RESUMO
The PIP3/PI3K network is a central regulator of metabolism and is frequently activated in cancer, commonly by loss of the PIP3/PI(3,4)P2 phosphatase, PTEN. Despite huge research investment, the drivers of the PI3K network in normal tissues and how they adapt to overactivation are unclear. We find that in healthy mouse prostate PI3K activity is driven by RTK/IRS signaling and constrained by pathway feedback. In the absence of PTEN, the network is dramatically remodeled. A poorly understood YXXM- and PIP3/PI(3,4)P2-binding PH domain-containing adaptor, PLEKHS1, became the dominant activator and was required to sustain PIP3, AKT phosphorylation, and growth in PTEN-null prostate. This was because PLEKHS1 evaded pathway-feedback and experienced enhanced PI3K- and Src-family kinase-dependent phosphorylation of Y258XXM, eliciting PI3K activation. hPLEKHS1 mRNA and activating Y419 phosphorylation of hSrc correlated with PI3K pathway activity in human prostate cancers. We propose that in PTEN-null cells receptor-independent, Src-dependent tyrosine phosphorylation of PLEKHS1 creates positive feedback that escapes homeostasis, drives PIP3 signaling, and supports tumor progression.
Assuntos
PTEN Fosfo-Hidrolase , Neoplasias da Próstata , Animais , Humanos , Masculino , Camundongos , Homeostase , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismoRESUMO
Severity of neurobehavioral deficits in children born from adverse pregnancies, such as maternal alcohol consumption and diabetes, does not always correlate with the adversity's duration and intensity. Therefore, biological signatures for accurate prediction of the severity of neurobehavioral deficits, and robust tools for reliable identification of such biomarkers, have an urgent clinical need. Here, we demonstrate that significant changes in the alternative splicing (AS) pattern of offspring lymphocyte RNA can function as accurate peripheral biomarkers for motor learning deficits in mouse models of prenatal alcohol exposure (PAE) and offspring of mother with diabetes (OMD). An aptly trained deep-learning model identified 29 AS events common to PAE and OMD as superior predictors of motor learning deficits than AS events specific to PAE or OMD. Shapley-value analysis, a game-theory algorithm, deciphered the trained deep-learning model's learnt associations between its input, AS events, and output, motor learning performance. Shapley values of the deep-learning model's input identified the relative contribution of the 29 common AS events to the motor learning deficit. Gene ontology and predictive structure-function analyses, using Alphafold2 algorithm, supported existing evidence on the critical roles of these molecules in early brain development and function. The direction of most AS events was opposite in PAE and OMD, potentially from differential expression of RNA binding proteins in PAE and OMD. Altogether, this study posits that AS of lymphocyte RNA is a rich resource, and deep-learning is an effective tool, for discovery of peripheral biomarkers of neurobehavioral deficits in children of diverse adverse pregnancies.
Assuntos
Diabetes Mellitus , Transtornos do Espectro Alcoólico Fetal , Efeitos Tardios da Exposição Pré-Natal , Camundongos , Animais , Criança , Humanos , Gravidez , Feminino , Processamento Alternativo , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Etanol , Diabetes Mellitus/induzido quimicamente , Biomarcadores/metabolismo , RNA/metabolismo , Transtornos do Espectro Alcoólico Fetal/genéticaRESUMO
A hallmark of fetal alcohol spectrum disorders (FASD) is neurobehavioral deficits that still do not have effective treatment. Here, we present that reduction of Apolipoprotein E (APOE) is critically involved in neurobehavioral deficits in FASD. We show that prenatal alcohol exposure (PAE) changes chromatin accessibility of Apoe locus, and causes reduction of APOE levels in both the brain and peripheral blood in postnatal mice. Of note, postnatal administration of an APOE receptor agonist (APOE-RA) mitigates motor learning deficits and anxiety in those mice. Several molecular and electrophysiological properties essential for learning, which are altered by PAE, are restored by APOE-RA. Our human genome-wide association study further reveals that the interaction of PAE and a single nucleotide polymorphism in the APOE enhancer which chromatin is closed by PAE in mice is associated with lower scores in the delayed matching-to-sample task in children. APOE in the plasma is also reduced in PAE children, and the reduced level is associated with their lower cognitive performance. These findings suggest that controlling the APOE level can serve as an effective treatment for neurobehavioral deficits in FASD.
