FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2040307459> ?p ?o ?g. }

• W2040307459 endingPage "10263" @default.
• W2040307459 startingPage "10252" @default.
• W2040307459 abstract "Organisms respond to available nutrient levels by rapidly adjusting metabolic flux, in part through changes in gene expression. A consequence of adaptations in metabolic rate is the production of mitochondria-derived reactive oxygen species. Therefore, we hypothesized that nutrient sensing could regulate the synthesis of the primary defense of the cell against superoxide radicals, manganese superoxide dismutase. Our data establish a novel nutrient-sensing pathway for manganese superoxide dismutase expression mediated through essential amino acid depletion concurrent with an increase in cellular viability. Most relevantly, our results are divergent from current mechanisms governing amino acid-dependent gene regulation. This pathway requires the presence of glutamine, signaling via the tricarboxylic acid cycle/electron transport chain, an intact mitochondrial membrane potential, and the activity of both the MEK/ERK and mammalian target of rapamycin kinases. Our results provide evidence for convergence of metabolic cues with nutrient control of antioxidant gene regulation, revealing a potential signaling strategy that impacts free radical-mediated mutations with implications in cancer and aging. Organisms respond to available nutrient levels by rapidly adjusting metabolic flux, in part through changes in gene expression. A consequence of adaptations in metabolic rate is the production of mitochondria-derived reactive oxygen species. Therefore, we hypothesized that nutrient sensing could regulate the synthesis of the primary defense of the cell against superoxide radicals, manganese superoxide dismutase. Our data establish a novel nutrient-sensing pathway for manganese superoxide dismutase expression mediated through essential amino acid depletion concurrent with an increase in cellular viability. Most relevantly, our results are divergent from current mechanisms governing amino acid-dependent gene regulation. This pathway requires the presence of glutamine, signaling via the tricarboxylic acid cycle/electron transport chain, an intact mitochondrial membrane potential, and the activity of both the MEK/ERK and mammalian target of rapamycin kinases. Our results provide evidence for convergence of metabolic cues with nutrient control of antioxidant gene regulation, revealing a potential signaling strategy that impacts free radical-mediated mutations with implications in cancer and aging. Nutrient availability relative to both carbohydrates and amino acids (AAs), 2The abbreviations used are: AA, amino acid; MnSOD, manganese superoxide dismutase; mTOR, mammalian target of rapamycin; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; ROS, reactive oxygen species; RT, reverse transcription; MEM, minimal essential medium; EBSS, Earle's balanced salt solution; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FBS, fetal bovine serum; siRNA, short interfering RNA; hGH, human growth hormone; IL, interleukin; PBS, phosphate-buffered saline; ASNS, asparagine synthetase; 2-DOG, 2-deoxy-d-glucose; 3-NPA, 3-nitropropionic acid; KMV, α-ke-to-β-methyl-n-valeric acid. 2The abbreviations used are: AA, amino acid; MnSOD, manganese superoxide dismutase; mTOR, mammalian target of rapamycin; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; ROS, reactive oxygen species; RT, reverse transcription; MEM, minimal essential medium; EBSS, Earle's balanced salt solution; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FBS, fetal bovine serum; siRNA, short interfering RNA; hGH, human growth hormone; IL, interleukin; PBS, phosphate-buffered saline; ASNS, asparagine synthetase; 2-DOG, 2-deoxy-d-glucose; 3-NPA, 3-nitropropionic acid; KMV, α-ke-to-β-methyl-n-valeric acid. in the mammalian diet, has potentially critical impacts on metabolic flux and ultimately the generation of ATP and its equivalents. With constantly changing constituents associated with the mammalian diet, organisms have adapted metabolic strategies to efficiently accommodate changes in the availability of critical nutrients. Extensive studies have addressed the importance of glucose excess (1Girard J. Ferre P. Foufelle F. Annu. Rev. Nutr. 1997; 17: 325-352Crossref PubMed Scopus (302) Google Scholar) and deprivation (2Lee A.S. Curr. Opin. Cell Biol. 1992; 4: 267-273Crossref PubMed Scopus (392) Google Scholar) as well as AA availability on metabolic and nuclear events (3Kilberg M.S. Pan Y.X. Chen H. Leung-Pineda V. Annu. Rev. Nutr. 2005; 25: 59-85Crossref PubMed Scopus (220) Google Scholar). For example, the levels of branch chain AA regulate intracellular signaling through the central regulator, mammalian target of rapamycin (mTOR), culminating in downstream impacts on overall protein synthesis (4Kim D.H. Sabatini D.M. Curr. Top. Microbiol. Immunol. 