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BBA - Molecular Cell Research (v.1773, #7)

Editorial Board (pp. ii).

Reduced expression of the NADPH oxidase NOX4 is a hallmark of adipocyte differentiation by Sarah Mouche; Sanae Ben Mkaddem; Wei Wang; Masa Katic; Yu-Hua Tseng; Stephanie Carnesecchi; Klaus Steger; Michelangelo Foti; Christoph A. Meier; Patrick Muzzin; C. Ronald Kahn; Eric Ogier-Denis; Ildiko Szanto (pp. 1015-1027).

Adipocyte differentiation is a complex process regulated among other factors by insulin and the production of reactive oxygen species (ROS). NOX4 is a ROS generating NADPH oxidase enzyme mediating insulin′s action in 3T3L1 adipocytes. In the present paper we show that NOX4 is expressed at high levels both in white and brown preadipocytes and that differentiation into adipocytes results in a decrease in their NOX4 mRNA content. These in vitro results were confirmed in vivo by demonstrating that in intact adipose tissue the majority of NOX4 expressing cells are localized within the preadipocyte containing stromal/vascular fraction, rather than in the portion consisting of mature adipocytes. In line with these observations, quantification of NOX4 mRNA in fat derived from different rodent models of insulin resistance indicated that alteration in NOX4 expression reflects changes in the ratio of adipocyte/interstitial fractions. In conclusion, we reveal that decreased NOX4 mRNA content is a hallmark of adipocyte differentiation and that NOX4 expression measured in whole adipose tissue is not an unequivocal indicator of intact or impaired insulin action.

Keywords: Reactive oxygen species; NADPH oxidase; NOX4; Insulin signaling; Adipocyte differentiation

MUC1 inhibits cell proliferation by a β-catenin-dependent mechanism by Erik P. Lillehoj; Wenju Lu; Timothy Kiser; Simeon E. Goldblum; K. Chul Kim (pp. 1028-1038).

β-Catenin binds to the cytoplasmic region of the type 1 membrane glycoprotein MUC1. In the current study, we utilized HEK293T cells expressing the full-length MUC1 protein, or a CD8/MUC1 fusion protein containing only the MUC1 cytoplasmic tail, to investigate the effects of β-catenin binding to MUC1 on downstream β-catenin-dependent events. Compared with HEK293T cells transfected with empty vector or CD8 alone, expression of the MUC1 cytoplasmic tail inhibited β-catenin binding to E-cadherin, decreased translocation of β-catenin into the nucleus, reduced activation of the LEF-1 transcription factor, and blocked expression of the cyclin D1 and c-Myc proteins. Furthermore, expression of MUC1 was associated with decreased cell proliferation, either in the context of the transfected HEK293T cells, or when comparing wild type (Muc1^+/+) vs. knockout (Muc1^−/−) mouse primary tracheal epithelial cells. We conclude that MUC1 inhibits cell proliferation through a β-catenin/LEF-1/cyclin D1/c-Myc pathway.

Keywords: Abbreviations; ALI; air–liquid interface; CT; cytoplasmic tail; GSK; glycogen synthase kinase; LEF; lymphoid enhancer factor; TCF; T cell factor; TCL; total cell lysate; TER; transepithelial electrical resistance; TSE; tracheal surface epithelialCyclin D1; c-Myc; E-cadherin; LEF-1; Cancer; Transcription; Nucleus; Cytoplasm; Membrane; Reepithelialization

Targeted disruption of the murine large nuclear KIAA1440/Ints1 protein causes growth arrest in early blastocyst stage embryos and eventual apoptotic cell death by Toshiyuki Hata; Manabu Nakayama (pp. 1039-1051).