RESUMO
The PI3K signaling pathway regulates cell growth and movement and is heavily mutated in cancer. Class I PI3Ks synthesize the lipid messenger PI(3,4,5)P3. PI(3,4,5)P3 can be dephosphorylated by 3- or 5-phosphatases, the latter producing PI(3,4)P2. The PTEN tumor suppressor is thought to function primarily as a PI(3,4,5)P3 3-phosphatase, limiting activation of this pathway. Here we show that PTEN also functions as a PI(3,4)P2 3-phosphatase, both in vitro and in vivo. PTEN is a major PI(3,4)P2 phosphatase in Mcf10a cytosol, and loss of PTEN and INPP4B, a known PI(3,4)P2 4-phosphatase, leads to synergistic accumulation of PI(3,4)P2, which correlated with increased invadopodia in epidermal growth factor (EGF)-stimulated cells. PTEN deletion increased PI(3,4)P2 levels in a mouse model of prostate cancer, and it inversely correlated with PI(3,4)P2 levels across several EGF-stimulated prostate and breast cancer lines. These results point to a role for PI(3,4)P2 in the phenotype caused by loss-of-function mutations or deletions in PTEN.
Assuntos
Neoplasias da Mama/enzimologia , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositóis/metabolismo , Neoplasias da Próstata/enzimologia , Sistemas do Segundo Mensageiro , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Fenótipo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Fatores de TempoRESUMO
The voltage-sensing phosphatase (VSP) is a unique protein that shows voltage-dependent phosphoinositide phosphatase activity. Here we report that VSP is activated in mice sperm flagellum and generates a unique subcellular distribution pattern of PtdIns(4,5)P2 Sperm from VSP-/- mice show more Ca2+ influx upon capacitation than VSP+/- mice and abnormal circular motion. VSP-deficient sperm showed enhanced activity of Slo3, a PtdIns(4,5)P2-sensitive K+ channel, which selectively localizes to the principal piece of the flagellum and indirectly enhances Ca2+ influx. Most interestingly, freeze-fracture electron microscopy analysis indicates that normal sperm have much less PtdIns(4,5)P2 in the principal piece than in the midpiece of the flagellum, and this polarized PtdIns(4,5)P2 distribution disappeared in VSP-deficient sperm. Thus, VSP appears to optimize PtdIns(4,5)P2 distribution of the principal piece. These results imply that flagellar PtdIns(4,5)P2 distribution plays important roles in ion channel regulation as well as sperm motility.
Assuntos
Canais Iônicos/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Motilidade dos Espermatozoides/fisiologia , Animais , Canais de Cálcio/metabolismo , Flagelos/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Knockout , Monoéster Fosfórico Hidrolases/genética , Cauda do Espermatozoide/metabolismo , Espermatozoides/metabolismoRESUMO
PIKfyve, VAC14, and FIG4 form a complex that catalyzes the production of PI(3,5)P2, a signaling lipid implicated in process ranging from lysosome maturation to neurodegeneration. While previous studies have identified VAC14 and FIG4 mutations that lead to both neurodegeneration and coat color defects, how PIKfyve regulates melanogenesis is unknown. In this study, we sought to better understand the role of PIKfyve in melanosome biogenesis. Melanocyte-specific PIKfyve knockout mice exhibit greying of the mouse coat and the accumulation of single membrane vesicle structures in melanocytes resembling multivesicular endosomes. PIKfyve inhibition blocks melanosome maturation, the processing of the melanosome protein PMEL, and the trafficking of the melanosome protein TYRP1. Taken together, these studies identify a novel role for PIKfyve in controlling the delivery of proteins from the endosomal compartment to the melanosome, a role that is distinct from the role of PIKfyve in the reformation of lysosomes from endolysosomes.
