Claire E. Monk

Regulation of the miR-212/132 locus by MSK1 and CREB in response to neurotrophins.

Neurotrophins are growth factors that are important in neuronal development and survival as well as synapse formation and plasticity. Many of the effects of neurotrophins are mediated by changes in protein expression as a result of altered transcription or translation. To determine whether neurotrophins regulate the production of microRNAs (miRNAs), small RNA species that modulate protein translation or mRNA stability, we used deep sequencing to identify BDNF (brain-derived neurotrophic factor)-induced miRNAs in cultured primary cortical mouse neurons. This revealed that the miR-212/132 cluster contained the miRNAs most responsive to BDNF treatment. This cluster was found to produce four miRNAs: miR-132, miR-132*, miR-212 and miR-212*. Using specific inhibitors, mouse models and promoter analysis we have shown that the regulation of the transcription of the miR-212/132 miRNA cluster and the miRNAs derived from it are regulated by the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway, via both MSK (mitogen and stress-activated kinase)-dependent and -independent mechanisms.

Carbon Monoxide-releasing Antibacterial Molecules Target Respiration and Global Transcriptional Regulators

Carbon monoxide, a classical respiratory inhibitor, also exerts vasodilatory, anti-inflammatory, and antiapoptotic effects. CO-releasing molecules have therapeutic value, increasing phagocytosis and reducing sepsis-induced lethality. Here we identify for the first time the bacterial targets of Ru(CO)3Cl(glycinate) (CORM-3), a ruthenium-based carbonyl that liberates CO rapidly under physiological conditions. Contrary to the expectation that CO would be preferentially inhibitory at low oxygen tensions or anaerobically, Escherichia coli cultures were also sensitive to CORM-3 at concentrations equimolar with oxygen. CORM-3, assayed as ruthenium, was taken up by bacteria and rapidly delivered CO intracellularly to terminal oxidases. Microarray analysis of CORM-3-treated cells revealed extensively modified gene expression, notably down-regulation of genes encoding key aerobic respiratory complexes. Genes involved in metal metabolism, homeostasis, or transport were also differentially expressed, and free intracellular zinc levels were elevated. Probabilistic modeling of transcriptomic data identified the global transcription regulators ArcA, CRP, Fis, FNR, Fur, BaeR, CpxR, and IHF as targets and potential CO sensors. Our discovery that CORM-3 is an effective inhibitor and global regulator of gene expression, especially under aerobic conditions, has important implications for administration of CO-releasing agents in sepsis and inflammation.

Regulation of miRNA Transcription in Macrophages in Response to Candida albicans

Macrophages detect pathogens via pattern recognition receptors (PRRs), which trigger several intracellular signaling cascades including the MAPK and NFκB pathways. These in turn mediate the up-regulation of pro-inflammatory cytokines that are essential to combat the pathogen. However as the over-production of pro-inflammatory cytokines results in tissue damage or septic shock, precise control of these signaling pathways is essential and achieved via the induction of multiple negative feedback mechanisms. miRNAs are small regulatory RNAs that are able to affect protein expression, via the regulation of either mRNA stability or translation. Up-regulation of specific miRNAs could have the potential to modulate PRR signaling, as has been shown for both miR-146 and miR-155. Here we have analysed which miRNAs are up-regulated in mouse macrophages in response to the fungal pathogen heat killed Candida albicans and compared the profile to that obtained with the TLR4 ligand LPS. We found that in addition to miR-146 and miR-155, both Candida albicans and LPS were also able to up-regulate miR-455 and miR-125a. Analysis of the signaling pathways required showed that NFκB was necessary for the transcription of all 4 pri-miRNAs, while the ERK1/2 and p38 MAPK pathways were also required for pri-miR-125a transcription. In addition the anti-inflammatory cytokine IL-10 was found to be able to induce miR-146a and b, but inhibited miR-155 induction. These results suggest that miR-455, miR-125, miR-146 and miR-155 may play important roles in regulating macrophage function following PRR stimulation.

Cross talk between the Akt and p38α pathways in macrophages downstream of Toll-like receptor signaling.

