scholarly journals Thrombospondin-1 Plays an Essential Role in Yes-Associated Protein Nuclear Translocation during the Early Phase of Trypanosoma cruzi Infection in Heart Endothelial Cells

2020 ◽  
Vol 21 (14) ◽  
pp. 4912 ◽  
Author(s):  
Ashutosh Arun ◽  
Kayla J. Rayford ◽  
Ayorinde Cooley ◽  
Girish Rachakonda ◽  
Fernando Villalta ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Trypanosoma Cruzi ◽  
Correlation Coefficient ◽  
Early Phase ◽  
Nuclear Translocation ◽  
Hippo Signaling ◽  
Vascular Endothelial ◽  
Wild Type ◽  
Hippo Signaling Pathway ◽  
Cruzi Infection

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease. This neglected tropical disease causes severe morbidity and mortality in endemic regions. About 30% of T. cruzi infected individuals will present with cardiac complications. Invasive trypomastigotes released from infected cells can be carried in the vascular endothelial system to infect neighboring and distant cells. During the process of cellular infection, the parasite induces host cells, to increase the levels of host thrombospondin-1 (TSP-1), to facilitate the process of infection. TSP-1 plays important roles in the functioning of vascular cells, including vascular endothelial cells with important implications in cardiovascular health. Many signal transduction pathways, including the yes-associated protein 1 (YAP)/transcriptional coactivator, with PDZ-binding motif (TAZ) signaling, which are upstream of TSP-1, have been linked to the pathophysiology of heart damage. The molecular mechanisms by which T. cruzi signals, and eventually infects, heart endothelial cells remain unknown. To evaluate the importance of TSP-1 expression in heart endothelial cells during the process of T. cruzi infection, we exposed heart endothelial cells prepared from Wild Type and TSP-1 Knockout mouse to invasive T. cruzi trypomastigotes at multiple time points, and evaluated changes in the hippo signaling cascade using immunoblotting and immunofluorescence assays. We found that the parasite turned off the hippo signaling pathway in TSP-1KO heart endothelial cells. The levels of SAV1 and MOB1A increased to a maximum of 2.70 ± 0.23 and 5.74 ± 1.45-fold at 3 and 6 h, respectively, in TSP-1KO mouse heart endothelial cells (MHEC), compared to WT MHEC, following a parasite challenge. This was accompanied by a significant continuous increase in the nuclear translocation of downstream effector molecule YAP, to a maximum mean nuclear fluorescence intensity of 10.14 ± 0.40 at 6 h, compared to wild type cells. Furthermore, we found that increased nuclear translocated YAP significantly colocalized with the transcription co-activator molecule pan-TEAD, with a maximum Pearson’s correlation coefficient of 0.51 ± 0.06 at 6 h, compared to YAP-Pan-TEAD colocalization in the WT MHEC, which decreased significantly, with a minimum Pearson’s correlation coefficient of 0.30 ± 0.01 at 6 h. Our data indicate that, during the early phase of infection, upregulated TSP-1 is essential for the regulation of the hippo signaling pathway. These studies advance our understanding of the molecular interactions occurring between heart endothelial cells and T. cruzi, in the presence and absence of TSP-1, providing insights into processes linked to parasite dissemination and pathogenesis.

scholarly journals Thrombospondin‐1 Plays an Essential Role in Hippo Signaling During the Early Phase of Trypanosoma cruzi Infection in Heart Endothelial Cells

2021 ◽  
Vol 35 (S1) ◽  
Author(s):  
Ashutosh Arun ◽  
Kayla Rayford ◽  
Ayorinde Cooley ◽  
Girish Rachakonda ◽  
Fernando Villalta ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Trypanosoma Cruzi ◽  
Early Phase ◽  
Essential Role ◽  
Hippo Signaling ◽  
Thrombospondin 1 ◽  
Cruzi Infection

scholarly journals Thrombospondin-1 expression and modulation of Wnt and hippo signaling pathways during the early phase of Trypanosoma cruzi infection of heart endothelial cells

2022 ◽  
Vol 16 (1) ◽  
pp. e0010074
Author(s):  
Ashutosh Arun ◽  
Kayla J. Rayford ◽  
Ayorinde Cooley ◽  
Tanu Rana ◽  
Girish Rachakonda ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Trypanosoma Cruzi ◽  
Signaling Pathways ◽  
Early Phase ◽  
Molecular Mechanisms ◽  
Glycogen Synthase ◽  
The Body ◽  
Mouse Heart ◽  
Hippo Signaling ◽  
Thrombospondin 1

