BBA - Molecular Cell Research (v.1813, #12)

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) during stress conditions causes activation of the unfolded protein response (UPR). If this adaptive response cannot restore ER homeostasis, cells undergo ER-mediated apoptosis. This study determines whether thermotolerance developed at a mild temperature (40 °C) can alter induction of ER-mediated stress and apoptosis by H2O2 in HeLa cells. Protein expression of PERK, p-PERK, eIF2α and p-eIF2α was increased in thermotolerant compared to non-thermotolerant cells. Thus, mild thermotolerance enhanced pro-survival effects of the PERK/eIF2α branch of the UPR. A short exposure (15 min) of cells to H2O2 (15–50 μM) activated the UPR: expression of p-PERK, p-eIF2α and p-IRE1α increased, and ATF6 cleavage occurred. Longer exposure (1–3 h) to H2O2 induced ER-mediated apoptosis, whereby CHOP expression increased, and enzymatic activity of calpain, caspase-7, -4, -12 and -9 also increased. These pro-apoptotic events and clonogenic cell killing were all diminished in thermotolerant cells. Activation of caspases-4/-12 was decreased by the calcium chelator BAPTA-AM, and by inhibitors of calpain and caspase-7, confirming the roles of calcium, calpain and caspase-7 in activation of ER-mediated apoptosis by H2O2. In thermotolerant cells with decreased levels of PERK by siRNA, there was partial reversal of resistance to H2O2-induced apoptosis. Hence, a causal connection exists between the ER stress response and resistance to H2O2-induced apoptosis. Mild thermotolerance plays a protective, anti-apoptotic role by increasing the threshold for induction of ER-mediated apoptosis by H2O2. Moreover, the adaptive response (UPR) dominates during milder H2O2 stress, whereas ER-mediated apoptosis occurs during more severe stress.► Mild thermotolerance (40 °C) activated the PERK/eIF2α branch of the UPR. ► Induction of ER stress by low dose H2O2 and apoptosis via the ER by higher doses. ► Calcium, calpain and caspase-7 were involved in caspase-4/12 activation. ► Mild thermotolerant decreased pro-apoptotic events induced by H2O2 at the ER. ► A causal connection exists between the ER stress response and resistance to H2O2-induced apoptosis.
Keywords: H2O2; Apoptosis; Unfolded protein response; Endoplasmic reticulum; Caspase; Calpain;

Recent evidence suggests that coupled leading and lagging strand DNA synthesis operates in mammalian mitochondrial DNA (mtDNA) replication, but the factors involved in lagging strand synthesis are largely uncharacterised. We investigated the effect of knockdown of the candidate proteins in cultured human cells under conditions where mtDNA appears to replicate chiefly via coupled leading and lagging strand DNA synthesis to restore the copy number of mtDNA to normal levels after transient mtDNA depletion. DNA ligase III knockdown attenuated the recovery of mtDNA copy number and appeared to cause single strand nicks in replicating mtDNA molecules, suggesting the involvement of DNA ligase III in Okazaki fragment ligation in human mitochondria. Knockdown of ribonuclease (RNase) H1 completely prevented the mtDNA copy number restoration, and replication intermediates with increased single strand nicks were readily observed. On the other hand, knockdown of neither flap endonuclease 1 (FEN1) nor DNA2 affected mtDNA replication. These findings imply that RNase H1 is indispensable for the progression of mtDNA synthesis through removing RNA primers from Okazaki fragments. In the nucleus, Okazaki fragments are ligated by DNA ligase I, and the RNase H2 is involved in Okazaki fragment processing. This study thus proposes that the mitochondrial replication system utilises distinct proteins, DNA ligase III and RNase H1, for Okazaki fragment maturation.► The data suggest that DNA ligase III is the replicative DNA ligase in human mitochondria. ► RNase H1 is indispensable for Okazaki fragment processing in human mtDNA replication. ► On the other hand, knockdown of neither DNA2 nor FEN1 affected mtDNA replication. ► Mitochondria utilise distinct proteins for Okazaki fragment maturation from nucleus.
Keywords: Mitochondrion; Mitochondrial DNA; DNA replication; Mitochondrial DNA replication factor; Okazaki fragment;

CITED2 controls the hypoxic signaling by snatching p300 from the two distinct activation domains of HIF-1α by Haejin Yoon; Ji-Hong Lim; Chung-Hyun Cho; L. Eric Huang; Jong-Wan Park (2008-2016).
HIF-1α plays a central role in cellular adaptation to hypoxia, and is closely related to the pathogeneses of life-threatening disorders. HIF-1α induces the expressions of numerous hypoxia-induced genes through two transactivation domains; N-terminal TAD (NAD) and C-terminal TAD (CAD). Furthermore, p300 is known to boost CAD-dependent transactivation, and CBP/p300-interacting transactivator with an ED-rich tail 2 (CITED2) inhibits HIF-1α-driven gene expression by interfering with the interaction between CAD and p300. However, few researches have focused on the role of CITED2 in the regulation of NAD activity, and thus, we addressed this point. CITED2 was found to attenuate the hypoxic activations of NAD-dependent and CAD-dependent genes, suggesting that CITED2 negatively regulates both CAD and NAD. Immunoprecipitation analyses showed that NAD interacts with the Cystein/Histidine region (CH) 1 and CH3 domains of p300. Moreover, CH1 and CH3 both were required for NAD-dependent transactivation. Furthermore, CITED2 was found to inactivate NAD by interfering with NAD binding to CH1, but not to CH3. These results indicate that CITED2 inactivates HIF-1α by blocking p300 recruitment by both NAD and CAD. We also found that pVHL inhibits NAD activity regardless of NAD degradation by blocking the interaction between p300 and NAD. Summarizing, NAD was activated by binding to p300, and this was blocked by either CITED2 or pVHL. We propose that pVHL controls NAD during normoxia and that CITED2 controls NAD during hypoxia. Our results provide a new strategy for controlling HIF-1α.► p300 CH1 and CH2 both participate in HIF-1α NAD-driven transcription. ► In hypoxia, CITED2 inactivates HIF-1α by interfering with NAD binding to CH1. ► In normoxia, pVHL inhibits NAD activity by interfering with NAD binding to CH3.
Keywords: Hypoxia-inducible factor 1α; Transactivation domains; CBP/p300-interacting transactivator with an ED-rich tail 2; von Hippel–Lindau protein; p300;

