RESUMEN
The GPR124/RECK/WNT7 pathway is an essential regulator of CNS angiogenesis and blood-brain barrier (BBB) function. GPR124, a brain endothelial adhesion seven-pass transmembrane protein, associates with RECK, which binds and stabilizes newly synthesized WNT7 that is transferred to frizzled (FZD) to initiate canonical ß-catenin signaling. GPR124 remains enigmatic: although its extracellular domain (ECD) is essential, the poorly conserved intracellular domain (ICD) appears to be variably required in mammals versus zebrafish, potentially via adaptor protein bridging of GPR124 and FZD ICDs. GPR124 ICD deletion impairs zebrafish angiogenesis, but paradoxically retains WNT7 signaling upon mammalian transfection. We thus investigated GPR124 ICD function using the mouse deletion mutant Gpr124ΔC. Despite inefficiently expressed GPR124ΔC protein, Gpr124ΔC/ΔC mice could be born with normal cerebral cortex angiogenesis, in comparison with Gpr124-/- embryonic lethality, forebrain avascularity and hemorrhage. Gpr124ΔC/ΔC vascular phenotypes were restricted to sporadic ganglionic eminence angiogenic defects, attributable to impaired GPR124ΔC protein expression. Furthermore, Gpr124ΔC and the recombinant GPR124 ECD rescued WNT7 signaling in culture upon brain endothelial Gpr124 knockdown. Thus, in mice, GPR124-regulated CNS forebrain angiogenesis and BBB function are exerted by ICD-independent functionality, extending the signaling mechanisms used by adhesion seven-pass transmembrane receptors.
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Barrera Hematoencefálica , Encéfalo , Neovascularización Fisiológica , Receptores Acoplados a Proteínas G , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/embriología , Neovascularización Fisiológica/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Ratones , Encéfalo/metabolismo , Encéfalo/embriología , Dominios Proteicos , Ratones Noqueados , Transducción de Señal , Proteínas Wnt/metabolismo , Proteínas Wnt/genética , Humanos , Células Endoteliales/metabolismo , Angiogénesis , Proteínas Ligadas a GPIRESUMEN
Early-onset preeclampsia, which occurrs before 34 weeks of gestation, is the most dangerous classification of preeclampsia, which is a pregnancy-specific disease that causes 1% of maternal deaths. G protein-coupled receptor 124 (GPR124) is significantly expressed at various stages of the human reproductive process, particularly during embryogenesis and angiogenesis. Our prior investigation demonstrated a notable decrease in GPR124 expression in the placentas of patients with early-onset preeclampsia compared to that in normal pregnancy placentas. However, there is a lack of extensive investigation into the molecular processes that contribute to the role of GPR124 in placenta development. This study aimed to examine the mechanisms by which GPR124 affects the occurrence of early-onset preeclampsia and its function in trophoblast. Proliferative, invasive, migratory, apoptotic, and inflammatory processes were identified in GPR124 knockdown, GPR124 overexpression, and normal HTR8/SVneo cells. The mechanism of GPR124-mediated cell function in GPR124 knockdown HTR8/SVneo cells was examined using inhibitors of the JNK or P38 MAPK pathway. Downregulation of GPR124 was found to significantly inhibit proliferation, invasion and migration, and promote apoptosis of HTR8/SVneo cells when compared to the control and GPR124 overexpression groups. This observation is consistent with the pathological characteristics of preeclampsia. In addition, GPR124 overexpression inhibits the secretion of pro-inflammatory cytokines interleukin (IL)-8 and interferon-γ (IFN-γ) while enhancing the secretion of the anti-inflammatory cytokine interleukin (IL)-4. Furthermore, GPR124 suppresses the activation of P-JNK and P-P38 within the JNK/P38 MAPK pathway. The invasion, apoptosis, and inflammation mediated by GPR124 were partially restored by suppressing the JNK and P38 MAPK pathways in HTR8/SVneo cells. GPR124 plays a crucial role in regulating trophoblast proliferation, invasion, migration, apoptosis, and inflammation via the JNK and P38 MAPK pathways. Furthermore, the effect of GPR124 on trophoblast suggests its involvement in the pathogenesis of early-onset preeclampsia.
