Abstract
The differentiation of stem cells into endothelial cells involves the modulation of highly interconnected metabolic and epigenetic processes. Therefore, the differentiation of endothelial ...cells is a tightly controlled process, which is adjusted at multiple levels, meaning that even the smallest variation can result in major consequences. Reactive oxygen species (ROS) represent a group of second messengers that can interfere with both metabolic and epigenetic processes. Besides their generation by mitochondria, ROS are produced in a controlled manner by the family of NADPH oxidases. The different members of the NADPH oxidase family produce superoxide anions or hydrogen peroxide. Due to the specific sub-cellular localization of the different NADPH oxidases, ROS are produced at diverse sites in the cell, such as the plasma membrane or the endoplasmic reticulum. Once produced, ROS interfere with proteins, lipids, and DNA to modulate intracellular signal cascades. Accordingly, ROS represent a group of readily available and specifically localized modulators of the highly sophisticated signalling network that eventually leads to the differentiation of stem cells into endothelial cells. This review focuses on the role of NADPH oxidases in the differentiation of stem cells into endothelial cells.
Graphical Abstract
Graphical Abstract
The culture of primary intestinal epithelia cells is not possible in a normal culture system. In 2009 a three-dimensional culture system of intestinal stem cells was established that shows many of ...the physiological features of the small intestine, such as crypt-villus structure, stem cell niche and all types of differentiated intestinal epithelial cells. These enteroids can be used to analyze biology of intestinal stem cells, gut homeostasis and the development of diseases. They also give the possibility to reduce animal numbers, as enteroids can be cryo-conserved and cultivated for many passages. To investigate the influence of genes such as NADPH oxidases on the gut homeostasis, transgenic approached are the method of choice. The generation of enteroids from knockout mice allows real-time observations of knockout effects. Often conditional knockout or overexpression strategies using inducible Cre recombinase are applied to avoid effects of adaption to the knockout. However, the Cre recombinase has many known caveats from unspecific binding and its endonuclease activity. In this study, we show that although NADPH oxidases are important for in vivo differentiation and proliferation of the intestine, their expression is drastically reduced in the organoid system. Activation of Cre recombinase by 4-hydroxy tamoxifen in freshly isolated enteroids, independently of floxed genes, leads to decreased diameter of organoids. This effect is concentration-dependent and is caused by reduced cell proliferation and induction of apoptosis and DNA damage. In contrast, constitutive expression of Cre has no impact on the enteroids. Therefore, reduction of tamoxifen concentration and treatment duration should be carefully titrated, and appropriate controls are necessary.
Epigenetic marks critically control gene expression and thus the cellular activity state. The functions of many epigenetic modifiers in the vascular system have not yet been studied. We screened for ...histone modifiers in endothelial cells and observed a fairly high expression of the histone plant homeodomain finger protein 8 (PHF8). Given its high expression, we hypothesize that this histone demethylase is important for endothelial cell function. Overexpression of PHF8 catalyzed the removal of methyl-groups from histone 3 lysine 9 (H3K9) and H4K20, whereas knockdown of the enzyme increased H3K9 methylation. Knockdown of PHF8 by RNAi also attenuated endothelial proliferation and survival. As a functional readout endothelial migration and tube formation was studied. PHF8 siRNA attenuated the capacity for migration and developing of capillary-like structures. Given the impact of PHF8 on cell cycle genes, endothelial E2F transcription factors were screened, which led to the identification of the gene repressor E2F4 to be controlled by PHF8. Importantly, PHF8 maintains E2F4 but not E2F1 expression in endothelial cells. Consistently, chromatin immunoprecipitation revealed that PHF8 reduces the H3K9me2 level at the E2F4 transcriptional start site, demonstrating a direct function of PHF8 in endothelial E2F4 gene regulation. Conclusion: PHF8 by controlling E2F4 expression maintains endothelial function.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Reactive oxygen species (ROS) produced by enzymes of the NADPH oxidase family serve as second messengers for cellular signaling. Processes such as differentiation and proliferation are regulated by ...NADPH oxidases. In the intestine, due to the exceedingly fast and constant renewal of the epithelium both processes have to be highly controlled and balanced. Nox1 is the major NADPH oxidase expressed in the gut, and its function is regulated by cytosolic subunits such as NoxO1. We hypothesize that the NoxO1-controlled activity of Nox1 contributes to a proper epithelial homeostasis and renewal in the gut.
NoxO1 is highly expressed in the colon. Knockout of NoxO1 reduces the production of superoxide in colon crypts and is not subsidized by an elevated expression of its homolog p47phox. Knockout of NoxO1 increases the proliferative capacity and prevents apoptosis of colon epithelial cells. In mouse models of dextran sulfate sodium (DSS)-induced colitis and azoxymethane/DSS induced colon cancer, NoxO1 has a protective role and may influence the population of natural killer cells.
NoxO1 affects colon epithelium homeostasis and prevents inflammation.
NADPH oxidases are important sources of reactive oxygen species (ROS). Several Nox homologues are present together in the vascular system but whether they exhibit crosstalk at the activity level is ...unknown. To address this, vessel function of knockout mice for the cytosolic Nox organizer proteins p47phox, NoxO1 and a p47phox-NoxO1-double knockout were studied under normal condition and during streptozotocin-induced diabetes.
