RESUMO
EphB1 is required for proper guidance of cortical axon projections during brain development, but how EphB1 regulates this process remains unclear. We show here that EphB1 conditional knockout (cKO) in GABAergic cells (Vgat-Cre), but not in cortical excitatory neurons (Emx1-Cre), reproduced the cortical axon guidance defects observed in global EphB1 KO mice. Interestingly, in EphB1 cKOVgat mice, the misguided axon bundles contained co-mingled striatal GABAergic and somatosensory cortical glutamatergic axons. In wild-type mice, somatosensory axons also co-fasciculated with striatal axons, notably in the globus pallidus, suggesting that a subset of glutamatergic cortical axons normally follows long-range GABAergic axons to reach their targets. Surprisingly, the ectopic axons in EphB1 KO mice were juxtaposed to major blood vessels. However, conditional loss of EphB1 in endothelial cells (Tie2-Cre) did not produce the axon guidance defects, suggesting that EphB1 in GABAergic neurons normally promotes avoidance of these ectopic axons from the developing brain vasculature. Together, our data reveal a new role for EphB1 in GABAergic neurons to influence proper cortical glutamatergic axon guidance during brain development.
Assuntos
Orientação de Axônios , Células Endoteliais , Animais , Camundongos , Axônios/fisiologia , Neurônios GABAérgicos , Camundongos Knockout , Receptores Proteína Tirosina Quinases , Receptor EphB1/metabolismoRESUMO
Social behavior starts with dynamic approach prior to the final consummation. The flexible processes ensure mutual feedback across social brains to transmit signals. However, how the brain responds to the initial social stimuli precisely to elicit timed behaviors remains elusive. Here, by using real-time calcium recording, we identify the abnormalities of EphB2 mutant with autism-associated Q858X mutation in processing long-range approach and accurate activity of prefrontal cortex (dmPFC). The EphB2-dependent dmPFC activation precedes the behavioral onset and is actively associated with subsequent social action with the partner. Furthermore, we find that partner dmPFC activity is responsive coordinately to the approaching WT mouse rather than Q858X mutant mouse, and the social defects caused by the mutation are rescued by synchro-optogenetic activation in dmPFC of paired social partners. These results thus reveal that EphB2 sustains neuronal activation in the dmPFC that is essential for the proactive modulation of social approach to initial social interaction.
Assuntos
Córtex Pré-Frontal , Receptor EphB2 , Comportamento Social , Animais , Camundongos , Encéfalo , Neurônios/fisiologia , Córtex Pré-Frontal/fisiologia , Receptor EphB2/genética , Receptor EphB2/fisiologiaRESUMO
Many brain diseases lead to a reduction in the number of functional neurons and it would be of value to be able to increase the number of neurons in the affected brain areas. In this study, we examined whether we can promote neural stem cells to produce mature neurons and whether an increase in the mature neurons can affect cognitive performance. We detected that the EphB2 receptor is localized in immature basolateral amygdala (BLA) neurons. We therefore aimed to increase the level of EphB2 activity in neural stem cells (NSCs) in the BLA and examine the effects on the production of mature neurons and cognition. Toward that end, we utilized a photoactivatable EphB2 construct (optoEphB2) to increase EphB2 forward signaling in NSCs in the BLA. We revealed that the activation of optoEphB2 in NSCs in the BLA increased the level of immature and mature neurons in the BLA. We further found that activation of optoEphB2 in BLA NSCs enhanced auditory, but not contextual, long-term fear memory formation. Impairing EphB2 forward signaling did not affect the level of immature and mature neurons in the BLA. This study provides evidence that NSCs can be promoted to produce mature neurons by activating EphB2 to enhance specific brain functions.
