Question: NFAT4 activation requires IP3R-mediated Ca2+ mobilization.

Title: Control of NFAT Isoform Activation and NFAT-Dependent Gene Expression through Two Coincident and Spatially Segregated Intracellular Ca2+ Signals Content: Excitation-transcription coupling, linking stimulation at the cell surface to changes in nuclear gene expression, is conserved throughout eukaryotes. How closely related coexpressed transcription factors are differentially activated remains unclear. Here, we show that two Ca2+-dependent transcription factor isoforms, NFAT1 and NFAT4, require distinct sub-cellular InsP3 and Ca2+ signals for physiologically sustained activation. NFAT1 is stimulated by sub-plasmalemmal Ca2+ microdomains, whereas NFAT4 additionally requires Ca2+ mobilization from the inner nuclear envelope by nuclear InsP3 receptors. NFAT1 is rephosphorylated (deactivated) more slowly than NFAT4 in both cytoplasm and nucleus, enabling a more prolonged activation phase. Oscillations in cytoplasmic Ca2+, long considered the physiological form of Ca2+ signaling, play no role in activating either NFAT protein. Instead, effective sustained physiological activation of NFAT4 is tightly linked to oscillations in nuclear Ca2+. Our results show how gene expression can be controlled by coincident yet geographically distinct Ca2+ signals, generated by a freely diffusible InsP3 message.

Title: Junctophilin-4, a component of the endoplasmic reticulum-plasma membrane junctions, regulates Ca2+ dynamics in T cells. Content: Orai1 and stromal interaction molecule 1 (STIM1) mediate store-operated Ca(2+) entry (SOCE) in immune cells. STIM1, an endoplasmic reticulum (ER) Ca(2+) sensor, detects store depletion and interacts with plasma membrane (PM)-resident Orai1 channels at the ER-PM junctions. However, the molecular composition of these junctions in T cells remains poorly understood. Here, we show that junctophilin-4 (JP4), a member of junctional proteins in excitable cells, is expressed in T cells and localized at the ER-PM junctions to regulate Ca(2+) signaling. Silencing or genetic manipulation of JP4 decreased ER Ca(2+) content and SOCE in T cells, impaired activation of the nuclear factor of activated T cells (NFAT) and extracellular signaling-related kinase (ERK) signaling pathways, and diminished expression of activation markers and cytokines. Mechanistically, JP4 directly interacted with STIM1 via its cytoplasmic domain and facilitated its recruitment into the junctions. Accordingly, expression of this cytoplasmic fragment of JP4 inhibited SOCE. Furthermore, JP4 also formed a complex with junctate, a Ca(2+)-sensing ER-resident protein, previously shown to mediate STIM1 recruitment into the junctions. We propose that the junctate-JP4 complex located at the junctions cooperatively interacts with STIM1 to maintain ER Ca(2+) homeostasis and mediate SOCE in T cells.

Title: A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function Content: Antigen stimulation of immune cells triggers Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, promoting the immune response to pathogens by activating the transcription factor NFAT. We have previously shown that cells from patients with one form of hereditary severe combined immune deficiency (SCID) syndrome are defective in store-operated Ca2+ entry and CRAC channel function. Here we identify the genetic defect in these patients, using a combination of two unbiased genome-wide approaches: a modified linkage analysis with single-nucleotide polymorphism arrays, and a Drosophila RNA interference screen designed to identify regulators of store-operated Ca2+ entry and NFAT nuclear import. Both approaches converged on a novel protein that we call Orai1, which contains four putative transmembrane segments. The SCID patients are homozygous for a single missense mutation in ORAI1, and expression of wild-type Orai1 in SCID T cells restores store-operated Ca2+ influx and the CRAC current (ICRAC). We propose that Orai1 is an essential component or regulator of the CRAC channel complex.

Title: Frontrunners of T cell activation: Initial, localized Ca2+ signals mediated by NAADP and the type 1 ryanodine receptor Content: The activation of T cells is the fundamental on switch for the adaptive immune system. Ca2+ signaling is essential for T cell activation and starts as initial, short-lived, localized Ca2+ signals. The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) forms rapidly upon T cell activation and stimulates early Ca2+ signaling. We developed a high-resolution imaging technique using multiple fluorescent Ca2+ indicator dyes to characterize these early signaling events and investigate the channels involved in NAADP-dependent Ca2+ signals. In the first seconds of activation of either primary murine T cells or human Jurkat cells with beads coated with an antibody against CD3, we detected Ca2+ signals with diameters close to the limit of detection and that were close to the activation site at the plasma membrane. In Jurkat cells in which the ryanodine receptor (RyR) was knocked down or in primary T cells from RyR1−/− mice, either these early Ca2+ signals were not detected or the number of signals was markedly reduced. Local Ca2+ signals observed within 20 ms upon microinjection of Jurkat cells with NAADP were also sensitive to RyR knockdown. In contrast, TRPM2 (transient receptor potential channel, subtype melastatin 2), a potential NAADP target channel, was not required for the formation of initial Ca2+ signals in primary T cells. Thus, through our high-resolution imaging method, we characterized early Ca2+ release events in T cells and obtained evidence for the involvement of RyR and NAADP in such signals.

Title: Local Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels stimulates production of an intracellular messenger and an intercellular pro-inflammatory signal. Content: Ca2+ entry through store-operated Ca2+ channels drives the production of the pro-inflammatory molecule leukotriene C4 (LTC4) from mast cells through a pathway involving Ca2+-dependent protein kinase C, mitogen-activated protein kinases ERK1/2, phospholipase A2, and 5-lipoxygenase. Here we examine whether local Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane stimulates this signaling pathway. Manipulating the amplitude and spatial extent of Ca2+ entry by altering chemical and electrical gradients for Ca2+ influx or changing the Ca2+ buffering of the cytoplasm all impacted on protein kinase C and ERK activation, generation of arachidonic acid and LTC4 secretion, with little change in the bulk cytoplasmic Ca2+ rise. Similar bulk cytoplasmic Ca2+ concentrations were achieved when CRAC channels were activated in 0.25 mm external Ca2+ versus 2 mm Ca2+ and 100 nm La3+, an inhibitor of CRAC channels. However, despite similar bulk cytoplasmic Ca2+, protein kinase C activation and LTC4 secretion were larger in 2 mm Ca2+ and La3+ than in 0.25 mm Ca2+, consistent with the central involvement of a subplasmalemmal Ca2+ rise. The nonreceptor tyrosine kinase Syk coupled CRAC channel opening to protein kinase C and ERK activation. Recombinant TRPC3 channels also activated protein kinase C, suggesting that subplasmalemmal Ca2+ rather than a microdomain exclusive to CRAC channels is the trigger. Hence a subplasmalemmal Ca2+ increase in mast cells is highly versatile in that it triggers cytoplasmic responses through generation of intracellular messengers as well as long distance changes through increased secretion of paracrine signals.

Title: Similar activation of signal transduction pathways by the herpesvirus-encoded chemokine receptors US28 and ORF74. Content: The virally encoded chemokine receptors US28 from human cytomegalovirus and ORF74 from human herpesvirus 8 are both constitutively active. We show that both receptors constitutively activate the transcription factors nuclear factor of activated T cells (NFAT) and cAMP response element binding protein (CREB) and that both pathways are modulated by their respective endogenous receptor ligands. By addition of specific pathway modulators against the G protein subunit Galphai, phospholipase C, protein kinase C, calcineurin, p38 MAP kinase, and MEK1, we find that the constitutive and ligand-dependent inductions are mediated by multiple yet similar pathways in both receptors. The NFAT and CREB transcription factors and their upstream activators are known inducers of host and virally encoded genes. We propose that the activity of these virally encoded chemokine receptors coordinates host and potentially viral gene expression similarly. As ORF74 is a known inducer of neoplasia, these findings may have important implications for cytomegalovirus-associated pathogenicity.

Title: Inhibition of receptor-mediated calcium influx in T cells by unsaturated non-esterified fatty acids. Content: The effect of omega-3, omega-6 and omega-9 unsaturated fatty acids (UFAs) on receptor-mediated Ca2+ entry was investigated in a T-cell line (JURKAT) by using anti-CD3 antibodies (OKT3) to induce intracellular Ca2+ [( Ca2+]i) increase and Ca2+ influx. All the UFAs, as well as Ni2+ ions and 12-O-tetradecanoylphorbol 13-acetate, decreased the OKT3-induced sustained [Ca2+]i increase to basal levels. Although non-esterified fatty acids activate protein kinase C (PKC) [McPhail, Clayton & Snyderman (1984) Science 224, 622-624; Murakami, Chan & Routtenberg (1986) J. Biol. Chem. 261, 15424-15429], studies using H-7 and analysis of the PKC-dependent phosphorylation of 19 and 80 kDa marker substrates ruled out the involvement of PKC in UFA-induced inhibition of Ca2+ entry. Flow-cytometry analysis showed that UFAs do not interfere with antibody-receptor binding. BSA (0.2%, w/v) reversed the effect of UFAs after these fatty acids have decreased the OKT3-induced [Ca2+]i increase to basal levels. The relevance of these findings and possible mechanisms for inhibition by UFAs of receptor-mediated Ca2+ influx were discussed.

Title: Ca2+-permeable AMPA receptors regulate growth of human glioblastoma via Akt activation. Content: Evidence has been accumulated that glioblastoma cells release and exploit glutamate for proliferation and migration by autocrine or paracrine loops through Ca2+-permeable AMPA-type glutamate receptors. Here, we show that Ca2+ signaling mediated by AMPA receptor regulates the growth and motility of glioblastoma cells via activation of Akt. Ca2+ supplied through Ca2+-permeable AMPA receptor phosphorylated Akt at Ser-473, thereby facilitating proliferation and mobility. A dominant-negative form of Akt inhibited cell proliferation and migration accelerated by overexpression of Ca2+-permeable AMPA receptor. In contrast, introduction of a constitutively active form of Akt rescued tumor cells from apoptosis induced by the conversion of Ca2+-permeable AMPA receptor to Ca2+-impermeable receptors by the delivery of GluR2 cDNA. Therefore, Akt functions as downstream effectors for Ca2+-signaling mediated by AMPA receptor in glioblastoma cells. The activation of the glutamate-AMPA receptor-Akt pathway may contribute to the high degree of anaplasia and invasive growth of human glioblastoma. This novel pathway might give an alternative therapeutic target.

Title: UCP1-independent signaling involving SERCA2b-mediated calcium cycling regulates beige fat thermogenesis and systemic glucose homeostasis Content: Uncoupling protein 1 (UCP1) plays a central role in nonshivering thermogenesis in brown fat; however, its role in beige fat remains unclear. Here we report a robust UCP1-independent thermogenic mechanism in beige fat that involves enhanced ATP-dependent Ca2+ cycling by sarco/endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) and ryanodine receptor 2 (RyR2). Inhibition of SERCA2b impairs UCP1-independent beige fat thermogenesis in humans and mice as well as in pigs, a species that lacks a functional UCP1 protein. Conversely, enhanced Ca2+ cycling by activation of α1- and/or β3-adrenergic receptors or the SERCA2b-RyR2 pathway stimulates UCP1-independent thermogenesis in beige adipocytes. In the absence of UCP1, beige fat dynamically expends glucose through enhanced glycolysis, tricarboxylic acid metabolism and pyruvate dehydrogenase activity for ATP-dependent thermogenesis through the SERCA2b pathway; beige fat thereby functions as a 'glucose sink' and improves glucose tolerance independently of body weight loss. Our study uncovers a noncanonical thermogenic mechanism through which beige fat controls whole-body energy homeostasis via Ca2+ cycling.

Title: Light Activation of Channelrhodopsin-2 in Excitable Cells of Caenorhabditis elegans Triggers Rapid Behavioral Responses Content: For studying the function of specific neurons in their native circuitry, it is desired to precisely control their activity. This often requires dissection to allow accurate electrical stimulation or neurotransmitter application , and it is thus inherently difficult in live animals, especially in small model organisms. Here, we employed channelrhodopsin-2 (ChR2), a directly light-gated cation channel from the green alga Chlamydomonas reinhardtii, in excitable cells of the nematode Caenorhabditis elegans, to trigger specific behaviors, simply by illumination. Channelrhodopsins are 7-transmembrane-helix proteins that resemble the light-driven proton pump bacteriorhodopsin , and they also utilize the chromophore all-trans retinal, but to open an intrinsic cation pore. In muscle cells, light-activated ChR2 evoked strong, simultaneous contractions, which were reduced in the background of mutated L-type, voltage-gated Ca2+-channels (VGCCs) and ryanodine receptors (RyRs). Electrophysiological analysis demonstrated rapid inward currents that persisted as long as the illumination. When ChR2 was expressed in mechanosensory neurons, light evoked withdrawal behaviors that are normally elicited by mechanical stimulation. Furthermore, ChR2 enabled activity of these neurons in mutants lacking the MEC-4/MEC-10 mechanosensory ion channel . Thus, specific neurons or muscles expressing ChR2 can be quickly and reversibly activated by light in live and behaving, as well as dissected, animals.

Title: Genome-wide RNAi screen of Ca(2+) influx identifies genes that regulate Ca(2+) release-activated Ca(2+) channel activity. Content: Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca(2+) influx and Ca(2+) release-activated Ca(2+) (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca(2+) influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca(2+) entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca(2+) pump sarco-/ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca(2+) depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.

Title: Nef is physically recruited into the immunological synapse and potentiates T cell activation early after TCR engagement. Content: The HIV-1 protein Nef enhances viral pathogenicity and accelerates disease progression in vivo. Nef potentiates T cell activation by an unknown mechanism, probably by optimizing the intracellular environment for HIV replication. Using a new T cell reporter system, we have found that Nef more than doubles the number of cells expressing the transcription factors NF-kappaB and NFAT after TCR stimulation. This Nef-induced priming of TCR signaling pathways occurred independently of calcium signaling and involved a very proximal step before protein kinase C activation. Engagement of the TCR by MHC-bound Ag triggers the formation of the immunological synapse by recruiting detergent-resistant membrane microdomains, termed lipid rafts. Approximately 5-10% of the total cellular pool of Nef is localized within lipid rafts. Using confocal and real-time microscopy, we found that Nef in lipid rafts was recruited into the immunological synapse within minutes after Ab engagement of the TCR/CD3 and CD28 receptors. This recruitment was dependent on the N-terminal domain of Nef encompassing its myristoylation. Nef did not increase the number of cell surface lipid rafts or immunological synapses. Recently, studies have shown a specific interaction of Nef with an active subpopulation of p21-activated kinase-2 found only in the lipid rafts. Thus, the corecruitment of Nef and key cellular partners (e.g., activated p21-activated kinase-2) into the immunological synapse may underlie the increased frequency of cells expressing transcriptionally active forms of NF-kappaB and NFAT and the resultant changes in T cell activation.

Title: Coordinated oscillations in cortical actin and Ca2+ correlate with cycles of vesicle secretion Content: The actin cortex both facilitates and hinders the exocytosis of secretory granules. How cells consolidate these two opposing roles was not well understood. Here we show that antigen activation of mast cells induces oscillations in Ca(2+) and PtdIns(4,5)P(2) lipid levels that in turn drive cyclic recruitment of N-WASP and cortical actin level oscillations. Experimental and computational analysis argues that vesicle fusion correlates with the observed actin and Ca(2+) level oscillations. A vesicle secretion cycle starts with the capture of vesicles by actin when cortical F-actin levels are high, followed by vesicle passage through the cortex when F-actin levels are low, and vesicle fusion with the plasma membrane when Ca(2+) levels subsequently increase. Thus, cells employ oscillating levels of Ca(2+), PtdIns(4,5)P(2) and cortical F-actin to increase secretion efficiency, explaining how the actin cortex can function as a carrier as well as barrier for vesicle secretion.

