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BBA - Molecular Cell Research (v.1823, #10)

Editorial Board (pp. i).

Russell body inducing threshold depends on the variable domain sequences of individual human IgG clones and the cellular protein homeostasis by Janelle Stoops; Samantha Byrd; Haruki Hasegawa (pp. 1643-1657).

Russell bodies are intracellular aggregates of immunoglobulins. Although the mechanism of Russell body biogenesis has been extensively studied by using truncated mutant heavy chains, the importance of the variable domain sequences in this process and in immunoglobulin biosynthesis remains largely unknown. Using a panel of structurally and functionally normal human immunoglobulin Gs, we show that individual immunoglobulin G clones possess distinctive Russell body inducing propensities that can surface differently under normal and abnormal cellular conditions. Russell body inducing predisposition unique to each immunoglobulin G clone was corroborated by the intrinsic physicochemical properties encoded in the heavy chain variable domain/light chain variable domain sequence combinations that define each immunoglobulin G clone. While the sequence based intrinsic factors predispose certain immunoglobulin G clones to be more prone to induce Russell bodies, extrinsic factors such as stressful cell culture conditions also play roles in unmasking Russell body propensity from immunoglobulin G clones that are normally refractory to developing Russell bodies. By taking advantage of heterologous expression systems, we dissected the roles of individual subunit chains in Russell body formation and examined the effect of non-cognate subunit chain pair co-expression on Russell body forming propensity. The results suggest that the properties embedded in the variable domain of individual light chain clones and their compatibility with the partnering heavy chain variable domain sequences underscore the efficiency of immunoglobulin G biosynthesis, the threshold for Russell body induction, and the level of immunoglobulin G secretion. We propose that an interplay between the unique properties encoded in variable domain sequences and the state of protein homeostasis determines whether an immunoglobulin G expressing cell will develop the Russell body phenotype in a dynamic cellular setting.► Full-length human IgGs with no gross structural defect induced Russell body (RB) formation during biosynthesis. ► Individual IgG clones have unique RB-inducing thresholds that are embedded in the variable domain sequences. ► Physicochemical properties encoded in the variable domain sequences influence multiple aspects of IgG biosynthesis. ► Interplay between intrinsic threshold and cellular protein homeostasis determines whether IgG expressing cells develop RBs.

Keywords: Abbreviations; CHO; Chinese hamster ovary; ER; endoplasmic reticulum; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; HC; heavy chain; HEK; human embryonic kidney; Ig; immunoglobulin; IgG; immunoglobulin G; LC; light chain; RB; Russell body; VL; light chain variable domain; VH; heavy chain variable domainRussell body; Immunoglobulin; Protein aggregation; Endoplasmic reticulum; Recombinant antibody; Protein folding

Sphingosine 1-phosphate induces MKP-1 expression via p38 MAPK- and CREB-mediated pathways in airway smooth muscle cells by Wenchi Che; Melanie Manetsch; Timo Quante; Md. Mostafizur Rahman; Brijeshkumar S. Patel; Qi Ge; Alaina J. Ammit (pp. 1658-1665).

Sphingosine 1-phosphate (S1P), a bioactive sphingolipid elevated in asthmatic airways, is increasingly recognized as playing an important role in respiratory disease. S1P activates receptor-mediated signaling to modulate diverse cellular functions and promote airway inflammation. Although many of the stimulatory pathways activated by S1P have been delineated, especially mitogen-activated protein kinases (MAPK), the question of whether S1P exerts negative feedback control on its own signaling cascade via upregulation of phosphatases remains unexplored. We show that S1P rapidly and robustly upregulates mRNA and protein expression of the MAPK deactivator–MAPK phosphatase 1 (MKP-1). Utilizing the pivotal airway structural cell, airway smooth muscle (ASM), we confirm that S1P activates all members of the MAPK family and, in part, S1P upregulates MKP-1 expression in a p38 MAPK-dependent manner. MKP-1 is a cAMP response element binding (CREB) protein-responsive gene and here, we reveal for the first time that an adenylate cyclase/PKA/CREB-mediated pathway also contributes to S1P-induced MKP-1. Thus, by increasing MKP-1 expression via parallel p38 MAPK- and CREB-mediated pathways, S1P temporally regulates MAPK signaling pathways by upregulating the negative feedback controller MKP-1. This limits the extent and duration of pro-inflammatory MAPK signaling and represses cytokine secretion in ASM cells. Taken together, our results demonstrate that S1P stimulates both kinases and the phosphatase MKP-1 to control inflammation in ASM cells and may provide a greater understanding of the molecular mechanisms responsible for the pro-asthmatic functions induced by the potent bioactive sphingolipid S1P in the lung.► S1P increases MKP-1 expression via parallel p38 MAPK- and CREB-mediated pathways. ► S1P controls MAPK signaling by upregulating the negative feedback controller MKP-1. ► MKP-1 limits the extent and duration of pro-inflammatory MAPK signaling. ► S1P controls pro-inflammatory cellular signaling in ASM cells via MKP-1.

Keywords: Abbreviation; ASM; airway smooth muscle; CREB; cAMP response element binding; DUSP; dual-specificity phosphatases; ERK; extracellular signal-regulated kinase; IL-6; interleukin 6; JNK; c-Jun N-terminal kinase; MAPK; mitogen-activated protein kinase; MKP; mitogen-activated protein kinase phosphatase; PKA; protein kinase A; SEM; standard error of the mean; S1P; sphingosine 1-phosphate; TNFα; tumor necrosis factor αSphingolipid; Cytokine; Adenylate cyclase; CREB; p38 MAPK; DUSP-1

KLF4 and SOX9 transcription factors antagonize β-catenin and inhibit TCF-activity in cancer cells by Hassan Sellak; Songwei Wu; Thomas M. Lincoln (pp. 1666-1675).

The transcriptional activator β-catenin is a key mediator of the canonical Wnt signaling pathway. β-catenin itself does not bind DNA but functions via interaction with T-cell factor (TCF)/lymphoid-enhancing factor (LEF) transcription factors. Thus, in the case of active Wnt signaling, β-catenin, in cooperation with TCF/LEF proteins family, activates the expression of a wide variety of genes. To date, the list of established β-catenin interacting targets is far from complete. In this study, we aimed to establish the interaction between β-catenin and transcription factors that might affect TCF activity. We took advantage of EMSA, using TCF as a probe, to screen oligonucleotides known to bind specific transcription factors that might dislodge or antagonize β-catenin/TCF binding. We found that Sox9 and KLF4 antagonize β-catenin/TCF binding in HEK293, A549, SW480, and T47D cells. This inhibition of TCF binding was concentration-dependent and correlated to the in vitro TCF-luciferase functional assays. Overexpression of Sox9 and KLF4 transcription factors in cancer cells shows a concentration-dependent reduction of TCF-luciferase as well as the TCF-binding activities. In addition, we demonstrated that both Sox9 and KLF4 interact with β-catenin in an immunoprecipitation assay and reduce its binding to TCF4. Together, these results demonstrate that Sox9 and KLF4 transcription factors antagonize β-catenin/TCF in cancer cells.► A search for beta-catenin-interacting partners was proposed. ► Sox9 and KLF4 transcription factors were revealed to be potent in inhibiting beta-catenin interaction with TCF probe. ► Sox9 and KLF4 reduced both TCF-binding and activity in many cancer cells lines. ► We are reporting that these transcription factors (Sox9/KLF4) bind to beta-catenin and reduce its interaction with TCF4. ► Sox9 and KLF4 are good candidates to suppress tumor progression.

Keywords: β-catenin; KLF4; Sox9; Cancer; Wnt signaling; T-cell factor

Vasoactive intestinal peptide (VIP) inhibits human renal cell carcinoma proliferation by Eva Vacas; Fernandez-Martinez Ana B. Fernández-Martínez; Ana M. Bajo; Sanchez-Chapado Manuel Sánchez-Chapado; Andrew V. Schally; Juan C. Prieto; María J. Carmena (pp. 1676-1685).

Clear renal cell carcinoma (cRCC) is an aggressive and fatal neoplasm. The present work was undertaken to investigate the antiproliferative potential of vasoactive intestinal peptide (VIP) exposure on non-tumoral (HK2) and tumoral (A498, cRCC) human proximal tubular epithelial cell lines. Reverse transcription and semiquantitative PCR was used at the VIP mRNA level whereas enzyme immunoanalysis was performed at the protein level. Both renal cell lines expressed VIP as well as VIP/pituitary adenylate cyclase-activating peptide (VPAC) receptors whereas only HK2 cells expressed formyl peptide receptor-like 1 (FPRL-1). Receptors were functional, as shown by VIP stimulation of adenylyl cyclase activity. Treatment with 0.1μM VIP (24h) inhibited proliferation of A498 but not HK2 cells as based on a reduction in the incorporation of [³H]-thymidine and BrdU (5′‐Br‐2′‐deoxyuridine), PCNA (proliferating‐cell nuclear antigen) expression and STAT3 (signal transducer and activator of transcription 3) expression and activation. VPAC₁-receptor participation was established using JV-1-53 antagonist and siRNA transfection. Growth-inhibitory response to VIP was related to the cyclic adenosine monophosphate (cAMP)/exchange protein directly activated by cAMP (EPAC)/phosphoinositide 3-kinase (PI3-K) signaling systems as shown by studies on adenylate cyclase stimulation, and using the EPAC-specific compound 8CPT-2Me-cAMP and specific kinase inhibitors such as H89, wortmannin and PD98059. The efficacy of VIP on the prevention of tumor progression was confirmed in vivo using xenografted athymic mouse. These actions support a potential role of this peptide and its agonists in new therapies for cRCC.► Clear renal cell carcinoma expresses vasoactive intestinal peptide (VIP) receptors. ► VIP inhibited tumoral renal cell proliferation through adenylyl cyclase stimulation. ► Growth-inhibitory response to VIP was related to EPAC/PI3-K signaling. ► VIP and its agonists have a potential therapeutic role in clear renal cell cancer.

Keywords: VIP; FPRL-1; cAMP; PI3-K; Proliferation; RCC

Lysine 394 is a novel Rad6B-induced ubiquitination site on beta-catenin by Brigitte Gerard; Matthew A. Sanders; Daniel W. Visscher; Larry Tait; Malathy P.V. Shekhar (pp. 1686-1696).

