scholarly journals LPHN2 controls vascular morphogenesis by inhibiting endothelial cell adhesion and YAP/TAZ signaling

2020 ◽  
Author(s):  
Chiara Camillo ◽  
Nicola Facchinello ◽  
Dafne Gays ◽  
Giulia Villari ◽  
Noemi Gioelli ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Molecular Mechanisms ◽  
Hippo Pathway ◽  
Intercellular Junctions ◽  
Zebrafish Embryos ◽  
Endothelial Barrier Function ◽  
Vascular Morphogenesis ◽  
The Hippo Pathway ◽  
G Protein Coupled

SUMMARYModulation of endothelial cell (EC) adhesion to extracellular matrix (ECM) in response to mechanostimuli is essential for blood vessel patterning and functioning, yet the underpinning molecular mechanisms are deciphered only in part. We identify the adhesion G protein-coupled receptor Latrophilin 2 (LPHN2) as a novel determinant of vascular morphogenesis and endothelial barrier function. In cultured ECs, endogenous LPHN2 localizes at ECM adhesions, signals through cAMP/Rap1, and negatively regulates ECM-elicited YAP/TAZ mechanosignaling and haptotaxis. ECs also express endogenous fibronectin-leucine-rich transmembrane 2 (FLRT2) ligand that promotes cAMP/Rap1 signaling and hinders haptotaxis via LPHN2. To validate these findings in vivo, we generated lphn2a mutant zebrafish embryos in which vascular ECs are abnormally stretched, display YAP/TAZ hyperactivation, and lack proper intercellular junctions. Consistently, intravascularly injected cancer cells extravasate more easily in lphn2a null embryos compared to control animals. Overall, we unveil in vascular ECs a novel crosstalk between LPHN2 and the Hippo pathway, that may be therapeutically exploited to interfere with cancer metastatic dissemination.

scholarly journals Downregulation of MYPT1 increases tumor resistance in ovarian cancer by targeting the Hippo pathway and increasing the stemness

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Sandra Muñoz-Galván ◽  
Blanca Felipe-Abrio ◽  
Eva M. Verdugo-Sivianes ◽  
Marco Perez ◽  
Manuel P. Jiménez-García ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Hippo Pathway ◽  
Ovarian Tumors ◽  
Tumor Resistance ◽  
Functional Link ◽  
The Hippo Pathway

Abstract Background Ovarian cancer is one of the most common and malignant cancers, partly due to its late diagnosis and high recurrence. Chemotherapy resistance has been linked to poor prognosis and is believed to be linked to the cancer stem cell (CSC) pool. Therefore, elucidating the molecular mechanisms mediating therapy resistance is essential to finding new targets for therapy-resistant tumors. Methods shRNA depletion of MYPT1 in ovarian cancer cell lines, miRNA overexpression, RT-qPCR analysis, patient tumor samples, cell line- and tumorsphere-derived xenografts, in vitro and in vivo treatments, analysis of data from ovarian tumors in public transcriptomic patient databases and in-house patient cohorts. Results We show that MYPT1 (PPP1R12A), encoding myosin phosphatase target subunit 1, is downregulated in ovarian tumors, leading to reduced survival and increased tumorigenesis, as well as resistance to platinum-based therapy. Similarly, overexpression of miR-30b targeting MYPT1 results in enhanced CSC-like properties in ovarian tumor cells and is connected to the activation of the Hippo pathway. Inhibition of the Hippo pathway transcriptional co-activator YAP suppresses the resistance to platinum-based therapy induced by either low MYPT1 expression or miR-30b overexpression, both in vitro and in vivo. Conclusions Our work provides a functional link between the resistance to chemotherapy in ovarian tumors and the increase in the CSC pool that results from the activation of the Hippo pathway target genes upon MYPT1 downregulation. Combination therapy with cisplatin and YAP inhibitors suppresses MYPT1-induced resistance, demonstrating the possibility of using this treatment in patients with low MYPT1 expression, who are likely to be resistant to platinum-based therapy.

scholarly journals MAML1/2 promote YAP/TAZ nuclear localization and tumorigenesis

2020 ◽  
Vol 117 (24) ◽  
pp. 13529-13540 ◽  
Author(s):  
Jiyoung Kim ◽  
Hyeryun Kwon ◽  
You Keun Shin ◽  
Gahyeon Song ◽  
Taebok Lee ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Messenger Rna ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Human Cancer ◽  
Hippo Pathway ◽  
Ectopic Expression ◽  
Dependent Manner ◽  
The Hippo Pathway

