RESUMEN

LPA3 receptors were expressed in TREx HEK 293 cells, and their signaling and phosphorylation were studied. The agonist, lysophosphatidic acid (LPA), increased intracellular calcium and ERK phosphorylation through pertussis toxin-insensitive processes. Phorbol myristate acetate, but not LPA, desensitizes LPA3-mediated calcium signaling, the agonists, and the phorbol ester-induced LPA3 internalization. Pitstop 2 (clathrin heavy chain inhibitor) markedly reduced LPA-induced receptor internalization; in contrast, phorbol ester-induced internalization was only delayed. LPA induced rapid ß-arrestin-LPA3 receptor association. The agonist and the phorbol ester-induced marked LPA3 receptor phosphorylation, and phosphorylation sites were detected using mass spectrometry. Phosphorylated residues were detected in the intracellular loop 3 (S221, T224, S225, and S229) and in the carboxyl terminus (S321, S325, S331, T333, S335, Y337, and S343). Interestingly, phosphorylation sites are within sequences predicted to constitute ß-arrestin binding sites. These data provide insight into LPA3 receptor signaling and regulation.

Asunto(s)

Lisofosfolípidos , Receptores del Ácido Lisofosfatídico , Transducción de Señal , Humanos , beta-Arrestinas/metabolismo , Sitios de Unión , Señalización del Calcio , Células HEK293 , Lisofosfolípidos/metabolismo , Fosforilación , Receptores del Ácido Lisofosfatídico/metabolismo

RESUMEN

BACKGROUND: Our previous studies have shown that lipoxin A4 (LXA4) can serve as a potential biomarker for assessing the efficacy of exercise therapy in knee osteoarthritis (KOA), and fibroblast-like synoviocytes (FLSs) may play a crucial role in KOA pain as well as in the progression of the pathology. OBJECTIVE: By analyzing the GSE29746 dataset and collecting synovial samples from patients with different Kellgren-Lawrence (KL) grades for validation, we focused on exploring the potential effect of LXA4 on ferroptosis in FLSs through the ESR2/LPAR3/Nrf2 axis to alleviate pain and pathological advancement in KOA. METHODS: The association between FLSs ferroptosis and chondrocyte matrix degradation was explored by cell co-culture. We overexpressed and knocked down LPAR3 in vitro to explore its potential mechanism in FLSs. A rat model of monosodium iodoacetate (MIA)-induced KOA was constructed and intervened with moderate-intensity treadmill exercise and intraperitoneal injection of PHTPP to investigate the effects of the LXA4 intracellular receptor ESR2 on exercise therapy. RESULTS: ESR2, LPAR3, and GPX4 levels in the synovium decreased with increasing KL grade. After LXA4 intervention in the co-culture system, GPX4, LPAR3, and ESR2 were upregulated in FLSs, collagen II was upregulated in chondrocytes, and MMP3 and ADAM9 were downregulated. LPAR3 overexpression upregulated the expression of GPX4, Nrf2, and SOD1 in FLSs, while downregulating the expression of MMP13 and MMP3; LPAR3 knockdown reversed these changes. Moderate-intensity platform training improved the behavioral manifestations of pain in KOA rats, whereas PHTPP treatment partially reversed the improvement in synovial and cartilage pathologies induced by platform training. CONCLUSION: LXA4 inhibited FLSs ferroptosis by activating the ESR2/LPAR3/Nrf2 axis, thereby alleviating the pain and pathological progression of KOA. This study brings a new target for the treatment of KOA and also leads to a deeper understanding of the potential mechanisms of exercise therapy for KOA.

