RESUMO
Retroviruses have been invading mammalian germlines for millions of years, accumulating in the form of endogenous retroviruses (ERVs) that account for nearly one-tenth of the mouse and human genomes. ERVs are epigenetically silenced during development, yet the cellular factors recognizing ERVs in a sequence-specific manner remain elusive. Here we demonstrate that ZFP809, a member of the Krüppel-associated box zinc finger protein (KRAB-ZFP) family, initiates the silencing of ERVs in a sequence-specific manner via recruitment of heterochromatin-inducing complexes. ZFP809 knockout mice display highly elevated levels of ZFP809-targeted ERVs in somatic tissues. ERV reactivation is accompanied by an epigenetic shift from repressive to active histone modifications but only slight destabilization of DNA methylation. Importantly, using conditional alleles and rescue experiments, we demonstrate that ZFP809 is required to initiate ERV silencing during embryonic development but becomes largely dispensable in somatic tissues. Finally, we show that the DNA-binding specificity of ZFP809 is evolutionarily conserved in the Muroidea superfamily of rodents and predates the endogenization of retroviruses presently targeted by ZFP809 in Mus musculus. In sum, these data provide compelling evidence that ZFP809 evolved to recognize foreign DNA and establish histone modification-based epigenetic silencing of ERVs.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Retrovirus Endógenos/genética , Epigênese Genética , Inativação Gênica , Animais , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos , Retrovirus Endógenos/fisiologia , Genoma , Histonas/metabolismo , Camundongos , Camundongos Knockout , Ligação Proteica , Ativação Viral/genética , Integração Viral/genéticaRESUMO
The nitric oxide (NO)/cGMP signaling cascade has an established role in synaptic plasticity. However, with conventional methods, the underlying cGMP signals were barely detectable. Here, we set out to confirm the well-known NMDA-induced cGMP increases, to test the impact of AMPA on those signals, and to identify the relevant phosphodiesterases (PDEs) using a more sensitive fluorescence resonance energy transfer (FRET)-based method. Therefore, a "knock-in" mouse was generated that expresses a FRET-based cGMP indicator (cGi-500) allowing detection of cGMP concentrations between 100 nM and 3 µM. Measurements were performed in cultured hippocampal and cortical neurons as well as acute hippocampal slices. In hippocampal and cortical neurons, NMDA elicited cGMP signals half as high as the ones elicited by exogenous NO. Interestingly, AMPA increased cGMP independently of NMDA receptors and dependent on NO synthase (NOS) activation. NMDA- and AMPA-induced cGMP signals were not additive indicating that both pathways converge on the level of NOS. Accordingly, the same PDEs, PDE1 and PDE2, were responsible for degradation of NMDA- as well as AMPA-induced cGMP signals. Mechanistically, AMPAR induced calcium influx through L-type voltage-gated calcium channels leading to NOS and finally NO-sensitive guanylyl cyclase activation. Our results demonstrate that in addition to NMDA also AMPA triggers endogenous NO formation and hence cGMP production.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Córtex Cerebral/metabolismo , GMP Cíclico/metabolismo , Hipocampo/metabolismo , Óxido Nítrico/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Cultura de ÓrgãosRESUMO
KEY POINTS: Normal pH is crucial for proper functioning of the brain, and disorders increasing the level of CO2 in the blood lead to a decrease in brain pH. CO2 can easily cross the barriers of the brain and will activate chemoreceptors leading to an increased exhalation of CO2 . The low pH, however, is harmful and bases such as HCO3- are imported across the brain barriers in order to normalize brain pH. We show that the HCO3- transporter NBCe2 in the choroid plexus of the blood-cerebrospinal fluid barrier is absolutely necessary for normalizing CSF pH during high levels of CO2 . This discovery represents a significant step in understanding the molecular mechanisms behind regulation of CSF pH during acid-base disturbances, such as chronic lung disease. ABSTRACT: The choroid plexus epithelium (CPE) is located in the brain ventricles where it produces the majority of the cerebrospinal fluid (CSF). The hypothesis that normal brain function is sustained by CPE-mediated CSF pH regulation by extrusion of acid-base equivalents was tested by determining the contribution of the electrogenic Na+ -HCO3- cotransporter NBCe2 to CSF pH regulation. A novel strain of NBCe2 (Slc4a5) knockout (KO) mice was generated and validated. The base extrusion rate after intracellular alkalization was reduced by 77% in NBCe2 KO mouse CPE cells compared to control mice. NBCe2 KO mice and mice with CPE-targeted NBCe2 siRNA knockdown displayed a reduction in CSF pH recovery during hypercapnia-induced acidosis of approximately 85% and 90%, respectively, compared to control mice. NBCe2 KO did not affect baseline respiration rate or tidal volume, and the NBCe2 KO and wild-type (WT) mice displayed similar ventilatory responses to 5% CO2 exposure. NBCe2 KO mice were not protected against pharmacological or heating-induced seizure development. In conclusion, we establish the concept that the CPE is involved in the regulation of CSF pH by demonstrating that NBCe2 is necessary for proper CSF pH recovery after hypercapnia-induced acidosis.