The protozoan parasite, Trypanosoma cruzi, causes severe morbidity and mortality in afflicted individuals. Approximately 30% of T. cruzi infected individuals present with cardiac pathology. The invasive forms of the parasite are carried in the vascular system to infect other cells of the body. During transportation, the molecular mechanisms by which the parasite signals and interact with host endothelial cells (EC) especially heart endothelium is currently unknown. The parasite increases host thrombospondin-1 (TSP1) expression and activates the Wnt/β-catenin and hippo signaling pathways during the early phase of infection. The links between TSP1 and activation of the signaling pathways and their impact on parasite infectivity during the early phase of infection remain unknown. To elucidate the significance of TSP1 function in YAP/β-catenin colocalization and how they impact parasite infectivity during the early phase of infection, we challenged mouse heart endothelial cells (MHEC) from wild type (WT) and TSP1 knockout mice with T. cruzi and evaluated Wnt signaling, YAP/β-catenin crosstalk, and how they affect parasite infection. We found that in the absence of TSP1, the parasite induced the expression of Wnt-5a to a maximum at 2 h (1.73±0.13), P< 0.001 and enhanced the level of phosphorylated glycogen synthase kinase 3β at the same time point (2.99±0.24), P<0.001. In WT MHEC, the levels of Wnt-5a were toned down and the level of p-GSK-3β was lowest at 2 h (0.47±0.06), P< 0.01 compared to uninfected control. This was accompanied by a continuous significant increase in the nuclear colocalization of β-catenin/YAP in TSP1 KO MHEC with a maximum Pearson correlation coefficient of (0.67±0.02), P< 0.05 at 6 h. In WT MHEC, the nuclear colocalization of β-catenin/YAP remained steady and showed a reduction at 6 h (0.29±0.007), P< 0.05. These results indicate that TSP1 plays an important role in regulating β-catenin/YAP colocalization during the early phase of T. cruzi infection. Importantly, dysregulation of this crosstalk by pre-incubation of WT MHEC with a β-catenin inhibitor, endo-IWR 1, dramatically reduced the level of infection of WT MHEC. Parasite infectivity of inhibitor treated WT MHEC was similar to the level of infection of TSP1 KO MHEC. These results indicate that the β-catenin pathway induced by the parasite and regulated by TSP1 during the early phase of T. cruzi infection is an important potential therapeutic target, which can be explored for the prophylactic prevention of T. cruzi infection.

scholarly journals Inhibition of Hippo Signaling Improves Skin Lesions in a Rosacea-Like Mouse Model

2021 ◽  
Vol 22 (2) ◽  
pp. 931
Author(s):  
Jihyun Lee ◽  
Yujin Jung ◽  
Seo won Jeong ◽  
Ga Hee Jeong ◽  
Gue Tae Moon ◽  
...  
Keyword(s):  
Mouse Model ◽  
Signaling Pathway ◽  
Normal Skin ◽  
Skin Lesions ◽  
Hippo Signaling ◽  
Vascular Endothelial ◽  
Expression Levels ◽  
Hippo Signaling Pathway ◽  
Endothelial Growth Factor

The Hippo signaling pathway plays a key role in regulating organ size and tissue homeostasis. Hippo and two of its main effectors, yes-associated protein (YAP) and WWTR1 (WW domain-containing transcription regulator 1, commonly listed as TAZ), play critical roles in angiogenesis. This study investigated the role of the Hippo signaling pathway in the pathogenesis of rosacea. We performed immunohistochemical analyses to compare the expression levels of YAP and TAZ between rosacea skin and normal skin in humans. Furthermore, we used a rosacea-like BALB/c mouse model induced by LL-37 injections to determine the roles of YAP and TAZ in rosacea in vivo. We found that the expression levels of YAP and TAZ were upregulated in patients with rosacea. In the rosacea-like mouse model, we observed that the clinical features of rosacea, including telangiectasia and erythema, improved after the injection of a YAP/TAZ inhibitor. Additionally, treatment with a YAP/TAZ inhibitor reduced the expression levels of YAP and TAZ and diminished vascular endothelial growth factor (VEGF) immunoreactivity in the rosacea-like mouse model. Our findings suggest that YAP/TAZ inhibitors can attenuate angiogenesis associated with the pathogenesis of rosacea and that both YAP and TAZ are potential therapeutic targets for patients with rosacea.

