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Originally published In Press as doi:10.1074/jbc.M401907200 on April 19, 2004

J. Biol. Chem., Vol. 279, Issue 26, 26839-26845, June 25, 2004
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Determinants of Human Plasma Glutathione Peroxidase (GPx-3) Expression*

Charlene Bierl{ddagger}§, Barbara Voetsch{ddagger}, Richard C. Jin{ddagger}, Diane E. Handy{ddagger}, and Joseph Loscalzo{ddagger}||

From the {ddagger}Whitaker Cardiovascular Institute and the Evans Department of Medicine and the §Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, Massachusetts 02118

Received for publication, February 20, 2004 , and in revised form, March 31, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasma glutathione peroxidase (GPx-3) is a selenocysteine-containing protein with antioxidant properties. GPx-3 deficiency has been associated with cardiovascular disease and stroke. The regulation of GPx-3 expression remains largely uncharacterized, however, and we studied its transcriptional and translational determinants in a cultured cell system. In transient transfections of a renal cell line (Caki-2), the published sequence cloned upstream of a luciferase reporter gene produced minimal activity (relative luminescence (RL) = 0.6 ± 0.4). Rapid amplification of cDNA ends was used to identify a novel transcription start site that is located 233 bp downstream (3') of the published site and that produced a >25-fold increase in transcriptional activity (RL = 16.8 ± 1.9; p < 0.0001). Analysis of the novel GPx-3 promoter identified Sp-1- and hypoxia-inducible factor-1-binding sites, as well as the redox-sensitive metal response element and antioxidant response element. Hypoxia was identified as a strong transcriptional regulator of GPx-3 expression, in part through the presence of the hypoxia-inducible factor-1-binding site, leading to an almost 3-fold increase in expression levels after 24 h compared with normoxic conditions (normalized RL = 3.5 ± 0.3 versus 1.2 ± 0.1; p < 0.001). We also investigated the role of the translational cofactors tRNASec, SECIS-binding protein-2, and SelD (selenophosphate synthetase D) in GPx-3 protein expression. tRNASec and SelD significantly enhanced GPx-3 expression, whereas SECIS-binding protein-2 showed a trend toward increased expression. These results demonstrate the presence of a novel functional transcription start site for the human GPx-3 gene with a promoter regulated by hypoxia, and identify unique translational determinants of GPx-3 expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasma glutathione peroxidase (GPx-3)1 is one of five known glutathione peroxidases (1) and one of at least 25 identified selenocysteine-containing enzymes in mammals (2). GPx-3 is unique among the members of the GPx family as the only extracellular isoform (3). It is a glycosylated tetramer of 23-kDa subunits that catalyzes the reduction of hydrogen peroxide and lipid peroxides at the expense of glutathione and is a major scavenger of reactive oxygen species (ROS) produced during normal metabolism or after oxidative insult (4, 5). The GPx-3 gene contains five exons that span ~10 kb in the q32 region of chromosome 5 (6). Although mRNA for GPx-3 has been found in most cell types studied (7–9), the mature protein is basolaterally secreted from renal proximal tubular cells and parietal epithelial cells of Bowman's capsule (10, 11). This localization was determined by in situ analysis of mouse kidney and supported by the clinical observation that anephric individuals (10) and patients with renal dysfuntion (12) have significantly lower levels of GPx-3 activity in plasma compared with healthy subjects. In addition to plasma, GPx-3 activity has been detected in lung lavage fluid (9) and breast milk (13).

Previous studies in our laboratory have shown an association between decreased levels of GPx-3 activity in plasma and familial childhood stroke (14, 15). Western blot analyses showed that this reduction in GPx-3 activity correlated with lower protein expression2; however, the mechanism for this down-regulation is unknown. The regulation of GPx-3 transcription and translation remains largely uncharacterized (16–19). A transcription start point (tsp) located 298 bp upstream of the translation start site has been proposed (6), yet no basal activity has been detected with promoter constructs based upon this tsp (20) despite synthesis of mRNA in the basal state (21). Only one study to date has reported regulation of the promoter of the GPx-3 gene: Comhair et al. (20) showed that transfection of a reporter gene construct containing 3.2 kb of the GPx-3 promoter into epithelial lung cells (BET1A) results in increased expression levels in response to treatment with a combination of pyrogallol and GSH. In addition, GPx-3 synthesis has been shown to be regulated in part by selenium levels (22), but whether this regulation occurs at the transcriptional or translational level remains to be determined. Thus, we sought to identify the determinants of GPx-3 expression by studying its transcriptional and translational regulation in vitro.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Media and fetal bovine serum (FBS) were purchased from Invitrogen. Complete medium refers to medium with FBS as described for each cell type. Except for human astrocytoma-derived cells (HTB13), cells were maintained at 37 °C in 95% O2 and 5% CO2. Caki-2 cells (American Type Culture Collection, Manassas, VA) were grown in McCoy's 5A medium supplemented with L-glutamine, 25 mM HEPES, and 10% FBS. Human embryonic kidney cells (HEK293) were grown in Dulbecco's modified essential medium supplemented with 4.5 g/liter glucose, 4.5 g/liter L-glutamine, 4.5 g/liter pyridoxine HCl, 110 mg/liter sodium pyruvate, and 10% FBS. Human vascular endothelial cells (Clonetics Corp., San Diego, CA) were maintained in EBM-2 medium (Clonetics Corp.), which consists of EBM endothelial cell basal medium supplemented with the EBM-bullet kit (final concentrations: 2% FBS, 10 ng/ml human recombinant epidermal growth factor, 1 µg/ml hydrocortisone, 50 µg/ml gentamycin, 50 ng/ml amphotericin B, and 12 µg/ml bovine brain extract; Clonetics Corp.). HTB13 cells (American Type Culture Collection) were grown at 37 °C without CO2 supplementation in Leibovitz L-15 medium containing 10% FBS.