Title: Calicum microdomains form within neutrophils at the neutrophil–tumor cell synapse: role in antibody-dependent target cell apoptosis Content: Ca2+ messages are broadly important in cellular signal transduction. In immune cells, Ca2+ signaling is an essential step in many forms of activation. Neutrophil-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) is one form of leukocyte activation that plays an important role in tumor cell killing in vitro and in patient care. Using fluorescence methodologies, we found that neutrophils exhibit Ca2+ signals during ADCC directed against breast fibrosarcoma cells. Importantly, these signals were localized to Ca2+ microdomains at the neutrophil-to-tumor cell interface where they display dynamic features such as movement, fusion, and fission. These signals were blocked by the intracellular Ca2+ buffer BAPTA. At the neutrophil–tumor cell synapse, the neutrophil’s cytoplasm was enriched in STIM1, a crucial mediator of Ca2+ signaling, whereas the Ca2+-binding proteins calbindin and parvalbumin were not affected. Our findings suggest that Ca2+ microdomains are due to an active signaling process. As Ca2+ signals within neutrophils were necessary for specific tumor cell apoptosis, a central role of microdomains in leukocyte-mediated tumor cell destruction is indicated.

Title: Differential requirement for CARMA1 in agonist-selected T-cell development. Content: Caspase recruitment domain-containing membrane-associated guanylate kinase protein-1 (CARMA1) is a critical component of the NF-kappaB signaling cascade mediated by TCR engagement. In addition to activation of naïve T cells, TCR signaling is important for the development of agonist-selected T-cell subsets such as Treg, NKT cells, and CD8-alpha alpha T cells. However, little is known about the role of CARMA1 in the development of these lineages. Here we show that CARMA1-deficient mice (CARMA1(-/-)) have altered populations of specific subsets of agonist-selected T cells. Specifically, CARMA1(-/-) mice have impaired natural and adaptive Treg development, whereas NKT cell numbers are normal compared with wild-type mice. Interestingly, CD8-alpha alpha T cells, which may also be able to develop through an extrathymic selection pathway, are enriched in the gut of CARMA1(-/-) mice, whereas memory-phenotype CD4(+) T cells (CD62L(low)/CD44(high)) are present at reduced numbers in the periphery. These results indicate that CARMA1 is essential for Treg development, but is not necessary for the development of other agonist-selected T-cell subsets. Overall, these data reveal an important but differential role for CARMA1-mediated TCR signaling in T-cell development.

Title: Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury. Content: Besides synthesizing nitric oxide (NO), purified neuronal NO synthase (nNOS) can produce superoxide (.O2-) at lower L-Arg concentrations. By using electron paramagnetic resonance spin-trapping techniques, we monitored NO and .O2- formation in nNOS-transfected human kidney 293 cells. In control transfected cells, the Ca2+ ionophore A23187 triggered NO generation but no .O2- was seen. With cells in L-Arg-free medium, we observed .O2- formation that increased as the cytosolic L-Arg levels decreased, while NO generation declined. .O2- formation was virtually abolished by the specific NOS blocker, N-nitro-L-arginine methyl ester (L-NAME). Nitrotyrosine, a specific nitration product of peroxynitrite, accumulated in L-Arg-depleted cells but not in control cells. Activation by A23187 was cytotoxic to L-Arg-depleted, but not to control cells, with marked lactate dehydrogenase release. The cytotoxicity was largely prevented by either superoxide dismutase or L-NAME. Thus, with reduced L-Arg availability NOS elicits cytotoxicity by generating .O2- and NO that interact to form the potent oxidant peroxynitrite. Regulating arginine levels may provide a therapeutic approach to disorders involving .O2-/NO-mediated cellular injury.

Title: Key role of Ubc5 and lysine-63 polyubiquitination in viral activation of IRF3. Content: The mitochondrial antiviral signaling protein (MAVS; also known as IPS-1, VISA, and CARDIF) is essential for innate immune response against RNA viruses. MAVS transduces signals from the cytosolic RIG-I-like receptors, which bind to viral RNAs. But how MAVS activates downstream transcription factors such as IRF3 to induce type-I interferons is not well understood. We have established a cell-free system in which mitochondria derived from virus-infected cells activate IRF3 in the cytosol. Fractionation of the cytosol led to the identification of Ubc5 as a ubiquitin-conjugating enzyme (E2) required for IRF3 activation. Using an inducible RNAi strategy, we demonstrate that catalytically active Ubc5 is required for IRF3 activation by viral infection. The activation of IRF3 also requires two ubiquitin-binding domains of NEMO. Furthermore, we show that replacement of endogenous ubiquitin with its K63R mutant abolishes viral activation of IRF3, demonstrating that K63 polyubiquitination plays a key role in IRF3 activation.

Title: Decoding of Cytoplasmic Ca2+ Oscillations through the Spatial Signature Drives Gene Expression Content: Cytoplasmic Ca(2+) oscillations are a universal signaling mode that activates numerous cellular responses [1, 2]. Oscillations are considered the physiological mechanism of Ca(2+) signaling because they occur at low levels of stimulus intensity [3]. Ca(2+) oscillations are proposed to convey information in their amplitude and frequency, leading to activation of specific downstream targets [4-6]. Here, we report that the spatial Ca(2+) gradient within the oscillation is key. Ca(2+) oscillations in mast cells evoked over a range of agonist concentrations in the presence of external Ca(2+) were indistinguishable from those in the absence of Ca(2+) when plasmalemmal Ca(2+) extrusion was suppressed. Nevertheless, only oscillations with accompanying Ca(2+) entry through store-operated CRAC channels triggered gene expression. Increased cytoplasmic Ca(2+) buffering prevented oscillations but not gene activation. Local Ca(2+) influx and not global Ca(2+) oscillations therefore drives gene expression at physiological levels of stimulation. Rather than serving to maintain Ca(2+) oscillations by replenishing stores, we suggest that the role of oscillations might be to activate CRAC channels, thereby ensuring the generation of spatially restricted physiological Ca(2+) signals driving gene activation. Furthermore, we show that the spatial profile of a Ca(2+) oscillation provides a novel mechanism whereby a pleiotropic messenger specifically activates gene expression.

Title: Vertebrate rod photoreceptors express both BK and IK calcium-activated potassium channels, but only BK channels are involved in receptor potential regulation. Content: In salamander rods, Ca(2+)-activated K(+) current (I(KCa)) provides an effective "clamp" of the dark membrane potential to its normal resting level. By a combination of electrophysiological, pharmacological, and immunohistochemical approaches, we show that salamander rods functionally express large-conductance Ca(2+)- and voltage-dependent potassium (BK) channel and intermediate-conductance Ca(2+)-dependent potassium (IK) channel, but not small-conductance Ca(2+)-dependent potassium channel (SK) subtypes. Application of 100 nM iberiotoxin and 100 nM clotrimazole reduced net I(KCa) to 36% and 63%, respectively, whereas the current was unaffected by application of 1 microM apamin. Consistently, anti- SK1, -SK2, and -SK3 antibodies were unable to stain rod photoreceptors, whereas both anti-BK and -SK4/ IK1 antibodies heavily stained the ellipsoid region of the inner segments of the rods. Moreover, by using current-clamp experiments, it was clearly seen that the strong clamping effect of the total I(KCa) was lost when IbTx, but not CLTZ, was applied to the bath. This behavior strongly suggests that of BK and IK channels, only the former are responsible for the clamping effect on the photoreceptor membrane potential.

Title: PI3K-Akt-mTORC1-S6K1/2 axis controls Th17 differentiation by regulating Gfi1 expression and nuclear translocation of RORγ. Content: The PI3K-Akt-mTORC1 axis contributes to the activation, survival, and proliferation of CD4(+) T cells upon stimulation through TCR and CD28. Here, we demonstrate that the suppression of this axis by deletion of p85α or PI3K/mTORC1 inhibitors as well as T cell-specific deletion of raptor, an essential component of mTORC1, impairs Th17 differentiation in vitro and in vivo in a S6K1/2-dependent fashion. Inhibition of PI3K-Akt-mTORC1-S6K1 axis impairs the downregulation of Gfi1, a negative regulator of Th17 differentiation. Furthermore, we demonstrate that S6K2, a nuclear counterpart of S6K1, is induced by the PI3K-Akt-mTORC1 axis, binds RORγ, and carries RORγ to the nucleus. These results point toward a pivotal role of PI3K-Akt-mTORC1-S6K1/2 axis in Th17 differentiation.

Title: Epigenetic regulation of NANOG by miR-302 cluster-MBD2 completes induced pluripotent stem cell reprogramming. Content: While most somatic cells undergoing induced pluripotent stem (iPS) cell reprogramming with Yamanaka factors accumulate at stable partially reprogrammed stages, the molecular mechanisms required to achieve full reprogramming are unknown. MicroRNAs (miRNAs) fine-tune mRNA translation and are implicated in reprogramming, but miRNA functional targets critical for complete iPS cell reprogramming remain elusive. We identified methyl-DNA binding domain protein 2 (MBD2) as an epigenetic suppressor, blocking full reprogramming of somatic to iPS cells through direct binding to NANOG promoter elements preventing transcriptional activation. When we overexpressed miR-302 cluster we observed a significant increase in conversion of partial to fully reprogrammed iPS cells by suppressing MBD2 expression, thereby increasing NANOG expression. Thus, expression of exogenous miR-302 cluster (without miR-367) is efficient in attaining a fully reprogrammed iPS state in partially reprogrammed cells by relieving MBD2-mediated inhibition of NANOG expression. Our studies provide a direct molecular mechanism involved in generating complete human iPS cell reprogramming to study disease pathogenesis, drug screening, and for potential cell-based therapies.

Title: 7α, 25-dihydroxycholesterol-mediated activation of EBI2 in immune regulation and diseases Content: EBI2, aka GPR183, is a G-couple receptor originally identified in 1993 as one of main genes induced in Burkitt's lymphoma cell line BL41 by Epstein-Barr virus (EBV) infection. After it was reported in 2009 that the receptor played a key role in regulating B cell migration and responses, we initiated an effort in looking for its endogenous ligand. In 2011 we and another group reported the identification of 7α, 25-dihydroxyxcholesterol (7α, 25-OHC), an oxysterol, as the likely physiological ligand of EBI2. A few subsequently published studies further elucidated how 7α, 25-OHC bound to EBI2, and how a gradient of 7α, 25-OHC could be generated in vivo and regulated migration, activation, and functions of B cells, T cells, dendritic cells (DCs), monocytes/macrophages, and astrocytes. The identification of 7α, 25-OHC as a G protein-coupled receptor ligand revealed a previously unknown signaling system of oxysterols, a class of molecules which exert profound biological functions. Dysregulation of the synthesis or functions of these molecules is believed to contribute to inflammation and autoimmune diseases, cardiovascular diseases, neurodegenerative diseases, cancer as well as metabolic diseases such as diabetes, obesity, and dyslipidemia. Therefore EBI2 may represent a promising target for therapeutic interventions for human diseases.

Title: E2F-dependent histone acetylation and recruitment of the Tip60 acetyltransferase complex to chromatin in late G1. Content: E2F proteins can either activate or repress transcription. Following mitogenic stimulation, repressive E2F4-p130-histone deacetylase complexes dissociate from, while activating species (E2F1, -2, and -3) associate with, target promoters. Histones H3 and H4 simultaneously become hyperacetylated, but it remains unclear whether this is a prerequisite or a consequence of E2F binding. Here, we show that activating E2F species are required for hyperacetylation of target chromatin in human cells. Overexpression of a dominant-negative (DN) E2F1 mutant in serum-stimulated T98G cells blocked all E2F binding, H4 acetylation, and, albeit partially, H3 acetylation. Target gene activation and S-phase entry were also blocked by DN E2F1. Conversely, ectopic activation of E2F1 rapidly induced H3 and H4 acetylation, demonstrating a direct role for E2F in these events. E2F1 was previously shown to bind the histone acetyltransferases (HATs) p300/CBP and PCAF/GCN5. In our hands, ectopically expressed E2F1 also bound the unrelated HAT Tip60 and induced recruitment of five subunits of the Tip60 complex (Tip60, TRRAP, p400, Tip48, and Tip49) to target promoters in vivo. Moreover, E2F-dependent recruitment of Tip60 to chromatin occurred in late G(1) following serum stimulation. We speculate that the activities of multiple HAT complexes account for E2F-dependent acetylation, transcription, and S-phase entry.

Title: The INO80 ATP-dependent chromatin remodeling complex is a nucleosome spacing factor. Content: The mobilization of nucleosomes by the ATP-dependent remodeler INO80 is quite different from another remodeler (SWI/SNF) that is also involved in gene activation. Unlike that recently shown for SWI/SNF, INO80 is unable to disassemble nucleosomes when remodeling short nucleosomal arrays. Instead, INO80 more closely resembles, although with notable exceptions, the nucleosome spacing activity of ISW2 and ISW1a, which are generally involved in transcription repression. INO80 required a minimum of 33 to 43 bp of extranucleosomal DNA for mobilizing nucleosomes, with 70 bp being optimal. INO80 prefers to move mononucleosomes to the center of DNA, like ISW2 and ISW1a, but does so with higher precision. Unlike ISW2/1a, INO80 does not require the H4 tail for nucleosome mobilization; instead, the H2A histone tail negatively regulates nucleosome movement by INO80. INO80 moved arrays of two or three nucleosomes with 50 or 79 bp of linker DNA closer together, with a final length of ∼30 bp of linker DNA or a repeat length of ∼177 bp. A minimum length of >30 bp of linker DNA was required for nucleosome movement and spacing by INO80 in arrays.

Title: Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Content: Transcription factor-based cellular reprogramming has opened the way to converting somatic cells to a pluripotent state, but has faced limitations resulting from the requirement for transcription factors and the relative inefficiency of the process. We show here that expression of the miR302/367 cluster rapidly and efficiently reprograms mouse and human somatic cells to an iPSC state without a requirement for exogenous transcription factors. This miRNA-based reprogramming approach is two orders of magnitude more efficient than standard Oct4/Sox2/Klf4/Myc-mediated methods. Mouse and human miR302/367 iPSCs display similar characteristics to Oct4/Sox2/Klf4/Myc-iPSCs, including pluripotency marker expression, teratoma formation, and, for mouse cells, chimera contribution and germline contribution. We found that miR367 expression is required for miR302/367-mediated reprogramming and activates Oct4 gene expression, and that suppression of Hdac2 is also required. Thus, our data show that miRNA and Hdac-mediated pathways can cooperate in a powerful way to reprogram somatic cells to pluripotency.

Title: Suppression of inflammatory and neuropathic pain by uncoupling CRMP-2 from the presynaptic Ca2+ channel complex Content: The use of N-type voltage-gated calcium channel (CaV2.2) blockers to treat pain is limited by many physiological side effects. Here we report that inflammatory and neuropathic hypersensitivity can be suppressed by inhibiting the binding of collapsin response mediator protein 2 (CRMP-2) to CaV2.2 and thereby reducing channel function. A peptide of CRMP-2 fused to the HIV transactivator of transcription (TAT) protein (TAT-CBD3) decreased neuropeptide release from sensory neurons and excitatory synaptic transmission in dorsal horn neurons, reduced meningeal blood flow, reduced nocifensive behavior induced by formalin injection or corneal capsaicin application and reversed neuropathic hypersensitivity produced by an antiretroviral drug. TAT-CBD3 was mildly anxiolytic without affecting memory retrieval, sensorimotor function or depression. At doses tenfold higher than that required to reduce hypersensitivity in vivo, TAT-CBD3 caused a transient episode of tail kinking and body contortion. By preventing CRMP-2–mediated enhancement of CaV2.2 function, TAT-CBD3 alleviated inflammatory and neuropathic hypersensitivity, an approach that may prove useful in managing chronic pain.