Secretagogues of lung surfactant increase annexin A7 localization with ABCA3 in alveolar type II cells by Tudevdagva Gerelsaikhan; Xiao-Liang Chen; Avinash Chander (2017-2025).
Membrane fusion between the lamellar bodies and plasma membrane is an obligatory event in the secretion of lung surfactant. Previous studies have postulated a role for annexin A7 (A7) in membrane fusion during exocytosis in some cells including alveolar type II cells. However, the intracellular trafficking of A7 during such fusion is not described. In this study, we investigated association of endogenous A7 with lamellar bodies in alveolar type II cells following treatment with several secretagogues of lung surfactant. Biochemical studies with specific antibodies showed increased membrane-association of cell A7 in type II cells stimulated with agents that increase secretion through different signaling mechanisms. Immuno-fluorescence studies showed increased co-localization of A7 with ABCA3, the lamellar body marker protein. Because these agents increase surfactant secretion through activation of PKC and PKA, we also investigated the effects of PKC and PKA inhibitors, bisindolylmaleimideI (BisI) and H89, respectively, on A7 partitioning. Western blot analysis showed that these inhibitors prevented secretagogue-mediated A7 increase in the membrane fractions. These inhibitors also blocked increased co-localization of A7 with ABCA3 in secretagogue-treated cells, as revealed by immuno-fluorescence studies. In vitro studies with recombinant A7 showed phosphorylation with PKC and PKA. The cell A7 was also phosphorylated in cells treated with surfactant secretagogues. Thus, our studies demonstrate that annexin A7 relocates to lamellar bodies in a phosphorylation-dependent manner. We suggest that activation of protein kinase promotes phosphorylation and membrane-association of A7 presumably to facilitate membrane fusion during lung surfactant secretion.► Specific antibody is used for annexin A7 trafficking in alveolar type II cells. ► Cell-stimulation increased annexin A7 in cell membranes and lamellar bodies. ► Membrane-association or co-localization required protein kinase activation. ► Stimulation increases annexin A7 phosphorylation at serine and threonine residues. ► Phosphorylation of annexin A7 may regulate its function during membrane fusion.
Keywords: Lung surfactant secretion; Exocytosis; Membrane fusion; Lamellar bodies; Protein kinase inhibitors; A7 phosphorylation;

Cross talk between smooth muscle cells and monocytes/activated monocytes via CX3CL1/CX3CR1 axis augments expression of pro-atherogenic molecules by Elena Dragomir Butoi; Ana Maria Gan; Ileana Manduteanu; Daniela Stan; Manuela Calin; Monica Pirvulescu; Rory Ryan Koenen; Christian Weber; Maya Simionescu (2026-2035).
In atherosclerotic lesions, fractalkine (CX3CL1) and its receptor (CX3CR1) expressed by smooth muscle cells (SMC) and monocytes/macrophages, mediate the heterotypic anchorage and chemotaxis of these cells. We questioned whether, during the close interaction of monocytes with SMC, the CX3CL1/CX3CR1 pair modulates the expression of pro-atherogenic molecules in these cells.SMC were co-cultured with monocytes or LPS-activated monocytes (18 h) and then the cells were separated and individually investigated for the gene and protein expression of TNFα, IL-1β, IL-6, CX3CR1 and metalloproteinases (MMP-2, MMP-9). We found that SMC–monocyte interaction induced, in each cell type, an increased mRNA and protein expression of TNFα, IL-1β, IL-6, CX3CR1, MMP-2 and MMP-9. Blocking the binding of fractalkine to CX3CR1 (by pre-incubation of monocytes with anti-CX3CR1 or by CX3CR1 siRNA transfection) before cell co-culture decreased the production of TNFα, CX3CR1 and MMP-9. Monocyte–SMC interaction induced the phosphorylation of p38MAPK and activation of AP-1 transcription factor. Silencing the p65 (NF-kB subunit) inhibited the IL-1β and IL-6 and silencing c-jun inhibited the TNFα, CX3CR1 and MMP-9 induced by SMC–monocyte interaction.The cross-talk between SMC and monocytes augments the inflammatory response in both cell types as revealed by the increased expression of TNFα, IL-1β, IL-6, CX3CR1 and MMPs. Up-regulation of TNFα, CX3CR1 and MMP-9 is further increased upon interaction of SMC with activated monocytes and is dependent on fractalkine/CXRCR1 pair. These data imply that the fractalkine/CX3RCR1 axis may represent a therapeutic target to impede the inflammatory process associated with atherosclerosis.► Cross-talk between SMC–monocytes induces an inflammatory response in both cell types. ► Up-regulation of TNFα, CX3CR1 and MMP-9 is dependent on fractalkine/CXRCR1 axis. ► IL-1β and IL6 up-regulation is independent of CX3CL1/CX3CR1 pair. ► Fractalkine–CX3CR1 binding triggers the activation of AP-1 transcription factor.
Keywords: Smooth muscle cell; Monocyte; Fractalkine; CX3CR1; Inflammatory cytokine; Metalloproteinase;

