Abstract 3678: Protein Kinase C-zeta (PKCζ) Is A Novel Mediator Of Endothelial Dysfunction By Inhibiting The ERK5/ Kruppel-like Factor 2 (KLF2) Pathway

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Patrizia Nigro ◽  
Chang Hoon Woo ◽  
Maria Antonietta Belisario ◽  
Carolyn McClain ◽  
Jun-ichi Abe ◽  
...  
Keyword(s):  
Shear Stress ◽  
Endothelial Dysfunction ◽  
Transcriptional Repression ◽  
Transcriptional Activity ◽  
Catalytic Domain ◽  
Mutational Analysis ◽  
Critical Role ◽  
Enos Expression ◽  
Interaction Domain ◽  
Pb1 Domain

Background: Shear stress-induced activation of ERK5 and KLF2 leads to eNOS expression maintaining normal endothelial cell (EC) function. PKCζ has emerged as a pathologic mediator of EC dysfunction based on 1) a positive correlation between PKCζ activity and disturbed flow pattern; and 2) PKCζ activation is essential for TNFα stimulation of JNK, caspase-3 and EC apoptosis. Therefore we hypothesized that TNFα and its pathologic mediator ONOO − would inhibit ERK5 transcriptional activity and eNOS expression induced by flow. Results: TNFα and ONOO − significantly inhibited ERK5 transcriptional activity, KLF2 promoter activity, and eNOS expression induced by flow (shear stress = 12 dyn/cm2, 24 hrs). Both TNFαand ONOO − increased PKCζ activity. Transfection of wild type PKCζ (WT-PKCζ) and catalytic domain of PKCζ (CATζ) significantly inhibited ERK5 transcriptional activity (38±1.4% and 57±2.8% respectively; p<0.01, p<0.005). Also, transfection of PKCζ siRNA reversed TNFα-mediated inhibition of ERK5 transcriptional activity (control vs PKCz siRNA, 28±2.5% vs 9±0.3%, p<0.05), suggesting a critical role for PKCζ in ERK5 transcriptional repression. Surprisingly, TNFα and ONOO − did not significantly decrease ERK5 phosphorylation, suggesting that inhibition occurred downstream of ERK5 phosphorylation. Previously we reported that ERK5-SUMOylation inhibited flow-mediated eNOS expression, but we could not detect increased ERK5-SUMOylation in WT-PKCζ or CATζ transfected cells. Importantly, we found that ONOO − significantly increased PKCζ-ERK5 interaction. PKCζ is known to contain a PB1 domain, a well studied protein-protein interaction domain. However, mutational analysis demonstrated that the ERK5 binding site in PKCζ was within the catalytic domain of PKCζ, not the PB1 domain. These data suggest that the PKCζ-ERK5 interaction likely inhibits ERK5 transcriptional activity by direct phosphorylation of ERK5 by PKCζ kinase. Conclusion: PKCζ is a novel mediator of TNFα and ONOO − induced endothelial dysfunction by inhibiting ERK5 transcriptional activity independent of kinase activity and ERK5-SUMOylation.

Exosomes from Patients with Sickle Cell Disease and History of Acute Chest Syndrome Alter Endothelial Integrity In Vitro

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 855-855
Author(s):  
Gabrielle Lapping-Carr ◽  
Abdelnaby Khalyfa ◽  
Wendy Darlington ◽  
Elizabeth Joyce ◽  
Joanna Gemel ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
Critical Role ◽  
Acute Chest Syndrome ◽  
Cell Disease ◽  
Enos Expression ◽  
History Of ◽  
Endothelial Integrity

