorly discernible morphologies. A recent study conducted species delineation of unionid mussels based on mitochondrial DNA variation, opening up a new avenue to grasp species diversity of the mussels. However, mtDNA-based classification may not align with species boundaries because mtDNA is prone to introgression and incomplete lineage sorting that cause discordance between species affiliation and gene phylogeny. In this study, we evaluated the validity of the mtDNA-based classification of unionid mussels Beringiana and Sinanodonta in Japan using mitochondrial sequence data, double digest restriction site-associated DNA library (ddRAD) sequencing, and morphological data. We found significant inconsistencies in the mitochondrial and nuclear DNA phylogenies, casting doubt on the reliability of the mtDNA-based classification in this group. In addition, nuclear DNA phylogeny revealed that there are at least two unionid lineages hidden in the mtDNA phylogeny. Although molecular dating technique indicates that Beringiana and Sinanodonta diverged >35 million years ago, their shell morphologies are often indistinguishable. Specifically, morphological analyses exhibited the parallel appearance of nearly identical ball-like shell forms in the two genera in Lake Biwa, which further complicates species identification and the morphological evolution of unionid mussels. Our study adds to a growing body of literature that accurate species identification of unionid mussels is difficult when using morphological characters alone. Although mtDNA-based classification is a simple and convenient way to classify unionid mussels, considerable caution is warranted for its application in ecological and evolutionary studies.
Assuntos
Bivalves , Unionidae , Animais , Bivalves/genética , DNA Mitocondrial/genética , Japão , Filogenia , Reprodutibilidade dos Testes , Unionidae/genéticaRESUMO
Oxidative stress is a deteriorating factor for pancreatic ß-cells under chronic hyperglycemia in diabetes. However, the molecular mechanism underlying the increase in oxidative stress in ß-cells under diabetic conditions remains unclear. We demonstrated previously that the selective alleviation of glucotoxicity ameliorated the downregulation of several ß-cell factors, including Cox6a2. Cox6a2 encodes a subunit of the respiratory chain complex IV in mitochondria. In this study, we analyzed the role of Cox6a2 in pancreatic ß-cell function and its pathophysiological significance in diabetes mellitus. Cox6a2-knockdown experiments in MIN6-CB4 cells indicated an increased production of reactive oxygen species as detected by CellROX Deep Red reagent using flow cytometry. In systemic Cox6a2-knockout mice, impaired glucose tolerance was observed under a high-fat high-sucrose diet. However, insulin resistance was reduced when compared with control littermates. This indicates a relative insufficiency of ß-cell function. To examine the transcriptional regulation of Cox6a2, ATAC-seq with islet DNA was performed and an open-chromatin area within the Cox6a2 enhancer region was detected. Reporter gene analysis using this area revealed that MafA directly regulates Cox6a2 expression. These findings suggest that the decreased expression of Cox6a2 increases the levels of reactive oxygen species and that Mafa is associated with decreased Cox6a2 expression under glucotoxic conditions.
Assuntos
Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Proteínas Musculares/deficiência , Espécies Reativas de Oxigênio/metabolismo , Animais , Linhagem Celular , Diabetes Mellitus Experimental/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Intolerância à Glucose/genética , Células HEK293 , Humanos , Insulina/metabolismo , Resistência à Insulina/genética , Fatores de Transcrição Maf Maior/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas Musculares/biossíntese , Proteínas Musculares/genética , Estresse Oxidativo , Transcrição GênicaRESUMO
Free fatty acid receptor 1 (FFAR1 or GPR40) has attracted attention for the treatment of type 2 diabetes mellitus, and various small-molecule agonists have been developed. However, most FFAR1 agonists as well as endogenous ligands, such as linoleic acids, have high lipophilicity, and their high lipophilicity is related to off-target toxicity. Therefore, we need to focus on new ligand candidates with less toxicity. In this study, we screened peptides with FFAR1 agonist activity as new ligand candidates. First, we used phage display to identify peptides with high affinity to FFAR1. Next, the agonist activities of peptides determined by the phage display were evaluated by the TGF-α shedding assay. Finally, to improve the FFAR1 agonist activity of the peptide, we performed an inclusive single amino acid substitution and sequence analysis. Logistic regression (LR) analysis using 120 physiochemical properties was performed to predict peptides with high FFAR1 agonist activity. STTGTQY determined by phage display promoted glucose-stimulated insulin secretion in pancreatic MIN6 cells. Furthermore, STKGTF predicted by the LR analysis showed high insulin secretion at low concentrations compared to STTGTQY. The results of this study suggest that peptides could be new candidates as FFAR1 agonists.
