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Transcriptional Regulation of γ-Glutamylcysteine Synthetase-Heavy Subunit by Oxidants in Human Alveolar Epithelial Cells
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
229, 832–837 (1996)
1888
Transcriptional Regulation of g-Glutamylcysteine Synthetase-Heavy Subunit by Oxidants in Human Alveolar Epithelial Cells Irfan Rahman,*,1 Agnes Bel,* Brigitte Mulier,* Mark F. Lawson,* David J. Harrison,† William MacNee,* and Christopher A. D. Smith† Rayne Laboratory, Respiratory Medicine Unit, *Department of Medicine, Department of the Royal Infirmary, and †Department of Pathology, University of Edinburgh, Teviot Place, Edinburgh EH8 9AG, Scotland, United Kingdom Received November 15, 1996 We studied the regulation of glutathione (GSH) synthesis and characterised the 5*-promoter region of the g-glutamylcysteine synthetase-heavy subunit (gGCS-HS) gene in human alveolar type II cells (A549) following exposure to menadione (MQ) and hydrogen peroxide (H2O2). Both MQ (100mM) and H2O2 (100mM) exposure increased intracellular GSH levels associated with increased gGCS activity. This was concomitant with enhanced expression of gGCS-HS mRNA. Transfection of deletion constructs of the gGCS-HS promoter (01050 to /82 bp) in a chloramphenicol acetyl transferase (CAT) reporter system revealed that an human antioxidant response element (hARE), present within the proximal region of the promoter (01050 to 0818 bp), is not required for oxidant-mediated gene induction. We conclude that oxidant stress-induced gGCS-HS mRNA expression is associated with AP-1 or AP-1 like responsive elements (0817 to /45 bp). q 1996 Academic Press
Reduced glutathione (GSH), a ubiquitous cellular non-protein sulfhydryl is important in maintaining intra-cellular redox balance and is involved in the detoxification of xenobiotics, electrophiles, organic peroxides and heavy metals, either through direct thiol conjugation or in enzyme-catalysed reactions (1). Glutathione is synthesised from its constituent amino acids in two sequential, ATP-dependent enzymatic reactions, catalysed by g-glutamylcysteine synthetase (gGCS) and GSH synthase (2). gGCS is the rate-limiting step in de novo GSH synthesis and is inhibited by a feedback mechanism involving GSH. The gGCS holoenzyme exists as a dimer composed of heavy (gGCS-HS; 73kDa) and light (gGCS-LS; 28kDa) subunits (3). The heavy subunit possesses all of the catalytic activity (4). Mulcahy and Jipp (5) and Yao et al (6) recently reported that the promoter (5*-flanking) region of human gGCS-HS contained a consensus fos/jun heterodimeric complex-activator protein (AP-1) sequence, AP-1 like binding sites; human antioxidant response element (hARE), and several SP-1 and AP-2 binding sites. However, there is no information so far available on the characteristics of the gGCS-HS promoter region in relation to the regulation of GSH synthesis under oxidative stress. Menadione (2-methyl-1,4 napthoquinone, MQ) is a quinone which imposes oxidative stress by generating reactive oxygen species (ROS) due to redox cycling. Recent evidence indicates that quinone compounds induce GSH synthesis in various cell lines (7,8). However, the exact molecular mechanism of increase in GSH synthesis is unclear. To test the hypothesis that oxidative stress, imposed by hydrogen peroxide (H2O2) and MQ, increases intracellular GSH by the induction of gGCS-HS gene expression, we investigated the effects of oxidants on the 1
