LEOPARD-type SHP2 mutant Gln510Glu attenuates cardiomyocyte differentiation and promotes cardiac hypertrophy via dysregulation of Akt/GSK-3β/β-catenin signaling

2011 ◽  
Vol 301 (4) ◽  
pp. H1531-H1539 ◽  
Author(s):  
Hidekazu Ishida ◽  
Shigetoyo Kogaki ◽  
Jun Narita ◽  
Hiroaki Ichimori ◽  
Nobutoshi Nawa ◽  
...  
Keyword(s):  
Late Stage ◽  
Fluorescent Protein ◽  
Glycogen Synthase ◽  
Morphological Changes ◽  
Cardiac Cells ◽  
The Other ◽  
Cardiomyocyte Differentiation ◽  
Cardiac Progenitors ◽  
P19cl6 Cells ◽  
Gsk 3Β

LEOPARD syndrome (LS) is an autosomal dominant inherited multisystemic disorder. Most cases involve mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase Src homology 2-containing protein phosphatase 2 (SHP2). LS frequently causes severe hypertrophic cardiomyopathy (HCM), even from the fetal period. However, the molecular pathogenesis has not been clearly elucidated. Here, we analyzed the roles of the LS-type SHP2 mutant Gln510Glu (Q510E), which showed the most severe type of HCM in LS, in cardiomyocyte differentiation, and in morphological changes. We generated mutant P19CL6 cell lines, the most convenient cardiomyocyte differentiation model, which continuously expressed SHP2-Q510E, SHP2-D61N (Noonan-type mutant), wild-type SHP2, and green fluorescent protein (native SHP2 expression only). SHP2-Q510E mutant P19CL6 cells showed significant attenuation of myofibrillogenesis, with increased proliferative activity. Mature cardiomyocytes from the SHP2-Q510E mutant were significantly larger than those of controls and the other mutants. However, expression of cardiac-specific transcriptional factors (Gata4, Tbx5, and Nkx2.5) did not differ significantly between the LS-type SHP2-Q510E mutants and the other mutants and controls. Our results indicate that SHP2-Q510E mutants can differentiate into cardiac progenitors but are inhibited from undergoing terminal differentiation into mature cardiomyocytes. In contrast, Akt and glycogen synthase kinase (GSK)-3β phosphorylation were upregulated, and nuclear β-catenin at the late stage of differentiation was highly accumulated in SHP2-Q510E mutant P19CL6 cells. Supplementation with the phosphoinositide 3-kinase/Akt inhibitor LY-294002 during the late stage of differentiation was found to partially restore myofibrillogenesis while suppressing the increase in size of individual mature cardiomyocytes derived from the SHP2-Q510E mutants. Our findings suggest that dysregulation of the Akt/GSK-3β/β-catenin pathway can contribute to the pathogenesis of HCM in LS patients, not only through hypertrophic changes in individual cardiac cells but also via the expansion of cardiac progenitors.

scholarly journals Invasion of Epithelial Cells and Proteolysis of Cellular Focal Adhesion Components by Distinct Types of Porphyromonas gingivalis Fimbriae

2006 ◽  
Vol 74 (7) ◽  
pp. 3773-3782 ◽  
Author(s):  
Ichiro Nakagawa ◽  
Hiroaki Inaba ◽  
Taihei Yamamura ◽  
Takahiro Kato ◽  
Shinji Kawai ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Porphyromonas Gingivalis ◽  
Focal Adhesion ◽  
Fluorescent Protein ◽  
Morphological Changes ◽  
Critical Role ◽  
Focal Adhesions ◽  
Cellular Migration ◽  
The Other ◽  
Type Ii

ABSTRACT Porphyromonas gingivalis fimbriae are classified into six types (types I to V and Ib) based on the fimA genes encoding FimA (a subunit of fimbriae), and they play a critical role in bacterial interactions with host tissues. In this study, we compared the efficiencies of P. gingivalis strains with distinct types of fimbriae for invasion of epithelial cells and for degradation of cellular focal adhesion components, paxillin, and focal adhesion kinase (FAK). Six representative strains with the different types of fimbriae were tested, and P. gingivalis with type II fimbriae (type II P. gingivalis) adhered to and invaded epithelial cells at significantly greater levels than the other strains. There were negligible differences in gingipain activities among the six strains; however, type II P. gingivalis apparently degraded intracellular paxillin in association with a loss of phosphorylation 30 min after infection. Degradation was blocked with cytochalasin D or in mutants with fimA disrupted. Paxillin was degraded by the mutant with Lys-gingipain disrupted, and this degradation was prevented by inhibition of Arg-gingipain activity by Nα-p-tosyl-l-lysine chloromethyl ketone. FAK was also degraded by type II P. gingivalis. Cellular focal adhesions with green fluorescent protein-paxillin macroaggregates were clearly destroyed, and this was associated with cellular morphological changes and microtubule disassembly. In an in vitro wound closure assay, type II P. gingivalis significantly inhibited cellular migration and proliferation compared to the cellular migration and proliferation observed with the other types. These results suggest that type II P. gingivalis efficiently invades epithelial cells and degrades focal adhesion components with Arg-gingipain, which results in cellular impairment during wound healing and periodontal tissue regeneration.