Assuntos
Bicarbonatos/metabolismo , Líquido Cefalorraquidiano/metabolismo , Plexo Corióideo/metabolismo , Simportadores de Sódio-Bicarbonato/fisiologia , Sódio/metabolismo , Acidose Respiratória/etiologia , Acidose Respiratória/patologia , Acidose Respiratória/prevenção & controle , Doença Aguda , Animais , Líquido Cefalorraquidiano/química , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Convulsões/etiologia , Convulsões/patologiaRESUMO
SORLA is a neuronal sorting receptor implicated both in sporadic and familial forms of AD. SORLA reduces the amyloidogenic burden by two mechanisms, either by rerouting internalized APP molecules from endosomes to the trans-Golgi network (TGN) to prevent proteolytic processing or by directing newly produced Aß to lysosomes for catabolism. Studies in cell lines suggested that the interaction of SORLA with cytosolic adaptors retromer and GGA is required for receptor sorting to and from the TGN. However, the relevance of anterograde or retrograde trafficking for SORLA activity in vivo remained largely unexplored. Here, we generated mouse models expressing SORLA variants lacking binding sites for GGA or retromer to query this concept in the brain. Disruption of retromer binding resulted in a retrograde-sorting defect with accumulation of SORLA in endosomes and depletion from the TGN, and in an overall enhanced APP processing. In contrast, disruption of the GGA interaction did not impact APP processing but caused increased brain Aß levels, a mechanism attributed to a defect in anterograde lysosomal targeting of Aß. Our findings substantiated the significance of adaptor-mediated sorting for SORLA activities in vivo, and they uncovered that anterograde and retrograde sorting paths may serve discrete receptor functions in amyloidogenic processes. SIGNIFICANCE STATEMENT: SORLA is a sorting receptor that directs target proteins to distinct intracellular compartments in neurons. SORLA has been identified as a genetic risk factor for sporadic, but recently also for familial forms of AD. To confirm the relevance of SORLA sorting for AD processes in the brain, we generated mouse lines, which express trafficking mutants instead of the wild-type form of this receptor. Studying neuronal activities in these mutant mice, we dissected distinct trafficking routes for SORLA guided by two cytosolic adaptors termed GGA and retromer. We show that these sorting pathways serve discrete functions in control of amyloidogenic processes and may represent unique therapeutic targets to interfere with specific aspects of neurodegenerative processes in the diseased brain.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Proteínas Relacionadas a Receptor de LDL/fisiologia , Proteínas de Membrana Transportadoras/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Endossomos/metabolismo , Feminino , Hipocampo/citologia , Proteínas Relacionadas a Receptor de LDL/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , RNA não Traduzido/genética , Proteínas Recombinantes de Fusão/metabolismo , Rede trans-Golgi/metabolismoRESUMO
Aquaporin 11 (AQP11) is a protein channel expressed intracellularly in multiple organs, yet its physiological function is unclear. Aqp11 knockout (KO) mice die early due to malfunction of the kidney, a result of hydropic degeneration of proximal tubule cells. Here we report the generation of liver-specific Aqp11 KO mice, allowing us to study the role of AQP11 protein in liver of mice with normal kidney function. The unchallenged liver-specific Aqp11 KO mice have normal longevity, their livers appeared normal, and the plasma biochemistries revealed only a minor defect in lipid handling. Fasting of the mice (24 h) induced modest dilatation of the rough endoplasmic reticulum (RER) in the periportal hepatocytes. Refeeding with standard mouse chow induced rapid generation of large RER-derived vacuoles in Aqp11 KO mice hepatocytes. Similar effects were observed following oral administration of pure protein or larger doses of various amino acids. The fasting/refeeding challenge is associated with increased expression of markers of ER stress Grp78 and GADD153 and decreased glutathione levels, suggesting that ER stress may play role in the development of vacuoles in the AQP11-deficient hepatocytes. NMR-based metabolome analysis of livers from mice subject to amino acid challenge showed decreased amount of extractable metabolites in the AQP11-deficient livers and particularly a decrease in glucose levels. In conclusion, in the liver, deletion of AQP11 results in disrupted RER homeostasis and increased sensitivity to RER injury upon metabolic challenge with amino acids.
