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1.
Immunity ; 50(5): 1232-1248.e14, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31027998

RESUMO

Regulatory T cells (Treg cells) are important for preventing autoimmunity and maintaining tissue homeostasis, but whether Treg cells can adopt tissue- or immune-context-specific suppressive mechanisms is unclear. Here, we found that the enzyme hydroxyprostaglandin dehydrogenase (HPGD), which catabolizes prostaglandin E2 (PGE2) into the metabolite 15-keto PGE2, was highly expressed in Treg cells, particularly those in visceral adipose tissue (VAT). Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ)-induced HPGD expression in VAT Treg cells, and consequential Treg-cell-mediated generation of 15-keto PGE2 suppressed conventional T cell activation and proliferation. Conditional deletion of Hpgd in mouse Treg cells resulted in the accumulation of functionally impaired Treg cells specifically in VAT, causing local inflammation and systemic insulin resistance. Consistent with this mechanism, humans with type 2 diabetes showed decreased HPGD expression in Treg cells. These data indicate that HPGD-mediated suppression is a tissue- and context-dependent suppressive mechanism used by Treg cells to maintain adipose tissue homeostasis.


Assuntos
Dinoprostona/análogos & derivados , Dinoprostona/metabolismo , Hidroxiprostaglandina Desidrogenases/metabolismo , Gordura Intra-Abdominal/imunologia , Linfócitos T Reguladores/enzimologia , Linfócitos T Reguladores/imunologia , Células 3T3 , Animais , Linhagem Celular , Diabetes Mellitus Tipo 2/metabolismo , Células HEK293 , Homeostase/imunologia , Humanos , Hidroxiprostaglandina Desidrogenases/genética , Resistência à Insulina/genética , Gordura Intra-Abdominal/citologia , Células Jurkat , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Knockout , Fator de Transcrição STAT5/metabolismo
2.
Blood ; 139(11): 1722-1742, 2022 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-34905596

RESUMO

Platelet ACKR3/CXCR7 surface expression is enhanced and influences prognosis in coronary artery disease (CAD) patients, who exhibit a distinct atherothrombotic platelet lipidome. Current investigation validates the potential of ACKR3/CXCR7 in regulating thromboinflammatory response through its impact on the platelet lipidome. CAD patients with enhanced platelet ACKR3/CXCR7 expression exhibited reduced aggregation. Pharmacological CXCR7 agonist (VUF11207) significantly reduced prothrombotic platelet response in blood from acute coronary syndrome patients ex vivo. CXCR7 agonist administration reduced thrombotic functions and thromboinflammatory plateletleukocyte interactions post-myocardial infarction and arterial injury in vivo. ACKR3/CXCR7 ligation did not affect surface availability of surface receptors, coagulation profile, bleeding time, plasma-dependent thrombin generation (thrombinoscopy), or clot formation (thromboelastography) but counteracted activation-induced phosphatidylserine exposure and procoagulant platelet-assisted thrombin generation. Targeted (micro-UHPLC-ESI-QTrap-MS/MS) and untargeted (UHPLCESI-QTOF-MS/MS) lipidomics analysis revealed that ACKR3/CXCR7 ligation favored generation of antithrombotic lipids (dihomo-γ-linolenic acid [DGLA], 12-hydroxyeicosatrienoic acid [12-HETrE]) over cyclooxygenase-1 (COX-1) or 12-lipoxygenase (12-LOX) metabolized prothrombotic and phospholipase-derived atherogenic lipids in healthy subjects and CAD patients, contrary to antiplatelet therapy. Through 12-HETrE, ACKR3/CXCR7 ligation coordinated with Gαs-coupled prostacyclin receptor to trigger cyclic adenosine monophosphate/protein kinase A-mediated platelet inhibition. ACKR3/CXCR7 ligation reduced generation of lipid agonists and lipid signaling intermediates, which affected calcium mobilization, intracellular signaling, and consequently platelet interaction with physiological matrices and thromboinflammatory secretome. This emphasized its functional dichotomy from prothrombotic CXCR4. Moreover, CXCR7 agonist regulated heparin-induced thrombocytopenia-sera/immunoglobulin G-triggered platelet and neutrophil activation, heparin-induced platelet aggregation, generation of thromboinflammatory lipids, platelet-neutrophil aggregate formation, and thromboinflammatory secretion ex vivo. Therefore, ACKR3/CXCR7 may offer a novel therapeutic strategy in acute/chronic thromboinflammation exaggerated cardiovascular pathologies and CAD.


