scholarly journals The Methyltransferase Smyd1 Mediates LPS-Triggered Up-Regulation of IL-6 in Endothelial Cells

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3515
Author(s):  
Ahmed Shamloul ◽  
Gustav Steinemann ◽  
Kerrin Roos ◽  
Celine Huajia Liem ◽  
Jonathan Bernd ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Biochemical Properties ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Set Domain ◽  
Luciferase Reporter Gene ◽  
Histone 3 ◽  
Lysine Methyltransferase ◽  
Ea.Hy926 Cells ◽  
Sarcomere Assembly

The lysine methyltransferase Smyd1 with its characteristic catalytic SET-domain is highly enriched in the embryonic heart and skeletal muscles, participating in cardiomyogenesis, sarcomere assembly and chromatin remodeling. Recently, significant Smyd1 levels were discovered in endothelial cells (ECs) that responded to inflammatory cytokines. Based on these biochemical properties, we hypothesized that Smyd1 is involved in inflammation-triggered signaling in ECs and therefore, investigated its role within the LPS-induced signaling cascade. Human endothelial cells (HUVECs and EA.hy926 cells) responded to LPS stimulation with higher intrinsic Smyd1 expression. By transfection with expression vectors containing gene inserts encoding either intact Smyd1, a catalytically inactive Smyd1-mutant or Smyd1-specific siRNAs, we show that Smyd1 contributes to LPS-triggered expression and secretion of IL-6 in EA.hy926 cells. Further molecular analysis revealed this process to be based on two signaling pathways: Smyd1 increased the activity of NF-κB and promoted the trimethylation of lysine-4 of histone-3 (H3K4me3) within the IL-6 promoter, as shown by ChIP-RT-qPCR combined with IL-6-promoter-driven luciferase reporter gene assays. In summary, our experimental analysis revealed that LPS-binding to ECs leads to the up-regulation of Smyd1 expression to transduce the signal for IL-6 up-regulation via activation of the established NF-κB pathway as well as via epigenetic trimethylation of H3K4.

Abstract 267: Hemoglobin, Endothelial Cells, and TLRs. Common Connection?

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Christina Lisk ◽  
David Irwin
Keyword(s):  
Endothelial Cells ◽  
Messenger Rna ◽  
Time Course ◽  
Vascular Endothelial Cells ◽  
Vascular Diseases ◽  
Luciferase Reporter ◽  
Signaling Cascade ◽  
Pulmonary Vascular Disease ◽  
Luciferase Reporter Gene ◽  
Pulmonary Arterial

Introduction: Patients suffering from chronic hereditary hemolytic anemic syndromes, such as sickle cell disease (SCD) and thalassemia, are often at risk for systemic and pulmonary vascular disease. It has been suggested that chronic exposure to cell free hemoglobin (CFH) may contribute to some vascular diseases associated with these syndromes such as pulmonary arterial hypertension. To date, the vasculotoxic effects of CFH have mostly been attributed to its pro-oxidant and nitric oxide scavenging characteristics. However, emerging evidence suggests CFH may contribute to inflammation by directly activating a signaling cascade event by binding to a pattern recognition receptor (PRR) or a toll like receptor (TLR) on vascular endothelial cells. Hypothesis: We hypothesized that CFH would increase the activity of transcription factors, NF-κb and HIF-1α, via a MyD88-dependent pathway. Methods: Human microvascular endothelial cells (HMEC) were transfected with either an NF-κB or HIF-1α luciferase reporter gene and treated with CFH (ferrous, ferric, and ferryl forms) in the presence or absence of SOD, catalase, dexamethasome, MyD88 inhibitor, or, the PHD inhibitor, DMOG. Messenger RNA for HIF-1α and HIF-2 were also measured after treatments. Results: All three states of hemoglobin increased NF-κB and HIF-1α activity in a dose response fashion, with ferryl inducing the greatest activity of both NF-κB and HIF-1α. Time course studies showed that NF-κB and HIF-1α activity tracked together. A unique synergy was noted with co-treatment of ferryl and DMOG. Co-treatment with SOD or catalase did not inhibit the CFH-induced NF-κB or HIF-1α response. Dexamthasome and MyD88 inhibition reduced the CFH-induced NF-κB and HIF-1α activity. Conclusion: Our results support the hypothesis, that CFH may activate a TLR or PRR signaling cascade subsequently activating MyD88-NF-κB and HIF-1α. Our data, that showed SOD and/or catalase did not block CFH effects, suggests that this event is not mediated by CFH pro-oxidant characteristics. CFH-induced HIF-1α was blocked by NF-κB inhibition with either, Dexamethasome or MyD88 inhibition emphasizing the importance of NF-κB in the HIF-1α pathway.