2004; 279: 259-270PubMed Google Scholar, 5Lindsley J.E. Rutter J. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2004; 139: 543-559Crossref PubMed Scopus (125) Google Scholar, 6Martin D.E. Hall M.N. Curr. Opin. Cell Biol. 2005; 17: 158-166Crossref PubMed Scopus (444) Google Scholar). Analogously, maintenance of tryptophan levels in fibroblast medium is critical to the regulation of the matrix-degrading enzymes, collagenase and stromelysin, by IL-1β (7Varga J. Li L. Mauviel A. Jeffrey J. Jimenez S.A. Lab. Investig. 1994; 70: 183-191PubMed Google Scholar, 8Varga J. Yufit T. Brown R.R. J. Clin. Investig. 1995; 96: 475-481Crossref PubMed Scopus (36) Google Scholar). On the other hand, tryptophan degradation mediates the inhibitory effects of interferon-γ-dependent increases in cellular indoleamine 2,3-dioxygenase mRNA (8Varga J. Yufit T. Brown R.R. J. Clin. Investig. 1995; 96: 475-481Crossref PubMed Scopus (36) Google Scholar, 9Varga J. Yufit T. Hitraya E. Brown R.R. Adv. Exp. Med. Biol. 1996; 398: 143-148Crossref PubMed Scopus (18) Google Scholar). Most relevant to the present study, deprivation of essential AA has been demonstrated to evoke responses at both the transcriptional and post-transcriptional levels for genes such as asparagine synthetase (ASNS), CCAAT/enhancer-binding protein homologous protein, cationic AA transporter (Cat-1Girard J. Ferre P. Foufelle F. Annu. Rev. Nutr. 1997; 17: 325-352Crossref PubMed Scopus (302) Google Scholar), sodium-coupled neutral AA transporter system A (SNAT2), and insulin-like growth factor-binding protein-1 (IGFBP-1) (3Kilberg M.S. Pan Y.X. Chen H. Leung-Pineda V. Annu. Rev. Nutr. 2005; 25: 59-85Crossref PubMed Scopus (220) Google Scholar). Fernandez et al. (10Fernandez J. Bode B. Koromilas A. Diehl J.A. Krukovets I. Snider M.D. Hatzoglou M. J. Biol. Chem. 2002; 277: 11780-11787Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) have also demonstrated the existence of an internal ribosome entry site within the 5′-untranslated region of the cat-1 gene that controls translation of this transport protein under conditions of AA depletion. Tissue and cellular adaptation to nutrient availability also affects carbon and nitrogen utilization through glycolysis, the tricarboxylic acid cycle, and ultimately the aerobic generation of ATP via electron transport. A critical consequence of nutrient availability and subsequent metabolism is the generation of reactive oxygen species (ROS) as by-products of normal metabolism (11Frank L. Massaro D. Am. J. Med. 1980; 69: 117-126Abstract Full Text PDF PubMed Scopus (247) Google Scholar). Previous estimates have indicated that under normal aerobic and nutrient conditions, 0.1% of consumed oxygen is released as superoxide radicals from mitochondrial electron transport (12Muller F.L. Liu Y. Abdul-Ghani M.A. Lustgarten M.S. Bhattacharya A. Jang Y.C. Van Remmen H. Biochem. J. 2008; 409: 491-499Crossref PubMed Scopus (127) Google Scholar). It has also been established that a significant increase in the levels of ROS occurs under pathological conditions involving the synthesis and action of many pro-inflammatory mediators (13Wong G.H. Elwell J.H. Oberley L.W. Goeddel D.V. Cell. 1989; 58: 923-931Abstract Full Text PDF PubMed Scopus (764) Google Scholar, 14Rogers R.J. Monnier J.M. Nick H.S. J. Biol. Chem. 2001; 276: 20419-20427Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Furthermore, the connection between nutrient levels and the generation of ROS is underscored when considering that caloric restriction can significantly delay the aging process (15Roth G.S. J. Am. Geriatr. Soc. 2005; 53: S280-S283Crossref PubMed Scopus (15) Google Scholar), which may be explained by a reduction in metabolic flux and a concomitant decline in ROS production (16Balaban R.S. Nemoto S. Finkel T. Cell. 2005; 120: 483-495Abstract Full Text Full Text PDF PubMed Scopus (3285) Google Scholar). This observation is also consistent with the mitochondrial theory of aging (17Harman D. J. Gerontol. 