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5-Lipoxygenase contributes to PPAR [gamma] activation in macrophages in response to apoptotic cells
(2012)
Background: One hallmark contributing to immune suppression during the late phase of sepsis is macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells (AC). Taking the important role of the nuclear receptor PPARγ for this phenotype switch into consideration, it remains elusive how AC activate PPARγ in macrophages. Therefore, we were interested to characterize the underlying principle.
Methods: Apoptosis was induced by treatment of Jurkat T cells for 3 hours with 0.5 μg/ml staurosporine. Necrotic cells (NC) were prepared by heating cells for 20 minutes to 65°C. PPARγ activation was followed by stably transducing RAW264.7 macrophages with a vector encoding the red fluorescent protein mRuby after PPARγ binding to 4 × PPRE sites downstream of the reporter gene sequence. This readout was established by treatment with the PPARγ agonist rosiglitazone (1 μM) and AC (5:1). Twenty-four hours after stimulation, mRuby expression was analysed by fluorescence microscopy. Lipid rafts of AC, NC, as well as living cells (LC) were enriched by sucrose gradient centrifugation. Fractions were analysed for lipid raft-associated marker proteins. Lipid rafts were incubated with transduced RAW264.7 macrophages as described above. 5-Lipoxygenase (5-LO) involvement was verified by pharmacological inhibition (MK-866, 1 μM) and overexpression.
Results: Assuming that the molecule responsible for PPARγ activation in macrophages is localized in the cell membrane of AC, most probably associated to lipid rafts, we isolated lipid rafts from AC, NC and LC. Mass spectrometric analysis of lipid rafts of AC showed the expression of 5-LO, whereas lipid rafts of LC did not. Moreover, incubating macrophages with lipid rafts of AC induced mRuby expression. In contrast, lipid rafts of NC and LC did not. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated for 30 minutes with the 5-LO inhibitor MK-866 (1 μM) before apoptosis induction. In line with our hypothesis, these AC did not induce mRuby expression. Finally, although living Jurkat T cells overexpressing 5-LO did not activate PPARγ in macrophages, mRuby expression was significantly increased when AC were generated from 5-LO overexpressing compared with wild-type Jurkat cells.
Conclusion: Our results suggest that induction of apoptosis activates 5-LO, localizing to lipid rafts, necessary for PPARγ activation in macrophages. Therefore, it will be challenging to determine whether 5-LO activity in AC, generated from other cell types, correlates with PPARγ activation, contributing to an immune-suppressed phenotype in macrophages.
We among others have recently demonstrated that normal cells produce “fusion mRNAs”. These fusion mRNAs do not derive from rearranged genomic loci, but rather they are derived from “early-terminated transcripts” (ETTs). Premature transcriptional termination takes place in intronic sequences that belong to “breakpoint cluster regions”. One important property of ETTs is that they exhibit an unsaturated splice donor site. This results in: (1) splicing to “cryptic exons” present in the final intron; (2) Splicing to another transcript of the same gene (intragenic trans-splicing), resulting in “exon repetitions”; (3) splicing to a transcript of another gene (intergenic trans-splicing), leading to “non-genomically encoded fusion transcripts” (NGEFTs). These NGEFTs bear the potential risk to influence DNA repair processes, since they share identical nucleotides with their DNA of origin, and thus, could be used as “guidance RNA” for DNA repair processes. Here, we present experimental data about four other genes. Three of them are associated with hemato-malignancies (ETV6, NUP98 and RUNX1), while one is associated with solid tumors (EWSR1). Our results demonstrate that all genes investigated so far (MLL, AF4, AF9, ENL, ELL, ETV6, NUP98, RUNX1 and EWSR1) display ETTs and produce transpliced mRNA species, indicating that this is a genuine property of translocating genes.
We present a computational method for the reaction-based de novo design of drug-like molecules. The software DOGS (Design of Genuine Structures) features a ligand-based strategy for automated ‘in silico’ assembly of potentially novel bioactive compounds. The quality of the designed compounds is assessed by a graph kernel method measuring their similarity to known bioactive reference ligands in terms of structural and pharmacophoric features. We implemented a deterministic compound construction procedure that explicitly considers compound synthesizability, based on a compilation of 25'144 readily available synthetic building blocks and 58 established reaction principles. This enables the software to suggest a synthesis route for each designed compound. Two prospective case studies are presented together with details on the algorithm and its implementation. De novo designed ligand candidates for the human histamine H4 receptor and γ-secretase were synthesized as suggested by the software. The computational approach proved to be suitable for scaffold-hopping from known ligands to novel chemotypes, and for generating bioactive molecules with drug-like properties.