Abstract Introduction: Acute Chest Syndrome (ACS) is the leading cause of death among children with sickle cell disease (SCD). While the phenotypic variability of ACS is incompletely understood, aberrant cell-cell interactions involving the endothelium are central to the pathophysiology. Recent studies suggest that circulating cell-derived extracellular vesicles, termed exosomes, can regulate endothelial integrity. We and others recently demonstrated that exosomes from SCD patients differentially affect endothelial integrity in vitro, but the relationship to specific complications of SCD, such as ACS, are unknown. Given the critical role of the endothelium in ACS, we hypothesized that exosomes from patients with a history of ACS induce increased endothelial damage compared to those without a previous ACS episodes. Methods: Plasma was isolated from 33 patients with SCD. Patients were >4 weeks since transfusion and had no new health-related complaints. Control plasma samples were from children without SCD or known medical problems, who had a BMI < 95%ile. Exosomes were isolated from plasma using established methodologies. The cellular origin of exosomes was determined using Image Stream flow cytometry. To determine the effects on endothelium, exosomes were added to cultures of human microvascular endothelial cells (HMVEC-D). Intercellular junctions were visualized by immunofluorescent microscopy for VE-cadherin. To quantify effects on endothelial barrier integrity, HMVEC-D endothelial cells were grown to confluence on an Electric Cell-substrate Impedance Sensing (ECIS) array, treated with exosomes and then continuously monitored for 36 hours. Endothelial Nitric Oxide Synthase (eNOS) mRNA expression was assessed in HMVEC-D cells 24 hours post-exposure by qRT-PCR. Results/Discussion: Flow cytometry demonstrated that the absolute exosome number was greatly increased in patients with SCD compared with controls. In contrast, no significant differences in total exosome numbers emerged between SCD patients based on ACS history. The origin of theexosomes was mainly erythroid (controls:9,661 ± 3,195 /100 uL vs. SCD: 31,338 ± 5,323 /100 uL, p<0.007), but significantly increased numbers of endothelial-, CD34+, lymphocyte-, and monocyte- derived exosomes were also detected. Although a minor population, ACS(+) patients had significantly more monocyte-derived exosomes than ACS(-) patients (monocyte exosomes ACS(-):45.89 ± 22.41 /100 uL vs. ACS(+): 477.4 ± 173.7 /100 uL, p=0.0218); exosomes from other sources did not differ. Immunolocalization of VE-cadherin showed that exosomes from SCD patients (especially ACS(+)) led to the formation of increased gaps between HMVECs as compared to untreated cells or cells treated with exosomes from control patients. ECIS recordings showed that samples from ACS(-) patients differed minimally from control patients, but exosomes from ACS(+)-patients greatly decreased monolayer resistance (relative resistance: ACS(+): 0.981±0.055 vs. ACS(-): 1.124±0.042; p = 0.006). Given its role in pulmonary endothelial dysfunction in ACS, we evaluated the ability of exosomes to induce eNOS expression. ACS(-) samples induced a ~50% increase in eNOS expression when compared to controls (controls;1.01± 0.00 versus ACS(-); 1.61 ± 0.06; p = 0.01) while ACS(+)-derived exosomes failed to induce any significant changes (ACS(+): 0.99 ± 0.05; p < 0.0001 vs. ACS(-)). Conclusions: The results confirm and expand on our prior observations that circulating exosomes are increased in patients with SCD and contribute to the maintenance of endothelial integrity. Exosomes from ACS(+) patients disrupt the endothelial monolayer in vitro (unlike exosomes from SCD patients who never experienced ACS). We speculate that the increased exosomes contribute to the vascular pathology in all SCD patients. However, some SCD patients are protected by specific exosome cargoes, as in the ACS(-) patients whose exosomes induced eNOS expression by the cultured endothelial cells in vitro, which plays a critical role in endothelial health. Together, the current findings suggest that exosomes are differentially generated based on ACS history, and that their function/cargo (as opposed to their number), modulates endothelial function. Thus, SCD exosomes that induce endothelial dysfunction likely contribute to the pathophysiology of ACS, and may serve as risk-related biomarkers. Disclosures No relevant conflicts of interest to declare.

scholarly journals Fluid shear stress stimulates phosphorylation-dependent nuclear export of HDAC5 and mediates expression of KLF2 and eNOS

Blood ◽  
2010 ◽  
Vol 115 (14) ◽  
pp. 2971-2979 ◽  
Author(s):  
Weiye Wang ◽  
Chang Hoon Ha ◽  
Bong Sook Jhun ◽  
Chelsea Wong ◽  
Mukesh K. Jain ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Nuclear Export ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Fluid Shear Stress ◽  
Inhibitory Effect ◽  
Fluid Shear ◽  
Enos Expression ◽  
Calcium Calmodulin Dependent

