Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M413103200 on June 9, 2005

J. Biol. Chem., Vol. 280, Issue 31, 28230-28240, August 5, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/31/28230    most recent
M413103200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Song, I.-S.
Right arrow Articles by Kuo, M. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Song, I.-S.
Right arrow Articles by Kuo, M. T.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Delayed Mechanism for Induction of {gamma}-Glutamylcysteine Synthetase Heavy Subunit mRNA Stability by Oxidative Stress Involving p38 Mitogen-activated Protein Kinase Signaling*

Im-Sook Song{ddagger}, Shigeru Tatebe, Wenping Dai, and M. Tien Kuo§

From the Department of Molecular Pathology, the University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

Received for publication, November 19, 2004 , and in revised form, May 27, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of the {gamma}-glutamylcysteine synthetase heavy subunit ({gamma}-GCSh), which encodes the rate-limiting enzymes for glutathione biosynthesis, is regulated by many cytotoxic agents. Moreover, {gamma}-GCSh mRNA expression is elevated in colorectal cancer, but how {gamma}-GCSh expression is regulated is not completely understood. By using actinomycin D, which inhibits new RNA synthesis, we showed that treatment of human colorectal cancer cells with the prooxidant sulindac increased the half-life of {gamma}-GCSh mRNA. By using a tetracycline-regulated {gamma}-GCSh mRNA assay system, we systematically dissected the cis-acting sequence and trans-acting factors that regulate the stability of {gamma}-GCSh by cytotoxic prooxidants. We demonstrated that a HuR recognition sequence, AUUUA, in the 3'-untranslated region is responsible for the decay of {gamma}-GCSh mRNA. Oxidative stress enhanced cytoplasmic content of HuR. Overexpression of HuR by transfection stabilized {gamma}-GCSh mRNA, whereas overexpression of a dominant-negative HuR mutant suppressed the induced stability. Furthermore, prooxidant-induced {gamma}-GCSh mRNA stabilization and HuR binding were blocked by p38 mitogen-activated protein kinase inhibitors. We provide the first evidence that reduction-oxidation regulation of {gamma}-GCSh expression, itself a reduction-oxidation sensor and regulator, is mediated at least in part by the p38 mitogen-activated protein kinase signaling through the HuR RNA-binding protein.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reactive oxygen species play important roles in the regulation of cell growth, differentiation, apoptosis, aging, and other physiological functions. Under normal physiological conditions, there is a balance between oxidants and antioxidants that constitutes reduction-oxidation (redox)1 homeostasis. Elevated production of reactive oxygen species exerts oxidative stress, leading to a host of pathologic consequences. One of the ways a cell regulates redox homeostasis is through the glutathione system that exists in both the reduced form (GSH) and the oxidized form (GSSG) (1). Under oxidative stress, GSH is oxidized by GSH peroxidase to GSSG that is eliminated by the MRP1 (multidrug resistance associated protein) efflux pump or is catalytically reduced back to GSH by the NADPH-dependent GSH reductase. GSH also regulates the activities and biosynthesis of other redox-regulating enzymes, such as superoxide dismutase and DT-diphorases (NADPH quinine oxidoreductases 1 and 2). Because of the intracellular abundance of glutathione (1–10 mM) (2, 3), the GSH/GSSG system is the main redox regulator of cells.

The biosynthesis of GSH is regulated mainly by the ratelimiting enzyme {gamma}-glutamylcysteine synthetase ({gamma}-GCS). The mammalian {gamma}-GCS is a heterodimer consisting of one 73-kDa heavy (or catalytic) subunit ({gamma}-GCSh) (4, 5) and one 28-kDa light (or regulatory) subunit (6, 7). Hereditary {gamma}-GCSh deficiency is associated with anemia, jaundice, and neurologic abnormalities (8), and total {gamma}-GCSh deficiency is embryonic lethal in knock-out mice (9).

We demonstrated previously that expression of {gamma}-GCSh could be induced by many cytotoxic agents, including antitumor agents (10, 11), heavy metals (11), carcinogens (12), and prooxidants (13–16). All of these treatments, to various extents, induce intracellular reactive oxygen species imbalance. Moreover, {gamma}-GCSh mRNA is frequently overexpressed in human colon cancers (17), which are associated with redox imbalance (18), suggesting that the GSH/{gamma}-GCS system is a molecular sensor of intracellular redox homeostasis. Although elevated expression of {gamma}-GCSh catalyzes enhanced expression of GSH, we observed that increased GSH expression provides feedback for the down-regulation of steady-state {gamma}-GCSh mRNA expression (16). In addition, high GSH expression has been reported previously to suppress {gamma}-GCSh enzymatic activities (19). This feedback mechanism underscores the importance of {gamma}-GCSh as a redox regulator.

Studies of {gamma}-GCSh gene regulation have focused on transcriptional levels. Transcriptional up-regulation of {gamma}-GCSh is mediated by oxidative stress-response elements (ORE) located within –3802 bp (20, 21), although other investigators have also reported the involvement of an AP-1 (activator protein-1)-binding site (22–24) at the 5' side of the {gamma}-GCSh gene. The oxidative stress-response element contains the consensus sequence 5'-TGAGTCA, which is a target of the leucine zipper transcription factor Nrf2 (22). Nrf2 is normally bound to keap1, which contains a cysteine-rich domain and is anchored to the cytoplasmic actin cytoskeleton (25, 26). Oxidative stress disrupts keap1-Nrf2 interactions by modifying the two critical cysteine residues of keapl (26), resulting in the release of Nrf2, which subsequently translocates into the nucleus. Nrf2 then associates with the small Maf proteins (MafK or MafG) (27–29), and the Nrf2-Maf complex transactivates {gamma}-GCSh expression (30–32). This mechanism has been shown to regulate many other so-called phase II detoxifying enzymes (NADPH quinine oxidoreductase 1, {gamma}-GCS light chain, glutathione S-transferase {alpha}1, and heme oxygenase-1) (33, 34).

We reported previously that steady-state expression of {gamma}-GCSh mRNA increases by severalfold in human glioma cells treated with the alkylating antitumor agent 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (10). However, nuclear run-on assays revealed less than a 0.5-fold increase in the transcriptional rate. These results suggest that post-transcriptional regulation is also involved in the increased steady-state expression, perhaps depending upon prooxidants and cell sources. The mechanism involved in this post-transcriptional regulation has yet to be elucidated. The objective of the current study was to gain insight into the post-transcriptional mechanism of redox-induced {gamma}-GCSh upregulation in a cultured cell system. We found that oxidative stress-induced {gamma}-GCSh expression is mediated by the mRNA-stabilizing protein HuR, which interacts with an AU-rich sequence in the 3'-untranslated region (UTR) of {gamma}-GCSh mRNA through the p38 mitogen-activated protein kinase (MAPK) signal transduction pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid DNA and Construction of Deletion Mutants—The –3802/GCSh5'-luc recombinant DNA (20), which contains nucleotides (nt) –3802 to +465 of the {gamma}-GCSh sequence in the luciferase reporter vector pGL3 basic (Promega, Madison, WI), was obtained from R. T. Mulcahy (University of Wisconsin Medical School, Madison, WI). A series of progressively deleted flanking sequences was created by PCR by using –3802/GCSh5'-luc DNA as a template and appropriate primer sets. The PCR products were cloned into the pGL3 basic vector, generating –814/GCSh5'-luc, –202/GCSh5'-luc, –149/GCSh5'-luc, and –22/GCSh5'-luc recombinants.

Wild-type and antisense HuR (35) were obtained from M. Gorospe (National Institute on Aging, Bethesda). Plasmids encoding the constitutively active and dominant-negative forms of apoptosis signal-regulating kinase 1 (ASK1), HA-ASK1-{Delta}N and HA-ASK1-KM (36), respectively, were obtained from H. Ichijo (The university of Tokyo, Tokyo, Japan), and the constitutively active and dominant-negative forms of MAPK-activated protein kinase 2 (MAPKAPK2), Aspx3, and Ala-207 (37), respectively, were obtained from C. Marshall (Institute of Cancer Research, London, UK). Plasmids encoding the wild-type and dominant-negative forms of p38 MAPK were provided by U. Rapp (Institut für Medizinische Strahlenkunde und Zellforschung, Universität Würzburg, Würzburg, Germany) and P. Scherer (Albert Einstein College of Medicine of Yeshiva University, New York). Plasmids encoding the wild-type and dominant-negative forms of MAPK kinase 3 (MKK3) were obtained from P. Chiao (University of Texas M. D. Anderson Cancer Center, Houston).