The ubiquitin conjugating enzyme Rad6B is overexpressed in breast cancer and induces β-catenin transcriptional activation and stabilization via K63-linked polyubiquitination. Here we identify β-catenin and Rad6B interacting regions, identify potential Rad6B ubiquitination sites in β-catenin, and characterize their breast cancer tissue expression. β-catenin and Rad6B colocalize in breast carcinoma and coimmunoprecipitate from MDA-MB-231 cells. Pull-down assays using GST-β-catenin and His-Rad6B deletion mutants identified amino acids 131–181 and 50–116, respectively, as necessary for their interaction. Ubiquitination assays using β-catenin deletion mutants mapped Rad6B-induced ubiquitination within β-catenin 181–422 encompassing Armadillo repeats 2–7. Lysine to arginine mutations within repeats 5–7 identified K394 as the major Rad6B ubiquitination site in vitro and in vivo, and confirmed by Rad6B ubiquitination of a β-catenin peptide encompassing K394. Ubiquitination of wild type- but not K394R-β-catenin was decreased by Rad6B silencing. Compared to wild type-, K312R-, K335R-, K345R-, or K354R-β-catenin, K394R mutation caused ~50% drop in TOP/Flash activity in Wnt-silent MCF-7 cells. Consistent with these data, expression of Rad6B, itself a β-catenin/TCF transcriptional target, was also reduced in K394R-β-catenin transfected cells. Steady-state K394R-β-catenin levels are decreased compared to wild type-β-catenin. The decreased expression is not due to proteasomal degradation as treatment with MG132 failed to rescue its levels. Lymph node-positive breast carcinomas express higher levels of Rad6 protein and Rad6 activity, and K63-linked ubiquitinated β-catenin than reduction mammoplasties. These data suggest that K394 is a novel site of β-catenin ubiquitination that may be important for the stability and activity of β-catenin in breast cancer.► Rad6B/β-catenin physical interactions and mapping of the interacting regions ► Identification of the lysine 394 in β-catenin that is ubiquitinated by Rad6B ► Functional involvement of K394 of β-catenin in its transcriptional activity ► In vivo relation between Rad6 expression/activity, and Ub-β-cat in breast cancer

Keywords: Breast cancer; Ubiquitination; Transcriptional activity; Wnt signaling

Real-time analysis of intracellular glucose and calcium in pancreatic beta cells by fluorescence microscopy by Martin Tobias Kaminski; Sigurd Lenzen; Simone Baltrusch (pp. 1697-1707).

Glucose is the physiological stimulus for insulin secretion in pancreatic beta cells. The uptake and phosphorylation of glucose initiate and control downstream pathways, resulting in insulin secretion. However, the temporal coordination of these events in beta cells is not fully understood. The recent development of the FLII¹²Pglu-700μ-δ6 glucose nanosensor facilitates real-time analysis of intracellular glucose within a broad concentration range. Using this fluorescence-based technique, we show the shift in intracellular glucose concentration upon external supply and removal in primary mouse beta cells with high resolution. Glucose influx, efflux, and metabolism rates were calculated from the time-dependent plots. Comparison of insulin-producing cells with different expression levels of glucose transporters and phosphorylating enzymes showed that a high glucose influx rate correlated with GLUT2 expression, but was largely also sustainable by high GLUT1 expression. In contrast, in cells not expressing the glucose sensor enzyme glucokinase glucose metabolism was slow. We found no evidence of oscillations of the intracellular glucose concentration in beta cells. Concomitant real-time analysis of glucose and calcium dynamics using FLII¹²Pglu-700μ-δ6 and fura-2-acetoxymethyl-ester determined a glucose threshold of 4mM for the [Ca²⁺]_i increase in beta cells. Indeed, a glucose concentration of 7mM had to be reached to evoke large amplitude [Ca²⁺]_i oscillations. The K_ATP channel closing agent glibenclamide was not able to induce large amplitude [Ca²⁺]_i oscillations in the absence of glucose. Our findings suggest that glucose has to reach a threshold to evoke the [Ca²⁺]_i increase and subsequently initiate [Ca²⁺]_i oscillations in a K_ATP channel independent manner.► We examine changes in the intracellular glucose concentration in pancreatic beta-cells by a FRET based technique. ► We show that the FLII¹²Pglu-700µ-δ6 sensor and fura-2 can be used to analyze glucose and calcium at the same time. ► Glucose is not a pacemaker of oscillations in pancreatic beta-cells. ► Glucose enables intracellular calcium oscillations in pancreatic beta-cells by a K_ATP channel independent pathway.

Keywords: Abbreviations; GK; glucokinase; ECFP; enhanced cyan fluorescent protein; FRET; fluorescence resonance energy transfer; HK; hexokinase; GLUT; glucose transporter; MH; mannoheptulose; 3-OMG; 3-O-methylglucose; FLIPglu; FLII; ¹²; Pglu-700μ-δ6; [Ca; ²⁺; ]; _i; intracellular calcium; G-6-P; glucose-6-phosphateGlucokinase; GLUT2; FLII; ¹²; Pglu-700μ-δ6; Glucose metabolism; Calcium imaging; Pancreatic beta cell

1α,25-Dihydroxyvitamin D₃ mechanism of action: Modulation of L-type calcium channels leading to calcium uptake and intermediate filament phosphorylation in cerebral cortex of young rats by Leila Zanatta; Paola Bez Goulart; Goncalves Renata Gonçalves; Paula Pierozan; Elisa C. Winkelmann-Duarte; Viviane Mara Woehl; Regina Pessoa-Pureur; Fátima Regina Mena Barreto Silva; Ariane Zamoner (pp. 1708-1719).

The involvement of calcium-mediated signaling pathways in the mechanism of action of 1α,25-dihydroxyvitamin D₃ (1,25D) is currently demonstrated. In this study we found that 1,25D induces nongenomic effects mediated by membrane vitamin D receptor (VDRm) by modulating intermediate filament (IF) phosphorylation and calcium uptake through L-type voltage-dependent calcium channels (L-VDCC) in cerebral cortex of 10day-old rats. Results showed that the mechanism of action of 1,25D involves intra- and extracellular calcium levels, as well as the modulation of chloride and potassium channels. The effects of L-VDCCs on membrane voltage occur over a broad potential range and could involve depolarizing or hyperpolarizing coupling modes, supporting a cross-talk among Ca2⁺ uptake and potassium and chloride channels. Also, the Na⁺/K⁺-ATPase inactivation by ouabain mimicked the 1,25D action on⁴⁵Ca2⁺ uptake. The Na⁺/K⁺-ATPase inhibition observed herein might lead to intracellular Na⁺ accumulation with subsequent L-VDCC opening and consequently increased⁴⁵Ca2⁺ (calcium, isotope of mass 45) uptake. Moreover, the 1,25D effect is dependent on the activation of the following protein kinases: cAMP‐dependent protein kinase (PKA), Ca2⁺/calmodulin‐dependent protein kinase (PKCaMII), phosphatidylinositol 3‐kinase (PI3K) and mitogen‐activated protein kinase p38 (p38^MAPK). The modulation of calcium entry into neural cells by the 1,25D we are highlighting, might take a role in the regulation of a plethora of intracellular processes. Considering that vitamin D deficiency can lead to brain illness, 1,25D may be a possible candidate to be used, at least as an adjuvant, in the pharmacological therapy of neuropathological conditions.Display Omitted► 1α,25-Dihydroxyvitamin D₃ induces Ca2⁺ uptake and cytoskeleton phosphorylation. ► The mechanism of hormone action involves the participation of K⁺ and Cl⁻ channels. ► Na⁺/K⁺ ATPase inhibition might lead to calcium uptake via L-VDCC. ► Protein kinases are involved in the L-VDCC opening and cytoskeleton phosphorylation. ► 1,25D-induced calcium entry might participate in modulation of neurotransmission.

Keywords: 1α,25-Dihydroxyvitamin D; ₃; Cerebral cortex; Calcium; Cytoskeleton; Protein kinase; VDRm

Molecular remodeling mechanisms of the neural somatodendritic compartment by Fletcher B. Moore; James D. Baleja (pp. 1720-1730).

Neuronal cells use the process of vesicle trafficking to manipulate the populations of neurotransmitter receptors and other membrane proteins. Long term potentiation (LTP) is a long-lived increase in synaptic strength between neurons and increases postsynaptic dendritic spine size and the concentration of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate‐type glutamate receptor (AMPAR) located in the postsynaptic density. AMPAR is removed from the cell surface via clathrin-mediated endocytosis. While the adaptor protein 2 (AP2) complex of endocytosis seems to have the components needed to allow temporal and spatial regulations of internalization, many accessory proteins are involved, such as epidermal growth factor receptor phosphorylation substrate 15 (Eps15). A sequence of repeats in the Eps15 protein is known as the Eps15 homology (EH) domain. It has affinity for asparagine–proline–phenylalanine (NPF) sequences that are contained within vesicle trafficking proteins such as epsin, Rab11 family interacting protein 2 (Rab11-FIP2), and Numb. After endocytosis, a pool of AMPAR is stored in the endosomal recycling compartment that can be transported to the dendritic spine surface upon stimulation during LTP for lateral diffusion into the postsynaptic density. Rab11 and the Eps15 homologue EHD1 are involved in receptor recycling. EHD family members are also involved in transcytosis of the neuronal cell adhesion molecule neuron-glia cell adhesion molecule (NgCAM) from the somatodendritic compartment to the axon. Neurons have a unique morphology comprising many projections of membrane that is constructed in part by the effects of the Eps15 homologue, intersectin. Morphogenesis in the somatodendritic compartment is becoming better understood, but there is still much exciting territory to explore, especially regarding the roles of various EH domain–NPF interactions in endocytic and recycling processes.► Neurons change their molecular composition in response to stimuli. ► A synapse strengthens via increasing glutamate receptors at the post-synaptic density. ► Molecular trafficking alters locations of proteins in the somatodendritic compartment.

Keywords: Neuronal plasticity; Eps15 homology domain; EH domain; Postsynaptic density; Endocytosis; Exocytosis

Connexin 43 phosphorylation and degradation are required for adipogenesis by Azadeh Yeganeh; Gerald L. Stelmack; Robert R. Fandrich; Andrew J. Halayko; Elissavet Kardami; Peter Zahradka (pp. 1731-1744).

Connexin-43 (Cx43) is a membrane phosphoprotein that mediates direct inter-cellular communication by forming gap junctions. In this way Cx43 can influence gene expression, differentiation and growth. Its role in adipogenesis, however, is poorly understood. In this study, we established that Cx43 becomes highly phosphorylated in early adipocyte differentiation and translocates to the plasma membrane from the endoplasmic reticulum. As preadipocytes differentiate, Cx43 phosphorylation declines, the protein is displaced from the plasma membrane, and total cellular levels are reduced via proteosomal degradation. Notably, we show that inhibiting Cx43 degradation or constitutively over-expressing Cx43 blocks adipocyte differentiation. These data reveal that transient activation of Cx43 via phosphorylation followed by its degradation is vital for preadipocyte differentiation and maturation of functional adipocytes.► Changes in Cx43 levels are required for adipogenic differentiation. ► Transient alterations in Cx43 subcellular localization occur during adipogenesis. ► Cx43 phosphorylation is transiently elevated during clonal induction of adipogenesis. ► Inhibition of proteasomal degradation of Cx43 blocks adipogenesis. ► Constitutive expression of Cx43 inhibits adipocyte maturation.

