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BBA - Molecular Cell Research (v.1783, #5)

Editorial Board (pp. ii).

Ca²⁺-permeable channels in the hepatocyte plasma membrane and their roles in hepatocyte physiology by Gregory J. Barritt; Jinglong Chen; Grigori Y. Rychkov (pp. 651-672).

Hepatocytes are highly differentiated and spatially polarised cells which conduct a wide range of functions, including intermediary metabolism, protein synthesis and secretion, and the synthesis, transport and secretion of bile acids. Changes in the concentrations of Ca²⁺ in the cytoplasmic space, endoplasmic reticulum (ER), mitochondria, and other intracellular organelles make an essential contribution to the regulation of these hepatocyte functions. While not yet fully understood, the spatial and temporal parameters of the cytoplasmic Ca²⁺ signals and the entry of Ca²⁺ through Ca²⁺-permeable channels in the plasma membrane are critical to the regulation by Ca²⁺ of hepatocyte function. Ca²⁺ entry across the hepatocyte plasma membrane has been studied in hepatocytes in situ, in isolated hepatocytes and in liver cell lines. The types of Ca²⁺-permeable channels identified are store-operated, ligand-gated, receptor-activated and stretch-activated channels, and these may vary depending on the animal species studied. Rat liver cell store-operated Ca²⁺ channels (SOCs) have a high selectivity for Ca²⁺ and characteristics similar to those of the Ca²⁺ release activated Ca²⁺ channels in lymphocytes and mast cells. Liver cell SOCs are activated by a decrease in Ca²⁺ in a sub-region of the ER enriched in type1 IP₃ receptors. Activation requires stromal interaction molecule type 1 (STIM1), and G_i2α, F-actin and PLCγ1 as facilitatory proteins. P_2x purinergic channels are the only ligand-gated Ca²⁺-permeable channels in the liver cell membrane identified so far. Several types of receptor-activated Ca²⁺ channels have been identified, and some partially characterised. It is likely that TRP (transient receptor potential) polypeptides, which can form Ca²⁺- and Na⁺-permeable channels, comprise many hepatocyte receptor-activated Ca²⁺-permeable channels. A number of TRP proteins have been detected in hepatocytes and in liver cell lines. Further experiments are required to characterise the receptor-activated Ca²⁺ permeable channels more fully, and to determine the molecular nature, mechanisms of activation, and precise physiological functions of each of the different hepatocyte plasma membrane Ca²⁺ permeable channels.

Keywords: Hepatocyte; Liver; Ca; ²⁺; channel; Plasma membrane; TRP channel; HormoneAbbreviations; ER; endoplasmic reticulum; SERCA; endoplasmic reticulum (Ca; ²⁺; +; Mg; ²⁺; ) ATP-ase; PM; plasma membrane; IP; ₃; R; inositol 1,4,5-trisphosphate receptor; SOC; store-operated Ca; ²⁺; channel; I; _SOC; store-operated channel current; CRAC; Ca; ²⁺; release activated Ca; ²⁺; channel; STIM1; stromal interaction molecule type 1; TRP; transient receptor potential; [Ca; ²⁺; ]; _cyt; cytoplasmic free Ca; ²⁺; concentration; [Ca; ²⁺; ]; _er; free Ca; ²⁺; concentration in the ER; [Ca; ²⁺; ]; _mt; free Ca; ²⁺; concentration in the mitochondria; Ca; ²⁺; _ext; extracellular Ca; ²⁺; PLC; phospholipase C; VOCC; voltage-operated Ca; ²⁺; channel; DBHQ; 2,5-di-(; tert; -butyl)1,4-benzohydro-quinone

Nucleotide- and nucleoside-converting ectoenzymes: Important modulators of purinergic signalling cascade by Gennady G. Yegutkin (pp. 673-694).

The involvement of extracellular nucleotides and adenosine in an array of cell-specific responses has long been known and appreciated, but the integrative view of purinergic signalling as a multistep coordinated cascade has emerged recently. Current models of nucleotide turnover include: (i) transient release of nanomolar concentrations of ATP and ADP; (ii) triggering of signalling events via a series of ligand-gated (P2X) and metabotropic (P2Y) receptors; (iii) nucleotide breakdown by membrane-bound and soluble nucleotidases, including the enzymes of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family, ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) family, ecto-5'-nucleotidase/CD73, and alkaline phosphatases; (iv) interaction of the resulting adenosine with own nucleoside-selective receptors; and finally, (v) extracellular adenosine inactivation via adenosine deaminase and purine nucleoside phosphorylase reactions and/or nucleoside uptake by the cells. In contrast to traditional paradigms that focus on purine-inactivating mechanisms, it has now become clear that “classical” intracellular ATP-regenerating enzymes, adenylate kinase, nucleoside diphosphate (NDP) kinase and ATP synthase can also be co-expressed on the cell surface. Furthermore, data on the ability of various cells to retain micromolar ATP levels in their pericellular space, as well as to release other related compounds (adenosine, UTP, dinucleotide polyphosphates and nucleotide sugars) gain another important insight into our understanding of mechanisms regulating a signalling cascade. This review summarizes recent advances in this rapidly evolving field, with particular emphasis on the nucleotide-releasing and purine-converting pathways in the vasculature.

Keywords: Purinergic signalling; Extracellular nucleotide release; Ecto-nucleotidase; Nucleotide kinase; Adenosine deaminase; Purine nucleoside phosphorylase

Posttranslational modifications on protein kinase c isozymes. Effects of epinephrine and phorbol esters by Martha Robles-Flores; Lennon Meléndez; Wendy García; Guillermo Mendoza-Hernández; Tukiet T. Lam; Cristina Castañeda-Patlán; Héctor González-Aguilar (pp. 695-712).

