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BBA - Molecular Cell Research (v.1783, #10)

Editorial Board (pp. ii).

ING2 recruits histone methyltransferase activity with methylation site specificity distinct from histone H3 lysines 4 and 9 by Frauke Goeman; Katja Otto; Sergiy Kyrylenko; Oliver Schmidt; Aria Baniahmad (pp. 1673-1680).

p33ING2 belongs to the ING-gene family that is involved in tumor suppression, DNA repair, cell cycle regulation, and cellular senescence. Most functions are dependent on the tumor suppressor p53. p33ING2 was also shown to bind to trimethylated lysine 4 of histone H3. Here, we show that p33ING2 contains a transferable silencing function, which is independent of p53. p33ING2-mediated gene silencing is resistant to the HDAC-inhibitor trichostatin A indicating that p33ING2 uses a non-HDAC class I or II pathway for gene repression in reporter assays. In line with that we show that p33ING2 is associated with histone methyltransferase (HMT) activity in vitro and in vivo, methylating specifically histone H3. Interestingly, the specificity is distinct from the MeCP2-recruited HMT. Mutation or methylation of lysine 9, a mark well known for repression, abrogates histone methylation by MeCP2 but not by the p33ING2 complex. Instead, the ING2-associated HMT shows an increased methylation activity if lysine 9 is methylated. In contrast, mutation or methylation of lysine 4, a methylation preferentially detected at active genes, led to a reduction of the ING2-associated HMT. Notably, also p33ING1 recruits HMT activity suggesting a more general biochemical interaction between members of p33ING family and HMT activity. Deletion analyses revealed that the ING2 C-terminus recruits HMT activity, which correlates with silencing function.

Keywords: ING2; p33ING2; p33ING1; Cellular senescence; Gene silencing; Tumor suppressor; Histone methyltransferase

FRET with multiply labeled HERG K⁺ channels as a reporter of the in vivo coarse architecture of the cytoplasmic domains by Pablo Miranda; Diego G. Manso; Francisco Barros; Luis Carretero; Thomas E. Hughes; Carlos Alonso-Ron; Dominguez Pedro Domínguez; de la Pena Pilar de la Peña (pp. 1681-1699).

The intracellular N-terminus of human ether-a-go-go-related gene (HERG) potassium channels constitutes a key determinant of activation and deactivation characteristics and is necessary for hormone-induced modifications of gating properties. However, the general organization of the long amino and carboxy HERG terminals remains unknown. In this study we performed fluorescence resonance energy transfer (FRET) microscopy with a library of fluorescent HERG fusion proteins obtained combining site-directed and transposon-based random insertion of GFP variants into multiple sites of HERG. Determinations of FRET efficiencies with functional HERG channels labeled in different combinations localize the fluorophores, introduced in the amino and carboxy ends, in two quadratic planes of 7.8 and 8.6 nm lateral size, showing a vertical separation of nearly 8 nm without major angular torsion between the planes. Similar analysis using labels at positions 345 and 905 of the amino and carboxy terminals, located them slightly above the planes delimited by the amino and carboxy end labels, respectively. Our data also indicate an almost vertical arrangement of the fluorophores introduced in the NH₂ and COOH ends and at position 905, but a near 45° angular rotation between the planes delimited by these labels and the 345-located fluorophores. Systematic triangulation using interfluorophore distances coming from multiply labeled channels provides an initial constraint on the overall in vivo arrangement of the HERG cytoplasmic domains, suggesting that the C-linker/CNBD region of HERG hangs centrally below the transmembrane core, with the initial portion of the amino terminus around its top and side surfaces directed towards the gating machinery.

Keywords: HERG; Potassium channel; Cytoplasmic domain architecture; FRET

The anti-apoptotic activity associated with phosphatidylinositol transfer protein α activates the MAPK and Akt/PKB pathway by Martijn Schenning; Joachim Goedhart; Theodorus W.J. Gadella Jr.; Diana Avram; Karel W.A. Wirtz; Gerry T. Snoek (pp. 1700-1706).

The conditioned medium (CM) from mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein α (PI-TPα; SPIα cells) demonstrates an increased anti-apoptotic activity compared with CM from wild type NIH3T3 (wtNIH3T3) cells. As previously shown, the anti-apoptotic activity acts by activating a G protein-coupled receptor, most probably a cannabinoid 1 (CB1)-like receptor as the activity was blocked by both pertussis toxin and rimonabant [M. Schenning, C.M. van Tiel, D. Van Manen, J.C. Stam, B.M. Gadella, K.W. Wirtz and G.T. Snoek, Phosphatidylinositol transfer protein alpha regulates growth and apoptosis of NIH3T3 cells: involvement of a cannabinoid 1-like receptor, J. Lipid Res. 45 (2004) 1555–1564]. The CB1 receptor appears to be expressed in mouse fibroblast cells, at levels in the order SPIα>wtNIH3T3>SPIβ cells (i.e. wild type cells overexpressing PI-TPβ). Upon incubation of SPIβ cells with the PI-TPα-dependent anti-apoptotic factors, both the ERK/MAP kinase and the Akt/PKB pathway are activated in a CB1 receptor dependent manner as shown by Western blotting. In addition, activation of ERK2 was also shown by EYFP-ERK2 translocation to the nucleus, as visualized by confocal laser scanning microscopy. The subsequent activation of the anti-apoptotic transcription factor NF-κB is in line with the increased resistance towards UV-induced apoptosis. On the other hand, receptor activation by CM from SPIα cells was not linked to phospholipase C activation as the YFP-labelled C2-domain of protein kinase C was not translocated to the plasma membrane of SPIβ cells as visualized by confocal laser scanning microscopy.

Keywords: Abbreviations; PI-TP; phosphatidylinositol transfer protein; PI; phosphatidylinositol; PC; phosphatidylcholine; PLA; phospholipase A; MAPK; mitogen-activated protein kinase; ERK; extracellular signalling related kinase; IAP; inhibitor of apoptosis protein; PKB; protein kinase B; PKA; protein kinase A; PKC; protein kinase C; YFP; yellow fluorescent protein; GFP; green fluorescent protein; DMEM; Dulbecco's modified Eagle medium; NCS; newborn calf serum; DBB; DMEM containing 0.1% bovine serum albumin; CM; conditioned medium; PBS; phosphate buffered saline; PMSF; phenylmethanesulphonylfluoride; CB1; cannabinoid 1; GPCR; G protein-coupled receptorPI-TPα; PI-TPβ; p42/p44 MAPK; NF-κB; Phospholipase C; Apoptosis

The optimized allotopic expression of ND1 or ND4 genes restores respiratory chain complex I activity in fibroblasts harboring mutations in these genes by Crystel Bonnet; Sébastien Augustin; Sami Ellouze; Benit Paule Bénit; Aicha Bouaita; Pierre Rustin; José-Alain Sahel; Marisol Corral-Debrinski (pp. 1707-1717).

Leber's Hereditary Optic Neuropathy (LHON) was the first maternally inherited mitochondrial disease identified and is now considered the most prevalent mitochondrial disorder. LHON patients harbor mutations in mitochondrial DNA (mtDNA). In about 90% of cases, the genes involved encode proteins of the respiratory chain complex I. Even though the molecular bases are known since 20 years almost all remains to be done regarding physiopathology and therapy. In this study, we report a severe decrease of complex I activity in cultured skin fibroblasts isolated from two LHON patients harboring mutations in ND4 or ND1 genes. Most importantly, we were able to restore sustainably (a) the ability to grow on galactose, (b) the ATP synthesis rate and (c) the complex I activity, initially impaired in these cells. Our strategy consisted of forcing mRNAs from nuclearly-encoded ND1 and ND4 genes to localize to the mitochondrial surface. The rescue of the respiratory chain defect observed was possible by discreet amounts of hybrid mRNAs and fusion proteins demonstrating the efficiency of their mitochondrial import. Hence, we confirmed here for two mitochondrial genes located in the organelle that the optimized allotopic expression approach represents a powerful tool that could ultimately be applied in human therapy for LHON.

Keywords: Complex I and V activities; Nuclearly-encoded; ND1; and; ND4; genes; Oxidative phosphorylation; LHON

Ceramide inhibition of MMP-2 expression and human cancer bronchial cell invasiveness involve decreased histone acetylation by Romain Debret; Sylvie Brassart-Pasco; Johanna Lorin; Alain Martoriati; Aurelie Deshorgue; François-Xavier Maquart; William Hornebeck; Irfan Rahman; Frank Antonicelli (pp. 1718-1727).

