152 research outputs found
Control of VEGF-A transcriptional programs by pausing and genomic compartmentalization.
Vascular endothelial growth factor A (VEGF-A) is a master regulator of angiogenesis, vascular development and function. In this study we investigated the transcriptional regulation of VEGF-A-responsive genes in primary human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using genome-wide global run-on sequencing (GRO-Seq). We demonstrate that half of VEGF-A-regulated gene promoters are characterized by a transcriptionally competent paused RNA polymerase II (Pol II). We show that transition into productive elongation is a major mechanism of gene activation of virtually all VEGF-regulated genes, whereas only ∼40% of the genes are induced at the level of initiation. In addition, we report a comprehensive chromatin interaction map generated in HUVECs using tethered conformation capture (TCC) and characterize chromatin interactions in relation to transcriptional activity. We demonstrate that sites of active transcription are more likely to engage in chromatin looping and cell type-specific transcriptional activity reflects the boundaries of chromatin interactions. Furthermore, we identify large chromatin compartments with a tendency to be coordinately transcribed upon VEGF-A stimulation. We provide evidence that these compartments are enriched for clusters of regulatory regions such as super-enhancers and for disease-associated single nucleotide polymorphisms (SNPs). Collectively, these findings provide new insights into mechanisms behind VEGF-A-regulated transcriptional programs in endothelial cells
Electrophiles modulate glutathione reductase activity via alkylation and upregulation of glutathione biosynthesis
Cells evolved robust homeostatic mechanisms to protect against oxidation or alkylation by electrophilic species.
Glutathione (GSH) is the most abundant intracellular thiol, protects cellular components from oxidation and is
maintained in a reduced state by glutathione reductase (GR). Nitro oleic acid (NO2-OA) is an electrophilic fatty
acid formed under digestive and inflammatory conditions that both reacts with GSH and induces its synthesis
upon activation of Nrf2 signaling. The effects of NO2-OA on intracellular GSH homeostasis were evaluated. In
addition to upregulation of GSH biosynthesis, we observed that NO2-OA increased intracellular GSSG in an
oxidative stress-independent manner. NO2-OA directly inhibited GR in vitro by covalent modification of the
catalytic Cys61, with kon of (3.45±0.04)×103 M−1 s−1, koff of (4.4±0.4)×10−4 s−1, and Keq of
(1.3±0.1)×10−7 M. Akin to NO2-OA, the electrophilic Nrf2 activators bardoxolone-imidazole (CDDO-Im),
bardoxolone-methyl (CDDO-Me) and dimethyl fumarate (DMF) also upregulated GSH biosynthesis while promoting GSSG accumulation, but without directly inhibiting GR activity. In vitro assays in which GR was treated
with increasing GSH concentrations and GSH depletion experiments in cells revealed that GR activity is finely
regulated via product inhibition, an observation further supported by theoretical (kinetic modeling of cellular
GSSG:GSH levels) approaches. Together, these results describe two independent mechanisms by which electrophiles modulate the GSH/GSSG couple, and provide a novel conceptual framework to interpret experimentally determined values of GSH and GSSG
Olives and olive oil are sources of electrophilic fatty acid nitroalkenes
Extra virgin olive oil (EVOO) and olives, key sources of unsaturated fatty acids in the Mediterranean diet, provide health benefits to humans. Nitric oxide (•NO) and nitrite (NO2-)-dependent reactions of unsaturated fatty acids yield electrophilic nitroalkene derivatives (NO 2-FA) that manifest salutary pleiotropic cell signaling responses in mammals. Herein, the endogenous presence of NO2-FA in both EVOO and fresh olives was demonstrated by mass spectrometry. The electrophilic nature of these species was affirmed by the detection of significant levels of protein cysteine adducts of nitro-oleic acid (NO2-OA-cysteine) in fresh olives, especially in the peel. Further nitration of EVOO by NO2- under acidic gastric digestive conditions revealed that human consumption of olive lipids will produce additional nitro-conjugated linoleic acid (NO2-cLA) and nitro-oleic acid (NO2-OA). The presence of free and protein-adducted NO2-FA in both mammalian and plant lipids further affirm a role for these species as signaling mediators. Since NO2-FA instigate adaptive anti-inflammatory gene expression and metabolic responses, these redox-derived metabolites may contribute to the cardiovascular benefits associated with the Mediterranean diet. © 2014 Fazzari et al
RNAi screen for NRF2 inducers identifies targets that rescue primary lung epithelial cells from cigarette smoke induced radical stress
Chronic Obstructive Pulmonary Disease (COPD) is a highly prevalent condition characterized by inflammation and progressive obstruction of the airways. At present, there is no treatment that suppresses the chronic inflammation of the disease, and COPD patients often succumb to the condition. Excessive oxidative stress caused by smoke inhalation is a major driving force of the disease. The transcription factor NRF2 is a critical player in the battle against oxidative stress and its function is impaired in COPD. Increasing NRF2 activity may therefore be a viable therapeutic option for COPD treatment. We show that down regulation of KEAP1, a NRF2 inhibitor, protects primary human lung epithelial cells from cigarette-smoke-extract (CSE) induced cell death in an established in vitro model of radical stress. To identify new potential drug targets with a similar effect, we performed a siRNA screen of the 'druggable' genome using a NRF2 transcriptional reporter cell line. This screen identified multiple genes that when down regulated increased NRF2 transcriptional activity and provided a survival benefit in the in vitro model. Our results suggest that inhibiting components of the ubiquitin-proteasome system will have the strongest effects on NRF2 transcriptional activity by increasing NRF2 levels. We also find that down regulation of the small GTPase Rab28 or the Estrogen Receptor ESRRA provide a survival benefit. Rab28 knockdown increased NRF2 protein levels, indicating that Rab28 may regulate NRF2 proteolysis. Conversely ESRRA down regulation increased NRF2 transcriptional activity without affecting NRF2 levels, suggesting a proteasome-independent mechanism
The importance of sedimenting organic matter, relative to oxygen and temperature, in structuring lake profundal macroinvertebrate assemblages
We quantified the role of a main food
resource, sedimenting organic matter (SOM), relative
to oxygen (DO) and temperature (TEMP) in structuring
profundal macroinvertebrate assemblages in
boreal lakes. SOM from 26 basins of 11 Finnish lakes
was analysed for quantity (sedimentation rates),
quality (C:N:P stoichiometry) and origin (carbon
stable isotopes, d13C). Hypolimnetic oxygen and
temperature were measured from each site during
summer stratification. Partial canonical correspondence
analysis (CCA) and partial regression analyses
were used to quantify contributions of SOM, DO and
TEMP to community composition and three macroinvertebrate
metrics. The results suggested a major
contribution of SOM in regulating the community
composition and total biomass. Oxygen best explained
the Shannon diversity, whereas TEMP had largest
contribution to the variation of Benthic Quality Index.
Community composition was most strongly related to d13C of SOM. Based on additional d13C and stoichiometric
analyses of chironomid taxa, marked differences
were apparent in their utilization of SOM and
body stoichiometry; taxa characteristic of oligotrophic
conditions exhibited higher C:N ratios and lower C:P
and N:P ratios compared to the species typical of
eutrophic lakes. The results highlight the role of SOM
in regulating benthic communities and the distributions
of individual species, particularly in oligotrophic
systems
Approaching the space issue in Nordic curriculum theory: national reflections of globalisation in social studies/citizenship textbook pictures in Sweden, England and Germany
β-Lapachone suppresses neuroinflammation by modulating the expression of cytokines and matrix metalloproteinases in activated microglia
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