RESUMO
Interaction of mast cells (MCs) with fibroblasts is essential for MC maturation within tissue microenvironments, although the underlying mechanism is incompletely understood. Through a phenotypic screening of >30 mouse lines deficient in lipid-related genes, we found that deletion of the lysophosphatidic acid (LPA) receptor LPA1, like that of the phospholipase PLA2G3, the prostaglandin D2 (PGD2) synthase L-PGDS, or the PGD2 receptor DP1, impairs MC maturation and thereby anaphylaxis. Mechanistically, MC-secreted PLA2G3 acts on extracellular vesicles (EVs) to supply lysophospholipids, which are converted by fibroblast-derived autotaxin (ATX) to LPA. Fibroblast LPA1 then integrates multiple pathways required for MC maturation by facilitating integrin-mediated MC-fibroblast adhesion, IL-33-ST2 signaling, L-PGDS-driven PGD2 generation, and feedforward ATX-LPA1 amplification. Defective MC maturation resulting from PLA2G3 deficiency is restored by supplementation with LPA1 agonists or PLA2G3-modified EVs. Thus, the lipid-orchestrated paracrine circuit involving PLA2G3-driven lysophospholipid, eicosanoid, integrin, and cytokine signaling fine-tunes MC-fibroblast communication, ensuring MC maturation.
Assuntos
Anafilaxia , Fibroblastos , Lisofosfolipídeos , Mastócitos , Camundongos Knockout , Comunicação Parácrina , Diester Fosfórico Hidrolases , Receptores de Ácidos Lisofosfatídicos , Transdução de Sinais , Animais , Mastócitos/imunologia , Mastócitos/metabolismo , Anafilaxia/imunologia , Anafilaxia/metabolismo , Camundongos , Fibroblastos/metabolismo , Lisofosfolipídeos/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Receptores de Ácidos Lisofosfatídicos/genética , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/genética , Prostaglandina D2/metabolismo , Vesículas Extracelulares/metabolismo , Interleucina-33/metabolismo , Oxirredutases Intramoleculares/metabolismo , Oxirredutases Intramoleculares/genética , Receptores de Prostaglandina/metabolismo , Receptores de Prostaglandina/genética , Diferenciação Celular , Camundongos Endogâmicos C57BL , Proteína 1 Semelhante a Receptor de Interleucina-1 , LipocalinasRESUMO
Gaucher disease (GD) is the most common lysosomal storage disease caused by recessive mutations in the degrading enzyme of ß-glucosylceramide (ß-GlcCer). However, it remains unclear how ß-GlcCer causes severe neuronopathic symptoms, which are not fully treated by current therapies. We herein found that ß-GlcCer accumulating in GD activated microglia through macrophage-inducible C-type lectin (Mincle) to induce phagocytosis of living neurons, which exacerbated Gaucher symptoms. This process was augmented by tumor necrosis factor (TNF) secreted from activated microglia that sensitized neurons for phagocytosis. This characteristic pathology was also observed in human neuronopathic GD. Blockade of these pathways in mice with a combination of FDA-approved drugs, minocycline (microglia activation inhibitor) and etanercept (TNF blocker), effectively protected neurons and ameliorated neuronopathic symptoms. In this study, we propose that limiting unrestrained microglia activation using drug repurposing provides a quickly applicable therapeutic option for fatal neuronopathic GD.