Assuntos
Complexo Nuclear Basolateral da Amígdala , Memória de Longo Prazo , Células-Tronco Neurais , Neurogênese , Receptor EphB2 , Animais , Receptor EphB2/metabolismo , Receptor EphB2/genética , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Memória de Longo Prazo/fisiologia , Masculino , Complexo Nuclear Basolateral da Amígdala/metabolismo , Complexo Nuclear Basolateral da Amígdala/citologia , Camundongos , Neurônios/metabolismo , Neurônios/citologia , Camundongos Endogâmicos C57BL , Medo/fisiologia , Transdução de SinaisRESUMO
BACKGROUND: The EphB receptor tyrosine kinase family participates in intricate signaling pathways that orchestrate neural networks, guide neuronal axon development, and modulate synaptic plasticity through interactions with surface-bound ephrinB ligands. Additionally, Kalirin, a Rho guanine nucleotide exchange factor, is notably expressed in the postsynaptic membrane of excitatory neurons and plays a role in synaptic morphogenesis. This study postulates that Kalirin may act as a downstream effector of EphB3 in epilepsy. This investigation focuses on understanding the link between EphB3 and epilepsy. MATERIALS AND METHODS: Chronic seizure models using LiCl-pilocarpine (LiCl/Pilo) and pentylenetetrazol were developed in rats. Neuronal excitability was gauged through whole-cell patch clamp recordings on rat hippocampal slices. Real-time PCR determined Kalirin's mRNA expression, and Western blotting was employed to quantify EphB3 and Kalirin protein levels. Moreover, dendritic spine density in epileptic rats was evaluated using Golgi staining. RESULTS: Modulation of EphB3 functionality influenced acute seizure severity, latency duration, and frequency of spontaneous recurrent seizures. Golgi staining disclosed an EphB3-driven alteration in dendritic spine density within the hippocampus of epileptic rats, underscoring its pivotal role in the reconfiguration of hippocampal neural circuits. Furthermore, our data propose Kalirin as a prospective downstream mediator of the EphB3 receptor. CONCLUSIONS: Our findings elucidate that EphB3 impacts the action potential dynamics in isolated rat hippocampal slices and alters dendritic spine density in the inner molecular layer of epileptic rat hippocampi, likely through Kalirin-mediated pathways. This hints at EphB3's significant role in shaping excitatory circuit loops and recurrent seizure activity via Kalirin.
Assuntos
Epilepsia , Neurônios , Ratos , Animais , Ratos Sprague-Dawley , Estudos Prospectivos , Neurônios/metabolismo , Epilepsia/metabolismo , Convulsões/metabolismoRESUMO
Recurrent spontaneous abortion, defined as at least three unexplained abortions occurring before the 20-24 week of pregnancy, has a great impact on women's quality of life. Ephrin receptor B4 has been associated with trophoblast function in preeclampsia. The present study aimed to verify the hypothesis that ephrin receptor B4 regulates the biological functions of trophoblasts in recurrent spontaneous abortion and to explore the upstream mechanism. Ephrin receptor B4 was overexpressed in mice with recurrent spontaneous abortion. Moreover, ephrin receptor B4 inhibited trophoblast proliferation, migration, and invasion while promoting apoptosis. Downregulation of early growth response protein 1 expression in mice with recurrent spontaneous abortion led to ephrin receptor B4 overexpression. Poor expression of WT1-associated protein in mice with recurrent spontaneous abortion reduced the modification of early growth response protein 1 mRNA methylation, resulting in decreased early growth response protein 1 mRNA stability and expression. Overexpression of WT1-associated protein reduced the incidence of recurrent spontaneous abortion in mice by controlling the phenotype of trophoblasts, which was reversed by early growth response protein 1 knockdown. All in all, our findings demonstrate that dysregulation of WT1-associated protein contributes to the instability of early growth response protein 1, thereby activating ephrin receptor B4-induced trophoblast dysfunction in recurrent spontaneous abortion. Our study provides novel insights into understanding the molecular pathogenesis of recurrent spontaneous abortion.
