Summary Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK is also a key regulator of TCA cycle flux. The TCA cycle integrates glucose, amino acid and lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. We show here an unexpected role for PEPCK in promoting cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. Unexpectedly, PEPCK also increased the synthesis of ribose from non-carbohydrate sources, such as glutamine, a phenomenon not previously described. Finally, we show that the effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Taken together, these data demonstrate a role for PEPCK that links metabolic flux and anabolic pathways to cancer cell proliferation.

This study reveals a new complexity in the cellular response to DNA damage: activation of interferon (IFN) signaling. The DNA damage response involves the rapid recruitment of repair enzymes, and the activation of signal transducers that regulate cell cycle checkpoints and cell survival. To understand the link between DNA damage and innate cellular defense that occurs in response to many viral infections, we evaluated the effects of agents such as etoposide that promote double-stranded DNA breaks. Treatment of human cells with etoposide led to the induction of IFN-stimulated genes, and the IFN-α and IFN-λ genes. The nuclear factor-κB (NF-κB), known to be activated in response to DNA damage, was shown to be a key regulator of this IFN gene induction. Expression of an NF-κB subunit, p65/RelA was sufficient for induction of the human IFN-λ1 gene. In addition, NF-κB was required for the induction of the IFN regulatory factors-1 and -7 that are able to stimulate expression of the IFN-α and IFN-λ genes. Cells that lack the NF-κB essential modulator (NEMO), lack the ability to induce the IFN genes following DNA damage. Breaks in DNA are generated during normal physiological processes of replication, transcription, and recombination, as well as by external genotoxic agents or infectious agents. The significant finding of IFN production as a stress response to DNA damage provides a new perspective on the role of IFN signaling.

During embryonic development, innervation induces the anatomical and biochemical specialization of a defined region of the muscle cell membrane immediately under the motor nerve ending. A prominent aspect of this specialization is the accumulation of high densities of nicotinic acetylcholine receptors (AChR) 1 at these sites (1, 2). The aggregation of AChR and other synaptic components is mediated by agrin, a heparansulfated proteoglycan that is synthesized by motor neurons and secreted into the synaptic cleft (3, 4). The recruitment of AChR into clusters in postsynaptic membranes ensures high efficiency synaptic transmission at neuromuscular junctions.Agrin-induced redistribution of surface AChR involves the co-clustering of multiple associated proteins, several of which have been identified to date (2, 5). These include the musclespecific receptor tyrosine kinase (MuSK) (6), the linker protein rapsyn (7), and the scaffolding proteins dystroglycan and utrophin (8). The clustering of MuSK upon its activation by agrin, the formation of AChR complexes with rapsyn, as well as the aggregation of these complexes and their stabilization upon the formation of dystroglycan-utrophin scaffolds appear to be sequential events that are to some extent independently regulated (9 -11). Although the signaling mechanisms that couple agrin activation of MuSK to the clustering of postsynaptic components are incompletely characterized, there is recent evidence for the participation of Src tyrosine kinases (12, 13), the Rho GTPases Rac and Cdc42 (14), and Dishevelled, a component of the Wnt signaling pathway (15).Focal changes in the peripheral actin-based cytoskeleton are thought to underlie the aggregation of AChR at neuromuscular junctions (16 -18). The monomeric G proteins Rac and Rho function to link extracellular signals to dynamic changes in actin cytoskeleton organization leading to the assembly of lamellipodia and actin-myosin filaments, respectively (19 -22). Rac activation induces actin polymerization at the plasma membrane, causing the appearance of lamellipodia with resultant stimulation of cell spreading and motility (23,24). Rho exerts the opposite effect by stimulating actin stress fiber appearance and focal adhesion complex formation to promote cell adhesion and contractility (25,26). As several recent studies have shown, Rac and Rho are mutually inhibitory in several cell types, and the balance between their antagonistic activities is responsible for the dynamic changes in cell morphology, adhesion, and motility (27,28). In other systems Rac serves as an upstream activator of Rho (29).We have recently shown that agrin triggers the activation of Rac and Cdc42 and that this activation is necessary but not sufficient for formation of full size AChR clusters (14). In this study we present evidence that Rho plays a crucial role in agrin-initiated signaling that is complementary to the contribution of Rac/Cdc42 and that together these Rho family GTPases serve to couple signaling initiated by extracellular agrin to the for...