The KIAA1440 protein contains no significant domains that allow for a prediction of its function, despite the fact that it is an extremely large protein comprising 2222 amino acids. In our current study, we show that the developing KIAA1440^−/− mouse embryo in a pure ICR background arrests its growth at the early blastocyst stage, whereas the majority of the KIAA1440^−/− embryos of mixed genetic backgrounds do not progress beyond the morula stage, approximately 0.5 days earlier. KIAA1440^−/− embryos exhibited no abnormal localization of E-cadherin or β-catenin and no obvious compaction abnormalities at the morula stage. In addition, E3.5 KIAA1440^−/− embryos are not viable even in in vitro cultures. Both TUNEL and FAM-caspase-3/7 assays performed on these embryos consistently showed that E3.5 KIAA1440^−/− embryos had activated caspase-3/7, which then induced an apoptotic response predominantly within the inner cell mass of the blastocyst. Moreover, qRT-PCR analysis showed that KIAA1440^−/− embryos had increased levels of the unprocessed, primary U2 snRNA transcript but decreased levels of the mature U2 snRNA transcript compared to heterozygotes. The impaired processing of U2 snRNA and the predominantly nuclear localization of KIAA1440 protein is also very consistent with recently reported data showing that it is the largest subunit of the integrator complex, which mediates U1 and U2 snRNA 3′-end processing. Large nuclear KIAA1440/Ints1 is thus suggested to play non-redundant roles in the cell such as the formation of a scaffold for the assembly of the integrator complex.

Keywords: Abbreviations; E; embryonic day; PBS; phosphate-buffered saline; PCR; polymerase chain reaction; SDS; sodium dodecyl sulfate; PFA; paraformaldehyde; BSA; bovine serum albumin; TUNEL; terminal dUTP nick-end labeling; DAPI; 4′, 6-diamidino-2-phenylindole; GFP; green fluorescent protein; RT; reverse transcription; snRNA; small nuclear RNA; ICM; inner cell mass; TE; trophectoderm; DIC; differential interference contrast; Ph; phase contrastIntegrator complex subunit 1; Large nuclear protein; Knockout mice; Embryonic lethality; Apoptosis; snRNA processing

The localization of nuclear exporters of the importin-β family is regulated by Snf1 kinase, nutrient supply and stress by XinXin Quan; Jennifer Yu; Howard Bussey; Ursula Stochaj (pp. 1052-1061).

In the budding yeast Saccharomyces cerevisiae, four members of the importin-β family of nuclear carriers, Xpo1p/Crm1p, Cse1p, Msn5p and Los1p, function as exporters of protein and tRNA. Under normal growth conditions GFP-tagged exporters are predominantly associated with nuclei. The presence of Snf1 kinase, a key regulator of cell growth and a metabolic sensor, controls the localization of GFP-exporters. Additional glucose-dependent, but Snf1-independent, mechanisms regulate carrier distribution and a switch from fermentable to non-fermentable carbon sources relocates all of the carriers, suggesting a link to the nutritional status of the cell. Moreover, stress controls the proper localization of GFP-exporters, which mislocalize upon exposure to heat, ethanol and starvation. Stress may activate the MAPK cell integrity cascade, and we tested the role of this pathway in exporter localization. Under non-stress conditions, the proper distribution of GFP-Cse1p and Xpo1p/Crm1p-GFP requires kinases of the cell integrity cascade. By contrast, Msn5p-GFP and Los1p-GFP rely on the MAPK module to relocate to the cytoplasm when cells are stressed with ethanol. Our results indicate that the association of nuclear exporters with nuclei is controlled by multiple mechanisms that are organized in a hierarchical fashion and linked to the physiological state of the cell.

Keywords: Abbreviations; BSA; bovine serum albumin; DAPI; 4',6-diamidino-2-phenylindole; DMSO; dimethyl sulfoxide; GFP; Aequorea victoria; green fluorescent protein; MAPK; mitogen-activated protein kinases; NE; nuclear envelope; PKC; protein kinase C; SNF1; sucrose non-fermenting; TOR; target of rapamycinNuclear export carriers; Snf1/AMP kinase; Stress; Cell integrity pathway

The small GTPases Rab5 and RalA regulate intracellular traffic of P-glycoprotein by Dong Fu; Ellen M. van Dam; Adam Brymora; Iain G. Duggin; Phillip J. Robinson; Basil D. Roufogalis (pp. 1062-1072).