To construct pTRE-{gamma}-GCSh (2983), we amplified the {gamma}-GCSh cDNA sequence from 400 to 2983 bp (using GenBankTM accession number M90656 [GenBank] as the reference) by PCR with primer pairs containing sequences 5'-CGACGCGTCTGAGTGTCCGTCTCGCGCC (sense; underscore sequence contains the MluI site) and 5'-CCATCGATCAGAGGGGAAAGCTTGGGGCA (antisense; underscore sequence contains ClaI site) using full pCMV-{gamma}-GCSh cDNA (20) as the template. The PCR product was digested with MluI and ClaI and inserted into the MluI/-ClaI sites of the pTRE vector (Clontech, Palo Alto, CA), which contains the tetracycline transactivator element-regulated promoter. Similar PCRs were carried out using the antisense primer sequences 5'-GCGTCGACGAGAAAATGTTTTTAAAGAGAAAAATT, 5'-GCGTCGACCATTTAGAAAACTGCTTAGACAGTAGGTTG, 5'-GCGTCGACTATGTACATGTACACTGTATAAACTCTAGA, and 5'-GCGTCGACGGCATGGTACTGTAGCCAGTTCGTC (underscores containe SalI site). These PCR products were digested with MluI/SalI and inserted into the pTRE vector to generate pTRE-{gamma}-GCSh(2781), pTRE-{gamma}-GCSh(2677), pTRE-{gamma}-GCSh(2571), and pTRE-{gamma}-GCSh(2431), respectively.

To generate site-directed mutations in the AU-rich sites, we used the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Briefly, pTRE-{gamma}-GCSh(2983) DNA was used as the template in PCRs using primers containing +2785AGGGA and +2595AGGGGA to replace +2785AUUUA (site II) and +2595AUUUUA (site I), respectively. The methylated, wild-type DNA (template) was digested by DpnI, and the nonmethylated, mutated DNA (PCR product) was transformed into XL1-Blue supercompetent cells (Stratagene). All the plasmid DNAs were confirmed by sequencing.

Cell Culture and Treatments with Prooxidants—HT-29 and HCT-15 human colorectal cancer cells were purchased from the American Type Culture Collection (Manassas, VA). The cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C in a 5% CO2 atmosphere. Cells at exponential growth conditions were treated with prooxidants at the following concentrations: 100 µM 2-acetylaminofluorene (2-AAF; Sigma), 50 µM menadione (Sigma), 100 µM pyrrolidine dithiocarbamate (PDTC; Sigma), 100 µM tert-butylhydroquinone (tBHQ; Sigma), and 800 µM sulindac (Sigma). In experiments where inhibitors were used, cells were treated with 25 mM N-acetylcysteine (Sigma) 2 h before the addition of sulindac, whereas 20 µM SB203580 (Calbiochem) and 20 µM PD98059 (Calbiochem) were added simultaneously with sulindac. Total RNA was extracted from the cells, and levels of {gamma}-GCSh mRNA were determined by the RNase protection assay.

Transient Transfection—Transient transfection was performed by using Lipofectamine (Invitrogen) according to the manufacturer's instructions. Briefly, HEK293T cells (1 x 106 cells) were seeded into 60-mm Petri dishes; 24 h later, recombinant pTRE-{gamma}-GCSh plasmid DNA and EC1214A plasmid, which expresses tTA regulation factor, were introduced into cells by using 8 µg of Lipofectamine (the amount of DNA was kept constant at 4.5 µg within experiments by adding empty vector as required). After 5 h of incubation, the medium was replaced with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. To shut off mRNA synthesis, tetracycline (2 µg/ml, Sigma) was added 24 h after transfection, and cells were harvested 0, 2, 4, 6, 9, and 12 h later in guanidine thiocyanate lysis buffer (STAT-60; Tel-Test, Friendswood, TX). RNA was extracted through phenol/chloroform extraction and isopropyl alcohol precipitation.

Luciferase Assay—HEK293T cells (2 x 105 cells) were seeded in 24-well plates, and 24 h later, 0.2 µg of {gamma}-GCSh reporter recombinant constructs and 10 ng of pRL-SV40 Renilla luciferase vector were cotranfected into cells using 4 µg of Lipofectamine. After 5 h of incubation, the medium was replaced with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. After 24 h of transfection, cells were treated with 100 µM tBHQ for 12 h, and the cells were washed twice with 1x phosphate-buffered saline and lysed with passive lysis buffer (Promega). Firefly and Renilla luciferase activities in aliquots (10 µl) of cell lysates were measured according to the manufacturer's instructions.

RNase Protection Assay—RNase protection was carried out as described previously (10). In brief, the template DNA was linearized by digestion with appropriate restriction enzymes and purified by phenol/chloroform extraction and ethanol precipitation. Antisense riboprobes were synthesized using T7 or Sp6 polymerase (Roche Applied Science) in the presence of 500 µCi of [{alpha}-32P]UTP (3,000 Ci/mmol; MP Biomedicals, Irvine, CA). The hybridization mixtures contained 20 µg of RNA and 2 x 105 cpm of riboprobe. Unhybridized RNA was degraded by incubation with RNase A (12 µg; Roche Applied Science) and RNase T (4 µg; Roche Applied Science). Protected RNA fragments were resolved by electrophoresis on denaturing 8% acrylamide, 8 M urea gels and were visualized by autoradiography. To determine the endogenous {gamma}-GCSh mRNA, we used a probe that generated a protected fragment of 268 nt. To determine exogenous {gamma}-GCSh transcripts from the transfected DNA, we constructed a plasmid that contains {gamma}-GCSh cDNA (+220 to 470 bp) and used it as a template. The riboprobe synthesized from this template annealed to the transfected (exogenous) {gamma}-GCSh mRNA and gave rise to a protected fragment of 70 nt. Under these conditions, this probe did not detect endogenous {gamma}-GCSh mRNA (data not shown). In all cases, the autoradiographic signals were quantified with an image analyzer (ImageQuant 5.0). The half-life (t1/2) of the {gamma}-GCSh mRNA was calculated from the exponential phase of the plot of remaining mRNA versus time. In all experiments, hybridization of 18 S rRNA was used as an internal control for sample loading. The intensities of the 18 S rRNA and {gamma}-GCSh mRNA bands were adjusted independently within the linear range of the signals.

Western Blot Analysis—HEK293T cells (2 x 106 cells) were seeded onto a 100-mm Petri dish. Twelve h later, cells were treated with sulindac (800 µM), PDTC (100 µM), or tBHQ (100 µM) alone or with SB203580 (20 µM) or PD98059 (20 µM) for 12 h. Cells were washed three times with phosphate-buffered saline and harvested. Cell were suspended in 200 µl of phosphate-buffered saline containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors (Complete; Roche Applied Science) and centrifuged at 12,000 rpm for 10 min at 4 °C. Protein concentrations were determined using a protein assay kit (Bio-Rad). Aliquots (40 µg) of protein were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) by using standard procedures. The membranes were then subjected to Western blotting, and the blots were developed with the enhanced chemiluminescence system (Amersham Biosciences).

Rabbit polyclonal antibodies against phospho-p38 MAPK (Thr-180/Tyr-182, 1:1,000 dilution), phospho-MKK3/6 (Ser-189/207, 1:1,000), phospho-MAPKAPK2 (Thr-334, 1:1,000), and those against nonphosphorylated p38 (1:500), MKK3/6 (1:500), and MAPKAPK2 (1:500 dilution) were purchased from Cell Signaling Technology (Beverly, MS). A mouse monoclonal antibody against {beta}-actin (Santa Cruz Biotechnology, Santa Cruz, CA) was used as a loading control. Phosphorylated and nonphosphorylated p38 MAPK control cell extracts from C6 glioma cells prepared with or without anisomycin treatment (Cell Signaling Technology) were used as negative and positive controls, respectively.

To investigate the effect of prooxidant on intracellular distribution of HuR between the cytoplasm and nucleus, we treated the cells with 800 µM sulindac or 100 µM tBHQ in the presence or absence of the inhibitors SB203580 or PD98059 for 12 h. Cytoplasmic and nucleic fractions were prepared from the treated cells using the NE-PER extraction kit (Pierce) and were processed by a standard Western blot using mouse monoclonal antibodies against HuR (Santa Cruz Biotechnology, 1:1,000), {alpha}-tubulin (Santa Cruz Biotechnology, 1:1,000), and lamin B (Calbiochem, 1:200).

RNA Gel Mobility Shift Assay—Single-stranded RNA oligonucleotides were synthesized by Dharmacon (Lafayette, CO). The sequences for the gene-specific oligonucleotides are site I WT (5'-UUAACAAUGUAUUUUAAUAACAUAU, underscore refers to site I), site I MT (5'-UUAACAAUGUAGGGGAAUAACAUAU), site II WT (5'-CUGGGACCUGAUUUAUUGAAAUUUU), and site II MT (5'-CUGGGACCUGAGGGAUUGAAAUUUU). 32P-Labeled RNA probes were prepared by kinase reaction using T4 polynucleotide kinase. Approximately 100 fmol of the radiolabeled RNA oligonucleotides (105 cpm/reaction) were incubated with 10 µg of cytoplasmic extract for 20 min at 22 °C in a buffer containing 10 mM Hepes (pH 7.6), 3 mM MgCl2, 40 mM KCl, 2 mM dithiothreitol, 10% glycerol, 0.5% Nonidet P-40, 1.5 µg/µl heparin, and 0.2 µg/µl yeast total RNA. RNA-protein complexes were separated in 6% nondenaturing polyacrylamide gel (60:1 acrylamide/bisacrylamide) in 0.5x TBE (Tris borate/EDTA) buffer at 4 °C and visualized by autoradiography.