Assuntos
Aborto Habitual , Aborto Espontâneo , Animais , Feminino , Humanos , Camundongos , Gravidez , Aborto Habitual/metabolismo , Aborto Espontâneo/genética , Movimento Celular , Proliferação de Células , Proteína 1 de Resposta de Crescimento Precoce , Efrinas/metabolismo , Qualidade de Vida , Trofoblastos/metabolismoRESUMO
Variants in EPHB4 (Ephrin type B receptor 4), a transmembrane tyrosine kinase receptor, have been identified in individuals with various vascular anomalies including Capillary Malformation-Arteriovenous Malformation syndrome 2 and lymphatic-related (non-immune) fetal hydrops (LRHF). Here, we identify two novel variants in EPHB4 that disrupt the SAM domain in two unrelated individuals. Proband 1 presented within the LRHF phenotypic spectrum with hydrops, and proband 2 presented with large nuchal translucency prenatally that spontaneously resolved in addition to dysmorphic features on exam postnatally. These are the first disease associated variants identified that do not disrupt EPHB4 protein expression or tyrosine-kinase activity. We identify that EPHB4 SAM domain disruptions can lead to aberrant downstream signaling, with a loss of the SAM domain resulting in elevated MAPK signaling in proband 1, and a missense variant within the SAM domain resulting in increased cell proliferation in proband 2. This data highlights that a functional SAM domain is required for proper EPHB4 function and vascular development.
Assuntos
Hidropisia Fetal , Motivo Estéril alfa , Feminino , Humanos , Hidropisia Fetal/diagnóstico por imagem , Hidropisia Fetal/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/genética , Receptor EphB4/genética , Receptor EphB4/metabolismoRESUMO
Ephrin-B-EphB signaling can promote pain through ligand-receptor interactions between peripheral cells, like immune cells expressing ephrin-Bs, and EphB receptors expressed by DRG neurons. Previous studies have shown increased ephrin-B2 expression in peripheral tissues like synovium of rheumatoid and osteoarthritis patients, indicating the clinical significance of this signaling. The primary goal of this study was to understand how ephrin-B2 acts on mouse and human DRG neurons, which express EphB receptors, to promote pain and nociceptor plasticity. We hypothesized that ephrin-B2 would promote nociceptor plasticity and hyperalgesic priming through MNK-eIF4E signaling, a critical mechanism for nociceptive plasticity induced by growth factors, cytokines and nerve injury. Both male and female mice developed dose-dependent mechanical hypersensitivity in response to ephrin-B2, and both sexes showed hyperalgesic priming when challenged with PGE2 injection either to the paw or the cranial dura. Acute nociceptive behaviors and hyperalgesic priming were blocked in mice lacking MNK1 (Mknk1 knockout mice) and by eFT508, a specific MNK inhibitor. Sensory neuron-specific knockout of EphB2 using Pirt-Cre demonstrated that ephrin-B2 actions require this receptor. In Ca2+-imaging experiments on cultured DRG neurons, ephrin-B2 treatment enhanced Ca2+ transients in response to PGE2 and these effects were absent in DRG neurons from MNK1-/- and EphB2-PirtCre mice. In experiments on human DRG neurons, ephrin-B2 increased eIF4E phosphorylation and enhanced Ca2+ responses to PGE2 treatment, both blocked by eFT508. We conclude that ephrin-B2 acts directly on mouse and human sensory neurons to induce nociceptor plasticity via MNK-eIF4E signaling, offering new insight into how ephrin-B signaling promotes pain.
Assuntos
Efrina-B2 , Fator de Iniciação 4E em Eucariotos , Hiperalgesia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor EphB2 , Transdução de Sinais , Animais , Hiperalgesia/metabolismo , Humanos , Masculino , Receptor EphB2/metabolismo , Receptor EphB2/genética , Feminino , Efrina-B2/metabolismo , Efrina-B2/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Gânglios Espinais/metabolismo , Gânglios Espinais/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Camundongos , Nociceptividade/efeitos dos fármacos , Células Cultivadas , Nociceptores/metabolismoRESUMO
BACKGROUND: Erythropoietin-producing hepatocellular (EPH) receptors are the largest known family of receptor tyrosine kinases characterized in humans. These proteins are involved in tissue organization, synaptic plasticity, vascular development and the progression of various diseases including cancer. The Erythropoietin-producing hepatocellular receptor tyrosine kinase member EphB6 is a pseudokinase which has not attracted an equivalent amount of interest as its enzymatically-active counterparts. The aim of this study was to assess the expression of EphB6 in pituitary tumors. METHODS AND RESULTS: Human normal pituitaries and pituitary tumors were examined for EphB6 mRNA expression using real-time PCR and for EphB6 protein by immunohistochemistry and Western blotting. EphB6 was highly expressed in non-functioning pituitary neuroendocrine tumors (NF-PitNETs) versus the normal pituitary and GH-secreting PitNETs. EphB6 mRNA expression was correlated with tumor size. CONCLUSIONS: Our results suggest EphB6 aberrant expression in NF-PitNETs. Future studies are warranted to determine the role and significance of EphB6 in NF-PitNETs tumorigenesis.
