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Elevation of glutathione and γ-glutamyl cycle enzyme activities and m RNA by naphthoquinones
Session 11: Regulation of Gene Expression in Oxidative Stress, Part II 11:1
REGULATION OF GENE EXPRESSION IN OXIDATIVE STRESS Nikki J. Holbrook, Jennifer D. Luethy. and Augustine M.K. Choi Laboratory of Molecular Genetics, National Institute on Aging, Baltimore. MD 21224 Oxidative stress plays an important role in the pathogenesis of many disease states and is likely to contribute directly to the aging process. All cells respond to oxidative stress with the rapid induction of numerous gene products, many of which have been shown to confer an adaptive advantage to the stressed cell. Several different regulons contribute to the response in bacteria, but in eucaryotes relatively little is known about the underlying mechanisms involved in the response. Recent studies have indicated that considerable overlap exists between the signal transduction pathways mediating gene activation in response to oxidative stress and DNA damage. This is not surprising since DNA is a major target of oxidant injury. However, oxidative damage to cellular components other than DNA may be the initiating signal responsible for gene activation following treatment of cells with certain genotoxic agents. Multiple pathways appear to be involved; the particular pathway followed being both gene specific and dependent on the stress-inducing agent. Tyrosine kinases, protein kinase C. other unidentified serine-tbreonine kinases, and the tumor suppressor protein ~53, have all been implicated in the response. Because of the presumed importance of oxidative stress and DNA damage in the aging process we have examined whether the molecular response to these stresses is altered with aging. Utilizing cultured WI-38 human diploid tibroblasts as a model of cellular aging, we have demonstrated that activation of activator protein 1 (AP-1) binding activity in response to treatment with either UV radiation or the alkylating agent methyl methanesulfonate is diminished in aged (late passage) cells relative to young (early passage) cells. This diminished AP-1 binding activity is associated with the reduced expression of the AP-1 dependent gene, collagenase, following DNA damage. It is likely that expression of other AP-1 dependent genes is similarly affected and could result in reduced tolerance of the aged cell to DNA damage,
11~3
G-PROTEINSCAN MODULATEANTIOXIDANTENZYMEGENE EXPRESSION. Jeri Ann Boose, Lung Biology Laboratory, Georgetown University School of Medicine,
Washington,
D.C.
20007,
USA.
Antioxidant enzymes (AOEs) protect cells from oxidant damage by scavenging harmful intermediates of oxygen metabolism. Superoxide dismutase (SODS) convert superoxide to hydrogen peroxide; catalase (CAT) and glutathione peroxidase (GP) each convert hydrogen peroxide to water. Heterotrimeric G-proteins transduce signals that mediate processes, such as ion transport, that make them important determinants of cellular oxygen metabolism. Using rat pulmonary epithelial-like (L2) cells we found G-protein activation with fluoride or a nonhydrolyzable analog of GTP (GTPyS) increased manganese-SOD (MnSOD), CAT and GP mRNAs. Tumor necrosis factor-u (TNF-a), an important inducer of resistance to oxygen stress, also increased the AOE mRNAs and cells treated with both TNF-o and GTPyS showed a synergistic increase in AOE mRNAs. Pertussis toxin (PTX), which inactivates the Gi\Go subfamily of Gproteins, blocked the TNF-a mediated elevation of the AOE mRNAs. Unlike the fluoride mediated increase in MnSOD mRNA which required both RNA and protein synthesis, the TNF-o mediated increase required transcription, but not translation. We conclude that G-protein activation elevates AOE gene expression and does so through at least two different pathways, one requiring and one not requiring protein synthesis. We suggest G-proteins link processes that are important determinants of cellular oxygen metabolism to the regulation of AOE gene expression. Coordination of these events would allow the cell to efficiently scavenge harmful oxygen metabolites as they are generated.