Delta-like 1-Lysine613 regulates notch signaling by Liguo Zhang; Ryan C. Widau; B. Paul Herring; Patricia J. Gallagher (2036-2043).
Delta ligands are important for regulating Notch signaling through transcellular stimulation of Notch receptors. The cytoplasmic tails of Delta ligands have multiple potential regulatory sites including several lysine residues that are putative targets for ubiquitination by the E3 ubiquitin ligases, Mind Bomb and Neuralized. To identify possible roles for specific lysine residues in the cytoplasmic tail of the Notch ligand Dll1 a mutational and functional analysis was performed. Examination of a panel of individual or clustered lysine mutants demonstrated that lysine 613 (K613) in the cytoplasmic tail of Dll1 is a key residue necessary for transcellular activation of Notch signaling. Multi-ubiquitination of the Dll1 mutant Dll1-K613R was altered compared to wild type Dll1, and the K613R mutation blocked the ability of Dll1 to interact with Notch1. Finally, mutation of K613 did not affect the stability of Dll1 or its ability to traffic to recycle to the plasma membrane, but did enhance the fraction associated with lipid rafts. Collectively these results suggest that the transcellular defect in Notch signaling attributed to residue K613 in cytoplasmic tail of Dll1 may result from altering its multi-ubiquitination and increasing its retention in lipid rafts.► Seventeen conserved lysine residues in the cytoplasmic tail of Delta like1 (Dll1) were identified and mutated. ► Notch signaling, ubiquitination, endocytic recycling, lipid raft localization and Notch binding functions were examined. ► K613 was identified as important for Notch signaling. ► K613R altered the multiubiquitination profile of Dll1. ► K613R did not alter endocytosis or stability of Dll1 but did alter binding to Notch1 and lipid raft localization.
Keywords: Notch; Delta; Ubiquitin; Endocytosis; Recycling; Lipid raft microdomain;

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a specific substrate of yeast metacaspase by A. Silva; B. Almeida; B. Sampaio-Marques; M.I.R. Reis; S. Ohlmeier; F. Rodrigues; A. do Vale; P. Ludovico (2044-2049).
Yeast metacaspase (Yca1p) is required for the execution of apoptosis upon a wide range of stimuli. However, the specific degradome of this yeast protease has not been unraveled so far. By combining different methodologies described as requisites for a protein to be considered a protease substrate, such as digestome analysis, cleavage of recombinant GAPDH by metacaspase and evaluation of protein levels in vivo, we show that upon H2O2-induced apoptosis, the metabolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a specific target of metacaspase. Nitric oxide (NO) signaling, which mediates H2O2-induced apoptosis, is required for metacaspase specific GAPDH cleavage. In conclusion, in this work we identified GAPDH as the first direct yeast metacaspase substrate described so far. Although mammalian caspases and yeast metacaspase apparently have distinct target cleavage sites, GAPDH arises as a common substrate for these proteases.► GAPDH as a substrate of yeast metacaspase during H2O2-induced apoptosis. ► GAPDH fragmentation by metacaspase, during H2O2-induced apoptosis, is dependent on NO levels. ► GAPDH as a common substrate from yeast to mammalian cells upon apoptotic conditions.
Keywords: H2O2-induced yeast apoptosis; Yeast metacaspase; Glyceraldehyde-3-phosphate dehydrogenase; Nitric oxide;

Zac1 functional interactions mediate AP-1 transcriptional activity by Wei-Ming Wang; Shu-Ting Liu; Shih-Ming Huang; Wei-Shiang Lin; Shyi-Gen Chen; Yung-Lung Chang (2050-2060).
A zinc-finger protein which regulates apoptosis and cell cycle arrest 1 (Zac1) is a novel seven-zinc-finger protein that can bind a specific GC-rich DNA element and has intrinsic transactivation activity; therefore, its role as a transcription factor has been proposed. Zac1 not only promotes cell cycle arrest and apoptosis but also acts as a transcriptional cofactor for nuclear receptors and p53. In this study, we examined the functional roles of mouse Zac1 (mZac1) in HeLa cells treated with 12-O-tetradecanoylphorbol-13-acetate (PMA), a potent Activator protein 1 (AP-1) activator. At first, we found that mZac1 prolonged and enhanced PMA-induced AP-1 activity in both HeLa and HeLa/p53 shRNA cells. We further identified physical and functional interactions between mZac1 and AP-1 proteins (either c-Jun, c-Fos or both). Finally, we showed that Zac1 might function as a selective coactivator of AP-1, demonstrated by AP-1-dependent transcriptional activation of collagenase, c-Fos and p21 WAF1/Cip1 promoter activities. Identification of AP-1 as a specific target for Zac1-mediated transcriptional events not only establishes a direct link between these two pivotal regulatory proteins but also raises the possibility that Zac1 contributes to certain AP-1-dependent biological effects.► Zac1 enhanced PMA-induced AP-1 activity in HeLa and HeLa/p53 shRNA cells. ► Zac1 physically and functionally interacted with AP-1 (c-Jun, c-Fos, or both) proteins. ► Zac1 functioned as a selective coactivator for AP-1-dependent transcription. ► Zac1 interplayed with PMA on the sub-G1 phase via the phosphorylation of H2A.X.
Keywords: Zac1 (a zinc-finger protein which regulates apoptosis and cell cycle arrest 1); AP-1 (activator protein 1); Gene regulation; c-Jun; c-Fos; p21;