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Apoptosis , Movimiento Celular , Proliferación Celular , Inflamación , Preeclampsia , Receptores Acoplados a Proteínas G , Trofoblastos , Proteínas Quinasas p38 Activadas por Mitógenos , Humanos , Trofoblastos/metabolismo , Trofoblastos/patología , Apoptosis/genética , Proliferación Celular/genética , Femenino , Movimiento Celular/genética , Embarazo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Preeclampsia/patología , Preeclampsia/genética , Preeclampsia/metabolismo , Inflamación/patología , Inflamación/genética , Inflamación/metabolismo , Sistema de Señalización de MAP Quinasas , Línea Celular , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Placenta/metabolismo , Placenta/patología , Receptores de EstrógenosRESUMEN
RECK has been described to modulate extracellular matrix components through negative regulation of MMP activities. Recently, RECK was demonstrated to bind to an orphan G protein-coupled receptor GPR124 to mediate WNT7 signaling in nontumor contexts. Here, we attempted to clarify the role of RECK in driving WNT signaling in cancer cells. RECK and GPR124 formed a complex in 293T cells, and when both were expressed, WNT signaling was significantly enhanced in a WNT7-dependent manner. This cooperation was abolished when RECK mutants unable to bind to GPR124 were transduced. RECK stimulated the growth of KRAS-mutated pancreatic ductal adenocarcinoma (PDAC) cells with increased sensitivity to WNT inhibitor in a GPR124-dependent manner. A gastric cancer cell line SH10TC endogenously expresses both RECK and GPR124 under regular culture conditions. In this cell line, inhibited cell growth and WNT signaling as well as increased apoptosis in the GPR124 depletion was dominantly found over those in the RECK deletion. These findings suggest that RECK promotes tumor cell growth by positively modulating WNT signaling through GPR124. This study proposes that the RECK/GPR124 complex might be a good therapeutic target in PDAC and gastric cancer.
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OBJECTIVES: Preeclampsia (PE) is a disease specific to pregnancy that causes 9-10â¯% of maternal deaths. Early-onset PE (<34 weeks' gestation) is the most dangerous category of PE. Wnt7a and GPR124 (G protein-coupled receptor 124) are widely expressed in the human reproductive process. Especially during embryogenesis and tumorigenesis, Wnt7a plays a crucial role. However, few studies have examined the association between Wnt7a-GPR124 and early-onset PE. The aim of this study was to examine the significance of Wnt7a and GPR124 in early-onset PE as well as Wnt7a's role in trophoblast cells. METHODS: Immunohistochemistry (IHC), real-time PCR, and western blotting (WB) were used to investigate Wnt7a and GPR124 expression in normal and early-onset PE placentas. Additionally, FACS, Transwell, and CCK-8 assays were used to diagnose Wnt7a involvement in migration, invasion, and proliferation. RESULTS: In the early-onset PE group, Wnt7a and GPR124 expression was significantly lower than in the normal group, especially in the area of syncytiotrophoblasts (STBs) and extravillous trophoblasts (EVTs). A negative correlation was found between Wnt7a RNA and GPR124 expression (r=-0.42, p<0.01). However, the Wnt7a RNA expression level was positive correlated with PE severity. In further cellular functional experiments, knockdown of Wnt7a inhibits HTR8/SVeno cells invasion and migration but has little effect on proliferation and apoptosis. CONCLUSIONS: Through the Wnt pathway, Wnt7a regulates trophoblast cell invasion and migration, and may contribute to early-onset preeclampsia pathogenesis. A molecular level study of Wnt7a will be needed to find downstream proteins and mechanisms of interaction.