Title: Activation by IKKα of a second, evolutionary conserved, NF-κB signaling pathway Content: In mammals, the canonical nuclear factor κB (NF-κB) signaling pathway activated in response to infections is based on degradation of IκB inhibitors. This pathway depends on the IκB kinase (IKK), which contains two catalytic subunits, IKKα and IKKβ. IKKβ is essential for inducible IκB phosphorylation and degradation, whereas IKKα is not. Here we show that IKKα is required for B cell maturation, formation of secondary lymphoid organs, increased expression of certain NF-κB target genes, and processing of the NF-κB2 (p100) precursor. IKKα preferentially phosphorylates NF-κB2, and this activity requires its phosphorylation by upstream kinases, one of which may be NF-κB–inducing kinase (NIK). IKKα is therefore a pivotal component of a second NF-κB activation pathway based on regulated NF-κB2 processing rather than IκB degradation.

Title: Canonical transient receptor potential 5 channel in conjunction with Orai1 and STIM1 allows Sr2+ entry, optimal influx of Ca2+, and degranulation in a rat mast cell line. Content: Degranulation of mast cells in response to Ag or the calcium mobilizing agent, thapsigargin, is dependent on emptying of intracellular stores of Ca(2+) and the ensuing influx of external Ca(2+), also referred to as store-operated calcium entry. However, it is unlikely that the calcium release-activated calcium channel is the sole mechanism for the entry of Ca(2+) because Sr(2+) and other divalent cations also permeate and support degranulation in stimulated mast cells. In this study we show that influx of Ca(2+) and Sr(2+) as well as degranulation are dependent on the presence of the canonical transient receptor potential (TRPC) channel protein TRPC5, in addition to STIM1 and Orai1, as demonstrated by knock down of each of these proteins by inhibitory RNAs in a rat mast cell (RBL-2H3) line. Overexpression of STIM1 and Orai1, which are known to be essential components of calcium release-activated calcium channel, allows entry of Ca(2+) but not Sr(2+), whereas overexpression of STIM1 and TRPC5 allows entry of both Ca(2+) and Sr(2+). These and other observations suggest that the Sr(2+)-permeable TRPC5 associates with STIM1 and Orai1 in a stoichiometric manner to enhance entry of Ca(2+) to generate a signal for degranulation.

Title: Full activation of the T cell receptor requires both clustering and conformational changes at CD3. Content: T cell receptor (TCR-CD3) triggering involves both receptor clustering and conformational changes at the cytoplasmic tails of the CD3 subunits. The mechanism by which TCRalphabeta ligand binding confers conformational changes to CD3 is unknown. By using well-defined ligands, we showed that induction of the conformational change requires both multivalent engagement and the mobility restriction of the TCR-CD3 imposed by the plasma membrane. The conformational change is elicited by cooperative rearrangements of two TCR-CD3 complexes and does not require accompanying changes in the structure of the TCRalphabeta ectodomains. This conformational change at CD3 reverts upon ligand dissociation and is required for T cell activation. Thus, our permissive geometry model provides a molecular mechanism that rationalizes how the information of ligand binding to TCRalphabeta is transmitted to the CD3 subunits and to the intracellular signaling machinery.

Title: Parathyroid hormone-induced E4BP4/NFIL3 down-regulates transcription in osteoblasts. Content: Parathyroid hormone (PTH), a major regulator of bone metabolism, activates the PTHR1 receptor on the osteoblast plasma membrane to initiate signaling and induce transcription of primary response genes. Subsequently, primary genes with transcriptional activity regulate expression of downstream PTH targets. We have identified the adenovirus E4 promoter-binding protein/nuclear factor regulated by IL-3 (E4bp4) as a PTH-induced primary gene in osteoblasts. E4BP4 is a basic leucine zipper (bZIP) transcription factor that represses or activates transcription in non-osteoblastic cells. We report here that PTH rapidly and transiently induced E4bp4 mRNA in osteoblastic cells and that this induction did not require protein synthesis. PTH also induced E4BP4 protein synthesis and E4BP4 binding to a consensus but not to a mutant E4BP4 response element (EBPRE). E4BP4 overexpression inhibited an EBPRE-containing promoter-reporter construct, whereas PTH treatment attenuated activity of the same construct in primary mouse osteoblasts. Finally, E4BP4 overexpression inhibited PTH-induced activity of a cyclooxygenase-2 promoter-reporter construct. Our data suggest a role for E4BP4 in attenuation of PTH target gene transcription in osteoblasts.

Title: Toll-like receptor-3 mediates HIV-1 transactivation via NFκB and JNK pathways and histone acetylation, but prolonged activation suppresses Tat and HIV-1 replication. Content: TLR3 has been implicated in the pathogenesis of several viral infections, including SIV- and HIV-1-induced inflammation and AIDS. However the molecular mechanisms of these TLR3-mediated effects are not known, and it is not known whether HIV interacts with cellular TLR3 to affect disease process. Here we investigate the effects of TLR3 ligands on HIV-1 transactivation using both primary human macrophages and cells containing integrated copies of the HIV-1 promoter. We demonstrate that TLR3 activation induced upregulation of transcription factors such as c-Jun, CCAAT/enhancer-binding protein alpha (CEBPA), signal transducer and activator of transcription (STAT)-1, STAT-2, RELB, and nuclear factor kappa-B1 (NFκB1), most of which are known to regulate the HIV promoter activity. We also demonstrate that TLR3 activation increased HIV-1 transactivation via the c-Jun N-terminal kinase (JNK) and NFκB pathways. This was associated with epigenetic modifications, including decreased histone deacetylase activity, increased histone acetyl transferase (HAT) activity, and increased acetylation of histones H3 and H4 at lysine residues in the nucleosome-0 and nucleosome-1 of the HIV-1 promoter. However, prolonged TLR3 activation decreased HIV-1 transactivation, decreased HAT activity and Tat transcription, and suppressed viral replication. Overall, data suggests that TLR3 can act as viral sensor to mediate viral transactivation, cellular signaling, innate immune response, and inflammation in HIV-infected humans. Our study provides novel insights into the molecular basis for these TLR3-mediated effects.

Title: Control of interneuron dendritic growth through NRG1/erbB4-mediated kalirin-7 disinhibition Content: Neuregulin 1 (NRG1) is a secreted trophic factor that activates the postsynaptic erbB4 receptor tyrosine kinase. Both NRG1 and erbB4 have been repeatedly associated with schizophrenia, but their downstream targets are not well characterized. ErbB4 is highly abundant in interneurons, and NRG1-mediated erbB4 activation has been shown to modulate interneuron function, but the role for NRG1-erbB4 signaling in regulating interneuron dendritic growth is not well understood. Here we show that NRG1/erbB4 promote the growth of dendrites in mature interneurons through kalirin, a major dendritic Rac1-GEF. Recent studies have shown associations of the KALRN gene with schizophrenia. Our data point to an essential role of phosphorylation in kalirin-7's C terminus as the critical site for these effects. As reduced interneuron dendrite length occurs in schizophrenia, understanding how NRG1-erbB4 signaling modulates interneuron dendritic morphogenesis might shed light on disease-related alterations in cortical circuits.

Title: The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Content: The rapamycin-sensitive TOR signalling pathway in Saccharomyces cerevisiae activates a cell-growth program in response to nutrients such as nitrogen and carbon. The TOR1 and TOR2 kinases (TOR) control cytoplasmic protein synthesis and degradation through the conserved TAP42 protein. Upon phosphorylation by TOR, TAP42 binds and possibly inhibits type 2A and type-2A-related phosphatases; however, the mechanism by which TOR controls nuclear events such as global repression of starvation-specific transcription is unknown. Here we show that TOR prevents transcription of genes expressed upon nitrogen limitation by promoting the association of the GATA transcription factor GLN3 with the cytoplasmic protein URE2. The binding of GLN3 to URE2 requires TOR-dependent phosphorylation of GLN3. Phosphorylation and cytoplasmic retention of GLN3 are also dependent on the TOR effector TAP42, and are antagonized by the type-2A-related phosphatase SIT4. TOR inhibits expression of carbon-source-regulated genes by stimulating the binding of the transcriptional activators MSN2 and MSN4 to the cytoplasmic 14-3-3 protein BMH2. Thus, the TOR signalling pathway broadly controls nutrient metabolism by sequestering several transcription factors in the cytoplasm.

Title: Maximal activation of transcription by statl and stat3 requires both tyrosine and serine phosphorylation Content: Stat1 and Stat3 are latent transcriptional factors activated initially through phosphorylation on single tyrosine residues induced by cytokine and growth factor occupation of cell surface receptors. Here we show that phosphorylation on a single serine (residue 727) in each protein is also required for maximal transcriptional activity. Both cytokines and growth factors are capable of inducing the serine phosphorylation of Stat1 and Stat3. These experiments show that gene activation by Stat1 and Stat3, which obligatorily require tyrosine phosphorylation to become active, also depends for maximal activation on one or more of the many serine kinases.

Title: Stepwise Histone Replacement by SWR1 Requires Dual Activation with Histone H2A.Z and Canonical Nucleosome Content: Histone variant H2A.Z-containing nucleosomes are incorporated at most eukaryotic promoters. This incorporation is mediated by the conserved SWR1 complex, which replaces histone H2A in canonical nucleosomes with H2A.Z in an ATP-dependent manner. Here, we show that promoter-proximal nucleosomes are highly heterogeneous for H2A.Z in Saccharomyces cerevisiae, with substantial representation of nucleosomes containing one, two, or zero H2A.Z molecules. SWR1-catalyzed H2A.Z replacement in vitro occurs in a stepwise and unidirectional fashion, one H2A.Z-H2B dimer at a time, producing heterotypic nucleosomes as intermediates and homotypic H2A.Z nucleosomes as end products. The ATPase activity of SWR1 is specifically stimulated by H2A-containing nucleosomes without ensuing histone H2A eviction. Remarkably, further addition of free H2A.Z-H2B dimer leads to hyperstimulation of ATPase activity, eviction of nucleosomal H2A-H2B, and deposition of H2A.Z-H2B. These results suggest that the combination of H2A-containing nucleosome and free H2A.Z-H2B dimer acting as both effector and substrate for SWR1 governs the specificity and outcome of the replacement reaction.

Title: N terminus of Swr1 binds to histone H2AZ and provides a platform for subunit assembly in the chromatin remodeling complex. Content: Variant histone H2AZ-containing nucleosomes are involved in the regulation of gene expression. In Saccharomyces cerevisiae, chromatin deposition of histone H2AZ is mediated by the fourteen-subunit SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. Previous work defined the role of seven SWR1 subunits (Swr1 ATPase, Swc2, Swc3, Arp6, Swc5, Yaf9, and Swc6) in maintaining complex integrity and H2AZ histone replacement activity. Here we examined the function of three additional SWR1 subunits, bromodomain containing Bdf1, actin-related protein Arp4 and Swc7, by analyzing affinity-purified mutant SWR1 complexes. We observed that depletion of Arp4 (arp4-td) substantially impaired the association of Bdf1, Yaf9, and Swc4. In contrast, loss of either Bdf1 or Swc7 had minimal effects on overall complex integrity. Furthermore, the basic H2AZ histone replacement activity of SWR1 in vitro required Arp4, but not Bdf1 or Swc7. Thus, three out of fourteen SWR1 subunits, Bdf1, Swc7, and previously noted Swc3, appear to have roles auxiliary to the basic histone replacement activity. The N-terminal region of the Swr1 ATPase subunit is necessary and sufficient to direct association of Bdf1 and Swc7, as well as Arp4, Act1, Yaf9 and Swc4. This same region contains an additional H2AZ-H2B specific binding site, distinct from the previously identified Swc2 subunit. These findings suggest that one SWR1 enzyme might be capable of binding two H2AZ-H2B dimers, and provide further insight on the hierarchy and interdependency of molecular interactions within the SWR1 complex.

Title: NSOM/QD-Based Direct Visualization of CD3-Induced and CD28-Enhanced Nanospatial Coclustering of TCR and Coreceptor in Nanodomains in T Cell Activation Content: Direct molecular imaging of nano-spatial relationship between T cell receptor (TCR)/CD3 and CD4 or CD8 co-receptor before and after activation of a primary T cell has not been reported. We have recently innovated application of near-field scanning optical microscopy (NSOM) and immune-labeling quantum dots (QD) to image Ag-specific TCR response during in vivo clonal expansion, and now up-graded the NSOM/QD-based nanotechnology through dipole-polarization and dual-color imaging. Using this imaging system scanning cell-membrane molecules at a best-optical lateral resolution, we demonstrated that CD3, CD4 or CD8 molecules were distinctly distributed as single QD-bound molecules or nano-clusters equivalent to 2-4 QD fluorescence-intensity/size on cell-membrane of un-stimulated primary T cells, and approximately 6-10% of CD3 were co-clustering with CD4 or CD8 as 70-110 nm nano-clusters without forming nano-domains. The ligation of TCR/CD3 on CD4 or CD8 T cells led to CD3 nanoscale co-clustering or interaction with CD4 or CD8 co-receptors forming 200-500 nm nano-domains or >500 nm micro-domains. Such nano-spatial co-clustering of CD3 and CD4 or CD3 and CD8 appeared to be an intrinsic event of TCR/CD3 ligation, not purely limited to MHC engagement, and be driven by Lck phosphorylation. Importantly, CD28 co-stimulation remarkably enhanced TCR/CD3 nanoscale co-clustering or interaction with CD4 co-receptor within nano- or micro-domains on the membrane. In contrast, CD28 co-stimulation did not enhance CD8 clustering or CD3-CD8 co-clustering in nano-domains although it increased molecular number and density of CD3 clustering in the enlarged nano-domains. These nanoscale findings provide new insights into TCR/CD3 interaction with CD4 or CD8 co-receptor in T-cell activation.

Title: Microtubule-associated histone deacetylase 6 supports the calcium store sensor STIM1 in mediating malignant cell behaviors. Content: Stromal-interaction molecule 1 (STIM1) is an endoplasmic reticulum Ca(2+) storage sensor that promotes cell growth, migration, and angiogenesis in breast and cervical cancers. Here, we report that the microtubule-associated histone deacetylase 6 (HDAC6) differentially regulates activation of STIM1-mediated store-operated Ca(2+) entry (SOCE) between cervical cancer cells and normal cervical epithelial cells. Confocal microscopy of living cells indicated that microtubule integrity was necessary for STIM1 trafficking to the plasma membrane and interaction with Orai1, an essential pore subunit of SOCE. Cancer cells overexpressed both STIM1 and Orai1 compared with normal cervical epithelial cells. HDAC6 upregulation in cancer cells was accompanied by hypoacetylated α-tubulin. Tubastatin-A, a specific HDAC6 inhibitor, inhibited STIM1 translocation to plasma membrane and blocked SOCE activation in cancer cells but not normal epithelial cells. Genetic or pharmacologic inhibition of HDAC6 blocked STIM1 membrane trafficking and downstream Ca(2+) influx, as evidenced by total internal reflection fluorescent images and intracellular Ca(2+) determination. In contrast, HDAC6 inhibition did not affect interactions between STIM1 and the microtubule plus end-binding protein EB1. Analysis of surgical specimens confirmed that most cervical cancer tissues overexpressed STIM1 and Orai1, accompanied by hypoacetylated α-tubulin. Together, our results identify HDAC6 as a candidate target to disrupt STIM1-mediated SOCE as a general strategy to block malignant cell behavior.

