Glyoxalase II, a Detoxifying Enzyme of Glycolysis Byproduct Methylglyoxal and a Target of p63 and p73, Is a Pro-survival Factor of the p53 Family
2006; Elsevier BV; Volume: 281; Issue: 36 Linguagem: Inglês
10.1074/jbc.m604758200
ISSN1083-351X
Autores Tópico(s)Folate and B Vitamins Research
ResumoThe p53 family proteins are transcription factors and have both common and distinct functions. p53 is a classic tumor suppressor, whereas p63 and p73 have fundamental functions in development. To gain an insight into the functional diversities among the p53 family, target genes specifically regulated by p63 and p73 were examined. Here, we found that the GLX2 gene, which encodes glyoxalase II enzyme, is up-regulated by p63 and p73. Accordingly, a specific responsive element was found in intron 1 of the GLX2 gene, which can be activated and bound by p63 and p73. We also found that, upon overexpression, the cytosolic, but not the mitochondrial, GLX2 inhibits the apoptotic response of a cell to methylglyoxal, a by-product of glycolysis. Likewise, we showed that cells deficient in GLX2 are hypersensitive to methylglyoxal-induced apoptosis. Interestingly, a deficiency in GLX2 also enhances the susceptibility of a cell to DNA damage-induced apoptosis in a p53-dependent manner. These observations reveal a novel link between the p53 family and the glyoxalase system. Given that methylglyoxal is frequently generated under both physiological and pathological conditions, we postulate that GLX2 serves as a pro-survival factor of the p53 family and plays a critical role in the normal development and in the pathogenesis of various human diseases, including cancer, diabetes, and neurodegenerative diseases. The p53 family proteins are transcription factors and have both common and distinct functions. p53 is a classic tumor suppressor, whereas p63 and p73 have fundamental functions in development. To gain an insight into the functional diversities among the p53 family, target genes specifically regulated by p63 and p73 were examined. Here, we found that the GLX2 gene, which encodes glyoxalase II enzyme, is up-regulated by p63 and p73. Accordingly, a specific responsive element was found in intron 1 of the GLX2 gene, which can be activated and bound by p63 and p73. We also found that, upon overexpression, the cytosolic, but not the mitochondrial, GLX2 inhibits the apoptotic response of a cell to methylglyoxal, a by-product of glycolysis. Likewise, we showed that cells deficient in GLX2 are hypersensitive to methylglyoxal-induced apoptosis. Interestingly, a deficiency in GLX2 also enhances the susceptibility of a cell to DNA damage-induced apoptosis in a p53-dependent manner. These observations reveal a novel link between the p53 family and the glyoxalase system. Given that methylglyoxal is frequently generated under both physiological and pathological conditions, we postulate that GLX2 serves as a pro-survival factor of the p53 family and plays a critical role in the normal development and in the pathogenesis of various human diseases, including cancer, diabetes, and neurodegenerative diseases. p53, a sequence-specific transcription factor, transactivates an array of target genes that mediate p53 functions, such as cell cycle arrest, apoptosis, and DNA repair (1Harms K. Nozell S. Chen X. Cell Mol. Life Sci. 2004; 61: 822-842Crossref PubMed Scopus (254) Google Scholar). The other two p53 family members, p63 and p73, share considerable structural homology with p53 and can also bind to p53-responsive elements (p53-REs) 2The abbreviations used are: RE, responsive element; GLX1, -2, glyoxalases I and II; MG, methylglyoxal; SLG, S-d-lactoylglutathione; AGE, advanced glycation end