Assuntos
Receptores CXCR/metabolismo , Trombose , Plaquetas/metabolismo , Humanos , Inflamação/metabolismo , Lipidômica , Lipídeos , Espectrometria de Massas em Tandem , Trombina/metabolismo , Tromboinflamação , Trombose/metabolismo
3.
J Neurosci ; 42(10): 1908-1929, 2022 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-34903569

RESUMO

The precise regulation of blood-brain barrier (BBB) permeability for immune cells and blood-borne substances is essential to maintain brain homeostasis. Sphingosine-1-phosphate (S1P), a lipid signaling molecule enriched in plasma, is known to affect BBB permeability. Previous studies focused on endothelial S1P receptors 1 and 2, reporting a barrier-protective effect of S1P1 and a barrier-disruptive effect of S1P2. Here, we present novel data characterizing the expression, localization, and function of the S1P receptor 4 (S1P4) on primary brain microvascular endothelial cells (BMECs). Hitherto, the receptor was deemed to be exclusively immune cell associated. We detected a robust expression of S1P4 in homeostatic murine BMECs (MBMECs), bovine BMECs (BBMECs), and porcine BMECs (PBMECs) and pinpointed its localization to abluminal endothelial membranes via immunoblotting of fractionated brain endothelial membrane fragments. Apical S1P treatment of BMECs tightened the endothelial barrier in vitro, whereas basolateral S1P treatment led to an increased permeability that correlated with S1P4 downregulation. Likewise, downregulation of S1P4 was observed in mouse brain microvessels (MBMVs) after stroke, a neurologic disease associated with BBB impairment. RNA sequencing and qPCR analysis of BMECs suggested the involvement of S1P4 in endothelial homeostasis and barrier function. Using S1P4 knock-out (KO) mice and S1P4 siRNA as well as pharmacological agonists and antagonists of S1P4 both in vitro and in vivo, we demonstrate an overall barrier-protective function of S1P4. We therefore suggest S1P4 as a novel target regulating BBB permeability and propose its therapeutic potential in CNS diseases associated with BBB dysfunction.SIGNIFICANCE STATEMENT Many neurologic diseases including multiple sclerosis and stroke are associated with blood-brain barrier (BBB) impairment and disturbed brain homeostasis. Sphingosine-1-phosphate receptors (S1PRs) are potent regulators of endothelial permeability and pharmacological S1PR modulators are already in clinical use. However, the precise role of S1P for BBB permeability regulation and the function of receptors other than S1P1 and S1P2 therein are still unclear. Our study shows both barrier-disruptive and barrier-protective effects of S1P at the BBB that depend on receptor polarization. We demonstrate the expression and novel barrier-protective function of S1P4 in brain endothelial cells and pinpoint its localization to abluminal membranes. Our work may contribute to the development of novel specific S1PR modulators for the treatment of neurologic diseases associated with BBB impairment.


Assuntos
Barreira Hematoencefálica , Receptores de Esfingosina-1-Fosfato , Acidente Vascular Cerebral , Animais , Barreira Hematoencefálica/metabolismo , Bovinos , Células Endoteliais/metabolismo , Homeostase , Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/farmacologia , Camundongos , Camundongos Knockout , Permeabilidade , Fenótipo , Receptores de Lisoesfingolipídeo/genética , Esfingosina/metabolismo , Esfingosina/farmacologia , Receptores de Esfingosina-1-Fosfato/metabolismo , Acidente Vascular Cerebral/metabolismo , Suínos
4.
Arterioscler Thromb Vasc Biol ; 42(8): 1023-1036, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35708027

RESUMO

BACKGROUND: Maladapted endothelial cells (ECs) secrete ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase 2; autotaxin)-a lysophospholipase D that generates lysophosphatidic acids (LPAs). ENPP2 derived from the arterial wall promotes atherogenic monocyte adhesion induced by generating LPAs, such as arachidonoyl-LPA (LPA20:4), from oxidized lipoproteins. Here, we aimed to determine the role of endothelial ENPP2 in the production of LPAs and atherosclerosis. METHODS: We quantified atherosclerosis in mice harboring loxP-flanked Enpp2 alleles crossed with Apoe-/- mice expressing tamoxifen-inducible Cre recombinase under the control of the EC-specific bone marrow X kinase promoter after 12 weeks of high-fat diet feeding. RESULTS: A tamoxifen-induced EC-specific Enpp2 knockout decreased atherosclerosis, accumulation of lesional macrophages, monocyte adhesion, and expression of endothelial CXCL (C-X-C motif chemokine ligand) 1 in male and female Apoe-/- mice. In vitro, ENPP2 mediated the mildly oxidized LDL (low-density lipoprotein)-induced expression of CXCL1 in aortic ECs by generating LPA20:4, palmitoyl-LPA (LPA16:0), and oleoyl-LPA (LPA18:1). ENPP2 and its activity were detected on the endothelial surface by confocal imaging. The expression of endothelial Enpp2 established a strong correlation between the plasma levels of LPA16:0, stearoyl-LPA (LPA18:0), and LPA18:1 and plaque size and a strong negative correlation between the LPA levels and ENPP2 activity in the plasma. Moreover, endothelial Enpp2 knockout increased the weight of high-fat diet-fed male Apoe-/- mice. CONCLUSIONS: We demonstrated that the expression of ENPP2 in ECs promotes atherosclerosis and endothelial inflammation in a sex-independent manner. This might be due to the generation of LPA20:4, LPA16:0, and LPA18:1 from mildly oxidized lipoproteins on the endothelial surface.