Ceramides have been proposed as potential therapeutic strategy with regard to their ability to induce cell death. We previously demonstrated that C2-ceramide generated apoptosis in bronchocarcinoma BZR cells. We here investigated whether ceramides also target other molecules involved in cell–cell or cell–matrix interactions during cancer progression. A SuperArray® analysis showed that ceramides modulate gene expression after 2 h. Among deregulated genes, we observed an inhibition of the transcript coding for the pro-metastatic enzyme MMP-2. The pharmacological inhibitor of caspases cascade, ZVAD-fmk, did not prevent C2-ceramide-induced down-regulation of MMP-2 ruling out apoptosis as a mediator of this event, whereas inhibition of oxidative stress using NAC confirmed a role for ROS. This effect of C2-ceramide was associated with changes in histone H3 acetylation. However, although histone deacetylase inhibitors are also currently under investigation for their anti-tumor activity, we demonstrated here that a combined treatment with trichostatin A abrogated both MMP-2 down-regulation and reduced invasive properties elicited by C2-ceramide alone. Hence, this study demonstrates that besides its apoptotic effect, C2-ceramide also exhibits anti-invasive properties, showing a dual beneficial effect against cancer progression, but casts some doubt on the use of HDAC inhibitors as combined treatment with drugs that trigger the ceramide pathway.

Keywords: MMP; Invasion; HDAC; Ceramide; Lung

IL-6 induces MUC4 expression through gp130/STAT3 pathway in gastric cancer cell lines by Mejias-Luque Raquel Mejías-Luque; Peiro Sandra Peiró; Audrey Vincent; Isabelle Van Seuningen; de Bolos Carme de Bolós (pp. 1728-1736).

The gastric mucosal levels of the pro-inflammatory cytokine Interleukin 6 (IL-6) have been reported to be increased in Helicobacter pylori-infected subjects and, in gastric adenocarcinomas, the up-regulation of intestinal mucin genes (MUC2 and MUC4) has been detected. To analyse the regulatory effects of IL-6 on the activation of intestinal mucins, six gastric cancer cell lines were treated for different times with several concentrations of IL-6, and the expression of MUC2 and MUC4 was evaluated. IL-6 induced MUC4 expression, detected by quantitative RT-PCR, Western blot and immunofluorescence, and MUC2 expression was not affected. MUC4 mRNA levels decreased after blocking the gp130/STAT3 pathway at the level of the receptor, and at the level of STAT3 activation using the AG490 specific inhibitor. MUC4 presents two putative binding sites for STAT factors that may regulate MUC4 transcription after a pro-inflammatory stimulus as IL-6. By EMSA, ChIP and site-directed mutagenesis we show that STAT3 binds to a cis-element at −123/−115, that conveys IL-6 mediated up-regulation of MUC4 transcriptional activity. We also demonstrated that p-STAT3 binds to MUC4 promoter and a three-fold increase in p-STAT3 binding was observed after treating GP220 cells with IL-6.In conclusion, IL-6 treatment induced MUC4 expression through the gp130/STAT3 pathway, indicating the direct role of IL-6 on the activation of the intestinal mucin gene MUC4 in gastric cancer cells.

Keywords: IL-6; STAT3; MUC4; Mucin; Gastric cell

Androgen regulates Cdc6 transcription through interactions between androgen receptor and E2F transcription factor in prostate cancer cells by Ipsita Mallik; Monica Davila; Tenekua Tapia; Brian Schanen; Ratna Chakrabarti (pp. 1737-1744).

Androgen receptor plays a critical role in the development and maintenance of cancers in the prostate. Earlier, we have shown that Cdc6, a regulatory protein for initiation of DNA replication, is down regulated in androgen-insensitive prostate cancer cells. In this report, we studied the involvement of androgen, mediated through androgen receptor (AR) in regulation of Cdc6 expression. Our results demonstrated that androgen treatment stimulated Cdc6 expression in xenograft tumors and androgen-sensitive prostate cancer cells. We also showed that androgen treatment stimulated Cdc6 transcription through possible interaction of AR with the ARE sequence in the Cdc6 promoter and that the stimulatory effect of androgen required intact E2F binding sites in the promoter. Androgen treatment differentially altered nuclear availability of E2F1 and E2F3, and increased the amount of hypophosphorylated retinoblastoma protein (pRb) in the nucleus in a time dependent fashion. We further showed that AR interacted with E2F transcription factors in a ligand-independent manner and that ligand-bound AR was less efficient in interacting with E2F proteins. DNA–protein interaction assays indicated that androgen treatment altered binding of E2F1 to the Cdc6 promoter in prostate cancer cells. We conclude that AR regulates Cdc6 transcription through interaction with the Cdc6 promoter, and complex formation with E2F1 and E2F3 in a differential manner.

Keywords: CDC6; Transcription regulation; Androgen; Androgen receptor; E2F transcription factor; Prostate cancer

Development of a pharmacologically improved peptide agonist of the leptin receptor by Laszlo Otvos Jr.; Marianna Terrasi; Sandra Cascio; Marco Cassone; Giovanni Abbadessa; Francesco De Pascali; Laura Scolaro; Daniel Knappe; Maciej Stawikowski; Predrag Cudic; John D. Wade; Ralf Hoffmann; Eva Surmacz (pp. 1745-1754).

Leptin, a hormone produced by adipose tissue, regulates energy balance in the hypothalamus and is involved in fertility, immune response and carcinogenesis. The existence of disorders related to leptin deficit and leptin overabundance calls for the development of drugs activating or inhibiting the leptin receptor (ObR). We synthesized four proposed receptor-binding leptin fragments (sites I, IIa and IIb, III), their reportedly antagonist analogs, and a peptide chimera composed of the two discontinuous site II arms. To assess the pharmacological utility of leptin fragments, we studied the peptides' ability to stimulate the growth of ObR-positive and ObR-negative cells. The combined site II construct and site III derivatives selectively reversed leptin-induced growth of ObR-positive cells at mid-nanomolar concentrations. However, these peptides appeared to be partial agonists/antagonists as they activated cell growth in the absence of exogenous leptin. A designer site III analog, featuring non-natural amino acids at terminal positions to decrease proteolysis and a blood–brain barrier (BBB) penetration-enhancing carbohydrate moiety, proved to be full agonist to ObR, i.e., stimulated proliferation of different ObR-positive but not ObR-negative cells in the presence or absence of leptin. This glycopeptide bound to isolated ObR on solid-phase assays and activated ERK-1/2 signaling in ObR-positive MCF-7 cells at 100–500 nM concentrations. The glycopeptide was stable in mouse serum, readily crossed endothelial/astrocyte cell layers in a cellular BBB model, and was distributed into the brain of Balb/c mice after intraperitoneal administration. These characteristics suggest a potential pharmaceutical utility of the designer site III glycopeptide in leptin-deficient diseases.

Keywords: Abbreviations; Ab; antibody; ANOVA; analysis of variance; ATCC; American Type Culture Collection; BBB; blood–brain barrier; BSA; bovine serum albumin; CNS; central nervous system; CRH; cytokine receptor homology; DMEM; Dulbecco's modified Eagle's medium; ELISA; enzyme-linked immunosorbent assay; ERK 1/2; extracellular signal-regulated kinases 1 and 2; Fmoc; 9-fluorenyl-methoxy-carbonyl; G-CSF; granulocyte-colony stimulating factor; GSK; glycogen synthase kinase; HRP; horseradish peroxidase; JAK; Janus kinase; MALDI-MS; matrix-assisted laser ionization/desorption mass spectroscopy; ObR; leptin receptor; PBST; phosphate buffered saline containing 0.5% Tween 20; PI-3K; phosphoinositide 3 kinase; PLC; phospholipase C; PKC; protein kinase C; RP-HPLC; reversed-phase high performance liquid chromatography; SDS-PAGE; sodium dodecyl sulfate – polyacrylamide gel electrophoresis; SFM; serum-free medium; SE; standard error; STAT; signal transducer and activator of transcription; TBST; Tris-buffered saline containing 0.1% Tween 20; TFA; trifluoroacetic acid; VEGF; vascular endothelial growth factorBlood–brain penetration; Leptin-deficient diseases; Neurohormone; Peptidomimetic; Serum stability; Signaling

Leukocyte Elastase Inhibitor, the precursor of L-DNase II, inhibits apoptosis by interfering with caspase-8 activation by Laura Padrón-Barthe; Jacqueline Courta; Chloé Leprêtre; Atf Nagbou; Alicia Torriglia (pp. 1755-1766).