Assuntos
Substituição de Aminoácidos , Avaliação Pré-Clínica de Medicamentos/métodos , Aprendizado de Máquina , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Sequência de Aminoácidos , Linhagem Celular , Clonagem Molecular , Glucose/farmacologia , Células HEK293 , Humanos , Insulina/metabolismo , Peptídeos/efeitos adversos , Peptídeos/genética , Ligação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Análise de Regressão , Fator de Crescimento Transformador alfa/metabolismoRESUMO
A pancreatic ß-cell line MIN6 was previously established in our lab from an insulinoma developed in an IT6 transgenic mouse expressing the SV40 T antigen in ß-cells. This cell line has been widely used for in vitro analysis of ß-cell function, but tends to lose the mature ß-cell features, including glucose-stimulated insulin secretion (GSIS), in long-term culture. The aim of this study was to develop a stable ß-cell line that retains the characteristics of mature ß-cells. Considering that mice derived from a cross between C3H and C57BL/6 strains are known to exhibit higher insulin secretory capacity than C57BL/6 mice, an IT6 male mouse of this hybrid background was used to isolate insulinomas, which were independently cultured. After 7 months of continuous culturing, we obtained the MIN6-CB4 ß-cell line, which stably maintains its GSIS. It has been noted that ß-cell lines express the glucagon (Gcg) gene at certain levels. MIN6-CB4 cells were utilized to assess the effects of differential Gcg expression on ß-cell function. Our data show the functional importance of Gcg expression and resulting basal activation of the GLP-1 receptor in ß-cells. MIN6-CB4 cells can serve as an invaluable tool for studying the regulatory mechanisms of insulin secretion, such as the GLP-1/cAMP signaling, in ß-cells.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Glucagon/fisiologia , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Insulinoma/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Feminino , Células Secretoras de Insulina/citologia , Insulinoma/patologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Pancreáticas/patologiaRESUMO
A common variant in the RAB27A gene in adults was recently found to be associated with the fractional exhaled nitric oxide level, a marker of eosinophilic airway inflammation. The small GTPase Rab27 is known to regulate intracellular vesicle traffic, although its role in allergic responses is unclear. We demonstrated that exophilin-5, a Rab27-binding protein, was predominantly expressed in both of the major IL-33 producers, lung epithelial cells, and the specialized IL-5 and IL-13 producers in the CD44hiCD62LloCXCR3lo pathogenic Th2 cell population in mice. Exophilin-5 deficiency increased stimulant-dependent damage and IL-33 secretion by lung epithelial cells. Moreover, it enhanced IL-5 and IL-13 production in response to TCR and IL-33 stimulation from a specific subset of pathogenic Th2 cells that expresses a high level of IL-33 receptor, which exacerbated allergic airway inflammation in a mouse model of asthma. Mechanistically, exophilin-5 regulates extracellular superoxide release, intracellular ROS production, and phosphoinositide 3-kinase activity by controlling intracellular trafficking of Nox2-containing vesicles, which seems to prevent the overactivation of pathogenic Th2 cells mediated by IL-33. This is the first report to our knowledge to establish the significance of the Rab27-related protein exophilin-5 in the development of allergic airway inflammation, and provides insights into the pathophysiology of asthma.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Asma/imunologia , Proteína 1 Semelhante a Receptor de Interleucina-1/imunologia , Interleucina-33/imunologia , Células Th2/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Asma/genética , Asma/patologia , Modelos Animais de Doenças , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Interleucina-33/genética , Camundongos , Camundongos Knockout , NADPH Oxidase 2/genética , NADPH Oxidase 2/imunologia , Espécies Reativas de Oxigênio/imunologia , Células Th2/patologiaRESUMO