The posttranslational modifications induced on PKC isozymes as result of their activation were investigated. Reciprocal immunoprecipitations followed by Western blot analysis demonstrated that all PKC isozymes expressed in rat hepatocytes are modified by tyrosine nitration and tyrosine phosphorylation in different ways upon exposure of cells to a direct PKC activator (TPA), or to an extracellular ligand known to activate PKC-dependent pathways (epinephrine). Our data demonstrate for the first time that all PKC isozymes are also dynamically modified by O-linked β- N-acetylglucosamine (O-GlcNAc); the presence of this modification was confirmed in part by FT-ICR mass spectrometry analysis. Interestingly, the O-GlcNAc modified Ser or Thr were mapped at similar positions in several PKC isozymes. The biochemical meaning of these posttranslational modifications was investigated for PKC alpha and delta. It was found that the PKC phosphorylation status of both isozymes in tyrosine and serine residues seems to regulate directly the enzyme activity since catalytic inactivation correlate with dephosphorylation of Ser at the C-terminus autophosphorylation sites of each PKC isozyme, and with an increase in the level of tyrosine phosphorylation. Whereas none of the other posttranslational modifications showed per se a direct effect in PKC delta activity, increased tyrosine nitration and O-GlcNAc modifications correlate negatively with PKCα activity.

Keywords: PKC; Posttranslational modification; O-GlcNAc glycosylation; Tyrosine nitration; Tyrosine phosphorylation; Protein phosphorylation

NF-κB/p65 antagonizes Nrf2-ARE pathway by depriving CBP from Nrf2 and facilitating recruitment of HDAC3 to MafK by Guang-Hui Liu; Jing Qu; Xun Shen (pp. 713-727).

Constitutively activated NF-κB occurs in many inflammatory and tumor tissues. Does it interfere with anti-inflammatory or anti-tumor signaling pathway? Here, we report that NF-κB p65 subunit repressed the Nrf2-antioxidant response element (ARE) pathway at transcriptional level. In the cells where NF-κB and Nrf2 were simultaneously activated, p65 unidirectionally antagonized the transcriptional activity of Nrf2. In the p65-overexpressing cells, the ARE-dependent expression of heme oxygenase-1 was strongly suppressed. However, p65 inhibited the ARE-driven gene transcription in a way that was independent of its own transcriptional activity. Two mechanisms were found to coordinate the p65-mediated repression of ARE: (1) p65 selectively deprives CREB binding protein (CBP) from Nrf2 by competitive interaction with the CH1-KIX domain of CBP, which results in inactivation of Nrf2. The inactivation depends on PKA catalytic subunit-mediated phosphorylation of p65 at S276. (2) p65 promotes recruitment of histone deacetylase 3 (HDAC3), the corepressor, to ARE by facilitating the interaction of HDAC3 with either CBP or MafK, leading to local histone hypoacetylation. This investigation revealed the participation of NF-κB p65 in the negative regulation of Nrf2-ARE signaling, and might provide a new insight into a possible role of NF-κB in suppressing the expression of anti-inflammatory or anti-tumor genes.

Keywords: NF-κB; Nrf2; ARE; MafK; CBP; HDAC; Transrepression

Cell cycle-dependent expression of Kv1.5 is involved in myoblast proliferation by Núria Villalonga; Ramón Martínez-Mármol; Meritxell Roura-Ferrer; Miren David; Carmen Valenzuela; Concepció Soler; Antonio Felipe (pp. 728-736).

Voltage-dependent K⁺ channels (Kv) are involved in the proliferation of many types of cells, but the mechanisms by which their activity is related to cell growth remain unclear. Kv antagonists inhibit the proliferation of mammalian cells, which is of physiological relevance in skeletal muscle. Although myofibres are terminally differentiated, some resident myoblasts may re-enter the cell cycle and proliferate. Here we report that the expression of Kv1.5 is cell-cycle dependent during myoblast proliferation. In addition to Kv1.5 other Kv, such as Kv1.3, are also up-regulated. However, pharmacological evidence mainly implicates Kv1.5 in myoblast growth. Thus, the presence of S0100176, a Kv antagonist, but not margatoxin and dendrotoxin, led to cell cycle arrest during the G₁-phase. The use of selective cell cycle blockers showed that Kv1.5 was transiently accumulated during the early G₁-phase. Furthermore, while myoblasts treated with S0100176 expressed low levels of cyclin A and D₁, the expression of p21^cip-1 and p27^kip1, two cyclin-dependent kinase inhibitors, increased. Our results indicate that the cell cycle-dependent expression of Kv1.5 is involved in skeletal muscle cell proliferation.

Keywords: Cell cycle; Myoblasts; Potassium channels; Proliferation; Skeletal muscle

Allosteric inhibition of the nonMyristoylated c-Abl tyrosine kinase by phosphopeptides derived from Abi1/Hssh3bp1 by Xiaoling Xiong; Ping Cui; Sajjad Hossain; Rong Xu; Brian Warner; Xinhua Guo; Xiuli An; Asim K. Debnath; David Cowburn; Leszek Kotula (pp. 737-747).

Here we report c-Abl kinase inhibition mediated by a phosphotyrosine located in trans in the c-Abl substrate, Abi1. The mechanism, which is pertinent to the nonmyristoylated c-Abl kinase, involves high affinity concurrent binding of the phosphotyrosine pY213 to the Abl SH2 domain and binding of a proximal PXXP motif to the Abl SH3 domain. Abi1 regulation of c-Abl in vivo appears to play a critical role, as demonstrated by inhibition of pY412 phosphorylation of the nonmyristoylated Abl by coexpression of Abi1. Pervanadate-induced c-Abl kinase activity was also reduced upon expression of the wild type Abi1 but not by expression of the Y213 to F213 mutant Abi1 in LNCaP cells, which are naturally deficient in the regulatory pY213. Our findings suggest a novel mechanism by which Abl kinase is regulated in cells.