product; HA, hemagglutinin; siRNA, small interference RNA; nt, nucleotide(s); ChIP, chromatin immunoprecipitation; PARP, poly(ADP-ribose) polymerase.2The abbreviations used are: RE, responsive element; GLX1, -2, glyoxalases I and II; MG, methylglyoxal; SLG, S-d-lactoylglutathione; AGE, advanced glycation end product; HA, hemagglutinin; siRNA, small interference RNA; nt, nucleotide(s); ChIP, chromatin immunoprecipitation; PARP, poly(ADP-ribose) polymerase. to transactivate some p53 target genes, such as p21, MDM2, FDXR, and POLH (2el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7890) Google Scholar, 3Wu X. Bayle J.H. Olson D. Levine A.J. Genes Dev. 1993; 7: 1126-1132Crossref PubMed Scopus (1621) Google Scholar, 4Liu G. Chen X. Oncogene. 2002; 21: 7195-7204Crossref PubMed Scopus (158) Google Scholar, 5Liu G. Chen X. Mol. Cell. Biol. 2006; 26: 1398-1413Crossref PubMed Scopus (76) Google Scholar). Thus, p63 and p73 appear to have overlapping functions with p53 (1Harms K. Nozell S. Chen X. Cell Mol. Life Sci. 2004; 61: 822-842Crossref PubMed Scopus (254) Google Scholar). However, unlike p53 as a classic tumor suppressor, p63 and p73 are rarely mutated in cancer and their roles in tumor suppression remain controversial (6Melino G. De Laurenzi V. Vousden K.H. Nat. Rev. Cancer. 2002; 2: 605-615Crossref PubMed Scopus (501) Google Scholar). Furthermore, mice deficient in either p63 or p73 exhibit profound developmental defects without increased susceptibility to spontaneous tumor formation (7Mills A.A. Zheng B. Wang X.J. Vogel H. Roop D.R. Bradley A. Nature. 1999; 398: 708-713Crossref PubMed Scopus (1687) Google Scholar, 8Yang A. Walker N. Bronson R. Kaghad M. Oosterwegel M. Bonnin J. Vagner C. Bonnet H. Dikkes P. Sharpe A. McKeon F. Caput D. Nature. 2000; 404: 99-103Crossref PubMed Scopus (878) Google Scholar). The diverse physiological functions of p63 and p73 suggest that a distinct set of transcriptional targets are regulated by p63 and p73.Glyoxalase II (GLX2), also known as hydroxyacylglutathione hydrolase, together with glyoxalase I (GLX1), constitutes the glyoxalase system (9Thornalley P.J. Biochem. Soc. Trans. 2003; 31: 1343-1348Crossref PubMed Google Scholar). The major function of the system is to detoxify α-ketoaldehydes, especially the potent and cytotoxic methylglyoxal (MG) (10Thornalley P.J. Gen. Pharmacol. 1996; 27: 565-573Crossref PubMed Scopus (517) Google Scholar). MG, a by-product of glycolysis, is produced through nonenzymatic phosphate elimination from the glycolytic intermediates, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. MG is converted to S-d-lactoylglutathione (SLG) by GLX1 with reduced glutathione as a cofactor, and SLG in turn is hydrolyzed to d-lactate along with regeneration of reduced glutathione by GLX2. GLX1, which uses MG as the preferred substrate, is found selectively overexpressed in leukemia cells, and GLX1 inhibitor is shown to be a potential anti-cancer agent (11Sakamoto H. Mashima T. Kizaki A. Dan S. Hashimoto Y. Naito M. Tsuruo T. Blood. 2000; 95: 3214-3218Crossref PubMed Google Scholar). Overexpression of GLX1 is capable of inhibiting the formation of hyperglycemia-induced advanced glycation end products (AGEs) in bovine endothelial cells, indicating that GLX1 has a protective role in diabetic microangiopathy (12Shinohara M. Thornalley P.J. Giardino I. Beisswenger P. Thorpe S.R. Onorato J. Brownlee M. J. Clin. Invest. 1998; 101: 1142-1147Crossref PubMed Scopus (422) Google Scholar). Recent studies revealed that GLX1 has additional roles in the pathogenesis of Alzheimer disease and anxiety (13Chen F. Wollmer M.A. Hoerndli F. Munch G. Kuhla B. Rogaev E.I. Tsolaki M. Papassotiropoulos A. Gotz J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7687-7692Crossref PubMed Scopus (141) Google Scholar, 14Hovatta I. Tennant R.S. Helton R. Marr R.A. Singer O. Redwine J.M. Ellison J.A. Schadt E.E. Verma I.M. Lockhart D.J. Barlow C. Nature. 