LEI (Leukocyte Elastase Inhibitor), the precursor of the pro-apoptotic molecule L-DNase II, belongs to the ovalbumin subgroup of serpins. Several serpins can inhibit apoptosis: the viral serpin Crm A inhibits Fas or TNFα-induced apoptosis, and overexpression of PAI-2 or PI-9 protects cells from TNFα or granzyme B induced apoptosis. We have previously shown that LEI overexpression protects cells from etoposide-induced apoptosis. The molecular reason of this anti-apoptotic activity is now investigated. We show that, in BHK-21 and HeLa cells, LEI anti-protease activity is essential for its anti-apoptotic effect. The protease inhibited is cathepsin D, released from the lysosome during etoposide treatment. Cathepsin D enhances caspase activity in the cell by cleaving procaspase-8 and LEI overexpression slows down this cleavage, protecting cells from apoptosis. This let us presume that high expression of LEI in tumor cells may reduce the efficiency of etoposide as a chemotherapeutic agent.

Keywords: Abbreviations; LEI, Leukocyte Elastase Inhibitor; L-DNase II, LEI-derived DNase II; HMA, Hexa-methylene-amiloride; Crm A, cytokine response modifier A; TNFα, tumor necrosis factor α; PAI-2, plasminogen activator inhibitor 2; PI-9, Proteinase Inhibitor-9; AP24, Apoptotic Protein of 24 kDa; VDAC, voltage-dependent ion selective channelProtease; Endonuclease; Apoptosis; Caspase-independent; Caspase-8; Cathepsin D; L-DNase II

Phosphorylation, lipid raft interaction and traffic of α-synuclein in a yeast model for Parkinson by Piotr Zabrocki; Ilse Bastiaens; Charlotte Delay; Tine Bammens; Ruben Ghillebert; Klaartje Pellens; Claudio De Virgilio; Fred Van Leuven; Joris Winderickx (pp. 1767-1780).

Parkinson's disease is a neurodegenerative disorder characterized by the formation of Lewy bodies containing aggregated α-synuclein. We used a yeast model to screen for deletion mutants with mislocalization and enhanced inclusion formation of α-synuclein. Many of the mutants were affected in functions related to vesicular traffic but especially mutants in endocytosis and vacuolar degradation combined inclusion formation with enhanced α-synuclein-mediated toxicity. The screening also allowed for identification of casein kinases responsible for α-synuclein phosphorylation at the plasma membrane as well as transacetylases that modulate the α-synuclein membrane interaction. In addition, α-synuclein was found to associate with lipid rafts, a phenomenon dependent on the ergosterol content. Together, our data suggest that toxicity of α-synuclein in yeast is at least in part associated with endocytosis of the protein, vesicular recycling back to the plasma membrane and vacuolar fusion defects, each contributing to the obstruction of different vesicular trafficking routes.

Keywords: α-Synuclein; Parkinson's disease; Yeast; Lipid raft; Casein kinase; N-terminal acetyltransferase; Vesicular trafficking; Endocytosis; Vesicular recycling

Involvement of Munc18 isoforms in the regulation of granule exocytosis in neutrophils by Cristiana Brochetta; Francesca Vita; Neeraj Tiwari; Lisa Scandiuzzi; Maria Rosa Soranzo; Claudine Guérin-Marchand; Giuliano Zabucchi; Ulrich Blank (pp. 1781-1791).

Human neutrophil granule exocytosis mobilizes a complex set of secretory granules. This involves different combinations of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins to facilitate membrane fusion. The control mechanisms governing the late fusion steps are still poorly understood. Here, we have analyzed SNARE-interacting Sec1/Munc18 (SM) family members. We found that human neutrophils express Munc18-2 and Munc18-3 isoforms and that Munc18-2 interacts with the target-SNARE syntaxin 3. Munc18-2 was associated preferentially with primary granules but could also be found with secondary and tertiary granules, while Munc18-3 was majorily associated with secondary and tertiary granules. Ultrastructural analysis showed that both Munc18-2 and Munc18-3 were often located in close proximity to their respective SNARE-binding partners syntaxin 3 and syntaxin 4. Both isoforms were also found in plasma membrane fractions and in the cytosol, where they associate with cytoskeletal elements. Upon stimulation, Munc18-2 and Munc18-3 redistributed and became enriched on granules and in the plasma membrane. Munc18-2 primary granule exocytosis can be blocked by introduction of Munc18-2-specific antibodies indicating a crucial role in primary granule fusion. Our results suggest that Munc18-2 acts as a regulator of primary granule exocytosis, while Munc18-3 may preferentially regulate the fusion of secondary granules.

Keywords: Neutrophils; Exocytosis; SNARE; SM proteins

The armadillo repeat domain of the APC tumor suppressor protein interacts with Striatin family members by Maya Breitman; Alona Zilberberg; Michal Caspi; Rina Rosin-Arbesfeld (pp. 1792-1802).

Adenomatous polyposis coli (APC) is a multifunctional tumor suppressor protein that negatively regulates the Wnt signaling pathway. The APC gene is ubiquitously expressed in various tissues, especially throughout the large intestine and central nervous system. Mutations in the gene encoding APC have been found in most colorectal cancers and in other types of cancer. The APC gene product is a large multidomain protein that interacts with a variety of proteins, many of which bind to the well conserved armadillo repeat domain of APC. Through its binding partners, APC affects a large number of important cellular processes, including cell–cell adhesion, cell migration, organization of the actin and microtubule cytoskeletons, spindle formation and chromosome segregation. The molecular mechanisms that control these diverse APC functions are only partly understood. Here we describe the identification of an additional APC armadillo repeat binding partner — the Striatin protein. The Striatin family members are multidomain molecules that are mainly neuronal and are thought to function as scaffolds. We have found that Striatin is expressed in epithelial cells and co-localizes with APC in the epithelial tight junction compartment and in neurite tips of PC12 cells. The junctional localization of APC and Striatin is actin-dependent. Depletion of APC or Striatin affected the localization of the tight junction protein ZO-1 and altered the organization of F-actin. These results raise the possibility that the contribution of APC to cell-cell adhesion may be through interaction with Striatin in the tight junction compartment of epithelial cells.

Keywords: Abbreviations; AJ; adherence junctions; APC; adenomatous polyposis coli; APCarm; armadillo repeat domain of APC; TJ; tight junctionsAPC; Striatin; Cell adhesion; ZO-1; Tight junction; Actin

HDAC1/HDAC3 modulates PPARG2 transcription through the sumoylated CEBPD in hepatic lipogenesis by Pei-Hua Lai; Wen-Lin Wang; Chiung-Yuan Ko; Yi-Chao Lee; Wen-Ming Yang; Tsung-Wei Shen; Wen-Chang Chang; Ju-Ming Wang (pp. 1803-1814).

CCAAT/Enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptors gamma (PPARG) play critical roles in the regulation of lipid metabolism genes. Overexpression of CEBPdelta (CEBPD) enhances lipid accumulation and specifically activates PPARG2 transcription in HepG2 cells. By using 5′-serial deletion reporter analysis, we show that the region comprising the −457 to +129 base pairs is required for CEBPD response of the PPARG2 promoter. Two critical CEBPD-binding motifs on the −324/−311 and −158/−145 of human PPARG2 promoter are identified. We previously have shown that the human CEBPD is sumoylated by small ubiquitin-related modifier-1 (SUMO1). We further demonstrated that the sumoylation of CEBPD lysine 120 is also detectable in HepG2 cells, and this modification functions for binding of the recruits, HDAC1 and HDAC3. Meanwhile, an in vivo chromatin IP assay demonstrated that the sumoylation mutant of CEBPD lost a significant portion of HDAC1 and HDAC3 interaction. Combining that the increasing amount of CEBPD and the sumoylated CEBPD (suCEBPD) consistently responded to lipogenic stimulation, these results suggest that the excess non-sumoylated CEBPD could be a critical activator which reverses suCEBPD/HDAC1/HDAC3-mediated PPARG2 gene inactivation and promotes hepatic lipogenesis. Taken together, we suggest that suCEBPD/HDAC1/HDAC3 complex inactivates PPARG2 transcription, and the induction of CEBPD expression transiently activates PPARG2 transcription which is involved in adipocyte-like lipogenesis in HepG2 cells.

Keywords: PPARG2; CEBPD; Lipogenesis; HDAC1/3; Sumoylation

Inclusion bodies: Specificity in their aggregation process and amyloid-like structure by Montse Morell; Ramona Bravo; Alba Espargaró; Xavier Sisquella; Francesc X. Avilés; Xavier Fernàndez-Busquets; Salvador Ventura (pp. 1815-1825).

