BBA - Molecular Cell Research (v.1803, #4)

Implication of RICTOR in the mTOR inhibitor-mediated induction of insulin-like growth factor-I receptor (IGF-IR) and human epidermal growth factor receptor-2 (Her2) expression in gastrointestinal cancer cells by Sven A. Lang; Christina Hackl; Christian Moser; Stefan Fichtner-Feigl; Gudrun E. Koehl; Hans J. Schlitt; Edward K. Geissler; Oliver Stoeltzing (435-442).
Inhibition of mTORC1 with the mTOR inhibitor rapamycin may lead to an induction of Akt phosphorylation in cancer cells via mTORC2 activation. Using gastric and pancreatic cancer cells, we further investigated this paradoxical signaling response and found that rapamycin additionally up-regulates both IGF-IR and Her2 expression. Using RNAi for down-regulating RICTOR, this induction of receptor kinase expression was identified to be mediated via an mTORC2-induced Akt activation. Moreover, mTORC2 inhibition reduced the phosphorylation of GSK-3 and NF-κB, and significantly impaired cancer cell motility. In conclusion, inhibition of mTORC2 may abrogate unfavorable signaling effects of mTOR inhibitors, hence providing a novel rationale for therapy.
Keywords: IGF-IR; mTOR; RICTOR; Gastrointestinal cancer; Her2;

The oncofetal H19 RNA connection: Hypoxia, p53 and cancer by Imad J. Matouk; Shaul Mezan; Aya Mizrahi; Patricia Ohana; Rasha Abu-lail; Yakov Fellig; Nathan deGroot; Eithan Galun; Abraham Hochberg (443-451).
Expression of the imprinted H19 gene is remarkably elevated in a large number of human cancers. Recently, we reported that H19 RNA is up-regulated in hypoxic stress and furthermore, it possesses oncogenic properties. However, the underlying mechanism(s) of these phenomena remain(s) unknown. Here we demonstrate a tight correlation between H19 RNA elevation by hypoxia and the status of the p53 tumor suppressor. Wild type p53 (p53wt) prevents the induction of H19 upon hypoxia, and upon its reconstitution in p53null cells. The last case is accompanied by a decrease in cell viability. The p53 effect is nuclear and seems independent of its tetramerization. Furthermore, using knockdown and over-expression approaches we identified HIF1-α as a critical factor that is responsible for H19 induction upon hypoxia. Knocking down HIF1-α abolishes H19 RNA induction, while its over-expression significantly enhances the H19 elevation in p53null hypoxic cells. In p53wt hypoxic cells simultaneous suppression of p53 and over-expression of HIF1-α are needed to induce H19 significantly, while each treatment separately resulting in a mild induction, indicating that the molecular mechanism of p53 suppression effect on H19 may at least in part involve interfering with HIF1-α activity. In vivo a significant increase in H19 expression occurred in tumors derived from p53null cells but not in p53wt cells. Taken together, our results indicate that a functional link exists between p53, HIF1-α and H19 that determines H19 elevation in hypoxic cancer cells. We suggest that this linkage plays a role in tumor development.
Keywords: Hypoxia; p53 tumor suppressor; H19 non-coding RNA; Hypoxia-inducible factor alpha; Cell viability;

Statin-triggered cell death in primary human lung mesenchymal cells involves p53-PUMA and release of Smac and Omi but not cytochrome c by Saeid Ghavami; Mark M. Mutawe; Kristin Hauff; Gerald L. Stelmack; Dedmer Schaafsma; Pawan Sharma; Karol D. McNeill; Tyler S. Hynes; Sam K. Kung; Helmut Unruh; Thomas Klonisch; Grant M. Hatch; Marek Los; Andrew J. Halayko (452-467).
Statins inhibit 3-hydroxy-3-methyl-glutarylcoenzyme CoA (HMG-CoA) reductase, the proximal enzyme for cholesterol biosynthesis. They exhibit pleiotropic effects and are linked to health benefits for diseases including cancer and lung disease. Understanding their mechanism of action could point to new therapies, thus we investigated the response of primary cultured human airway mesenchymal cells, which play an effector role in asthma and chronic obstructive lung disease (COPD), to simvastatin exposure. Simvastatin induced apoptosis involving caspase-9, -3 and -7, but not caspase-8 in airway smooth muscle cells and fibroblasts. HMG-CoA inhibition did not alter cellular cholesterol content but did abrogate de novo cholesterol synthesis. Pro-apoptotic effects were prevented by exogenous mevalonate, geranylgeranyl pyrophosphate and farnesyl pyrophosphate, downstream products of HMG-CoA. Simvastatin increased expression of Bax, oligomerization of Bax and Bak, and expression of BH3-only p53-dependent genes, PUMA and NOXA. Inhibition of p53 and silencing of p53 unregulated modulator of apoptosis (PUMA) expression partly counteracted simvastatin-induced cell death, suggesting a role for p53-independent mechanisms. Simvastatin did not induce mitochondrial release of cytochrome c, but did promote release of inhibitor of apoptosis (IAP) proteins, Smac and Omi. Simvastatin also inhibited mitochondrial fission with the loss of mitochondrial Drp1, an essential component of mitochondrial fission machinery. Thus, simvastatin activates novel apoptosis pathways in lung mesenchymal cells involving p53, IAP inhibitor release, and disruption of mitochondrial fission.
Keywords: Apoptosis; Statin; Caspase; Airway smooth muscle; Fibroblast; Mitochondria;