Assuntos
Aterosclerose , Células Endoteliais , Diester Fosfórico Hidrolases , Animais , Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Feminino , Lisofosfolipídeos , Masculino , Camundongos , Camundongos Knockout para ApoE , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Tamoxifeno
5.
Cell Mol Life Sci ; 78(21-22): 7025-7041, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34626204

RESUMO

Hepatocellular carcinoma (HCC) is one of the most difficult cancer types to treat. Liver cancer is often diagnosed at late stages and therapeutic treatment is frequently accompanied by development of multidrug resistance. This leads to poor outcomes for cancer patients. Understanding the fundamental molecular mechanisms leading to liver cancer development is crucial for developing new therapeutic approaches, which are more efficient in treating cancer. Mice with a liver specific UDP-glucose ceramide glucosyltransferase (UGCG) knockout (KO) show delayed diethylnitrosamine (DEN)-induced liver tumor growth. Accordingly, the rationale for our study was to determine whether UGCG overexpression is sufficient to drive cancer phenotypes in liver cells. We investigated the effect of UGCG overexpression (OE) on normal murine liver (NMuLi) cells. Increased UGCG expression results in decreased mitochondrial respiration and glycolysis, which is reversible by treatment with EtDO-P4, an UGCG inhibitor. Furthermore, tumor markers such as FGF21 and EPCAM are lowered following UGCG OE, which could be related to glucosylceramide (GlcCer) and lactosylceramide (LacCer) accumulation in glycosphingolipid-enriched microdomains (GEMs) and subsequently altered signaling protein phosphorylation. These cellular processes lead to decreased proliferation in NMuLi/UGCG OE cells. Our data show that increased UGCG expression itself does not induce pro-cancerous processes in normal liver cells, which indicates that increased GlcCer expression leads to different outcomes in different cancer types.


Assuntos
Biomarcadores Tumorais/metabolismo , Metabolismo Energético/fisiologia , Glucosilceramidas/metabolismo , Fígado/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Resistência a Múltiplos Medicamentos/fisiologia , Glucosiltransferases/metabolismo , Glicólise/fisiologia , Glicoesfingolipídeos/metabolismo , Neoplasias Hepáticas/metabolismo , Camundongos , Mitocôndrias/metabolismo , Transdução de Sinais/fisiologia
6.
Proc Natl Acad Sci U S A ; 115(43): E10022-E10031, 2018 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-30305425

RESUMO

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that depletes cellular dNTPs in noncycling cells to promote genome stability and to inhibit retroviral and herpes viral replication. In addition to being substrates, cellular nucleotides also allosterically regulate SAMHD1 activity. Recently, it was shown that high expression levels of SAMHD1 are also correlated with significantly worse patient responses to nucleotide analog drugs important for treating a variety of cancers, including acute myeloid leukemia (AML). In this study, we used biochemical, structural, and cellular methods to examine the interactions of various cancer drugs with SAMHD1. We found that both the catalytic and the allosteric sites of SAMHD1 are sensitive to sugar modifications of the nucleotide analogs, with the allosteric site being significantly more restrictive. We crystallized cladribine-TP, clofarabine-TP, fludarabine-TP, vidarabine-TP, cytarabine-TP, and gemcitabine-TP in the catalytic pocket of SAMHD1. We found that all of these drugs are substrates of SAMHD1 and that the efficacy of most of these drugs is affected by SAMHD1 activity. Of the nucleotide analogs tested, only cladribine-TP with a deoxyribose sugar efficiently induced the catalytically active SAMHD1 tetramer. Together, these results establish a detailed framework for understanding the substrate specificity and allosteric activation of SAMHD1 with regard to nucleotide analogs, which can be used to improve current cancer and antiviral therapies.


Assuntos
Sítio Alostérico/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Interações Medicamentosas/fisiologia , Leucemia Mieloide Aguda/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X/métodos , Células HL-60 , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Nucleotídeos/farmacologia , Especificidade por Substrato
7.
Int J Mol Sci ; 22(21)2021 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-34769126

RESUMO

Acute respiratory inflammation, most commonly resulting from bacterial or viral infection, is one of the leading causes of death and disability worldwide. The inflammatory lipid mediator prostaglandin D2 (PGD2) and its rate-limiting enzyme, hematopoietic PGD synthase (hPGDS), are well-known drivers of allergic pulmonary inflammation. Here, we sought to investigate the source and role of hPGDS-derived PGD2 in acute pulmonary inflammation. Murine bronchoalveolar monocytes/macrophages from LPS- but not OVA-induced lung inflammation released significant amounts of PGD2. Accordingly, human monocyte-derived macrophages expressed high basal levels of hPGDS and released significant levels of PGD2 after LPS/IFN-γ, but not IL-4 stimulation. Human peripheral blood monocytes secreted significantly more PGD2 than monocyte-derived macrophages. Using human precision-cut lung slices (PCLS), we observed that LPS/IFN-γ but not IL-4/IL-13 drive PGD2 production in the lung. HPGDS inhibition prevented LPS-induced PGD2 release by human monocyte-derived macrophages and PCLS. As a result of hPGDS inhibition, less TNF-α, IL-6 and IL-10 could be determined in PCLS-conditioned medium. Collectively, this dataset reflects the time-dependent release of PGD2 by human phagocytes, highlights the importance of monocytes and macrophages as PGD2 sources and suggests that hPGDS inhibition might be a potential therapeutic option for acute, non-allergic lung inflammation.