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Preeclampsia , Embarazo , Femenino , Humanos , Preeclampsia/genética , Línea Celular , Placenta/metabolismo , Trofoblastos/fisiología , ARN/metabolismo , Proliferación CelularRESUMEN
A hitherto unidentified N-ethyl-N-nitrosourea (ENU)-induced mutation affects dorsal root ganglia (DRG) formation in ouchless mutant zebrafish larvae. In contrast to previous findings assigning the ouchless phenotypes to downregulated sorbs3 transcript levels, this work re-attributes the phenotypes to an essential splice site mutation affecting adgra2 (gpr124) splicing and function. Accordingly, ouchless mutants fail to complement previously characterized adgra2 mutants and exhibit highly penetrant cerebrovascular defects. The aberrantly spliced adgra2 transcript found in ouchless mutants encodes a receptor lacking a single leucine-rich repeat (LRR) within its N-terminus.
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Mutación , Empalme del ARN/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiología , Pez Cebra , Animales , Animales Modificados Genéticamente , Trastornos Cerebrovasculares/embriología , Trastornos Cerebrovasculares/genética , Embrión no Mamífero , Malformaciones Arteriovenosas Intracraneales/genética , Malformaciones del Sistema Nervioso/genética , Fenotipo , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
BACKGROUND/AIMS: Endothelial cell dysfunction is the principal pathological process underlying atherosclerotic cardiovascular disease. G protein-coupled receptor 124 (GPR124), an orphan receptor in the adhesion GPCR subfamily, promotes angiogenesis in the brain. In the present study, we explored the role of endothelial GPR124 in the development and progression of atherosclerosis in adult mice. METHODS: Using tetracycline-inducible transgenic systems, we generated mice expressing GPR124 specifically under control of the Tie-2 promoter. The animal model of atherosclerosis was constructed by intravenously injecting AAV-PCSK9DY into tetracycline-regulated mice and feeding the mice a high-fat diet for 16 consecutive weeks. Biochemical analysis and immunohistochemistry methods were used to address the role and mechanism of GPR124 in the pathological process of atherosclerosis. RESULTS: Higher TC (total cholesterol) and LDL-C (low density lipoprotein cholesterol) levels in serum and greater lipid deposition in the aortic sinus were found in atherosclerotic mice with GPR124 overexpression, coincident with the elevated proliferation of smooth muscle cells. We observed an elevation of ONOO- in the aortic sinus in this model by using immunofluorescence, and the experiments showed that the specific overexpression of GPR124 in the endothelium induced the up-regulation of CD68, NLRP3 and caspase-1 levels in the aortic sinus. CONCLUSION: The above results indicate that manipulating GPR124 in the endothelium may contribute to delayed pathological progression of atherosclerosis.
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Aterosclerosis/patología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Aterosclerosis/metabolismo , Caspasa 1/metabolismo , Colesterol/sangre , LDL-Colesterol/sangre , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Humanos , Inflamación/etiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Ácido Peroxinitroso/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Receptores Acoplados a Proteínas G/genética , Seno Aórtico/metabolismo , Seno Aórtico/patologíaRESUMEN
Hypertension (HBP) is a chronic disease characterized by increased blood pressure, which despite several treatments maintains a high morbi-mortality, which suggests that there are other mechanisms involved in this pathology, within which the orphan receptors could be candidates for the treatment of the HBP; these receptors are called orphan receptors because their ligand is unknown. These receptors have been suggested to participate in some pathologies because they are associated with various systems such as GPR88, which has been linked to the dopaminergic system, and GPR124 with angiogenesis, suggesting that these receptors could take part in HBP. Hence, the aim of this work was to study the expression of orphan receptors GPR88 and GPR124 in various tissues of normotensive and hypertensive rats. We used Wistar Kyoto (WKY) and spontaneously hypertensive rat (SHR) of 6-8 and 10-12 weeks of age and we determined systolic blood pressure (SBP), heart rate, as well as mRNA of GPR88 and GPR124 receptors by reverse transcription polymerase chain reaction (RT-PCR) in the aorta, heart, kidney, and brain. Our results showed that GPR88 and GPR124 were expressed in all analyzed tissues, but their expression is dependent on the age and development of HBP because their expression tends to be modified as HBP is established. Therefore, we conclude that GPR88 and GPR124 receptors may be involved in the development or maintenance of high blood pressure.
