scholarly journals Nuclear Factor of Activated T Cells Is Activated in the Endothelium of Retinal Microvessels in Diabetic Mice

2015 ◽  
Vol 2015 ◽  
pp. 1-14 ◽  
Author(s):  
Anna V. Zetterqvist ◽  
Fabiana Blanco ◽  
Jenny Öhman ◽  
Olga Kotova ◽  
Lisa M. Berglund ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Microvascular Complications ◽  
Cerebral Arteries ◽  
Extracellular Nucleotides ◽  
Nuclear Accumulation ◽  
Luciferase Reporter ◽  
Retinal Vessels ◽  
Diabetic Mice ◽  
Activated T Cells

The pathogenesis of diabetic retinopathy (DR) remains unclear but hyperglycemia is an established risk factor. Endothelial dysfunction and changes in Ca2+signaling have been shown to precede the onset of DR. We recently demonstrated that high extracellular glucose activates the Ca2+/calcineurin-dependent transcription factor NFAT in cerebral arteries and aorta, promoting the expression of inflammatory markers. Here we show, using confocal immunofluorescence, that NFAT is expressed in the endothelium of retinal microvessels and is readily activated by high glucose. This was inhibited by the NFAT blocker A-285222 as well as by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. Acute hyperglycemia induced by an IP-GTT (intraperitoneal glucose tolerance test) resulted in increased NFATc3 nuclear accumulation and NFAT-dependent transcriptional activity in retinal vessels of NFAT-luciferase reporter mice. In both Akita (Ins2+/−) and streptozotocin- (STZ-) induced diabetic mice, NFAT transcriptional activity was elevated in retinal vessels.In vivoinhibition of NFAT with A-285222 decreased the expression ofOPNandICAM-1mRNA in retinal vessels, prevented a diabetes driven downregulation of anti-inflammatory IL-10 in retina, and abrogated the increased vascular permeability observed in diabetic mice. Results identify NFAT signaling as a putative target for treatment of microvascular complications in diabetes.

scholarly journals Novel blocker of NFAT activation inhibits IL-6 production in human myometrial arteries and reduces vascular smooth muscle cell proliferation

2007 ◽  
Vol 292 (3) ◽  
pp. C1167-C1178 ◽  
Author(s):  
Lisa M. Nilsson ◽  
Zheng-Wu Sun ◽  
Jenny Nilsson ◽  
Ina Nordström ◽  
Yung-Wu Chen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Synthesis ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Transcriptional Activity ◽  
Cell Types ◽  
Nuclear Accumulation ◽  
Luciferase Reporter ◽  
Activated T Cells ◽  
Endothelin 1

The calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway has been found to play a role in regulating growth and differentiation in several cell types. However, the functional significance of NFAT in the vasculature is largely unclear. Here we show that NFATc1, NFATc3, and NFATc4 are expressed in human myometrial arteries. Confocal immunofluorescence and Western blot analysis revealed that endothelin-1 efficiently increases NFATc3 nuclear accumulation in native arteries. Endothelin-1 also stimulates NFAT-dependent transcriptional activity, as shown by a luciferase reporter assay. Both the agonist-induced NFAT nuclear accumulation and transcriptional activity were prevented by the calcineurin inhibitor CsA and by the novel NFAT blocker A-285222. Chronic inhibition of NFAT significantly reduced IL-6 production in intact myometrial arteries and inhibited cell proliferation in vascular smooth muscle cells cultured from explants from the same arteries. Furthermore, by using small interfering RNA-mediated reduction of NFATc3, we show that this isoform is involved in the regulation of cell proliferation. Protein synthesis in intact arteries was investigated using autoradiography of [35S]methionine incorporation in serum-free culture. Inhibition of NFAT signaling did not affect overall protein synthesis or specifically the synthesis rates of major proteins associated with the contractile/cytoskeletal system. An intact contractile phenotype under these conditions was also shown by unchanged force response to depolarization or agonist stimulation. Our results demonstrate NFAT expression and activation in native human vessels and point out A-285222 as a powerful pharmacological blocker of NFAT signaling in the vasculature.

scholarly journals In vivo inhibition of Nuclear Factor of Activated T‐cells (NFAT) results in enhanced levels of anti‐inflammatory IL‐10 in the retina of diabetic mice

