BBA - Molecular Cell Research (v.1745, #1)

Acid sensing ionic channels: Modulation by redox reagents by Fedorenko Andrey; Timur Tsintsadze; Tatyana Volkova; Natalia Lozovaya; Oleg Krishtal (1-6).
Acid-sensing ion channels (ASICs) are widely expressed in mammalian sensory neurons and supposedly play a role in nociception and acid sensing. In the course of functioning the redox status of the tissue is subjected to changes. Using whole-cell patch-clamp/concentration clamp techniques we have investigated the effect of redox reagents on the ASIC-like currents in the sensory ganglia and hippocampal neurons of rat. The reducing agent dithiothreitol (DTT), when applied in the concentrations 1–2 mM, reversibly potentiates proton-activated currents, while the oxidizing reagent 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) causes their inhibition. The EC50 and Hill coefficient for the activation of ASIC-like currents by protons are not affected by DTT. Redox modulation of proton-activated currents is independent on the membrane potential and on the level of pH used for the current activation. The endogenous antioxidant tripeptide glutathione (its reduced form, g-l-glutamyl-l-cysteinyl-glycine, GSH) also potentiates proton-activated currents. Our results indicate that ASIC-like currents are susceptible to regulation by redox agents.

The distribution of the cation-independent mannose 6-phosphate and 78 kDa receptors was studied in postnuclear subcellular fractions from two rat liver cell lines. ELISA assays revealed that the mannose 6-phosphate receptor is enriched in the light buoyant Percoll fractions that contain Golgi structures and early endosomes. Most of the 78 kDa receptor is localized in a heavy fraction at the bottom of the Percoll gradient and smaller amounts in the endosomal fractions. The high-density compartment is denser than lysosomes, contains LAMP2 but not LIMPII or acid hydrolases, and is not disrupted with glycyl-l-phenylalanine 2-naphthylamide, a substrate for cathepsin C that selectively disrupts lysosomes. Immunofluorescence microscopy studies indicate no colocalization of the 78 kDa receptor with the mannose 6-phosphate receptor or LIMPII. Mannose 6-phosphate-independent endocytosed β-glucuronidase was found in the lysosomal, the early and late endosomal fractions. These fractions were immunoadsorbed in columns containing antibodies against the 78 kDa receptor. Only the endocytosed β-glucuronidase present in the early and late endosomal fractions is associated to immunoadsorbed vesicles. In these vesicles, LAMP2 was detected but no LIMPII or the mannose 6-phosphate receptor. Results obtained suggest that the 78 kDa receptor is found along the endocytic pathway, but in vesicles different from the cation-independent mannose 6-phosphate receptor.
Keywords: Mannose 6-phosphate-independient endocytosis; Mannose 6-phosphate receptor; 78 kDa receptor; Lysosomal targeting;

Effector-induced Syk-mediated phosphorylation in human erythrocytes by Luciana Bordin; Florina Ion-Popa; Anna Maria Brunati; Giulio Clari; Philip S. Low (20-28).
Band 3 (AE1), the most prominent polypeptide of the human erythrocyte membrane, becomes heavily tyrosine phosphorylated following treatment of intact cells with protein tyrosine phosphatase inhibitors such as diamide, pervanadate, vanadate, or N-ethylmaleimide (NEM). The mechanism underlying this tyrosine phosphorylation is thought to involve the sequential action of two protein tyrosine kinases, Syk (p72syk) and Lyn (p53/56lyn). While Lyn catalysed phosphorylation appears to be strictly dependent on prior phosphorylation of Tyr8 and 21 of band 3 by Syk, little is known about the mechanism of induction of Syk phosphorylation. Data presented here show that both the fraction of Syk that associates with the membrane and the extent of phosphorylation of band 3 differ in response to the above inhibitors. While diamide and NEM stimulate syk translocation to the membrane during their induction of band 3 tyrosine phosphorylation, pervanadate and vanadate induce no change in kinase distribution. Moreover, diamide and NEM-induced Syk recruitment to the membrane are phosphotyrosine independent and involve their preferential association with Triton X-100-insoluble membrane skeletons. Together these data reveal a complex process controlling the association and catalytic activity of protein tyrosine kinases syk and lyn with the human erythrocyte membrane.
Keywords: Syk; Tyrosine-phosphorylation; Human erythrocyte; Band 3;

