MEK-1 [MAPK (mitogen-activated protein kinase) kinase-1] is an important signal transducing enzyme that is implicated in many aspects of cellular functions. In the present paper, we report that cellular polyamines regulate MEK-1 expression at the post-transcriptional level through the RNA-binding protein HuR (Hu-antigen R) in IECs (intestinal epithelial cells). Decreasing the levels of cellular polyamines by inhibiting ODC (ornithine decarboxylase) stabilized MEK-1 mRNA and promoted its translation through enhancement of the interaction between HuR and the 3′-untranslated region of MEK-1 mRNA, whereas increasing polyamine levels by ectopic ODC overexpression destabilized the MEK-1 transcript and repressed its translation by reducing the abundance of HuR–MEK-1 mRNA complex; neither intervention changed MEK-1 gene transcription via its promoter. HuR silencing rendered the MEK-1 mRNA unstable and inhibited its translation, thus preventing increases in MEK-1 mRNA and protein in polyamine-deficient cells. Conversely, HuR overexpression increased MEK-1 mRNA stability and promoted its translation. Inhibition of MEK-1 expression by MEK-1 silencing or HuR silencing prevented the increased resistance of polyamine-deficient cells to apoptosis. Moreover, HuR overexpression did not protect against apoptosis if MEK-1 expression was silenced. These results indicate that polyamines destabilize the MEK-1 mRNA and repress its translation by inhibiting the association between HuR and the MEK-1 transcript. Our findings indicate that MEK-1 is a key effector of the HuR-elicited anti-apoptotic programme in IECs.

INTRODUCTION

The epithelium of mammalian intestinal mucosa is a rapidly self-renewing tissue in the body, and maintenance of its integrity depends on a dynamic balance between cell proliferation and apoptosis [1,2]. In response to stress, rapid changes in gene expression patterns in IECs (intestinal epithelial cells) control cell division and survival, thereby preserving the epithelial homoeostasis [3]. Although gene expression is critically regulated at the transcription level in IECs, the essential contribution of post-transcriptional events, particularly altered mRNA turnover and translation, is becoming increasingly recognized [2,46]. The post-transcriptional fate of a given mRNA is primarily controlled by the interaction of specific mRNA sequences (cis-elements) with specific trans-acting factors such as RBPs (RNA-binding proteins) and non-coding regulatory RNAs (such as microRNAs) [710]. The most common cis-elements responsible for rapid regulation of mRNA decay and translation in mammalian cells are AREs (AU-rich elements) located in the 3′-UTRs (3′-untranslated regions) of many mRNAs [1114]. RNP (ribonucleoprotein) associations either increase or decrease mRNA stability or/and translation depending on the particular mRNA sequence, cellular growth conditions and the stimulus type [5,15,17]. Among the RBPs that regulate specific subsets of mRNAs are several RBPs that modulate mRNA turnover [HuR (Hu-antigen R), NF90 (nuclear factor 90), AUF1 (A- and U-rich RNA-binding factor), BRF1 (butyrate response factor 1), TTP (tristetraprolin) and KSRP (KH domain-containing RBP)] and RBPs that modulate translation {HuR, TIAR [TIA (T-cell intracellular antigen)-related], NF90 and TIA-1]}, collectively known as TTR (translation and turnover-regulatory)-RBPs [1618].

The HuR protein is the ubiquitously expressed member of the ELAV (embryonic lethal abnormal visual)-like family of TTR-RBPs. HuR has two N-terminal RRMs (RNA-recognition motifs), followed by a nucleocytoplasmic shuttling sequence and a C-terminal RRM [1922]. HuR is predominantly located in the nucleus in unstimulated cells, but it rapidly translocates to the cytoplasm, where it directly interacts with and regulates target mRNA stability and/or translation in response to specific stimuli [20,23]. Recently, HuR was shown to play an important role in the regulation of intestinal epithelial homoeostasis by modulating IEC proliferation and apoptosis [6,2427]. The subcellular localization of HuR and its binding affinity for specific target transcripts in IECs is tightly regulated by numerous factors, including cellular polyamines [6,26]. The natural polyamines spermidine and spermine, and their precursor putrescine, are ubiquitous small basic molecules that are intimately involved in the control of epithelial homoeostasis [2729]. Normal IEC proliferation in the mucosa depends on the supply of polyamines to the dividing cells in the crypts [2,4,5,30]; polyamines also regulate IEC apoptosis [31,32]. Decreasing cellular polyamines by inhibiting ODC (ornithine decarboxylase), the first rate-limiting step for polyamine biosynthesis, is found to increase cytoplasmic levels of p53 and NPM (nucleophosmin) mRNAs via HuR-stabilizing, thus contributing to the inhibition of IEC proliferation [6,26]. Polyamines are also necessary for HuR phosphorylation in IECs, and polyamine depletion represses c-Myc translation by reducing the abundance of the HuR–c-Myc mRNA complex through inhibition of Chk2-dependent HuR phosphorylation [25]. Indeed, HuR is emerging as a pivotal post-transcriptional regulator essential for maintaining the intestinal epithelial integrity.

MEK-1 [MAPK (mitogen-activated protein kinase) kinase-1] is a dual-specificity kinase that plays a role in the regulation of various cellular functions including proliferation, development, differentiation, migration and apoptosis by activating MAPK signals [3335]. Whereas a single MEK gene is present in C. elegans, Drosophila and Xenopus, there are two MEK homologues, MEK-1 and MEK-2, in mammalian systems [33]. Studies analysing the protein sequence differences between MEK-1 and MEK-2 suggest that MEK-2 diverged from MEK-1, probably to achieve unique functions in mammalian systems [36,37]. Gene disruption of MEK-1 is lethal at early stages of embryonic development [38], but MEK-2−/− mice are normal in their overall general behaviour [39], suggesting that MEK-1 can compensate for the absence of MEK-2 protein, but that MEK-2 fails to substitute for MEK-1. The cellular MEK-1 protein level and its kinase activity in IECs are highly regulated and have been shown to be crucial for maintaining epithelial homoeostasis [40,41]; however, the exact regulatory mechanisms involved in controlling MEK-1 expression remain elusive. In the present study, we set out to investigate whether polyamines regulate MEK-1 expression at the post-transcriptional level through the RBP HuR. The results presented indicate that polyamine depletion increased the stability and translation of MEK-1 mRNA by inducing the association of HuR with MEK-1 mRNA, whereas elevating the cellular levels of polyamines reduced the abundance of HuR–MEK-1 mRNA complexes, thus decreasing the steady-state level of MEK-1. Furthermore, HuR-mediated elevation of MEK-1 levels suppressed IEC apoptosis, whereas silencing MEK-1 or HuR sensitized IECs to apoptotic cell death.

EXPERIMENTAL

Chemicals and supplies

Tissue culture medium and dialysed FBS (fetal bovine serum) were from Invitrogen. Biochemicals were from Sigma–Aldrich. The antibodies recognizing HuR, MEK-1 and caspase-3 were from Santa Cruz Biotechnology and the secondary antibody conjugated to horseradish peroxidase was purchased from Sigma–Aldrich. DFMO (D,L-α-difluoromethylornithine) was from Genzyme. L-[1-14C]ornithine (specific radioactivity of 51.6 Ci/mmol) was purchased from NEN Radiopharmaceutical.

Cell culture and stable ODC gene transfection

The IEC-6 cell line, derived from normal rat intestinal crypt cells [42], was purchased from the American Type Culture Collection at passage 13 and used at passages 15–20 [5,6]. Cells were maintained in DMEM (Dulbecco's modified Eagle's medium) supplemented with 5 % (v/v) inactivated FBS, 10 μg/ml insulin and 50 μg/ml gentamicin. IEC-ODC+ cells (IEC-6-derived cell-line stably overexpressing ODC) were developed as described in our previous studies [30] and expressed a more stable ODC variant with full enzyme activity [43].

Reporter plasmids and luciferase assays

The MEK-1 promoter sequence was predicted by the Genomatrix software and amplified with primers of sequence 5′-GACCTGCGTGCTAGAACCTC-3′ (sense) and 5′-TCTGGACGCTTGTAGCAGAG-3′ (antisense) using rat genomic DNA (Clontech) as a template. PCR products were sequenced (see Supplementary Figure S1 available at http://www.BiochemJ.org/bj/426/bj4260293add.htm) to confirm that no mutations were introduced by PCR and then cloned into pGL3-basic Luciferase vector (Promega). Transient transfections were performed using the Lipofectamine™ reagent (Invitrogen) according to the manufacturer's instructions. The promoter constructs were transfected into cells along with phRL-null, a Renilla luciferase control reporter vector (Promega), to monitor transfection efficiencies as described previously [44]. The transfected cells were lysed for assays of promoter activity using the Dual Luciferase Assay System (Promega) 48 h after the transfection. The levels of luciferase activity were normalized to the Renilla-driven luciferase activity in every experiment. The chimaeric firefly luciferase reporter construct with the MEK-1 3′-UTR was generated as described previously [25,45,46]. Briefly, the 924-bp ARE fragment from the MEK-1 3′-UTR was amplified and subcloned into the pGL3-Luc plasmid (Promega) at the XbaI site to generate the chimaeric pGL3-Luc-MEK1-3′UTR plasmid. The sequence and orientation of the fragment in the luciferase reporter was confirmed by DNA sequencing and enzyme digestion. Luciferase activity was measured using the Dual Luciferase Assay System. To measure translational changes (translation efficiency), the firefly and Renilla luciferase activities were compared with firefly and Renilla normalized for RNA levels.

