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Institute
Recently, we reported that in crude enzyme preparations, a monocyte-derived soluble protein (M-DSP) renders 5-lipoxygenase (5-LO) activity Ca2+-dependent. Here we provide evidence that this M-DSP is glutathione peroxidase (GPx)-1. Thus, the inhibitory effect of the M-DSP on 5-LO could be overcome by the GPx-1 inhibitor mercaptosuccinate and by the broad spectrum GPx inhibitor iodoacetate, as well as by addition of 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-HPODE). Also, the chromatographic characteristics and the estimated molecular mass (80-100 kDa) of the M-DSP fit to GPx-1 (87 kDa), and GPx-1, isolated from bovine erythrocytes, mimicked the effects of the M-DSP. Intriguingly, only a trace amount of thiol (10 micro M GSH) was required for reduction of 5-LO activity by GPx-1 or the M-DSP. Moreover, the requirement of Ca2+ allowing 5-LO product synthesis in various leukocytes correlated with the respective GPx-1 activities. Mutation of the Ca2+ binding sites within the C2-like domain of 5-LO resulted in strong reduction of 5-LO activity by M-DSP and GPx-1, also in the presence of Ca2+. In summary, our data suggest that interaction of Ca2+ at the C2-like domain of 5-LO protects the enzyme against the effect of GPx-1. Apparently, in the presence of Ca2+, a low lipid hydroperoxide level is sufficient for 5-LO activation.
5-lipoxygenase (5-LO), the key enzyme in leukotriene biosynthesis, is expressed in a tissue- and cell differentiation-specific manner. The 5-LO core promoter required for basal promoter activity has a unique (G+C)-rich sequence that contains five tandem Sp1 consensus sequences. The mechanisms involved in the regulation of cell type-specific 5-LO expression are unknown. Here we show that 5-LO expression is regulated by DNA methylation. Treatment of the 5-LO-negative cell lines U937 and HL-60TB with the demethylating agent 5-aza-2'-deoxycytidine (AdC) up-regulated expression of 5-LO primary transcripts and mature mRNA in a similar fashion, indicating that AdC stimulates 5-LO gene transcription. Analysis of the methylation status of the 5-LO promoter revealed that the core promoter region was methylated in U937 and HL-60TB cells, whereas it was unmethylated in the 5-LO-positive parent HL-60 cell line. Reporter gene assays with 5-LO promoter constructs gave up to 68- and 655-fold repression of 5-LO promoter activity in HeLa and Mono Mac 6 cells by methylation. 1,25-dihydroxyvitamin D(3) and transforming growth factor-beta (TGFbeta), potent inducers of the 5-LO pathway in myeloid cell lines, increased 5-LO RNA expression in HL-60TB and U937 cells, but co-treatment with AdC was required to achieve 5-LO expression levels in HL-60TB cells that were comparable with wild-type HL-60 cells. In reporter gene assays, 1,25-dihydroxyvitamin D(3) and TGFbeta were unable to induce promoter activity when the 5-LO promoter constructs were methylated, which suggests that 5-LO promoter demethylation is a prerequisite for the high level induction of 5-LO gene expression by 1,25-dihydroxyvitamin D(3) and TGFbeta and that the effects of both agents on 5-LO mRNA expression are not related to DNA methylation.
Designed multitarget ligands are a popular approach to generating efficient and safe drugs, and fragment-based strategies have been postulated as a versatile avenue to discover multitarget ligand leads. To systematically probe the potential of fragment-based multiple ligand discovery, we have employed a large fragment library for comprehensive screening on five targets chosen from proteins for which multitarget ligands have been successfully developed previously (soluble epoxide hydrolase, leukotriene A4 hydrolase, 5-lipoxygenase, retinoid X receptor, farnesoid X receptor). Differential scanning fluorimetry served as primary screening method before fragments hitting at least two targets were validated in orthogonal assays. Thereby, we obtained valuable fragment leads with dual-target engagement for six out of ten target combinations. Our results demonstrate the applicability of fragment-based approaches to identify starting points for polypharmacological compound development with certain limitations.
5-Lipoxygenase (5-LO) is the key enzyme in the formation of pro-inflammatory leukotrienes (LT) which play an important role in a number of inflammatory diseases. Accordingly, 5-LO inhibitors are frequently used to study the role of 5-LO and LT in models of inflammation and cancer. Interestingly, the therapeutic efficacy of these inhibitors is highly variable. Here we show that the frequently used 5-LO inhibitors AA-861, BWA4C, C06, CJ-13,610 and the FDA approved compound zileuton as well as the pan-LO inhibitor nordihydroguaiaretic acid interfere with prostaglandin E2 (PGE2) release into the supernatants of cytokine-stimulated (TNFα/IL-1β) HeLa cervix carcinoma, A549 lung cancer as well as HCA-7 colon carcinoma cells with similar potencies compared to their LT inhibitory activities (IC50 values ranging from 0.1–9.1 µM). In addition, AA-861, BWA4C, CJ-13,610 and zileuton concentration-dependently inhibited bacterial lipopolysaccharide triggered prostaglandin (PG) release into human whole blood. Western Blot analysis revealed that inhibition of expression of enzymes involved in PG synthesis was not part of the underlying mechanism. Also, liberation of arachidonic acid which is the substrate for PG synthesis as well as PGH2 and PGE2 formation were not impaired by the compounds. However, accumulation of intracellular PGE2 was found in the inhibitor treated HeLa cells suggesting inhibition of PG export as major mechanism. Further, experiments showed that the PG exporter ATP-binding cassette transporter multidrug resistance protein 4 (MRP-4) is targeted by the inhibitors and may be involved in the 5-LO inhibitor-mediated PGE2 inhibition. In conclusion, the pharmacological effects of a number of 5-LO inhibitors are compound-specific and involve the potent inhibition of PGE2 export. Results from experimental models on the role of 5-LO in inflammation and pain using 5-LO inhibitors may be misleading and their use as pharmacological tools in experimental models has to be revisited. In addition, 5-LO inhibitors may serve as new scaffolds for the development of potent prostaglandin export inhibitors.