In vivo delivery of small interfering RNAs (siRNAs) to target cells via the extracellular space has been hampered by dilution effects and immune responses. Gap junction-mediated transfer between cells avoids the extracellular space and its associated limitations. Because of these advantages cell based delivery via gap junctions has emerged as a viable alternative for siRNA or miRNA delivery. Here we discuss the advantages and disadvantages of extracellular delivery and cell to cell delivery via gap junction channels composed of connexins. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

BackgroundTitanium dioxide (TiO2) is one of the most common nanoparticles found in industry ranging from food additives to energy generation. Approximately four million tons of TiO2 particles are produced worldwide each year with approximately 3000 tons being produced in nanoparticulate form, hence exposure to these particles is almost certain.ResultsEven though TiO2 is also used as an anti-bacterial agent in combination with UV, we have found that, in the absence of UV, exposure of HeLa cells to TiO2 nanoparticles significantly increased their risk of bacterial invasion. HeLa cells cultured with 0.1 mg/ml rutile and anatase TiO2 nanoparticles for 24 h prior to exposure to bacteria had 350 and 250 % respectively more bacteria per cell. The increase was attributed to bacterial polysaccharides absorption on TiO2 NPs, increased extracellular LDH, and changes in the mechanical response of the cell membrane. On the other hand, macrophages exposed to TiO2 particles ingested 40 % fewer bacteria, further increasing the risk of infection.ConclusionsIn combination, these two factors raise serious concerns regarding the impact of exposure to TiO2 nanoparticles on the ability of organisms to resist bacterial infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0184-y) contains supplementary material, which is available to authorized users.

We examined whether coupling of a ventricular myocyte to a non-myocyte cell expressing HCN2 could create a two-cell syncytium capable of generating sustained pacing. Three non-myocyte cell types were transfected with the mHCN2 gene and used as sources of mHCN2-induced currents. They were human mesenchymal stem cells and HEK293 cells, both of which express connexin43 (Cx43), and HeLa cells transfected with Cx43. Cell-cell coupling between heterologous pairs increased with time in co-culture, and hyperpolarization of the myocyte induced HCN2 currents, indicating current transfer from the mHCN2-expressing cell to the myocyte via gap junctions. The magnitude of the HCN2 currents recorded in myocytes increased with increasing junctional conductance. Once a critical level of electrical cell-cell coupling between myocytes and mHCN2 transfected cells was exceeded spontaneous action potentials were generated at frequencies of ∼0.6 to 1.7 Hz (1.09 ± 0.05 Hz). Addition of carbenoxolone (200 μm), a gap junction channel blocker, to the media stopped spontaneous activity in heterologous cell pairs. Carbenoxolone washout restored activity. Blockade of HCN2 currents by 100 μm 9-amino-1,2,3,4-tetrahydroacridine (THA) stopped spontaneous activity and subsequent washout restored it. Neither THA nor carbenoxolone affected electrically stimulated action potentials in isolated single myocytes. In summary, the inward current evoked in the genetically engineered (HCN2-expressing) cell was delivered to the cardiac myocyte via gap junctions and generated action potentials such that the cell pair could function as a pacemaker unit. This finding lays the groundwork for understanding cell-based biological pacemakers in vivo once an understanding of delivery and target cell geometry is defined.

