RESUMO
The concept of immune cell exhaustion/dysfunction has developed mainly to understand impaired type 1 immune responses especially by CD8 T cells against tumors or virus-infected cells and has been applied to other lymphocytes. Natural killer (NK) cells and CD4 T cells support the efficient activation of CD8 T cells but exhibit a dysfunctional phenotype in tumor microenvironments and in chronic virus infections. In contrast, the concept of type 2 immune cell exhaustion/dysfunction is poorly established. Group 2 innate lymphoid cells (ILC2s) and T-helper 2 (Th2) cells are the major lymphocyte subsets that initiate and expand type 2 immune responses for antiparasitic immunity or allergy. In mouse models of chronic parasitic worm infections, Th2 cells display impaired type 2 immune responses. Chronic airway allergy induces exhausted-like ILC2s that quickly fall into activation-induced cell death to suppress exaggerated inflammation. Thus, the modes of exhaustion/dysfunction are quite diverse and rely on the types of inflammation and the cells. In this review, we summarize current knowledge of lymphocyte exhaustion/dysfunction in the context of type 1 and type 2 immune responses and discuss ILC2-specific regulatory mechanisms during chronic allergy.
RESUMO
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
Group 2 innate lymphoid cells (ILC2s) reside in peripheral tissues such as the lungs, skin, nasal cavity, and gut and provoke innate type 2 immunity against allergen exposure, parasitic worm infection, and respiratory virus infection by producing TH2 cytokines. Recent advances in understanding ILC2 biology revealed that ILC2s can be trained by IL-33 or allergic inflammation, are long-lived, and mount memory-like type 2 immune responses to any other allergens afterwards. In contrast, IL-33, together with retinoic acid, induces IL-10-producing immunosuppressive ILC2s. In this review, we discuss how the allergic cytokine milieu and other immune cells direct the generation of trained ILC2s with immunostimulatory or immunosuppressive recall capability in allergic diseases and infections associated with type 2 immunity. The molecular mechanisms of trained immunity by ILCs and the physiological relevance of trained ILC2s are also discussed.
Assuntos
Hipersensibilidade/imunologia , Imunidade Inata , Linfócitos/imunologia , Alarminas/imunologia , Animais , Comunicação Celular/imunologia , Humanos , Mediadores da Inflamação/imunologia , Interleucina-10/imunologia , Interleucina-33/imunologia , Lipídeos/imunologia , Neurônios/imunologia , Viroses/imunologiaRESUMO
TMEM55a (also known as PIP4P2) is an enzyme that dephosphorylates the phosphatidylinositol (PtdIns) PtdIns(4,5)P2 to form PtdIns(5)P in vitro However, the in vivo conversion of the polyphosphoinositide into PtdIns(5)P by the phosphatase has not yet been demonstrated, and the role of TMEM55a remains poorly understood. Here, we found that mouse macrophages (Raw264.7) deficient in TMEM55a showed an increased engulfment of large particles without affecting the phagocytosis of Escherichia coli Transfection of a bacterial phosphatase with similar substrate specificity to TMEM55a, namely IpgD, into Raw264.7 cells inhibited the engulfment of IgG-erythrocytes in a manner dependent on its phosphatase activity. In contrast, cells transfected with PIP4K2a, which catalyzes PtdIns(4,5)P2 production from PtdIns(5)P, increased phagocytosis. Fluorescent TMEM55a transfected into Raw264.7 cells was found to mostly localize to the phagosome. The accumulation of PtdIns(4,5)P2, PtdIns(3,4,5)P3 and F-actin on the phagocytic cup was increased in TMEM55a-deficient cells, as monitored by live-cell imaging. Phagosomal PtdIns(5)P was decreased in the knockdown cells, but the augmentation of phagocytosis in these cells was unaffected by the exogenous addition of PtdIns(5)P. Taken together, these results suggest that TMEM55a negatively regulates the phagocytosis of large particles by reducing phagosomal PtdIns(4,5)P2 accumulation during cup formation.