The accumulation of aggregated protein in the cell is associated with the pathology of many diseases and constitutes a major concern in protein production. Intracellular aggregates have been traditionally regarded as nonspecific associations of misfolded polypeptides. This view is challenged by studies demonstrating that, in vitro, aggregation often involves specific interactions. However, little is known about the specificity of in vivo protein deposition. Here, we investigate the degree of in vivo co-aggregation between two self-aggregating proteins, Aβ42 amyloid peptide and foot-and-mouth disease virus VP1 capsid protein, in prokaryotic cells. In addition, the ultrastructure of intracellular aggregates is explored to decipher whether amyloid fibrils and intracellular protein inclusions share structural properties. The data indicate that in vivo protein aggregation exhibits a remarkable specificity that depends on the establishment of selective interactions and results in the formation of oligomeric and fibrillar structures displaying amyloid-like properties. These features allow prokaryotic Aβ42 intracellular aggregates to act as effective seeds in the formation of Aβ42 amyloid fibrils. Overall, our results suggest that conserved mechanisms underlie protein aggregation in different organisms. They also have important implications for biotechnological and biomedical applications of recombinant polypeptides.

Keywords: Abbreviations; AFM; Atomic Force Microscopy; BFP; Blue Fluorescent Protein; CHC; Central Hydrophobic Cluster; FRET; Fluorescence Resonance Energy Transfer; FT-IR; Fourier Transform Infrared; GuHCl; Guanidinium Hydrochloride; IBs; Inclusion Bodies; MALDI-TOFMS; Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry; PK; proteinase K; TEM; Transmission Electron Microscopy; Th-T; Thioflavin TProtein aggregation; Inclusion bodies; Conformational diseases; Amyloid fibrils; Protein folding

Ubiquitin–proteasome system mediates heme oxygenase-1 degradation through endoplasmic reticulum-associated degradation pathway by Pu-Hua Lin; Ming-Tsai Chiang; Lee-Young Chau (pp. 1826-1834).

The present study investigated the cellular mechanism underlying the degradation of heme oxygenase-1 (HO-1), an endoplasmic reticulum (ER)-anchored protein. The turnover of HO-1 induced in vascular smooth muscle cells (VSMCs) was significantly attenuated by proteasome inhibitors, suggesting the involvement of a proteasome-mediated pathway. High molecular weight ubiquitin conjugates were co-immunoprecipitated with HO-1 from VSMCs after proteasome inhibition, and HO-1 ubiquitination was confirmed in HEK293 cells overexpressing His-tagged HO-1 and HA-tagged ubiquitin. Endogenous p97, an ATPase, and Ufd1, both implicated as essential components in the ER-associated degradation pathway (ERAD), were co-eluted with His-tagged HO-1 from metal affinity resin. Knockdown of either p97 or Ufd1 in HEK293 cells using specific siRNA significantly prolonged the half-life of endogenously induced HO-1 and slowed the degradation of ubiquitinated HO-1. HO-1 ubiquitination in HEK293 cells was enhanced by zinc chloride, but suppressed with a zinc chelator ( N, N, N′, N′-tetrakis(2-pyridylmethyl)ethyl-enediamine), suggesting the involvement of a RING-E3 ligase in this process. Collectively, these data indicate that HO-1 protein turnover is regulated by the ubiquitin–proteasome system through the ERAD pathway.

Keywords: Heme oxygenase-1; Proteasome; Endoplasmic reticulum-associated degradation; p97; Ufd1; RING-E3

Novel function of neuron-restrictive silencer factor (NRSF) for posttranscriptional regulation by Chun Sung Kim ⁎; Cheol Kyu Hwang; Kyu Young Song; Hack Sun Choi; Do Kyung Kim; Ping-Yee Law; Li-Na Wei; Horace H. Loh (pp. 1835-1846).

The neuron-restrictive silencer factor (NRSF) functions as a transcriptional repressor of neuronal genes in nonneuronal cells. However, it is expressed in certain mature neurons in adults, suggesting that it might have complex and novel roles depending on its cellular and physiological context. Overexpression of NRSF led to both increased opioid ligand-binding activity of the endogenous MOR and MOR–GFP fusion protein expression. In RNA immunoprecipitation and gel-shift assays, NRSF specifically interacted with the NRSE sequence of MOR mRNA. When MOR and NRSF genes were coexpressed, the specific ligand-binding activity of MOR was increased in neuroblastoma NMB cells, but decreased in PC12 cells result from its localization. Indeed, after overexpressing NRSF in NMB cells, the target RNA moved to the translationally active polysomal fraction. Overexpression of NRSF also led to enhanced phosphorylation of eIF4G. In contrast, knockdown of NRSF by siRNA transfection significantly decreased eIF4G phosphorylation. These findings indicate that NRSF may deliver the target MOR transcripts to the polyribosomal complex and activate eIF4G phosphorylation, resulting in translational activation. We report here a novel function of NRSF that enhance the translation of the mu opioid receptor (MOR) gene through its RNA binding sequence, the neuron-restrictive silencer element (NRSE).

Keywords: NRSF; NRSE; Mu opioid receptor; Translational regulation; eIF4G

Heterodimerization with small Maf proteins enhances nuclear retention of Nrf2 via masking the NESzip motif by Wenge Li; Siwang Yu; Tong Liu; Jung-Hwan Kim; Volker Blank; Hong Li; A.-N. Tony Kong (pp. 1847-1856).

Nrf2 is the key transcription factor regulating the antioxidant response. When exposed to oxidative stress, Nrf2 translocates to cell nucleus and forms heterodimer with small Maf proteins (sMaf). Nrf2/sMaf heterodimer binds specifically to a cis-acting enhancer called antioxidant response element and initiates transcription of a battery of antioxidant and detoxification genes. Nrf2 possesses a NESzip motif (nuclear export signal co-localized with the leucine zipper (ZIP) domain). Heterodimerization with MafG via ZIP–ZIP binding enhanced Nrf2 nuclear retention, which could be abrogated by the deletion of the ZIP domain or site-directed mutations targeting at the ZIP domain. In addition, dimerization with MafG precluded Nrf2zip/CRM1 binding, suggesting that Nrf2/MafG heterodimerization may simultaneously mask the NESzip motif. MafG-mediated nuclear retention may enable Nrf2 proteins to evade cytosolic proteasomal degradation and consequently stabilize Nrf2 signaling. For the first time, we show that under the physiological condition, the NESzip motif can be switched-off by heterodimerization.

Keywords: Nrf2; MafG; ZIP; CRM1; FRET

Isolation and characterization of mutant animal cell line defective in alkyl-dihydroxyacetonephosphate synthase: Localization and transport of plasmalogens to post-Golgi compartments by Masanori Honsho; Yuichi Yagita; Naohiko Kinoshita; Yukio Fujiki (pp. 1857-1865).

We herein isolated plasmalogen-deficient Chinese hamster ovary (CHO) mutant, ZPEG251, with a phenotype of normal import of peroxisomal matrix and membrane proteins. In ZPEG251, plasmenylethanolamine (PlsEtn) was severely reduced. Complementation analysis by expression of genes responsible for the plasmalogen biogenesis suggested that alkyl-dihydroxyacetonephosphate synthase (ADAPS), catalyzing the second step of plasmalogen biogenesis, was deficient in ZPEG251. ADAPS mRNA was barely detectable as verified by Northern blot and reverse transcription-PCR analyses. Defect of ADAPS expression was also assessed by immunoblot. As a step toward delineating functional roles of PlsEtn, we investigated its subcellular localization. PlsEtn was localized to post-Golgi compartments and enriched in detergent-resistant membranes. Transport of PlsEtn to post-Golgi compartments was apparently affected by lowering cellular ATP, but not by inhibitors of microtubule assembly and vesicular transport. Partitioning of cholesterol and sphingomyelin, a typical feature of lipid rafts, was not impaired in plasmalogen-deficient cells, including peroxisome assembly-defective mutants, hence suggesting that PlsEtn was not essential for lipid-raft architecture in CHO cells.

