RESUMO
Exposure of bone-marrow-derived dendritic cells (BMDC) to high-dose ultrapure lipopolysaccharide for 24 hr (LPS-primed BMDC) enhances their potency in preventing inter-photoreceptor retinoid binding protein: complete Freund's adjuvant-induced experimental autoimmune uveoretinitis (EAU). LPS-primed BMDC are refractory to further exposure to LPS (= endotoxin tolerance), evidenced here by decreased phosphorylation of TANK-binding kinase 1, interferon regulatory factor 3 (IRF3), c-Jun N-terminal kinase and p38 mitogen-activated protein kinase as well as impaired nuclear translocation of nuclear factor κB (NF-κB) and IRF3, resulting in reduced tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-12 and interferon-ß secretion. LPS-primed BMDC also show reduced surface expression of Toll-like receptor-4 and up-regulation of CD14, followed by increased apoptosis, mediated via nuclear factor of activated T cells (NFATc)-2 signalling. LPS-primed BMDC are not only homotolerant to LPS but are heterotolerant to alternative pathogen-associated molecular pattern ligands, such as mycobacterial protein extract (Mycobacterium tuberculosis). Specifically, while M. tuberculosis protein extract induces secretion of IL-1ß, TNF-α and IL-6 in unprimed BMDC, LPS-primed BMDC fail to secrete these cytokines in response to M. tuberculosis. We propose that LPS priming of BMDC, by exposure to high doses of LPS for 24 hr, stabilizes their tolerogenicity rather than promoting immunogenicity, and does so by multiple mechanisms, namely (i) generation of tolerogenic apoptotic BMDC through CD14:NFATc signalling; (ii) reduction of NF-κB and IRF3 signalling and downstream pro-inflammatory cytokine production; and (iii) blockade of inflammasome activation.
Assuntos
Doenças Autoimunes/imunologia , Células da Medula Óssea/imunologia , Células Dendríticas/imunologia , Fator Regulador 3 de Interferon/metabolismo , Fatores de Transcrição NFATC/metabolismo , Retinite/imunologia , Uveíte/imunologia , Animais , Células Cultivadas , Citocinas/metabolismo , Tolerância Imunológica , Mediadores da Inflamação/metabolismo , Fator Regulador 3 de Interferon/genética , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/genética , Transdução de Sinais , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Although many pathogenic copy number variations (CNVs) are associated with neuropsychiatric diseases, few of them have been functionally characterised. Here we report multiple schizophrenia cases with CNV abnormalities specific to unc-51-like kinase 4 (ULK4), a serine/threonine kinase gene. Deletions spanning exons 21-34 of ULK4 were present in 4 out of 3391 schizophrenia patients from the International Schizophrenia Consortium, but absent in 3181 controls. Deletions removing exons 33 and 34 of the large splice variant of ULK4 also were enriched in Icelandic schizophrenia and bipolar patients compared with 98,022 controls (Pâ=â0.0007 for schizophrenia plus bipolar disorder). Combining the two cohorts gives a P-value less than 0.0001 for schizophrenia, or for schizophrenia plus bipolar disorder. The expression of ULK4 is neuron-specific and developmentally regulated. ULK4 modulates multiple signalling pathways that include ERK, p38, PKC and JNK, which are involved in stress responses and implicated in schizophrenia. Knockdown of ULK4 disrupts the composition of microtubules and compromises neuritogenesis and cell motility. Targeted Ulk4 deletion causes corpus callosum agenesis in mice. Our findings indicate that ULK4 is a rare susceptibility gene for schizophrenia.
