RESUMEN
Lipid biosynthesis is recently studied its functions in a range of cellular physiology including differentiation and regeneration. However, it still remains to be elucidated in its precise function. To reveal this, we evaluated the roles of lysophosphatidic acid (LPA) signaling in alveolar bone formation using the LPA type 2 receptor (LPAR2) antagonist AMG-35 (Amgen Compound 35) using tooth loss without periodontal disease model which would be caused by trauma and usually requires a dental implant to restore masticatory function. In this study, in vitro cell culture experiments in osteoblasts and periodontal ligament fibroblasts revealed cell type-specific responses, with AMG-35 modulating osteogenic differentiation in osteoblasts in vitro. To confirm the in vivo results, we employed a mouse model of tooth loss without periodontal disease. Five to 10 days after tooth extraction, AMG-35 facilitated bone formation in the tooth root socket as measured by immunohistochemistry for differentiation markers KI67, Osteocalcin, Periostin, RUNX2, transforming growth factor beta 1 (TGF-ß1) and SMAD2/3. The increased expression and the localization of these proteins suggest that AMG-35 elicits osteoblast differentiation through TGF-ß1 and SMAD2/3 signaling. These results indicate that LPAR2/TGF-ß1/SMAD2/3 represents a new signaling pathway in alveolar bone formation and that local application of AMG-35 in traumatic tooth loss can be used to facilitate bone regeneration and healing for further clinical treatment.
Asunto(s)
Lisofosfolípidos , Osteogénesis , Receptores Lisofosfolípidos , Pérdida de Diente , Animales , Ratones , Diferenciación Celular/fisiología , Lisofosfolípidos/metabolismo , Osteoblastos/metabolismo , Ligamento Periodontal/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Receptores Lisofosfolípidos/metabolismoRESUMEN
Dysregulation of the autotaxin (ATX, Enpp2)-lysophosphatidic acid (LPA) signaling in cancerous cells contributes to tumorigenesis and therapy resistance. We previously found that ATX activity was elevated in p53-KO mice compared to wild-type (WT) mice. Here, we report that ATX expression was upregulated in mouse embryonic fibroblasts from p53-KO and p53R172H mutant mice. ATX promoter analysis combined with yeast one-hybrid testing revealed that WT p53 directly inhibits ATX expression via E2F7. Knockdown of E2F7 reduced ATX expression and chromosome immunoprecipitation showed that E2F7 promotes Enpp2 transcription through cooperative binding to two E2F7 sites (promoter region -1393 bp and second intron 996 bp). Using chromosome conformation capture, we found that chromosome looping brings together the two E2F7 binding sites. We discovered a p53 binding site in the first intron of murine Enpp2, but not in human ENPP2. Binding of p53 disrupted the E2F7-mediated chromosomal looping and repressed Enpp2 transcription in murine cells. In contrast, we found no disruption of E2F7-mediated ENPP2 transcription via direct p53 binding in human carcinoma cells. In summary, E2F7 is a common transcription factor that upregulates ATX in human and mouse cells but is subject to steric interference by direct intronic p53 binding only in mice.