Keywords: Adipogenesis; Connexin-43; Gap junction

DNA and nuclear aggregates of polyamines by Giuseppe Iacomino; Gianluca Picariello; Luciano D'Agostino (pp. 1745-1755).

Polyamines (PAs) are linear polycations that are involved in many biological functions. Putrescine, spermidine and spermine are highly represented in the nucleus of eukaryotic cells and have been the subject of decades of extensive research. Nevertheless, their capability to modulate the structure and functions of DNA has not been fully elucidated. We found that polyamines self-assemble with phosphate ions in the cell nucleus and generate three forms of compounds referred to as Nuclear Aggregates of Polyamines (NAPs), which interact with genomic DNA. In an in vitro setting that mimics the nuclear environment, the assembly of PAs occurs within well-defined ratios, independent of the presence of the DNA template. Strict structural and functional analogies exist between the in vitro NAPs ( ivNAPs) and their cellular homologues. Atomic force microscopy showed that ivNAPs, as theoretically predicted, have a cyclic structure, and in the presence of DNA, they form a tube-like arrangement around the double helix. Features of the interaction between ivNAPs and genomic DNA provide evidence for the decisive role of “natural” NAPs in regulating important aspects of DNA physiology, such as conformation, protection and packaging, thus suggesting a new vision of the functions that PAs accomplish in the cell nucleus.Display Omitted► Polyamines self-assemble with phosphate, both in vivo and in vitro, generating NAP. ► NAP have a basic cyclic structure. ► The hierarchical growth of NAP generates filamentous structures. ► NAP protect DNA and assist its conformational transitions. ► NAP/DNA complexes arrange in long and repetitive structures.

Keywords: Nuclear aggregates of polyamines; Genomic DNA; DNA packaging; Self-assembly; Supramolecular chemistry; Molecular recognition

A nuclear export sequence in GPN-loop GTPase 1, an essential protein for nuclear targeting of RNA polymerase II, is necessary and sufficient for nuclear export by Humberto Reyes-Pardo; Angel A. Barbosa-Camacho; Perez-Mejia Ana E. Pérez-Mejía; Lara-Chacon Bárbara Lara-Chacón; Leslie A. Salas-Estrada; Angélica Y. Robledo-Rivera; Montero-Moran Gabriela M. Montero-Morán; Lara-Gonzalez Samuel Lara-González; Mónica R. Calera; Sanchez-Olea Roberto Sánchez-Olea (pp. 1756-1766).

XAB1/Gpn1 is a GTPase that associates with RNA polymerase II (RNAPII) in a GTP-dependent manner. Although XAB1/Gpn1 is essential for nuclear accumulation of RNAPII, the underlying mechanism is not known. A XAB1/Gpn1-EYFP fluorescent protein, like endogenous XAB1/Gpn1, localized to the cytoplasm but it rapidly accumulated in the cell nucleus in the presence of leptomycin B, a chemical inhibitor of the nuclear transport receptor Crm1. Crm1 recognizes short peptides in substrate proteins called nuclear export sequences (NES). Here, we employed site-directed mutagenesis and fluorescence microscopy to assess the functionality of all six putative NESs in XAB1/Gpn1. Mutating five of the six putative NESs did not alter the cytoplasmic localization of XAB1/Gpn1-EYFP. However, a V302A/L304A double mutant XAB1/Gpn1-EYFP protein was clearly accumulated in the cell nucleus, indicating the disruption of a functional NES. This functional XAB1/Gpn1 NES displays all features present in most common and potent NESs, including, in addition to Φ1–Φ4, a critical fifth hydrophobic amino acid Φ0. Therefore, in human Gpn1 this NES spans amino acids 292-LERLRKDMGSVAL-304. XAB1/Gpn1 NES is remarkably conserved during evolution. XAB1/Gpn1 NES was sufficient for nuclear export activity, as it caused a complete exclusion of EYFP from the cell nucleus. Molecular modeling of XAB1/Gpn1 provided a mechanistic reason for NES selection, as functionality correlated with accessibility, and it also suggested a mechanism for NES inhibition by intramolecular masking. In conclusion, we have identified a highly active, evolutionarily conserved NES in XAB1/Gpn1 that is critical for nucleo-cytoplasmic shuttling and steady-state cytoplasmic localization of XAB1/Gpn1.► The GTPase Gpn1 is essential for the nuclear accumulation of RNA polymerase II. ► Although Gpn1 is cytoplasmic, it shuttles between the cytoplasm and cell nucleus. ► We identify the nuclear export sequence (NES) that mediates nuclear export of Gpn1 ► Gpn1 NES is highly conserved and both, necessary and sufficient, for nuclear export. ► Molecular modeling of Gpn1 revealed that NES accessibility equals functionality.

Keywords: GPN-loop GTPase 1 (Gpn1); XAB1; Nuclear export sequence; Nucleocytoplasmic transport; RNA polymerase II nuclear targeting

Glutathione and modulation of cell apoptosis by Magdalena L. Circu; Tak Yee Aw (pp. 1767-1777).

Apoptosis is a highly organized form of cell death that is important for tissue homeostasis, organ development and senescence. To date, the extrinsic (death receptor mediated) and intrinsic (mitochondria derived) apoptotic pathways have been characterized in mammalian cells. Reduced glutathione, is the most prevalent cellular thiol that plays an essential role in preserving a reduced intracellular environment. glutathione protection of cellular macromolecules like deoxyribose nucleic acid proteins and lipids against oxidizing, environmental and cytotoxic agents, underscores its central anti-apoptotic function. Reactive oxygen and nitrogen species can oxidize cellular glutathione or induce its extracellular export leading to the loss of intracellular redox homeostasis and activation of the apoptotic signaling cascade. Recent evidence uncovered a novel role for glutathione involvement in apoptotic signaling pathways wherein post-translational S-glutathiolation of protein redox active cysteines is implicated in the potentiation of apoptosis. In the present review we focus on the key aspects of glutathione redox mechanisms associated with apoptotic signaling that includes: (a) changes in cellular glutathione redox homeostasis through glutathione oxidation or GSH transport in relation to the initiation or propagation of the apoptotic cascade, and (b) evidence for S-glutathiolation in protein modulation and apoptotic initiation.► Death receptor-mediated and mitochondria-mediated pathways as modes of apoptosis. ► Distinct GSH redox pools exist in subcellular organelles within cells. ► Disrupted cellular GSH balance contributes to activation of the apoptotic cascade. ► Formation of S-glutathiolated proteins is important in apoptotic signaling.

Keywords: Abbreviations; ∆Ψ; mitochondrial membrane potential; ANT; adenine nucleotide translocase; Apaf-1; apoptotic protease-activating factor-1; ASK-1; apoptosis signal-regulating kinase-1; ATO; arsenic trioxide; AVD; apoptotic volume decrease; BSO; l; -buthionine (; S; ,; R; )-sulfoximine; Cyt; c; cytochrome; c; DEM; diethyl maleate; Diablo; direct inhibitor of apoptosis binding protein with low pI; DIC; dicarboxylate carrier; DISC; death-inducing signaling complex; DR; death receptor; EndoG; endonuclease G; ER; endoplasmic reticulum; Ero1; ER-resident endoplasmic oxidoreductin 1; ERK; extracellular signal-regulated kinase; FADD; Fas-associated death domain; FLIP; FLICE (caspase-8)-inhibitory protein; GCL; glutamate cysteine ligase; GGT; γ-glutamyl transpeptidase; GR; glutathione reductase; GS; GSH synthase; IAP; inhibitors of apoptotic proteins; IMAC; inner membrane anion channel; JNK; c-Jun N-terminal kinase; MAPK; mitogen-activated protein kinase; MAPKKK or MAP3K; MAPK kinase kinase; MAPK kinase or MAP2K; MAPK kinase; MQ; menadione; MRP1; multidrug resistance protein-1; mtDNA; mitochondrial DNA; mtGSH; mitochondrial GSH; OATP; organic anion transporting polypeptide; OGC; 2-oxoglutarate carrier; OMM; outer mitochondrial membrane; PDI; protein disulfide isomerase; PEITC; β-phenethyl isothiocyanate; Pr-SH; protein containing a reduced sulfhydryl group; Pr-SSG; S-glutathiolated protein; PS; phosphatydyl serine; PTP; permeability transition pore; RIP1; receptor-interacting protein 1; Smac; second mitochondria-derived activator of caspases; SOD; superoxide dismutase; TRADD; TNF receptor type 1-associated death domain; TRAF2; TNF receptor-associated factor 2; UPR; unfolding protein response; VDAC; voltage dependent anion channelGSH oxidation and apoptosis; GSH efflux and apoptosis; S-glutathiolation potentiation of apoptotic cascade; GSH compartmentation

The endocytic adaptor protein Disabled-2 is required for cellular uptake of fibrinogen by Wei-Shan Hung; Chien-Ling Huang; Jui-Ting Fan; Ding-Yuan Huang; Chun-Fung Yeh; Ju-Chien Cheng; Ching-Ping Tseng (pp. 1778-1788).

Endocytosis is pivotal for uptake of fibrinogen from plasma into megakaryocytes and platelet α-granules. Due to the complex adaptor and cargo contents in endocytic vehicles, the underlying mechanism of fibrinogen uptake is not yet completely elucidated. In this study, we investigated whether the endocytic adaptor protein Disabled-2 (DAB2) mediates fibrinogen uptake in an adaptor-specific manner. By employing primary megakaryocytes and megakaryocytic differentiating human leukemic K562 cells as the study models, we found that fibrinogen uptake is associated with the expression of integrin αIIbβ3 and DAB2 and is mediated through clathrin-dependent manner. Accordingly, constitutive and inducible knockdown of DAB2 by small interfering RNA reduced fibrinogen uptake for 53.2±9.8% and 59.0±10.7%, respectively. Culturing the cells in hypertonic solution or in the presence of clathrin inhibitor chlorpromazine abrogated clathrin-dependent endocytosis and diminished the uptake of fibrinogen. Consistent with these findings, 72.2±0.2% of cellular DAB2 was colocalized with clathrin, whereas 56.4±4.1% and 54.6±2.0% of the internalized fibrinogen were colocalized with clathrin and DAB2, respectively. To delineate whether DAB2 mediates fibrinogen uptake in an adaptor-specific manner, K562 stable cell lines with knockdown of the adaptor protein-2 (AP-2) or double knockdown of AP-2/DAB2 were established. The AP-2 knockdown cells elicited normal fibrinogen uptake activity but the uptake of collagen was diminished. In addition, collagen uptake was further reduced in DAB2/AP-2 knockdown cells. These findings thereby define an adaptor-specific mechanism in the control of fibrinogen uptake and implicate that DAB2 is the key adaptor in the clathrin-associated endocytic complexes to mediate fibrinogen internalization.► Fibrinogen uptake is associated with the expression of DAB2 and is clathrin-dependent. ► Knockdown of DAB2 reduces fibrinogen uptake. ► Fibrinogen is colocalized with clathrin and DAB2. ► Knockdown of AP-2 has no effect on fibrinogen uptake but diminishes collagen uptake. ► DAB2 is the key adaptor mediating fibrinogen uptake.