Assuntos
Aminoácidos/farmacologia , Aquaporinas/genética , Aquaporinas/fisiologia , Retículo Endoplasmático Rugoso/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/ultraestrutura , Vacúolos/efeitos dos fármacos , Animais , Compostos Azo , Western Blotting , Corantes , DNA/genética , Chaperona BiP do Retículo Endoplasmático , Retículo Endoplasmático Rugoso/ultraestrutura , Jejum/fisiologia , Glicogênio/metabolismo , Glicosilação , Hepatócitos/ultraestrutura , Homeostase/efeitos dos fármacos , Imuno-Histoquímica , Fígado/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Knockout , RNA/biossíntese , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Fixação de Tecidos , Vacúolos/ultraestruturaRESUMO
SEPTIN9 (SEPT9) is a filament-forming protein involved in numerous cellular processes. We have used a conditional knock out allele of Sept9 to specifically delete Sept9 in T-cells. As shown by fluorescence-activated cell sorting, loss of Sept9 at an early thymocyte stage in the thymus results in increased numbers of double-negative cells indicating that SEPT9 is involved in the transition from the double-negative stage during T-cell development. Accordingly, the relative numbers of mature T-cells in the periphery are decreased in mice with a T-cell-specific deletion of Sept9. Proliferation of Sept9-deleted CD8(+) T-cells from the spleen is decreased upon stimulation in culture. The altered T-cell homeostasis caused by the loss of Sept9 results in an increase of CD8(+) central memory T-cells.
Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Homeostase/imunologia , Septinas/metabolismo , Animais , Contagem de Células , Diferenciação Celular/genética , Proliferação de Células , Regulação para Baixo/genética , Memória Imunológica/genética , Integrases/metabolismo , Depleção Linfocítica , Tecido Linfoide/citologia , Tecido Linfoide/metabolismo , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Septinas/genética , Regulação para Cima/genéticaRESUMO
The identification of the mutation causing the phenotype of the amyotrophic lateral sclerosis (ALS) model mouse, wobbler, has linked motor neuron degeneration with retrograde vesicle traffic. The wobbler mutation affects protein stability of Vps54, a ubiquitously expressed vesicle-tethering factor and leads to partial loss of Vps54 function. Moreover, the Vps54 null mutation causes embryonic lethality, which is associated with extensive membrane blebbing in the neural tube and is most likely a consequence of impaired vesicle transport. Investigation of cells derived from wobbler and Vps54 null mutant embryos demonstrates impaired retrograde transport of the Cholera-toxin B subunit to the trans-Golgi network and mis-sorting of mannose-6-phosphate receptors and cargo proteins dependent on retrograde vesicle transport. Endocytosis assays demonstrate no difference between wobbler and wild type cells, indicating that the retrograde vesicle traffic to the trans-Golgi network, but not endocytosis, is affected in Vps54 mutant cells. The results obtained on wobbler cells were extended to test the use of cultured skin fibroblasts from human ALS patients to investigate the retrograde vesicle traffic. Analysis of skin fibroblasts of ALS patients will support the investigation of the critical role of the retrograde vesicle transport in ALS pathogenesis and might yield a diagnostic prospect.