Periostin mediates human adipose tissue-derived mesenchymal stem cell-stimulated tumor growth in a xenograft lung adenocarcinoma model by Soon Chul Heo; Kook One Lee; Sang Hun Shin; Yang Woo Kwon; Young Mi Kim; Chang Hun Lee; Yeong Dae Kim; Min Ki Lee; Man-Soo Yoon; Jae Ho Kim (2061-2070).
Mesenchymal stem cells stimulate tumor growth in vivo through a lysophosphatidic acid (LPA)-dependent mechanism. However, the molecular mechanism by which mesenchymal stem cells stimulate tumorigenesis is largely elusive. In the present study, we demonstrate that conditioned medium from A549 human lung adenocarcinoma cells (A549 CM) induces expression of periostin, an extracellular matrix protein, in human adipose tissue-derived mesenchymal stem cells (hASCs). A549 CM-stimulated periostin expression was abrogated by pretreatment of hASCs with the LPA receptor 1 (LPA1) inhibitor Ki16425 or short hairpin RNA-mediated silencing of LPA1, suggesting a key role of the LPA–LPA1 signaling axis in A549 CM-stimulated periostin expression. Using a xenograft transplantation model of A549 cells, we demonstrated that co-injection of hASCs potentiated tumor growth of A549 cells in vivo and that co-transplanted hASCs expressed not only periostin but also α-smooth muscle actin (α-SMA), a marker of carcinoma-associated fibroblasts. Small interfering RNA- or short hairpin RNA-mediated silencing of periostin resulted in blockade of LPA-induced α-SMA expression in hASCs. In addition, silencing of periostin resulted in blockade of hASC-stimulated growth of A549 xenograft tumors and in vivo differentiation of transplanted hASCs to α-SMA-positive carcinoma-associated fibroblasts. Conditioned medium derived from LPA-treated hASCs (LPA CM) potentiated proliferation and adhesion of A549 cells and short interfering RNA-mediated silencing or immunodepletion of periostin from LPA CM abrogated proliferation and adhesion of A549 cells. These results suggest a pivotal role for hASC-secreted periostin in growth of A549 xenograft tumors within the tumor microenvironment.► A549 cells stimulate periostin expression in mesenchymal stem cells through lysophosphatidic acid-mediated manner. ► Mesenchymal stem cells-derived periostin stimulates in vivo growth of A549 xenograft tumor. ► Mesenchymal stem cells-derived periostin stimulates adhesion and proliferation of A549 cells. ► Periostin is involved in differentiation of mesenchymal stem cells to carcinoma-associated fibroblasts.
Keywords: Periostin; Lysophosphatidic acid; Mesenchymal stem cells; Adhesion; Tumorigenesis;

The Pax5 and its isoforms influence proliferation of B- and T-cells, during development and oncogenesis but molecular mechanism and host–tumor relationship is not clear. This report describes status of Pax5 isoforms and co-regulation of molecular markers of ascite cells causing Dalton's lymphoma in murine. Higher expressions of Pax5, CD19, CD3, Ras and Raf were observed in DLA cells. The levels of transcripts as well as p53 protein were also higher in DLA cells. The transcript of p53 from DLA cells was a variant of p53 having deletion of 50 bp as compared to control. On annotation, it reflects transformation related protein p53 pseudogene mRNA. Lower level of superoxide dismutase (SOD) indicates oxidative stress and higher level of LDH5 in DLA cells reflects hypoxia in cancerous condition. The expression of Pax5d/e isoforms in DLA cells suggests presence of resting B-cells. Thus, isoforms of Pax5 and co-regulation of T- and B-cells associated genes influence phenotypic traits of ascetic cells causing Dalton's lymphoma.► Pax5 isoforms are expressed in Dalton's lymphoma ascite (DLA) cells. ► Co-regulation of T- and B-cells associated genes influence phenotypic traits of ascetic cells causing Dalton's lymphoma. ► Transcript of p53 from DLA cells represents a variant of p53.
Keywords: B-cells; Dalton's ascite cells; Lymphoma; Pax5; T-cells;

Susceptibility to simvastatin-induced toxicity is partly determined by mitochondrial respiration and phosphorylation state of Akt by Peter J. Mullen; Anja Zahno; Peter Lindinger; Swarna Maseneni; Andrea Felser; Stephan Krähenbühl; Karin Brecht (2079-2087).
Statins are widely used to prevent cardiovascular diseases. They are well-tolerated, with side-effects mainly seen in skeletal muscle. How these side-effects are caused is unknown. We compared isolated primary mouse skeletal muscle myocytes, C2C12 myotubes and liver HepG2 cells to detect differences that could uncover why statins are toxic in skeletal muscle but less so in the liver. 10 μM simvastatin caused a decrease in mitochondrial respiration in the primary mouse myocytes and C2C12 myotubes, but had no effect in the HepG2 cells. Mitochondrial integrity is maintained by multiple signaling pathways. One of these pathways, Igf-1/Akt signaling, is also heavily implicated in causing statin-induced toxicity by upregulating atrogin-1. We found that phosphorylated Akt was reduced in C2C12 myotubes but not in HepG2 cells. HepG2 mitochondrial respiration became susceptible to simvastatin-treatment after Akt inhibition, and mitochondrial respiration was rescued in Igf-1-treated C2C12 myotubes. These results suggest that disruption of Igf-1/Akt signaling is a causative factor in simvastatin-induced mitochondrial dysfunction in C2C12 myotubes, whereas HepG2 cells are protected by maintaining Igf-1/Akt signaling. We conclude that phosphorylation of Akt is a key indicator of susceptibility to statin-induced toxicity. How statins can disrupt Igf-1/Akt signaling is unknown. Statins reduce geranylgeranylation of small GTPases, such as Rap1. Previous studies implicate Rap1 as a link between cAMP/Epac and Igf-1/Akt signaling. Transient transfection of constitutively active Rap1 into C2C12 myotubes led to a partial rescue of simvastatin-induced inhibition of mitochondrial respiration, providing a novel link between signaling and respiration.► Statin-induced myotoxicity is a major side-effect. ► Impairment of mitochondrial respiration is one sign of sensitivity to statins. ► Phosphorylation of Akt is reduced by simvastatin treatment in sensitive cells. ► Inhibiting Akt causes mitochondrial dysfunction and non-sensitive HepG2 cells. ► Rap1 could link statins to mitochondrial dysfunction.
Keywords: Statin; Toxicity; Mitochondrion; Akt; Rap1;