To deplete HuR from the cell extracts, 10 µg of extract was incubated with 200 ng of anti-HuR antibody followed by an addition of 5 µl of protein A/G plus-agarose (Santa Cruz Biotechnology) for 2 h at 4 °C. The reaction mixture was centrifuged for 2 min at 14,000 rpm at 4 °C. The supernatant was incubated with radiolabeled site II wild-type RNA oligonucleotide and assayed by gel mobility shift assay. As a control, the cytoplasmic fraction was incubated with protein A/G plus-agarose without anti-HuR antibody.

In Vivo Cross-linking—RNA and protein were cross-linked by the method of Cook et al. (66) with minor modifications. HEK293T cells (2 x 106 cells) were seeded onto a 100-mm Petri dish. Twelve h later, cells were treated with sulindac (800 µM) alone or with SB203580 (20 µM) or PD98059 (20 µM) for 12 h. Cells were washed twice with ice-cold phosphate-buffered saline and resuspended in 2 ml of phosphate-buffered saline in a 100-mm Petri dish. Cells were irradiated on ice with 254-nm UV light using a transilluminator (UVP, San Gabriel, CA) without filter at a distance of 4 cm for 15 min. The cell pellets were resuspended by vortexing in 200 µl of lysis buffer (0.5% SDS, 50 mM Tris-Cl (pH 8), 1 mM EDTA, and 1 mM dithiothreitol) and boiled at 95 °C for 5 min. After dilution with 800 µl of correction buffer (1.25% Nonidet P-40, 0.625% sodium deoxycholate, 62.5 mM Tris-Cl (pH 8), 1.75 mM EDTA, 187.5 mM NaCl) containing protease inhibitor mixture (Roche Applied Science) and 150 units of RNase inhibitor (Roche Applied Science), the sample was gently sonicated two or three times and centrifuged for 90 min. The supernatants (1 ml total) were incubated with anti-HuR antibody (2 µg) followed by 50 µl of protein A/G plus-agarose (Santa Cruz Biotechnology) for 2 h at 4 °C. The samples were divided into 2 aliquots; 1 aliquot of 800 µl was centrifuged for 2 min at 14,000 rpm at 4 °C. Pellets were washed four times with NET-2 and digested with proteinase K with tumbling at 37 °C for 1 h. Following guanidine isothiocyanate extraction and ethanol precipitation, mRNA was analyzed by the RNase protection assay with probes for {gamma}-GCSh described above. The other aliquot of 200 µl was washed four times with NET-2, suspended in electrophoresis loading buffer (15 µl), boiled at 95 °C for 5 min, and centrifuged. The supernatants were subjected to Western blotting using anti-HuR antibody as described above.



View larger version (48K):
[in this window]
[in a new window]
  		FIG. 1.
Induction of {gamma}-GCSh mRNA expression and analysis of {gamma}-GCSh promoter sequence. A, up-regulation of {gamma}-GCSh mRNA by cytotoxic agents is a late event. HT-29 cells were treated with sulindac (400 µM), 2-AAF (100 µM), tBHQ (100 µM), and PDTC (200 µM), for different time intervals as indicated. Levels of {gamma}-GCSh mRNA were determined by the RNase protection assay using 18 S RNA as loading control. B, promoter sequence contributes minimally to the transcriptional up-regulation of {gamma}-GCSh expression. Left, HT-29 cells were co-transfected with a series of {gamma}-GCSh-luciferase reporter recombinant plasmids containing various lengths of {gamma}-GCSh promoter sequences and control pRL-SV40 vector for 24 h. Right, luciferase activity was normalized to control Renilla luciferase activity and was presented as a fold induction. Open bars represent control group, and hatched bars represent tBHQ treatment (100 µM) group. Each value represents mean ± S.D. of triplicate experiments.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Post-transcriptional Regulation of {gamma}-GCSh mRNA Expression Is Induced by Prooxidants—Fig. 1A shows the time course induction of {gamma}-GCSh expression by various prooxidants, including sulindac, 2-AAF, tBHQ, and PDTC. Increased steady-state levels of {gamma}-GCSh mRNA were observed 4–6 h and continued throughout the 10–24-h treatment time. Levels of induction ranged from 3- to 8.5-fold, depending upon the prooxidants. These results are consistent with our previous reports (14–16) that induction of {gamma}-GCSh expression by prooxidants was not an early event.

To investigate whether the induced up-regulation of {gamma}-GCSh by prooxidants is related to the transcriptional control, we analyzed the promoter activity of {gamma}-GCSh using a –3802/GCSh5'-luc reporter construct in response to the prooxidant tBHQ. –3802/GCSh5'-luc plasmid contains ORE (20, 21) and AP-1 (23–24). Fig. 1B shows that treatment of 100 µM tBHQ failed to increase an appreciable amount of luciferase activities. Further analyses using recombinants with progressive deletions showed that treatment with tBHQ enhanced luciferase activity by no more than 0.5-fold. Similar results were obtained with sulindac (data not shown). Given the severalfold induction of steady-state {gamma}-GCSh mRNA by tBHQ in cultured cells (Fig. 1A), we conclude that transcriptional regulation by the proximal region of {gamma}-GCSh is relatively minimal.



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 2.
Enhanced {gamma}-GCSh mRNA stability by the sulindac. HT-29 (A) and HCT-15 (B) cells were treated with 5 µg/ml actinomycin D (Act D) or 10 µM cycloheximide (CHX) alone or simultaneously with 800 µM sulindac for up to 24 h. Total RNA was prepared from treated cells, and {gamma}-GCSh mRNA expression was measured by RNase protection assay followed by densitometric analysis of mRNA stability. The t1/2 of {gamma}-GCSh mRNA was calculated from the exponential decay of the percentage of mRNA remaining versus time and expressed as mean ± S.D. of three independent experiments.

 
To investigate whether a post-transcriptional regulation mechanism is involved in {gamma}-GCSh expression, we used actinomycin D at the concentration (5 µg/ml) that inhibits RNA polymerase II activities (mRNA synthesis), but not RNA polymerase I activities (ribosomal RNA synthesis), to shut down new RNA synthesis, and we then measured the stability of {gamma}-GCSh mRNA in the presence or absence of the prooxidant sulindac. The t1/2 of {gamma}-GCSh mRNA in HT-29 and HCT-15 colorectal cancer cells treated by actinomycin D alone was 6.3 ± 1.6 and 6.8 ± 1.0 h, respectively (Fig. 2, top). In the presence of sulindac, however, the respective values increased almost 2-fold to 12 ± 1.4 and 13 ± 1.3 h, respectively. These results suggested that post-transcriptional regulation is involved in the up-regulation of {gamma}-GCSh expression induced by sulindac.

To investigate the effects of protein synthesis inhibitors on {gamma}-GCSh mRNA expression, we treated HT-29 and HCT-15 cells with 10 µM cycloheximide with or without sulindac. In both cell lines, cycloheximide alone reduced {gamma}-GCSh mRNA levels over time, with t1/2 values of 6.5 ± 1.2 and 8.4 ± 0.7 h, respectively (Fig. 2, bottom). Simultaneous treatment with cycloheximide and sulindac initially decreased {gamma}-GCSh mRNA expression, but at 6 h and thereafter, the levels increased such that the t1/2 was >20 h. These results suggested a delayed mechanism of enhanced {gamma}-GCSh steady-state mRNA expression by sulindac.

To rule out the possibility of nonspecific inhibitor effects and to investigate post-transcriptional involvement in the regulation of {gamma}-GCSh mRNA expression, we constructed a tetracycline-regulated {gamma}-GCSh recombinant plasmid (pTRE-{gamma}-GCSh(2983)) that produces a transcript of 2543 nt (83 nt in a 5'-UTR, 1914 nt in a coding region, and 588 nt in a 3'-UTR) (Fig. 3A). In the presence of tetracycline, binding of tTA (encoded by the cotransfected EC1214A plasmid) to the promoter is blocked, and transcription of exogenously transfected {gamma}-GCSh is inhibited (38, 39). We transfected pTRE-{gamma}-GCSh(2983) and EC1214 plasmid DNA into HEK293T cells. After 24 h, tetracycline alone or with sulindac was added into the culture medium. Cellular RNA was prepared at different time intervals thereafter. {gamma}-GCSh transcripts from the transfected DNA were determined by RNase protection assay using a probe that detected transcript from the transfected plasmid DNA but not from the endogenous counterpart. The t1/2 of the transcript was 7.8 ± 0.7 and 8.4 ± 1.0 h, respectively (Fig. 3, B and C). However, when sulindac was added 12 h before the addition of tetracycline, the t1/2 increased to >20 h (Fig. 3, B and C). We excluded the possibility that the concentration of tetracycline used here affected endogenous {gamma}-GCSh mRNA stability (data not shown). These results confirmed that sulindac enhances {gamma}-GCSh mRNA stability through a delayed mechanism.