Freshwater mussels (Bivalvia: Unionidae) is a diverse family with around 700 species being widespread in the Northern Hemisphere and Africa. These animals fulfill key ecological functions and provide important services to humans. Unfortunately, populations have declined dramatically over the last century, rendering Unionidae one of the world's most imperiled taxonomic groups. In Far East Asia (comprising Japan, Korea, and Eastern Russia), conservation actions have been hindered by a lack of basic information on the number, identity, distribution and phylogenetic relationships of species. Available knowledge is restricted to studies on national and sub-national levels. The present study aims to resolve the diversity, biogeography and evolutionary relationships of the Far East Asian Unionidae in a globally comprehensive phylogenetic and systematic context. We reassessed the systematics of all Unionidae species in the region, including newly collected specimens from across Japan, South Korea, and Russia, based on molecular (including molecular species delineation and a COIâ¯+â¯28S phylogeny) and comparative morphological analyses. Biogeographical patterns were then assessed based on available species distribution data from the authors and previous reference works. We revealed that Unionidae species richness in Far East Asia is 30% higher than previously assumed, counting 43 species (41 nativeâ¯+â¯2 alien) within two Unionidae subfamilies, the Unioninae (32â¯+â¯1) and Gonideinae (9â¯+â¯1). Four of these species are new to science, i.e. Beringiana gosannensissp. nov., Beringiana fukuharaisp. nov., Buldowskia kamiyaisp. nov., and Koreosolenaia sitgyensisgen. & sp. nov. We also propose a replacement name for Nodularia sinulata, i.e. Nodularia breviconchanom. nov. and describe a new tribe (Middendorffinaiini tribe nov.) within the Unioninae subfamily. Biogeographical patterns indicate that this fauna is related to that from China south to Vietnam until the Mekong River basin. The Japanese islands of Honshu, Shikoku, Kyushu, Hokkaido, and the Korean Peninsula were identified as areas of particularly high conservation value, owing to high rates of endemism, diversity and habitat loss. The genetically unique species within the genera Amuranodonta, Obovalis, Koreosolenaiagen. nov., and Middendorffinaia are of high conservation concern.
Assuntos
Unionidae/classificação , Animais , Evolução Biológica , Água Doce , Japão , Coreia (Geográfico) , Filogenia , Filogeografia , Federação Russa , Unionidae/genéticaRESUMO
Lymphangiogenesis plays important roles in normal fetal development and postnatal growth. However, its molecular regulation remains unclear. Here, we have examined the function of forkhead box protein O1 (FOXO1) transcription factor, a known angiogenic factor, in developmental dermal lymphangiogenesis using endothelial cell-specific FOXO1-deficient mice. FOXO1-deficient mice showed disconnected and dilated lymphatic vessels accompanied with increased proliferation and decreased apoptosis in the lymphatic capillaries. Comprehensive DNA microarray analysis of the causes of in vivo phenotypes in FOXO1-deficient mice revealed that the gene encoding C-X-C chemokine receptor 4 (CXCR4) was the most drastically downregulated in FOXO1-deficient primary lymphatic endothelial cells (LECs). CXCR4 was expressed in developing dermal lymphatic capillaries in wild-type mice but not in FOXO1-deficient dermal lymphatic capillaries. Furthermore, FOXO1 suppression impaired migration toward the exogenous CXCR4 ligand, C-X-C chemokine ligand 12 (CXCL12), and coordinated proliferation in LECs. These results suggest that FOXO1 serves an essential role in normal developmental lymphangiogenesis by promoting LEC migration toward CXCL12 and by regulating their proliferative activity. This study provides valuable insights into the molecular mechanisms underlying developmental lymphangiogenesis.