Asunto(s)

Ferroptosis , Lipoxinas , Factor 2 Relacionado con NF-E2 , Osteoartritis de la Rodilla , Sinoviocitos , Animales , Osteoartritis de la Rodilla/metabolismo , Osteoartritis de la Rodilla/terapia , Osteoartritis de la Rodilla/patología , Ratas , Lipoxinas/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Sinoviocitos/metabolismo , Humanos , Masculino , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Transducción de Señal , Ratas Sprague-Dawley , Membrana Sinovial/metabolismo , Progresión de la Enfermedad

RESUMEN

Lysophosphatidic acid (LPA) type 3 (LPA3) receptor mutants were generated in which the sites detected phosphorylated were substituted by non-phosphorylatable amino acids. Substitutions were made in the intracellular loop 3 (IL3 mutant), the carboxyl terminus (Ctail), and both domains (IL3/Ctail). The wild-type (WT) receptor and the mutants were expressed in T-REx HEK293 cells, and the consequences of the substitutions were analyzed employing different functional parameters. Agonist- and LPA-mediated receptor phosphorylation was diminished in the IL3 and Ctail mutants and essentially abolished in the IL3/Ctail mutant, confirming that the main phosphorylation sites are present in both domains and their role in receptor phosphorylation eliminated by substitution and distributed in both domains. The WT and mutant receptors increased intracellular calcium and ERK 1/2 phosphorylation in response to LPA and PMA. The agonist, Ki16425, diminished baseline intracellular calcium, which suggests some receptor endogenous activity. Similarly, baseline ERK1/2 phosphorylation was diminished by Ki16425. An increase in baseline ERK phosphorylation was detected in the IL3/Ctail mutant. LPA and PMA-induced receptor interaction with ß-arrestin 2 and LPA3 internalization were severely diminished in cells expressing the mutants. Mutant-expressing cells also exhibit increased baseline proliferation and response to different stimuli, which were inhibited by the antagonist Ki16425, suggesting a role of LPA receptors in this process. Migration in response to different attractants was markedly increased in the Ctail mutant, which the Ki16425 antagonist also attenuated. Our data experimentally show that receptor phosphorylation in the distinct domains is relevant for LPA3 receptor function.

Asunto(s)

Lisofosfolípidos , Receptores del Ácido Lisofosfatídico , Transducción de Señal , Humanos , Fosforilación , Receptores del Ácido Lisofosfatídico/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Células HEK293 , Lisofosfolípidos/metabolismo , Calcio/metabolismo , Endocitosis , Mutación

RESUMEN

Ferroptosis, a unique form of programmed cell death trigged by lipid peroxidation and iron accumulation, has been implicated in embryonic erythropoiesis and aging. Our previous research demonstrated that lysophosphatidic acid receptor 3 (LPA3) activation mitigated oxidative stress in progeria cells and accelerated the recovery of acute anemia in mice. Given that both processes involve iron metabolism, we hypothesized that LPA3 activation might mediate cellular ferroptosis. In this study, we used an LPA3 agonist, 1-Oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT), to activate LPA3 and examine its effects on the ferroptosis process. OMPT treatment elevated anti-ferroptosis gene protein expression, including solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), heme oxygenase-1 (HO-1), and ferritin heavy chain (FTH1), in erastin-induced cells. Furthermore, OMPT reduced lipid peroxidation and intracellular ferrous iron accumulation, as evidenced by C11 BODIPY™ 581/591 Lipid Peroxidation Sensor and FerroOrange staining. These observations were validated by applying LPAR3 siRNA in the experiments mentioned above. In addition, the protein expression level of nuclear factor erythroid 2-related factor (NRF2), a key regulator of oxidative stress, was also enhanced in OMPT-treated cells. Lastly, we verified that LPA3 plays a critical role in erastin-induced ferroptotic human erythroleukemia K562 cells. OMPT rescued the erythropoiesis defect caused by erastin in K562 cells based on a Gly A promoter luciferase assay. Taken together, our findings suggest that LPA3 activation inhibits cell ferroptosis by suppressing lipid oxidation and iron accumulation, indicating that ferroptosis could potentially serve as a link among LPA3, erythropoiesis, and aging.