The survival effect of mitochondrial Higd-1a is associated with suppression of cytochrome C release and prevention of caspase activation by Hyun-Jung An; Hyojung Shin; Sang-Geun Jo; Young Jin Kim; Jie-Oh Lee; Sang-Gi Paik; Hayyoung Lee (2088-2098).
Higd-1a (hypoxia induced gene domain family-1a) is a mitochondrial inner membrane protein with a conformation of N-terminal outside-C-terminal outside and loop inside. There are four Higd genes, Higd-1a, -1b, -1c and -2a, in the mouse. Higd-1a and -2a are expressed primarily in the brain, heart, kidney and leukocytes. HIF (hypoxia-inducible factor) overexpression induced the endogenous expression and promoter activity of Higd-1a. Mutation of the HRE (hypoxia-response element) site at − 32 bp in the Higd-1a promoter reduced the promoter activity, suggesting that transcription of Higd-1a is regulated by binding of the transcription factor HIF to the HRE. Higd-1a promoted cell survival under hypoxia. RAW264.7 cells stably transfected with Higd-1a underwent less apoptosis than control cells in a hypoxic condition, and hypoxia-induced apoptosis was strongly enhanced when endogenous Higd-1a was silenced by siRNA. The survival effect of Higd-1a was completely abolished by deletion of the 26 N-terminal amino acids, and we showed that Higd-1a increased survival by inhibiting cytochrome C release and reducing the activities of caspases. However, expression of Bcl-2, Bax, Bad, and BNIP3 and translocation of AIF were unaffected under the same conditions. Higd-2a also enhanced cell survival under hypoxia. Cells transfected with Higd-2a underwent less apoptosis than control cells in hypoxic conditions, and hypoxia-induced apoptosis increased when endogenous Higd-2a was depleted. Together these observations indicate that Higd-1a is induced by hypoxia in a HIF-dependent manner and its anti-apoptotic effect results from inhibiting cytochrome C release and reducing caspase activities.► Here we have shown that Higd-1a is induced by hypoxia in a HIF-dependent manner. ► Higd-1a enhances survival by suppressing cytochrome C release and caspase activation. ► The N-terminal 26 a.a region of Higd-1a is indispensable for this survival effect. ► Higd-2a also promoted cell survival under hypoxic conditions. ► This is the first report concerning the mechanisms of action and induction of Higd.
Keywords: Higd; HIF; HRE; Apoptosis; Cytochrome C; Caspase;

The epithelial-to-mesenchymal transition (EMT) is a program of cellular development associated with loss of cell–cell contacts, a decreased cell adhesion and substantial morphological changes. Besides its importance for numerous developmental processes, EMT has also been held responsible for the development and progression of tumors and formation of metastases. The influence of the cytokine transforming growth factor β1 (TGF-β1) induced EMT on structure, migration, cytoskeletal dynamics and long-term correlations of the mammalian epithelial cell lines NMuMG, A549 and MDA-MB231 was investigated with time-resolved impedance analysis. The three cell lines show important differences in concentration dependency, cellular morphology and dynamics upon their response to TGF-β1. A549 cells and the non-tumor mouse epithelial cell line NMuMG show a substantial change in morphology mirrored in stepwise changes of their phenotype upon cytokine treatment. Impedance based measurements of micromotility reveal a complex dynamic response to TGF-β1 exposure which leads to a transient increase in fluctuation amplitude and long-term correlation. These changes in fluctuation amplitude are also detectable for MDA-MB231 cells, whereas the long-term correlation remains unvaried. We were able to distinguish three time domains during EMT. Initially, all cell lines display an increase in micromotion lasting 4 to 9 h termed transitional state I. This regime is followed by transitional state II lasting approximately 20 h, where cellular dynamics are diminished and, in case of the NMuMG cell line, a loss of cell–cell contacts occurs. Finally, the transformation into the mesenchymal-like phenotype occurs 24–30 h after exposure to TGF-β1.► Influence of cytokine TGF-β1 induced EMT on different cell lines. ► Three epithelial cell lines were investigated by time-resolved impedance analysis. ► Temporal changes in structure, cytoskeletal dynamics and long-term correlations. ► Distinction of different time regimes during EMT.
Keywords: Electric Cell-Substrate Impedance Sensing; Variance; Detrended fluctuation analysis; Epithelial-to-mesenchymal transition;