scholarly journals CD14 Protein Acts as an Adaptor Molecule for the Immune Recognition of Salmonella Curli Fibers

2013 ◽  
Vol 288 (20) ◽  
pp. 14178-14188 ◽  
Author(s):  
Glenn J. Rapsinski ◽  
Tiffanny N. Newman ◽  
Gertrude O. Oppong ◽  
Jos P. M. van Putten ◽  
Çagla Tükel
Keyword(s):  
Bone Marrow ◽  
Cancer Cell Line ◽  
Enteric Bacteria ◽  
Fiber Formation ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Cervical Cancer Cell Line ◽  
Immune Recognition ◽  
Luciferase Reporter Gene ◽  
Membrane Bound

Amyloids, protein aggregates with a cross β-sheet structure, contribute to inflammation in debilitating disorders, including Alzheimer's disease. Enteric bacteria also produce amyloids, termed curli, contributing to inflammation during infection. It has been demonstrated that curli and β-amyloid are recognized by the immune system via the Toll-like receptor (TLR) 2/TLR1 complex. Here we investigated the role of CD14 in the immune recognition of bacterial amyloids. We used HeLa 57A cells, a human cervical cancer cell line containing a luciferase reporter gene under the control of an NF-κB promoter. When HeLa 57A cells were transiently transfected with combinations of human expression vectors containing genes for TLR2, TLR1, and CD14, membrane-bound CD14 enhanced NF-κB activation through the TLR2/TLR1 complex stimulated with curli fibers or recombinant CsgA, the curli major subunit. Similarly, soluble CD14 augmented the TLR2/TLR1 response to curli fibers in the absence of membrane-bound CD14. We further revealed that IL-6 and nitric oxide production were significantly higher by wild-type (C57BL/6) bone marrow-derived macrophages compared with TLR2-deficient or CD14-deficient bone marrow-derived macrophages when stimulated with curli fibers, recombinant CsgA, or synthetic CsgA peptide, CsgA-R4–5. Binding assays demonstrated that recombinant TLR2, TLR1, and CD14 bound purified curli fibers. Interestingly, CD14-curli interaction was specific to the fibrillar form of the amyloid, as demonstrated by using synthetic CsgA peptides proficient and deficient in fiber formation, respectively. Activation of the TLR2/TLR1/CD14 trimolecular complex by amyloids provides novel insights for innate immunity with implications for amyloid-associated diseases.

scholarly journals Endothelial-specific gene expression directed by the tie gene promoter in vivo

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1828-1835 ◽  
Author(s):  
J Korhonen ◽  
I Lahtinen ◽  
M Halmekyto ◽  
L Alhonen ◽  
J Janne ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transgenic Mice ◽  
Reporter Gene ◽  
Promoter Activity ◽  
Cultured Cells ◽  
Luciferase Reporter ◽  
Specific Gene ◽  
Luciferase Reporter Gene ◽  
Human And Mouse

The tie gene encodes a receptor tyrosine kinase that is expressed in the endothelium of blood vessels, particularly during embryonic development and angiogenesis in adults. We have cloned and characterized the mouse tie gene and isolated the human and mouse tie promoters. The promoter activities of human and mouse tie were analyzed using luciferase reporter gene constructs in transfected cell lines and beta-galactosidase constructs in transgenic mice. In transfection assays of cultured cells, both human and mouse promoter DNA fragments showed activity that was not restricted to endothelial cells. In contrast, in transgenic mice both promoters directed expression of the reporter gene to endothelial cells undergoing vasculogenesis and angiogenesis. In adult mice, tie promoter activity in lung and many vessels of the kidney was as high as in the vessels of the corresponding embryonic tissues, whereas in the heart, brain and liver, tie promoter activity was downregulated and restricted to coronaries, cusps, capillaries, and arteries. Our results show that the endothelial cell-type specificity of the tie promoter in vivo can be transferred to heterologous genes by using relatively short promoter fragments. The tie promoter, thus, has useful properties for potential gene therapy.

scholarly journals Induction of P-Glycoprotein by Antiretroviral Drugs in Human Brain Microvessel Endothelial Cells

2013 ◽  
Vol 57 (9) ◽  
pp. 4481-4488 ◽  
Author(s):  
Gary N. Y. Chan ◽  
Rucha Patel ◽  
Carolyn L. Cummins ◽  
Reina Bendayan
Keyword(s):  
Endothelial Cells ◽  
Human Brain ◽  
Pregnane X Receptor ◽  
Antiretroviral Drugs ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
P Glycoprotein ◽  
Microvessel Endothelial Cells ◽  
Brain Microvessel ◽  
The Brain