Keywords: c-Abl; Tyrosine kinase activity; Abi1; Hssh3bp1; Allosteric mechanism; SH3 and SH2 domain

A lysophosphatidic acid receptor lacking the PDZ-binding domain is constitutively active and stimulates cell proliferation by Shinya Shano; Kazuki Hatanaka; Shinsuke Ninose; Ryutaro Moriyama; Toshifumi Tsujiuchi; Nobuyuki Fukushima (pp. 748-759).

Lysophosphatidic acid (LPA) is an extracellular signaling lipid that regulates cell proliferation, survival, and motility of normal and cancer cells. These effects are produced through G protein–coupled LPA receptors, LPA₁ to LPA₅. We generated an LPA₁ mutant lacking the SerValVal sequence of the C-terminal PDZ-binding domain to examine the role of this domain in intracellular signaling and other cellular functions. B103 neuroblastoma cells expressing the mutant LPA₁ showed rapid cell proliferation and tended to form colonies under serum-free conditions. The enhanced cell proliferation of the mutant cells was inhibited by exogenous expression of the plasmids inhibiting G proteins including G_βγ, G_αi and G_αq or G_α12/13, or treatment with pertussis toxin, phosphoinositide 3-kinase (PI3K) inhibitors or a Rho inhibitor. We confirmed that the PI3K-Akt and Rho pathways were intrinsically activated in mutant cells by detecting increases in phosphorylated Akt in western blot analyses or by directly measuring Rho activity. Interestingly, expression of the mutant LPA₁ in non-tumor mouse fibroblasts induced colony formation in a clonogenic soft agar assay, indicating that oncogenic pathways were activated. Taken together, these observations suggest that the mutant LPA₁ constitutively activates the G protein signaling leading to PI3K-Akt and Rho pathways, resulting in enhanced cell proliferation.

Keywords: Lysophosphatidic acid receptor; PDZ-binding domain; Phosphoinositide 3-kinase; Akt; Rho; Oncogenic signal

Peroxisome proliferation in Hansenula polymorpha requires Dnm1p which mediates fission but not de novo formation by Shirisha Nagotu; Ruchi Saraya; Marleen Otzen; Marten Veenhuis; Ida J. van der Klei (pp. 760-769).

We show that the dynamin-like proteins Dnm1p and Vps1p are not required for re-introduction of peroxisomes in Hansenula polymorpha pex3 cells upon complementation with PEX3-GFP. Instead, Dnm1p, but not Vps1p, plays a crucial role in organelle proliferation via fission. In H. polymorpha DNM1 deletion cells ( dnm1) a single peroxisome is present that forms long extensions, which protrude into developing buds and divide during cytokinesis. Budding pex11.dnm1 double deletion cells lack these peroxisomal extensions, suggesting that the peroxisomal membrane protein Pex11p is required for their formation. Life cell imaging revealed that fluorescent Dnm1p-GFP spots fluctuate between peroxisomes and mitochondria. On the other hand Pex11p is present over the entire organelle surface, but concentrates during fission at the basis of the organelle extension in dnm1 cells.Our data indicate that peroxisome fission is the major pathway for peroxisome multiplication in H. polymorpha.

Keywords: Peroxisome; Yeast; Dynamin-like protein; Dnm1p; Pex11p

Regulation of platelet Rac1 and Cdc42 activation through interaction with calmodulin by Sherif M. Elsaraj; Rajinder P. Bhullar (pp. 770-778).

Rac1 and Cdc42 are members of the Rho family of small GTPases and have been shown to induce lamellipodia and filopodia formation, respectively. This leads to changes in cytoskeleton organization and as a consequence affects cell migration. In the present work we demonstrate that endogenous Rac1 and Cdc42 interact with calmodulin (CaM) in a Ca²⁺-dependent fashion. The interaction of Rac1 and Cdc42 with CaM was shown to be direct. This novel interaction was further confirmed in platelets using co-immunoprecipitation studies. Using CaM database analysis and in vitro peptide competition assays we have identified a 14 amino acid region in Rac1 that is essential for CaM binding. The scrambled form of the peptide did not bind CaM demonstrating specificity of the predicted CaM binding region in Rac1. A similar region capable of binding CaM exists in Cdc42. Furthermore, using the optimal activation time-point for each GTPase, the role of CaM in the function of Rac1 and Cdc42 was examined. Results demonstrate that in human platelets, thrombin caused maximal activation of Rac1 and Cdc42 at ~60 s and ~25 s respectively. The potent CaM antagonist W7 abolished thrombin-mediated activation of Rac1. However, addition of W7 resulted in the activation of Cdc42 over basal and W7 did not inhibit thrombin-mediated activation of Cdc42. The less potent CaM inhibitor, W5, did not have any effect on Rac1 and Cdc42 activation. The results demonstrate that in platelets, binding of CaM to Rac1 increases its activation while its binding to Cdc42 reduces the activation of this GTPase. This suggests an important role for CaM in coordinating Rac1 and Cdc42 activation and in the regulation of cytoskeleton remodeling.