Assuntos
Fagocitose/genética , Fagossomos/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatases de Fosfoinositídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas de Transporte Vesicular/metabolismo , Animais , Membrana Celular/metabolismo , Macrófagos/metabolismo , Camundongos , Fagossomos/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositóis/metabolismo , Ligação Proteica , Células RAW 264.7RESUMO
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
Macrophages endocytose modified low-density lipoproteins (LDL) vigorously via scavenger receptor A (SR-A) to become foam cells. In the present study, we found that Sac1, a member of the Sac family of phosphoinositide phosphatases, increases the protein level of SR-A and upregulates foam cell formation. Mouse macrophages (RAW264.7) were transfected with short hairpin RNAs (shRNAs) against Sac1. Sac1 knockdown decreased cell surface SR-A levels and impaired acetylated LDL-induced foam cell formation. Transfection of Sac1-knockdown cells with shRNA-resistant flag-Sac1 effectively rescued the expression of SR-A. Glycosylation of SR-A was largely attenuated by Sac1 knockdown, but neither mRNA expression nor protein degradation of SR-A were affected. These results suggest that Sac1 maintains SR-A protein levels by modulating SR-A glycosylation.
Assuntos
Células Espumosas/metabolismo , Proteínas de Membrana/metabolismo , Fosfatases de Fosfoinositídeos/metabolismo , Receptores Depuradores Classe A/metabolismo , Animais , Lipoproteínas LDL/metabolismo , Proteínas de Membrana/genética , Camundongos , Fosfatases de Fosfoinositídeos/genética , Células RAW 264.7 , RNA Mensageiro , RNA Interferente Pequeno , Receptores Depuradores Classe A/genéticaRESUMO
The findings of this study suggest that the phosphoinositide phosphatase Sac3 maintains the protein level of scavenger receptor A (SR-A) and regulates foam cell formation. RAW264.7 macrophages were transfected with short hairpin RNAs that target Sac3. The knockdown decreased the level of the cell surface SR-A and suppressed the acetylated low density lipoprotein-induced foam cell formation. The associated regulator of PIKfyve (ArPIKfyve) is a scaffold protein that protects Sac3 from proteasome-dependent degradation. The knockdown of ArPIKfyve decreased Sac3, cell surface SR-A, and foam cell formation. The knockdown of PIKfyve had no effect on SR-A protein levels. These results suggest that the ArPIKfyve-Sac3 complex regulates SR-A protein levels independently of its effect on PIKfyve activity.
Assuntos
Flavoproteínas/metabolismo , Gotículas Lipídicas/metabolismo , Macrófagos/metabolismo , Fosfatases de Fosfoinositídeos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Receptores Depuradores/metabolismo , Animais , Membrana Celular/metabolismo , Flavoproteínas/genética , Técnicas de Silenciamento de Genes/métodos , Humanos , Camundongos , Fosfatases de Fosfoinositídeos/genética , Monoéster Fosfórico Hidrolases/genética , Células RAW 264.7 , Receptores Depuradores Classe A/metabolismoRESUMO
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
Phosphoinositide 5'-phosphatases have been implicated in the regulation of phagocytosis. However, their precise roles in the phagocytic process are poorly understood. We prepared RAW264.7 macrophages deficient in Inpp5e (shInpp5e) to clarify the role of this lipid phosphatase. In the shInpp5e cells, the uptake of solid particles was increased and the rate of phagosome acidification was accelerated. As expected, levels of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 were increased and decreased respectively, on the forming phagocytic cups of these cells. Unexpectedly, the most prominent consequence of the Inpp5e deficiency was the decreased accumulation of PtdIns3P and Rab5 on the phagosome. The expression of a constitutively active form of Rab5b in the shInpp5e cells rescued the PtdIns3P accumulation. Rab20 has been reported to regulate the activity of Rabex5, a guanine nucleotide exchange factor for Rab5. The association of Rab20 with the phagosome was remarkably abrogated in the shInpp5e cells. Over-expression of Rab20 increased phagosomal PtdIns3P accumulation and delayed its elimination. These results suggest that Inpp5e, through functional interactions with Rab20 on the phagosome, activates Rab5, which, in turn, increases PtdIns3P and delays phagosome acidification.