Depletion of the AP-1 repressor JDP2 induces cell death similar to apoptosis by M. Lerdrup; C. Holmberg; N. Dietrich; E. Shaulian; T. Herdegen; M. Jäättelä; T. Kallunki (29-37).
JDP2 is a ubiquitously expressed nuclear protein that efficiently represses the activity of the transcription factor AP-1. Thus far, all studies of JDP2 function have relied on the ectopic expression of the protein. In this study, we use a different approach: depletion of JDP2 from cells. Specific depletion of JDP2 resulted in p53-independent cell death that resembles apoptosis and was evident at 72 h. The death mechanism was caspase dependent as the cells could be rescued by treatment with caspase inhibitor zVAD. Our studies suggest that JDP2 functions as a general survival protein, not only following UV-irradiation, as reported earlier, but also under normal culture conditions. Thus, our data support that JDP2 is a cellular survival protein whose presence is necessary for normal cellular function.
Keywords: JDP2; AP-1; Programmed cell death; Caspase; Antisense;

CCAAT/enhancer binding protein α maintains the ability of insulin-stimulated GLUT4 translocation in 3T3-C2 fibroblastic cells by Muneya Fujimoto; Hiroaki Masuzaki; Yuji Yamamoto; Nobuyoshi Norisada; Makoto Imori; Masako Yoshimoto; Tsutomu Tomita; Tomohiro Tanaka; Kayoko Okazawa; Junji Fujikura; Hideki Chusho; Ken Ebihara; Tatsuya Hayashi; Kiminori Hosoda; Gen Inoue; Kazuwa Nakao (38-47).
In 3T3-L1 preadipocytes, hormonal induction causes adipose conversion and facilitates the expression of insulin-sensitive glucose transporter, GLUT4. Evidence has accumulated that, in 3T3-L1 preadipocytes, the formation of GLUT4 storage vesicle and its translocation to plasma membrane precede both lipid accumulation and expression of GLUT4 and C/EBPα, a key transcription factor for adipose differentiation. On the other hand, 3T3-C2 fibroblastic cells, a subline of 3T3-L1, follow adipogenic process till mitotic clonal expansion stage (2 days after hormonal induction), but do not proceed to terminal differentiation stage (8 days after the induction), resulting in a lack of adipose conversion and GLUT4 expression. Here we show that, when myc-tagged GLUT4 was retrovirally expressed in 3T3-C2 cells, insulin-stimulated GLUT4 translocation did occur on day 2 after the induction. On day 8 after the induction, however, neither GLUT4 translocation nor the expression of C/EBPα was observed. We also created 3T3-C2 cells stably expressing both myc-tagged GLUT4 and C/EBPα, demonstrating that co-expressed cells showed insulin-stimulated GLUT4 translocation on day 8 after the induction, as well as adipose conversion coupling with PPARγ expression. Our results provide evidence that C/EBPα has the potential to maintain the ability of insulin-stimulated GLUT4 translocation in C/EBPα-deficient 3T3-C2 fibroblastic cells.
Keywords: GLUT4 translocation; 3T3-C2 fibroblast; C/EBPα; Peroxisome proliferator-activated receptor γ; Adipogenesis;

Regulation of Bcl-2 and Bcl-xL anti-apoptotic protein expression by nuclear receptor PXR in primary cultures of human and rat hepatocytes by Nathalie Zucchini; Georges de Sousa; Béatrice Bailly-Maitre; Jean Gugenheim; Rémi Bars; Géraldine Lemaire; Roger Rahmani (48-58).
The pregnane X receptor (PXR) plays a major role in the protection of the body by regulating the genes involved in the metabolism and elimination of potentially toxic xeno- and endobiotics. We previously described that PXR activator dexamethasone protects hepatocytes from spontaneous apoptosis. We hypothesise a PXR-dependent co-regulation process between detoxication and programmed cell death. Using primary cultured human and rat hepatocytes, we investigated to determine if PXR is implicated in the regulation of Bcl-2 and Bcl-xL, two crucial apoptosis inhibitors. In the present study we demonstrated that the treatment of primary cultured hepatocytes with PXR agonists increased hepatocyte viability and protects them from staurosporine-induced apoptosis. The anti-apoptotic capacity of PXR activation was correlated with Bcl-2 and Bcl-xL induction at both the transcriptional and protein levels in man and rats, respectively. The inhibition of PXR expression by antisense oligonucleotide abolished PXR activators Bcl-xL induction. Accordingly, PXR overexpression in HepG2 cells led to bcl-2 induction upon clotrimazole treatment and protects cells against Fas-induced apoptosis. Our results demonstrate that PXR expression is required for Bcl-2 and Bcl-xL up-regulation upon PXR activators treatment in human and rat hepatocytes. They also suggest that PXR may protect the liver against chemicals by simultaneously regulating detoxication and the apoptotic pathway.
Keywords: Primary human and rat hepatocyte; PXR; Bcl-2; Bcl-xL; CYP3A;

Regulation of the release and function of tumor cell-derived soluble CD44 by Joanna Cichy; Paulina Kulig; Ellen Puré (59-64).
CD44, a major receptor for glycosaminoglycan hyaluronan (HA), is a broadly distributed cell surface glycoprotein implicated in multiple functions, including tumor growth and dissemination. The affinity of surface CD44 for HA is subject to regulation at several levels. CD44 is found in multiple phases, including as an integral transmembrane protein and as soluble fragment of the extracellular domain found in the circulation and other body fluids. Transmembrane CD44 and its ability to interact with HA have been a focus of numerous studies in the past, but the function of soluble CD44 remains obscure. Interestingly, malignant diseases are often associated with an increase in the plasma level of CD44. The delineation of the HA binding capacity of tumor-derived soluble CD44 is an important step toward understanding the biological function of this molecule. In this study, we demonstrate that tumor cells activated to bind HA by cytokines rapidly release CD44 upon treatment with phorbol ester (PMA). The affinity for HA of the soluble CD44 released in response to PMA varied depending on the cytokine pretreatment. These results suggest that the function of tumor-derived soluble CD44, like the transmembrane form of the receptor, can be regulated.
Keywords: Shedding; Tumor; Adhesion; Oncostatin M; TGF; Phorbol ester;

Y+ and y+L arginine transporters in neuronal cells expressing tyrosine hydroxylase by S.Y. Bae; Q. Xu; D. Hutchinson; C.A. Colton (65-73).
Arginine is a semi-essential amino acid that serves as sole substrate for enzymes involved in diverse cell processes including redox balance via nitric oxide synthase (NOS) and cell proliferation via arginase. Neurons that express nNOS require intracellular arginine to generate nitric oxide (NO). Using a TH+ neuronal cell line (CAD cells), we show that neuronal NO production is largely dependent on extracellular arginine. Although a small intracellular pool exists in CAD cells, the lack of mRNA for argininosuccinate synthase (AS), a rate limiting enzyme for arginine recycling, suggests that intracellular pools are not re-supplied by this mechanism in this sub-class of neurons. Rather, arginine is taken up from the extracellular media by two primary transport systems, the y+ and the y+L systems. The expression of CAT1, CAT3, y+LAT1 and y+LAT2 mRNAs supports the presence of each system. CAD cell arginine transport is depressed by increased extracellular K+ levels and demonstrates that variations in membrane potential control neuronal arginine uptake. Short term exposure to the oxidizing agents, rotenone and Angeli's salt, but not FeSO4, increases arginine transport. The regulation of arginine uptake by physiological factors suggests that arginine supply adapts in a moment-to-moment fashion to the changing needs of the neuron.
Keywords: Arginine transport; Neuronal nitric oxide synthase; Cationic amino acid transporter; Citrulline–NO cycle; Rotenone; Nitroxyl;

Human myotonic dystrophy protein kinase effect in S. cerevisiae by Renata Del Carratore; Francesca Ciucci; Pascale Beffy; Marta Casella; Mariarita Puntoni; Silvana Simi; Marcella Simili (74-83).
Human myotonic dystrophy protein kinase (DMPK), the product of the myotonic dystrophy (DM) locus, is a member of a novel class of multidomain serine–threonine protein kinases, which interacts with members of the Rho family of small GTPases. DMPK has been shown to affect the cell growth, size and shape in different organisms, from fission yeast to man, but its physiological role is still unclear. We examined the effect of the overexpression of two forms of human DMPK, full-length (DMFL) and a C-terminal truncated form (DMT) on the growth and cell morphology of S. cerevisiae, which possesses a DMPK homologous gene (CBK1) important for polarized growth and cell division. We report that the overexpression of either forms of human DMPK did not complement the CBK1 function in the haploid strain WR208-1a, deleted for CBK1. The truncated form, but not the full length one, slowed down growth rate and induced elongation of the haploid wild type strain CBK1. Similar results were obtained in the diploid wild type strain RS112 of S. cerevisiae where also the full-length form was effective. These effects were abolished when either DMFL or DMT were mutated in the ATP binding site (K100R mutation), suggesting that the kinase activity of DMPK is required. Interestingly, DMPK localization in yeast is similar to that of Cbk1 protein suggesting that it might affect a pathway, which regulates cell morphogenesis and progression through cell cycle, possibly involving CBK1.
Keywords: S. cerevisiae; DMPK; CBK; Polarized growth;

Alsin is partially associated with centrosome in human cells by Stéphanie Millecamps; Benoît J. Gentil; François Gros-Louis; Guy Rouleau; Jean-Pierre Julien (84-100).
Mutations in the ALS2 gene has recently been linked to cases of juvenile amyotrophic lateral sclerosis, juvenile primary lateral sclerosis and ascending hereditary spastic paralysis. All reported mutations predict the production of truncated forms of Alsin suggesting a loss of function mechanism for these motor neuron disorders. Here we used the tetracycline-regulated expression system to overexpress the full-length and truncated forms of Alsin in different cell lines. Alsin overexpression caused severe phenotypic changes in monkey COS-7 cells including the enlargement and accumulation of early endosomes, impairment of mitochondria trafficking and fragmentation of the Golgi apparatus. Our results further demonstrate the requirement of the Alsin VPS9 domain for occurrence of the vacuolation process and the role of Alsin as a guanine nucleotide exchange factor for Rab5. Transfected human SW13 cells exhibited an unexpected centrosomal localization for Alsin that was linked to the presence of the c-terminal part of the protein. Immunofluorescence staining revealed a colocalization of Alsin with the centrosomal markers γ-tubulin and A kinase anchoring protein (AKAP-450). Similar results were obtained with human LA-N-2 and SK-N-SH neuronal cells. Moreover endogenous Alsin was detected in a centrosome preparation purified from human cortical brain. Considering the crucial role of centrosome in the production of microtubules required for intracellular transport, these findings are of potential relevance for unravelling the disease mechanisms linked to Alsin mutations.
Keywords: Amyotrophic lateral sclerosis; ALS2; Alsin; Endosome; Centrosome; Microtubule;

Bcl-xL/Bax ratio is altered by IFNγ in TNFα- but not in TRAIL-induced apoptosis in colon cancer cell line by Gilbert Baillat; Francoise Garrouste; Maryse Remacle-Bonnet; Jacques Marvaldi; Gilbert Pommier (101-110).
Apoptosis is a crucial mechanism to eliminate harmful cells in which growth factors and cytokines are key regulators. In HT29-D4 cells, a model of human colon carcinoma, IFNγ presensitization is essential to induce an apoptotic response to TNFα whereas it only slightly enhances TRAIL-induced apoptosis. To compare the transcriptional profiles induced by TNFα and TRAIL and their regulation by IFNγ, we optimized a cDNA array analysis on targeted signaling pathways and confirmed the gene expression modulations by comparative RT-PCR. Although the two TNFSF ligands induced a same strong up-expression of pro-apoptotic Bax gene, the expression of anti-apoptotic Bcl-xL gene was more strongly up-regulated in TNFα- than in TRAIL-stimulated cells. Thus, TRAIL but not TNFα induced apoptotic mitochondrial cascade as highlighted by cytochrome c release into cytosol. IFNγ presensitization of TRAIL-stimulated cells did not induce any change in cytochrome c release, suggesting that the increase of IFNγ/TRAIL-induced apoptosis is independent of this pathway. In contrast, IFNγ pretreatment prevented Bcl-xL gene up-expression in TNFα-stimulated cells and allowed cytochrome c release. Thus, we hypothesize that the Bcl-xL/Bax ratio can block the apoptotic response in TNFα-stimulated cells but allows cell death initiation when it is altered by a crosstalk between IFNγ presensitization and TNFα induced signalings.
Keywords: Apoptosis; Colon cancer; Tumor necrosis factor superfamily; IFNγ; Bcl-2 family; cDNA array;