Title: Wdr5 Mediates Self-Renewal and Reprogramming via the Embryonic Stem Cell Core Transcriptional Network Content: The embryonic stem (ES) cell transcriptional and chromatin-modifying networks are critical for self-renewal maintenance. However, it remains unclear whether these networks functionally interact and, if so, what factors mediate such interactions. Here, we show that WD repeat domain 5 (Wdr5), a core member of the mammalian Trithorax (trxG) complex, positively correlates with the undifferentiated state and is a regulator of ES cell self-renewal. We demonstrate that Wdr5, an "effector" of H3K4 methylation, interacts with the pluripotency transcription factor Oct4. Genome-wide protein localization and transcriptome analyses demonstrate overlapping gene regulatory functions between Oct4 and Wdr5. The Oct4-Sox2-Nanog circuitry and trxG cooperate in activating transcription of key self-renewal regulators, and furthermore, Wdr5 expression is required for the efficient formation of induced pluripotent stem (iPS) cells. We propose an integrated model of transcriptional and epigenetic control, mediated by select trxG members, for the maintenance of ES cell self-renewal and somatic cell reprogramming.

Title: Parathyroid hormone induces E4bp4 messenger ribonucleic acid expression primarily through cyclic adenosine 3',5'-monophosphate signaling in osteoblasts. Content: PTH binding to its receptor activates protein kinase A (PKA), protein kinase C (PKC), and calcium signaling to induce transcription of primary response genes in osteoblasts. Adenovirus E4 promoter-binding protein/nuclear factor regulated by IL-3 (E4BP4/NFIL3), a transcriptional repressor, is a PTH-induced primary response gene in primary mouse osteoblasts (MOBs). Here we investigate the signaling pathway(s) that lead to PTH induction of E4bp4 mRNA expression. Ten and 100 nm PTH induced maximum E4bp4 expression in MOBs. Forskolin (FSK), an adenylate cyclase inducer, 8-bromo-cAMP, a cAMP analog, and phorbol myristate acetate, a PKC activator, increased E4bp4 mRNA levels, whereas ionomycin, a calcium ionophore, had no effect. Pretreatment of cells with 30 microm H89, a PKA inhibitor, strongly inhibited PTH- and FSK-induced E4bp4 expression. In contrast, overnight pretreatment with 1 microm phorbol myristate acetate to down-regulate PKC signaling did not alter PTH and FSK effects. Moreover, PTH (3-34) that does not activate cAMP signaling did not increase E4bp4 expression. Prostaglandin E(2), which signals through cAMP, increased E4bp4 mRNA at all doses, whereas prostaglandin F(2alpha) that primarily activates PKC and calcium signaling, induced E4bp4 only at high doses and fluprostenol that only activates PKC and calcium signaling, had no effect. Finally, 80 microg/kg PTH (1-34) ip injection induced E4bp4 mRNA expression at 1 h in mice. In contrast, 80 microg/kg PTH (3-34) had no effect. Our data suggest that PTH-induced E4bp4 mRNA expression is mediated primarily through cAMP-PKA signaling in vitro and in vivo. In conjunction with our previous report, we hypothesize that E4bp4 attenuates transcription of osteoblastic genes possessing E4bp4 promoter binding sites.

Title: Human CYP3A4 and murine Cyp3A11 are regulated by equol and genistein via the pregnane X receptor in a species-specific manner. Content: Pregnane X receptor (PXR) is an important component of the body's adaptive defense system responsible for the elimination of various toxic xenobiotics. PXR activation by endogenous and exogenous chemicals, including steroids, antibiotics, bile acids, and herbal compounds, results in induction of drug metabolism. We investigated the ability of the isoflavones genistein, daidzein, and the daidzein metabolite equol to activate human and mouse PXR in vitro using cell-based transient transfection studies and primary hepatocytes and in vivo in a mouse model. In transient transfection assays, the isoflavones genistein and daidzein activate full-length, wild-type mouse PXR, but not a mutant form, with genistein being the most potent. In contrast, equol was a more potent activator of human PXR than genistein or daidzein. In a mammalian 2-hybrid assay, isoflavones induced recruitment of the coactivator steroid receptor coactivator 1 to PXR. When tested against the native human Cytochrome P450 3A4 (CYP3A4) promoter, equol was the more potent activator and treatment of human hepatocytes with equol increased CYP3A4 mRNA and immunoreactive protein expression. Treatment of wild-type, but not PXR(-/-), mouse hepatocytes showed that genistein and daidzein induced the expression of Cytochrome P450 3A11 (Cyp3A11) mRNA, whereas equol had no effect. Cyp3A11 mRNA was also induced in vivo in mice fed a soy protein-containing diet. The results presented herein demonstrate that there is a species-specific difference in the activation of PXR by isoflavones and equol.

Title: Targeting TLR4 signaling by TLR4 Toll/IL-1 receptor domain-derived decoy peptides: identification of the TLR4 Toll/IL-1 receptor domain dimerization interface. Content: Agonist-induced dimerization of TLR4 Toll/IL-1R (TIR) domains initiates intracellular signaling. Therefore, identification of the TLR4-TIR dimerization interface is one key to the rational design of therapeutics that block TLR4 signaling. A library of cell-permeating decoy peptides, each of which represents a nonfragmented patch of the TLR4 TIR surface, was designed such that the peptides entirely encompass the TLR4 TIR surface. Each peptide was synthesized in tandem with a cell-permeating Antennapedia homeodomain sequence and tested for the ability to inhibit early cytokine mRNA expression and MAPK activation in LPS-stimulated primary murine macrophages. Five peptides--4R1, 4R3, 4BB, 4R9, and 4αE--potently inhibited all manifestations of TLR4, but not TLR2 signaling. When tested for their ability to bind directly to TLR4 TIR by Förster resonance energy transfer using time-resolved fluorescence spectroscopy, Bodipy-TMR-X-labeled 4R1, 4BB, and 4αE quenched fluorescence of TLR4-Cerulean expressed in HeLa or HEK293T cells, whereas 4R3 was partially active, and 4R9 was least active. These findings suggest that the area between the BB loop of TLR4 and its fifth helical region mediates TLR4 TIR dimerization. Moreover, our data provide direct evidence for the utility of the decoy peptide approach, in which peptides representing various surface-exposed segments of a protein are initially probed for the ability to inhibit protein function, and then their specific targets are identified by Förster resonance energy transfer to define recognition sites in signaling proteins that may be targeted therapeutically to disrupt functional transient protein interactions.

Title: PHD3-dependent hydroxylation of HCLK2 promotes the DNA damage response. Content: The DNA damage response (DDR) is a complex regulatory network that is critical for maintaining genome integrity. Posttranslational modifications are widely used to ensure strict spatiotemporal control of signal flow, but how the DDR responds to environmental cues, such as changes in ambient oxygen tension, remains poorly understood. We found that an essential component of the ATR/CHK1 signaling pathway, the human homolog of the Caenorhabditis elegans biological clock protein CLK-2 (HCLK2), associated with and was hydroxylated by prolyl hydroxylase domain protein 3 (PHD3). HCLK2 hydroxylation was necessary for its interaction with ATR and the subsequent activation of ATR/CHK1/p53. Inhibiting PHD3, either with the pan-hydroxylase inhibitor dimethyloxaloylglycine (DMOG) or through hypoxia, prevented activation of the ATR/CHK1/p53 pathway and decreased apoptosis induced by DNA damage. Consistent with these observations, we found that mice lacking PHD3 were resistant to the effects of ionizing radiation and had decreased thymic apoptosis, a biomarker of genomic integrity. Our identification of HCLK2 as a substrate of PHD3 reveals the mechanism through which hypoxia inhibits the DDR, suggesting hydroxylation of HCLK2 is a potential therapeutic target for regulating the ATR/CHK1/p53 pathway.

Title: CD28 delivers a unique signal leading to the selective recruitment of RelA and p52 NF-kappaB subunits on IL-8 and Bcl-xL gene promoters. Content: CD28 is one of the most important costimulatory receptors necessary for full T lymphocyte activation. The CD28 receptor can enhance T cell antigen receptor (TCR) signals, as well as deliver independent signals. Indeed, CD28 engagement by B7 can generate TCR-independent signals leading to IkappaB kinase and NF-kappaB activation. Here we demonstrate that the TCR-independent CD28 signal leads to the selective transcription of survival (Bcl-xL) and inflammatory (IL-8 and B cell activation factor, but not proliferative (IL-2), genes, in a NF-kappaB-dependent manner. CD28-stimulated T cells actively secrete IL-8, and Bcl-xL up-regulation protects T cells from radiation-induced apoptosis. The transcription of CD28-induced genes is mediated by the specific recruitment of RelA and p52 NF-kappaB subunits to target promoters. In contrast, p50 and c-Rel, which preferentially bind NF-kappaB sites on the IL-2 gene promoter after anti-CD3 stimulation, are not involved. Thus, we identify CD28 as a key regulator of genes important for both survival and inflammation.

Title: in Content: Transglutaminase 2 (TGase 2) is a Ca+2-dependent enzyme that catalyzes both intracellular and extracellular cross-linking reactions by transamidation of specific glutamine residues. TGase 2 is known to be involved in the membrane-mediated events required for glucose-stimulated insulin release from the pancreatic beta cells. Here we show that targeted disruption of TGase 2 impairs glucose-stimulated insulin secretion. TGase 2-/- mice show glucose intolerance after intraperitoneal glucose loading. TGase 2-/- mice manifest a tendency to develop hypoglycemia after administration of exogenous insulin as a consequence of enhanced insulin receptor substrate 2 (IRS-2) phosphorylation. We suggest that the increased peripheral sensitivity to insulin partially compensates for the defective secretion in this animal model. TGase 2-/- mouse phenotype resembles that of the maturity-onset diabetes of young (MODY) patients. In the course of screening for human TGase 2 gene in Italian subjects with the clinical features of MODY, we detected a missense mutation (N333S) in the active site of the enzyme. Collectively, these results identify TGase 2 as a potential candidate gene in type 2 diabetes.

Title: PTEN Regulates PI(3,4)P2 Signaling Downstream of Class I PI3K Content: The PI3K signaling pathway regulates cell growth and movement and is heavily mutated in cancer. Class I PI3Ks synthesize the lipid messenger PI(3,4,5)P3. PI(3,4,5)P3 can be dephosphorylated by 3- or 5-phosphatases, the latter producing PI(3,4)P2. The PTEN tumor suppressor is thought to function primarily as a PI(3,4,5)P3 3-phosphatase, limiting activation of this pathway. Here we show that PTEN also functions as a PI(3,4)P2 3-phosphatase, both in vitro and in vivo. PTEN is a major PI(3,4)P2 phosphatase in Mcf10a cytosol, and loss of PTEN and INPP4B, a known PI(3,4)P2 4-phosphatase, leads to synergistic accumulation of PI(3,4)P2, which correlated with increased invadopodia in epidermal growth factor (EGF)-stimulated cells. PTEN deletion increased PI(3,4)P2 levels in a mouse model of prostate cancer, and it inversely correlated with PI(3,4)P2 levels across several EGF-stimulated prostate and breast cancer lines. These results point to a role for PI(3,4)P2 in the phenotype caused by loss-of-function mutations or deletions in PTEN.

Title: Germinal center T follicular helper cell IL-4 production is dependent on signaling lymphocytic activation molecule receptor (CD150). Content: CD4 T cell help is critical for the generation and maintenance of germinal centers (GCs), and T follicular helper (T(FH)) cells are the CD4 T cell subset required for this process. Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP [SH2D1A]) expression in CD4 T cells is essential for GC development. However, SAP-deficient mice have only a moderate defect in T(FH) differentiation, as defined by common T(FH) surface markers. CXCR5(+) T(FH) cells are found within the GC, as well as along the boundary regions of T/B cell zones. In this study, we show that GC-associated T follicular helper (GC T(FH)) cells can be identified by their coexpression of CXCR5 and the GL7 epitope, allowing for phenotypic and functional analysis of T(FH) and GC T(FH) populations. GC T(FH) cells are a functionally discrete subset of further polarized T(FH) cells, with enhanced B cell help capacity and a specialized ability to produce IL-4 in a T(H)2-independent manner. Strikingly, SAP-deficient mice have an absence of the GC T(FH) cell subset and SAP(-) T(FH) cells are defective in IL-4 and IL-21 production. We further demonstrate that SLAM (Slamf1, CD150), a surface receptor that uses SAP signaling, is specifically required for IL-4 production by GC T(FH) cells. GC T(FH) cells require IL-4 and -21 production for optimal help to B cells. These data illustrate complexities of SAP-dependent SLAM family receptor signaling, revealing a prominent role for SLAM receptor ligation in IL-4 production by GC CD4 T cells but not in T(FH) cell and GC T(FH) cell differentiation.

Title: Activated AMPK boosts the Nrf2/HO-1 signaling axis—A role for the unfolded protein response Content: In light of the emerging interplay between redox and metabolic signaling pathways we investigated the potential cross talk between nuclear factor E2-related factor 2 (Nrf2) and AMP-activated kinase (AMPK), central regulators of the cellular redox and energy balance, respectively. Making use of xanthohumol (XN) as an activator of both the AMPK and the Nrf2 signaling pathway we show that AMPK exerts a positive influence on Nrf2/heme oxygenase (HO)-1 signaling in mouse embryonic fibroblasts. Genetic ablation and pharmacological inhibition of AMPK blunts Nrf2-dependent HO-1 expression by XN already at the mRNA level. XN leads to AMPK activation via interference with mitochondrial function and activation of liver kinase B1 as upstream AMPK kinase. The subsequent AMPK-mediated enhancement of the Nrf2/HO-1 response does not depend on inhibition of the mammalian target of rapamycin, inhibition of glycogen synthase kinase 3β, or altered abundance of Nrf2 (total and nuclear). However, reduced endoplasmic reticulum stress was identified and elaborated as a step in the AMPK-augmented Nrf2/HO-1 response. Overall, we shed more light on the hitherto incompletely understood cross talk between the LKB1/AMPK and the Nrf2/HO-1 axis revealing for the first time involvement of the unfolded protein response as an additional player and suggesting tight cooperation between signaling pathways controlling cellular redox, energy, or protein homeostasis.

Title: R93W mutation in Orai1 causes impaired calcium influx in platelets. Content: The intracellular Ca(2+) concentration of many nonexcitable cells is regulated by calcium store release and store-operated calcium entry (SOCE). In platelets, STIM1 was recently identified as the main calcium sensor expressed in the endoplasmic reticulum. To evaluate the role of the SOC channel moiety, Orai1, in platelet SOCE, we generated mice expressing a mutated, inactive form of Orai1 in blood cells only (Orai1(R93W)). Platelets expressing Orai1(R93W) were characterized by markedly reduced SOCE and impaired agonist-induced increases in [Ca(2+)](i). Orai1(R93W) platelets showed reduced integrin activation and impaired degranulation when stimulated with low agonist concentrations under static conditions. This defect, however, did not significantly affect the ability of Orai1(R93W) platelets to aggregate or to adhere to collagen under arterial flow conditions ex vivo. In contrast, these adherent Orai1(R93W) platelets were defective in surface phosphatidylserine exposure, suggesting that Orai1 is crucial for the platelets' procoagulant response rather than for other Ca(2+)-dependent cellular responses.

Title: Kruppel-like factor 2 is a transcriptional regulator of chronic and acute inflammation. Content: Although myeloid cell activation is requisite for an optimal innate immune response, this process must be tightly controlled to prevent collateral host tissue damage. Kruppel-like factor 2 (KLF2) is a potent regulator of myeloid cell proinflammatory activation. As an approximately 30% to 50% reduction in KLF2 levels has been observed in human subjects with acute or chronic inflammatory disorders, we studied the biological response to inflammation in KLF2(+/-) mice. Herein, we show that partial deficiency of KLF2 modulates the in vivo response to acute (sepsis) and subacute (skin) inflammatory challenge. Mechanistically, we link the anti-inflammatory effects of KLF2 to the inhibition of NF-κB transcriptional activity. Collectively, the observations provide biologically relevant insights into KLF2-mediated modulation of these inflammatory processes that could potentially be manipulated for therapeutic gain.