Keywords: Abbreviations; AP-2; adaptor protein-2; ApoER2; apolipoprotein E receptor 2; ARH; autosomal recessive hypercholesterolemia; CPZ; chlorpromazine; DAB2; Disabled-2; DAPI; 4′,6-diamidino-2-phenylindole; HRP; horseradish peroxidase; LDLR; low density lipoprotein receptor; LF2000; Lipofectamine 2000; LRP1; low density lipoprotein receptor-related protein 1; MFI; mean fluorescence index; PTB; phosphotyrosine binding; TR; transferrin receptor; TPO; thrombopoietin; TPA; 12-O-tetradecanoylphorbol-13-acetateDisabled-2; Endocytosis; Fibrinogen; Integrin αIIbβ3; Megakaryocytic differentiation

Seminal plasma induces angiogenic chemokine expression in cervical cancer cells and regulates vascular function by Kurt J. Sales; Jason R. Sutherland; Henry N. Jabbour; Arieh A. Katz (pp. 1789-1795).

Cervical cancer is one of the leading gynecological malignancies in women. We have recently shown that seminal plasma (SP) can regulate the inflammatory cyclooxygenase-prostaglandin pathway and enhance the growth of cervical epithelial tumours in vivo by promoting cellular proliferation and alteration of vascular function. This study investigated the molecular mechanism whereby SP regulates vascular function using an in vitro model system of HeLa cervical adenocarcinoma cells and human umbilical vein endothelial cells (HUVECs). We found that SP rapidly enhanced the expression of the angiogenic chemokines, interleukin (IL)-8 and growth regulated oncogene alpha (GRO) in HeLa cells in a time-dependent manner. We investigated the molecular mechanism of SP-mediated regulation of IL-8 and GRO using a panel of chemical inhibitors of cell signalling. We found that treatment of HeLa cells with SP elevated expression of IL-8 and GRO by transactivation of the epidermal growth factor receptor, activation of extracellular signal-regulated kinase and induction of cyclooxygenase enzymes and nuclear factor kappa B. We investigated the impact of IL-8 and GRO, released from HeLa cells after treatment with SP, on vascular function using a co-culture model system of conditioned medium (CM) from HeLa cells, treated with or without SP, and HUVECs. We found that CM from HeLa cells induced the arrangement of endothelial cells into a network of tube-like structures via the CXCR2 receptor on HUVECs. Taken together our data outline a molecular mechanism whereby SP can alter vascular function in cervical cancers via the pro-angiogenic chemokines, IL-8 and GRO.► The impact of seminal plasma on cervical tumour growth was assessed in vitro. ► Seminal plasma induces expression of chemokines in cervical cancer cells. ► Chemokines mediated by seminal plasma alter vascular function. ► Seminal plasma activates extracellular signal regulated kinase (ERK1/2). ► ERK1/2 activation is dependent on the epidermal growth factor receptor.

Keywords: Inflammation; Seminal fluid; Cyclooxygenase; Prostaglandin; Signalling; Cervical cancer

Annexin A7 and SNAP23 interactions in alveolar type II cells and in vitro: A role for Ca²⁺ and PKC by Tudevdagva Gerelsaikhan; Pavan Kumar Vasa; Avinash Chander (pp. 1796-1806).

Lung surfactant secretion involves lamellar body docking and fusion with the plasma membrane in alveolar type II cells. Annexin A7 (A7) is postulated to play a role in membrane fusion during exocytosis. Our recent studies demonstrated increased co-localization of A7 with ABCA3 in lamellar bodies in type II cells stimulated with established secretagogues of lung surfactant. In this study, we investigated in vivo and in vitro interactions of A7 with the t-SNARE protein, SNAP23. Immuno-fluorescence studies showed time-dependent increases in co-localization of A7 with SNAP23 in PMA- and in A23187-stimulated cells. PMA and A23187 also caused a time-dependent increase in co-localization of ABCA3 with SNAP23. The relocation of A7 to SNAP23 domains was inhibited in the presence of PKC inhibitor, similar to that previously reported for co-localization of A7 with ABCA3. The interaction of A7 and SNAP23 was confirmed by affinity binding and by in vitro interaction of recombinant A7 and SNAP23 proteins. The in vitro binding of recombinant A7 (rA7) to GST-SNAP23 fusion protein was calcium-dependent. Phosphorylation of rA7 with PKC increased its in vitro binding to SNAP23 suggesting that a similar mechanism may operate during A7 relocation to t-SNARE domains. Thus, our studies demonstrate that annexin A7 may function in co-ordination with SNARE proteins and that protein kinase activation may be required for annexin A7 trafficking to the interacting membranes (lamellar bodies and plasma membrane) to facilitate membrane fusion during surfactant secretion.► Interactions of annexin A7 with SNARE protein SNAP23 are demonstrated. ► Surfactant secretagogues increase annexin A7 and SNAP23 interactions in lung cells. ► The intracellular co-localization and in vitro binding is PKC dependent. ► The trafficking of lamellar body to SNAP23 domains is regulated by PKC.

Keywords: Lung surfactant secretion; t-SNARE protein; Exocytosis; Membrane fusion; Lamellar body; Protein kinase C

Pdcd4 knockdown up-regulates MAP4K1 expression and activation of AP-1 dependent transcription through c-Myc by Qing Wang; Yan Zhang; Hsin-Sheng Yang (pp. 1807-1814).

Programme d cell death 4 (Pdcd4) is a novel tumor suppressor, whose expression is frequently down-regulated in several types of cancers. In the present study, we demonstrated that Pdcd4 knockdown up-regulates MAP kinase kinase kinase kinase 1 (MAP4K1) expression and increases phosphorylation of c-Jun. Over-expression of c-Myc in HEK293 cells increases the levels of MAP4K1, MAP4K1 promoter activity, and phospho-c-Jun. Mutation analysis showed that the c-Myc binding site at −536bp (relative to the initiation ATG) of map4k1 promoter responds to c-Myc regulation. In addition, chromatin immunoprecipitation demonstrated that c-Myc directly binds to map4k1 promoter at this site. Down-regulation of c-Myc reverses MAP4K1 expression and AP-1 activation in Pdcd4 knockdown cells. Moreover, over-expression of dominant negative Tcf4 decreases expression of c-Myc and MAP4K1, JNK activation, and AP-1 dependent transcription. Thus, activation of β-catenin/Tcf dependent transcription in Pdcd4 knockdown cells up-regulates MAP4K1 expression and AP-1 activity via c-Myc. The study presented here further reveals in detail the mechanism of how Pdcd4 inhibits tumor cell invasion and provides a functional connection between β-catenin/Tcf and AP-1 dependent transcription.Display Omitted► The c-Myc binds to MAP4K1 promoter and regulates MAP4K1 promoter activity. ► Down-regulation of c-Myc inhibits MAP4K1 expression and AP-1 activation. ► The dnTcf4 inhibits c-Myc and MAP4K1 expression. ► Pdcd4 knockdown up-regulates MAP4K1 expression and activation of AP-1 via c-Myc.

Keywords: Abbreviations; Pdcd4; programmed cell death 4; MAP4K1; mitogen-activated protein kinase kinase kinase kinase 1; AP-1; activator protein-1; TPA; 12-O-tetradecanoylphorbol-13-acetate; DMBA; 7,12-dimethylbenz(a)anthracene; TAK1; transforming growth factor β‐activated kinase 1; MKK4; mitogen‐activated kinase kinase 4; JNK; Jun N-terminal kinase; ERK; extracellular signal-regulated kinases; ChIP; chromatin immunoprecipitation; siRNA; small interfering RNAPdcd4; MAP4K1; JNK signaling pathway; AP-1; c-Myc

Pyruvate dehydrogenase E1α phosphorylation is induced by glucose but does not control metabolism-secretion coupling in INS-1E clonal β-cells by Dmitry Akhmedov; Umberto De Marchi; Claes B. Wollheim; Andreas Wiederkehr (pp. 1815-1824).

Glucose-induced insulin secretion from pancreatic β-cells depends on mitochondrial activation. In the organelle, glucose-derived pyruvate is metabolised along the oxidative and anaplerotic pathway to generate downstream signals leading to insulin granule exocytosis. Entry into the oxidative pathway is catalysed by pyruvate dehydrogenase (PDH) and controlled in part by phosphorylation of the PDH E1α subunit blocking enzyme activity. We find that glucose but not other nutrient secretagogues induce PDH E1α phosphorylation in INS-1E cells and rat islets. INS-1E cells and primary β-cells express pyruvate dehydrogenase kinase (PDK) 1, 2 and 3, which mediate the observed phosphorylation. In INS-1E cells, suppression of the two main isoforms, PDK1 and PDK3, almost completely prevented PDH E1α phosphorylation. Under basal glucose conditions, phosphorylation was barely detectable and therefore the enzyme almost fully active (90% of maximal). During glucose stimulation, PDH is only partially inhibited (to 78% of maximal). Preventing PDH phosphorylation in situ after suppression of PDK1, 2 and 3 neither enhanced pyruvate oxidation nor insulin secretion. In conclusion, although glucose stimulates E1α phosphorylation and therefore inhibits PDH activity, this control mechanism by itself does not alter metabolism-secretion coupling in INS-1E clonal β-cells.► Glucose induces phosphorylation of PDH in INS-1E cells and islets. ► Glucose-induced PDH E1α phosphorylation is mainly mediated by PDK1 and PDK3. ► Glucose stimulation causes a partial inhibition of pyruvate dehydrogenase activity. ► Preventing PDH E1α phosphorylation does not alter glucose-induced insulin secretion.

Keywords: Beta-cell; Insulin; Pyruvate dehydrogenase; Pyruvate dehydrogenase kinase

The molecular mechanism of apoptosis upon caspase-8 activation: Quantitative experimental validation of a mathematical model by Katsuya Kominami; Jun Nakabayashi; Takeharu Nagai; Yuki Tsujimura; Kumiko Chiba; Haruna Kimura; Atsushi Miyawaki; Tatsuya Sawasaki; Hideo Yokota; Noboru Manabe; Kazuhiro Sakamaki (pp. 1825-1840).

