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Elevating cytosolic Ca2+stimulates glucose uptake in skeletal muscle, but how Ca2+affects intracellular traffic of GLUT4 is unknown. In tissue, changes in Ca2+leading to contraction preclude analysis of the impact of individual, Ca2+-derived signals. In L6 muscle cells stably expressing GLUT4 myc, the Ca2+ionophore ionomycin raised cytosolic Ca2+and caused a gain in cell surface GLUT4 myc. Extra- and intracellular Ca2+chelators (EGTA, BAPTA-AM) reversed this response. Ionomycin activated calcium calmodulin kinase II (CaMKII), AMPK, and PKCs, but not Akt. Silencing CaMKIIδ or AMPKα1/α2 partly reduced the ionomycin-induced gain in surface GLUT4 myc, as did peptidic or small molecule inhibitors of CaMKII (CN21) and AMPK (Compound C). Compared with the conventional isoenzyme PKC inhibitor Gö6976, the conventional plus novel PKC inhibitor Gö6983 lowered the ionomycin-induced gain in cell surface GLUT4 myc. Ionomycin stimulated GLUT4 myc exocytosis and inhibited its endocytosis in live cells. siRNA-mediated knockdown of CaMKIIδ or AMPKα1/α2 partly reversed ionomycin-induced GLUT4 myc exocytosis but did not prevent its reduced endocytosis. Compared with Gö6976, Gö6983 markedly reversed the slowing of GLUT4 myc endocytosis triggered by ionomycin. In summary, rapid Ca2+influx into muscle cells accelerates GLUT4 myc exocytosis while slowing GLUT4 myc endocytosis. CaMKIIδ and AMPK stimulate GLUT4 myc exocytosis, whereas novel PKCs reduce endocytosis. These results identify how Ca2+-activated signals selectively regulate GLUT4 exocytosis and endocytosis in muscle cells.

Real-time fluorescence imaging of the activity of nucleases in living cells has been a difficult issue because of unintended degradation of the natural oligonucleotides by nontarget nucleases or interactions with other proteins. In this work, we demonstrate two types of highly selective, sensitive, and robust oligonucleotide probes for simultaneous imaging of the activities of two different nucleases in living cells. The probes consist of the desired substrate structure of the target nuclease and partially phosphorothioate modified backbone labeled with fluorophore and quencher for protection from undesired degradation by other nucleases and signal transduction. Upon reaction with the target nuclease, the initially fluorescence quenched probe was cleaved and the fluorophore was separated from the quencher, giving out strong fluorescence signals. Two nucleases, DNase I and Exonuclease III, were employed as model enzymes to demonstrate the concept. In vitro studies proved that the two probes could discriminate their respective target nucleases in serum with high resistance to other coexisting enzymes. The lower limits of detection for DNase I and Exonuclease III were observed to be 40 U/L and 2.0 U/L, respectively. By labeling the two probes with different fluorophores and quenchers, simultaneous visualization of the activities of DNases and 3' exonucleases was achieved in both HeLa cells and the suspension cells of Arabidopsis thaliana. The developed approaches may greatly facilitate the studies on the intracellular functions of the two nucleases and other related biological processes. The probe design concept may also be further adapted to the detection of many other nucleases.

To characterize how signaling by TLR ligands can be modulated by non-TLR ligands, murine RAW 264.7 cells were treated with LPS, IFN-γ, 2-methyl-thio-ATP (2MA), PGE2, and isoproterenol (ISO). Ligands were applied individually and in combination with LPS, for 1, 2, and 4 h, and transcriptional changes were measured using customized oligo arrays. We used nonadditive transcriptional responses to dual ligands (responses that were reproducibly greater or less than the expected additive responses) as a measure of pathway interaction. Our analysis suggests that cross-talk is limited

We examined the major patterns of changes in gene expression in mouse splenic B cells in response to stimulation with 33 single ligands for 0.5, 1, 2, and 4 h. We found that ligands known to directly induce or costimulate proliferation, namely, anti-IgM (anti-Ig), anti-CD40 (CD40L), LPS, and, to a lesser extent, IL-4 and CpG-oligodeoxynucleotide (CpG), induced significant expression changes in a large number of genes. The remaining 28 single ligands produced changes in relatively few genes, even though they elicited measurable elevations in intracellular Ca2+ and cAMP concentration and/or protein phosphorylation, including cytokines, chemokines, and other ligands that interact with G protein-coupled receptors. A detailed comparison of gene expression responses to anti-Ig, CD40L, LPS, IL-4, and CpG indicates that while many genes had similar temporal patterns of change in expression in response to these ligands, subsets of genes showed unique expression patterns in response to IL-4, anti-Ig, and CD40L.