Assuntos
Melanossomas/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Animais , Flavoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melaninas/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Knockout , Organelas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatases de Fosfoinositídeos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Transporte ProteicoRESUMO
Lysophosphatidylinositol-acyltransferase-1 (LPIAT1) specifically catalyzes the transfer of arachidonoyl-CoA to lysophosphoinositides. LPIAT-/- mice have been shown to have severe defects in the brain and liver; however, the exact molecular mechanisms behind these conditions are not well understood. As immune cells have been implicated in liver inflammation based on disfunction of LPIAT1, we generated Raw264.7 macrophages deficient in LPIAT1, using shRNA and CRISPR/Cas9. The amount of C38:4 species in phosphoinositides, especially in PtdInsP2 , was remarkably decreased in these cells. Unlike in wild-type cells, LPIAT1-deficient cells showed prolonged oscillations of intracellular Ca2+ upon UDP stimulation, which is known to activate phospholipase Cß through the Gq-coupled P2Y6 receptor, even in the absence of extracellular Ca2+ . It is speculated that the prolonged Ca2+ response may be relevant to the increased risk of liver inflammation induced by LPIAT1 disfunction.
Assuntos
Aciltransferases/metabolismo , Sinalização do Cálcio , Aciltransferases/genética , Animais , Camundongos , Células RAW 264.7RESUMO
Endocytosis mediates the internalization and ingestion of a variety of endogenous or exogenous substances, including virus particles, under the control of intracellular signaling pathways. We have previously reported that the complex formed between the small GTPase Ras and phosphoinositide 3-kinase (PI3K) translocates from the plasma membrane to endosomes, signaling from which thereby regulates clathrin-independent endocytosis, endosome maturation, influenza virus internalization, and infection. However, the molecular mechanism by which the Ras-PI3K complex is recruited to endosomes remains unclear. Here, we have identified the amino acid sequence responsible for endosomal localization of the Ras-PI3K complex. PI3K lacking this sequence failed to translocate to endosomes, and expression of the peptide comprising this PI3K-derived sequence inhibited clathrin-independent endocytosis, influenza virus internalization, and infection. Moreover, treatment of cells with this peptide in an arginine-rich, cell-penetrating form successfully suppressed influenza virus infection in vitro and ex vivo, making this peptide a potential therapeutic agent against influenza virus infection.Key words: signal transduction, endocytosis, endosome, imaging, influenza virus.
Assuntos
Endocitose/efeitos dos fármacos , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/fisiologia , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol 3-Quinase/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Humanos , Fragmentos de Peptídeos/química , Transporte Proteico/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Proteínas ras/metabolismoRESUMO
Docosahexaenoic acid (DHA) is one of the essential ω-3 polyunsaturated fatty acids with a wide range of physiological roles important for human health. For example, DHA renders cell membranes more flexible and is therefore important for cellular function, but information on the mechanisms that control DHA levels in membranes is limited. Specifically, it is unclear which factors determine DHA incorporation into cell membranes and how DHA exerts biological effects. We found that lysophosphatidic acid acyltransferase 3 (LPAAT3) is required for producing DHA-containing phospholipids in various tissues, such as the testes and retina. In this study, we report that LPAAT3-KO mice display severe male infertility with abnormal sperm morphology. During germ cell differentiation, the expression of LPAAT3 was induced, and germ cells obtained more DHA-containing phospholipids. Loss of LPAAT3 caused drastic reduction of DHA-containing phospholipids in spermatids that led to excess cytoplasm around its head, which is normally removed by surrounding Sertoli cells via endocytosis at the final stage of spermatogenesis. In vitro liposome filtration assay raised the possibility that DHA in phospholipids promotes membrane deformation that is required for the rapid endocytosis. These data suggest that decreased membrane flexibility in LPAAT3-KO sperm impaired the efficient removal of sperm content through endocytosis. We conclude that LPAAT3-mediated enrichment of cell membranes with DHA-containing phospholipids endows these membranes with physicochemical properties needed for normal cellular processes, as exemplified by spermatogenesis.