Assuntos
Derme/metabolismo , Proteína Forkhead Box O1/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Linfangiogênese/genética , Receptores CXCR4/genética , Cauda/metabolismo , Regulação para Cima/genética , Animais , Animais Recém-Nascidos , Antígenos CD/metabolismo , Apoptose , Sequência de Bases , Caderinas/metabolismo , Morte Celular , Proliferação de Células , Quimiocina CXCL12/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Elementos Facilitadores Genéticos/genética , Deleção de Genes , Integrases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Receptores CXCR4/metabolismoRESUMO
The splenic white pulp is underpinned by poorly characterized stromal cells that demarcate distinct immune cell microenvironments. Here we establish fibroblastic reticular cell (FRC)-specific fate-mapping in mice to define their embryonic origin and differentiation trajectories. Our data show that all reticular cell subsets descend from multipotent progenitors emerging at embryonic day 19.5 from periarterial progenitors. Commitment of FRC progenitors is concluded during the first week of postnatal life through occupation of niches along developing central arterioles. Single cell transcriptomic analysis facilitated deconvolution of FRC differentiation trajectories and indicated that perivascular reticular cells function both as adult lymphoid organizer cells and mural cell progenitors. The lymphotoxin-ß receptor-independent sustenance of postnatal progenitor stemness unveils that systemic immune surveillance in the splenic white pulp is governed through subset specification of reticular cells from a multipotent periarterial progenitor cell. In sum, the finding that discrete signaling events in perivascular niches determine the differentiation trajectories of reticular cell networks explains the development of distinct microenvironmental niches in secondary and tertiary lymphoid tissues that are crucial for the induction and regulation of innate and adaptive immune processes.
Assuntos
Linhagem da Célula , Microambiente Celular , Fibroblastos/fisiologia , Animais , Diferenciação Celular , Perfilação da Expressão Gênica , Vigilância Imunológica , Linfócitos , Camundongos , BaçoRESUMO
The evolutionary history of eight-barbel loaches of the genus Lefua was investigated to resolve the faunal formation and evolutionary history of Japanese and East Asian freshwater fishes. In the present study, we found that seven Lefua specimens obtained from western Japan could not be assigned genetically to any population recognized so far, although they were morphologically and ecologically indistinguishable from Lefua sp 1. We tentatively designated the specimens as the Nihonkai population. Sequencing of the mitochondrial D-loop region showed that five specimens of the Nihonkai population were allied with the Kinki and Tokai populations of L. echigonia and other two with Lefua sp. 2, indicating a discrepancy between morphological-ecological and genetic traits. In order to determine whether the population experienced parallel evolution or introgression of mitochondrial DNA, we sequenced the nuclear ribosomal S7 subunit, and demonstrated that the Nihonkai population is included within Lefua sp. 1. These results indicate that the Nihonkai population can be identified as Lefua sp. 1, possessing introgressive mitochondrial DNA, and suggest that the evolutionary history and faunal formation of Japanese and East Asian freshwater fishes are more complicated than previously thought.
Assuntos
Núcleo Celular/genética , Cipriniformes/genética , DNA Mitocondrial/genética , Fluxo Gênico/genética , Filogenia , Animais , Japão , Análise de Sequência de DNARESUMO
In mammalian pancreatic ß cells, the IRE1α-XBP1 pathway is constitutively and highly activated under physiological conditions. To elucidate the precise role of this pathway, we constructed ß cell-specific Ire1α conditional knockout (CKO) mice and established insulinoma cell lines in which Ire1α was deleted using the Cre-loxP system. Ire1α CKO mice showed the typical diabetic phenotype including impaired glycemic control and defects in insulin biosynthesis postnatally at 4-20 weeks. Ire1α deletion in pancreatic ß cells in mice and insulinoma cells resulted in decreased insulin secretion, decreased insulin and proinsulin contents in cells, and decreased oxidative folding of proinsulin along with decreased expression of five protein disulfide isomerases (PDIs): PDI, PDIR, P5, ERp44, and ERp46. Reconstitution of the IRE1α-XBP1 pathway restored the proinsulin and insulin contents, insulin secretion, and expression of the five PDIs, indicating that IRE1α functions as a key regulator of the induction of catalysts for the oxidative folding of proinsulin in pancreatic ß cells.