In the mouse aorta, mRNA expression for NoxO1 was predominant in smooth muscle and endothelial cells, whereas p47phox was markedly expressed in adventitial cells comprising leukocytes and tissue resident macrophages. Knockout of either NoxO1 or p47phox resulted in lower basal blood pressure. Deletion of any of the two subunits also prevented diabetes-induced vascular dysfunction. mRNA expression analysis by MACE (Massive Analysis of cDNA ends) identified substantial gene expression differences between the mouse lines and in response to diabetes. Deletion of p47phox induced inflammatory activation with increased markers of myeloid cells and cytokine and chemokine induction. In contrast, deletion of NoxO1 resulted in an attenuated interferon gamma signature and reduced expression of genes related to antigen presentation. This aspect was also reflected by a reduced number of circulating lymphocytes in NoxO1-/- mice.
ROS production stimulated by NoxO1 and p47phox limit endothelium-dependent relaxation and maintain blood pressure in mice. However, NoxO1 and p47phox cannot substitute each other despite their similar effect on vascular function. Deletion of NoxO1 induced an anti-inflammatory phenotype, whereas p47phox deletion rather elicited a hyper-inflammatory response.
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The intestinal epithelium is one of the most rapidly renewing tissues in the body. Low constitutive ROS formation is needed for cellular differentiation in different tissues. In the intestine, Nox1 ...is the predominant Nox and the cytosolic scaffolding protein NoxO1 enables its constitutive activity. We hypothesize that NoxO1 by enabling a constitutive low Reactive Oxygen Species (ROS) formation impacts on proliferation of intestinal stem cells.
In the mouse colon Nox1 mRNA was expressed at the bottom of the crypts where NoxO1 mRNA was expressed throughout the whole epithelium with reduced abundance toward the top of the crypt. Interestingly NoxO1 protein expression was restricted to the bottom of the crypts, indicating that Nox1 may impact on the protein stability of NoxO1. In Noxo1 knockout mice, Nox1 expression was increased in the crypts whereas the expression of a potential substitute for NoxO1, namely p47phox, was not changed. Accordingly superoxide anion production in intestinal crypts isolated from NoxO1 knockout mice as measured by LO12 chemiluminescence was reduced. In cells isolated from the crypt bottom of NoxO1-/- mice PCNA was decreased, indicating a decreased cell proliferation. Interestingly, Hes-1 was significantly reduced in colons of NoxO1-/- mice.
We conclude that loss of NoxO1 may impact on the proliferation of epithelial cells in the gut, mediated by the formation of superoxide anions.
Nox4 is the only constitutively active NADPH oxidase, producing H2O2. It is highly expressed in endothelial cells, where it plays an important role in differentiation. It is therefore hypothesized ...that Nox4 induces differentiation in endothelial cells and by deficiency preserves stemness.
Using Yamanaka factors, MEFs from wildtype and Nox4-/- mice were reprogramed into iPSCs and then differentiated into endothelial cells. In the course of differentiation, Nox4 expression increased in wildtype cells. Absence of Nox4 resulted in a prolonged expression of stem cell markers and in a diminished expression of endothelial markers in differentiated cells. On the functional level a lower tube formation and sprouting capacity of Nox4-deficient ECs was observed. Using an in vivo matrigel plug assay, a lower capacity of Nox4-/- iPSC-ECs integrated in a newly formed vascular network. As a potential mechanism we observed, increased H3K27me3 in Nox4-/-, which leads to decreased CD31 and VEGFR2 expression. Demethylation of this histone site is mediated by JmjD3, which is not differentially expressed in both cell strains. A BIAM switch assay revealed that JmjD3 was less oxidized in Nox4-/- than in WT cells. Therefore we conclude that Nox4 oxidizes and activates JmjD3. In conclusion Nox4 via an epigenetic modification promotes the differentiation of endothelial cells out of iPSCs.
NADPH oxidases of the Nox family are important sources of reactive oxygen species. Only a limited number of Nox-differentially oxidized proteins have been identified so far. We set out to identify ...redox-targets of Nox4 by redox-proteomics. Tetracycline-inducible HEK293 cells overexpressing Nox4 (HEK-TET-Nox4) and podocytes of WT and Nox4-/- mice were used. Redox-modified proteins were identified by the BIAM switch assay in combination with mass spectrometry and western blot analysis.
Increased Nox4 expression in response to TGFβ-1 was detected by western blot analysis in podocytes of WT mice. In response to TGFβ-1, oxidation of 138 proteins increased in podocytes of wildtype but not in podocytes of Nox4-/- mice. Identified proteins clustered in different groups of cellular processes like “cellular oxidant detoxification” and “activation of protein kinase activity”. As a second approach, HEK-TET-Nox4 cells were used in which Nox4 overexpression and Reactive Oxygen Species (ROS) production can be stimulated by tetracycline. In this protocol, an overlap in the oxidized proteins to the WT/KO system was found for proteins with antioxidant capacity as well for some interesting novel redox-targets of Nox4.
The BIAM-Switch assay coupled to Mass spec is a powerful and versatile tool to identify differentially oxidized proteins. Nox4 is a source of ROS which changes the redox-state of numerous proteins.
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