Recombinant viral construction and infection

Recombinant adenoviral plasmids containing human HuR cDNA were constructed by using the Adeno-X Expression System (Clontech) according to the protocol provided by the manufacturer. Briefly, the full-length cDNA of human wild-type HuR was cloned into the pShuttle by digesting with BamHI and HindIII, and ligating the resulting fragments into the XbaI site of the pShuttle vector [24]. AdHuR (pAdeno-HuR) was constructed by digesting the pShuttle construct with PI-SceI and I-CeuI and ligating the resulting fragment into the PI-SceI and I-CeuI sites of the pAdeno-X adenoviral vector. Recombinant adenoviral plasmids were packaged into infectious adenoviral particles by transfecting HEK (human embryonic kidney)-293 cells using Lipofectamine™ Plus reagent (Invitrogen). The adenoviral particles were propagated in HEK-293 cells and purified by caesium chloride ultracentrifugation according to the protocol provided by Clontech. Titres of the adenoviral stock were determined by a standard plaque assay. Recombinant adenoviruses were screened for the expression of the introduced gene by Western blotting using the anti-HuR antibody. pAdeno-X, which was the recombinant replication-incompetent adenovirus carrying no HuR cDNA insert (Adnull), was grown and purified as described above and served as a control adenovirus. Cells were infected with AdHuR or Adnull, and expression of HuR was assayed at 48 h after infection.

RNAi (RNA interference)

The silencing RNA duplexes that were designed to specifically target HuR and MEK-1 mRNAs were synthesized and transfected into cells as described previously [6,24]. The sequences of siRNA (small interfering RNA) that specifically targetted the CR (coding region) of HuR (siHuR) and MEK-1 (siMEK-1) mRNA were 5′-AACACACTGAACGGCTTGAGG-3′ and 5′-CAGAAGGTGGGAGAGTTGAAGGATG-3′ respectively, whereas the sequence of control siRNA (C-siRNA) was 5′-AAGTGTAGTAGATCACCAGGC-3′. The siRNA was introduced into cells by transient transfection as described previously [24]. Briefly, for each 60-mm cell culture dish, 15 μl of the 20 μM stock duplex was mixed with 500 μl of Opti-MEM medium (Invitrogen). This mixture was gently added to a solution containing 15 μl of Lipofectamine™ 2000 in 500 μl of Opti-MEM. The solution was incubated for 20 min at room temperature (25 °C) and gently overlaid on to monolayers of cells in 4 ml of medium, and cells were harvested for various assays after 48 h as described previously [4].

Analysis of newly translated protein

New synthesis of MEK-1 was measured by incubating IEC-6 cells with 1 mCi L-[35S]methionine and L-[35S]cysteine per 60-mm plate for 20 min, then cells were lysed using RIPA buffer [47]. IPs (immunoprecipitations) were carried out for 1 h at 4 °C using either a polyclonal antibody recognizing MEK-1 or IgG1 (BD Pharmingen). Following extensive washes in TNN buffer [50 mM Tris/HCl, pH 7.5, containing 250 mM NaCl, 5 mM EDTA and 0.5% NP40 (Nonidet P40)], the immunoprecipitated material was resolved by SDS/PAGE (10% gels), transferred on to PVDF filters and visualized with a PhosphorImager (Molecular Dynamics).

Western blot analysis

Whole-cell lysates were prepared using 2% SDS and were sonicated using a 60 Sonic Dismembrator (Fisher Scientific). The sonication was performed in ice at less than 5 W for 2–5 s. The lysates were centrifuged (15000 g) at 4 °C for 15 min. The supernatants were boiled for 5 min and size-fractionated by SDS/PAGE (7.5% gels). After transferring proteins on to nitrocellulose filters, the blots were incubated with primary antibodies recognizing MEK-1, HuR or caspase-3. After incubations with secondary antibodies, immunocomplexes were developed by using chemiluminescence.

RT (reverse transcription)–PCR and qRT-PCR (quantitative real-time PCR) analysis

Total RNA was isolated by using an RNeasy mini kit (Qiagen) and used in RT–PCR amplification reactions as described previously [24]. PCR primers for rat MEK-1 were 5′-CAGAAGAAGCTGGAGGAGCT-3′ (sense) and 5′-GCTTCTCTCGTAGATATGTCAGG-3′ (antisense), yielding a 450-bp fragment. Primers for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were 5′-TACTAGCGGTTTTACGGGCG-3′ and 5′-TCGAACAGGAGGAGCAGAGAGCGA-3′. Primers for luciferase were 5′-TCAAAGAGGCGAACTGTGTG-3′ and 5′-GGTGTTGGAGCAAGATGGAT-3′. The levels of the β-actin PCR product were assessed to monitor the even input of RNA in RT–PCR samples. qRT-PCR was performed using the 7500-Fast Real-Time PCR System (Applied Biosystems) with specific primers, probes and software (Applied Biosystems). The levels of MEK-1 mRNA were quantified by qRT-PCR analysis and normalized with respect to GAPDH levels.

Preparation of synthetic RNA transcripts

cDNA from IECs was used as a template for PCR amplification of the CR and 3′-UTR of MEK-1. The 5′-primers contained the T7 RNA polymerase promoter sequence (T7): 5′-CCAAGCTTCTAATACGACTCACTATAGGGAGA-3′. To prepare a transcript of the CR of MEK-1 (spanning positions 27–1228), oligonucleotides 5′-CAAGATGCCCAAGAAGAAGC-3′ and 5′-GCTTCCCAAAGGCTCAGAT-3′ were used. To prepare the MEK-1 3′-UTR template (spanning positions 1215–2108), oligonucleotides 5′-CTTTGGGAAGCAGCAGAGAG-3′ and 5′-ACTTTAACTTGATAGTATTTT-3′ were used. To prepare the MEK-1 3′-UTR fragments F1, F2, F3 and F7 (spanning positions 1212–1359, 1327–1430, 1400–1686 and 1856–2136 respectively), the following oligonucleotides were used: 5′-GTTGCTTTCAGGCCTCTCC-3′ and 5′-GACACAAGTACGATTTGGCACA-3′; 5′-AGAACACAGCATGTGCCAAA-3′ and 5′-ACAAATAGCCCCAAGCACAA-3′; 5′-AGTGGATTGGCTTTGTGCTT-3′ and 5′-CGTTCTGGTGCTCATTTCAG-3′; and 5′-AGTGGATTGGCTTTGTGCTT-3′ and 5′-TTTGATAAACATCTTGAGTAAAGTGG-3′. PCR-amplified products were used as templates to transcribe biotinylated RNAs by using T7 RNA polymerase in the presence of biotin–cytidine 5′-triphosphate as described previously [6,24]. Various short RNA probes for MEK-1 3′-UTR fragments, including F2, F3, F4 (spanning positions 1680–1790), F5 (spanning positions 1769–1833), F6 (spanning positions 1861–1877) and F7, were synthesized in the Biopolymer Laboratory at the University of Maryland Baltimore, MA, U.S.A.

RNA–protein binding assays

For biotin pull-down assays, 6 μg of biotinylated transcripts were incubated with 120 μg of cytoplasmic lysate for 30 min at room temperature. Complexes were isolated with paramagnetic streptavidin-conjugated Dynabeads (Dynal) and analysed by Western blotting. To assess the association of endogenous HuR with endogenous MEK-1 mRNAs, IPs of HuR–mRNA complexes were performed as described previously [6]. IEC-6 cells were collected (approx. 20×106 per sample) and lysates were incubated for 4 h at room temperature in the presence of excess (30 μg) IP antibody (anti-IgG or anti-HuR antibody). RNA in IP materials was used for RT–PCR analysis to detect the presence of MEK-1 mRNA.

Immunofluorescence staining

Immunofluorescence was performed as described previously [48] with minor changes [6]. Cells were fixed using 3.7% (w/v) formaldehyde, and the rehydrated samples were incubated overnight at 4 °C with the primary anti-MEK-1 antibody diluted 1:300 in blocking buffer [1% (w/v) BSA in PBS] and then incubated with secondary antibody conjugated with Alexa Fluor®-594 (Molecular Probes) for 2 h at room temperature. After rinsing in PBS, slides were incubated with 1 μM TO-PRO3 (Molecular Probes) for 10 min to stain nuclei, rinsed in PBS again, mounted and viewed with a Zeiss confocal microscope (model LSM410). Images were processed using PhotoShop software (Adobe). Annexin-V staining, to measure apoptosis levels, was carried out by using a commercial apoptosis detection kit (BD Biosciences) and performed according to the protocol recommended by the manufacturer.

Assays for ODC enzyme activity and cellular polyamine content

ODC activity was determined by a radiometric technique in which the amount of 14CO2 liberated from L-[1-14C]ornithine was estimated [30] and enzymatic activity was expressed as pmol of CO2 released per mg of protein per hour. The cellular polyamine content was analysed by HPLC analysis as described previously [4]. After 0.5 M perchloric acid was added, the cells were frozen at −80 °C until ready for extraction, dansylation and HPLC analysis. The standard curve encompassed 0.31–10 μM. Values that fell >25% below the curve were considered undetectable. The results were expressed as nmol of polyamines per mg of protein.

Statistics

Values are means±S.E.M. for between three and six samples. Autoradiographic and immunoblotting results were repeated three times. The significance of the difference between means was determined by analysis of variance. The level of significance was determined using Duncan's multiple range test [49].

RESULTS

Polyamine depletion increases MEK-1 expression post-transcriptionally

To determine the role of cellular polyamines in the regulation of MEK-1 expression, we examined changes in the levels of MEK-1 mRNA and protein following polyamine depletion. Consistent with our previous studies [5,24] and works of others [40,41], inhibition of ODC activity by treatment with 5 mM DFMO almost completely depleted cellular polyamines in IEC-6 cells. The levels of putrescine and spermidine were undetectable at 6 days after treatment with DFMO and spermine was decreased by approx. 60% (results not shown). As shown in Figure 1(A), depletion of cellular polyamines by DFMO increased the steady-state levels of MEK-1 mRNA and protein, but this induction was totally prevented by addition of 10 μM exogenous putrescine together with DFMO. Spermidine (5 μM) given together with DFMO had an effect equal to that of putrescine on levels of MEK-1 mRNA and protein expression when it was added to cultures that contained DFMO (results not shown). Immunofluorescence staining revealed further that the induced MEK-1 was predominantly located in the cytoplasm after polyamine depletion (Figure 1B), whereas combined treatment with DFMO plus putrescine completely prevented the increased MEK-1 immunostaining, with similar subcellular localization patterns to those observed in control cells.