2010 ◽  
Vol 24 (S1) ◽  
Author(s):  
Anna V Zetterqvist ◽  
Jenny Nilsson‐Öhman ◽  
Olga Kotova ◽  
Lisa M Nilsson‐Berglund ◽  
Sergio Frutos Garcia ◽  
...  
Keyword(s):  
T Cells ◽  
Nuclear Factor ◽  
Diabetic Mice ◽  
Activated T Cells ◽  
Anti Inflammatory

In vivo regulation of β-MHC gene in rodent heart: role of T3 and evidence for an upstream enhancer

1999 ◽  
Vol 276 (4) ◽  
pp. C883-C891 ◽  
Author(s):  
Carola E. Wright ◽  
F. Haddad ◽  
A. X. Qin ◽  
P. W. Bodell ◽  
K. M. Baldwin
Keyword(s):  
Gene Expression ◽  
Thyroid Hormone ◽  
Reporter Gene ◽  
Normal Control ◽  
Transcriptional Activity ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Promoter Construct ◽  
Hormone Responsiveness

Cardiac β-myosin heavy chain (β-MHC) gene expression is mainly regulated through transcriptional processes. Although these results are based primarily on in vitro cell culture models, relatively little information is available concerning the interaction of key regulatory factors thought to modulate MHC expression in the intact rodent heart. Using a direct gene transfer approach, we studied the in vivo transcriptional activity of different-length β-MHC promoter fragments in normal control and in altered thyroid states. The test β-MHC promoter was fused to a firefly luciferase reporter gene, whereas the control α-MHC promoter was fused to the Renilla luciferase reporter gene and was used to account for variations in transfection efficiency. Absolute reporter gene activities showed that β- and α-MHC genes were individually and reciprocally regulated by thyroid hormone. The β-to-α ratios of reporter gene expression demonstrated an almost threefold larger β-MHC gene expression in the longest than in the shorter promoter fragments in normal control animals, implying the existence of an upstream enhancer. A mutation in the putative thyroid response element of the −408-bp β-MHC promoter construct caused transcriptional activity to drop to null. When studied in the −3,500-bp β-MHC promoter, construct activity was reduced (∼100-fold) while thyroid hormone responsiveness was retained. These findings suggest that, even though the bulk of the thyroid hormone responsiveness of the gene is contained within the first 215 bp of the β-MHC promoter sequence, the exact mechanism of triiodothyronine (T3) action remains to be elucidated.

scholarly journals Neuritin Up-regulates Kv4.2 α-Subunit of Potassium Channel Expression and Affects Neuronal Excitability by Regulating the Calcium-Calcineurin-NFATc4 Signaling Pathway

2016 ◽  
Vol 291 (33) ◽  
pp. 17369-17381 ◽  
Author(s):  
Jin-jing Yao ◽  
Qian-Ru Zhao ◽  
Dong-Dong Liu ◽  
Chi-Wing Chow ◽  
Yan-Ai Mei
Keyword(s):  
Signaling Pathway ◽  
Neural Development ◽  
Cortical Neurons ◽  
Molecular Mechanisms ◽  
Neuronal Excitability ◽  
Outward Current ◽  
Nuclear Accumulation ◽  
Luciferase Reporter ◽  
Activated T Cells ◽  
Α Subunit

Neuritin is an important neurotrophin that regulates neural development, synaptic plasticity, and neuronal survival. Elucidating the downstream molecular signaling is important for potential therapeutic applications of neuritin in neuronal dysfunctions. We previously showed that neuritin up-regulates transient potassium outward current (IA) subunit Kv4.2 expression and increases IA densities, in part by activating the insulin receptor signaling pathway. Molecular mechanisms of neuritin-induced Kv4.2 expression remain elusive. Here, we report that the Ca2+/calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) c4 axis is required for neuritin-induced Kv4.2 transcriptional expression and potentiation of IA densities in cerebellum granule neurons. We found that neuritin elevates intracellular Ca2+ and increases Kv4.2 expression and IA densities; this effect was sensitive to CaN inhibition and was eliminated in Nfatc4−/− mice but not in Nfatc2−/− mice. Stimulation with neuritin significantly increased nuclear accumulation of NFATc4 in cerebellum granule cells and HeLa cells, which expressed IR. Furthermore, NFATc4 was recruited to the Kv4.2 gene promoter loci detected by luciferase reporter and chromatin immunoprecipitation assays. More importantly, data obtained from cortical neurons following adeno-associated virus-mediated overexpression of neuritin indicated that reduced neuronal excitability and increased formation of dendritic spines were abrogated in the Nfatc4−/− mice. Together, these data demonstrate an indispensable role for the CaN/NFATc4 signaling pathway in neuritin-regulated neuronal functions.