Assuntos
Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Ácidos/metabolismo , Animais , Células Cultivadas , Macrófagos/metabolismo , Camundongos , Fagocitose/genética , Ligação Proteica , TransfecçãoRESUMO
Voltage-sensing phosphatases (VSPs) consist of a voltage-sensor domain and a cytoplasmic region with remarkable sequence similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase. VSPs dephosphorylate the 5' position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] upon voltage depolarization. However, it is unclear whether VSPs also have 3' phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities of Ciona intestinalis VSP (Ci-VSP) and transmembrane phosphatase with tensin homology (TPTE) and PTEN homologous inositol lipid phosphatase (TPIP; one human ortholog of VSP) with radiolabeled PI(3,4,5)P(3). TLC assay showed that the 3' phosphate of PI(3,4,5)P(3) was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] was removed by VSPs. Monitoring of PI(3,4)P(2) levels with the pleckstrin homology (PH) domain from tandem PH domain-containing protein (TAPP1) fused with GFP (PH(TAPP1)-GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5' phosphatase activity of VSP toward PI(3,4,5)P(3). However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P(3) is dephosphorylated at the 5' position, PI(3,4)P(2) is then dephosphorylated at the 3' position. These results suggest that substrate specificity of the VSP changes with membrane potential.
Assuntos
Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia em Camada Fina , Humanos , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/química , Homologia de Sequência de AminoácidosRESUMO
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
While group-2 innate lymphoid cells (ILC2s) are highly proliferative in allergic inflammation, the removal of overactivated ILC2s in allergic diseases has not been investigated. We previously showed that chronic airway allergy induces "exhausted-like" dysfunctional ILC2s expressing T cell immunoreceptor with Ig and ITIM domains (TIGIT). However, the physiological relevance of these cells in chronic allergy remains elusive. To precisely identify and monitor TIGIT+ ILC2s, we generated TIGIT lineage tracer mice. Chronic allergy stably induced TIGIT+ ILC2s, which were highly activated, apoptotic, and were quickly removed from sites of chronic allergy. Transcripts from coding genes were globally suppressed in the cells, possibly due to reduced chromatin accessibility. Cell death in TIGIT+ ILC2s was enhanced by interactions with CD155 expressed on macrophages, whereas genetic ablation of Tigit or blockade by anti-TIGIT antagonistic antibodies promoted ILC2 survival, thereby deteriorating chronic allergic inflammation. Our work demonstrates that TIGIT shifts the fate of ILC2s toward activation-induced cell death, which could present a new therapeutic target for chronic allergies.
Assuntos
Hipersensibilidade , Imunidade Inata , Receptores Imunológicos , Animais , Camundongos , Morte Celular , Inflamação , Linfócitos , Receptores Imunológicos/genéticaRESUMO
The membrane phospholipid phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P(2)] is a critical signal transducer in eukaryotic cells. However, the physiological roles of the type I phosphatidylinositol phosphate kinases (PIPKIs) that synthesize PI(4,5)P(2) are largely unknown. Here, we show that the alpha isozyme of PIPKI (PIPKIalpha) negatively regulates mast cell functions and anaphylactic responses. In vitro, PIPKIalpha-deficient mast cells exhibited increased degranulation and cytokine production after Fcepsilon receptor-I cross-linking. In vivo, PIPKIalpha(-/-) mice displayed enhanced passive cutaneous and systemic anaphylaxis. Filamentous actin was diminished in PIPKIalpha(-/-) mast cells, and enhanced degranulation observed in the absence of PIPKIalpha was also seen in wild-type mast cells treated with latrunculin, a pharmacological inhibitor of actin polymerization. Moreover, the association of FcepsilonRI with lipid rafts and FcepsilonRI-mediated activation of signaling proteins was augmented in PIPKIalpha(-/-) mast cells. Thus, PIPKIalpha is a negative regulator of FcepsilonRI-mediated cellular responses and anaphylaxis, which functions by controlling the actin cytoskeleton and dynamics of FcepsilonRI signaling. Our results indicate that the different PIPKI isoforms might be functionally specialized.