Normoxic conditions corresponded to pO2 = 150 mm Hg. To induce hypoxic conditions (pO2 = 35 mm Hg), cells were placed in a modular incubation chamber (Billups-Rothenberg, Del Mar, CA) and exposed to 95% N2 and 5% CO2 for 30 min; the chamber was then sealed and returned to the incubator. Experiments with hypoxic cells were performed inside an inflatable glove bag purged with 95% N2 and 5% CO2 to maintain hypoxic conditions.

5'-Rapid Amplification of cDNA Ends (5'-RACE)—Human kidney RACE-ready cDNA (Ambion Inc., Austin, TX) was amplified using three GPx-3 gene-specific antisense primers, 5'-GAGGACGTATTTGCCAGCAT-3' (located in exon 2), 5'-GAGACCCTTGCAGCCAAATC-3' (located 152 bp downstream of the published tsp), and 5'-ACAGCGGTCTCCATTACAGC-3' (located 24 bp upstream of the published tsp), in combination with the manufacturer's 5'-RACE sense primer, 5'-GCTGATGGCGATGAATGAACACTG-3', which anneals to an adapter region ligated immediately 5' to the cDNA end. PCR was performed in a 50-µl volume with 0.5 ng of cDNA, 0.4 µM sense and antisense primers, 0.2 mM dNTP, 4.5 mM MgCl2, 1 unit of AmpliTaq Gold (Applied Biosystems, Foster City, CA), and 10x buffer components. The PCR product was excised from a 1% low melting point agarose gel, purified using a Wizard PCR prep DNA purification system (Promega, Madison, WI), and ligated into a TA-cloning vector (Invitrogen) for sequencing.

GPx-3 Reporter Constructs—A reporter construct containing ~1300 bp of the GPx-3 5'-region (positions –1237 to +35) was generated by PCR amplification of genomic DNA using the sense primer 5'-ATATGGTACCGTCAGAGAGATTTGAGACTGATTCC-3' and the antisense primer 5'-CCTAAAGCTTTGAGCCGCTGCCTGATCCC-3', which contain a KpnI site and a HindIII site, respectively (underlined). This construct was designated the full-length construct (construct A). Four deletion constructs (constructs B–E) were generated with the same antisense primer described above and the sense primers shown in Table I, each of which included a KpnI site. The PCR mixtures contained ~200 ng of genomic DNA, 0.2 µM each primer, 0.2 mM dNTPs, 1.5 mM MgCl2, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, and 1 unit of high fidelity Taq polymerase (Roche Diagnostics) in a final volume of 25 µl. After initial denaturation at 95 °C for 2 min, PCR was carried out for 35 cycles, each composed of denaturation at 95 °C for 30 s, annealing at 62 °C for 30 s, and extension at 72 °C for 1 min, with a final extension time of 7 min at 72 °C. All of the above fragments were constructed so that the 3'-end terminated downstream of the newly proposed tsp and upstream of the ATG site. To obtain an additional construct lacking the new tsp (construct F), the full-length fragment was digested at an XmaI site in the GPx-3 sequence, located at position –147. Each of the fragments was digested and cloned into the corresponding sites (KpnI and HindIII sites for constructs A–E and KpnI and XmaI sites for construct F) of the pGL3-basic reporter vector (Promega) with T4 DNA ligase (Invitrogen) and transformed into competent HB101 cells (Promega). Plasmids were amplified using endotoxin-free maxiprep kits (EndoFree plasmid maxi kit, QIAGEN Inc., Valencia, CA). Restriction enzymes were purchased from New England Biolabs Inc. (Beverly, MA).