The Hippo pathway plays a pivotal role in tissue homeostasis and tumor suppression. YAP and TAZ are downstream effectors of the Hippo pathway, and their activities are tightly suppressed by phosphorylation-dependent cytoplasmic retention. However, the molecular mechanisms governing YAP/TAZ nuclear localization have not been fully elucidated. Here, we report that Mastermind-like 1 and 2 (MAML1/2) are indispensable for YAP/TAZ nuclear localization and transcriptional activities. Ectopic expression or depletion of MAML1/2 induces nuclear translocation or cytoplasmic retention of YAP/TAZ, respectively. Additionally, mutation of the MAML nuclear localization signal, as well as its YAP/TAZ interacting region, both abolish nuclear localization and transcriptional activity of YAP/TAZ. Importantly, we demonstrate that the level ofMAML1messenger RNA (mRNA) is regulated by microRNA-30c (miR-30c) in a cell-density-dependent manner. In vivo and clinical results suggest that MAML potentiates YAP/TAZ oncogenic function and positively correlates with YAP/TAZ activation in human cancer patients, suggesting pathological relevance in the context of cancer development. Overall, our study not only provides mechanistic insight into the regulation of YAP/TAZ subcellular localization, but it also strongly suggests that the miR30c–MAML–YAP/TAZ axis is a potential therapeutic target for developing novel cancer treatments.

scholarly journals Activated MST2 kinase is free of kinetic regulation

2022 ◽  
Author(s):  
Thomas J Koehler ◽  
Thao Tran ◽  
Jennifer M Kavran
Keyword(s):  
Kinetic Analysis ◽  
Molecular Mechanisms ◽  
Hippo Pathway ◽  
Kinase Domain ◽  
Caspase Cleavage ◽  
Downstream Signaling ◽  
Steady State Kinetic ◽  
Signaling Events ◽  
The Hippo Pathway

Canonically, MST1/2 functions as a core kinase of the Hippo pathway and non-canonically is both activated during apoptotic signaling and acts in concert with RASSFs in T-cells. Faithful signal transduction relies on both appropriate activation and regulated substrate phosphorylation by the activated kinase. Considerable progress has been made understanding the molecular mechanisms regulating activation of MST1/2 and identifying downstream signaling events. Here we present a kinetic analysis analyzing how the ability of MST1/2 to phosphorylate substrates is regulated. Using a steady state kinetic system, we parse the contribution of different factors including the domains of MST2, phosphorylation, caspase cleavage, and complex formation to MST2 activity. In the unphosphorylated state, we find the SARAH domain stabilizes substrate binding. Phosphorylation, we also determine, drives activation of MST2 and that once activated the kinase domain is free of regulation. The binding partners SAV1, MOB1A, and RASSF5 do not alter the kinetics of phosphorylated MST2. We also show that the caspase cleaved MST2 fragment is as active as full-length suggesting that the linker region of MST2 does not inhibit the catalytic activity of the kinase domain but instead regulates MST2 activity through non-catalytic mechanisms. This kinetic analysis helps establish a framework for interpreting how signaling events, mutations, and post-translational modifications contribute to signaling of MST2 in vivo.

scholarly journals Hippo-YAP/MCP-1 mediated tubular maladaptive repair promote inflammation in renal failed recovery after ischemic AKI

2021 ◽  
Vol 12 (8) ◽  
Author(s):  
Zhihuang Zheng ◽  
Chuanlei Li ◽  
Guangze Shao ◽  
Jinqing Li ◽  
Kexin Xu ◽  
...  
Keyword(s):  
Chronic Inflammation ◽  
Ischemia Reperfusion ◽  
Hippo Pathway ◽  
Glucose Deprivation ◽  
Macrophage Infiltration ◽  
Renal Inflammation ◽  
Monocyte Chemoattractant Protein 1 ◽  
The Hippo Pathway