1956; 11: 298-300Crossref PubMed Scopus (6497) Google Scholar), which implicates continuous generation of ROS as a critical factor for damage to mitochondrial DNA as well as oxidative reactions with components of the cytosol and nucleus. Mutations in genes from the insulin-like signaling network in Caenorhabditis elegans, such as age-1, a homologue of the mammalian phosphatidylinositol-3-OH kinase, and daf-2, a homologue of the insulin or insulin-like growth factor receptor family, cause a life span extension phenotype, with animals living twice as long as the wild type (18Honda Y. Honda S. Ann. N. Y. Acad. Sci. 2002; 959: 466-474Crossref PubMed Scopus (95) Google Scholar). These mutations may propagate their effects by conferring resistance to oxidative stress, possibly through increases in the antioxidant enzyme, manganese superoxide dismutase (MnSOD) (18Honda Y. Honda S. Ann. N. Y. Acad. Sci. 2002; 959: 466-474Crossref PubMed Scopus (95) Google Scholar). Studies in yeast (19Harris N. Costa V. MacLean M. Mollapour M. Moradas-Ferreira P. Piper P.W. Free Radic. Biol. Med. 2003; 34: 1599-1606Crossref PubMed Scopus (80) Google Scholar) and Drosophila (20Tower J. Mech. Ageing Dev. 2000; 118: 1-14Crossref PubMed Scopus (80) Google Scholar, 21Sun J. Folk D. Bradley T.J. Tower J. Genetics. 2002; 161: 661-672Crossref PubMed Google Scholar) have also implicated overexpression of MnSOD with an increase in organism life span. Furthermore, Kokoszka et al. (22Kokoszka J.E. Coskun P. Esposito L.A. Wallace D.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2278-2283Crossref PubMed Scopus (379) Google Scholar) have demonstrated that MnSOD heterozygous mice displayed increased mitochondrial dysfunction marked by increased proton leakage, inhibition of respiration, and the accumulation of mitochondrial oxidative damage. These studies also provided a link between chronic oxidative stress in the heterozygous mice and a premature induction of apoptosis, thus implicating the importance of MnSOD in cell death and aging. These observations are also consistent with data demonstrating the potent anti-apoptotic activity associated with overexpression of this mitochondrial localized antioxidant enzyme in a variety of mitochondria-dependent cell death pathways (23Cox G. Oberley L.W. Hunninghake G.W. Am. J. Respir. Cell Mol. Biol. 1994; 10: 493-498Crossref PubMed Scopus (22) Google Scholar, 24Keller J.N. Kindy M.S. Holtsberg F.W. St Clair D.K. Yen H.C. Germeyer A. Steiner S.M. Bruce-Keller A.J. Hutchins J.B. Mattson M.P. J. Neurosci. 1998; 18: 687-697Crossref PubMed Google Scholar, 25Bruce-Keller A.J. Geddes J.W. Knapp P.E. McFall R.W. Keller J.N. Holtsberg F.W. Parthasarathy S. Steiner S.M. Mattson M.P. J. Neuroimmunol. 1999; 93: 53-71Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 26Zwacka R.M. Dudus L. Epperly M.W. Greenberger J.S. Engelhardt J.F. Hum. Gene Ther. 1998; 9: 1381-1386Crossref PubMed Scopus (110) Google Scholar). Additionally, the significant induction of MnSOD gene expression by pro-inflammatory cytokines (13Wong G.H. Elwell J.H. Oberley L.W. Goeddel D.V. Cell. 1989; 58: 923-931Abstract Full Text PDF PubMed Scopus (764) Google Scholar, 27Wong G.H. Goeddel D.V. Science. 1988; 242: 941-944Crossref PubMed Scopus (836) Google Scholar, 28Visner G.A. Dougall W.C. Wilson J.M. Burr I.A. Nick H.S. J. Biol. Chem. 1990; 265: 2856-2864Abstract Full Text PDF PubMed Google Scholar) is also in line with a cell survival strategy that accompanies increased levels of ROS associated with the inflammatory response. Therefore, we postulated that nutrient availability, in the form of either glucose or AA deprivation, may have relevant metabolic and cell survival benefits through elevation of MnSOD levels. Our results establish a nutrient-dependent regulation pathway for MnSOD that is novel when compared with the cellular mechanisms that are currently proposed to orchestrate the induction of gene expression via essential AA deprivation (3Kilberg M.S. Pan Y.X. Chen H. Leung-Pineda