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Expresión Génica , Hipertensión/genética , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/genética , Animales , Aorta/metabolismo , Presión Sanguínea , Encéfalo/metabolismo , Frecuencia Cardíaca , Riñón/metabolismo , Masculino , Miocardio/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
The transformation suppressor gene RECK was isolated by cDNA expression cloning (1998), and GPR124/TEM5 was detected as a tumor endothelial marker by differential screening (2000). The importance of Wnt7a/b and Gpr124 in brain angiogenesis was demonstrated by reverse genetics in mice (2008-2010). A series of recent studies using genetically engineered mice and zebrafish as well as luciferase reporter assays in cultured cells led to the discovery of functional interactions among Reck, Gpr124, and Wnt7a/b in triggering canonical Wnt signaling with relevance to embryonic brain angiogenesis and blood-brain barrier formation.
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Proteínas Ligadas a GPI/metabolismo , Neovascularización Fisiológica , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Animales , Humanos , Proteínas Proto-Oncogénicas/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Retinal microangiopathies, such as neovascularization and preretinal and vitreous hemorrhages, are the primary pathological features of diabetic retinopathy (DR). These conditions can worsen visual impairment and may result in blindness. Furthermore, multiple metabolic pathways are associated with microangiopathy in DR. However, the specific underlying pathological mechanisms remain unclear. Several studies have demonstrated the important role of G protein-coupled receptor 124 (Gpr124) in cerebral vascular endothelial cells, but its effect on the retinal endothelium has not been elucidated. In this study, we found that Gpr124 is expressed in both pathological retinal fibrous vascular membranes of DR patients and retinal blood vessels of mice, with elevated protein expression specifically observed in the retinas of DR model mice. Furthermore, Gpr124 expression was elevated after high-glucose treatment of human retinal microvascular endothelial cells (HRMECs). Inhibition of Gpr124 expression affected the high glucose-induced proliferation, migration, and tube-forming ability of HRMECs. We concluded that Gpr124 expression was upregulated in DR and promoted HRMECs angiogenesis in a high-glucose environment. This finding may help to elucidate the pathogenesis of DR and provide a critical research basis for identifying effective treatments.
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Spinal cord injury (SCI) causes blood-spinal cord barrier (BSCB) disruption, leading to secondary damage, such as hemorrhagic infiltration, inflammatory response, and neuronal cell death. It is of great significance to rebuild the BSCB at the early stage of SCI to alleviate the secondary injury for better prognosis. Yet, current research involved in the reconstruction of BSCB is insufficient. Accordingly, we provide a thermosensitive hydrogel-based G protein-coupled receptor 124 (GPR124) delivery strategy for rebuilding BSCB. Herein, we firstly found that the expression of GPR124 decreased post-SCI and demonstrated that treatment with recombinant GPR124 could partially alleviate the disruption of BSCB post-SCI by restoring tight junctions (TJs) and promoting migration and tube formation of endothelial cells. Interestingly, GPR124 could also boost the energy metabolism of endothelial cells. However, the absence of physicochemical stability restricted the wide usage of GPR124. Hence, we fabricated a thermosensitive heparin-poloxamer (HP) hydrogel that demonstrated sustained GPR124 production and maintained the bioactivity of GPR124 (HP@124) for rebuilding the BSCB and eventually enhancing functional motor recovery post-SCI. HP@124 hydrogel can encapsulate GPR124 at the lesion site by injection, providing prolonged release, preserving wounded tissues, and filling injured tissue cavities. Consequently, it induces synergistically efficient integrated regulation by blocking BSCB rupture, decreasing fibrotic scar formation, minimizing inflammatory response, boosting remyelination, and regenerating axons. Mechanistically, giving GPR124 activates energy metabolism via elevating the expression of phosphoenolpyruvate carboxykinase 2 (PCK2), and eventually restores the poor state of endothelial cells. This research demonstrated that early intervention by combining GPR124 with bioactive multifunctional hydrogel may have tremendous promise for restoring locomotor recovery in patients with central nervous system disorders, in addition to a translational approach for the medical therapy of SCI.