ABSTRACTThe membrane-associated drug transporter P-glycoprotein (P-gp) plays an essential role in drug efflux from the brain. Induction of this protein at the blood-brain barrier (BBB) could further affect the ability of a drug to enter the brain. At present, P-gp induction mediated by antiretroviral drugs at the BBB has not been fully investigated. Since P-gp expression is regulated by ligand-activated nuclear receptors, i.e., human pregnane X receptor (hPXR) and human constitutive androstane receptor (hCAR), these receptors could represent potential pathways involved in P-gp induction by antiretroviral drugs. The aims of this study were (i) to determine whether antiretroviral drugs currently used in HIV pharmacotherapy are ligands for hPXR or hCAR and (ii) to examine P-gp function and expression in human brain microvessel endothelial cells treated with antiretroviral drugs identified as ligands of hPXR and/or hCAR. Luciferase reporter gene assays were performed to examine the activation of hPXR and hCAR by antiretroviral drugs. The hCMEC/D3 cell line, which is known to display several morphological and biochemical properties of the BBB in humans, was used to examine P-gp induction following 72 h of exposure to these agents. Amprenavir, atazanavir, darunavir, efavirenz, ritonavir, and lopinavir were found to activate hPXR, whereas abacavir, efavirenz, and nevirapine were found to activate hCAR. P-gp expression and function were significantly induced in hCMEC/D3 cells treated with these drugs at clinical concentrations in plasma. Together, our data suggest that P-gp induction could occur at the BBB during chronic treatment with antiretroviral drugs identified as ligands of hPXR and/or hCAR.

scholarly journals Genomic structure and transcriptional regulation of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene

2003 ◽  
Vol 31 (1) ◽  
pp. 105-121 ◽  
Author(s):  
A Nandy ◽  
S Jenatschke ◽  
B Hartung ◽  
K Milde-Langosch ◽  
AM Bamberger ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Binding Sites ◽  
Biological Activities ◽  
Genomic Structure ◽  
Start Codon ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Flanking Sequence ◽  
Luciferase Reporter Gene ◽  
Mobility Shift

The NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is a catabolic enzyme that controls the biological activities of prostaglandins by converting them into inactive keto-metabolites. Here we report the genomic organisation of the complete human PGDH gene and characterise its transcriptional regulation. The PGDH gene spans about 31 kb on chromosome 4 and contains 7 exons. Within 2.4 kb of the 5'-flanking sequence we identified two regions with clustered putative transcription factor binding sites. The distal promoter element PGDH-DE (positions-2152/-1944 relative to the start codon) contains binding sites for Ets and activating protein-1 (AP-1) flanked by two cAMP-responsive element-binding protein binding sites (CREB1, CREB2), whereas the proximal element PGDH-PE (-235/-153) includes an Ets and an AP-1 binding sequence. By electrophoretic mobility shift assay, no high affinity binding of Ets or AP-1 factors was observed with PGDH-PE, whereas we confirmed interaction of members of the Ets, AP-1 and CREB families of transcription factors with PGDH-DE. Transcriptional control of the PGDH promoter was assessed by transiently transfecting JEG-3 choriocarcinoma cells. A luciferase reporter gene construct containing the PGDH-PE was not induced by c-jun/c-fos in the absence or presence of co-expressed Ets-1. A construct carrying the PGDH-DE in front of the minimal homologous promoter was activated by co-transfection of expression vectors for AP-1 proteins. Mutation of the AP-1 or CREB2 site reduced the response to c-jun/c-fos, whereas mutation of the Ets site of the distal element reduced basal promoter activity. CREB activated the PGDH-DE construct through the CREB1 site. These results defined the distal element as an integrator of transcriptional regulation by AP-1, Ets and CREB proteins.

scholarly journals Transcriptional Regulation of Human 11β-Hydroxylase (hCYP11B1)

Endocrinology ◽  
2000 ◽  
Vol 141 (10) ◽  
pp. 3587-3594 ◽  
Author(s):  
Xiao-Li Wang ◽  
Mary Bassett ◽  
Yin Zhang ◽  
Su Yin ◽  
Colin Clyne ◽  
...  
Keyword(s):  
Adrenal Cortex ◽  
Electrophoretic Mobility ◽  
Binding Protein ◽  
Regulatory Elements ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Maximal Induction ◽  
Electrophoretic Mobility Shift Assays