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regulation of gGCS-HS mRNA in alveolar epithelial cells. We also characterised the 5*-region of the gGCS-HS gene using partial deletion constructs to analyse the role of various putative transcription factors binding sites in the regulation of gGCS-HS promoter in response to oxidants. MATERIALS AND METHODS Unless otherwise stated, all of the biochemical reagents used in this study were purchased from the Sigma Chemical Co., Poole, England; cell culture media from GIBCO-BRL (Paisley, Scotland); molecular biology reagents from GIBCO BRL, Paisley, Scotland. A549 epithelial cells. The type II alveolar epithelial cell line, A549 (ECACC No. 86012804), which was mycoplasma free, was maintained in continuous culture at 377C, 5% CO2 in Dulbecco’s modified minumum essential medium (DMEM, GIBCO) containing penicillin/streptomycin mixture, L-glutamine, sodium bicarbonate, and 10% fetal bovine serum (FBS) (GIBCO, Paisley, Scotland). A549 epithelial cell exposure to oxidants. Confluent monolayers of A549 cells were rinsed twice with DMEM and exposed to H2O2 (100mM) or MQ (100mM) for 1 hour in 5 ml of DMEM with 10% FBS at 377C, 5% CO2 . After incubation, the monolayers were washed with fresh medium and incubated for a further 24 hours. The monolayers were then washed twice with cold sterile PBS (Ca2/ and Mg2/ free) (pH 7.4), trypsinised and used for the GSH, gGCS enzyme and mRNA assays. GSH and gGCS activity assays. Acid extracts of A549 cells, were spun and the supernatant immediately used in the soluble GSH assay by the 5,5*-dithiobis-(2-nitrobenzoic acid) DTNB-GSSG reductase recycling method described by Tietze (9). gGCS activity was measured by the method described by Seelig and Meister (10). Isolation of RNA and reverse transcription. RNA was isolated from A549 cells by the acid-guanidine method described by Chomczynski and Sacchi (11). Total RNA was reverse transcribed using Superscript II according to the manufacturers instructions (GIBCO-BRL). The resultant cDNA was stored at 0207C, until required. Primers and polymerase chain reaction (PCR). To quantitate gGCS mRNA expression a reverse transcriptase-PCR assay was used as previously described (13). Oligonucleotide primers were selected from the published sequence of gGCS-HS cDNA (12), and b-actin (Stratagene, Cambridge, UK). The primers for gGCS-HS and the details of the PCR conditions have been described previously (13). Amplified PCR products were applied to a 2% agarose gel containing ethidium bromide and electrophoresed in 0.51Tris-borate-EDTA buffer. Bands were visualised by uv transillumination and photograph negatives were scanned using a LKB-ultrascan XL enhanced laser densitometer. The level of gGCS-HS mRNA was compared with b-actin mRNA and results were expressed as a percentage ratio. Promoter deletion constructs. The gGCS-HS promoter was isolated by PCR from human genomic DNA using the upstream oligonucleotide 5*- (/82) GGCGACATCCAATATGAAGGCTGTG-3* and downstream oligonucleotides 5*- (01050) TTCCTACTTGTGACCAAAACCTGCG-3*. The resulting promoter fragment (01050 to /82 bp) was cloned into pCRII cloning vector (Invitrogen, USA) and a Hind III and Sph 1 fragment (1138 bp) containing the promoter was isolated and subcloned into polylinker of the promoterless plasmid pCAT Basic Vector (Promega, USA). This construct was denoted pCBGCS. Deletion fragments of the gGCS-HS promoter were generated using Kpn 1 restriction site present within the gGCS-HS promoter which resulted in a short fragment containing hARE (01050 to 0818 bp) (pCBGCSA) and remaining large fragment (0817 to /45 bp) (pCBGCSDK) containing AP-1 and AP-1 like sites. Both the fragments were subcloned into pCAT Basic vector. Transient transfection and CAT assay. A549 cells (Ç0.8 1106) per well were seeded into 6-well tissue culture plates and cultured at 377C until 70% confluent. Plasmid DNA transfections were performed using lipofectAMINE reagent (GIBCO), as per manufacturers instructions. After H2O2 (100mM) or MQ (100mM) treatment, cell extracts were prepared and assayed for protein content using BCA reagent (Pierce, Rockford, IL, USA). Chloramphenicol acetyl transferase (CAT) activity was quantitated by the CAT enzyme-linked immunosorbent assay (ELISA). bgalactosidase expression plasmid (PSVgal, Promega) was co-transfected as an internal control to normalise transfection efficiency. In all transfection experiments, pCAT-Basic, pCAT-Control were used as negative and positive controls, respectively. Statistical analysis. Results were expressed as mean{SEM. Differences between values were compared by Duncan’s multiple range test.