Polyamine depletion increases MEK-1 expression

Figure 1
Polyamine depletion increases MEK-1 expression

(A) Levels of MEK-1 mRNA and protein in cells exposed to 5 mM DFMO alone or DFMO plus putrescine (Put, 10 μM) for 6 days. Panel (a): changes in MEK-1 mRNA as measured by RT–PCR analysis. The first-strand cDNAs, synthesized from total cellular RNA, were amplified with the specific sense and antisense primers, and PCR-amplified products separated on a agarose gel. The size of the fragments in base pairs is indicated on the left. Panel (b): representative MEK-1 protein levels by Western blot analysis. To measure levels of MEK-1 protein, 20 μg of total proteins were applied to each lane, and immunoblots were hybridized with the antibody specific for MEK-1. Actin immunoblotting was performed as an internal control for equal loading. The approx. size of the proteins is indicated on the left. (B) Cellular distribution of MEK-1. Panel (a): control. Panel (b): DFMO-treated cells. Panel (c): cells treated with DMFO plus putrescine (Put, 10 μM). Cells were permeabilized and incubated with the anti-MEK-1 antibody and then with anti-IgG antibody conjugated with Alexa Fluor®. Nuclei were stained with TO-PRO3. Green, MEK-1 signals; Red, nuclei. (C) Levels of MEK-1-promoter activity in cells described in (A). Panel (a): schematic of MEK-1-promoter luciferase (Luc) reporter gene construct (pMEK1-Luc). Panel (b): levels of luciferase reporter activity after polyamine depletion. After cells were treated with DFMO or DFMO plus putrescine (Put) for 4 days, they were transfected with the pMEK1-Luc or control vector (pGL3); luciferase activity was examined 48 h after transfection. In a separate study, cells were co-transfected with the pMEK1-luc and the c-Jun expression vector (Adc-jun) or control vector (Adnull) and luciferase activity was assayed after 48 h. Results were normalized to the Renilla-driven luciferase activity and expressed as means±S.E.M. for three separate experiments. *P<0.05 compared with controls and cells infected with Adnull. (D) Half-life of MEK-1 mRNA in cells as described in (A). After cells were incubated with actinomycin D for the indicated times, total cellular RNA was isolated, and the levels of remaining MEK-1 and GAPDH mRNAs were measured by qRT-PCR analysis. Values are the means±S.E.M for three samples. (E) Newly translated MEK-1 protein in cells as described in (A). MEK-1 translation was measured by incubating cells with L-[35S]methionine and L-[35S]cysteine for 20 min, followed by IP by using an anti-MEK-1 antibody. Samples were resolving by SDS/PAGE and transferred for visualization of signals using a PhosphorImager. The translation of housekeeping control GAPDH was measured similarly. The approx. molecular mass in kDa is indicated on the left. (F) Schematic of plasmids. Panel (a): control (pGL3-Luc). Panel (b): chimaeric firefly luciferease (Luc)-MEK-1 3′UTR (pGL3-Luc-MEK1ARE). (G) Changes in MEK-1 translation efficiency as measured by using pGL3-Luc-MEK1ARE reporter assays in cells as described in (A). The pGL3-Luc-MEK1ARE or pGL3-Luc (negative control) was co-transfected with a Renilla luciferase reporter. Firefly and Renilla luciferase activities were assayed after 24 h. Luciferase values were normalized to the mRNA levels to obtain translation efficiencies and expressed as means±S.E.M. for three separate experiments. *P<0.05 compared with controls and cells treated with DFMO plus putrescine.

Figure 1
Polyamine depletion increases MEK-1 expression

(A) Levels of MEK-1 mRNA and protein in cells exposed to 5 mM DFMO alone or DFMO plus putrescine (Put, 10 μM) for 6 days. Panel (a): changes in MEK-1 mRNA as measured by RT–PCR analysis. The first-strand cDNAs, synthesized from total cellular RNA, were amplified with the specific sense and antisense primers, and PCR-amplified products separated on a agarose gel. The size of the fragments in base pairs is indicated on the left. Panel (b): representative MEK-1 protein levels by Western blot analysis. To measure levels of MEK-1 protein, 20 μg of total proteins were applied to each lane, and immunoblots were hybridized with the antibody specific for MEK-1. Actin immunoblotting was performed as an internal control for equal loading. The approx. size of the proteins is indicated on the left. (B) Cellular distribution of MEK-1. Panel (a): control. Panel (b): DFMO-treated cells. Panel (c): cells treated with DMFO plus putrescine (Put, 10 μM). Cells were permeabilized and incubated with the anti-MEK-1 antibody and then with anti-IgG antibody conjugated with Alexa Fluor®. Nuclei were stained with TO-PRO3. Green, MEK-1 signals; Red, nuclei. (C) Levels of MEK-1-promoter activity in cells described in (A). Panel (a): schematic of MEK-1-promoter luciferase (Luc) reporter gene construct (pMEK1-Luc). Panel (b): levels of luciferase reporter activity after polyamine depletion. After cells were treated with DFMO or DFMO plus putrescine (Put) for 4 days, they were transfected with the pMEK1-Luc or control vector (pGL3); luciferase activity was examined 48 h after transfection. In a separate study, cells were co-transfected with the pMEK1-luc and the c-Jun expression vector (Adc-jun) or control vector (Adnull) and luciferase activity was assayed after 48 h. Results were normalized to the Renilla-driven luciferase activity and expressed as means±S.E.M. for three separate experiments. *P<0.05 compared with controls and cells infected with Adnull. (D) Half-life of MEK-1 mRNA in cells as described in (A). After cells were incubated with actinomycin D for the indicated times, total cellular RNA was isolated, and the levels of remaining MEK-1 and GAPDH mRNAs were measured by qRT-PCR analysis. Values are the means±S.E.M for three samples. (E) Newly translated MEK-1 protein in cells as described in (A). MEK-1 translation was measured by incubating cells with L-[35S]methionine and L-[35S]cysteine for 20 min, followed by IP by using an anti-MEK-1 antibody. Samples were resolving by SDS/PAGE and transferred for visualization of signals using a PhosphorImager. The translation of housekeeping control GAPDH was measured similarly. The approx. molecular mass in kDa is indicated on the left. (F) Schematic of plasmids. Panel (a): control (pGL3-Luc). Panel (b): chimaeric firefly luciferease (Luc)-MEK-1 3′UTR (pGL3-Luc-MEK1ARE). (G) Changes in MEK-1 translation efficiency as measured by using pGL3-Luc-MEK1ARE reporter assays in cells as described in (A). The pGL3-Luc-MEK1ARE or pGL3-Luc (negative control) was co-transfected with a Renilla luciferase reporter. Firefly and Renilla luciferase activities were assayed after 24 h. Luciferase values were normalized to the mRNA levels to obtain translation efficiencies and expressed as means±S.E.M. for three separate experiments. *P<0.05 compared with controls and cells treated with DFMO plus putrescine.

To define the mechanism by which decreasing the levels of cellular polyamines induced MEK-1 expression, three sets of experiments were performed. First, we examined the effect of polyamine depletion by DFMO on MEK-1 gene transcription. The MEK-1 promoter fragment [which contains several potential AP-1 (activator protein 1) and CREB (cAMP-response-element-binding protein) sites (see Supplementary Figure S1)] was cloned from human genomic DNA, and the MEK-1 promoter luciferase reporter gene construct was made as illustrated in Figure 1(C), panel (a). Polyamine depletion by DFMO did not change MEK-1 gene transcription, as judged by the lack of any significant differences in the activity level of the MEK-1 promoter luciferase reporter gene construct between control cells and cells exposed to DFMO alone or DFMO plus putrescine (Figure 1C, panel b). Analysis of the kinetics of adding exogenous putrescine or spermidine on the MEK-1 promoter activity in control cells also revealed that exposure of normal IEC-6 cells (without DFMO) to 10 μM putrescine or 5 μM spermidine for 2 and 4 h similarly failed to alter the activity level of the MEK-1 promoter luciferase reporter gene construct (results not shown). On the other hand, ectopic overexpression of the AP-1 transcription factor c-Jun by infection with an adenoviral vector containing the human c-Jun cDNA significantly increased the levels of MEK-1 promoter activity (Figure 1C, panel b). These results indicate that polyamine depletion does not influence MEK-1 gene transcription via its promoter in IECs and that the increase in steady-state levels of MEK-1 mRNA following polyamine depletion is probably not related to its synthesis.

Secondly, we determined if the induction of MEK-1 mRNA levels by polyamine depletion was instead influenced by changes in mRNA turnover. As shown in Figure 1(D), depletion of cellular polyamines by DFMO increased the stability of MEK-1 mRNA. In control cells, the mRNA levels declined gradually after gene transcription was inhibited by the addition of 5 μg/ml actinomycin D, displaying an apparent half-life of approx. 247 min. However, the stability of MEK-1 mRNA was dramatically increased following polyamine depletion with a half-life of >480 min, an increase that was totally prevented when exogenous putrescine was given together with DFMO. The half-life of MEK-1 mRNA in cells exposed to DFMO and putrescine was approx. 255 min, similar to that of control cells (without DFMO). These findings indicate that polyamines regulate MEK-1 expression post-transcriptionally and that depletion of cellular polyamines increases the MEK-1 mRNA levels primarily by enhancing MEK-1 mRNA stability.

