Selenoprotein H Is a Redox-sensing High Mobility Group Family DNA-binding Protein That Up-regulates Genes Involved in Glutathione Synthesis and Phase II Detoxification

Abstract

Selenoprotein H is a recently identified member of the selenoprotein family whose function is not fully known. Previous studies from our laboratory and others showed that Drosophila melanogaster selenoprotein H is essential for viability and antioxidant defense. In this study we investigated the function of human selenoprotein H in murine hippocampal HT22 cells engineered to stably overexpress the protein. After treatment of cells with l-buthionine-(S,R)-sulfoximine to deplete glutathione, selenoprotein H-overexpressing cells exhibited higher levels of total glutathione, total antioxidant capacities, and glutathione peroxidase enzymatic activity than did vector control cells. Overexpression of selenoprotein H also up-regulated the mRNA levels of endogenous selenoprotein H, glutamylcysteine synthetase heavy and light chains, and glutathione S-transferases Alpha 2, Alpha 4, and Omega 1. The amino acid sequence of selenoprotein H contains four putative nuclear localization sequences and an AT-hook motif, a small DNA-binding domain first identified in high mobility group proteins. Chromatin immunoprecipitation using a green fluorescent protein-selenoprotein H fusion revealed binding to sequences containing heat shock and/or stress response elements. Thus, selenoprotein H is a redox-responsive DNA-binding protein of the AT-hook family and functions in regulating expression levels of genes involved in de novo glutathione synthesis and phase II detoxification in response to redox status. Selenoprotein H is a recently identified member of the selenoprotein family whose function is not fully known. Previous studies from our laboratory and others showed that Drosophila melanogaster selenoprotein H is essential for viability and antioxidant defense. In this study we investigated the function of human selenoprotein H in murine hippocampal HT22 cells engineered to stably overexpress the protein. After treatment of cells with l-buthionine-(S,R)-sulfoximine to deplete glutathione, selenoprotein H-overexpressing cells exhibited higher levels of total glutathione, total antioxidant capacities, and glutathione peroxidase enzymatic activity than did vector control cells. Overexpression of selenoprotein H also up-regulated the mRNA levels of endogenous selenoprotein H, glutamylcysteine synthetase heavy and light chains, and glutathione S-transferases Alpha 2, Alpha 4, and Omega 1. The amino acid sequence of selenoprotein H contains four putative nuclear localization sequences and an AT-hook motif, a small DNA-binding domain first identified in high mobility group proteins. Chromatin immunoprecipitation using a green fluorescent protein-selenoprotein H fusion revealed binding to sequences containing heat shock and/or stress response elements. Thus, selenoprotein H is a redox-responsive DNA-binding protein of the AT-hook family and functions in regulating expression levels of genes involved in de novo glutathione synthesis and phase II detoxification in response to redox status. Selenium has long been known for its antioxidant properties, and accumulated evidence indicates that many of the beneficial effects of this trace element in our diet are attributable to selenoenzymes. The functions of selenoenzymes include protecting cell membranes, proteins, and nucleic acids from cumulative oxidative damage and maintaining cellular redox balance. Selenium is highly retained in neuronal tissue during selenium deficiency (1Behne D. Hilmert H. Scheid S. Gessner H. Elger W. Biochim. Biophys. Acta. 1988; 996: 12-21Crossref Scopus (390) Google Scholar), and the functions of selenoproteins in the brain are highlighted by the development of neurological defects in mice that underwent targeted disruption of selenoprotein P, a selenium transport protein whose functions may also include antioxidant defense and heavy metal chelation (2Schomburg L. Schweizer U. Holtmann B. Flohe L. Sendtner M. Kohrle J. Biochem. J. 2003; 370: 397-402Crossref PubMed Scopus (342) Google Scholar, 3Hill K.E. Zhou J. McMahan W.J. Motley A.K. Atkins J.F. Gesteland R.F. Burk R.F. J. Biol. Chem. 2003; 278: 13640-13646Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). To date, 25 selenoprotein genes have been identified in the human genome (4Kryukov G.V. Castellano S. Novoselov S.V. Lobanov A.V. Zehtab O. Guigo R. Gladyshev V.N. Science. 