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  		TABLE I
Primers used for amplification of inserts for GPx-3 deletion constructs

Underlined sequences in the sense primers indicate a KpnI site, and the underlined sequence in the antisense primer indicates a HindIII site.

 
DNA Sequencing—All constructs and 5'-RACE products were sequenced following a fluorescence dideoxy chain termination method using an automated sequencer (Big Dyes Model ABI 377-96, Applied Biosystems). DNA sequencing was performed by the Molecular Genetics Core Laboratory at the Boston University School of Medicine.

Transient Transfections of GPx-3 Reporter Constructs—Four different cell types were transfected with the GPx-3-luciferase constructs: Caki-2, human vascular endothelial, HTB13, and HEK293 cells. The transfection methods were optimized for each cell type. Caki-2 cells were seeded in 12-well plates and the remaining cell types in 24-well plates and allowed to reach 70–90% confluence. For Caki-2 cells, 2 µg of luciferase construct, 20 ng of pRL-CMV (cytomegalovirus-Renilla luciferase construct, Promega), and 3 µl of Superfect (QIAGEN Inc.) were combined with serum-free medium for a total volume of 75 µl. This mixture was incubated for 10 min before adding 400 µl of prewarmed complete medium. Cells were washed twice with serum-free medium, and the transfection mixture was added to the cells for 2 h at 37 °C. The transfection mixture was removed by washing twice with phosphate-buffered saline and replaced with complete medium until cells were either treated with conditioned medium or harvested.

For human vascular endothelial cells, 0.5 µg of luciferase construct, 0.133 µg of pRL-CMV, and 2.5 µl of Superfect were combined with serum-free medium for a total volume of 37.5 µl and incubated for 10 min. Complete medium (200 µl) was then added, and the transfection mixture was incubated with the previously washed cells. The transfection was stopped after 2 h by washing twice with phosphate-buffered saline and adding complete medium for 22 h.

For HTB13 and HEK293 cells, a transfection mixture containing 0.105 µg of pRL-TK (thymidine kinase-Renilla luciferase construct, Promega), 0.21 µg of luciferase construct, 2.1 µl of LipofectAMINE (Invitrogen), and 17 µl of Opti-MEM I (Invitrogen) was incubated at room temperature for 15–45 min according to the manufacturers' instructions. Immediately prior to use, 170 µl of prewarmed (37 °C) Opti-MEM I was added to the mixture. The cells were washed twice with serum-free medium, and the transfection mixture was incubated with the cells for 5 h at 37 °C. The transfection mixture was removed and replaced with complete medium for 22 h.

Luciferase Assays—Cells were harvested by washing twice with phosphate-buffered saline without calcium (Invitrogen) and then rocked in the presence of 100 µl (for 24-well plates) or 250 µl (for 12-well plates) of passive lysis buffer (Promega) for 1–2 h. All luciferase measurements (Dual-Reporter assay, Promega) were made using a Turner TD-20e luminometer according to the manufacturer's instructions. Firefly luciferase luminescence measurements were normalized to Renilla luciferase luminescence measurements prior to any additional comparison or analysis.

Generation of GPx-3 cDNA—GPx-3 cDNA was generated by reverse transcriptase-PCR on RNA purified from Caki-2 cells by standard methods using 5'-AGGGATCAGGCAGCGGCTCAG-3' as the sense primer and 5'-CTGCAGAAAGGCTTTTACTGGGCAGA-3' as the antisense primer. The PCR conditions were as follows: 1-min denaturing at 94 °C, 1-min annealing at 65 °C, and 2-min extension at 72 °C, lengthened by 15 s every cycle. The final product was gel-purified, directly cloned into a pCR-XL-TOPO vector (Invitrogen), sequenced, transferred to a pCR2.1 vector (Invitrogen), and resequenced. From this vector, the cDNA was digested with HindIII and NotI and ligated into the same sites of the pcDNA3.1 vector (Invitrogen).

GPx-3 Protein Expression—HEK293 cells were plated in either 100-mm dishes or 6-well plates and grown until 60–70% confluent prior to treatment. Experimental conditions are described for 100-mm dishes. Selenium incorporation was performed after overnight recovery from transfection using published procedures (23). The transfection medium included 75 µl LipofectAMINE with the GPx-3 plasmid (12.5 µg). When noted, cofactors were present at 2.5 µg. The cells were transfected for 8 h (7.2-ml total volume), after which the transfection medium was replaced with complete medium for 16 h. The cells were then incubated with 5 ml of serum-free medium containing 15 µCi of 75Se (3 µCi/ml; University of Missouri Reactor) and 100 nM sodium selenite for an additional 24 h. The harvested medium was combined with lithium dodecyl sulfate sample buffer and reducing agent (Invitrogen) according to the manufacturer's recommendations. Samples were denatured and electrophoresed through NuPAGE® BisTris gels (12%, pH 6.4) in the presence of MOPS buffer (NuPAGE® system, Invitrogen). Gels were stained with Coomassie Blue, dried, and exposed to x-ray film. Constructs for SelD (selenophosphate synthetase D) (GenBankTM/EBI accession number U34044 [GenBank] ), selenocysteine-specific tRNASec from Xenopus laevis (accession number M34507 [GenBank] ), and SECIS-binding protein-2 (SBP2) (accession number NM_024002 [GenBank] ) were kind gifts from Drs. Marla Berry, Dolph Hatfield, and Paul Copeland, respectively.