Assuntos
Endorribonucleases/metabolismo , Células Secretoras de Insulina/enzimologia , Insulina/metabolismo , Proinsulina/metabolismo , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Fator 6 Ativador da Transcrição/metabolismo , Animais , Sítios de Ligação , Glicemia/metabolismo , Linhagem Celular Tumoral , Diabetes Mellitus/sangue , Diabetes Mellitus/enzimologia , Diabetes Mellitus/genética , Endorribonucleases/deficiência , Endorribonucleases/genética , Insulina/genética , Insulinoma/enzimologia , Insulinoma/genética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Oxirredução , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Fosforilação , Proinsulina/química , Proinsulina/genética , Regiões Promotoras Genéticas , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Proteína 1 de Ligação a X-Box/genética , eIF-2 Quinase/metabolismoRESUMO
The piRNA pathway is a piRNA-guided retrotransposon silencing system which includes processing of retrotransposon transcripts by PIWI-piRNAs in secondary piRNA biogenesis. Although several proteins participate in the piRNA pathway, the ones crucial for the cleavage of target RNAs by PIWI-piRNAs have not been identified. Here, we show that GTSF1, an essential factor for retrotransposon silencing in male germ cells in mice, associates with both MILI and MIWI2, mouse PIWI proteins that function in prospermatogonia. GTSF1 deficiency leads to a severe defect in the production of secondary piRNAs, which are generated from target RNAs of PIWI-piRNAs. Furthermore, in Gtsf1 mutants, a known target RNA of PIWI-piRNAs is left unsliced at the cleavage site, and the generation of secondary piRNAs from this transcript is defective. Our findings indicate that GTSF1 is a crucial factor for the slicing of target RNAs by PIWI-piRNAs and thus affects secondary piRNA biogenesis in prospermatogonia.
Assuntos
Regulação da Expressão Gênica , Proteínas/metabolismo , RNA Interferente Pequeno/genética , Transcrição Gênica , Células-Tronco Germinativas Adultas/metabolismo , Animais , Núcleo Celular/metabolismo , Amplificação de Genes , Inativação Gênica , Genes de Partícula A Intracisternal , Peptídeos e Proteínas de Sinalização Intracelular , Elementos Nucleotídeos Longos e Dispersos , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas/genética , Interferência de RNA , Proteínas Recombinantes de Fusão , Retroelementos , Testículo/metabolismoRESUMO
Olfactory receptors (ORs) mediate olfactory chemo-sensation in OR neurons. Herein, we have demonstrated that the OR chemo-sensing machinery functions in pancreatic ß-cells and modulates insulin secretion. First, we found several OR isoforms, including OLFR15 and OLFR821, to be expressed in pancreatic islets and a ß-cell line, MIN6. Immunostaining revealed OLFR15 and OLFR821 to be uniformly expressed in pancreatic ß-cells. In addition, mRNAs of Olfr15 and Olfr821 were detected in single MIN6 cells. These results indicate that multiple ORs are simultaneously expressed in individual ß-cells. Octanoic acid, which is a medium-chain fatty acid contained in food and reportedly interacts with OLFR15, potentiated glucose-stimulated insulin secretion (GSIS), thereby improving glucose tolerance in vivo. GSIS potentiation by octanoic acid was confirmed in isolated pancreatic islets and MIN6 cells and was blocked by OLFR15 knockdown. While Gα olf expression was not detectable in ß-cells, experiments using inhibitors and siRNA revealed that the pathway dependent on phospholipase C-inositol triphosphate, rather than cAMP-protein kinase A, mediates GSIS potentiation via OLFR15. These findings suggest that the OR system in pancreatic ß-cells has a chemo-sensor function allowing recognition of environmental substances obtained from food, and potentiates insulin secretion in a cell-autonomous manner, thereby modulating systemic glucose metabolism.
Assuntos
Glucose/metabolismo , Células Secretoras de Insulina/química , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , Receptores Odorantes/análise , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/análise , Receptores Odorantes/genéticaRESUMO
Forkhead box protein O1 (FoxO1) is a transcription factor and a critical regulator of angiogenesis. Various environmental stimuli, including growth factors, nutrients, shear stress, oxidative stress and hypoxia, affect FoxO1 subcellular localization and strongly influence its transcriptional activity; however, FoxO1-localization patterns in endothelial cells (ECs) during development have not been clarified in vivo. Here, we reported that FoxO1 expression was observed in three layers of angiogenic vessels in developing mouse retinas and that among these layers, the front layer showed high levels of FoxO1 expression in the nuclei of most tip ECs. Because tip ECs migrate toward the avascular hypoxic area, we focused on hypoxia as a major stimulus regulating FoxO1 subcellular localization in tip cells. In cultured ECs, FoxO1 accumulated into the nucleus under hypoxic conditions, with hypoxia also inducing expression of tip-cell-specific genes, including endothelial-specific molecule 1 (ESM1), which was suppressed by FoxO1 knockdown. Additionally, in murine models, EC-specific FoxO1 deletion resulted in reduced ESM1 expression and suppressed tip-cell migration during angiogenesis. These findings indicated roles for FoxO1 in tip-cell migration and that its transcriptional activity is regulated by hypoxia.