ELEVATION OF GLUTATHIONE AND T-GLUTAMYL CYCLE ENZYME ACTIVITIES AND MRNA BY NAPHTHOQUINONES Henry Jay Forman, Ming Shi, Amir Kugelman and Hemy A. Choy. Insitute for Toxicology and Depts. of Molec. Pharm. t Tax., Pediat. and Path., Univ. Southern California, Los Angeles, CA 90033 USA
543 11:2
Extracellular glutathione (GSH) can be used as a source of amino acids for de nova GSH synthesis after degradation by y-glutamyl transpeptidaae (yGT), an exoenzyme. Synthesis of GSH is initiated by y-glutamylcysteine synthetaae (yGCS). Several studies have indicated that inhibition of either enzyme increases susceptibility to oxidant injury while yGT-cDNA transfected fibroblasts have increased capacity to use extracellular GSH to resist oxidant injury This resistance correlated with an increased rate of GSH synthesis compared with control cells. An increase in GSH that occurs in cells exposed to sublethal oxidant stress has been proposed as an adaptive response. We hypothesized that the activities of @T or flCS may increase as part of this response. Increased flT activity, protein and rnRNA were observed in an alveolar epithelial cell line (L-2) exposed to menadione @IQ). Redox cycling of MQ generates H,O,. MQ can also deplete GSH through conjugation. Increased flCS activity and mRNA were observed in bovine pulmonary artery endothelial cells (BPAEC) exposed to MQ or 2,3-dimethoxy-1,4naphthoquinone (DMNQ). DMNQ generates H,O, but cannot conjugate with GSH. In BPAEC, sublethal doses of MQ caused a transient increase in GSSG and a transient decrease in total intracellular glutathione. This was followed by an increase in GSH to a sustained elevated concentration. While DMNQ also produced a sustained elevation of GSH after a lag period, it did not produce a transient decrease in total glutathione. DMNQ also caused an initial elevation in GSSG that remained fairly constant. The results suggest that increased GSH content of oxidant exposed cells was due to increased yCrCS and that the increased expression of yGCS was stimulated by oxidant stress that did not require a transient decrease in total glutathione content. Supported by
NIH HL37556.
INHIBITION OF GLUTATHIONE REDUCTASE ACTIVITY IN CHO CELLS BY ANTISENSE GENE TRANSFECTION INCREASES THEIR SENSITIVITY TO OXIDANT INJURY. T.N. Hansen. H. Tonoki, H. McMicken, and C. V. Smith, Dept. of Pediatrics/Cell Biology, Baylor College of Med Houston, TX. 77030 Glutathione reductase (GR) protects tissues from Introduction: oxidant injury by catalyzing the reduction of glutathione disulfide (GSSG) to ghttathione (GSH). Inhibition of GR activity with BCNU has been used to study the antioxidant role of GR but these studies may be confounded by effects of BCNU other than GR inhibition. Purpose: The purpose of these experiments was to transfect cells with antisense cDNA to human GR and create a stably transformed cell line that was GR deficient and to use this cell line to study the role of GR in protecting cells from oxidant injury. Methods: We constructed an expression vector using a full length cDNA for human GR, cloned in our laboratory, positioned antisense and downstream from the human metallothionein IIa promoter. We co-transfected this vector with a neomycin resistance gene into Chinese Hamster Ovary (CHO) cells to obtain stable cell lines. Results: One clone (G17) had decreased cytosolic and mitochondrial GR activities (all data mean+SD; activities in mU/mg protein; cytosol: 8.3i0.3 in G17 and 16.9*1.6 in control; mitochondria: Baseline GSSG 1.81+0.19 in G17 and 3.94H.76 in control). concentrations were higher in G17 cells than in controls (0.92+0.19 vs 0.22&0.16 nmoVmg protein) while GSH concentrations were similar. Treatment of G17 and control cells with increasing doses (1 to IO mM) of t-butyl hydroperoxide (TOOH) increased inaacelhdar GSSG concentrations and decreased GSH. Concentrations of GSH were lower in G17 cells than in controls at all doses of TOOH while GSSG concentrations were higher at all concentrations except 10 mM. Exposure to 10 mM TOOH for 4 h resulted in greater LDH release in G17 than in controls (50% vs 15 %). Partial GR deficiency impaired the G17 cell lines Conclusions: ability to recycle GSSG resulting in loss of GSH and increased sensitivity to injury by TOOH.