V. Annu. Rev. Nutr. 2005; 25: 59-85Crossref PubMed Scopus (220) Google Scholar). We have demonstrated that, in contrast to all previously studied AA-regulated genes, the induction of MnSOD by histidine depletion, a representative essential AA, requires the presence of millimolar levels of glutamine (Gln). We have shown that this regulatory pathway requires intracellular signals through the tricarboxylic acid cycle and electron transport chain with a dependence on mitochondrial membrane potential but not ATP levels. Our results also demonstrate that this unique AA-dependent regulatory pathway necessitates normal signaling through both the MEK/ERK and mTOR pathways. Reagents and Antibodies—2-Methoxyestradiol, KMV, 3-nitropropionic acid, malonate, actinomycin-b, cycloheximide, antimycin, oligomycin, and 2,4-dinitrophenol were obtained from Sigma. SB202190, SB203580, PD98059, JNK (c-Jun N-terminal kinase) inhibitor II, and U0126 were purchased from Calbiochem. Fluoroacetate and rapamycin were obtained from Fluka, St. Louis, and LC Laboratories, Woburn, MA, respectively. Phospho-p70 S6 kinase (Thr-389), phospho-4E-BP1 (Thr-37/46), and phospho-p70 S6 kinase (Thr-421/Ser-424) antibodies were purchased from Cell Signaling, Danvers, MA, and MnSOD was from StressGen, San Diego. Cell Culture and Treatment Conditions—HepG2 (human hepatoma) cells were maintained in minimal essential medium (MEM) (Sigma), pH 7.4, supplemented with 25 mm NaHCO3, 4 mm Gln, antibiotic/antimycotic (Invitrogen), and 10% fetal bovine serum (FBS) (Invitrogen) at 37 °C with 5% CO2. All data involve the use of this cell line. Cells were grown to 50–65% confluency on 10-cm dishes and split 1:8 into 60-mm dishes for 24 h. In all experiments, medium was changed 12 h before the start of the experiment to ensure the AA levels in general are not depleted. The cells were washed three times with phosphate-buffered saline (PBS), incubated in 3 ml of the appropriate medium, and collected at the indicated times for either Northern or immunoblot analysis. All experiments were performed with 10% dialyzed FBS. For experiments where AA other than histidine are depleted, Earle's Balanced Salt Solution (EBSS) (Sigma) was used and supplemented with antibiotic/antimycotic, a vitamin mixture (Invitrogen), and 10% dialyzed FBS. Each AA was then added individually to the medium at the same levels as in the MEM while omitting the AA being tested. For the “AA add back” experiments, the same EBSS base and supplements were used, and then only the indicated AA was added at a concentration of 5 mm. The pH of the medium was maintained at 7.2–7.4 and adjusted (when necessary) with 0.1 m NaOH or HCl. Stock solutions of inhibitors were adjusted to a neutral pH before treatments. RNA Isolation and Northern Analysis—Total RNA was isolated from cells as described by the Chomczynski and Sacchi method with modifications (28Visner G.A. Dougall W.C. Wilson J.M. Burr I.A. Nick H.S. J. Biol. Chem. 1990; 265: 2856-2864Abstract Full Text PDF PubMed Google Scholar). Five to 10 μg of total RNA were fractionated on 1% agarose, 6% formaldehyde gels, electrotransferred to a Zeta-Probe membrane (Bio-Rad), and UV cross-linked. Membranes were then incubated for 1 h in a pre-hybridization buffer and incubated with a 32P-radiolabeled gene-specific probe generated by random primer extension overnight at 62 °C. Membranes were then washed with a high stringency buffer at 66 °C and exposed to x-ray film. Generation of cDNA—To generate cDNA for real time PCR analysis SuperScript™ first strand synthesis kit from Invitrogen was used. 1 μg of total RNA isolated as described was used as the template. Prior to further analysis, double distilled H2O was added to a final reaction volume of 100 μl. Real Time RT-PCR—2 μl of cDNA generated from first strand synthesis (as described above) was used as the template for real time PCR. To this, 0.3 μm of each primer was added, 12.5 μl of iTaq™ SYBR® Green Supermix with ROX (Bio-Rad), and water to a final volume of 25 μl. The Applied Biosystems 7000 sequence detection system was used with the following parameters: cycle 1 (95 °C for 10 min) 1 time; cycle 2 (95 °C for 15 s, 60 °C for 1 min) for 40 cycles. The ΔΔCT method was used to determine the relative fold changes, normalized to the cyclophilin A gene, and is described in Ref. 29Livak K.J. Schmittgen T.D. Methods (San Diego). 