Caspase-8 (CASP8) is a cysteine protease that plays a pivotal role in the extrinsic apoptotic signaling pathway via death receptors. The kinetics, dynamics, and selectivity with which the pathway transmits apoptotic signals to downstream molecules upon CASP8 activation are not fully understood. We have developed a system for using high-sensitivity FRET-based biosensors to monitor the protease activity of CASP8 and its downstream effector, caspase-3, in living single cells. Using this system, we systematically investigated the caspase cascade by regulating the magnitude of extrinsic signals received by the cell. Furthermore, we determined the molar concentration of five caspases and Bid required for hierarchical transmission of apoptotic signals in a HeLa cell. Based on these quantitative experimental data, we validated a mathematical model suitable for estimation of the kinetics and dynamics of caspases, which predicts the minimal concentration of CASP8 required to act as an initiator. Consequently, we found that less than 1% of the total CASP8 proteins are sufficient to set the apoptotic program in motion if activated. Taken together, our findings demonstrate the precise cascade of CASP8-mediated apoptotic signals through the extrinsic pathway.► A monitoring system with high sensitivity FRET-based biosensors has been developed. ► The established system enabled us to detect the most minute dynamics of caspase activation in single cells. ► The kinetics, dynamics, and selectivity in apoptotic signaling pathways with activation of caspase-8 were redefined. ► The actual number of pro-apoptotic molecules required for extrinsic apoptotic signal transduction was evaluated. ► A mathematical model predicted the minimal concentration of caspase-8 required to act as an initiator.

Keywords: Abbreviations; ARK5; AMPK-related protein kinase 5; Bid; BH3-interacting domain death agonist; CARP; caspases-8- and -10-associated RING protein; CASP; caspase; CHX; cycloheximide; DED; death effector domain; DISC; death-inducing signaling complex; EGFP; enhanced green fluorescent protein; FADD; Fas-associated death domain protein; FasL; Fas ligand; FCS; fetal calf serum; FRB; FKBP rapamycin-binding domain of the FKBP-associated protein; FRET; fluorescence resonance energy transfer; Fv; FK506-binding protein containing a F36V mutation; mRFP1; monomeric red fluorescent protein 1; PARP; poly(ADP-ribose) polymerase; proCASP; procaspase; seCFP; super-enhanced cyan fluorescent protein; siRNA; small interfering RNA; TRAIL; tumor necrosis factor-related apoptosis-inducing ligand; XIAP; X-chromosome linked inhibitor of apoptosis protein; z-IETD-fmk; carbobenzoyl-Ile-Glu-Thr-Asp-fluoromethylketoneApoptosis; Caspase cascade; FRET; Mathematical model; Positive-feedback loop

The small MAF transcription factors MAFF, MAFG and MAFK: Current knowledge and perspectives by Meenakshi B. Kannan; Vera Solovieva; Volker Blank (pp. 1841-1846).

The small MAFs, MAFF, MAFG and MAFK have emerged as crucial regulators of mammalian gene expression. Previous studies have linked small MAF function, by virtue of their heterodimerization with the Cap ‘n’ Collar (CNC) family of transcription factors, to the stress response and detoxification pathways. Recent analyses have revealed a complex regulatory network involving small MAF transcription factors and other cellular proteins. The expression and activity of small MAFs are tightly regulated at multiple levels. With regard to their clinical importance, small MAFs have been linked to various diseases, such as diabetes, neuronal disorders, thrombocytopenia and carcinogenesis. A better understanding of the molecular mechanisms governing the activity of small MAFs will provide novel insights into the control of mammalian transcription and may lead to the development of novel therapeutic strategies to treat common human disorders.► Small MAFs have emerged as crucial regulators of mammalian gene expression. ► Small MAF expression and activity is tightly regulated. ► Small MAFs may play a role a series of diseases. ► Diseases include diabetes, neuronal disorders, thrombocytopenia and cancer.

Keywords: MAF; bZIP; CNC; Gene regulation; Stress signaling; Cancer

The Ras-ERK MAPK regulatory network controls dedifferentiation in Caenorhabditis elegans germline by Dong Seok Cha; Udaya Sree Datla; Sarah E. Hollis; Judith Kimble; Myon-Hee Lee (pp. 1847-1855).

How a committed cell can be reverted to an undifferentiated state is a central question in stem cell biology. This process, called dedifferentiation, is likely to be important for replacing stem cells as they age or get damaged. Tremendous progress has been made in understanding this fundamental process, but its mechanisms are poorly understood. Here we demonstrate that the aberrant activation of Ras-ERK MAPK signaling promotes cellular dedifferentiation in the Caenorhabditis elegans germline. To activate signaling, we removed two negative regulators, the PUF-8 RNA-binding protein and LIP-1 dual specificity phosphatase. The removal of both of these two regulators caused secondary spermatocytes to dedifferentiate and begin mitotic divisions. Interestingly, reduction of Ras-ERK MAPK signaling, either by mutation or chemical inhibition, blocked the initiation of dedifferentiation. By RNAi screening, we identified RSKN-1/P90_RSK as a downstream effector of MPK-1/ERK that is critical for dedifferentiation: rskn-1 RNAi suppressed spermatocyte dedifferentiation and instead induced meiotic divisions. These regulators are broadly conserved, suggesting that similar molecular circuitry may control cellular dedifferentiation in other organisms, including humans.Display Omitted► PUF-8 and LIP-1 normally repress dedifferentiation by inhibiting MPK-1/ERK MAPK signaling. ► Active MPK-1/ERK controls initiation of dedifferentiation. ► RSKN-1/P90_RSK, a downstream effector of MPK-1/ERK, is critical for dedifferentiation.

Keywords: Ras-ERK MAPK signaling; PUF RNA-binding protein; Dedifferentiation; RSKN-1/P90; _RSK; Caenorhabditis elegans; Spermatocytes

Role of mitochondrial uncoupling protein 2 in cancer cell resistance to gemcitabine by Elisa Dalla Pozza; Claudia Fiorini; Ilaria Dando; Marta Menegazzi; Anna Sgarbossa; Chiara Costanzo; Marta Palmieri; Massimo Donadelli (pp. 1856-1863).

Cancer cells exhibit an endogenous constitutive oxidative stress higher than that of normal cells, which renders tumours vulnerable to further reactive oxygen species (ROS) production. Mitochondrial uncoupling protein 2 (UCP2) can mitigate oxidative stress by increasing the influx of protons into the mitochondrial matrix and reducing electron leakage and mitochondrial superoxide generation. Here, we demonstrate that chemical uncouplers or UCP2 over-expression strongly decrease mitochondrial superoxide induction by the anticancer drug gemcitabine (GEM) and protect cancer cells from GEM-induced apoptosis. Moreover, we show that GEM IC₅₀ values well correlate with the endogenous level of UCP2 mRNA, suggesting a critical role for mitochondrial uncoupling in GEM resistance. Interestingly, GEM treatment stimulates UCP2 mRNA expression suggesting that mitochondrial uncoupling could have a role also in the acquired resistance to GEM. Conversely, UCP2 inhibition by genipin or UCP2 mRNA silencing strongly enhances GEM-induced mitochondrial superoxide generation and apoptosis, synergistically inhibiting cancer cell proliferation. These events are significantly reduced by the addition of the radical scavenger N-acetyl-l-cysteine or MnSOD over-expression, demonstrating a critical role of the oxidative stress. Normal primary fibroblasts are much less sensitive to GEM/genipin combination. Our results demonstrate for the first time that UCP2 has a role in cancer cell resistance to GEM supporting the development of an anti-cancer therapy based on UCP2 inhibition associated to GEM treatment.► Mitochondrial uncoupling reduces GEM effect in cancer cells ► GEM and UCP2 inhibition synergistically inhibit cancer cell proliferation ► GEM and UCP2 inhibition induce ROS-mediated apoptosis in cancer cells ► UCP2 expression is a potential predictive biomarker of cancer resistance to GEM ► UCP2 is an efficient molecular target to selectively inhibit cancer cell proliferation

Keywords: Cancer; Gemcitabine; Uncoupling protein 2; Oxidative stress; Apoptosis; Resistance

ERK1/2-dependent bestrophin-3 expression prevents ER-stress-induced cell death in renal epithelial cells by reducing CHOP by Wing-Kee Lee; Prabir K. Chakraborty; Eleni Roussa; Natascha A. Wolff; Thevenod Frank Thévenod (pp. 1864-1876).

Upon endoplasmic reticulum (ER) stress induction, cells endeavor to survive by engaging the adaptive stress response known as the unfolded protein response or by removing aggregated proteins via autophagy. Chronic ER stress culminates in apoptotic cell death, which involves induction of pro-apoptotic CHOP. Here, we show that bestrophin-3 (Best-3), a protein previously associated with Ca²⁺-activated Cl⁻ channel activity, is upregulated by the ER stressors, thapsigargin (TG), tunicamycin (TUN) and the toxic metal Cd²⁺. In cultured rat kidney proximal tubule cells, ER stress, CHOP and cell death were induced after 6h by Cd²⁺ (25μM), TG (3μM) and TUN (6μM), were associated with increased cytosolic Ca²⁺ and downstream formation of reactive oxygen species and attenuated by the Ca²⁺ chelator BAPTA-AM (10μM), the antioxidant α-tocopherol (100μM), or overexpression of catalase (CAT). Immunofluorescence staining showed Best-3 expression in the plasma membrane, nuclei and intracellular compartments, though not in the ER, in cultured cells and rat kidney cortex sections. Best-3 mRNA was augmented by ER stress and signaled through increased Ca²⁺, oxidative stress and ERK1/2 phosphorylation, because it was attenuated by α-tocopherol, CAT expression, BAPTA-AM, calmodulin kinase inhibitor calmidazolium (40μM), ERK1/2 inhibitor U0126 (10μM), and ERK1/2 RNAi. Knockdown of Best-3 resulted in decreased cell number consequentially of cell death, as determined by nuclear staining and PARP-1 cleavage. Furthermore, reduced ER stress-cell death by Best-3 overexpression is attributed to diminished CHOP. Since Best-3 overexpression did not affect upstream signaling pathways, we hypothesize that Best-3 possibly interferes with CHOP transcription. From our novel observations, we conclude that ERK1/2-dependent Best-3 activation regulates cell fate decisions during ER stress by suppressing CHOP induction and death.► In kidney cells, Bestrophin-3 is expressed in plasma membrane, nuclei and intracellular compartments. ► Renal Bestrophin-3 is increased by ER stress. ► ER stress-induced Bestrophin-3 expression depends on Ca²⁺, ROS and ERK. ► Bestrophin-3 enhances cell survival during ER stress. ► Bestrophin 3-dependent cell survival occurs through reduction of pro-apoptotic CHOP.

Keywords: Abbreviations; CAT; catalase; ER; endoplasmic reticulum; PERK; RNA‐dependent protein kinase-like ER kinase; IRE1; inositol-requiring ER-to-nucleus signal kinase 1; CHOP; CCAAT/enhancer-binding protein-homologous protein; ERK; extracellular signal-regulated kinase; TUN; tunicamycin; TG; thapsigargin; SERCA; sarcoplasmic/endoplasmic reticulum Ca; ²⁺; -ATPase; Cd; ²⁺; cadmium; Best; bestrophin; UPR; unfolded protein response; PT; proximal tubuleReactive oxygen species; ERK; Calcium; Apoptosis; Unfolded protein response

The major isoforms of Bim contribute to distinct biological activities that govern the processes of autophagy and apoptosis in interleukin-7 dependent lymphocytes by Shannon M. Ruppert; Wenqing Li; Ge Zhang; Adina L. Carlson; Arati Limaye; Scott K. Durum; Annette R. Khaled (pp. 1877-1893).