Statistics—Statistical analysis was performed with SigmaStat Version 3.0.1 (SPSS Inc., Chicago, IL). Comparison between two groups was performed by unpaired two-tailed Student's t test. Multiple groups were compared by one-way analysis of variance, followed by post hoc analysis with the Student-Newman-Keuls test for pairwise comparison. All values are given as means ± S.E.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
5'-RACE—Previously published chromosomal alignment and primer extension studies have suggested that the tsp of the GPx-3 gene is located 298 bp upstream of its translation start site (6). Preliminary work with this start site showed little promoter activity in a luciferase-based reporter construct in Caki-2 cells, a cell line derived from renal proximal tubules, and prompted the use of a RACE procedure to identify potential alternative start sites in these cells. We used human kidney RACE-ready cDNA with an adapter sequence ligated to its 5'-end. A sense primer specifically designed against the adapter sequence was used in combination with three different antisense primers specific to GPx-3: upstream of the published tsp, in exon 1 relative to the published tsp, and in exon 2. The only combination of primers that resulted in a reproducible RACE product was that with the antisense primer based in exon 2 (Fig. 1). Sequencing of this product confirmed that it aligns with the published GPx-3 cDNA sequence and showed that it contains 64 bp of 5'-untranslated sequence. This result suggested the location of a novel tsp (Fig. 2) 233 bp downstream of the tsp published previously by Yoshimura et al. (6).



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  		FIG. 1.
5'-RACE. RACE-ready cDNA prepared from human kidney was amplified using a sense primer against an adapted 5'-end in combination with an antisense primer specific to exon 2 (lane D). Lane A, 1-kb plus DNA weight marker; lane B, no cDNA; lane C, no adapter primer.

 



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  		FIG. 2.
Plasma glutathione peroxidase promoter sequence. The GPx-3 promoter sequence located between the previously reported and the newly proposed transcription start sites is shown. All numbering is relative to the new transcription start site (+1), indicated by the black arrowhead. The previously published transcription start site (position –233) is indicated by the white arrowhead. Coding sequence is shown in boldface uppercase letters (position +65). Sequences showing homology to consensus binding sites of known transcription factors are underlined. The CCAAT box is in reverse orientation and partially overlaps with the MRE.

 
Sequence Analysis—Three sequences for the GPx-3 promoter are available in the GenBankTM/EBI Data Bank: accession numbers D16360 [GenBank] (6), AF285633 [GenBank] (20), and AY310878 [GenBank] (24). Our sequence (accession number AY552097 [GenBank] ) is 100% homologous to that reported most recently by Rieder et al. (accession number AY310878 [GenBank] ) (24). We analyzed the sequence located between the published tsp and our newly identified downstream tsp for conserved sequence motifs and potential transcription factor-binding sites by identifying consensus sequences (with the Web-based program MatInspector (25) and with MacVector Version 7 (Accelyrs, San Diego, CA)). We found a classical CCAAT box in reverse orientation at positions –77 to –81, but no TATTA box. In addition, we identified a metal response element (MRE) from positions –73 to –79, a GC-rich region from positions –104 to –109 corresponding to an Sp-1 site, an antioxidant response element (ARE) from positions –148 to –158, and a hypoxia-inducible factor-1 (HIF-1)-binding site from positions –203 to –209. The positions of these newly described elements are shown in Fig. 2.

Promoter Function—Caki-2 cells have previously been shown to express GPx-3 in greater quantities compared with cell lines from other source organs (21). To test whether the newly identified promoter sequence could drive basal expression, luciferase constructs containing varying lengths of the GPx-3 promoter were transfected into Caki-2 cells. Experiments were run in triplicate and performed a minimum of three times. Expression levels measured after 24 h as the ratio of firefly-to-Renilla luciferase luminescence are shown in Fig. 3. All constructs that included the newly identified tsp (constructs A–E) resulted in ~25-fold higher activity compared with the construct based on the published tsp alone (construct F) (p < 0.0001). Expression levels were similar for constructs A–E, suggesting that elements critical for promoter function lie within the 180 bp immediately upstream of the newly proposed tsp.