Cytosolic phospholipase A2-α enhances induction of endoplasmic reticulum stress by Guohui Ren; Tomoko Takano; Joan Papillon; Andrey V. Cybulsky (468-481).
Induction of endoplasmic reticulum (ER) stress by the complement membrane attack complex is enhanced by activation of cytosolic phospholipase A2-α (cPLA2). To address mechanisms by which cPLA2 may modulate ER stress, we produced a mutant cPLA2, containing an ER targeting domain (cPLA2-ERmut). After transfection and fractionation of COS-1 cells, cPLA2-ERmut was present mainly in the membrane fraction, whereas wild type (wt) cPLA2 was principally in the cytosol. By fluorescence microscopy, cPLA2-ERmut was enriched in a perinuclear distribution under basal conditions, colocalizing with the ER protein, calnexin, while cPLA2-wt was mainly cytosolic. Both forms of cPLA2 transiently expressed in COS cells showed basal phosphorylation at serine505, which correlates with catalytic activity. Expression of cPLA2-wt was ∼ 5-fold greater, compared with cPLA2-ERmut, but both enzymes produced comparable increases in free arachidonic acid, implying that cPLA2-ERmut effectively hydrolyzed ER membrane phospholipids. Although transfection of cPLA2-ERmut or wt did not induce ER stress independently, cPLA2-ERmut and wt enhanced the induction of ER stress by tunicamycin, dithiothreitol and ionomycin (monitored by induction of grp94 and C/EBP homologous protein-10), and the effect was dependent on the catalytic activity. cPLA2-ERmut enhanced production of superoxide. Induction of ER stress in tunicamycin-treated cells expressing cPLA2-ERmut was attenuated in the presence of the antioxidant, N-acetyl cysteine, and reduced glutathione, and was exacerbated by dl-buthionine-(S,R)-sulfoximine (which depletes glutathione). Expression of cPLA2-ERmut exacerbated tunicamycin-induced apoptosis. Thus, induction of ER stress is facilitated by the activation of cPLA2 at the ER. The mechanism involves ER membrane phospholipid hydrolysis, and accumulation of reactive oxygen species.
Keywords: Apoptosis; Arachidonic acid; Reactive oxygen species; Unfolded protein response;

Redox signaling via lipid raft clustering in homocysteine-induced injury of podocytes by Chun Zhang; Jun-Jun Hu; Min Xia; Krishna M. Boini; Christopher Brimson; Pin-Lan Li (482-491).
Our recent studies have indicated that hyperhomocysteinemia (hHcys) may induce podocyte damage, resulting in glomerulosclerosis. However, the molecular mechanisms mediating hHcys-induced podocyte injury are still poorly understood. In the present study, we first demonstrated that an intact NADPH oxidase system is present in podocytes as shown by detection of its membrane subunit (gp91 phox ) and cytosolic subunit (p47 phox ). Then, confocal microscopy showed that gp91 phox and p47 phox could be aggregated in lipid raft (LR) clusters in podocytes treated with homocysteine (Hcys), which were illustrated by their colocalization with cholera toxin B, a common LR marker. Different mechanistic LR disruptors, either methyl-β-cyclodextrin (MCD) or filipin abolished such Hcys-induced formation of LR-gp91 phox or LR-p47 phox transmembrane signaling complexes. By flotation of detergent-resistant membrane fractions we found that gp91 phox and p47 phox were enriched in LR fractions upon Hcys stimulation, and such enrichment of NADPH oxidase subunits and increase in its enzyme activity were blocked by MCD or filipin. Functionally, disruption of LR clustering significantly attenuated Hcys-induced podocyte injury, as shown by their inhibitory effects on Hcys-decreased expression of slit diaphragm molecules such as nephrin and podocin. Similarly, Hcys-increased expression of desmin was also reduced by disruption of LR clustering. In addition, inhibition of such LR-associated redox signaling prevented cytoskeleton disarrangement and apoptosis induced by Hcys. It is concluded that NADPH oxidase subunits aggregation and consequent activation of this enzyme through LR clustering is an important molecular mechanism triggering oxidative injury of podocytes induced by Hcys.
Keywords: Membrane microdomains; Oxidative injury; Homocysteine; Podocytes; Glomerulosclerosis;