Keywords: Abbreviations; Calmodulin; CaM; SDS; sodium dodecyl sulfate; PAGE; polyacrylamide gel electrophoresis; PVDF; polyvinylidene difluoride; GST; glutathione; S; -transferase; Pak1; p21-activated kinase; PMSF; phenylmethylsulfonyl fluoride; AEBSF; 4-[(2-aminoethyl)]-benzenesulfonyl fluoride; EGTA; Ethylene glycol-bis (2-aminoethyl ether)-; N; ,; N; ,; N′; ,; N′; )-tetraacetic acid; GEF; guanine nucleotide exchange factorRac1; Cdc42; GTPases; Calmodulin; Platelet

CFTR in a lipid raft-TNFR1 complex modulates gap junctional intercellular communication and IL-8 secretion by Tecla Dudez; Florence Borot; Song Huang; Brenda R. Kwak; Marc Bacchetta; Mario Ollero; Bruce A. Stanton; Marc Chanson (pp. 779-788).

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause a chronic inflammatory response in the lung of patients with Cystic Fibrosis (CF). We have showed that TNF-α signaling through the Src family tyrosine kinases (SFKs) was defective as determined by an inability of TNF-α to regulate gap junctional communication (GJIC) in CF cells. Here, we sought to elucidate the mechanisms linking TNF-α signaling to the functions of CFTR at the molecular level. In a MDCKI epithelial cell model expressing wild-type (WtCFTR) or mutant CFTR lacking its PDZ-interacting motif (CFTR-ΔTRL), TNF-α increased the amount of WtCFTR but not CFTR-ΔTRL in detergent-resistant membrane microdomains (DRMs). This recruitment was modulated by SFK activity and associated with DRM localization of TNFR1 and c-Src. Activation of TNFR1 signaling also decreased GJIC and markedly stimulated IL-8 production in WtCFTR cells. In contrast, the absence of CFTR in DRMs was associated with abnormal TNFR1 signaling as revealed by no recruitment of TNFR1 and c-Src to lipid rafts in CFTR-ΔTRL cells and loss of regulation of GJIC and IL-8 secretion. These results suggest that localization of CFTR in lipid rafts in association with c-Src and TNFR1 provides a responsive signaling complex to regulate GJIC and cytokine signaling.

Keywords: Cystic fibrosis; CFTR; Lipid rafts; Tumor necrosis factor; Src tyrosine kinase; Inflammation; IL-8; Gap junctions

Reactive oxygen species produced up- or downstream of calcium influx regulate proinflammatory mediator release from mast cells: Role of NADPH oxidase and mitochondria by Toshio Inoue; Yoshihiro Suzuki; Tetsuro Yoshimaru; Chisei Ra (pp. 789-802).

Earlier studies have demonstrated that mast cells produce reactive oxygen species (ROS), which play a role in regulating Ca²⁺ influx, while in other cell types ROS are produced in a Ca²⁺-dependent manner. We sought to determine whether ROS are produced downstream of the extracellular Ca²⁺ entry in mast cells. Thapsigargin (TG), a receptor-independent agonist, could evoke a robust burst of intracellular ROS. However, this response was distinct from the antigen-induced burst of ROS with respect to time course and dependence on Ca²⁺ and phosphatidylinositol-3-kinase (PI3K). The antigen-induced ROS generation occurred immediately, while the TG-induced ROS generation occurred with a significant lag time (∼2 min). Antigen but not TG caused extracellular release of superoxide (O₂⁻)/hydrogen peroxide (H₂O₂), which was blocked by diphenyleneiodonium, apocynin, and wortmannin. A capacitative Ca²⁺ entry resulted in the generation of O₂⁻ in the mitochondria in a PI3K-independent manner. Blockade of ROS generation inhibited TG-induced mediator release. Finally, when used together, antigen and TG evoked the release of leukotriene C₄, tumor necrosis factor-α, and interleukin-13 as well as ROS generation synergistically. These results suggest that ROS produced upstream of Ca²⁺ influx by NADPH oxidase and downstream of Ca²⁺ influx by the mitochondria regulate the proinflammatory response of mast cells.

Keywords: Mast cells; Antigen; Thapsigargin; Reactive oxygen species; Calcium; Mediator release

Modulation by insulin-like growth factor I of the phosphatase PTEN in astrocytes by Silvia Fernández; Miguel García-García; Ignacio Torres-Alemán (pp. 803-812).

Characterization of intracellular pathways underlying the pleiotropic actions of insulin-like growth factor-I (IGF-I) on brain cells is incomplete. We analyzed IGF-I signalling on astrocytes through the canonical phosphatidylinositol 3-kinase (PI3K)/Akt pathway and focused on possible changes in PTEN, a phosphatase that modulates IGF-I signalling by inhibiting Akt activation and, in turn is positively regulated by PI3K. After exposure of astrocytes to IGF-I, PTEN mRNA and protein levels were reduced and its phosphatase activity diminished. Inhibition of PTEN involved activation of a PI3K/protein kinase C (PKC) pathway that decreased in a proteasome-dependent step the levels of the transcription factor Egr-1, a key regulator of PTEN levels in astrocytes, causing decreased binding of Egr-1 to the PTEN promoter. Enhanced mitogenesis in PTEN siRNA-transduced astrocytes after IGF-I suggested that reduced PTEN may be a permissive factor for the mitogenic activity of IGF-I. Subsequent recovery of reduced PTEN required also activation by IGF-I of PI3K to recruit in this case protein kinase A (PKA) which stimulated Egr-1 levels and, consequently PTEN synthesis. Because basal levels of PTEN in astrocytes are also governed by PI3K, IGF-I appears to modulate PTEN in astrocytes by redirecting its homeostasic control through PI3K in a timed fashion.

Keywords: IGF-I; Astrocyte; PI; ₃; K; PTEN; PKA; Egr-1; PKC

Metabolic transformation of rabbit skeletal muscle cells in primary culture in response to low glucose by Nina Hanke; Joachim D. Meissner; Renate J. Scheibe; Volker Endeward; Gerolf Gros; Hans-Peter Kubis (pp. 813-825).