Assuntos
Variações do Número de Cópias de DNA/genética , Neuritos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Esquizofrenia/genética , Animais , Movimento Celular/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Neuritos/patologia , Neurônios/metabolismo , Neurônios/patologia , Esquizofrenia/etiologia , Esquizofrenia/patologia , Deleção de SequênciaRESUMO
Restoration of immunological tolerance to self antigens has been a major drive in understanding the mechanisms of, and developing new treatments for, autoimmune and autoinflammatory disease. Sessile dendritic cells (DC) are considered the main instruments underpinning immunological tolerance particularly the CD205+ (DEC205+) cDC1 subset in contrast to DCIR2+ cDC2 which mediate immunogenicity. Targeting DC using autoantigen peptide-antibody fusion proteins has been a well explored methodology for inducing tolerance. Here we show that subcutaneous (s.c.) inoculation of hen-egg lysozyme (HEL)-DEC205 Ig fusion prevents the development of spontaneous uveoretinitis (experimental autoimmune uveoretinitis, EAU) in a transgenic mouse model generated by crossing interphotoreceptor retinol binding protein (IRBP)-HEL (sTg HEL) with HEL specific TCR (sTg TCR) mice. Prolonged suppression of EAU required injections of HEL-DEC205 Ig once weekly, reflecting the half life of s.c. DC. Interestingly, HEL-DCIR2 Ig also had a suppressive effect on development of EAU but less so than DEC205 Ig while it had minimal effect on preventing the retinal atrophy associated with EAU. In addition, HEL-DEC205 Ig was only effective when administered s.c. rather than systemically and had no effect on EAU induced by adoptive transfer of HEL-activated T cells. These data demonstrate the importance of systemic (lymph node) rather than local (eye) antigen presentation in the development of EAU as well as suggest a potential therapeutic approach to controlling sight-threatening immune-mediated uveitis provided relevant antigen(s) can be identified.
Assuntos
Anticorpos , Autoantígenos , Animais , Camundongos , Transferência Adotiva , Células Dendríticas , Receptores de Antígenos de Linfócitos TRESUMO
The mechanisms that co-ordinate centrosome maturation and the migration of human cells remain elusive. Protein phosphatase 4 (Ppp4) is a ubiquitous protein serine/threonine phosphatase in eukaryotes that is enriched at centrosomes. HEK293 cells cultures depleted to 30% Ppp4c levels by lentivirus-delivered stable gene silencing were delayed in mitosis at the prometaphase/metaphase boundary and displayed cells with aberrant chromosome organisation and microtubules unconnected to the centrosomes. The levels of alpha- and gamma-tubulin and aurora A were decreased; in mitotic cells, the cytological localisations of polo-like kinase 1, alpha- and gamma-tubulin and aurora A were aberrant and the phosphorylation of Aurora A-Thr 288 was decreased. The novel localisation of endogenous Ppp4 regulatory subunit, R3A, to centrosomes in human mitotic cells suggests that a Ppp4c-R2-R3 trimeric complex mediates centrosome maturation. We demonstrate for the first time that human cells depleted to 30% Ppp4c showed severely decreased migration and exhibit decreased levels of both total beta-actin and filamentous actin in cell extensions, filopodia and lamellopodia-like structures. Our studies show that Ppp4c is required for the organisation of the actin cytoskeleton at the leading edge of human cells during migration. We also demonstrate that the active forms of the RhoGTPases, Rac1 and Cdc42, are substantially decreased in the presence and absence of growth factor in Ppp4c depleted cells, implicating Ppp4c in the regulation of these GTPases. The results suggest that Ppp4c-R2-R3 complexes may co-ordinate centrosome maturation and cell migration via regulation of RhoGTPases and that Ppp4 may be a useful anticancer target.
Assuntos
Movimento Celular , Centrossomo/enzimologia , Fosfoproteínas Fosfatases/deficiência , Proteínas rho de Ligação ao GTP/metabolismo , Aurora Quinases , Caspases/metabolismo , Contagem de Células , Morte Celular , Linhagem Celular , Forma Celular , Centríolos/enzimologia , Humanos , Lentivirus/metabolismo , Mitose , Modelos Biológicos , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Quaternária de Proteína , Transporte Proteico , Tubulina (Proteína)/metabolismoRESUMO
The uncharacterized Saccharomyces cerevisiae open reading frame (ORF) YIL064w is predicted to encode a cytoplasmic 28 kDa protein, recognized by sequence similarity as a putative S-adenosyl-L-methionine methyltransferase. A micro-scale screening performed in our laboratory with the EUROSCARF S. cerevisiae BY4741 deletion mutant collection identified YIL064w deletion as negatively affecting secretory production of reporter alpha-amylase. The work presented here corroborates the later observations of the yil064w mutant in a larger-scale assay and shows that Yil064p is necessary for the efficient secretory production of two reporter proteins, murine alpha-amylase and fungal polygalacturonase. Further, we analysed endocytosis in the yil064w mutant strain and observed defects at both very early and later stages of endocytic transport in cells in the late logarithmic phase. The defects at very early stages may decisively account for the low transfection (DNA uptake by endocytosis) efficiency that we also observed in the yil064w mutant. These are the first in vivo data reporting a functional role for the protein encoded by ORF YIL064w and identify Yil064p, named here secretion and early endocytosis 1 protein (See1p), as a novel component of intracellular transport.