Thirdly, we examined the effect of polyamine depletion on MEK-1 translation. To directly investigate whether increased MEK-1 expression might also result from the induction of MEK-1 translation following polyamine depletion, we compared the rate of new MEK-1 protein synthesis between control cells and cells treated with DFMO alone or DFMO plus putrescine. Cells were incubated in the presence of L-[35S]methionine and L-[35S]cysteine for 20 min, when newly translated MEK-1 was visualized by IP. The brief incubation period was chosen to minimize the contribution of MEK-1 degradation in our analysis. As shown in Figure 1(E), newly synthesized MEK-1 was markedly increased in DFMO-treated cells, whereas when exogenous putrescine was given together with DFMO the rate of new MEK-1 protein synthesis restored to normal levels. Polyamine depletion did not affect nascent GAPDH translation, as the rates of newly synthesized GAPDH protein in control cells were similar to those in cells treated with DFMO alone or with DFMO plus putrescine.

To further confirm these findings and to examine whether the translational effect of polyamines was exerted through the ARE, we used a firefly luciferase reporter gene construct containing the MEK-1 3′-UTR ARE (pGL3-Luc-MEK1ARE) and the negative control vector pGL3-Luc (Figure 1F). A plasmid expressing Renilla luciferase was also co-transfected as an internal control for normalization of firefly luciferase. To distinguish translational output from mRNA turnover, the luciferase assays were normalized to the luciferase-reporter mRNA levels to obtain the translation efficiency [10]. As shown in Figure 1(G), polyamine depletion by DFMO induced the MEK-1 translation via the ARE within its 3′-UTR, as indicated by an increase in the activity level of the MEK-1 ARE luciferase reporter gene construct. The combined DFMO and putrescine treatment prevented the increase in MEK-1 translation, rendering the rate of MEK-1 ARE-mediated translation similar to that observed in control cells. In contrast, no change in the luciferase reporter gene construct activity was seen in response to polyamine depletion when testing a control construct without the MEK-1 ARE (results not shown). These results indicate that decreasing the levels of cellular polyamines increases MEK-1 mRNA translation through the MEK-1 3′-UTR ARE. We also examined changes in the half-life of MEK-1 protein and demonstrated that MEK-1 protein stability [as measured by incubating cells with CHX (cycloheximide) to block de novo protein synthesis] was not affected by polyamine depletion (see Supplementary Figure S2 available at http://www.BiochemJ.org/bj/426/bj4260293add.htm), supporting the view that polyamine depletion increases MEK-1 expression by stabilizing MEK-1 mRNA and promoting its translation.

Increasing cellular polyamines represses MEK-1 expression by destabilizing MEK-1 mRNA and inhibiting its translation

To determine the effect of increasing the levels of cellular polyamines on MEK-1 expression, two clonal populations of IEC-ODC+ cells [30] were used in this study. As reported previously [6], IEC-ODC+ cells exhibited very high levels of ODC protein and there was a greater than 50-fold increase in ODC enzyme activity in the cells. Consistently, the levels of putrescine, spermidine and spermine in stable IEC-ODC+ cells were increased, by approx. 12-fold, 2-fold and 1.25-fold respectively, when compared with cells transfected with the control vector lacking the ODC coding region (results not shown). As shown in Figure 2(A), increasing cellular polyamines by ODC overexpression repressed MEK-1 expression, as shown by a significant decrease in the levels of MEK-1 mRNA and protein in IEC-ODC+ cells when compared with those observed in cells infected with control vector. As increasing the levels of cellular polyamines did not affect levels of the MEK-1-promoter activity (Figure 2B), this repression in MEK-1 expression by ODC overexpression also occurs at the post-transcriptional level. Furthermore, the results presented in Figure 2(C) show that the half-life of MEK-1 mRNA was decreased in IEC-ODC+ cells when compared with that in cells infected with control vector. This inhibition of MEK-1 expression by increasing cellular polyamines was also partially due to a reduction in MEK-1 mRNA translation, because levels of newly synthesized MEK-1 protein (Figure 2D) and the activity of the MEK-1 ARE luciferase reporter gene construct (Figure 2E) were significantly decreased in IEC-ODC+ cells. The inhibitory effects of ODC overexpression on MEK-1 expression were not simply due to clonal variation, as the two different clonal populations, ODC-C1 and ODC-C2, showed similar responses. The decrease in MEK-1 expression in IEC-ODC+ cells did not result from a reduction in MEK-1 protein stability, as the half-life of MEK-1 protein was not significantly different in control cells when compared with IEC-ODC+ cells (see Supplementary Figure S3 http://www.BiochemJ.org/bj/426/bj4260293add.htm). Together, these results indicate that increasing the levels of cellular polyamines destabilizes MEK-1 mRNA and represses its translation.

Increasing cellular polyamines represses MEK-1 expression

Figure 2
Increasing cellular polyamines represses MEK-1 expression

(A) Changes in MEK-1 mRNA and protein in IEC-ODC+ cells (clones ODC-C1 and ODC-C2) and control cells (C-vector). IEC-6 cells were infected with either the retroviral vector containing the sequence encoding mouse ODC cDNA or control retroviral vector lacking ODC cDNA. Clones resistant to the selection medium were isolated and screened for ODC expression. The levels of MEK-1 mRNA and protein were assessed by RT–PCR analysis and Western immunoblotting respectively. (B) Levels of MEK-1-promoter activity in cells as described in (A). Luciferase activity was examined 48 h after transfection with pMEK1-Luc or the control vector (pGL3). Results were normalized to the Renilla-driven luciferase activity and expressed as means±S.E.M. for three separate experiments. (C) Half-life of MEK-1 mRNA in cells as described in (A). After cells were incubated with actinomycin D for the indicated times, total cellular RNA was isolated, and the levels of remaining MEK-1 and GAPDH mRNAs were measured by qRT-PCR analysis. Values are the means±S.E.M for three samples. (D) Newly synthesized MEK-1 protein in cells as described in (A). After cells were incubated with L-[35S]methionine and L-[35S]cysteine for 20 min, cell lysates were prepared and immunoprecipitated using anti-MEK-1 antibody, resolved by SDS/PAGE and transferred for visualization of signals by using a PhosphorImager. The translation of housekeeping control GAPDH was measured similarly. (E) Changes in MEK-1 translation efficiency as measured by using pGL3-Luc-MEK1ARE reporter assays in cells as described in (A). The pGL3-Luc-MEK1ARE or pGL3-Luc (negative control) was co-transfected with a Renilla luciferase reporter, and firefly and Renilla luciferase activities were assayed after 24 h. Luciferase values were normalized to the mRNA levels to obtain translation efficiencies and expressed as means±S.E.M for three separate experiments. *P<0.05 compared with controls.

Figure 2
Increasing cellular polyamines represses MEK-1 expression

(A) Changes in MEK-1 mRNA and protein in IEC-ODC+ cells (clones ODC-C1 and ODC-C2) and control cells (C-vector). IEC-6 cells were infected with either the retroviral vector containing the sequence encoding mouse ODC cDNA or control retroviral vector lacking ODC cDNA. Clones resistant to the selection medium were isolated and screened for ODC expression. The levels of MEK-1 mRNA and protein were assessed by RT–PCR analysis and Western immunoblotting respectively. (B) Levels of MEK-1-promoter activity in cells as described in (A). Luciferase activity was examined 48 h after transfection with pMEK1-Luc or the control vector (pGL3). Results were normalized to the Renilla-driven luciferase activity and expressed as means±S.E.M. for three separate experiments. (C) Half-life of MEK-1 mRNA in cells as described in (A). After cells were incubated with actinomycin D for the indicated times, total cellular RNA was isolated, and the levels of remaining MEK-1 and GAPDH mRNAs were measured by qRT-PCR analysis. Values are the means±S.E.M for three samples. (D) Newly synthesized MEK-1 protein in cells as described in (A). After cells were incubated with L-[35S]methionine and L-[35S]cysteine for 20 min, cell lysates were prepared and immunoprecipitated using anti-MEK-1 antibody, resolved by SDS/PAGE and transferred for visualization of signals by using a PhosphorImager. The translation of housekeeping control GAPDH was measured similarly. (E) Changes in MEK-1 translation efficiency as measured by using pGL3-Luc-MEK1ARE reporter assays in cells as described in (A). The pGL3-Luc-MEK1ARE or pGL3-Luc (negative control) was co-transfected with a Renilla luciferase reporter, and firefly and Renilla luciferase activities were assayed after 24 h. Luciferase values were normalized to the mRNA levels to obtain translation efficiencies and expressed as means±S.E.M for three separate experiments. *P<0.05 compared with controls.

Polyamines inhibit the association between HuR and MEK-1 mRNA

Our previous observations [6,26] have demonstrated that polyamine depletion by DFMO enhanced the cytoplasmic abundance of HuR, whereas increasing cellular polyamines by ODC overexpression decreased cytoplasmic HuR, although neither intervention changed whole-cell HuR levels (results not shown). Given the predicted affinity of HuR for the 3′-UTR of the MEK-1 mRNA (Figure 3A), we hypothesized that HuR would bind to the MEK-1 3′-UTR and further postulated that this association could be regulated by cellular polyamines. Three sets of experiments were performed to test this hypothesis. First, we examined if there was an association between MEK-1 mRNA and HuR in IECs by performing RNP IP assays using an anti-HuR antibody under conditions that preserved RNP integrity [50]. This interaction was examined by isolating RNA from the IP material and subjecting it to RT–PCR or qRT–PCR analyses. MEK-1 PCR products were highly enriched in HuR-immunoprecipitated samples compared with control (IgG1-immunoprecipitated) samples (Figures 3B and 3C), indicating that MEK-1 mRNA is a target of HuR. The enrichment of the p53 PCR product was also examined and served as a positive control (results not shown) as HuR is known to bind to p53 mRNA [6]. The amplification of GAPDH PCR products, found in all samples as a low-level contaminating transcript (i.e. not a HuR target), served to monitor sample input as reported previously [51].

