Kontrollmechanismen der endothelialen NO-Synthase-Expression
Final Report Abstract
A single nucleotide polymorphism (SNP) within the promoter of the endothelial NO synthase (NOS-3) gene (T-786C) negatively affects its responsiveness to unidirectional fluid shear stress (FSS) and the anti-inflammatory cytokine interleukin-10 (IL-10). In Caucasians homozygosity for this polymorphism is a predictor for chronic inflammatory diseases including coronary heart disease (CHD) and rheumatoid arthritis (RA). A decoy oligodesoxynucleotide mimicking the mutant C-type promoter around position -786 restores normal gene function. However, no DNA-interacting protein that sterically prevents binding of a FSS-responsive transcription factor could be identified, nor could we establish a role of the FSS-sensitive KLF2 signalling pathway. An initially found FSS-dependent differential demethylation of the promoter in TT- vs. CC-genotype endothelial cells (ECs), and the fact that generic methyltransferase inhibition differentially affected FSS-dependent NOS3 transcription in TT- vs. CC-genotype ECs led us to hypothesize that hypermethylation of the C-type promoter at its unique CpG-dinucleotide (position -786 to -785) prevents a FSS-responsive transcription factor from binding to the DNA. However, bisulfite sequencing of the C-type vs. T-type NOS3 promoter revealed no methylation at this particular position, but only a methylated CpG-dinucleotide in the T-type promoter proximal to the SNP, disproving our working hypothesis. Alternative data base screening (ENCODE project) revealed a hotspot for histone marks around position -786 in human ECs. Inhibition of deacetylation of H3K27 with trichostatin A (TSA) attenuated FSS-dependent NOS3 expression in TT-genotype but disinhibited it in CC-genotype ECs, particularly after exposure to FSS for 4 instead of 24 hours. In mice, which do not have a T-786C SNP, TSA had no differential effect on basal vs. FSS-dependent Nos3 expression. Specific inhibition of class I histone deacetylases with MS275 as well as their siRNA-based knockdown reduced both basal and FSS-dependent up-regulation of NOS-3 expression in TT- and CC- genotype human ECs. Differences in chromatin remodelling may thus be responsible for reduced expression of the NOS3 gene in CC-genotype ECs. This hypothesis agrees with our finding that a STAT consensus cis-element upstream of the SNP mediates stimulation of NOS3 expression by FSS and IL-10. Chromatin remodelling, particularly monomethylation of H3K4, may additionally affect sensitivity of the gene to these stimuli. Two mechanisms identified by us compensate for the relative loss of NO in homozygous carriers of the SNP, and prevent an early onset of CHD or RA. In CC- but not TT-genotype ECs exposed to FSS, expression of the NO-sensitive transcription factor EGR1 is up-regulated, which boosts expression of superoxide dismutase-2, which in turn quenches superoxide anions to maintain a critical level of bioavailable NO. In CC-genotype ECs, FSS stimulates cyclooxygenase-2 (COX2) and lipocalin-type prostaglandin D synthase (PTGDS) expression, leading to an increased release of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), a potent anti-inflammatory prostanoid. Consequently, human monocytes (THP-1 cells) transmigrate less through CC- vs. TT-genotype ECs exposed to FSS and do not express pro-inflammatory cytokines. Silencing of PTGDS expression enhanced THP-1 cell transmigration through CC-genotype ECs while exposure to authentic 15d-PGJ2 inhibited their transmigration through TT-genotype ECs and down-regulated their expression of the CD40 and IL1B gene. This suggests that 15d-PGJ2 released by CC-genotype ECs compensates for the relative lack of NO and its anti-inflammatory properties. It markedly reduces the capacity of Keap1 to trap NRF2 in the cytoplasm, which thus accumulates in the nucleus where it binds to antioxidant response elements (ARE) in the promoter of the IL1B gene hence suppressing its transcription. Moreover, plasma 15d- PGJ2 levels are 8-fold higher in patients with CHD than in age-matched controls. For a proper subgroup analysis, we are currently acquiring further samples from patients with CHD and RA. Since the T-786C SNP only occurs in humans, we are currently establishing humanized mouse models in which the full-length mouse Nos3 promoter including exon 1 has been exchanged against the human C- or T-type promoter plus exon 1. They will be crossed, e.g. with ApoE-null mice to study functional consequences of the T-786C SNP in the context of atherosclerosis.
Publications
- Disinhibition of SOD-2 expression to compensate for a genetically determined NO deficit in endothelial cells - brief report. Arterioscler Thromb Vasc Biol. 2009;29(11):1890-3
Asif AR, Hecker M, Cattaruzza M
- T-786C polymorphism of the NOS-3 gene and the endothelial cell response to fluid shear stress - a proteome analysis. J Proteome Res. 2009;8(6):3161-8
Asif AR, Oellerich M, Armstrong VW, Hecker M, Cattaruzza M
- Shear stress-induced 15-deoxy-Δ12,14- prostaglandin J2 production reinforces the anti-inflammatory capacity of endothelial cells with a genetically determined nitric oxide deficit. Clin Res Cardiol. 2014;103, Suppl 1:P756
Kadiyska I, Yakubenia S, Cattaruzza M, Hecker M
- Endothelial cells counteract a genetically determined nitric oxide deficit by enhancing the release of the anti-inflammatory prostanoid 15-deoxy-Δ12,14-prostaglandin J2. Acta Physiol. 2015;213, Suppl 699:P069
Kadiyska I, Gehrmann S, Wagner A, Hecker M
- T-786C single nucleotide polymorphism of the endothelial nitric oxide synthase gene as a risk factor for endothelial dysfunction in polymyalgia rheumatica. Clin Exp Rheumatol. 2015;33(5):726-30
Löffers C, Heilig B, Hecker M