Assuntos
Endocitose , Deleção de Genes , Metiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Regulação da Expressão Gênica , Metiltransferases/genética , Camundongos , Poligalacturonase/genética , Poligalacturonase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Transfecção , Proteínas de Transporte Vesicular/genética , alfa-Amilases/genética , alfa-Amilases/metabolismoRESUMO
Dendritic cells (DC) are the major antigen-presenting cells bridging innate and adaptive immunity, a function they perform by converting quiescent DC to active, mature DC with the capacity to activate naïve T cells. They do this by migrating from the tissues to the T cell area of the secondary lymphoid tissues. Here, we demonstrate that myeloid cell-specific genetic deletion of PTP1B (LysM PTP1B) leads to defects in lipopolysaccharide-driven bone marrow-derived DC (BMDC) activation associated with increased levels of phosphorylated Stat3. We show that myeloid cell-specific PTP1B deletion also causes decreased migratory capacity of epidermal DC, as well as reduced CCR7 expression and chemotaxis to CCL19 by BMDC. PTP1B deficiency in BMDC also impairs their migration in vivo. Further, immature LysM PTP1B BMDC display fewer podosomes, increased levels of phosphorylated Src at tyrosine 527, and loss of Src localization to podosome puncta. In co-culture with T cells, LysM PTP1B BMDC establish fewer and shorter contacts than control BMDC. Finally, LysM PTP1B BMDC fail to present antigen to T cells as efficiently as control BMDC. These data provide first evidence for a key regulatory role for PTP1B in mediating a central DC function of initiating adaptive immune responses in response to innate immune cell activation.
Assuntos
Células Dendríticas/imunologia , Ativação Linfocitária , Podossomos/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/fisiologia , Linfócitos T/imunologia , Animais , Células da Medula Óssea , Diferenciação Celular , Movimento Celular/fisiologia , Células Cultivadas , Quimiocina CCL19/metabolismo , Técnicas de Cocultura , Feminino , Camundongos , Camundongos Knockout , Células Mieloides/enzimologia , Coativador 1 de Receptor Nuclear/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Receptores CCR7/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
Significance: Skin wounds cause great distress and are a huge economic burden, particularly with an increasingly aging population that heals poorly. There is an urgent need for better therapies that improve repair. Intracellular signaling pathways that regulate wound repair are activated by growth factors, hormones, and cytokines released at the wound. In addition, endogenous electric fields (EFs) are generated by epithelia in response to injury and are an important cue that coordinates cell behavior at wounds. Electrical stimulation (ES), therefore, holds the potential to be effective therapeutically in treating wounds. Recent Advances: ES of wounds is an old idea based on observations of the natural occurrence of EF at wound sites. However, it is now receiving increasing attention, because (1) the underpinning mechanisms are being clarified; (2) devices that measure skin wound currents are in place; and (3) medical devices that apply EF to poorly healing wounds are in clinical use with promising results. Critical Issues: Several signaling proteins transduce the EF influence to cells. However, a bigger picture of the EF-proteome is needed in order to understand this complex process and target it in a controlled manner. Future Directions: Dissecting the signaling pathways driving electrical wound healing will allow further identification of key molecular switches that control the cellular response to EFs. These findings herald the development of a new concept, the use of hydrogel electrodes impregnated with small molecules that target signaling pathways to explore the potential of dual electric-pharmacological therapies to repair wounds.