Changes in binding of HuR to the MEK-1 mRNA after altering the levels of cellular polyamines

Figure 3
Changes in binding of HuR to the MEK-1 mRNA after altering the levels of cellular polyamines

(A) Schematic representative of the MEK-1 mRNA. Sequences matching the signature HuR-binding motif in the 3′-UTR are underlined. (B) Levels of association of endogenous HuR with endogenous MEK-1 mRNA after polyamine depletion. IECs were exposed to DFMO alone or DFMO plus putrescine (Put) for 6 days. After IP of RNA–protein complexes from cell lysates using either the anti-HuR antibody (+HuR) or control anti-IgG1 antibody (+IgG). RNA was isolated and used in RT reactions. Left panel: representative RT–PCR products visualized in stained agarose gels; low-level amplification of the housekeeping GAPDH mRNA (which is not a HuR target) served as a negative control. Right panel: fold differences in MEK-1 transcript abundance in IP with anti-HuR antibody compared with anti-IgG antibody, as measured by qRT–PCR analysis. Values were means±S.E.M. for three samples. *P<0.05 compared with controls and cells treated with DFMO plus putrescine. (C) Changes in levels of MEK-1 mRNA in a HuR IP from clonal populations of cells overexpressing ODC cells (ODC-C1 and ODC-C2) and control cells (C-vector). Left panel: representative RT–PCR products of MEK-1 and GAPDH mRNAs. Right panel: fold differences in MEK-1 mRNA in an anti-HuR antibody IP compared with an anti-IgG antibody IP as measured by qRT–PCR analysis. Values were means±S.E.M. for three samples. *P<0.05 compared with controls.

Figure 3
Changes in binding of HuR to the MEK-1 mRNA after altering the levels of cellular polyamines

(A) Schematic representative of the MEK-1 mRNA. Sequences matching the signature HuR-binding motif in the 3′-UTR are underlined. (B) Levels of association of endogenous HuR with endogenous MEK-1 mRNA after polyamine depletion. IECs were exposed to DFMO alone or DFMO plus putrescine (Put) for 6 days. After IP of RNA–protein complexes from cell lysates using either the anti-HuR antibody (+HuR) or control anti-IgG1 antibody (+IgG). RNA was isolated and used in RT reactions. Left panel: representative RT–PCR products visualized in stained agarose gels; low-level amplification of the housekeeping GAPDH mRNA (which is not a HuR target) served as a negative control. Right panel: fold differences in MEK-1 transcript abundance in IP with anti-HuR antibody compared with anti-IgG antibody, as measured by qRT–PCR analysis. Values were means±S.E.M. for three samples. *P<0.05 compared with controls and cells treated with DFMO plus putrescine. (C) Changes in levels of MEK-1 mRNA in a HuR IP from clonal populations of cells overexpressing ODC cells (ODC-C1 and ODC-C2) and control cells (C-vector). Left panel: representative RT–PCR products of MEK-1 and GAPDH mRNAs. Right panel: fold differences in MEK-1 mRNA in an anti-HuR antibody IP compared with an anti-IgG antibody IP as measured by qRT–PCR analysis. Values were means±S.E.M. for three samples. *P<0.05 compared with controls.

Secondly, we determined changes in the levels of HuR–MEK-1 mRNA complexes after altering the levels of cellular polyamines. As shown in Figure 3(B), polyamine depletion significantly enhanced the amount of HuR–MEK-1 mRNA binding, as indicated by an increase in the levels of MEK-1 mRNA in the HuR-immunoprecipitated material from DFMO-treated cells compared with control cells; this induction was prevented by putrescine given together with DFMO. In contrast, the increased levels of cellular polyamines in IEC-ODC+ cells repressed the formation of HuR–MEK-1 mRNA complexes; the levels of MEK-1 mRNA were decreased in the HuR-immunoprecipitated materials from IEC-ODC+ cells when compared with those observed in control cells (Figure 3C). MEK-1 mRNA was undetectable when using the non-specific anti-IgG1 antibody in IPs for all treatment groups (Figures 3B and 3C).

Thirdly, we tested if polyamines regulate the formation of the HuR–MEK-1 mRNA complex through the MEK-1 3′-UTR or CR by using biotinylated transcripts spanning the MEK-1 mRNA regions shown in Figure 4(A). The MEK-1 3′-UTR transcript readily associated with cytoplasmic HuR, as can be seen in a Western blot analysis of the pull-down material (Figure 4B, panel a); the binding intensity increased significantly when using lysates prepared from cells that were treated with DFMO, but was reduced when cells had been treated with DFMO plus putrescine. This increase in the levels of HuR binding to MEK-1 3′-UTR is specific, because transcripts corresponding to the MEK-1 CR did not bind to HuR in controls and cells exposed to DFMO alone or DFMO plus putrescine (Figure 4B, panel b). On the other hand, increasing the levels of cellular polyamines by ODC overexpression inhibited the association of HuR with the MEK-1 3′-UTR, as indicated by a decrease in the levels of HuR in the MEK-1 3′-UTR pull-down materials from IEC-ODC+ cells (Figure 4C, panel a). Consistently, HuR was undetectable in the MEK-1 CR pull-down materials from control cells and IEC-ODC+ cells (Figure 4C, panel b). To examine whether binding of HuR to the MEK-1 3′-UTR is mediated through the specific sites containing the predicted HuR-binding motif, biotinylated transcripts partially spanning the MEK-1 3′-UTR were prepared (Figure 4D, upper panel) and their association with HuR was tested in pull-down assays. HuR was found to bind to the F3, F4, F6 and F7 transcripts, four transcripts that contained potential HuR-binding motifs, but it did not bind to the F1, F2 and F5 transcripts, although they also contained HuR-binding motifs. Together, these findings indicate that cytoplasmic HuR specifically binds to the 3′-UTR of MEK-1 mRNA and that this binding is negatively regulated by cellular polyamines.

Levels of HuR binding to the 3′-UTR and CR of MEK-1 mRNA after altering the levels of cellular polyamines

Figure 4
Levels of HuR binding to the 3′-UTR and CR of MEK-1 mRNA after altering the levels of cellular polyamines

(A) Schematic representation of the MEK-1 biotinylated transcripts (CR and 3′-UTR) used in this study. (B) Representative HuR immunoblots using the pull-down materials by the different regions of MEK-1 mRNA after polyamine depletion. Panel (a): HuR binding to the 3′-UTR. Panel (b): HuR binding to the CR. Cytoplasmic lysates prepared from control cells and cells exposed to DFMO alone or DFMO plus putrescine (Put) for 6 days were incubated with 6 μg of biotinylated MEK-1 3′-UTR or CR for 30 min at 25 °C. The resulting RNP complexes were pulled down by using streptavidin-coated beads. The presence of HuR in the pull-down material was assayed by Western blotting. β-actin in the pull-down material was also examined and served as a negative control. (C) Representative HuR immunoblots using the pull-down materials in clonal populations of ICE-ODC+ (ODC-C1 and ODC-C2) and control cells (C-vector). Panel a: HuR binding to 3′-UTR. Panel b: HuR binding to the CR. (D) Upper panel: schematic representation of the MEK-1 3′-UTR biotinylated transcripts used in this study. Lower panel: representative HuR immunoblots in the material pulled down by the different biotinylated fractions of the MEK-1 mRNA 3′-UTR (F1–F7). Three experiments were performed that showed similar results.

Figure 4
Levels of HuR binding to the 3′-UTR and CR of MEK-1 mRNA after altering the levels of cellular polyamines

(A) Schematic representation of the MEK-1 biotinylated transcripts (CR and 3′-UTR) used in this study. (B) Representative HuR immunoblots using the pull-down materials by the different regions of MEK-1 mRNA after polyamine depletion. Panel (a): HuR binding to the 3′-UTR. Panel (b): HuR binding to the CR. Cytoplasmic lysates prepared from control cells and cells exposed to DFMO alone or DFMO plus putrescine (Put) for 6 days were incubated with 6 μg of biotinylated MEK-1 3′-UTR or CR for 30 min at 25 °C. The resulting RNP complexes were pulled down by using streptavidin-coated beads. The presence of HuR in the pull-down material was assayed by Western blotting. β-actin in the pull-down material was also examined and served as a negative control. (C) Representative HuR immunoblots using the pull-down materials in clonal populations of ICE-ODC+ (ODC-C1 and ODC-C2) and control cells (C-vector). Panel a: HuR binding to 3′-UTR. Panel b: HuR binding to the CR. (D) Upper panel: schematic representation of the MEK-1 3′-UTR biotinylated transcripts used in this study. Lower panel: representative HuR immunoblots in the material pulled down by the different biotinylated fractions of the MEK-1 mRNA 3′-UTR (F1–F7). Three experiments were performed that showed similar results.

HuR silencing prevents the increased stability of MEK-1 mRNA and inhibits its translation in polyamine-deficient cells

To directly examine the putative role of HuR in the increased MEK-1 mRNA stability and translation in polyamine-deficient cells, siRNA targeting the HuR mRNA (siHuR) was used to reduce HuR levels. With >95% cells transfected (results not shown), siHuR potently and specifically silenced HuR expression in polyamine-deficient cells (Figure 5A). As shown in Figure 5(B), silencing HuR completely prevented the increased MEK-1 mRNA levels in polyamine-deficient cells. This reduction in MEK-1 mRNA by HuR silencing resulted primarily from the destabilization of the MEK-1 transcript, because the half-life of MEK-1 mRNA in DFMO-treated cells transfected with siHuR was similar to that measured in control cells (Figure 5C). HuR silencing also abolished the polyamine depletion-induced MEK-1 translation as indicated by a decrease in the activity of the MEK-1 ARE luciferase reporter gene construct (Figure 5D). Furthermore, in HuR-silenced populations, the increase in MEK-1 protein levels after polyamine depletion was also prevented (Figure 5A) and was reduced to levels similar to those obtained from control cells. Transfection with control siRNA had no effect on the MEK-1 mRNA stability and translation or the levels of MEK-1 protein in polyamine-deficient cells (Figure 5). These findings strongly suggest that the increased MEK-1 mRNA stability and its translation in IECs following polyamine depletion result from the enhanced interaction of HuR with the MEK-1 3′-UTR.