Assuntos
Doença de Gaucher , Camundongos , Animais , Humanos , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/genética , Doença de Gaucher/patologia , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Glucosilceramidase/uso terapêutico , Glucosilceramidas/metabolismo , Glucosilceramidas/uso terapêutico , Microglia/metabolismo , Neurônios/metabolismo , FagocitoseRESUMO
BACKGROUND AND AIMS: The common genetic variant rs641738 C>T is a risk factor for metabolic dysfunction-associated steatotic liver disease and metabolic dysfunction-associated steatohepatitis (MASH), including liver fibrosis, and is associated with decreased expression of the phospholipid-remodeling enzyme MBOAT7 (LPIAT1). However, whether restoring MBOAT7 expression in established metabolic dysfunction-associated steatotic liver disease dampens the progression to liver fibrosis and, importantly, the mechanism through which decreased MBOAT7 expression exacerbates MASH fibrosis remain unclear. APPROACH AND RESULTS: We first showed that hepatocyte MBOAT7 restoration in mice with diet-induced steatohepatitis slows the progression to liver fibrosis. Conversely, when hepatocyte-MBOAT7 was silenced in mice with established hepatosteatosis, liver fibrosis but not hepatosteatosis was exacerbated. Mechanistic studies revealed that hepatocyte-MBOAT7 restoration in MASH mice lowered hepatocyte-TAZ (WWTR1), which is known to promote MASH fibrosis. Conversely, hepatocyte-MBOAT7 silencing enhanced TAZ upregulation in MASH. Finally, we discovered that changes in hepatocyte phospholipids due to MBOAT7 loss-of-function promote a cholesterol trafficking pathway that upregulates TAZ and the TAZ-induced profibrotic factor Indian hedgehog (IHH). As evidence for relevance in humans, we found that the livers of individuals with MASH carrying the rs641738-T allele had higher hepatocyte nuclear TAZ, indicating higher TAZ activity and increased IHH mRNA. CONCLUSIONS: This study provides evidence for a novel mechanism linking MBOAT7-LoF to MASH fibrosis, adds new insight into an established genetic locus for MASH, and, given the druggability of hepatocyte TAZ for MASH fibrosis, suggests a personalized medicine approach for subjects at increased risk for MASH fibrosis due to inheritance of variants that lower MBOAT7.
RESUMO
Asthma is a chronic inflammatory disease of the airways characterized by recurrent episodes of airway obstruction, hyperresponsiveness, remodeling, and eosinophilia. Phospholipase A2 s (PLA2 s), which release fatty acids and lysophospholipids from membrane phospholipids, have been implicated in exacerbating asthma by generating pro-asthmatic lipid mediators, but an understanding of the association between individual PLA2 subtypes and asthma is still incomplete. Here, we show that group III-secreted PLA2 (sPLA2 -III) plays an ameliorating, rather than aggravating, role in asthma pathology. In both mouse and human lungs, sPLA2 -III was expressed in bronchial epithelial cells and decreased during the asthmatic response. In an ovalbumin (OVA)-induced asthma model, Pla2g3-/- mice exhibited enhanced airway hyperresponsiveness, eosinophilia, OVA-specific IgE production, and type 2 cytokine expression as compared to Pla2g3+/+ mice. Lipidomics analysis showed that the pulmonary levels of several lysophospholipids, including lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidic acid (LPA), were decreased in OVA-challenged Pla2g3-/- mice relative to Pla2g3+/+ mice. LPA receptor 2 (LPA2 ) agonists suppressed thymic stromal lymphopoietin (TSLP) expression in bronchial epithelial cells and reversed airway hyperresponsiveness and eosinophilia in Pla2g3-/- mice, suggesting that sPLA2 -III negatively regulates allergen-induced asthma at least by producing LPA. Thus, the activation of the sPLA2 -III-LPA pathway may be a new therapeutic target for allergic asthma.
Assuntos
Asma , Eosinofilia , Fosfolipases A2 Secretórias , Hipersensibilidade Respiratória , Humanos , Animais , Camundongos , Lisofosfolipídeos , Fosfolipases A2 Secretórias/genética , CitocinasRESUMO
Functional mass spectrometry imaging (fMSI) is a potent tool for elucidating the spatial distribution of enzyme activities in tissues at high resolution. In this study, we applied fMSI to probe the intricate biosynthesis of phospholipids, which exist as thousands of molecular species in tissues and exhibit a unique distribution specific to cell type. By using deuterium- and 13C-labeled substrates, we visualized the activities of key enzymes involved in phospholipid synthesis, including glycerol 3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferases (LPAAT), lysophospholipid acyltransferases (LPLAT), and long-chain acyl-CoA synthetase (ACSL). Additionally, we were able to visualize a two-step sequential enzyme reaction involving ACSL and LPLAT. This novel approach unveiled significant variations in enzyme activity distribution depending on the type of fatty acids used as substrates. It will also help to reveal the mechanisms underlying the formation of numerous phospholipid species.