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Brain metastasis of lung cancer causes high mortality, but the exact mechanisms underlying the metastasis remain unclear. Here we report that vascular pericytes derived from CD44+ lung cancer stem cells (CSCs) in lung adenocarcinoma (ADC) potently cause brain metastases through the G-protein-coupled receptor 124 (GPR124)-enhanced trans-endothelial migration (TEM). CD44+ CSCs in perivascular niches generate the majority of vascular pericytes in lung ADC. CSC-derived pericyte-like cells (Cd-pericytes) exhibit remarkable TEM capacity to effectively intravasate into the vessel lumina, survive in the circulation, extravasate into the brain parenchyma, and then de-differentiate into tumorigenic CSCs to form metastases. Cd-pericytes uniquely express GPR124 that activates Wnt7-ß-catenin signaling to enhance TEM capacity of Cd-pericytes for intravasation and extravasation, two critical steps during tumor metastasis. Furthermore, selective disruption of Cd-pericytes, GPR124, or the Wnt7-ß-catenin signaling markedly reduces brain and liver metastases of lung ADC. Our findings uncover an unappreciated cellular and molecular paradigm driving tumor metastasis.
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Adenocarcinoma del Pulmón , Neoplasias Encefálicas , Neoplasias Pulmonares , Humanos , Adenocarcinoma del Pulmón/secundario , beta Catenina , Neoplasias Encefálicas/secundario , Cadmio , Receptores de Hialuranos , Pulmón , Neoplasias Pulmonares/patología , Pericitos , Receptores Acoplados a Proteínas GRESUMEN
Within the viral genome, short stretches of homologous host pathogen sequences (SSHHPS) span the protease cleavage sites. To identify host proteins that may be cleaved during infection, we searched the human proteome for viral protease cleavage sites (~20 amino acids). We developed a sequence-to-symptom tool, automating the search and pairing process. We used the viral protein sequence, PHI-BLAST, and UniProt database for gene ontologies and disease relationships. We applied the tool to nine neuroinvasive viruses: Venezuelan and Eastern Equine encephalitis virus (VEEV, EEEV); severe acute respiratory syndrome (SARS, SARS-CoV-2); Middle East respiratory syndrome (MERS); EV-71; Japanese encephalitis virus (JEV); West Nile (WNV); and Zika (ZIKV). A comparison of the hits identified a protein common to all nine viruses called ADGRA2 (GPR124). ADGRA2 was a predicted hit of the 3CL main protease and papain-like protease (PLpro) of SARS-CoV-2. ADGRA2 is an adhesion G protein-coupled receptor and a key endothelial regulator of brain-specific angiogenesis. It is a Wnt7A/Wnt7B specific coactivator of beta-catenin signaling and is essential for blood-brain barrier (BBB) integrity in central nervous system (CNS) diseases. We show the cleavage of the predicted sequences in MYOM1, VWF by the SARS-CoV-2 PLpro; DNAH8 (dynein) by the MERS PLpro; ADGRA2 by the alphaviral VEEV nsP2 protease; and POT1 by the SARS-CoV-2 and MERS PLpro.
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COVID-19 , Infección por el Virus Zika , Virus Zika , Caballos , Animales , Humanos , SARS-CoV-2/genética , Endopeptidasas , Péptido HidrolasasRESUMEN
Functional embryo-maternal interactions occur during the embryo implantation and placentation. Extracellular vesicles with microRNA (miR) between cells have been considered of critical importance for embryo implantation and the programming of human pregnancy. MiR-138-5p functions as the transcriptional regulator of G protein-coupled receptor 124 (GPR124). However, the signaling pathway of miR138-5p- and GPR124-adjusted NLRP3 inflammasome activation remains unclear. In this study, we examine the roles of the miR138-5p and GPR124-regulated inflammasome in embryo implantation and early pregnancy. Human decidual stromal cells were isolated from the abortus tissue and collected by curettage from missed abortion patients and normal pregnant women at 6- to 12-week gestation, after informed consent. Isolated extracellular vesicles from decidua and decidual stromal cells were confirmed by transmission electron microscopy (TEM). Next-Generation Sequencing (NGS) and microarray were performed for miR analysis. The predicated target genes of the differentially expressed miR were analyzed to identify the target genes and their pathway. We demonstrated the down-regulation of miR-138-5p and the overexpression of GPR124 in spontaneous miscarriage compared to normal pregnancy. We also showed the excessive activation of the NLRP3 inflammasome in spontaneous miscarriage compared to normal pregnancy. Here, we newly demonstrate that the miR-138-5p and GPR124-adjusted NLRP3 inflammasome were expressed in extracellular vesicles derived from decidua and decidual stromal cells, indicating that the miR-138-5p, GPR124 and NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome have a potential modulatory role on the decidual programming and placentation of human pregnancy. Our findings represent a new concept regarding the role of extracellular vesicles, miR-138-5p, GPR124, and the NLRP3 inflammasome in normal early pregnancy and spontaneous miscarriage.