Assuntos
Lesão Pulmonar Aguda/imunologia , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/metabolismo , Macrófagos Alveolares/metabolismo , Monócitos/metabolismo , Prostaglandina D2/metabolismo , Animais , Humanos , Camundongos
8.
J Autoimmun ; 115: 102528, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32768245

RESUMO

Autoimmune diseases are defined by an immune response against a specific autoantigen, driven by antigen-specific T cells or antibodies. While the mechanisms resolving brief episodes of acute inflammation elicited by microbial components or tissue injury are well understood, the mechanisms resolving tissue inflammation in autoimmune diseases are still largely elusive. We have, therefore, addressed the mechanisms of resolution in IgG-mediated autoimmune diseases using a mouse model of the pemphigoid disease "bullous pemphigoid-like epidermolysis bullosa acquisita" (BP-like EBA) as prototypical example. We found that 12/15-LO is induced in skin lesions of BP-like EBA and is predominantly expressed in eosinophils. Dependent on the expression of 12/15-LO, large amounts of proresolving lipid mediators, are biosynthesized in the skin by the point disease peaks. Their production is timely correlated to the gradual reversal of tissue inflammation. Genetic deficiency in Alox15, the gene encoding 12/15-LO, disrupts this process significantly protracting and aggravating disease. This protraction is associated reduced recruitment of regulatory T cells (Tregs) into lesional skin. Intriguingly, Alox15-/- mice also exhibit reduced recruitment of eosinophils into the skin, and the chemotaxis of cultured Alox15-/- eosinophils towards CCL11/eotaxin-1 is compromised. Finally, we demonstrate that 15-lipoxygenase-1, the human homologue of 12/15-LO is induced in granulocytes in lesional skin of patients suffering from a pemphigoid disease. Collectively, our result uncover key mechanisms resolving IgG-mediated skin inflammation. These mechanisms are orchestrated by 12/15-LO expressed in eosinophils promoting the recruitment of eosinophils and Tregs, which in turn inhibit neutrophils.


Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/metabolismo , Eosinófilos/enzimologia , Epidermólise Bolhosa Adquirida/imunologia , Penfigoide Bolhoso/imunologia , Animais , Araquidonato 12-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/análise , Araquidonato 15-Lipoxigenase/genética , Biópsia , Modelos Animais de Doenças , Eosinófilos/imunologia , Epidermólise Bolhosa Adquirida/patologia , Humanos , Imunoglobulina G/metabolismo , Camundongos , Camundongos Knockout , Penfigoide Bolhoso/patologia , Pele/citologia , Pele/imunologia , Pele/patologia , Linfócitos T Reguladores/imunologia
9.
J Allergy Clin Immunol ; 143(6): 2202-2214.e5, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30578872

RESUMO

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) play a key role in the initiation and maintenance of type 2 immune responses. The prostaglandin (PG) D2-chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) receptor axis potently induces cytokine production and ILC2 migration. OBJECTIVE: We set out to examine PG production in human ILC2s and the implications of such endogenous production on ILC2 function. METHODS: The effects of the COX-1/2 inhibitor flurbiprofen, the hematopoietic prostaglandin D2 synthase (HPGDS) inhibitor KMN698, and the CRTH2 antagonist CAY10471 on human ILC2s were determined by assessing receptor and transcription factor expression, cytokine production, and gene expression with flow cytometry, ELISA, and quantitative RT-PCR, respectively. Concentrations of lipid mediators were measured by using liquid chromatography-tandem mass spectrometry and ELISA. RESULTS: We show that ILC2s constitutively express HPGDS and upregulate COX-2 upon IL-2, IL-25, and IL-33 plus thymic stromal lymphopoietin stimulation. Consequently, PGD2 and its metabolites can be detected in ILC2 supernatants. We reveal that endogenously produced PGD2 is essential in cytokine-induced ILC2 activation because blocking of the COX-1/2 or HPGDS enzymes or the CRTH2 receptor abolishes ILC2 responses. CONCLUSION: PGD2 produced by ILC2s is, in a paracrine/autocrine manner, essential in cytokine-induced ILC2 activation. Hence we provide the detailed mechanism behind how CRTH2 antagonists represent promising therapeutic tools for allergic diseases by controlling ILC2 function.