Asunto(s)
Fibroblastos , Proteína p53 Supresora de Tumor , Humanos , Ratones , Animales , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Transducción de Señal , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Cromosomas , Lisofosfolípidos/metabolismo , Factor de Transcripción E2F7/genética , Factor de Transcripción E2F7/metabolismoRESUMEN
Lysophosphatidic acid (LPA) is a bioactive phospholipid mediator that has been found to ameliorate nonsteroidal anti-inflammatory drug (NSAID)-induced gastric injury by acting on lysophosphatidic acid type 2 receptor (LPAR2). In this study, we investigated whether LPAR2 signaling was implicated in the development of NSAID-induced small intestinal injury (enteropathy), another major complication of NSAID use. Wild-type (WT) and Lpar2 deficient (Lpar2-/-) mice were treated with a single, large dose (20 or 30 mg/kg, i.g.) of indomethacin (IND). The mice were euthanized at 6 or 24 h after IND treatment. We showed that IND-induced mucosal enteropathy and neutrophil recruitment occurred much earlier (at 6 h after IND treatment) in Lpar2-/- mice compared to WT mice, but the tissue levels of inflammatory mediators (IL-1ß, TNF-α, inducible COX-2, CAMP) remained at much lower levels. Administration of a selective LPAR2 agonist DBIBB (1, 10 mg/kg, i.g., twice at 24 h and 30 min before IND treatment) dose-dependently reduced mucosal injury and neutrophil activation in enteropathy, but it also enhanced IND-induced elevation of several proinflammatory chemokines and cytokines. By assessing caspase-3 activation, we found significantly increased intestinal apoptosis in IND-treated Lpar2-/- mice, but it was attenuated after DBIBB administration, especially in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Finally, we showed that IND treatment reduced the plasma activity and expression of autotaxin (ATX), the main LPA-producing enzyme, and also reduced the intestinal expression of Lpar2 mRNA, which preceded the development of mucosal damage. We conclude that LPAR2 has a dual role in NSAID enteropathy, as it contributes to the maintenance of mucosal integrity after NSAID exposure, but also orchestrates the inflammatory responses associated with ulceration. Our study suggests that IND-induced inhibition of the ATX-LPAR2 axis is an early event in the pathogenesis of enteropathy.
Asunto(s)
Diabetes Mellitus Tipo 2 , Enfermedades Intestinales , Lisofosfolípidos , Ratones , Animales , Receptores del Ácido Lisofosfatídico/genética , Receptores del Ácido Lisofosfatídico/metabolismo , Ratones Endogámicos NOD , Ratones SCID , Antiinflamatorios no Esteroideos , Indometacina/efectos adversos , Enfermedades Intestinales/inducido químicamenteRESUMEN
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.
Asunto(s)
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
Sphingolipids are important biological mediators both in health and disease. We investigated the vascular effects of enhanced sphingomyelinase (SMase) activity in a mouse model of type 2 diabetes mellitus (T2DM) to gain an understanding of the signaling pathways involved. Myography was used to measure changes in the tone of the thoracic aorta after administration of 0.2 U/mL neutral SMase in the presence or absence of the thromboxane prostanoid (TP) receptor antagonist SQ 29,548 and the nitric oxide synthase (NOS) inhibitor L-NAME. In precontracted aortic segments of non-diabetic mice, SMase induced transient contraction and subsequent weak relaxation, whereas vessels of diabetic (Leprdb/Leprdb, referred to as db/db) mice showed marked relaxation. In the presence of the TP receptor antagonist, SMase induced enhanced relaxation in both groups, which was 3-fold stronger in the vessels of db/db mice as compared to controls and could not be abolished by ceramidase or sphingosine-kinase inhibitors. Co-administration of the NOS inhibitor L-NAME abolished vasorelaxation in both groups. Our results indicate dual vasoactive effects of SMase: TP-mediated vasoconstriction and NO-mediated vasorelaxation. Surprisingly, in spite of the general endothelial dysfunction in T2DM, the endothelial NOS-mediated vasorelaxant effect of SMase was markedly enhanced.
Asunto(s)
Diabetes Mellitus Tipo 2 , Óxido Nítrico Sintasa de Tipo III , Ratones , Animales , Óxido Nítrico Sintasa de Tipo III/metabolismo , Vasodilatación , Esfingomielina Fosfodiesterasa/metabolismo , NG-Nitroarginina Metil Éster/farmacología , NG-Nitroarginina Metil Éster/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Óxido Nítrico/metabolismo , Modelos Animales de Enfermedad , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/metabolismoRESUMEN
The ATX-LPA-LPAR1 signaling pathway plays a universal role in stimulating diverse cellular responses, including cell proliferation, migration, survival, and invasion in almost every cell type. The ATX-LPAR1 axis is linked to several metabolic and inflammatory diseases including cancer, fibrosis, and rheumatoid arthritis. Numerous selective ATX or LPAR1 inhibitors have been developed and so far, their clinical efficacy has only been evaluated in idiopathic pulmonary fibrosis. None of the ATX and LPAR1 inhibitors have advanced to clinical trials for cancer and rheumatoid arthritis. Nonetheless, several research groups, including ours, have shown considerable benefit of simultaneous ATX and LPAR1 inhibition through combination therapy. Recent research suggests that dual-targeting therapies are superior to combination therapies that use two selective inhibitors. However, limited reports are available on ATX-LPAR1 dual inhibitors, potentially due to co-expression of multiple different LPARs with close structural similarities at the same target. In this review, we discuss rational design and future directions of dual ATX-LPAR1 inhibitors.