Effect of HuR silencing on MEK-1 mRNA stability and its translation in polyamine-deficient cells

Figure 5
Effect of HuR silencing on MEK-1 mRNA stability and its translation in polyamine-deficient cells

(A) Representative HuR and MEK-1 immunoblots. After cells were cultured in the presence of DFMO for 4 days, they were transfected with either siRNA targeting the HuR mRNA coding region (siHuR) or control siRNA (C-siRNA) and whole-cell lysates were harvested 48 h thereafter. The levels of HuR and MEK-1 proteins were measured by Western blot anaylsis and equal loading was monitored by β-actin immunoblotting. (B) Levels of MEK-1 mRNA in cells that were processed as described in (A). Total RNA from each group was harvested and levels of MEK-1 mRNA were measured by RT followed by qRT–PCR analysis. The results were normalized to the amount of GAPDH mRNA and the values represented as the means±S.E.M for three experiments. *P<0.05 compared with controls (Con) and cells transfected with C-siRNA. (C) Half-life of the MEK-1 mRNA in cells that were transfected and treated as described in (A). Total cellular RNA was isolated at the indicated times after administration of actinomycin D, and the remaining levels of MEK-1 and GAPDH mRNAs were measured by qRT-PCR analysis. Values are means±S.E.M. for three samples. (D) Changes in MEK-1 translation efficiency as measured by using pGL3-Luc-MEK1ARE reporter assays in cells as described in (A). The pGL3-Luc-MEK1ARE or pGL3-Luc (negative control) was co-transfected with a Renilla luciferase reporter, and firefly and Renilla luciferase activities were assayed after 24 h. Luciferase values were normalized to the mRNA levels to obtain translation efficiencies and expressed as means±S.E.M. for three separate experiments. *P<0.05 compared with control cells and DFMO-treated cells transfected with siHuR.

Figure 5
Effect of HuR silencing on MEK-1 mRNA stability and its translation in polyamine-deficient cells

(A) Representative HuR and MEK-1 immunoblots. After cells were cultured in the presence of DFMO for 4 days, they were transfected with either siRNA targeting the HuR mRNA coding region (siHuR) or control siRNA (C-siRNA) and whole-cell lysates were harvested 48 h thereafter. The levels of HuR and MEK-1 proteins were measured by Western blot anaylsis and equal loading was monitored by β-actin immunoblotting. (B) Levels of MEK-1 mRNA in cells that were processed as described in (A). Total RNA from each group was harvested and levels of MEK-1 mRNA were measured by RT followed by qRT–PCR analysis. The results were normalized to the amount of GAPDH mRNA and the values represented as the means±S.E.M for three experiments. *P<0.05 compared with controls (Con) and cells transfected with C-siRNA. (C) Half-life of the MEK-1 mRNA in cells that were transfected and treated as described in (A). Total cellular RNA was isolated at the indicated times after administration of actinomycin D, and the remaining levels of MEK-1 and GAPDH mRNAs were measured by qRT-PCR analysis. Values are means±S.E.M. for three samples. (D) Changes in MEK-1 translation efficiency as measured by using pGL3-Luc-MEK1ARE reporter assays in cells as described in (A). The pGL3-Luc-MEK1ARE or pGL3-Luc (negative control) was co-transfected with a Renilla luciferase reporter, and firefly and Renilla luciferase activities were assayed after 24 h. Luciferase values were normalized to the mRNA levels to obtain translation efficiencies and expressed as means±S.E.M. for three separate experiments. *P<0.05 compared with control cells and DFMO-treated cells transfected with siHuR.

HuR overexpression stabilizes MEK-1 mRNA and promotes its translation

To further define the role of HuR in regulating MEK-1 post-transcriptionally, we examined the effect of overexpressing wild-type HuR on MEK-1 mRNA stability and its translation in control IEC-6 cells (without DFMO). We utilized an adenoviral vector containing human HuR cDNA under the control of the human cytomegalovirus immediate-early gene promoter (AdHuR) as described previously [24,25]. The adenoviral vectors used in the present study infect IECs with near 100% efficiency [30]; >95% of IEC-6 cells were positive when they were infected for 24 h with an adenoviral vector encoding green fluorescent protein (results not shown). As noted in Figure 6(A), the levels of HuR protein increased with viral load and reached approx. 2-fold, 6-fold, 12-fold and 15-fold higher levels than control cells when AdHuR was used at 50, 100, 150 and 200 pfu (plaque-forming units) per cell respectively. A control adenovirus that lacked exogenous HuR cDNA (Adnull) failed to induce HuR. Transient infection with AdHuR (100 pfu/cell) for 48 h increased the levels of MEK-1 mRNA (Figure 6B); this induction in MEK-1 mRNA was due to increased stabilization of MEK-1 mRNA, as indicated by a significant increase in its half-life in IEC-6 cells (Figure 6C). In addition, HuR overexpression also enhanced MEK-1 translation as shown by an increase in the level of MEK-1 ARE luciferase reporter gene construct activity (Figure 6D). Consistently, the increased MEK-1 mRNA stability and its translation were associated with an increase in the steady-state levels of MEK-1 protein after the infection with AdHuR compared with that observed in control cells and cells infected with Adnull (Figure 6A). These results indicate that HuR overexpression enhances MEK-1 expression by stabilizing MEK-1 mRNA and enhancing its translation.

Changes in MEK-1 mRNA stability and translational efficiency after ectopic HuR overexpression

Figure 6
Changes in MEK-1 mRNA stability and translational efficiency after ectopic HuR overexpression

(A) Representative immunoblots of HuR and MEK-1 proteins after ectopic HuR expression. Cells were infected with the recombinant adenoviral vector encoding HuR cDNA (AdHuR) or adenoviral vector lacking HuR cDNA (Adnull) at a multiplicity of infection of 50–200 pfu/cell; the expression of HuR and MEK-1 proteins was analysed 48 h after the infection. The approx. molecular mass in kDa is indicated on the left. (B) Levels of MEK-1 mRNA as measured by qRT-PCR analysis in cells infected with AdHuR or Adnull at the concentration of 100 pfu/cell for 48 h. Results were normalized to the amount of GAPDH mRNA and values are means±S.E.M for three experiments. *P<0.05 compared with cells infected with Adnull. (C) Half-life of the MEK-1 mRNA as measured by qRT–PCR analysis by using actinomycin D in cells as described in (B). Values are the means±S.E.M. for three samples. (D) Changes in MEK-1 translation efficiency as measured by using pGL3-Luc-MEK1ARE reporter assays in cells as described in (B). Cells were transfected with the pGL3-Luc-MEK1ARE or pGL3-Luc (negative control) and the levels of luciferase activity were measured after 24 h. Values were normalized to the mRNA levels to obtain translation efficiencies and are expressed as means±S.E.M. for three separate experiments. *P<0.05 compared with cells transfected with Adnull.

Figure 6
Changes in MEK-1 mRNA stability and translational efficiency after ectopic HuR overexpression

(A) Representative immunoblots of HuR and MEK-1 proteins after ectopic HuR expression. Cells were infected with the recombinant adenoviral vector encoding HuR cDNA (AdHuR) or adenoviral vector lacking HuR cDNA (Adnull) at a multiplicity of infection of 50–200 pfu/cell; the expression of HuR and MEK-1 proteins was analysed 48 h after the infection. The approx. molecular mass in kDa is indicated on the left. (B) Levels of MEK-1 mRNA as measured by qRT-PCR analysis in cells infected with AdHuR or Adnull at the concentration of 100 pfu/cell for 48 h. Results were normalized to the amount of GAPDH mRNA and values are means±S.E.M for three experiments. *P<0.05 compared with cells infected with Adnull. (C) Half-life of the MEK-1 mRNA as measured by qRT–PCR analysis by using actinomycin D in cells as described in (B). Values are the means±S.E.M. for three samples. (D) Changes in MEK-1 translation efficiency as measured by using pGL3-Luc-MEK1ARE reporter assays in cells as described in (B). Cells were transfected with the pGL3-Luc-MEK1ARE or pGL3-Luc (negative control) and the levels of luciferase activity were measured after 24 h. Values were normalized to the mRNA levels to obtain translation efficiencies and are expressed as means±S.E.M. for three separate experiments. *P<0.05 compared with cells transfected with Adnull.

HuR-mediated MEK-1 expression plays a critical role in the regulation of IEC apoptosis

To investigate the biological consequences of inducing endogenous MEK-1 levels following polyamine depletion, we examined its possible involvement in regulating IEC apoptosis. Our previous studies [31,32] and the work of other groups [29,41], have shown that polyamines regulate apoptosis through multiple signalling pathways and that depletion of cellular polyamines promotes the resistance of normal IECs to apoptosis. The results presented in Figure 7 further show that inhibition of MEK-1 expression by HuR-silencing, or MEK-1-silencing by transfection with siRNA targeting MEK-1 mRNA (siMEK-1), altered the susceptibility of polyamine-deficient cells to apoptosis induced by 20 ng/ml TNF-α (tumour necrosis factor-α) and 25 μg/ml CHX. As shown in Figures 7B and 7C, control cells exposed to TNF-α and CHX for 4 h, showed morphological features characteristic of programmed cell death. The assessments of apoptosis were confirmed by an increase in levels of active caspase-3 (Figure 7D) after treatment with TNF-α and CHX. Consistent with our previous studies, exposure of polyamine-deficient cells to the same doses of TNF-α and CHX caused no apoptosis. In keeping with our earlier findings [31,32], there were no differences in the morphological features or number of apoptotic cells when comparing DFMO-treated cells with DFMO-treated cells exposed to TNF-α and CHX for 4 h (results not shown). This increased resistance to TNF-α and CHX-induced apoptosis was not affected when polyamine-deficient cells were transfected with control siRNA (Figure 7B, panel b), but it was lost when MEK-1 expression was inhibited by HuR silencing (Figure 7B, panel c) or MEK-1 silencing (Figure 7B, panel d). The percentages of apoptotic cells (Figure 7C) and the levels of the active caspase-3 protein (Figure 7D) in DFMO-treated cells transfected with siHuR or siMEK-1 increased significantly when compared with those observed in DFMO-treated cells transfected with control siRNA.