scholarly journals NPHP4, a cilia-associated protein, negatively regulates the Hippo pathway

2011 ◽  
Vol 193 (4) ◽  
pp. 633-642 ◽  
Author(s):  
Sandra Habbig ◽  
Malte P. Bartram ◽  
Roman U. Müller ◽  
Ricarda Schwarz ◽  
Nikolaos Andriopoulos ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cellular Proliferation ◽  
Nuclear Translocation ◽  
Hippo Pathway ◽  
Negative Regulator ◽  
Hippo Signaling ◽  
Transcriptional Coactivator ◽  
Hippo Signaling Pathway ◽  
The Hippo Pathway ◽  
Potent Negative Regulator

The conserved Hippo signaling pathway regulates organ size in Drosophila melanogaster and mammals and has an essential role in tumor suppression and the control of cell proliferation. Recent studies identified activators of Hippo signaling, but antagonists of the pathway have remained largely elusive. In this paper, we show that NPHP4, a known cilia-associated protein that is mutated in the severe degenerative renal disease nephronophthisis, acts as a potent negative regulator of mammalian Hippo signaling. NPHP4 directly interacted with the kinase Lats1 and inhibited Lats1-mediated phosphorylation of the Yes-associated protein (YAP) and TAZ (transcriptional coactivator with PDZ-binding domain), leading to derepression of these protooncogenic transcriptional regulators. Moreover, NPHP4 induced release from 14-3-3 binding and nuclear translocation of YAP and TAZ, promoting TEA domain (TEAD)/TAZ/YAP-dependent transcriptional activity. Consistent with these data, knockdown of NPHP4 negatively affected cellular proliferation and TEAD/TAZ activity, essentially phenocopying loss of TAZ function. These data identify NPHP4 as a negative regulator of the Hippo pathway and suggest that NPHP4 regulates cell proliferation through its effects on Hippo signaling.

scholarly journals Toxoplasma gondii Dysregulates Barrier Function and Mechanotransduction Signaling in Human Endothelial Cells

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Armond L. Franklin-Murray ◽  
Sharmila Mallya ◽  
Allen Jankeel ◽  
Suhas Sureshchandra ◽  
Ilhem Messaoudi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Toxoplasma Gondii ◽  
Molecular Interactions ◽  
Barrier Function ◽  
Hippo Signaling ◽  
Vascular Endothelial ◽  
Human Endothelial Cells ◽  
Content Type

ABSTRACT Toxoplasma gondii can infect and replicate in vascular endothelial cells prior to entering host tissues. However, little is known about the molecular interactions at the parasite-endothelial cell interface. We demonstrate that T. gondii infection of primary human umbilical vein endothelial cells (HUVEC) altered cell morphology and dysregulated barrier function, increasing permeability to low-molecular-weight polymers. T. gondii disrupted vascular endothelial cadherin (VE-cadherin) and β-catenin localization to the cell periphery and reduced VE-cadherin protein expression. Notably, T. gondii infection led to reorganization of the host cytoskeleton by reducing filamentous actin (F-actin) stress fiber abundance under static and microfluidic shear stress conditions and by reducing planar cell polarity. RNA sequencing (RNA-Seq) comparing genome-wide transcriptional profiles of infected to uninfected endothelial cells revealed changes in gene expression associated with cell-cell adhesion, extracellular matrix reorganization, and cytokine-mediated signaling. In particular, genes downstream of Hippo signaling and the biomechanical sensor and transcriptional coactivator Yes-associated protein (YAP) were downregulated in infected endothelial cells. Interestingly, T. gondii infection activated Hippo signaling by increasing phosphorylation of LATS1, leading to cytoplasmic retention of YAP, and reducing YAP target gene expression. These findings suggest that T. gondii infection triggers Hippo signaling and YAP nuclear export, leading to an altered transcriptional profile of infected endothelial cells. IMPORTANCE Toxoplasma gondii is a foodborne parasite that infects virtually all warm-blooded animals and can cause severe disease in individuals with compromised or weakened immune systems. During dissemination in its infected hosts, T. gondii breaches endothelial barriers to enter tissues and establish the chronic infections underlying the most severe manifestations of toxoplasmosis. The research presented here examines how T. gondii infection of primary human endothelial cells induces changes in cell morphology, barrier function, gene expression, and mechanotransduction signaling under static conditions and under the physiological conditions of shear stress found in the bloodstream. Understanding the molecular interactions occurring at the interface between endothelial cells and T. gondii may provide insights into processes linked to parasite dissemination and pathogenesis.