2005; 438: 662-666Crossref PubMed Scopus (394) Google Scholar). All these studies indicate that the glyoxalase system plays an important role in the pathogenesis of various human diseases. However, very little is known about GLX2, including its role in the glyoxalase system and its physiological significance in the stress response.In this study, we showed that GLX2 is a novel target of p63 and p73. We also showed that the cytosolic, but not the mitochondrial, GLX2 protects cells from MG-induced cell death, whereas a deficiency in GLX2 promotes MG-induced cell death. Furthermore, we showed that GLX2 knockdown sensitizes cells to DNA damage-induced apoptosis in a p53-dependent manner, implying that GLX2 has a novel function in the DNA damage response. Taken together, our data reveal a novel link between the p53 family and the glyoxalase system, which plays an important role in the normal development and in the pathogenesis of various human diseases, such as cancer, diabetes and Alzheimer disease.EXPERIMENTAL PROCEDURESPlasmids—The cDNAs encoding both the cytosolic and mitochondrial GLX2 were generated by reverse transcription-PCR using total RNAs purified from MCF7 cells. The primers used to generate the cytosolic GLX2 were forward primer cGLX2-F (5′-AAGCTTGCCACCATGAAGGTAGAGGTGCTGCCTGCCCTG-3′) and reverse primer GLX2-R (5′-CTCGAGTCAGTCCCGGGGCATCTTGAACTGGTCCTT-3′). The primers used to generate the mitochondrial GLX2 were forward primer mGLX2-F (5′-AAGCTTGCCACCATGGTGGTGGGCCGAGGGCTGCTCGGC-3′) and reverse primer GLX2-R. For inducible expression of GLX2 tagged with hemagglutinin (HA) epitope at the N or C termini, cDNA was cloned into a pcDNA4 tetracycline-inducible expression vector. The cDNA encoding the N-terminally HA-tagged cytosolic GLX2 was amplified with forward primer GLX2-5HA-F (5′-AAGCTTGCCACCATGTACCCATACGATGTTCCAGATTACGCTAAGGTAGAGGTGCTGCTGCCCTGACC-3′) and reverse primer GLX2-R. The cDNA encoding the C-terminally HA-tagged cytosolic GLX2 was amplified with forward primer cGLX2-F and reverse primer GLX2-3HA-R (5′-CTCGAGTCAAGCGTAATCTGGAACATCGTATGGGTAGTCCCGGGGCATCTTGAACTGGTCCTT-3′). The cDNA encoding the C-terminally HA-tagged mitochondrial GLX2 was amplified with forward primer mGLX2-F and reverse primer GLX2-3HA-R. All GLX2 cDNA constructs were confirmed by DNA sequencing.To generate a construct that expresses a small interfering RNA (siRNA) against GLX2 under the control of the tetracycline-regulated H1 promoter, two oligonucleotides targeting the region of +955 to +975 in the GLX2 transcript (NM_005326) were annealed and cloned into pBabe-H1 at the HindIII and BglII sites, and the resulting construct was designated pBabe-H1-siGLX2. pBabe-H1 is a polymerase III promoter-driven vector with a tetracycline operon sequence inserted before the transcription start site (15van de Wetering M. Oving I. Muncan V. Pon Fong M.T. Brantjes H. van Leenen D. Holstege F.C. Brummelkamp T.R. Agami R. Clevers H. EMBO Rep. 2003; 4: 609-615Crossref PubMed Scopus (461) Google Scholar). The sequence for sense oligonucleotide (targeting region shown in bold) was 5′-GATCCCCAAGGGACTGCGGATGAGATGTTTCAAGAGAACATCTCATCCGCAGTCCCTTTTTTTGGAAA-3′, and that for antisense oligonucleotide was 5′-AGCTTTTCCAAAAAAAGGGACTGCGGATGAGATGTTCTCTTGAAACATCTCATCCGCAGTCCCTTGGG-3′. The construct that expresses siRNA against p53 was used as described previously (16Liu G. Xia T. Chen X. J. Biol. Chem. 2003; 278: 17557-17565Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar).For generation of a luciferase reporter under the control of a potential p53-response element in the GLX2 gene, a 709-bp DNA fragment from intron 1 (nt +3419 to +4127) was amplified from the genomic DNA purified from MCF7 cells with forward primer GAin1 +3419F (5′-AGATCTGCAGGGGGTAGAATGTCGCCAT-3′) and reverse primer GAin1 +4127R (5′-AAGCTTGCAGACACAGAGCACAACTCAGCG-3′). The fragment was confirmed by sequencing and then cloned into a promoterless luciferase reporter vector pGL2 (Promega). The resulting plasmid was designated as pGL2-GLX2(+3419/+4127). Other luciferase reporters were also generated by PCR using pGL2-GLX2(+3419/+4127) as a template. The primers used to generate pGL2-GLX2(+3850/+4127) were forward primer GAin1 +3850F (5′-AGATCTAGCCTGGGCGACAGAGCGAGAC-3′) and reverse primer GAin1 +4127R. The primers used to generate pGL2-GLX2(+3954/+4127) were forward primer GAin1 +3954F (5′-AGATCTAGTCCTCCCATCTCAGTCTCTTA-3′) and reverse primer GAin1 +4127R. The primers used to generate pGL2-GLX2(Δ+3879/+3935) were forward primer GAin1(Δ+3879/+3935)F (5′-TCTAGACTCCTGCCCTCAAGCAGTCCTCCCATC-3′) and reverse primer GAin1(Δ+3879/+3935)R (5′-TCTAGACGGAGTCTCGCTCTGTCGCCCAGGC-3′). The primers used to generate pGL2-GLX2(+3901/+4127) were forward primer GAin1 +3901F (5′-AGATCTAGACAAGGTCTTGCTATGTTGCCCA-3′) and reverse primer GAin1 +4127R. The primers used to generate pGL2-GLX2(+3901/+4127)M2 were forward primer GAin1 +3901M2F (point mutations shown in bold) (5′-AGATCTAGATAAGGTCTTGCTATGTTGCCCAGGATGGTCTCAAACTCCTG-3′) and reverse primer GAin1 +4127R. The primers used to generate pGL2-GLX-2(+3901/+4127)M4 are forward primer GAin1 +3901M4F (5′-AGATCTAGATAATGTCTTGCTATGTTGCCCAGGATGA- TCTCAAACTCCTG-3′) and reverse primer GAin1 +4127R.Cell Culture—MCF7 cell lines, which inducibly express various isoforms of the p53 family proteins, were cultured as described previously (17Helton E.S. Zhu J. Chen X. J. Biol. Chem. 2006; 281: 2533-2542Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 18Nozell S. Wu Y. McNaughton K. Liu G. Willis A. Paik J.C. Chen X. Oncogene. 2003; 22: 4333-4347Crossref PubMed Scopus (30) Google Scholar). RKO-GLX2 cell lines, which inducibly express GLX2 under the control of the tetracycline-inducible promoter, were generated as described previously (19Dohn M. Nozell S. Willis A. Chen X. Methods Mol. Biol. 2003; 223: 221-235PubMed Google Scholar). To generate inducible GLX2 knockdown cell lines, pBabe-H1-siGLX2 was transfected into RKO or MCF7 cells in which a tetracycline repressor was expressed by pcDNA6 (5Liu G. Chen X. Mol. Cell. Biol. 2006; 26: 1398-1413Crossref PubMed Scopus (76) Google Scholar). GLX2 knockdown cell lines were selected with puromycin and confirmed by Western blot analysis. To generate cell lines in which p53 is stably knocked down and GLX2 is inducibly knocked down, pBabe-H1-siGLX2 and pBabe-U6-sip53 were cotransfected into RKO cells and then selected with puromycin; p53 and GLX2 double knockdown RKO cell lines were confirmed by Western blot analysis.Affymetrix GeneChip Assay and Northern Blot Analysis—Total RNAs were isolated from MCF7 cells using TRIzol reagent (Invitrogen). U133 plus GeneChip was purchased from Affymetrix (Santa Clara, CA), which contains oligonucleotides representing 37,000 unique human transcripts. GeneChip analysis was performed according to the manufacturer's instructions. Northern blot analysis and preparation of p21 and glyceraldehyde-3-phosphate dehydrogenase probes were as described previously (20Chen X. Bargonetti J. Prives C. Cancer Res. 1995; 55: 4257-4263PubMed Google Scholar). The GLX2 probe was prepared from an expressed sequence tag clone (GenBank™ BC000840).Luciferase Assay—The dual luciferase assay was performed in triplicate according to the manufacturer's instructions (Promega, Madison, WI). Briefly, 0.25 μg of a luciferase reporter, 0.25 μg of pcDNA3 or pcDNA3 that expresses a p53 family protein, and 5 ng of Renilla luciferase assay vector pRL-CMV (Promega) were cotransfected into H1299 cells. The -fold increase in relative luciferase activity is a product of the luciferase activity induced by a p53 family protein divided by that induced by an empty pcDNA3 vector. The pGL2-p21A luciferase reporter under the control of the two p53-responsive elements in the p21 promoter was used as a positive control (21Nozell S. Chen X. Oncogene. 2002; 21: 1285-1294Crossref PubMed Scopus (60) Google Scholar).Chromatin Immunoprecipitation Assay—ChIP assay was performed as previously described (22Harms K.L. Chen X. Mol. Cell. Biol. 2005; 25: 2014-2030Crossref PubMed Scopus (88) Google Scholar). After induction (+) or no induction (-) of p63γ or p73β for 24 h, chromatins in MCF7 cells were cross-linked with 1% formaldehyde for 10 min at room temperature, and then sonicated to generate 500- to 1000-bp DNA fragments and immunoprecipitated with anti-Myc antibody for p63γ or anti-HA antibody for p73β. After reverse cross-linking and phenol-chloroform extraction, the bound DNA fragments were purified by using a Qiagen column. PCR was performed to visualize the enriched DNA fragments. Primers designed to amplify the region from nt +3779 to +3969 in the GLX2 intron 1 were forward primer (5′-CTGAGACAGGAGAACGGCGTGAACC-3′) and reverse primer (5′-CTGAGATGGGAGGACTGCTTGAGGG-3′). Primers designed to amplify the region from nt -2312 to -2131 in the p21 promoter were forward primer (5′-CAGGCTGTGGCTCTGATTGG-3′) and reverse primer (5′-TTCAGAGTAACAGGCTAAGG-3′).Colony Formation Assay—RKO cells seeded at 300 per well and MCF7 cells seeded at 500 per well in a 6-well plate were incubated in the absence or presence of tetracycline (1.0 μg/ml) for 72 h, then treated with various concentrations of MG for 1 h followed by repetitive wash with phosphate-buffered saline to remove MG. The cells were maintained in fresh medium for next 12-14 days and then stained with crystal violet.Western Blot Analysis—Chicken anti-GLX2 polyclonal IgY antibody was purchased from GenWay Biotech (San Diego, CA). Mouse anti-PARP monoclonal antibody was purchased from BD Pharmingen. Antibodies against p53, p21, HA, and Myc epitopes and actin were described previously (23Zhu J. Zhou W. Jiang J. Chen X. J. Biol. Chem. 1998; 273: 13030-13036Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Whole cell extracts were prepared by lysing cells with 2× SDS sample buffer. Proteins were separated on 7-10% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with the indicated antibodies followed by ECL detection.DNA Histogram Analysis—Cells were seeded at 2 × 105 per 6-cm plate with or without tetracycline for 72 h, and then treated with MG or a DNA damage agent for various times. Both floating dead cells in the medium and live cells on the plate were collected and fixed with 1 ml of 100% ethanol for at least 30 min. The fixed cells were centrifuged and re-suspended in 0.5 ml of phosphate-buffered saline solution containing 50 μg/ml each of RNase A and propidium iodide (Sigma). The stained cells were analyzed in a fluorescence-activated cell sorter within 4 h. The percentages of cells in the sub-G1,G1,S, and G2-M phases were determined using the CellQuest program (BD Biosciences).Immunofluorescence Microscopy—Cells grown on slides were fixed with 3% paraformaldehyde in phosphate-buffered saline for 45 min at room temperature and treated with 0.5% Triton X-100 for 5 min, blocked with 1% bovine serum albumin for 1 h, and stained with anti-HA to detect HA-tagged GLX2 or with chicken anti-GLX2 to detect endogenous GLX2 at room temperature for 1 h. Slides were washed and then incubated with fluorescein isothiocyanate-conjugated secondary antibodies against mouse or chicken immunoglobulin G (Jackson ImmunoResearch and Molecular Probes), followed by treatment with 4′,6-diamidino-2-phenylindole (Sigma) to stain nuclei. For MitoTracker (Molecular Probes) staining, cells were incubated in 50 nm MitoTracker for 30 min and then washed with prewarmed growth medium followed by paraformaldehyde fixation.RESULTSIdentification of GLX2 as a Target Gene of p63 and p73—To identify novel target genes regulated by the p53 family, an Affymetrix GeneChip assay was performed. Total RNAs were isolated from MCF7 cells that were uninduced and induced to express p63γ by withdrawal of tetracycline from the culture medium and used to prepare probes for the Affymetrix U133+ GeneChip. We found that GLX2 was highly induced in cells expressing p63γ. To confirm and extend the microarray result, we performed Northern blot analysis and found that GLX2 was highly induced by p63γ and p73β and little if any by p63α, ΔNp63α, ΔNp63γ, and ΔNp73β (Fig. 1A). The level of p21 was examined as a positive control and found to be induced by p63γ and p73β. The level of glyceraldehyde-3-phosphate dehydrogenase was determined as a loading control. We also found that GLX2 was only slightly induced by p53 but not significantly by DNA damage in cells that carry endogenous wild-type p53 (Fig. 1B). Furthermore, consistent with the increase in the mRNA level, Western blot analysis showed that GLX2 protein was significantly induced by p63γ and p73β but little if any by p63α, ΔNp63α, ΔNp63γ, ΔNp73β, wild-type p53, and mutant p53 in MCF7 cells (Fig. 1, C and D). These results suggest that both GLX2 mRNA and protein are preferentially up-regulated by p63γ and p73β but little if any by p53.As members of the p53 family, p63 and p73 are capable of transactivating a promoter that contains a p53-responsive element (p53-RE), RRRC(A/T)(A/T)GYYY, in which R represents purine, whereas Y represents pyrimidine (24el-Deiry W.S. Kern S.E. Pietenpol J.A. Kinzler K.W. Vogelstein B. Nat. Genet. 1992; 1: 45-49Crossref PubMed Scopus (1736) Google Scholar). Thus, we searched for and found a potential p53-RE in the first intron (from nt +3901 to +3934) of the GLX2 gene (Fig. 2A). The localization of p53-RE in the intron of a target gene is not unusual, because several well established p53-REs are found in the introns of p53 targets, such as PML and PUMA (25de Stanchina E. Querido E. Narita M. Davuluri R.V. Pandolfi P.P. Ferbeyre G. Lowe S.W. Mol. Cell. 2004; 13: 523-535Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 26Nakano K. Vousden K.H. Mol. Cell. 2001; 7: 683-694Abstract Full Text Full Text PDF PubMed Scopus (1854) Google Scholar). To determine whether this potential p53-RE is functional, we cloned the intron fragment as well as several mutant fragments in front of a luciferase reporter (Fig. 2A). We showed that the fragment from nt +3419 to +4127 was highly responsive to p63γ and p73β (Fig. 2, B and C). Similarly, the fragment from nt +3850 to +4127 was also responsive (Fig. 2, B and C). However, the fragment from nt +3954 to +4127 and the internal deletion mutant, Δ+3879/+3935, were only weakly responsive (Fig. 2, B and C), suggesting that the potential p53-RE from nt +3901 to +3934 is necessary. Next, point mutations at the sites predicted to be critical for p53 binding were made in the potential p53-RE. Two nucleotide substitutions, C3904T and C3928A, were made for GLX2-(+3910/+4127)M2, whereas four nucleotide substitutions, C3904T, G3907T, C3928A, and G3931A, were made for GLX2-(+3910/+4127)M4 (Fig. 2A). We found that, like the deletion of the potential p53-RE, mutations also made the p53-RE less responsive to p63 and p73 (Fig. 2, D and E). These data imply that the p53-RE from nt +3901 to +3934 is responsible for p63γ and p73β induction of the GLX2 gene.FIGURE 2GLX2 is a direct target of p63 and p73. A, schematic presentation of the GLX2 genomic locus containing intron 1 and the pGL2 luciferase reporter constructs. A potential p53-responsive element is located between nt +3901 and +3934 in intron 1 of the GLX2 gene. The reporter constructs, +3901/+4127 M2 and +3901/+4127 M4, carry two and four nucleotide substitutions at the critical nucleotides (in bold) in the potential p53-responsive element, respectively. B and C, the potential p53-responsive element in intron 1 is responsive to p63γ and p73β. The luciferase assay was carried out as described under "Experimental Procedures." D and E, mutations at the critical nucleotides abrogate the potential p53-responsive element in intron 1. F, schematic presentation of the GLX2 intron 1 and p21 promoter with the location of the p53-responsive element and PCR primers used for ChIP assay. G and H, p63γ and p73β bind directly to the p53-responsive element in the GLX2 intron 1 and p21 promoter in vivo.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether p63γ and p73β directly bind to the p53-RE in the GLX2 gene in vivo, we performed ChIP assays. The Myc-tagged p63γ-DNA complexes were immunoprecipitated with anti-Myc antibody, whereas anti-HA was used as a control. The HA-tagged p73β-DNA complexes were immunoprecipitated with anti-HA antibody, whereas anti-Myc was used as a control. To visualize the enriched DNA fragments, PCR was performed to amplify the region spanning the p53-RE in the GLX2 gene along with the p53-RE1 within the p21 promoter as a positive control with primers shown in Fig. 2F.We found that the captured fragment containing the p53-RE in the GLX2 gene was highly enriched upon inducible expression of p63γ or p73β (Fig. 2, G and H, GLX2 panels). No DNA fragment was enriched by the control antibody (Fig. 2, G and H). As a positive control, the fragment containing the p53-RE1 in the p21 gene was also enriched upon inducible expression of p63γ or p73β (Fig. 2, G and H, p21 panels). Taken together, these data indicate that GLX2 is a direct transcriptional target of p63 and p73.Subcellular Localization of GLX2 Protein—In yeast and higher plants, the cytosolic and mitochondrial isoforms of GLX2 are encoded by two separate genes, whereas in human both isoforms are encoded by the single GLX2 gene (27Cordell P.A. Futers T.S. Grant P.J. Pease R.J. J. Biol. Chem. 2004; 279: 28653-28661Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). As shown in Fig. 3A, the human GLX2 gene encodes two isoforms of the protein through alternate translational start sites: the mitochondrial GLX2 translated from the first ATG codon located in exon 1 and the cytosolic GLX2 from the second ATG codon, an internal ribosome entry site in exon 2. To characterize the function of both isoforms, we generated multiple RKO cell lines that inducibly express GLX2 under the control of the tetracycline-inducible promoter. The cytosolic GLX2 was tagged with an HA epitope at either the N or C termini. The mitochondrial GLX2 was tagged with HA only at the C terminus, because an N-terminal HA might interfere with the mitochondrial targeting signal located at the N terminus. One representative clone from each cell line, that is, RKO-GLX2-5HA-3, which inducibly expresses the N-terminally HA-tagged cytosolic GLX2, RKO-GLX2-3HA-16, which inducibly expresses the C-terminally HA-tagged cytosolic GLX2, and RKO-mGLX2-3HA-4, which inducibly expresses the C-terminally HA-tagged mitochondrial GLX2, are shown in Fig. 3B. Next, the intracellular localizations of these GLX2 isoforms were determined by immunofluorescence microscopy. We found that endogenous GLX2 detected by anti-GLX2 was localized primarily in cytosol with some mitochondrial distribution (Fig. 3C, top panel). The HA-tagged cytosolic GLX2 was localized primarily in cytosol (Fig. 3C, second and third panels). However, the C-terminally HA-tagged mitochondrial GLX2 was found to be localized exclusively in mitochondria as it colocalized well with the Mito-Tracker Red (Fig. 3C, fourth panel). Interestingly, the N-terminally HA-tagged GLX2 was localized in cytosol (Fig. 3C, bottom panel), suggesting that the N-terminal HA tag masked the mitochondrial localization signal.FIGURE 3Subcellular localization of GLX2. A, schematic presentation of the human GLX2 genomic locus with exons and two translational start sites. The mitochondrial GLX2 is translated from the first ATG codon in exon 1. The cytosolic GLX2 is translated from the second ATG codon, an internal ribosomal entry site in exon 2. B, generation of RKO cell lines that inducibly express GLX2. Western blots were prepared using extracts from RKO cells that were uninduced (-) or induced (+) to express the N-terminally HA-tagged cytosolic GLX2 (GLX2-5HA), the C-terminally HA-tagged cytosolic GLX2 (GLX2-3HA), or the C-terminally HA-tagged mitochondrial GLX2 (mGLX2-3HA) for 24 h. The blots were analyzed with anti-GLX2 and anti-actin antibodies, respectively. C, subcellular localization of endogenous GLX2 (first panel), the N-terminally HA-tagged cytosolic GLX2 (second panel), the C-terminally HA-tagged cytosolic GLX2 (third panel), the C-terminally HA-tagged mitochondrial GLX2(fourthpanel), and the N-terminally HA-tagged mitochondrial GLX2(bottom panel)in RKO cells. Endogenous GLX2 was detected by anti-GLX2 antibody (GenWay Biotech, San Diego, CA), and HA-tagged GLX2 was detected by anti-HA antibody. Immunofluorescence microscopy was performed as described under "Experimental Procedures." GLX2 is stained as green, nuclei as blue (4′,6-diamidino-2-phenylindole), and mitochondria as red (MitoTracker).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Cytosolic GLX2 Protects Cells from MG-induced Cell Death—MG is the preferred substrate of GLX1, and the product of GLX1 enzymatic reaction, SLG, is the substrate for GLX2. Currently, it is not clear whether GLX2 may impact on the detoxification process of MG, and even less is known about which isoform is active in the MG detoxification pathway. Thus, we performed a colony formation assay to determine the effect of GLX2 on MG-induced growth inhibition (Fig. 4A). For cells without MG treatment, GLX2 overexpression had little effect on cell proliferation (Fig. 4A, control panel). Following MG treatment, most cells died as only a few colonies formed (Fig. 4A, bottom panel, no GLX2 expression). Upon induction of GLX2, the number of colonies was substantially higher than the group without GLX2 expression, regardless of GLX2 that was tagged with HA at its N or C termini (Fig. 4A, bottom panel and first and second columns). In contrast, there was no difference in colony formation between control cells and cells induced to express the mitochondrial GLX2 (Fig. 4A, bottom panel and third column). These data indicate that the cytosolic but not mitochondrial GLX2 confers cell resistance to MG-induced growth inhibition.FIGURE 4Overexpression of the cytosolic GLX2 inhibits MG-induced cell death. A, colony formation assay was performed using RKO cells
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