Assuntos
Aciltransferases/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Infertilidade Masculina/enzimologia , Espermatogênese , Espermatozoides/metabolismo , Testículo/metabolismo , Aciltransferases/genética , Animais , Ácidos Docosa-Hexaenoicos/análise , Ácidos Docosa-Hexaenoicos/química , Endocitose , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Lipossomos , Masculino , Fluidez de Membrana , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Cabeça do Espermatozoide/metabolismo , Cabeça do Espermatozoide/patologia , Cabeça do Espermatozoide/ultraestrutura , Espermátides/metabolismo , Espermátides/patologia , Espermátides/ultraestrutura , Espermatozoides/patologia , Espermatozoides/ultraestrutura , Testículo/patologia , Testículo/ultraestruturaRESUMO
Docosahexaenoic acid (DHA) has essential roles in photoreceptor cells in the retina and is therefore crucial to healthy vision. Although the influence of dietary DHA on visual acuity is well known and the retina has an abundance of DHA-containing phospholipids (PL-DHA), the mechanisms associated with DHA's effects on visual function are unknown. We previously identified lysophosphatidic acid acyltransferase 3 (LPAAT3) as a PL-DHA biosynthetic enzyme. Here, using comprehensive phospholipid analyses and imaging mass spectroscopy, we found that LPAAT3 is expressed in the inner segment of photoreceptor cells and that PL-DHA disappears from the outer segment in the LPAAT3-knock-out mice. Dynamic light-scattering analysis of liposomes and molecular dynamics simulations revealed that the physical characteristics of DHA reduced membrane-bending rigidity. Following loss of PL-DHA, LPAAT3-knock-out mice exhibited abnormalities in the retinal layers, such as incomplete elongation of the outer segment and decreased thickness of the outer nuclear layers and impaired visual function, as well as disordered disc morphology in photoreceptor cells. Our results indicate that PL-DHA contributes to visual function by maintaining the disc shape in photoreceptor cells and that this is a function of DHA in the retina. This study thus provides the reason why DHA is required for visual acuity and may help inform approaches for overcoming retinal disorders associated with DHA deficiency or dysfunction.
Assuntos
Aciltransferases/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Transtornos da Visão/metabolismo , Aciltransferases/genética , Animais , Biomarcadores/metabolismo , Cruzamentos Genéticos , Ácidos Docosa-Hexaenoicos/análise , Ácidos Docosa-Hexaenoicos/química , Eletrorretinografia , Lipossomos , Fluidez de Membrana , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Simulação de Dinâmica Molecular , Imagem Multimodal , Imagem Óptica , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Células Fotorreceptoras de Vertebrados/ultraestrutura , Fenômenos Físicos , Retina/metabolismo , Retina/patologia , Retina/ultraestrutura , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/patologia , Segmento Externo das Células Fotorreceptoras da Retina/ultraestrutura , Transtornos da Visão/patologiaRESUMO
OBJECTIVE: Neutrophil extracellular traps (NETs) are peculiar structures composed of the externalized chromatin with intracellular proteins and formed by activated neutrophils in a reactive oxygen species (ROS)-dependent manner. Aberrant NETs are considered to be autoantigens for anti-neutrophil cytoplasmic antibodies (ANCAs) underling the development of microscopic polyangiitis (MPA). However, little is known regarding the therapeutic efficacy of in vivo inhibition of NET formation (NETosis) on MPA pathogenesis. This study determines whether reducing NETosis prevents ANCA production and improves characteristic involvement. METHODS: A mouse model of MPA induced by administering a novel extract from Candida albicans was devised. By applying this method to mice lacking phosphoinositide 3-kinase gamma (PI3K-gamma), which is indispensable for ROS production in neutrophils, we investigated the levels of in vivo NETs, ANCA titers and histological damage. RESULTS: Our model exhibited accumulation of NETs in vivo, elevation of ANCA titers and characteristic pathologies mimicking human MPA, including small-vessel vasculitis and crescentic glomerulonephritis. Strikingly, these abnormalities were reduced by genetically and/or pharmacologically blocking PI3K-gamma. Moreover, a pharmacological PI3K-gamma blockade decreased the levels of human NETs. CONCLUSION: Our results suggest that in vivo inhibition of NETosis by blocking PI3K-gamma could be a promising therapeutic strategy for the pathogenesis of MPA.