P-glycoprotein (P-gp) is a plasma membrane glycoprotein that can cause multidrug resistance (MDR) of cancer cells by acting as an ATP-dependent drug efflux pump. The regulatory effects of the small GTPases Rab5 and RalA on the intracellular trafficking of P-gp were investigated in HeLa cells. As expected, overexpressed enhanced green fluorescent protein (EGFP)-tagged P-gp (P-gp-EGFP) is mainly localised to the plasma membrane. However, upon cotransfection of either dominant negative Rab5 (Rab5-S34N) or constitutively active RalA (RalA-G23V) the intracellular P-gp-EGFP levels increased approximately 9 and 13 fold, respectively, compared to control P-gp-EGFP cells. These results suggest that Rab5 and RalA regulate P-gp trafficking between the plasma membrane and an intracellular compartment. In contrast, coexpression of constitutively active Rab5 (Rab5-Q79L) or dominant negative RalA (RalA-S28N) had no effect on the localisation of P-gp-EGFP. Furthermore, the intracellular accumulation of daunorubicin, a substrate for P-gp, increased significantly with an increased intracellular localisation of P-gp-EGFP. These results imply that it may be possible to overcome MDR by controlling the plasma membrane localisation of P-gp.

Keywords: P-glycoprotein; endocytosis; exocytosis; Rab5; RalA; multidrug resistance in cancer

Baicalein inhibition of hydrogen peroxide-induced apoptosis via ROS-dependent heme oxygenase 1 gene expression by Hui-Yi Lin; Shing-Chuan Shen; Cheng-Wei Lin; Liang-Yo Yang; Yen-Chou Chen (pp. 1073-1086).

In the present study, baicalein (BE) but not its glycoside, baicalin (BI), induced heme oxygenase-1 (HO-1) gene expression at both the mRNA and protein levels, and the BE-induced HO-1 protein was blocked by adding cycloheximide (CHX) or actinomycin D (Act D). Activation of ERK, but not JNK or p38, proteins via induction of phosphorylation in accordance with increasing intracellular peroxide levels was detected in BE-treated RAW264.7 macrophages. The addition of the ERK inhibitor, PD98059, (but not the p38 inhibitor, SB203580, or the JNK inhibitor, SP600125) and the chemical antioxidant, N-acetyl cysteine (NAC), significantly reduced BE-induced HO-1 protein expression by respectively blocking ERK protein phosphorylation and intracellular peroxide production. Additionally, BE but not BI effectively protected RAW264.7 cells from hydrogen peroxide (H₂O₂)-induced cytotoxicity, and the preventive effect was attenuated by the addition of the HO inhibitor, SnPP, and the ERK inhibitor, PD98059. H₂O₂-induced apoptotic events including hypodiploid cells, DNA fragmentation, activation of caspase 3 enzyme activity, and a loss in the mitochondrial membrane potential with the concomitant release of cytochrome c from mitochondria to the cytosol were suppressed by the addition of BE but not BI. Blocking HO-1 protein expression by the HO-1 antisense oligonucleotide attenuated the protective effect of BE against H₂O₂-induced apoptosis by suppressing HO-1 gene expression in macrophages. Overexpression of the HO-1 protein inhibited H₂O₂-induced apoptotic events such as DNA fragmentation and hypodiploid cells by reducing intracellular peroxide production induced by H₂O₂, compared with those events in neo-control (neo-RAW264.7) cells. In addition, CO, but not bilirubin and biliverdin, addition inhibits H₂O₂-induced cytotoxicity in macrophages. It suggests that CO can be responsible for the protective effect associated with HO-1 overexpression. The notion of induction of HO-1 gene expression through a ROS-dependent manner suppressing H₂O₂-induced cell death is identified in the present study.

Keywords: Abbreviations; BE; baicalein; BI; baicalin; ERK; extracellular regulated kinases; JNK; c-Jun N-terminal kinases; HO-1; heme oxygenase 1; NAC; N-acetyl cysteine; CHX; cycloheximide; Act D; actinomycin D; H; ₂; O; ₂; hydrogen peroxide; ROS; reactive oxygen species; MTT; (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide); DCHF-DA; 2′,7′-dichlorodihydrofluorescein-diacetate; PI; propidium iodine; DiOC6(3); NBT, 3,3′-dihexyloxacarbocyanine iodide nitroblue tetrazolium; BCIP; 5-bromo-4-chloro-3-indolyl phosphate; SnPP; tin protoporphyrin; LDH; lactate dehydrogenase; CO; carbon monoxideHeme oxygenase 1; Flavonoids; ROS; Apoptosis; ERKs; CO

Bombesin attenuates pre-mRNA splicing of glucocorticoid receptor by regulating the expression of serine-arginine protein p30c (SRp30c) in prostate cancer cells by Jin Zhu; Jun Y. Gong; Oscar B. Goodman Jr.; Luca Cartegni; David M. Nanus; Ruoqian Shen (pp. 1087-1094).

