scholarly journals Stability of Smyd1 in endothelial cells is controlled by PML-dependent SUMOylation upon cytokine Stimulation

2020 ◽  
Author(s):  
Samuel Becker ◽  
Gustav Steinemann ◽  
Weronika Karle ◽  
Kerrin Roos ◽  
Celine Huajia Liem ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Mrna Level ◽  
Feedback Mechanism ◽  
Nuclear Bodies ◽  
Expression Vectors ◽  
Dependent Manner ◽  
Molecular Control ◽  
Pml Nuclear Bodies ◽  
Ea.Hy926 Cells ◽  
Ifn Γ

Smyd1 is an epigenetic modulator of gene expression that has been well-characterized in muscle cells. It was recently reported that Smyd1 levels are modulated by inflammatory processes. Since inflammation affects the vascular endothelium, this study aimed to characterize Smyd1 expression in endothelial cells. We detected Smyd1 in human endothelial cells (HUVEC and EA.hy926 cells), where the protein was largely localized in PML nuclear bodies (PML-NBs). By transfection of EA.hy926 cells with expression vectors encoding Smyd1, PML, SUMO1, active or mutant forms of the SUMO protease SuPr1 and/or the SUMO-conjugating UBC9, as well as Smyd1- or PML-specific siRNAs, in the presence or absence of the translation blocker cycloheximide or the proteasome-inhibitor MG132, and supported by computational modeling, we show that Smyd1 is SUMOylated in a PML-dependent manner and thereby addressed for degradation in proteasomes. Furthermore, transfection with Smyd1-encoding vectors led to PML up-regulation at the mRNA level, while PML transfection lowered Smyd1 protein stability. Incubation of EA.hy926 cells with the pro-inflammatory cytokine TNF-α resulted in a constant increase in Smyd1 mRNA and protein over 24 h, while incubation with IFN-γ induced a transient increase of Smyd1 expression, which peaked at 6 h and decreased to control values within 24 h. The IFN-γ-induced increase of Smyd1 was accompanied by more Smyd1 SUMOylation and more/larger PML-NBs. In conclusion, our data indicate that in endothelial cells, Smyd1 levels are regulated through a negative feedback mechanism based on SUMOylation and PML availability. This molecular control loop is stimulated by various cytokines.

PML nuclear bodies are recruited to persistent DNA damage lesions in an RNF168-53BP1 dependent manner and contribute to DNA repair

DNA Repair ◽  
2019 ◽  
Vol 78 ◽  
pp. 114-127 ◽  
Author(s):  
Marketa Vancurova ◽  
Hana Hanzlikova ◽  
Lucie Knoblochova ◽  
Jan Kosla ◽  
Dusana Majera ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Nuclear Bodies ◽  
Dependent Manner ◽  
Pml Nuclear Bodies

scholarly journals Arsenic-Induced SUMO-Dependent Recruitment of RNF4 into PML Nuclear Bodies

2010 ◽  
Vol 21 (23) ◽  
pp. 4227-4239 ◽  
Author(s):  
Marie-Claude Geoffroy ◽  
Ellis G. Jaffray ◽  
Katherine J. Walker ◽  
Ronald T. Hay
Keyword(s):  
Retinoic Acid Receptor ◽  
E3 Ligase ◽  
Nuclear Body ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Dependent Manner ◽  
Nuclear Trafficking ◽  
Sumo Modification ◽  
Ubiquitin E3 Ligase ◽  
Pml Nuclear Bodies