We have investigated the mechanism of the changes in the profile of metabolic enzyme expression that occur in association with fast-to-slow transformation of rabbit skeletal muscle. The hypotheses assessed are: do 1) lowered intracellular ATP concentration or 2) reduction of the muscular glycogen stores act as triggers of metabolic transformation? We find that 3 days of decreased cytosolic ATP content have no impact on the investigated metabolic markers, whereas incubation of the cells with little or no glucose leads to decreases in glycogen in conjunction with decreases in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter activity, GAPDH mRNA and specific GAPDH enzyme activity (indicators of the anaerobic glycolytic pathway), and furthermore to increases in mitochondrial acetoacetyl-CoA thiolase (MAT, also known as ACAT) promoter activity, peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) expression and citrate synthase (CS) specific enzyme activity (all indicators of oxidative metabolic pathways). The AMP-activated protein kinase (AMPK) activity under these conditions is reduced compared to controls. In experiments with two inhibitors of glycogen degradation we show that the observed metabolic transformation caused by low glucose takes place even if intracellular glycogen content is high. These findings for the first time provide evidence that metabolic adaptation of skeletal muscle cells from rabbit in primary culture can be induced not only by elevation of intracellular calcium concentration or by a rise of AMPK activity, but also by reduction of glucose supply. Contrary to expectations, neither an increase in phospho-AMPK nor a reduction of muscular glycogen content are crucial events in the glucose-dependent induction of metabolic transformation in the muscle cell culture system studied.

Keywords: Muscle plasticity; AMP-activated protein kinase (AMPK); Glycogen; Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α); Glucose-dependent gene regulation

Involvement of Ymer in suppression of NF-κB activation by regulated interaction with lysine-63-linked polyubiquitin chain by Miyuki Bohgaki; Tadasuke Tsukiyama; Ayako Nakajima; Satoru Maruyama; Masashi Watanabe; Takao Koike; Shigetsugu Hatakeyama (pp. 826-837).

It is known that the cytoplasmic zinc finger protein A20 functionally dampens inflammatory signals and apoptosis via inhibition of NF-κB activation and biochemically acts as a unique ubiquitin-modifying protein with deubiquitinating activity and ubiquitin ligase activity. However, the molecular mechanisms of A20-modulated signal transduction that influence normal immune responses or tumor immunity have not been fully elucidated. Using a yeast two-hybrid system to search for proteins interacting with A20, we identified one novel binding protein, Ymer. Ymer, which has been reported to be highly phosphorylated on tyrosine residues via EGF stimulation, bound to lysine (K)-63-linked polyubiquitin chain on receptor-interacting serine/threonine-protein kinase 1 (RIP1), which is essential for NF-κB signaling in collaboration with A20. A luciferase assay showed that NF-κB signaling was down-regulated by overexpression of Ymer, whereas knock-down of Ymer up-regulated NF-κB signaling even without stimulation. These findings demonstrate that Ymer is likely to be a negative regulator for the NF-κB signaling pathway.

Keywords: NF-κB; A20; Ymer; RIP1; Ubiquitin

VDUP1 mediates nuclear export of HIF1α via CRM1-dependent pathway by Daesung Shin; Jun-Ho Jeon; Mira Jeong; Hyun-Woo Suh; Seyl Kim; Hyoung-Chin Kim; Og-Sung Moon; Yong-Sung Kim; Jin Woong Chung; Suk Ran Yoon; Woo-Ho Kim; Inpyo Choi (pp. 838-848).

Hypoxia-inducible factor 1α (HIF1α) is a critical transcriptional factor for inducing tumor metastasis, and stabilized under hypoxia but degraded by von Hippel–Lindau protein (pVHL) under normoxia. For the maximal degradation of HIF1α, it must be exported to the cytoplasm via an unidentified transporter. Here, we demonstrate that vitamin D3 up-regulated protein 1 (VDUP1) associates with the β-domain of pVHL and enhances the interaction between pVHL and HIF1α to promote the nuclear export and degradation of HIF1α hypoxia-independently. Blocking of VDUP1 translocation either by leptomycin B or by nuclear export signal mutation inhibited the nuclear export of pVHL/HIF1α and relieved the destabilization of HIF1α. VDUP1 suppressed cell invasiveness and tumor metastasis, which were also recovered by blocking of nuclear export. Taken together, these findings indicate that VDUP1 is a novel tumor suppressor which mediates the nuclear export of pVHL/HIF1α complex to destabilize HIF1α.

Keywords: Abbreviations; CRM1; chromosome region maintenance 1; HDAC; histone deacetylase; HIF1α; hypoxia-inducible factor 1α; NES; nuclear export signal; pVHL; von Hippel–Lindau protein; SAHA; suberoylanilide hydroxamic acid; VDUP1; vitamin D; ₃; up-regulated protein 1HIF1α; pVHL; VDUP1; CRM1; Transport

Mitoparan and target-selective chimeric analogues: Membrane translocation and intracellular redistribution induces mitochondrial apoptosis by Sarah Jones; Cecile Martel; Anne-Sophie Belzacq-Casagrande; Catherine Brenner; John Howl (pp. 849-863).