AbstractAcute kidney injury (AKI) is associated with significant morbidity and its chronic inflammation contributes to subsequent chronic kidney disease (CKD) development. Yes-associated protein (YAP), the major transcriptional coactivator of the Hippo pathway, has been shown associated with chronic inflammation, but its role and mechanism in AKI-CKD transition remain unclear. Here we aimed to investigate the role of YAP in AKI-induced chronic inflammation. Renal ischemia/reperfusion (I/R) was used to induce a mouse model of AKI-CKD transition. We used verteporfin (VP), a pharmacological inhibitor of YAP, to treat post-IRI mice for a period, and evaluated the influence of YAP inhibition on long-term outcomes of AKI. In our results, severe IRI led to maladaptive tubular repair, macrophages infiltration, and progressive fibrosis. Following AKI, the Hippo pathway was found significantly altered with YAP persistent activation. Besides, tubular YAP activation was associated with the maladaptive repair, also correlated with interstitial macrophage infiltration. Monocyte chemoattractant protein 1 (MCP-1) was found notably upregulated with YAP activation. Of note, pharmacological inhibition of YAP in vivo attenuated renal inflammation, including macrophage infiltration and MCP-1 overexpression. Consistently, in vitro oxygen-glucose deprivation and reoxygenation (OGD/R) induced YAP activation and MCP-1 overproduction whereas these could be inhibited by VP. In addition, we modulated YAP activity by RNA interference, which further confirmed YAP activation enhances MCP-1 expression. Together, we concluded tubular YAP activation with maladaptive repair exacerbates renal inflammation probably via promoting MCP-1 production, which contributes to AKI-CKD transition.

scholarly journals Mdm2-Mediated Downmodulation of GRK2 Restricts Centrosome Separation for Proper Chromosome Congression

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 729
Author(s):  
Clara Reglero ◽  
Belén Ortiz del Castillo ◽  
Verónica Rivas ◽  
Federico Mayor ◽  
Petronila Penela
Keyword(s):  
Hippo Pathway ◽  
Growth Factor Receptor ◽  
Receptor Kinase ◽  
Egfr Signaling ◽  
Polyubiquitin Chains ◽  
Centrosome Separation ◽  
Chromosome Congression ◽  
Epidermal Growth ◽  
The Hippo Pathway ◽  
G Protein Coupled

The timing of centrosome separation and the distance moved apart influence the formation of the bipolar spindle, affecting chromosome stability. Epidermal growth factor receptor (EGFR) signaling induces early centrosome separation through downstream G protein-coupled receptor kinase GRK2, which phosphorylates the Hippo pathway component MST2 (Mammalian STE20-like protein kinase 2), in turn allowing NIMA kinase Nek2A activation for centrosomal linker disassembly. However, the mechanisms that counterbalance centrosome disjunction and separation remain poorly understood. We unveil that timely degradation of GRK2 by the E3 ligase Mdm2 limits centrosome separation in the G2. Both knockout expression and catalytic inhibition of Mdm2 result in GRK2 accumulation and enhanced centrosome separation before mitosis onset. Phosphorylation of GRK2 on residue S670 enables a complex pattern of non-K48-linked polyubiquitin chains assembled by Mdm2, which correlate with kinase protein degradation. Remarkably, GRK2-S670A protein fails to phosphorylate MST2 despite overcoming Mdm2-dependent degradation, which results in defective centrosome separation, shorter spindles, and abnormal chromosome congression. Conversely, extra levels of wild-type kinase in the G2 cause increased inter-centrosome distances with longer spindles, also converging in congression issues. Our findings show that the signals enabling activity of the GRK2/MST2/Nek2A axis for separation also switches on Mdm2 degradation of GRK2 to ensure accurate centrosome dynamics and proper mitotic spindle functionality.

Polyphyllin D, a steroidal saponin from Paris polyphylla, inhibits endothelial cell functions in vitro and angiogenesis in zebrafish embryos in vivo

2011 ◽  
Vol 137 (1) ◽  
pp. 64-69 ◽  
Author(s):  
Judy Yuet-Wa Chan ◽  
Johnny Chi-Man Koon ◽  
Xiaozhou Liu ◽  
Michael Detmar ◽  
Biao Yu ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Zebrafish Embryos ◽  
Steroidal Saponin ◽  
Cell Functions ◽  
Paris Polyphylla ◽  
Polyphyllin D

Abstract P279: YAP, a Transcriptional Cofactor of the Hippo Pathway, Regulates Cardiomyocyte Growth, Survival, and Proliferation

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Yanfei Yang ◽  
Noritsugu Nakano ◽  
Junichi Sadoshima
Keyword(s):  
Cardiac Hypertrophy ◽  
Hippo Pathway ◽  
Neonatal Rat ◽  
Border Zone ◽  
Luciferase Reporter ◽  
Tunel Staining ◽  
H2o2 Treatment ◽  
Rat Hearts ◽  
The Hippo Pathway