Abstract Fluid shear stress generated by steady laminar blood flow protects vessels from atherosclerosis. Krüppel-like factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS) are fluid shear stress–responsive genes and key mediators in flow anti-inflammatory and antiatherosclerotic actions. However, the molecular mechanisms underlying flow induction of KLF2 and eNOS remain largely unknown. Here, we show a novel role of histone deacetylase 5 (HDAC5) in flow-mediated KLF2 and eNOS expression. We found for the first time that fluid shear stress stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a calcium/calmodulin-dependent pathway. Consequently, flow induced the dissociation of HDAC5 and myocyte enhancer factor-2 (MEF2) and enhanced MEF2 transcriptional activity, which leads to expression of KLF2 and eNOS. Adenoviral overexpression of a HDAC5 phosphorylation–defective mutant (Ser259/Ser498 were replaced by Ala259/Ala498, HDAC5-S/A), which shows resistance to flow-induced nuclear export, suppressed flow-mediated MEF2 transcriptional activity and expression of KLF2 and eNOS. Importantly, HDAC5-S/A attenuated the flow-inhibitory effect on monocyte adhesion to endothelial cells. Taken together, our results reveal that phosphorylation-dependent derepression of HDAC5 mediates flow-induced KLF2 and eNOS expression as well as flow anti-inflammation, and suggest that HDAC5 could be a potential therapeutic target for the prevention of atherosclerosis.

The extreme COOH terminus of the retinoblastoma tumor suppressor protein pRb is required for phosphorylation on Thr-373 and activation of E2F

2008 ◽  
Vol 295 (5) ◽  
pp. C1151-C1160 ◽  
Author(s):  
Laura L. Gorges ◽  
Nathan H. Lents ◽  
Joseph J. Baldassare
Keyword(s):  
Transcriptional Repression ◽  
Cell Cycle Progression ◽  
Mutational Analysis ◽  
Human Cancer ◽  
Critical Role ◽  
S Phase ◽  
Terminal Region ◽  
Phase Cell ◽  
Terminal Domain ◽  
Multiple Domains

The retinoblastoma protein pRb plays a pivotal role in G1- to S-phase cell cycle progression and is among the most frequently mutated gene products in human cancer. Although much focus has been placed on understanding how the A/B pocket and COOH-terminal domain of pRb cooperate to relieve transcriptional repression of E2F-responsive genes, comparatively little emphasis has been placed on the function of the NH2-terminal region of pRb and the interaction of the multiple domains of pRb in the full-length context. Using “reverse mutational analysis” of RbΔCDK (a dominantly active repressive allele of Rb), we have previously shown that restoration of Thr-373 is sufficient to render RbΔCDK sensitive to inactivation via cyclin-CDK phosphorylation. This suggests that the NH2-terminal region plays a more critical role in pRb regulation than previously thought. In the present study, we have expanded this analysis to include additional residues in the NH2-terminal region of pRb and further establish that the mechanism of pRb inactivation by Thr-373 phosphorylation is through the dissociation of E2F. Most surprisingly, we further have found that removal of the COOH-terminal domain of either RbΔCDK+T373 or wild-type pRb yields a functional allele that cannot be inactivated by phosphorylation and is repressive of E2F activation and S-phase entry. Our data demonstrate a novel function for the NH2-terminal domain of pRb and the necessity for cooperation of multiple domains for proper pRb regulation.

scholarly journals The helix bundle domain of primase RepB’ is required for dinucleotide formation and extension

2020 ◽  
Author(s):  
Sofia Banchenko ◽  
Chris Weise ◽  
Erich Lanka ◽  
Wolfram Saenger ◽  
Sebastian Geibel
Keyword(s):  
Dna Replication ◽  
Transfer Reaction ◽  
Catalytic Domain ◽  
Mutational Analysis ◽  
Dna Polymerases ◽  
Critical Role ◽  
Helix Bundle ◽  
Dna Strand ◽  
Α Helix ◽  
Template Dna

ABSTRACTDuring DNA replication, primases synthesize oligonucleotide primers on single-stranded template DNA, which are then extended by DNA polymerases to synthesize a complementary DNA strand. Primase RepB’ of plasmid RSF1010 initiates DNA replication on two 40 nucleotide long inverted repeats, termed ssiA and ssiB, within the oriV of RSF1010. RepB’ consists of a catalytic domain and a helix bundle domain which are connected by long α-helix 6 and an unstructured linker. Previous work has demonstrated that RepB’ requires both domains for initiation of dsDNA synthesis in DNA replication assays. However, the precise functions of these two domains in primer synthesis have been unknown. Here we report that both domains of RepB’ are required to synthesizes a 10–12 nucleotide long DNA primer whereas the isolated domains are inactive. Mutational analysis of the catalytic domain indicates that the solvent-exposed W50 plays a critical role in resolving a hairpin structures formed by ssiA and ssiB. Three structurally conserved aspartates (D77, D78 and D134) of RepB’ catalyse the nucleotidyl transfer reaction. Mutations on the helix bundle domain are identified that either reduce the primer length to a dinucleotide (R285A) or abolish primer synthesis (D238A) indicating that the helix bundle domain is required to form and extend the initial dinucleotide synthesized by the catalytic domain.