Assuntos
Células Endoteliais/metabolismo , Proteína Forkhead Box O1/metabolismo , Regulação da Expressão Gênica , Hipóxia/metabolismo , Retina/crescimento & desenvolvimento , Neovascularização Retiniana/metabolismo , Animais , Células Endoteliais/patologia , Proteína Forkhead Box O1/genética , Técnicas de Silenciamento de Genes , Humanos , Hipóxia/genética , Hipóxia/patologia , Camundongos , Camundongos Transgênicos , Retina/patologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologiaRESUMO
BACKGROUND: Alveolar bone is a critical tissue for tooth retention; however, once alveolar bone is lost, it may not spontaneously regenerate. Currently, bone grafts or artificial bone is commonly used for alveolar bone regeneration therapy. However, these therapies require surgical procedures, which present risks, particularly in elderly patients. Therefore, development of alveolar bone regeneration techniques that do not require surgical procedures is critical. It is well known that stem cells present in the periosteal and periodontal ligament may be induced to differentiate into osteogenic cells. This study hypothesizes that transfer of the bone morphogenetic protein-2/7 (BMP-2/7) gene into periodontal tissues via in vivo electroporation induces exogenous BMP production and causes stem cells in periodontal tissues to differentiate into osteogenic cells, enabling generation of new alveolar bone. METHOD: The BMP-2/7 gene expression vector was introduced via electroporation into the target site in periodontal tissues of the first molar of rat maxillae. RESULTS: Exogenous BMP-2 and -7 were detected in the target areas, and growth of new alveolar bone tissue was observed 5 days after gene transfer. On day 7, new alveolar bone tissues were found to connect to the original bone tissues. Moreover, mineral apposition rates of the alveolar bone after BMP-2/7 gene transfer were significantly higher than those in the control group after LacZ gene transfer. CONCLUSION: The present findings indicate that a combination of the BMP-2/7 non-viral vector and in vivo electroporation represents a promising non-surgical option for alveolar bone regeneration therapy.
Assuntos
Proteína Morfogenética Óssea 2 , Regeneração Óssea , Idoso , Animais , Terapia Genética , Humanos , Osteogênese , Ligamento Periodontal , Periodonto , Ratos , RegeneraçãoRESUMO
The Cys2/His2-type zinc finger protein Zfp296 has been implicated in stem cell pluripotency and tumor pathogenesis. However, its mechanisms remain elusive. Here, we demonstrated that a Zfp296 deficiency in mice impairs germ-cell development and embryonic growth. Zfp296 was intracellularly localized to heterochromatin in embryos. A GST-Zfp296 pull-down experiment using ES cell nuclear extract followed by LC-MS/MS showed that Zfp296 interacts with component proteins of heterochromatin (such as HP1, Dnmt1, Dnmt3b, and ATRX) and the NuRD complex. We focused on H3K9 methylation as a hallmark of heterochromatin, and found that Zfp296 overexpression in cultured cells reduces the Suv39h1-mediated H3K9 methylation. Consistent with this finding, in Zfp296 -/- mouse embryos, we observed a global increase in H3K9 methylation in a developmental stage-dependent manner, and showed, by ChIP-qPCR, that the H3K9me3 levels at major satellite repeats were elevated in Zfp296 -/- embryos. Our results demonstrate that Zfp296 is a component of heterochromatin that affects embryonic development by negatively regulating H3K9 methylation.
