Death receptor-induced signaling pathways are differentially regulated by gamma interferon upstream of caspase 8 processing

FasL and gamma interferon (IFN-gamma) are produced by activated T cells and NK cells and synergistically induce apoptosis. Although both cytokines can also elicit proinflammatory responses, a possible cross talk of these ligands with respect to nonapoptotic signaling has been poorly addressed. Here, we show that IFN-gamma sensitizes KB cells for apoptosis induction by facilitating death-inducing signaling complex (DISC)-mediated caspase 8 processing. Moreover, after protection against death receptor-induced apoptosis by caspase inhibition or Bcl2 overexpression, IFN-gamma also sensitized for Fas- and TRAIL death receptor-mediated NF-kappaB activation leading to synergistic upregulation of a variety of proinflammatory genes. In contrast, Fas-mediated activation of JNK, p38, and p42/44 occurred essentially independent from IFN-gamma sensitization, indicating that the apoptosis- and NF-kappaB-related FasL-IFN-gamma cross talk was not due to a simple global enhancement of Fas signaling. Overexpression of FLIP(L) and FLIP(S) inhibited Fas- as well as TRAIL-mediated NF-kappaB activation and apoptosis induction in IFN-gamma-primed cells suggesting that both responses are coregulated at the level of the DISC.     

Cytotoxicity of myeloperoxidase/nitrite-oxidized low-density lipoprotein toward endothelial cells is due to a high 7beta-hydroxycholesterol to 7-ketocholesterol ratio

Oxygenated cholesterols (oxysterols) formed during oxidation of low-density lipoprotein (LDL) are associated with endothelial dysfunction and atherogenesis. We compared the profile of oxysterols in modified human LDL obtained on reaction with myeloperoxidase/H2O2 plus nitrite (MPO/H2O2/nitrite-oxLDL) with that on Cu2+ -catalyzed oxidation. The 7beta-hydroxycholesterol/7-ketocholesterol ratio was markedly higher in MPO/H2O2/nitrite-oxLDL than in Cu2+ -oxidized LDL (7.9 +/- 3.0 versus 0.94 +/- 0.10). Like MPO/H2O2/nitrite-oxLDL, 7beta-hydroxycholesterol was cytotoxic toward endothelial cells through eliciting oxidative stress. Cytotoxicity was accompanied by DNA fragmentation and was prevented by the NADPH oxidase inhibitor apocynin, suggesting stimulation of NADPH oxidase-mediated O2-* formation. 7-Ketocholesterol was only cytotoxic when added alone, whereas a 1:1-mixture with 7beta-hydroxycholesterol surprisingly was noncytotoxic. We conclude from our data that (i) 7beta-hydroxycholesterol is a pivotal cytotoxic component of oxidized LDL, (ii) 7-ketocholesterol protects against 7beta-hydroxycholesterol in oxysterol mixtures or oxLDL, (iii) the 7beta-hydroxycholesterol/7-ketocholesterol ratio is a crucial determinant for cytotoxicity of oxidized LDL species and oxysterol mixtures, and (iv) the low share of 7-ketocholesterol explains the higher cytotoxicity of MPO/H2O2/nitrite-oxLDL than other forms of oxidized LDL. The dietary polyphenol (-)-epicatechin inhibited not only formation but also cytotoxic actions of both oxLDL and oxysterols.     

An application programming interface for CellNetAnalyzer

CellNetAnalyzer (CNA) is a MATLAB toolbox providing computational methods for studying structure and function of metabolic and cellular signaling networks. In order to allow non-experts to use these methods easily, CNA provides GUI-based interactive network maps as a means of parameter input and result visualization. However, with the availability of high-throughput data, there is a need to make CNA's functionality also accessible in batch mode for automatic data processing. Furthermore, as some algorithms of CNA are of general relevance for network analysis it would be desirable if they could be called as sub-routines by other applications. For this purpose, we developed an API (application programming interface) for CNA allowing users (i) to access the content of network models in CNA, (ii) to use CNA's network analysis capabilities independent of the GUI, and (iii) to interact with the GUI to facilitate the development of graphical plugins.Here we describe the organization of network projects in CNA and the application of the new API functions to these projects. This includes the creation of network projects from scratch, loading and saving of projects and scenarios, and the application of the actual analysis methods. Furthermore, API functions for the import/export of metabolic models in SBML format and for accessing the GUI are described. Lastly, two example applications demonstrate the use and versatile applicability of CNA's API. CNA is freely available for academic use and can be downloaded from http://www.mpi-magdeburg.mpg.de/projects/cna/cna.html.     