Assuntos
Perda do Embrião/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular/deficiência , Animais , Western Blotting , Catepsina B/metabolismo , Membrana Celular/metabolismo , Toxina da Cólera/metabolismo , Perda do Embrião/patologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Endocitose , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Transporte Proteico , Receptor IGF Tipo 2/metabolismo , Pele/patologia , Vesículas Transportadoras/ultraestrutura , Proteínas de Transporte Vesicular/metabolismoRESUMO
The occurrence of NO/cGMP signalling in cardiac cells is a matter of debate. Recent measurements with a FRET-based cGMP indicator in isolated cardiac cells revealed NO-induced cGMP signals in cardiac fibroblasts while cardiomyocytes were devoid of these signals. In a fibroblast/myocyte co-culture model though, cGMP formed in fibroblasts in response to NO entered cardiomyocytes via gap junctions. Here, we demonstrate gap junction-mediated cGMP transfer from cardiac fibroblasts to myocytes in intact tissue. In living cardiac slices of mice with cardiomyocyte-specific expression of a FRET-based cGMP indicator (αMHC/cGi-500), NO-dependent cGMP signals were shown to occur in myocytes, to depend on gap junctions and to be degraded mainly by PDE3. Stimulation of NO-sensitive guanylyl cyclase enhanced Forskolin- and Isoproterenol-induced cAMP and phospholamban phosphorylation. Genetic inactivation of NO-GC in Tcf21-expressing cardiac fibroblasts abrogated the synergistic action of NO-GC stimulation on Iso-induced phospholamban phosphorylation, identifying fibroblasts as cGMP source and substantiating the necessity of cGMP-transfer to myocytes. In sum, NO-stimulated cGMP formed in cardiac fibroblasts enters cardiomyocytes in native tissue where it exerts an inhibitory effect on cAMP degradation by PDE3, thereby increasing cAMP and downstream effects in cardiomyocytes. Hence, enhancing ß-receptor-induced contractile responses appears as one of NO/cGMP's functions in the non-failing heart.
Assuntos
Coração , Células Cultivadas , Animais , Camundongos , Fibroblastos/metabolismo , GMP Cíclico/metabolismo , Óxido Nítrico/metabolismo , Células Musculares/metabolismo , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Transdução de Sinais , Sobrevivência CelularRESUMO
Biochemical and biomechanical signals regulate stem cell function in the niche environments in vivo. Current in vitro culture of mouse embryonic stem cells (mESC) uses laminin (LN-511) to provide mimetic biochemical signaling (LN-521 for human systems) to maintain stemness. Alternative approaches propose topographical cues to provide biomechanical cues, however combined biochemical and topographic cues may better mimic the in vivo environment, but are largely unexplored for in vitro stem cell expansion. In this study, we directly compare in vitro signals from LN-511 and/or topographic cues to maintain stemness, using systematically-varied submicron pillar patterns or flat surfaces with or without preadsorbed LN-511. The adhesion of cells, colony formation, expression of the pluripotency marker,octamer-binding transcription factor 4 (Oct4), and transcriptome profiling were characterized. We observed that either biochemical or topographic signals could maintain stemness of mESCs in feeder-free conditions, indicated by high-level Oct4 and gene profiling by RNAseq. The combination of LN-511 with nanotopography reduced colony growth, while maintaining stemness markers, shifted the cellular phenotype indicating that the integration of biochemical and topographic signals is antagonistic. Overall, significantly faster (up to 2.5 times) colony growth was observed at nanotopographies without LN-511, suggesting for improved ESC expansion.
Assuntos
Células-Tronco Embrionárias , Células-Tronco Embrionárias Murinas , Animais , Camundongos , Humanos , Células Cultivadas , Ligantes , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fenótipo , Diferenciação Celular/fisiologiaRESUMO
BACKGROUND: Disturbances in pH affect artery function, but the mechanistic background remains controversial. We investigated whether Na(+), HCO3- transporter NBCn1, by regulating intracellular pH(pH1), influences artery function and blood pressure regulation. METHODS AND RESULTS: Knockout of NBCn1 in mice eliminated Na+, HCO3â» cotransport and caused a lower steady-state pH(i) in mesenteric artery smooth muscle and endothelial cells in situ evaluated by fluorescence microscopy. Using myography, arteries from NBCn1 knockout mice showed reduced acetylcholine-induced NO-mediated relaxations and lower rho-kinase-dependent norepinephrine-stimulated smooth muscle Ca²âº sensitivity. Acetylcholine-stimulated NO levels (electrode measurements) and N-nitro-l-arginine methyl ester-sensitive l-arginine conversion (radioisotope measurements) were reduced in arteries from NBCn1 knockout mice, whereas relaxation to NO-donor S-nitroso-N-acetylpenicillamine, acetylcholine-induced endothelial Ca²âº responses (fluorescence microscopy), and total and Ser-1177 phosphorylated endothelial NO-synthase expression (Western blot analyses) were unaffected. Reduced NO-mediated relaxations in arteries from NBCn1 knockout mice were not rescued by superoxide scavenging. Phosphorylation of myosin phosphatase targeting subunit at Thr-850 was reduced in arteries from NBCn1 knockout mice. Evaluated by an in vitro assay, rho-kinase activity was reduced at low pH. Without CO2/HCO3â», no differences in pH(i), contraction or relaxation were observed between arteries from NBCn1 knockout and wild-type mice. Based on radiotelemetry and tail-cuff measurements, NBCn1 knockout mice were mildly hypertensive at rest, displayed attenuated blood pressure responses to NO-synthase and rho-kinase inhibition and were resistant to developing hypertension during angiotensin-II infusion. CONCLUSIONS: Intracellular acidification of smooth muscle and endothelial cells after knockout of NBCn1 inhibits NO-mediated and rho-kinase-dependent signaling in isolated arteries and perturbs blood pressure regulation.