Title: NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations. Content: Phosphatidylinositol-3-kinase (PI3K) pathway deregulation is a common event in human cancer, either through inactivation of the tumor suppressor phosphatase and tensin homologue deleted from chromosome 10 or activating mutations of p110-alpha. These hotspot mutations result in oncogenic activity of the enzyme and contribute to therapeutic resistance to the anti-HER2 antibody trastuzumab. The PI3K pathway is, therefore, an attractive target for cancer therapy. We have studied NVP-BEZ235, a dual inhibitor of the PI3K and the downstream mammalian target of rapamycin (mTOR). NVP-BEZ235 inhibited the activation of the downstream effectors Akt, S6 ribosomal protein, and 4EBP1 in breast cancer cells. The antiproliferative activity of NVP-BEZ235 was superior to the allosteric selective mTOR complex inhibitor everolimus in a panel of 21 cancer cell lines of different origin and mutation status. The described Akt activation due to mTOR inhibition was prevented by higher doses of NVP-BEZ235. NVP-BEZ235 reversed the hyperactivation of the PI3K/mTOR pathway caused by the oncogenic mutations of p110-alpha, E545K, and H1047R, and inhibited the proliferation of HER2-amplified BT474 cells exogenously expressing these mutations that render them resistant to trastuzumab. In trastuzumab-resistant BT474 H1047R breast cancer xenografts, NVP-BEZ235 inhibited PI3K signaling and had potent antitumor activity. In treated animals, there was complete inhibition of PI3K signaling in the skin at pharmacologically active doses, suggesting that skin may serve as surrogate tissue for pharmacodynamic studies. In summary, NVP-BEZ235 inhibits the PI3K/mTOR axis and results in antiproliferative and antitumoral activity in cancer cells with both wild-type and mutated p110-alpha.

Title: The inflammasome component NLRP3 impairs antitumor vaccine by enhancing the accumulation of tumor-associated myeloid-derived suppressor cells. Content: The inflammasome is a proteolysis complex that generates the active forms of the proinflammatory cytokines interleukin (IL)-1β and IL-18. Inflammasome activation is mediated by NLR proteins that respond to microbial and nonmicrobial stimuli. Among NLRs, NLRP3 senses the widest array of stimuli and enhances adaptive immunity. However, its role in antitumor immunity is unknown. Therefore, we evaluated the function of the NLRP3 inflammasome in the immune response using dendritic cell vaccination against the poorly immunogenic melanoma cell line B16-F10. Vaccination of Nlrp3(-/-) mice led to a relative 4-fold improvement in survival relative to control animals. Immunity depended on CD8(+) T cells and exhibited immune specificity and memory. Increased vaccine efficacy in Nlrp3(-/-) hosts did not reflect differences in dendritic cells but rather differences in myeloid-derived suppressor cells (MDSC). Although Nlrp3 was expressed in MDSCs, the absence of Nlrp3 did not alter either their functional capacity to inhibit T cells or their presence in peripheral lymphoid tissues. Instead, the absence of Nlrp3 caused a 5-fold reduction in the number of tumor-associated MDSCs found in host mice. Adoptive transfer experiments also showed that Nlrp3(-/-) MDSCs were less efficient in reaching the tumor site. Depleting MDSCs with an anti-Gr-1 antibody increased the survival of tumor-bearing wild-type mice but not Nlrp3(-/-) mice. We concluded that Nlrp3 was critical for accumulation of MDSCs in tumors and for inhibition of antitumor T-cell immunity after dendritic cell vaccination. Our findings establish an unexpected role for Nlrp3 in impeding antitumor immune responses, suggesting novel approaches to improve the response to antitumor vaccines by limiting Nlrp3 signaling.

Title: Regulation of ISW2 by concerted action of histone H4 tail and extranucleosomal DNA. Content: The stable contact of ISW2 with nucleosomal DNA approximately 20 bp from the dyad was shown by DNA footprinting and photoaffinity labeling using recombinant histone octamers to require the histone H4 N-terminal tail. Efficient ISW2 remodeling also required the H4 N-terminal tail, although the lack of the H4 tail can be mostly compensated for by increasing the incubation time or concentration of ISW2. Similarly, the length of extranucleosomal DNA affected the stable contact of ISW2 with this same internal nucleosomal site, with the optimal length being 70 to 85 bp. These data indicate the histone H4 tail, in concert with a favorable length of extranucleosomal DNA, recruits and properly orients ISW2 onto the nucleosome for efficient nucleosome remodeling. One consequence of this property of ISW2 is likely its previously observed nucleosome spacing activity.

Title: Calcium store sensor stromal-interaction molecule 1-dependent signaling plays an important role in cervical cancer growth, migration, and angiogenesis. Content: Store-operated Ca(2+) entry (SOCE) is the principal Ca(2+) entry mechanism in nonexcitable cells. Stromal-interaction molecule 1 (STIM1) is an endoplasmic reticulum Ca(2+) sensor that triggers SOCE activation. However, the role of STIM1 in regulating cancer progression remains controversial and its clinical relevance is unclear. Here we show that STIM1-dependent signaling is important for cervical cancer cell proliferation, migration, and angiogenesis. STIM1 overexpression in tumor tissue is noted in 71% cases of early-stage cervical cancer. In tumor tissues, the level of STIM1 expression is significantly associated with the risk of metastasis and survival. EGF-stimulated cancer cell migration requires STIM1 expression and EGF increases the interaction between STIM1 and Orai1 in juxta-membrane areas, and thus induces Ca(2+) influx. STIM1 involves the activation of Ca(2+)-regulated protease calpain, as well as Ca(2+)-regulated cytoplasmic kinase Pyk2, which regulate the focal-adhesion dynamics of migratory cervical cancer cells. Because of an increase of p21 protein levels and a decrease of Cdc25C protein levels, STIM1-silencing in cervical cancer cells significantly inhibits cell proliferation by arresting the cell cycle at the S and G2/M phases. STIM1 also regulates the production of VEGF in cervical cancer cells. Interference with STIM1 expression or blockade of SOCE activity inhibits tumor angiogenesis and growth in animal models, confirming the crucial role of STIM1-mediated Ca(2+) influx in aggravating tumor development in vivo. These results make STIM1-dependent signaling an attractive target for therapeutic intervention.

Title: Arachidonic acid cytochrome P450 epoxygenase pathway. Content: Cytochrome P450 (CYP) epoxygenases convert arachidonic acid to four epoxyeicosatrienoic acid (EET) regioisomers, 5,6-, 8,9-, 11,12-, and 14,15-EET, that function as autacrine and paracrine mediators. EETs produce vascular relaxation by activating smooth muscle large-conductance Ca2+-activated K+ channels (BKCa). In addition, they have anti-inflammatory effects on blood vessels and in the kidney, promote angiogenesis, and protect ischemic myocardium and brain. CYP epoxygenases also convert eicosapentaenoic acid to vasoactive epoxy-derivatives, and endocannabinoids containing 11,12- and 14,15-EET are formed. Many EET actions appear to be initiated by EET binding to a membrane receptor that activates ion channels and intracellular signal transduction pathways. However, EETs also are taken up by cells, are incorporated into phospholipids, and bind to cytosolic proteins and nuclear receptors, suggesting that some functions may occur through direct interaction of the EET with intracellular effector systems. Soluble epoxide hydrolase (sEH) converts EETs to dihydroxyeicosatrienoic acids (DHETs). Because this attenuates many of the functional effects of EETs, sEH inhibition is being evaluated as a mechanism for increasing and prolonging the beneficial actions of EETs.

Title: Cdk1 is sufficient to drive the mammalian cell cycle Content: Unicellular organisms such as yeasts require a single cyclin-dependent kinase, Cdk1, to drive cell division. In contrast, mammalian cells are thought to require the sequential activation of at least four different cyclin-dependent kinases, Cdk2, Cdk3, Cdk4 and Cdk6, to drive cells through interphase, as well as Cdk1 to proceed through mitosis. This model has been challenged by recent genetic evidence that mice survive in the absence of individual interphase Cdks. Moreover, most mouse cell types proliferate in the absence of two or even three interphase Cdks. Similar results have been obtained on ablation of some of the activating subunits of Cdks, such as the D-type and E-type cyclins. Here we show that mouse embryos lacking all interphase Cdks (Cdk2, Cdk3, Cdk4 and Cdk6) undergo organogenesis and develop to midgestation. In these embryos, Cdk1 binds to all cyclins, resulting in the phosphorylation of the retinoblastoma protein pRb and the expression of genes that are regulated by E2F transcription factors. Mouse embryonic fibroblasts derived from these embryos proliferate in vitro, albeit with an extended cell cycle due to inefficient inactivation of Rb proteins. However, they become immortal on continuous passage. We also report that embryos fail to develop to the morula and blastocyst stages in the absence of Cdk1. These results indicate that Cdk1 is the only essential cell cycle Cdk. Moreover, they show that in the absence of interphase Cdks, Cdk1 can execute all the events that are required to drive cell division.

Title: Abi1 is essential for the formation and activation of a WAVE2 signalling complex Content: WAVE2 belongs to a family of proteins that mediates actin reorganization by relaying signals from Rac to the Arp2/3 complex, resulting in lamellipodia protrusion. WAVE2 displays Arp2/3-dependent actin nucleation activity in vitro, and does not bind directly to Rac. Instead, it forms macromolecular complexes that have been reported to exert both positive and negative modes of regulation. How these complexes are assembled, localized and activated in vivo remains to be established. Here we use tandem mass spectrometry to identify an Abi1-based complex containing WAVE2, Nap1 (Nck-associated protein) and PIR121. Abi1 interacts directly with the WHD domain of WAVE2, increases WAVE2 actin polymerization activity and mediates the assembly of a WAVE2–Abi1–Nap1–PIR121 complex. The WAVE2–Abi1–Nap1–PIR121 complex is as active as the WAVE2–Abi1 sub-complex in stimulating Arp2/3, and after Rac activation it is re-localized to the leading edge of ruffles in vivo. Consistently, inhibition of Abi1 by RNA interference (RNAi) abrogates Rac-dependent lamellipodia protrusion. Thus, Abi1 orchestrates the proper assembly of the WAVE2 complex and mediates its activation at the leading edge in vivo.

Title: The histone demethylases Jhdm1a/1b enhance somatic cell reprogramming in a vitamin-C-dependent manner. Content: Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) resets the epigenome to an embryonic-like state. Vitamin C enhances the reprogramming process, but the underlying mechanisms are unclear. Here we show that the histone demethylases Jhdm1a/1b are key effectors of somatic cell reprogramming downstream of vitamin C. We first observed that vitamin C induces H3K36me2/3 demethylation in mouse embryonic fibroblasts in culture and during reprogramming. We then identified Jhdm1a/1b, two known vitamin-C-dependent H3K36 demethylases, as potent regulators of reprogramming through gain- and loss-of-function approaches. Furthermore, we found that Jhdm1b accelerates cell cycle progression and suppresses cell senescence during reprogramming by repressing the Ink4/Arf locus. Jhdm1b also cooperates with Oct4 to activate the microRNA cluster 302/367, an integral component of the pluripotency machinery. Our results therefore reveal a role for H3K36me2/3 in cell fate determination and establish a link between histone demethylases and vitamin-C-induced reprogramming.

Title: Non-canonical PI3K-Cdc42-Pak-Mek-Erk Signaling Promotes Immune-Complex-Induced Apoptosis in Human Neutrophils Content: Neutrophils are peripheral blood leukocytes that represent the first line of immune cell defense against bacterial and fungal infections but are also crucial players in the generation of the inflammatory response. Many neutrophil cell surface receptors regulate important cellular processes via activation of agonist-activated PI3Ks. We show here that activation of human neutrophils with insoluble immune complexes drives a previously uncharacterized, PI3K-dependent, non-canonical, pro-apoptotic signaling pathway, FcγR-PI3Kβ/δ-Cdc42-Pak-Mek-Erk. This is a rare demonstration of Ras/Raf-independent activation of Erk and of PI3K-mediated activation of Cdc42. In addition, comparative analysis of immune-complex- and fMLF-induced signaling uncovers key differences in pathways used by human and murine neutrophils. The non-canonical pathway we identify in this study may be important for the resolution of inflammation in chronic inflammatory diseases that rely on immune-complex-driven neutrophil activation.

Title: Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells. Content: Although TNF is a major proinflammatory cytokine, increasing evidence indicates that TNF also has immunosuppressive feedback effects. We have demonstrated in this study that, in both resting and activated states, mouse peripheral CD4(+)CD25(+) T regulatory cells (Tregs) expressed remarkably higher surface levels of TNFR2 than CD4(+)CD25(-) T effector cells (Teffs). In cocultures of Tregs and Teffs, inhibition of proliferation of Teffs by Tregs was initially transiently abrogated by exposure to TNF, but longer exposure to TNF restored suppressive effects. Cytokine production by Teffs remained continually suppressed by Tregs. The profound anergy of Tregs in response to TCR stimulation was overcome by TNF, which expanded the Treg population. Furthermore, in synergy with IL-2, TNF expanded Tregs even more markedly up-regulated expression of CD25 and FoxP3 and phosphorylation of STAT5, and enhanced the suppressive activity of Tregs. Unlike TNF, IL-1beta and IL-6 did not up-regulate FoxP3-expressing Tregs. Furthermore, the number of Tregs increased in wild-type mice, but not in TNFR2(-/-) mice following sublethal cecal ligation and puncture. Depletion of Tregs significantly decreased mortality following cecal ligation and puncture. Thus, the stimulatory effect of TNF on Tregs resembles the reported costimulatory effects of TNF on Teffs, but is even more pronounced because of the higher expression of TNFR2 by Tregs. Moreover, our study suggests that the slower response of Tregs than Teffs to TNF results in delayed immunosuppressive feedback effects.

Title: Regulation of Hippo pathway transcription factor TEAD by p38 MAPK-induced cytoplasmic translocation Content: The Hippo pathway controls organ size and tissue homeostasis, with deregulation leading to cancer. The core Hippo components in mammals are composed of the upstream serine/threonine kinases Mst1/2, MAPK4Ks and Lats1/2. Inactivation of these upstream kinases leads to dephosphorylation, stabilization, nuclear translocation and thus activation of the major functional transducers of the Hippo pathway, YAP and its paralogue TAZ. YAP/TAZ are transcription co-activators that regulate gene expression primarily through interaction with the TEA domain DNA-binding family of transcription factors (TEAD). The current paradigm for regulation of this pathway centres on phosphorylation-dependent nucleocytoplasmic shuttling of YAP/TAZ through a complex network of upstream components. However, unlike other transcription factors, such as SMAD, NF-κB, NFAT and STAT, the regulation of TEAD nucleocytoplasmic shuttling has been largely overlooked. In the present study, we show that environmental stress promotes TEAD cytoplasmic translocation via p38 MAPK in a Hippo-independent manner. Importantly, stress-induced TEAD inhibition predominates YAP-activating signals and selectively suppresses YAP-driven cancer cell growth. Our data reveal a mechanism governing TEAD nucleocytoplasmic shuttling and show that TEAD localization is a critical determinant of Hippo signalling output.