Keywords: Abbreviations; AOx; acyl-CoA oxidase; ADAPS; alkyl-dihydroxyacetonephosphate synthase; BFA; brefeldin A; β-MCD; β-methyl cyclodextrin; CHO; Chinese hamster ovary; DHAPAT; dihydroxyacetonephosphate acyltransferase; DRM; detergent-resistant membranes; EGFP; “enhanced” green fluorescent protein; ER; endoplasmic reticulum; HB; homogenizing buffer; lyso-PE; 2-acylglycerophosphoethanolamine; HG; sn; -1-hexadecylglycerol; NDGA; nordihydroguaiaretic acid; P450R; cytochrome P450 reductase; P9OH/UV; 9-(1′-pyrene)nonanol/ultraviolet; PE; phosphatidylethanolamine; PlsEtn; plasmenylethanolamine; PM; plasma membranes; PNS; post-nuclear supernatant; PTS1 and PTS2; peroxisome targeting signal types 1 and 2; RT; reverse transcription; SM; sphingomyelin; TCA; trichloroacetic acid; TLC; thin-layer chromatography; TfR; transferrin receptorPlasmalogen; Peroxisome; CHO cell mutant; Alkyl-dihydroxyacetonephosphate synthase; Post-Golgi compartments

Caveolae are an essential component of the pathway for endothelial cell signaling associated with abrupt reduction of shear stress by Tatyana Milovanova; Shampa Chatterjee; Brian J. Hawkins; NanKang Hong; Elena M. Sorokina; Kris DeBolt; Jonni S. Moore; Muniswamy Madesh; Aron B. Fisher (pp. 1866-1875).

Abrupt cessation of flow representing the acute loss of shear stress (simulated ischemia) to flow-adapted pulmonary microvascular endothelial cells (PMVEC) leads to reactive oxygen species (ROS) generation that signals for EC proliferation. We evaluated the role of caveolin-1 on this cellular response with mouse PMVEC that were preconditioned for 72 h to laminar flow at 5 dyn/cm² followed by stop of flow (“ischemia”). Preconditioning resulted in a 2.7-fold increase in cellular expression of K_ATP (K_IR 6.2) channels but no change in expression level of caveolin-1, gp91^phox, or MAP kinases. The initial response to ischemia in wild type cells was cell membrane depolarization that was abolished by gene targeting of K_IR 6.2. The subsequent response was increased ROS production associated with activation of NADPH oxidase (NOX2) and then phosphorylation of MAP kinases (Erk, JNK). After 24 h of ischemia in wild type cells, the cell proliferation index increased 2.5 fold and the % of cells in S+G₂/M phases increased 6-fold. This signaling cascade (cell membrane depolarization, ROS production, MAP kinase activation and cell proliferation) was abrogated in caveolin-1 null PMVEC or by treatment of wild type cells with filipin. These studies indicate that caveolin-1 functions as a shear sensor in flow-adapted EC resulting in ROS-mediated cell signaling and endothelial cell proliferation following the abrupt reduction in flow.

Keywords: Abbreviations; ROS; reactive oxygen species; MPMVEC; mouse pulmonary microvascular endothelial cells; DPI; diphenyleneiodonium chloride; FITC; fluorescein isothiocyanate; DCF; dichlorofluoresceinShear stress; Mechanotransduction; K; _ATP; channel; NADPH oxidase; Pulmonary microvascular endothelium; Reactive oxygen species; Cell proliferation

YY1 restrained cell senescence through repressing the transcription of p16 by Xiuli Wang; Yunpeng Feng; Liang Xu; Yuli Chen; Yu Zhang; Dongmei Su; Guoling Ren; Jun Lu; Baiqu Huang (pp. 1876-1883).

The transcription factor YY1 has been implicated to play a role in cell growth control. In this report, we demonstrate that YY1 was able to suppress NCI-H460 cell senescence through regulating the expression of p16^INK4a, a cyclin-dependent kinase inhibitor. We also show that YY1 participated in the repression of p16^INK4a expression in 293T cells through an epigenetic mechanism involving histone acetylation modification. Specifically, HDAC3 and HDAC4 inhibited the p16^INK4a promoter activity. The chromatin immunoprecipitation (ChIP) assays verified that HDAC3 and HDAC4 were recruited to p16^INK4a promoter by YY1. Moreover, co-immunoprecipitation assays revealed that these three protein factors formed a complex. Furthermore, knockdown of these factors induced cell enlargement and flattened morphology and significantly increased the SA-β-gal activity, a biochemical marker of cell senescence. Overall, data from this study suggest that YY1, HDAC3 and HDAC4 restrained cell senescence by repressing p16^INK4a expression through an epigenetic modification of histones.

Keywords: YY1; Cell senescence; p16; HDAC3; HDAC4

Identification of a functional interaction between Kv4.3 channels and c-Src tyrosine kinase by Pedro Gomes; Tomoaki Saito; Cris del Corsso; Abderrahmane Alioua; Mansoureh Eghbali; Ligia Toro; Enrico Stefani (pp. 1884-1892).

Voltage-gated K⁺ (Kv) channels are key determinants of cardiac and neuronal excitability. A substantial body of evidence has accumulated in support of a role for Src family tyrosine kinases in the regulation of Kv channels. In this study, we examined the possibility that c-Src tyrosine kinase participates in the modulation of the transient voltage-dependent K⁺ channel Kv4.3. Supporting a mechanistic link between Kv4.3 and c-Src, confocal microscopy analysis of HEK293 cells stably transfected with Kv4.3 showed high degree of co-localization of the two proteins at the plasma membrane. Our results further demonstrate an association between Kv4.3 and c-Src by co-immunoprecipitation and GST pull-down assays, this interaction being mediated by the SH2 and SH3 domains of c-Src. Furthermore, we show that Kv4.3 is tyrosine phosphorylated under basal conditions. The functional relevance of the observed interaction between Kv4.3 and c-Src was established in patch-clamp experiments, where application of the Src inhibitor PP2 caused a decrease in Kv4.3 peak current amplitude, but not the inactive structural analogue PP3. Conversely, intracellular application of recombinant c-Src kinase or the protein tyrosine phosphatase inhibitor bpV(phen) increased Kv4.3 peak current amplitude. In conclusion, our findings provide evidence that c-Src-induced Kv4.3 channel activation involves their association in a macromolecular complex and suggest a role for c-Src-Kv4.3 pathway in regulating cardiac and neuronal excitability.

Keywords: Abbreviations; Kv; voltage-dependent K; ⁺; HEK293-Kv4.3; human embryonic kidney cell line stably expressing Kv4.3 channels; SH2 and SH3; Src homology domains 2 and 3; GST; glutathione; S; -transferase; PAGE; polyacrylamide gel electrophoresisPotassium channels; Kv4.3; Protein tyrosine kinases; c-Src; Phosphorylation; Protein-protein interactions

Mechanism of thrombin mediated eNOS phosphorylation in endothelial cells is dependent on ATP levels after stimulation by Brynhildur Thors; Haraldur Halldórsson; Gudbjorg Jónsdóttir; Gudmundur Thorgeirsson (pp. 1893-1902).

Conflicting results have been reported concerning the role of AMP-activated protein kinase (AMPK) in mediating thrombin stimulation of endothelial NO-synthase (eNOS). We examined the involvement of two upstream kinases in AMPK activation in cultured human umbilical endothelial cells, LKB1 stimulated by a rise in intracellular AMP/ATP ratio, and Ca⁺²/CaM kinase kinase (CaMKK) responding to elevation of intracellular Ca⁺². We also studied the effects of AMPK activation on the downstream target eNOS. In culture medium 1640 the level of intracellular ATP was unchanged after thrombin stimulation and the CaMKK inhibitor STO-609 totally inhibited phosphorylation of AMPK and acetyl coenzyme A carboxylase (ACC) but not eNOS. In Morgan's medium 199 thrombin caused a significant lowering of intracellular ATP and STO-609 only partially inhibited the phosphorylation of AMPK, ACC and eNOS. Inhibition of AMPK by Compound C or AMPK downregulation using siRNA partially inhibited the phosphorylation of eNOS in medium 199 but not in 1640, underscoring a clear difference in the pathways mediating thrombin-stimulated eNOS phosphorylation in different culture media. Thus, conditions subjecting endothelial cells to a fall in ATP after thrombin stimulation facilitate activation of pathways partly dependent on AMPK causing downstream phosphorylation of eNOS. In contrast, under culture conditions that do not facilitate a fall in ATP after stimulation, AMPK activation is exclusively mediated by CaMKK and does not contribute to the phosphorylation of eNOS.

Keywords: Abbreviations; ACC; acetyl coenzyme A carboxylase; AICAR; 5-aminoimidazole-4-carboxamide-1-β-4 ribofuranoside; AMPK; AMP-activated protein kinase; CaMKK; Ca; ⁺²; /CaM kinase kinase; eNOS; endothelial NO-synthase; HUVEC; human umbilical vein endothelial cells; l; -NA; N; (G)-nitro-; l; -arginine; NO; nitric oxideeNOS; AMPK; ATP; CaMKK; Thrombin; Endothelial cells

Intracellular signaling dynamics during apoptosis execution in the presence or absence of X-linked-inhibitor-of-apoptosis-protein by Carla L. O'Connor; Sergio Anguissola; Heinrich J. Huber; Heiko Dussmann; Jochen H.M. Prehn; Markus Rehm (pp. 1903-1913).