Assuntos
Cálcio/fisiologia , Hipertensão/prevenção & controle , Músculo Liso Vascular/fisiopatologia , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/fisiologia , Simportadores de Sódio-Bicarbonato/deficiência , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Vasodilatação/fisiologia , Animais , Sinalização do Cálcio/genética , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Hipertensão/genética , Hipertensão/metabolismo , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Simportadores de Sódio-Bicarbonato/biossíntese , Simportadores de Sódio-Bicarbonato/genética , Trocadores de Sódio-Hidrogênio/biossíntese , Trocadores de Sódio-Hidrogênio/genética , Vasodilatação/efeitos dos fármacosRESUMO
Overexpression of α-synuclein with tyrosine mutated to phenylalanine at position 125 leads to a severe phenotype with motor impairment and neuropathology in Drosophila. Here, we hypothesized that tyrosine mutations would similarly lead to impaired motor performance with neuropathology in a rodent model. In transgenic mice (ASO), tyrosines at positions 125, 133, and 136 in human α-synuclein were mutated to phenylalanine and cloned into a Thy1.2 expression vector, which was used to create transgenic mouse lines on a mixed genetic background TgN(Thy-1-SNCA-YF)4Emfu (YF). The YF mice had a decreased lifespan and displayed a dramatic motor phenotype with paralysis of both hind- and forelegs. Post-translational modification of α-synuclein due to phosphorylation of serine 129 is often seen in inclusions in the brains of patients with α-synucleinopathies. We observed a slight but significant increase in phosphorylation of serine 129 in the cytosol in YF mice compared to age-matched human α-synuclein transgenic mice (ASO). Conversely, significantly decreased phosphorylation of serine 129 was seen in synaptosomes of YF mice that also contained higher amounts of soluble oligomers. YF mice deposited full-length α-synuclein aggregates in neurons widespread in the CNS with the main occurrence in the forebrain structures of the cerebral cortex, the basal ganglia, and limbic structures. Full-length α-synuclein labeling was also prominent in many nuclear regions of the brain stem, deep cerebellar nuclei, and cerebellar cortex. The study shows that the substitution of tyrosines to phenylalanine in α-synuclein at positions 125, 133, and 136 leads to severe toxicity in vivo. An insignificant change upon tyrosine substitution suggests that the phosphorylation of serine 129 is not the cause of the toxicity.
Assuntos
Síndromes Neurotóxicas , alfa-Sinucleína , Humanos , Animais , Camundongos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Camundongos Transgênicos , Tirosina , Mutação/genética , Serina/genética , FenilalaninaRESUMO
Septin9 (Sept9) is a member of the filament-forming septin family of structural proteins and is associated with a variety of cancers and with hereditary neuralgic amyotrophy. We have generated mice with constitutive and conditional Sept9 knockout alleles. Homozygous deletion of Sept9 results in embryonic lethality around day 10 of gestation whereas mice homozygous for the conditional allele develop normally. Here we report the consequences of homozygous loss of Sept9 in immortalized murine embryonic fibroblasts. Proliferation rate was not changed but cells without Sept9 had an altered morphology compared to normal cells, particularly under low serum stress. Abnormal, fragmented, and multiple nuclei were more frequent in cells without Sept9. Cell migration, as measured by gap-filling and filter-invasion assays, was impaired, but individual cells did not move less than wild-type cells. Sept9 knockout cells showed a reduced resistance to hypo-osmotic stress. Stress fiber and vinculin staining at focal adhesion points was less prominent. Long septin filaments stained for Sept7 disappeared. Instead, staining was found in short, often curved filaments and rings. Furthermore, Sept7 was no longer localized to the mitotic spindle. Together, these data reveal the importance of Sept9 for septin filament formation and general cell stability.