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  		FIG. 3.
Basal expression of GPx-3 constructs in Caki-2 cells. GPx-3 promoter fragments of varying length were cloned in front of the firefly luciferase reporter gene and transfected into Caki-2 cells. Cells were cotransfected with a Renilla luciferase vector. Expression levels were measured after 24 h as the ratio of firefly to Renilla luciferase luminescence. All constructs are numbered relative to the new tsp (position +1). Constructs A–E contain the new tsp, whereas construct F isolates the published tsp (position –233). All constructs are in the pGL3-basic vector. Experiments were performed in triplicate and repeated a minimum of three times. Ratios of firefly to Renilla luciferase are presented with their respective S.E. values. *, p < 0.0001 compared with construct F.

 
To determine whether this new tsp functions in other cell types, we transfected three additional cell types: HEK293 cells (another kidney-derived cell line), human vascular endothelial cells (because of their contact with plasma), and HTB13 cells (as selenoproteins are reported to be most stable in the brain) (18). To account for differences in transfection efficiency among cell types, the average ratio of firefly-to-Renilla luciferase luminescence was normalized to the average ratio produced by the pGL3 enhancer vector. As observed in the Caki-2 cells, the constructs containing the new downstream start site produced activity that was significantly higher than that of the construct lacking the new start site. The relative level of expression of all GPx-3 constructs in Caki-2 cells was, however, ~10-fold higher than that in the other cell types (data not shown); for this reason, all subsequent experiments were performed in Caki-2 cells.

Treatment with Oxidants—Comhair et al. (20) reported an increase in GPx-3 promoter activity and in GPx-3 mRNA expression in BET1A cells (a human bronchial epithelial cell line that expresses GPx-3) in response to a combination of 100 µM pyrogallol (a superoxide generator) and 10 mM GSH. Aside from this study, no other work has provided evidence for transcriptional regulation of the GPx-3 gene. There is evidence, however, that GPx-1, the cellular isoform of GPx, is regulated by oxidant stress; and thus, we postulated that GPx-3 might have similar transcriptional determinants. We treated Caki-2 cells transfected with the full-length construct A with 100 µM hydrogen peroxide (relative luminescence (RL) = 13.2 ± 2.1), 100 µM tert-butyl hydroperoxide (RL = 16.3 ± 0.6), or 100 µM pyrogallol (RL = 18.7 ± 1.9) for 2 h and found no significant change in expression levels compared with untreated cells (RL = 15.8 ± 1.7). In addition, we were unable to reproduce the significant stimulation of promoter activity with GSH and pyrogallol described by Comhair et al. (20) using this new promoter in Caki-2 cells (data not shown).

Modulation by Hypoxia—As hypoxia has been shown to induce antioxidant enzyme expression and as the proposed new promoter sequence contains an HIF-1-binding site, we exposed Caki-2 cells transfected with the full-length promoter construct A to hypoxic conditions for 6-h intervals up to 24 h. Values were normalized to the expression levels of the full-length construct A in normoxia at 6 h. The transcriptional activity of the GPx-3 construct in normoxia remained relatively stable during the 24-h period. In contrast, a progressive increase in GPx-3 transcriptional activity was observed under hypoxic conditions; for each time point, the GPx-3 construct maintained in hypoxia showed higher expression than its normoxic counterpart, with a maximal 2.9-fold increase reached at 24 h (normalized RL = 3.5 ± 0.3 versus 1.2 ± 0.1; p < 0.001) (Fig. 4A). This induction of GPx-3 expression by hypoxia was consistent for all constructs, with the exception of construct E (normalized RL = 2.0 ± 0.1), which lacks the HIF-1 response element (Fig. 4B).



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  		FIG. 4.
Regulation of GPx-3 expression by hypoxia. A, Caki-2 cells transfected with the full-length GPx-3 promoter construct were maintained in normoxia (gray bars) or exposed to 95% N2 and 5% CO2 for 6-h intervals up to 24 h (hatched bars). Expression levels were measured as the ratio of firefly-to-Renilla luciferase luminescence and normalized to the full-length construct in normoxia at 6 h. *, p < 0.01; **, p < 0.001 compared with expression levels at the same time point in normoxia. B, luciferase constructs containing varying lengths of the GPx-3 promoter (constructs A–E) were transfected into Caki-2 cells and maintained in hypoxia for 24 h (hatched bars). Expression levels were normalized to the full-length construct A in normoxia at 24 h (gray bar). All GPx-3 constructs showed a similar induction of expression under hypoxic conditions, with the exception of construct E. *, p < 0.01 compared with construct A in hypoxia. All experiments were run in triplicate and performed a minimum of three times.