scholarly journals Tripterine Treatment Improves Endothelial Progenitor Cell Function via Integrin-Linked Kinase

2015 ◽  
Vol 37 (3) ◽  
pp. 1089-1103 ◽  
Author(s):  
Chenhui Lu ◽  
Xixiang Yu ◽  
Keqiang Zuo ◽  
Xiaoping Zhang ◽  
Chuanwu Cao ◽  
...  
Keyword(s):  
Vascular Function ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Glycogen Synthase ◽  
Tube Formation ◽  
Dominant Negative ◽  
Endothelial Progenitor ◽  
Integrin Linked Kinase ◽  
Gsk 3Β ◽  
Green Fluorescent

Background/Aims: Atherosclerosis is associated with dysfunction of endothelial progenitor cells (EPCs). Tripterine, a chemical compound derived from the Chinese medicinal plant Tripterygium wilfordii Hook, displays anti-inflammatory properties in several animal models. We hypothesized that tripterine can improve EPC function and thus the efficiency of EPC transplantation. Methods and Results: Tripterine preconditioning (2.5 μM, 4 h) improved EPC proliferation, tube formation, migration, and adhesion, and reduced apoptosis in cells cultured in ox-LDL (200 µg/ml). Tripterine restored integrin-linked kinase (ILK) levels downregulated by ox-LDL in EPCs, suggesting the involvement of the ILK/Akt pathway. Small interfering RNA-mediated depletion of ILK and dominant-negative ILK transduction inhibited the phosphorylation of the ILK downstream signaling targets protein kinase B/Akt and glycogen synthase kinase 3-beta (GSK-3β), and reduced β-catenin and cyclin D1 expression. In atherosclerotic mice injected with green fluorescent protein-labeled EPCs to evaluate EPC function, tripterine decreased aortic lesions and plaque deposition, and injection of tripterine-treated EPCs restored ILK levels. Conclusion: The present results suggest that tripterine improves vascular function in atherosclerosis by enhancing EPC function through a mechanism involving the ILK signaling pathway.

scholarly journals Glycogen Synthase Kinase-3β Inhibition with 9-ING-41 Attenuates the Progression of Pulmonary Fibrosis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ann Jeffers ◽  
Wenyi Qin ◽  
Shuzi Owens ◽  
Kathleen B. Koenig ◽  
Satoshi Komatsu ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Signaling Pathway ◽  
Glycogen Synthase ◽  
Ex Vivo ◽  
Morphological Changes ◽  
Alveolar Epithelial Cell ◽  
Lung Fibroblasts ◽  
Myofibroblast Differentiation ◽  
Gsk 3Β

AbstractIdiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with a median survival of 3 years after diagnosis. Although the etiology of IPF is unknown, it is characterized by extensive alveolar epithelial cell apoptosis and proliferation of myofibroblasts in the lungs. While the origins of these myofibroblast appear to be diverse, fibroblast differentiation contributes to expansion of myofibroblasts and to disease progression. We found that agents that contribute to neomatrix formation and remodeling in pulmonary fibrosis (PF); TGF-β, Factor Xa, thrombin, plasmin and uPA all induced fibroblast/myofibroblast differentiation. These same mediators enhanced GSK-3β activation via phosphorylation of tyrosine-216 (p-Y216). Inhibition of GSK-3β signaling with the novel inhibitor 9-ING-41 blocked the induction of myofibroblast markers; α-SMA and Col-1 and reduced morphological changes of myofibroblast differentiation. In in vivo studies, the progression of TGF-β and bleomycin mediated PF was significantly attenuated by 9-ING-41 administered at 7 and 14 days respectively after the establishment of injury. Specifically, 9-ING-41 treatment significantly improved lung function (compliance and lung volumes; p < 0.05) of TGF-β adenovirus treated mice compared to controls. Similar results were found in mice with bleomycin-induced PF. These studies clearly show that activation of the GSK-3β signaling pathway is critical for the induction of myofibroblast differentiation in lung fibroblasts ex vivo and pulmonary fibrosis in vivo. The results offer a strong premise supporting the continued investigation of the GSK-3β signaling pathway in the control of fibroblast-myofibroblast differentiation and fibrosing lung injury. These data provide a strong rationale for extension of clinical trials of 9-ING-41 to patients with IPF.