RESUMO
Protein tyrosine phosphatase-1B (PTP1B) negatively regulates insulin and leptin signaling, rendering it an attractive drug target for treatment of obesity-induced insulin resistance. However, some studies suggest caution when targeting macrophage PTP1B, due to its potential anti-inflammatory role. We assessed the role of macrophage PTP1B in inflammation and whole-body metabolism using myeloid-cell (LysM) PTP1B knockout mice (LysM PTP1B). LysM PTP1B mice were protected against lipopolysaccharide (LPS)-induced endotoxemia and hepatic damage associated with decreased proinflammatory cytokine secretion in vivo. In vitro, LPS-treated LysM PTP1B bone marrow-derived macrophages (BMDMs) displayed increased interleukin (IL)-10 mRNA expression, with a concomitant decrease in TNF-α mRNA levels. These anti-inflammatory effects were associated with increased LPS- and IL-10-induced STAT3 phosphorylation in LysM PTP1B BMDMs. Chronic inflammation induced by high-fat (HF) feeding led to equally beneficial effects of macrophage PTP1B deficiency; LysM PTP1B mice exhibited improved glucose and insulin tolerance, protection against LPS-induced hyperinsulinemia, decreased macrophage infiltration into adipose tissue, and decreased liver damage. HF-fed LysM PTP1B mice had increased basal and LPS-induced IL-10 levels, associated with elevated STAT3 phosphorylation in splenic cells, IL-10 mRNA expression, and expansion of cells expressing myeloid markers. These increased IL-10 levels negatively correlated with circulating insulin and alanine transferase levels. Our studies implicate myeloid PTP1B in negative regulation of STAT3/IL-10-mediated signaling, highlighting its inhibition as a potential anti-inflammatory and antidiabetic target in obesity.
Assuntos
Gorduras na Dieta/efeitos adversos , Hiperinsulinismo/induzido quimicamente , Inflamação/induzido quimicamente , Lipopolissacarídeos/toxicidade , Células Mieloides/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/patologia , Animais , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas , Endotoxemia/induzido quimicamente , Regulação Enzimológica da Expressão Gênica/fisiologia , Glucose/metabolismo , Homeostase , Inflamação/patologia , Interleucina-10/genética , Interleucina-10/metabolismo , Janus Quinases/genética , Janus Quinases/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Células Mieloides/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Baço/citologia , Baço/metabolismoRESUMO
Since the plasticity and the potential for re-programming cells has become widely accepted, there has been great interest in cell-based therapies. These are being applied to a range of diseases, not least ocular diseases, where it is assumed that there is a reduced risk of immune rejection although this may be more perceived than real. There are two broad classes of cell-based therapies: those aimed at restoring structure and function of specific tissues and cells; and those directed towards restoring immunological homeostasis by controlling the damaging effects of inflammatory disease. Stem cells of all types represent the first group and prototypically have been used with the aim of regenerating failing cells. In contrast, immune cells have been suggested as potential modulators of inflammation. However, there is functional overlap in these two applications, with some types of stem cells, such as mesenchymal stem cells, demonstrating a potent immunomodulatory effect. This review summarises recent information on cell based therapies for ocular disease, with special emphasis on ocular inflammatory disease, and explores current uses, potential and limitations.
Assuntos
Oftalmopatias/terapia , Inflamação/terapia , Transplante de Células-Tronco , Humanos , Transplante de Células-Tronco/métodos , Uveíte/terapiaRESUMO
Many pharmaceuticals used to treat cancer target the cell cycle or mitotic spindle dynamics, such as the anti-tumor drug, paclitaxel, which stabilizes microtubules. Here we show that, in cells arrested in mitosis with the spindle toxins, nocodazole, or paclitaxel, the endogenous protein phosphatase 4 (Ppp4) complex Ppp4c-R2-R3A is phosphorylated on its regulatory (R) subunits, and its activity is inhibited. The phosphorylations are blocked by roscovitine, indicating that they may be mediated by Cdk1-cyclin B. Endogenous Ppp4c is enriched at the centrosomes in the absence and presence of paclitaxel, nocodazole, or roscovitine, and the activity of endogenous Ppp4c-R2-R3A is inhibited from G 1/S to the G 2/M phase of the cell cycle. Endogenous γ-tubulin and its associated protein, γ-tubulin complex protein 2, both of which are essential for nucleation of microtubules at centrosomes, interact with the Ppp4 complex. Recombinant γ-tubulin can be phosphorylated by Cdk1-cyclin B or Brsk1 and dephosphorylated by Ppp4c-R2-R3A in vitro. The data indicate that Ppp4c-R2-R3A regulates microtubule organization at centrosomes during cell division in response to stress signals such as spindle toxins, paclitaxel, and nocodazole, and that inhibition of the Ppp4 complex may be advantageous for treatment of some cancers.