RESULTS
Effects of MQ and H2O2 on GSH Levels and gGCS Activity in Alveolar Epithelial Cells MQ (100mM) exposure depleted GSH levels by 37% (põ0.01) at 1 hour, followed by a 73% increase in GSH levels 24 hours after exposure (figure 1 A, põ0.001 compared with control values). This was concomitant with a 75% increase in gGCS activity (figure 1 B). 833
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FIG. 1. Effect of 100 mM menadione (MQ) on GSH levels (A) and gGCS activity (B) in A549 type II alveolar epithelial cells. Each histogram represents mean{SEM of 8 experiments. **põ0.01, ***põ0.001 compared with control (C) values.
H2O2 (100mM) exposure produced a 82% increase in GSH levels (põ0.01) and a 90% increase in gGCS activity (põ0.001) 24 hours post exposure (figures 2 A,B). Effects of MQ and H2O2 on gGCS-HS mRNA Expression gGCS-HS mRNA expression did not change 1 hour post exposure in response to either MQ (100mM) or H2O2 (100mM) (data not shown). However, gGCS-HS mRNA increased 24 hours after the cells were washed and re-cultured in fresh medium (figure 3 A,B).
Effects of MQ and H2O2 on gGCS-HS Promoter Construct-Derived Chloramphenicol Acetyl Transferase (CAT) Activity MQ (100mM) or H2O2 (100mM) exposure of cells transfected with the full promoter linked to the CAT reporter system (pCBGCS) produced 171% and 295% increase in CAT activity,
FIG. 2. Effect of 100 mM H2O2 on GSH levels (A) and gGCS activity (B) in A549 epithelial cells. Each histogram represents mean{SEM of 4 experiments. **põ0.01, ***põ0.001 compared with control (C) values. 834
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FIG. 3. Effects of MQ and H2O2 on gGCS-HS mRNA expression in A549 cells. Total RNA was isolated from control cells and cells exposed to (top, A) MQ (100mM) for 24 hours, (top, B) H2O2 (100mM for 24 hours. RNA was reversed transcribed and used for PCR analysis of gGCS-HS mRNA. Bottom: The gel was scanned on a LKB ultrascan enhanced laser densitometer and numeric estimates of gGCS-HS mRNA levels were compared with the subsequent b-actin bands from the same sample.
respectively. Deletion of a 232 bp fragment containing the hARE responsive element, did not affect this induction (figure 4). Furthermore, when the putative hARE fragment was linked directly with pCB vector, exposure of cells transfected with this construct to oxidants showed no significant increase in the CAT activity compared to control pCB vector. DISCUSSION
We have shown that after initial depletion of intracellular GSH by MQ and H2O2 , prolonged re-culture (i.e. 24 hrs) results in an increase in GSH levels and a concomitant with an increase in gGCS activity. We also show that these events are associated with an increase in the relative level of gGCS-HS mRNA. gGCS-HS is induced by various redox-regulating agents (6,13-16). Recently we have demonstrated that cigarette smoke, which contains 1014-1016 free radicals/puff also induces gGCSHS expression in human alveolar type II cells (13), but the precise molecular mechanism and the involvement of ROS in the induction of gGCS-HS mRNA is not clear. ROS generated by MQ and H2O2 may modulate AP-1(17,18) and hARE transcription factors (19) promote their binding to the oligonucleotide consensus region of the gGCS-HS promoter so inducing transcription. In order to assess the mechanism of the transcriptional upregulation of gGCS-HS in response to oxidants, we carried out gGCS-HS promoter deletion studies. The full proximal promoter (01050 to /82 bp) linked to the CAT reporter system (pCBGCS) produced increased CAT activity on exposure to MQ or H2O2 . However, deletion of a 232 bp fragment containing the hARE responsive element, did not affect this induction. In addition, the fragment of the 5* region encompassing the putative hARE contained no inate promoter 835
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FIG. 4. Transient expression analysis of the human gGCS-HS gene. The line diagram at the top left is a restriction map of the promoter region cloned in pCRII vector. The dotted line on the left indicate an additional 50 bp from multiple cloning sites of the pCRII vector. Transcriptional start site is indicated by bent arrow. The structure of the gGCS-CAT plasmids are shown below on the left. Deletion mutants were ligated to upstream of the CAT gene in pCAT Basic vector (pCB), transfected into A549 cells and exposed to MQ or H2O2 . After 24-hours incubation the cells were harvested and assayed for CAT activity. The activities of different constructs are shown on the right are expressed as means{SEM of triplicate transfection experiments, each performed in duplicate with the activity of pCBGCS set at 100%. ***põ0.001 compared to pCBGCS.