Assuntos
Espectrometria de Massas , Fosfolipídeos , Fosfolipídeos/metabolismo , Fosfolipídeos/análise , Animais , CamundongosRESUMO
Activated microglia contribute to many neuroinflammatory diseases in the central nervous system. In this study, we attempted to identify an anti-inflammatory compound that could suppress microglial activation. We performed high-throughput screening with a chemical library developed at our institute. We performed a luciferase assay of nuclear factor-kappa B (NF-κB) reporter stable HT22 cells and identified a compound that was confirmed to inhibit the anti-inflammatory response in BV2 microglial cells. The selected dihydropyridine derivative can suppress the expression response of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor (TNF), as well as NF-κB phosphorylation and nuclear translocation, and reduce the intracellular calcium level. Thus, our identified compound has a potential role in suppressing microglial activation and may contribute to the development of a new therapeutic molecule against neuroinflammatory diseases.
Assuntos
Cálcio , Di-Hidropiridinas , Microglia , NF-kappa B , Animais , Microglia/efeitos dos fármacos , Microglia/metabolismo , Camundongos , NF-kappa B/metabolismo , Cálcio/metabolismo , Linhagem Celular , Di-Hidropiridinas/farmacologia , Fosforilação/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/efeitos dos fármacosRESUMO
Lysophosphatidic acid (LPA) exerts various biological activities through six characterized G protein-coupled receptors (LPA1-6 ). While LPA-LPA1 signaling contributes toward the demyelination and retraction of C-fiber and induces neuropathic pain, the effects of LPA-LPA1 signaling on acute nociceptive pain is uncertain. This study investigated the role of LPA-LPA1 signaling in acute nociceptive pain using the formalin test. The pharmacological inhibition of the LPA-LPA1 axis significantly attenuated formalin-induced nociceptive behavior. The LPA1 mRNA was expressed in satellite glial cells (SGCs) in dorsal root ganglion (DRG) and was particularly abundant in SGCs surrounding large DRG neurons, which express neurofilament 200. Treatment with LPA1/3 receptor (LPA1/3 ) antagonist inhibited the upregulation of glial markers and inflammatory cytokines in DRG following formalin injection. The LPA1/3 antagonist also attenuated phosphorylation of extracellular signal-regulated kinase, especially in SGCs and cyclic AMP response element-binding protein in the dorsal horn following formalin injection. LPA amounts after formalin injection to the footpad were quantified by liquid chromatography/tandem mass spectrometry, and LPA levels were found to be increased in the innervated DRGs. Our results indicate that LPA produced in the innervated DRGs promotes the activation of SGCs through LPA1 , increases the sensitivity of primary neurons, and modulates pain behavior. These results facilitate our understanding of the pathology of acute nociceptive pain and demonstrate the possibility of the LPA1 on SGCs as a novel target for acute pain control.
Assuntos
Isoxazóis/farmacologia , Lisofosfolipídeos/metabolismo , Neuroglia/efeitos dos fármacos , Dor Nociceptiva/prevenção & controle , Propionatos/farmacologia , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Feminino , Gânglios Espinais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/metabolismo , Dor Nociceptiva/etiologia , Dor Nociceptiva/metabolismo , Dor Nociceptiva/patologia , Fosforilação , Transdução de SinaisRESUMO
Our group has reported various derivatives of lysophosphatidylserine (LysoPS) as potent and subtype-selective agonists for G-protein-coupled receptors (GPCRs). However, the ester linkage between the glycerol moiety and fatty acid or fatty acid surrogate is present in all of them. In order to develop these LysoPS analogs as drug candidates, appropriate pharmacokinetic consideration is essential. Here, we found that the ester bond of LysoPS is highly susceptible to metabolic degradation in mouse blood. Accordingly, we examined isosteric replacement of the ester linkage with heteroaromatic rings. The resulting compounds showed excellent retention of potency and receptor subtype selectivity, as well as increased metabolic stability in vitro.
Assuntos
Lisofosfolipídeos , Receptores Acoplados a Proteínas G , Camundongos , Animais , Receptores de Lisofosfolipídeos/agonistas , Receptores de Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/química , Lisofosfolipídeos/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Ácidos Graxos/metabolismo , Glicerol/químicaRESUMO
Autotaxin, encoded by the ENPP2 gene, is a known key element of neuropathic pain; however, its involvement in nociceptive pain processing remains unclear. We explored the associations between postoperative pain intensity, 24-h postoperative opioid dose requirements, and 93 ENNP2-gene single-nucleotide polymorphisms (SNPs) in 362 healthy patients who underwent cosmetic surgery using the dominant, recessive, and genotypic models. Next, we validated the associations between relevant SNPs on the one hand and pain intensity and daily opioid dosages on the other in 89 patients with cancer-related pain. In this validation study, a Bonferroni correction for multiplicity was applied on all relevant SNPs of the ENPP2 gene and their respective models. In the exploratory study, three models of two SNPs (rs7832704 and rs2249015) were significantly associated with postoperative opioid doses, although the postoperative pain intensity was comparable. In the validation study, the three models of the two SNPs were also significantly associated with cancer pain intensity (p < 0.017). Patients with a minor allele homozygosity complained of more severe pain compared with patients with other genotypes when using comparable daily opioid doses. Our findings might suggest that autotaxin is associated with nociceptive pain processing and the regulation of opioid requirements.
Assuntos
Dor do Câncer , Dor Nociceptiva , Humanos , Analgésicos Opioides/efeitos adversos , Medição da Dor , Polimorfismo de Nucleotídeo Único , Dor do Câncer/tratamento farmacológico , Dor do Câncer/genética , Dor Pós-Operatória/etiologia , Dor Pós-Operatória/genéticaRESUMO
The main fatty acids at the sn-1 position of phospholipids (PLs) are saturated or monounsaturated fatty acids such as palmitic acid (C16:0), stearic acid (C18:0), and oleic acid (C18:1) and are constantly replaced, like unsaturated fatty acids at the sn-2 position. However, little is known about the molecular mechanism underlying the replacement of fatty acids at the sn-1 position, i.e., the sn-1 remodeling. Previously, we established a method to evaluate the incorporation of fatty acids into the sn-1 position of lysophospholipids (lyso-PLs). Here, we used this method to identify the enzymes capable of incorporating fatty acids into the sn-1 position of lyso-PLs (sn-1 lysophospholipid acyltransferase [LPLAT]). Screenings using siRNA knockdown and recombinant proteins for 14 LPLATs identified LPLAT7/lysophosphatidylglycerol acyltransferase 1 (LPGAT1) as a candidate. In vitro, we found LPLAT7 mainly incorporated several fatty acids into the sn-1 position of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE), with weak activities toward other lyso-PLs. Interestingly, however, only C18:0-containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were specifically reduced in the LPLAT7-mutant cells and tissues from knockout mice, with a concomitant increase in the level of C16:0- and C18:1-containing PC and PE. Consistent with this, the incorporation of deuterium-labeled C18:0 into PLs dramatically decreased in the mutant cells, while deuterium-labeled C16:0 and C18:1 showed the opposite dynamic. Identifying LPLAT7 as an sn-1 LPLAT facilitates understanding the biological significance of sn-1 fatty acid remodeling of PLs. We also propose to use the new nomenclature, LPLAT7, for LPGAT1 since the newly assigned enzymatic activities are quite different from the LPGAT1s previously reported.
Assuntos
1-Acilglicerofosfocolina O-Aciltransferase , Fosfatidiletanolaminas , Camundongos , Animais , 1-Acilglicerofosfocolina O-Aciltransferase/genética , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Lisofosfatidilcolinas , RNA Interferente Pequeno , Deutério , Lisofosfolipídeos/metabolismo , Ácidos Graxos/metabolismo , Fosfatidilcolinas/metabolismo , Ácidos Esteáricos , Ácido Palmítico/metabolismo , Ácidos Graxos Insaturados , Proteínas Recombinantes , Ácidos Oleicos , Ácidos Graxos MonoinsaturadosRESUMO
Some patients with diabetic kidney disease (DKD) show a fast progression of kidney dysfunction and are known as a "fast decliner" (FD). Therefore, it is critical to understand pathomechanisms specific for fast decline. Here, we performed a comprehensive metabolomic analysis of patients with stage G3 DKD and identified increased urinary lysophosphatidylcholine (LPC) in fast decline. This was confirmed by quantification of urinary LPC using mass spectrometry and identified urinary LPC containing saturated fatty acids palmitic (16:0) and stearic (18:0) acids was increased in FDs. The upsurge in urinary LPC levels was correlated with a decline in estimated glomerular filtration rate after 2.5 years. To clarify a pathogenic role of LPC in FD, we studied an accelerated rat model of DKD and observed an increase in LPC (16:0) and (18:0) levels in the urine and kidney tubulointerstitium as the disease progressed. These findings suggested that local dysregulation of lipid metabolism resulted in excessive accumulation of this LPC species in the kidney. Our in vitro studies also confirmed LPC-mediated lipotoxicity in cultured proximal tubular cells. LPC induced accumulation of lipid droplets via activation of peroxisome proliferator-activated receptor-δ followed by upregulation of the lipid droplet membrane protein perilipin 2 and decreased autophagic flux, thereby inducing organelle stress and subsequent apoptosis. Thus, LPC (16:0) and (18:0) may mediate a fast progression of DKD and may serve as a target for novel therapeutic approaches.
Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Insuficiência Renal , Animais , Diabetes Mellitus/metabolismo , Nefropatias Diabéticas/patologia , Taxa de Filtração Glomerular , Humanos , Rim/patologia , Lisofosfatidilcolinas/metabolismo , RatosRESUMO
BACKGROUND: Among various complications of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), renal complications, namely COVID-19-associated kidney injuries, are related to the mortality of COVID-19. METHODS: In this retrospective cross-sectional study, we measured the sphingolipids and glycerophospholipids, which have been shown to possess potent biological properties, using liquid chromatography-mass spectrometry in 272 urine samples collected longitudinally from 91 COVID-19 subjects and 95 control subjects without infectious diseases, to elucidate the pathogenesis of COVID-19-associated kidney injuries. RESULTS: The urinary levels of C18:0, C18:1, C22:0, and C24:0 ceramides, sphingosine, dihydrosphingosine, phosphatidylcholine, lysophosphatidylcholine, lysophosphatidic acid, and phosphatidylglycerol decreased, while those of phosphatidylserine, lysophosphatidylserine, phosphatidylethanolamine, and lysophosphatidylethanolamine increased in patients with mild COVID-19, especially during the early phase (day 1-3), suggesting that these modulations might reflect the direct effects of infection with SARS-CoV-2. Generally, the urinary levels of sphingomyelin, ceramides, sphingosine, dihydrosphingosine, dihydrosphingosine L-phosphate, phosphatidylcholine, lysophosphatidic acid, phosphatidylserine, lysophosphatidylserine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylglycerol, lysophosphatidylglycerol, phosphatidylinositol, and lysophosphatidylinositol increased, especially in patients with severe COVID-19 during the later phase, suggesting that their modulations might result from kidney injuries accompanying severe COVID-19. CONCLUSIONS: Considering the biological properties of sphingolipids and glycerophospholipids, an understanding of their urinary modulations in COVID-19 will help us to understand the mechanisms causing COVID-19-associated kidney injuries as well as general acute kidney injuries and may prompt researchers to develop laboratory tests for predicting maximum severity and/or novel reagents to suppress the renal complications of COVID-19.
Assuntos
COVID-19 , Esfingolipídeos , Humanos , COVID-19/complicações , Glicerofosfolipídeos , Esfingosina , Fosfatidiletanolaminas , SARS-CoV-2 , Fosfatidilserinas , Estudos Retrospectivos , Estudos Transversais , Ceramidas , Rim , Fosfatidilgliceróis , FosfatidilcolinasRESUMO
Lysophosphatidic acid (LPA) is a potent signaling lipid, and state-dependent alterations in plasma LPA make it a promising diagnostic marker for various diseases. However, plasma LPA concentrations vary widely among reports, even under normal conditions. These variations can be attributed, at least in part, to the artificial metabolism of LPA after blood collection. Here, we aimed to develop an optimized plasma preparation method that reflects the concentration of LPA in the circulating blood. The main features of the devised method were suppression of both LPA production and degradation after blood collection by keeping whole blood samples at low temperature followed by the addition of an autotaxin inhibitor to plasma samples. Using this devised method, the LPA level did not change for 30 min after blood collection. Also, human and mouse LPA levels were found to be much lower than those previously reported, ranging from 40 to 50 nM with minimal variation across the individual. Finally, the increased accuracy made it possible to detect circadian rhythms in the levels of certain LPA species in mouse plasma. These results demonstrate the usefulness of the devised plasma preparation method to determine accurate plasma LPA concentrations.
Assuntos
LisofosfolipídeosRESUMO
Two common conjugated linoleic acids (LAs), cis-9, trans-11 CLA (c9,t11 CLA) and trans-10, cis-12 CLA (t10,c12 CLA), exert various biological activities. However, the effect of CLA on the generation of neurotoxic amyloid-ß (Aß) protein remains unclear. We found that c9,t11 CLA significantly suppressed the generation of Aß in mouse neurons. CLA treatment did not affect the level of ß-site APP-cleaving enzyme 1 (BACE1), a component of active γ-secretase complex presenilin 1 amino-terminal fragment, or Aß protein precursor (APP) in cultured neurons. BACE1 and γ-secretase activities were not directly affected by c9,t11 CLA. Localization of BACE1 and APP in early endosomes increased in neurons treated with c9,t11 CLA; concomitantly, the localization of both proteins was reduced in late endosomes, the predominant site of APP cleavage by BACE1. The level of CLA-containing phosphatidylcholine (CLA-PC) increased dramatically in neurons incubated with CLA. Incorporation of phospholipids containing c9,t11 CLA, but not t10,c12 CLA, into the membrane may affect the localization of some membrane-associated proteins in intracellular membrane compartments. Thus, in neurons treated with c9,t11 CLA, reduced colocalization of APP with BACE1 in late endosomes may decrease APP cleavage by BACE1 and subsequent Aß generation. Our findings suggest that the accumulation of c9,t11 CLA-PC/LPC in neuronal membranes suppresses the production of neurotoxic Aß in neurons.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Ácido Linoleico/farmacologia , Ácidos Linoleicos Conjugados/farmacologia , Neurônios/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/toxicidade , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Células Cultivadas , Suplementos Nutricionais , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Fosfatidilcolinas/metabolismoRESUMO
During pregnancy, up-regulation of heparin-binding (HB-) EGF and cyclooxygenase-2 (COX-2) in the uterine epithelium contributes to decidualization, a series of uterine morphological changes required for placental formation and fetal development. Here, we report a key role for the lipid mediator lysophosphatidic acid (LPA) in decidualization, acting through its G-protein-coupled receptor LPA3 in the uterine epithelium. Knockout of Lpar3 or inhibition of the LPA-producing enzyme autotaxin (ATX) in pregnant mice leads to HB-EGF and COX-2 down-regulation near embryos and attenuates decidual reactions. Conversely, selective pharmacological activation of LPA3 induces decidualization via up-regulation of HB-EGF and COX-2. ATX and its substrate lysophosphatidylcholine can be detected in the uterine epithelium and in pre-implantation-stage embryos, respectively. Our results indicate that ATX-LPA-LPA3 signaling at the embryo-epithelial boundary induces decidualization via the canonical HB-EGF and COX-2 pathways.
Assuntos
Decídua/crescimento & desenvolvimento , Embrião de Mamíferos/fisiologia , Lisofosfolipídeos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais , Útero/fisiologia , Animais , Ciclo-Oxigenase 2/metabolismo , Desenvolvimento Embrionário , Feminino , Técnicas de Inativação de Genes , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Camundongos , Camundongos Knockout , Receptores de Ácidos Lisofosfatídicos/deficiênciaRESUMO
Matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) is an emerging label-free method for mapping the distribution of diverse molecular species in tissue sections. Despite recent progress in MALDI-MSI analyses of lipids, it is still difficult to visualize minor bioactive lipids including lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). Here, we have developed a novel on-tissue derivatization method using Phos-tag, a zinc complex that specifically binds to a phosphate monoester group. MALDI-MSI with Phos-tag derivatization made it possible to image LPA and S1P in the murine brain. Furthermore, we were able to visualize other low-abundance lipids containing phosphate monoester, such as phosphatidic acid and ceramide-1-phosphate. Compared with conventional MALDI-MS, this derivatization produced LPA images with high spatial accuracy discriminating LPA artificially produced during MALDI-MS analysis. In mice with deficiencies in enzymes that degrade LPA and S1P, we observed marked S1P and/or LPA accumulation in specific regions of the brain. Thus, the present study provides a simple and optimal way to reveal the spatial localization of potent bioactive lipid phosphates such as LPA and S1P in tissues.
Assuntos
Lipídeos , Fosfatos , Animais , Camundongos , Piridinas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: Lysophosphatidic acid (LPA) is involved in numerous biological processes, including neurodevelopment, chronic inflammation, and immunologic response in the central nervous system. Autotaxin (ATX) is a secreted enzyme that produces LPA from lysophosphatidylcholine (LPC). Previous studies have demonstrated decreased protein levels of ATX in cerebrospinal fluid (CSF) of patients with major depressive disorder (MDD). Based on those studies, the current study investigated the levels of lysophospholipids species including LPA and related metabolic enzymes, in CSF of patients with MDD and schizophrenia (SCZ). METHODS: The levels of lysophospholipids species and related metabolic enzymes were measured with either liquid chromatography-tandem mass spectrometry or enzyme-linked immunosorbent assay. Japanese patients were diagnosed with DSM-IV-TR. CSF was obtained from age- and sex-matched healthy controls (n = 27) and patients with MDD (n = 26) and SCZ (n = 27). RESULTS: Of all lysophospholipids species, the levels of LPA 22:6 (LPA - docosahexaenoic acid) were significantly lower in patients with MDD and SCZ than in healthy controls. These levels were negatively correlated with several clinical symptomatic scores of MDD, but not those of SCZ. In addition, the levels of LPA 22:6 were significantly correlated with the levels of LPC 22:6 among all 3 groups. On the other hand, the levels of LPA 22:6 were not correlated with ATX activity in patients with MDD and SCZ. CONCLUSION: The lower levels of LPA 22:6 in patients with MDD and SCZ suggest an abnormality of LPA 22:6 metabolism. In addition, several depressive symptoms in patients with MDD were significantly associated with the lower levels of LPA 22:6, suggesting an involvement of LPA 22:6 in the pathophysiology of MDD.
Assuntos
Transtorno Depressivo Maior/líquido cefalorraquidiano , Ácidos Docosa-Hexaenoicos/líquido cefalorraquidiano , Lisofosfolipídeos/líquido cefalorraquidiano , Esquizofrenia/líquido cefalorraquidiano , Adulto , Estudos de Casos e Controles , Cromatografia Líquida , Feminino , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Diester Fosfórico Hidrolases/líquido cefalorraquidianoRESUMO
In vitro mouse spermatogenesis using a classical organ culture method became possible by supplementing basal culture medium with only the product of bovine serum albumin purified by chromatography (AlbuMAX), which indicated that AlbuMAX contained every chemical factor necessary for mouse spermatogenesis. However, since the identity of these factors was unclear, improvements in culture media and our understanding of the nutritional and signal substances required for spermatogenesis were hindered. In the present study, chemically defined media (CDM) without AlbuMAX was used to evaluate each supplementary factor and their combinations for the induction of spermatogenesis. Similar to in vivo conditions, retinoic acid, triiodothyronine (T3 ), and testosterone (T) were needed. Based on differences in spermatogenic competence between AlbuMAX, fetal bovine serum, and adult bovine serum, we identified α-tocopherol, which strongly promoted spermatogenesis when combined with ascorbic acid and glutathione. Differences were also observed in the abilities of lipids extracted from AlbuMAX using two different methods to induce spermatogenesis. This led to the identification of lysophospholipids, particularly lysophosphatidylcholine, lysophosphatidic acid, and lysophosphatidylserine, as important molecules for spermatogenesis. New CDM formulated based on these results induced and promoted spermatogenesis as efficiently as AlbuMAX-containing medium. In vitro spermatogenesis with CDM may provide a unique experimental system for research on spermatogenesis that cannot be performed in in vivo experiments.