Title: Effective Use of PI3K and MEK Inhibitors to Treat Mutant K-Ras G12D and PIK3CA H1047R Murine Lung Cancers Content: Somatic mutations that activate phosphoinositide 3-kinase (PI3K) have been identified in the p110-alpha catalytic subunit (encoded by PIK3CA). They are most frequently observed in two hotspots: the helical domain (E545K and E542K) and the kinase domain (H1047R). Although the p110-alpha mutants are transforming in vitro, their oncogenic potential has not been assessed in genetically engineered mouse models. Furthermore, clinical trials with PI3K inhibitors have recently been initiated, and it is unknown if their efficacy will be restricted to specific, genetically defined malignancies. In this study, we engineered a mouse model of lung adenocarcinomas initiated and maintained by expression of p110-alpha H1047R. Treatment of these tumors with NVP-BEZ235, a dual pan-PI3K and mammalian target of rapamycin (mTOR) inhibitor in clinical development, led to marked tumor regression as shown by positron emission tomography-computed tomography, magnetic resonance imaging and microscopic examination. In contrast, mouse lung cancers driven by mutant Kras did not substantially respond to single-agent NVP-BEZ235. However, when NVP-BEZ235 was combined with a mitogen-activated protein kinase kinase (MEK) inhibitor, ARRY-142886, there was marked synergy in shrinking these Kras-mutant cancers. These in vivo studies suggest that inhibitors of the PI3K-mTOR pathway may be active in cancers with PIK3CA mutations and, when combined with MEK inhibitors, may effectively treat KRAS mutated lung cancers.

Title: Oct4-Induced Pluripotency in Adult Neural Stem Cells Content: The four transcription factors Oct4, Sox2, Klf4, and c-Myc can induce pluripotency in mouse and human fibroblasts. We previously described direct reprogramming of adult mouse neural stem cells (NSCs) by Oct4 and either Klf4 or c-Myc. NSCs endogenously express Sox2, c-Myc, and Klf4 as well as several intermediate reprogramming markers. Here we report that exogenous expression of the germline-specific transcription factor Oct4 is sufficient to generate pluripotent stem cells from adult mouse NSCs. These one-factor induced pluripotent stem cells (1F iPS) are similar to embryonic stem cells in vitro and in vivo. Not only can these cells can be efficiently differentiated into NSCs, cardiomyocytes, and germ cells in vitro, but they are also capable of teratoma formation and germline transmission in vivo. Our results demonstrate that Oct4 is required and sufficient to directly reprogram NSCs to pluripotency.

Title: Dissection of signaling cascades through gp130 in vivo: reciprocal roles for STAT3- and SHP2-mediated signals in immune responses. Content: We generated a series of knockin mouse lines, in which the cytokine receptor gp130-dependent STAT3 and/or SHP2 signals were disrupted, by replacing the mouse gp130 gene with human gp130 mutant cDNAs. The SHP2 signal-deficient mice (gp130F759/F759 were born normal but displayed splenomegaly and lymphadenopathy and an enhanced acute phase reaction. In contrast, the STAT3 signal-deficient mice (gp130FXQ/FXXQ) died perinatally, like the gp130-deficient mice (gp130D/D). The gp130F759/F759 mice showed prolonged gp130-induced STAT3 activation, indicating a negative regulatory role for SHP2. Th1-type cytokine production and IgG2a and IgG2b production were increased in the gp130F759/F759 mice, while they were decreased in the gp130FXXQ/FXXQ immune system. These results indicate that the balance of positive and negative signals generated through gp130 regulates the immune responses.

Title: TLR-Mediated Innate Production of IFN-γ by CD8+ T Cells Is Independent of Glycolysis. Content: CD8(+) T cells can respond to unrelated infections in an Ag-independent manner. This rapid innate-like immune response allows Ag-experienced T cells to alert other immune cell types to pathogenic intruders. In this study, we show that murine CD8(+) T cells can sense TLR2 and TLR7 ligands, resulting in rapid production of IFN-γ but not of TNF-α and IL-2. Importantly, Ag-experienced T cells activated by TLR ligands produce sufficient IFN-γ to augment the activation of macrophages. In contrast to Ag-specific reactivation, TLR-dependent production of IFN-γ by CD8(+) T cells relies exclusively on newly synthesized transcripts without inducing mRNA stability. Furthermore, transcription of IFN-γ upon TLR triggering depends on the activation of PI3K and serine-threonine kinase Akt, and protein synthesis relies on the activation of the mechanistic target of rapamycin. We next investigated which energy source drives the TLR-induced production of IFN-γ. Although Ag-specific cytokine production requires a glycolytic switch for optimal cytokine release, glucose availability does not alter the rate of IFN-γ production upon TLR-mediated activation. Rather, mitochondrial respiration provides sufficient energy for TLR-induced IFN-γ production. To our knowledge, this is the first report describing that TLR-mediated bystander activation elicits a helper phenotype of CD8(+) T cells. It induces a short boost of IFN-γ production that leads to a significant but limited activation of Ag-experienced CD8(+) T cells. This activation suffices to prime macrophages but keeps T cell responses limited to unrelated infections.

Title: Tumor-derived exosomes induce N2 polarization of neutrophils to promote gastric cancer cell migration Content: BACKGROUND Exosomes are extracellular vesicles that mediate cellular communication in health and diseases. Neutrophils could be polarized to a pro-tumor phenotype by tumor. The function of tumor-derived exosomes in neutrophil regulation remains unclear. METHODS We investigated the effects of gastric cancer cell-derived exosomes (GC-Ex) on the pro-tumor activation of neutrophils and elucidated the underlying mechanisms. RESULTS GC-Ex prolonged neutrophil survival and induced expression of inflammatory factors in neutrophils. GC-Ex-activated neutrophils, in turn, promoted gastric cancer cell migration. GC-Ex transported high mobility group box-1 (HMGB1) that activated NF-κB pathway through interaction with TLR4, resulting in an increased autophagic response in neutrophils. Blocking HMGB1/TLR4 interaction, NF-κB pathway, and autophagy reversed GC-Ex-induced neutrophil activation. Silencing HMGB1 in gastric cancer cells confirmed HMGB1 as a key factor for GC-Ex-mediated neutrophil activation. Furthermore, HMGB1 expression was upregulated in gastric cancer tissues. Increased HMGB1 expression was associated with poor prognosis in patients with gastric cancer. Finally, gastric cancer tissue-derived exosomes acted similarly as exosomes derived from gastric cancer cell lines in neutrophil activation. CONCLUSION We demonstrate that gastric cancer cell-derived exosomes induce autophagy and pro-tumor activation of neutrophils via HMGB1/TLR4/NF-κB signaling, which provides new insights into mechanisms for neutrophil regulation in cancer and sheds lights on the multifaceted role of exosomes in reshaping tumor microenvironment.

Title: A neural basis for melanocortin-4 receptor regulated appetite Content: Pro-opiomelanocortin (POMC)- and agouti-related peptide (AgRP)-expressing neurons of the arcuate nucleus of the hypothalamus (ARC) are oppositely regulated by caloric depletion and coordinately stimulate and inhibit homeostatic satiety, respectively. This bimodality is principally underscored by the antagonistic actions of these ligands at downstream melanocortin-4 receptors (MC4R) in the paraventricular nucleus of the hypothalamus (PVH). Although this population is critical to energy balance, the underlying neural circuitry remains unknown. Using mice expressing Cre recombinase in MC4R neurons, we demonstrate bidirectional control of feeding following real-time activation and inhibition of PVH(MC4R) neurons and further identify these cells as a functional exponent of ARC(AgRP) neuron-driven hunger. Moreover, we reveal this function to be mediated by a PVH(MC4R)→lateral parabrachial nucleus (LPBN) pathway. Activation of this circuit encodes positive valence, but only in calorically depleted mice. Thus, the satiating and appetitive nature of PVH(MC4R)→LPBN neurons supports the principles of drive reduction and highlights this circuit as a promising target for antiobesity drug development.

Title: Tet and TDG mediate DNA demethylation essential for mesenchymal-to-epithelial transition in somatic cell reprogramming. Content: Tet-mediated DNA oxidation is a recently identified mammalian epigenetic modification, and its functional role in cell-fate transitions remains poorly understood. Here, we derive mouse embryonic fibroblasts (MEFs) deleted in all three Tet genes and examine their capacity for reprogramming into induced pluripotent stem cells (iPSCs). We show that Tet-deficient MEFs cannot be reprogrammed because of a block in the mesenchymal-to-epithelial transition (MET) step. Reprogramming of MEFs deficient in TDG is similarly impaired. The block in reprogramming is caused at least in part by defective activation of key miRNAs, which depends on oxidative demethylation promoted by Tet and TDG. Reintroduction of either the affected miRNAs or catalytically active Tet and TDG restores reprogramming in the knockout MEFs. Thus, oxidative demethylation to promote gene activation appears to be functionally required for reprogramming of fibroblasts to pluripotency. These findings provide mechanistic insight into the role of epigenetic barriers in cell-lineage conversion.

Title: Expression profiles of Toll-like receptors in non-small cell lung cancer and idiopathic pulmonary fibrosis. Content: Patients with idiopathic pulmonary fibrosis (IPF) have a higher incidence of lung cancer. The role of Toll-like receptors (TLRs), a key component of the innate immunity, in interstitial lung diseases (ILDs) and lung cancer pathogenesis is not clarified. TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9 mRNA expression was quantitatively measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR) in bronchoalveolar lavage fluid (BALF) of 16 IPF patients, 16 non-small cell lung cancer (NSCLC) patients and 9 control subjects. TLR2, TLR3, TLR4 and TLR9 protein expression was assessed on BALF T-lymphocytes using flow cytometry. TLR3 mRNA expression was significantly higher in NSCLC compared to IPF (p=0.023) and controls (p=0.001). TLR7 mRNA expression levels were significantly higher in both NSCLC and IPF groups compared to controls (p=0.029, p=0.009). TLR9 expression at the mRNA level was significantly higher in both NSCLC and IPF groups compared to controls (p=0.01, p=0.001). Finally, TLR2 mRNA expression was significantly higher in IPF patients compared to controls (p=0.042). Flow cytometry revealed decreased TLR3 and TLR9 expression in IPF patients compared to the NSCLC group (p=0.02, p=0.014) and decreased TLR9 expression in IPF compared with the controls (p=0.04). TLR2 protein expression was significantly higher in IPF patients compared to NSCLC (p=0.04). Increased expression of endosomal TLRs in NSCLC patients and elevated expression of TLR2 in pulmonary fibrosis are the main results of this study. These results do not provide support for a common TLR pathway hypothesis between NSCLC and IPF.

Title: CCR7 provides localized access to IL-2 and defines homeostatically distinct regulatory T cell subsets Content: Immune tolerance and activation depend on precise control over the number and function of immunosuppressive Foxp3(+) regulatory T (T reg) cells, and the importance of IL-2 in maintaining tolerance and preventing autoimmunity is clear. However, the homeostatic requirement for IL-2 among specific populations of peripheral T reg cells remains poorly understood. We show that IL-2 selectively maintains a population of quiescent CD44(lo)CD62L(hi) T reg cells that gain access to paracrine IL-2 produced in the T cell zones of secondary lymphoid tissues due to their expression of the chemokine receptor CCR7. In contrast, CD44(hi)CD62L(lo)CCR7(lo) T reg cells that populate nonlymphoid tissues do not access IL-2-prevalent regions in vivo and are insensitive to IL-2 blockade; instead, their maintenance depends on continued signaling through the co-stimulatory receptor ICOS (inducible co-stimulator). Thus, we define a fundamental homeostatic subdivision in T reg cell populations based on their localization and provide an integrated framework for understanding how T reg cell abundance and function are controlled by unique signals in different tissue environments.

Title: Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4. Content: The transcription factors HNF3 (FoxA) and GATA-4 are the earliest known to bind the albumin gene enhancer in liver precursor cells in embryos. To understand how they access sites in silent chromatin, we assembled nucleosome arrays containing albumin enhancer sequences and compacted them with linker histone. HNF3 and GATA-4, but not NF-1, C/EBP, and GAL4-AH, bound their sites in compacted chromatin and opened the local nucleosomal domain in the absence of ATP-dependent enzymes. The ability of HNF3 to open chromatin is mediated by a high affinity DNA binding site and by the C-terminal domain of the protein, which binds histones H3 and H4. Thus, factors that potentiate transcription in development are inherently capable of initiating chromatin opening events.

Title: TCR stimulation with modified anti-CD3 mAb expands CD8+ T cell population and induces CD8+CD25+ Tregs. Content: Modified anti-CD3 mAbs are emerging as a possible means of inducing immunologic tolerance in settings including transplantation and autoimmunity such as in type 1 diabetes. In a trial of a modified anti-CD3 mAb [hOKT3gamma1(Ala-Ala)] in patients with type 1 diabetes, we identified clinical responders by an increase in the number of peripheral blood CD8+ cells following treatment with the mAb. Here we show that the anti-CD3 mAb caused activation of CD8+ T cells that was similar in vitro and in vivo and induced regulatory CD8+CD25+ T cells. These cells inhibited the responses of CD4+ cells to the mAb itself and to antigen. The regulatory CD8+CD25+ cells were CTLA4 and Foxp3 and required contact for inhibition. Foxp3 was also induced on CD8+ T cells in patients during mAb treatment, which suggests a potential mechanism of the anti-CD3 mAb immune modulatory effects involving induction of a subset of regulatory CD8+ T cells.

Title: Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Content: Somatic cells can be induced into pluripotent stem cells (iPSCs) with a combination of four transcription factors, Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28. This provides an enabling platform to obtain patient-specific cells for various therapeutic and research applications. However, several problems remain for this approach to be therapeutically relevant due to drawbacks associated with efficiency and viral genome integration. Recently, it was shown that neural progenitor cells (NPCs) transduced with Oct4/Klf4 can be reprogrammed into iPSCs. However, NPCs express Sox2 endogenously, possibly facilitating reprogramming in the absence of exogenous Sox2. In this study, we identified a small-molecule combination, BIX-01294 and BayK8644, that enables reprogramming of Oct4/Klf4-transduced mouse embryonic fibroblasts, which do not endogenously express the factors essential for reprogramming. This study demonstrates that small molecules identified through a phenotypic screen can compensate for viral transduction of critical factors, such as Sox2, and improve reprogramming efficiency.

Title: A link between inflammation and metastasis: serum amyloid A1 and A3 induce metastasis, and are targets of metastasis-inducing S100A4 Content: S100A4 is implicated in metastasis and chronic inflammation, but its function remains uncertain. Here we establish an S100A4-dependent link between inflammation and metastatic tumor progression. We found that the acute-phase response proteins serum amyloid A (SAA) 1 and SAA3 are transcriptional targets of S100A4 via Toll-like receptor 4 (TLR4)/nuclear factor-κB signaling. SAA proteins stimulated the transcription of RANTES (regulated upon activation normal T-cell expressed and presumably secreted), G-CSF (granulocyte-colony-stimulating factor) and MMP2 (matrix metalloproteinase 2), MMP3, MMP9 and MMP13. We have also shown for the first time that SAA stimulate their own transcription as well as that of proinflammatory S100A8 and S100A9 proteins. Moreover, they strongly enhanced tumor cell adhesion to fibronectin, and stimulated migration and invasion of human and mouse tumor cells. Intravenously injected S100A4 protein induced expression of SAA proteins and cytokines in an organ-specific manner. In a breast cancer animal model, ectopic expression of SAA1 or SAA3 in tumor cells potently promoted widespread metastasis formation accompanied by a massive infiltration of immune cells. Furthermore, coordinate expression of S100A4 and SAA in tumor samples from colorectal carcinoma patients significantly correlated with reduced overall survival. These data show that SAA proteins are effectors for the metastasis-promoting functions of S100A4, and serve as a link between inflammation and tumor progression.