Assuntos
Carcinoma Hepatocelular , Eritropoetina , Neoplasias Hepáticas , Tumores Neuroendócrinos , Neoplasias Hipofisárias , Humanos , Neoplasias Hipofisárias/genética , Receptores da Eritropoetina , Tumores Neuroendócrinos/genética , Linhagem Celular Tumoral , Neoplasias Hepáticas/genética , RNA Mensageiro/genéticaRESUMO
Capillary malformation-arteriovenous malformation is a rare autosomal dominant disorder associated with EPHB4 loss-of-function mutations. We report the unique presentation of a 6-year-old girl with multiple capillary malformations in a unilateral segmental distribution affecting the right hemiface, right upper chest, and right arm associated with overgrowth. Targeted next-generation sequencing on a tissue sample revealed a novel heterozygotic variant in the EPHB4 gene (NM_004444.5 (EPHB4): c.715T>A, p.[Cys239Ser]). This case highlights a distinct presentation of CM-AVM type 2 and showcases a new variant in EPHB4 not previously reported in the literature.
Assuntos
Malformações Arteriovenosas , Capilares/anormalidades , Mancha Vinho do Porto , Feminino , Humanos , Criança , Proteína p120 Ativadora de GTPase/genética , Mancha Vinho do Porto/genética , Malformações Arteriovenosas/diagnóstico , Malformações Arteriovenosas/genética , MutaçãoRESUMO
Previous studies have demonstrated that the synaptic EphB1 receptor tyrosine kinase is a major mediator of neuropathic pain, suggesting that targeting the activity of this receptor might be a viable therapeutic option. Therefore, we set out to determine if any FDA-approved drugs can act as inhibitors of the EphB1 intracellular catalytic domain. An in silico screen was first used to identify a number of tetracycline antibiotics which demonstrated potential docking to the ATP-binding catalytic domain of EphB1. Kinase assays showed that demeclocycline, chlortetracycline, and minocycline inhibit EphB1 kinase activity at low micromolar concentrations. In addition, we cocrystallized chlortetracycline and EphB1 receptor, which confirmed its binding to the ATP-binding domain. Finally, in vivo administration of the three-tetracycline combination inhibited the phosphorylation of EphB1 in the brain, spinal cord, and dorsal root ganglion (DRG) and effectively blocked neuropathic pain in mice. These results indicate that demeclocycline, chlortetracycline, and minocycline can be repurposed for treatment of neuropathic pain and potentially for other indications that would benefit from inhibition of EphB1 receptor kinase activity.
Assuntos
Sistema Nervoso Central/enzimologia , Clortetraciclina , Neuralgia , Inibidores de Proteínas Quinases , Receptor EphB1 , Animais , Clortetraciclina/química , Clortetraciclina/farmacologia , Cristalografia por Raios X , Humanos , Masculino , Camundongos , Neuralgia/tratamento farmacológico , Neuralgia/enzimologia , Domínios Proteicos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Receptor EphB1/antagonistas & inibidores , Receptor EphB1/química , Receptor EphB1/metabolismoRESUMO
OBJECTIVE: To investigate the expression of ephrin type B receptor 3 (EphB3) in thyroid tumors and its usage as an ancillary diagnostic biomarker for thyroid tumors. METHODS: Formalin-fixed and paraffin-embedded (FFPE) tissue samples (78 cases) and FNAC samples (57 cases) were assessed with the EphB3 antibody using immunohistochemistry. PTC and other thyroid follicular tumors were compared regarding their EphB3 expression. Sanger sequencing was used to assess for the presence of a BRAF V600E mutation. RESULTS: EphB3 was positive in 81.8 % (27/33) of papillary thyroid carcinoma (PTC), 83.3 % (5/6) of medullary thyroid carcinoma (MTC), 25 % (1/4) of hyperplastic/adenomatoid nodule (HN), 14.3 % (1/7) of follicular adenoma (FA), and negative in follicular tumors of uncertain malignant potential (FT-UMP) (0/13), noninvasive follicular neoplasm with papillary-like nuclear features (NIFTP) (0/7), thyroid follicular carcinoma (TFC) (0/4), Hashimoto's thyroiditis (0/4), and normal thyroid follicular tissues (0/33). In cellular blocks, EphB3 was positive in 87.1 % (20/23) of PTC, 75 % (3/4) of MTC, 20 % (2/10) of HN, and negative in atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) (0/20) and normal thyroid follicular cells (0/10). CONCLUSION: EphB3 is expressed in the majority of PTC, but less so in benign follicular nodules. EphB3 expression in fine needle aspiration cytology (FNAC) specimens can be used as a diagnostic tool to differentiate thyroid cancer from other follicular lesions in its differential diagnosis, especially AUS/FLUS and PTC.