scholarly journals Mutational Analysis of a Histone Deacetylase in Drosophila melanogaster: Missense Mutations Suppress Gene Silencing Associated With Position Effect Variegation

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 657-668 ◽  
Author(s):  
Randy Mottus ◽  
Richard E Sobel ◽  
Thomas A Grigliatti
Keyword(s):  
Drosophila Melanogaster ◽  
Histone Deacetylase ◽  
Transcriptional Activity ◽  
Mutational Analysis ◽  
Position Effect ◽  
Transcriptional Regulator ◽  
Position Effect Variegation ◽  
Missense Mutations ◽  
Effect Variegation ◽  
New Mutations

Abstract For many years it has been noted that there is a correlation between acetylation of histones and an increase in transcriptional activity. One prediction, based on this correlation, is that hypomorphic or null mutations in histone deacetylase genes should lead to increased levels of histone acetylation and result in increased levels of transcription. It was therefore surprising when it was reported, in both yeast and fruit flies, that mutations that reduced or eliminated a histone deacetylase resulted in transcriptional silencing of genes subject to telomeric and heterochromatic position effect variegation (PEV). Here we report the first mutational analysis of a histone deacetylase in a multicellular eukaryote by examining six new mutations in HDAC1 of Drosophila melanogaster. We observed a suite of phenotypes accompanying the mutations consistent with the notion that HDAC1 acts as a global transcriptional regulator. However, in contrast to recent findings, here we report that specific missense mutations in the structural gene of HDAC1 suppress the silencing of genes subject to PEV. We propose that the missense mutations reported here are acting as antimorphic mutations that “poison” the deacetylase complex and propose a model that accounts for the various phenotypes associated with lesions in the deacetylase locus.

scholarly journals Crystallization of and selenomethionine phasing strategy for a SETMAR–DNA complex

2016 ◽  
Vol 72 (9) ◽  
pp. 713-719 ◽  
Author(s):  
Qiujia Chen ◽  
Millie Georgiadis
Keyword(s):  
Dna Binding ◽  
Catalytic Domain ◽  
Specific Binding ◽  
Critical Role ◽  
Binding Activity ◽  
Structural Basis ◽  
Unexpected Finding ◽  
Terminal Inverted Repeat ◽  
Dna Binding Activity ◽  
New Genes

Transposable elements have played a critical role in the creation of new genes in all higher eukaryotes, including humans. Although the chimeric fusion protein SETMAR is no longer active as a transposase, it contains both the DNA-binding domain (DBD) and catalytic domain of theHsmar1transposase. The amino-acid sequence of the DBD has been virtually unchanged in 50 million years and, as a consequence, SETMAR retains its sequence-specific binding to the ancestralHsmar1terminal inverted repeat (TIR) sequence. Thus, the DNA-binding activity of SETMAR is likely to have an important biological function. To determine the structural basis for the recognition of TIR DNA by SETMAR, the design of TIR-containing oligonucleotides and SETMAR DBD variants, crystallization of DBD–DNA complexes, phasing strategies and initial phasing experiments are reported here. An unexpected finding was that oligonucleotides containing two BrdUs in place of thymidines produced better quality crystals in complex with SETMAR than their natural counterparts.

scholarly journals Polyamine-modulated c-Myc expression in normal intestinal epithelial cells regulates p21Cip1 transcription through a proximal promoter region

2006 ◽  
Vol 398 (2) ◽  
pp. 257-267 ◽  
Author(s):  
Lan Liu ◽  
Xin Guo ◽  
Jaladanki N. Rao ◽  
Tongtong Zou ◽  
Bernard S. Marasa ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Transcriptional Repression ◽  
Critical Role ◽  
Ectopic Expression ◽  
Proximal Region ◽  
Proximal Promoter ◽  
Transcriptional Level ◽  
Intestinal Epithelial ◽  
Major Player ◽  
Stimulation Of