11:4
REGULATION OF GENE EXPRESSION IN OXIDATIVE STRESS Nikki J. Holbrook, Jennifer D. Luethy. and Augustine M.K. Choi Laboratory of Molecular Genetics, National Institute on Aging, Baltimore. MD 21224 Oxidative stress plays an important role in the pathogenesis of many disease states and is likely to contribute directly to the aging process. All cells respond to oxidative stress with the rapid induction of numerous gene products, many of which have been shown to confer an adaptive advantage to the stressed cell. Several different regulons contribute to the response in bacteria, but in eucaryotes relatively little is known about the underlying mechanisms involved in the response. Recent studies have indicated that considerable overlap exists between the signal transduction pathways mediating gene activation in response to oxidative stress and DNA damage. This is not surprising since DNA is a major target of oxidant injury. However, oxidative damage to cellular components other than DNA may be the initiating signal responsible for gene activation following treatment of cells with certain genotoxic agents. Multiple pathways appear to be involved; the particular pathway followed being both gene specific and dependent on the stress-inducing agent. Tyrosine kinases, protein kinase C. other unidentified serine-tbreonine kinases, and the tumor suppressor protein ~53, have all been implicated in the response. Because of the presumed importance of oxidative stress and DNA damage in the aging process we have examined whether the molecular response to these stresses is altered with aging. Utilizing cultured WI-38 human diploid tibroblasts as a model of cellular aging, we have demonstrated that activation of activator protein 1 (AP-1) binding activity in response to treatment with either UV radiation or the alkylating agent methyl methanesulfonate is diminished in aged (late passage) cells relative to young (early passage) cells. This diminished AP-1 binding activity is associated with the reduced expression of the AP-1 dependent gene, collagenase, following DNA damage. It is likely that expression of other AP-1 dependent genes is similarly affected and could result in reduced tolerance of the aged cell to DNA damage,
11~3
G-PROTEINSCAN MODULATEANTIOXIDANTENZYMEGENE EXPRESSION. Jeri Ann Boose, Lung Biology Laboratory, Georgetown University School of Medicine,
Washington,
D.C.
20007,
USA.
Antioxidant enzymes (AOEs) protect cells from oxidant damage by scavenging harmful intermediates of oxygen metabolism. Superoxide dismutase (SODS) convert superoxide to hydrogen peroxide; catalase (CAT) and glutathione peroxidase (GP) each convert hydrogen peroxide to water. Heterotrimeric G-proteins transduce signals that mediate processes, such as ion transport, that make them important determinants of cellular oxygen metabolism. Using rat pulmonary epithelial-like (L2) cells we found G-protein activation with fluoride or a nonhydrolyzable analog of GTP (GTPyS) increased manganese-SOD (MnSOD), CAT and GP mRNAs. Tumor necrosis factor-u (TNF-a), an important inducer of resistance to oxygen stress, also increased the AOE mRNAs and cells treated with both TNF-o and GTPyS showed a synergistic increase in AOE mRNAs. Pertussis toxin (PTX), which inactivates the Gi\Go subfamily of Gproteins, blocked the TNF-a mediated elevation of the AOE mRNAs. Unlike the fluoride mediated increase in MnSOD mRNA which required both RNA and protein synthesis, the TNF-o mediated increase required transcription, but not translation. We conclude that G-protein activation elevates AOE gene expression and does so through at least two different pathways, one requiring and one not requiring protein synthesis. We suggest G-proteins link processes that are important determinants of cellular oxygen metabolism to the regulation of AOE gene expression. Coordination of these events would allow the cell to efficiently scavenge harmful oxygen metabolites as they are generated.
ELEVATION OF GLUTATHIONE AND T-GLUTAMYL CYCLE ENZYME ACTIVITIES AND MRNA BY NAPHTHOQUINONES Henry Jay Forman, Ming Shi, Amir Kugelman and Hemy A. Choy. Insitute for Toxicology and Depts. of Molec. Pharm. t Tax., Pediat. and Path., Univ. Southern California, Los Angeles, CA 90033 USA
543 11:2
Extracellular glutathione (GSH) can be used as a source of amino acids for de nova GSH synthesis after degradation by y-glutamyl transpeptidaae (yGT), an exoenzyme. Synthesis of GSH is initiated by y-glutamylcysteine synthetaae (yGCS). Several studies have indicated that inhibition of either enzyme increases susceptibility to oxidant injury while yGT-cDNA transfected fibroblasts have increased capacity to use extracellular GSH to resist oxidant injury This resistance correlated with an increased rate of GSH synthesis compared with control cells. An increase in GSH that occurs in cells exposed to sublethal oxidant stress has been proposed as an adaptive response. We hypothesized that the activities of @T or flCS may increase as part of this response. Increased flT activity, protein and rnRNA were observed in an alveolar epithelial cell line (L-2) exposed to menadione @IQ). Redox cycling of MQ generates H,O,. MQ can also deplete GSH through conjugation. Increased flCS activity and mRNA were observed in bovine pulmonary artery endothelial cells (BPAEC) exposed to MQ or 2,3-dimethoxy-1,4naphthoquinone (DMNQ). DMNQ generates H,O, but cannot conjugate with GSH. In BPAEC, sublethal doses of MQ caused a transient increase in GSSG and a transient decrease in total intracellular glutathione. This was followed by an increase in GSH to a sustained elevated concentration. While DMNQ also produced a sustained elevation of GSH after a lag period, it did not produce a transient decrease in total glutathione. DMNQ also caused an initial elevation in GSSG that remained fairly constant. The results suggest that increased GSH content of oxidant exposed cells was due to increased yCrCS and that the increased expression of yGCS was stimulated by oxidant stress that did not require a transient decrease in total glutathione content. Supported by
NIH HL37556.
INHIBITION OF GLUTATHIONE REDUCTASE ACTIVITY IN CHO CELLS BY ANTISENSE GENE TRANSFECTION INCREASES THEIR SENSITIVITY TO OXIDANT INJURY. T.N. Hansen. H. Tonoki, H. McMicken, and C. V. Smith, Dept. of Pediatrics/Cell Biology, Baylor College of Med Houston, TX. 77030 Glutathione reductase (GR) protects tissues from Introduction: oxidant injury by catalyzing the reduction of glutathione disulfide (GSSG) to ghttathione (GSH). Inhibition of GR activity with BCNU has been used to study the antioxidant role of GR but these studies may be confounded by effects of BCNU other than GR inhibition. Purpose: The purpose of these experiments was to transfect cells with antisense cDNA to human GR and create a stably transformed cell line that was GR deficient and to use this cell line to study the role of GR in protecting cells from oxidant injury. Methods: We constructed an expression vector using a full length cDNA for human GR, cloned in our laboratory, positioned antisense and downstream from the human metallothionein IIa promoter. We co-transfected this vector with a neomycin resistance gene into Chinese Hamster Ovary (CHO) cells to obtain stable cell lines. Results: One clone (G17) had decreased cytosolic and mitochondrial GR activities (all data mean+SD; activities in mU/mg protein; cytosol: 8.3i0.3 in G17 and 16.9*1.6 in control; mitochondria: Baseline GSSG 1.81+0.19 in G17 and 3.94H.76 in control). concentrations were higher in G17 cells than in controls (0.92+0.19 vs 0.22&0.16 nmoVmg protein) while GSH concentrations were similar. Treatment of G17 and control cells with increasing doses (1 to IO mM) of t-butyl hydroperoxide (TOOH) increased inaacelhdar GSSG concentrations and decreased GSH. Concentrations of GSH were lower in G17 cells than in controls at all doses of TOOH while GSSG concentrations were higher at all concentrations except 10 mM. Exposure to 10 mM TOOH for 4 h resulted in greater LDH release in G17 than in controls (50% vs 15 %). Partial GR deficiency impaired the G17 cell lines Conclusions: ability to recycle GSSG resulting in loss of GSH and increased sensitivity to injury by TOOH.
11:4