Intersecting Chemical Genomic and Genetic Screens Identifies Glycogen Synthase Kinase-3α (GSK-3α) as a Modulator of Differentiation In Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1000-1000
Author(s):  
Versha Banerji ◽  
Kenneth N. Ross ◽  
Loretta S. Li ◽  
Stacey M. Frumm ◽  
Anna C. Schinzel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Small Molecule ◽  
High Throughput ◽  
Glycogen Synthase ◽  
Morphological Changes ◽  
Gene Expression Signature ◽  
Gsk 3Β ◽  
Shrna Screen

Abstract Abstract 1000 The treatment of acute myeloid leukemia (AML) poses a vexing challenge despite an improved understanding of its molecular pathogenesis. A two-hit theory has been proposed for the pathogenesis of AML where the first hit imparts a proliferation defect and the second a block in differentiation. Drug discovery efforts for AML, however, have largely focused on the proliferation defect. Using the intersection of chemical biology and high-throughput genetic screening we sought to identify new AML differentiation targets by measuring the induction of a complex gene expression signature of myeloid maturation. We performed two independent small molecule library screens and a high-throughput shRNA screen for perturbations that induce differentiation in AML cells. We measured this differentiation signature using the previously described gene expression-based high-throughput screening (GE-HTS) approach in which gene expression signatures serve as surrogates for different biological states. Glycogen Synthase Kinase-3 (GSK-3) emerged as a target at the intersection of these three screens. GSK-3 is a multifunctional serine threonine kinase involved in diverse cellular processes including differentiation, signal transduction, cell cycle regulation and proliferation, with an emerging role in human leukemia. We demonstrate that the GSK-3 inhibitors scoring in the primary screens indeed induce the differentiation signature with a dose-response in AML cell lines. In order to further validate GSK-3 as a target, we extended testing to lithium chloride and SB216763, two commonly used GSK-3 inhibitors not in the original screens. Both of these molecules induced AML differentiation as measured by gene expression and morphological changes in multiple AML cell lines and in primary patient blasts in vitro. GSK-3 is expressed as two highly homologous but non-redundant isoforms, GSK-3α and GSK-3β, with small molecule inhibitors non-selectively reported to target both. In order to further validate the findings of the small molecule library screens, we performed a high-throughput shRNA screen targeting the human kinome for shRNAs that induce differentiation. Multiple hairpins against GSK-3α scored. In secondary testing of four AML cell lines, we found that genetic loss of GSK-3α induced differentiation as measured by induction of the complex gene expression signature, alterations in genome-wide expression, and morphological changes associated with maturation. Moreover, colony formation in methylcellulose was impeded. These effects could be rescued with a GSK-3α cDNA immune to the effects of the shRNA, further supporting the on-target activity of the hairpin. In contrast, GSK-3β-directed hairpins induced minimal differentiation. We next extended testing to an in vivo U937 orthotopic model of AML. While pan-GSK-3 inhibition with lithium chloride treatment did not demonstrate efficacy in this model, inhibition of GSK-3α with shRNA attenuated development of disease compared to an shRNA control. We hypothesize that the rise in β-catenin with pan-inhibition of GSK-3 may attenuate the response to lithium in vivo. In contrast, isolated knockdown of GSK-3α does not induce β-catenin. While much of the prior cancer literature has focused on the role of GSK-3β in human malignancy, these studies suggest a role for GSK-3α in AML differentiation and support a role for GSK-3α-directed targeted therapy. Disclosures: No relevant conflicts of interest to declare.

Synthesis of Coumarin Derivatives as Versatile Scaffolds for GSK-3β Enzyme Inhibition

2020 ◽  
Vol 20 (2) ◽  
pp. 153-160 ◽  
Author(s):  
Carla S. Francisco ◽  
Clara L. Javarini ◽  
Iatahanderson de S. Barcelos ◽  
Pedro A.B. Morais ◽  
Heberth de Paula ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Glycogen Synthase ◽  
Synthetic Methods ◽  
Kinase Assay ◽  
Crystallographic Structure ◽  
Coumarin Derivatives ◽  
Enzymatic Production ◽  
Docking Simulations ◽  
In Silico Studies ◽  
Gsk 3Β