2003; 300: 1439-1443Crossref PubMed Scopus (1777) Google Scholar), but the functions of many of them are yet to be fully defined. Selenoprotein H (SelH) 3The abbreviations used are: SelH, selenoprotein H; ABTS, 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid); AOP, antioxidant potential; ARE/EpRE, antioxidant response elements/electrophile response elements; BSO, l-buthionine-(S,R)-sulfoximine; FRAP, ferric-reducing ability of plasma; GCS, glutamylcysteine synthetase; GPx, glutathione peroxidase; GR, glutathione reductase; GST, glutathione S-transferase; HMG, high mobility group; HSE, heat shock element; mGAPDH, mouse glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; STRE, stress response element; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; TEAC, Trolox equivalent antioxidant capacity; TPTZ, 2,4,6-tri-pyridyl-s-triazine; GFP, green fluorescent protein; ANOVA, analysis of variance. 3The abbreviations used are: SelH, selenoprotein H; ABTS, 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid); AOP, antioxidant potential; ARE/EpRE, antioxidant response elements/electrophile response elements; BSO, l-buthionine-(S,R)-sulfoximine; FRAP, ferric-reducing ability of plasma; GCS, glutamylcysteine synthetase; GPx, glutathione peroxidase; GR, glutathione reductase; GST, glutathione S-transferase; HMG, high mobility group; HSE, heat shock element; mGAPDH, mouse glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; STRE, stress response element; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; TEAC, Trolox equivalent antioxidant capacity; TPTZ, 2,4,6-tri-pyridyl-s-triazine; GFP, green fluorescent protein; ANOVA, analysis of variance. was initially identified in the Drosophila melanogaster genome and subsequently in the human and mouse genomes, where expression is high in the brain. In previous studies we and others showed that D. melanogaster SelH is required for viability, and overexpression of the protein increased antioxidant capacity in Drosophila embryo-derived Schneider S2 cells when exposed to homocysteic acid-induced GSH depletion (5Morozova N. Forry E.P. Shahid E. Zavacki A.M. Harney J.W. Kraytsberg Y. Berry M.J. Genes Cells. 2003; 8: 963-971Crossref PubMed Scopus (41) Google Scholar, 6Kwon S.Y. Badenhorst P. Martin-Romero F.J. Carlson B.A. Paterson B.M. Gladyshev V.N. Lee B.J. Hatfield D.L. Mol. Cell. Biol. 2003; 23: 8495-8504Crossref PubMed Scopus (12) Google Scholar). Two recent studies have begun to investigate the functions of human SelH. We reported that overexpression of human SelH protects against UV-induced cell death via a decrease in superoxide levels (7Ben Jilani K.E. Panee J. He Q. Berry M.J. Li P.A. Int. J. Biol. Sci. 2007; 3: 198-204Crossref PubMed Google Scholar). A study employing bioinformatic analysis and gene silencing identified a thioredoxin-fold motif in SelH and demonstrated a protective role for the protein against hydrogen peroxide-induced cell death (8Novoselov S.V. Kryukov G.V. Xu X.M. Carlson B.A. Hatfield D.L. Gladyshev V.N. J. Biol. Chem. 2007; 282: 11960-11968Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In the present study, we investigated the function of SelH in neuronal cells against oxidative stress induced by GSH depletion. Murine hippocampal neuronal HT22 cells were engineered to stably overexpress human SelH and subjected to l-buthionine-(S,R)-sulfoximine (BSO) treatment to inactivate γ-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis. The effects of BSO treatment on antioxidant parameters and gene expression were assessed in SelH-overexpressing and vector control HT22 cells. These studies revealed that expression of SelH protected intracellular GSH and antioxidant levels and increased expression of key enzymes in GSH biosynthesis and in phase II detoxification. To gain insight into how SelH might up-regulate expression of these genes, the deduced amino acid sequence was analyzed using ExPASy analysis tools. The SelH sequence contains multiple nuclear localization signals, and we show that the protein localizes predominantly to the nucleus. Alignment with known members of the high mobility group (HMG) family of DNA-binding proteins revealed the presence of an AT-hook DNA-binding motif. Chromatin immunoprecipitation using a GFP-SelH fusion protein and antibodies to GFP revealed binding to sequences containing multiple heat shock or stress response elements. These findings identify SelH as a redox-sensing DNA-binding protein that up-regulates transcription of genes involved in antioxidant defense and phase II detoxification. Cell Maintenance and Sample Harvesting—All of the cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 15 μg/ml gentamicin, 50 μg/ml ampicillin, with 4 mml-glutamine supplement at 5% CO2 and ∼50% relative humidity. The selenium concentration in the serum-supplemented medium used in this study was ∼56 nm, as determined by inductively coupled plasma mass spectrometry analysis of the serum. Cells in 100 mm cell culture dishes were subjected to BSO treatment when the optical confluency reached ∼90%. After treatment, the dishes were transferred onto ice, and the medium was aspirated. The cells were washed once with cold phosphate-buffered saline (PBS), trypsinized, and then suspended in 2 ml of fetal bovine serum-free Dulbecco’s modified Eagle’s medium. 10 μl of cell suspension was taken out in duplicate for counting cell numbers, and 1900 μlof cell suspension were transferred into centrifuge tubes. The cell pellets were harvested by centrifuging at 5000 × g for 5 min at 4 °C. After the supernatant was removed, suitable amounts of cold PBS were added to pellets to give a concentration of 5.0 × 107 cells/ml. The samples were then sonicated on ice and centrifuged at 11,000 × g for 10 min at 4 °C. The supernatants were removed and stored at –80 °C until assay. The samples were used for assays of total intracellular GSH, total antioxidant capacity, and antioxidant enzyme activity. For reverse transcription real time PCR assays, HT22-hSelH and HT22-vector cells were maintained in normal cell culture conditions until 80–90% optical confluency and then either harvested for RNA extraction or treated with BSO before RNA harvesting. RNeasy mini kits (Qiagen) were used for RNA extraction. hSelH Transfection—The hSelH clone was purchased from Invitrogen and subcloned into pMSCVpuro retroviral vector purchased from BD Biosciences. Packaging cells (RetroPack PT67, BD Biosciences) were plated in 60-mm dishes at 70% optical confluency 12 h before transfection. The cells were transfected with FuGENE 6 transfection reagent (Roche Applied Science) mixed with 2 μg of hSelH-MSCV retroviral vector or empty MSCV vector. Culture medium was aspirated 4 h after transfection. PT67 cells were washed twice with PBS and allowed to grow for 24 h in 3 ml of complete medium. The cells were selected in puromycin, with the concentration of puromycin optimized to kill the nontransfected control PT67 cells in 7–10 days. Transfected PT67 cells that survived the puromycin selection stably produced virus. For viral infection of target cells, HT22 cells were plated 12 h before infection at ∼70% optical confluency. The medium from transfected PT67 cells containing virus was collected, filtered through a 0.45-μm filter, and added to the HT22 cells in the presence of 4 μg/ml Polybrene. Virus-containing medium was replaced after 24 h of incubation. After infection for 48 h, the target cells were subjected to puromycin selection, with uninfected cells as selection control. Selection lasted for ∼10 days, until all of the uninfected control cells were killed. The cells that survived the selection made up the HT22-hSelH and HT22-vector cell lines. 75Se Labeling of SelH—HT22-hSelH and HT22-vector cell lines were labeled by the addition of neutralized [75Se]selenious acid (3 μCi/ml; specific activity, 1,000 Ci/mmol), followed by incubation for 36 h. The cells were harvested, and nuclear and cytoplasmic lysates were prepared using a compartment protein extraction kit (Chemicon) according to the manufacturer’s instructions. The proteins were separated on a 10–14.5% gradient SDS-polyacrylamide gel and transferred to nitrocellulose Hybond C-extra membranes (Amersham Biosciences), followed by phosphorimaging using a CycloneTM Storage phosphor screen (PerkinElmer Life Sciences). Reverse Transcription Real Time PCR—cDNAs were synthesized from RNA extracts using SuperScript III First-Strand synthesis system (Invitrogen). Sequences of forward and reverse primers for real time PCR of the housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (mGAPDH) and hypoxanthine phosphoribsyl-transferase (mHPRT) and for target genes are given in Table 1. All of the PCRs were performed in 10-μl reaction volumes using a Platinum SYBR Green qPCR SuperMix-UDG kit from Invitrogen. Amplification and detection were carried out using a LightCycler 2.0 real time PCR machine (Roche Applied Science).TABLE 1Reverse transcription real time-PCR primers used for cDNA amplificationmGAPDH forward5′-TGACATCAAGAAGGTGGTGAAGC-3′mGAPDH reverse5′-CCCTGTTGCTGTAGCCGTATTC-3′mHPRT1 forward5′-TCCTCCTCAGACCGCTTTT-3′mHPRT1 reverse5′-CCTGGTTCATCATCGCTAATC-3′hSelH forward5′-GCTTCCAGTAAAGGTGAACCCG-3′hSelH reverse5′-ACCCAAATCTCCCTACGACAGG-3′mSelH forward5′-GGAAGAAAGCGTAAGGCGGG-3′mSelH reverse5′-GGTTTGGACGGGTTCACTTGC-3′mGCS-HC forward5′-ATGATAGAACACGGGAGGAGAG-3′mGCS-HC reverse5′-TGATCCTAAAGCGATTGTTCTTC-3′mGCS-LC forward5′-TGACTCACAATGACCCGAAA-3′mGCS-LC reverse5′-GATGCTTTCTTGAAGAGCTTCCT-3′mGSTo1 forward5′-CAGCGATGTCGGGAGAAT-3′mGSTo1 reverse5′-GGCAGAACCTCATGCTGTAGA-3′mGSTa2 forward5′-TCTGACCCCTTTCCCTCTG-3′mGSTa2 reverse5′-GCTGCCAGGATGTAGGAACT-3′mGSTa4 forward5′-CCCCTGTACTGTCCGACTTC-3′mGSTa4 reverse5′-GGAATGTTGCTGATTCTTGTCTT-3′ Open table in a new tab GSH and Total Antioxidant Capacity Measurements—Total intracellular GSH assays were carried out using the GSH/GSSG-412 TM assay kit from Oxis following the instructions of the manufacturer. The Fe3+-reducing ability was measured by the ferric-reducing ability of plasma (FRAP) procedure with modification (9Benzie I.F.F. Strain J.J. Anal. Biochem. 1996; 239: 70-76Crossref PubMed Scopus (14582) Google Scholar). The reagent contained 0.83 mm TPTZ (2, 4, 6-tri-pyridyl-s-triazine) and mm in A of was mixed with μl of reagent and at for 10 of to was by the at by Trolox, a was used as The are as Trolox equivalent antioxidant capacity The 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic ability was measured by the by N. R. M. Scopus Google Scholar). To the an was in by for h in the and then in PBS to an of at A of was added to μl of followed by incubation at for 10 of the was then by the decrease at Trolox was used as and the were as The ability of samples was measured using an antioxidant assay kit The manufacturer’s instructions were followed that Trolox was used as and the were as Antioxidant activity assay was on the D. W. J. Google with to the HT22 cell The used in this study was the concentration of GSH was to and the of the assay was increased to The of activity was as of activity assay measured the of to GSH, using as the A of reagent containing in 5 mm at was mixed with 10 μl of and at °C for 3 and then was added to the The of and were and The decrease of at was for 3 and the was The of activity was as that of on were in medium containing to and to was performed using an with an and Chromatin immunoprecipitation was carried out using an of the of and PubMed Scopus Google Scholar). SelH was to GFP in the vector GFP-SelH or was in cells by transfection using reagent the cells were with at °C for and the were with at for 5 After with PBS, the cells were harvested and with ml of mm mm mm mm mm with complete (Roche Applied The lysates were for min on ice with to in of The nuclear was in nuclear mm 10 mm complete (Roche Applied Science) and on ice for 10 The nuclear lysates were sonicated for four of 10 and in of to a of was used for immunoprecipitation with antibodies using For an of was with normal of a specific was by and used for in vector 4 was used for and assay. A was as of hSelH in Cells after expression levels of hSelH and were by reverse transcription real time PCR in HT22-hSelH and HT22-vector cells in normal culture and the are in The hSelH gene was highly following with expression levels higher than the expression levels of endogenous the mRNA of hSelH in HT22-vector cells was not The mRNA of endogenous is higher in HT22-hSelH cells than in HT22-vector cells. expression as by with [75Se]selenious acid is in Total GSH cells were maintained in normal culture conditions at optical confluency of the total intracellular GSH of HT22-hSelH was cells, and that of HT22-vector cells was cells from was after treatment with of BSO from to 10 mm for 15 h, the GSH was maintained in the HT22-hSelH cells than in the HT22-vector cells Antioxidant are selenoproteins that using GSH as the is an enzyme that to The of SelH expression on the of these enzymes was Reverse transcription real time PCR analysis indicates that the for and are in HT22 cells, the for and are the of detection not Thus, the of the activity measured in the present study from and The in HT22-hSelH and HT22-vector cells did not from After treatment with BSO for 15 h, the activity in increased to of the control at mm BSO, that in cells to from in the cells were not after treatment with higher BSO not when the incubation time was to 4, or 12 h, mm BSO treatment in higher in HT22-hSelH cells than in HT22-vector cells is the enzyme that to GSH, the for to GPx, the of were in the cell lines. After treatment with mm BSO for 15 h, the activity in cells increased to of the that in the cells was of the control Total Antioxidant the ferric-reducing ability of samples at when GSH and are as and proteins are Thus, the the total antioxidant capacity from normal cell culture the of HT22-hSelH and HT22-vector cells were not when the cells were with or 10 mm BSO for 15 h, the of the increased to and of the control of the HT22-vector cells to and of the control not The assay the of a to in the of BSO, in the of the cell lines was GSH synthesis was with mm BSO for 15 h, the as the BSO concentration increased and maintained but higher in HT22-hSelH cells in with the vector control not is a of ability of cell In the of BSO, in was the cell lines. After treatment with mm BSO for 15 h, the of the HT22-hSelH cells was to of the control the that of HT22-vector was to the of when the BSO concentration was increased from to 10 mm with mm BSO for h produced a decrease in in the HT22-hSelH cells to the of of that of the HT22-vector cells was maintained at ∼70% of the control Overexpression of SelH the of is the rate-limiting enzyme involved in de novo GSH synthesis. of and the and and mRNA levels were measured in the and HT22-vector cell lines after treatment with of BSO for 15 h 4, A and Overexpression of SelH in in the mRNA levels for the of BSO are enzymes involved in phase II detoxification. We the of SelH overexpression on the mRNA levels of Alpha and Omega expression levels in HT22-hSelH cells were than twice the levels in HT22-vector cells but were not by treatment with mm BSO The for and were higher in cells than in HT22-vector cells not After treatment with mm BSO for 15 h, endogenous expression was in the cell lines In the of BSO, the mRNA expression was higher in the HT22-hSelH cells than in the control cells. concentration of BSO mRNA levels in HT22-hSelH cells to the levels as in the HT22-vector cells. higher of BSO, HT22-hSelH cells exhibited higher expression levels than HT22-vector cells. Selenoprotein H an DNA-binding increased levels of the for the and murine SelH in HT22-vector cells to the deduced amino acid sequences of SelH genes for that might these The of sequences and are in putative nuclear localization signals, at amino acids and in the human were identified using the II and to be all expression of a GFP-SelH fusion protein in predominantly nuclear localization ExPASy analysis and with the known members of the family of DNA-binding proteins, we identified an AT-hook motif that high the and SelH sequences The AT-hook is a small DNA-binding protein motif of a amino acid of the small of the AT-hook are to by sequence L. D. PubMed Scopus Google Scholar). To the DNA-binding domain of SelH exhibited in we performed immunoprecipitation using a GFP-SelH fusion protein and antibodies to transfection of cells, GFP-SelH and transfected cells were and the were The nuclear lysates were sonicated to and were subcloned for After with were were and were to the or heat shock or stress response multiple multiple and and The containing were all small in from 10 to in Table Thus, than of the or and the that did not of small The control with in of 10 were contained a HSE, contained and the contained with immunoprecipitation of heat shock and stress response with selenoprotein of 10 Open table in a new tab The is known to be by and in its Y. N. E. J. PubMed Scopus Google Scholar), and of these sequences with the and sequences by GFP-SelH revealed in these a role for SelH in regulating expression of via these elements. HT22 is a neuronal cell that is highly to GSH depletion Y. P. D. Full Text Full Text PDF PubMed Scopus Google Scholar). The expression of endogenous was in HT22 the hSelH gene in overexpression of SelH. In the of induced oxidative in total intracellular GSH, total antioxidant capacity, and and were SelH-overexpressing and vector control cells, of control of that the redox of the cells, as of the enzymatic activity of GCS, the key enzyme involved in GSH through multiple The heavy is by and PubMed Scopus Google Scholar). of GSH also to the enzymatic activity of GCS, to control GSH synthesis J. Biol. Chem. Full Text PDF PubMed Google Scholar). The intracellular of a of total antioxidant capacity multiple in the we used assays J. Chem. PubMed Scopus Google Scholar). SelH overexpression maintained higher total antioxidant capacity during BSO treatment, but the of the of total antioxidant capacity that SelH overexpression a on of antioxidant oxidative study that SelH gene function and that this function is by cellular redox GSH depletion. in 4 and the expression levels of and are by SelH overexpression and oxidative The of via response HSE, STRE, response and antioxidant response also response J.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is also by and Scopus Google Scholar). The of an AT-hook in SelH, and the via immunoprecipitation that SelH and in with the of and by SelH in response to BSO, a function for SelH as a redox-responsive DNA-binding protein of the The AT-hook motif to the of via a acid containing this motif are in and as transcription Thus, SelH may as a cellular redox that functions in with of the many transcription that to in redox status. GSH depletion and oxidative stress have been reported in the of and Selenium was to the GSH levels in the in mice treated with M. S. J. 2003; PubMed Scopus Google Scholar). show that the mRNA levels of in HT22 cells are highly up-regulated by selenium a of the gene function of SelH be in beneficial effects of selenium in of oxidative