Assuntos
Hipersensibilidade/tratamento farmacológico , Linfócitos/imunologia , Prostaglandina D2/metabolismo , Antialérgicos/farmacologia , Antialérgicos/uso terapêutico , Carbazóis/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Comunicação Celular , Células Cultivadas , Citocinas/metabolismo , Flurbiprofeno/farmacologia , Humanos , Oxirredutases Intramoleculares/antagonistas & inibidores , Lipocalinas/antagonistas & inibidores , Ativação Linfocitária , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Sulfonamidas/farmacologia , Células Th2/imunologia
10.
Int J Mol Sci ; 21(17)2020 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-32872326

RESUMO

Emerging evidence suggests a complex relationship between sphingosine 1-phosphate (S1P) signaling and stroke. Here, we show the kinetics of S1P in the acute phase of ischemic stroke and highlight accompanying changes in immune cells and S1P receptors (S1PR). Using a C57BL/6 mouse model of middle cerebral artery occlusion (MCAO), we assessed S1P concentrations in the brain, plasma, and spleen. We found a steep S1P gradient from the spleen towards the brain. Results obtained by qPCR suggested that cells expressing the S1PR type 1 (S1P1+) were the predominant population deserting the spleen. Here, we report the cerebral recruitment of T helper (TH) and regulatory T (TREG) cells to the ipsilateral hemisphere, which was associated with differential regulation of cerebral S1PR expression patterns in the brain after MCAO. This study provides insight that the S1P-S1PR axis facilitates splenic T cell egress and is linked to the cerebral recruitment of S1PR+ TH and TREG cells. Further insights by which means the S1P-S1PR-axis orchestrates neuronal positioning may offer new therapeutic perspectives after ischemic stroke.


Assuntos
Encéfalo/imunologia , AVC Isquêmico/metabolismo , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Modelos Animais de Doenças , AVC Isquêmico/etiologia , AVC Isquêmico/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Baço/metabolismo
11.
J Biol Chem ; 293(25): 9685-9695, 2018 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-29752406

RESUMO

Prostaglandin (PG) E2 is an important lipid mediator that is involved in several pathophysiological processes contributing to fever, inflammation, and pain. Previous studies have shown that early and continuous application of nonsteroidal anti-inflammatory drugs significantly reduces pain behavior in the spared nerve injury (SNI) model for trauma-induced neuropathic pain. However, the role of PGE2 and its receptors in the development and maintenance of neuropathic pain is incompletely understood but may help inform strategies for pain management. Here, we sought to define the nociceptive roles of the individual PGE2 receptors (EP1-4) in the SNI model using EP knockout mice. We found that PGE2 levels at the site of injury were increased and that the expression of the terminal synthase for PGE2, cytosolic PGE synthase was up-regulated in resident positive macrophages located within the damaged nerve. Only genetic deletion of the EP3 receptor affected nociceptive behavior and reduced the development of late-stage mechanical allodynia as well as recruitment of immune cells to the injured nerve. Importantly, EP3 activation induced the release of CC-chemokine ligand 2 (CCL2), and antagonists against the CCL2 receptor reduced mechanical allodynia in WT but not in EP3 knockout mice. We conclude that selective inhibition of EP3 might present a potential approach for reducing chronic neuropathic pain.


Assuntos
Quimiocina CCL2/toxicidade , Hiperalgesia/prevenção & controle , Neuralgia/prevenção & controle , Receptores de Prostaglandina E Subtipo EP3/fisiologia , Nervo Isquiático/fisiopatologia , Animais , Células Cultivadas , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuralgia/etiologia , Neuralgia/metabolismo , Neuralgia/patologia , Medição da Dor , Pirrolidinas/farmacologia , Receptores CCR2/antagonistas & inibidores , Receptores CCR2/metabolismo , Nervo Isquiático/lesões
12.
J Biol Chem ; 292(15): 6123-6134, 2017 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-28242764

RESUMO

Sensitization of the heat-activated ion channel transient receptor potential vanilloid 1 (TRPV1) through lipids is a fundamental mechanism during inflammation-induced peripheral sensitization. Leukotriene B4 is a proinflammatory lipid mediator whose role in peripheral nociceptive sensitization is not well understood to date. Two major G-protein-coupled receptors for leukotriene B4 have been identified: the high-affinity receptor BLT1 and the low-affinity receptor BLT2. Transcriptional screening for the expression G-protein-coupled receptors in murine dorsal root ganglia showed that both receptors were among the highest expressed in dorsal root ganglia. Calcium imaging revealed a sensitization of TRPV1-mediated calcium increases in a relative narrow concentration range for leukotriene B4 (100-200 nm). Selective antagonists and neurons from knock-out mice demonstrated a BLT1-dependent sensitization of TRPV1-mediated calcium increases. Accordingly, leukotriene B4-induced thermal hyperalgesia was mediated through BLT1 and TRPV1 as shown using the respective knock-out mice. Importantly, higher leukotriene B4 concentrations (>0.5 µm) and BLT2 agonists abolished sensitization of the TRPV1-mediated calcium increases. Also, BLT2 activation inhibited protein kinase C- and protein kinase A-mediated sensitization processes through the phosphatase calcineurin. Consequently, a selective BLT2-receptor agonist increased thermal and mechanical withdrawal thresholds during zymosan-induced inflammation. In accordance with these data, immunohistochemical analysis showed that both leukotriene B4 receptors were expressed in peripheral sensory neurons. Thus, the data show that the two leukotriene B4 receptors have opposing roles in the sensitization of peripheral sensory neurons forming a self-restricting system.


Assuntos
Sinalização do Cálcio/fisiologia , Gânglios Espinais/metabolismo , Leucotrieno B4/metabolismo , Receptores do Leucotrieno B4/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Calcineurina/genética , Calcineurina/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Hiperalgesia/induzido quimicamente , Hiperalgesia/genética , Hiperalgesia/metabolismo , Leucotrieno B4/farmacologia , Camundongos , Camundongos Knockout , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Receptores do Leucotrieno B4/genética , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo
13.
Int J Cancer ; 142(1): 121-132, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28875496

RESUMO

The putative cannabinoid receptor GPR55 has been shown to play a tumor-promoting role in various cancers, and is involved in many physiological and pathological processes of the gastrointestinal (GI) tract. While the cannabinoid receptor 1 (CB1 ) has been reported to suppress intestinal tumor growth, the role of GPR55 in the development of GI cancers is unclear. We, therefore, aimed at elucidating the role of GPR55 in colorectal cancer (CRC), the third most common cancer worldwide. Using azoxymethane (AOM)- and dextran sulfate sodium (DSS)-driven CRC mouse models, we found that GPR55 plays a tumor-promoting role that involves alterations of leukocyte populations, i.e. myeloid-derived suppressor cells and T lymphocytes, within the tumor tissues. Concomitantly, expression levels of COX-2 and STAT3 were reduced in tumor tissue of GPR55 knockout mice, indicating reduced presence of tumor-promoting factors. By employing the experimental CRC models to CB1 knockout and CB1 /GPR55 double knockout mice, we can further show that GPR55 plays an opposing role to CB1 . We report that GPR55 and CB1 mRNA expression are differentially regulated in the experimental models and in a cohort of 86 CRC patients. Epigenetic methylation of CNR1 and GPR55 was also differentially regulated in human CRC tissue compared to control samples. Collectively, our data suggest that GPR55 and CB1 play differential roles in colon carcinogenesis where the former seems to act as oncogene and the latter as tumor suppressor.


Assuntos
Carcinogênese/metabolismo , Neoplasias Colorretais/patologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Neoplasias Colorretais/metabolismo , Humanos , Camundongos , Camundongos Knockout , Receptor CB1 de Canabinoide/metabolismo , Receptores de Canabinoides/metabolismo
14.
Cell Physiol Biochem ; 45(6): 2516-2528, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29587249

RESUMO

BACKGROUND/AIMS: Signaling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear. METHODS: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA. RESULTS: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors. CONCLUSION: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.


Assuntos
Adenilil Ciclases/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina/metabolismo , Animais , AMP Cíclico/metabolismo , Ativação Enzimática , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Células PC12 , Ratos , Transdução de Sinais
15.
Clin Sci (Lond) ; 132(17): 1963-1976, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30219773

RESUMO

Ceramide synthases (CerS) synthesize chain length specific ceramides (Cer), which mediate cellular processes in a chain length-dependent manner. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed that the genetic deletion of CerS2 suppresses EAE pathology by interaction with granulocyte-colony stimulating factor (G-CSF) signaling and CXC motif chemokine receptor 2 (CXCR2) expression, leading to impaired neutrophil migration. In the present study, we investigated the importance of Cers and their synthesizing/metabolizing enzymes in MS. For this purpose, a longitudinal study with 72 MS patients and 25 healthy volunteers was performed. Blood samples were collected from healthy controls and MS patients over 1- or 3-year periods, respectively. Immune cells were counted using flow cytometry, ceramide levels were determined using liquid chromatography-tandem mass spectrometry, and mRNA expression was analyzed using quantitative PCR. In white blood cells, C16-LacCer and C24-Cer were down-regulated in MS patients in comparison with healthy controls. In plasma, C16-Cer, C24:1-Cer, C16-GluCer, and C24:1-GluCer were up-regulated and C16-LacCer was down-regulated in MS patients in comparison with healthy controls. Blood samples from MS patients were characterized by an increased B-cell number. However, there was no correlation between B-cell number and Cer levels. mRNA expression of Cer metabolizing enzymes and G-CSF signaling enzymes was significantly increased in MS patients. Interestingly, G-CSF receptor (G-CSFR) and CXCR2 mRNA expression correlated with CerS2 and UDP-glucose Cer glucosyltransferase (UGCG) mRNA expression. In conclusion, our results indicate that Cer metabolism is linked to G-CSF signaling in MS.


