Nitric oxide, the new architect of epigenetic landscapes.

Nitric oxide (NO) is an endogenously produced signaling molecule with multiple regulatory functions in physiology and disease. The most studied molecular mechanisms underlying the biological functions of NO include its reaction with heme proteins and regulation of protein activity via modification of thiol residues. A significant number of transcriptional responses and phenotypes observed in NO microenvironments, however, still lack mechanistic understanding. Recent studies shed new light on NO signaling by revealing its influence on epigenetic changes within the cell. Epigenetic alterations are important determinants of transcriptional responses and cell phenotypes, which can relay heritable information during cell division. As transcription across the genome is highly sensitive to these upstream epigenetic changes, this mode of NO signaling provides an alternate explanation for NO-mediated gene expression changes and phenotypes. This review will provide an overview of the interplay between NO and epigenetics as well as emphasize the unprecedented importance of these pathways to explain phenotypic effects associated with biological NO synthesis.

The compromise of macrophage functions by hyperoxia is attenuated by ethacrynic acid via inhibition of NF-κB-mediated release of high-mobility group box-1.

The prolonged exposure to hyperoxia can compromise macrophage functions and contribute to the development of ventilator-associated pneumonia. High levels of extracellular high-mobility group box-1 (HMGB1) in the airways of mice exposed to hyperoxia can directly cause macrophage dysfunction. Hence, inhibition of the release of nuclear HMGB1 into the extracellular milieu may help to maintain macrophage functions under hyperoxic conditions. The present study investigates whether ethacrynic acid (EA) affects hyperoxia-induced HMGB1 release from macrophages and improves their functions. Macrophage-like RAW 264.7 cells and bone marrow-derived macrophages were exposed to different concentrations of EA for 24 hours in the presence of 95% O2. EA significantly decreased the accumulation of extracellular HMGB1 in cultured media. Importantly, the phagocytic activity and migration capability of macrophages were significantly enhanced in EA-treated cells. Interestingly, hyperoxia-induced NF-κB activation was also inhibited in these cells. To determine whether NF-κB plays a role in hyperoxia-induced HMGB1 release, BAY 11-7082, an inhibitor of NF-κB activation, was used. Similar to EA, BAY 11-7082 significantly inhibited the accumulation of extracellular HMGB1 and improved hyperoxia-compromised macrophage migration and phagocytic activity. Furthermore, 24-hour hyperoxic exposure of macrophages caused hyperacetylation of HMGB1 and its subsequent cytoplasmic translocation and release, which were inhibited by EA and BAY 11-7082. Together, these results suggest that EA enhances hyperoxia-compromised macrophage functions by inhibiting HMGB1 hyperacetylation and its release from macrophages, possibly through attenuation of the NF-κB activation. Therefore, the activation of NF-κB could be one of the underlying mechanisms that mediate hyperoxia-compromised macrophage functions.

Nitric oxide modifies global histone methylation by inhibiting Jumonji C domain-containing demethylases.

Methylation of lysine residues on histone tails is an important epigenetic modification that is dynamically regulated through the combined effects of methyltransferases and demethylases. The Jumonji C domain Fe(II) α-ketoglutarate family of proteins performs the majority of histone demethylation. We demonstrate that nitric oxide ((•)NO) directly inhibits the activity of the demethylase KDM3A by forming a nitrosyliron complex in the catalytic pocket. Exposing cells to either chemical or cellular sources of (•)NO resulted in a significant increase in dimethyl Lys-9 on histone 3 (H3K9me2), the preferred substrate for KDM3A. G9a, the primary methyltransferase acting on H3K9me2, was down-regulated in response to (•)NO, and changes in methylation state could not be accounted for by methylation in general. Furthermore, cellular iron sequestration via dinitrosyliron complex formation correlated with increased methylation. The mRNA of several histone demethylases and methyltransferases was also differentially regulated in response to (•)NO. Taken together, these data reveal three novel and distinct mechanisms whereby (•)NO can affect histone methylation as follows: direct inhibition of Jumonji C demethylase activity, reduction in iron cofactor availability, and regulation of expression of methyl-modifying enzymes. This model of (•)NO as an epigenetic modulator provides a novel explanation for nonclassical gene regulation by (•)NO.

