BBA - Molecular Cell Research (v.1642, #1-2)

Multiple signal transduction pathways are generally triggered simultaneously by a single extracellular stimulus. As a result, multiple transcription factors (TFs) can be activated downstream to mediate the inducible expression of target genes. Profiling the activation of all TFs will aid in the dissection of the numerous pathways of signal transduction. Tumor necrosis factor alpha (TNFα) and phorbol 12-myristate 13-acetate (PMA) mediate many biological functions, including cell proliferation and apoptosis, by stimulating signaling pathways. Two TFs, nuclear factor kappaB (NFκB) and activating factor 1 (AP1), have been identified as targets of both TNFα and PMA activation. Here, we describe the use of a protein/DNA array system to identify additional TFs activated by TNFα and PMA in HeLa cells. From a total of 150 targeted TFs, six—CREB, E2F, CETP/CRE, c-Rel, MSP1, and Pax6—were identified whose activities, like NFκB and AP1, were regulated by both TNFα- and PMA-induced pathways. Interestingly, the TF E47 was shown to be specifically activated by TNFα but was not affected by treatment with PMA. In addition, GATA, NF-E1, and ISRE were shown to be specifically activated by PMA but not TNFα. These findings suggest that TNFα and PMA both stimulate unique signaling pathways while mediating transcriptional activation through common pathways.
Keywords: Protein/DNA interaction; TNF; PMA; Transcription factor; Signal transduction;

Acquisition of heat shock tolerance by regulation of intracellular redox states by Jeonghoon Ueom; Soonhyun Kwon; Sunhee Kim; Youngkee Chae; Kyunghee Lee (9-16).
In the yeast Saccharomyces cerevisiae, a mild heat treatment strongly induces Hsp104p which provides acquisition of thermotolerance. The mechanism by which Hsp104p protects cells from the severe heat shock has not yet been completely elucidated. In this study, a pivotal role of Hsp104p as an efficient scavenger of the reactive oxygen species (ROS) is investigated. In our previous study, we reported that Hsp104p acted as a regulator in the mitochondrial respiration pathway. In this report, the recombinant wild-type and hypersensitive ras mutants (ira2Δ) with the extrachromosomal plasmids wild-type and mutant hsp104 genes were studied. The resulting strains successfully expressed both wild-type and mutant Hsp104p and showed the thermotolerance phenotype in the strain with the functional wild-type Hsp104p expressed. Upon treatment with H2O2 and menadione, the strains with the functional Hsp104p expressed showed higher survival rates than the other mutants, indicating the protective role of Hsp104p from the oxidative stress. Fluorescence measurement of the oxidation-dependent probe, 2′,7′-dichlorofluoroscein diacetate (H2DCFDA), also indicated that Hsp104p significantly reduced the amount of ROS. Resistance to the oxidative stress was independent of the amount of the glutathione in the hyperactivated ras mutants. Taken all together, this study confirms that Hsp104p plays a crucial role in keeping cells from being damaged by the oxidative stress, thus acting as a modulator of the intracellular redox state.
Keywords: Hsp104p; Thermotolerance; Oxidative stress; Reactive oxygen species (ROS); Ras/cAMP pathway;

A cell-free binding assay maps the LSP1 cytoskeletal binding site to the COOH-terminal 30 amino acids by Michael J. Wong; Irish A. Malapitan; Barbara A. Sikorski; Jan Jongstra (17-24).
The leukocyte specific protein 1 or LSP1 is a multi functional protein involved in such divers biological processes as the regulation of neutrophil motility, chemotaxis, adhesion and membrane immunoglobulin M (mIgM) mediated apoptosis of B-lymphocytes. The 330-amino-acid mouse LSP1 protein contains a high-affinity F-actin binding site and in intact cells localizes to the F-actin filament containing cytoskeleton. Here we use a high-speed F-actin co sedimentation assay and transfection experiments in the LSP1 T-lymphoma cell line BW5147 to show that LSP1 interacts with F-actin and the cytoskeleton through residues downstream of amino acid residue 230. We then designed a novel cell-free cytoskeleton binding assay in which a set of GST–LSP1 fusion proteins are allowed to bind to the cytoskeleton in NP-40 soluble lysates of BW5147 cells and are recovered in the low-speed detergent insoluble pellet. Using this assay the cytoskeleton binding site of mouse LSP1 maps to the 300–330 interval. These results will allow the design of LSP1 mutants that do not bind to the cytoskeleton to determine the importance of LSP1 cytoskeleton binding for the diverse functions of LSP1.
Keywords: Leukocyte specific protein 1; Cytoskeleton; F-actin; Cell-free cytoskeleton binding assay; Adhesion; Motility; Apoptosis;