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/metabolismo , Armadilhas Extracelulares/metabolismo , Poliangiite Microscópica/metabolismo , Fosfatidilinositol 3-Quinases/deficiência , Animais , Produtos Biológicos/toxicidade , Candida/química , Armadilhas Extracelulares/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Poliangiite Microscópica/etiologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Phosphorylated derivatives of phosphatidylinositol, collectively referred to as phosphoinositides, occur in the cytoplasmic leaflet of cellular membranes and regulate activities such as vesicle transport, cytoskeletal reorganization and signal transduction. Recent studies have indicated an important role for phosphoinositide metabolism in the aetiology of diseases such as cancer, diabetes, myopathy and inflammation. Although the biological functions of the phosphatases that regulate phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) have been well characterized, little is known about the functions of the phosphatases regulating the closely related molecule phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P(2)). Here we show that inositol polyphosphate phosphatase 4A (INPP4A), a PtdIns(3,4)P(2) phosphatase, is a suppressor of glutamate excitotoxicity in the central nervous system. Targeted disruption of the Inpp4a gene in mice leads to neurodegeneration in the striatum, the input nucleus of the basal ganglia that has a central role in motor and cognitive behaviours. Notably, Inpp4a(-/-) mice show severe involuntary movement disorders. In vitro, Inpp4a gene silencing via short hairpin RNA renders cultured primary striatal neurons vulnerable to cell death mediated by N-methyl-d-aspartate-type glutamate receptors (NMDARs). Mechanistically, INPP4A is found at the postsynaptic density and regulates synaptic NMDAR localization and NMDAR-mediated excitatory postsynaptic current. Thus, INPP4A protects neurons from excitotoxic cell death and thereby maintains the functional integrity of the brain. Our study demonstrates that PtdIns(3,4)P(2), PtdIns(3,4,5)P(3) and the phosphatases acting on them can have distinct regulatory roles, and provides insight into the unique aspects and physiological significance of PtdIns(3,4)P(2) metabolism. INPP4A represents, to our knowledge, the first signalling protein with a function in neurons to suppress excitotoxic cell death. The discovery of a direct link between PtdIns(3,4)P(2) metabolism and the regulation of neurodegeneration and involuntary movements may aid the development of new approaches for the treatment of neurodegenerative disorders.
Assuntos
Ácido Glutâmico/toxicidade , Neurônios/citologia , Neurônios/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Regulação para Baixo , Discinesias/genética , Discinesias/patologia , Discinesias/fisiopatologia , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Neostriado/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia , Neurônios/enzimologia , Neurônios/patologia , Monoéster Fosfórico Hidrolases/deficiência , Monoéster Fosfórico Hidrolases/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Taxa de Sobrevida , Sinapses/metabolismo , Redução de PesoRESUMO
The metabolism of membrane phosphoinositides is critical for a variety of cellular processes. Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P(2)] controls multiple steps of the intracellular membrane trafficking system in both yeast and mammalian cells. However, other than in neuronal tissues, little is known about the physiological functions of PtdIns(3,5)P(2) in mammals. Here, we provide genetic evidence that type III phosphatidylinositol phosphate kinase (PIPKIII), which produces PtdIns(3,5)P(2), is essential for the functions of polarized epithelial cells. PIPKIII-null mouse embryos die by embryonic day 8.5 because of a failure of the visceral endoderm to supply the epiblast with maternal nutrients. Similarly, although intestine-specific PIPKIII-deficient mice are born, they fail to thrive and eventually die of malnutrition. At the mechanistic level, we show that PIPKIII regulates the trafficking of proteins to a cell's apical membrane domain. Importantly, mice with intestine-specific deletion of PIPKIII exhibit diarrhea and bloody stool, and their gut epithelial layers show inflammation and fibrosis, making our mutants an improved model for inflammatory bowel diseases. In summary, our data demonstrate that PIPKIII is required for the structural and functional integrity of two different types of polarized epithelial cells and suggest that PtdIns(3,5)P(2) metabolism is an unexpected and critical link between membrane trafficking in intestinal epithelial cells and the pathogenesis of inflammatory bowel disease.