Asunto(s)
Artritis Reumatoide , Fibrosis Pulmonar Idiopática , Neoplasias , Humanos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Lisofosfolípidos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismoRESUMEN
Sphingosine-1-phosphate (S1P) plays important roles as a signaling lipid in a variety of physiological and pathophysiological processes. S1P signals via a family of G-protein-coupled receptors (GPCRs) (S1P1-5) and intracellular targets. Here, we report on photoswitchable analogs of S1P and its precursor sphingosine, respectively termed PhotoS1P and PhotoSph. PhotoS1P enables optical control of S1P1-3, shown through electrophysiology and Ca2+ mobilization assays. We evaluated PhotoS1P in vivo, where it reversibly controlled S1P3-dependent pain hypersensitivity in mice. The hypersensitivity induced by PhotoS1P is comparable to that induced by S1P. PhotoS1P is uniquely suited for the study of S1P biology in cultured cells and in vivo because it exhibits prolonged metabolic stability compared to the rapidly metabolized S1P. Using lipid mass spectrometry analysis, we constructed a metabolic map of PhotoS1P and PhotoSph. The formation of these photoswitchable lipids was found to be light dependent, providing a novel approach to optically probe sphingolipid biology.
Asunto(s)
Lisofosfolípidos/metabolismo , Esfingosina/análogos & derivados , Animales , Lisofosfolípidos/química , Ratones , Modelos Moleculares , Estructura Molecular , Imagen Óptica , Procesos Fotoquímicos , Esfingosina/química , Esfingosina/metabolismoRESUMEN
The tight junction (TJ) and barrier function of colonic epithelium is highly sensitive to ionizing radiation. We evaluated the effect of lysophosphatidic acid (LPA) and its analog, Radioprotein-1, on γ-radiation-induced colonic epithelial barrier dysfunction using Caco-2 and m-ICC12 cell monolayers in vitro and mice in vivo. Mice were subjected to either total body irradiation (TBI) or partial body irradiation (PBI-BM5). Intestinal barrier function was assessed by analyzing immunofluorescence localization of TJ proteins, mucosal inulin permeability, and plasma lipopolysaccharide (LPS) levels. Oxidative stress was analyzed by measuring protein thiol oxidation and antioxidant mRNA. In Caco-2 and m-ICC12 cell monolayers, LPA attenuated radiation-induced redistribution of TJ proteins, which was blocked by a Rho-kinase inhibitor. In mice, TBI and PBI-BM5 disrupted colonic epithelial tight junction and adherens junction, increased mucosal permeability, and elevated plasma LPS; TJ disruption by TBI was more severe in Lpar2-/- mice compared to wild-type mice. RP1, administered before or after irradiation, alleviated TBI and PBI-BM5-induced TJ disruption, barrier dysfunction, and endotoxemia accompanied by protein thiol oxidation and downregulation of antioxidant gene expression, cofilin activation, and remodeling of the actin cytoskeleton. These data demonstrate that LPAR2 receptor activation prevents and mitigates γ-irradiation-induced colonic mucosal barrier dysfunction and endotoxemia.