2001; 25: 402-408Crossref PubMed Scopus (123392) Google Scholar. Primers for cyclophilin A are as follows: forward, 5′-CAT CCT AAA GCA TAC GGG TCC-3′, and reverse, 5′-GCT GGT CTT GCC ATT CCT G-3′. De Novo Transcriptional Analysis of MnSOD—RNA was isolated as described previously with the addition of DNase I treatment. Heterogeneous nuclear RNA levels were then quantified by real time RT-PCR. Growth Hormone Reporter Constructs—Regions of the MnSOD gene were cloned into a human growth hormone (hGH) reporter plasmid (30Selden R.F. Howie K.B. Rowe M.E. Goodman H.M. Moore D.D. Mol. Cell. Biol. 1986; 6: 3173-3179Crossref PubMed Scopus (471) Google Scholar). A phage artificial chromosome clone, obtained from the Sanger Institute (RP1-56L9), containing the entire human MnSOD gene was used to clone regions of interest into a promoterless growth hormone reporter plasmid (pØGH). A 3.6-kb BamHI fragment of the human promoter was digested from the phage artificial chromosome vector and separated on a 0.7% agarose gel, gel-purified, and cloned into the BamHI site of pØGH. Unique restriction enzyme sites were used to generate 1.4-, 1.3-, 1.1-, and 0.83-kb promoter fragments. To generate the promoter constructs in conjunction with the human MnSOD enhancer, a 488-bp fragment was digested from a previously generated construct (31Rogers R.J. Chesrown S.E. Kuo S. Monnier J.M. Nick H.S. Biochem. J. 2000; 347: 233-242Crossref PubMed Google Scholar). The 488-bp human enhancer fragment was subcloned into the HindIII site of the hGH constructs containing the indicated human MnSOD promoter. A plasmid containing a minimal viral thymidine kinase promoter coupled to the pØGH plasmid was used with the human MnSOD enhancer fragment as described previously (31Rogers R.J. Chesrown S.E. Kuo S. Monnier J.M. Nick H.S. Biochem. J. 2000; 347: 233-242Crossref PubMed Google Scholar). Transient Transfection of Reporter Constructs—HepG2 cells were cultured as described previously and transfected at ∼50% confluency in a 10-cm dish. The reporter plasmid containing different regions of the MnSOD gene were transiently transfected using a FuGENE 6 transfection reagent (Roche Applied Science). For each 10-cm dish, 15 μl of the FuGENE 6 transfection reagent was diluted to a final volume of 600 μl in serum-free MEM. 5 μg of reporter plasmid was then added, and the reaction was incubated at room temperature for 30 min and then transferred to HepG2 cells. After 24 h, the cells were split 1:10 into 35-mm dishes and incubated for another 12 h after which the cells were then incubated in either FED or –His media. pØGH was also transfected to ensure that the transfection itself or the hGH plasmid did not have an effect on the MnSOD message. Northern analyses were used to evaluate the effect of histidine deprivation on the hGH message. A fragment from the hGH cDNA was used to generate a radiolabeled probe for Northern analysis. Protein Isolation and Immunoblot Analysis—HepG2 cells were washed twice with ice-cold PBS and lysed with a buffer containing both protease (Roche Applied Science) and phosphatase inhibitors (Calbiochem), in 1 m Tris, pH 7.5, 5 m NaCl, 0.5 m EDTA pH 8, and Triton X-100 (TBST). Protein concentrations were determined by bicinchoninic acid (BCA) assay (Pierce). 10–20 μg of total cellular protein was separated on a Tris-HCl, SDS-polyacrylamide gel and transferred to a Hybond ECL nitrocellulose membrane (Amersham Biosciences). The membranes were then blocked for 1 h with 5% nonfat dry milk in TBST at room temperature. The membranes were incubated at 4 °C with primary antibody overnight, washed three times with TBST, incubated with a secondary for 2 h, washed again three times, and subjected to ECL chemiluminescence (Amersham Biosciences). Cell Viability—Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Invitrogen). 