3'-UTR Sequence of {gamma}-GCSh mRNA Is Important for Prooxidant-induced Delayed Stabilization—Sequences that control mRNA degradation can be located at the 5'-UTR, coding region, and 3'-UTR. We investigated whether the 3'-UTR of {gamma}-GCSh mRNA contains an unstable sequence that is a target of prooxidants. To this end, we constructed a 3'-UTR deletion mutant, pTRE-{gamma}-GCSh(2431), that lacked nt 2431–2983 (Fig. 4). This recombinant plasmid was transfected into HEK293T cells, and the stability of the transcript was similarly measured. The t1/2 was >20 h with or without sulindac (Fig. 4C). These results strongly suggested that the 3'-UTR from nt 2431 to 2983 contains unstable sequence(s).



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 3.
Delayed induction of {gamma}-GCSh mRNA stability by treatment with sulindac. A, schematic diagram of pTRE-{gamma}-GCSh(2983). UTR, untranslated region; ATG, translation start codon; CR, coding region; TGA, translation stop codon. The location of riboprobe sequence is also indicated. B, plasmid pTRE-{gamma}-GCSh(2983) (2.25 µg) was cotransfected with EC1214A (2.25 µg) into HEK293T cells. Cells were treated with 1 µg/ml tetracycline alone (top), 800 µM tetracycline and sulindac simultaneously (middle), or sulindac 12 h prior to the addition of tetracycline (bottom). Total RNA was prepared, and {gamma}-GCSh mRNA level was determined by RNase protection assay. The expression level of 18 S RNA was also measured as a control for sample loading. C, graphical representation of data from B. {gamma}-GCSh/18 S ratios were plotted as percentage of the maximum value at the time of tetracycline addition. Values in B are mean ± S.D. of three independent experiments. Because of differences in abundance, signals for {gamma}-GCSh and 18 S RNA were independently adjusted to be in the linear ranges by appropriate exposure times.

 



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 4.
3'-UTR sequence of {gamma}-GCSh mRNA is important for prooxidant-induced delayed stability. A, schematic diagram showing a series of {gamma}-GCSh mRNA 3'-UTR deletion constructs. B, the recombinant plasmids (2.25 µg) were cotransfected with EC1214A (2.25 µg) into HEK293T cells, which were then treated with tetracycline alone (untreated) or with sulindac and, 12 h later, tetracycline (sulindac). Total RNA was prepared, and the stability of {gamma}-GCSh mRNA was measured by RNase protection assay. The expression level of 18 S RNA was also measured as a control for sample loading. C, graphical representation of the experiments shown in B. Each experiment was performed at least twice. {gamma}-GCSh/18 S ratios were plotted as percentage of the maximum value at the time of tetracycline addition.

 
To determine more precisely the unstable sequence, we constructed three additional recombinants, pTRE-{gamma}-GCSh(2781), pTRE-{gamma}-GCSh(2677), and pTRE-{gamma}-GCSh(2517), whose 3'-UTR sequences ended at nt 2781, 2677, and 2517, respectively (Fig. 4A). These recombinant plasmids were transfected into HEK293T cells, and the t1/2 of the corresponding mRNA was determined. Deletion of nt 2781–2983 from the 3'-UTR stabilized {gamma}-GCSh mRNA expression in the transfection assay, as the t1/2 increased from 7.8 ± 0.8 to >20 h (Fig. 4B). These results suggest that nt 2781–2983 of {gamma}-GCSh mRNA contains unstable sequence(s). Furthermore, when sulindac was included in the transfection assay, the t1/2 of the transcript increased to >20 h (Fig. 4B, top). This result indicated that the sequence responsible for sulindac-induced mRNA stabilization is located in the same region.

HuR Is Responsible for Sulindac-induced mRNA Stabilization—Several trans-acting factors have been identified that control mRNA stabilization and destabilization The most notable of these is the HuR factor, which recognizes AU-rich sequences (30, 40–42). By examining the sequence from nt 2781 to 2983 in the 3'-UTR of {gamma}-GCSh, we found a putative HuR-binding AU-rich sequence, +2785AUUUA (site II). To investigate whether this sequence is involved in {gamma}-GCSh mRNA destabilization and is also the target for the sulindac-induced stabilization, we used site-directed mutagenesis in pTRE-{gamma}-GCSh(2983) to replace this sequence with +2785AGGGA. For comparison, we also replaced a sequence outside that region, site I (+2595AUUUUA) with +2595AGGGGA, or replaced both wild-type sites with mutant sequences (Fig. 5A). We then transfected the respective plasmids under tetracycline-regulated conditions and analyzed the stabilities of the transcripts. Mutation of site I did not enhance stabilization of the {gamma}-GCSh transcript, whereas mutations of site II or both sites I and II did (Fig. 5, B and C, untreated), suggesting that the site II sequence is the mRNA-degrading element. More importantly, site II was also the target of sulindac-induced {gamma}-GCSh mRNA stability, because only WT site II was responsive to sulindac induction (Fig. 5, B and C, sulindac). Site II contains the HuR-responsive sequence because in the cotransfection experiment with HuR expression vector, only the wild-type and site I sequence conferred {gamma}-GCSh mRNA stability (Fig. 5, B and C, HuR). Taken together, these results strongly suggested that sulindac-induced {gamma}-GCSh mRNA stability is mediated by the HuR-interacting +2785AUUUA (site II) sequence.



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 5.
HuR binding controls {gamma}-GCSh mRNA stability. A, wild-type and three recombinant pTRE-{gamma}-GCSh(2983) constructs. Name of construct indicates which site was mutated. B, the plasmids were cotransfected with EC1214A or empty vector (1.5 µg each) into HEK293T cells, which were then treated with tetracycline alone (untreated) or with sulindac 12 h before tetracycline (sulindac). In addition, HuR plasmid (1.5 µg) was cotransfected with the respective vectors (1.5 µg) and EC1214A (1.5 µg) into HEK 293T cells, which were then treated with tetracycline alone (HuR). Total RNA was prepared, and the stability of {gamma}-GCSh mRNA expression was measured by the RNase protection assay. C, graphical representation of the data shown in B. Each data point represents mean ± S.D. of three independent experiments. {gamma}-GCSh/18 S ratios were plotted as percentage of the maximum value at the time of tetracycline addition. Because of differences in abundance, signals for {gamma}-GCSh and 18 S were independently adjusted to be in the linear ranges by appropriate exposure times.

 
Other Prooxidants Induce {gamma}-GCSh mRNA Stability—To investigate whether prooxidants other than sulindac can induce stabilization of {gamma}-GCSh mRNA, we used the tetracycline-regulated system to determine the t1/2 of pTRE-{gamma}-GCSh transcripts in transfected HEK293T cells treated with 2-AAF, menadione, or t-BHQ, following the procedure as described in Fig. 3. All four prooxidants, like sulindac, induced stabilization of {gamma}-GCSh transcripts in delayed mechanism (Fig. 6). Because these agents can induce redox imbalance, our results are consistent with the idea of redox regulation of {gamma}-GCSh mRNA stability.

p38 MAPK Pathway Is Involved in Induction of {gamma}-GCSh mRNA Stability—To investigate the signal transduction mechanism by which redox conditions influence {gamma}-GCSh mRNA stability, we used an activator and several inhibitors of various signal transduction pathways (Fig. 7A). We envisioned that inhibitors of the involved pathways would suppress the induction of {gamma}-GCSh mRNA stability. To this end, HT-29 colorectal cancer cells were treated with a prooxidant (sulindac, PDTC, or tBHQ) and/or the activator or inhibitors. Expression levels of {gamma}-GCSh mRNA were determined by the RNase protection assay and quantified by densitometry, using the signal from 18 S RNA as a control for sample loading. The expression of endogenous {gamma}-GCSh mRNA was increased by treatment with prooxidants alone (Fig. 7B). This increase was not suppressed by the addition of genistein, H7, or staurosporine, which inhibit protein kinases A and C, and protein kinase A, C, and G, and calmodulin kinase pathways, respectively. The induction of {gamma}-GCSh expression was moderately suppressed by sodium azide, an activator of AMP-activated protein kinase, and by PD98059, an inhibitor of MAPK/ERK kinase signals. SB203580, a specific inhibitor of the p38 MAPK pathway, was the most potent inhibitor overall. None of the inhibitors by themselves, at the concentrations used, enhanced the expression of {gamma}-GCSh. These results suggest that p38 MAPK is an important signaling pathway involved in the induction of {gamma}-GCSh expression by prooxidants, although the results also suggested other signaling mechanisms may be involved to a lesser degree.