Inhibition of 5-lipoxygenase triggers apoptosis in prostate cancer cells via down-regulation of protein kinase C-epsilon by Sivalokanathan Sarveswaran; Vijayalakshmi Thamilselvan; Chaya Brodie; Jagadananda Ghosh (2108-2117).
Previous studies have shown that human prostate cancer cells constitutively generate 5-lipoxygenase (5-LOX) metabolites from arachidonic acid, and inhibition of 5-LOX blocks production of 5-LOX metabolites and triggers apoptosis in prostate cancer cells. This apoptosis is prevented by exogenous metabolites of 5-LOX, suggesting an essential role of 5-LOX metabolites in the survival of prostate cancer cells. However, downstream signaling mechanisms which mediate the survival-promoting effects of 5-LOX metabolites in prostate cancer cells are still unknown. Recently, we reported that MK591, a specific inhibitor of 5-LOX activity, induces apoptosis in prostate cancer cells without inhibition of Akt, or ERK, two well-characterized regulators of pro-survival mechanisms, suggesting the existence of an Akt and ERK-independent survival mechanism in prostate cancer cells regulated by 5-LOX. Here, we report that 5-LOX inhibition-induced apoptosis in prostate cancer cells occurs via rapid inactivation of protein kinase C-epsilon (PKCε), and that exogenous 5-LOX metabolites prevent both 5-LOX inhibition-induced down-regulation of PKCε and induction of apoptosis. Interestingly, pre-treatment of prostate cancer cells with diazoxide (a chemical activator of PKCε), or KAE1-1 (a cell-permeable, octa-peptide specific activator of PKCε) prevents 5-LOX inhibition-induced apoptosis, which indicates that inhibition of 5-LOX triggers apoptosis in prostate cancer cells via down-regulation of PKCε. Altogether, these findings suggest that metabolism of arachidonic acid by 5-LOX activity promotes survival of prostate cancer cells via signaling through PKCε, a pro-survival serine/threonine kinase.► Inhibition of 5-LOX triggers apoptosis in prostate cancer cells. ► Apoptosis involves annexin V binding, PARP-cleavage, and degradation of DNA. ► Apoptosis is associated with inhibition of PKC-epsilon, a known oncogene. ► Inhibition of PKC-epsilon and induction of apoptosis are prevented by 5-oxoETE. ► Metabolites of 5-LOX may promote prostate cancer cell survival via PKC-epsilon.
Keywords: 5-Lipoxygenase; Prostate cancer; Apoptosis; PKC-epsilon;

Cardiotonic steroids trigger non-classical testosterone signaling in Sertoli cells via the α4 isoform of the sodium pump by Lutz Konrad; Raimund Dietze; Ulrike Kirch; Herbert Kirch; Alexander Eva; Georgios Scheiner-Bobis (2118-2124).
The α4 isoform of the Na+,K+-ATPase (sodium pump) is known to be expressed in spermatozoa and to be critical for their motility. In the investigation presented here, we find that the rat-derived Sertoli cell line 93RS2 also expresses considerable amounts of the α4 isoform in addition to the α1 isoform. Since Sertoli cells are not motile, one can assume that the function of the α4 isoform in these cells must differ from that in spermatozoa. Thus, we assessed a potential involvement of this isoform in signaling pathways that are activated by the cardiotonic steroid (CTS) ouabain, a highly specific sodium pump ligand. Treatment of 93RS2 cells with ouabain leads to activation of the c-Src/c-Raf/Erk1/2 signaling cascade. Furthermore, we show for the first time that the activation of this cascade by ouabain results in phosphorylation and activation of the transcription factor CREB. This signaling cascade is induced at low nanomolar concentrations of ouabain, consistent with the involvement of the α4 isoform. This is further supported by experiments involving siRNA: silencing of α4 expression entirely blocks ouabain-induced activation of Erk1/2 whereas silencing of α1 has no effect. The findings of this study unveil new aspects in CTS/sodium pump interactions by demonstrating for the first time ouabain-induced signaling through the α4 isoform. The c-Src/c-Raf/Erk1/2/CREB cascade activated by ouabain is identical to the so-called non-classical signaling cascade that is normally triggered in Sertoli cells by testosterone. Taking into consideration that CTS are produced endogenously, our results may help to gain new insights into the physiological mechanisms associated with male fertility and reproduction.► The α4 isoform of the sodium pump alpha subunit is expressed in Sertoli cells. ► This subunit mediates signaling cascades that are induced by ouabain. ► The ouabain-triggered Src/cRaf/Erk1/2 signaling cascade results in CREB activation. ► This cascade matches the non-classical signaling cascade induced by testosterone. ► The results may help to unveil novel mechanisms associated with male fertility.
Keywords: Cardiotonic steroid; Ouabain; Sertoli cell; CREB; Erk1/2; Alpha4 isoform;

Aldosterone increases VEGF-A production in human neutrophils through PI3K, ERK1/2 and p38 pathways by Cécile Walczak; Fanny Gaignier; Alexandre Gilet; Feng Zou; Simon N. Thornton; Armelle Ropars (2125-2132).
Aldosterone is now recognised as an important actor in inflammation processes. Neoangiogenesis plays a crucial role in this complex process and immune cells, such as neutrophils, appear to be able to secrete different forms of (pro)angiogenic molecules, especially VEGF-A. The present work was undertaken to investigate whether aldosterone was able to regulate VEGF-A production in human neutrophils. The HL-60 (progranulocytic) cell line and human polymorphonuclear leukocytes were incubated for different time periods with aldosterone. Total cellular RNA extraction, submitted to reverse transcription and real time semi-quantitative PCR, was used to study VEGF-A mRNA expression. Cell supernatants were collected and ELISA tests were performed to analyse VEGF-A protein production. Aldosterone increased VEGF-A mRNA and protein expression in a dose- and time-dependent manner in both cell types. Inhibitors of PI3 kinases, ERK1/2, and to a lesser extent of p38 MAPK, decreased this aldosterone-induced immune cell activation. Western-blot performed with HL-60 cells confirmed that ERK1/2 and p38 MAPK pathways were stimulated by aldosterone. Mineralocorticoid receptors are implicated in this VEGF-A up-regulation because HL-60 cells pre-treated with spironolactone, an aldosterone receptor antagonist, diminished the effects of aldosterone. Aldosterone was also able to increase VEGF-A production of phagocytic cells such as neutrophils. These results suggest that this hormone could play an active role in the neovascularisation process by favouring entry of plasma proteins and fluids into the vascular wall, cell proliferation and tissue rebuilding.► Aldosterone is implicated in inflammation processes. ► Little data exist about aldosterone actions on immune cells and especially in angiogenesis. ► VEGF-A is strongly implicated in angiogenesis and synthesized by different cells notably neutrophils. ► We investigate if aldosterone was able to regulate their VEGF-A production. ► Aldosterone uses mineralocorticoid receptors, activates Pi3K, ERK1/2 and p38 pathways to increase VEGF-A production.
Keywords: Aldosterone; Neutrophil; VEGF-A; Angiogenesis; Signal transduction;