Assuntos
Adenocarcinoma Folicular , Adenoma , Carcinoma Neuroendócrino , Carcinoma Papilar , Neoplasias da Glândula Tireoide , Nódulo da Glândula Tireoide , Humanos , Adenocarcinoma Folicular/patologia , Biomarcadores , Carcinoma Papilar/diagnóstico , Carcinoma Papilar/patologia , Hiperplasia , Estudos Retrospectivos , Câncer Papilífero da Tireoide/diagnóstico , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Nódulo da Glândula Tireoide/patologia , Receptor EphB3RESUMO
EphB2-ephrinB signalling, which plays a major role in cell segregation during embryonic development and tissue homeostasis, induces an important reorganization of the cortical actin network. We have previously reported that myosin 1b contributes to reorganization of the cortical actin network upon EphB2 signalling. In this report, we identify Plekhh1 as a new partner of members of the myosin 1 family and EphB2 receptors. Plekhh1 interacts with myosin 1b via its N-terminal domain and with EphB2 via its C-terminal domain. Furthermore, Plekhh1 is tyrosine phosphorylated, and this depends on EphB2 kinase activity. Similar to the effects of manipulating levels of myosin 1b and myosin 1c, manipulation of Plekhh1 expression levels alters the formation of filopodia, the length of focal adhesions and the formation of blebs. Furthermore, binding of the Plekhh1 interacting domain to myosin 1b increases the motor activity of myosin 1b in vitro. Taken together, our data show that Plekhh1 is an effector of EphB2 and suggest that Plekhh1 regulates the cortical actin network via the interaction of its N-terminal domain with myosin 1 upon EphB2-ephrinB signalling.
Assuntos
Actinas , Receptor EphB2 , Actinas/genética , Comunicação Celular , Fosforilação , Receptor EphB2/genética , Transdução de SinaisRESUMO
Intracranial vascular malformations manifest on a continuum ranging from predominantly arterial to predominantly venous in pathology. Cerebral cavernous malformations (CCMs) are capillary malformations that exist at the midpoint of this continuum. The axon guidance factor Ephrin B2 and its receptor EphB4 are critical regulators of vasculogenesis in the developing central nervous system. Ephrin B2/EphB4 dysregulation has been implicated in the pathogenesis of arterial-derived arteriovenous malformations and vein-based vein of Galen malformations. Increasing evidence supports the hypothesis that aberrant Ephrin B2/EphB4 signaling may contribute to developing vascular malformations, but their role in CCMs remains largely uncharacterized. Evidence of Ephrin dysregulation in CCMs would be important to establish a common link in the pathogenic spectrum of EphrinB2/Ephb4 dysregulation. By studying patient-derived primary CCM endothelial cells (CCMECs), we established that CCMECs are functionally distinct from healthy endothelial cell controls; CCMECs demonstrated altered patterns of migration, motility, and impaired tube formation. In addition to the altered phenotype, the CCMECs also displayed an increased ratio of EphrinB2/EphB4 compared to the healthy endothelial control cells. Furthermore, whole exome sequencing identified mutations in both EphrinB2 and EphB4 in the CCMECs. These findings identify functional alterations in the EphrinB2/EphB4 ratio as a feature linking pathophysiology across the spectrum of arterial, capillary, and venous structural malformations in the central nervous system while revealing a putative therapeutic target.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central , Receptor EphB2 , Receptor EphB4 , Humanos , Receptor EphB4/genética , Receptor EphB2/genética , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Células Endoteliais/patologia , Cultura Primária de Células , Sequenciamento do Exoma , Masculino , Feminino , Pré-Escolar , Criança , AdolescenteRESUMO