Assuntos
Anafilaxia/metabolismo , Sinalização do Cálcio/fisiologia , Degranulação Celular/fisiologia , Mastócitos/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Actinas/metabolismo , Anafilaxia/genética , Animais , Sinalização do Cálcio/genética , Degranulação Celular/genética , Células Cultivadas , Isoenzimas/genética , Isoenzimas/metabolismo , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Knockout , Antígenos de Histocompatibilidade Menor , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Receptores de IgE/metabolismo , Tiazóis/farmacologiaRESUMO
Acyl-CoA:monoacylglycerol acyltransferase (MGAT) plays a predominant role in the resynthesis of triacylglycerol in the small intestine, but its contribution to triacylglycerol synthesis in other tissues, such as the liver, is not clear. In this study, we identified a novel MGAT gene, which is identical with lysophosphatidylglycerol acyltransferase1 (LPGAT1). Mouse LPGAT1 is expressed in a number of tissues and most highly expressed in the liver. Hepatic LPGAT1 expression in diabetic db/db mice is higher than that in the control db/m mouse, which is consistent with increased hepatic MGAT activity in db/db mouse. To elucidate the role of LPGAT1 gene in lipid metabolism in db/db mice, we constructed an adenovirus of short hairpin RNA (shRNA) targeting LPGAT1 to selectively knockdown LPGAT1 gene expression in the liver. Hepatic MGAT activity and LPGAT1 expression in db/db mice infected with LPGAT1 shRNA adenovirus were significantly lower than those in mice infected with the control virus. Notably, treatment with LPGAT1 shRNA adenovirus caused a marked reduction in serum triacylglycerol and cholesterol levels and a significant increase in hepatic cholesterol level. These findings indicate that LPGAT1, a newly identified MGAT enzyme, plays a significant role in hepatic triacylglycerol synthesis and secretion in db/db mice.
Assuntos
Aciltransferases/metabolismo , Fígado/metabolismo , Triglicerídeos/metabolismo , Aciltransferases/deficiência , Aciltransferases/genética , Adenoviridae/genética , Animais , Células CHO , Cricetinae , Cricetulus , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Sequências Repetidas Invertidas , Metabolismo dos Lipídeos/genética , Fígado/enzimologia , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triglicerídeos/sangueRESUMO
PIKfyve phosphorylates PtdIns(3)P to PtdIns(3, 5)P2. One of the best characterized effector downstream of PtdIns(3, 5)P2 is a lysosomal Ca2+ channel, TRPML1. Although it has been reported that TRPML1 is involved in phagosome-lysosome fusion, the relevance of the Ca2+ channel in phagosome acidification has been denied. In this article, however, we demonstrated that the phagosome acidification was dependent on TRPML1. Based on the classical idea that Fluorescein isothiocyanate (FITC)-fluorescence is highly sensitive to acidic pH, we could estimate the phagosome acidification by time laps imaging. FITC-zymosan fluorescence that was engulfed by macrophages, decreased immediately after the uptake while the extinction of FITC-zymosan fluorescence was delayed in PIKfyve-deficient cells. The acidification arrest was completely rescued in the presence of Ca2+ ionophore A23187. Cells treated with a PIKfyve inhibitor, apilimod, also showed delayed phagosome acidification but were rescued by the overexpression of TRPML1. Additionally, TRPML1 agonist, ML-SA1 was effective to acidify the phagosome in PIKfyve-deficient cells. Another phenotype observed in PIKfyve-deficient cells is vacuole formation. Unexpectedly, enlarged vacuole formation in PIKfyve-deficient cells was not rescued by Ca2+ or over expression of TRPML1. It is likely that the acidification and vacuolation arrest is bifurcating downstream of PIKfyve.
Assuntos
Ácidos/metabolismo , Canais de Cálcio/metabolismo , Endossomos/metabolismo , Fagossomos/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Canais de Potencial de Receptor Transitório/metabolismo , Vacúolos/metabolismo , Animais , Cálcio/metabolismo , Inibidores Enzimáticos/farmacologia , Fluoresceína-5-Isotiocianato/química , Fluorescência , Concentração de Íons de Hidrogênio , Ionóforos/administração & dosagem , Macrolídeos/farmacologia , Camundongos , Inibidores de Fosfoinositídeo-3 Quinase , Células RAW 264.7 , Imagem com Lapso de Tempo , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidoresRESUMO
TMEM55B is first identified as phosphatidylinositol-4,5-P24-phosphatases (PtdIns-4,5-P24-phosphatases) that catalyse dephosphorylation of PtdIns-4,5-P2 to PtdIns-5-P. We demonstrate for the first time that TMEM55B is phosphorylated by Erk/MAPK and that this mechanism participates in regulation of lysosomal clustering. Exposure of RAW264.7 macrophages to various stimuli induces phosphorylation of TMEM55B on Ser76 and Ser169, sites corresponding to consensus sequences (PX(S/T)P) for phosphorylation by MAPK. Of these stimuli, Toll-like receptor ligands most strongly induce TMEM55B phosphorylation, and this is blocked by the MEK1/2 inhibitor U0126. However, phosphorylation does not impact intrinsic phosphatase activity of TMEM55B. TMEM55B has recently been implicated in starvation induced lysosomal translocation. Amino acid starvation induces perinuclear lamp1 clustering in RAW264.7 macrophages, which was attenuated by shRNA-mediated knock-down or CRISPR/Cas9-mediated knock-out of TMEM55B. Cells exposed to U0126 also exhibit attenuated lamp1 clustering. Overexpression of TMEM55B but not TMEM55A notably enhances lamp1 clustering, with TMEM55B mutants (lacking phosphorylation sites or mimicking the phosphorylated state) exhibiting lower and higher efficacies (respectively) than wild-type TMEM55B. Collectively, results suggest that phosphorylation of TMEM55B by Erk/MAPK impacts lysosomal dynamics.
Assuntos
Lisossomos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatases de Fosfoinositídeos/química , Fosfatases de Fosfoinositídeos/metabolismo , Animais , Camundongos , Fosforilação , Células RAW 264.7RESUMO
Phosphoinositides (PIPs) participate in many cellular processes, including cancer progression; however, the metabolic features of PIPs associated with prostate cancer (PCa) are unknown. We investigated PIPs profiles in PTEN-deficient prostate cancer cell lines, human prostate tissues obtained from patients with PCa and benign prostate hyperplasia (BPH) specimens using mass spectrometry. In immortalized normal human prostate PNT1B cells, PTEN deficiency increased phosphatidylinositol tris-phosphate (PIP3) and decreased phosphatidylinositol mono- and bis-phosphate (PIP1 and PIP2), consistent with PTEN's functional role as a PI(3,4,5)P3 3-phosphatase. In human prostate tissues, levels of total (sum of all acyl variants) phosphatidylinositol (PI) and PIP1 in PCa were significantly higher than in BPH, whereas PIP2 and PIP3 contents were significantly lower than in BPH. PCa patients had significantly higher proportion of PI, PIP1, and PIP2 with 0-2 double bonds in acyl chains than BPH patients. In subgroup analyses based on PCa aggressiveness, mean total levels of PI with 0-2 double bonds in acyl chains were significantly higher in patients with pathological stage T3 than in those with pathological stage T2. These data indicate that alteration of PIPs level and the saturation of acyl chains may be associated with the development and aggressiveness of prostate cancer, although it is unknown whether this alteration is causative.
Assuntos
PTEN Fosfo-Hidrolase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Próstata/patologia , Hiperplasia Prostática/patologia , Neoplasias da Próstata/patologia , Acilação , Células Cultivadas , Progressão da Doença , Humanos , Masculino , PTEN Fosfo-Hidrolase/genética , Próstata/metabolismo , Hiperplasia Prostática/metabolismo , Neoplasias da Próstata/metabolismoRESUMO
Hypertrophic cardiomyopathy (HCM) is a common heart disease with a prevalence of 1 in 500 in the general population. Several mutations in genes encoding cardiac proteins have been found in HCM patients, but these changes do not predict occurrence or prognosis and the molecular mechanisms underlying HCM remain largely elusive. Here we show that cardiac expression of vacuolar protein sorting 34 (Vps34) is reduced in a subset of HCM patients. In a mouse model, muscle-specific loss of Vps34 led to HCM-like manifestations and sudden death. Vps34-deficient hearts exhibited abnormal histopathologies, including myofibrillar disarray and aggregates containing αB-crystallin (CryAB). These features result from a block in the ESCRT-mediated proteolysis that normally degrades K63-polyubiquitinated CryAB. CryAB deposition was also found in myocardial specimens from a subset of HCM patients whose hearts showed decreased Vps34. Our results identify disruption of the previously unknown Vps34-CryAB axis as a potentially novel etiology of HCM.