Assuntos
Proteína Quinase CDC2/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fuso Acromático/metabolismo , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Ciclo Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Nocodazol/farmacologia , Fosfoproteínas Fosfatases/química , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Subunidades Proteicas/metabolismo , Fuso Acromático/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
Electrical gradients are present in many developing and regenerating tissues and around tumours. Mimicking endogenous electric fields in vitro has profound effects on the behaviour of many cell types. Intriguingly, specific cell types migrate cathodally, others anodally and some polarise with their long axis perpendicular to the electric vector. These striking phenomena are likely to have in vivo relevance since one of the determining factors during cancer metastasis is the ability to switch between attractive and repulsive migration in response to extracellular guidance stimuli. We present evidence that the cervical cancer cell line HeLa migrates cathodally in a direct current electric field of physiological intensity, while the strongly metastatic prostate cancer cell line PC-3-M migrates anodally. Notably, genetic disruption of protein serine/threonine phosphatase-1 (PP1) and its regulator NIPP1 decrease directional migration in these cell lines. Conversely, the inducible expression of NIPP1 switched the directional response of HeLa cells from cathodal to slightly anodal in a PP1-dependent manner. Remarkably, induction of a hyperactive PP1/NIPP1 holoenzyme, further shifted directional migration towards the anode. We show that PP1 association with NIPP1 upregulates signalling by the GTPase Cdc42 and demonstrate that pharmacological inhibition of Cdc42 in cells overexpressing NIPP1 recovered cathodal migration. Taken together, we provide the first evidence for regulation of directional cell migration by NIPP1. In addition, we identify PP1/NIPP1 as a novel molecular compass that controls directed cell migration via upregulation of Cdc42 signalling and suggest a way by which PP1/NIPP1 may contribute to the migratory properties of cancer cells.
Assuntos
Movimento Celular , Endorribonucleases/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Centrossomo/efeitos dos fármacos , Centrossomo/metabolismo , Eletricidade , Eletrodos , Genes Neoplásicos , Humanos , Modelos Biológicos , Ligação Proteica/efeitos dos fármacos , Tetraciclina/farmacologia , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Activation of 5'-AMP-activated protein kinase (AMPK) is believed to be the mechanism by which the pharmaceuticals, metformin and phenformin, exert their beneficial effects for treatment of type 2 diabetes. These biguanide drugs elevate 5'-AMP, which allosterically activates AMPK and promotes phosphorylation on Thr172 of AMPK catalytic α subunits. Although kinases phosphorylating this site have been identified, phosphatases that dephosphorylate it are unknown. The aim of this study is to identify protein phosphatase(s) that dephosphorylate AMPKα-Thr172 within cells. Our initial data indicated that members of the protein phosphatase Mg/Mn(2+)-dependent [corrected] (PPM) family and not those of the PPP family of protein serine/threonine phosphatases may be directly or indirectly inhibited by phenformin. Using antibodies raised to individual Ppm phosphatases that facilitated the assessment of their activities, phenformin stimulation of cells was found to decrease the Mg(2+)/Mn(2+)-dependent [corrected] protein serine/threonine phosphatase activity of Ppm1E and Ppm1F, but not that attributable to other PPM family members, including Ppm1A/PP2Cα. Depletion of Ppm1E, but not Ppm1A, using lentiviral-mediated stable gene silencing, increased AMPKα-Thr172 phosphorylation approximately three fold in HEK293 cells. In addition, incubation of cells with low concentrations of phenformin and depletion of Ppm1E increased AMPK phosphorylation synergistically. Ppm1E and the closely related Ppm1F interact weakly with AMPK and assays with lysates of cells stably depleted of Ppm1F suggest [corrected] that this phosphatase contributes to dephosphorylation of AMPK. The data indicate that Ppm1E and probably PpM1F are in cellulo AMPK phosphatases and that Ppm1E is a potential anti-diabetic drug target.