Title: Chemokine receptor trio: CXCR3, CXCR4 and CXCR7 crosstalk via CXCL11 and CXCL12. Content: Although chemokines are well established to function in immunity and endothelial cell activation and proliferation, a rapidly growing literature suggests that CXC Chemokine receptors CXCR3, CXCR4 and CXCR7 are critical in the development and progression of solid tumors. The effect of these chemokine receptors in tumorigenesis is mediated via interactions with shared ligands I-TAC (CXCL11) and SDF-1 (CXCL12). Over the last decade, CXCR4 has been extensively reported to be overexpressed in most human solid tumors and has earned considerable attention toward elucidating its role in cancer metastasis. To enrich the existing armamentarium of anti-cancerous agents, many inhibitors of CXCL12-CXCR4 axis have emerged as additional or alternative agents for neo-adjuvant treatments and even many of them are in preclinical and clinical stages of their development. However, the discovery of CXCR7 as another receptor for CXCL12 with rather high binding affinity and recent reports about its involvement in cancer progression, has questioned the potential of "selective blockade" of CXCR4 as cancer chemotherapeutics. Interestingly, CXCR7 can also bind another chemokine CXCL11, which is an established ligand for CXCR3. Recent reports have documented that CXCR3 and their ligands are overexpressed in different solid tumors and regulate tumor growth and metastasis. Therefore, it is important to consider the interactions and crosstalk between these three chemokine receptors and their ligand mediated signaling cascades for the development of effective anti-cancer therapies. Emerging evidence also indicates that these receptors are differentially expressed in tumor endothelial cells as well as in cancer stem cells, suggesting their direct role in regulating tumor angiogenesis and metastasis. In this review, we will focus on the signals mediated by this receptor trio via their shared ligands and their role in tumor growth and progression.

Title: Activation of AMPKα2 in adipocytes is essential for nicotine-induced insulin resistance in vivo Content: Cigarette smoking promotes body weight reduction in humans while paradoxically also promoting insulin resistance (IR) and hyperinsulinemia. However, the mechanisms behind these effects are unclear. Here we show that nicotine, a major constituent of cigarette smoke, selectively activates AMP-activated protein kinase α2 (AMPKα2) in adipocytes, which in turn phosphorylates MAP kinase phosphatase-1 (MKP1) at serine 334, initiating its proteasome-dependent degradation. The nicotine-dependent reduction of MKP1 induces the aberrant activation of both p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, leading to increased phosphorylation of insulin receptor substrate 1 (IRS1) at serine 307. Phosphorylation of IRS1 leads to its degradation, protein kinase B inhibition, and the loss of insulin-mediated inhibition of lipolysis. Consequently, nicotine increases lipolysis, which results in body weight reduction, but this increase also elevates the levels of circulating free fatty acids and thus causes IR in insulin-sensitive tissues. These results establish AMPKα2 as an essential mediator of nicotine-induced whole-body IR in spite of reductions in adiposity.

Title: Kruppel-like factor KLF10 targets transforming growth factor-beta1 to regulate CD4(+)CD25(-) T cells and T regulatory cells. Content: CD4(+)CD25(+) regulatory T cells (T regs) play a major role in the maintenance of self-tolerance and immune suppression, although the mechanisms controlling T reg development and suppressor function remain incompletely understood. Herein, we provide evidence that Kruppel-like factor 10 (KLF10/TIEG1) constitutes an important regulator of T regulatory cell suppressor function and CD4(+)CD25(-) T cell activation through distinct mechanisms involving transforming growth factor (TGF)-beta1 and Foxp3. KLF10 overexpressing CD4(+)CD25(-) T cells induced both TGF-beta1 and Foxp3 expression, an effect associated with reduced T-Bet (Th1 marker) and Gata3 (Th2 marker) mRNA expression. Consistently, KLF10(-/-) CD4(+)CD25(-) T cells have enhanced differentiation along both Th1 and Th2 pathways and elaborate higher levels of Th1 and Th2 cytokines. Furthermore, KLF10(-/-) CD4(+)CD25(-) T cell effectors cannot be appropriately suppressed by wild-type T regs. Surprisingly, KLF10(-/-) T reg cells have reduced suppressor function, independent of Foxp3 expression, with decreased expression and elaboration of TGF-beta1, an effect completely rescued by exogenous treatment with TGF-beta1. Mechanistic studies demonstrate that in response to TGF-beta1, KLF10 can transactivate both TGF-beta1 and Foxp3 promoters, implicating KLF10 in a positive feedback loop that may promote cell-intrinsic control of T cell activation. Finally, KLF10(-/-) CD4(+)CD25(-) T cells promoted atherosclerosis by approximately 2-fold in ApoE(-/-)/scid/scid mice with increased leukocyte accumulation and peripheral pro-inflammatory cytokines. Thus, KLF10 is a critical regulator in the transcriptional network controlling TGF-beta1 in both CD4(+)CD25(-) T cells and T regs and plays an important role in regulating atherosclerotic lesion formation in mice.

Title: Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells. Content: TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Here, we show that Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem cells (ESCs) and are induced concomitantly with 5hmC during reprogramming of fibroblasts to induced pluripotent stem cells. ESCs depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1 and display hyperactive Nodal signaling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in vitro. In Fgf4- and heparin-supplemented culture conditions, Tet1-depleted ESCs activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in midgestation embryo chimeras. Consistent with these findings, Tet1-depleted ESCs form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm, and ectopic appearance of trophoblastic giant cells. Thus, 5hmC is an epigenetic modification associated with the pluripotent state, and Tet1 functions to regulate the lineage differentiation potential of ESCs.

Title: Nicotinic receptor-mediated neuroprotection in neurodegenerative disease models. Content: Multiple lines of evidence, from molecular and cellular to epidemiological, have implicated nicotinic transmission in the pathology of Alzheimer's disease (AD) and Parkinson's disease (PD). This review article presents evidence for nicotinic acetylcholine receptor (nAChR)-mediated protection and the signal transduction involved in this mechanism. The data is based mainly on our studies using rat-cultured primary neurons. Nicotine-induced protection was blocked by an alpha7 nAChR antagonist, a phosphatidylinositol 3-kinase (PI3K) inhibitor, and an Src inhibitor. Levels of phosphorylated Akt, an effector of PI3K, Bcl-2 and Bcl-x were increased by nicotine administration. From these experimental data, our hypothesis for the mechanism of nAChR-mediated survival signal transduction is that the alpha7 nAChR stimulates the Src family, which activates PI3K to phosphorylate Akt, which subsequently transmits the signal to up-regulate Bcl-2 and Bcl-x. Up-regulation of Bcl-2 and Bcl-x could prevent cells from neuronal death induced by beta-amyloid (Abeta), glutamate and rotenone. These findings suggest that protective therapy with nAChR stimulation could delay the progress of neurodegenerative diseases such as AD and PD.

Title: Human TRPML1 channel structures in open and closed conformations Content: Transient receptor potential mucolipin 1 (TRPML1) is a Ca2+-releasing cation channel that mediates the calcium signalling and homeostasis of lysosomes. Mutations in TRPML1 lead to mucolipidosis type IV, a severe lysosomal storage disorder. Here we report two electron cryo-microscopy structures of full-length human TRPML1: a 3.72-Å apo structure at pH 7.0 in the closed state, and a 3.49-Å agonist-bound structure at pH 6.0 in an open state. Several aromatic and hydrophobic residues in pore helix 1, helices S5 and S6, and helix S6 of a neighbouring subunit, form a hydrophobic cavity to house the agonist, suggesting a distinct agonist-binding site from that found in TRPV1, a TRP channel from a different subfamily. The opening of TRPML1 is associated with distinct dilations of its lower gate together with a slight structural movement of pore helix 1. Our work reveals the regulatory mechanism of TRPML channels, facilitates better understanding of TRP channel activation, and provides insights into the molecular basis of mucolipidosis type IV pathogenesis.

Title: Fasting Activation of AgRP Neurons Requires NMDA Receptors and Involves Spinogenesis and Increased Excitatory Tone Content: AgRP neuron activity drives feeding and weight gain whereas that of nearby POMC neurons does the opposite. However, the role of excitatory glutamatergic input in controlling these neurons is unknown. To address this question, we generated mice lacking NMDA receptors (NMDARs) on either AgRP or POMC neurons. Deletion of NMDARs from AgRP neurons markedly reduced weight, body fat and food intake whereas deletion from POMC neurons had no effect. Activation of AgRP neurons by fasting, as assessed by c-Fos, Agrp and Npy mRNA expression, AMPA receptor-mediated EPSCs, depolarization and firing rates, required NMDARs. Furthermore, AgRP but not POMC neurons have dendritic spines and increased glutamatergic input onto AgRP neurons caused by fasting was paralleled by an increase in spines, suggesting fasting induced synaptogenesis and spinogenesis. Thus glutamatergic synaptic transmission and its modulation by NMDARs play key roles in controlling AgRP neurons and determining the cellular and behavioral response to fasting.

Title: MUC1 oncoprotein activates the IκB kinase β complex and constitutive NF-κB signalling Content: Nuclear factor-κB (NF-κB) is constitutively activated in diverse human malignancies by mechanisms that are not understood. The MUC1 oncoprotein is aberrantly overexpressed by most human carcinomas and, similarly to NF-κB, blocks apoptosis and induces transformation. This study demonstrates that overexpression of MUC1 in human carcinoma cells is associated with constitutive activation of NF-κB p65. We show that MUC1 interacts with the high-molecular-weight IκB kinase (IKK) complex in vivo and that the MUC1 cytoplasmic domain binds directly to IKKβ and IKKγ. Interaction of MUC1 with both IKKβ and IKKγ is necessary for IKKβ activation, resulting in phosphorylation and degradation of IκBα. Studies in non-malignant epithelial cells show that MUC1 is recruited to the TNF-R1 complex and interacts with IKKβ–IKKγ in response to TNFα stimulation. TNFα-induced recruitment of MUC1 is dependent on TRADD and TRAF2, but not the death-domain kinase RIP1. In addition, MUC1-mediated activation of IKKβ is dependent on TAK1 and TAB2. These findings indicate that MUC1 is important for physiological activation of IKKβ and that overexpression of MUC1, as found in human cancers, confers sustained induction of the IKKβ–NF-κB p65 pathway.

Title: Mobilization of intracellular copper stores by the ctr2 vacuolar copper transporter. Content: Copper plays an essential role in processes including signaling to the transcription and protein trafficking machinery, oxidative phosphorylation, iron mobilization, neuropeptide maturation, and normal development. Whereas much is known about intracellular mobilization of ions such as calcium, little information is available on how eukaryotic cells mobilize intracellular copper stores. We describe a mechanism by which the Saccharomyces cerevisiae Ctr2 protein provides bioavailable copper via mobilization of intracellular copper stores. Whereas Ctr2 exhibits structural similarity to the Ctr1 plasma membrane copper importer, microscopic and biochemical fractionation studies localize Ctr2 to the vacuole membrane. We demonstrate that Ctr2 mobilizes vacuolar copper stores in a manner dependent on amino acid residues conserved between the Ctr1 and Ctr2 copper transport family and that ctr2 Delta mutants hyper-accumulate vacuolar copper. Furthermore, a Ctr2 mutant that is mislocalized to the plasma membrane stimulates extracellular copper uptake, supporting a direct role for Ctr2 in copper transport across membranes. These studies identify a novel mechanism for copper mobilization and suggest that organisms cope with copper deprivation via the use of intracellular vesicular stores.

Title: Autophagy and mTORC1 regulate the stochastic phase of somatic cell reprogramming Content: We describe robust induction of autophagy during the reprogramming of mouse fibroblasts to induced pluripotent stem cells by four reprogramming factors (Sox2, Oct4, Klf4 and c-Myc), henceforth 4F. This process occurs independently of p53 activation, and is mediated by the synergistic downregulation of mechanistic target of rapamycin complex 1 (mTORC1) and the induction of autophagy-related genes. The 4F coordinately repress mTORC1, but bifurcate in their regulation of autophagy-related genes, with Klf4 and c-Myc inducing them but Sox2 and Oct4 inhibiting them. On one hand, inhibition of mTORC1 facilitates reprogramming by promoting cell reshaping (mitochondrial remodelling and cell size reduction). On the other hand, mTORC1 paradoxically impairs reprogramming by triggering autophagy. Autophagy does not participate in cell reshaping in reprogramming but instead degrades p62, whose accumulation in autophagy-deficient cells facilitates reprogramming. Our results thus reveal a complex signalling network involving mTORC1 inhibition and autophagy induction in the early phase of reprogramming, whose delicate balance ultimately determines reprogramming efficiency.

Title: Activation of the NLRP3 inflammasome in dendritic cells induces IL-1β–dependent adaptive immunity against tumors Content: The therapeutic efficacy of anticancer chemotherapies may depend on dendritic cells (DCs), which present antigens from dying cancer cells to prime tumor-specific interferon-γ (IFN-γ)–producing T lymphocytes. Here we show that dying tumor cells release ATP, which then acts on P2X7 purinergic receptors from DCs and triggers the NOD-like receptor family, pyrin domain containing-3 protein (NLRP3)-dependent caspase-1 activation complex ('inflammasome'), allowing for the secretion of interleukin-1β (IL-1β). The priming of IFN-γ–producing CD8+ T cells by dying tumor cells fails in the absence of a functional IL-1 receptor 1 and in Nlpr3-deficient (Nlrp3−/−) or caspase-1–deficient (Casp-1−/−) mice unless exogenous IL-1β is provided. Accordingly, anticancer chemotherapy turned out to be inefficient against tumors established in purinergic receptor P2rx7−/− or Nlrp3−/− or Casp1−/− hosts. Anthracycline-treated individuals with breast cancer carrying a loss-of-function allele of P2RX7 developed metastatic disease more rapidly than individuals bearing the normal allele. These results indicate that the NLRP3 inflammasome links the innate and adaptive immune responses against dying tumor cells.

Title: Angiopoietin-2 at high concentration can enhance endothelial cell survival through the phosphatidylinositol 3′-kinase/Akt signal transduction pathway Content: The angiopoietin-Tie2 system in endothelial cells is an important regulator of vasculogenesis and vascular integrity. High levels of angiopoietin-2 (Ang2) mRNA are observed in vascular activation during tumorigenesis. Although Ang2 is known to be a naturally occurring antagonist of angiopoietin-1 (Ang1) in vivo, the exact function of Ang2 itself is not known. Here, we found that a high concentration of Ang2 (800 ng/ml) acts as an apoptosis survival factor for endothelial cells during serum deprivation apoptosis. The survival effect of high concentration Ang2 was blocked by pre-treatment with soluble Tie2 receptor and the PI 3′-kinase-specific inhibitors, wortmannin and LY294002. Accordingly, 800 ng/ml of Ang2 induced phosphorylation of Tie2, the p85 subunit of phosphatidylinositol 3′-kinase (PI 3′-kinase), and serine-threonine kinase Akt at Ser473 in the human umbilical vein endothelial cells; lower concentrations of Ang2 (50–400 ng/ml) did not produce notable effects. These findings indicate that at high concentrations, Ang2, like Ang1, can be an apoptosis survival factor for endothelial cells through the activation of the Tie2 receptor, PI 3′-kinase and Akt, and thus may be a positive regulator of tumor angiogenesis.

Title: The Proto-Oncogene c-maf Is Responsible for Tissue-Specific Expression of Interleukin-4 Content: The molecular basis for the distinctive cytokine expression of CD4+ T helper 1 (Th1) and T helper 2 (Th2) subsets remains elusive. Here, we report that the proto-oncogene c-maf, a basic region/leucine zipper transcription factor, controls tissue-specific expression of IL-4. c-Maf is expressed in Th2 but not Th1 clones and is induced during normal precursor cell differentiation along a Th2 but not Th1 lineage. c-Maf binds to a c-Maf response element (MARE) in the proximal IL-4 promoter adjacent to a site footprinted by extracts from Th2 but not Th1 clones. Ectopic expression of c-Maf transactivates the IL-4 promoter in Th1 cells, B cells, and nonlymphoid cells, a function that maps to the MARE and Th2-specific footprint. Furthermore, c-Maf acts in synergy with the nuclear factor of activated T cells (NF-ATp) to initiate endogeneous IL-4 production by B cells. Manipulation of c-Maf may alter Th subset ratios in human disease.