scholarly journals The Pseudomonas aeruginosa product pyochelin interferes with Trypanosoma cruzi infection and multiplication in vitro

2020 ◽  
Vol 114 (7) ◽  
pp. 492-498 ◽  
Author(s):  
Gabriele Sass ◽  
Laura C Miller Conrad ◽  
Terrence-Thang H Nguyen ◽  
David A Stevens
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Iron Acquisition ◽  
Vero Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Current Standard ◽  
Cruzi Infection

Abstract Background Bacteria are sources of numerous molecules used in treatment of infectious diseases. We investigated effects of molecules produced by 26 Pseudomonas aeruginosa strains against infection of mammalian cell cultures with Trypanosoma cruzi, the aetiological agent of Chagas disease. Methods Vero cells were infected with T. cruzi in the presence of wild-type P. aeruginosa supernatants or supernatants of mutants with defects in the production of various virulence, quorum sensing and iron acquisition factors. Quantification of T. cruzi infection (percentage of infected cells) and multiplication (number of amastigotes per infected cell) was performed and cell viability was determined. Results Wild-type P. aeruginosa products negatively affected T. cruzi infection and multiplication in a dose-dependent manner, without evident toxicity for mammalian cells. PvdD/pchE mutation (loss of the P. aeruginosa siderophores pyoverdine and pyochelin) had the greatest impact on anti–T. cruzi activity. Negative effects on T. cruzi infection by pure pyochelin, but not pyoverdine, or other P. aeruginosa exoproducts studied, were quantitatively similar to the effects of benznidazole, the current standard therapy against T. cruzi. Conclusions The P. aeruginosa product pyochelin showed promising activity against T. cruzi and might become a new lead molecule for therapy development.

scholarly journals MST4 kinase suppresses gastric tumorigenesis by limiting YAP activation via a non-canonical pathway

2020 ◽  
Vol 217 (6) ◽  
Author(s):  
Liwei An ◽  
Pingping Nie ◽  
Min Chen ◽  
Yang Tang ◽  
Hui Zhang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Signaling Pathway ◽  
Poor Prognosis ◽  
Human Gastric Cancer ◽  
Hippo Signaling ◽  
Wild Type ◽  
Hippo Signaling Pathway ◽  
Phosphorylation Level ◽  
Signaling Axis ◽  
Importin Α

Hyperactivation of YAP has been commonly associated with tumorigenesis, and emerging evidence hints at multilayered Hippo-independent regulations of YAP. In this study, we identified a new MST4–YAP axis, which acts as a noncanonical Hippo signaling pathway that limits stress-induced YAP activation. MST4 kinase directly phosphorylated YAP at Thr83 to block its binding with importin α, therefore leading to YAP cytoplasmic retention and inactivation. Due to a consequential interplay between MST4-mediated YAP phospho-Thr83 signaling and the classical YAP phospho-Ser127 signaling, the phosphorylation level of YAP at Thr83 was correlated to that at Ser127. Mutation of T83E mimicking MST4-mediated alternative signaling restrained the activity of both wild-type YAP and its S127A mutant mimicking loss of classical Hippo signal. Depletion of MST4 in mice promoted gastric tumorigenesis with diminished Thr83 phosphorylation and hyperactivation of YAP. Moreover, loss of MST4–YAP signaling was associated with poor prognosis of human gastric cancer. Collectively, our study uncovered a noncanonical MST4–YAP signaling axis essential for suppressing gastric tumorigenesis.

scholarly journals The Effect of Sodium Bicarbonate, a Beneficial Adjuvant Molecule in Cystic Fibrosis, on Bronchial Epithelial Cells Expressing a Wild-Type or Mutant CFTR Channel

2020 ◽  
Vol 21 (11) ◽  
pp. 4024 ◽  
Author(s):  
Ilona Gróf ◽  
Alexandra Bocsik ◽  
András Harazin ◽  
Ana Raquel Santa-Maria ◽  
Gaszton Vizsnyiczai ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Endothelial Cells ◽  
Epithelial Cells ◽  
Sodium Bicarbonate ◽  
Vascular Endothelial Cells ◽  
Bronchial Epithelium ◽  
Barrier Properties ◽  
Vascular Endothelial ◽  
Wild Type ◽  
Bronchial Epithelial