Extreme calorie restriction and energy source starvation in Saccharomyces cerevisiae represent distinct physiological states by Léonie G.M. Boender; Marinka J.H. Almering; Madelon Dijk; Antonius J.A. van Maris; Johannes H. de Winde; Jack T. Pronk; Pascale Daran-Lapujade (2133-2144).
Cultivation methods used to investigate microbial calorie restriction often result in carbon and energy starvation. This study aims to dissect cellular responses to calorie restriction and starvation in Saccharomyces cerevisiae by using retentostat cultivation. In retentostats, cells are continuously supplied with a small, constant carbon and energy supply, sufficient for maintenance of cellular viability and integrity but insufficient for growth. When glucose-limited retentostats cultivated under extreme calorie restriction were subjected to glucose starvation, calorie-restricted and glucose-starved cells were found to share characteristics such as increased heat-shock tolerance and expression of quiescence-related genes. However, they also displayed strikingly different features. While calorie-restricted yeast cultures remained metabolically active and viable for prolonged periods of time, glucose starvation resulted in rapid consumption of reserve carbohydrates, population heterogeneity due to appearance of senescent cells and, ultimately, loss of viability. Moreover, during starvation, calculated rates of ATP synthesis from reserve carbohydrates were 2–3 orders of magnitude lower than steady-state ATP-turnover rates calculated under extreme calorie restriction in retentostats. Stringent reduction of ATP turnover during glucose starvation was accompanied by a strong down-regulation of genes involved in protein synthesis. These results demonstrate that extreme calorie restriction and carbon starvation represent different physiological states in S. cerevisiae.► CR and ST yeast cells were compared using retentostat cultivation. ► CR and ST cells share features: quiescence transcripts, heat-shock tolerance. ► CR and ST cells are different in population heterogeneity and glycogen metabolism. ► Glycogen consumption is fine-tuned in ST cells by a yet unknown mechanism. ► Retentostat is a powerful tool to study CR and ST cultures and chronological aging.
Keywords: Retentostat; Saccharomyces cerevisiae; Calorie restriction; Carbon starvation; Reserve carbohydrates; Population heterogeneity;

The novel PKCθ isoform is highly expressed in T-cells, brain and skeletal muscle and originally thought to have a restricted distribution. It has been extensively studied in T-cells and shown to be important for apoptosis, T-cell activation and proliferation. Recent studies showed its presence in other tissues and importance in insulin signaling, lung surfactant secretion, intestinal barrier permeability, platelet and mast-cell functions. However, little information is available for PKCθ activation by gastrointestinal (GI) hormones/neurotransmitters and growth factors. In the present study we used rat pancreatic acinar cells to explore their ability to activate PKCθ and the possible interactions with important cellular mediators of their actions. Particular attention was paid to cholecystokinin (CCK), a physiological regulator of pancreatic function and important in pathological processes affecting acinar function, like pancreatitis. PKCθ-protein/mRNA was present in the pancreatic acini, and T538-PKCθ phosphorylation/activation was stimulated only by hormones/neurotransmitters activating phospholipase C. PKCθ was activated in time- and dose-related manner by CCK, mediated 30% by high-affinity CCKA-receptor activation. CCK stimulated PKCθ translocation from cytosol to membrane. PKCθ inhibition (by pseudostrate-inhibitor or dominant negative) inhibited CCK- and TPA-stimulation of PKD, Src, RafC, PYK2, p125FAK and IKKα/β, but not basal/stimulated enzyme secretion. Also CCK- and TPA-induced PKCθ activation produced an increment in PKCθ's direct association with AKT, RafA, RafC and Lyn. These results show for the first time the PKCθ presence in pancreatic acinar cells, its activation by some GI hormones/neurotransmitters and involvement in important cell signaling pathways mediating physiological responses (enzyme secretion, proliferation, apoptosis, cytokine expression, and pathological responses like pancreatitis and cancer growth).► Establishes PKCθ is expressed in pancreatic acinar cells. ► PKCθ is activated by stimulants of PLC or PKC, but not by those activating PKA or by pancreatic growth factors. ► CCK PKCθ maximal stimulation requires both high (30%) and low-affinity (70%) CCKAR state activation. ► PKCθ activation by CCK and TPA produces an increment in PKCθ’s direct association with AKT, RafA, RafC and Lyn. ► PKCθ inhibition reduces CCK- and TPA-stimulation of PKD1, Src, RafC, PYK2, p125FAK and IKKα/β, but not enzyme secretion.
Keywords: PKCθ activation; Pancreatic acini; CCK; Signaling; Pancreatic growth factors; PKC;