To investigate whether the p38 MAPK pathway regulates {gamma}-GCSh mRNA stability, we again used the tetracycline-regulated system in the transfection assay. Fig. 8 shows that sulindac-induced mRNA stability was inhibited by cotreatment with SB203580 but not with PD98059. N-Acetylcysteine, an inhibitor of oxidative stress, was also a potent inhibitor of sulindac-induced {gamma}-GCSh mRNA stability. These results, together with those described above, strongly suggest that prooxidant-induced {gamma}-GCSh mRNA stability is regulated by p38 MAPK signaling through the HuR-binding site located at +2785AUUUA of the {gamma}-GCSh 3'-UTR.



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 6.
Enhanced stability of {gamma}-GCSh mRNA by various prooxidants. pTRE-{gamma}-GCSh(2983) (2.25 µg) was cotransfected with EC1214A (2.25 µg) into HEK293T cells. Twelve h later, cells were treated with 100 µM 2-AAF, 50 µM menadione, 800 µM sulindac, 100 µM tBHQ for an additional 12 h and followed by 1 µg/ml tetracycline. Total RNA was prepared, and the stability of {gamma}-GCSh mRNA expression was measured by RNase protection assay. Values shown represent mean ± S.D. A, graphical representation of the data in B. Each experiment was performed at least twice. {gamma}-GCSh/18 S ratios were plotted as percentage of the maximum value at the time of tetracycline addition.

 



View larger version (47K):
[in this window]
[in a new window]
  		FIG. 7.
Signal transduction pathways involved in prooxidant-induced stabilization of {gamma}-GCSh expression. A, function, target, and dose of activator and inhibitors used. B, HT-29 cells were treated with 800 µM sulindac, 100 µM PDTC, or 100 µM tBHQ for 12 h with or without indicated inhibitors. Total RNA was prepared, and the stability of {gamma}-GCSh mRNA expression was measured by RNase protection assay. +, prooxidant treatment; –, no prooxidant treatment.

 
Prooxidants Increase Cytoplasmic HuR and Binding to {gamma}-GCSh mRNA—To strengthen the involvement of HuR in prooxidant-induced, p38 MAPK-mediated stabilization of {gamma}-GCS mRNA, we treated HEK293T cells with sulindac or tBHQ in the presence of SB203580 or PD98059. Cytoplasmic and nuclear fractions were prepared from the treated cells and subjected to Western blotting using nuclear (lamin B) and cytoplasmic ({alpha}-tubulin) markers. The amounts of cytoplasmic and nuclear HuR were determined by Western blotting. No detectable cytoplasmic contamination of the nuclear fraction was evident (Fig. 9). Treatments with sulindac or tBHQ increased cytoplasmic content of HuR in the absence of any inhibitors. This increase was suppressed by SB203580 but not by PD98059. These results demonstrated that the prooxidant-induced increase in cytoplasmic HuR is specifically mediated by p38 MAPK signaling.

To demonstrate that the increase in HuR was accompanied by an increase in its binding to the {gamma}-GCSh 3'-UTR sequence, we performed gel mobility shift assays by incubating various cytoplasmic extracts with 32P-labeled probes containing site I, site II, and their mutated sequences. Fig. 10A shows that sulindac-treated extract could only support mobility shift of site II WT probe but not with probes containing site I and their mutated sequences. This mobility shift was not seen in the extracts from SB203580-treated cells. Extract from PD98059-treated cells did not diminish the mobility shift. These results demonstrated that the induced binding activity to site II sequence involved p38 MAPK signaling. Binding of site II probe could be competed efficiently by its cognate (2785AUUUA) sequence but not by the mutant (2785AGGGA) sequence within the same concentration range, demonstrating the sequence specificity of protein binding (Fig. 10B). Moreover, depleting the cell extract by using an anti-HuR antibody diminished the binding (Fig. 10C), demonstrating the involvement of HuR in the binding to site II sequence.

The in vivo interaction between {gamma}-GCSh mRNA and HuR protein was further confirmed by using UV cross-linking. Treatment of sulindac increased the amount of {gamma}-GCSh mRNA bound to HuR (Fig. 11, compare lanes 3 and lane 1), and cotreatment with SB203580 (lane 7), but not PD98059 (lane 5), reduced the amount of bound {gamma}-GCSh mRNA to the control level (lane 1). Only marginal amounts of {gamma}-GCSh mRNA were detected when UV was not used (Fig. 11, lanes 2, 4, 6, and 8). Taken together, these results demonstrated that the prooxidant-induced increase in the cytoplasmic distribution of HuR is mediated by p38 MAPK signaling and that HuR interacts specifically with the site II sequence of {gamma}-GCSh mRNA.

p38 Signal Transduction Pathway in Prooxidant-induced {gamma}-GCSh mRNA Stability—The p38 MAPK signaling pathway is composed of three sequentially activated kinase families as follows: MAPK, MAPK kinase (MKK), and MKK kinase families. MAPK phosphorylates substrates upon activation through phosphorylation by MKKs, which are themselves activated by MKK kinases (43). To demonstrate further the involvement of the p38 pathway in prooxidant-induced {gamma}-GCSh mRNA stability, we investigated whether sulindac, PDTC, and tBHQ can activate p38 and MKK activities. Treating HT-29 cells with these prooxidants indeed activated p38, MKK3/6, and MAPK activating protein kinase 2 (MAPKAPK2), as evidenced by Western blotting (Fig. 12). Activation of p38 MAPK was suppressed by SB203580 but not by PD98059, consistent with the results shown above that SB203580 but not PD98059 suppressed sulindac-induced {gamma}-GCSh mRNA stability. Activation of MAPKAPK2 by these prooxidants was suppressed by SB203580 but not by PD98059, consistent with the results that MAPKAPK2 is a downstream effector of p38 signaling (43). SB203580 did not suppress MKK3/6 activation because this kinase is upstream of p38 MAPK.



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 8.
Inhibition of sulindac-induced {gamma}-GCSh mRNA stabilization. pTRE-{gamma}-GCSh(2983) (2.25 µg) was co-transfected with EC1214A (2.25 µg) into HEK293T cells, which were then treated with 1 µg/ml tetracycline alone (control) or with 800 µM sulindac (alone or with 25 mM N-acetylcysteine, 20 µM SB203580, or 20 µM PD98059) 12 h before 1 µg/ml tetracycline. Total RNA was prepared, and the stability of {gamma}-GCSh mRNA expression was measured by RNase protection assay. Values shown represent mean ± S.D. of three independent experiments. A, graphical representation of the data shown in B.

 



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 9.
Increase cytoplasmic HuR by prooxidant treatments. HEK293T cells were treated with 800 µM sulindac or 100 µM tBHQ for 12 h in the presence of 20 µM SB203580 or 20 µM PD98059. Nuclear (N) and cytoplasmic (C) fractions were prepared, and proteins (40 µg/lane) were separated by 12% SDS-PAGE and sequentially probed with anti-HuR, anti-lamin B, and anti-{alpha}-tubulin antibodies.

 
The results presented thus far are consistent with the following signal transduction mechanisms for the induction of {gamma}-GCSh mRNA stability: prooxidants -> MKK kinase (ASK1) -> MKK (MKK3/6) -> p38 MAPK -> MAPKAPK2 -> HuR -> {gamma}-GCSh mRNA stabilization. To provide support for this proposition, we cotransfected constitutively active and dominant-negative recombinants encoding various kinases with the tetracycline-regulated {gamma}-GCSh mRNA stability assay system. As shown in Fig. 13, cotransfection with plasmid DNA encoding constitutively active ASK1, MKK3, p38 MAPK, and MAPKAPK2 stabilized {gamma}-GCSh mRNA (Fig. 13A), whereas co-transfection with plasmid DNA encoding dominant-negative forms of these kinases suppressed the induced stabilization of {gamma}-GCSh mRNA (Fig. 13B). Similarly, cotransfection of HuR expression plasmid induced {gamma}-GCSh mRNA stability, whereas cotransfection of antisense HuR mutant suppressed the induction (Fig. 13B, bottom). Thus, we conclude that induction of {gamma}-GCSh mRNA stability by prooxidants is controlled by the p38 MAPK pathway through HuR, which interacts with an AU-rich sequence located at the 3'-UTR of the mRNA.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Post-transcriptional Regulation of {gamma}-GCSh Expression by Prooxidants—Oxidative stress is known to regulate the expression of a number of genes (reviewed in Ref. 44). Recent studies demonstrated that changes in redox conditions can transcriptionally activate many cellular genes without effects on mRNA stability, including those encoding angiotensin II receptor (45), mitochondrial transporter (UCP2) (46), insulin-like growth factor binding protein-1 (47), and Rac1 GTPase (48). Moreover, redox-regulated gene expression can be controlled at the post-transcriptional levels by modulating mRNA stability. Oxidative stress induced by glucose deprivation in cultured cells increases vascular endothelial growth factor mRNA stability (49) but reduces insulin growth factor-1 mRNA stability (50). Increased stability of extracellular superoxide dismutase mRNA, but not manganese superoxide dismutase mRNA, is associated with changes of cellular redox conditions by the treatment of 17{beta}-estradiol (51). These observations, collectively, suggest multiple effects on mRNA stability by oxidative stress, perhaps depending upon the context of genes, cell types, and redox-modulating agents. However, mechanisms that regulate mRNA stability within these contexts are not well understood.