Assuntos
Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Diferenciação Celular , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas de Ligação a DNA/deficiência , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/química , Histonas/genética , Masculino , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Ovário/anormalidades , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Cultura Primária de Células , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Testículo/anormalidades , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo , DNA Metiltransferase 3BRESUMO
Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.
Assuntos
Comunicação Celular , Transformação Celular Neoplásica/metabolismo , Metabolismo Energético , Células Epiteliais/metabolismo , Animais , Linhagem Celular Transformada , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Técnicas de Cocultura , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Cães , Feminino , Genes ras , Glucose/metabolismo , Glicólise , Ácido Láctico/metabolismo , Células Madin Darby de Rim Canino , Masculino , Potencial da Membrana Mitocondrial , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Interferência de RNA , Transdução de Sinais , Técnicas de Cultura de Tecidos , TransfecçãoRESUMO
A promising approach to new diabetes therapies is to generate ß cells from other differentiated pancreatic cells in vivo. Because the acinar cells represent the most abundant cell type in the pancreas, an attractive possibility is to reprogram acinar cells into ß cells. The transcription factor Pdx1 (Pancreas/duodenum homeobox protein 1) is essential for pancreatic development and cell lineage determination. Our objective is to examine whether exogenous expression of Pdx1 in acinar cells of adult mice might induce reprogramming of acinar cells into ß cells. We established a transgenic mouse line in which Pdx1 and EGFP (enhanced green fluorescent protein) could be inducibly expressed in the acinar cells. After induction of Pdx1, we followed the acinar cells for their expression of exocrine and endocrine markers using cell-lineage tracing with EGFP. The acinar cell-specific expression of Pdx1 in adult mice reprogrammed the acinar cells as endocrine precursor cells, which migrated into the pancreatic islets and differentiated into insulin-, somatostatin-, or PP (pancreatic polypeptide)-producing endocrine cells, but not into glucagon-producing cells. When the mice undergoing such pancreatic reprogramming were treated with streptozotocin (STZ), the newly generated insulin-producing cells were able to ameliorate STZ-induced diabetes. This paradigm of in vivo reprogramming indicates that acinar cells hold promise as a source for new islet cells in regenerative therapies for diabetes.
Assuntos
Células Acinares/citologia , Transdiferenciação Celular/genética , Pâncreas/citologia , Transativadores/genética , Células Acinares/metabolismo , Animais , Glicemia/metabolismo , Reprogramação Celular , Glândulas Exócrinas/citologia , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Insulina/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
Elucidating the regulation of glucose-stimulated insulin secretion (GSIS) in pancreatic ß cells is important for understanding and treating diabetes. The pancreatic ß cell line, MIN6, retains GSIS but gradually loses it in long-term culture. The MIN6 subclone, MIN6c4, exhibits well-regulated GSIS even after prolonged culture. We previously used DNA microarray analysis to compare gene expression in the parental MIN6 cells and MIN6c4 cells and identified several differentially regulated genes that may be involved in maintaining GSIS. Here we investigated the potential roles of six of these genes in GSIS: Tmem59l (Transmembrane protein 59 like), Scgn (Secretagogin), Gucy2c (Guanylate cyclase 2c), Slc29a4 (Solute carrier family 29, member 4), Cdhr1 (Cadherin-related family member 1), and Celsr2 (Cadherin EGF LAG seven-pass G-type receptor 2). These genes were knocked down in MIN6c4 cells using lentivirus vectors expressing gene-specific short hairpin RNAs (shRNAs), and the effects of the knockdown on insulin expression and secretion were analyzed. Suppression of Tmem59l, Scgn, and Gucy2c expression resulted in significantly decreased glucose- and/or KCl-stimulated insulin secretion from MIN6c4 cells, while the suppression of Slc29a4 expression resulted in increased insulin secretion. Tmem59l overexpression rescued the phenotype of the Tmem59l knockdown MIN6c4 cells, and immunostaining analysis indicated that the TMEM59L protein colocalized with insulin and GM130, a Golgi complex marker, in MIN6 cells. Collectively, our findings suggested that the proteins encoded by Tmem59l, Scgn, Gucy2c, and Slc29a4 play important roles in regulating GSIS. Detailed studies of these proteins and their functions are expected to provide new insights into the molecular mechanisms involved in insulin secretion.