 
In an attempt to modulate the effects of hypoxia on GPx-3 gene expression, we treated hypoxic cells transfected with the full-length construct A with the nitric oxide donors S-nitrosoglutathione and diethylenetriamine/nitric oxide as well as with the thiol N-acetylcysteine and the thiol precursor oxothiazolidinecarboxylic acid; these latter two species increase intracellular levels of reduced glutathione and therefore have antioxidant properties. All treatments were performed for 2 h after the cells had been maintained in hypoxia for 22 h. Cells treated with either of the NO donors (normalized RL after treatment with 0.5 mM S-nitrosoglutathione = 2.5 ± 0.2 and with 5 mM diethylenetriamine/nitric oxide = 2.7 ± 0.2) showed some reduction in transcriptional activity compared with untreated cells (normalized RL = 3.0 ± 0.4), yet this effect was not significant. In contrast, treatment with 20 mM N-acetylcysteine (normalized RL = 1.5 ± 0.5) or 1 mM oxothiazolidinecarboxylic acid (normalized RL = 1.4 ± 0.03) significantly attenuated the transcriptional activation induced by hypoxia, reducing the expression levels almost to that of the normoxic construct (Fig. 5). Treatment of transfected cells maintained in normoxia with these redox-active substances did not significantly influence translational activity (data not shown). These findings indicate that cellular redox status modulates the response of the GPx-3 promoter to hypoxia.



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  		FIG. 5.
Treatment with thiols. Hypoxic cells transfected with the full-length construct A were maintained in hypoxia for 22 h and then treated for 2 h with 0.5 mM S-nitrosoglutathione (SNO-Glu), 5 mM diethylenetriamine/nitric oxide (DETA-NO), 20 mM N-acetylcysteine (NAC), or 1 mM oxothiazolidinecarboxylic acid (OTC) (hatched bars). Expression levels were normalized to the untreated full-length construct in normoxia at 24 h (gray bar). Cells treated with the NO donors showed a non-significant reduction in transcriptional activity compared with hypoxic untreated cells. However, treatment with the thiols N-acetylcysteine and oxothiazolidinecarboxylic acid reduced the expression levels almost to that of the normoxic control construct. *, p < 0.01 compared with untreated construct A in hypoxia.

 
Overexpression of GPx-3—To determine the effects of translational cofactors on GPx-3 expression, we used 75Se to radiolabel newly synthesized selenocysteine proteins. This assay detected only recombinant GPx-3 in the medium. Transfection of the renal cell line HEK293 with cDNA for GPx-3 and the translational cofactors tRNASec and SelD resulted in clearly detectable protein (Fig. 6, lanes 1 and 2), whereas no band was detected when only these cofactors were transfected without GPx-3 cDNA (lanes 3 and 4). It has previously been shown that overexpression of the known translational cofactors can enhance expression of (other) selenoproteins (23, 26, 27). To test the effect of known cofactors in the GPx-3 cell system, we cotransfected HEK293 cells with plasmids containing SelD, SBP2, and tRNASec each over a range of concentrations. To account for any changes in expression from increases in the total DNA transfected, parallel experiments included a green fluorescent protein cDNA-containing plasmid that was cotransfected with the GPx-3 cDNA-containing plasmid. Increases in expression, relative to parallel transfections with green fluorescent protein cDNA, were observed with cotransfection of the tRNASec, SelD, and SBP2 constructs (Fig. 7). In the presence of the tRNASec cDNA (Fig. 7A) or the SelD cDNA (Fig. 7B), a statistically significant increase in expression of GPx-3 protein was apparent. In the presence of the SBP2 cDNA (Fig. 7C), a trend toward increased GPx-3 protein expression was observed. These results show dependence of GPx-3 protein expression on translational cofactors unique to selenocysteine-containing proteins.



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  		FIG. 6.
Overexpression of GPx-3. Cells transfected with GPx-3 cDNA, X. laevis tRNASec, and SelD were labeled with 3 µCi/ml 75Se as sodium selenide for 24 h (lanes 1 and 2). Control cells were transfected with the X. laevis selenocysteine-specific tRNASec and SelD, but not with GPx-3 (lanes 3 and 4). Equal loading of samples and size were determined by Coomassie Blue staining of the gel (not shown) prior to drying and exposure to x-ray film.