Asunto(s)
Colon/efectos de la radiación , Mucosa Intestinal/efectos de la radiación , Radiación Ionizante , Receptores del Ácido Lisofosfatídico/genética , Uniones Estrechas/efectos de la radiación , Uniones Adherentes/efectos de los fármacos , Uniones Adherentes/metabolismo , Uniones Adherentes/efectos de la radiación , Animales , Células CACO-2 , Línea Celular , Colon/efectos de los fármacos , Colon/metabolismo , Humanos , Uniones Intercelulares/efectos de los fármacos , Uniones Intercelulares/metabolismo , Uniones Intercelulares/efectos de la radiación , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Lisofosfolípidos/farmacología , Ratones Noqueados , Permeabilidad/efectos de los fármacos , Permeabilidad/efectos de la radiación , Receptores del Ácido Lisofosfatídico/metabolismo , Proteínas de Uniones Estrechas/genética , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismoRESUMEN
Lysophosphatidic acid (LPA) is a phospholipid that acts as an extracellular signaling molecule and activates the family of lysophosphatidic acid receptors (LPA1-6). These G protein-coupled receptors (GPCRs) are broadly expressed and are particularly important in development as well as in the nervous, cardiovascular, reproductive, gastrointestinal, and pulmonary systems. Here, we report on a photoswitchable analogue of LPA, termed AzoLPA, which contains an azobenzene photoswitch embedded in the acyl chain. AzoLPA enables optical control of LPA receptor activation, shown through its ability to rapidly control LPA-evoked increases in intracellular Ca2+ levels. AzoLPA shows greater activation of LPA receptors in its light-induced cis-form than its dark-adapted (or 460 nm light-induced) trans-form. AzoLPA enabled the optical control of neurite retraction through its activation of the LPA2 receptor.
Asunto(s)
Lisofosfolípidos/metabolismo , Humanos , Lisofosfolípidos/química , Procesos Fotoquímicos , Receptores del Ácido Lisofosfatídico/química , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de SeñalRESUMEN
The lysophospholipase D autotaxin (ATX) generates lysophosphatidic acid (LPA) that activates six cognate G-protein coupled receptors (GPCR) in cancerous cells, promoting their motility and invasion. Four novel compounds were generated aided by molecular docking guided design and synthesis techniques to obtain new dual inhibitors of ATX and the lysophosphatidic acid receptor subtype 1 (LPAR1). Biological evaluation of these compounds revealed two compounds, 10 and 11, as new ATX enzyme inhibitors with potencies in the range of 218-220 nM and water solubility (>100 µg/mL), but with no LPAR1 inhibitory activity. A QSAR model was generated that included four newly designed compounds and twenty-one additional compounds that we have reported previously. The QSAR model provided excellent predictability of the pharmacological activity and potency among structurally related drug candidates. This model will be highly useful in guiding the synthesis of new ATX inhibitors in the future.
Asunto(s)
Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Piranos/farmacología , Animales , Línea Celular Tumoral , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de Fosfodiesterasa/síntesis química , Inhibidores de Fosfodiesterasa/metabolismo , Unión Proteica , Piranos/síntesis química , Piranos/metabolismo , Relación Estructura-Actividad Cuantitativa , Ratas , Receptores del Ácido Lisofosfatídico/metabolismoRESUMEN
The growth factor-like lipid mediator, lysophosphatidic acid (LPA), is a potent signaling molecule that influences numerous physiologic and pathologic processes. Manipulation of LPA signaling is of growing pharmacotherapeutic interest, especially because LPA resembles compounds with drug-like features. The action of LPA is mediated through activation of multiple types of molecular targets, including six G protein-coupled receptors that are clear targets for drug development. However, the LPA signaling has been linked to pathological responses that include promotion of fibrosis, atherogenesis, tumorigenesis, and metastasis. Thus, a question arises: Can we harness, in an LPA-like drug, the many beneficial activities of this lipid without eliciting its dreadful actions? We developed octadecyl thiophosphate (OTP; subsequently licensed as Rx100), an LPA mimic with higher stability in vivo than LPA. This article highlights progress made toward developing analogs like OTP and exploring prosurvival and regenerative LPA signaling. We determined that LPA prevents cell death triggered by various cellular stresses, including genotoxic stressors, and rescues cells condemned to apoptosis. LPA2 agonists provide a new treatment option for secretory diarrhea and reduce gastric erosion caused by nonsteroidal anti-inflammatory drugs. The potential uses of LPA2 agonists like OTP and sulfamoyl benzoic acid-based radioprotectins must be further explored for therapeutic uses.