Clinical and experimental results with inhaled sodium bicarbonate as an adjuvant therapy in cystic fibrosis (CF) are promising due to its mucolytic and bacteriostatic properties, but its direct effect has not been studied on respiratory epithelial cells. Our aim was to establish and characterize co-culture models of human CF bronchial epithelial (CFBE) cell lines expressing a wild-type (WT) or mutant (deltaF508) CF transmembrane conductance regulator (CFTR) channel with human vascular endothelial cells and investigate the effects of bicarbonate. Vascular endothelial cells induced better barrier properties in CFBE cells as reflected by the higher resistance and lower permeability values. Activation of CFTR by cAMP decreased the electrical resistance in WT but not in mutant CFBE cell layers confirming the presence and absence of functional channels, respectively. Sodium bicarbonate (100 mM) was well-tolerated by CFBE cells: it slightly reduced the impedance of WT but not that of the mutant CFBE cells. Sodium bicarbonate significantly decreased the more-alkaline intracellular pH of the mutant CFBE cells, while the barrier properties of the models were only minimally changed. These observations indicate that sodium bicarbonate is beneficial to deltaF508-CFTR expressing CFBE cells. Thus, sodium bicarbonate may have a direct therapeutic effect on the bronchial epithelium.

scholarly journals The TDP43 CFTS Affect Brain Endothelial Cell Functions by Regulating YAP and Tight Junction Proteins in Cerebral Ischemic Injury.

2021 ◽  
Author(s):  
Xiaotian Xu ◽  
Changwen Zhang ◽  
Jianxiong Jiang ◽  
Mei Xin ◽  
Jiukuan Hao
Keyword(s):  
Endothelial Cells ◽  
Ischemic Stroke ◽  
Protein Expression ◽  
Tight Junction ◽  
Signaling Pathway ◽  
Ischemic Injury ◽  
Hippo Signaling ◽  
Brain Endothelial Cells ◽  
Hippo Signaling Pathway ◽  
Cerebral Ischemic

Abstract Background: As important components of the blood-brain barrier (BBB), brain endothelial cells (ECs) interact with pericytes, astrocytes, neurons, microglia and extracellular matrix in the neurovascular unit to maintain central nerve system (CNS) homeostasis and regulate neurological functions. Pathological changes in brain endothelium plays an important role in progression of ischemic stroke. The compromised BBB under ischemic stroke condition causes neuronal damage. However, the pathophysiological mechanisms of BBB in normal and under ischemic stroke condition has not been fully elucidated.Methods: C57bl/6 mice were subjected to 1-hour transient middle cerebral occlusion (tMCAO) model, and collected the brain samples after reperfusion for 24hours, 72hours and 1week. Lentivirus (YAP/TAZ shRNA), adenovirus (YAPS112A), TDP43 siRNA, TDP43-CTFS35 overexpression plasmid, Oxygen-glucose deprivation (OGD), and lipopolysaccharides (LPS) were applied to brain endothelial cells in vitro experiments. Brain endothelial cells (ECs) functions were tested by cell proliferation, migration and cell viability. Hippo signaling pathway was examined by immunofluorescence and western blotting.Results: The present study demonstrates that TDP43 is an essential gene to regulate brain ECs normal function, and knockdown of TDP43 reduces tight junction protein expression and inhibits brain ECs migration. Furthermore, ischemic injury and inflammation induce cytoplasmic TDP43-CTFS35 aggregation brain ECs, which weaken TDP43 full-length’s function leading to impairing tight junction (TJ) protein expression and cell migration. The expression of cytoplasmic TDP43-CTFS35 in brain ECs increased at 24 hours and 72 hours after MCAO, but disappeared at 1 week after MCAO. The expressions of TJ proteins, ZO-1 and claudin-5, and expression of P-YAP are associated with the dynamic changes of TDP43-CTFS35 expression in brain ECs after MCAO. The underlying mechanism of TDP43-CTFS35’s effects on brain ECs is that TDP43-CTFS35 turns off the Hippo signaling pathway by inhibition of PMST1/2 phosphorylation leading to de-phosphorylate YAP, and subsequently causes brain ECs functional changes.Conclusions: The present study provides new insight knowledge regarding the mechanisms of brain vascular ECs regulation and pathological change in the BBB after cerebral ischemic injury.

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