Background: Glycogen synthase kinase-3 (GSK-3) is involved in the phosphorylation and inactivation of glycogen synthase. GSK-3 inhibitors have been associated with a variety of diseases, including Alzheimer´s disease (AD), diabetes type II, neurologic disorders, and cancer. The inhibition of GSK-3β isoforms is likely to represent an effective strategy against AD. Objective: The present work aimed to design and synthesize coumarin derivatives to explore their potential as GSK-3β kinase inhibitors. Method: The through different synthetic methods were used to prepare coumarin derivatives. The GSK-3β activity was measured through the ADP-Glo™ Kinase Assay, which quantifies the kinasedependent enzymatic production of ADP from ATP, using a coupled-luminescence-based reaction. A docking study was performed by using the crystallographic structure of the staurosporine/GSK-3β complex [Protein Data Bank (PDB) code: 1Q3D]. Results: The eleven coumarin derivatives were obtained and evaluated as potential GSK-3β inhibitors. Additionally, in silico studies were performed. The results revealed that the compounds 5c, 5d, and 6b inhibited GSK-3β enzymatic activity by 38.97–49.62% at 1 mM. The other coumarin derivatives were tested at 1 mM, 1 µM, and 1 nM concentrations and were shown to be inhibitor candidates, with significant IC50 (1.224–6.875 µM) values, except for compound 7c (IC50 = 10.809 µM). Docking simulations showed polar interactions between compound 5b and Lys85 and Ser203, clarifying the mechanism of the most potent activity. Conclusion: The coumarin derivatives 3a and 5b, developed in this study, showed remarkable activity as GSK-3β inhibitors.

scholarly journals GSK-3β in Pancreatic Cancer: Spotlight on 9-ING-41, Its Therapeutic Potential and Immune Modulatory Properties

Biology ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 610
Author(s):  
Robin Park ◽  
Andrew L. Coveler ◽  
Ludimila Cavalcante ◽  
Anwaar Saeed
Keyword(s):  
Pancreatic Cancer ◽  
Therapeutic Potential ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3 ◽  
In Vivo Models ◽  
Gsk 3Β ◽  
Early Phase Clinical Trials

Glycogen synthase kinase-3 beta is a ubiquitously and constitutively expressed molecule with pleiotropic function. It acts as a protooncogene in the development of several solid tumors including pancreatic cancer through its involvement in various cellular processes including cell proliferation, survival, invasion and metastasis, as well as autophagy. Furthermore, the level of aberrant glycogen synthase kinase-3 beta expression in the nucleus is inversely correlated with tumor differentiation and survival in both in vitro and in vivo models of pancreatic cancer. Small molecule inhibitors of glycogen synthase kinase-3 beta have demonstrated therapeutic potential in pre-clinical models and are currently being evaluated in early phase clinical trials involving pancreatic cancer patients with interim results showing favorable results. Moreover, recent studies support a rationale for the combination of glycogen synthase kinase-3 beta inhibitors with chemotherapy and immunotherapy, warranting the evaluation of novel combination regimens in the future.

scholarly journals Structural-Based Optimizations of the Marine-Originated Meridianin C as Glucose Uptake Agents by Inhibiting GSK-3β

Marine Drugs ◽  
2021 ◽  
Vol 19 (3) ◽  
pp. 149
Author(s):  
Shuwen Han ◽  
Chunlin Zhuang ◽  
Wei Zhou ◽  
Fener Chen
Keyword(s):  
Glucose Uptake ◽  
Therapeutic Potential ◽  
Glycogen Synthase ◽  
Molecular Target ◽  
Optimization Strategy ◽  
Lead Compounds ◽  
Significant Toxicity ◽  
Treatment Of Diabetes ◽  
Gsk 3Β ◽  
Positive Compound

Glycogen synthase kinase 3β (GSK-3β) is a widely investigated molecular target for numerous diseases, and inhibition of GSK-3β activity has become an attractive approach for the treatment of diabetes. Meridianin C, an indole-based natural product isolated from marine Aplidium meridianum, has been reported as a potent GSK-3β inhibitor. In the present study, applying the structural-based optimization strategy, the pyrimidine group of meridianin C was modified by introducing different substituents based on the 2-aminopyrimidines-substituted pyrazolo pyridazine scaffold. Among them, compounds B29 and B30 showed a much higher glucose uptake than meridianin C (<5%) and the positive compound 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8, 16%), with no significant toxicity against HepG2 cells at the same time. Furthermore, they displayed good GSK-3β inhibitory activities (IC50 = 5.85; 24.4 μM). These results suggest that these meridianin C analogues represent novel lead compounds with therapeutic potential for diabetes.

scholarly journals Resveratrol Inhibits Venezuelan Equine Encephalitis Virus Infection by Interfering with the AKT/GSK Pathway