Mst1 and Lats2, components of the mammalian Hippo pathway, stimulate apoptosis and inhibit hypertrophy of cardiomyocytes (CMs), thereby mediating reperfusion injury and heart failure. YAP, a transcription factor co-factor, is negatively regulated by the Hippo pathway, and controls cell survival, proliferation and tissue regeneration. The role of YAP in regulating growth and death of CMs is poorly understood. YAP overexpression in CMs induced cardiac hypertrophy, as indicated by increases in cell size (+1.2 fold, p<0.01), protein content (+1.1 fold, p<0.01) and ANF (luciferase reporter activity +1.7 fold, mRNA +2.2 fold, and staining +2.7 fold, p<0.01). Lats2 phosphorylates YAP at Serine 127, which induces cytoplasmic translocation of YAP, whereas YAP(S127A) is localized constitutively in the nucleus. Expression of YAP(S127A) enhanced hypertrophy in cultured CMs compared to that of wild type YAP (+1.87 fold ANF staining, p<0.05), suggesting that the Mst1/Hippo pathway negatively regulates cardiac hypertrophy through YAP. YAP inhibited cell death induced by H2O2 treatment, as evaluated with TUNEL staining (-65%, p<0.05) and CellTiter Blue assays (+34.9%, p<0.01), indicating that YAP plays an essential role in mediating CM survival. Interestingly, YAP also significantly increased Ki67 positive cells in cultured CMs compared to LacZ (+2.65 fold, p<0.05). We used a mouse model of chronic myocardial infarction (MI) to evaluate the function of YAP in the heart in vivo. Although YAP is diffusely localized both in the nucleus and cytosol in CMs in control hearts, CMs in the border zone of MI exhibited nuclear localization of YAP whereas YAP was excluded from the nucleus in CMs in the remodeling area four days after MI (+6.52 fold and +1.28 fold). Some of the YAP positive CMs in the border zone exhibited positive co-staining with Ki67, suggesting that YAP potentially induces CM proliferation. A significant increase in nuclear YAP and Ki67 positive CMs (+2.95 fold, p<0.01 and +2.18 fold, p<0.05) was also observed in neonatal rat hearts whose apex was surgically resected three days before euthanasia. These results suggest that YAP plays an important role in mediating not only hypertrophy and survival, but also proliferation of CMs in response to myocardial injury.

Abstract 116: Arterial Endothelial Cell Has Stronger Angiogenic Potential Compared To Venous Endothelial Cell Through Up-regulation Of Endogenous FGF2 And FGF5 Expression In 3D Microfluidic System

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Ha-Rim Seo ◽  
Hyo Eun Jeong ◽  
Hyung Joon Joo ◽  
Seung-Cheol Choi ◽  
Jong-Ho Kim ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Molecular Mechanisms ◽  
Umbilical Vein ◽  
Assay System ◽  
Vascular Density ◽  
Angiogenic Potential ◽  
Angiogenesis Assay

Background: Human body contains many kinds of different type of endothelial cells (EC). However, cellular difference of their angiogenic potential has been hardly understood. We compared in vitro angiogenic potential between arterial EC and venous EC and investigated its underlying molecular mechanisms. Method: Used human aortic endothelial cells (HAEC) which was indicated from arterial EC and human umbilical vein endothelial cells (HUVEC) indicated from venous EC. To explore angiogenic potential in detail, we adopted a novel 3D microfluidic angiogenesis assay system, which closely mimic in vivo angiogenesis. Results: In 3D microfluidic angiogenesis assay system, HAEC demonstrated stronger angiogenic potential compared to HUVEC. HAEC maintained its profound angiogenic property under different biophysical conditions. In mRNA microarray sorted on up- regulated or down-regulated genes, HAEC demonstrated significantly higher expression of gastrulation brain homeobox 2 (GBX2), fibroblast grow factor 2 (FGF2), FGF5 and collagen 8a1. Angiogenesis-related protein assay revealed that HAEC has higher secretion of endogenous FGF2 than HUVEC. HAEC has only up-regulated FGF2 and FGF5 in this part of FGF family. Furthermore, FGF5 expression under vascular endothelial growth factor-A (VEGF-A) stimulation was higher in HAEC compared to HUVEC although VEGF-A augmented FGF5 expression in both HAEC and HUVEC. Those data suggested that FGF5 expression in both HAEC and HUVEC is partially dependent to VEGF-A stimulate. HUVEC and HAEC reduced vascular density after FGF2 and FGF5 siRNA treat. Conclusion: HAEC has stronger angiogenic potential than HUVEC through up-regulation of endogenous FGF2 and FGF5 expression

scholarly journals LPHN2 inhibits vascular permeability by differential control of endothelial cell adhesion