Maintenance of intestinal mucosal epithelial integrity requires cellular polyamines that regulate expression of various genes involved in cell proliferation, growth arrest and apoptosis. Our previous studies have shown that polyamines are essential for expression of the c-myc gene and that polyamine-induced c-Myc plays a critical role in stimulation of normal IEC (intestinal epithelial cell) proliferation, but the exact downstream targets of induced c-Myc are still unclear. The p21Cip1 protein is a major player in cell cycle control, which is primarily regulated at the transcriptional level. The current study was designed to determine whether induced c-Myc stimulates normal IEC proliferation by repressing p21Cip1 transcription following up-regulation of polyamines. Overexpression of the ODC (ornithine decarboxylase) gene increased levels of cellular polyamines, induced c-Myc expression and inhibited p21Cip1 transcription, as indicated by repression of p21Cip1 promoter activity and a decrease in p21Cip1 protein levels. In contrast, depletion of cellular polyamines by inhibiting ODC enzyme activity with α-difluoromethylornithine decreased c-Myc, but increased p21Cip1 transcription. Ectopic expression of wild-type c-myc not only inhibited basal levels of p21Cip1 transcription in control cells, but also prevented increased p21Cip1 in polyamine-deficient cells. Experiments using different p21Cip1 promoter mutants showed that transcriptional repression of p21Cip1 by c-Myc was mediated through Miz-1- and Sp1-binding sites within the proximal region of the p21Cip1 promoter in normal IECs. These findings confirm that p21Cip1 is one of the direct mediators of induced c-Myc following increased polyamines and that p21Cip1 repression by c-Myc is implicated in stimulation of normal IEC proliferation.

Mutational analysis of a ras catalytic domain

1986 ◽  
Vol 6 (7) ◽  
pp. 2646-2654
Author(s):  
B M Willumsen ◽  
A G Papageorge ◽  
H F Kung ◽  
E Bekesi ◽  
T Robins ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Mutational Analysis ◽  
Nucleotide Binding ◽  
Membrane Localization ◽  
Wide Range ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Transforming Activity ◽  
Type V

We used linker insertion-deletion mutagenesis to study the catalytic domain of the Harvey murine sarcoma virus v-rasH transforming protein, which is closely related to the cellular rasH protein. The mutants displayed a wide range of in vitro biological activity, from those that induced focal transformation of NIH 3T3 cells with approximately the same efficiency as the wild-type v-rasH gene to those that failed to induce any detectable morphologic changes. Correlation of transforming activity with the location of the mutations enabled us to identify three nonoverlapping segments within the catalytic domain that were dispensable for transformation and six other segments that were required for transformation. Segments that were necessary for guanosine nucleotide (GDP) binding corresponded to three of the segments that were essential for transformation; two of the three segments share strong sequence homology with other purine nucleotide-binding proteins. Loss of GDP binding was associated with apparent instability of the protein. Lesions in two of the three other required regions significantly reduced GDP binding, while small lesions in the last required region did not impair GDP binding or membrane localization. We speculate that this latter region interacts with the putative cellular target of ras. The results suggest that transforming ras proteins require membrane localization, guanosine nucleotide binding, and an additional undefined function that may represent interaction with their target.

A model of anemic tissue perfusion after blood transfusion shows critical role of endothelial response to shear stress stimuli

2021 ◽  
Author(s):  
Weiyu Li ◽  
Amy G. Tsai ◽  
Marcos Intaglietta ◽  
Daniel M. Tartakovsky
Keyword(s):  
Blood Flow ◽  
Shear Stress ◽  
Blood Transfusion ◽  
Critical Role ◽  
Cardiovascular Responses ◽  
Arterial Blood Flow ◽  
Microvascular Blood Flow ◽  
No Production ◽  
Arterial Blood ◽  
Transfusion Requirements

­­ ­Although some of the cardiovascular responses to changes in hematocrit (Hct) are not fully quantified experimentally, available information is sufficient to build a mathematical model of the consequences of treating anemia by introducing RBCs into the circulation via blood transfusion. We present such a model, which describes how the treatment of normovolemic anemia with blood transfusion impacts oxygen (O2) delivery (DO2, the product of blood O2 content and arterial blood flow) by the microcirculation. Our analysis accounts for the differential response of the endothelium to the wall shear stress (WSS) stimulus, changes in nitric oxide (NO) production due to modification of blood viscosity caused by alterations of both hematocrit (Hct) and cell free layer thickness, as well as for their combined effects on microvascular blood flow and DO2. Our model shows that transfusions of 1- and 2-unit of blood have a minimal effect on DO2 if the microcirculation is unresponsive to the WSS stimulus for NO production that causes vasodilatation increasing blood flow and DO2. Conversely, in a fully WSS responsive organism, blood transfusion significantly enhances blood flow and DO2, because increased viscosity stimulates endothelial NO production causing vasodilatation. This finding suggests that evaluation of a patients' pre-transfusion endothelial WSS responsiveness should be beneficial in determining the optimal transfusion requirements for treating anemic patients.

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