TRPC channels are a family of nonselective cation channels that regulate ion homeostasis and intracellular Ca2+ signaling in numerous cell types. Important physiological functions such as vasoregulation, neuronal growth, and pheromone recognition have been assigned to this class of ion channels. Despite their physiological relevance, few selective pharmacological tools are available to study TRPC channel function. We, therefore, screened a selection of pharmacologically active compounds for TRPC modulating activity. We found that the synthetic gestagen norgestimate inhibited diacylglycerol-sensitive TRPC3 and TRPC6 with IC50s of 3–5 µM, while half-maximal inhibition of TRPC5 required significantly higher compound concentrations (>10 µM). Norgestimate blocked TRPC-mediated vasopressin-induced cation currents in A7r5 smooth muscle cells and caused vasorelaxation of isolated rat aorta, indicating that norgestimate could be an interesting tool for the investigation of TRP channel function in native cells and tissues. The steroid hormone progesterone, which is structurally related to norgestimate, also inhibited TRPC channel activity with IC50s ranging from 6 to 18 µM but showed little subtype selectivity. Thus, TRPC channel inhibition by high gestational levels of progesterone may contribute to the physiological decrease of uterine contractility and immunosuppression during pregnancy.
Interleukin-22 predicts severity and death in advanced liver cirrhosis: a prospective cohort study
(2012)
Background: Interleukin-22 (IL-22), recently identified as a crucial parameter of pathology in experimental liver damage, may determine survival in clinical end-stage liver disease. Systematic analysis of serum IL-22 in relation to morbidity and mortality of patients with advanced liver cirrhosis has not been performed so far.
Methods: This is a prospective cohort study including 120 liver cirrhosis patients and 40 healthy donors to analyze systemic levels of IL-22 in relation to survival and hepatic complications.
Results: A total of 71% of patients displayed liver cirrhosis-related complications at study inclusion. A total of 23% of the patients died during a mean follow-up of 196 +/- 165 days. Systemic IL-22 was detectable in 74% of patients but only in 10% of healthy donors (P <0.001). Elevated levels of IL-22 were associated with ascites (P = 0.006), hepatorenal syndrome (P <0.0001), and spontaneous bacterial peritonitis (P = 0.001). Patients with elevated IL-22 (>18 pg/ml, n = 57) showed significantly reduced survival compared to patients with regular ([less than or equal to]18 pg/ml) levels of IL-22 (321 days versus 526 days, P = 0.003). Other factors associated with overall survival were high CRP ([greater than or equal to]2.9 mg/dl, P = 0.005, hazard ratio (HR) 0.314, confidence interval (CI) (0.141 to 0.702)), elevated serum creatinine (P = 0.05, HR 0.453, CI (0.203 to 1.012)), presence of liver-related complications (P = 0.028, HR 0.258 CI (0.077 to 0.862)), model of end stage liver disease (MELD) score [greater than or equal to]20 (P = 0.017, HR 0.364, CI (0.159 to 0.835)) and age (P = 0.011, HR 1.047, CI (1.011 to 1.085)). Adjusted multivariate Cox proportional-hazards analysis identified elevated systemic IL-22 levels as independent predictors of reduced survival (P = 0.007, HR 0.218, CI (0.072 to 0.662)).
Conclusions: In patients with liver cirrhosis, elevated systemic IL-22 levels are predictive for reduced survival independently from age, liver-related complications, CRP, creatinine and the MELD score. Thus, processes that lead to a rise in systemic interleukin-22 may be relevant for prognosis of advanced liver cirrhosis.
BACKGROUND: The endocannabinoid 2-arachidonoyl glycerol (2-AG) acts as a retrograde messenger and modulates synaptic signaling e. g. in the hippocampus. 2-AG also exerts neuroprotective effects under pathological situations. To better understand the mechanism beyond physiological signaling we used Organotypic Entorhino-Hippocampal Slice Cultures (OHSC) and investigated the temporal regulation of 2-AG in different cell subsets during excitotoxic lesion and dendritic lesion of long range projections in the enthorhinal cortex (EC), dentate gyrus (DG) and the cornu ammonis region 1 (CA1).
RESULTS: 2-AG levels were elevated 24 h after excitotoxic lesion in CA1 and DG (but not EC) and 24 h after perforant pathway transection (PPT) in the DG only. After PPT diacylglycerol lipase alpha (DAGL) protein, the synthesizing enzyme of 2-AG was decreased when Dagl mRNA expression and 2-AG levels were enhanced. In contrast to DAGL, the 2-AG hydrolyzing enzyme monoacylglycerol lipase (MAGL) showed no alterations in total protein and mRNA expression after PPT in OHSC. MAGL immunoreaction underwent a redistribution after PPT and excitotoxic lesion since MAGL IR disappeared in astrocytes of lesioned OHSC. DAGL and MAGL immunoreactions were not detectable in microglia at all investigated time points. Thus, induction of the neuroprotective endocannabinoid 2-AG might be generally accomplished by down-regulation of MAGL in astrocytes after neuronal lesions.
CONCLUSION: Increase in 2-AG levels during secondary neuronal damage reflects a general neuroprotective mechanism since it occurred independently in both different lesion models. This intrinsic up-regulation of 2-AG is synergistically controlled by DAGL and MAGL in neurons and astrocytes and thus represents a protective system for neurons that is involved in dendritic reorganisation.
TO THE EDITOR: We read an interesting paper by Palta et al. in a recent issue of the Korean Journal of Hematology titled, "ZBTB16-RARA variant of acute promyelocytic leukemia with tuberculosis: a case report and review of literature" [1]. We would like to add some comments to their article and suggest additional molecular methods to confirm variant translocations in acute promyelocytic leukemia (APL)....