Assuntos
Endoderma/metabolismo , Mucosa Intestinal/metabolismo , Fosfatidilinositol 3-Quinases/genética , Vísceras/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/ultraestrutura , Células-Tronco Embrionárias/metabolismo , Endoderma/embriologia , Endoderma/ultraestrutura , Feminino , Perfilação da Expressão Gênica , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Intestinos/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Vísceras/embriologia , Vísceras/ultraestruturaRESUMO
The ability to perceive noxious stimuli is critical for an animal's survival in the face of environmental danger, and thus pain perception is likely to be under stringent evolutionary pressure. Using a neuronal-specific RNAi knock-down strategy in adult Drosophila, we recently completed a genome-wide functional annotation of heat nociception that allowed us to identify α2δ3 as a novel pain gene. Here we report construction of an evolutionary-conserved, system-level, global molecular pain network map. Our systems map is markedly enriched for multiple genes associated with human pain and predicts a plethora of novel candidate pain pathways. One central node of this pain network is phospholipid signaling, which has been implicated before in pain processing. To further investigate the role of phospholipid signaling in mammalian heat pain perception, we analysed the phenotype of PIP5Kα and PI3Kγ mutant mice. Intriguingly, both of these mice exhibit pronounced hypersensitivity to noxious heat and capsaicin-induced pain, which directly mapped through PI3Kγ kinase-dead knock-in mice to PI3Kγ lipid kinase activity. Using single primary sensory neuron recording, PI3Kγ function was mechanistically linked to a negative regulation of TRPV1 channel transduction. Our data provide a systems map for heat nociception and reinforces the extraordinary conservation of molecular mechanisms of nociception across different species.
Assuntos
Drosophila , Redes Reguladoras de Genes , Dor Nociceptiva , Fosfolipídeos , Transdução de Sinais , Animais , Capsaicina/toxicidade , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Classe Ib de Fosfatidilinositol 3-Quinase/fisiologia , Drosophila/genética , Drosophila/fisiologia , Temperatura Alta , Humanos , Hipersensibilidade/genética , Camundongos , Neurônios Aferentes/metabolismo , Dor Nociceptiva/induzido quimicamente , Dor Nociceptiva/genética , Dor Nociceptiva/fisiopatologia , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Fosfolipídeos/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/fisiologiaRESUMO
[Purpose] To investigate the effect of Tai Chi on cognitive and physical function in the elderly. [Subjects and Methods] A randomized trial design was used. A total 150 subjects were enrolled and were divided into Tai Chi and control groups. Subjects in the Tai Chi group participated Tai Chi for 6 months, and subjects in the control group participated in other non-athletic activities. [Results] There were no differences between the groups in the one leg standing time with eyes open, left grip strength, or the Frontal Assessment Battery at bedside after 3 and 6 months of intervention. The Mini-Mental State Examination scores after 3 and 6 months were higher in the Tai Chi group than in the control group. The right grip strength after 3 months increased more in the Tai Chi group than in the control group. Both the 5-m high walking speed and 10-m normal walking speed were significantly lower after 3 and 6 months of Tai Chi practice. [Conclusion] These results suggest that regular Tai Chi practice may improve cognitive and physical function in the elderly.