Prostate cancer-associated SPOP mutations confer resistance to BET inhibitors through stabilization of BRD4.

The bromodomain and extraterminal (BET) family of proteins comprises four members-BRD2, BRD3, BRD4 and the testis-specific isoform BRDT-that largely function as transcriptional coactivators and play critical roles in various cellular processes, including the cell cycle, apoptosis, migration and invasion. BET proteins enhance the oncogenic functions of major cancer drivers by elevating the expression of these drivers, such as c-Myc in leukemia, or by promoting the transcriptional activities of oncogenic factors, such as AR and ERG in prostate cancer. Pathologically, BET proteins are frequently overexpressed and are clinically linked to various types of human cancer; they are therefore being pursued as attractive therapeutic targets for selective inhibition in patients with cancer. To this end, a number of bromodomain inhibitors, including JQ1 and I-BET, have been developed and have shown promising outcomes in early clinical trials. Although resistance to BET inhibitors has been documented in preclinical models, the molecular mechanisms underlying acquired resistance are largely unknown. Here we report that cullin-3SPOP earmarks BET proteins, including BRD2, BRD3 and BRD4, for ubiquitination-mediated degradation. Pathologically, prostate cancer-associated SPOP mutants fail to interact with and promote the degradation of BET proteins, leading to their elevated abundance in SPOP-mutant prostate cancer. As a result, prostate cancer cell lines and organoids derived from individuals harboring SPOP mutations are more resistant to BET-inhibitor-induced cell growth arrest and apoptosis. Therefore, our results elucidate the tumor-suppressor role of SPOP in prostate cancer in which it acts as a negative regulator of BET protein stability and also provide a molecular mechanism for resistance to BET inhibitors in individuals with prostate cancer bearing SPOP mutations.

Nitric Oxide Regulates Gene Expression in Cancers by Controlling Histone Posttranslational Modifications.

Altered nitric oxide (•NO) metabolism underlies cancer pathology, but mechanisms explaining many •NO-associated phenotypes remain unclear. We have found that cellular exposure to •NO changes histone posttranslational modifications (PTM) by directly inhibiting the catalytic activity of JmjC-domain containing histone demethylases. Herein, we describe how •NO exposure links modulation of histone PTMs to gene expression changes that promote oncogenesis. Through high-resolution mass spectrometry, we generated an extensive map of •NO-mediated histone PTM changes at 15 critical lysine residues on the core histones H3 and H4. Concomitant microarray analysis demonstrated that exposure to physiologic •NO resulted in the differential expression of over 6,500 genes in breast cancer cells. Measurements of the association of H3K9me2 and H3K9ac across genomic loci revealed that differential distribution of these particular PTMs correlated with changes in the level of expression of numerous oncogenes, consistent with epigenetic code. Our results establish that •NO functions as an epigenetic regulator of gene expression mediated by changes in histone PTMs.

Insights into the diverse effects of nitric oxide on tumor biology.

Among its many roles in cellular biology, nitric oxide (·NO) has long been associated with cancers both as a protumorigenic and as an antitumorigenic agent. The dual nature of this signaling molecule in varied settings is attributable to its temporal and concentration-dependent effects that produce different phenotypes. The steady-state ·NO concentration within the cell is a balance between its rate of enzymatic synthesis from the three nitric oxide synthase (NOS) isoforms and consumption via numerous metabolic pathways and demonstrates strong dependence on the tissue oxygen concentration. NOS expression and ·NO production are often deregulated and associated with numerous types of cancers with dissimilar prognostic outcomes. ·NO influences several facets of tumor initiation and progression including DNA damage, chronic inflammation, angiogenesis, epithelial-mesenchymal transition, and metastasis, to name a few. The role of ·NO as an epigenetic modulator has also recently emerged and has potentially important mechanistic implications in regulating transcription of oncogenes and tumor-suppressor genes. ·NO-derived cellular adducts such as dinitrosyliron complexes and the formation of higher nitrogen oxides further alter its cellular behavior. Among anticancer strategies, the use of NOS as a prognostic biomarker and modulation of ·NO production for therapeutic benefit have gained importance over the past decade. Numerous ·NO-releasing drugs and NOS inhibitors have been evaluated in preclinical and clinical settings to arrest tumor growth. Taken together, ·NO affects various arms of cancer signaling networks. An overview of this complex interplay is provided in this chapter.