Effects of MEK-1 or HuR silencing on apoptosis in polyamine-deficient cells

Figure 7
Effects of MEK-1 or HuR silencing on apoptosis in polyamine-deficient cells

(A) Cells were cultured with DFMO for 4 days and then transfected with either siRNA specifically targeting the MEK-1 mRNA coding region (siMEK-1), HuR (siHuR) or with control siRNA (C-siRNA). The levels of MEK-1 protein were measured by Western blot analysis 48 h after transfection; equal loading was monitored by β-actin immunoblotting. The approx. molecular mass in kDa is indicated on the left. (B) TNFα and CHX-induced apoptosis in cells treated as described in (A). Panel (a): control cells. Panel (b): DFMO-treated cells transfected with C-siRNA. Panel (c): DFMO-treated cells transfected with siHuR. Panel (d): DFMO-treated cells transfected with siMEK-1. Apoptosis was measured by morphological analysis (middle images) and ApoAlert annexin-V (A-V) staining (right-hand images) 4 h after treatment with TNFα and CHX compared with no treatment (no-TNFα/CHX). (C) Percentage of apoptotic cells in cultures as described in (B). Values are the means±S.E.M. for six samples. *P<0.05 compared with cells that were not treated with TNFα and CHX. +P<0.05 compared with DFMO-treated cells that were transfected with C-siRNA and then treated with TNFα and CHX for 4 h. (D) Representative immunoblots for pro-caspase-3 and caspase-3 in cells that were processed as described in (B). Whole-cell lysates were harvested 4 h after treatment with TNFα and CHX, and the levels of pro-caspase-3 and caspase-3 were examined by Western blot analysis. Three experiments were performed that showed similar results.

Figure 7
Effects of MEK-1 or HuR silencing on apoptosis in polyamine-deficient cells

(A) Cells were cultured with DFMO for 4 days and then transfected with either siRNA specifically targeting the MEK-1 mRNA coding region (siMEK-1), HuR (siHuR) or with control siRNA (C-siRNA). The levels of MEK-1 protein were measured by Western blot analysis 48 h after transfection; equal loading was monitored by β-actin immunoblotting. The approx. molecular mass in kDa is indicated on the left. (B) TNFα and CHX-induced apoptosis in cells treated as described in (A). Panel (a): control cells. Panel (b): DFMO-treated cells transfected with C-siRNA. Panel (c): DFMO-treated cells transfected with siHuR. Panel (d): DFMO-treated cells transfected with siMEK-1. Apoptosis was measured by morphological analysis (middle images) and ApoAlert annexin-V (A-V) staining (right-hand images) 4 h after treatment with TNFα and CHX compared with no treatment (no-TNFα/CHX). (C) Percentage of apoptotic cells in cultures as described in (B). Values are the means±S.E.M. for six samples. *P<0.05 compared with cells that were not treated with TNFα and CHX. +P<0.05 compared with DFMO-treated cells that were transfected with C-siRNA and then treated with TNFα and CHX for 4 h. (D) Representative immunoblots for pro-caspase-3 and caspase-3 in cells that were processed as described in (B). Whole-cell lysates were harvested 4 h after treatment with TNFα and CHX, and the levels of pro-caspase-3 and caspase-3 were examined by Western blot analysis. Three experiments were performed that showed similar results.

To further define the role of MEK-1 in the HuR-mediated anti-apoptotic effect in IECs, we also examined changes in TNF-α and CHX-induced apoptosis after ectopic HuR overexpression in MEK-1-silenced populations of cells. As shown in Figure 8, HuR overexpression protected IEC-6 cells against TNF-α and CHX-induced apoptosis, but this protective effect was prevented by MEK-1 silencing. Numbers of apoptotic cells (Figures 8B, panel b and 8C) and the active caspase-3 protein levels (Figure 8D) decreased remarkably in HuR-expressing cells when compared with those observed in cells infected with Adnull after exposure to TNF-α and CHX. This HuR-mediated protection was abolished by MEK-1 silencing (Figure 8B, panel d) and not altered when HuR-expressing cells were transfected with control siRNA (Figure 8B, panel c). The percentage of apoptotic cells (Figure 8C) and active caspase-3 protein levels (Figure 8D) in MEK-1-silenced cells infected with AdHuR significantly increased after exposure to TNF-α and CHX. These results implicate HuR-mediated MEK-1 expression in the regulation of intestinal epithelial apoptosis and indicate that the elevation in MEK-1 levels promotes an increase in resistance to apoptosis following polyamine depletion.

Effect of MEK-1 silencing on apoptotic sensitivity in cells overexpressing HuR

Figure 8
Effect of MEK-1 silencing on apoptotic sensitivity in cells overexpressing HuR

(A) Representative HuR and MEK-1 immunoblots. Cells were transfected with either siRNA specifically targeting the MEK-1 mRNA coding region (siMEK-1) or with control siRNA (C-siRNA) for 24 h and then infected with recombinant adenoviral vector encoding HuR cDNA (AdHuR) or adenoviral vector lacking HuR cDNA (Adnull) at 100 pfu/cell. HuR and MEK-1 protein levels were examined by Western blot analysis 24 h after infection and equal loading was monitored by β-actin immunoblotting. (B) TNFα and CHX-induced apoptosis in cells described in (A). Panel (a): cells infected with Adnull. Panel (b): cells infected with AdHuR. Panel (c): cells infected with AdHuR and transfected with C-siRNA. Panel (d): cells infected with AdHuR and transfected with siMEK-1. Apoptosis was measured by morphological analysis (middle images) and ApoAlert annexin-V (A-V) staining (right-hand images) 4 h after treatment with TNFα and CHX. (C) Percentage of apoptotic cells as described in (B). Values are means±S.E.M. for six samples. *P<0.05 compared with groups that were not treated with TNFα and CHX. +P<0.05 compared with cells infected with Adnull and then treated with TNFα and CHX. (D) Representative immunoblots for pro-caspase-3 and caspase-3 in cells that were processed as described in (B). Whole-cell lysates were harvested 4 h after treatment with TNFα and CHX and the levels of pro-caspase-3 and caspase-3 proteins were examined by Western blot analysis. Three experiments were performed that showed similar results.

Figure 8
Effect of MEK-1 silencing on apoptotic sensitivity in cells overexpressing HuR

(A) Representative HuR and MEK-1 immunoblots. Cells were transfected with either siRNA specifically targeting the MEK-1 mRNA coding region (siMEK-1) or with control siRNA (C-siRNA) for 24 h and then infected with recombinant adenoviral vector encoding HuR cDNA (AdHuR) or adenoviral vector lacking HuR cDNA (Adnull) at 100 pfu/cell. HuR and MEK-1 protein levels were examined by Western blot analysis 24 h after infection and equal loading was monitored by β-actin immunoblotting. (B) TNFα and CHX-induced apoptosis in cells described in (A). Panel (a): cells infected with Adnull. Panel (b): cells infected with AdHuR. Panel (c): cells infected with AdHuR and transfected with C-siRNA. Panel (d): cells infected with AdHuR and transfected with siMEK-1. Apoptosis was measured by morphological analysis (middle images) and ApoAlert annexin-V (A-V) staining (right-hand images) 4 h after treatment with TNFα and CHX. (C) Percentage of apoptotic cells as described in (B). Values are means±S.E.M. for six samples. *P<0.05 compared with groups that were not treated with TNFα and CHX. +P<0.05 compared with cells infected with Adnull and then treated with TNFα and CHX. (D) Representative immunoblots for pro-caspase-3 and caspase-3 in cells that were processed as described in (B). Whole-cell lysates were harvested 4 h after treatment with TNFα and CHX and the levels of pro-caspase-3 and caspase-3 proteins were examined by Western blot analysis. Three experiments were performed that showed similar results.

DISCUSSION

The regulation of MEK-1 activity through its phosphorylation has been extensively investigated [33,34,52,53], but little is known about the control of MEK-1 protein abundance, particularly at the post-transcriptional level. In the present study, we highlight a novel function of the RBP HuR and cellular polyamines in the regulation of MEK-1 mRNA stability and translation, thus advancing our understanding of the mechanisms that control the expression of MEK-1 and the biological function of cellular polyamines. Our studies aimed at characterizing the molecular aspects of this process revealed that HuR bound to the MEK-1 mRNA by interacting with its 3′-UTR and affected the turnover and translation of the MEK-1 transcript. Depletion of cellular polyamines enhanced the association of HuR with the MEK-1 mRNA, thereby stabilizing the MEK-1 mRNA and promoting its translation. Conversely, increasing the levels of cellular polyamines decreased the abundance of the HuR–MEK-1 mRNA complex, leading to a reduction in the steady-state level of MEK-1 by destabilizing the MEK-1 mRNA and repressing its translation. The present studies also implicated the HuR-mediated changes in MEK-1 levels as contributing to the regulation of IEC apoptosis, and therefore to intestinal epithelial homoeostasis.