10 μl of 12 mm MTT dissolved in PBS was added to the culture medium and allowed to incorporate into the cells for 4 h at 37°C. The media were then removed and the cells rinsed with PBS. Cells were solubilized in Me2SO diluted in PBS and incubated for 10 min at 37 °C. The cells were then mixed and the plates read at 540 nm in a Spectramax plus 384 plate reader. siRNA—HepG2 cells were transfected with a final concentration of 100 nm SMARTpool® MnSOD siRNA (Dharmacon) using DharmaFECT™ 4 siRNA transfection reagent (Dharmacon). A cyclophilin-specific siRNA (Dharmacon) was employed as a control for off-target effects of siRNA. ATP Measurements—ATP levels were measured from HepG2 cells utilizing the ATP bioluminescent kit from Sigma. After 12 h of treatment cells were trypsinized; 3 ml of medium was added, the cells were mixed thoroughly, and 1 ml was transferred to a 1.5-ml microcentrifuge tube. A 100-μl aliquot of ATP assay solution (diluted 1:25) was added to a fresh tube and incubated at room temperature for 3 min, allowing for endogenous ATP to be hydrolyzed and decreasing the background. In a separate 1.5-ml microcentrifuge tube, the following was mixed: 100 μl of releasing agent (FL-SAR), 50 μl of water, and 50 μl of the cell sample. A 100-μl aliquot of this mix was added to the 100-μl ATP assay solution, and relative light units were measured using a Berthold SIRUS luminometer version 3.0. Densitometry and Statistical Analysis—All densitometry was quantified from autoradiography films using a Microtek scan maker 9600XL and analyzed with the UN-SCAN-IT program, Silk Scientific Corp., version 5.1. Densitometric quantification of the autoradiographs for MnSOD employed the intensity of the 4-kb mRNA. The relative fold induction was determined from FED or EBSS levels, normalized to the internal control, ribosomal protein L7a. Data points are the means from independent experiments. An asterisk denotes significance as determined by a Student's t test to a value of p ≤ 0.05. Nutrient availability in the mammalian diet has a potentially critical impact on metabolic flux, the generation of ATP, and as a consequence, the generation of mitochondrially derived ROS. Given the role of MnSOD in the detoxification of mitochondrially derived ROS, we postulated that nutrient availability might have direct effects on the levels of MnSOD gene expression. To test this hypothesis, the response of MnSOD in cell culture to the exclusion of a single essential AA, histidine, was evaluated by Northern analysis in the human hepatoma cell line, HepG2, and compared with the documented response to pro-inflammatory mediators. It has been previously established that lipopolysaccharide, interleukins 1 and 6 (IL-1β and IL-6), tumor necrosis factor-α, and interferon-γ induce MnSOD at the mRNA and protein levels (27Wong G.H. Goeddel D.V. Science. 1988; 242: 941-944Crossref PubMed Scopus (836) Google Scholar, 32Visner G.A. Chesrown S.E. Monnier J. Ryan U.S. Nick H.S. Biochem. Biophys. Res. Commun. 1992; 188: 453-462Crossref PubMed Scopus (65) Google Scholar, 33Dougall W.C. Nick H.S. Endocrinology. 1991; 129: 2376-2384Crossref PubMed Scopus (115) Google Scholar, 34Del Vecchio P.J. Shaffer J.B. Curr. Eye Res. 1991; 10: 919-925Crossref PubMed Scopus (8) Google Scholar, 35Raineri I. Huang T.T. Epstein C.J. Epstein L.B. J. Interferon Cytokine Res. 1996; 16: 61-68Crossref PubMed Scopus (13) Google Scholar). The top panel of Fig. 1A shows MnSOD mRNA levels in HepG2 cells incubated for 12 h in complete MEM medium (FED), MEM lacking histidine (–His), or treated with either that lipopolysaccharide, tumor necrosis factor-α, IL-1β, or IL-6. Two mRNA species are produced from the human MnSOD gene because of alternative polyadenylation (36Hurt J. Hsu J.L. Dougall W.C. Visner G.A. Burr I.M. Nick H.S. Nucleic Acids Res. 1992; 20: 2985-2990Crossref PubMed Scopus (66) Google Scholar, 37Melendez J.A. Baglioni C. Free Radic. Biol. Med. 1993; 14: 601-608Crossref PubMed Scopus (21) Google Scholar). Steady state MnSOD mRNA levels are induced in response to –His, relative to FED, and addition of 5 mm histidine to FED conditions (1st lane of Fig. 1A (+His)) further reduced basal levels, demonstrating the depletion of histidine over the 12-h incubation time and subsequent increases in MnSOD mRNA levels (zero and FED). This observation could have profound effects because most conventional cell culture procedures do not recommend feeding of cells for up to 48 h. To ensure that all experiments have similar MnSOD basal levels, fresh medium was given to the cells 12 h before the start of each experiment. Because HepG2 cells preferentially produce the 4-kb message in response to –His, this is the species referred to and utilized in densitometry for all experiments. In