Asunto(s)
Descubrimiento de Drogas/métodos , Receptores del Ácido Lisofosfatídico/agonistas , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Humanos , Receptores del Ácido Lisofosfatídico/química , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Stem cells are a rare subpopulation defined by the potential to self-renew and differentiate into specific cell types. A population of stem-like cells has been reported to possess the ability of self-renewal, invasion, metastasis, and engraftment of distant tissues. This unique cell subpopulation has been designated as cancer stem cells (CSC). CSC were first identified in leukemia, and the contributions of CSC to cancer progression have been reported in many different types of cancers. The cancer stem cell hypothesis attempts to explain tumor cell heterogeneity based on the existence of stem cell-like cells within solid tumors. The elimination of CSC is challenging for most human cancer types due to their heightened genetic instability and increased drug resistance. To combat these inherent abilities of CSC, multi-pronged strategies aimed at multiple aspects of CSC biology are increasingly being recognized as essential for a cure. One of the most challenging aspects of cancer biology is overcoming the chemotherapeutic resistance in CSC. Here, we provide an overview of autotaxin (ATX), lysophosphatidic acid (LPA), and their signaling pathways in CSC. Increasing evidence supports the role of ATX and LPA in cancer progression, metastasis, and therapeutic resistance. Several studies have demonstrated the ATX-LPA axis signaling in different cancers. This lipid mediator regulatory system is a novel potential therapeutic target in CSC. In this review, we summarize the evidence linking ATX-LPA signaling to CSC and its impact on cancer progression and metastasis. We also provide evidence for the efficacy of cancer therapy involving the pharmacological inhibition of this signaling pathway.
Asunto(s)
Neoplasias/enzimología , Neoplasias/patología , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Hidrolasas Diéster Fosfóricas/metabolismo , Animales , Humanos , Lisofosfolípidos/metabolismoRESUMEN
Sphingosine-1-phosphate (S1P) has been implicated recently in the physiology and pathology of the cardiovascular system including regulation of vascular tone. Pilot experiments showed that the vasoconstrictor effect of S1P was enhanced markedly in the presence of phenylephrine (PE). Based on this observation, we hypothesized that S1P might modulate α1-adrenergic vasoactivity. In murine aortas, a 20-minute exposure to S1P but not to its vehicle increased the Emax and decreased the EC50 of PE-induced contractions indicating a hyperreactivity to α1-adrenergic stimulation. The potentiating effect of S1P disappeared in S1P2 but not in S1P3 receptor-deficient vessels. In addition, smooth muscle specific conditional deletion of G12/13 proteins or pharmacological inhibition of the Rho-associated protein kinase (ROCK) by Y-27632 or fasudil abolished the effect of S1P on α1-adrenergic vasoconstriction. Unexpectedly, PE-induced contractions remained enhanced markedly as late as three hours after S1P-exposure in wild-type (WT) and S1P3 KO but not in S1P2 KO vessels. In conclusion, the S1P-S1P2-G12/13-ROCK signaling pathway appears to have a major influence on α1-adrenergic vasoactivity. This cooperativity might lead to sustained vasoconstriction when increased sympathetic tone is accompanied by increased S1P production as it occurs during acute coronary syndrome and stroke.
Asunto(s)
Lisofosfolípidos/farmacología , Receptores Adrenérgicos alfa 1/fisiología , Transducción de Señal/efectos de los fármacos , Esfingosina/análogos & derivados , Vasoconstricción/efectos de los fármacos , Quinasas Asociadas a rho/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Amidas/farmacología , Animales , Sinergismo Farmacológico , Ratones Endogámicos C57BL , Ratones Noqueados , Fenilefrina/farmacología , Piridinas/farmacología , Esfingosina/farmacología , Receptores de Esfingosina-1-Fosfato/genética , Receptores de Esfingosina-1-Fosfato/metabolismo , Vasoconstrictores/farmacología , Vasodilatadores/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Lysophosphatidic acid (LPA) has been recognized recently as an endothelium-dependent vasodilator, but several lines of evidence indicate that it may also stimulate vascular smooth muscle cells (VSMCs), thereby contributing to vasoregulation and remodeling. In the present study, mRNA expression of all 6 LPA receptor genes was detected in murine aortic VSMCs, with the highest levels of LPA1, LPA2, LPA4, and LPA6 In endothelium-denuded thoracic aorta (TA) and abdominal aorta (AA) segments, 1-oleoyl-LPA and the LPA1-3 agonist VPC31143 induced dose-dependent vasoconstriction. VPC31143-induced AA contraction was sensitive to pertussis toxin (PTX), the LPA1&3 antagonist Ki16425, and genetic deletion of LPA1 but not that of LPA2 or inhibition of LPA3, by diacylglycerol pyrophosphate. Surprisingly, vasoconstriction was also diminished in vessels lacking cyclooxygenase-1 [COX1 knockout (KO)] or the thromboxane prostanoid (TP) receptor (TP KO). VPC31143 increased thromboxane A2 (TXA2) release from TA of wild-type, TP-KO, and LPA2-KO mice but not from LPA1-KO or COX1-KO mice, and PTX blocked this effect. Our findings indicate that LPA causes vasoconstriction in VSMCs, mediated by LPA1-, Gi-, and COX1-dependent autocrine/paracrine TXA2 release and consequent TP activation. We propose that this new-found interaction between the LPA/LPA1 and TXA2/TP pathways plays significant roles in vasoregulation, hemostasis, thrombosis, and vascular remodeling.-Dancs, P. T., Ruisanchez, E., Balogh, A., Panta, C. R., Miklós, Z., Nüsing, R. M., Aoki, J., Chun, J., Offermanns, S., Tigyi, G., Benyó, Z. LPA1 receptor-mediated thromboxane A2 release is responsible for lysophosphatidic acid-induced vascular smooth muscle contraction.
Asunto(s)
Lisofosfolípidos/farmacología , Contracción Muscular , Músculo Liso Vascular/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Tromboxano A2/metabolismo , Vasoconstricción , Animales , Aorta/citología , Aorta/fisiología , Ciclooxigenasa 1/genética , Ciclooxigenasa 1/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Receptores del Ácido Lisofosfatídico/agonistas , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Receptores del Ácido Lisofosfatídico/genéticaRESUMEN
Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of nature's simplest phospholipids, is found in cells from slime mold to humans and has a largely unknown function. We find here that CPA is generated in mammalian cells in a stimulus-coupled manner by phospholipase D2 (PLD2) and binds to and inhibits the nuclear hormone receptor PPARgamma with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPARgamma target-gene transcription that normally drives adipocytic differentiation of 3T3-L1 cells, lipid accumulation in RAW264.7 cells and primary mouse macrophages, and arterial wall remodeling in a rat model in vivo. Inhibition of PLD2 by shRNA, a dominant-negative mutant, or a small molecule inhibitor blocks CPA production and relieves PPARgamma inhibition. We conclude that CPA is a second messenger and a physiological inhibitor of PPARgamma, revealing that PPARgamma is regulated by endogenous agonists as well as by antagonists.
Asunto(s)
Adipocitos/metabolismo , Macrófagos/metabolismo , PPAR gamma/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/metabolismo , Células 3T3-L1 , Animales , Diferenciación Celular/fisiología , Ratones , Co-Represor 2 de Receptor Nuclear/genética , Co-Represor 2 de Receptor Nuclear/metabolismo , PPAR gamma/genética , Ácidos Fosfatidicos/genética , Fosfolipasa D/genética , Ratas , Transcripción Genética/fisiologíaRESUMEN
Lysophosphatidic acid (LPA) is a pleiotropic lipid signaling molecule associated with asthma pathobiology. LPA elicits its effects by binding to at least six known cell surface G protein-coupled receptors (LPA1-6) that are expressed in the lung in a cell type-specific manner. LPA2 in particular has emerged as an attractive therapeutic target in asthma because it appears to transduce inhibitory or cell-protective signals. We studied a novel and specific small molecule LPA2 agonist (2-[4-(1,3-dioxo-1H,3H-benzoisoquinolin-2-yl)butylsulfamoyl] benzoic acid [DBIBB]) in a mouse model of house dust mite-induced allergic airway inflammation. Mice injected with DBIBB developed significantly less airway and lung inflammation compared with vehicle-treated controls. Levels of lung Th2 cytokines were also significantly attenuated by DBIBB. We conclude that pharmacologic activation of LPA2 attenuates Th2-driven allergic airway inflammation in a mouse model of asthma. Targeting LPA receptor signaling holds therapeutic promise in allergic asthma.
Asunto(s)
Antiasmáticos/farmacología , Antiinflamatorios/farmacología , Asma/prevención & control , Pulmón/efectos de los fármacos , Naftalimidas/farmacología , Neumonía/prevención & control , Receptores del Ácido Lisofosfatídico/agonistas , Sulfonamidas/farmacología , Alérgenos , Animales , Antígenos Dermatofagoides , Proteínas de Artrópodos , Asma/inmunología , Asma/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Ratones Endogámicos BALB C , Hidrolasas Diéster Fosfóricas/metabolismo , Neumonía/inmunología , Neumonía/metabolismo , Receptores del Ácido Lisofosfatídico/inmunología , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de Señal/efectos de los fármacos , Células Th2/efectos de los fármacos , Células Th2/inmunología , Células Th2/metabolismo , Factores de TiempoRESUMEN
Erythrocytes and megakaryocytes (MK) are derived from a common progenitor that undergoes lineage specification. Lysophosphatidic acid (LPA), a lipid growth factor was previously shown to be a regulator for erythropoietic process through activating LPA receptor 3 (LPA3). However, whether LPA affects megakaryopoiesis remains unclear. In this study, we used K562 leukemia cell line as a model to investigate the roles of LPA in MK differentiation. We demonstrated that K562 cells express both LPA2 and LPA3, and the expression levels of LPA2 are higher than LPA3. Treatment with phorbol 12-myristate 13-acetate, a commonly used inducer of megakaryopoiesis, reciprocally regulates the expressions of LPA2 and LPA3. By pharmacological blockers and knockdown experiments, we showed that activation of LPA2 suppresses whereas, LPA3 promotes megakaryocytic differentiation in K562. The LPA2-mediated inhibition is dependent on ß-catenin translocation, whereas reactive oxygen species (ROS) generation is a downstream signal for activation of LPA3. Furthermore, the hematopoietic transcriptional factors GATA-1 and FLI-1, appear to be involved in these regulatory mechanisms. Taken together, our results suggested that LPA2 and LPA3 may function as a molecular switch and play opposing roles during megakaryopoiesis of K562 cells.
Asunto(s)
Leucemia Eritroblástica Aguda/metabolismo , Megacariocitos/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Trombopoyesis , Factor de Transcripción GATA1/metabolismo , Humanos , Integrina beta3/metabolismo , Células K562 , Leucemia Eritroblástica Aguda/genética , Megacariocitos/efectos de los fármacos , Proteínas de Microfilamentos/metabolismo , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Receptores del Ácido Lisofosfatídico/genética , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología , Trombopoyesis/efectos de los fármacos , Factores de Tiempo , Transactivadores , Transfección , beta Catenina/metabolismoRESUMEN
The goals of this study were to evaluate the effects of ionizing radiation on apical junctions in colonic epithelium and mucosal barrier function in mice in vivo. Adult mice were subjected to total body irradiation (4 Gy) with or without N-acetyl-l-cysteine (NAC) feeding for 5 days before irradiation. At 2-24 h postirradiation, the integrity of colonic epithelial tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton was assessed by immunofluorescence microscopy and immunoblot analysis of detergent-insoluble fractions for TJ and AJ proteins. The barrier function was evaluated by measuring vascular-to-luminal flux of fluorescein isothiocyanate (FITC)-inulin in vivo and luminal-to-mucosal flux in vitro. Oxidative stress was evaluated by measuring protein thiol oxidation. Confocal microscopy showed that radiation caused redistribution of occludin, zona occludens-1, claudin-3, E-cadherin, and ß-catenin, as well as the actin cytoskeleton as early as 2 h postirradiation, and this effect was sustained for at least 24 h. Feeding NAC before irradiation blocked radiation-induced disruption of TJ, AJ, and the actin cytoskeleton. Radiation increased mucosal permeability to inulin in colon, which was blocked by NAC feeding. The level of reduced-protein thiols in colon was depleted by radiation with a concomitant increase in the level of oxidized-protein thiol. NAC feeding blocked the radiation-induced protein thiol oxidation. These data demonstrate that radiation rapidly disrupts TJ, AJ, and the actin cytoskeleton by an oxidative stress-dependent mechanism that can be prevented by NAC feeding.