The results reported in the present paper show that polyamines negatively regulate expression of the MEK-1 gene post-transcriptionally. As observed in a range of tissues [33], expression of the MEK-1 gene is constitutive in IECs and its basal level is relatively high. Decreasing cellular polyamines by inhibiting ODC with DFMO led to additional increases in the steady-state levels of MEK-1 mRNA and protein in IECs (Figure 1), whereas increasing cellular polyamines by ectopic ODC overexpression repressed the expression of the MEK-1 (Figure 2). Our results further indicate that altering the levels of cellular polyamines did not influence MEK-1 gene transcription, because there were no significant differences in the activity level of a MEK-1 promoter construct between control cells, polyamine-deficient cells and cells overexpressing ODC. Instead, depletion of cellular polyamines not only increased the half-life of MEK-1 mRNA but also enhanced its translation, through AREs located in the MEK-1 3′-UTR. The increases in MEK-1 mRNA stability and translation in DFMO-treated cells were completely prevented by the addition of exogenous putrescine, indicating that the observed post-transcriptional changes in MEK-1 gene expression are due to the polyamine depletion rather than to non-specific effects of DFMO. Consistent with our current findings, it has been reported that MEK-1 expression is increased by IL (interleukin)-4 following treatment with lysophosphatidic acid in human mast cells [54], but its expression is repressed during the process of postnatal heart development in rats [55]. However, the exact mechanism underlying the regulation of MEK-1 gene expression in response to specific stimuli remains elusive.

The present study also indicates that the MEK-1 mRNA is a target of the RBP HuR and that altering the levels of cellular polyamines affects the association of HuR with MEK-1 mRNA. Although the precise mechanisms by which HuR mediates its effect in post-transcriptional regulation remains to be uncovered, an increasing body of evidence shows that HuR-mediated transcript stabilization and translation are closely linked to its cytoplasmic presence [23,56]. Our previous studies [6,26] have demonstrated that depletion of cellular polyamines increases the cytoplasmic abundance of HuR by inhibiting the AMPK (AMP-activated protein kinase)-regulated phosphorylation and acetylation of importin-α1, whereas increased levels of polyamines decrease cytoplasmic HuR content by activating the AMPK-driven nuclear import via importin-α1. The results presented in Figure 3 show that polyamine depletion increased binding of HuR to the MEK-1 mRNA and the levels of the HuR–MEK-1 mRNA complex decreased in the presence of elevated cellular polyamines (in cells overexpressing ODC). The present study also showed that the cytoplasmic HuR binds to the 3′-UTR of MEK-1 mRNA rather than its CR and that this binding affinity is primarily mediated through four specific regions containing the signature HuR-binding motif (Figure 4). These findings are consistent with previous observations showing that HuR binds to AREs commonly located in the 3′-UTRs of labile mRNAs (in particular in mRNAs bearing a specific motif) [7,21,24,45,47]. However, the sequence spanning positions 1212–1430 (F1 and F2) and 1769–1833 (F5) of the MEK-1 3′-UTR also contained the computationally predicted HuR-binding motif, but failed to bind to HuR in polyamine-deficient cells, as measured by the biotin pull-down assays. This suggests that it is possible that these sequences were inaccessible to HuR, perhaps because they were targeted by other RBPs with a higher binding affinity than HuR.

Increased association of HuR with the MEK-1 mRNA in polyamine-deficient cells stabilizes MEK-1 mRNA and enhances its translation in IECs. As shown in Figure 5, depletion of cellular polyamines by DFMO increased the half-life of MEK-1 mRNA and enhanced its translation, but these effects were completely abolished in cells in which HuR expression was silenced; in turn these cells showed a marked reduction in MEK-1 protein levels. Consistent with the present findings, increased cytoplasmic HuR has also been shown to interact with and stabilize p53, NPM, ATF2 (activating transcription factor 2, and XIAP (X-linked inhibitor of apoptosis protein) mRNAs in polyamine-deficient cells [6,24,47]. On the other hand, ectopic HuR overexpression stabilized MEK-1 mRNA and induced its translation, thereby increasing the steady-state level of MEK-1 protein (Figure 6). Although the exact mechanisms whereby cytoplasmic HuR alters the post-transcriptional regulation of MEK-1 after changes in cellular polyamines levels are still unknown, several studies suggest that HuR acts by protecting the body of the mRNAs from degradation, rather than by slowing the rate of deadenylation [7,8]. Ongoing experiments are being conducted with the aim of testing if the stimulatory effects of HuR on the MEK-1 mRNA and translation depend on HuR phosphorylation, which was shown to regulate both HuR subcellular localization and its binding affinity for specific target mRNAs [51,56,57].

The HuR-mediated MEK-1 expression plays an important role in the regulation of IEC apoptosis and is thus implicated in maintaining homoeostasis of the intestinal epithelium. The epithelium of the intestinal mucosa is continuously renewed from the proliferative zone within the crypts, and this dynamic process is counterbalanced by apoptosis [58]. Apoptosis occurs in the crypt area, where it maintains the critical balance in cell number between newly divided and surviving cells, and at the luminal surface of the intestinal mucosa, where differentiated cells are lost. Our previous studies [31,32] and studies from other laboratories [29] have demonstrated that polyamines are crucial for the maintenance of epithelial homoeostasis and that depletion of cellular polyamines promotes the resistance of IECs to apoptosis through multiple signalling pathways. For example, polyamines negatively regulate NF-κB (nuclear factor κB) and Akt signals, and polyamine depletion increases NF-κB transcriptional activity [31,59] and activates Akt kinase [32]. Polyamines also inhibit the expression of ATF2 and XIAP genes at the post-transcriptional level, as decreasing the levels of cellular polyamines increases the steady-state levels of ATF2 [24] and XIAP [27] through stabilization of their mRNAs. The present study shows further that HuR-mediated increased levels of endogenous MEK-1 in polyamine-deficient cells also contribute to the increased resistance of IECs to apoptosis, as this tolerance was significantly abrogated by silencing MEK-1 or HuR (Figure 7). In support of this finding, the anti-apoptotic influence induced by ectopic HuR overexpression was also blocked by MEK-1 silencing in normal IEC-6 cells (Figure 8). These results suggest that MEK-1 is a critical downstream effector of the prosurvival programme elicited by HuR.

In summary, these results indicate that polyamines down-regulate MEK-1 expression at the post-transcriptional level through HuR-mediated events. Depletion of cellular polyamines increases MEK-1 expression by increasing the half-life of MEK-1 mRNA and enhancing its translation without affecting its gene transcription, whereas increasing the levels of cellular polyamines reduces the steady-state level of MEK-1 by destabilizing MEK-1 transcript and repressing its translation. Experiments investigating the mechanism of polyamine action in this process show further that increases in the cytoplasmic HuR abundances following polyamine depletion were linked to the increased binding of HuR to the MEK-1 3′-UTR through specific RNA regions containing predicted HuR-binding motifs and to increased stability and translation of the MEK-1 mRNA. The present study also indicates that MEK-1 is a novel downstream effector of the HuR-mediated anti-apoptotic effect and that increased levels of endogenous MEK-1 promote the survival of IECs and elevate their resistance to apoptosis after polyamine depletion. These findings suggest that polyamine-regulated MEK-1 expression, through HuR, is of physiological significance and is crucial for maintaining intestinal epithelium homoeostasis.

Abbreviations

     
  • AMPK

    AMP-activated protein kinase

  •  
  • AP-1

    activator protein 1

  •  
  • ARE

    AU-rich element

  •  
  • ATF2

    activating transcription factor 2

  •  
  • CHX

    cycloheximide

  •  
  • CR

    coding region

  •  
  • CREB

    cAMP-response-element-binding protein

  •  
  • DFMO

    D,L-α-difluoromethylornithine

  •  
  • FBS

    fetal bovine serum

  •  
  • GAPDH

    glyceraldehyde-3-phosphate dehydrogenase

  •  
  • HEK

    human embryonic kidney

  •  
  • HuR

    Hu-antigen R

  •  
  • IEC

    intestinal epithelial cell

  •  
  • IP

    immunoprecipitation

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MEK-1

    MAPK kinase-1

  •  
  • NF90

    nuclear factor 90

  •  
  • NF-κB

    nuclear factor κB

  •  
  • NPM

    nucleophosmin

  •  
  • ODC

    ornithine decarboxylase

  •  
  • IEC-ODC+

    IEC-6-derived cell-line stably expressing ODC

  •  
  • pfu

    plaque-forming units

  •  
  • qRT-PCR

    quantitative real-time PCR

  •  
  • RBP

    RNA-binding protein

  •  
  • RNP

    ribonucleoprotein

  •  
  • RRM

    RNA-recognition motif

  •  
  • RT

    reverse transcription

  •  
  • siRNA

    small interfering RNA

  •  
  • TIA

    T-cell intracellular antigen

  •  
  • TNF-α

    tumour necrosis factor-α

  •  
  • TTR

    translation and turnover-regulatory

  •  
  • 3′-UTR

    3′-untranslated region

  •  
  • XIAP

    X-linked inhibitor of apoptosis protein

AUTHOR CONTRIBUTION

Peng-Yuan Wang performed most experiments, summarized data and contributed to manuscript preparation. Jaladanki Rao performed some experiments and analysed results. Tongtong Zou participated in the immunohistochemical analysis. Lan Liu contributed to the studies involving RNA-binding assays. Lan Xiao was involved in studies related to apoptosis. Ting-Xi Yu performed the siRNA experiments. Douglas Turner contributed to data analysis. Myriam Gorospe contributed to experimental design and data analysis. Jian-Ying Wang designed experiments, analysed data and was involved in manuscript preparation.

FUNDING

This work was supported by the Department of Veterans Affairs, U.S.A. [Merit Review Grant (to J.-Y. W.)]; the National Institutes of Health [grant numbers DK-57819, DK-61972 and DK-68491 (to J.-Y. W.)]. P.-Y. W. is a visiting scientist from Peking University First Hospital, Beijing, China. J.-Y. W. is a Research Career Scientist, Medical Research Service, Department of Veterans Affairs. M. G. is supported by the National Institute on Aging Intramural Research Program.

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Author notes

1

Present address: Peking University First Hospital, Peking University, Beijing 100034, People's Republic of China.

Supplementary data