scholarly journals A Splice Variant of NCOR2, BQ323636.1, Confers Chemoresistance in Breast Cancer by Altering the Activity of NRF2

Cancers ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 533
Author(s):  
Man-Hong Leung ◽  
Ho Tsoi ◽  
Chun Gong ◽  
Ellen PS Man ◽  
Stefania Zona ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Oxidative Stress ◽  
Splice Variant ◽  
Primary Breast Cancer ◽  
Transcriptional Activity ◽  
Control Cell ◽  
Luciferase Reporter ◽  
Nrf2 Signaling Pathway

Breast cancer is the most common type of female cancer. Reactive oxygen species (ROS) are vital in regulating signaling pathways that control cell survival and cell proliferation. Chemotherapeutic drugs such as anthracyclines induce cell death via ROS induction. Chemoresistance development is associated with adaptive response to oxidative stress. NRF2 is the main regulator of cytoprotective response to oxidative stress. NRF2 can enhance cell growth, antioxidant expression, and chemoresistance by providing growth advantage for malignant cells. Previously, we identified BQ323636.1 (BQ), a novel splice variant of nuclear co-repressor NCOR2, which can robustly predict tamoxifen resistance in primary breast cancer. In this study, we found that BQ was overexpressed in epirubicin-resistant cells and demonstrated that BQ overexpression could reduce the levels of epirubicin-induced ROS and confer epirubicin resistance. In vivo analysis using tissue microarray of primary breast cancer showed direct correlation between BQ expression and chemoresistance. In vitro experiments showed BQ could modulate NRF2 transcriptional activity and upregulate antioxidants. Luciferase reporter assays showed that although NCOR2 repressed the transcriptional activity of NRF2, the presence of BQ reduced this repressive activity. Co-immunoprecipitation confirmed that NCOR2 could bind to NRF2 and that this interaction was compromised by BQ overexpression, leading to increased transcriptional activity in NRF2. Our findings suggest BQ can regulate the NRF2 signaling pathway via interference with NCOR2 suppressive activity and reveals a novel role for BQ as a modulator of chemoresistance in breast cancer.

scholarly journals Modulating PKCα Activity to Target Wnt/β-Catenin Signaling in Colon Cancer

Cancers ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 693 ◽  
Author(s):  
Sébastien Dupasquier ◽  
Philippe Blache ◽  
Laurence Picque Lasorsa ◽  
Han Zhao ◽  
Jean-Daniel Abraham ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Target Genes ◽  
Tumor Development ◽  
Orphan Receptor ◽  
Nuclear Accumulation ◽  
Kinase C ◽  
Extracellular Signal

Inactivating mutations of the tumor suppressor Adenomatosis Polyposis Coli (APC), which are found in familial adenomatosis polyposis and in 80% of sporadic colorectal cancers (CRC), result in constitutive activation of the Wnt/β-catenin pathway and tumor development in the intestine. These mutations disconnect the Wnt/β-catenin pathway from its Wnt extracellular signal by inactivating the APC/GSK3-β/axin destruction complex of β-catenin. This results in sustained nuclear accumulation of β-catenin, followed by β-catenin-dependent co-transcriptional activation of Wnt/β-catenin target genes. Thus, mechanisms acting downstream of APC, such as those controlling β-catenin stability and/or co-transcriptional activity, are attractive targets for CRC treatment. Protein Kinase C-α (PKCα) phosphorylates the orphan receptor RORα that then inhibits β-catenin co-transcriptional activity. PKCα also phosphorylates β-catenin, leading to its degradation by the proteasome. Here, using both in vitro (DLD-1 cells) and in vivo (C57BL/6J mice) PKCα knock-in models, we investigated whether enhancing PKCα function could be beneficial in CRC treatment. We found that PKCα is infrequently mutated in CRC samples, and that inducing PKCα function is not deleterious for the normal intestinal epithelium. Conversely, di-terpene ester-induced PKCα activity triggers CRC cell death. Together, these data indicate that PKCα is a relevant drug target for CRC treatment.

scholarly journals CircFOXO3 promotes glioblastoma progression by acting as a competing endogenous RNA for NFAT5

2019 ◽  
Vol 21 (10) ◽  
pp. 1284-1296 ◽  
Author(s):  
Shuai Zhang ◽  
Keman Liao ◽  
Zengli Miao ◽  
Qing Wang ◽  
Yifeng Miao ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Noncoding Rna ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Competing Endogenous Rna ◽  
Activated T Cells ◽  
Reverse Transcription Pcr ◽  
Proliferation And Invasion