Bim is a BH3-only member of the Bcl-2 family that enables the death of T-cells. Partial rescue of cytokine-deprived T-cells occurs when Bim and the receptor for the T-cell growth factor, interleukin-7, are deleted, implicating Bim as a possible target of interleukin-7-mediated signaling. Alternative splicing yields three major isoforms: BimEL, BimL and BimS. To study the effect of Bim deficiency and define the function of the major isoforms, Bim-containing and Bim-deficient T-cells, dependent on interleukin-7 for growth, were used. Loss of total Bim in interleukin-7-deprived T-cells resulted in delayed apoptosis. However, loss of Bim also impeded the later degradative phase of autophagy. p62, an autophagy-adaptor protein which is normally degraded, accumulated in Bim deficient cells. To explain this, BimL was found to support acidification of lysosomes that later may associate with autophagic vesicles. Key findings showed that inhibition of lysosomal acidification accelerated death upon interleukin-7 withdrawal only in Bim-containing T-cells. intereukin-7 dependent T-cells lacking Bim were less sensitive to inhibition of lysosomal acidification. BimL co-immunoprecipitated with dynein and Lamp1-containing vesicles, indicating BimL could be an adaptor for dynein to facilitate loading of lysosomes. In Bim deficient T-cells, lysosome-tracking probes revealed vesicles of less acidic pH. Over-expression of BimL restored acidic vesicles in Bim deficient T-cells, while other isoforms, BimEL and BimS, promoted intrinsic cell death. These results reveal a novel role for BimL in lysosomal positioning that may be required for the formation of degradative autolysosomes.► Bim-mediated activities contribute to IL-7-driven T-cell homeostasis. ► Bim deficiency delays apoptosis and impairs the degradative phase of autophagy. ► Bim deficiency makes T-cells resistant to autophagy inhibition. ► Supported by IL-7, BimL interacts with dynein to enable lysosome acidification. ► BimEL and BimS promote apoptosis during IL-7 withdrawal.

Keywords: Bcl-2; Cytokine; Lysosome; Fluorescence; Acidification; Dynein

Dual function of Pin1 in NR4A nuclear receptor activation: Enhanced activity of NR4As and increased Nur77 protein stability by Claudia M. van Tiel; Kondababu Kurakula; Duco S. Koenis; Erik van der Wal; Carlie J.M. de Vries (pp. 1894-1904).

Nur77, Nurr1 and NOR-1 form the NR4A subfamily of the nuclear receptor superfamily and have been shown to regulate various biological processes among which are cell survival and differentiation, apoptosis, inflammation and metabolism. These nuclear receptors have been proposed to act in a ligand-independent manner and we aim to gain insight in the regulation of NR4A activity. A yeast two-hybrid screen identified the peptidyl-prolyl isomerase Pin1 as a novel binding partner of NR4As, which was confirmed by co-immunoprecipitation. Pin1 enhances the transcriptional activity of all three NR4A nuclear receptors and increases protein stability of Nur77 through inhibition of its ubiquitination. Enhanced transcriptional activity of NR4As requires the WW-domain of Pin1 that interacts with the N-terminal transactivation domain and the DNA-binding domain of Nur77. Most remarkably, this enhanced activity is independent of Pin1 isomerase activity. A systematic mutation analysis of all 17 Ser/Thr-Pro-motifs in Nur77 revealed that Pin1 enhances protein stability of Nur77 in an isomerase-dependent manner by acting on phosphorylated Nur77 involving protein kinase CK2-mediated phosphorylation of the Ser¹⁵²-Pro¹⁵³ motif in Nur77. Given the role of Nur77 in vascular disease and metabolism, this novel regulation mechanism provides perspectives to manipulate Nur77 activity to attenuate these processes.► NR4A nuclear orphan receptors are important in vascular disease and metabolism. ► Pin1 binds NR4A receptors and enhances their transcriptional activity. ► Pin1 increases Nur77, but not Nurr1 or NOR-1 protein stability. ► Serine152 is crucial in Pin1-mediated Nur77 protein stability. ► This novel regulation mechanism offers new possibilities to manipulate NR4A activity.

Keywords: Abbreviations; CHX; cycloheximide; CK2; protein kinase CK2; DBD; DNA-binding domain; DCOH; dimerization cofactor for hepatocyte nuclear factor 1; GST; glutathione S-transferase; LBD; ligand-binding domain; Pin1; peptidyl-prolyl; cis/trans; isomerase 1; PPIase; Peptidyl-prolyl isomerase; pSer/pThr-Pro; phosphorylated proline-directed serine/threonine; TBB; 4,5,6,7-tetrabromobenzotriazoleProtein–protein interaction; Protein stability; Transcription factor; NR4A nuclear receptors

Monocyte chemotactic protein-1-induced protein-1 (MCPIP1) is a novel multifunctional modulator of inflammatory reactions by Jolanta Jura; Lukasz Skalniak; Aleksander Koj (pp. 1905-1913).

The generalized inflammatory response leads to activation of hundreds of genes transcribed in an established sequence in specialized cells. Transcriptome analysis of human monocyte-derived cells stimulated with IL-1beta or with monocyte chemotactic protein-1 (MCP-1) has led to the identification of a new inflammation-related gene ZC3H12A encoding a chain of 599 amino acids corresponding to a 66-kDa protein. The protein, given a provisional name of MCPIP1 (monocyte chemotactic protein-induced protein-1), is expressed in several human and murine tissues such as bone marrow, spleen, heart and placenta. In in vivo studies, mice with inactivated MCPIP1-encoding gene showed growth retardation, lymphadenopathy, splenomegaly and enhanced inflammatory symptoms. Principal molecular features of MCPIP1 include a single zinc finger motif, an RNase-like PIN domain and ubiquitin-binding domain. Reports from independent laboratories suggest that MCPIP1 may function also as a deubiquitinase. Although MCPIP1 is regarded by some authors as a new transcription factor or cell differentiation factor modulating angiogenesis or adipogenesis, its principal function appears to be downregulation of inflammatory responses through at least two independent mechanisms: increased degradation of cytokine mRNAs and inhibition of LPS- and IL-1-induced NF-kappaB signaling pathway. The interference with NF-kappaB activation is highly complex and includes TRAF6 and TANK interaction with the ubiquitin-associated (UBA) domain of MCPIP1. Purified MCPIP1 protein was reported to degrade specific mRNA and cleave K48- and K63-linked polyubiquitin chains. Although some structural features and the mechanism of action of MCPIP1 are not fully explained yet, its importance in the regulation of inflammatory reactions has been firmly established.► MCPIP1 acts as an RNase regulating stability of transcripts coding for IL-1β, IL-6, IL-12 but also affecting miRNA synthesis. ► It functions also as a negative regulator of transcription factors NF-κB and AP-1. ► In vivo studies on mice indicate MCPIP1 as an important regulator of inflammatory response.

Keywords: mRNA stability and decay; RNase; Proinflammatory cytokines; NF-kappaB signaling cascade; Ubiquitin-binding domains; Deubiquitinase

TrxR1 and GPx2 are potently induced by isothiocyanates and selenium, and mutually cooperate to protect Caco-2 cells against free radical-mediated cell death by Lawrence N. Barrera; Aedin Cassidy; Wei Wang; Taotao Wei; Nigel J. Belshaw; Ian T. Johnson; Brigelius-Flohe Regina Brigelius-Flohé; Yongping Bao (pp. 1914-1924).

Currently, there is significant interest in the field of diet–gene interactions and the mechanisms by which food compounds regulate gene expression to modify cancer susceptibility. From a nutrition perspective, two key components potentially exert cancer chemopreventive effects: isothiocyanates (ITCs), present in cruciferous vegetables, and selenium (Se) which, as selenocysteine, is an integral part of selenoproteins. However, the role of these compounds in the expression of key selenoenzymes once the cancer process has been initiated still needs elucidation. Therefore, this investigation examined the effect of two forms of selenium, selenium-methylselenocysteine and sodium selenite, both individually and in combination with two ITCs, sulforaphane or iberin, on the expression of the two selenoenzymes, thioredoxin reductase 1 (TrxR1) and gastrointestinal glutathione peroxidase (GPx2), which are targets of ITCs, in Caco-2 cells. Co-treatment with both ITCs and Se induced expression of TrxR1 and GPx2 more than either compound alone. Moreover, pre-treatment of cells with ITC+Se enhanced cytoprotection against H₂O₂-induced cell death through a ROS-dependent mechanism. Furthermore, a single and double knockdown of TrxR1 and/or GPx2 suggested that both selenoproteins were responsible for protecting against H₂O₂-induced cell death. Together, these data shed new light on the mechanism of interactions between ITC and Se in which translational expression of the enhanced transcripts by the former is dependent on an adequate Se supply, resulting in a cooperative antioxidant protective effect against cell death.Display Omitted► ITC+Se protects colon cancer cells against H₂O₂-induced cell death. ► Protection is mediated through increased expression of TrxR1 and GPx2. ► Such increase enhances ROS detoxification mechanisms and Caco-2 growth advantage. ► Protection is abolished after silencing TrxR1 and GPx2 by siRNA. ► Se is the limiting factor for protein synthesis from increased ITC-induced selenoprotein transcription.

Keywords: Thioredoxin reductase; Gastrointestinal glutathione peroxidase; Nrf2; Isothiocyanate; Selenium; Colon cancer

The miR-126 regulates Angiopoietin-1 signaling and vessel maturation by targeting p85β by Roberto Sessa; Giorgio Seano; Laura di Blasio; Paolo Armando Gagliardi; Claudio Isella; Enzo Medico; Franco Cotelli; Federico Bussolino; Luca Primo (pp. 1925-1935).

Blood vessel formation depends on the highly coordinated actions of a variety of angiogenic regulators. Vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang-1) are both potent and essential proangiogenic factors with complementary roles in vascular development and function. Whereas VEGF is required for the formation of the initial vascular plexus, Ang-1 contributes to the stabilization and maturation of growing blood vessels.Here, we provide evidence of a novel microRNA (miRNA)-dependent molecular mechanism of Ang-1 signalling modulation aimed at stabilizing adult vasculature. MiRNAs are short non-coding RNA molecules that post-trascriptionally regulate gene expression by translational suppression or in some instances by cleavage of the respective mRNA target. Our data indicate that endothelial cells of mature vessels express high levels of miR-126, which primarily targets phosphoinositide-3-kinase regulatory subunit 2 (p85β). Down-regulation of miR-126 and over-expression of p85β in endothelial cells inhibit the biological functions of Ang-1. Additionally, knockdown of miR-126 in zebrafish resulted in vascular remodelling and maturation defects, reminiscent of the Ang-1 loss-of-function phenotype. Our findings suggest that miR-126-mediated phosphoinositide-3-kinase regulation, not only fine-tunes VEGF-signaling, but it strongly enhances the activities of Ang-1 on vessel stabilization and maturation.► Mature blood vessels express high levels of miR-126. ► Endothelial miR-126 targets the PI3K regulatory subunit 2 (p85β). ► High levels of p85β inhibit endothelial cells survival and sprouting induced by Ang1.