X-linked-inhibitor-of-apoptosis-protein (XIAP) is the most potent intracellular inhibitor of caspases-9, -3 and -7. While highly elevated XIAP levels reduce the apoptotic response to various stimuli, the potency of physiological XIAP expression to control caspase activation and the consequences of XIAP deficiency on apoptosis execution remain controversial. We therefore analyzed parental and XIAP-deficient DLD-1 and HCT-116 colon cancer cells by employing fluorescence-based single-cell imaging of mitochondrial permeabilisation and effector caspase activation. Our results demonstrate that physiological XIAP expression maintains a transient “off”-state for effector caspase activation following mitochondrial permeabilisation. Loss of XIAP expression instead accelerated the caspase activation response, but did not enhance the measured caspase activity. Apoptosis execution kinetics were independent of activating the intrinsic or extrinsic pathway by either staurosporine or TRAIL, and corresponded to computational systems analyses of caspase activation dynamics. We confirmed a protective role of XIAP upstream of mitochondrial permeabilisation during TRAIL-induced apoptosis, however, once mitochondria permeabilised ultimately no cell could escape effector caspase activation, regardless of potential cell-to-cell variability within the populations or the presence of XIAP. Our study provides comprehensive kinetic and mechanistic insight into the rapid molecular dynamics during apoptosis execution in the presence or absence of physiological XIAP expression.

Keywords: Apoptosis; Systems biology; Single-cell analysis; Fluorescence resonance energy transfer; XIAP; Caspase

Agonist-dependent internalization and trafficking of the human prostacyclin receptor: A direct role for Rab5a GTPase by Martina B. O'Keeffe; Helen M. Reid; B. Therese Kinsella (pp. 1914-1928).

The human prostacyclin receptor (hIP) undergoes rapid agonist-induced internalization by largely unknown mechanism(s). Herein the involvement of Rab5 in regulating cicaprost-induced internalization of the hIP expressed in human embryonic kidney 293 cells was investigated. Over-expression of Rab5a significantly increased agonist-induced hIP internalization. Additionally, the hIP co-localized to Rab5a-containing endocytic vesicles in response to cicaprost stimulation and there was a coincident net translocation of Rab5 from the cytosol/soluble fraction of the cell. Co-immunoprecipitation studies confirmed a direct physical interaction between the hIP and Rab5a that was augmented by cicaprost. Whilst the dominant negative Rab5a^S34N did not show decreased interaction with the hIP or fully impair internalization, it prevented hIP sorting to endocytic vesicles. Moreover, the GTPase deficient Rab5a^Q79L significantly increased internalization and co-localized with the hIP in enlarged endocytic vesicles. While deletion of the carboxyl terminal (C)-tail domain of the hIP did not inhibit agonist-induced internalization, co-localization or co-immunoprecipitation with Rab5a per se, receptor trafficking was altered suggesting that it contains structural determinant(s) for hIP sorting post Rab5-mediated endocytosis. Taken together, data herein and in endothelial EA.hy 926 cells demonstrate a direct role for Rab5a in agonist-internalization and trafficking of the hIP and increases knowledge of the factors regulating prostacyclin signaling.

Keywords: Abbreviations; C-tail; carboxyl-terminal tail; CCV; clathrin coated vesicles; FBS; foetal bovine serum; GFP; green fluorescent protein; GPCR; G protein-coupled receptor; GRK; GPCR receptor kinase; HA; hemagglutinin; HEK; human embryonic kidney; hIP; human IP; IP; prostacyclin receptorProstacyclin; Receptor; Agonist; Internalization; Rab5; Human

PKCepsilon mediates glucose-regulated insulin production in pancreatic beta-cells by Nasim Warwar; Avital Dov; Eva Abramovitch; Ren Wu; Marina Jmoudiak; Esther Haber; Erol Cerasi; Rafael Nesher (pp. 1929-1934).

Endocrine cells produce large amounts of one or more peptides. The post-translational control of selective production of a single protein is often unknown. We used 3 unrelated approaches to diminish PKCε in rat islets to evaluate its role in preferential glucose-mediated insulin production. Transfection with siRNA (siR-PKCε) or expression of inactive PKCε (PKCε-KD) resulted in a significant reduction in insulin response to glucose (16.7 mmol/l). Glucose stimulation resulted in concentration of PKCε in the perinuclear region, an area known to be rich in ER–Golgi systems, associated with insulin-containing structures. ß’COP1 (RACK2) is the anchoring protein for PKCε. Glucose-stimulated proinsulin production was diminished by 50% in islets expressing PKCε-KD, and 60% in islets expressing RACK2 binding protein (εV1-2); total protein biosynthesis was not affected. In islets expressing εV1-2, a chase period following glucose stimulus resulted in a reduced proinsulin conversion to mature insulin. We propose that PKCε plays a specific role in mediating the glucose-signal into insulin production: binding to ß’COP1 localizes the activated enzyme to the RER where it modulates the shuttling of proinsulin to the TGN. Subsequently the enzyme may be involved in anterograde trafficking of the prohormone or in its processing within the TGN.

Keywords: Abbreviations; ER; endoplasmic reticulum; PKC; protein kinase C; RACK2; receptor for activated C kinase 2; COP1; coatomer protein 1; AUC; area under the curve; siRNA; small interfering RNA; DAG; diacylglycerol; PIP3; phosphatidylinositol 3,4,5-triphosphate; TGN; trans Golgi network; RP-HPLC; reverse-phase high pressure liquid chromatographyβ′COP1; Anchoring proteins; Translocation inhibiting peptides; siRNA; Proinsulin biosynthesis

Staufen1 is expressed in preimplantation mouse embryos and is required for embryonic stem cell differentiation by Hannah Gautrey; Josie McConnell; Majlinda Lako; Judith Hall; John Hesketh (pp. 1935-1942).

Pluripotent mouse embryonic stem (mES) cells derived from the blastocyst of the preimplantation embryo can be induced to differentiate in vitro along different cell lineages. However the molecular and cellular factors that signal and/or determine the expression of key genes, and the localisation of the encoded proteins, during the differentiation events are poorly understood. One common mechanism by which proteins can be targeted to specific regions of the cell is through the asymmetric localisation of mRNAs and Staufen, a double-stranded RNA binding protein, is known to play a direct role in mRNA transport and localisation. The aims of the present study were to describe the expression of Staufen in preimplantation embryos and mES cells and to use RNA interference (RNAi) to investigate the roles of Staufen1 in mES cell lineage differentiation. Western blotting and immunocytochemistry demonstrated that Staufen is present in the preimplantation mouse embryo, pluripotent mES cells and mES cells stimulated to differentiate into embryoid bodies, but the Staufen staining patterns did not support asymmetric distribution of the protein. Knockdown of Staufen1 gene expression in differentiating mES cells reduced the synthesis of lineage-specific markers including Brachyury, α-fetoprotein (AFP), PAX-6, and Vasa. There was however no significant change in either the gene expression of Nanog and Oct4, or in the synthesis of SSEA-1, all of which are key markers of pluripotency. These data indicate that inhibition of Staufen1 gene expression by RNAi affects an early step in mES cell differentiation and suggest a key role for Staufen in the cell lineage differentiation of mES cells.

Keywords: Staufen; Differentiation; Embryonic stem cell; RNA binding protein; RNA interference

PAK1 interacts with β-catenin and is required for the regulation of the β-catenin signalling pathway by gastrins by Hong He; Arthur Shulkes; Graham S. Baldwin (pp. 1943-1954).

β-catenin regulates cell–cell adhesion by binding to E-cadherin at the cell membrane and, when translocated into the nucleus, mediates signalling by activation of transcription factors such as TCF4. Mutations of the components of the Wnt/β-catenin pathway are found in many gastrointestinal cancers. Gastrins, including amidated (Gamide) and glycine-extended (Ggly) gastrin₁₇, stimulate the proliferation of gastrointestinal cancer cells. Gastrins also regulate β-catenin signalling through multiple pathways which seem to converge on p21-activated kinase 1 (PAK1). In this study, we have investigated the role of PAK1 in the regulation of β-catenin signalling by gastrins. Here we report for the first time that PAK1 associated with β-catenin. Both Gamide and Ggly stimulated the phosphorylation and activation of β-catenin in a PAK1-dependent manner. A kinase-inactive mutant PAK1^K299A blocked the gastrin-stimulated dissociation of β-catenin from E-cadherin, translocation of β-catenin from the cell membrane to the nucleus, and association of β-catenin with the transcription factor TCF4. The PAK1^K299A mutant also inhibited the stimulation of the expression of c-myc and cyclin D1, and of cell proliferation and migration, by gastrins. The results indicate that gastrins regulate β-catenin signalling through a PAK1-dependent pathway. PAK1 seems to be the point of convergence of multiple signalling pathways activated by gastrins.