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 346
Author(s):  
Caitlin W. Lehman ◽  
Kylene Kehn-Hall ◽  
Megha Aggarwal ◽  
Nicole R. Bracci ◽  
Han-Chi Pan ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Fluorescent Protein ◽  
Glycogen Synthase ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Host Cells ◽  
Dynamics Simulation ◽  
Luciferase Reporter ◽  
Venezuelan Equine Encephalitis ◽  
Equine Encephalitis Virus

The host proteins Protein Kinase B (AKT) and glycogen synthase kinase-3 (GSK-3) are associated with multiple neurodegenerative disorders. They are also important for the replication of Venezuelan equine encephalitis virus (VEEV), thereby making the AKT/GSK-3 pathway an attractive target for developing anti-VEEV therapeutics. Resveratrol, a natural phytochemical, has been shown to substantially inhibit the AKT pathway. Therefore, we attempted to explore whether it exerts any antiviral activity against VEEV. In this study, we utilized green fluorescent protein (GFP)- and luciferase-encoding recombinant VEEV to determine the cytotoxicity and antiviral efficacy via luciferase reporter assays, flow cytometry, and immunofluorescent assays. Our results indicate that resveratrol treatment is capable of inhibiting VEEV replication, resulting in increased viability of Vero and U87MG cells as well as reduced virion production and viral RNA contents within host cells for at least 48 h with a single treatment. Furthermore, the suppression of apoptotic signaling adaptors, caspase-3, caspase-7, and annexin V may also be implicated in resveratrol-mediated antiviral activity. We found that decreased phosphorylation of the AKT/GSK-3 pathway, mediated by resveratrol, can be triggered during the early stages of VEEV infection, suggesting that resveratrol disrupts the viral replication cycle and consequently promotes cell survival. Finally, molecular docking and dynamics simulation studies revealed that resveratrol can directly bind to VEEV glycoproteins, which may interfere with virus attachment and entry. In conclusion, our results suggest that resveratrol exerts inhibitory activity against VEEV infection and upon further modification could be a useful compound to study in neuroprotective research and veterinary sciences.

scholarly journals Fisetin inhibits lipopolysaccharide-induced inflammatory response by activating β-catenin, leading to a decrease in endotoxic shock

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ilandarage Menu Neelaka Molagoda ◽  
Jayasingha Arachchige Chathuranga C Jayasingha ◽  
Yung Hyun Choi ◽  
Rajapaksha Gedara Prasad Tharanga Jayasooriya ◽  
Chang-Hee Kang ◽  
...  
Keyword(s):  
Glycogen Synthase ◽  
Endotoxic Shock ◽  
Inflammatory Activity ◽  
Prostaglandin E ◽  
Necrosis Factor Alpha ◽  
Zebrafish Larvae ◽  
Proinflammatory Mediators ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Gsk 3Β

AbstractFisetin is a naturally occurring flavonoid that possesses several pharmacological benefits including anti-inflammatory activity. However, its precise anti-inflammatory mechanism is not clear. In the present study, we found that fisetin significantly inhibited the expression of proinflammatory mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), and cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Additionally, fisetin attenuated LPS-induced mortality and abnormalities in zebrafish larvae and normalized the heart rate. Fisetin decreased the recruitment of macrophages and neutrophils to the LPS-microinjected inflammatory site in zebrafish larvae, concomitant with a significant downregulation of proinflammatory genes, such as inducible NO synthase (iNOS), cyclooxygenase-2a (COX-2a), IL-6, and TNF-α. Fisetin inhibited the nuclear localization of nuclear factor-kappa B (NF-κB), which reduced the expression of pro-inflammatory genes. Further, fisetin inactivated glycogen synthase kinase 3β (GSK-3β) via phosphorylation at Ser9, and inhibited the degradation of β-catenin, which consequently promoted the localization of β-catenin into the nucleus. The pharmacological inhibition of β-catenin with FH535 reversed the fisetin-induced anti-inflammatory activity and restored NF-κB activity, which indicated that fisetin-mediated activation of β-catenin results in the inhibition of LPS-induced NF-κB activity. In LPS-microinjected zebrafish larvae, FH535 promoted the migration of macrophages to the yolk sac and decreased resident neutrophil counts in the posterior blood island and induced high expression of iNOS and COX-2a, which was accompanied by the inhibition of fisetin-induced anti-inflammatory activity. Altogether, the current study confirmed that the dietary flavonoid, fisetin, inhibited LPS-induced inflammation and endotoxic shock through crosstalk between GSK-3β/β-catenin and the NF-κB signaling pathways.

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