Transcription factors of the Forkhead Box, class O (FOXO) family promote cell-cycle arrest and/or apoptosis in a variety of cell types. Mitogenic stimuli inactivate FOXO function by way of an evolutionarily conserved pathway involving the activation of phosphoinositide 3-kinase (PI3K) and its downstream effector, Akt. Although PI3K activation is required for B-lymphocyte proliferation, it is not known whether PI3K-dependent inactivation of FOXO proteins is important for cell-cycle progression and survival of these cells. Here, we show that B-cell receptor (BCR) engagement triggers PI3K-dependent phosphorylation and nuclear export of FOXO1. Furthermore, forced expression of PI3K-independent variants of FOXO1 or FOXO3a in activated B cells induces partial arrest in G1 phase of the cell cycle and increases apoptosis. These findings establish that FOXO inactivation is a functionally important consequence of PI3K signaling in primary B cells.

Enzymes containing 3'-5' exonuclease activities play vital roles in maintaining genome stability. Though a wide variety of methods have been developed for detection of these enzymes, few of them can be directly applied for in situ and real-time monitoring of the secretion of these active substances by living cells. Taking advantages of the free 3'-end of stacked guanine-quenched photoinduced electron transfer fluorescent probes, here we demonstrate a novel assay capable of in situ and real-time monitoring of the 3'-5' exonucleases secreted by living cells. The detection limit of the new method achieved as low as 0.04 U/mL, allowing direct monitoring of the target enzymes in an extracellular environment without preconcentration steps. False positive signals caused by other nonspecific enzymes were easily ruled out by the use of a control probe with the 3'-end modified with exonuclease-resistant phosphorothioate guanines. Using Alexa Fluor 488 as the fluorophore, the probe is adaptable to a wide range of pH conditions. The approach was successfully applied for in situ, real-time monitoring of the 3'-5' exonucleases secreted by suspension cells of Arabidopsis thaliana. It also holds great potential for in situ and real-time detection of many other DNA end-processing enzymes produced by other types of cells.

The activation of macrophages through Toll-like receptor (TLR) pathways leads to the production of a broad array of cytokines and mediators that coordinate the immune response. The inflammatory potential of this response can be reduced by compounds, such as prostaglandin E(2), that induce the production of cyclic adenosine monophosphate (cAMP). Through experiments with cAMP analogs and multigene RNA interference (RNAi), we showed that key anti-inflammatory effects of cAMP were mediated specifically by cAMP-dependent protein kinase (PKA). Selective inhibitors of PKA anchoring, time-lapse microscopy, and RNAi screening suggested that differential mechanisms of PKA action existed. We showed a specific role for A kinase-anchoring protein 95 in suppressing the expression of the gene encoding tumor necrosis factor-alpha, which involved phosphorylation of p105 (also known as Nfkb1) by PKA at a site adjacent to the region targeted by inhibitor of nuclear factor kappaB kinases. These data suggest that crosstalk between the TLR4 and cAMP pathways in macrophages can be coordinated through PKA-dependent scaffolds that localize specific pools of the kinase to distinct substrates.

Background Effective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems. Results We have developed a flexible platform using RNA polymerase II promoter-driven expression of microRNA-like short hairpin RNAs which permits robust depletion of multiple target genes from a single transcript. Recombination-based subcloning permits expression of multi-shRNA transcripts from a comprehensive range of plasmid or viral vectors. Retroviral delivery of transcripts targeting isoforms of cAMP-dependent protein kinase in the RAW264.7 murine macrophage cell line emphasizes the utility of this approach and provides insight to cAMP-dependent transcription. Conclusion We demonstrate functional consequences of depleting multiple endogenous target genes using miR-shRNAs, and highlight the versatility of the described vector platform for multiple target gene knockdown in mammalian cells.