Although glucocorticoids are frequently administered to patients with hormone refractory prostate cancer, their therapeutic effectiveness is limited by the development of glucocorticoid resistance. The molecular mechanisms of glucocorticoid resistance are unknown but are believed to involve neuropeptide growth factors and cytokines. We examined the functional interaction between bombesin and dexamethasone in PC-3 cells and found that bombesin could act as a survival factor by interfering with dexamethasone-mediated growth inhibition. Because glucocorticoids exert their effects through glucocorticoid receptors (GRs), we measured the expression of GRα and GRβ isoforms in the presence of bombesin. Western blotting and real time PCR revealed bombesin induced expression of GRβ, but not GRα. Because GR isoforms are generated by alternative splicing of a common GR gene, we examined the expression of serine-arginine (SR) proteins involved in alternative splicing, and found that the expression of SRp30 was induced by bombesin in PC-3 cells. To characterize the role of SRp30 in splicing of GR isoforms, siRNAs specific to various SRp30 isoforms were transfected into PC-3 cells. We found that suppression of SRp30c expression by siRNA specifically antagonized bombesin's effect on glucocorticoid-mediated inhibition of PC cells, suggesting that bombesin-induced expression of SRp30c affects GR pre-mRNA splicing, leading to increased GRβ expression and contributing to glucocorticoid resistance in PC cells.

Keywords: Glucocorticoids; Glucocorticoid receptors; SRp30 proteins; Bombesin; Neuropeptides; Prostate cancer

Synergistic inhibition of pancreatic adenocarcinoma cell growth by trichostatin A and gemcitabine by Massimo Donadelli; Chiara Costanzo; Stefania Beghelli; Maria Teresa Scupoli; Mario Dandrea; Antonio Bonora; Paolo Piacentini; Alfredo Budillon; Michele Caraglia; Aldo Scarpa; Marta Palmieri (pp. 1095-1106).

We investigated the ability of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) to interact with gemcitabine (GEM) in inducing pancreatic cancer cell death. The combined treatment with TSA and GEM synergistically inhibited growth of four pancreatic adenocarcinoma cell lines and induced apoptosis. This effect was associated with the induction of reactive oxygen species (ROS) by GEM, increased expression of the pro-apoptotic BIM gene by both TSA and GEM and downregulation of the 5′-nucleotidase UMPH type II gene by TSA. The expression of other genes critical for GEM resistance (nucleoside transporters, deoxycytidine kinase, cytidine deaminase, and ribonucleotide reductase genes) was not affected by TSA. The functional role of ROS in cell growth inhibition by GEM was supported by (i) a significantly reduced GEM-associated growth inhibition by the free radical scavenger N-acetyl-l-cysteine, and (ii) a positive correlation between the basal level of ROS and sensitivity to GEM in 10 pancreatic cancer cell lines. The functional role of both Bim and 5′-nucleotidase UMPH type II in cell growth inhibition by TSA and GEM was assessed by RNA interference assays. In vivo studies on xenografts of pancreatic adenocarcinoma cells in nude mice showed that the association of TSA and GEM reduced to 50% the tumour mass and did not cause any apparent form of toxicity, while treatments with TSA or GEM alone were ineffective. In conclusion, the present study demonstrates a potent anti-tumour activity of TSA/GEM combination against pancreatic cancer cells in vitro and in vivo, strongly supporting the use of GEM in combination with an HDAC inhibitor for pancreatic cancer therapy.

Keywords: Pancreatic cancer; Trichostatin A; Gemcitabine; Apoptosis; Oxidative stress; RNA interference

Lithium toxicity and expression of stress-related genes or proteins in A549 cells by M.S. Allagui; C. Vincent; A. El feki; Y. Gaubin; F. Croute (pp. 1107-1115).

To unveil some molecular mechanisms underlying lithium toxicity, expression changes of stress-related genes or proteins were analysed in A549 cells, cultured for 3 days in presence of lithium. A dose-dependant cell-growth inhibition was found for concentrations ranging from 2 (toxicity threshold) to 12 mM (lethality threshold). cDNA arrays technology was used to analyse effects of 5 and 10 mM lithium. Among genes involved in cell cycle regulation, proliferating cell nuclear antigen (PCNA) was down-regulated and cyclin kinase inhibitor p21 (CDKN1A), up-regulated. Genes of paraoxonase 2, known to prevent LDL lipid peroxidation, and of catalase and SOD were found to be down-regulated whereas genes of cytochrome P450 (CYP2F1, CYP2E1) were up-regulated. This probably results in higher intracellular levels of reactive oxygen species and account for increased levels of lipid peroxidation commonly associated with lithium exposure. Moreover, lithium was found to down-regulate genes coding for anti-apoptotic gene BAG-1 and for most of the molecular chaperones (HSP, GRP). This might account for lithium toxicity since these proteins are critical for cell survival. At translational level, a 105 kDa protein was found to be over-expressed. This protein was recognized by the anti-GRP94, anti-KDEL and anti-phosphoserine monoclonal antibodies suggesting that, lithium could induce post-translational modifications of GRP94 phosphorylation. Using tunicamycin and thapsigargin, it was concluded that lithium effects are not related to defect in N-linked glycosylation and/or to changes in calcium homeostasis.

Keywords: Lithium; oxidative stress; Cytochrome P450; HSP; GRP94

Curcumin reverses breast tumor exosomes mediated immune suppression of NK cell tumor cytotoxicity by Huang-Ge Zhang; Helen Kim; Cunren Liu; Shaohua Yu; Jianhua Wang; William E. Grizzle; Robert P. Kimberly; Stephen Barnes (pp. 1116-1123).

An important characteristic of tumors is that they at some point in their development overcome the surveillance of the immune system. Tumors secrete exosomes, multivesicular bodies containing a distinct set of proteins that can fuse with cells of the circulating immune system. Purified exosomes from TS/A breast cancer cells, but not non-exosomal fractions, inhibit (at concentrations of nanograms per ml protein) IL-2-induced natural killer (NK) cell cytotoxicity. The dietary polyphenol, curcumin (diferuloylmethane), partially reverses tumor exosome-mediated inhibition of natural killer cell activation, which is mediated through the impairment of the ubiquitin–proteasome system. Exposure of mouse breast tumor cells to curcumin causes a dose-dependent increase in ubiquitinated exosomal proteins compared to those in untreated TS/A breast tumor cells. Furthermore, exosomes isolated from tumor cells pretreated with curcumin have a much attenuated inhibition of IL-2 stimulated NK cell activation. Jak3-mediated activation of Stat5 is required for tumor cytotoxicity of IL-2 stimulated NK cells. TS/A tumor exosomes strongly inhibit activation of Stat5, whereas the tumor exosomes isolated from curcumin-pretreated tumor cells have a lowered potency for inhibition of IL-2 stimulated NK cell cytotoxicity. These data suggest that partial reversal of tumor exosome-mediated inhibition of NK cell tumor cytotoxicity may account for the anti-cancer properties of curcumin.

Keywords: Breast tumor exosomes; Ubiquitination; NK immunosuppression; Curcumin

Probing the mechanism of FET3 repression by Izh2p overexpression by Brian R. Kupchak; Ibon Garitaonandia; Nancy Y. Villa; Matthew B. Mullen; Marilee G. Weaver; Lisa M. Regalla; Elizabeth A. Kendall; Thomas J. Lyons (pp. 1124-1132).

We previously reported a role for the IZH2 gene product in metal ion metabolism. Subsequently, Izh2p was also identified as a member of the PAQR family of receptors and, more specifically, as the receptor for the plant protein osmotin. In this report, we investigate the effect of Izh2p on iron homeostasis. We show that overproduction of Izh2p prevents the iron-dependent induction of the Fet3p component of the high-affinity iron-uptake system and is deleterious for growth in iron-limited medium. We demonstrate that the effect of Izh2p requires cAMP-dependent kinase and AMP-dependent kinase and is not mediated by general inhibition of the Aft1p iron-responsive transcriptional activator. We also show that Izh2p-overproduction negatively regulates Nrg1p/Nrg2p- and Msn2p/Msn4p-dependent reporters. Furthermore, we show that the Nrg1p/Nrg2p and Msn2p/Msn4p pairs are epistatic to each other with respect to their effects on FET3 expression. Finally, we show that the mechanism by which PAQR receptors activate signal transduction pathways is likely to be conserved from yeast to humans.

Keywords: FET3; IZH2; Osmotin; PAQR; Adiponectin

Prostasin induces protease-dependent and independent molecular changes in the human prostate carcinoma cell line PC-3 by Mengqian Chen; Ya-Yuan Fu; Chen-Yong Lin; Li-Mei Chen; Karl X. Chai (pp. 1133-1140).

Expression of prostasin in the PC-3 human prostate carcinoma cells inhibited in vitro invasion, but the molecular mechanisms are unknown. Wild-type human prostasin or a serine active-site mutant prostasin was expressed in the PC-3 cells. Molecular changes were measured at the mRNA and the protein levels. Cell signaling changes were evaluated by measuring phosphorylation of the extracellular signal-regulated kinases (Erk1/2) following epidermal growth factor (EGF) treatment of the cells. Protein expression of the EGF receptor (EGFR) was differentially down-regulated by the wild-type and the active-site mutant prostasin. The mRNA expression of EGFR and the transcription repressor SLUG was reduced in cells expressing wild-type prostasin but not the active-site mutant. Phosphorylation of Erk1/2 in response to EGF was greatly reduced by the wild-type prostasin but not by the active-site mutant. The mRNA expression of the urokinase-type plasminogen activator (uPA), the uPA receptor (uPAR), cyclooxygenase-2 (COX-2), and the inducible nitric oxide synthase (iNOS) was decreased by the wild-type and the active-site mutant prostasin. The mRNA or protein expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), matriptase, and E-cadherin was greatly increased by the active-site mutant prostasin. In conclusion, prostasin expression elicits both protease-dependent and independent molecular changes in the PC-3 cells.

Keywords: Serine Protease; Matriptase; Epidermal Growth Factor Receptor; Prostate Cancer

Functional characterization of two missense mutations in Pex5p—C11S and N526K by Andreia F. Carvalho; Cláudia P. Grou; Manuel P. Pinto; Inês S. Alencastre; João Costa-Rodrigues; Marc Fransen; Clara Sá-Miranda; Jorge E. Azevedo (pp. 1141-1148).

Most newly synthesized peroxisomal proteins are targeted to the organelle by Pex5p, the peroxisomal cycling receptor. Pex5p interacts with these proteins in the cytosol, transports them to the peroxisomal docking/translocation machinery and promotes their translocation across the organelle membrane. Finally, Pex5p is recycled back to the cytosol in order to catalyse additional rounds of transportation. Although several properties of this protein sorting pathway have been recently uncovered, we are still far from comprehending many of its basic principles. Here, we describe the mechanistic implications of two single-amino acid substitutions in Pex5p. The first mutation characterized, Cys11Ser, blocks the recycling of Pex5p back into the cytosol at the step in which stage 2 Pex5p is converted into stage 3 Pex5p. The mutation Asn526Lys, previously described in a child with neonatal adrenoleukodystrophy and shown to abolish the PTS1-binding capacity of Pex5p, results in a Pex5p protein exhibiting import capacity. Protease assays suggest that the Asn526Lys mutation causes conformational alterations at the N-terminal half of Pex5p mimicking the ones induced by binding of a PTS1-containing peptide to the normal peroxin. The implications of these findings on the mechanism of protein translocation across the peroxisomal membrane are discussed.

Keywords: Abbreviations; PTS; peroxisomal targeting signal; PNS; postnuclear supernatant; MOPS; 4-morpholinepropanesulfonic acid; DTT; dithiothreitol; ATPγS; adenosine 5′-; O; - (thiotriphosphate); TPR; tetratricopeptide repeatsPex5p; Peroxisomes; Protein translocation; Peroxisomal matrix proteins

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BBA - Molecular Cell Research (v.1773, #7)