ABSTRACT The stimulation of Toll-like receptors (TLRs) on macrophages by pathogen-associated molecular patterns (PAMPs) results in the activation of intracellular signaling pathways that are required for initiating a host immune response. Both phosphatidylinositol 3-kinase (PI3K)–Akt and p38 mitogen-activated protein kinase (MAPK) signaling pathways are activated rapidly in response to TLR activation and are required to coordinate effective host responses to pathogen invasion. In this study, we analyzed the role of the p38-dependent kinases MK2/3 in the activation of Akt and show that lipopolysaccharide (LPS)-induced phosphorylation of Akt on Thr308 and Ser473 requires p38α and MK2/3. In cells treated with p38 inhibitors or an MK2/3 inhibitor, phosphorylation of Akt on Ser473 and Thr308 is reduced and Akt activity is inhibited. Furthermore, BMDMs deficient in MK2/3 display greatly reduced phosphorylation of Ser473 and Thr308 following TLR stimulation. However, MK2/3 do not directly phosphorylate Akt in macrophages but act upstream of PDK1 and mTORC2 to regulate Akt phosphorylation. Akt is recruited to phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the membrane, where it is activated by PDK1 and mTORC2. Analysis of lipid levels in MK2/3-deficient bone marrow-derived macrophages (BMDMs) revealed a role for MK2/3 in regulating Akt activity by affecting availability of PIP3 at the membrane. These data describe a novel role for p38α-MK2/3 in regulating TLR-induced Akt activation in macrophages.

Oxygen- and NssR-dependent Globin Expression and Enhanced Iron Acquisition in the Response of Campylobacter to Nitrosative Stress

Pathogenic bacteria experience nitrosative stress from NO generated in the host and from nitrosating species such as S-nitrosoglutathione. The food-borne pathogen Campylobacter jejuni responds by activating gene expression from a small regulon under the control of the NO-sensitive regulator, NssR. Here, we describe the full extent of the S-nitrosoglutathione response using transcriptomic and proteomic analysis of batch- and chemostat-cultured C. jejuni. In addition to the NssR regulon, which includes two hemoglobins (Cgb and Ctb), we identify more than 90 other up-regulated genes, notably those encoding heat shock proteins and proteins involved in oxidative stress tolerance and iron metabolism/transport. Up-regulation of a subset of these genes, including cgb, is also elicited by NO-releasing compounds. Mutation of the iron-responsive regulator Fur results in insensitivity of growth to NO, suggesting that derepression of iron-regulated genes and augmentation of iron acquisition is a physiological response to nitrosative damage. We describe the effect of oxygen availability on nitrosative stress tolerance; cells cultured at higher rates of oxygen diffusion have elevated levels of hemoglobins, are more resistant to inhibition by NO of both growth and respiration, and consume NO more rapidly. The oxygen response is mediated by NssR. Thus, in addition to NO detoxification catalyzed by the hemoglobins Cgb and possibly Ctb, C. jejuni mounts an extensive stress response. We suggest that inhibition of respiration by NO may increase availability of oxygen for Cgb synthesis and function.

The NO-responsive hemoglobins of Campylobacter jejuni: Concerted responses of two globins to NO and evidence in vitro for globin regulation by the transcription factor NssR

Campylobacter jejuni possesses NO-responsive and -detoxifying mechanisms to survive NO during transmission and pathogenesis. C. jejuni possesses two hemoglobins. The first (Cgb) is a single-domain (non-flavo)hemoglobin encoded by gene Cj1586 (cgb), mutation of which leads to hypersensitivity to S-nitrosoglutathione and NO. Transcription of cgb is induced by nitrosative stress and confers resistance to NO, presumably via a Cgb-catalyzed dioxygenase or denitrosylase reaction that converts NO and oxygen to nitrate. Expression of Cgb in response to NO is mediated via the positively-acting transcription factor NssR, which regulates expression of a small regulon that includes cgb and ctb (Cj0465c), the latter encoding the truncated hemoglobin, Ctb. The role of Ctb is unclear: it is not directly involved in NO detoxification but is implicated in oxygen delivery or metabolism. Here, we describe attempts to define a function for Ctb by examining the effects of a ctb mutation on the NO transcriptome and cgb gene expression during normoxia and hypoxia. Mutation of ctb does not elicit major compensatory transcriptomic changes but relatively minor changes in genes involved in intermediary metabolism, solute transport and signal transduction. We present and test the hypothesis that, by binding NO or O(2), Ctb dampens the response to NO under hypoxic conditions and limits cgb expression, perhaps because Cgb function (i.e. NO detoxification) requires O(2)-dependent chemistry. We report the purification of NssR and specific binding to the ctb promoter. GSNO does not affect the high affinity of NssR for the ctb promoter.

Microbial responses to nitric oxide and nitrosative stress: growth, "omic," and physiological methods.