Keywords: Abbreviations; EC; endothelial cells; VEGF; Vascular endothelial growth factor-A; Ang-1; Angiopoietin 1; miRNA; microRNA; 3′UTR; 3′ untranslated region; PI3K; phosphoinositide-3-kinase; p85β; PI3K regulatory subunit 2; HUVEC; human umbilical vein EC; MAR; murine aortic rings; ECM; extracellular matrixmicroRNA; Phosphoinositide-3-kinase; Angiopoietin-1; Endothelial cell

Synergistic roles of scleraxis and Smads in the regulation of collagen 1α2 gene expression by Rushita A. Bagchi; Michael P. Czubryt (pp. 1936-1944).

Cardiac fibrosis is marked by increased deposition of extracellular matrix components including fibrillar collagens, leading to impaired cardiac contractility and function. We recently demonstrated that the transcription factor scleraxis is expressed in collagen-producing cardiac fibroblasts and myofibroblasts, is up-regulated in the collagen-rich scar following myocardial infarction and is sufficient to transactivate the human collagen 1α2 (COL1A2) gene, suggesting a central role in fibrosis. Here we describe the mechanism of scleraxis-mediated regulation of the COL1A2 promoter. Using chromatin immunoprecipitation in primary human cardiac fibroblasts in combination with luciferase assays, we demonstrate that two E box sequences within the proximal COL1A2 promoter are required for scleraxis-mediated transactivation. Expression of scleraxis itself was induced by receptor Smad3, an effector of the pro-fibrotic growth factor TGF-β₁, and attenuated by inhibitory Smad7. TGF-β₁ augmented the effect of scleraxis on COL1A2 transactivation, an effect which was due to synergy between scleraxis and Smad3. Mutation of the COL1A2 Smad-binding element significantly attenuated the ability of scleraxis to transactivate the promoter, while mutation of the scleraxis-interacting E boxes attenuated the effect of Smad3, suggesting that these factors form a common signaling complex at the promoter. COL1A2 promoter transactivation and Col1α2 gene expression in cardiac fibroblasts were completely abrogated by a scleraxis basic domain deletion mutant in a dominant negative fashion, blocking the ability of TGF-β₁ to activate collagen synthesis and suggesting that scleraxis–DNA interaction is absolutely required for this process. Scleraxis thus appears to play a key role in the transcriptional regulation of type I collagen synthesis.► Scleraxis regulates collagen 1α2 gene expression in cardiac fibroblasts. ► Scleraxis interacts with E box sites in the human proximal collagen 1α2 promoter. ► Collagen 1α2 expression is synergistically regulated by scleraxis and Smad3. ► A dominant negative scleraxis mutant blocks collagen gene expression by TGF-β1.

Keywords: Abbreviations; bHLH; basic helix–loop–helix; ECM; extracellular matrix; EMSA; electrophoretic mobility shift assay; qPCR; quantitative real-time polymerase chain reactionGene expression; Collagen; bHLH transcription factor; Cardiac fibroblast; Fibrosis

Mitochondrial ABC transporters function: The role of ABCB10 (ABC-me) as a novel player in cellular handling of reactive oxygen species by Marc Liesa; Wei Qiu; Orian S. Shirihai (pp. 1945-1957).

Mitochondria are one of the major sources of reactive oxygen species (ROS) in the cell. When exceeding the capacity of antioxidant mechanisms, ROS production may lead to different pathologies, such as ischemia–reperfusion injury, neurodegeneration, anemia and ageing. As a consequence of the endosymbiotic origin of mitochondria, eukaryotic cells have developed different transport mechanisms that coordinate mitochondrial function with other cellular compartments. Four mitochondrial ATP-binding cassette (ABC) transporters have been described to date in mammals: ABCB6, ABCB8, ABCB7 and ABCB10. ABCB10 is located in the inner mitochondrial membrane forming homodimers, with the ATP binding domain facing the mitochondrial matrix. ABCB10 expression is highly induced during erythroid differentiation and its overexpression increases hemoglobin synthesis in erythroid cells. However, ABCB10 is also expressed in nonerythroid tissues, suggesting a role not directly related to hemoglobin synthesis. Recent evidence points toward ABCB10 as an important player in the protection from oxidative stress in mammals. In this regard, ABCB10 is required for normal erythropoiesis and cardiac recovery after ischemia–reperfusion, processes intimately related to mitochondrial ROS generation. Here, we review the current knowledge on mitochondrial ABC transporters and ABCB10 and discuss the potential mechanisms by which ABCB10 and its transport activity may regulate oxidative stress. We discuss ABCB10 as a potential therapeutic target for diseases in which increased mitochondrial ROS production and oxidative stress play a major role.► ABCB10 is a mitochondrial transporter and its substrates have not been indentified yet. ► ABCB10 is an important player in the protection from oxidative stress. ► ABCB10 is essential for erythropoiesis and normal recovery from cardiac ischemia-reperfusion. ► Antioxidant treatment ameliorates the defects caused by ABCB10 deletion. ► Hypotheses about how ABCB10 transport function protects from oxidative stress are presented.

Keywords: Mitochondria; Oxidative stress; ABCB10; ABC-me; Erythropoiesis; Ischemia–reperfusion

Heat shock induces a massive but differential inactivation of SUMO-specific proteases by Manuel P. Pinto; Andreia F. Carvalho; Cláudia P. Grou; Rodriguez-Borges José E. Rodríguez-Borges; Sa-Miranda Clara Sá-Miranda; Jorge E. Azevedo (pp. 1958-1966).

Covalent conjugation of the small ubiquitin-like modifier (SUMO) to proteins is a highly dynamic and reversible process. Cells maintain a fine-tuned balance between SUMO conjugation and deconjugation. In response to stress stimuli such as heat shock, this balance is altered resulting in a dramatic increase in the levels of SUMO conjugates. Whether this reflects an activation of the conjugation cascade, a decrease in the activity of SUMO-specific proteases (SENPs), or both, remains unknown. Here, we show that from the five human SENPs detected in HeLa cells (SENP1/2/3/6/7) the activities of all but one (SENP6) were largely diminished after 30min of heat shock. The decreased activity is not due to changes in their steady-state levels. Rather, in vitro experiments suggest that these SENPs are intrinsically heat-sensitive, a property most likely emerging from their catalytic domains. Heat shock inactivation seems to be a specific property of SENPs because numerous members of the related deubiquitinase family of cysteine proteases are not affected by this stress condition. Overall, our results suggest that SENPs are particularly sensitive to heat shock, a property that may be important for the adaptation of cells to this stress condition.► Heat shock induces a massive accumulation of SUMO2/3 protein conjugates. ► The mechanism of this response remains unknown. ► The activities, but not the levels, of most SENPs are diminished during heat shock. ► SENP inactivation may provide an advantage in the adaptation of cells to heat stress.

Keywords: Abbreviations; AMC; 7-amino-4-methyl coumarin; IMAC; immobilized metal ion affinity chromatography; NEM; N-ethylmaleimide; SENP; SUMO-specific protease; cSENP; SENP catalytic domain; SUMO; small ubiquitin-like modifier; Ub; ubiquitin; VME; vinyl methyl esterSUMO-specific protease; SENP; SUMO; Heat shock; Activity-based probe

Enzyme-coupled assays for simultaneous detection of nanomolar ATP, ADP, AMP, adenosine, inosine and pyrophosphate concentrations in extracellular fluids by Mikko Helenius; Sirpa Jalkanen; Gennady G. Yegutkin (pp. 1967-1975).

Purinergic signaling cascade includes the release of endogenous ATP and other agonists by chemical and mechanical stimuli, modulation of diverse cellular functions and subsequent ectoenzymatic inactivation. Basal release of extracellular purines and its physiological relevance remain controversial. Here we employed a combination of enzyme-coupled approaches for simultaneous bioluminescent (ATP, ADP, PP_i) and fluorometric (AMP, adenosine, inosine, hypoxanthine) measurements of ATP and its metabolites without additional manipulations or derivatization of sampled biological fluids. By using these sensing techniques, extracellular purines were determined in various cells and tissues both at resting and pro-inflammatory conditions. The results obtained revealed the ability of endothelial, lymphoid and tumor cells to maintain extracellular ATP, ADP and adenosine at certain characteristic nanomolar levels. By quantifying the amounts of endogenously released and/or exogenously applied purines and their metabolites, these sensing techniques may be applied for evaluating purine-converting pathways on the cell surfaces and also for ex vivo analysis of purine homeostasis in the intact tissues. Furthermore, we provide novel insight into the mechanisms underlying tumorigenic effects of ATP by demonstrating the ability of metastatic prostate carcinoma PC3 and breast cancer MDA-MB-231 cells to maintain PP_i, which derives from extracellular ATP in the course of nucleotide pyrophosphatase/phosphodiesterase reaction. Collectively, the results imply a complex pattern of nucleotide turnover where extracellular ATP, ADP and adenosine are maintained at steady-state levels via counterbalanced release and inactivation of ATP and other purines, and further suggest the importance of basal agonist release for continuous activation and/or desensitization of purinergic receptors.Display Omitted► Novel enzyme-coupled fluorometric AMP- and adenosine-sensing assay is developed. ► ATP and its metabolites were simultaneously determined in extracellular fluids. ► Endothelial and tumor cells maintain low nanomolar ATP, ADP and adenosine levels. ► Prostate and breast cancer cells convert ATP into PP_i via pyrophosphatase reaction. ► Basal purine levels may be involved in the control of purinergic signaling cascade.

Keywords: Abbreviations; ADA; adenosine deaminase; APS; adenosine 5′-phosphosulfate; BSS; basal salt solution; HDMEC; human dermal microvascular endothelial cells; HRP; horseradish peroxidase; HUVEC; human umbilical vein endothelial cells; KRPG; Krebs Ringer phosphate glucose; NBT; S-(4-nitrobenzyl)-6-thioinosine; NDPK; nucleoside diphosphate kinase; NPP; nucleotide pyrophosphatase/phosphodiesterase; NTPDase; nucleoside triphosphate diphosphohydrolase; PNP; purine nucleoside phosphorylase; TLC; thin-layer chromatography; XO; xanthine oxidaseAMP; Adenosine and inosine sensors; Extracellular ATP; ADP and pyrophosphate; Endothelial and tumor cells; Purine-converting enzymes

Two-pore channel 2 (TPC2) modulates store-operated Ca2⁺ entry by Lopez José J. López; Natalia Dionisio; Alejandro Berna-Erro; Galan Carmen Galán; Ginés M. Salido; Juan A. Rosado (pp. 1976-1983).

