Subverting sterols: rerouting an oxysterol-signaling pathway to promote tumor growth.

Oxysterols are oxidized derivatives of cholesterol that are generated enzymatically or through autoxidation. Initially identified as important lipid signaling molecules in the context of atherosclerosis and inflammation, accumulated evidence indicates that these lipid-signaling molecules can have pleiotropic effects on the fate and function of the immune system. These effects range from the regulation of immune cell survival and proliferation to chemotaxis and antiviral immunity. New studies now indicate that tumor-derived oxysterols can serve to subvert the immune system by recruiting protumorigenic neutrophils into the tumor microenvironment. The consequence of this recruitment is the generation of proangiogenic factors and matrix metalloproteinase proteins that provide a tumor a significant growth and survival advantage. In combination with other recent studies, these data highlight the ongoing cross talk between sterol metabolism and the immune system, and they raise the intriguing possibility that targeting oxysterol pathways could serve as a novel therapeutic approach in the war on cancer

Doxycycline alters metabolism and proliferation of human cell lines.

The tetracycline antibiotics are widely used in biomedical research as mediators of inducible gene expression systems. Despite many known effects of tetracyclines on mammalian cells-including inhibition of the mitochondrial ribosome-there have been few reports on potential off-target effects at concentrations commonly used in inducible systems. Here, we report that in human cell lines, commonly used concentrations of doxycycline change gene expression patterns and concomitantly shift metabolism towards a more glycolytic phenotype, evidenced by increased lactate secretion and reduced oxygen consumption. We also show that these concentrations are sufficient to slow proliferation. These findings suggest that researchers using doxycycline in inducible expression systems should design appropriate controls to account for potential confounding effects of the drug on cellular metabolism

Subverting sterols: rerouting an oxysterol-signaling pathway to promote tumor growth

Oxysterols are oxidized derivatives of cholesterol that are generated enzymatically or through autoxidation. Initially identified as important lipid signaling molecules in the context of atherosclerosis and inflammation, accumulated evidence indicates that these lipid-signaling molecules can have pleiotropic effects on the fate and function of the immune system. These effects range from the regulation of immune cell survival and proliferation to chemotaxis and antiviral immunity. New studies now indicate that tumor-derived oxysterols can serve to subvert the immune system by recruiting protumorigenic neutrophils into the tumor microenvironment. The consequence of this recruitment is the generation of proangiogenic factors and matrix metalloproteinase proteins that provide a tumor a significant growth and survival advantage. In combination with other recent studies, these data highlight the ongoing cross talk between sterol metabolism and the immune system, and they raise the intriguing possibility that targeting oxysterol pathways could serve as a novel therapeutic approach in the war on cancer

Cooperative activity of cdk8 and GCN5L within Mediator directs tandem phosphoacetylation of histone H3

The human Mediator complex is generally required for expression of protein-coding genes. Here, we show that the GCN5L acetyltransferase stably associates with Mediator together with the TRRAP polypeptide. Yet, contrary to expectations, TRRAP/GCN5L does not associate with the transcriptionally active core Mediator but rather with Mediator that contains the cdk8 subcomplex. Consequently, this derivative ‘T/G-Mediator' complex does not directly activate transcription in a reconstituted human transcription system. However, within T/G-Mediator, cdk8 phosphorylates serine-10 on histone H3, which in turn stimulates H3K14 acetylation by GCN5L within the complex. Tandem phosphoacetylation of H3 correlates with transcriptional activation, and ChIP assays demonstrate co-occupancy of T/G-Mediator components at several activated genes in vivo. Moreover, cdk8 knockdown causes substantial reduction of global H3 phosphoacetylation, suggesting that T/G-Mediator is a major regulator of this H3 mark. Cooperative H3 modification provides a mechanistic basis for GCN5L association with cdk8-Mediator and also identifies a biochemical means by which cdk8 can indirectly activate gene expression. Indeed our results suggest that T/G-Mediator directs early events—such as modification of chromatin templates—in transcriptional activation

Doxycycline alters metabolism and proliferation of human cell lines.

The tetracycline antibiotics are widely used in biomedical research as mediators of inducible gene expression systems. Despite many known effects of tetracyclines on mammalian cells-including inhibition of the mitochondrial ribosome-there have been few reports on potential off-target effects at concentrations commonly used in inducible systems. Here, we report that in human cell lines, commonly used concentrations of doxycycline change gene expression patterns and concomitantly shift metabolism towards a more glycolytic phenotype, evidenced by increased lactate secretion and reduced oxygen consumption. We also show that these concentrations are sufficient to slow proliferation. These findings suggest that researchers using doxycycline in inducible expression systems should design appropriate controls to account for potential confounding effects of the drug on cellular metabolism

Cellular Stress-Induced Metabolites in <i>Escherichia coli</i>

Escherichia coli isolates commonly inhabit the human microbiota, yet the majority of E. coli’s small-molecule repertoire remains uncharacterized. We previously employed erythromycin-induced translational stress to facilitate the characterization of autoinducer-3 (AI-3) and structurally related pyrazinones derived from “abortive” tRNA synthetase reactions in pathogenic, commensal, and probiotic E. coli isolates. In this study, we explored the “missing” tryptophan-derived pyrazinone reaction and characterized two other families of metabolites that were similarly upregulated under erythromycin stress. Strikingly, the abortive tryptophanyl-tRNA synthetase reaction leads to a tetracyclic indole alkaloid metabolite (1) rather than a pyrazinone. Furthermore, erythromycin induced two naphthoquinone-functionalized metabolites (MK-hCys, 2; and MK-Cys, 3) and four lumazines (7–10). Using genetic and metabolite analyses coupled with biomimetic synthesis, we provide support that the naphthoquinones are derived from 4-dihydroxy-2-naphthoic acid (DHNA), an intermediate in the menaquinone biosynthetic pathway, and the amino acids homocysteine and cysteine. In contrast, the lumazines are dependent on a flavin intermediate and α-ketoacids from the aminotransferases AspC and TyrB. We show that one of the lumazine members (9), an indole-functionalized analogue, possesses antioxidant properties, modulates the anti-inflammatory fate of isolated TH17 cells, and serves as an aryl-hydrocarbon receptor (AhR) agonist. These three systems described here serve to illustrate that new metabolic branches could be more commonly derived from well-established primary metabolic pathways

Toll-Like Receptors Induce Signal-Specific Reprogramming of the Macrophage Lipidome

Macrophages reprogram their lipid metabolism in response to activation signals. However, a systems-level understanding of how different pro-inflammatory stimuli reshape the macrophage lipidome is lacking. Here, we use complementary "shotgun" and isotope tracer mass spectrometry approaches to define the changes in lipid biosynthesis, import, and composition of macrophages induced by various Toll-like receptors (TLRs) and inflammatory cytokines. "Shotgun" lipidomics data revealed that different TLRs and cytokines induce macrophages to acquire distinct lipidomes, indicating their specificity in reshaping lipid composition. Mechanistic studies showed that differential reprogramming of lipid composition is mediated by the opposing effects of MyD88- and TRIF-interferon-signaling pathways. Finally, we applied these insights to show that perturbing reprogramming of lipid composition can enhance inflammation and promote host defense to bacterial challenge. These studies provide a framework for understanding how inflammatory stimuli reprogram lipid composition of macrophages while providing a knowledge platform to exploit differential lipidomics to influence immunity