The study of bacterial responses to nitric oxide (NO), nitrosating agents, and other agents of nitrosative stress has a short history but has rapidly produced important insights into the interactions of these agents with model microbial systems as well as pathogenic species. Several methodological problems arise in attempting to define the global responses to these agents, whether in simply measuring growth or performing omic experiments in which the objective is to determine the genome-wide (transcriptomic) or proteome-wide responses. The first problem is the relatively long timescale over which the experiments are conducted--minutes, hours, or days in the case of slow-growing cultures. The second problem is not unique to NO and its congeners but concerns the difficulties encountered when sensitive and comprehensive analytical techniques (such as transcriptomics) are applied to cultures whose growth and physiology are perturbed by an inhibitor. In essence, the problem is seeing the wood for the trees. This chapter reviews briefly the state of knowledge of NO responses and mechanisms in bacteria, particularly Escherichia coli and Campylobacter jejuni. Continuous culture has several advantages for investigating the consequences of NO exposure, and this approach is outlined with examples of recent results and conclusions. The major advantage of the chemostat is establishment of a reproducible quasi-steady state in growth, in which the growth rate can be controlled and maintained. Contrary to common belief, neither the concept nor the apparatus is difficult. Commercially available and homemade systems are described with practical advice. Establishing continuous cultures paves the way for other omic approaches, particularly proteomics and metabolomics, which are not covered here, as their application to the field of NO biology is in its infancy. A key to the literature describing methods suitable for assessing toxicity to microbes of NO and reactive nitrogen species is given.

Do Globins in Microaerophilic Campylobacter jejuni Confer Nitrosative Stress Tolerance Under Oxygen Limitation

The microaerophilic pathogen Campylobacter jejuni possesses inducible systems for resisting NO. Two globins--Cgb (a single-domain globin) and Ctb (a truncated globin)--are up-regulated in response to NO via the positively acting transcription factor NssR. Our aims were to determine whether these oxygen-binding globins also function in severely oxygen-limited environments, as in the host. At growth-limiting oxygen transfer rates, bacteria were more S-nitrosoglutathione (GSNO) sensitive, irrespective of the presence of Cgb, Ctb, or NssR. Pregrowth of cells with GSNO enhanced GSNO resistance, even in nssR and cgb mutants, but transcriptomic profiling of oxygen-limited, NO-exposed cells failed to reveal the NssR regulon. Nevertheless, globin expression in an Escherichia coli mutant lacking the NO-detoxifying flavohemoglobin Hmp showed that Cgb and Ctb consume NO aerobically or anoxically and offer some protection to respiratory inhibition by NO. The constitutively expressed nitrite reductase NrfA does not provide resistance under oxygen-limited conditions. We, therefore, hypothesize that, although Cgb and NrfA can detoxify NO, even anoxically, they are neither up-regulated nor functional under physiologically relevant oxygen-limited conditions and, second, responses to NO do not stem from trancriptional regulation.

Beta Interferon Production Is Regulated by p38 Mitogen-Activated Protein Kinase in Macrophages via both MSK1/2- and Tristetraprolin-Dependent Pathways

ABSTRACT Autocrine or paracrine signaling by beta interferon (IFN-β) is essential for many of the responses of macrophages to pathogen-associated molecular patterns. This feedback loop contributes to pathological responses to infectious agents and is therefore tightly regulated. We demonstrate here that macrophage expression of IFN-β is negatively regulated by mitogen- and stress-activated kinases 1 and 2 (MSK1/2). Lipopolysaccharide (LPS)-induced expression of IFN-β was elevated in both MSK1/2 knockout mice and macrophages. Although MSK1 and -2 promote the expression of the anti-inflammatory cytokine interleukin 10, it did not strongly contribute to the ability of MSKs to regulate IFN-β expression. Instead, MSK1 and -2 inhibit IFN-β expression via the induction of dual-specificity phosphatase 1 (DUSP1), which dephosphorylates and inactivates the mitogen-activated protein kinases p38 and Jun N-terminal protein kinase (JNK). Prolonged LPS-induced activation of p38 and JNK, phosphorylation of downstream transcription factors, and overexpression of IFN-β mRNA and protein were similar in MSK1/2 and DUSP1 knockout macrophages. Two distinct mechanisms were implicated in the overexpression of IFN-β: first, JNK-mediated activation of c-jun, which binds to the IFN-β promoter, and second, p38-mediated inactivation of the mRNA-destabilizing factor tristetraprolin, which we show is able to target the IFN-β mRNA.