Abstract Background Circular RNAs (circRNAs), a newly discovered type of endogenous noncoding RNA, have been proposed to mediate the progression of diverse types of tumors. Systematic studies of circRNAs have just begun, and the physiological roles of circRNAs remain largely unknown. Here, we focused on elucidating the potential role and molecular mechanism of circular forkhead box O3 (circFOXO3) in glioblastoma (GBM) progression. Methods First, we analyzed circFOXO3 alterations in GBM and noncancerous tissues through real-time quantitative reverse transcription PCR (qRT-PCR). Next, we used loss- and gain-of-function approaches to evaluate the effect of circFOXO3 on GBM cell proliferation and invasion. Mechanistically, fluorescent in situ hybridization, RNA pull-down, dual luciferase reporter, and RNA immunoprecipitation assays were performed to confirm the interaction between circFOXO3 and miR-138-5p/miR-432-5p in GBM. An animal model was used to verify the in vitro experimental findings. Results CircFOXO3 expression was significantly higher in GBM tissues than in noncancerous tissues. GBM cell proliferation and invasion were reduced by circFOXO3 knockdown and enhanced by circFOXO3 overexpression. Further biochemical analysis showed that circFOXO3 exerted its pro-tumorigenic activity by acting as a competing endogenous RNA (ceRNA) to increase expression of nuclear factor of activated T cells 5 (NFAT5) via sponging both miR-138-5p and miR-432-5p. Notably, tumor inhibition by circFOXO3 downregulation could be reversed by miR-138-5p/miR-432-5p inhibitors in GBM cells. Moreover, GBM cells with lower circFOXO3 expression developed less aggressive tumors in vivo. Conclusions Our data demonstrate that circFOXO3 can exert regulatory functions in GBM and that ceRNA-mediated microRNA sequestration might be a potential strategy for GBM therapy.

scholarly journals Xiao Yao San against Corticosterone-Induced Stress Injury via Upregulating Glucocorticoid Receptor Reaction Element Transcriptional Activity

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Guoping Cao ◽  
Shenglan Gong ◽  
Fengxue Zhang ◽  
Wenjun Fu
Keyword(s):  
Hippocampal Neurons ◽  
Transcriptional Activity ◽  
Nuclear Translocation ◽  
Luciferase Reporter ◽  
Potential Candidate ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Stress Injury ◽  
Induced Stress

Previous studies have revealed that uncontrollable stress can impair the synaptic plasticity and firing property of hippocampal neurons, which influenced various hippocampal-dependent tasks including memory, cognition, behavior, and mood. In this work, we had investigated the effects and mechanisms of the Chinese herbal medicine Xiao Yao San (XYS) against corticosterone-induced stress injury in primary hippocampal neurons (PHN) cells. We found that XYS and RU38486 could increase cell viabilities and decrease cell apoptosis by MTT, immunofluorescence, and flow cytometry assays. In addition, we observed that XYS notably inhibited the nuclear translocation of GR and upregulated the mRNA and protein expressions levels of Caveolin-1, GR, BDNF, TrkB, and FKBP4. However, XYS downregulated the FKBP51 expressions. Furthermore, the results of the electrophoretic mobility shift assay (EMSA) and double luciferase reporter gene detection indicated that FKBP4 promotes the transcriptional activity of GR reaction element (GRE) by binding with GR, and FKBP51 processed the opposite action. Thein vivoexperiment also proved the functions of XYS. These results suggested that XYS showed an efficient neuroprotection against corticosterone-induced stress injury in PHN cells by upregulating GRE transcriptional activity, which should be developed as a potential candidate for treating stress injury in the future.

scholarly journals Crosstalk between SUMOylated PPARγ1 and FOXO1 Exerts a Positive-feedback Effect on Vascular Endothelial Insulin Resistance

2021 ◽  
Author(s):  
Ying Kong ◽  
Ailin Niu ◽  
Wanwan Yuan ◽  
Min Xia ◽  
Xiaowei Xiong ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Positive Feedback ◽  
Transcriptional Activity ◽  
Feedback Effect ◽  
Nuclear Accumulation ◽  
Akt Pathway ◽  
Vascular Endothelial ◽  
Positive Feedback Effect