Mastoparan, and structurally-related amphipathic peptides, may induce cell death by augmentation of necrotic and/or apoptotic pathways. To more precisely delineate cytotoxic mechanisms, we determined that [Lys^5,8Aib¹⁰]mastoparan (mitoparan) specifically induces apoptosis of U373MG and ECV304 cells, as demonstrated by endonuclease and caspase-3 activation and phosphatidylserine translocation. Live cell imaging confirmed that, following translocation of the plasma membrane, mitoparan specifically co-localizes with mitochondria. Complementary studies indicated that mitoparan induces swelling and permeabilization of isolated mitochondria, through cooperation with a protein of the permeability transition pore complex VDAC, leading to the release of the apoptogenic factor, cytochrome c. N-terminal acylation of mitoparan facilitated the synthesis of chimeric peptides that incorporated target-specific address motifs including an integrin-specific RGD sequence and a Fas ligand mimetic. Significantly, these sychnologically-organised peptides demonstrated further enhanced cytotoxic potencies. We conclude that the cell penetrant, mitochondriotoxic and apoptogenic properties of mitoparan, and its chimeric analogues, offer new insights to the study and therapeutic induction of apoptosis.

Keywords: Abbreviations; MP; mastoparan; mitP; mitoparan; TUNEL; terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling; VDAC; voltage-dependent anion channel; CPP; cell penetrating peptide; PTPC; permeability transition pore complex.Cell penetrating peptides; Apoptosis; Mitochondria; Chimerism; Mastoparan

Comparison of the PTS1- and Rab8b-binding properties of Pex5p and Pex5Rp/TRIP8b by Marc Fransen; Leen Amery; Andreas Hartig; Chantal Brees; Anja Rabijns; Guy P. Mannaerts; Paul P. Van Veldhoven (pp. 864-873).

Tetratricopeptide (TPR)-domain proteins are involved in various cellular processes. The TPR domain is known to be responsible for interaction with other proteins commonly recognizing sequence motifs at the C-termini. One such TPR-protein, TRIP8b, was originally identified in rat as an interaction partner of Rab8b, and its human orthologue as a protein related to the peroxisomal targeting signal 1 (PTS1) receptor Pex5p (Pex5Rp). Somewhat later, the mouse orthologue was reported to bind the hyperpolarization-activated, cyclic nucleotide-regulated HCN channels, and, very recently, the rat orthologue was shown to interact with latrophilin 1, the calcium-independent receptor of α-latrotoxin. Here we employed various methodological approaches to investigate and compare the binding specificities of the human PTS1 receptor Pex5p and the related protein Pex5Rp/TRIP8b towards a subset of targets, including Rab8b and various C-termini resembling PTS1. The results show that the TPR domains of Pex5p and Pex5Rp/TRIP8b have distinct but overlapping substrate specificities. This suggests that selectivity in the recognition of substrates by the TPR domains of Pex5p and Pex5Rp/TRIP8b is a matter of considerable complexity, and that no single determinant appears to be sufficient in unambiguously defining a binding target for either protein. This idea is further corroborated by our observations that changes in the surrounding residues or the conformational state of one of the binding partners can profoundly alter their binding activities. The implications of these findings for the possible peroxisome-related functions of Pex5Rp/TRIP8b are discussed.

Keywords: TPR proteins; Pex5p; Pex5Rp/TRIP8b; Rab8b; PTS1; Peroxisomes

Store-operated Ca²⁺ channels and Stromal Interaction Molecule 1 (STIM1) are targets for the actions of bile acids on liver cells by Edoardo C. Aromataris; Joel Castro; Grigori Y. Rychkov; Greg J. Barritt (pp. 874-885).

Cholestasis is a significant contributor to liver pathology and can lead to primary sclerosis and liver failure. Cholestatic bile acids induce apoptosis and necrosis in hepatocytes but these effects can be partially alleviated by the pharmacological application of choleretic bile acids. These actions of bile acids on hepatocytes require changes in the release of Ca²⁺ from intracellular stores and in Ca²⁺ entry. However, the nature of the Ca²⁺ entry pathway affected is not known. We show here using whole cell patch clamp experiments with H4-IIE liver cells that taurodeoxycholic acid (TDCA) and other choleretic bile acids reversibly activate an inwardly-rectifying current with characteristics similar to those of store-operated Ca²⁺ channels (SOCs), while lithocholic acid (LCA) and other cholestatic bile acids inhibit SOCs. The activation of Ca²⁺ entry was observed upon direct addition of the bile acid to the incubation medium, whereas the inhibition of SOCs required a 12 h pre-incubation. In cells loaded with fura-2, choleretic bile acids activated a Gd³⁺-inhibitable Ca²⁺ entry, while cholestatic bile acids inhibited the release of Ca²⁺ from intracellular stores and Ca²⁺ entry induced by 2,5-di-( tert-butyl)-1,4-benzohydro-quinone (DBHQ). TDCA and LCA each caused a reversible redistribution of stromal interaction molecule 1 (STIM1, the endoplasmic reticulum Ca²⁺ sensor required for the activation of Ca²⁺ release-activated Ca²⁺ channels and some other SOCs) to puncta, similar to that induced by thapsigargin. Knockdown ofStim1 using siRNA caused substantial inhibition of Ca²⁺-entry activated by choleretic bile acids. It is concluded that choleretic and cholestatic bile acids activate and inhibit, respectively, the previously well-characterised Ca²⁺-selective hepatocyte SOCs through mechanisms which involve the bile acid-induced redistribution of STIM1.

Keywords: Liver cell; Cholestasis; Ca; ²⁺; channel; STIM1; Bile acid; Patch clamp recording

Visfatin enhances ICAM-1 and VCAM-1 expression through ROS-dependent NF-κB activation in endothelial cells by Su-Ryun Kim; Yun-Hee Bae; Soo-Kyung Bae; Kyu-Sil Choi; Kwon-Ha Yoon; Tae Hyeon Koo; Hye-Ock Jang; Il Yun; Kyu-Won Kim; Young-Guen Kwon; Mi-Ae Yoo; Moon-Kyoung Bae (pp. 886-895).

Visfatin has recently been identified as a novel visceral adipokine which may be involved in obesity-related vascular disorders. However, it is not known whether visfatin directly contributes to endothelial dysfunction. Here, we investigated the effect of visfatin on vascular inflammation, a key step in a variety of vascular diseases. Visfatin induced leukocyte adhesion to endothelial cells and the aortic endothelium by induction of the cell adhesion molecules, ICAM-1 and VCAM-1. Promoter analysis revealed that visfatin-mediated induction of CAMs is mainly regulated by nuclear factor-κB (NF-κB). Visfatin stimulated IκBα phosphorylation, nuclear translocation of the p65 subunit of NF-κB, and NF-κB DNA binding activity in HMECs. Furthermore, visfatin increased ROS generation, and visfatin-induced CAMs expression and NF-κB activation were abrogated in the presence of the direct scavenger of ROS. Taken together, our results demonstrate that visfatin is a vascular inflammatory molecule that increases expression of the inflammatory CAMs, ICAM-1 and VCAM-1, through ROS-dependent NF-κB activation in endothelial cells.

Keywords: Cell adhesion molecules; NF-κB; Reactive oxygen species; Vascular inflammation; Visfatin

The epidermal growth factor receptor (EGFR) is proteolytically modified by the Matriptase–Prostasin serine protease cascade in cultured epithelial cells by Mengqian Chen; Li-Mei Chen; Chen-Yong Lin; Karl X. Chai (pp. 896-903).

Prostasin is expressed at the apical surface of normal epithelial cells and suppresses in vitro invasion of cancer cells. Prostasin re-expression in the PC-3 prostate carcinoma cells down-regulated the epidermal growth factor receptor (EGFR) protein expression and EGF-induced phosphorylation of the extracellular signal-regulated kinases (Erk1/2). We report here that prostasin and its activating enzyme matriptase are capable of inducing proteolytic cleavages in the EGFR extracellular domain (ECD) when co-expressed in the FT-293 cells, generating two amino-terminally truncated fragments EGFR135 and EGFR110, at 135 and 110 kDa. Prostasin's role in EGFR cleavage is dependent on the serine active-site but not the GPI-anchor. The modifications of EGFR were confirmed to be on the primary structure by deglycosylation. EGFR135 and EGFR110 are not responsive to EGF stimulation, indicating loss of the ligand-binding domains. EGFR110 is constitutively phosphorylated and in its presence Erk1/2 phosphorylation is increased in the absence of EGF. The protease-induced EGFR cleavages are not dependent on EGFR phosphorylation. The EGFR ECD proteolytic modification by matriptase–prostasin is also observed in the BEAS-2B normal lung epithelial cells, the BPH-1 benign prostate hyperplasia and the MDA-MB-231 breast cancer cell lines; and represents a novel mechanism for epithelial cells to modulate EGF-EGFR signaling.

Keywords: ErbB receptor tyrosine kinase; GPI-anchor; Transmembrane glycoprotein; Extracellular signal-regulated kinase; MT-SP1; PRSS8

SLP-2 interacts with prohibitins in the mitochondrial inner membrane and contributes to their stability by Sandrine Da Cruz; Philippe A. Parone; Philippe Gonzalo; Willy V. Bienvenut; Daniel Tondera; Alexis Jourdain; Manfredo Quadroni; Jean-Claude Martinou (pp. 904-911).

Stomatin is a member of a large family of proteins including prohibitins, HflK/C, flotillins, mechanoreceptors and plant defense proteins, that are thought to play a role in protein turnover. Using different proteomic approaches, we and others have identified SLP-2, a member of the stomatin gene family, as a component of the mitochondria. In this study, we show that SLP-2 is strongly associated with the mitochondrial inner membrane and that it interacts with prohibitins. Depleting HeLa cells of SLP-2 lead to increased proteolysis of prohibitins and of subunits of the respiratory chain complexes I and IV. Further supporting the role of SLP-2 in regulating the stability of specific mitochondrial proteins, we found that SLP-2 is up-regulated under conditions of mitochondrial stress leading to increased protein turnover. These data indicate that SLP-2 plays a role in regulating the stability of mitochondrial proteins including prohibitins and subunits of respiratory chain complexes.

Keywords: Mitochondria; Stomatin; Prohibitin; Chaperone; AAA protease; Protein turnover

Overexpression of phospholipase D suppresses taxotere-induced cell death in stomach cancer cells by Ju Hwan Cho; Seong-Kweon Hong; Eun-Young Kim; Shin-Young Park; Chang-Hwan Park; Jung Mogg Kim; Oh Jung Kwon; Sung-Joon Kwon; Ki-Sung Lee; Joong-Soo Han (pp. 912-923).

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid (PA) and choline. There are at least two PLD isozymes, PLD1 and PLD2. Genetic and pharmacological approaches implicate both PLD isozymes in a diverse range of cellular processes, including receptor signaling, membrane transport control, and actin cytoskeleton reorganization. Several recent studies reported that PLD has a role in signaling pathways that oppose apoptosis and promote cell survival in cancer. In this study, we examined the role of PLD in taxotere-induced apoptosis in stomach cell lines; normal stomach (NSC) and stomach cancer cells (SNU 484). Taxotere treatment resulted in increase of PLD activity. To confirm the role of PLD in taxotere-induced apoptosis, PLDs were transfected into SNU 484 cells. Overexpression of PLD isozymes resulted in inhibition of taxotere-induced apoptotic cell death, evidenced by decreased degradation of chromosomal DNA, and increased cell viability. Concurrently, Bcl-2 expression was upregulated, and taxotere-induced activation of procaspase 3 was inhibited after PLD's transfection. However, when PLD was selectively inhibited by specific siRNA-PLD1 or -PLD2, taxotere-induced apoptosis was exacerbated in SNU 484 cells. On top of this, PA—the product of PLDs, also resulted in upregulation of Bcl-2 in SNU 484. Although PA-induced Bcl-2 expression was blocked by mepacrine, an inhibitor of phospholipase A₂ (PLA₂), increased Bcl-2 expression by PA was not abrogated by propranolol, an inhibitor of PA phospholyhydrolase (PAP). Taken together, PLD1 and PLD2 are closely related with Bcl-2 expression together with PLA₂, but not with PAP, during taxotere-induced apoptosis in SNU 484 cells.