Abstract Steroid 11β-hydroxylase is a mitochondrial enzyme that catalyzes the conversion of deoxycortisol to cortisol. The gene encoding human 11β-hydroxylase (hCYP11B1) is expressed in the adrenal cortex under the control of circulating levels of ACTH. The current study was undertaken to define the cis-regulatory elements and transacting factors that regulate hCYP11B1 transcription. The hCYP11B1 5′-flanking DNA was studied using transient transfection of luciferase reporter constructs in NCI-H295R human adrenocortical cells. A cAMP analogue ((Bu)2cAMP) increased expression of a construct containing −1102 bp of hCYP11B1 5′-flanking DNA (pB1–1102). An element at position −71/−64 (TGACGTGA, previously termed Ad1) resembling a consensus cAMP response element (CRE) was required for maximal induction by cAMP. The Ad1 element bound several transcriptional factors in electrophoretic mobility shift assays, including CRE-binding protein, activating transcription factor-1 (ATF-1), and ATF-2, but only the ATF-2 complex migrated similarly to a complex seen using H295R nuclear extract. In addition, Western analysis of H295R and adrenal lysates demonstrated expression of high levels of ATF-2 and ATF-1. CRE-binding protein levels varied among the strains of H295R cells tested. Transcription of CYP11B1 also appeared to be regulated by steroidogenic factor-1 (SF-1). Luciferase reporter gene activity was increased after cotransfection with expression vectors containing SF-1. An element in hCYP11B1 at positions −242/−234 (CCAAGGCTC), previously termed Ad4, was required for maximal induction by SF-1 and was found to bind SF-1 in electrophoretic mobility shift assays. The key role for SF-1 in hCYP11B1 transcription is in contrast to its lack of an effect on expression of the hCYP11B2 (aldosterone synthase) isozyme. The differential effects of SF-1 on transcription of hCYP11B1 and hCYP11B2 may be one of the mechanisms controlling differential expression of these isozymes within the zonae fasciculata and glomerulosa of the human adrenal cortex.

Increased mitochondrial H2O2 production promotes endothelial NF-κB activation in aged rat arteries

2007 ◽  
Vol 293 (1) ◽  
pp. H37-H47 ◽  
Author(s):  
Zoltan Ungvari ◽  
Zsuzsanna Orosz ◽  
Nazar Labinskyy ◽  
Aracelie Rivera ◽  
Zhao Xiangmin ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Endothelial Cells ◽  
Vascular System ◽  
Vascular Inflammation ◽  
Luciferase Reporter ◽  
H2o2 Production ◽  
Luciferase Reporter Gene ◽  
Mitochondrial Oxidative Stress ◽  
Gene Assay

Previous studies have shown that the aging vascular system undergoes pro-atherogenic phenotypic changes, including increased oxidative stress and a pro-inflammatory shift in endothelial gene expression profile. To elucidate the link between increased oxidative stress and vascular inflammation in aging, we compared the carotid arteries and aortas of young and aged (24 mo old) Fisher 344 rats. In aged vessels there was an increased NF-κB activity (assessed by luciferase reporter gene assay and NF-κB binding assay), which was attenuated by scavenging H2O2. Aging did not alter the vascular mRNA and protein expression of p65 and p50 subunits of NF-κB. In endothelial cells of aged vessels there was an increased production of H2O2 (assessed by 5,6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate-acetyl ester fluorescence), which was attenuated by the mitochondrial uncoupler FCCP. In young arteries and cultured endothelial cells, antimycin A plus succinate significantly increased FCCP-sensitive mitochondrial H2O2 generation, which was associated with activation of NF-κB. In aged vessels inhibition of NF-κB (by pyrrolidenedithiocarbamate, resveratrol) significantly attenuated inflammatory gene expression and inhibited monocyte adhesiveness. Thus increased mitochondrial oxidative stress contributes to endothelial NF-κB activation, which contributes to the pro-inflammatory phenotypic alterations in the aged vaculature. Our model predicts that by reducing mitochondrial H2O2 production and/or directly inhibiting NF-κB novel anti-aging pharmacological treatments (e.g., calorie restriction mimetics) will exert significant anti-inflammatory and vasoprotective effects.

Abstract 234: Defining Domains In The Transcription Factor Grainyhead-like 3 Required For Protective Functions In The Endothelium

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Sabrina Farrokh ◽  
Niloofar Ale-Agha ◽  
Judith Haendeler ◽  
Joachim Altschmied
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Large Scale ◽  
Target Genes ◽  
Intracellular Localization ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Deletion Mutants ◽  
Nuclear Localization Signals ◽  
Apoptosis Level

Important aspects during aging of human endothelial cells (EC) are an increased apoptosis level and a reduced migratory capacity. In a screening for anti-apoptotic genes we have identified the transcription factor Grainyhead-like 3 (GRHL3) and demonstrated its anti-apoptotic and pro-migratory effects after overexpression and knockdown in EC. To define the domains in GRHL3 responsible for these - potentially also extranuclear - functions we have cloned large scale deletion mutants and mutants with deletions of putative nuclear localization signals (NLS) and analyzed them for their intracellular localization and functional properties. Immunostaining of transfected EC were used to examine the subcellular distribution. Two mutants with deletions in a bioinformatically predicted bipartite NLS were localized predominantly in the cytoplasm. To corroborate these data, lysates of the cells were fractionated biochemically. Western blotting confirmed the immunostaining data, indicating that we have identified the major NLS in GRHL3. To analyze the transcriptional properties of these mutants, we constructed a GRHL3-specific luciferase reporter containing a tandem GRHL3 binding sites in front of a minimal promoter and cotransfected it with expression vectors for the GRHL3 deletion mutants. Besides the previously described activation domain we identified another region required for transcriptional upregulation of target genes in the N-terminal half of the protein. We will now use these mutants to further dissect the regions in GRHL3 necessary for its pro-migratory and anti-apoptotic activities in endothelial cells. This will also allow us to investigate if all of these functions are mediated solely by the activation of target genes or by other mechanisms coupled to a non-nuclear function of GRHL3.

scholarly journals MiR-34a regulates blood–tumor barrier function by targeting protein kinase Cε

2015 ◽  
Vol 26 (10) ◽  
pp. 1786-1796 ◽  
Author(s):  
Wei Zhao ◽  
Ping Wang ◽  
Jun Ma ◽  
Yun-Hui Liu ◽  
Zhen Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Protein Expression ◽  
Tight Junction ◽  
Target Genes ◽  
Vascular Endothelial Cells ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Expression Levels ◽  
Related Proteins

MicroRNA-34a (miR-34a) functions to regulate protein expression at the posttranscriptional level by binding the 3′ UTR of target genes and regulates functions of vascular endothelial cells. However, the role of miR-34a in regulating blood–tumor barrier (BTB) permeability remains unknown. In this study, we show that miR-34a overexpression leads to significantly increased permeability of BTB, whereas miR-34a silencing reduces the permeability of the BTB. In addition, miR-34a overexpression significantly down-regulates the expression and distribution of tight junction–related proteins in glioma endothelial cells (GECs), paralleled by protein kinase Cε (PKCε) reduction. Moreover, luciferase reporter gene analysis shows that PKCε is the target gene of miR-34a. We also show that cotransfection of miR-34a and PKCε inversely coregulates BTB permeability and protein expression levels of tight junction–related proteins. Pretreatment of ψεRACK, a PKCε-specific activator, decreases BTB permeability in miR-34a–overexpressed GECs and up-regulates expression levels of tight junction proteins. In contrast, pretreatment of εV1-2, a specific PKCε inhibitor, gives opposite results. Collectively, our findings indicate that miR-34a regulates BTB function by targeting PKCε; after phosphorylation, PKCε is activated and contributes to regulation of the expression of tight junction–related proteins, ultimately altering BTB permeability.

scholarly journals Late temporal gene expression from the human cytomegalovirus pp28US (UL99) promoter when integrated into the host cell chromosome

2001 ◽  
Vol 82 (5) ◽  
pp. 1147-1155 ◽  
Author(s):  
Jun Wu ◽  
Joseph O’Neill ◽  
Miguel S. Barbosa
Keyword(s):  
Human Cytomegalovirus ◽  
Gene Promoter ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Promoter Activation ◽  
Luciferase Reporter Gene ◽  
Temporal Regulation ◽  
Temporal Gene Expression ◽  
Simplex Virus

Toward understanding the temporal regulation of human cytomegalovirus (HCMV) late genes, we studied the regulation of the late gene promoter (pp28US, UL99) when outside the context of the viral genome and its response to the immediate early (IE) proteins. Expression of the luciferase reporter gene, regulated by the pp28US promoter, was synchronous with that of the endogenous viral pp28 gene, independently of whether the reporter was episomal or integrated into the glioblastoma cell line U373MG. Cotransfection of the reporter with expression vectors for each of the three major IE genes, IE72, IE86 and IE55, indicated that only IE86 transactivated the pp28US promoter. However, the magnitude of the promoter activation upon HCMV infection suggested that additional factors are also required for higher promoter activity. The promoter activation was specific to HCMV, as herpes simplex virus type 1 infection did not induce luciferase expression.

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