In acute promyelocytic leukemia (APL), the promyelocytic leukemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). Arsenic is an effective treatment for this disease as it induces SUMO-dependent ubiquitin-mediated proteasomal degradation of the PML-RAR fusion protein. Here we analyze the nuclear trafficking dynamics of PML and its SUMO-dependent ubiquitin E3 ligase, RNF4 in response to arsenic. After administration of arsenic, PML immediately transits into nuclear bodies where it undergoes SUMO modification. This initial recruitment of PML into nuclear bodies is not dependent on RNF4, but RNF4 quickly follows PML into the nuclear bodies where it is responsible for ubiquitylation of SUMO-modified PML and its degradation by the proteasome. While arsenic restricts the mobility of PML, FRAP analysis indicates that RNF4 continues to rapidly shuttle into PML nuclear bodies in a SUMO-dependent manner. Under these conditions FRET studies indicate that RNF4 interacts with SUMO in PML bodies but not directly with PML. These studies indicate that arsenic induces the rapid reorganization of the cell nucleus by SUMO modification of nuclear body-associated PML and uptake of the ubiquitin E3 ligase RNF4 leading to the ubiquitin-mediated degradation of PML.

scholarly journals Transcriptional Regulation Is Affected by Subnuclear Targeting of Reporter Plasmids to PML Nuclear Bodies

2006 ◽  
Vol 26 (23) ◽  
pp. 8814-8825 ◽  
Author(s):  
Gregory J. Block ◽  
Christopher H. Eskiw ◽  
Graham Dellaire ◽  
David P. Bazett-Jones
Keyword(s):  
Transcriptional Regulation ◽  
Transcriptional Activation ◽  
Plasmid Dna ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Nuclear Bodies ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Reporter Plasmid ◽  
Pml Nuclear Bodies

ABSTRACT Whereas the PML protein has been reported to have both transcriptional coactivator and corepressor potential, the contribution of the PML nuclear body (PML NB) itself to transcriptional regulation is not well understood. Here we demonstrate that plasmid DNA artificially tethered to PML or the PML NB-targeting domain of Sp100 is preferentially localized to PML NBs. Using the tethering technique, we targeted a simian virus 40 promoter-driven luciferase reporter plasmid to PML NBs, resulting in the repression of the transgene transcriptional activity. Conversely, the tethering of a cytomegalovirus promoter-containing reporter plasmid resulted in activation. Targeting a minimal eukaryotic promoter did not affect its activity. The expression of targeted promoters could be modulated by altering the cellular concentration of PML NB components, including Sp100 and isoforms of the PML protein. Finally, we demonstrate that ICP0, the promiscuous herpes simplex virus transactivator, increases the level of transcriptional activation of plasmid DNA tethered to the PML NB. We conclude that when PML NB components are artificially tethered to reporter plasmids, the PML NB contributes to the regulation of the tethered DNA in a promoter-dependent manner. Our findings demonstrate that transient transcription assays are sensitive to the subnuclear localization of the transgene plasmid.

scholarly journals The Disruption of ND10 during Herpes Simplex Virus Infection Correlates with the Vmw110- and Proteasome-Dependent Loss of Several PML Isoforms

1998 ◽  
Vol 72 (8) ◽  
pp. 6581-6591 ◽  
Author(s):  
Roger D. Everett ◽  
Paul Freemont ◽  
Hisato Saitoh ◽  
Mary Dasso ◽  
Anne Orr ◽  
...  
Keyword(s):  
Virus Infection ◽  
Regulatory Protein ◽  
Biochemical Properties ◽  
Nuclear Bodies ◽  
Dependent Manner ◽  
Pml Nuclear Bodies ◽  
Protease Enzyme ◽  
Specific Protease ◽  
Nuclear Structures ◽  
Simplex Virus