The human fatty-acid synthase (HFAS) is a potential target for anti-tumor drug discovery. As a prelude to the design of compounds that target the enoyl reductase (ER) component of HFAS, the recognition of NADPH and exogenous substrates by the ER active site has been investigated. Previous studies demonstrate that modification of Lys-1699 by pyridoxal 5-phosphate results in a specific decrease in ER activity. For the overall HFAS reaction, the K1699A and K1699Q mutations reduced k cat and k cat /K NADPH by 8-and 600-fold, respectively (where K NADPH indicates the K m value for NADPH). Thus, Lys-1699 contributes 4 kcal/mol to stabilization of the rate-limiting transition state following NADPH binding, while also stabilizing the most stable ground state after NADPH binding by 3 kcal/mol. A similar effect of the mutations on the ER partial reaction was observed, in agreement with the proposal that Lys-1699 is located in the ER NADPH-binding site. Most unexpectedly, however, both k cat and k cat /K NADPH for the ␤-ketoacyl reductase (BKR) reaction were also impacted by the Lys-1699 mutations, raising the possibility that the ER and BKR activities share a single active site. However, based on previous data indicating that the two reductase activities utilize distinct cofactor binding sites, mutagenesis of Lys-1699 is hypothesized to modulate BKR activity via allosteric effects between the ER and BKR NADPH sites.Fatty acid synthesis generates important intermediates for the construction of cell membranes and for energy storage (1). In humans the reactions resulting in the production of fatty acids are catalyzed by a multifunctional enzyme complex (HFAS), 3 consisting of the following seven catalytic activities: acetyl/malonyl transacylase, ␤-ketoacyl synthase, ␤-ketoacyl reductase (BKR), ␤-hydroxyacyl dehydratase (DH), enoyl reductase (ER), and thioesterase (2-10). In addition, there is an acyl carrier protein (ACP) domain, which carries fatty acyl intermediates in the form of fatty acyl thioesters (5). Palmitate, a C 16 -saturated fatty acid, is the major product produced (5, 11). Fatty acid synthesis, although important evolutionarily for survival during famines (1), was thought to have very little significance for today's modern society (1,9,10,12). This is because of the dietary intake of lipids, which causes HFAS to be down-regulated in normal cells (6,7,13,14). However, HFAS overexpression in many cancers, such as in breast, prostate, and colon tumors, and also in many pre-malignant growths, has led to the hypothesis that HFAS is an important target for the study of tumor biology (7,13,14).Our laboratory has a long standing interest in the development of antibacterial compounds that target prokaryotic fatty acid synthesis. Thus, an initial interest in HFAS was as a control system for validating the specificity of our bacterial FAS inhibitors. However, as described above, HFAS is now thought to be a bona fide target in its own right as a putative anti-tumor target. There have also been suggestions that HFAS inhibi...

Cellular delivery of small interfering RNAs to target cells of a tissue has the potential to travel by two intercellular pathways. For intimately apposed cells gap junctions allow transport exclusive of the extracellular space. For cells not in intimate contact, exocytotic release of vesicular contents and subsequent retrieval via endocytosis of exosomes and other vesicular contents represent an alternative intercellular delivery system that utilizes the extracellular space. Previous studies have shown siRNA/miRNA transfer from a delivery cell to a target cell via gap junction channels. We hypothesized that siRNA can be delivered via gap junctions and downregulate the expression of a reporter gene, the cyclic nucleotide-gated cation channel gene (mHCN2), in the recipient cells of cell pairs. Whole-cell patch clamp was used to measure the mHCN2-induced current and junctional conductance. The target cells were HEK293 cells that endogenously express Cx43 or HeLaCx43 cells, both transfected with mHCN2. The source cells were HEK293 or HeLaCx43 cells transfected with fluorescent-labeled siRNA targeting mHCN2. We found that siRNA targeting mHCN2 resulted in significant downregulation of mHCN2 currents both in single cells and the recipient cell of a cell pair. In addition we also documented downregulation in target cells that were not in contact with source cells suggesting an extracellular-mediated delivery. To test further for extracellular delivery HEK293/HCN2 or HeLaCx43/HCN2 cells were cultured in medium collected from HEK293 or HeLaCx43 cells transfected with fluorescent-labeled siRNA or fluorescent-labeled morpholino designed to target HCN2. After 24 h single HEK293/HCN2 or HeLaCx43cells showed accumulation of siRNA. The mHCN2 currents were also down regulated in cells with siRNA uptake. Application of 200 nmol/L Bafilomycin A1, which has been shown to affect endosome acidification and endocytotic activity, resulted in a smaller accumulation of fluorescent-labeled siRNA in single target cells. In distinction to siRNA, morpholinos targeting HCN2 exhibited greatly reduced extracellularly mediated transfer while in cell pairs, target cells exhibited reduced HCN2 currents consistent with effective gap junction-mediated delivery.