OBJECTIVES: Human-derived pulp stem cells play key roles during dentinogenesis. Erythropoietin is reportedly involved in osteoblastogenesis and facilitates bone formation. However, the mechanism is still unknown. This research was to study the potential of erythropoietin in enhancing odontoblastic differentiation of human-derived pulp stem cells and to determine the underlying mechanism. METHODS: The human-derived pulp stem cells were treated with erythropoietin, EphB4 inhibitor, and MAPK inhibitors, and the odontoblastic differentiation was measured by ALP staining, ALP activity assay, alizarin red S staining, and their quantitative analysis, and RT-qPCR of DSPP, DMP1, OCN, and RUNX2. The direct pulp capping model was established to evaluate the formation of tertiary dentin after treatment with erythropoietin. Western blot assay was conducted to assess relevant protein expressions in the phosphorylated EphB4 and MAPK pathway. RESULTS: The results showed that erythropoietin promoted odontoblastic differentiation of human-derived pulp stem cells at 20 U/ml. Erythropoietin induced tertiary dentin formation in vivo. The potential mechanism of this was upregulating phosphorylated EphB4 and phosphorylated MAPK; furthermore, this effect could be decreased by EphB4 inhibitors, which inhibited MAPK phosphorylation. Blockage of MAPK pathways attenuated human-derived pulp stem cells' odontoblastic differentiation, suggesting that MAPK pathways are involved. CONCLUSION: Erythropoietin induced tertiary dentin formation in vivo. And erythropoietin enhanced human-derived pulp stem cells' odontoblastic differentiation via the EphB4-mediated MAPK signaling pathway.
Assuntos
Eritropoetina , Transdução de Sinais , Humanos , Sistema de Sinalização das MAP Quinases , Diferenciação Celular , Odontoblastos , Polpa Dentária , Eritropoetina/farmacologia , Eritropoetina/metabolismo , Células-Tronco , Células CultivadasRESUMO
BACKGROUND: Osteoblasts suppress osteoclastogenesis during the reversal phase of bone remodelling and the mechanism needs to be further investigated. Here, we investigated the role of histone demethylase Jumonji domain-containing 3 (Jmjd3) in osteoblasts on regulating osteoclastogenesis. METHODS: Jmjd3 expression was silenced in osteoblasts. Osteoblasts and osteoclasts were co-cultured in direct or indirect contact ways, and osteoclastogenesis was determined by tartrate-resistant acid phosphatase (TRAP) staining and Western blotting. Additionally, Ephrin receptor B4 (EphB4) and receptor activator of nuclear factor-kappa Β ligand (RANKL) expression were quantified in osteoblasts via real-time PCR, Western blotting, and enzyme-linked immunosorbent assay. Subsequently, EphB4 was overexpressed in osteoblasts and RANKL expression and osteoclastogenesis was quantified. RESULTS: Osteoclastogenesis and marker protein expression levels was promoted when osteoclasts were co-cultured with Jmjd3-silenced osteoblasts. Silencing of Jmjd3 expression in osteoblasts decreased EphB4 expression, owing to suppression of demethylation of H3K27me3 on the promoter region of EphB4. Whereas RANKL expression was upregulated in Jmjd3-silenced osteoblasts. Overexpression of EphB4 in osteoblasts inhibited osteoclastogenesis and RANKL expression. CONCLUSION: Jmjd3 in osteoblasts is a crucial regulator of osteoblast-to-osteoclast communication through EphB4-EphrinB2, RANKL-RANK and EphB4-RANKL signalling axes, suggesting the pivotal role of Jmjd3 in bone remodelling process in bone destruction disease such as chronic apical periodontitis.