Novel chalcone-based fluorescent human histamine H 3 receptor ligands as pharmacological tools
(2012)
Novel fluorescent chalcone-based ligands at human histamine H(3) receptors (hH(3)R) have been designed, synthesized, and characterized. Compounds described are non-imidazole analogs of ciproxifan with a tetralone motif. Tetralones as chemical precursors and related fluorescent chalcones exhibit affinities at hH(3)R in the same concentration range like the reference antagonist ciproxifan (hH(3)R pK(i) value of 7.2). Fluorescence characterization of our novel ligands shows emission maxima about 570 nm for yellow fluorescent chalcones and ≥600 nm for the red fluorescent derivatives. Interferences to cellular autofluorescence could be excluded. All synthesized chalcone compounds could be used to visualize hH(3)R proteins in stably transfected HEK-293 cells using confocal laser scanning fluorescence microscopy. These novel fluorescent ligands possess high potential to be used as pharmacological tools for hH(3)R visualization in different tissues.
Synaptic long-term potentiation (LTP) at spinal neurons directly communicating pain-specific inputs from the periphery to the brain has been proposed to serve as a trigger for pain hypersensitivity in pathological states. Previous studies have functionally implicated the NMDA receptor-NO pathway and the downstream second messenger, cGMP, in these processes. Because cGMP can broadly influence diverse ion-channels, kinases, and phosphodiesterases, pre- as well as post-synaptically, the precise identity of cGMP targets mediating spinal LTP, their mechanisms of action, and their locus in the spinal circuitry are still unclear. Here, we found that Protein Kinase G1 (PKG-I) localized presynaptically in nociceptor terminals plays an essential role in the expression of spinal LTP. Using the Cre-lox P system, we generated nociceptor-specific knockout mice lacking PKG-I specifically in presynaptic terminals of nociceptors in the spinal cord, but not in post-synaptic neurons or elsewhere (SNS-PKG-I−/− mice). Patch clamp recordings showed that activity-induced LTP at identified synapses between nociceptors and spinal neurons projecting to the periaqueductal grey (PAG) was completely abolished in SNS-PKG-I−/− mice, although basal synaptic transmission was not affected. Analyses of synaptic failure rates and paired-pulse ratios indicated a role for presynaptic PKG-I in regulating the probability of neurotransmitter release. Inositol 1,4,5-triphosphate receptor 1 and myosin light chain kinase were recruited as key phosphorylation targets of presynaptic PKG-I in nociceptive neurons. Finally, behavioural analyses in vivo showed marked defects in SNS-PKG-I−/− mice in several models of activity-induced nociceptive hypersensitivity, and pharmacological studies identified a clear contribution of PKG-I expressed in spinal terminals of nociceptors. Our results thus indicate that presynaptic mechanisms involving an increase in release probability from nociceptors are operational in the expression of synaptic LTP on spinal-PAG projection neurons and that PKG-I localized in presynaptic nociceptor terminals plays an essential role in this process to regulate pain sensitivity.
Introduction: Despite the excellent anti-inflammatory and immunosuppressive action of glucocorticoids (GCs), their use for the treatment of inflammatory bowel disease (IBD) still carries significant risks in terms of frequently occurring severe side effects, such as the impairment of intestinal tissue repair. The recently-introduced selective glucocorticoid receptor (GR) agonists (SEGRAs) offer anti-inflammatory action comparable to that of common GCs, but with a reduced side effect profile.
Methods: The in vitro effects of the non-steroidal SEGRAs Compound A (CpdA) and ZK216348, were investigated in intestinal epithelial cells and compared to those of Dexamethasone (Dex). GR translocation was shown by immunfluorescence and Western blot analysis. Trans-repressive effects were studied by means of NF-κB/p65 activity and IL-8 levels, trans-activation potency by reporter gene assay. Flow cytometry was used to assess apoptosis of cells exposed to SEGRAs. The effects on IEC-6 and HaCaT cell restitution were determined using an in vitro wound healing model, cell proliferation by BrdU assay. In addition, influences on the TGF-β- or EGF/ERK1/2/MAPK-pathway were evaluated by reporter gene assay, Western blot and qPCR analysis.
Results: Dex, CpdA and ZK216348 were found to be functional GR agonists. In terms of trans-repression, CpdA and ZK216348 effectively inhibited NF-κB activity and IL-8 secretion, but showed less trans-activation potency. Furthermore, unlike SEGRAs, Dex caused a dose-dependent inhibition of cell restitution with no effect on cell proliferation. These differences in epithelial restitution were TGF-β-independent but Dex inhibited the EGF/ERK1/2/MAPK-pathway important for intestinal epithelial wound healing by induction of MKP-1 and Annexin-1 which was not affected by CpdA or ZK216348.
Conclusion: Collectively, our results indicate that, while their anti-inflammatory activity is comparable to Dex, SEGRAs show fewer side effects with respect to wound healing. The fact that SEGRAs did not have a similar effect on cell restitution might be due to a different modulation of EGF/ERK1/2 MAPK signalling.