Effects of agmatine accumulation in human colon carcinoma cells on polyamine metabolism, DNA synthesis and the cell cycle by Camille Mayeur; Géraldine Veuillet; Marie Michaud; Francis Raul; Hervé M. Blottière; François Blachier (111-123).
Putrescine, spermidine and spermine are low molecular polycations that play important roles in cell growth and cell cycle progression of normal and malignant cells. Agmatine (1-amino-4-guanidobutane), another polyamine formed through arginine decarboxylation, has been reported to act as an antiproliferative agent in several non-intestinal mammalian cell models. Using the human colon adenocarcinoma HT-29 Glc−/+ cell line, we demonstrate that agmatine, which markedly accumulated inside the cells without being metabolised, exerted a strong cytostatic effect with an IC50 close to 2 mM. Agmatine decreased the rate of l-ornithine decarboxylation and induced a 70% down-regulation of ornithine decarboxylase (ODC) expression. Agmatine caused a marked decrease in putrescine and spermidine cell contents, an increase in the N1-acetylspermidine level without altering the spermine pool. We show that agmatine induced the accumulation of cells in the S and G2/M phases, reduced the rate of DNA synthesis and decreased cyclin A and B1 expression. We conclude that the anti-metabolic action of agmatine on HT-29 cells is mediated by a reduction in polyamine biosynthesis and induction in polyamine degradation. The decrease in intracellular polyamine contents, the reduced rate of DNA synthesis and the cell accumulation in the S phase are discussed from a causal perspective.
Keywords: Agmatine; Polyamine; Cell cycle; Cyclin A; Cyclin B1; Metabolism;

Aluminum exposure affects transferrin-dependent and -independent iron uptake by K562 cells by Gladys Pérez; Nicolás Pregi; Daniela Vittori; Cecilia Di Risio; Graciela Garbossa; Alcira Nesse (124-130).
Aluminum (Al) and iron (Fe) share several physicochemical characteristics and they both bind to transferrin (Tf), entering the cell via Tf receptors (TfR). Previously, we found similar values of affinity constant for the binding of TfR to Tf carrying either Al or Fe. The competitive interaction between both metals prevented normal Fe incorporation into K562 cells and triggered the upregulation of Fe transport. In the present work we demonstrated that Al modified Fe uptake without affecting the expression of Tf receptors. Both TfR and TfR2 mRNA levels, evaluated by RT-PCR, and TfR antigenic sites, analyzed by flow cytometry, were found unchanged after Al exposure. In turn, Al did induce upregulation of non-Tf bound Fe (NTBI) uptake. This modulation was not due to intracellular Fe decrease since NTBI transport proved not to be regulated by Fe depletion. Unlike its behavior in the presence of Tf, Al was unable to compete with NTBI uptake, suggesting that both metals do not share the same alternative transport pathway. We propose that Al interference with TfR-mediated Fe incorporation might trigger the upregulation of NTBI uptake, an adaptation aimed at incorporating the essential metal required for cellular metabolism without allowing the simultaneous access of a potentially toxic metal.
Keywords: Aluminum; Iron metabolism; Transferrin receptor; Transferrin-mediated iron uptake; Non-transferrin bound iron transport; K562 cell line;

The membrane-associated guanylate kinase protein, MAGI-1, has been shown to be a component of epithelial tight junctions in both Madin–Darby canine kidney cells and in intestinal epithelium. Because we have previously observed MAGI-1 expression in glomerular visceral epithelial cells (podocytes) of the kidney, we screened a glomerular cDNA library to identify the potential binding partners of MAGI-1 and isolated a partial cDNA encoding a novel protein. The partial cDNA exhibited a high degree of identity to an uncharacterized human cDNA clone, KIAA0989, which encodes a protein of 780 amino acids and contains a predicted coiled-coil domain in the middle of the protein. In vitro binding assays using the partial cDNA as a GST fusion protein confirm the binding to full-length MAGI-1 expressed in HEK293 cells, as well as endogenous MAGI-1, and also identified the first WW domain of MAGI-1 as the domain responsible for binding to this novel protein. Although a conventional PPxY binding motif for WW domains was not present in the partial cDNA clone, a variant WW binding motif was identified, LPxY, and found to be necessary for interacting with MAGI-1. When expressed in Madin–Darby canine kidney cells, the full-length novel protein was found to colocalize with MAGI-1 at the tight junction of these cells and the coiled-coil domain was found to be necessary for this localization. Because of its interaction with MAGI-1 and its localization to cell–cell junctions, this novel protein has been given the name MAGI-1-associated coiled-coil tight junction protein (MASCOT).
Keywords: MAGI-1; Tight junction; WW domain; Coiled-coil; MAGI-1-associated coiled-coil tight junction protein; AMOTL2;