ERK Activity Imaging During Migration of Living Cells In Vitro and In Vivo

Hirata, Eishu; Kiyokawa, Etsuko

doi:10.3390/ijms20030679

Cited by 19 publications

(13 citation statements)

References 77 publications

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“…We further explored the underlying mechanism and found Erk1/2 phosphorylation was significantly inhibited in the APOH-exos group compared with the control group, whereas Erk1/2, p38 and P-p38 levels were equivalent in both groups, suggesting that the P-Erk signaling pathway was inhibited by APOH. P-Erk is a member of mitogen-activated protein kinase (MAPK) signaling pathway, and plays an important role in cell growth, migration and angiogenesis (25)(26)(27)(28)(29)(30)(31)(32)(33). APOH-exos may inhibit P-Erk1/2, then inhibit cell migration and tube formation, which means the inhibit of angiogenesis.…”

Section: Discussionmentioning

confidence: 99%

Exosome-Contained APOH Associated With Antiphospholipid Syndrome

et al. 2021

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BackgroundAntiphospholipid syndrome (APS) is a systemic autoimmune disease that can lead to thrombosis and/or pregnancy complications. Exosomes, membrane-encapsulated vesicles that are released into the extracellular environment by many types of cells, can carry signals to recipient cells to affect angiogenesis, apoptosis, and inflammation. There is increasing evidence suggesting that exosomes play critical roles in pregnancy. However, the contribution of exosomes to APS is still unknown.MethodsPeripheral plasma was collected from healthy early pregnancy patients (NC-exos) and early pregnancy patients with APS (APS-exos) for exosome extraction and characterization. The effect of exosomes from different sources on pregnancy outcomes was determined by establishing a mouse pregnancy model. Following the coincubation of exosomes and human umbilical vein endothelial cells (HUVECs), functional tests examined the features of APS-exos. The APS-exos and NC-exos were analyzed by quantitative proteomics of whole protein tandem mass tag (TMT) markers to explore the different compositions and identify key proteins. After incubation with HUVECs, functional tests investigated the characteristics of key exosomal proteins. Western blot analysis was used to identify the key pathways.ResultsIn the mouse model, APS-exos caused an APS-like birth outcome. In vitro experiments showed that APS-exos inhibited the migration and tube formation of HUVECs. Quantitative proteomics analysis identified 27 upregulated proteins and 9 downregulated proteins in APS-exos versus NC-exos. We hypothesized that apolipoprotein H (APOH) may be a core protein, and the analysis of clinical samples was consistent with finding from the proteomic TMT analysis. APOH-exos led to APS-like birth outcomes. APOH-exos directly enter HUVECs and may play a role through the phospho-extracellular signal-regulated kinase pathway.ConclusionsOur study suggests that both APS-exos and APOH-exos impair vascular development and lead to pregnancy complications. APOH-exos may be key actors in the pathogenesis of APS. This study provides new insights into the pathogenesis of APS and potential new targets for therapeutic intervention.

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Section: Discussionmentioning

confidence: 99%

Exosome-Contained APOH Associated With Antiphospholipid Syndrome

et al. 2021

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“…A variety of biosensors for the Ras/MAPK signaling are used in vivo (i.e., rodents, insects, and zebrafish; Hirata and Kiyokawa, 2019) to capture spatiotemporally cell dynamics, depending on ERK activity in the context of cell growth, differentiation, and migration. Classical fluorescence resonance energy transfer (FRET)-based extracellular-regulated kinase activity reporter (EKAR)-types biosensors exploit the ability of activated ERK to phosphorylate a substrate that triggers a conformational change of the sensor, such as to bring the FRET pair of fluorophores in close physical proximity (FRET pairs classically employing a GFP-like fluorescent protein as a donor and a redemitting protein as an acceptor).…”

Section: Ras/mapk Biosensors and Actuatorsmentioning

confidence: 99%

In vivo Functional Genomics for Undiagnosed Patients: The Impact of Small GTPases Signaling Dysregulation at Pan-Embryo Developmental Scale

Lauri

Fasano

Venditti

et al. 2021

Front. Cell Dev. Biol.

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While individually rare, disorders affecting development collectively represent a substantial clinical, psychological, and socioeconomic burden to patients, families, and society. Insights into the molecular mechanisms underlying these disorders are required to speed up diagnosis, improve counseling, and optimize management toward targeted therapies. Genome sequencing is now unveiling previously unexplored genetic variations in undiagnosed patients, which require functional validation and mechanistic understanding, particularly when dealing with novel nosologic entities. Functional perturbations of key regulators acting on signals’ intersections of evolutionarily conserved pathways in these pathological conditions hinder the fine balance between various developmental inputs governing morphogenesis and homeostasis. However, the distinct mechanisms by which these hubs orchestrate pathways to ensure the developmental coordinates are poorly understood. Integrative functional genomics implementing quantitative in vivo models of embryogenesis with subcellular precision in whole organisms contribute to answering these questions. Here, we review the current knowledge on genes and mechanisms critically involved in developmental syndromes and pediatric cancers, revealed by genomic sequencing and in vivo models such as insects, worms and fish. We focus on the monomeric GTPases of the RAS superfamily and their influence on crucial developmental signals and processes. We next discuss the effectiveness of exponentially growing functional assays employing tractable models to identify regulatory crossroads. Unprecedented sophistications are now possible in zebrafish, i.e., genome editing with single-nucleotide precision, nanoimaging, highly resolved recording of multiple small molecules activity, and simultaneous monitoring of brain circuits and complex behavioral response. These assets permit accurate real-time reporting of dynamic small GTPases-controlled processes in entire organisms, owning the potential to tackle rare disease mechanisms.

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“…The entire signaling pathway utilizes various kinases, such as Ras/Raf/MAPK-ERK (MEK), ribosomal s6 kinases, MAP kinase-interacting serine/threonine-protein kinases, mitogen- and stress-activated protein kinases, and cytosolic phospholipase A2 89 . This pathway is a known modulator of bispecific phosphatases 90 , 91 , subcellular localization of cascade components 92 , 93 , cellular motility 94 , 95 , cytokeratins 78 , 96 , 97 , 98 , and other scaffolding proteins 99 , 100 . The ERK 1/2 signaling pathway has been implicated in several cancer subtypes 89 , 101 , 102 ; therefore, abnormal manipulation of this pathway by GABRP can result in carcinogenesis.…”

Section: Aberrant Gabrp Regulation In Different Types Of Cancermentioning

confidence: 99%