Assuntos
Septinas/metabolismo , Animais , Western Blotting , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Citoesqueleto/genética , Citoesqueleto/metabolismo , Imunofluorescência , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , Septinas/genéticaRESUMO
Transcription of retroviruses is initiated at the U3-R region boundary in the integrated provirus and continues unidirectionally to produce genomic and mRNA products of positive polarity. Several studies have recently demonstrated the existence of naturally occurring protein-encoding transcripts of negative polarity in complex retroviruses. We report here on the identification of transcripts of negative polarity in simple murine leukemia virus (MLV). In T-cell and B-cell lymphomas induced by SL3-3 and Akv MLV, antisense transcripts initiated in the U3 region of the proviral 5' long terminal repeat (LTR) and continued into the cellular proto-oncogenes Jdp2 and Bach2 to create chimeric transcripts consisting of viral and host sequence. The phenomenon was validated in vivo using a knock-in mouse model homozygous for a single LTR at a position known to activate Nras in B-cell lymphomas. A 5' rapid amplification of cDNA ends (RACE) analysis indicated a broad spectrum of initiation sites within the U3 region of the 5' LTR. Our data show for the first time transcriptional activity of negative polarity initiating in the U3 region of simple retroviruses and suggest a novel mechanism of insertional activation of host genes. Elucidation of the nature and potential regulatory role of 5' LTR antisense transcription will be relevant to the design of therapeutic vectors and may contribute to the increasing recognition of pervasive eukaryotic transcription.
Assuntos
Genes Virais , Vírus da Leucemia Murina/genética , Mutagênese Insercional , Provírus/genética , RNA Antissenso/biossíntese , RNA Viral/biossíntese , Transcrição Gênica , Animais , Camundongos , RNA Antissenso/genética , RNA Viral/genética , Sítio de Iniciação de TranscriçãoRESUMO
MicroRNAs are important regulators of cellular functions. MiR-302/367 is a polycistronic miRNA cluster that can induce and maintain pluripotency. Here we investigate the transcriptional control and the processing of the miR-302 host-gene in mice. Our results indicate that the mmu-miR-302 host-gene is alternatively spliced, polyadenylated and exported from the nucleus. The regulatory sequences extend at least 2 kb upstream of the transcription start site and contain several conserved binding sites for both transcriptional activators and repressors. The gene structure and regulatory elements are highly conserved between mouse and human. So far, regulating miR-302 expression is the only known function of the miR-302 host-gene. Even though we here only provide one example, regulation of microRNA transcription might be a so far little recognized function of long non-coding RNA genes.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Biologia Computacional , Camundongos , MicroRNAs/genética , RNA Longo não Codificante/genéticaRESUMO
BACKGROUND: Cancer stem cells are believed to be a major reason for long-term therapy failure because they are multi-drug resistant and able to rest mitotically inactive in the hypoxic center of tumors. Due to their variable number and their often low proliferation rate, cancer stem cells are difficult to purify in decent quantities and to grow in cell culture systems, where they are easily outcompeted by faster growing more 'differentiated', i.e., less stem cell-like tumor cells. METHODS: Here we present a proof of principle study based on the idea to select cancer stem cells by means of the expression of a stem cell-specific gene. A selectable egfp-neo coding sequence was inserted in the last exon of the non-coding murine miR-302 host gene. As a stem cell specific regulatory element, 2.1 kb of the genomic region immediately upstream of the miR-302 host gene transcription start site was used. Stable transgenic CJ7 embryonic stem cells were used to induce teratomas. RESULTS: After three weeks, tumors were removed for analysis and primary cultures were established. Stem cell-like cells were selected from these culture based on G418 selection. When the selection was removed, stem cell morphology and miR-302 expression were rapidly lost, indicating that it was not the original ES cells that had been isolated. CONCLUSIONS: We show the possibility to use drug resistance expressed from a regulatory sequence of a stem cell-specific marker, to isolate and propagate cancer stem cells that otherwise might be hidden in the majority of tumor cells.