SK&F 96365 induces apoptosis and autophagy by inhibiting Akt–mTOR signaling in A7r5 cells by Eun-Jung Park; Sang-Yeob Kim; Su-Hwa Kim; Chae Ryun Lee; In-San Kim; Jong Kwan Park; Sung Won Lee; Byung Joo Kim; Jung Nyeo Chun; Insuk So; Ju-Hong Jeon (2157-2164).
SK&F 96365 has been widely used as an inhibitor of transient receptor potential (TRP) calcium channels in various physiological settings. However, growing evidence suggests that SK&F 96365 affects several cellular and molecular processes via uncharacterized off-target mechanisms. In this study, we showed that SK&F 96365 induces apoptosis and autophagy in A7r5 vascular smooth muscle cells. The combined suppression of apoptosis and autophagy provoked necrosis rather than rescued cell death in the cells treated with SK&F 96365. In addition, we found that SK&F 96365 inhibits Akt–mTOR signaling pathways, which is comparable with the efficacy of other known Akt inhibitors. Our findings suggest that SK&F 96365 can be a useful agent for delineating the molecular mechanisms underlying crosstalk among cell death pathways.► SK&F 96365 induces apoptosis and autophagy. ► Combined suppression of SK&F 96365-induced apoptosis and autophagy provokes necrosis. ► SK&F 96365 inhibits Akt–mTOR signaling pathways.
Keywords: SK&F 96365; Apoptosis; Autophagy; Akt–mTOR signaling; TRP channel;

Signal integration, crosstalk mechanisms and networks in the function of inflammatory cytokines by M. Lienhard Schmitz; Axel Weber; Thomas Roxlau; Matthias Gaestel; Michael Kracht (2165-2175).
Infection or cell damage triggers the release of pro-inflammatory cytokines such as interleukin(IL)-1α or β and tumor necrosis factor (TNF)α which are key mediators of the host immune response. Following their identification and the elucidation of central signaling pathways, recent results show a highly complex crosstalk between various cytokines and their signaling effectors. The molecular mechanisms controlling signaling thresholds, signal integration and the function of feed-forward and feedback loops are currently revealed by combining methods from biochemistry, genetics and in silico analysis. Increasing evidence is mounted that defects in information processing circuits or their components can be causative for chronic or overshooting inflammation. As progress in biosciences has always benefitted from the use of well-studied model systems, research on inflammatory cytokines may function as a paradigm to reveal general principles of signal integration, crosstalk mechanisms and signaling networks.► Inflammatory cytokines use complex signaling mechanisms during innate immune reactions. ► Intracellular components and interactions are organized in signaling networks. ► These networks contain multiple feed-forward and feedback loops to boost or terminate reactions. ► Disturbances in information flow may cause chronic inflammatory disease.
Keywords: Cytokine signaling network; IL-1; TNF; Feed-forward loop; Feedback loop; Inflammation;

Signal-dependent targeting of proteins into and out of the nucleus is mediated by members of the importin (IMP) family of transport receptors, which recognise targeting signals within a cargo protein and mediate passage through the nuclear envelope-embedded nuclear pore complexes. Regulation of this process is paramount to processes such as cell division and differentiation, but is also critically important for viral replication and pathogenesis; phosphorylation appears to play a major role in regulating viral protein nucleocytoplasmic trafficking, along with other posttranslational modifications. This review focuses on viral proteins that utilise the host cell IMP machinery in order to traffic into/out of the nucleus, and in particular those where trafficking is critical to viral replication and/or pathogenesis, such as simian virus SV40 large tumour antigen (T-ag), human papilloma virus E1 protein, human cytomegalovirus processivity factor ppUL44, and various gene products from RNA viruses such as Rabies. Understanding of the mechanisms regulating viral protein nucleocytoplasmic trafficking is paramount to the future development of urgently needed specific and effective anti-viral therapeutics. This article was originally intended for the special issue “Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import”. The Publisher apologizes for any inconvenience caused.► Nucleocytoplasmic trafficking of viral proteins is central to viral infection. ► Posttranslational modification is a key means to regulate viral protein trafficking. ► Nuclear trafficking of viral proteins can be a target for development of anti-virals.
Keywords: Simian virus 40 T-ag; Human cytomegalovirus ppUL44; Human papillomavirus E1; Rabies virus P; Phosphorylation; Nuclear import;

Malignant hyperthermia — Human stress triggering by Gerald A. Gronert; Joseph R. Tobin; Sheila Muldoon (2191-2192).
Letter to the Editor concerns the question of a discussion of awake porcine malignant hyperthermia that erroneously omits the awake human stress reaction of malignant hyperthermia.
Keywords: malignant hyperthermia; muscle rigidity; hypercarbia; genetic analysis;

Corrigendum to “Primary effect of 1α,25(OH)2D3 on IL-10 expression in monocytes is short-term down-regulation” [Biochim. Biophys. Acta 1803 (2010) 1276–86] by Juha M. Matilainen; Tiia Husso; Sari Toropainen; Sabine Seuter; Mikko P. Turunen; Petra Gynther; Seppo Ylä-Herttuala; Carsten Carlberg; Sami Väisänen (2195).

Corrigendum to “iPLA2, a novel determinant in Ca2+- and phosphorylation-dependent S100A8/A9 regulated NOX2 activity” [Biochim. Biophys. Acta 1803 (2010) 840–847] by Véronique Schenten; Sabrina Bréchard; Sébastien Plançon; Chantal Melchior; Jean-Pol Frippiat; Eric J. Tschirhart (2196).