Assuntos
Osteoblastos , Osteogênese , Diferenciação Celular , Células Cultivadas , Ligantes , NF-kappa B/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Disruption of intestinal barrier function and an imbalance in intestinal immunity are crucial for the occurrence and development of ulcerative colitis. Because of their important roles in regulating inflammation and immunity, exosomes (Exos) released from bone marrow mesenchymal stem cells (BMSCs) may be useful for treating ulcerative colitis. The EphB/EphrinB signaling pathway plays a crucial role in the inflammatory process and the development and function of immune cells, and can mediate long-distance intercellular communication through extracellular vesicles. This study was conducted to explore the effects of pre-modified BMSC-Exos expressing EphB2 (EphB2-Exos) on immunoregulation in vitro. METHODS: We transfected a lentivirus vector encoding EphB2 into BMSCs and isolated EphB2-Exos from the culture supernatant. Inflammation and oxidative damage in the human colon adenocarcinoma cell line (Caco-2) were induced by dextran sulfate sodium/hydrogen peroxide. In addition, spleen CD4+ T lymphocytes of rats were sorted in vitro. We conducted a series of experiments to explore the biological functions of EphB2-Exos. RESULTS: EphB2-Exos were successfully isolated and were found to significantly protect the activity, proliferation, and migration of Caco-2 cells that were inhibited by dextran sulfate sodium. EphB2-Exos alleviated inflammation and apoptosis and increased the activity of antioxidant enzymes while inhibiting oxidative stress in Caco-2 cells. EphB2-Exos restored intestinal barrier function by inhibiting the RhoA/ROCK pathway and regulated the polarization of CD4+T cells. CONCLUSION: EphB2-Exos enhanced intestinal barrier function and regulated the immune balance by inhibiting the RhoA/ROCK pathway in vitro. These findings suggest that EphB2-Exos can be applied as a cell-free therapy for ulcerative colitis.
Assuntos
Adenocarcinoma , Colite Ulcerativa , Neoplasias do Colo , Exossomos , Células-Tronco Mesenquimais , Ratos , Humanos , Animais , Exossomos/metabolismo , Células CACO-2 , Colite Ulcerativa/metabolismo , Adenocarcinoma/metabolismo , Sulfato de Dextrana/metabolismo , Neoplasias do Colo/metabolismo , Inflamação/metabolismoRESUMO
Cavernous malformations (CM) that arise in the central nervous system have long been considered congenital, while there are many reports of de novo non-familial-type CM adjacent to developmental venous anomalies (DVA) or after radiation. The mechanisms that cause de novo formations of sporadic cavernous malformation (CM) still remain unknown and purely speculative. We report a case of de novo cerebral CM in a child with multiple developmental venous anomalies and cutaneous vascular malformations. Histological examination and whole-exome sequencing (WES) was performed on a fresh-frozen tissue sample of the CM. WES revealed 2 missense non-synonymous variants in two genes, EPHB4 and PIK3CA. The mutant allele of EPHB4 (NM_004444.4: c.1840 T > C, p.Y614H) appeared in 248/469 WES reads (allele frequency, 52.88%), which suggested the mutation a germline one. PIK3CA (NM_006218.2) somatic mutations were found in exon 9: c.1624G > A (p.Glu542Lys) with variant frequency of 2.2% (2/89 WES reads). We did not find any non-synonymous mutations of the three CCM genes (KRIT1, CCM2, and PDCD10) in this patient. Our findings suggested that the combination of gain of function in PIK3CA and loss of function in EPHB4 may play an important role in the pathogenesis of CM, which can develop in acquired form like tumorigenesis.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central , Criança , Humanos , Proteínas de Transporte/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Mutação em Linhagem Germinativa , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Proteínas de Membrana/genética , Mutação , Proteínas Proto-Oncogênicas/genéticaRESUMO
OBJECTIVE: The aim of this study was to investigate the role of ephrinB2-EphB4 signalling in alveolar bone remodelling on the tension side during orthodontic tooth movement (OTM). MATERIALS AND METHODS: An OTM model was established on sixty 8-week-old male Wistar rats. They were randomly divided into the experimental group and the control group. The animals in the experimental group were administrated with subcutaneous injection of EphB4 inhibitor NVP-BHG712 every other day, whereas the control group received only the vehicle. Samples containing the maxillary first molar and the surrounding bone were collected after 0, 3, 7, 14 and 21 days of tooth movement. RESULTS: EphrinB2-EphB4 signalling was actively expressed on the tension side during tooth movement. Micro-CT analysis showed the distance of tooth movement in the experimental group was significantly greater than that of the control group (P < .05) with significantly increased trabecular separation (Tb. Sp) and decreased trabecular number (Tb. N) from day 14 to day 21. The number of osteoclasts significantly increased in the experimental group compared with the control group after 3 and 7 days of tooth movement (P < .05). The expressions of alkaline phosphatase (ALP) and osteopontin (OPN) were significantly reduced by inhibition of EphB4 (P < .05). CONCLUSION: The inhibition of EphB4 suppressed bone formation and enhanced bone resorption activities on the tension side of tooth movement. The ephrinB2-EphB4 signalling might play an important role in alveolar bone remodelling during OTM.