Two-pore channels (TPCs) are NAADP-sensitive receptor channels that conduct Ca2⁺ efflux from the intracellular stores. Discharge of the internal Ca2⁺ pools results in the activation of store-operated Ca2⁺ entry (SOCE); however, the role of TPCs in the modulation of SOCE remains unexplored. Mammalian cells express three TPCs: TPC1, TPC2 and TPC3, a pseudogene in humans. Here we report that MEG01 and HEK293 cells endogenously express TPC1 and TPC2. Silencing TPC2 expression results in attenuation of the rate and extent of thapsigargin (TG)-evoked SOCE both in MEG01 and HEK293 cells, without having any effect on the ability of cells to accumulate Ca2⁺ into the TG-sensitive stores. Similarly, silencing of native TPC2 expression reduced thrombin-induced Ca2⁺ entry in MEG01 cells. In contrast, silencing of TPC1 expression was without effect either on TG or thrombin-stimulated Ca2⁺ entry both in MEG01 and HEK293 cells. Biotinylation analysis revealed that TPC1 and TPC2 are expressed in internal membranes. Finally, co-immunoprecipitation experiments indicated that endogenously expressed TPC2, but not TPC1, associates with STIM1 and Orai1, but not with TRPC1, in MEG01 cells with depleted intracellular Ca2⁺ stores, but not in resting cells. These results provide strong evidence for the modulation of SOCE by TPC2 involving de novo association between TPC2 and STIM1, as well as Orai1, in human cells.► ShRNA-based TPC2 silencing decreases agonist-induced Ca2⁺ entry in MEG-01 and HEK-293 cells. ► TPC1 silencing had no effect on agonist-evoked Ca2⁺ entry. ► TPC1 and TPC2 are expressed in internal membranes. ► TPC2 associates with STIM1 and Orai1, but not with TRPC1, upon Ca2⁺ store depletion in MEG-01 cells.

Keywords: Abbreviations; [Ca; 2; ⁺; ]; _i; intracellular free calcium concentration; BSA; bovine serum albumin; ER; endoplasmic reticulum; HBS; HEPES-buffered saline; TRPC1; canonical TRP1; IP; ₃; inositol 1,4,5-trisphosphate; NAADP; nicotinic acid adenine dinucleotide phosphate; PBS; phosphate-buffered saline; PM; plasma membrane; PMSF; phenyl methyl sulphonyl fluoride; SERCA; sarco/endoplasmic reticulum Ca; 2; ⁺; ATPase; SOCE; store-operated calcium entry; SOCs; store-operated channels; STIM1; stromal interaction molecule 1; TBST; tris-buffered saline with 0.1% Tween 20; TG; thapsigargin; TPC; two-pore channelsTPC1; TPC2; STIM1; Orai1; Store-operated Ca; 2; ⁺; entry

TNF-α influences the lateral dynamics of TNF receptor I in living cells by Meike Heidbreder; Christin Zander; Sebastian Malkusch; Darius Widera; Barbara Kaltschmidt; Christian Kaltschmidt; Deepak Nair; Daniel Choquet; Jean-Baptiste Sibarita; Mike Heilemann (pp. 1984-1989).

In mammalian cells, inflammation is mainly mediated by the binding of tumor necrosis factor alpha to tumor necrosis factor receptor 1. In this study, we investigated lateral dynamics of TNF-R1 before and after ligand binding using high-density single-particle tracking in combination with photoactivated localization microscopy. Our single-molecule data indicates the presence of tumor necrosis factor receptor 1 with different mobilities in the plasma membrane, suggesting different molecular organizations. Cholesterol depletion led to a decrease of slow receptor species and a strong increase in the average diffusion coefficient. Moreover, as a consequence of tumor necrosis factor-alpha treatment, the mean diffusion coefficient moderately increased while its distribution narrowed. Based on our observation, we propose a refined mechanism on the structural arrangement and activation of tumor necrosis factor receptor 1 in the plasma membrane.► Single-molecule fluorescence microscopy of TNF-R1 in living cells ► Long-term observation through selective photoactivation of fluorophores ► TNF-R1 species with different mobilities revealed which indicates clustering ► TNF-R1 mobility increases upon cholesterol depletion and induction by TNF-α. ► Whole-cell diffusion maps with single-molecule resolution

Keywords: TNF receptor I; Receptor multimerization; Photoactivation; Single-particle tracking; Single-molecule fluorescence

Leukotriene B₄ inhibits neutrophil apoptosis via NADPH oxidase activity: Redox control of NF-κB pathway and mitochondrial stability by Pedro Barcellos-de-Souza; Cláudio Canetti; Christina Barja-Fidalgo; Maria Augusta Arruda (pp. 1990-1997).

Leukotriene B₄, an arachidonic acid-derived lipid mediator, is a known proinflammatory agent that has a direct effect upon neutrophil physiology, inducing reactive oxygen species generation by the NADPH oxidase complex and impairing neutrophil spontaneous apoptosis, which in turn may corroborate to the onset of chronic inflammation. Despite those facts, a direct link between inhibition of neutrophil spontaneous apoptosis and NADPH oxidase activation by leukotriene B₄ has not been addressed so far. In this study, we aim to elucidate the putative role of NADPH oxidase-derived reactive oxygen species in leukotriene B₄-induced anti-apoptotic effect. Our results indicate that NADPH oxidase-derived reactive oxygen species are critical to leukotriene B₄ pro-survival effect on neutrophils. This effect also relies on redox modulation of nuclear factor kappaB signaling pathway. We have also observed that LTB₄-induced Bad degradation and mitochondrial stability require NADPH oxidase activity. All together, our results strongly suggest that LTB₄-induced anti-apoptotic effect in neutrophils occurs in a reactive oxygen species-dependent manner. We do believe that a better knowledge of the molecular mechanisms underlying neutrophil spontaneous apoptosis may contribute to the development of more successful strategies to control chronic inflammatory conditions such as rheumatoid arthritis.► LTB₄ inhibits neutrophil apoptosis via NADPHox-derived ROS signaling. ► Redox modulation of NF-κB activity is a key event in LTB₄ anti-apoptotic effect. ► NADPHox and NF-κB activities are involved in LTB₄-induced Bad degradation and mitochondrial stability.

Keywords: Abbreviations; 12-HHT; 12(S)-hydroxyheptadeca-5Z; 8E; 10E-trienoic acid; BLT; leukotriene B; ₄; receptor; DPI; diphenyleneiodonium; ΔΨ; _m; mitochondrial transmembrane potential; ECL; enhanced chemiluminescence system; ERK; extracellular signal-regulated kinase; FITC; fluorescein isothiocyanate; H; ₂; O; ₂; hydrogen peroxide; HBSS; Hank's balanced salt solution; IκB; inhibitor of κB; IKK; IκB kinase; LTB; ₄; Leukotriene B4; NADPH; nicotinamide adenine dinucleotide phosphate; NADPHox; NADPH oxidase; NF-κB; nuclear factor kappa-B; O; ₂; ⁻; superoxide; PBS; phosphate buffered saline; peg-SOD; polyethylenoglycol-superoxide dismutase; PDTC; pyrrolidine dithiocarbamate; PI3K; phosphoinositide 3-kinase; PMA; phorbol myristate acetate; PMSF; phenylmethylsulphonyl fluoride; PS; phosphatidylserine; redox; reduction–oxidation reaction; ROS; reactive oxygen species; SDS; sodium dodecyl sulfate; SDS-PAGE; SDS-polyacrylamide gel electrophoresis; SEM; standard error meanReactive oxygen species; Redox signaling; NF-κB; Mitochondrial transmembrane potential; Bcl-2 family protein

Interaction of the human prostacyclin receptor and the NHERF4 family member intestinal and kidney enriched PDZ protein (IKEPP) by Helen M. Reid; Elizebeth C. Turner; Eamon P. Mulvaney; Paula B. Hyland; Caitriona McLean; B. Therese Kinsella (pp. 1998-2012).

Prostacyclin and its I prostanoid receptor, the IP, play central roles in hemostasis and in re-endothelialization in response to vascular injury. Herein, intestinal and kidney enriched PDZ protein (IKEPP) was identified as an interactant of the human (h) IP mediated through binding of PDZ domain 1 (PDZ^D1) and, to a lesser extent, PDZ^D2 of IKEPP to a carboxyl-terminal Class I ‘PDZ ligand’ within the hIP. While the interaction is constitutive, agonist-activation of the hIP leads to cAMP-dependent protein kinase (PK) A and PKC‐phosphorylation of IKEPP, coinciding with its increased interaction with the hIP. Ectopic expression of IKEPP increases functional expression of the hIP, enhancing its ligand binding and agonist-induced cAMP generation. Originally thought to be restricted to renal and gastrointestinal tissues, herein, IKEPP was also found to be expressed in vascular endothelial cells where it co-localizes and complexes with the hIP. Furthermore, siRNA-disruption of IKEPP expression impaired hIP-induced endothelial cell migration and in vitro angiogenesis, revealing the functional importance of the IKEPP:IP interaction within the vascular endothelium. Identification of IKEPP as a functional interactant of the IP reveals novel mechanistic insights into the role of these proteins within the vasculature and, potentially, in other systems where they are co-expressed.► Prostacyclin plays a central role in hemostasis and vascular re-endothelialization. ► IKEPP was discovered as a direct functional interactant of the prostacyclin receptor. ► We provide the first evidence that IKEPP is expressed in the vascular endothelium. ► IKEPP co-localizes and is associated with the hIP in vascular endothelial cells. ► IKEPP regulates prostacyclin-induced endothelial migration and in vitro angiogenesis.

Keywords: Abbreviations; DDO; double drop-out; FTI; farnesyl transferase inhibitor; GPCR; G protein-coupled receptor; HA; hemagglutinin; hIP; human prostacyclin receptor; HUVEC; human umbilical vein endothelial cell; IKEPP; intestinal and kidney enriched PDZ protein; IP; prostacyclin receptor; NHERF; NA; ⁺; /H; ⁺; exchange regulatory factor; PKA; cAMP-dependent protein kinase A; PL; phospholipase; PDZ; Postsynaptic density-95, disks large, zonula occludens-1; PDZK1; PDZ-domain-containing protein 1; QDO; quadruple drop-out; RBD; Rab11 binding domain; RLBA; radioligand binding assay; Y2H; yeast-two-hybridProstacyclin receptor; IKEPP; Interaction; Phosphorylation; NHERF; GPCR

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BBA - Molecular Cell Research (v.1823, #10)