Abstract Background: PPARγ and FOXO1 are key regulators of transcription factors that mediate insulin sensitivity. We previously showed that a small ubiquitin-related modifier of PPARγ1 at K77 (SUMOylation) favors endothelial insulin resistance (IR) induced by high-fat/high-glucose (HF/HG) administration. However, whether and how the crosstalk between SUMOylated PPARγ1 and FOXO1 mediates the development of IR remains unclear. Here, we place emphasis on elucidating how PPARγ1-K77 SUMOylation interacts with FOXO1 and participates in the development of endothelial IR.Methods: Adenovirus or adeno-associated virus carrying a truncated PPARγ1 containing AF1 and DBD domains fused with SUMO-1 (PPARγ1[1-182 aa]-SUMO-1 fusion protein) was utilized to simulate PPARγ1-K77 SUMOylation. Furthermore, we carried out PPARγ1-K77 SUMOylation imitating-IR and worsening-IR experiments in vitro and in vivo. The vascular diastolic function and levels of p-IKK, IKK, p-PI3K, PI3K, p-Akt, Akt, p-eNOS, and eNOS were measured. To elucidate the underlying mechanism, the interaction of PPARγ1-K77 SUMOylation and FOXO1 was examined by co-immunoprecipitation. The recruitment of PPARγ1 or FOXO1 to PPRE was analyzed by chromatin immunoprecipitation, followed by measuring the PPARγ1 transcriptional activity and translocation of FOXO1.Results: Our results show that like HF/HG, PPARγ1-K77 SUMOylation imitates endothelial IR and dysfunction, presenting decreased NO levels and elevated ET-1 levels, with PI3K/Akt/eNOS pathway inhibited, and endothelium-dependent vasodilation function impaired. Moreover, combination of HF/HG and PPARγ1-K77 SUMOylation exhibits a synergistic worsening effect on endothelial IR. Mechanistically, the results reveal that PPARγ1-K77 SUMOylation readily interacts with FOXO1, and the PPRE binding site of PI3K is competitively blocked by FOXO1, which represses PPARγ1 transcriptional activity and downregulates the PI3K-Akt pathway. Inhibition of the PI3K-Akt pathway promotes the nuclear accumulation of FOXO1, which interacts with SUMOylated PPARγ1 in the nucleus, exerting a positive feedback effect on IR pathogenesis.Conclusion: These results reveal a novel association between PPARγ1-K77 SUMOylation and FOXO1, which inhibits PPARγ1 transcriptional activity and contributes to vascular endothelial IR. These findings will be beneficial for better understanding the pathogenesis of endothelial IR and providing novel pharmacological targets for diabetic angiopathy.

scholarly journals NULP1 Alleviates Cardiac Hypertrophy by Suppressing NFAT3 Transcriptional Activity

2020 ◽  
Vol 9 (16) ◽  
Author(s):  
Xin Zhang ◽  
Fang Lei ◽  
Xiao‐Ming Wang ◽  
Ke‐Qiong Deng ◽  
Yan‐Xiao Ji ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Transcriptional Activity ◽  
Therapeutic Strategy ◽  
Aortic Banding ◽  
Activated T Cells ◽  
Rat Cardiomyocytes ◽  
Helix Loop Helix ◽  
Pathological Cardiac Hypertrophy ◽  
Transcription Activators

Background The development of pathological cardiac hypertrophy involves the coordination of a series of transcription activators and repressors, while their interplay to trigger pathological gene reprogramming remains unclear. NULP1 (nuclear localized protein 1) is a member of the basic helix‐loop‐helix family of transcription factors and its biological functions in pathological cardiac hypertrophy are barely understood. Methods and Results Immunoblot and immunostaining analyses showed that NULP1 expression was consistently reduced in the failing hearts of patients and hypertrophic mouse hearts and rat cardiomyocytes. Nulp1 knockout exacerbates aortic banding‐induced cardiac hypertrophy pathology, which was significantly blunted by transgenic overexpression of Nulp1 . Signal pathway screening revealed the nuclear factor of activated T cells (NFAT) pathway to be dramatically suppressed by NULP1. Coimmunoprecipitation showed that NULP1 directly interacted with the topologically associating domain of NFAT3 via its C‐terminal region, which was sufficient to suppress NFAT3 transcriptional activity. Inactivation of the NFAT pathway by VIVIT peptides in vivo rescued the aggravated pathogenesis of cardiac hypertrophy resulting from Nulp1 deficiency. Conclusions NULP1 is an endogenous suppressor of NFAT3 signaling under hypertrophic stress and thus negatively regulates the pathogenesis of cardiac hypertrophy. Targeting overactivated NFAT by NULP1 may be a novel therapeutic strategy for the treatment of pathological cardiac hypertrophy and heart failure.

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