ABSTRACT The small nuclear structures known as ND10 or PML nuclear bodies have been implicated in a variety of cellular processes including response to stress and interferons, oncogenesis, and viral infection, but little is known about their biochemical properties. Recently, a ubiquitin-specific protease enzyme (named HAUSP) and a ubiquitin-homology family protein (PIC1) have been found associated with ND10. HAUSP binds strongly to Vmw110, a herpesvirus regulatory protein which has the ability to disrupt ND10, while PIC1 was identified as a protein which interacts with PML, the prototype ND10 protein. We have investigated the role of ubiquitin-related pathways in the mechanism of ND10 disruption by Vmw110 and the effect of virus infection on PML stability. The results show that the disruption of ND10 during virus infection correlates with the loss of several PML isoforms and this process is dependent on active proteasomes. The PML isoforms that are most sensitive to virus infection correspond closely to those which have recently been identified as being covalently conjugated to PIC1. In addition, a large number of PIC1-protein conjugates can be detected following transfection of a PIC1 expression plasmid, and many of these are also eliminated in a Vmw110-dependent manner during virus infection. These observations provide a biochemical mechanism to explain the observed effects of Vmw110 on ND10 and suggest a simple yet powerful mechanism by which Vmw110 might function during virus infection.

Stimulation of Tetrahydrobiopterin Synthesis by Basic Fibroblast Growth Factor in Vascular Endothelial Cells

Pteridines ◽  
2003 ◽  
Vol 14 (1) ◽  
pp. 9-12 ◽  
Author(s):  
Shunichi Shimizu ◽  
Yoshiyuki Miyasaka ◽  
Shinichiro Yamamoto ◽  
Masakazu Ishii ◽  
Yuji Kiuchi
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Basic Fibroblast Growth Factor ◽  
De Novo ◽  
Mrna Level ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Fibroblast Growth ◽  
Concentration Dependent Manner

Abstract The purpose of this study was to examine whether basic fibroblast growth factor (bFGF) stimulates tetrahydrobiopterin (BH4) synthesis in mouse brain microvascular endothelial cells. BH4 content was determined by oxidation under acidic conditions as biopterin and analysed with reversed-phase high Performance liquid chromatography. Measurement of the mRNA level of QTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme of the de novo pathway of BH4 synthesis. The addition of bFGF to endothelial cells increased the BH4 content and GTPCH mRNA levels in an incubation period- and a concentration-dependent manner. 2,4-Diamino-6- hydroxypyrimidine, an inhibitor of GTPCH, strongly reduced the bFGF-induced increase in BH4 content. These findings suggest that bFGF stimulates BH4 synthesis via a de novo pathway with the induction of GTPCH.

scholarly journals Analysis of Endogenous LRP6 Function Reveals a Novel Feedback Mechanism by Which Wnt Negatively Regulates Its Receptor

2007 ◽  
Vol 27 (20) ◽  
pp. 7291-7301 ◽  
Author(s):  
Zahid Khan ◽  
Sapna Vijayakumar ◽  
Teresa Villanueva de la Torre ◽  
Sabrina Rotolo ◽  
Anna Bafico
Keyword(s):  
Wnt Signaling ◽  
Wnt Pathway ◽  
Mrna Level ◽  
Cellular Membrane ◽  
Feedback Mechanism ◽  
Bone Diseases ◽  
Dependent Manner ◽  
Direct Role ◽  
Initial Stimulus ◽  
Canonical Wnt

ABSTRACT The canonical Wnt pathway plays a crucial role in embryonic development, and its deregulation is involved in human diseases. The LRP6 single-span transmembrane coreceptor is essential for transmission of canonical Wnt signaling. However, due to the lack of immunological reagents, our understanding of LRP6 structure and function has relied on studies involving its overexpression, and regulation of the endogenous receptor by the Wnt ligand has remained unexplored. Using a highly sensitive and specific antibody to LRP6, we demonstrate that the endogenous receptor is modified by N-glycosylation and is phosphorylated in response to Wnt stimulation in a sustained yet ligand-dependent manner. Moreover, following triggering by Wnt, endogenous LRP6 is internalized and recycled back to the cellular membrane within hours of the initial stimulus. Finally, we have identified a novel feedback mechanism by which Wnt, acting through β-catenin, negatively regulates LRP6 at the mRNA level. Together, these findings contribute significantly to our understanding of LRP6 function and uncover a new level of regulation of Wnt signaling. In light of the direct role that the Wnt pathway plays in human bone diseases and malignancies, our findings may support the development of novel therapeutic approaches that target Wnt signaling through LRP6.