{gamma}-GCSh mRNA is an important system for studying redox-regulated mRNA stability. It encodes the rate-limiting enzyme for the de novo biosynthesis of GSH. The increased GSH levels preserve cytoprotective functions under oxidative stress (52). Thus, {gamma}-GCSh, like glutathione S-transferase {alpha}2 and NADPH quinine oxidoreductase 1, can be considered as a phase II enzyme in the detoxification system of drug metabolism (33, 34). Previous studies on the regulation of genes encoding phase II enzymes have been mostly focused on transcriptional mechanisms (53, 54). Post-transcriptional regulation of the phase II genes has not been well studied.

The present demonstration that increased {gamma}-GCSh mRNA stability under oxidative stress conditions underscores the importance of the post-transcriptional mechanism in the regulation of this phase II gene expression. Actinomycin D inhibitor experiments demonstrated that sulindac treatment resulted in an ~2-fold increase in the t1/2 values of {gamma}-GCSh mRNA stability. Results from transient transfection assays using the tetracycline-regulated system are consistent with these results. More importantly, we have demonstrated that this post-transcriptional regulation was activated by many oxidative stress-inducing agents, including sulindac, PDTC, tBHQ, 2-AAF, and menadione; and some of these agents are not known for transcriptional activation of the phase II enzymes-encoded genes. Thus, the post-transcriptional regulation mechanism described here is not spurious and represents an important mechanism of {gamma}-GCSh gene regulation under stress conditions.

One of the important findings presented in this work is the identification of HuR as a target of prooxidant-induced {gamma}-GCSh mRNA stability. We first identified an AU-rich sequence (2785AUUUA) located in the 3'-UTR of {gamma}-GCSh mRNA that is responsible for the instability of {gamma}-GCSh mRNA. Many AU-rich binding proteins have been identified, but HuR functions as an mRNA-stabilizing factor (35, 40–42). Indeed, overexpression of HuR by transfection stabilizes {gamma}-GCSh mRNA degradation. HuR is a ubiquitously expressed, predominantly nucleus-located member of the elav (embryonic-lethal abnormal visual in Drosophila melanogaster) family of RNA-binding proteins (56). It has been shown that HuR translocates to the cytoplasm and stabilizes ARE-containing mRNAs (35, 57). Consistent with this mechanism, we demonstrated that upon treatment of prooxidants, the cytoplasmic contents of HuR are increased. This is associated with the enhanced binding of HuR to {gamma}-GCSh mRNA as demonstrated by the in vivo cross-linking procedure. Another AU-rich RNA binding factor hnRNP D has also been reported to up-regulate mRNA stability in a cell type-specific manner (55). Whether hnRNP D plays a role in prooxidant-induced {gamma}-GCSh mRNA stabilization remains to be investigated.



View larger version (45K):
[in this window]
[in a new window]
  		FIG. 10.
Gel mobility shift analysis of HuR binding to {gamma}-GCSh 3'-UTR sequences. A, cytoplasmic proteins (10 µg) prepared from sulindac-treated HEK293 T cells in the presence or absence of SB20358 or PD98059 were incubated with 32P-labeled probes (100 fmol) containing wild-type (WT), site I, site II, and their mutated (MT) sequences. Mobility shift was determined by gel electrophoresis. B, analysis of protein binding specificity by competition assay. Cytoplasmic extracts from sulindac-treated cells were incubated with wild-type site II sequence probe in the presence of increased amounts of unlabeled oligoribonucleotides containing wild-type (2785AUUUA) or mutated (2785AGGGA) sequence followed by mobility shift assay. C, cytoplasmic extracts (10 µg) prepared from sulindac-treated HEK293 T cells were incubated with anti-HuR antibody (200 ng) followed by 5 µl of protein A/G plus-agarose to pull-down HuR. The HuR-depleted supernatants were incubated with radiolabeled wild-type site II RNA oligonucleotide and assayed by gel mobility shift assay. As a control, the cytoplasmic fraction was incubated with protein A/G plus-agarose without anti-HuR antibody.

 



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 11.
In vivo interaction between {gamma}-GCSh mRNA and HuR. Sulindac-treated HEK 293T cells, in the presence or absence of SB203580 or PD98059, were irradiated with 254-nm UV light to cross-link RNA and protein in vivo (lanes 1, 3, 5, and 7) or without cross-linking (lanes 2, 4, 6, and 8). Cell extracts were prepared and divided into 2 aliquots; one was used for immunoprecipitation of HuR-bound {gamma}-GCSh mRNA using anti-HuR antibody, and the other was used for Western blotting of HuR as reference of sample loading. {gamma}-GCSh mRNA levels were measured by RNase protection assay.

 



View larger version (76K):
[in this window]
[in a new window]
  		FIG. 12.
Activation of p38 MAPK pathway by various prooxidants. HT-29 cells were treated with 800 µM sulindac, 100 µM PDTC, or100 µM tBHQ for 12 h in the presence or absence of 20 µM SB203580 or 20 µM PD98059. Cell lysates were prepared, and proteins (40 µg/lane) were separated by 10% SDS-PAGE and probed by the indicated antibodies. Negative (–) and positive (+) controls were cell lysates provided by the vendor for each antibody used. {beta}-Actin was used as a loading control. Phosphoproteins are indicated by p-.

 
Redox Regulation of p38 MAPK Kinase Signaling in the Post-transcriptional Regulation of {gamma}-GCSh mRNA Stability— Another important finding from the present study is the identification that the p38 MAPK pathway is involved in the oxidative stress-induced {gamma}-GCSh mRNA stability. This was first demonstrated by using a panel of inhibitors to various signal transduction pathways, including SB203580 at concentration (20 µM) that has been demonstrated to be highly selective to the p38 MAPK signaling (57–59). This initial observation led to the formulation of prooxidant-induced {gamma}-GCSh mRNA stabilization mechanism as depicted in Fig. 14.

Fig. 14 shows that activation of p38 MAPK initiates its downstream signal, MAPKAPK2 (39, 57, 61), and we demonstrated that cotransfection of expression vector for MAP-KAPK2 enhanced {gamma}-GCSh mRNA stabilization, whereas dominant-negative recombinant suppressed prooxidant-induced stabilization. Previous studies have shown that overexpression of MAPKAPK2 increases cytoplasmic HuR accumulation, which is associated with dramatic changes in the formation of HuR and ARE complexes (42). How MAKPAPK2 regulates cytoplasmic HuR accumulation remains to be elucidated, although the Hsp20 (39) and hnRNP A0 (62) have been implicated as targets of MAPKAPK2.



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 13.
Enhanced {gamma}-GCSh mRNA stability by cotransfection of recombinant plasmid DNA encoding members of the p38 MAPK pathway. pTRE-{gamma}-GCSh(2983) (1.5 µg) and EC1214 (1.5 µg) were cotransfected with recombinant plasmids (1.5 µg) expressing constitutively active ASK1 and MAPKAPK2 (A) or wild-type (WT) MKK3, p38 MAPK, and HuR (also A), dominant-negative recombinant forms of all components (B). Empty vector (1.5 µg) was used as control. Cells were then treated with 1 µg/ml tetracycline alone (A) or with 800 µM sulindac 12 h before tetracycline (B). Total RNA was prepared, and the stability of {gamma}-GCSh mRNA expression was measured by RNase protection assay. C, graphical representation of data shown in B. Data in B and C represent mean ± S.D. of three independent experiments.

 



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 14.
Proposed pathway for redox regulation of {gamma}-GCSh mRNA stability.

 
It has been demonstrated that under normal physiologic conditions, the reduced form of thioredoxin is complexed with ASK1 (also known as MKK kinase 5) that inhibits ASK1 activity (63). Upon oxidative stress, thioredoxin is oxidized and dissociated from ASK1, which subsequently activates downstream signaling, by sequential phosphorylation of MKK3/6 and p38 MAPK (64). This mechanism also explains the association between cellular oxidative stress and the MAPK pathway activation as shown in Fig. 14.

This signal transduction pathway emphasizes the sequential events for redox-induced mRNA stabilization. The involvement of many steps of activation in the process may explain the delayed mechanism of induction as described here. The increased steady-state {gamma}-GCSh mRNA expression induced by various prooxidants is a late event; stabilization of {gamma}-GCSh transcripts can only be observed when the prooxidants are added before the switch to the tetracycline-off system; and induction was delayed in the time course analysis of {gamma}-GCSh mRNA expression in cells treated with cycloheximide and sulindac. However, this delayed effect was not seen when actinomycin D was used. This discrepancy might be attributable to unknown side effects of actinomycin D.