 



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  		FIG. 7.
Effect of cotransfection of cofactors on GPx-3 protein expression. Increasing concentrations of tRNASec (A), SelD (B), and SBP2 (C) cDNAs were combined with 1 µg of GPx-3 cDNA. Parallel transfections with the same amount of green fluorescent protein were included as a negative control (not shown). The density of each band was measured, and the relative intensity to the no-cofactor control was calculated. These values were then compared with green fluorescent protein and are presented relative to the latter. Representative blots are presented for each concentration above the graphs of densitometric values. The presence of SBP2 resulted in an overall trend (p = 0.104) toward increased expression, whereas the presence of tRNASec (p = 0.024) and SelD (p = 0.031) both significantly increased expression of GPx-3. *, p < 0.05 relative to the no-cofactor control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The regulation of expression of the human GPx-3 gene is a complex process and involves transcriptional and translational events that are incompletely understood. Although the kidney is the primary source of GPx-3, most regulation studies have focused on GPx-3 expression in the lung, with inconsistent results. Pulmonary epithelial cells exposed to hyperoxia and lung lavage fluid from patients exposed to ozone both showed a decrease in GPx-3 activity compared with controls (28); in contrast, an increase in GPx-3 activity in lavage fluid was observed in mice exposed to hyperoxia (29). Another study showed no change in GPx-3 mRNA expressed in the airway epithelia and alveolar macrophages of patients given 12 h of 100% oxygen (30). These same investigators reported an induction of GPx-3 mRNA in response to cigarette smoke and, in a later study, found that patients with asthma have higher levels of GPx-3 mRNA in their airway epithelia than do healthy controls (20). The variety of responses reported by three different investigators using similar induction parameters in related cell types underscores the need for further understanding of the transcriptional and translational regulation of GPx-3.

To study the regulation of GPx-3 expression at the transcriptional level, we initially cloned the 5'-flanking sequence of the GPx-3 gene based on the published tsp located 298 bp upstream of the translation start site (6). Previous studies have detected no basal activity with promoter constructs based upon this site (20); however, unstimulated cells have been shown to contain GPx-3 mRNA (21), suggesting that basal enzyme activity should be detectable. We confirmed the lack of basal activity of luciferase constructs with the published tsp in renal Caki-2 cells and, by 5'-RACE analysis of human kidney extracts, identified a novel tsp located 233 bp downstream of the published tsp. The novel promoter based on this newly identified tsp (GenBankTM/EBI Accession Number AY552097 [GenBank] ) has basal activity in Caki-2 cells, shows cell-type specificity, and is strongly regulated by hypoxia.

Our analysis of this novel GPx-3 promoter identified a number of sequence motifs and consensus transcription factor-binding sites that had not been previously identified. Unlike the previously described promoter, which does not contain any conserved sequence motifs, the novel promoter has a classical CCAAT box 80 bp upstream of the tsp. A GC-rich region corresponding to a binding site for Sp-1, a classical transcriptional activator, was found ~100 bp upstream of the tsp. In addition, two response elements (MRE and ARE) that are susceptible to regulation by ROS and/or antioxidants were identified within the first 160 bp of the promoter. The MRE is responsive mainly to heavy metals, but can also be induced by oxidative stimuli such as hydrogen peroxide and tert-butylhydroquinone (31). Similarly, the ARE (also referred to as electrophile response element) represents a cis-acting regulatory element that is responsive to hydrogen peroxide and tert-butylhydroquinone. The ARE is best characterized in the promoters of phase II drug-metabolizing enzyme genes, but has also been identified in genes that are transcriptionally activated to protect eukaryotic cells against oxidative stress, such as genes encoding glutathione S-transferase, quinone reductase, and {gamma}-glutamylcysteine synthetase (32).

In addition to these response elements, we identified a binding site for HIF-1 located ~200 bp upstream of the tsp. HIF-1 is a transcription factor involved in the global regulation of oxygen homeostasis and the physiological responses to hypoxia and is known to regulate transcription of >50 downstream target genes involved in glucose and energy metabolism, erythropoiesis, cell proliferation, and vascular development and remodeling (33). HIF-1 is a heterodimer composed of HIF-1{alpha} and HIF-1{beta} subunits; the stability of HIF-1{alpha} is tightly regulated by cellular O2 concentration, being degraded more rapidly as the O2 concentration increases. Among the glutathione peroxidases, the GPx-3 gene is the only one in which an HIF-1-binding site has been identified. The cellular isoform of GPx (GPx-1) has recently been reported to be regulated by oxygen tension, but through the presence of two oxygen-responsive elements that, in contrast to HIF-1, activate transcription in response to increasing O2 levels (34). The strong increase in expression of all GPx-3 constructs during hypoxia, with the exception of the construct lacking the HIF-1-binding site, supports the importance of this transcription factor in regulating GPx-3 levels. Expression levels were nevertheless increased even by this construct, which is most likely due to the presence of the other redox-sensitive elements, the ARE and MRE sites.

Hypoxia leads to the formation of ROS, mainly generated by mitochondria (35–37), and there is strong evidence that these ROS play a central role in promoting ischemic injury in vitro and in vivo (38). Thus, susceptibility to these toxic compounds depends in part on the ability to up-regulate ROS-scavenging enzymes such as GPx-3. Oxidative stress and/or variations in the intracellular redox state are well established modulators of the transcription of several genes (39). Indeed, our results support that the up-regulation of GPx-3 in the setting of hypoxia is, at least in part, redox-mediated, as the effect was modulated by treatment with thiols.

The association between decreased GPx-3 activity, arterial thrombosis, and the clinical manifestations of ischemic stroke (14) and coronary artery disease (40–42) indicates that the normal function of this enzyme is important for vascular homeostasis. Nevertheless, some controversy has surrounded the role of plasma GPx-3 as an antioxidant enzyme in plasma because of the relatively low plasma concentration of GSH. Thioredoxin and glutaredoxin have been shown to function effectively as electron donors for GPx-3 in in vitro assays (43) at concentrations well below their normal plasma levels (nanomolar range) and have been proposed to serve as reducing cofactors. In addition, a recent study has indicated that the apparent requirement for high concentrations of GSH was an artifact of assay conditions that resulted from the use of Tris buffer (44), thus suggesting that plasma GSH levels may after all be sufficient to act as a reductant for this enzyme.

Regulation of selenoproteins is unique in that their translation machinery enables an opal codon (UGA) to be read as coding for selenocysteine. Several translational cofactors have been described as necessary, but not alone sufficient, for synthesis of these proteins. SelD is responsible for the biosynthesis of selenocysteine from an L-serine bound to a special tRNASec that can recognize the UGA codon (45). Actual incorporation of selenocysteine into the nascent polypeptide involves the presence of a stem-loop structure formed by a selenocysteine insertion sequence (SECIS) in the 3'-untranslated region of the mRNA and SBP2, a SECIS-binding protein that recruits other factors necessary for incorporation (26). The fact that translation of these proteins requires a complex of translational cofactor proteins inherently leads to multiple potential levels of regulation: transcriptional, post-transcriptional, and translational. For example, work with cellular glutathione peroxidase (GPx-1) has shown that its mRNA is subject to regulation by selenium at the level of degradation (46). Because of the complexity of translation, it is possible that significant regulation occurs post-transcriptionally; experiments are currently underway to address this issue.

To examine potential regulation at the translational level, we have successfully overexpressed GPx-3 and present results showing regulation by these translational cofactors. Overexpression of selenoproteins is difficult. Barkats et al. (47) showed that overexpression of GPx-1 results in, at most, a 2-fold increase in activity when being driven by an SV40 promoter in an adenoviral system. Brigelius-Flohe et al. (27) transfected an endothelial cell line with GPx-4 and SelD and observed no increase in activity; however, when SelD was transfected into cells that had been stably transfected with the GPx-4 cDNA, a 5-fold increase in expression was detectable. Another study provided evidence for a contribution of SBP2 to selective up-regulation of constructs containing SECIS elements specific for GPx-4 and SelP (26). Here, we have presented data suggesting modulation of GPx-3 translation by SelD, tRNASec, and SBP2.

In summary, we have identified a novel and functional transcription start site that results in significantly higher basal transcriptional activity in Caki-2 cells compared with the previously characterized promoter. Alternative transcription has also recently been described for another member of the glutathione peroxidase family: phospholipid GPx (48, 49). For the first time, hypoxia has been identified as a strong transcriptional regulator of GPx-3 expression, likely through an HIF-1-dependent mechanism. In addition, we have successfully over-expressed GPx-3 protein with evidence that the presence of SBP2, tRNASec, and SelD regulates protein expression. These data represent novel findings in this complex field of selenocysteine-containing protein expression.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants HL55993, HL58976, and HL61795 (to J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY552097 [GenBank] . Back

Both authors contributed equally to this work. Back

|| To whom correspondence should be addressed: Whitaker Cardiovascular Inst., Boston University School of Medicine, 715 Albany St., W507, Boston, MA 02118. Tel.: 617-638-4890; Fax: 617-638-4066; E-mail: jloscalz{at}bu.edu.

1 The abbreviations used are: GPx-3, plasma glutathione peroxidase; ROS, reactive oxygen species; tsp, transcription start point; FBS, fetal bovine serum; RACE, rapid amplification of cDNA ends; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MOPS,4-morpholinepropanesulfonic acid; SECIS, selenocysteine insertion sequence; SBP2, SECIS-binding protein-2; MRE, metal response element; ARE, antioxidant response element; HIF-1, hypoxia-inducible factor-1; RL, relative luminescence. Back

2 J. Freedman, N. Avissar, and J. Loscalzo, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Ying-Yi Zhang, Shelley Russek, and Norbert Weiss for expert advice and thoughtful criticism.



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