RESUMO
The lens of the eye is composed of fiber cells, which differentiate from epithelial cells and undergo programmed organelle degradation during terminal differentiation. Although autophagy, a major intracellular degradation system, is constitutively active in these cells, its physiological role has remained unclear. We have previously shown that Atg5-dependent macroautophagy is not necessary for lens organelle degradation, at least during the embryonic period. Here, we generated lens-specific Atg5 knock-out mice and showed that Atg5 is not required for lens organelle degradation at any period of life. However, deletion of Atg5 in the lens results in age-related cataract, which is accompanied by accumulation of polyubiquitinated and oxidized proteins, p62, and insoluble crystallins, suggesting a defect in intracellular quality control. We also produced lens-specific Pik3c3 knock-out mice to elucidate the possible involvement of Atg5-independent alternative autophagy, which is proposed to be dependent on Pik3c3 (also known as Vps34), in lens organelle degradation. Deletion of Pik3c3 in the lens does not affect lens organelle degradation, but it leads to congenital cataract and a defect in lens development after birth likely due to an impairment of the endocytic pathway. Taken together, these results suggest that clearance of lens organelles is independent of macroautophagy. These findings also clarify the physiological role of Atg5 and Pik3c3 in quality control and development of the lens, respectively.
Assuntos
Catarata/embriologia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Cápsula do Cristalino/embriologia , Proteínas Associadas aos Microtúbulos/metabolismo , Organelas/metabolismo , Animais , Autofagia/genética , Proteína 5 Relacionada à Autofagia , Catarata/genética , Catarata/patologia , Classe III de Fosfatidilinositol 3-Quinases/genética , Cristalinas/genética , Cristalinas/metabolismo , Endocitose/genética , Cápsula do Cristalino/patologia , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Organelas/genética , Organelas/patologia , Proteínas Ubiquitinadas/genética , Proteínas Ubiquitinadas/metabolismoRESUMO
Proper neutrophil migration into inflammatory sites ensures host defense without tissue damage. Phosphoinositide 3-kinase (PI(3)K) and its lipid product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) regulate cell migration, but the role of PtdIns(3,4,5)P(3)-degrading enzymes in this process is poorly understood. Here, we show that Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1), a PtdIns(3,4,5)P(3) phosphatase, is a key regulator of neutrophil migration. Genetic inactivation of SHIP1 led to severe defects in neutrophil polarization and motility. In contrast, loss of the PtdIns(3,4,5)P(3) phosphatase PTEN had no impact on neutrophil chemotaxis. To study PtdIns(3,4,5)P(3) metabolism in living primary cells, we generated a novel transgenic mouse (AktPH-GFP Tg) expressing a bioprobe for PtdIns(3,4,5)P(3.) Time-lapse footage showed rapid, localized binding of AktPH-GFP to the leading edge membrane of chemotaxing ship1(+/+)AktPH-GFP Tg neutrophils, but only diffuse localization in ship1(-/-)AktPH-GFP Tg neutrophils. By directing where PtdIns(3,4,5)P(3) accumulates, SHIP1 governs the formation of the leading edge and polarization required for chemotaxis.
Assuntos
Movimento Celular , Polaridade Celular , Quimiotaxia , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/fisiologia , Animais , Células Cultivadas , Classe Ib de Fosfatidilinositol 3-Quinase , Humanos , Inositol Polifosfato 5-Fosfatases , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Macrófagos/fisiologia , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Neutrófilos/fisiologia , Proteína Oncogênica v-akt/genética , Proteína Oncogênica v-akt/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismoRESUMO
Obesity and insulin resistance, the key features of metabolic syndrome, are closely associated with a state of chronic, low-grade inflammation characterized by abnormal macrophage infiltration into adipose tissues. Although it has been reported that chemokines promote leukocyte migration by activating class IB phosphoinositide-3 kinase (PI3Kγ) in inflammatory states, little is known about the role of PI3Kγ in obesity-induced macrophage infiltration into tissues, systemic inflammation, and the development of insulin resistance. In the present study, we used murine models of both diet-induced and genetically induced obesity to examine the role of PI3Kγ in the accumulation of tissue macrophages and the development of obesity-induced insulin resistance. Mice lacking p110γ (Pik3cg(-/-)), the catalytic subunit of PI3Kγ, exhibited improved systemic insulin sensitivity with enhanced insulin signaling in the tissues of obese animals. In adipose tissues and livers of obese Pik3cg(-/-) mice, the numbers of infiltrated proinflammatory macrophages were markedly reduced, leading to suppression of inflammatory reactions in these tissues. Furthermore, bone marrow-specific deletion and pharmacological blockade of PI3Kγ also ameliorated obesity-induced macrophage infiltration and insulin resistance. These data suggest that PI3Kγ plays a crucial role in the development of both obesity-induced inflammation and systemic insulin resistance and that PI3Kγ can be a therapeutic target for type 2 diabetes.
Assuntos
Inflamação/tratamento farmacológico , Resistência à Insulina , Obesidade/complicações , Inibidores de Fosfoinositídeo-3 Quinase , Tecido Adiposo/citologia , Animais , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Citometria de Fluxo , Perfilação da Expressão Gênica , Técnicas Histológicas , Inflamação/etiologia , Fígado/citologia , Macrófagos/fisiologia , Camundongos , Camundongos Knockout , Quinoxalinas/farmacologia , Tiazolidinedionas/farmacologiaRESUMO
Background: Numerous studies demonstrated the risk factors for urological complications in patients with diabetes before sodium-glucose co-transporter 2 inhibitor (SGLT2i) became commercially available. This study aimed to comprehensively investigate urological characteristics in patients with type 2 diabetes (T2DM) after SGLT2i became commercially available. Methods: We examined 63 outpatients with T2DM suspected of bacteriuria based on urinary sediment examinations. Urine cultures were performed, and lower urinary tract symptoms (LUTS) were assessed via questionnaires. Patients with bacteriuria were assessed using ultrasonography to measure post-void residual volume (PVR). Utilizing demographic and laboratory data, a random forest algorithm predicted LUTS, bacteriuria, and symptomatic bacteriuria (SB). Results: Thirty-two patients had LUTS and 31 had bacteriuria. High-density lipoprotein cholesterol level was crucial in predicting LUTS, while age was crucial in predicting bacteriuria. In predicting SB among patients with bacteriuria, creatinine level and estimated glomerular filtration rate were crucial. Our models had high predictive accuracy for LUTS (area under the curve [AUC] = 0.846), followed by bacteriuria (AUC = 0.770) and SB (AUC = 0.938) in receiver operating characteristic curve analysis. These predictors were previously reported as risk factors for urological complications. Although SGLT2i use was not an important predictor in our study, all SGLT2i users with bacteriuria had SB and exhibited higher PVR compared to non-SGLT2i users with bacteriuria. Conclusion: This study's random forest model highlighted distinct essential predictors for each urological condition. The predictors were consistent before and after SGLT2i became commercially available. Supplementary Information: The online version contains supplementary material available at 10.1007/s13340-023-00687-1.
RESUMO
H3.1 histone is predominantly synthesized and enters the nucleus during the G1/S phase of the cell cycle, as a new component of duplicating nucleosomes. Here, we found that p53 is necessary to secure the normal behavior and modification of H3.1 in the nucleus during the G1/S phase, in which p53 increases C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1) levels and decreases enhancer of zeste homolog 2 (EZH2) levels in the H3.1 interactome. In the absence of p53, H3.1 molecules tended to be tethered at or near the nuclear envelope (NE), where they were predominantly trimethylated at lysine 27 (H3K27me3) by EZH2, without forming nucleosomes. This accumulation was likely caused by the high affinity of H3.1 toward phosphatidic acid (PA). p53 reduced nuclear PA levels by increasing levels of CTDNEP1, which activates lipin to convert PA into diacylglycerol. We moreover found that the cytosolic H3 chaperone HSC70 attenuates the H3.1-PA interaction, and our molecular imaging analyses suggested that H3.1 may be anchored around the NE after their nuclear entry. Our results expand our knowledge of p53 function in regulation of the nuclear behavior of H3.1 during the G1/S phase, in which p53 may primarily target nuclear PA and EZH2.