Asunto(s)

Ferroptosis , Receptores del Ácido Lisofosfatídico , Ratones , Animales , Humanos , Receptores del Ácido Lisofosfatídico/genética , Receptores del Ácido Lisofosfatídico/metabolismo , Apoptosis , Estrés Oxidativo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Hierro/metabolismo

RESUMEN

Sepsis consists of life-threatening organ dysfunction resulting from a dysregulated response to infection. Recent studies have found that excessive neutrophil extracellular traps (NETs) contribute to the pathogenesis of sepsis, thereby increasing morbidity and mortality. Lysophosphatidic acid (LPA) is a small glycerophospholipid molecule that exerts multiple functions by binding to its receptors. Although LPA has been functionally identified to induce NETs, whether and how LPA receptors, especially lysophosphatidic acid receptor 3 (LPA3), play a role in the development of sepsis has never been explored. A comprehensive understanding of the impact of LPA3 on sepsis is essential for the development of medical therapy. After intraperitoneal injection of lipopolysaccharide (LPS), Lpar3-/-mice showed a substantially higher mortality, more severe injury, and more fibrinogen content in the lungs than wild-type (WT) mice. The values of blood coagulation markers, plasma prothrombin time (PT) and fibrinogen (FIB), indicated that the Lpar3-/- mice underwent a severe coagulation process, which resulted in increased thrombosis. The levels of NETs in Lpar3-/- mice were higher than those in WT mice after LPS injection. The mortality rate and degree of lung damage in Lpar3-/- mice with sepsis were significantly reduced after the destruction of NETs by DNaseI treatment. Furthermore, in vitro experiments with co-cultured monocytes and neutrophils demonstrated that monocytes from Lpar3-/- mice promoted the formation of NETs, suggesting that LPA3 acting on monocytes inhibits the formation of NETs and plays a protective role in sepsis. Mechanistically, we found that the amount of CD14, an LPS co-receptor, expressed by monocytes in Lpar3-/-mice was significantly elevated after LPS administration, and the MyD88-p65-NFκB signaling axis, downstream of toll-like receptor 4 signaling, in monocytes was overactivated. Finally, after an injection of the LPA3 agonist (2S)-1-oleoyl-2-methylglycero-3-phosphothionate (OMPT), the survival rate of mice with sepsis was improved, organ damage was reduced, and the production of NETs was decreased. This suggested the possible translational value and application prospects of (2S)-OMPT in the treatment of sepsis. Our study confirms an important protective role of LPA3 in curbing the development of sepsis by suppressing NETs production and thrombosis and provides new ideas for sepsis treatment strategies.

Asunto(s)

Trampas Extracelulares , Sepsis , Trombosis , Animales , Trampas Extracelulares/metabolismo , Fibrinógeno/metabolismo , Lipopolisacáridos/metabolismo , Lipopolisacáridos/toxicidad , Ratones , Receptores del Ácido Lisofosfatídico/genética , Receptores del Ácido Lisofosfatídico/metabolismo , Sepsis/metabolismo , Trombosis/metabolismo

RESUMEN

We developed a mouse model for central post-stroke pain (CPSP), a centrally-originated neuropathic pain (NeuP). In this mode, mice were first injected with Rose Bengal, followed by photo-irradiation of left middle cerebral artery (MCA) to generate thrombosis. Although the MCA thrombosis was soon dissolved, the reduced blood flow remained for more than 24 h due to subsequent occlusion of microvessels. This photochemically induced thrombosis (PIT) model showed a hypersensitivity to the electrical stimulation of both sides of paw, but did not show any abnormal pain in popular thermal or mechanical nociception tests. When tissue-type plasminogen activator (tPA) was injected 6 h after the PIT stress, tPA-dependent hypersensitivity to the electrical paw stimulation and stable thermal and mechanical hyperalgesia on both sides for more than 17 or 18 days after the PIT treatment. These hyperalgesic effects were abolished in lysophosphatidic acid receptor 1 (LPA1)- and lysophosphatidic acid receptor 3 (LPA3)-deficient mice. When Ki-16425, an LPA1 and LPA3 antagonist was treated twice daily for 6 days consecutively, the thermal and mechanical hyperalgesia at day 17 and 18 were significantly reversed. The liquid chromatography-mass spectrometry (LC-MS/MS) analysis revealed that there is a significant increase in several species of LPA molecules in somatosensory S-I and medial dorsal thalamus (MD), but not in striatum or ventroposterior thalamus. All these results suggest that LPA1 and LPA3 signaling play key roles in the development and maintenance of CPSP.