Assuntos
Efrina-B2 , Técnicas de Movimentação Dentária , Animais , Masculino , Ratos , Remodelação Óssea , Efrina-B2/metabolismo , Osteoclastos/metabolismo , Ratos Wistar , Efrinas/metabolismo , Transdução de SinaisRESUMO
Liver fibrosis is a pathological process characterized by the excessive synthesis and accumulation of extracellular matrix proteins (ECMs) contributed mainly by the activated hepatic stellate cells (HSCs). Currently, no direct and effective anti-fibrotic agents have been approved for clinical use worldwide. Although the dysregulation of Eph receptor tyrosine kinase EphB2 has been reported to associate with the development of liver fibrosis, the involvement of other Eph family members in liver fibrosis remains underexplored. In this study, we found that the expression of EphB1 is significantly increased accompanying remarkable neddylation in activated HSCs. Mechanistically, this neddylation enhanced the kinase activity of EphB1 by the prevention of its degradation, thereby promoting the proliferation, migration, and activation of HSCs. Our findings revealed the involvement of EphB1 in the development of liver fibrosis through its neddylation, which provides new insights into the Eph receptor signaling and a potential target for the treatment of liver fibrosis.
Assuntos
Cirrose Hepática , Transdução de Sinais , Humanos , Células Estreladas do Fígado/metabolismo , Fígado/metabolismo , Cirrose Hepática/metabolismo , Fosforilação , Receptor EphB1RESUMO
EphB4 angiogenic kinase over-expression in Mesothelioma cells relies upon a degradation rescue signal provided by autocrine IGF-II activation of Insulin Receptor A. However, the identity of the molecular machinery involved in EphB4 rapid degradation upon IGF-II signal deprivation are unknown. Using targeted proteomics, protein-protein interaction methods, PCR cloning, and 3D modeling approaches, we identified a novel ubiquitin E3 ligase complex recruited by the EphB4 C tail upon autocrine IGF-II signal deprivation. We show this complex to contain a previously unknown N-Terminal isoform of Deltex3 E3-Ub ligase (referred as "DTX3c"), along with UBA1(E1) and UBE2N(E2) ubiquitin ligases and the ATPase/unfoldase Cdc48/p97. Upon autocrine IGF-II neutralization in cultured MSTO211H (a Malignant Mesothelioma cell line that is highly responsive to the EphB4 degradation rescue IGF-II signal), the inter-molecular interactions between these factors were enhanced and their association with the EphB4 C-tail increased consistently with the previously described EphB4 degradation pattern. The ATPase/unfoldase activity of Cdc48/p97 was required for EphB4 recruitment. As compared to the previously known isoforms DTX3a and DTX3b, a 3D modeling analysis of the DTX3c Nt domain showed a unique 3D folding supporting isoform-specific biological function(s). We shed light on the molecular machinery associated with autocrine IGF-II regulation of oncogenic EphB4 kinase expression in a previously characterized IGF-II+/EphB4+ Mesothelioma cell line. The study provides early evidence for DTX3 Ub-E3 ligase involvement beyond the Notch signaling pathway.