Conserved amino acid residues in C-terminus of PERIOD 2 are involved in interaction with CRYPTOCHROME 1 by Tatsunosuke Tomita; Koyomi Miyazaki; Yoshiaki Onishi; Shinya Honda; Norio Ishida; Katsutaka Oishi (492-498).
We investigated the amino acid sequences of rat PERIOD2 (rPER2) that are required for interaction with CRYPTOCHROME1 (CRY1) to understand the molecular mechanism of the circadian clock. Co-immunoprecipitation assays using various C-terminal fragments of rPER2 with internal deletions revealed that amino acid residues 1179–1198 are necessary for interaction with CRY1. To identify precisely which amino acid residues are responsible for the interaction, we substituted alanine for residues conserved among PER isoforms and species. We found that more than three mutations of conserved PER2 residues impaired not only binding to CRY1 but also subsequent nuclear translocation, although mutations of non-conserved residues did not affect interaction with CRY1. Thus, the conserved amino acid residues of 1179–1198 in PER2 are apparently responsible for binding to CRY1.
Keywords: Circadian rhythm; Clock genes; PERIOD; CRYPTOCHROME; Interaction;

Ageing and eating by Patrick Rockenfeller; Frank Madeo (499-506).
Epidemiological studies propose that extension of the human lifespan or the reduction of age associated diseases may be achieved by physical exercise, caloric restriction, and by consumption of certain substances such as resveratrol, selenium, flavonoids, zinc, omega 3 unsaturated fatty acids, vitamins E and C, Ginkgo biloba extracts, aspirin, green tea catechins, antioxidants in general, and even by light caffeine or alcohol consumption. Though intriguing, these studies only show correlative (not causative) effects between the application of the particular substance and longevity. On the other hand, obesity is yet a strong menace to the western society and it will emerge even more so throughout the next decades according to the prediction of the WHO. Although obesity is considered a severe problem, very little is known about the molecular mechanisms causing the associated degeneration of organs and finally death. Nutrient related adverse consequences for health and thus ageing may be due to a high sugar or high fat diet, excessive alcohol consumption and cigarette smoke amongst others. In this article we examine the interdependencies of eating and ageing and suggest yeast, one of the most successful ageing models, as an easy tool to elucidate the molecular pathways from eating to ageing. The conservation of most ageing pathways in yeast and their easy genetic tractability may provide a chance to discriminate between the correlative and causative effects of nutrition on ageing.
Keywords: Ageing; Lifespan; Longevity; Lipotoxicity; Calorie restriction; Nutrition;

Viruses as vesicular carriers of the viral genome: A functional module perspective by Bastian Thaa; Klaus Peter Hofmann; Michael Veit (507-519).
Enveloped viruses and cellular transport vesicles share obvious morphological and functional properties. Both are composed of a closed membrane, which is lined with coat proteins and encases cargo. Transmembrane proteins inserted into the membrane define the target membrane area with which the vesicle or virus is destined to fuse. Here we discuss recent insight into the functioning of enveloped viruses in the framework of the “functional module” concept. Vesicular transport is an exemplary case of a functional module, as defined as a part of the proteome that assembles to perform a specific autonomous function in a living cell. Cellular vesicles serve to transport cargo between membranous organelles inside the cell, while enveloped viruses can be seen as carriers of the viral genome delivering their cargo from an infected to an uninfected cell. The turnover of both vesicles and viruses involves an analogous series of submodular events. This comprises assembly of elements, budding from the donor compartment, uncoating and/or maturation, docking to and finally fusion with the target membrane to release the cargo. This modular perception enables us to define submodular building blocks so that mechanisms and elements can be directly compared. It will be analyzed where viruses have developed their own specific strategy, where they share functional schemes with vesicles, and also where they even have “hijacked” complete submodular schemes from the cell. Such a perspective may also include new and more specific approaches to pharmacological interference with virus function, which could avoid some of the most severe side effects.
Keywords: Functional module; Vesicular transport; Virus assembly; Budding; Membrane fusion; Influenza virus;

Moonlighting proteins: An intriguing mode of multitasking by Daphne H.E.W. Huberts; Ida J. van der Klei (520-525).
Proteins are macromolecules, which perform a large variety of functions. Most of them have only a single function, but an increasing number of proteins are being identified as multifunctional. Moonlighting proteins form a special class of multifunctional proteins. They perform multiple autonomous and often unrelated functions without partitioning these functions into different domains of the protein. Striking examples are enzymes, which in addition to their catalytic function are involved in fully unrelated processes such as autophagy, protein transport or DNA maintenance. In this contribution we present an overview of our current knowledge of moonlighting proteins and discuss the significant implications for biomedical and fundamental research.
Keywords: Multifunctional protein; Enzyme; Moonlighting; Gene sharing;