Keywords: Phospholipase D; Apoptosis; Bcl-2; Phosphatidic acid; SNU 484 cell

Galectin-3 but not galectin-1 induces mast cell death by oxidative stress and mitochondrial permeability transition by Yoshihiro Suzuki; Toshio Inoue; Tetsuro Yoshimaru; Chisei Ra (pp. 924-934).

Galectin-1 and galectin-3 are the most ubiquitously expressed members of the galectin family and more importantly, these two molecules are shown to have opposite effects on pro-inflammatory responses and/or apoptosis depending on the cell type. Herein, we demonstrate for the first time that galectin-3 induces mast cell apoptosis. Mast cells expressed substantial levels of galectin-3 and galectin-1 and to a lesser extent the receptor for advanced glycation end products (RAGE) on their surfaces. Treatment of cells with galectin-3 at concentrations of ≥100 nM for 18–44 h resulted in cell death by apoptosis. Galectin-3-induced apoptosis was completely prevented by lactose, neutralizing antibody to RAGE, and the caspase-3 inhibitor z-DEVD-fmk. Galectin-3-induced apoptosis was also completely abolished by dithiothreitol and superoxide dismutase, but not inhibited by catalase. Moreover, galectin-3 but not galectin-1 induced the release of superoxide, which was blocked by lactose, anti-RAGE, and dithiothreitol. Finally, galectin-3-induced apoptosis was blocked by bongkrekic acid, an antagonist of the mitochondrial permeability transition pore (PTP), while atractyloside, an agonist of the PTP, greatly facilitated galectin-1-induced apoptosis. These data suggest that galectin-3 induces oxidative stress, PTP opening, and the caspase-dependent death pathway by binding to putative surface receptors including RAGE via the carbohydrate recognition domain.

Keywords: Mast cell; Galectin-3; Galectin-1; Reactive oxygen species; Apoptosis; Mitochondria

Induction of p21 by p65 in p53 null cells treated with Doxorubicin by Shenglin Ma; Juanjuan Tang; Jianguo Feng; Yaping Xu; Xinmin Yu; Qinghua Deng; Yanjun Lu (pp. 935-940).

NFκB/p65 is a transcription factor that can protect or contribute to cell death. Here we show that knockdown of p65 by IκBSR or p65 siRNA decreased the cytotoxic effect of DOX on HCT116 (p53+/+) cells, correlating with increased induction of p21. In previous work, we demonstrated that p21 suppressed cell death via its CDK-inhibitory activity. Thus, we propose that the p65 activity is required for p53-dependent cell death through limitation of p53-induced p21 expression. In HCT116 (p53−/−) cells, downregulation of p65 expression enhanced the cytotoxic effect of DOX, due to decreased p21 expression levels. We present evidence that in p53-null tumor cells treated with DOX, p65 was involved in induction of p21 expression by directly binding to the p21 promoter. Our data suggested that both p53 and p65 limited each other's ability to stimulate p21 induction and this mutual repression mechanism was consistent with a model in which both factors were competing for limiting pool of p300/CBP coactivator protein complexes. These findings indicate an association between p21 expression and resistance to cell death through p65, a novel regulatory mechanism in which p21 bridges a transcriptional crosstalk between p53 and p65.

Keywords: p21; NFκB/p65; p53; CBP/p300; Cell death

IEX-1 directly interferes with RelA/p65 dependent transactivation and regulation of apoptosis by Alexander Arlt; Philip Rosenstiel; Marie-Luise Kruse; Frauke Grohmann; Jörg Minkenberg; Neil D. Perkins; Ulrich R. Fölsch; Stefan Schreiber; Heiner Schäfer (pp. 941-952).

The early response gene IEX-1 plays a complex role in the regulation of apoptosis. Depending on the cellular context and the apoptotic stimulus, IEX-1 is capable to either enhance or suppress apoptosis. To further dissect the molecular mechanisms involved in the modulation of apoptosis by IEX-1, we analysed the molecular crosstalk between IEX-1 and the NF-κB pathway. Using GST-pulldown assays, a direct interaction of IEX-1 with the C-terminal region of the subunit RelA/p65 harbouring the transactivation domain of the NF-κB transcription factor was shown. This interaction negatively regulates RelA/p65 dependent transactivation as shown by GAL4-and luciferase assay and was confirmed for the endogenous proteins by co-immunoprecipitation experiments. Using deletion constructs, we were able to map the C-terminal region of IEX-1 as the critical determinant of the interaction with RelA/p65. We could further show, that IEX-1 mediated NF-κB inhibition accounts for the reduced expression of the anti-apoptotic NF-κB target genes Bcl-2, Bcl-xL, cIAP1 and cIAP2, thereby sensitizing cells for apoptotic stimuli. Finally, ChIP-assays revealed that IEX-1 associates with the promoter of these genes. Altogether, our findings suggest a critical role of IEX-1 in the NF-κB dependent regulation of apoptotic responses.

Keywords: NF-κB; apoptosis; Signal transduction; IEX-1; IAP; Chromatin

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BBA - Molecular Cell Research (v.1783, #5)