Expansion of Lox-1+CD15+ myeloid-derived suppressor cells in hepatocellular carcinoma patients.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 124-124 ◽  
Author(s):  
Xing Li ◽  
Qing-Jian Ye ◽  
Yan-Fang Xing ◽  
Jin-Xiang Lin ◽  
Qu Lin ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
T Cell ◽  
Whole Blood ◽  
Mrna Level ◽  
Suppressor Cells ◽  
Suppressor Cell ◽  
T Cell Proliferation ◽  
Dependent Manner ◽  
Ifn Γ

124 Background: The top issue in the field of myeloid deprived suppressor cell (MDSC) was lack of specific markers. Lox-1 was reported to be a novel marker for polymorphonuclear MDSC (PMN-MDSC) in whole blood of head and neck cancer and lung cancer patients. The present study is aimed to detecting the lox-1 PMN-MDSC in whole blood. Methods: In the present study, a series of 24 hepatocellular carcinoma (HCC) patients and 12 healthy donors were analyzed investigating frequencies of PMN-MDSC (Lox-1+CD15+) in whole blood. The immunosuppressive function of MDSC were evaluated using T cell proliferation and activation tests. The underly mechnisms were determined using inhibitors, genes expression and activity tests. The association between MDSC and clinical parameters were determined retrospectively. Results: Patients presented significantly higher level of PMN-MDSCs. In order to confirm immune suppressive capacity of PMN-MDSCs in HCC patients, circulative PMN-MDSCs and T cells were purified using flow sorting and cocultured. T cell proliferation was abrogated by the addition of PMN-MDSC with a dosage dependent manner, as well as the production of IFN-γ. Besides, the suppression on T cell proliferation and IFN-γ production was partially reversed by reactive oxygen species (ROS) inhibitor and Arginase inhibitor. The ROS level were higher in PMN-MDSC than their normal controls. The mRNA level of NOX2, the key protein complex responsible for ROS productin in MDSC, and Arginase I were higher in PMN-MDSCs. Finally, the frequencies of PMN-MDSCs was positively associated with tumor volume. Conclusions: The present study found that Lox-1+CD15+ were novel markers for PMN-MDSCs in whole blood and very easily to be standardized between institutions.

Transcriptional regulation of endothelial constitutive PGHS-1 expression by phorbol ester

1996 ◽  
Vol 270 (1) ◽  
pp. C259-C264 ◽  
Author(s):  
X. M. Xu ◽  
J. L. Tang ◽  
A. Hajibeigi ◽  
D. S. Loose-Mitchell ◽  
K. K. Wu
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Mrna Level ◽  
Northern Analysis ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Polymerase Chain ◽  
Prostaglandin H ◽  
Umbilical Vein Endothelial Cells

Human endothelial cells contain two isoforms of prostaglandin H synthase (PGHS). PGHS-1 is constitutively expressed, whereas PGHS-2 is inducible. To determine whether expression of PGHS-1 is regulated, we treated cultured human umbilical vein endothelial cells (HUVEC) with phorbol 12-myristate 13-acetate (PMA) or its inactive analogue and measured PGHS-1 mRNA levels by Northern analysis and competitive polymerase chain reaction. PMA increased PGHS-1 mRNA levels determined by both techniques in a time- and concentration-dependent manner. The mRNA level was increased about twofold over the basal level after 4-6 h of PMA (10-50 nM) treatment. The level of PGHS-1 protein was similarly increased by PMA. Stimulation of PGHS-1 mRNA levels was abrogated by cycloheximide, actinomycin D, staurosporine, or calphostin C. The 5'-promoter activity of human PGHS-1 gene was increased twofold over the basal level by PMA in NS-20 cells. These results indicate that the constitutive PGHS-1 in HUVEC is transcriptionally stimulated by PMA in a protein kinase C-dependent manner.

scholarly journals Gamma-linolenic and pinolenic acids exert anti-inflammatory effects in cultured human endothelial cells through their elongation products

2020 ◽  
Vol 79 (OCE2) ◽  
Author(s):  
Ella Baker ◽  
Elizabeth Miles ◽  
Philip Calder
Keyword(s):  
Fatty Acids ◽  
Endothelial Cells ◽  
Linolenic Acid ◽  
Inflammatory Responses ◽  
Cell Surface Expression ◽  
Dependent Manner ◽  
Epa And Dha ◽  
Ea.Hy926 Cells ◽  
Anti Inflammatory ◽  
Pre Treatment

AbstractIt is recommended that humans consume fatty fish twice a week to increase dietary intake of eicosapentaenoic acid (EPA) and docosapentaenoic acid (DHA) to achieve long-term health benefits. However current stocks of fish are likely insufficient to meet the needs of humans. Plant-derived polyunsaturated fatty acids (PUFAs) gamma-linolenic acid (GLA) and pinolenic acid (PIN) may provide sustainable land-based sources of bioactive fatty acids.Anti-inflammatory effects of GLA and PIN were compared to EPA and DHA in cultured EA.hy926 cells. Cells were treated with PUFAs (10, 25 and 50 μM) for 48 hours prior to stimulation with tumour necrosis factor for 24 hours. Incorporation of PUFA was measured by gas chromatography; inflammatory responses were measured by ELISA and flow cytometry.All fatty acids were incorporated into EA.hy926 cells, after 48 hours, in a dose dependent manner (10 and 50 μM). Pre-treatment with GLA and PIN (50 μM) resulted in significant increases in their elongation products, dihomo-γ-linolenic acid (DGLA) (p < 0.0001) from GLA and eicosatrienoic (ETrA) (p < 0.0001) from PIN.Pre-treatment with GLA, PIN, EPA or DHA (50 μM) had differential effects depending on fatty acid and cytokine examined. Pre-treatment of EA.hy926 cells with both GLA and PIN resulted in a lower concentration of soluble ICAM-1 (p < 0.01); however EPA and DHA showed greater reduction (p < 0.0001). MCP-1 production was significantly lower after treatment with PIN (p < 0.05), again to a lesser extent than EPA and DHA (p < 0.0001). Pre-treatment with EPA and DHA (50 μM) resulted in lower cell surface expression of ICAM-1 (p < 0.001, p < 0.0001), an effect not observed with GLA or PIN.Anti-inflammatory effects of GLA and PIN were possibly due to their elongation products, and therefore silencing of elongase 5 (ELOVL5) was explored. ELOVL5 siRNA significantly inhibited the production of DGLA and ETrA in EA.hy926 cells pre-treated with GLA and PIN (50 μM). Furthermore significant decreases in sICAM-1 and MCP-1 were not seen after pre-treatment with GLA or PIN in ELOVL5 siRNA silenced EA.hy926 cells.Plant PUFAs (GLA and PIN) demonstrate anti-inflammatory effects in this model using endothelial cells, but are less potent than EPA or DHA. Anti-inflammatory effects of GLA and PIN may be due to their elongation metabolites; DGLA and ETrA.

Stability of HTLV-2 antisense protein is controlled by PML nuclear bodies in a SUMO-dependent manner

Oncogene ◽  
2018 ◽  
Vol 37 (21) ◽  
pp. 2806-2816 ◽  
Author(s):  
Louise Dubuisson ◽  
Florence Lormières ◽  
Stefania Fochi ◽  
Jocelyn Turpin ◽  
Amandine Pasquier ◽  
...  
Keyword(s):  
Nuclear Bodies ◽  
Dependent Manner ◽  
Pml Nuclear Bodies
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