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Osteoporosis occurs frequently in women after menopause and old age, and it is very easy to cause osteoporotic fractures, resulting in disability and death. In osteoporosis patients, the potential of bone marrow mesenchymal stem cells (BMSCs) to differentiate into osteoblasts gradually is inhibited, leading to decreased new bone formation. In the current study, the potential effect of G-protein-coupled receptor 124 (GPR124) on the osteoblastic differentiation of BMSCs was determined. BMSCs were isolated and cultured in osteogenic media to induced osteogenic differentiation. Then, osteogenic differentiation was evaluated by Alizarin Red staining and ALP activity. The expression of osteogenic differentiation biomarkers, and Wnt/ß-catenin signaling were determined by qRT-PCR and Western blotting. The results indicated that the expression of GPR124 was significantly increased during osteogenic differentiation of BMSCs. Moreover, GPR124 knockdown significantly inhibited osteoblastic differentiation and GPR124 overexpression promoted osteoblastic differentiation of BMSCs. GPR124 knockdown suppressed the activation of Wnt/ß-catenin signaling pathway. What's more, the increased osteogenic differentiation induced by GPR124 overexpression was abolished by the inhibitor of Wnt/ß-catenin pathway and Wnt7a knockdown. Taken together, GPR124 promotes osteogenic differentiation of BMSCs through the Wnt/ß-catenin pathway and may serve as a potential target for enhancing osteogenesis of osteoporosis patients.
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Células Madre Mesenquimatosas , Osteoporosis , Animales , Biomarcadores/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Femenino , Humanos , Osteogénesis/genética , Osteoporosis/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Within the central nervous system, Wnt7a/b are unambiguously discriminated from other Wnt ligands by an endothelial receptor complex made of the glycosylphosphatidylinositol (GPI)-anchored Reck and the adhesion G protein-coupled receptor (GPCR) Gpr124. Reck is a Wnt7a/b-specific receptor, while Gpr124 facilitates the delivery of Reck-bound Wnt7a/b ligands to Frizzled, through partially characterized mechanisms. We report that, in zebrafish, the Gpr124-Frizzled interactions are dominated by intracellular scaffolds that exploit the striking molecular mimicry between Gpr124 and Frizzled intracellular domains (ICDs): an internal Dvl-binding motif and a C-terminal ETTV motif that recruits Dlg4 and Magi3. By contrast, mammalian Gpr124 receptors exhibit an ICD-independent interaction mechanism governed by species-specific attributes of their transmembrane and extracellular domains. This mechanism seemingly evolved to replace the Dvl-mediated mechanism. By contrasting zebrafish, mouse, and human Gpr124, this study provides insights into the evolution of Gpr124/Reck function across the vertebrate clade, a receptor complex uniquely implicated in Wnt ligand-specific cellular responses.