Lipoprotein (a) downregulates lysosomal acid lipase and induces interleukin-6 in human blood monocytes by Christa Buechler; Heidrun Ullrich; Charalampos Aslanidis; Salim Maa Bared; Arno Lingenhel; Mirko Ritter; Gerd Schmitz (25-31).
The association of elevated lipoprotein (a) (Lp(a)) with an increased risk for coronary events is clearly established. This increased risk may in part be due to the activation of monocytes as major cells involved in atherogenesis. High concentrations of plasma Lp(a) were shown to influence the gene expression of human blood monocytes and in the present study we demonstrate a reduced abundance of the lysosomal acid lipase (LAL) mRNA in monocytes of patients with coronary disease and selective Lp(a) hyperlipidemia. This is also supported by in vitro studies where purified Lp(a) but not low-density lipoprotein (LDL) was shown to downregulate mRNA levels of the LAL in control monocytes. A correlation of Lp(a) serum levels and the proinflammatory cytokine IL-6 was recently also described. Therefore, we investigated whether Lp(a) is capable to enhance the release of this acute phase cytokine from human blood monocytes. Purified Lp(a) led to an increased secretion of IL-6, but not TNF-α arguing against a general activation of these cells. The association of reduced LAL activity with the premature development of coronary artery disease has been demonstrated in patients with hypercholesterolemia, and in the present study we show for the first time that LAL expression is suppressed in monocytes from patients with Lp(a) hyperlipidemia and by purified Lp(a). In addition, increased levels of IL-6 also predict future cardiovascular events and IL-6 secretion was also induced by purified Lp(a).
Keywords: Lipoprotein (a); Lysosomal acid lipase; Interleukin-6; Blood monocyte; Atherogenesis;

Activation of NF-κB nuclear transcription factor by flow in human endothelial cells by David C. Hay; Catherine Beers; Vicky Cameron; Lesley Thomson; Frederick W. Flitney; Ronald T. Hay (33-44).
The tractive force generated by blood flow, called fluid shear stress, is an important regulator of endothelial cell gene expression. Several transcription factors are activated by shear stress, including members of the NF-κB/Rel family. The nature of the upstream-signaling components involved in the activation of NF-κB by flow has been studied in human endothelial cells. Flow rapidly increased endogenous IKK1/2 activity and transiently degraded IκBα and IκBβ1, but not p105/p50. Nuclear translocation of the p65 subunit was induced by flow in wild-type (w/t) cells and in cells overexpressing w/t NIK, IKK1 or IKK2, but not in cells transiently transfected with kinase-inactive mutants of these enzymes. Nuclear translocation of p65 in response to flow was not affected by overexpressing a dominant-negative mutant of a MAPKKK related to NIK, called TPL2 kinase, nor by pretreating cells with the selective PKC inhibitor bisindoylmaleimide-1. Gel shift assays showed that the binding of p50/p65 heterodimer to radiolabeled oligonucleotide containing a shear-stress response element was increased by flow. The activity of a 3κB conA-luciferase reporter was also increased, confirming that NF-κB activated by flow was transcriptionally active. We conclude that shear stress induces gene transactivation by NF-κB (p50/p65) via the NIK-IKK1/2 pathway and proteosome-dependent degradation of IκB and that induction by flow does not involve TPL-2 kinase or PKC.
Keywords: Endothelial cell; NF-κB transcription factor; Flow mechanotransduction; Shear stress; Gene regulation; Atherogenesis;

Lysosomal traffic of liganded endothelin B receptor by Natasha Foster; To Ha Loi; Robert Owe-Young; Keith K. Stanley (45-52).
The endothelin B receptor (ETB) is an endothelial cell receptor found in caveolae. Studies with GFP-tagged ETB have suggested that the protein is constitutively endocytosed and targeted to lysosomes where it is rapidly degraded. We report that iodinated endothelin-1 ligand (ET-1) is taken up by cells transfected with ETB and remains undegraded for at least 17 h. Analysis of the intracellular traffic of endocytosed ET-1 on isotonic Ficoll gradients shows that it is rapidly internalised to lysosomes by a chloroquine sensitive and cholesterol dependent pathway. Low-temperature nonreducing SDS gels show that the ET-1 initially binds to full-length GFP-tagged ETB, which is rapidly clipped at the amino-terminus and is then stable for at least 6 h. Analysis of GFP tagged ETB on reducing SDS gels shows that it is proteolytically cleaved with a half time of approximately 3 h. However, nonreducing gels show that the receptor is virtually intact, suffering only a similar cleavage to the liganded receptor. We conclude that the ETB receptor shows remarkable stability in lysosomes, held together by disulfide bonds, and maintaining ligand binding for long periods of time.
Keywords: Chloroquine; Cyclodextrin; Degradation; Endothelin receptor; Lysosome; Traffic;

2,5-Anhydro-d-mannitol increases hepatocyte sodium: transduction of a hepatic hunger stimulus? by Mark I. Friedman; Grazyna Graczyk-Millbrandt; Hong Ji; Nancy E. Rawson; Mary D. Osbakken (53-58).
To test the hypothesis that decreased hepatocyte ATP is transduced into a hepatic neuronal signal via a change in sodium pump activity, we examined the effect of 2,5-anhydro-d-mannitol (2,5-AM), which stimulates feeding behavior in rats, on intracellular sodium levels using 23Na nuclear magnetic resonance (NMR) spectroscopy. Isolated hepatocytes suspended in agarose beads were superfused with either 2.5 mM 2,5-AM or fructose in the presence of the paramagnetic shift reagent, thulium(III)(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonate)). Superfusion with 2,5-AM decreased hepatocyte ATP and increased intracellular sodium levels compared with superfusion with either fructose or shift reagent alone starting within 15 min of exposure, reaching a maximum level of 120% of baseline by 30 min and declining gradually thereafter over the next 90 min. Superfusion with fructose, which also decreased hepatocyte ATP but by less than half the amount seen with 2,5-AM, had no significant effect on cellular sodium levels. The results support the hypothesis that changes in sodium pump activity could participate in transducing a hunger stimulus associated with hepatocyte energy status into a signal for hunger.
Keywords: Liver; Intracellular sodium; Food intake; Nuclear magnetic resonance spectroscopy; 2,5-Anhydro-d-mannitol; Fructose;

The fructose analogue, 2,5-anhydro-d-mannitol (2,5-AM), triggers feeding in rats via a mechanism linked to its ability to trap phosphate and deplete hepatic ATP. This metabolic inhibitor is particularly useful in the study of the role of the liver in initiation of feeding as its effects are preferentially localized to the liver, and its metabolic consequences have been extensively characterized. To determine whether changes in intracellular calcium may participate in a mechanism conveying information about hepatic energy status to the nervous system, we studied the effects of 2,5-AM on intracellular calcium in isolated hepatocytes using the ratiometric indicator, fura-2. 2,5-AM elicited a marked elevation of intracellular calcium within 2–3 min of exposure that returned to baseline upon removal of the agent. Removal of external calcium failed to prevent this response, while emptying intracellular stores prevented it. These data are consistent with the hypothesis that hepatic energy status may be conveyed to the nervous system via a calcium-mediated secretion event.
Keywords: 2,5-Anhdyro-d-mannitol; Hepatocyte calcium; Appetite;

Coupling to Gs and Gq/11 of histamine H2 receptors heterologously expressed in adult rat atrial myocytes by Marie-Cécile Wellner-Kienitz; Kirsten Bender; Thomas Meyer; Lutz Pott (67-77).
The predominant histamine receptor subtype in the supraventricular and ventricular tissue of various mammalian species is the H2 receptor (H2-R) subtype, which is known to couple to stimulatory G proteins (Gs), i.e. the major effects of this autacoid are an increase in sinus rate and in force of contraction. To investigate histamine effects in H2-R-transfected rat atrial myocytes, endogenous GIRK currents and L-type Ca2+ currents were used as functional assays. In H2-R-transfected myocytes, exposure to His resulted in a reversible augmentation of L-type Ca2+ currents, consistent with the established coupling of this receptor to the Gs–cAMP–PKA signalling pathway. Mammalian K+ channels composed of GIRK (Kir3.x) subunits are directly controlled by interaction with βγ subunits released from G proteins, which couple to seven-helix receptors. In mock-transfected atrial cardiomyocytes, activation of muscarinic K+ channels (IK(ACh)) was limited to Gi-coupled receptors (M2R, A1R). In H2-R-overexpressing cells, histamine activated IK(ACh) via Gs-derived βγ subunits since the histamine-induced current was insensitive to pertussis toxin. These data indicate that overexpression of Gs-coupled H2-R results in a loss of target specificity due to an increased agonist-induced release of Gs-derived βγ subunits. When IK(ACh) was maximally activated by GTP-γ–S, histamine induced an irreversible inhibition of the inward current in a fraction of H2-R-transfected cells. This inhibition is supposed to be mediated via a Gq/11-PLC-mediated depletion of PIP2, suggesting a partial coupling of overexpressed H2-R to Gq/11. Dual coupling of H2-Rs to Gs and Gq is demonstrated for the first time in cardiac myocytes. It represents a novel mechanism to augment positive inotropic effects by activating two different signalling pathways via one type of histamine receptor. Activation of the Gs–cAMP–PKA pathway promotes Ca2+ influx through phosphorylation of L-type Ca2+ channels. Simultaneous activation of Gq-signalling pathways might result in phosphoinositide turnover and Ca2+ release from intracellular stores, thereby augmenting H2-induced increases in [Ca2+]i.
Keywords: Histamine; H2 receptor; Atrial cell; GIRK current; G protein;

Dexamethasone induces caspase activation in murine osteoblastic MC3T3-E1 cells by Chu Chang Chua; Balvin H.L. Chua; Zhongyi Chen; Cathy Landy; Ronald C. Hamdy (79-85).
Glucocorticoids are widely used as anti-inflammatory and chemotherapeutic agents. However, prolonged use of glucocorticoids leads to osteoporosis. This study was designed to examine the mechanism of dexamethasone (DEX)-induced apoptosis in murine osteoblastic MC3T3-E1 cells. Total RNA was extracted from MC3T3-E1 cells treated with 10−7 M DEX for 6 h. DEX exerted a variety of effects on apoptotic gene expression in osteoblasts. Ribonuclease protection assays (RPA) revealed that DEX upregulated mRNA levels of caspases-1, -3, -6, -8, -11, -12, and bcl-XL. Western blot analysis showed enhanced processing of these caspases, with the appearance of their activated enzymes 8 h after DEX treatment. In addition, DEX also induced the activation of caspase-9. DEX elevated the levels of cleaved poly(ADP-ribose) polymerase and lamin A, a caspase-3 and a caspase-6 substrate, respectively. Expression of bcl-XL protein level was upregulated by DEX. Cytochrome c release was detected in the cytosol of DEX-treated cells. Furthermore, caspase-3 enzyme activity was elevated by 2-fold after DEX treatment for 7 h. Finally, early apoptotic cells were detected in cells treated with DEX for 3 h. Our results demonstrate that DEX-induced apoptosis involves gene activation of a number of caspases.
Keywords: Dexamethasone; Caspase; Apoptosis; Osteoblast;

VDAC is a conserved element of death pathways in plant and animal systems by A. Godbole; J. Varghese; A. Sarin; M.K. Mathew (87-96).
Programmed cell death (PCD) is very much a part of plant life, although the underlying mechanisms are not so well understood as in animals. In animal cells, the voltage-dependent anion channel (VDAC), a major mitochondrial outer membrane transporter, plays an important role in apoptosis by participating in the release of intermembrane space proteins. To characterize plant PCD pathways by investigating the function of putative components in a mammalian apoptotic context, we have overexpressed a rice VDAC (osVDAC4) in the Jurkat T-cell line. Overexpression of osVDAC4 induces apoptosis, which can be blocked by Bcl-2 and the VDAC inhibitor DIDS. Modifying endogenous VDAC function by DIDS and hexokinase II (HxKII) in Jurkat cells inhibits mitochondria-mediated apoptotic pathways. Finally, we show that DIDS also abrogates heat-induced PCD in cucumber cotyledons. Our data suggest that VDAC is a conserved mitochondrial element of the death machinery in both plant and animal cells.
Keywords: Programmed cell death; Apoptosis; Mitochondrion; Permeability transition pore complex; Voltage-dependent anion channel;

Injection of FGF6 accelerates regeneration of the soleus muscle in adult mice by Anne-Sophie Armand; Thierry Launay; Claude Pariset; Bruno Della Gaspera; Frédéric Charbonnier; Christophe Chanoine (97-105).
FGF6, a member of the fibroblast growth factor (FGF) family, accumulated almost exclusively in the myogenic lineage, supporting the finding that FGF6 could specifically regulate myogenesis. Using FGF6 (−/−) mutant mice, important functions in muscle regeneration have been proposed for FGF6 but remain largely controversial. Here, we examined the effect of a single injection of recombinant FGF6 (rhFGF6) on the regeneration of mouse soleus subjected to cardiotoxin injection, specifically looking for molecular and morphological phenotypes. The injection of rhFGF6 has two effects. First, there is an up-regulation of cyclin D1 mRNA, accounting for the regulating role of a high FGF6 concentration on proliferation, and second, differentiation markers such as CdkIs and MHC I and Tn I increase and cellular differentiation is accelerated. We also show a down-regulation of endogenous FGF6, acceleration of FGFR1 receptor expression and deceleration of the FGFR4 receptor expression, possibly accounting for biphasic effects of exogenous FGF6 on muscle regeneration.
Keywords: FGF; FGF6; Growth factor; Muscle regeneration; Soleus;

Differential expression of versican isoforms is a component of the human melanoma cell differentiation process by Clelia Domenzain; María José Docampo; Montserrat Serra; Laia Miquel; Anna Bassols (107-114).
Versican is a large chondroitin sulfate proteoglycan produced by human melanoma cell lines and malignant melanocytic lesions. In the present work, we have analyzed the expression of versican spliced variants V0, V1, V2 and V3 in human melanoma cell lines at several differentiation degrees. The isoform expression pattern depends on the degree of cell differentiation. Differentiated cell lines do not produce any of the versican isoforms as analyzed by Western blot, Northern blot and RT-PCR. All cell lines with an early or intermediate degree of differentiation (AX3, SK-mel-37, Rider, SK-mel-1.36-1–5 and SK-mel-3.44) expressed V0 and V1 transcripts, whereas V2 and V3 expression was shown only by the undifferentiated cell lines SK-mel-1.36-1–5 and Rider. Furthermore, we have analyzed the expression of versican isoforms in SK-mel-3.44 and SK-mel-1.36-1–5 cells induced to differentiate by TPA treatment. The expression of the large V0, V1 and V2 isoforms practically disappears in differentiated cells, whereas V3 remains detectable by RT-PCR analysis.
Keywords: Versican; Melanoma; Cell differentiation; Proteoglycan;

Changes of mitochondrial respiration, mitochondrial content and cell size after induction of apoptosis in leukemia cells by Kathrin Renner; Albert Amberger; Günther Konwalinka; Reinhard Kofler; Erich Gnaiger (115-123).
Mitochondrial damage with release of cytochrome c is implicated in cell death signalling pathways. To examine mitochondrial function in apoptotic cells, we applied high-resolution respirometry to human leukemia cells arrested in the G1- and S-phase by exposure to the glucocorticoid dexamethasone and nucleotide analogue gemcitabine. At 30% apoptosis, opposite effects were observed on respiratory capacity (71% and 131% of controls, respectively). These changes correlated with alterations in cell size, cytosolic, and mitochondrial marker enzymes. Mitochondrial ATP production and membrane potential were maintained in all treatments, as deduced from high respiratory uncoupling control ratios (UCR). Bcl-2 over-expression did not prevent apoptosis after gemcitabine-treatment, but protected dexamethasone-treated cells from apoptosis, without fully preventing the decline of respiration and cell size. These results, therefore, provide conclusive evidence that alterations in respiratory capacity and enzyme activities per cell are mainly caused by opposite changes in cell size, occurring upon cell cycle arrest triggered by dexamethasone and gemcitabine in the early phase of apoptosis.
Keywords: Apoptosis; Dexamethasone; Gemcitabine; Mitochondrial respiratory control; Cell cycle;