Cutting edge: microRNA regulation of macrophage fusion into multinucleated giant cells

Cellular fusion of macrophages into multinucleated giant cells is a distinguishing feature of the granulomatous response to inflammation, infection, and foreign bodies (Kawai and Akira. 2011. Immunity 34: 637-650). We observed a marked increase in fusion of macrophages genetically deficient in Dicer, an enzyme required for canonical microRNA (miRNA) biogenesis. Gene expression profiling of miRNA-deficient macrophages revealed an upregulation of the IL-4-responsive fusion protein Tm7sf4, and analyses identified miR-7a-1 as a negative regulator of macrophage fusion, functioning by directly targeting Tm7sf4 mRNA. miR-7a-1 is itself an IL-4-responsive gene in macrophages, suggesting feedback control of cellular fusion. Collectively, these data indicate that miR-7a-1 functions to regulate IL-4-directed multinucleated giant cell formation.     

LINE-1 retrotransposition events affect endothelial proliferation and migration

Long interspersed nuclear element-1 (LINE-1, L1) is a retrotransposon which affects the human genome by a variety of mechanisms. While LINE-1 expression is suppressed in the most somatic human cells, LINE-1 elements are activated in human cancer. Recently, high accumulation of LINE-1-encoded ORF1p and ORF2p in endothelial cells of mature human blood vessels was described. Here, we demonstrate that LINE-1 de novo retrotransposition events lead to a reduction of endothelial cell proliferation and migration in a porcine aortic endothelial (PAE) cell model. Cell cycle studies show a G0/G1 arrest in PAE cells harboring LINE-1 de novo retrotransposition events. Remarkably, in in situ analysis LINE-1-encoded ORF2p was not detectable in tumor blood vessels of different human organs while vascular endothelial cells of corresponding normal organs strongly expressed LINE-1 ORF2p. Quantitative RT-PCR analysis revealed that LINE-1 de novo retrotransposition influences selectively the expression of some angiogenic factors such as VEGF and Tie-2. Thus, our data suggest that LINE-1 de novo retrotransposition events might suppress angiogenesis and tumor vascularisation by reducing the angiogenic capacity of vascular endothelial cells.     

Control of plant growth by nitrogen and phosphorus in mesotrophic fens

A fertilization experiment was carried out in 3 mesotrophic fens to investigate whether plant growth in these systems is controlled by the availability of N, P or K. The fens are located in an area with high N inputs from precipitation. They are annually mown in the summer to prevent succession to woodland. Above-ground plant biomass increased significantly upon N fertilization in the two mid-succession fens studied. In the late-succession fen that had been mown for at least 60 years, however, plant biomass increased significantly upon P fertilization. The mowing regime depletes the P pool in the soil, while it keeps N inputs and outputs in balance. A long-term shift occurs from limitation of plant production by N toward limitation by P. Hence, mowing is a suitable management tool to conserve the mesothrophic character of the fens.     

Automated scanning for phylogenetically informative transposed elements in rodents

Transposed elements constitute an attractive, useful source of phylogenetic markers to elucidate the evolutionary history of their hosts. Frequent and successive amplifications over evolutionary time are important requirements for utilizing their presence or absence as landmarks of evolution. Although transposed elements are well distributed in rodent taxa, the generally high degree of genomic sequence divergence among species complicates our access to presence/absence data. With this in mind we developed a novel, high-throughput computational strategy, called CPAL (Conserved Presence/Absence Locus-finder), to identify genome-wide distributed, phylogenetically informative transposed elements flanked by highly conserved regions. From a total of 232 extracted chromosomal mouse loci we randomly selected 14 of these plus 2 others from previous test screens and attempted to amplify them via PCR in representative rodent species. All loci were amplifiable and ultimately contributed 31 phylogenetically informative markers distributed throughout the major groups of Rodentia.