We demonstrated previously that expression of {gamma}-GCSh is itself regulated by redox conditions (16). The pathway described in Fig. 14 can also explain the redox-induced feedback inhibition of {gamma}-GCSh biosynthesis. Stabilization of {gamma}-GCSh mRNA may increase the biosynthesis of GSH, resulting in a reduced intracellular redox status. This in turn may suppress the oxidation of thioredoxin and its downstream p38 MAPK kinase pathway, thereby suppressing the induction of {gamma}-GCSh mRNA stability.

Post-transcriptional Regulation of {gamma}-GCSh Expression in Cancer—We observed previously that {gamma}-GCSh mRNA expression is correlated with colorectal tumor progression, being low in adenomas and high in carcinomas (15, 17). The post-transcriptional regulation mechanism is likely to involve the increased {gamma}-GCSh mRNA levels in colon cancer from the following considerations. First, it has been suggested that increased oxidative stress is associated with colorectal carcinogenesis (18). Second, by using immunohistochemical staining, it has been shown that the cytoplasmic abundance of HuR increases with malignancy, particularly in colorectal carcinomas (65). Moreover, the development of colon cancer in experimental animals correlates with HuR content. Colon cancer cells overexpressing HuR produced significantly larger tumors than those arising from control cells, whereas those with reduced HuR levels through small interference RNA- or antisense HuR-based approaches developed significantly more slowly (65). Third, constitutively elevated p38 MAPK expression is frequently associated with cancer progression (57). These findings, together with those described in this study, suggest that the pathway elicited in Fig. 14 may be the underlying mechanism for the enhanced expression of {gamma}-GCSh mRNA in colon carcinomas. We have observed previously that expression of MRP1, an ATP-dependent efflux pump for eliminating diverse anticancer drugs, is tightly associated with that of {gamma}-GCSh (13–16), including colon cancers (15, 17). It is plausible that the post-transcriptional regulation mechanism described here may be involved in the regulation of this important drug resistance transporter.

Taken together, our results have important implications in the redox regulation of colorectal carcinogenesis and the evolution of drug resistance during disease development. Further investigations are required to establish the clinical relevance of these observations. These studies may eventually lead to the development of intervention strategies for the management of colon cancer.


   FOOTNOTES
 
* This work was supported in part by NCI, National Institutes of Health Grants CA79085, CA72404 (to M. T. K.), and CA16672 (Cancer Center Core) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported in part by the post-doctoral fellowship program of the Korean Science and Engineering Foundation. Back

§ To whom correspondence should be addressed: Dept. of Molecular Pathology, Unit 89, the University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-3214; Fax: 713-792-8424; E-mail: tkuo{at}mdanderson.org.

