RESUMEN
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.
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Anafilaxia , Fibroblastos , Lisofosfolípidos , Mastocitos , Ratones Noqueados , Comunicación Paracrina , Hidrolasas Diéster Fosfóricas , Receptores del Ácido Lisofosfatídico , Transducción de Señal , Animales , Mastocitos/inmunología , Mastocitos/metabolismo , Anafilaxia/inmunología , Anafilaxia/metabolismo , Ratones , Fibroblastos/metabolismo , Lisofosfolípidos/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Prostaglandina D2/metabolismo , Vesículas Extracelulares/metabolismo , Interleucina-33/metabolismo , Oxidorreductasas Intramoleculares/metabolismo , Oxidorreductasas Intramoleculares/genética , Receptores de Prostaglandina/metabolismo , Receptores de Prostaglandina/genética , Diferenciación Celular , Ratones Endogámicos C57BL , Proteína 1 Similar al Receptor de Interleucina-1 , LipocalinasRESUMEN
Fasting is associated with improved outcomes in cancer. Here, we investigated the impact of fasting on natural killer (NK) cell anti-tumor immunity. Cyclic fasting improved immunity against solid and metastatic tumors in an NK cell-dependent manner. During fasting, NK cells underwent redistribution from peripheral tissues to the bone marrow (BM). In humans, fasting also reduced circulating NK cell numbers. NK cells in the spleen of fasted mice were metabolically rewired by elevated concentrations of fatty acids and glucocorticoids, augmenting fatty acid metabolism via increased expression of the enzyme CPT1A, and Cpt1a deletion impaired NK cell survival and function in this setting. In parallel, redistribution of NK cells to the BM during fasting required the trafficking mediators S1PR5 and CXCR4. These cells were primed by an increased pool of interleukin (IL)-12-expressing BM myeloid cells, which improved IFN-γ production. Our findings identify a link between dietary restriction and optimized innate immune responses, with the potential to enhance immunotherapy strategies.
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Ayuno , Células Asesinas Naturales , Ratones Endogámicos C57BL , Animales , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Ratones , Humanos , Neoplasias/inmunología , Médula Ósea/inmunología , Médula Ósea/metabolismo , Ratones Noqueados , Interferón gamma/metabolismo , Interferón gamma/inmunología , Bazo/inmunología , Bazo/metabolismo , Inmunidad Innata/inmunología , Interleucina-12/metabolismo , Interleucina-12/inmunología , Receptores CXCR4/metabolismoRESUMEN
Lipid biology continues to emerge as an area of significant therapeutic interest, particularly as the result of an enhanced understanding of the wealth of signaling molecules with diverse physiological properties. This growth in knowledge is epitomized by lysophosphatidic acid (LPA), which functions through interactions with at least six cognate G protein-coupled receptors. Herein, we present three crystal structures of LPA1 in complex with antagonist tool compounds selected and designed through structural and stability analyses. Structural analysis combined with molecular dynamics identified a basis for ligand access to the LPA1 binding pocket from the extracellular space contrasting with the proposed access for the sphingosine 1-phosphate receptor. Characteristics of the LPA1 binding pocket raise the possibility of promiscuous ligand recognition of phosphorylated endocannabinoids. Cell-based assays confirmed this hypothesis, linking the distinct receptor systems through metabolically related ligands with potential functional and therapeutic implications for treatment of disease.
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Cristalografía por Rayos X , Sitios de Unión , Cromatografía en Gel , Humanos , Ligandos , Modelos Moleculares , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Receptores de Lisoesfingolípidos/química , Bibliotecas de Moléculas PequeñasRESUMEN
The lymph node periphery is an important site for many immunological functions, from pathogen containment to the differentiation of helper T cells, yet the cues that position cells in this region are largely undefined. Here, through the use of a reporter for the signaling lipid S1P (sphingosine 1-phosphate), we found that cells sensed higher concentrations of S1P in the medullary cords than in the T cell zone and that the S1P transporter SPNS2 on lymphatic endothelial cells generated this gradient. Natural killer (NK) cells are located at the periphery of the lymph node, predominantly in the medulla, and we found that expression of SPNS2, expression of the S1P receptor S1PR5 on NK cells, and expression of the chemokine receptor CXCR4 were all required for NK cell localization during homeostasis and rapid production of interferon-γ by NK cells after challenge. Our findings elucidate the spatial cues for NK cell organization and reveal a previously unknown role for S1P in positioning cells within the medulla.
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Proteínas de Transporte de Anión/metabolismo , Células Endoteliales/inmunología , Células Asesinas Naturales/inmunología , Ganglios Linfáticos/inmunología , Lisofosfolípidos/metabolismo , Receptores CXCR4/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/análogos & derivados , Animales , Proteínas de Transporte de Anión/genética , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Quimiotaxis , Homeostasis , Interferón gamma/metabolismo , Activación de Linfocitos/genética , Lisofosfolípidos/química , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CXCR4/genética , Receptores de Lisoesfingolípidos/genética , Transducción de Señal , Esfingosina/química , Esfingosina/metabolismo , Linfocitos T Colaboradores-Inductores/fisiologíaRESUMEN
In this Article, the top label in Fig. 5d should read 'DISH 3/16' instead of 'DISH 3/17'. This error has been corrected online.
RESUMEN
RATIONALE: Myocardial infarction (MI) is one of the most dangerous adverse cardiovascular events. Our previous study found that lysophosphatidic acid (LPA) is increased in human peripheral blood after MI, and LPA has a protective effect on the survival and proliferation of various cell types. However, the role of LPA and its receptors in MI is less understood. OBJECTIVES: To study the unknown role of LPA and its receptors in heart during MI. METHODS AND RESULTS: In this study, we found that mice also had elevated LPA level in peripheral blood, as well as increased cardiac expression of its receptor LPA2 in the early stages after MI. With adult and neonate MI models in global Lpar2 knockout (Lpar2-KO) mice, we found Lpar2 deficiency increased vascular leak leading to disruption of its homeostasis, so as to impaired heart function and increased early mortality. Histological examination revealed larger scar size, increased fibrosis, and reduced vascular density in the heart of Lpar2-KO mice. Furthermore, Lpar2-KO also attenuated blood flow recovery after femoral artery ligation with decreased vascular density in gastrocnemius. Our study revealed that Lpar2 was mainly expressed and altered in cardiac endothelial cells during MI, and use of endothelial-specific Lpar2 knockout mice phenocopied the global knockout mice. Additionally, adenovirus-Lpar2 and pharmacologically activated LPA2 significantly improved heart function, reduced scar size, increased vascular formation, and alleviated early mortality by maintaining vascular homeostasis owing to protecting vessels from leakage. Mechanistic studies demonstrated that LPA-LPA2 signaling could promote endothelial cell proliferation through PI3K-Akt/PLC-Raf1-Erk pathway and enhanced endothelial cell tube formation via PKD1-CD36 signaling. CONCLUSIONS: Our results indicate that endothelial LPA-LPA2 signaling promotes angiogenesis and maintains vascular homeostasis, which is vital for restoring blood flow and repairing tissue function in ischemic injuries. Targeting LPA-LPA2 signal might have clinical therapeutic potential to protect the heart from ischemic injury.
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Infarto del Miocardio , Receptores del Ácido Lisofosfatídico , Animales , Cicatriz , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Homeostasis , Humanos , Lisofosfolípidos , Ratones , Ratones Noqueados , Infarto del Miocardio/genética , Fosfatidilinositol 3-Quinasas , Receptores del Ácido Lisofosfatídico/genética , Receptores del Ácido Lisofosfatídico/metabolismo , Remodelación VentricularRESUMEN
Plasticity related gene-1 (PRG-1) is a brain-specific membrane protein related to lipid phosphate phosphatases, which acts in the hippocampus specifically at the excitatory synapse terminating on glutamatergic neurons. Deletion of prg-1 in mice leads to epileptic seizures and augmentation of EPSCs, but not IPSCs. In utero electroporation of PRG-1 into deficient animals revealed that PRG-1 modulates excitation at the synaptic junction. Mutation of the extracellular domain of PRG-1 crucial for its interaction with lysophosphatidic acid (LPA) abolished the ability to prevent hyperexcitability. As LPA application in vitro induced hyperexcitability in wild-type but not in LPA(2) receptor-deficient animals, and uptake of phospholipids is reduced in PRG-1-deficient neurons, we assessed PRG-1/LPA(2) receptor-deficient animals, and found that the pathophysiology observed in the PRG-1-deficient mice was fully reverted. Thus, we propose PRG-1 as an important player in the modulatory control of hippocampal excitability dependent on presynaptic LPA(2) receptor signaling.
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Proteoglicanos/metabolismo , Sinapsis/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Electroencefalografía , Hipocampo/química , Hipocampo/citología , Hipocampo/metabolismo , Lisofosfolípidos/metabolismo , Ratones , Ratones Noqueados , Proteoglicanos/análisis , Proteoglicanos/genética , Receptores AMPA/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de Señal , Proteínas de Transporte Vesicular/análisis , Proteínas de Transporte Vesicular/genéticaRESUMEN
The diversity and complexity of the human brain are widely assumed to be encoded within a constant genome. Somatic gene recombination, which changes germline DNA sequences to increase molecular diversity, could theoretically alter this code but has not been documented in the brain, to our knowledge. Here we describe recombination of the Alzheimer's disease-related gene APP, which encodes amyloid precursor protein, in human neurons, occurring mosaically as thousands of variant 'genomic cDNAs' (gencDNAs). gencDNAs lacked introns and ranged from full-length cDNA copies of expressed, brain-specific RNA splice variants to myriad smaller forms that contained intra-exonic junctions, insertions, deletions, and/or single nucleotide variations. DNA in situ hybridization identified gencDNAs within single neurons that were distinct from wild-type loci and absent from non-neuronal cells. Mechanistic studies supported neuronal 'retro-insertion' of RNA to produce gencDNAs; this process involved transcription, DNA breaks, reverse transcriptase activity, and age. Neurons from individuals with sporadic Alzheimer's disease showed increased gencDNA diversity, including eleven mutations known to be associated with familial Alzheimer's disease that were absent from healthy neurons. Neuronal gene recombination may allow 'recording' of neural activity for selective 'playback' of preferred gene variants whose expression bypasses splicing; this has implications for cellular diversity, learning and memory, plasticity, and diseases of the human brain.
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Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/genética , Variación Genética/genética , Neuronas/citología , Neuronas/patología , Recombinación Genética , Empalme Alternativo/genética , Animales , ADN Complementario/análisis , ADN Complementario/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Exones/genética , Femenino , Humanos , Intrones/genética , Masculino , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Especificidad de Órganos , Mutación Puntual/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Análisis de Secuencia de ADN , Eliminación de Secuencia/genéticaRESUMEN
Down syndrome (DS), trisomy of human chromosome 21 (HSA21), is characterized by lifelong cognitive impairments and the development of the neuropathological hallmarks of Alzheimer's disease (AD). The cellular and molecular modifications responsible for these effects are not understood. Here we performed single-nucleus RNA sequencing (snRNA-seq) employing both short- (Illumina) and long-read (Pacific Biosciences) sequencing technologies on a total of 29 DS and non-DS control prefrontal cortex samples. In DS, the ratio of inhibitory-to-excitatory neurons was significantly increased, which was not observed in previous reports examining sporadic AD. DS microglial transcriptomes displayed AD-related aging and activation signatures in advance of AD neuropathology, with increased microglial expression of C1q complement genes (associated with dendritic pruning) and the HSA21 transcription factor gene RUNX1 Long-read sequencing detected vast RNA isoform diversity within and among specific cell types, including numerous sequences that differed between DS and control brains. Notably, over 8,000 genes produced RNAs containing intra-exonic junctions, including amyloid precursor protein (APP) that had previously been associated with somatic gene recombination. These and related results illuminate large-scale cellular and transcriptomic alterations as features of the aging DS brain.
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Envejecimiento/fisiología , Síndrome de Down/metabolismo , Isoformas de ARN/metabolismo , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Cromosomas Humanos Par 21 , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Síndrome de Down/genética , Expresión Génica , Humanos , Microglía , ARN/metabolismo , Análisis de Secuencia de ARN , Regulación hacia ArribaRESUMEN
We have previously reported that, in aortic rings, 18:1 lysophosphatidic acid (LPA) can induce both vasodilation and vasoconstriction depending on the integrity of the endothelium. The predominant molecular species generated in blood serum are poly-unsaturated LPA species, yet the vascular effects of these species are largely unexplored. We aimed to compare the vasoactive effects of seven naturally occurring LPA species in order to elucidate their potential pathophysiological role in vasculopathies. Vascular tone was measured using myography, and thromboxane A2 (TXA2) release was detected by ELISA in C57Bl/6 mouse aortas. The Ca2+-responses to LPA-stimulated primary isolated endothelial cells were measured by Fluo-4 AM imaging. Our results indicate that saturated molecular species of LPA elicit no significant effect on the vascular tone of the aorta. In contrast, all 18 unsaturated carbon-containing (C18) LPAs (18:1, 18:2, 18:3) were effective, with 18:1 LPA being the most potent. However, following inhibition of cyclooxygenase (COX), these LPAs induced similar vasorelaxation, primarily indicating that the vasoconstrictor potency differed among these species. Indeed, C18 LPA evoked a similar Ca2+-signal in endothelial cells, whereas in endothelium-denuded aortas, the constrictor activity increased with the level of unsaturation, correlating with TXA2 release in intact aortas. COX inhibition abolished TXA2 release, and the C18 LPA induced vasoconstriction. In conclusion, polyunsaturated LPA have markedly increased TXA2-releasing and vasoconstrictor capacity, implying potential pathophysiological consequences in vasculopathies.
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Aorta , Lisofosfolípidos , Ratones Endogámicos C57BL , Tromboxano A2 , Vasoconstricción , Animales , Tromboxano A2/metabolismo , Vasoconstricción/efectos de los fármacos , Ratones , Lisofosfolípidos/metabolismo , Lisofosfolípidos/farmacología , Aorta/efectos de los fármacos , Aorta/metabolismo , Masculino , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Calcio/metabolismoRESUMEN
Ponesimod is a sphingosine 1-phosphate (S1P) receptor (S1PR) modulator that was recently approved for treating relapsing forms of multiple sclerosis (MS). Three other FDA-approved S1PR modulators for MS-fingolimod, siponimod, and ozanimod-share peripheral immunological effects via common S1P1 interactions, yet ponesimod may access distinct central nervous system (CNS) mechanisms through its selectivity for the S1P1 receptor. Here, ponesimod was examined for S1PR internalization and binding, human astrocyte signaling and single-cell RNA-seq (scRNA-seq) gene expression, and in vivo using murine cuprizone-mediated demyelination. Studies confirmed ponesimod's selectivity for S1P1 without comparable engagement to the other S1PR subtypes (S1P2,3,4,5 ). Ponesimod showed pharmacological properties of acute agonism followed by chronic functional antagonism of S1P1 . A major locus of S1P1 expression in the CNS is on astrocytes, and scRNA-seq of primary human astrocytes exposed to ponesimod identified a gene ontology relationship of reduced neuroinflammation and reduction in known astrocyte disease-related genes including those of immediate early astrocytes that have been strongly associated with disease progression in MS animal models. Remarkably, ponesimod prevented cuprizone-induced demyelination selectively in the cingulum, but not in the corpus callosum. These data support the CNS activities of ponesimod through S1P1 , including protective, and likely selective, effects against demyelination in a major connection pathway of the brain, the limbic fibers of the cingulum, lesions of which have been associated with several neurologic impairments including MS fatigue.
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Astrocitos/metabolismo , Sistema Nervioso Central/efectos de los fármacos , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismo , Sustancias Protectoras/farmacología , Receptores de Esfingosina-1-Fosfato/metabolismo , Tiazoles/farmacología , Animales , Astrocitos/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Sistema Nervioso Central/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacosRESUMEN
RATIONALE: Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P1 modulation in stroke. OBJECTIVE: To address roles and mechanisms of engagement of endothelial cell S1P1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy. METHODS AND RESULTS: Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P1 in the mouse brain. With an S1P1 signaling reporter, we reveal that abluminal polarization shields S1P1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P1-selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion. CONCLUSIONS: This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P1 agonists.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Arterias Cerebrales/metabolismo , Células Endoteliales/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Ataque Isquémico Transitorio/metabolismo , Accidente Cerebrovascular Isquémico/metabolismo , Lisofosfolípidos/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Esfingosina/análogos & derivados , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/fisiopatología , Arterias Cerebrales/efectos de los fármacos , Arterias Cerebrales/patología , Arterias Cerebrales/fisiopatología , Circulación Cerebrovascular , Modelos Animales de Enfermedad , Células Endoteliales/patología , Femenino , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/fisiopatología , Infarto de la Arteria Cerebral Media/prevención & control , Ataque Isquémico Transitorio/patología , Ataque Isquémico Transitorio/fisiopatología , Ataque Isquémico Transitorio/prevención & control , Accidente Cerebrovascular Isquémico/patología , Accidente Cerebrovascular Isquémico/fisiopatología , Accidente Cerebrovascular Isquémico/prevención & control , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Microcirculación , Fármacos Neuroprotectores/farmacología , Transducción de Señal , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/agonistas , Receptores de Esfingosina-1-Fosfato/genética , Grado de Desobstrucción VascularRESUMEN
Fingolimod (FTY720, Gilenya) was the first US Food and Drug Administration-approved oral therapy for relapsing forms of multiple sclerosis (MS). Research on modified fungal metabolites converged with basic science studies that had identified lysophospholipid (LP) sphingosine 1-phosphate (S1P) receptors, providing mechanistic insights on fingolimod while validating LP receptors as drug targets. Mechanism of action (MOA) studies identified receptor-mediated processes involving the immune system and the central nervous system (CNS). These dual actions represent a more general theme for S1P and likely other LP receptor modulators. Fingolimod's direct CNS activities likely contribute to its efficacy in MS, with particular relevance to treating progressive disease stages and forms that involve neurodegeneration. The evolving understanding of fingolimod's MOA has provided strategies for developing next-generation compounds with superior attributes, suggesting new ways to target S1P as well as other LP receptor modulators for novel therapeutics in the CNS and other organ systems.
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Clorhidrato de Fingolimod/farmacología , Clorhidrato de Fingolimod/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Animales , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Humanos , Lisofosfolípidos/metabolismo , Esclerosis Múltiple/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismoRESUMEN
Inflammatory and neuropathic-like components underlie rheumatoid arthritis (RA)-associated pain, and lysophosphatidic acid (LPA) is linked to both joint inflammation in RA patients and to neuropathic pain. Thus, we investigated a role for LPA signalling using the collagen antibody-induced arthritis (CAIA) model. Pain-like behavior during the inflammatory phase and the late, neuropathic-like phase of CAIA was reversed by a neutralizing antibody generated against LPA and by an LPA1/3 receptor inhibitor, but joint inflammation was not affected. Autotaxin, an LPA synthesizing enzyme was upregulated in dorsal root ganglia (DRG) neurons during both CAIA phases, but not in joints or spinal cord. Late-phase pronociceptive neurochemical changes in the DRG were blocked in Lpar1 receptor deficient mice and reversed by LPA neutralization. In vitro and in vivo studies indicated that LPA regulates pain-like behavior via the LPA1 receptor on satellite glia cells (SGCs), which is expressed by both human and mouse SGCs in the DRG. Furthermore, CAIA-induced SGC activity is reversed by phospholipid neutralization and blocked in Lpar1 deficient mice. Our findings suggest that the regulation of CAIA-induced pain-like behavior by LPA signalling is a peripheral event, associated with the DRGs and involving increased pronociceptive activity of SGCs, which in turn act on sensory neurons.
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Artritis Experimental , Neuralgia , Animales , Anticuerpos , Colágeno , Ganglios Espinales , Humanos , Lisofosfolípidos , Ratones , Neuroglía , Células Receptoras SensorialesRESUMEN
S1P and its receptors have been reported to play important roles in the development of renal fibrosis. Although S1P5 has barely been investigated so far, there are indications that it can influence inflammatory and fibrotic processes. Here, we report the role of S1P5 in renal inflammation and fibrosis. Male S1P5 knockout mice and wild-type mice on a C57BL/6J background were fed with an adenine-rich diet for 7 days or 14 days to induce tubulointerstitial fibrosis. The kidneys of untreated mice served as respective controls. Kidney damage, fibrosis, and inflammation in kidney tissues were analyzed by real-time PCR, Western blot, and histological staining. Renal function was assessed by plasma creatinine ELISA. The S1P5 knockout mice had better renal function and showed less kidney damage, less proinflammatory cytokine release, and less fibrosis after 7 days and 14 days of an adenine-rich diet compared to wild-type mice. S1P5 knockout ameliorates tubular damage and tubulointerstitial fibrosis in a model of adenine-induced nephropathy in mice. Thus, targeting S1P5 might be a promising goal for the pharmacological treatment of kidney diseases.
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Adenina , Insuficiencia Renal Crónica , Adenina/efectos adversos , Animales , Fibrosis , Inflamación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Insuficiencia Renal Crónica/patología , Esfingosina/farmacología , Receptores de Esfingosina-1-FosfatoRESUMEN
A new form of somatic gene recombination (SGR) has been identified in the human brain that affects the Alzheimer's disease gene, amyloid precursor protein (APP). SGR occurs when a gene sequence is cut and recombined within a single cell's genomic DNA, generally independent of DNA replication and the cell cycle. The newly identified brain SGR produces genomic complementary DNAs (gencDNAs) lacking introns, which integrate into locations distinct from germline loci. This brief review will present an overview of likely related recombination mechanisms and genomic cDNA-like sequences that implicate evolutionary origins for brain SGR. Similarities and differences exist between brain SGR and VDJ recombination in the immune system, the first identified SGR form that now has a well-defined enzymatic machinery. Both require gene transcription, but brain SGR uses an RNA intermediate and reverse transcriptase (RT) activity, which are characteristics shared with endogenous retrotransposons. The identified gencDNAs have similarities to other cDNA-like sequences existing throughout phylogeny, including intron-less genes and inactive germline processed pseudogenes, with likely overlapping biosynthetic processes. gencDNAs arise somatically in an individual to produce multiple copies; can be functional; appear most frequently within postmitotic cells; have diverse sequences; change with age; and can change with disease state. Normally occurring brain SGR may represent a mechanism for gene optimization and long-term cellular memory, whereas its dysregulation could underlie multiple brain disorders and, potentially, other diseases like cancer. The involvement of RT activity implicates already Food and Drug Administration-approved RT inhibitors as possible near-term interventions for managing SGR-associated diseases and suggest next-generation therapeutics targeting SGR elements.
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Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Encéfalo/metabolismo , Elementos de Nucleótido Esparcido Largo , ADN Polimerasa Dirigida por ARN , Recombinación Genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Humanos , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismoRESUMEN
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.
Asunto(s)
Decidua/crecimiento & desarrollo , Embrión de Mamíferos/fisiología , Lisofosfolípidos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de Señal , Útero/fisiología , Animales , Ciclooxigenasa 2/metabolismo , Desarrollo Embrionario , Femenino , Técnicas de Inactivación de Genes , Factor de Crecimiento Similar a EGF de Unión a Heparina/metabolismo , Ratones , Ratones Noqueados , Receptores del Ácido Lisofosfatídico/deficienciaRESUMEN
LPA1 is one of six known receptors (LPA1-6) for lysophosphatidic acid (LPA). Constitutive Lpar1 null mutant mice have been instrumental in identifying roles for LPA-LPA1 signaling in neurobiological processes, brain development, and behavior, as well as modeling human neurological diseases like neuropathic pain. Constitutive Lpar1 null mutant mice are protected from partial sciatic nerve ligation (PSNL)-induced neuropathic pain, however, the cell types that are functionally responsible for mediating this protective effect are unknown. Here, we report the generation of an Lpar1flox/flox conditional null mutant mouse that allows for cre-mediated conditional deletion, combined with a PSNL pain model. Lpar1flox/flox mice were crossed with cre transgenic lines driven by neural gene promoters for nestin (all neural cells), synapsin (neurons), or P0 (Schwann cells). CD11b-cre transgenic mice were also used to delete Lpar1 in microglia. PSNL-initiated pain responses were reduced following cre-mediated Lpar1 deletion with all three neural promoters as well as the CD11b promoter, supporting involvement of Schwann cells, central and/or peripheral neurons, and microglia in mediating pain. Interestingly, rescue responses were nonidentical, implicating distinct roles for Lpar1-expressing cell types. Our results with a new Lpar1 conditional mouse mutant expand an understanding of LPA1 signaling in the PSNL model of neuropathic pain.
Asunto(s)
Microglía/patología , Neuralgia/patología , Neuronas/patología , Receptores del Ácido Lisofosfatídico/fisiología , Células de Schwann/patología , Nervio Ciático/cirugía , Animales , Femenino , Marcación de Gen , Ligadura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/metabolismo , Neuralgia/etiología , Neuralgia/metabolismo , Neuronas/metabolismo , Células de Schwann/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that acts through its six cognate G protein-coupled receptors. As a family, lysophospholipids have already produced medicines (e.g., sphingosine 1-phosphate) as is being pursued for LPA through the use of specific antibodies that reduce ligand availability. METHODS: The binding properties of a commercially available, reportedly specific, monoclonal LPA antibody named 504B3 that is related to the clinical candidate Lpathomab/LT3015 were reexamined using a free solution assay (FSA) measured in a compensated interferometric reader (CIR). RESULTS: Measurement of 504B3 binding properties with an FSA-CIR approach revealed similar binding affinities for 504B3 against LPA as well as the non-LPA lipids, phosphatidic acid (PA) and lysophosphatidylcholine (LPC). CONCLUSIONS: Antibody binding specificity and sensitivity, particularly involving lipid ligands, can be assessed in solution and without labels using FSA-CIR. These findings could affect interpretations of both current and past basic and clinical studies employing 504B3 and related anti-LPA antibodies.