2021 ◽  
Vol 220 (11) ◽  
Author(s):  
Chiara Camillo ◽  
Nicola Facchinello ◽  
Giulia Villari ◽  
Giulia Mana ◽  
Noemi Gioelli ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Focal Adhesion ◽  
Nuclear Localization ◽  
Molecular Mechanisms ◽  
Transcriptional Regulators ◽  
Dependent Manner ◽  
Knock Out ◽  
Dynamic Modulation ◽  
Differential Control ◽  
G Protein Coupled

Dynamic modulation of endothelial cell-to-cell and cell–to–extracellular matrix (ECM) adhesion is essential for blood vessel patterning and functioning. Yet the molecular mechanisms involved in this process have not been completely deciphered. We identify the adhesion G protein–coupled receptor (ADGR) Latrophilin 2 (LPHN2) as a novel determinant of endothelial cell (EC) adhesion and barrier function. In cultured ECs, endogenous LPHN2 localizes at ECM contacts, signals through cAMP/Rap1, and inhibits focal adhesion (FA) formation and nuclear localization of YAP/TAZ transcriptional regulators, while promoting tight junction (TJ) assembly. ECs also express an endogenous LPHN2 ligand, fibronectin leucine-rich transmembrane 2 (FLRT2), that prevents ECM-elicited EC behaviors in an LPHN2-dependent manner. Vascular ECs of lphn2a knock-out zebrafish embryos become abnormally stretched, display a hyperactive YAP/TAZ pathway, and lack proper intercellular TJs. Consistently, blood vessels are hyperpermeable, and intravascularly injected cancer cells extravasate more easily in lphn2a null animals. Thus, LPHN2 ligands, such as FLRT2, may be therapeutically exploited to interfere with cancer metastatic dissemination.

scholarly journals Antioxidant, Anti-Inflammatory and Anti-Angiogenic Properties of Citrus lumia Juice

2020 ◽  
Vol 11 ◽  
Author(s):  
Antonella Smeriglio ◽  
Marcella Denaro ◽  
Valeria D’Angelo ◽  
Maria Paola Germanò ◽  
Domenico Trombetta
Keyword(s):  
Ascorbic Acid ◽  
Acid Content ◽  
Molecular Mechanisms ◽  
Biological Effects ◽  
Ascorbic Acid Content ◽  
Zebrafish Embryos ◽  
Anti Inflammatory ◽  
Dose Dependent

Citrus juices are a rich source of bioactive compounds with various and well-known health benefits. The aim of this study was to investigate the polyphenols and ascorbic acid content as well as to investigate the antioxidant, anti-inflammatory and anti-angiogenic properties of the juice of an ancient Mediterranean species, Citrus lumia Risso (CLJ). The antioxidant and anti-inflammatory activities were evaluated by several in vitro cell-free and cell-based assays, whereas two different in vivo models, the chick chorioallantoic membrane (CAM) and the zebrafish embryos, were used to characterize the anti-angiogenic properties. Twenty-eight polyphenols were identified by RP-LC-DAD-ESI-MS analysis (flavonoids 68.82% and phenolic acids 31.18%) with 1-caffeoyl-5-feruloylquinic acid and kaempferol 3′-rhamnoside, which represent the most abundant compounds (25.70 and 23.12%, respectively). HPLC-DAD analysis showed a high ascorbic acid content (352 mg/kg of CLJ), which contributes with polyphenols to the marked and dose-dependent antioxidant and anti-inflammatory properties observed. CLJ showed strong and dose-dependent anti-angiogenic activity as highlighted by the inhibition of blood vessel formation on CAMs and the decrease of endogenous alkaline phosphatase on zebrafish embryos. Moreover, within the concentration range tested, no dead or malformed embryos were recorded. Certainly, further studies are needed to investigate the molecular mechanisms underlying these promising biological effects, but considering the evidence of the present study, the use of CLJ as a ready-to drink safe prevention strategy for inflammatory-based diseases correlated to angiogenesis could be justified.

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