Title: Angiopoietin 2 is a partial agonist/antagonist of Tie2 signaling in the endothelium. Content: Angiopoietin 2 (Ang2) was originally shown to be a competitive antagonist for Ang1 of the receptor tyrosine kinase Tie2 in endothelial cells (ECs). Since then, reports have conflicted on whether Ang2 is an agonist or antagonist of Tie2. Here we show that Ang2 functions as an agonist when Ang1 is absent but as a dose-dependent antagonist when Ang1 is present. Exogenous Ang2 activates Tie2 and the promigratory, prosurvival PI3K/Akt pathway in ECs but with less potency and lower affinity than exogenous Ang1. ECs produce Ang2 but not Ang1. This endogenous Ang2 maintains Tie2, phosphatidylinositol 3-kinase, and Akt activities, and it promotes EC survival, migration, and tube formation. However, when ECs are stimulated with Ang1 and Ang2, Ang2 dose-dependently inhibits Ang1-induced Tie2 phosphorylation, Akt activation, and EC survival. We conclude that Ang2 is both an agonist and an antagonist of Tie2. Although Ang2 is a weaker agonist than Ang1, endogenous Ang2 maintains a level of Tie2 activation that is critical to a spectrum of EC functions. These findings may reconcile disparate reports of Ang2's effect on Tie2, impact our understanding of endogenous receptor tyrosine kinase signal transduction mechanisms, and affect how Ang2 and Tie2 are targeted under conditions such as sepsis and cancer.

Title: AMPA receptor ligand binding domain mobility revealed by functional cross linking. Content: Glutamate receptors mediate the majority of excitatory synaptic transmission in the CNS. The AMPA-subtype has rapid kinetics, with activation, deactivation and desensitization proceeding on the millisecond timescale or faster. Crystallographic, biochemical, and functional studies suggest that GluR2 Cys mutants which form intermolecular disulfide cross-links between the lower D2 lobes of the ligand binding cores can be trapped in a conformation that represents the desensitized state. We used multi-channel rapid perfusion techniques to examine the state dependence of cross-linking in these mutants. Under reducing conditions, both wild-type GluR2 and the G725C and S729C mutants have normal activation and desensitization kinetics, but the Cys mutants can be efficiently trapped in nonconducting states when oxidized. In contrast the I664C mutant is only partially inactivated under oxidizing conditions. For S729C, disulfide cross-links form rapidly when receptors are desensitized in the presence of glutamate, but receptors also become trapped at rest, in the absence of agonist. We assessed such spontaneous trapping in various conditions, including CNQX, a competitive antagonist; kainate, a weak partial agonist; or when desensitization was blocked by the L483Y mutation that stabilizes the D1 dimer interface. These experiments suggest that trapping in the absence of glutamate is due to two motions: Spontaneous breaking of the D1 dimer interface and hyperextension of the lower lobes of the ligand binding core. These data show that the glutamate binding domains are surprisingly mobile in the absence of ligand, which could influence receptor activity in the brain.

Title: Reprogramming factor expression initiates widespread targeted chromatin remodeling. Content: Despite rapid progress in characterizing transcription factor-driven reprogramming of somatic cells to an induced pluripotent stem cell (iPSC) state, many mechanistic questions still remain. To gain insight into the earliest events in the reprogramming process, we systematically analyzed the transcriptional and epigenetic changes that occur during early factor induction after discrete numbers of divisions. We observed rapid, genome-wide changes in the euchromatic histone modification, H3K4me2, at more than a thousand loci including large subsets of pluripotency-related or developmentally regulated gene promoters and enhancers. In contrast, patterns of the repressive H3K27me3 modification remained largely unchanged except for focused depletion specifically at positions where H3K4 methylation is gained. These chromatin regulatory events precede transcriptional changes within the corresponding loci. Our data provide evidence for an early, organized, and population-wide epigenetic response to ectopic reprogramming factors that clarify the temporal order through which somatic identity is reset during reprogramming.

Title: Single-Cell Expression Analyses during Cellular Reprogramming Reveal an Early Stochastic and a Late Hierarchic Phase Content: During cellular reprogramming, only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of the previously suggested reprogramming markers Fbxo15, Fgf4, and Oct4. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc, and Nanog, can activate the pluripotency circuitry.

Title: AIR-2: An Aurora/Ipl1-related Protein Kinase Associated with Chromosomes and Midbody Microtubules Is Required for Polar Body Extrusion and Cytokinesis in Caenorhabditis elegans Embryos Content: An emerging family of kinases related to the Drosophila Aurora and budding yeast Ipl1 proteins has been implicated in chromosome segregation and mitotic spindle formation in a number of organisms. Unlike other Aurora/Ipl1-related kinases, the Caenorhabditis elegans orthologue, AIR-2, is associated with meiotic and mitotic chromosomes. AIR-2 is initially localized to the chromosomes of the most mature prophase I–arrested oocyte residing next to the spermatheca. This localization is dependent on the presence of sperm in the spermatheca. After fertilization, AIR-2 remains associated with chromosomes during each meiotic division. However, during both meiotic anaphases, AIR-2 is present between the separating chromosomes. AIR-2 also remains associated with both extruded polar bodies. In the embryo, AIR-2 is found on metaphase chromosomes, moves to midbody microtubules at anaphase, and then persists at the cytokinesis remnant. Disruption of AIR-2 expression by RNA- mediated interference produces entire broods of one-cell embryos that have executed multiple cell cycles in the complete absence of cytokinesis. The embryos accumulate large amounts of DNA and microtubule asters. Polar bodies are not extruded, but remain in the embryo where they continue to replicate. The cytokinesis defect appears to be late in the cell cycle because transient cleavage furrows initiate at the proper location, but regress before the division is complete. Additionally, staining with a marker of midbody microtubules revealed that at least some of the components of the midbody are not well localized in the absence of AIR-2 activity. Our results suggest that during each meiotic and mitotic division, AIR-2 may coordinate the congression of metaphase chromosomes with the subsequent events of polar body extrusion and cytokinesis.

Title: Emerging functional cross-talk between the Keap1-Nrf2 system and mitochondria Content: Nuclear factor erythroid-derived 2-related factor 2 (Nrf2) was originally identified as a positive regulator of drug detoxifying enzyme gene expression during exposure to environmental electrophiles. Currently, Nrf2 is known to regulate the expression of hundreds of cytoprotective genes to counteract endogenously or exogenously generated oxidative stress. Furthermore, when activated in human tumors by somatic mutations, Nrf2 confers growth advantages and chemoresistance by regulating genes involved in various processes such as the pentose phosphate pathway and nucleotide synthesis in addition to antioxidant proteins. Interestingly, increasing evidence shows that Nrf2 is associated with mitochondrial biogenesis during environmental stresses in certain tissues such as the heart. Furthermore, SKN-1, a functional homolog of Nrf2 in C. elegans, is activated by mitochondrial reactive oxygen species and extends life span by promoting mitochondrial homeostasis (i.e., mitohormesis). Similarly, Nrf2 activation was recently observed in the heart of surfeit locus protein 1 (Surf1) -/- mice in which cellular respiration was decreased due to cytochrome c oxidase defects. In this review, we critically examine the relationship between Nrf2 and mitochondria and argue that the Nrf2 stress pathway intimately communicates with mitochondria to maintain cellular homeostasis during oxidative stress.

Title: Proteomic and genomic approaches reveal critical functions of H3K9 methylation and Heterochromatin Protein-1γ in reprogramming to pluripotency Content: Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) involves a marked reorganization of chromatin. To identify post-translational histone modifications that change in global abundance during this process, we have applied a quantitative mass-spectrometry-based approach. We found that iPSCs, compared with both the starting fibroblasts and a late reprogramming intermediate (pre-iPSCs), are enriched for histone modifications associated with active chromatin, and depleted for marks of transcriptional elongation and a subset of repressive modifications including H3K9me2/me3. Dissecting the contribution of H3K9 methylation to reprogramming, we show that the H3K9 methyltransferases Ehmt1, Ehmt2 and Setdb1 regulate global H3K9me2/me3 levels and that their depletion increases iPSC formation from both fibroblasts and pre-iPSCs. Similarly, we find that inhibition of heterochromatin protein-1γ (Cbx3), a protein known to recognize H3K9 methylation, enhances reprogramming. Genome-wide location analysis revealed that Cbx3 predominantly binds active genes in both pre-iPSCs and pluripotent cells but with a strikingly different distribution: in pre-iPSCs, but not in embryonic stem cells, Cbx3 associates with active transcriptional start sites, suggesting a developmentally regulated role for Cbx3 in transcriptional activation. Despite largely non-overlapping functions and the predominant association of Cbx3 with active transcription, the H3K9 methyltransferases and Cbx3 both inhibit reprogramming by repressing the pluripotency factor Nanog. Together, our findings demonstrate that Cbx3 and H3K9 methylation restrict late reprogramming events, and suggest that a marked change in global chromatin character constitutes an epigenetic roadblock for reprogramming.

Title: Mechanistic characterization of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at tRNA wobble positions Content: The wobble modification in tRNAs, 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U), is required for the proper decoding of NNR codons in eukaryotes. The 2-thio group confers conformational rigidity of mcm(5)s(2)U by largely fixing the C3'-endo ribose puckering, ensuring stable and accurate codon-anticodon pairing. We have identified five genes in Saccharomyces cerevisiae, YIL008w (URM1), YHR111w (UBA4), YOR251c (TUM1), YNL119w (NCS2) and YGL211w (NCS6), that are required for 2-thiolation of mcm(5)s(2)U. An in vitro sulfur transfer experiment revealed that Tum1p stimulated the cysteine desulfurase of Nfs1p, and accepted persulfide sulfurs from Nfs1p. URM1 is a ubiquitin-related modifier, and UBA4 is an E1-like enzyme involved in protein urmylation. The carboxy-terminus of Urm1p was activated as an acyl-adenylate (-COAMP), then thiocarboxylated (-COSH) by Uba4p. The activated thiocarboxylate can be utilized in the subsequent reactions for 2-thiouridine formation, mediated by Ncs2p/Ncs6p. We could successfully reconstitute the 2-thiouridine formation in vitro using recombinant proteins. This study revealed that 2-thiouridine formation shares a pathway and chemical reactions with protein urmylation. The sulfur-flow of eukaryotic 2-thiouridine formation is distinct mechanism from the bacterial sulfur-relay system which is based on the persulfide chemistry.

Title: A Temporarily Distinct Subpopulation of Slow-Cycling Melanoma Cells Is Required for Continuous Tumor Growth Content: Melanomas are highly heterogeneous tumors, but the biological significance of their different subpopulations is not clear. Using the H3K4 demethylase JARID1B (KDM5B/PLU-1/RBP2-H1) as a biomarker, we have characterized a small subpopulation of slow-cycling melanoma cells that cycle with doubling times of >4 weeks within the rapidly proliferating main population. Isolated JARID1B-positive melanoma cells give rise to a highly proliferative progeny. Knockdown of JARID1B leads to an initial acceleration of tumor growth followed by exhaustion which suggests that the JARID1B-positive subpopulation is essential for continuous tumor growth. Expression of JARID1B is dynamically regulated and does not follow a hierarchical cancer stem cell model because JARID1B-negative cells can become positive and even single melanoma cells irrespective of selection are tumorigenic. These results suggest a new understanding of melanoma heterogeneity with tumor maintenance as a dynamic process mediated by a temporarily distinct subpopulation.

Title: Pituitary adenylate cyclase-activating polypeptide (PACAP(1-38)) enhances N-methyl-D-aspartate receptor function and brain-derived neurotrophic factor expression via RACK1. Content: We recently identified a novel mechanism for modulation of the phosphorylation state and function of the N-methyl-d-aspartate (NMDA) receptor via the scaffolding protein RACK1. We found that RACK1 binds both the NR2B subunit of the NMDA receptor and the nonreceptor protein-tyrosine kinase, Fyn. RACK1 inhibits Fyn phosphorylation of NR2B and decreases NMDA receptor-mediated currents in CA1 hippocampal slices (Yaka, R., Thornton, C., Vagts, A. J., Phamluong, K., Bonci, A., and Ron, D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5710-5715). Here, we identified the signaling cascade by which RACK1 is released from the NMDA receptor complex and identified the consequences of the dissociation. We found that activation of the cAMP/protein kinase A pathway in hippocampal slices induced the release of RACK1 from NR2B and Fyn. This resulted in the induction of NR2B phosphorylation and the enhancement of NMDA receptor-mediated activity via Fyn. We identified the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP(1-38)), as a ligand that induced phosphorylation of NR2B and enhanced NMDA receptor potentials. Finally, we found that activation of the cAMP/protein kinase A pathway induced the movement of RACK1 to the nuclear compartment in dissociated hippocampal neurons. Nuclear RACK1 in turn was found to regulate the expression of brain-derived neurotrophic factor induced by PACAP(1-38). Taken together our results suggest that activation of adenylate cyclase by PACAP(1-38) results in the release of RACK1 from the NMDA receptor and Fyn. This in turn leads to NMDA receptor phosphorylation, enhanced activity mediated by Fyn, and to the induction of brain-derived neurotrophic factor expression by RACK1.

Title: Mitochondria as sensors and regulators of calcium signalling Content: During the past two decades calcium (Ca2+) accumulation in energized mitochondria has emerged as a biological process of utmost physiological relevance. Mitochondrial Ca2+ uptake was shown to control intracellular Ca2+ signalling, cell metabolism, cell survival and other cell-type specific functions by buffering cytosolic Ca2+ levels and regulating mitochondrial effectors. Recently, the identity of mitochondrial Ca2+ transporters has been revealed, opening new perspectives for investigation and molecular intervention.

Title: Extrasynaptic Glutamate Spillover in the Hippocampus: Dependence on Temperature and the Role of Active Glutamate Uptake Content: At excitatory synapses on CA1 pyramidal cells of the hippocampus, a larger quantal content is sensed by N-methyl-D-aspartic acid receptors (NMDARs) than by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). A novel explanation for this discrepancy is that glutamate released from terminals presynaptic to one cell can diffuse to and activate NMDARs, but not AMPARs, on a neighboring cell. If this occurs in the living brain, it could invalidate the view that glutamatergic synapses function as private communication channels between neurons. Here, we show that the discrepancy in quantal content mediated by the two receptors is greatly decreased at physiological temperature, compared with conventional recording conditions. This effect of temperature is not due to changes in release probability or uncovering of latent AMPARs. It is, however, partially reversed by the glutamate uptake inhibitor dihydrokainate. The results suggest that glutamate transporters play a critical role in limiting the extrasynaptic diffusion of glutamate, thereby minimizing cross-talk between neighboring excitatory synapses.

Title: The myeloid transcription factor KLF2 regulates the host response to polymicrobial infection and endotoxic shock. Content: Precise control of myeloid cell activation is required for optimal host defense. However, this activation process must be under exquisite control to prevent uncontrolled inflammation. Herein, we identify the Kruppel-like transcription factor 2 (KLF2) as a potent regulator of myeloid cell activation in vivo. Exposure of myeloid cells to hypoxia and/or bacterial products reduced KLF2 expression while inducing hypoxia inducible factor-1α (HIF-1α), findings that were recapitulated in human septic patients. Myeloid KLF2 was found to be a potent inhibitor of nuclear factor-kappaB (NF-κB)-dependent HIF-1α transcription and, consequently, a critical determinant of outcome in models of polymicrobial infection and endotoxemia. Collectively, these observations identify KLF2 as a tonic repressor of myeloid cell activation in vivo and an essential regulator of the innate immune system.