Graded levels of molecular oxygen (O2) exist within developing mammalian embryos and can differentially regulate cellular specification pathways. During differentiation, cells acquire distinct epigenetic landscapes, which determine their function, however the mechanisms which regulate this are poorly understood. The demethylation of 5-methylcytosine (5mC) is achieved via successive oxidation reactions catalysed by the Ten-Eleven-Translocation (Tet) enzymes, yielding the 5-hydroxymethylcytosine (5hmC) intermediate. These require O2 as a co-factor, and hence may link epigenetic processes directly to O2 gradients during development. We demonstrate that the activities of Tet enzymes display distinct patterns of [O2]-dependency, and that Tet1 activity, specifically, is subject to differential regulation within a range of O2 which is physiologically relevant in embryogenesis. Further, differentiating embryonic stem cells displayed a transient burst of 5hmC, which was both dependent upon Tet1 and inhibited by low (1%) [O2]. A GC-rich promoter region within the Tet3 locus was identified as a significant target of this 5mC-hydroxylation. Further, this region was shown to associate with Tet1, and display the histone epigenetic marks, H3K4me3 and H3K27me3, which are characteristic of a bivalent, developmentally ‘poised’ promoter. We conclude that Tet1 activity, determined by [O2] may play a critical role in regulating cellular differentiation and fate in embryogenesis.
The gasotransmitter, hydrogen sulfide (H2S) is recognized as an important mediator of endothelial cell homeostasis and function that impacts upon vascular tone and blood pressure. Cystathionine-γ-lyase (CSE) is the predominant endothelial generator of H2S, and recent evidence suggests that its transcriptional expression is regulated by the reactive oxygen species, H2O2. However, the cellular source of H2O2 and the redox-dependent molecular signaling pathway that modulates this is not known. We aimed to investigate the role of Nox4, an endothelial generator of H2O2, in the regulation of CSE in endothelial cells. Both gain- and loss-of-function experiments in human endothelial cells in vitro demonstrated Nox4 to be a positive regulator of CSE transcription and protein expression. We demonstrate that this is dependent upon a heme-regulated inhibitor kinase/eIF2α/activating transcription factor 4 (ATF4) signaling module. ATF4 was further demonstrated to bind directly to cis-regulatory sequences within the first intron of CSE to activate transcription. Furthermore, CSE expression was also increased in cardiac microvascular endothelial cells, isolated from endothelial-specific Nox4 transgenic mice, compared with wild-type littermate controls. Using wire myography we demonstrate that endothelial-specific Nox4 transgenic mice exhibit a hypo-contractile phenotype in response to phenylephrine that was abolished when vessels were incubated with a CSE inhibitor, propargylglycine. We, therefore, conclude that Nox4 is a positive transcriptional regulator of CSE in endothelial cells and propose that it may in turn contribute to the regulation of vascular tone via the modulation of H2S production.
Reactive oxygen species have emerged as key participants in a broad range of physiological and pathophysiological processes, not least within the vascular system. Diverse cellular functions which have been attributed to some of these pro-oxidants within the vasculature include the regulation of blood pressure, neovascularisation and vascular inflammation. We here highlight the emerging roles of the enzymatically-generated reaction oxygen species, O2- and H2O2, in the regulation of the functions of the gaseous signalling molecules: nitric oxide (NO), carbon monoxide (CO), and hydrogen sulphide (H2S). These gasotransmitters are produced on demand from distinct enzymatic sources and in recent years it has become apparent that they are capable of mediating a number of homeostatic processes within the cardiovascular system including enhanced vasodilation, angiogenesis, wound healing and improved cardiac function following myocardial infarction. In common with O2- and/or H2O2 they signal by altering the functions of target proteins, either by the covalent modification of thiol groups or by direct binding to metal centres within metalloproteins, most notably haem proteins. The regulation of the enzymes which generate NO, CO and H2S have been shown to be influenced at both the transcriptional and post-translational levels by redox-dependent mechanisms, while the activity and bioavailability of the gasotransmitters themselves are also subject to oxidative modification. Within vascular cells, the family of nicotinamide adenine dinucleotide phosphate oxidases (NAPDH oxidases/Noxs) have emerged as functionally significant sources of regulated O2- and H2O2 production and accordingly, direct associations between Nox-generated oxidants and the functions of specific gasotransmitters are beginning to be identified. This review focuses on the current knowledge of the redox-dependent mechanisms which regulate the generation and activity of these gases, with particular reference to their roles in angiogenesis.
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