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Receptores Acoplados a Proteínas G , Vía de Señalización Wnt , Animales , Sistema Nervioso Central , Humanos , Ligandos , Ratones , Receptores Acoplados a Proteínas G/metabolismo , Pez CebraRESUMEN
Prolonged occlusion of multiple microvessels causes microvascular injury. G protein-coupled receptor 124 (GPR124) has been reported to be required for maintaining central nervous system (CNS) angiogenesis and blood-brain barrier integrity. However, the molecular mechanisms by which GPR124 regulates pericytes during ischemia have remained elusive. Methods: A microsphere embolism-induced ischemia model was used to evaluate the expression of GPR124 following microsphere embolism. Immunocytochemistry and stochastic optical reconstruction microscopy imaging were used to assess the expression and distribution of GPR124 in human brain vascular pericytes (HBVPs) and after the treatment with 3-morpholino-sydnonimine (SIN-1) or oxygen-glucose deprivation (OGD). The effect of GPR124 knockdown or overexpression on HBVP migration was analyzed in vitro using wound healing assays and a microfluidic device. GPR124 loss-of-function studies were performed in HBVPs and HEK293 cells using CRISPR-Cas9-mediated gene deletion. Time-lapse imaging was used to assess dynamic changes in the formation of filopodia in an individual cell. Finally, to explore the functional domains required for GPR124 activity, deletion mutants were constructed for each of the N-terminal domains. Results: GPR124 expression was increased in pericytes following microsphere embolism. Morphological analysis showed localization of GPR124 to focal adhesions where GPR124 bound directly to the actin binding protein vinculin and upregulated Cdc42. SIN-1 or OGD treatment redistributed GPR124 to the leading edges of HBVPs where GPR124 signaling was required for pericyte filopodia formation and directional migration. Partial deletion of GPR124 domains decreased SIN-1-induced filopodia formation and cell migration. Conclusion: Taken together, our results provide the first evidence for a role of GPR124 in pericyte migration under ischemic conditions and suggest that GPR124 was essential for Cdc42 activation and filopodia formation.
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Isquemia Encefálica/metabolismo , Polaridad Celular/fisiología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Pericitos/citología , Pericitos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Western Blotting , Línea Celular , Polaridad Celular/genética , Adhesiones Focales/metabolismo , Células HEK293 , Humanos , Inmunohistoquímica , Inmunoprecipitación , Lentivirus/genética , Masculino , Ratones , Plásmidos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Acoplados a Proteínas G/genética , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiologíaRESUMEN
The molecular basis of Wnt-Frizzled specificity is a central question in developmental biology. Reck, a multi-domain and multi-functional glycosylphosphatidylinositol-anchored protein, specifically enhances beta-catenin signaling by Wnt7a and Wnt7b in cooperation with the 7-transmembrane protein Gpr124. Among amino acids that distinguish Wnt7a and Wnt7b from other Wnts, two clusters are essential for signaling in a Reck- and Gpr124-dependent manner. Both clusters are far from the site of Frizzled binding: one resides at the amino terminus and the second resides in a protruding loop. Within Reck, the fourth of five tandem repeats of an unusual domain with six-cysteines (the CC domain) is essential for Wnt7a stimulation: substitutions P256A and W261A in CC4 eliminate this activity without changing protein abundance or surface localization. Mouse embryos carrying ReckP256A,W261A have severe defects in forebrain angiogenesis, providing the strongest evidence to date that Reck promotes CNS angiogenesis by specifically stimulating Wnt7a and Wnt7b signaling.