Assuntos
Ceramidas/sangue , Proteínas de Membrana/metabolismo , Esclerose Múltipla/sangue , Esclerose Múltipla/metabolismo , Esfingosina N-Aciltransferase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Linfócitos B/metabolismo , Ceramidas/química , Ceramidas/metabolismo , Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , Contagem de Leucócitos , Leucócitos/metabolismo , Estudos Longitudinais , Proteínas de Membrana/genética , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Esclerose Múltipla/genética , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Transdução de Sinais , Esfingosina N-Aciltransferase/genética , Proteínas Supressoras de Tumor/genética
16.
Chirality ; 30(5): 632-641, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29406583

RESUMO

Flurbiprofen (F) is a nonsteroidal anti-inflammatory drug (NSAID) used therapeutically as the racemate of (R)-enantiomer and (S)-enantiomer. The inversion of RF to SF and vice versa was investigated in C57Bl/6 and SJL mice and Dark Agouti and Lewis rats. The enzyme α-methylacyl-CoA racemase (AMACR) is involved in the chiral inversion pathway that converts members of the 2-arylpropionic acid NSAIDs from the R-enantiomer to the S-enantiomer. We studied C57Bl/6 mice deficient in AMACR postulating that they should show reduced inversion of RF to SF. In line with the data of others in mice, (R)-inversion to (S)-inversion was relatively high in both the C57Bl/6 and SJL mice (fraction inverted, FI  = 37.7% and 24.7%, respectively). In contrast, in AMACR deficient mice, there was no measurable peak for SF after administration of RF. The results in both rat strains (Dark Agouti and Lewis rats, FI  = 1.4% and 4.1%, respectively) confirm the low chiral inversion of the enantiomers of flurbiprofen in the rat, as observed by other authors in the Sprague-Dawley strain (<5%). From the present results, we conclude that for the study of flurbiprofen enantiomers, the rat is more suitable than the mouse as a model for the human in which (R)-inversion to (S)-inversion is negligible.


Assuntos
Flurbiprofeno/química , Flurbiprofeno/farmacocinética , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacocinética , Feminino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Racemases e Epimerases/genética , Ratos Endogâmicos Lew , Estereoisomerismo
17.
J Lipid Res ; 58(2): 386-392, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27913583

RESUMO

Nonsteroidal anti-inflammatory drugs are the most widely used medicine to treat pain and inflammation, and to inhibit platelet function. Understanding the expression regulation of enzymes of the prostanoid pathway is of great medical relevance. Histone acetylation crucially controls gene expression. We set out to identify the impact of histone deacetylases (HDACs) on the generation of prostanoids and examine the consequences on vascular function. HDAC inhibition (HDACi) with the pan-HDAC inhibitor, vorinostat, attenuated prostaglandin (PG)E2 generation in the murine vasculature and in human vascular smooth muscle cells. In line with this, the expression of the key enzyme for PGE2 synthesis, microsomal PGE synthase-1 (PTGES1), was reduced by HDACi. Accordingly, the relaxation to arachidonic acid was decreased after ex vivo incubation of murine vessels with HDACi. To identify the underlying mechanism, chromatin immunoprecipitation (ChIP) and ChIP-sequencing analysis were performed. These results suggest that HDACs are involved in the recruitment of the transcriptional activator p300 to the PTGES1 gene and that HDACi prevented this effect. In line with the acetyltransferase activity of p300, H3K27 acetylation was reduced after HDACi and resulted in the formation of heterochromatin in the PTGES1 gene. In conclusion, HDAC activity maintains PTGES1 expression by recruiting p300 to its gene.


Assuntos
Proteína p300 Associada a E1A/genética , Histona Desacetilase 1/genética , Prostaglandina-E Sintases/genética , Transcrição Gênica/efeitos dos fármacos , Acetilação , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Dinoprostona/biossíntese , Dinoprostona/genética , Proteína p300 Associada a E1A/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Histona Desacetilase 1/antagonistas & inibidores , Inibidores de Histona Desacetilases/administração & dosagem , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Camundongos , Prostaglandina-E Sintases/biossíntese , Processamento de Proteína Pós-Traducional/genética , Vorinostat
18.
Mol Pain ; 13: 1744806917703111, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28381108

RESUMO

Background: Caloric restriction is associated with broad therapeutic potential in various diseases and an increase in health and life span. In this study, we assessed the impact of caloric restriction on acute and inflammatory nociception in mice, which were either fed ad libitum or subjected to caloric restriction with 80% of the daily average for two weeks. Results: The behavioral tests revealed that inflammatory nociception in the formalin test and in zymosan-induced mechanical hypersensitivity were significantly decreased when mice underwent caloric restriction. As potential mediators of the diet-induced antinociception, we assessed genes typically induced by inflammatory stimuli, AMP-activated kinase, and the endocannabinoid system which have all already been associated with nociceptive responses. Zymosan-induced inflammatory markers such as COX-2, TNFα, IL-1ß, and c-fos in the spinal cord were not altered by caloric restriction. In contrast, AMPKα2 knock-out mice showed significant differences in comparison to C57BL/6 mice and their respective wild type littermates by missing the antinociceptive effects after caloric restriction. Endocannabinoid levels of anandamide and 2-arachidonyl glyceroldetermined in serum by LC-MS/MS were not affected by either caloric restriction alone or in combination with zymosan treatment. However, cannabinoid receptor type 1 expression in the spinal cord, which was not altered by caloric restriction in control mice, was significantly increased after caloric restriction in zymosan-induced paw inflammation. Since increased cannabinoid receptor type 1 signaling might influence AMP-activated kinase activity, we analyzed effects of anandamide on AMP-activated kinase in cell culture and observed a significant activation of AMP-activated kinase. Thus, endocannabionoid-induced AMP-activated kinase activation might be involved in antinociceptive effects after caloric restriction. Conclusion: Our data suggest that caloric restriction has an impact on inflammatory nociception which might involve AMP-activated kinase activation and an increased activity of the endogenous endocannabinoid system by caloric restriction-induced cannabinoid receptor type 1 upregulation.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Ácidos Araquidônicos/farmacologia , Endocanabinoides/farmacologia , Inflamação/tratamento farmacológico , Nociceptividade/efeitos dos fármacos , Alcamidas Poli-Insaturadas/farmacologia , Analgésicos/farmacologia , Animais , Restrição Calórica/métodos , Masculino , Camundongos Endogâmicos C57BL
19.
Kidney Int ; 91(4): 818-829, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28040266

RESUMO

Deletion of cyclooxygenase-2 (COX-2) causes impairment of postnatal kidney development. Here we tested whether the renin angiotensin system contributes to COX-2-dependent nephrogenesis in mice after birth and whether a rescue of impaired renal development and function in COX-2-/- mice was achievable. Plasma renin concentration in mouse pups showed a birth peak and a second peak around day P8 during the first 10 days post birth. Administration of the angiotensin II receptor AT1 antagonist telmisartan from day P1 to P3 did not result in cortical damage. However, telmisartan treatment from day P3 to P8, the critical time frame of renal COX-2 expression, led to hypoplastic glomeruli, a thinned subcapsular cortex and maturational arrest of superficial glomeruli quite similar to that observed in COX-2-/- mice. In contrast, AT2 receptor antagonist PD123319 was without any effect on renal development. Inhibition of the renin angiotensin system by aliskiren and enalapril caused similar glomerular defects as telmisartan. Administration of the AT1 receptor agonist L162313 to COX-2-/- pups improved kidney growth, ameliorated renal defects, but had no beneficial effect on reduced cortical mass. L162313 rescued impaired renal function by reducing serum urea and creatinine and mitigated pathologic albumin excretion. Moreover, glomerulosclerosis in the kidneys of COX-2-/- mice was reduced. Thus, angiotensin II-AT1-receptor signaling is necessary for COX-2-dependent normal postnatal nephrogenesis and maturation.


Assuntos
Angiotensina II/metabolismo , Ciclo-Oxigenase 2/metabolismo , Néfrons/enzimologia , Receptor Tipo 1 de Angiotensina/metabolismo , Sistema Renina-Angiotensina , Transdução de Sinais , Fatores Etários , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 2 de Angiotensina II/farmacologia , Animais , Animais Recém-Nascidos , Creatinina/sangue , Ciclo-Oxigenase 2/deficiência , Ciclo-Oxigenase 2/genética , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Feminino , Genótipo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Néfrons/efeitos dos fármacos , Néfrons/crescimento & desenvolvimento , Néfrons/patologia , Fenótipo , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Receptor Tipo 2 de Angiotensina/efeitos dos fármacos , Receptor Tipo 2 de Angiotensina/metabolismo , Renina/sangue , Sistema Renina-Angiotensina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ureia/sangue
20.
Prostaglandins Other Lipid Mediat ; 133: 53-59, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28583890

RESUMO

The CD200/CD200R signalling pathway downregulates the synthesis of proinflammatory mediators and induces the synthesis of antiinflammatory mediators in macrophages and microglia. However, very little is known about the effect of this immunosuppressive pathway on the synthesis of lipid mediators. Therefore, we determined the synthesis of 35 lipids spanning 5 different lipid families in bone marrow-derived macrophages, which were treated with interleukin (IL) 4, IL10, lipopolysaccharide (LPS), or interferon γ (IFNγ) in absence and presence of CD200. Out of these conditions the only significant effect of CD200 was an increased synthesis of prostaglandin (PG) E2 and D2 in the presence of LPS. Accordingly, mRNA levels of cyclooxygenase-2, microsomal PGE2 synthase-1 and hematopoietic PGD synthase were upregulated by CD200 in presence of LPS. During Complete Freund's Adjuvant (CFA-) induced inflammation mPGES-1 was expressed in monocyte-derived macrophages and its expression was stronger in CD200R-positive than in CD200R-negative macrophages.


Assuntos
Antígenos CD/farmacologia , Dinoprostona/biossíntese , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Prostaglandina D2/biossíntese , Regulação para Cima/efeitos dos fármacos , Animais , Camundongos , Camundongos Endogâmicos C57BL
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