Assuntos
Antioxidantes/farmacologia , Lisofosfolipídeos/farmacologia , Técnicas de Cultura de Órgãos/métodos , Espermatogênese , Testículo/citologia , Vitaminas/farmacologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Testículo/efeitos dos fármacos , Testículo/metabolismoRESUMO
RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs) accompanying increased production of inflammatory factors and adaptation of the mitochondrial metabolism to a hyperproliferative state. However, all the drugs in clinical use target pulmonary vascular dilatation, which may not be effective for patients with advanced PAH. OBJECTIVE: We aimed to discover a novel drug for PAH that inhibits PASMC proliferation. METHODS AND RESULTS: We screened 5562 compounds from original library using high-throughput screening system to discover compounds which inhibit proliferation of PASMCs from patients with PAH (PAH-PASMCs). We found that celastramycin, a benzoyl pyrrole-type compound originally found in a bacteria extract, inhibited the proliferation of PAH-PASMCs in a dose-dependent manner with relatively small effects on PASMCs from healthy donors. Then, we made 25 analogs of celastramycin and selected the lead compound, which significantly inhibited cell proliferation of PAH-PASMCs and reduced cytosolic reactive oxygen species levels. Mechanistic analysis demonstrated that celastramycin reduced the protein levels of HIF-1α (hypoxia-inducible factor 1α), which impairs aerobic metabolism, and κB (nuclear factor-κB), which induces proinflammatory signals, in PAH-PASMCs, leading to reduced secretion of inflammatory cytokine. Importantly, celastramycin treatment reduced reactive oxygen species levels in PAH-PASMCs with increased protein levels of Nrf2 (nuclear factor erythroid 2-related factor 2), a master regulator of cellular response against oxidative stress. Furthermore, celastramycin treatment improved mitochondrial energy metabolism with recovered mitochondrial network formation in PAH-PASMCs. Moreover, these celastramycin-mediated effects were regulated by ZFC3H1 (zinc finger C3H1 domain-containing protein), a binding partner of celastramycin. Finally, celastramycin treatment ameliorated pulmonary hypertension in 3 experimental animal models, accompanied by reduced inflammatory changes in the lungs. CONCLUSIONS: These results indicate that celastramycin ameliorates pulmonary hypertension, reducing excessive proliferation of PAH-PASMCs with less inflammation and reactive oxygen species levels, and recovered mitochondrial energy metabolism. Thus, celastramycin is a novel drug for PAH that targets antiproliferative effects on PAH-PASMCs.
Assuntos
Miócitos de Músculo Liso/efeitos dos fármacos , Naftoquinonas/farmacologia , Hipertensão Arterial Pulmonar/tratamento farmacológico , Pirróis/farmacologia , Resorcinóis/farmacologia , Animais , Células Cultivadas , Citocinas/biossíntese , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Metabolismo Energético/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Hipóxia/complicações , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Indóis/toxicidade , Masculino , Metaboloma/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Monocrotalina/toxicidade , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/biossíntese , Naftoquinonas/uso terapêutico , Estresse Oxidativo , Hipertensão Arterial Pulmonar/induzido quimicamente , Artéria Pulmonar/citologia , Pirróis/uso terapêutico , Pirróis/toxicidade , Ratos , Espécies Reativas de Oxigênio/metabolismo , Resorcinóis/uso terapêutico , Fatores de Transcrição/fisiologiaRESUMO
The ATP-binding cassette subfamily G member 2 (ABCG2) transporter is involved in the development of multidrug resistance in cancer patients. Many inhibitors of ABCG2 have been reported to enhance the chemosensitivity of cancer cells. However, none of these inhibitors are being used clinically. The aim of this study was to identify novel ABCG2 inhibitors by high-throughput screening of a chemical library. Among the 5812 compounds in the library, 23 compounds were selected in the first screening, using a fluorescent plate reader-based pheophorbide a (PhA) efflux assay. Thereafter, to validate these compounds, a flow cytometry-based PhA efflux assay was performed and 16 compounds were identified as potential inhibitors. A cytotoxic assay was then performed to assess the effect these 16 compounds had on ABCG2-mediated chemosensitivity. We found that the phenylfurocoumarin derivative (R)-9-(3,4-dimethoxyphenyl)-4-((3,3-dimethyloxiran-2-yl)methoxy)-7H-furo [3,2-g]chromen-7-one (PFC) significantly decreased the IC50 of SN-38 in HCT-116/BCRP colon cancer cells. In addition, PFC stimulated ABCG2-mediated ATP hydrolysis, suggesting that this compound interacts with the substrate-binding site of ABCG2. Furthermore, PFC reversed the resistance to irinotecan without causing toxicity in the ABCG2-overexpressing HCT-116/BCRP cell xenograft mouse model. In conclusion, PFC is a novel inhibitor of ABCG2 and has promise as a therapeutic to overcome ABCG2-mediated MDR, to improve the efficiency of cancer chemotherapy.