Keywords: PAK1; β-catenin; E-cadherin; TCF4; Gastrins

ERp57-associated mitochondrial μ-calpain truncates apoptosis-inducing factor by Taku Ozaki; Tetsuro Yamashita; Sei-ichi Ishiguro (pp. 1955-1963).

Calpains, calcium-dependent neutral cystein proteases, are involved in a variety of cellular processes. We have previously shown the characteristics of mitochondrial μ-calpain even though calpastatin, a specific endogenous inhibitor of cytosolic calpains, was not present in the mitochondria. This suggested that the regulatory system of mitochondrial calpains differs from that of cytosolic calpains, and endogenous regulatory molecule(s) must exist in the mitochondria. In this study, we have identified ERp57 in partially purified mitochondrial μ-calpain using peptide mass fingerprinting based on MALDI-TOFMS. ERp57 is a member of the protein-disulfide isomerase (PDI) family and functions as a molecular chaperone within the ER. We showed that ERp57 was present in the mitochondria and was associated with mitochondrial μ-calpain. PDI inhibitors, such as DTNB and PAO, caused a degradation of the mitochondrial μ-calpain large subunit. The release of apoptosis-inducing factor (AIF) from the mitochondrial inner membrane was inhibited by treatment of the isolated mitochondria with DTNB and immunoprecipitation of ERp57-associated mitochondrial μ-calpain. Mitochondrial μ-calpain band in casein zymography disappeared by treatment with anti-ERp57 antibody. Our results demonstrate that ERp57 forms complexes with mitochondrial μ-calpain, and ERp57-associated mitochondrial μ-calpain cleaves AIF to a truncated form.

Keywords: Abbreviations; SDS; sodium dodecyl sulfate; PAGE; polyacrylamide gel electrophoresisMitochondrial μ-calpain; ERp57; Apoptosis-inducing factor (AIF); MALDI-TOFMS; Mitochondrial intermembrane space

Overexpression of functional TrkA receptors after internalisation in human airway smooth muscle cells by Véronique Freund-Michel; Nelly Frossard (pp. 1964-1971).

Trafficking of the TrkA receptor after stimulation by NGF is of emerging importance in structural cells in the context of airway inflammatory diseases. We have recently reported the expression of functional TrkA receptors in human airway smooth muscle cells (HASMC). We have here studied the TrkA trafficking mechanisms in these cells. TrkA disappearance from the cell membrane was induced within 5 min of NGF (3pM) stimulation. Co-immunoprecipitation of clathrin-TrkA was revealed, and TrkA internalisation inhibited either by clathrin inhibitors or by siRNA inducing downregulation of endogenous clathrin. TrkA internalised receptors were totally degraded in lysosomes, with no recycling phenomenon. Newly synthesized TrkA receptors were thereafter re-expressed at the cell membrane within 10 h. TrkA re-synthesis was inhibited by blockade of clathrin-dependent internalisation, but not of TrkA receptors lysosomal degradation. Finally, we observed that NGF multiple stimulations progressively increased TrkA expression in HASMC, which was associated with an increase in NGF/TrkA-dependent proliferation. In conclusion, we show here the occurrence of clathrin-dependent TrkA internalisation and lysosomal degradation in the airway smooth muscle, followed by upregulated re-synthesis of functional TrkA receptors and increased proliferative effect in the human airway smooth muscle. This may have pathophysiological consequences in airway inflammatory diseases.

Keywords: Abbreviations; CHC; clathrin heavy chain; Chloro; chloroquine; CPZ; chlorpromazine; FBS; foetal bovine serum; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; HASMC; human airway smooth muscle cells; MDC; monodansylcadaverine; NGF; nerve growth factor; PBS; phosphate-buffered saline; RIPA; RadioImmunoPrecipitation Assay; RT; reverse transcription; SiRNA C; control non-specific small interfering RNA; SiRNA Clat; small interfering siRNA against clathrin; Sulfo-NHS-biotin; Sulfosuccinimidobiotin; TrkA; tropomyosin-related kinase A; XTT; 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilideAirway smooth muscle; Clathrin; Lysosomes; Nerve growth factor (NGF); Trafficking; TrkA receptor

Differential subnuclear localisation of hnRNPs A/B is dependent on transcription and cell cycle stage by Lexie R. Friend; Siew Ping Han; Joseph A. Rothnagel; Ross Smith (pp. 1972-1980).

The heterogeneous nuclear ribonucleoproteins A1, A2/B1 and A3 (hnRNPs A/B) are involved in many nuclear functions that are confined to distinct regions within the nucleus. To characterise and compare the distribution of the hnRNPs A/B in these subnuclear compartments, their colocalisation with spliceosomal components, nascent transcripts and other nuclear markers in HeLa cells was investigated by immunostaining and transfection of GFP constructs. The mechanisms of this localisation were further explored by treating cells with detergent, nucleases and transcription inhibitors. We have also examined the dynamics of A2/B1 throughout the cell cycle. Our results show that hnRNPs A/B have different subnuclear localisations, with A1 differentially localised to the nuclear envelope, and A2/B1 and A3 enriched around nucleoli. This pattern of distribution was dependent on RNA integrity and active transcription. The hnRNPs A/B preferentially colocalised with a subset of splicing factors. Significantly, only rarely did transcription factories colocalise with high levels of these hnRNPs. Moreover, localisation of A2/B1 changed with cell cycle stage. Our findings show that the subnuclear localisation of the hnRNPs A/B is differentially, spatially and temporally regulated, and suggest that this localisation may be relevant to their nuclear functions.

Keywords: Abbreviations; hnRNPs; heterogeneous nuclear ribonuclear proteins; RRM; RNA recognition motif; snRNPs; small nuclear ribonuclear proteinshnRNP A/B; Spliceosome; Splicing factor; Transcription inhibition; Immunoprecipitation; Cell cycle

The iron-regulated metastasis suppressor, Ndrg-1: Identification of novel molecular targets by Zaklina Kovacevic; Dong Fu; Des R. Richardson (pp. 1981-1992).

A recently identified metastasis suppressor, N-myc downstream regulated gene-1 ( Ndrg-1), has been shown to reduce the invasion and metastasis of breast, colon, prostate and pancreatic cancer. Among its many functions, Ndrg-1 is involved in modulating differentiation, proliferation and angiogenesis. However, knowledge of the molecular targets of Ndrg-1 is limited. The current study has focused on examining the functions of Ndrg-1 in a number of different cancer cell models including prostate, colon, lung and pancreatic cancer to elucidate the known pleiotropic nature of this protein. Furthermore, the potential gene targets of Ndrg-1 were analyzed using whole genome gene array revealing a substantial number of genes whose expression was affected by this metastasis suppressor. Significantly, Ndrg-1 up-regulated thiamine triphosphatase ( Thtpa) expression in three of the four cell models. Thtpa is known to decrease the levels of the energy currency molecule, thiamine triphosphate, suggesting a potential pathway for the anti-proliferative effects of Ndrg-1. Furthermore, Ndrg-1 reduced the protein levels of cathepsin C which plays a role in invasion, indicating a potential mechanism of its anti-metastatic role in pancreatic cancer cells. These findings provide a potential link between the observed functions of Ndrg-1 and its molecular targets, further demonstrating its anti-metastatic effect.

Keywords: Iron chelator; Iron; Desferioxamine; Ndrg-1; Metastasis suppressor

Bestrophin 1 and 2 are components of the Ca²⁺ activated Cl⁻ conductance in mouse airways by René Barro-Soria; Rainer Schreiber; Karl Kunzelmann (pp. 1993-2000).