RESUMO
BACKGROUND AND PURPOSE: The intracellular signalling molecule cGMP, formed by NO-sensitive GC (NO-GC), has an established function in the vascular system. Despite numerous reports about NO-induced cGMP effects in the heart, the underlying cGMP signals are poorly characterized. EXPERIMENTAL APPROACH: Therefore, we analysed cGMP signals in cardiac myocytes and fibroblasts isolated from knock-in mice expressing a FRET-based cGMP indicator. KEY RESULTS: Whereas in cardiac myocytes, none of the known NO-GC-activating substances (NO, GC activators, and GC stimulators) increased cGMP even in the presence of PDE inhibitors, they induced substantial cGMP increases in cardiac fibroblasts. As cardiac myocytes and fibroblasts are electrically connected via gap junctions, we asked whether cGMP can take the same route. Indeed, in cardiomyocytes co-cultured on cardiac fibroblasts, NO-induced cGMP signals were detectable, and two groups of unrelated gap junction inhibitors abolished these signals. CONCLUSION AND IMPLICATION: We conclude that NO-induced cGMP formed in cardiac fibroblasts enters cardiac myocytes via gap junctions thereby turning cGMP into an intercellular signalling molecule. The findings shed new light on NO/cGMP signalling in the heart and will potentially broaden therapeutic opportunities for cardiac disease.
Assuntos
GMP Cíclico/metabolismo , Fibroblastos/metabolismo , Junções Comunicantes/metabolismo , Miócitos Cardíacos/metabolismo , Óxido Nítrico/farmacologia , Animais , Células Cultivadas , Técnicas de Cocultura , GMP Cíclico/genética , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Junções Comunicantes/efeitos dos fármacos , Técnicas de Introdução de Genes , Camundongos , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacosRESUMO
The cell surface protein Stem Cell Antigen-1 (Sca-1) marks stem or progenitor cells in several murine tissues and is normally upregulated during cancer development. Although the specific function of Sca-1 remains unknown, Sca-1 seems to play a role in proliferation, differentiation and cell migration in a number of tissues. In the skin epithelium, Sca-1 is highly expressed in the interfollicular epidermis but is absent in most compartments of the hair follicle; however, the function of Sca-1 in the skin has not been investigated. To explore the role of Sca-1 in normal and malignant skin development we generated transgenic mice that express Sca-1 in the hair follicle stem cells that are normally Sca-1 negative. Development of hair follicles and interfollicular epidermis appeared normal in Sca-1 mutant mice; however, follicular induction of Sca-1 expression in bulge region and isthmus stem cells reduced the overall yield of papillomas in a chemical carcinogenesis protocol. Despite that fewer papillomas developed in transgenic mice a higher proportion of the papillomas underwent malignant conversion. These findings suggest that overexpression of Sca-1 in the hair follicle stem cells contributes at different stages of tumour development. In early stages, overexpression of Sca-1 decreases tumour formation while at later stages overexpression of Sca-1 seems to drive tumours towards malignant progression.
Assuntos
Antígenos Ly/metabolismo , Carcinogênese/patologia , Progressão da Doença , Folículo Piloso/patologia , Proteínas de Membrana/metabolismo , Transgenes , 9,10-Dimetil-1,2-benzantraceno , Animais , Células Clonais , Ensaio de Unidades Formadoras de Colônias , Feminino , Integrases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Morfogênese , Pele/patologia , Acetato de TetradecanoilforbolRESUMO
Autophagy-mediated degradation of synaptic components maintains synaptic homeostasis but also constitutes a mechanism of neurodegeneration. It is unclear how autophagy of synaptic vesicles and components of presynaptic active zones is regulated. Here, we show that Pleckstrin homology containing family member 5 (Plekhg5) modulates autophagy of synaptic vesicles in axon terminals of motoneurons via its function as a guanine exchange factor for Rab26, a small GTPase that specifically directs synaptic vesicles to preautophagosomal structures. Plekhg5 gene inactivation in mice results in a late-onset motoneuron disease, characterized by degeneration of axon terminals. Plekhg5-depleted cultured motoneurons show defective axon growth and impaired autophagy of synaptic vesicles, which can be rescued by constitutively active Rab26. These findings define a mechanism for regulating autophagy in neurons that specifically targets synaptic vesicles. Disruption of this mechanism may contribute to the pathophysiology of several forms of motoneuron disease.