Upgrading dilute ethanol from syngas fermentation to n-caproate with reactor microbiomes.

Fermentation of syngas from renewable biomass, which is part of the syngas platform, is gaining momentum. Here, the objective was to evaluate a proof-of-concept bioprocessing system with diluted ethanol and acetic acid in actual syngas fermentation effluent as the substrate for chain elongation into the product n-caproic acid, which can be separated with less energy input than ethanol. Chain elongation is performed with open cultures of microbial populations (reactor microbiomes) as part of the carboxylate platform. The highest concentration of n-caproic acid of ~1 g L(-1) was produced at a pH of 5.44 and a production rate of 1.7 g L(-1) day(-1). A higher n-butyrate production rate of 20 g L(-1) day(-1) indicated that product toxicity was limiting the chain elongation step from n-butyric acid to n-caproic acid. This result shows that the syngas and carboxylate platforms can be integrated within a biorefinery, but that product separation is necessary.

Oxygen dependence of nitric oxide-mediated signaling.

Nitric oxide (•NO) is a biologically important short-lived free radical signaling molecule. Both the enzymatic synthesis and the predominant forms of cellular metabolism of •NO are oxygen-dependent. For these reasons, changes in local oxygen concentrations can have a profound influence on steady-state •NO concentrations. Many proteins are regulated by •NO in a concentration-dependent manner, but their responses are elicited at different thresholds. Using soluble guanylyl cyclase (sGC) and p53 as model •NO-sensitive proteins, we demonstrate that their concentration-dependent responses to •NO are a function of the O2 concentration. p53 requires relatively high steady-state •NO concentrations (>600 nM) to induce its phosphorylation (P-ser-15), whereas sGC responds to low •NO concentrations (<100 nM). At a constant rate of •NO production (liberation from •NO-donors), decreasing the O2 concentration (1%) lowers the rate of •NO metabolism. This raises steady-state •NO concentrations and allows p53 activation at lower doses of the •NO donor. Enzymatic •NO production, however, requires O2 as a substrate such that decreasing the O2 concentration below the K m for O2 for nitric oxide synthase (NOS) will decrease the production of •NO. We demonstrate that the amount of •NO produced by RAW 264.7 macrophages is a function of the O2 concentration. Differences in rates of •NO production and •NO metabolism result in differential sGC activation that is not linear with respect to O2. There is an optimal O2 concentration (≈5-8%) where a balance between the synthesis and metabolism of •NO is established such that both the •NO concentration and sGC activation are maximal.

Is S-nitrosocysteine a true surrogate for nitric oxide?

S-Nitrosothiol (RSNO) formation is one manner by which nitric oxide (•NO) exerts its biological effects. There are several proposed mechanisms of formation of RSNO in vivo: auto-oxidation of •NO, transnitrosation, oxidative nitrosylation, and from dinitrosyliron complexes (DNIC). Both free •NO, generated by •NO donors, and S-nitrosocysteine (CysNO) are widely used to study •NO biology and signaling, including protein S-nitrosation. It is assumed that the cellular effects of both compounds are analogous and indicative of in vivo •NO biology. A quantitative comparison was made of formation of DNIC and RSNO, the major •NO-derived cellular products. In RAW 264.7 cells, both •NO and CysNO were metabolized, leading to rapid intracellular RSNO and DNIC formation. DNIC were the dominant products formed from physiologic •NO concentrations, however, and RSNO were the major product from CysNO treatment. Chelatable iron was necessary for DNIC assembly from either •NO or CysNO, but not for RSNO formation. These profound differences in RSNO and DNIC formation from •NO and CysNO question the use of CysNO as a surrogate for physiologic •NO. Researchers designing experiments intended to elucidate the biological signaling mechanisms of •NO should be aware of these differences and should consider the biological relevance of the use of exogenous CysNO.