all Northern analyses, the large ribosomal subunit 7a (L7a) was used as an internal loading control. The immunoblot analysis at the bottom of Fig. 1A also demonstrates associated increases of MnSOD protein by 48 h. This induction is also reproducible in another human hepatoma cell line, HuH7, as well as, less robustly, in human lung fibroblast and epithelial cells (data not shown). Many other genes known to be regulated by AA deprivation also respond to glucose starvation (3Kilberg M.S. Pan Y.X. Chen H. Leung-Pineda V. Annu. Rev. Nutr. 2005; 25: 59-85Crossref PubMed Scopus (220) Google Scholar). In addition, glucose metabolism may have profound downstream effects on the production of mitochondrially derived ROS. To evaluate the specificity of changes in MnSOD mRNA levels, cells were starved for glucose (–Glc) and compared with –His. As shown in Fig. 1B, MnSOD mRNA levels are induced only in response to AA deprivation. As a positive control for glucose deprivation, the membranes were reprobed for glucose-regulated protein (GRP78) (38Lee A.S. Methods (San Diego). 2005; 35: 373-381Crossref PubMed Scopus (775) Google Scholar). Fig. 1C is a summary of densitometric data from three independent experiments. Two other known inducers of the endoplasmic reticulum stress response (39Lievremont J.P. Rizzuto R. Hendershot L. Meldolesi J. J. Biol. Chem. 1997; 272: 30873-30879Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar), thapsigargin and tunicamycin, were also tested and, similar to glucose deprivation, showed no induction of MnSOD mRNA (data not shown). To further characterize the MnSOD gene induction in response to AA deprivation, mRNA levels were evaluated when each individual AA was omitted from complete medium and incubated for 2, 6, and 12 h. As shown in the representative examples in Fig. 1D, depletion of essential AA other than His also causes a similar increase in MnSOD mRNA levels, with the exception of tryptophan. On the other hand, depletion of nonessential AA from culture medium had no effect. A densitometric and statistical summary is shown in Fig. 1E. Given the uniqueness of the induction of MnSOD by essential amino acid starvation (–His condition), we next evaluated a potential physiological role for MnSOD during amino acid deprivation. Cell viability was measured using an MTT assay. As illustrated in Fig. 2A, incubation in –His medium reproducibly causes an increase in cell viability (∼48%) as compared with the FED condition. To evaluate the role of MnSOD in the increased cell survival, cells were also treated with 2-methoxyestradiol, which has been shown to inhibit MnSOD (40Gao N. Rahmani M. Shi X. Dent P. Grant S. Blood. 2006; 107: 241-249Crossref PubMed Scopus (34) Google Scholar, 41Golab J. Nowis D. Skrzycki M. Czeczot H. Baranczyk-Kuzma A. Wilczynski G.M. Makowski M. Mroz P. Kozar K. Kaminski R. Jalili A. Kopec M. Grzela T. Jakobisiak M. J. Biol. Chem. 2003; 278: 407-414Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). These data (Fig. 2A) also demonstrate that 10 μm 2-methoxyestradiol eliminates the increased cell survival associated with the –His conditions, whereas the inhibitor had no effect on cells grown in the FED condition. To determine whether –His conditions offer a cytoprotective advantage during stress conditions, cells were preincubated for 24 h in –His medium followed by exposure to sub-lethal levels of UV light. The cell survival curves in Fig. 2B demonstrate that –His conditions can reduce cell death following UV exposure. The level of cytoprotection was most significant at 1 and 2 min of UV exposure with an ∼50 and 70% reduction in cell death at these time points, respectively. To further address the specific role of MnSOD in the increase of cell survival associated with depletion of essential AA, we exposed cells to an siRNA specific to MnSOD as compared with a non" @default.
• W2040307459 created "2016-06-24" @default.
• W2040307459 creator A5024447801 @default.
• W2040307459 creator A5034022874 @default.
• W2040307459 creator A5056981082 @default.
• W2040307459 creator A5067555607 @default.
• W2040307459 date "2008-04-01" @default.
• W2040307459 modified "2023-09-29" @default.
• W2040307459 title "Metabolic Regulation of Manganese Superoxide Dismutase Expression via Essential Amino Acid Deprivation" @default.