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Encéfalo/irrigación sanguínea , Encéfalo/crecimiento & desarrollo , Receptores Frizzled/metabolismo , Proteínas Ligadas a GPI/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/patología , Receptores Frizzled/química , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Células HEK293 , Humanos , Ligandos , Luciferasas/metabolismo , Ratones , Mutación/genética , Neovascularización Fisiológica , Dominios Proteicos , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/químicaRESUMEN
The purpose of the present study was to investigate the anticancer effects of ß-elemene and paclitaxel for bone neoplasms. MTT assay, reverse transcription-quantitative polymerase chain reaction, western blotting, flow cytometry and immunostaining were used to analyze the combined effects of ß-elemene and paclitaxel both in vitro and in vivo. The results demonstrated that combined treatment of ß-elemene and paclitaxel (ß-elemene-paclitaxel) significantly inhibited growth and aggressiveness of U-2OS cells compared with either ß-elemene or paclitaxel treatment alone. It was demonstrated that ß-elemene promoted paclitaxel-induced apoptosis of U-2OS cells. Anti-apoptosis B-cell lymphoma (Bcl)-2 and Bcl-w genes were downregulated and pro-apoptotic Bcl-2-associated agonist of cell death and caspase-3 genes were upregulated in U-2OS cells following treatment with ß-elemene-paclitaxel. Treatment of ß-elemene-paclitaxel arrested the cell cycle and decreased cyclin-dependent kinase, cyclin-B1, P21 and P27 expression levels and decreased resistant genes alterations of ATP binding cassette subfamily B member 1, LDL receptor related protein and TS in U-2OS cells. Results demonstrated that ß-elemene-paclitaxel decreased G-protein coupled receptor 124 (GPR124), vascular endothelial growth factor receptor, matrix metallopeptidase (MMP)-3, MMP-9 expression levels and increased endostatin, TIMP metallopeptidase inhibitor (TIMP)-1, TIMP-2 expression in U-2OS cells. In vivo assay demonstrated that ß-elemene-paclitaxel treatment inhibited tumor growth of BALB/c-nu/nu nude mice and prolonged survival rate of tumor-bearing mice. Immunostaining demonstrated that ß-elemene-paclitaxel treatment increased apoptotic bodies, GPR124 and increased endostatin, TIMP-1 and TIMP-2 expression in tumor tissues. In conclusion, these results suggest that the combined treatment of ß-elemene-paclitaxel is more effective at inhibiting bone neoplasm growth than ß-elemene or paclitaxel single treatment GPR124.
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The adhesion G protein-coupled receptor A2 (Adgra2) is a seven transmembrane receptor that has been described to be a regulator for angiogenesis in mice. Furthermore, the zebrafish ouchless mutant is unable to develop dorsal root ganglia through a disrupted trafficking of Adgra2. Besides RNA sequencing data, nothing is reported about Adgra2 in the south African crawled frog Xenopus laevis. In this study, we investigated for the first time the spatio-temporal expression of adgra2 during early Xenopus embryogenesis in detail. In silico approaches showed that the genomic adgra2 region as well as the Adgra2 protein sequence is highly conserved among different species including Xenopus. RT-PCR experiments confirmed that embryonic adgra2 expression is primarily detected at the beginning of neurulation and is then present throughout the whole Xenopus embryogenesis until stage 42. Whole mount in situ hybridization approaches visualized adgra2 expression in many tissues during Xenopus embryogenesis such as the cardiovascular system including the heart, the migrating neural crest cells and the developing eye including the periocular mesenchyme. Our results indicate a role of Adgra2 for embryogenesis and are a good starting point for further functional studies during early vertebrate development.
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Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Animales , Embrión no Mamífero/citología , Desarrollo Embrionario , Receptores Acoplados a Proteínas G/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/crecimiento & desarrolloRESUMEN
WNT7A and WNT7B control CNS angiogenesis and blood-brain barrier formation by activating endothelial Wnt/ß-catenin signaling. The GPI-anchored protein RECK and adhesion G protein-coupled receptor GPR124 critically regulate WNT7-specific signaling in concert with FZD and LRP co-receptors. Here, we demonstrate that primarily the GPR124 ectodomain, but not its transmembrane and intracellular domains, mediates RECK/WNT7-induced canonical Wnt signaling. Moreover, RECK is the predominant binding partner of GPR124 in rat brain blood vessels in situ. WNT7A and WNT7B, but not WNT3A, directly bind to purified recombinant soluble RECK, full-length cell surface RECK, and the GPR124:RECK complex. Chemical cross-linking indicates that RECK and WNT7A associate with 1:1 stoichiometry, which stabilizes short-lived, active, monomeric, hydrophobic WNT7A. In contrast, free WNT7A rapidly converts into inactive, hydrophilic aggregates. Overall, RECK is a selective WNT7 receptor that mediates GPR124/FZD/LRP-dependent canonical Wnt/ß-catenin signaling by stabilizing active cell surface WNT7, suggesting isoform-specific regulation of Wnt bioavailability.