RESUMEN

Objective To investigate the effects of ovarian induction with raloxifene(RAL)versus clomi-phene citrate(CC)on the endometrial receptivity in mouse endometrium during perimplantation period. Methods 48 female Kun-ming mouse were randomly divided into four groups in equal number:RAL 240 mg group,RAL 180 mg group,CC group,natural conception group(NC),all treated with ovulation induction after drug administration.Successfully mated female mouse were killed,and uterus samples were collected for HE stain-ing and immunohistochemistry. Results HE staining showed that the endometrial morphology in the RAL 180 mg group and RAL 240 mg group and NC group were better than that of CC group.The expressions of COX-2 and LP-AR3 in the RAL 180 mg group and RAL 240 mg group were similar to NC group,without significant difference among the three groups(P > 0.05). But in the CC group,it was statistically significantly lower than other three groups(P<0.05),indicating ovarian induction with RAL did not decrease the expressions of COX-2 and LPAR3 in endometrium. Conclusion The mechanism of ovulation induction with RAL is similar to CC,but RAL has fewer adverse effects on the endometrial receptivity compared with CC.

RESUMEN

Lysophosphatidylethanolamine (LPE) is a lyso-type metabolite of phosphatidylethanolamine (a plasma membrane component), and its intracellular Ca(2+) ([Ca(2+)]i) increasing actions may be mediated through G-protein-coupled receptor (GPCR). However, GPCRs for lysophosphatidic acid (LPA), a structurally similar representative lipid mediator, have not been implicated in LPE-mediated activities in SK-OV3 or OVCAR-3 ovarian cancer cells or in receptor over-expression systems. In the present study, LPE-induced [Ca(2+)]i increase was observed in MDA-MB-231 cells but not in other breast cancer cell lines. In addition, LPE- and LPA-induced responses showed homologous and heterologous desensitization. Furthermore, VPC32183 and Ki16425 (antagonists of LPA1 and LPA3) inhibited LPE-induced [Ca(2+)]i increases, and knockdown of LPA1 by transfection with LPA1 siRNA completely inhibited LPE-induced [Ca(2+)]i increases. Furthermore, the involvement of CD97 (an adhesion GPCR) in the action of LPA1 in MDA-MB-231 cells was demonstrated by siRNA transfection. Pertussis toxin (a specific inhibitor of Gi/o proteins), edelfosine (an inhibitor of phospholipase C), or 2-APB (an inhibitor of IP3 receptor) completely inhibited LPE-induced [Ca(2+)]i increases, whereas HA130, an inhibitor of autotaxin/lysophospholipase D, did not. Therefore, LPE is supposed to act on LPA1-CD97/Gi/o proteins/phospholipase C/IP3/Ca(2+) rise in MDA-MB-231 breast cancer cells.

Asunto(s)

Antígenos CD/genética , Calcio/metabolismo , Regulación Neoplásica de la Expresión Génica , Lisofosfolípidos/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Antígenos CD/metabolismo , Compuestos de Boro/farmacología , Línea Celular Tumoral , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Isoxazoles/farmacología , Especificidad de Órganos , Organofosfatos/farmacología , Toxina del Pertussis/farmacología , Propionatos/farmacología , Piridinas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores Acoplados a Proteínas G , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de Señal , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismo