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J. Biol. Chem., Vol. 276, Issue 40, 37186-37193, October 5, 2001

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Identification of a Mouse Thiamine Transporter Gene as a Direct Transcriptional Target for p53^*

Pang-Kuo Lo, Jeou-Yuan Chen^§, Pi-Pei Tang, Jiayuh Lin^¶, Chi-Hung Lin, Li-Ting Su, Chia-Hui Wu, Tse-Ling Chen, Yin Yang, and Fung-Fang Wang^**

From the Institutes of  Biochemistry and  Microbiology and Immunology, National Yang Ming University, Shih-Pai, Taipei 112, Taiwan, the ^§ Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, and the ^¶ Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, Michigan 48109

Received for publication, May 23, 2001, and in revised form, July 19, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

p53 tumor suppressor is a transcription factor that functions, in part, through many of its downstream target genes. We have identified a p53-inducible gene by performing mRNA differential display on IW32 murine erythroleukemia cells containing a temperature-sensitive p53 mutant allele, tsp53^Val-135. Sequence analysis of the full-length cDNA revealed its identity as the mouse homologue of the human thiamine transporter 1 (THTR-1). Induction of the mouse THTR-1 (mTHTR-1) mRNA was detectable as early as 1 h at 32.5 °C; upon shifting back to 38.5 °C, mTHTR-1 transcript was rapidly degraded with a half-life of less than 2 h. Elevation of mTHTR-1 expression was found in DNA damage-induced normal mouse embryonic fibroblast cells, but not in p53^/ mouse embryonic fibroblast cells, suggesting that mTHTR-1 induction was p53-dependent. A region within the first intron of the mTHTR-1 gene bound to p53 and conferred the p53-mediated transactivation. Furthermore, increased thiamine transporter activities were found in cells overexpressing mTHTR-1 and under conditions of DNA damage or p53 activation. Our findings indicate that p53 may be involved in maintaining thiamine homeostasis through transactivation of THTR-1.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mutation on the p53 tumor suppressor gene is one of the most frequent events in human cancers. p53 has been shown to inhibit cell cycle progression, promote apoptosis, and suppress tumor cell growth (reviewed in Refs. 1-4). Numerous studies have demonstrated that p53 is an important factor for maintaining the integrity of the genome (5, 6). When cells are exposed to DNA-damaging agents, such as actinomycin D, adriamycin, UV radiation, and -irradiation (7-9), under hypoxia (10) or upon deprivation of ribonucleotides (11), stabilization and activation of p53 are noted, which then lead to cell cycle arrest or elicit apoptosis. Accumulating evidence has also indicated that p53 may be involved in DNA repair (4, 12). These functions of p53 are thought to prevent the replication of damaged DNA and protect the organism from accumulating genetic damages.

One well studied characteristic of p53 is its ability to act as a sequence-specific transcription factor. It binds to distinct DNA motif containing the consensus sequence 5'-PuPuPuC (A/T)(A/T)GPyPyPy-3' and induces the transcription of genes residing in the vicinity of the response elements (13, 14). Most of the p53 mutations found in human cancers are associated with inactivation of its DNA binding ability. Extensive effort has therefore been directed to search for the p53 downstream target genes in the hope of gaining insight into the mechanisms mediating its function.

An increasing number of p53 target genes have been identified, and the best characterized among these is p21^Waf1/Cip1. p21^Waf1/Cip1 inhibits cyclin-dependent kinases (15) and blocks the proliferating cell nuclear antigen-dependent DNA polymerase activity (16) and is vital in mediating the p53-induced G₁ arrest. p53 has also been suggested to play a role in regulating the G₂/M progression. The recent identification of a number of p53-inducible genes further substantiates its role in G₂/M checkpoint control responding to various stress conditions. B99 is a p53-inducible gene that encodes a microtubule localized protein and is specifically expressed in G₂ phase (17). BTG2 (18) and 14-3-3 (19) are other p53 target genes that have been implicated in the control of G₂/M progression. BAX (20), NOXA (21), and PUMA (22, 23) are p53 transcriptional targets that mediate the apoptotic promoting activity of p53 through their association with and inhibition of BCL2, and p53AIP1 (24) is a mitochondrial protein that, by disrupting the mitochondrial membrane potential, plays a pivotal role for the p53-induced apoptosis. Induction of a specific ribonucleotide reductase gene after DNA damage has recently been documented (25), suggesting a role for p53 in DNA repair. The growing list of p53-regulated genes explains why p53 can respond to divergent environmental stresses to protect cells against neoplastic transformation. As well as the genes that are directly involved in growth suppression, p53 also induces the expression of growth-promoting or anti-apoptotic genes. For example, p53 up-regulates the expression of TRID (26) and TRUNDD (27), the decoy death receptors that, upon overexpression, inhibit apoptosis. Although the contribution of the p53-dependent induction of these anti-apoptotic molecules in p53 signaling remains unclear, it is becoming apparent that the cellular response to p53 depends on the intricate coordination of an abundance of genes. Characterization of these genes will be crucial in understanding the p53 signaling network.

We have introduced a temperature-sensitive p53, tsp53^Val-135 (28), into the p53-null IW32 murine erythroleukemia cells (29). Several transfectants that stably express the temperature-sensitive p53 mutant protein were obtained. At permissive temperature, these cells were growth-arrested and underwent either differentiation and/or apoptosis. In the present study, we have identified a direct transcriptional target gene of p53 by the method of mRNA differential display (30). Sequence analysis revealed its identity to be the mouse orthologue of the newly identified human THTR-1¹ (31-34). We have shown that THTR-1 gene was induced under DNA damage in a p53-dependent manner; moreover, increased thiamine uptake activity correlated with p53 activation. Thus, in addition to the currently identified p53 target genes known to be involved in growth regulation and apoptosis, our findings demonstrate that p53 also regulates the expression of a molecule that is critical to thiamine homeostasis and mitochondria integrity.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture-- The IW32 murine erythroleukemia cell line and its stable transfectants expressing tsp53^Val-135 allele (29) and H1299 non-small cell lung carcinoma cells were grown in RPMI medium containing 10% fetal bovine serum and 50 µg/ml gentamycin in 5% CO₂. Temperature shift was performed by transferring subconfluent flasks to a preequilibrated incubator. Wild-type and p53-knockout mouse embryonic fibroblast (MEF) cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin; the human embryonic kidney (HEK) 293 cells and its SV40 T antigen-transformed subline (293T) were cultured in the same medium containing 10% fetal bovine serum. NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium containing 10% calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin; cells were maintained in 5% CO₂ at 37 °C and subcultured every 3 days.

RNA Isolation-- Total RNA was isolated by lysing the cells in Tri reagent (Molecular Research Center, Inc.) according to the manufacturer's recommendation. Poly(A)⁺ RNA was isolated from total RNA preparations using an Oligotex mRNA kit (Qiagen) according to the procedures described by the manufacturer.

PCR-based Differential Display-- We utilized the RNAimage kit from GenHunter for mRNA differential display (30). The parental IW32 cells and tsp53^Val-135-expressing clone 1-5 cells were cultured at 0, 5, and 9 h at 32.5 °C; RNA was isolated and resuspended in diethyl pyrocarbonate-treated H₂O. The RNA (0.4 µg) was incubated with 100 units of Moloney murine leukemia virus reverse transcriptase, 40 units of RNasin, and 0.2 µM H-T11G (A or C) primer in 20 µl of reverse transcription buffer (25 mM Tris-HCl, pH 8.3, 37.6 mM KCl, 1.5 mM MgCl₂, 5 mM dithiothreitol, and 20 µM dNTP) at 37 °C for 1 h. After heat inactivation of the reverse transcriptase at 75 °C for 5 min, 2 µl of the sample was used for PCR in 20 µl of PCR buffer (10 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl₂, 0.001% gelatin, and 2 µM dNTP) with 0.2 µM arbitrary primer (H-AP1 to H-AP16), 0.2 µM H-T11G (A or C) primer, 1 µl of [-³⁵S]dATP (1000 Ci/mmol) and 1 unit of AmpliTaq polymerase (PerkinElmer Life Sciences). The parameters for PCR were as follows: 40 cycles of cycling step (94 °C for 30 s, 40 °C for 2 min, 72 °C for 30 s) followed by 72 °C elongation step for 5 min. The amplified cDNAs were separated by electrophoresis on a 6% sequencing gel. The positive bands were excised from the dried gel and boiled in 100 µl of H₂O for 15 min, and DNA was precipitated with ethanol and resuspended in 10 µl of H₂O. Four µl of recovered cDNAs were used for reamplification by PCR in 40 µl of reaction volume with the same primers used for initial amplification except with a higher concentration of dNTP (20 µM). The parameters for PCR were the same as in the initial amplification. Reamplified cDNAs were purified from agarose gel and subcloned into pGEM-T TA cloning vector (Promega) for sequencing.

Cloning of the Mouse DDA1/THTR-1 Gene-- The differentially expressed DDA1 cDNA fragment was sequenced using a Thermo Sequenase radiolabeled terminator cycle sequencing kit (Amersham Pharmacia Biotech) as described in the manufacturer's directions. To extend the 5' cDNA sequences, 5'-RACE was performed with the Marathon cDNA amplification kit (CLONTECH) using poly(A)⁺ RNA prepared from clone 1-5 cells cultured at 32.5 °C. Alternatively, the mouse liver Marathon-ready cDNA (CLONTECH) was used as template for 5'-RACE reaction. Adaptor primer (AP-1, 5'-CCATCCTAATACGACTCACTATAGGGC-3') and gene-specific primer (GSP-1d, 5'-GCGTGGTAAAATGTGGATACAGTC-3') complementary to the 3' end of DDA1 were used for the PCRs. A 3.3-kb partial cDNA was obtained from PCR, and repeated 5'-RACE cloning did not result in additional 5' sequence. To explore the possibility that there are extremely GC-rich sequences in the 5' region of the gene that prevents the extension, 5'-RACE was performed again using Advantage-GC cDNA polymerase (CLONTECH), which has the benefits of efficient amplification of GC-rich templates. The amplified fragment extended the 5' sequence 237 nucleotides farther upstream. Sequence compilation revealed a cDNA of 3554 bp that contained a noninterrupted open reading frame of 498 amino acids. TBLASTN was used to compare DDA1 protein sequence with the GenBank^TM data base. Search results indicate that DDA1 is the mouse orthologue of the human thiamine transporter gene THTR-1 (31-34); we therefore renamed DDA1 as mTHTR-1 (mouse THTR-1). Using the mouse liver cDNA library as template with primers homologous to mTHTR-1, PCR products of 3.5 kb in length were obtained. These cDNAs were subcloned into pGEM-T (pGEM-T-mTHTR-1), and sequencing from both directions was performed on several clones. Multiple sequence alignment was performed with CLUSTALW (www2.ebi.ac.uk/clustalw/). The mTHTR-1 genome was cloned from ICR Swiss mouse GenomeWalker genomic libraries (CLONTECH). Primer pairs specific for mTHTR-1 cDNA were synthesized for genome walking PCRs according to the protocols provided.

Northern Blot Analysis-- Twenty-five µg of total RNA per lane was separated by electrophoresis on 1.2% MOPS-formaldehyde agarose gel. RNA was then blotted to the Hybond-N nylon membrane (Amersham Pharmacia Biotech) by capillary transfer. After UV radiation, the blot was prehybridized in hybridization buffer (5× saline/sodium phosphate/EDTA, 50% formamide, 10× Denhardt's solution, 0.3% SDS, and 200 µg/ml of denatured salmon sperm DNA) at 42 °C for 3-4 h. Hybridization was performed by incubating the blot with hybridization buffer containing ³²P-labeled probe at 42 °C for 20-24 h, followed by washing in 0.2× SSC and 0.1% SDS at 55 °C. Probes were gel-purified cDNA fragments labeled with [-³²P]dCTP by the Rediprime kit (Amersham Pharmacia Biotech).

Luciferase Assay-- Sense and antisense oligonucleotides for p53RE(2D) -p53RE(4D) and p53MRE(4D), with GATC added to the 5'-end for directional cloning, were synthesized,. After annealing of the oligonucleotides, they were cloned into pGUP.PA.8 luciferase-expressing vector (14) that had been predigested with BamHI and SmaI. To perform the reporter assay, 1 × 10⁵ of H1299 cells were seeded per well in 12-well culture plates for 24 h, the reporter construct (0.5 µg) was cotransfected with p53-expressing plasmid (0.5 µg) by LipofectAMINE PLUS (Life Technologies, Inc.) according to the manufacturer's directions; pCMV--galactosidase (0.5 µg) was included for calibrating the transfection efficiency. Twenty-four hours after transfection, cells were harvested and analyzed for luciferase and -galactosidase activities.

Gel Mobility Shift Assay-- Nuclear extract was prepared according to the protocols described (35). H1299 cells with or without p53 transfection were washed in ice-cold phosphate-buffered saline, and lysed in 100 µl of ice-cold hypotonic solution (10 mM HEPES, 10 mM KCl, 1 mM Mg(OAc)₂, 1 mM dithiothreitol, 10% glycerol, and 1 mM phenylmethylsulfonyl fluoride) for 10 min. The mixture was centrifuged at 12,000 × g for 5 min, and the pellet was extracted with 40 µl of hypertonic solution (hypotonic solution containing 0.5 M KCl) with continuous agitation for 30 min. After centrifuging at 12,000 × g for 5 min, the supernatant containing the nuclear extract was used for electrophoresis mobility shift assay.

p53RE(4D) was radiolabeled with [-³²P]ATP and T4 polynucleotide kinase, and the probe was incubated with nuclear extracts (7.5 µg) prepared from H1299 cells transfected with or without wild-type p53 DNA in a final reaction volume of 12 µl containing 50 mM NaCl, 1.5 mM MgCl₂, 5 mM dithiothreitol, 10% glycerol, and 0.8 µg poly(dI-dC) in 20 mM HEPES buffer, pH 7.5. p53-specific antibody PAb421 (0.15 µg) was added where indicated. Unlabeled oligonucleotides in 50-fold molar excess (5 pmol) were added as competitors for p53-DNA complex formation. After incubating at room temperature for 30 min, the mixture was chilled on ice and analyzed by electrophoresis in a native 4% polyacryamide gel. The p53Con used in the competition assay corresponds to the p53 response element from the human p21^Waf1/Cip1 gene (15) and contains the sequence 5'-GAACATGTCCCAACATGTTG-3'.

Plasmid Construction-- To construct the pCMV-mTHTR-1, pGEM-T-mTHTR-1 was digested with NotI, and the NotI fragment containing the entire open reading frame of mTHTR-1 cDNA was ligated into pRc-CMV (Invitrogen) through the NotI site. Mouse reduced folate carrier 1 (mRFC-1) cDNA containing the entire open reading frame was obtained by PCR using the mouse liver cDNAs as templates. It was ligated to the NotI site of pRc-CMV for expression. The SalI and ClaI mTHTR-1 cDNA fragment was cloned into the EcoRI site of pTRE plasmid (CLONTECH) for establishment of the Tet-on-inducible mTHTR-1 expression cells.

Thiamine Uptake Assay-- Human 293T cells were seeded at 2 × 10⁵ cells per well in 24-well culture dishes in 0.5 ml of vitamin-free RPMI (Select-Amine kit, Life Technologies, Inc.) supplemented with 10% dialyzed fetal bovine serum for 24 h. The cells were then transfected by LipofectAMINE 2000 (Life Technologies, Inc.) with 1 µg of pRc-CMV, pCMV-mTHTR-1 or pCMV-mRFC-1. After 24 h, cells were washed twice with vitamin-free RPMI medium, followed by a 30-min incubation with 0.4 ml of vitamin-free RPMI medium containing 25 nM [³H]thiamine (10 Ci/mmol) (American Radiolabeled Chemicals Inc.) at 37 °C. After being washed three times with ice-cold phosphate-buffered saline, the cells were solubilized by overnight incubation in 0.2 N NaOH followed by neutralization with 0.2 N HCl. Radioactivity was determined by liquid scintillation counting. For measuring thiamine uptake in tsp53^Val-135 transfected IW32 cells (clone 1-5), 2 × 10⁶ cells were seeded per well in 12-well dishes and cultured at 32.5 °C for 0, 6, and 12 h. Cells were harvested and assayed for [³H]thiamine uptake as described. To assay thiamine uptake in DNA damage-induced cells, 1 × 10⁵ NIH3T3 cells were seeded per well in 12-well culture dishes and cultured at 37 °C for 24 h. The cells were treated with 400 ng/ml of adriamycin for 16 h and processed for thiamine uptake assay as described. Nonspecific uptake of [³H]thiamine was determined by performing the reaction in the presence of 10 mM unlabeled thiamine.

mTHTR-1 Antibody Production-- A nucleotide with sequence corresponding to amino acids 209-294 of the mTHTR-1 cDNA was cloned into pGEX-5X-3. The GST fusion protein was expressed in Escherichia coli and purified with a glutathione-Sepharose 4B column. The protein (100 µg) was mixed with Freund's adjuvant and injected subcutaneously into rabbits. Rabbits were boosted three times at 4-week intervals, and samples of serum were collected 2 weeks after injection.

Establishment of mTHTR-1-expressing Cells and Detection of mTHTR-1 Expression-- To establish the mTHTR-1-expressing cells under the control of a tetracycline promoter, the pTRE-mTHTR-1 plasmid was transfected into the tet-on HEK 293 cells (CLONTECH). Cells were cultured in the presence of 0.15 mg/ml hygromycin for 3 weeks, and the resistant clones were selected. Expression of mTHTR-1 was confirmed by reverse transcription-PCR using mTHTR-1-specific primer pairs.

For immunofluorescence assay, the tet-on HEK 293 cells stably transfected with pTRE-mTHTR-1 or the control pTRE-vector were seeded on glass slides for 16 h and treated with or without doxycycline (5 µg/ml) for 24 h. The cells were incubated with anti-mTHTR-1 antibodies (1: 1000 dilution) for 16 h followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:1000 dilution) and viewed under a confocal microscope.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Identification of a p53-activated Gene in IW32 Erythroleukemia Cells Stably Transfected with tsp53^Val-135-- We have cotransfected the p53-null IW32 murine erythroleukemia cells with pSV2neo and a temperature-sensitive p53 mutant DNA, tsp53^Val-135 (28); several stable clones containing the mutant p53 allele were obtained (29). These cells were growth-arrested when cultured at 32.5 °C; some clones predominantly underwent apoptosis, whereas others were viably arrested and differentiated along the erythroid pathway. In order to search for p53-regulated genes, we performed mRNA differential display on one of the tsp53^Val-135 transfectants (clone 1-5) cultured at 32.5 and 38.5 °C. Clone 1-5 cells when grown at permissive temperature, arrested in G₁ phase of the cell cycle and matured along the erythroid pathway, as indicated by increased hemoglobin mRNA and protein synthesis (29). Upon differential display with a total of 16 arbitrary 10-mers and 3 poly(T) primers, 5 cDNA fragments were identified that showed increased levels of expression upon p53 induction. The activation of two of these genes, provisionally named DDA1 and DDA3 (36), as they were identified by differential display and were activated by p53, was further verified by Northern blot analysis. The 346-base pair DDA1 differential display fragment hybridized to a transcript corresponding to 3.8 kb in size that showed a pronounced rise in clone 1-5 cells cultured at 32.5 °C (Fig. 1A). The induction was first observed at 1 h following temperature downshifting, earlier than that of p21^Waf1/Cip1 (Fig. 1A). No induction of DDA1 could be detected in pSV2neo-alone transfected IW32 cells (Neo) cultured at 32.5 °C, ruling out the possibility that up-regulation of DDA1 transcript was a nonspecific temperature effect. Moreover, the p53-dependent activation of DDA1 was consistently observed in each of the five sublines of p53 transfectants tested (data not shown), indicating that the induction of DDA1 was not merely a positional effect due to site-specific p53 integration.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   DDA1 mRNA is transcriptionally induced by p53. A, the clone 1-5 cells harboring the tsp53Val-135 allele and the IW32 cells transfected with neo-control vector (Neo) were cultured at 32.5 °C for the indicated times, and total RNA was extracted and separated on agarose gel by electrophoresis as described under "Materials and Methods." The RNA was transferred to a Hybond-N membrane and hybridized with the 32P-labeled 346-bp DDA1 fragment cloned from differential display and the 32P-labeled mouse p21Waf1/Cip1 cDNA. The same blot was reprobed with DNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for equal amount of RNA loading. B, cells were treated with (+) or without (-) cycloheximide (CHX) (10 µg/ml) for 1 h at 38.5 °C; the cells were then incubated for 4 h at 32.5 °C as indicated. C, the clone 1-5 cells were cultured in the presence (+) or absence (-) of actinomycin D (Ac D) (5 µg/ml) for 30 min at 38.5 °C; cells were then cultured at 32.5 °C for the indicated times. Total RNA was extracted and analyzed by Northern blot analysis for DDA1 and glyceraldehyde-3-phosphate dehydrogenase mRNA expression.

We further examined the mechanisms underlying p53-dependent activation of DDA1 mRNA expression. Clone 1-5 cells were pretreated with or without protein synthesis inhibitor cycloheximide for 1 h before shifting to 32.5 °C, and DDA1 induction was analyzed. As can be seen in Fig. 1B, induction of DDA1 mRNA was not inhibited by cycloheximide pretreatment, indicating that protein synthesis is not required for the activation of DDA1 by p53. Control experiments with the Neo-transfected IW32 cells showed that cycloheximide had no significant effect on DDA1 mRNA levels. To confirm whether p53 activated DDA1 at the transcriptional level, cells were incubated with the RNA synthesis inhibitor actinomycin D for 30 min before temperature downshift, and DDA1 expression was monitored by Northern blotting. As can be seen in Fig. 1C, induction of DDA1 mRNA was abolished when cells were pretreated with actinomycin D. These results, together with the robust expression and fast kinetics of its induction, suggest that DDA1 is a potential p53 target gene.

Cloning of Mouse DDA1 cDNA, an Orthologue of the Human THTR-1-- 5'-RACE was adopted to clone the full-length cDNA of DDA1. After several rounds of RACE experiments using the cDNAs prepared from clone 1-5 cells and a mouse liver cDNA library as templates, a compilation of sequence data established a DDA1 sequence of 3554 nucleotides. The sequence contains an open reading frame of 1497 bp, with an ATG initiation codon starting at nucleotide 199 and a termination codon stopping at nucleotide 1695. The most upstream ATG is likely to be the translation initiation codon because it is preceded by two in-frame stop codons located at nucleotides 28 and 73, and the nucleotide sequence surrounding it resembles the Kozak consensus sequence (37). The open reading frame is followed by a 3'-untranslated region of 1859 bp that contains a polyadenylation signal. It predicts a protein of 498 amino acids with two potential N-glycosylation sites at asparagines 63 and 415. The deduced amino acid sequence, containing 12 transmembrane domains, has 40% identity to the mRFC-1 (38) and 90% identity to the human THTR-1 (31-34). DDA1 was hereinafter named mTHTR-1. Fig. 2 represents the homology alignment of the predicted amino acid sequences of THTR-1 from mouse and human (31-34) and those of RFC-1 from human (39-41), mouse (38), rat, and hamster (42). The most conserved regions are the 12 hydrophobic stretches that are clustered into two groups, each spanning the N-terminal and C-terminal halves of the protein, and are separated by a hydrophilic loop of about 90 amino acids. There is no significant homology in the loop regions of THTR-1 and RFC-1 from various species.

View larger version (66K): [in this window] [in a new window]   Fig. 2.   Comparison of the predicted amino acid sequence of mTHTR-1 with human THTR-1 and the RFC-1 family proteins. The predicted amino acid sequence of mTHTR-1 is compared with that of human THTR-1 (hTHTR-1) (31-34) and the RFCs from human (hRFC-1) (39-41), mouse (mRFC-1) (38), rat (rRFC-1), and hamster (chRFC-1) (42). Gaps (indicated by dashed lines) are introduced for maximal alignment, and residues identical to mTHTR-1 are indicated by dots. The boxed regions are the predicted transmembrane domains, and asterisks indicate the end of a sequence.

Induction of mTHTR-1 Is Dependent on p53-- To examine whether the expression of mTHTR-1 required the continuous presence of p53, the following experiment was performed. After incubating at 32.5 °C for 4 h, clone 1-5 cells were divided into two parts. Half of the cells were shifted to 38.5 °C, whereas the remaining half were maintained at 32.5 °C. The expression of mTHTR-1 in these two batches of cells was monitored by Northern blot analysis at different time points after temperature shifting. As shown in Fig. 3, mTHTR-1 mRNA was rapidly down-regulated upon shifting to 38.5 °C, and its expression was undetectable within 2 h. On the contrary, cells that were cultured at 32.5 °C maintained stable levels of mTHTR-1 mRNA throughout the time course analyzed. These results strongly support that mTHTR-1 expression is dependent on the presence of functional p53.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   mTHTR-1 mRNA expression is dependent on the continuous presence of p53. The clone 1-5 tsp53Val-135 cells were cultured at 32.5 °C for 4 h. Half of the cells were shifted to 38.5 °C, and the remaining half were maintained at 32.5 °C. At the indicated times, RNA was isolated and analyzed by Northern blotting for mTHTR-1 expression. The same blot was reprobed for 18 S rRNA expression to ensure equal loading.

The possibility that mTHTR-1 could be activated by endogenous p53 in cells undergoing DNA damage was also examined. We found that mTHTR-1 transcript was significantly induced in adriamycin- or mitomycin C-treated NIH3T3 cells harboring wild-type p53 allele (data not shown). To further examine whether DNA damage-induced activation of mTHTR-1 was p53-dependent, MEF cells from normal and p53-knockout mice were exposed to UV radiation or treated with adriamycin for various time periods, and Northern blotting was performed to monitor the mRNA expression of mTHTR-1. As seen in Fig. 4, elevated levels of mTHTR-1 and p21^Waf1/Cip1 transcripts were observed in normal MEF cells (p53^+/+) undergoing UV or drug-induced DNA damage; in contrast, no induction of mTHTR-1 could be seen in p53-knockout MEF cells (p53^/). Taken together, these results indicate that mTHTR-1 is induced in a p53-dependent manner under DNA damage.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Induction of mTHTR-1 upon DNA damage is dependent on p53. MEF cells from normal (p53+/+) and p53-knockout (p53/) mice were treated with UV (60 J/m2) for 5 and 10 h, with adriamycin (AD) (400 ng/ml) for 8 and 16 h, or with vehicle only (Con) for 16 h, and total RNA was extracted and analyzed for mTHTR-1 mRNA expression. As a p53-inducible control, the blot was reprobed for the expression of p21Waf1/Cip1. Equal amount of RNA loading was verified by reprobing the blot with glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

mTHTR-1 Contains an Intronic p53 Response Element-- To determine whether mTHTR-1 is a direct transcriptional target gene of p53, we cloned the mTHTR-1 genome using the PCR-based Genome Walking kits (CLONTECH) as templates. By comparing the mTHTR-1 cDNA sequence to the genomic DNA, the mTHTR-1 genomic structure was deduced to contain 6 exons and 5 introns, spanning a 16-kb genomic region (data not shown). An examination of the mTHTR-1 gene revealed the presence of putative p53 binding motifs at nucleotides +1649 to +1693 within the first intron (p53RE, Fig. 5A). As shown in Fig. 5B, the region spanning nucleotides +1649 to +1668 (p53RE(2D)) contains two contiguous decamers matching the previously defined p53 binding site consensus, except at the four residues indicated in the figure by lowercase letters. A third decamer with perfect match to consensus p53 binding motif is located seven bases downstream of p53RE(2D) (p53RE(3D)), and a fourth half-site that overlaps in two bases with the third site is located farther downstream (p53RE(4D)).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   The intronic p53 response element binds p53 and confers p53-mediated transactivation. A, structure of the 5' region of the mTHTR-1 gene containing the potential p53 response element (p53RE). E1 and E2 represent exons 1 and 2, respectively. Number refers to length in nucleotide pairs. Locations of the translation start site and the putative p53RE are indicated. B, sequences of the putative p53RE on mTHTR-1 (p53RE(2D)-(4D)) and the consensus p53 binding motif (p53Con) are shown; mismatches are indicated by lowercase letters. p53RE(2D) contains two adjacent half-sites with four mismatches, and an additional half-site was found 7 bases downstream of p53RE(2D), as indicated by the underlined sequence in p53RE(3D). The p53RE(4D) has a fourth half-site (underlined) that overlaps in two bases with the third site, and mutations were introduced into p53MRE(4D) at critical residues as denoted by arrowheads. C, the intronic p53RE is responsive to p53 activation. The putative p53 response elements (p53RE(2D) to p53RE(4D)) and p53MRE(4D) were cloned upstream to a luciferase reporter plasmid pGUP.PA.8 (14). These reporter constructs (0.5 µg) were cotransfected with vectors (0.5 µg) expressing either wild-type (wtp53) or mutant p53 (mp53R175H) or its vector control (pRc-CMV) into H1299 cells. Luciferase activities were analyzed 24 h after transfection. The luciferase activity was normalized with the activity of -galactosidase introduced as a transfection efficiency control. The relative luciferase activity from the cells cotransfected with pGUP.PA.8 and pRc-CMV was arbitrarily set as 1 for calculation of fold induction. Results are means ± S.D. from three independent experiments, each done in triplicate. D, the p53RE in intron 1 binds p53. The oligonucleotides corresponding to the p53RE(4D) were synthesized and radiolabeled. The probe was incubated with nuclear extracts (7.5 µg) prepared from H1299 cells transfected with (+) or without (-) wild-type p53 or in the presence (+) or absence (-) of PAb421 (0.15 µg) as indicated, and the reaction mixtures were resolved by electrophoresis using a 4% native polyacrylamide gel. Specific complex was formed when p53 containing extracts and PAb421 were both present (arrow). The ternary complex could be displaced in the presence of a 50-fold molar excess of unlabeled p53RE(4D) but not p53MRE(4D). Similarly, oligonucleotides to the p53 binding site in the p21Waf1/Cip1 gene (p53Con) were able to abolish the ternary complex formation.

To examine whether these putative p53 binding sites contained p53-dependent transcriptional activity, oligonucleotides corresponding to these sequences were synthesized and cloned upstream to a vector containing the luciferase gene driven by a minimal promoter (pGUP.PA.8). Cotransfection of the reporter plasmids containing the putative p53 binding sites with wild-type p53 resulted in significant activation of the luciferase activity (Fig. 5C). The highest p53-dependent activation was observed with p53RE(4D). In contrast, p53MRE(4D), which contained mutations introduced into the critical residues (Fig. 5B), was, as expected, not responsive to wild-type p53 activation. As a control, cells cotransfected with vector control (pRc-CMV) or the transcriptionally inactive mutant p53, mp53^R175H, showed no activation of luciferase activities.

The possibility that p53 binds directly to the intronic response sequence was next examined. Complementary oligonucleotides corresponding to p53RE(4D) were synthesized and radiolabeled. The probe was incubated with nuclear extracts prepared from p53-null H1299 lung carcinoma cells transfected with or without wild-type p53 DNA. The p53-specific antibody PAb421 was present in the reaction mixture to activate p53 DNA binding activity, as previously reported (43). As demonstrated by electrophoretic mobility shift analysis (Fig. 5D), ternary complex formation was observed in the presence of PAb421 and p53-containing extract (arrow). Formation of the complex was inhibited in the presence of excess unlabeled oligonucleotides p53RE(4D) or p53Con, the p53 binding sequence of human p21^Waf1/Cip1 gene. On the other hand, the mutated oligonucleotides p53MRE(4D) had no appreciable effect on the ternary complex formation (Fig. 5D). These results indicate that the intronic p53 response element of mTHTR-1 is a functional p53 binding site. Together with the finding that induction of mTHTR-1 was inhibited by transcription inhibitor actinomycin D but not protein synthesis inhibitor cycloheximide, these data establish mTHTR-1 as an immediate-early p53-inducible gene.

mTHTR-1 Encodes a Thiamine Transporter-- We next examined whether expression of mTHTR-1 would lead to increased thiamine uptake. The HEK 293 cells transformed with SV40 T antigen were transiently transfected with a plasmid expressing mTHTR-1 (pCMV-mTHTR-1), and the ability of cells to uptake [³H]thiamine was measured. As shown in Fig. 6, increased thiamine influx was seen in mTHTR-1 transfected cells in comparison with vector-transfected control cells (pRc-CMV). On the other hand, transfection with the plasmid expressing mRFC-1 did not significantly change the ability of cells to uptake [³H]thiamine (Fig. 6). These results demonstrated a direct association between mTHTR-1 expression and [³H]thiamine transport. The subcellular localization of mTHTR-1 was also examined. The HEK 293 tet-on cells stably expressing mTHTR-1 under the control of the tetracycline-responsive promoter (TRE-mTHTR-1) were established. We also generated a rabbit antiserum against the loop region of mTHTR-1. Immunofluorescence analysis using a confocal microscope demonstrated that mTHTR-1 was clearly located to the plasma membrane (Fig. 7). Together, these results indicate that mTHTR-1 encodes a thiamine transporter.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Expression of mTHTR-1 leads to increased thiamine influx. The HEK 293 cells transformed with SV40 T antigen were transfected with DNA expressing mTHTR-1, mRFC-1, or the vector control pRc-CMV as described under "Materials and Methods." After 24 h, cells were washed with vitamin-free medium and incubated with 25 nM [3H]thiamine for 30 min. The cells were washed to remove the unincorporated thiamine and the imported thiamine determined by liquid scintillation counting. Error bars represent the standard deviations from three independent experiments.

View larger version (76K): [in this window] [in a new window]   Fig. 7.   Immunofluorescence showing the plasma membrane localization of mTHTR-1. The HEK 293 tet-on cells expressing mTHTR-1 under the control of the tetracycline-responsive promoter were treated with (+) or without (-) 5 µg/ml doxycycline for 24 h. Cells were fixed, reacted with mTHTR-1-specific polyclonal antiserum (1:1000 dilution) followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:1000 dilution), and viewed under a confocal microscope.

Increased Thiamine Transporting Activity Correlates with p53 Activation in Cells-- To examine whether p53 expression would lead to increased thiamine uptake, we measured changes in thiamine transporting activity of clone 1-5 cells harboring the tsp53^Val-135 allele cultured at 32.5 °C for various time periods. As indicated in Fig. 8A, time-dependent increases in [³H]thiamine uptake were seen after these cells were transferred to 32.5 °C. As a control, we showed that temperature downshift had no significant effect on [³H]thiamine influx in pSV2neo alone transfected cells (Neo). We next investigated whether DNA damage would affect the ability of cells to uptake thiamine. [³H]Thiamine transport was measured in NIH3T3 cells treated with adriamycin for 16 h; under such circumstances, activation of p53 and accumulation of mTHTR-1 transcript were demonstrated (data not shown). As indicated in Fig. 8B, a greater than 2-fold increase in [³H]thiamine transport was seen in cells undergoing adriamycin-induced DNA damage. Together, we have shown that increased thiamine uptake correlated with p53 activation in cells.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Increased thiamine influx correlates with p53 activation. A, wild-type p53 overexpression results in elevated thiamine uptake. The p53-null IW32 cells stably transfected with pSV2neo alone (Neo) and the clone 1-5 cells harboring tsp53Val-135 DNA were shifted to 32.5 °C for 0, 6, and 12 h as indicated, and thiamine uptake was determined as described under "Materials and Methods." B, increased thiamine influx is associated with cells undergoing DNA damage. NIH3T3 cells were treated with (+) or without (-) 400 ng/ml adriamycin for 16 h, cells were harvested, and the [3H]thiamine uptake activity was determined. Error bars represent the standard deviations from three independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The ability of p53 to transactivate genes containing specific binding motifs is central to its role as a tumor suppressor. Many of the p53-activated genes have been shown to encode proteins that are critical in mediating p53-induced growth arrest or apoptosis. We now report the identification of the mouse thiamine transporter mTHTR-1 as a direct transcriptional target of p53, and we suggest that p53 may be involved in maintaining thiamine homeostasis and mitochondria integrity.

Several lines of evidence shown in the current study indicate that induction of mTHTR-1 is p53-dependent. First, the induction of mTHTR-1 was robust; it could be detected as early as 1 h after the expression of the wild-type p53. Moreover, mTHTR-1 expression required the continuous presence of p53, and its level returned to the noninduced state in less than 2 h following p53 inactivation. Finally, the p53-dependent activation of mTHTR-1 is supported by the finding that induction of mTHTR-1 was abolished in p53^/ MEF cells undergoing UV- and adriamycin-induced DNA damages. Our results showing that activation of mTHTR-1 was dependent on RNA synthesis but not on new protein synthesis, together with the identification of a p53 binding region within the first intron of mTHTR-1 gene that responded to p53-mediated transactivation in a transient reporter assay, indicate that mTHTR-1 is a direct transcriptional target gene of p53.

Although mTHTR-1 was identified from a subline of tsp53^Val-135 transfectant undergoing erythroid differentiation upon p53 expression, no correlation was found between the induction of mTHTR-1 and erythroleukemia cell differentiation. IW32 cells could be induced to proceed along erythroid differentiation by VM26, camptothecin, and sodium butyrate (44); however, induction of mTHTR-1 was not observed in the differentiating cells treated with any of these chemicals (data not shown). On the contrary, up-regulation of mTHTR-1 was observed in all the sublines of the tsp53^Val-135 transfectants cultured at permissive temperature, regardless of the cellular fate upon p53 expression, suggesting that mTHTR-1 is not a differentiation-associated gene for erythroid lineage. The findings that mouse THTR-1 mRNA expression could be activated by the endogenously expressed p53 in DNA damage-induced cells strongly support the notion that THTR-1 is a general downstream target of p53.

Thiamine is a vitamin required in the diet and imported through specific transporters in eukaryotic cells; thiamine transporter is therefore critical for proper functioning of cells. Thiamine is a cofactor for many enzymes of various metabolic pathways, including the mitochondrial enzymes pyruvate dehydrogenase and -ketoglutarate dehydrogenase; the latter is a key tricarboxylic acid cycle enzyme. Being the common end process in the oxidation of fatty acids, carbohydrates, and amino acids, the tricarboxylic acid cycle plays a vital part in the metabolism of almost all aerobic creatures. The high affinity thiamine transporter hTHTR-1 has recently shown to be mutated in patients of the thiamine-responsive megaloblastic anemia (31, 33, 34), an early onset, autosomal recessive disorder manifesting megaloblastic anemia, diabetes mellitus, and sensorineural deafness. Fibroblasts from these patients lack the high affinity transport system for thiamine, and treatment with thiamine alleviates the symptoms of megaloblastic anemia and diabetes mellitus. No symptoms or signs comparable to those of thiamine-responsive megaloblastic anemia patients were reported in the characterization of the p53-knockout mice; it is possible that mTHTR-1 expression is also regulated by p53-independent pathway. Consistent with this notion, we have found significant basal levels of mTHTR-1 mRNA expression in p53-knockout MEF and in several p53-null cell lines. We have shown by multiple tissue blot analysis that mTHTR-1 mRNA was most abundantly expressed in mouse liver and was absent or only modestly expressed in most tissues, including those with high levels of oxidative capacity, such as the heart and brain (data not shown). This finding, coupled with the fact that thiamine-responsive megaloblastic anemia patients are responsive to thiamine treatment, suggests that other types of thiamine transporters may exist to support the thiamine-mediated functions of mammalian cells in the absence of environmental stress.

THTR-1 belongs to the superfamily of reduced folate carrier proteins, the mTHTR-1 shares 40% identity in amino acid sequence with mRFC-1. We have examined the possibility that mTHTR-1 may function as a carrier for reduced folate by transiently transfecting mTHTR-1 cDNA to the methotrexate-resistant cell line MTX^RZR-75-1, which did not express detectable levels of folate receptor and RFC activity (45, 46). Consistent with data previously reported for human THTR-1 (32), we found no significant increase in the ability of cells to transport [³H]methotrexate, a folate analogue (data not shown). Similarly, the present results also showed that mRFC-1 did not carry any residual thiamine transporter activity. Thus, despite significant amino acid sequence homology between these two vitamin transporters, they are highly specific toward their substrates. Whereas our evidence supports that p53 activates mTHTR-1 expression at the transcriptional level, Ding et al. (47) have recently shown that p53 suppressed the hRFC gene expression. The physiological significance of the differential regulation of these vitamin transporters by p53 remains to be established.

The findings that both the expression and thiamine transporting activity of mTHTR-1 are activated by p53 suggest a role of p53 in maintaining thiamine homeostasis in cells. In the case of thiamine deficiency, oxidation through tricarboxylic acid cycle is inhibited, and cells therefore must depend on anaerobic glycolysis for energy supply; this often leads to increased production of reactive oxygen species (ROS) (48, 49). Increased ROS production has also been reported in cells exposed to ionizing irradiation (50, 51), UV radiation (52), DNA damage drugs (53), and hypoxia (54, 55); under these conditions, elevated p53 levels are consistently noted. These observations are consistent with the notion that p53 participates in orchestrating the cellular responses to oxidative stresses. Several lines of evidence indicate that perturbation of the cellular redox balance is also a downstream event of p53 activation; overexpression of p53 in DLD-1 colorectal cancer cells is reported to be accompanied by an increased production of ROS, leading to oxidative damage to mitochondria and apoptosis (56). Polyak et al. (56) have demonstrated that p53 transcriptionally induces redox-controlling genes, including PIG3, a homologue of quinone oxidoreductase, and PIG5, a proline oxidase. It is speculated that the combined effect of these enzymes may account for increased ROS generation in p53-overexpressed DLD-1 cells. On the other hand, up-regulation of antioxidant enzymes by p53 has also been reported. Expression of PIG12 (56), a novel member of the microsomal glutathione S-transferase family involved in detoxification of lipid peroxides, and glutathione peroxidase (57), an antioxidant enzyme that scavenges hydrogen peroxide and phospholipid hydroperoxides in cells, is activated by p53. Together, these findings suggest that p53 may play a crucial role in maintaining ROS balance in cells. It is conceivable that transactivation of the mTHTR-1 by p53 increases thiamine influx to ensure the proper functioning of tricarboxylic acid cycle and alleviate the deleterious effect of mitochondrial dysfunction, which aids in suppressing the intracellular ROS levels. This is especially important for cells temporarily arrested in growth because they will eventually reenter the cell cycle. In summary, it appears that p53 may induce the expression of genes, the gene products of which are important in protecting the cells from oxidative damage. The net effect of p53 expression on cellular ROS levels that may ultimately affect cellular fate is therefore dependent on the complex interplay among the proteins encoded by the myriad of p53 target genes.

	ACKNOWLEDGEMENT

We thank Dr. M. Oren (Weizmann Institute of Science, Rehovot, Israel) for kindly providing us with the tsp53^Val-135 cDNA.

	FOOTNOTES

^* This work was supported by Grants NSC88-2316-B010-022-M46 and NSC 89-2320-B010-142-M46 from the National Science Council, Taiwan, Republic of China.The costs of publication of this article were defrayed in part by the payment of page charges. The 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 GenBank^TM/EMBL Data Bank with accession number(s) AF179403 (cDNA) and AF224341 (genomic).

^** To whom correspondence should be addressed. Tel.: 022-826-7126; Fax: 022-826-4843; E-mail: ffwang@ym.edu.tw.

Published, JBC Papers in Press, July 31, 2001, DOI 10.1074/jbc.M104701200

	ABBREVIATIONS

The abbreviations used are: THTR, thiamine transporter; m, mouse; h, human; r, rat; RFC, reduced folate carrier; RACE, rapid amplification of cDNA ends; bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction; p53RE, p53 response element; ROS, reactive oxygen species; MEF, mouse embryonic fibroblast; CMV, cytomegalovirus; MOPS, 4-morpholinepropanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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