• W2040307459 cites W1496861468 @default.
• W2040307459 cites W1528951702 @default.
• W2040307459 cites W158023586 @default.
• W2040307459 cites W1596685255 @default.
• W2040307459 cites W1963723155 @default.
• W2040307459 cites W1963943602 @default.
• W2040307459 cites W1970121621 @default.
• W2040307459 cites W1976641851 @default.
• W2040307459 cites W1989395105 @default.
• W2040307459 cites W1992865315 @default.
• W2040307459 cites W1995707631 @default.
• W2040307459 cites W1995717781 @default.
• W2040307459 cites W2002265929 @default.
• W2040307459 cites W2002725367 @default.
• W2040307459 cites W2008239768 @default.
• W2040307459 cites W2011123924 @default.
• W2040307459 cites W2013333305 @default.
• W2040307459 cites W2018396699 @default.
• W2040307459 cites W2018735766 @default.
• W2040307459 cites W2019447617 @default.
• W2040307459 cites W2020075481 @default.
• W2040307459 cites W2020761570 @default.
• W2040307459 cites W2022825683 @default.
• W2040307459 cites W2022879400 @default.
• W2040307459 cites W2029312295 @default.
• W2040307459 cites W2033039016 @default.
• W2040307459 cites W2035500640 @default.
• W2040307459 cites W2036713864 @default.
• W2040307459 cites W2038440449 @default.
• W2040307459 cites W2044494908 @default.
• W2040307459 cites W2045471846 @default.
• W2040307459 cites W2046102852 @default.
• W2040307459 cites W2048553024 @default.
• W2040307459 cites W2051867686 @default.
• W2040307459 cites W2054943275 @default.
• W2040307459 cites W2060131781 @default.
• W2040307459 cites W2060574598 @default.
• W2040307459 cites W2069333165 @default.
• W2040307459 cites W2070885735 @default.
• W2040307459 cites W2072780644 @default.
• W2040307459 cites W2074027455 @default.
• W2040307459 cites W2079795909 @default.
• W2040307459 cites W2082067798 @default.
• W2040307459 cites W2086415396 @default.
• W2040307459 cites W2087561593 @default.
• W2040307459 cites W2090419702 @default.
• W2040307459 cites W2099364141 @default.
• W2040307459 cites W2106090797 @default.
• W2040307459 cites W2107277218 @default.
• W2040307459 cites W2115074336 @default.
• W2040307459 cites W2117104323 @default.
• W2040307459 cites W2119949419 @default.
• W2040307459 cites W2121966499 @default.
• W2040307459 cites W2128600167 @default.
• W2040307459 cites W2133593669 @default.
• W2040307459 cites W2135090837 @default.
• W2040307459 cites W2137261869 @default.
• W2040307459 cites W2145532527 @default.
• W2040307459 cites W2145968380 @default.
• W2040307459 cites W2146415848 @default.
• W2040307459 cites W2148046166 @default.
• W2040307459 cites W2155739395 @default.
• W2040307459 cites W2160082612 @default.
• W2040307459 cites W2161857920 @default.
• W2040307459 cites W2171142773 @default.
• W2040307459 cites W2315512556 @default.
• W2040307459 cites W4211054414 @default.
• W2040307459 doi "https://doi.org/10.1074/jbc.m709944200" @default.
• W2040307459 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2447627" @default.
• W2040307459 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18187411" @default.
• W2040307459 hasPublicationYear "2008" @default.
• W2040307459 type Work @default.
• W2040307459 sameAs 2040307459 @default.
• W2040307459 citedByCount "14" @default.
• W2040307459 countsByYear W20403074592012 @default.
• W2040307459 countsByYear W20403074592013 @default.
• W2040307459 countsByYear W20403074592018 @default.
• W2040307459 countsByYear W20403074592020 @default.
• W2040307459 countsByYear W20403074592022 @default.
• W2040307459 crossrefType "journal-article" @default.
• W2040307459 hasAuthorship W2040307459A5024447801 @default.
• W2040307459 hasAuthorship W2040307459A5034022874 @default.
• W2040307459 hasAuthorship W2040307459A5056981082 @default.
• W2040307459 hasAuthorship W2040307459A5067555607 @default.
• W2040307459 hasBestOaLocation W20403074591 @default.
• W2040307459 hasConcept C178790620 @default.
• W2040307459 hasConcept C181199279 @default.
• W2040307459 hasConcept C185592680 @default.
• W2040307459 hasConcept C2775838275 @default.

• next