activity as measured by CAT ELISA (figure 4). This suggests that this hARE region of the proximal promoter is not involved in the regulation of gGCS-HS activity in response to these oxidants. Rather, downstream sequences, i.e. 0817 to /1 bp containing AP-1/AP-1 like responsive elements may be required. Since we and others have evidenced increased AP-1 activity by cigarette smoke (13) and oxidants (17,18) which are also known to induce gGCSHS expression (7,8,13). In addition, this is in agreement with an earlier indirect observation that c-jun/c-jun homodimers (AP-1) regulate gGCS-HS gene expression induced by cisplatin (6) and cadmium (20). Further studies are in progress to characterise and identify other response elements upstream of this region of the gene as well as the specific elements of the gGCSHS promoter which regulate basal gGCS-HS gene expression. ACKNOWLEDGMENTS This work was supported by the Normal Salvesen Emphysema Research Trust and the British Lung Foundation.
REFERENCES 1. 2. 3. 4. 5. 6.
Reed, D. J. (1990) Annu. Rev. Pharmacol Toxicol. 30, 603–631. Seelig, G. F., and Meister, A. (1984) J. Biol. Chem. 259, 3534–3538. Seelig, G. F., Simondsen, R. P., and Meister, A. (1984) J. Biol. Chem. 259, 9345–9347. Huang, C. S., Chang, L. S., Anderson, M. E., and Meister, A. (1993) J. Biol. Chem. 268, 19675–19680. Mulcahy, R. T., and Gipp, J. J. (1995) Biochem. Biophys. Res. Commun. 209, 227–233. Yao, K. S., Godwin, A. K., Johnson, S. W., Ozols, R. F., O’Dwyer, P. J., and Hamilton, T. C. (1995) Cancer Res. 55, 4367–4374. 7. Shi, M. M., Kugelman, A., Takeo, I., Tian, L., and Forman, H. J. (1994) J. Biol. Chem. 269, 26512–26517. 8. Shi, M. M., Iwamoto, T., and Forman, H. J. (1994) Am. J. Physiol. 267, L414–421. 9. Tietze, F. (1969) Anal. Biochem. 27, 502–522. 836
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Seelig, G. F., and Meister, A. (1984) J. Biol. Chem. 259, 3534–3538. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156–159. Gipp, J. J., Chang, C., and Mulcahy, R. T. (1992) Biochem. Biophys. Res. Commun. 185, 29–35. Rahman, I., Smith, C. A. D., Lawson, M. F., Harrison, D. J., and MacNee, W. (1996) FEBS Letts. 396, 21–25. Barroz, K, I., Buetler, T. M., and Eaton, D. L. (1994) Toxicol. Appl. Pharmacol. 126, 150–155. Liu, R. M., Vasiliou, V., Zhu, H., Duh, J. L., Tabor, M. W., Puga, A., Nebert, D. W., Sainsbury, M., and Shertzer, H. G. (1994) Carcinogenesis. 15, 2347–2352. Hatcher, E. L., Chen, Y., and Kang, Y. J. (1995) Free Rad. Biol. Med. 19, 805–812. Bergelson, S., Pinkus, R., and Daniel, V. (1994) Oncogene. 9, 565–571. Pinkus, R., Weiner, L. M., and Daniel, V. (1995) Biochemistry. 34, 81–88. Jaiswal, A. K. (1994) Biochem. Pharmacol. 48, 439–444. Wu, A. L., and Moye-Rowley, W. S. (1994) Mol. Cell Biol. 14, 5832–5873.
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Transcriptional Regulation of g-Glutamylcysteine Synthetase-Heavy Subunit by Oxidants in Human Alveolar Epithelial Cells Irfan Rahman,*,1 Agnes Bel,* Brigitte Mulier,* Mark F. Lawson,* David J. Harrison,† William MacNee,* and Christopher A. D. Smith† Rayne Laboratory, Respiratory Medicine Unit, *Department of Medicine, Department of the Royal Infirmary, and †Department of Pathology, University of Edinburgh, Teviot Place, Edinburgh EH8 9AG, Scotland, United Kingdom Received November 15, 1996 We studied the regulation of glutathione (GSH) synthesis and characterised the 5*-promoter region of the g-glutamylcysteine synthetase-heavy subunit (gGCS-HS) gene in human alveolar type II cells (A549) following exposure to menadione (MQ) and hydrogen peroxide (H2O2). Both MQ (100mM) and H2O2 (100mM) exposure increased intracellular GSH levels associated with increased gGCS activity. This was concomitant with enhanced expression of gGCS-HS mRNA. Transfection of deletion constructs of the gGCS-HS promoter (01050 to /82 bp) in a chloramphenicol acetyl transferase (CAT) reporter system revealed that an human antioxidant response element (hARE), present within the proximal region of the promoter (01050 to 0818 bp), is not required for oxidant-mediated gene induction. We conclude that oxidant stress-induced gGCS-HS mRNA expression is associated with AP-1 or AP-1 like responsive elements (0817 to /45 bp). q 1996 Academic Press
Reduced glutathione (GSH), a ubiquitous cellular non-protein sulfhydryl is important in maintaining intra-cellular redox balance and is involved in the detoxification of xenobiotics, electrophiles, organic peroxides and heavy metals, either through direct thiol conjugation or in enzyme-catalysed reactions (1). Glutathione is synthesised from its constituent amino acids in two sequential, ATP-dependent enzymatic reactions, catalysed by g-glutamylcysteine synthetase (gGCS) and GSH synthase (2). gGCS is the rate-limiting step in de novo GSH synthesis and is inhibited by a feedback mechanism involving GSH. The gGCS holoenzyme exists as a dimer composed of heavy (gGCS-HS; 73kDa) and light (gGCS-LS; 28kDa) subunits (3). The heavy subunit possesses all of the catalytic activity (4). Mulcahy and Jipp (5) and Yao et al (6) recently reported that the promoter (5*-flanking) region of human gGCS-HS contained a consensus fos/jun heterodimeric complex-activator protein (AP-1) sequence, AP-1 like binding sites; human antioxidant response element (hARE), and several SP-1 and AP-2 binding sites. However, there is no information so far available on the characteristics of the gGCS-HS promoter region in relation to the regulation of GSH synthesis under oxidative stress. Menadione (2-methyl-1,4 napthoquinone, MQ) is a quinone which imposes oxidative stress by generating reactive oxygen species (ROS) due to redox cycling. Recent evidence indicates that quinone compounds induce GSH synthesis in various cell lines (7,8). However, the exact molecular mechanism of increase in GSH synthesis is unclear. To test the hypothesis that oxidative stress, imposed by hydrogen peroxide (H2O2) and MQ, increases intracellular GSH by the induction of gGCS-HS gene expression, we investigated the effects of oxidants on the 1
To whom correspondence should be addressed. Fax: 44 131 650 4384. 832
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regulation of gGCS-HS mRNA in alveolar epithelial cells. We also characterised the 5*-region of the gGCS-HS gene using partial deletion constructs to analyse the role of various putative transcription factors binding sites in the regulation of gGCS-HS promoter in response to oxidants. MATERIALS AND METHODS Unless otherwise stated, all of the biochemical reagents used in this study were purchased from the Sigma Chemical Co., Poole, England; cell culture media from GIBCO-BRL (Paisley, Scotland); molecular biology reagents from GIBCO BRL, Paisley, Scotland. A549 epithelial cells. The type II alveolar epithelial cell line, A549 (ECACC No. 86012804), which was mycoplasma free, was maintained in continuous culture at 377C, 5% CO2 in Dulbecco’s modified minumum essential medium (DMEM, GIBCO) containing penicillin/streptomycin mixture, L-glutamine, sodium bicarbonate, and 10% fetal bovine serum (FBS) (GIBCO, Paisley, Scotland). A549 epithelial cell exposure to oxidants. Confluent monolayers of A549 cells were rinsed twice with DMEM and exposed to H2O2 (100mM) or MQ (100mM) for 1 hour in 5 ml of DMEM with 10% FBS at 377C, 5% CO2 . After incubation, the monolayers were washed with fresh medium and incubated for a further 24 hours. The monolayers were then washed twice with cold sterile PBS (Ca2/ and Mg2/ free) (pH 7.4), trypsinised and used for the GSH, gGCS enzyme and mRNA assays. GSH and gGCS activity assays. Acid extracts of A549 cells, were spun and the supernatant immediately used in the soluble GSH assay by the 5,5*-dithiobis-(2-nitrobenzoic acid) DTNB-GSSG reductase recycling method described by Tietze (9). gGCS activity was measured by the method described by Seelig and Meister (10). Isolation of RNA and reverse transcription. RNA was isolated from A549 cells by the acid-guanidine method described by Chomczynski and Sacchi (11). Total RNA was reverse transcribed using Superscript II according to the manufacturers instructions (GIBCO-BRL). The resultant cDNA was stored at 0207C, until required. Primers and polymerase chain reaction (PCR). To quantitate gGCS mRNA expression a reverse transcriptase-PCR assay was used as previously described (13). Oligonucleotide primers were selected from the published sequence of gGCS-HS cDNA (12), and b-actin (Stratagene, Cambridge, UK). The primers for gGCS-HS and the details of the PCR conditions have been described previously (13). Amplified PCR products were applied to a 2% agarose gel containing ethidium bromide and electrophoresed in 0.51Tris-borate-EDTA buffer. Bands were visualised by uv transillumination and photograph negatives were scanned using a LKB-ultrascan XL enhanced laser densitometer. The level of gGCS-HS mRNA was compared with b-actin mRNA and results were expressed as a percentage ratio. Promoter deletion constructs. The gGCS-HS promoter was isolated by PCR from human genomic DNA using the upstream oligonucleotide 5*- (/82) GGCGACATCCAATATGAAGGCTGTG-3* and downstream oligonucleotides 5*- (01050) TTCCTACTTGTGACCAAAACCTGCG-3*. The resulting promoter fragment (01050 to /82 bp) was cloned into pCRII cloning vector (Invitrogen, USA) and a Hind III and Sph 1 fragment (1138 bp) containing the promoter was isolated and subcloned into polylinker of the promoterless plasmid pCAT Basic Vector (Promega, USA). This construct was denoted pCBGCS. Deletion fragments of the gGCS-HS promoter were generated using Kpn 1 restriction site present within the gGCS-HS promoter which resulted in a short fragment containing hARE (01050 to 0818 bp) (pCBGCSA) and remaining large fragment (0817 to /45 bp) (pCBGCSDK) containing AP-1 and AP-1 like sites. Both the fragments were subcloned into pCAT Basic vector. Transient transfection and CAT assay. A549 cells (Ç0.8 1106) per well were seeded into 6-well tissue culture plates and cultured at 377C until 70% confluent. Plasmid DNA transfections were performed using lipofectAMINE reagent (GIBCO), as per manufacturers instructions. After H2O2 (100mM) or MQ (100mM) treatment, cell extracts were prepared and assayed for protein content using BCA reagent (Pierce, Rockford, IL, USA). Chloramphenicol acetyl transferase (CAT) activity was quantitated by the CAT enzyme-linked immunosorbent assay (ELISA). bgalactosidase expression plasmid (PSVgal, Promega) was co-transfected as an internal control to normalise transfection efficiency. In all transfection experiments, pCAT-Basic, pCAT-Control were used as negative and positive controls, respectively. Statistical analysis. Results were expressed as mean{SEM. Differences between values were compared by Duncan’s multiple range test.
RESULTS
Effects of MQ and H2O2 on GSH Levels and gGCS Activity in Alveolar Epithelial Cells MQ (100mM) exposure depleted GSH levels by 37% (põ0.01) at 1 hour, followed by a 73% increase in GSH levels 24 hours after exposure (figure 1 A, põ0.001 compared with control values). This was concomitant with a 75% increase in gGCS activity (figure 1 B). 833
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FIG. 1. Effect of 100 mM menadione (MQ) on GSH levels (A) and gGCS activity (B) in A549 type II alveolar epithelial cells. Each histogram represents mean{SEM of 8 experiments. **põ0.01, ***põ0.001 compared with control (C) values.
H2O2 (100mM) exposure produced a 82% increase in GSH levels (põ0.01) and a 90% increase in gGCS activity (põ0.001) 24 hours post exposure (figures 2 A,B). Effects of MQ and H2O2 on gGCS-HS mRNA Expression gGCS-HS mRNA expression did not change 1 hour post exposure in response to either MQ (100mM) or H2O2 (100mM) (data not shown). However, gGCS-HS mRNA increased 24 hours after the cells were washed and re-cultured in fresh medium (figure 3 A,B).
Effects of MQ and H2O2 on gGCS-HS Promoter Construct-Derived Chloramphenicol Acetyl Transferase (CAT) Activity MQ (100mM) or H2O2 (100mM) exposure of cells transfected with the full promoter linked to the CAT reporter system (pCBGCS) produced 171% and 295% increase in CAT activity,
FIG. 2. Effect of 100 mM H2O2 on GSH levels (A) and gGCS activity (B) in A549 epithelial cells. Each histogram represents mean{SEM of 4 experiments. **põ0.01, ***põ0.001 compared with control (C) values. 834
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FIG. 3. Effects of MQ and H2O2 on gGCS-HS mRNA expression in A549 cells. Total RNA was isolated from control cells and cells exposed to (top, A) MQ (100mM) for 24 hours, (top, B) H2O2 (100mM for 24 hours. RNA was reversed transcribed and used for PCR analysis of gGCS-HS mRNA. Bottom: The gel was scanned on a LKB ultrascan enhanced laser densitometer and numeric estimates of gGCS-HS mRNA levels were compared with the subsequent b-actin bands from the same sample.
respectively. Deletion of a 232 bp fragment containing the hARE responsive element, did not affect this induction (figure 4). Furthermore, when the putative hARE fragment was linked directly with pCB vector, exposure of cells transfected with this construct to oxidants showed no significant increase in the CAT activity compared to control pCB vector. DISCUSSION
We have shown that after initial depletion of intracellular GSH by MQ and H2O2 , prolonged re-culture (i.e. 24 hrs) results in an increase in GSH levels and a concomitant with an increase in gGCS activity. We also show that these events are associated with an increase in the relative level of gGCS-HS mRNA. gGCS-HS is induced by various redox-regulating agents (6,13-16). Recently we have demonstrated that cigarette smoke, which contains 1014-1016 free radicals/puff also induces gGCSHS expression in human alveolar type II cells (13), but the precise molecular mechanism and the involvement of ROS in the induction of gGCS-HS mRNA is not clear. ROS generated by MQ and H2O2 may modulate AP-1(17,18) and hARE transcription factors (19) promote their binding to the oligonucleotide consensus region of the gGCS-HS promoter so inducing transcription. In order to assess the mechanism of the transcriptional upregulation of gGCS-HS in response to oxidants, we carried out gGCS-HS promoter deletion studies. The full proximal promoter (01050 to /82 bp) linked to the CAT reporter system (pCBGCS) produced increased CAT activity on exposure to MQ or H2O2 . However, deletion of a 232 bp fragment containing the hARE responsive element, did not affect this induction. In addition, the fragment of the 5* region encompassing the putative hARE contained no inate promoter 835
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FIG. 4. Transient expression analysis of the human gGCS-HS gene. The line diagram at the top left is a restriction map of the promoter region cloned in pCRII vector. The dotted line on the left indicate an additional 50 bp from multiple cloning sites of the pCRII vector. Transcriptional start site is indicated by bent arrow. The structure of the gGCS-CAT plasmids are shown below on the left. Deletion mutants were ligated to upstream of the CAT gene in pCAT Basic vector (pCB), transfected into A549 cells and exposed to MQ or H2O2 . After 24-hours incubation the cells were harvested and assayed for CAT activity. The activities of different constructs are shown on the right are expressed as means{SEM of triplicate transfection experiments, each performed in duplicate with the activity of pCBGCS set at 100%. ***põ0.001 compared to pCBGCS.
activity as measured by CAT ELISA (figure 4). This suggests that this hARE region of the proximal promoter is not involved in the regulation of gGCS-HS activity in response to these oxidants. Rather, downstream sequences, i.e. 0817 to /1 bp containing AP-1/AP-1 like responsive elements may be required. Since we and others have evidenced increased AP-1 activity by cigarette smoke (13) and oxidants (17,18) which are also known to induce gGCSHS expression (7,8,13). In addition, this is in agreement with an earlier indirect observation that c-jun/c-jun homodimers (AP-1) regulate gGCS-HS gene expression induced by cisplatin (6) and cadmium (20). Further studies are in progress to characterise and identify other response elements upstream of this region of the gene as well as the specific elements of the gGCSHS promoter which regulate basal gGCS-HS gene expression. ACKNOWLEDGMENTS This work was supported by the Normal Salvesen Emphysema Research Trust and the British Lung Foundation.
REFERENCES 1. 2. 3. 4. 5. 6.
Reed, D. J. (1990) Annu. Rev. Pharmacol Toxicol. 30, 603–631. Seelig, G. F., and Meister, A. (1984) J. Biol. Chem. 259, 3534–3538. Seelig, G. F., Simondsen, R. P., and Meister, A. (1984) J. Biol. Chem. 259, 9345–9347. Huang, C. S., Chang, L. S., Anderson, M. E., and Meister, A. (1993) J. Biol. Chem. 268, 19675–19680. Mulcahy, R. T., and Gipp, J. J. (1995) Biochem. Biophys. Res. Commun. 209, 227–233. Yao, K. S., Godwin, A. K., Johnson, S. W., Ozols, R. F., O’Dwyer, P. J., and Hamilton, T. C. (1995) Cancer Res. 55, 4367–4374. 7. Shi, M. M., Kugelman, A., Takeo, I., Tian, L., and Forman, H. J. (1994) J. Biol. Chem. 269, 26512–26517. 8. Shi, M. M., Iwamoto, T., and Forman, H. J. (1994) Am. J. Physiol. 267, L414–421. 9. Tietze, F. (1969) Anal. Biochem. 27, 502–522. 836
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Vol. 229, No. 3, 1996 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
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Seelig, G. F., and Meister, A. (1984) J. Biol. Chem. 259, 3534–3538. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156–159. Gipp, J. J., Chang, C., and Mulcahy, R. T. (1992) Biochem. Biophys. Res. Commun. 185, 29–35. Rahman, I., Smith, C. A. D., Lawson, M. F., Harrison, D. J., and MacNee, W. (1996) FEBS Letts. 396, 21–25. Barroz, K, I., Buetler, T. M., and Eaton, D. L. (1994) Toxicol. Appl. Pharmacol. 126, 150–155. Liu, R. M., Vasiliou, V., Zhu, H., Duh, J. L., Tabor, M. W., Puga, A., Nebert, D. W., Sainsbury, M., and Shertzer, H. G. (1994) Carcinogenesis. 15, 2347–2352. Hatcher, E. L., Chen, Y., and Kang, Y. J. (1995) Free Rad. Biol. Med. 19, 805–812. Bergelson, S., Pinkus, R., and Daniel, V. (1994) Oncogene. 9, 565–571. Pinkus, R., Weiner, L. M., and Daniel, V. (1995) Biochemistry. 34, 81–88. Jaiswal, A. K. (1994) Biochem. Pharmacol. 48, 439–444. Wu, A. L., and Moye-Rowley, W. S. (1994) Mol. Cell Biol. 14, 5832–5873.
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