Ca²⁺ activated Cl⁻ transport is found in airways and other organs and is abnormal in cystic fibrosis, polycystic kidney disease and infectious diarrhea. The molecular identity of Ca²⁺ activated Cl⁻ channels (CaCC) in the airways is still obscure. Bestrophin proteins were described to form CaCC and to regulate voltage gated Ca²⁺ channels. The present Ussing chamber recordings on tracheas of bestrophin 1 knockout (vmd2^−/−) mice indicate a reduced Cl⁻ secretion when activated by the purinergic agonist ATP (0.1–1 μM). As two paralogs, best1 and best2, are present in mouse tracheal epithelium, we examined the contribution of each paralog to Ca²⁺ activated Cl⁻ secretion. In whole cell patch-clamp measurements on primary airway epithelial cells from vmd2^−/− tracheas, ATP activated Cl⁻ currents were reduced by 50%. Additional knockdown of mbest2 in vmd2^−/− cells by short interfering RNA further suppressed ATP-induced Cl⁻ currents down to 20% of that observed in cells from vmd2^+/+ animals. Moreover, RNAi-suppression of both mbest1 and mbest2 reduced CaCC in vmd2^+/+ cells. Direct activation of CaCC by increase of intracellular Ca²⁺ was also reduced in whole cell recordings of vmd2^−/− cells. These results clearly suggest a role of bestrophin 1 and 2 for Ca²⁺ dependent Cl⁻ secretion in mouse airways.

Keywords: Ca; ²⁺; activated Cl; ⁻; channel; Bestrophin 1; Bestrophin 2; Vmd2; Cl; ⁻; conductance; Airways; Epithelium

Cytosolic prion protein is the predominant anti-Bax prion protein form: Exclusion of transmembrane and secreted prion protein forms in the anti-Bax function by David T.S. Lin; Julie Jodoin; Michaël Baril; Cynthia G. Goodyer; Andréa C. LeBlanc (pp. 2001-2012).

Prion protein (PrP) prevents Bax-mediated cell death by inhibiting the initial Bax conformational change that converts cytosolic Bax into a pro-apoptotic protein. PrP is mostly a glycophosphatidylinositol-anchored cell surface protein but it is also retrotranslocated into cytosolic PrP (CyPrP) or can become a type 1 or type 2 transmembrane protein. To determine the form and subcellular location of the PrP that has anti-Bax function, we co-expressed various Syrian hamster PrP (SHaPrP) mutants that favour specific PrP topologies and subcellular localization with N-terminally green fluorescent protein tagged pro-apoptotic Bax (EGFP-Bax) in MCF-7 cells and primary human neurons. Mutants that generate both CyPrP and secreted PrP (^SecPrP) or only CyPrP have anti-Bax activity. Mutants that produce^CtmPrP or^NtmPrP lose the anti-Bax activity, despite their ability to also make^SecPrP. Transmembrane-generating mutants do not produce CyPrP and both normal and cognate mutant forms of CyPrP rescue against the loss of anti-Bax activity.^SecPrP-generating constructs also produce non-membrane attached^SecPrP. However, this form of PrP has minimal anti-Bax activity. We conclude that CyPrP is the predominant form of PrP with anti-Bax function. These results imply that the retrotranslocation of PrP encompasses a survival function and is not merely a pathway for the proteasomal degradation of misfolded protein.

Keywords: Abbreviations; PrP; prion protein; Bax; Bcl-2 associated protein X; SHaPrP; Syrian hamster prion protein; EGFP; enhanced green fluorescence protein; EGFP-Bax; N-terminally EGFP-tagged-Bax; CyPrP; cytosolic prion protein; GPI; glycosylphosphatidylinositol; SP-CyPrP; signal peptide-attached CyPrP; WT; wild type; Endo H; endoglycosidase H; PNGaseF; peptide: N-glycosidase F; rPrP; recombinant PrP; CMV; cytomegalovirus; Opn; osteopontin; Prl; prolactin; STE; stop transfer effector; TMD; transmembrane domain; DF; degrees of freedom; F-value; Fisher valueBax; Prion; Transmembrane prion protein; Cytosolic prion protein; Secreted prion protein; Neuroprotection; Apoptosis

Marked mitochondrial alterations upon starvation without cell death, caspases or Bcl-2 family members by Artemis Kosta; Marie-Françoise Luciani; Willie J.C. Geerts; Pierre Golstein (pp. 2013-2019).

Dictyostelium HMX44A cells can withstand starvation under monolayer conditions for a few days without dying. They die only when the differentiation factor DIF-1 is exogenously added. Still, when HMX44A were subjected to starvation without addition of DIF-1 they showed, by electron microscopy and electron tomography, gross mitochondrial lesions including marked cristae alterations with frequent “holes” probably originating from dilated cristae. Since these cells did not die as shown for instance by FACS analysis, these results showed unexpected resilience of cells bearing markedly altered mitochondria, and thus showed that apparently destructive mitochondrial alterations may not lead to cell death. Also, these marked mitochondrial lesions could not be caused by caspases or bcl-2 family members, which these cells do not encode.

Keywords: Mitochondria; Dictyostelium; Programmed cell death; Electron microscopy; Electron tomography

Glutathione peroxidase 1-deficient mice are more susceptible to doxorubicin-induced cardiotoxicity by Jinping Gao; Ye Xiong; Ye-Shih Ho; Xuwan Liu; Chu Chang Chua; Xingshun Xu; Hong Wang; Ronald Hamdy; Balvin H.L. Chua (pp. 2020-2029).

Doxorubicin (DOX)-induced cardiotoxicity is thought to be mediated by the generation of superoxide anion radicals (superoxide) from redox cycling of DOX in cardiomyocyte mitochondria. Reduction of superoxide generates H₂O₂, which diffuses throughout the cell and potentially contributes to oxidant-mediated cardiac injury. The mitochondrial and cytosolic glutathione peroxidase 1 (Gpx1) primarily functions to eradicate H₂O₂. In this study, we hypothesize that Gpx1 plays a pivotal role in the clearance of H₂O₂ generated by DOX. To test this hypothesis, we compared DOX-induced cardiac dysfunction, mitochondrial injury, protein nitration, and apoptosis in Gpx1-deficient and wild type mouse hearts. The Gpx1-deficient hearts showed increased susceptibility to DOX-induced acute functional derangements than wild type hearts, including impaired contractility and diastolic properties, decreased coronary flow rate, and reduced heart rate. In addition, DOX treatment impaired the mitochondrial function of Gpx1-deficient hearts. Specifically, Gpx1-deficient hearts treated with DOX demonstrated an increased rate of NAD-linked state 4 respiration and a decline in the P/O ratio relative to wild type hearts, suggesting that DOX uncouples the electron transfer chain and oxidative phosphorylation in Gpx1-deficient hearts. Finally, apoptosis and protein nitration were significantly increased in Gpx1-deficient mouse hearts compared to wild type hearts. These studies suggest that Gpx1 plays significant roles in protecting DOX-induced mitochondrial impairment and cardiac dysfunction in the acute phase.

Keywords: Doxorubicin; Glutathione peroxidase deficiency; Mitochondrial function; Cardiac function; Apoptosis; Protein nitration

HDAC-class II specific inhibition involves HDAC proteasome-dependent degradation mediated by RANBP2 by Annamaria Scognamiglio; Angela Nebbioso; Fabio Manzo; Sergio Valente; Antonello Mai; Lucia Altucci (pp. 2030-2038).

Discovered for their ability to deacetylate histones and repress transcription, HDACs are a promising target for therapy of human diseases. The class II HDACs are mainly involved in developmental and differentiation processes, such as myogenesis. We report here that class I and class II HDAC inhibitors such as SAHA or the class II selective inhibitor MC1568 induce down-regulation of class II HDACs in human cells. In particular, both SAHA and MC1568 induce HDAC 4 down-regulation by increasing its specific sumoylation followed by activation of proteasomal pathways of degradation. Sumoylation that corresponds to HDAC 4 nuclear localization results in a transient increase of the HDAC 4 repressive action on target genes such as RARα and TNFα. The HDAC 4 degradation that follows to its sumoylation results in gene target activation. Silencing of the RANBP2 E3 ligase reverts HDAC 4 repression by blocking its own sumoylation. These findings identify a crosstalk occurring between acetylation, deacetylation and sumoylation pathways and suggest that class II specific HDAC inhibitors may affect different epigenetic pathways.

Keywords: HDACs; Cancer; Epigenetic; Signal transduction; Sumoylation; Epidrugs

Corrigendum to “Cellular binding, motion, and internalization of synthetic gene delivery polymers” [Biochim. Biophys. Acta 1773 (2007) 1583–1588] by Gaelen T. Hess; William H. Humphries IV; Nicole C. Fay; Christine K. Payne (pp. 2039-2039).

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BBA - Molecular Cell Research (v.1783, #10)

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BBA - Molecular Cell Research (v.1783, #10)