Human 5-lipoxygenase (5-LO) is the key enzyme of leukotriene biosynthesis, mostly expressed in leukocytes and thus a crucial component of the innate immune system.
In this study, we show that 5-LO, besides its canonical function as an arachidonic acid metabolizing enzyme, is a regulator of gene expression associated with euchromatin. By Crispr-Cas9-mediated 5-LO knockout (KO) in MonoMac6 (MM6) cells and subsequent RNA-Seq analysis, we identified 5-LO regulated genes which could be clustered to immune/defense response, cell adhesion, transcription and growth/developmental processes. Analysis of differentially expressed genes identified cyclooxygenase-2 (COX2, PTGS2) and kynureninase (KYNU) as strongly regulated 5-LO target genes. 5-LO knockout affected MM6 cell adhesion and tryptophan metabolism via inhibition of the degradation of the immunoregulator kynurenine. By subsequent FAIRE-Seq and 5-LO ChIP-Seq analyses, we found an association of 5-LO with euchromatin, with prominent 5-LO binding to promoter regions in actively transcribed genes. By enrichment analysis of the ChIP-Seq results, we identified potential 5-LO interaction partners. Furthermore, 5-LO ChIP-Seq peaks resemble patterns of H3K27ac histone marks, suggesting that 5-LO recruitment mainly takes place at acetylated histones.>
In summary, we demonstrate a noncanonical function of 5-LO as transcriptional regulator in monocytic cells.
Formation of specialized pro-resolving lipid mediators (SPMs) such as lipoxins or resolvins usually involves arachidonic acid 5-lipoxygenase (5-LO, ALOX5) and different types of arachidonic acid 12- and 15-lipoxygenating paralogues (15-LO1, ALOX15; 15-LO2, ALOX15B; 12-LO, ALOX12). Typically, SPMs are thought to be formed via consecutive steps of oxidation of polyenoic fatty acids such as arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid. One hallmark of SPM formation is that reported levels of these lipid mediators are much lower than typical pro-inflammatory mediators including the monohydroxylated fatty acid derivatives (e.g., 5-HETE), leukotrienes or certain cyclooxygenase-derived prostaglandins. Thus, reliable detection and quantification of these metabolites is challenging. This paper is aimed at critically evaluating i) the proposed biosynthetic pathways of SPM formation, ii) the current knowledge on SPM receptors and their signaling cascades and iii) the analytical methods used to quantify these pro-resolving mediators in the context of their instability and their low concentrations. Based on current literature it can be concluded that i) there is at most, a low biosynthetic capacity for SPMs in human leukocytes. ii) The identity and the signaling of the proposed G-protein-coupled SPM receptors have not been supported by studies in knock-out mice and remain to be validated. iii) In humans, SPM levels were neither related to dietary supplementation with their ω-3 polyunsaturated fatty acid precursors nor were they formed during the resolution phase of an evoked inflammatory response. iv) The reported low SPM levels cannot be reliably quantified by means of the most commonly reported methodology. Overall, these questions regarding formation, signaling and occurrence of SPMs challenge their role as endogenous mediators of the resolution of inflammation.
Epigenetic control of microsomal prostaglandin E synthase-1 by HDAC-mediated recruitment of p300
(2017)
Nonsteroidal anti-inflammatory drugs are the most widely used medicine to treat pain and inflammation, and to inhibit platelet function. Understanding the expression regulation of enzymes of the prostanoid pathway is of great medical relevance. Histone acetylation crucially controls gene expression. We set out to identify the impact of histone deacetylases (HDACs) on the generation of prostanoids and examine the consequences on vascular function. HDAC inhibition (HDACi) with the pan-HDAC inhibitor, vorinostat, attenuated prostaglandin (PG)E2 generation in the murine vasculature and in human vascular smooth muscle cells. In line with this, the expression of the key enzyme for PGE2 synthesis, microsomal PGE synthase-1 (PTGES1), was reduced by HDACi. Accordingly, the relaxation to arachidonic acid was decreased after ex vivo incubation of murine vessels with HDACi. To identify the underlying mechanism, chromatin immunoprecipitation (ChIP) and ChIP-sequencing analysis were performed. These results suggest that HDACs are involved in the recruitment of the transcriptional activator p300 to the PTGES1 gene and that HDACi prevented this effect. In line with the acetyltransferase activity of p300, H3K27 acetylation was reduced after HDACi and resulted in the formation of heterochromatin in the PTGES1 gene. In conclusion, HDAC activity maintains PTGES1 expression by recruiting p300 to its gene.