1 The abbreviations used are: redox, reduction-oxidation; {gamma}-GCS, {gamma}-glutamylcysteine synthetase; {gamma}-GCSh, {gamma}-glutamylcysteine synthetase heavy subunit, UTR, untranslated region; MAPK, mitogen-activated protein kinase; ASK1, apoptosis signal-regulating kinase 1; MAP-KAPK2, MAPK-activated protein kinase 2; MKK3, MAPK kinase 3; PDTC, pyrrolidine dithiocarbamate; tBHQ, tert-butylhydroquinone; 2-AAF, 2-acetyl aminofluorene; nt, nucleotide; hnRNP, heterogeneous nuclear ribonucleoprotein; ORE, oxidative stress-response elements; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank Dr. A. B. Shyu, University of Texas Health Science Center at Houston Medical School, for advice on the use of the tetracycline-regulated system for the analysis of mRNA stability, and Pierrette Lo, Scientific Publications, M. D. Anderson Cancer Center, for editorial help with the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Smith, C. V., Jones, D. P., Guenthner, T. M., Lash, L. H., and Lauterburg, B. H. (1996) Toxicol. Appl. Pharmacol. 140, 1–12[CrossRef][Medline] [Order article via Infotrieve]
  2. Gipp, J. J., Chang, C., and Mulcahy, R. T. (1992) Biochem. Biophys. Res. Commun. 185, 29–35[CrossRef][Medline] [Order article via Infotrieve]
  3. Hwang, C., Sinskey, A. J., and Lodish, H. F. (1992) Science 257, 1496–1502[Abstract/Free Full Text]
  4. Hamilton, D., Wu, J. H., Alaoui-Jamali, M., and Batist, G. (2003) Blood 102, 725–730[Abstract/Free Full Text]
  5. Huang, C. S., Chang, L. S., Anderson, M. E., and Meister, A. (1993) J. Biol. Chem. 268, 19675–19680[Abstract/Free Full Text]
  6. Gipp, J. J., Bailey, H. H., and Mulcahy, R. T. (1995) Biochem. Biophys. Res. Commun. 206, 584–589[CrossRef][Medline] [Order article via Infotrieve]
  7. Huang, C. S., Anderson, M. E., and Meister, A. (1993) J. Biol. Chem. 268, 20578–20583[Abstract/Free Full Text]
  8. Ristoff, E., Augustson, C., Geissler, J., de Rijk, T., Carlsson, K., Luo, J. L., Andersson, K., Weening, R. S., van Zwieten, R., Larsson, A., and Roos, D. (2000) Blood 95, 2193–2196[Abstract/Free Full Text]
  9. Dalton, T. P., Dieter, M. Z., Yang, Y., Schertzer, H. G., and Nebert, D. W. (2000) Biochem. Biophys. Res. Commun. 279, 324–329[CrossRef][Medline] [Order article via Infotrieve]
  10. Gomi, A., Shinoda, S., Masuzawa, T., Ishikawa, T., and Kuo, M. T. (1997) Cancer Res. 57, 5292–5299[Abstract/Free Full Text]
  11. Ishikawa, T., Bao, J. J., Yamane, Y., Akimaru, K., Frindrich, K., Wright, C. D., and Kuo, M. T. (1996) J. Biol. Chem. 271, 14981–14988[Abstract/Free Full Text]
  12. Deng, L., Lin-Lee, Y. C., Claret, F. X., and Kuo, M. T. (2001) J. Biol. Chem. 276, 413–420[Abstract/Free Full Text]
  13. Ikegami, Y., Tatebe, S., Lin-Lee, Y. C., Xie, Q. W., Ishikawa, T., and Kuo, M. T. (2000) J. Cell. Physiol. 185, 293–301[CrossRef][Medline] [Order article via Infotrieve]
  14. Lin-Lee, Y. C., Tatebe, S., Savaraj, N., Ishikawa, T., and Kuo, M. T. (2001) Biochem. Pharmacol. 61, 555–563[CrossRef][Medline] [Order article via Infotrieve]
  15. Tatebe, S., Sinicrope, F. A., and Kuo, M. T. (2002) Biochem. Biophys. Res. Commun. 290, 1427–1433[CrossRef][Medline] [Order article via Infotrieve]
  16. Yamane, Y., Furuichi, M., Song, R., Van, N. T., Mulcahy, R. T., Ishikawa, T., and Kuo, M. T. (1998) J. Biol. Chem. 273, 31075–31085[Abstract/Free Full Text]
  17. Kuo, M. T., Bao, J. J., Curley, S. A., Ikeguchi, M., Johnston, D. A., and Ishikawa, T. (1996) Cancer Res. 56, 3642–3644[Abstract/Free Full Text]
  18. Cerutti, P. A. (1994) Lancet 344, 862–863[CrossRef][Medline] [Order article via Infotrieve]
  19. Meister, A. (1995) Methods Enzymol. 251, 3–7[Medline] [Order article via Infotrieve]
  20. Mulcahy, R. T., Wartman, M. A., Bailey, H. H., and Gipp, J. J. (1997) J. Biol. Chem. 272, 7445–7454[Abstract/Free Full Text]
  21. Wild, A. C., Moinova, H. R., and Mulcahy, R. T. (1999) J. Biol. Chem. 274, 33627–33636[Abstract/Free Full Text]
  22. Rahman, I., Smith, C. A., Lawson, M. F., Harrison, D. J., and MacNee, W. (1996) FEBS Lett. 396, 21–25[CrossRef][Medline] [Order article via Infotrieve]
  23. Tomonari, A., Nishio, K., Kurokawa, H., Arioka, H., Ishida, T., Fukumoto, H., Fukuoka, K., Nomoto, T., Iwamoto, Y., Heike, Y., Itakura, M., and Saijo, N. (1997) Biochem. Biophys. Res. Commun. 232, 522–527[CrossRef][Medline] [Order article via Infotrieve]
  24. Urata, Y., Honma, S., Goto, S., Todoroki, S., Iida, T., Cho, S., Honma, K., and Kondo, T. (1999) Free Radic. Biol. Med. 27, 838–847[CrossRef][Medline] [Order article via Infotrieve]
  25. Itoh, K., Wakabayashi, N., Katoh, Y., Ishii, T., Igarash, K., and Yamamoto, M. (1999) Genes Dev. 13, 76–86[Abstract/Free Full Text]
  26. Wakabayashi, N., Dinkova-Kostova, A. T., Holtzclaw, W. D., Kang, M. I., Kobayashi, A., Yamamoto, M., Kensler, T. W., and Talalay, P. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 2040–2045[Abstract/Free Full Text]
  27. Jeyapaul, J., and Jaiswal, A. K. (2000) Biochem. Pharmacol. 59, 1433–1439[CrossRef][Medline] [Order article via Infotrieve]
  28. Moran, J. A., Dahl, E. L., and Mulcahy, R. T. (2001) Biochem. J. 361, 371–377
  29. Nguyen, T., Huang, H. C., and Pickett, C. B. (2000) J. Biol. Chem. 275, 15466–15473[Abstract/Free Full Text]
  30. Nieto, N., Mari, M., and Cederbaum, A. I. (2003) Hepatology 37, 96–106[CrossRef][Medline] [Order article via Infotrieve]
  31. Ray, S., Watkins, D. N., Misso, N. L., and Thompson, P. J. (2002) Clin. Exp. Allergy 32, 571–577[CrossRef][Medline] [Order article via Infotrieve]
  32. Zipper, L. M., and Mulcahy, R. T. (2002) J. Biol. Chem. 277, 36544–36552[Abstract/Free Full Text]
  33. Buckley, B. J., Marshall, Z. M., and Whorton, A. R. (2003) Biochem. Biophys. Res. Commun. 307, 973–979[CrossRef][Medline] [Order article via Infotrieve]
  34. Wild, A. C., and Mulcahy, R. T. (2000) Free Radic. Res. 32, 281–301[Medline] [Order article via Infotrieve]
  35. Wang, W., Furneaux, H., Cheng, H., Caldwell, M. C., Hutter, D., Liu, Y., Holbrook, N., and Gorospe, M. (2000) Mol. Cell. Biol. 20, 760–769[Abstract/Free Full Text]
  36. Saitoh, M., Nishitoh, H., Fujii, M., Takeda, K., Tobiume, K., Sawada, Y., Kawabata, M., Miyazono, K., and Ichijo, H. (1998) EMBO J. 17, 2596–2606[CrossRef][Medline] [Order article via Infotrieve]
  37. Ben-Levy, R., Leighton, I. A., Doza, Y. N., Attwood, P., Morrice, N., Marshall, C. J., and Cohen, P. (1995) EMBO J. 14, 5920–5930[Medline] [Order article via Infotrieve]
  38. Gossen, M., and Bujard, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5547–5551[Abstract/Free Full Text]
  39. Lasa, M., Mahtani, K. R., Finch, A., Brewer, G., Saklatvala, J., and Clark, A. R. (2000) Mol. Cell. Biol. 20, 4265–4274[Abstract/Free Full Text]
  40. Dean, J. L., Wait, R., Mahtani, K. R., Sully, G., Clark, A. R., and Saklatvala, J. (2001) Mol. Cell. Biol. 21, 721–730[Abstract/Free Full Text]
  41. Fan, X. C., and Steitz, J. A. (1998) EMBO J. 17, 3448–3460[CrossRef][Medline] [Order article via Infotrieve]
  42. Peng, S. S., Chen, C. Y., Xu, N., and Shyu, A. B. (1998) EMBO J. 17, 3461–3470[CrossRef][Medline] [Order article via Infotrieve]
  43. Johnson, G. L., and Lapadat, R. (2002) Science 298, 1911–1912[Abstract/Free Full Text]
  44. Allen, R. G., and Tresini, M. (2002) Free Radic. Biol. Med. 28, 463–499
  45. Wassmann, S., Stumpf, M., Strehlow, K., Schmid, A., Schieffer, B., Böhm, M., and Nickenig, G. (2004) Circ. Res. 94, 534–541[Abstract/Free Full Text]
  46. Nakatani, T., Tsuboyama-Kasaoka, T., Takahashi, M., Miura, S., and Ezaki, O. (2002) J. Biol. Chem. 277, 9562–9569[Abstract/Free Full Text]
  47. Lang, C. H., Nystrom, G. J., and Frost R. A. (1999) Am. J. Physiol. 276, G719–G727[Medline] [Order article via Infotrieve]
  48. Laufs, U., Adam, O., Strehlow, K., Wassmann, S., Kondol, C., Laufs, K., Schmidt, W., Bohm, M., and Nickenig, G. (2003) J. Biol. Chem. 278, 5956–5962[Abstract/Free Full Text]
  49. Yun, H., Lee, M., Kim, S.-S., and Ha, J. (2005) J. Biol. Chem. 280, 9963–9972[Abstract/Free Full Text]
  50. Wang, L., Yang, H., and Admo, M. L. (2000) Biochem. Biophys. Res. Commun. 269, 336–346[CrossRef][Medline] [Order article via Infotrieve]
  51. Strehlow, K., Rotter, S., Wassmann, S., Adam, O., Grohe, C., Laufs, K., Bohm, M., and Nickenig, G. (2003) Circ. Res. 93, 170–177[Abstract/Free Full Text]
  52. Evers, R., de Haas, M., Sparidans, R., Beijnen, J., Wielinga, P. R., Lankelma, J., and Borst, P. (2000) Br. J. Cancer 83, 375–383[CrossRef][Medline] [Order article via Infotrieve]
  53. Kobayashi, A., Ohta, T., and Yamamoto, M. (2004) Methods Enzymol. 378, 273–286[Medline] [Order article via Infotrieve]
  54. Nguyen, T., Sherratt, P. J., and Pickett, C. B. (2003) Annu. Rev. Pharmacol. Toxicol. 43, 233–260[CrossRef][Medline] [Order article via Infotrieve]
  55. Xu, N., Chen, C. Y., and Shyu, A. B. (2001) Mol. Cell. Biol. 21, 6960–6971[Abstract/Free Full Text]
  56. Wilson, G. M., and Brewer, G. (1999) Prog. Nucleic Acid Res. 62, 257–291[Medline] [Order article via Infotrieve]
  57. Tran, H., Maurer, F., and Nagamine, Y. (2003) Mol. Cell. Biol. 23, 7177–7188[Abstract/Free Full Text]
  58. Dean, J. L., Brook, M., Clark, A. R., and Saklatvala, J. (1999) J. Biol. Chem. 274, 264–269[Abstract/Free Full Text]
  59. Miyazawa, K., Mori, A., Miyata, H., Akahane, M., Ajisawa, Y., and Okudaira, H. (1998) J. Biol. Chem. 273, 24832–24838[Abstract/Free Full Text]
  60. Deleted in proof
  61. Winzen, R., Kracht, M., Ritter, B., Wilhelm, A., Chen, C. Y. A., Shyu, A. B., Muller, M., Gaestel, M., Resch, K., and Holtmann, H. (1999) EMBO J. 18, 4969–4980[CrossRef][Medline] [Order article via Infotrieve]
  62. Rousseau, S., Morrice N., Peggie, N., Campbell, D. G., Gaestel, M., and Cohen, P. (2002) EMBO J. 21, 6505–6514[CrossRef][Medline] [Order article via Infotrieve]
  63. Ichijo, H., Nishida, E., Irie, K., ten Dijke, P., Saitoh, M., Moriguchi, T., Takagi, M., Matsumoto, K., Miyazono, K., and Gotoh, Y. (1997) Science 275, 90–94[Abstract/Free Full Text]
  64. Gotoh, Y., and Cooper, J. A. (1998) J. Biol. Chem. 273, 17477–17482[Abstract/Free Full Text]
  65. Lopez de Silanes, I., Fan, J., Yang, X., Zonderman, A. B., Potapova, O., Pizer, E. S., and Gorospe, M. (2003) Oncogene 22, 7146–7154[CrossRef][Medline] [Order article via Infotrieve]
  66. Cook, H. L., Mischo, H. E., and Steitz, J. A. (2004) Mol. Cell. Biol. 24, 4522–4533[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol Hum ReprodHome page
C. E. Bell, M. D. Calder, and A. J. Watson
Genomic RNA profiling and the programme controlling preimplantation mammalian development
Mol. Hum. Reprod., December 1, 2008; 14(12): 691 - 701.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
H. H. W. Chen, I.-S. Song, A. Hossain, M.-K. Choi, Y. Yamane, Z. D. Liang, J. Lu, L. Y.-H. Wu, Z. H. Siddik, L. W. J. Klomp, et al.
Elevated Glutathione Levels Confer Cellular Sensitization to Cisplatin Toxicity by Up-Regulation of Copper Transporter hCtr1
Mol. Pharmacol., September 1, 2008; 74(3): 697 - 704.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
E. J. Quann, F. Khwaja, and D. Djakiew
The p38 MAPK Pathway Mediates Aryl Propionic Acid Induced Messenger RNA Stability of p75NTR in Prostate Cancer Cells
Cancer Res., December 1, 2007; 67(23): 11402 - 11410.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/31/28230    most recent
M413103200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Song, I.-S.
Right arrow Articles by Kuo, M. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Song, I.-S.
Right arrow Articles by Kuo, M. T.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement