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J. Biol. Chem., Vol. 277, Issue 47, 44631-44637, November 22, 2002

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Translational Regulation of Prostaglandin Endoperoxide H Synthase-1 mRNA in Megakaryocytic MEG-01 Cells

SPECIFIC PROTEIN BINDING TO A CONSERVED 20-NUCLEOTIDE CIS ELEMENT IN THE 3'-UNTRANSLATED REGION^*

Maryse Duquette and Odette Laneuville^§

From the Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, 451 Smyth Road, Ottawa K1H 8M5, Canada

Received for publication, July 12, 2002, and in revised form, August 23, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Prostaglandin endoperoxide H synthase-1 (PGHS-1) is an abundant enzyme in platelets, where it plays a key role in the cascade of prostanoid formation. In platelets, the primary site of PGHS-1 synthesis is in precursor megakaryocytic cells. We have previously shown that in megakaryocytic MEG-01 cells, TPA induces an increase of PGHS-1 mRNA within a few hours, whereas protein increase occurs after several days of treatment. We now report that the delayed increase in PGHS-1 protein is caused by translational regulation. De novo PGHS-1 synthesis, measured using [³⁵S]methionine pulse labeling followed by immunoprecipitation, was detected at day 4 after TPA treatment but not at day 1. To identify a potential element of PGHS-1 mRNA controlling translation, we compared the 3'-untranslated region from different species and identified a 20-nt segment perfectly conserved. The 20-nt segment was used as a probe in RNA gel mobility-shift assays using MEG-01 extracts from control cells or from TPA-treated cells. Four complexes were formed with extracts from control cells or cells treated with TPA for 1 day but were not observed with extracts from cells treated for 4 days. Of the 4 complexes, one was sequence-specific and binding involved uridylate residues and interactions with a 45-kDa protein and a protein doublet of 116 kDa. Binding of this 45/116-kDa complex to the 20-nt conserved cis element most likely regulates negatively PGHS-1 protein accumulation. We have provided evidence that the PGHS-1 gene is regulated at the translational level.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The formation of prostanoids results from a cascade of enzymatic reactions in which the enzyme prostaglandin endoperoxide H synthase (PGHS)¹ plays a central role (1). Initially, the first substrate, arachidonic acid, is released from membrane phospholipids by phospholipases. The enzyme PGHS, also known as cyclooxygenase, catalyzes the bis oxygenation of arachidonate to generate prostaglandin H₂. Finally, prostaglandin H₂ serves as a common substrate for the various isomerases which catalyze the final step in the formation of prostaglandins and thromboxane A₂ (TxA₂).

Much attention has been devoted to the step catalyzed by PGHS since it is the target of aspirin and other nonsteroidal anti-inflammatory drugs (2). Two distinct PGHS enzymes are known, PGHS-1 and PGHS-2, and both catalyze the formation of prostaglandin H₂ from arachidonic acid (3). The two PGHS have different profiles of expression; PGHS-1 is present in almost all tissues, and stimulation with hormones or mitogenic agents does not affect its basal levels significantly (4). In contrast, PGHS-2 enzyme becomes detectable in certain contexts such as inflammation, cancer, or on addition of mitogenic agents (5). Although we have a great understanding of the regulatory mechanisms leading to the induction of PGHS-2 gene, the response elements and factors regulating the expression of PGHS-1 gene are essentially unknown.

PGHS-1 gene has a TATA-less promoter, is CG rich, and contains multiple potential start sites for transcription (6). A promoter study, using human umbilical vein endothelial cells, has shown that the 916-nucleotides sequence located upstream from the transcriptional start site had very low promoter activity and a modest increase of 1.8-fold was reported after TPA treatment (6). A few studies have shown variations in PGHS-1 mRNA and protein levels, notably, during the development of ovine lung (7), as well as during TPA-induced differentiation of monocytes (THP-1) cells to a macrophage phenotype (8). Thus far, mechanisms regulating the basal expression of PGHS-1 gene as well as its induction during development and differentiation have not been described.

To study the regulation of expression of the PGHS-1 gene, we have chosen the megakaryocytic cell line MEG-01 induced to differentiate into platelet-like structures on addition of TPA (9). Using this model, we have monitored the levels of PGHS-1 protein and mRNA during MEG-01 differentiation; the highest levels of PGHS-1 protein were measured in the most differentiated cells, the enucleated platelet-like population, whereas PGHS-1 mRNA levels were greatest in the nucleated adherent population (10). We also performed a time-course study and noted that only mRNA levels were increased at day 1 after addition of TPA whereas both mRNA and protein increased at day 4 after stimulation (11). The delay in PGHS-1 enzyme accumulation we observed is not unique to MEG-01 cells. Similar to our observation, the levels of PGHS-1 mRNA and protein were maximal at 24 h and 48 h, respectively, after treatment of human astrocyte cells with histone deacetylase inhibitors (12). PGHS-1 was also up-regulated by retinoic acid during neuronal differentiation in neuroblastoma cell lines, and PGHS-1 protein increase occurred 1 day after mRNA increase (13). These reports of a delayed accumulation of PGHS-1 enzyme relative to the increase in mRNA levels suggest that PGHS-1 protein synthesis could be under the control of a mechanism yet to be defined.

In the current study, we report that although the steady-state levels of PGHS-1 mRNA are comparable at days 1 and 4, synthesis of PGHS-1 protein occurred at day 4 only. To elucidate the mechanism involved in the delay of PGHS-1 protein synthesis, we have identified a conserved 20-nt segment in the 3'UTR and have used it in RNA gel shift assays. We provide evidences for specific protein interactions with the 20-nt conserved segment of the PGHS-1 3'UTR in control or in 1-day-TPA-treated cells. The sequence-specific interaction was not observed using extracts from cells treated for 4 days that contain high levels of PGHS-1 protein. Our experiments indicate the presence of RNA-binding proteins (complex C) that bind to the 20-nt conserved cis element of the 3'UTR of PGHS-1 transcript, likely silencing its translation early after TPA stimulation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Cultures and TPA treatment-- MEG-01 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum without antibiotics as previously published by our laboratory (11). TPA was added to each culture at a final concentration of 16 nM. Control cells were treated with the same (CH₃)₂SO concentration that TPA-treated cells received (0.0001% v/v). Cells were incubated (37 °C, air/CO₂ (19:1)) for 1 or 4 days and were harvested for RNA or protein analysis, enzymatic activity, metabolic labeling, or gel mobility-shift assays.

RNA Extraction and Analysis-- Total RNA was extracted from MEG-01 cells using Trizol reagent (Invitrogen), mainly as we have described previously (11). For Northern blot analysis, after gel separation and transfer, nylon membranes were prehybridized in 5× SSC, 1× Denhardt's solution, 50% formamide, 1% SDS, 10% dextran sulfate, 20 mM Tris-Cl for 30 min. at 42 °C with 30 µg/ml of herring sperm DNA. Hybridization was done at 42 °C overnight using random-primed ³²P-labeled PGHS-1 cDNA corresponding to the entire open reading frame, as a probe. The membranes were washed twice in 2× SSC/0.05% SDS at room temperature and then once in 0.1× SSC/0.1% SDS at 65 °C for 30 min. The membranes were then exposed on phosphorscreen to visualize mRNA. The ³²P-labeled human -actin cDNA was used as control for the quantity of RNA loaded in each lane. For analysis of ribosomal RNA, total RNA was isolated from 1 × 10⁷ MEG-01 cells, resolved on 1% agarose gels, and stained with ethidium bromide.

Western Blot Analysis-- Protein isolation, quantification, and Western blotting were done as described previously (11). Samples (10 µg of total protein) were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes. The primary anti-PGHS-1 antibody is directed against residues Leu²⁷²-Gln²⁸³ of hPGHS-1 (gift of Dr. W. L. Smith, Michigan State University), and detected with anti-rabbit IgG linked to horseradish peroxidase (Promega). Protein bands were visualized using the Roche Molecular Biochemicals Chemiluminescence Blotting Substrate detection system (POD) and photographed on Polaroid black and white Polapan 667 film. The membranes were incubated simultaneously with an anti-actin antibody developed in rabbit (Sigma) (loading control). For the MG-132 experiment, after stripping, membranes were incubated with monoclonal anti-c-Myc (Sigma) as a positive control for the proteasome inhibitor treatment; c-Myc was detected with an anti-mouse IgG horseradish peroxidase conjugate (Promega). Sheep PGHS-1 purified from vesicular glands was used as a standard (1 µg) (provided from Dr. W. L. Smith).

Enzymatic Activity-- Thromboxane synthesis by intact MEG-01 cells was measured at 1 day and 4 days after TPA treatment. Cells (10⁶) were removed from culture dishes, collected by centrifugation, and resuspended in 0.5 ml of serum-free RPMI 1640 medium per 25 ml of culture. The substrate, [1-¹⁴C]arachidonic acid (final concentration of 10 µM; 42 mCi/mM) was added, and the cell suspension was incubated at 37 °C for 30 min. To terminate the reaction, the cells were centrifuged for 5 min at 1000 × g, and the medium was removed. Proteins from the medium were precipitated by adding 1 volume of ice-cold acetone. The supernatant containing the radioactive prostanoid products, mainly TxB₂, and unreacted arachidonic acid was acidified with 1 volume of 0.1 M HCl and extracted with 3 volumes of ethyl acetate/methanol/0.2 M citric acid (30/4/1). The organic phase was evaporated to dryness under a stream of N₂ and resuspended in 50 µl of the thin-layer chromatography (TLC) solvent: ethyl acetate/2,2,4-trimethylpentane/acetic acid, saturated with water. Samples were applied to a silica gel 60 TLC plate (VWR). The lipid products were separated by chromatography and detected by exposure to x-ray film for 40 h. Radioactive TxB₂ was identified by comparison with an authentic TxB₂ standard and quantified by densitometry analysis.

Proteolytic Systems Inhibition Analysis-- MEG-01 cells were cultured as described above and treated or not with TPA for 1 or 4 days. Drugs were added 8 h before harvesting at the following concentrations: MG-132 (Sigma), 100 µM; chloroquine (Sigma), 100 µM; and PD150606 (Calbiochem), 20 µM. Both MG-132 and PD150606 were dissolved in (CH₃)₂SO at 100 mM and 20 mM, respectively, and chloroquine was dissolved in water at 100 mM.

De Novo Synthesis and Immunoprecipitation-- MEG-01 cells (2 × 10⁷ cells) were treated with TPA for 1 or 4 days. The cells were collected by centrifugation at 7 000 × g, washed in methionine-free medium, and resuspended in 4 ml of methionine-free RPMI. The cells were incubated 15 min at 37 °C to deplete the intracellular pools of methionine and metabolically labeled by incubation for 30 min with [³⁵S]methionine (50 µCi/ml). To prepare lysates, the cells were suspended in a mixture of 20 mM Tris pH 8.0, 137 mM NaCl, 1% Triton X-100, 10% glycerol, and incubated at 4 °C for 1 h. Before immunoprecipitation, an aliquot (2.5% of total volume) of the lysates were kept and run on a 7.5% SDS-PAGE. Newly synthesized ³⁵S-labeled PGHS-1 was immunoprecipitated from lysates by using rabbit anti-human PGHS-1 IgG and protein A-Sepharose in the lysis buffer. Proteins were resolved by 7.5% SDS-PAGE. Gels were dried and allowed to expose onto MR film (Eastman Kodak Co., Rochester, NY). PGHS-1 standard was run in parallel and stained with Coomassie Blue to confirm band identity.

Plasmids and in Vitro Transcription-- Expression vector pGEM-3Z (Promega) was previously digested with EcoRI and HindIII and isolated from agarose gel. The following oligonucleotides were designed to anneal to each other (annealing: 65 °C for 5 min and 15 min on ice) and to be ligated into the linear pGEM-3Z vector: oligo 1) 5'-AATTCCTCGAGTTCATTTTCCTGTTCAGTGAA-3' and oligo 2) 5'-AGCTTTCACTGAACAGGAAAATGAACTCGAGG-3'. The bold characters represent the conserved 20-nt sequence; the 5' and 3' extremities correspond either to the EcoRI or HindIII cohesive ends of the linear plasmid; the underlined sequence is the XhoI site used to identify positive clones. The same approach was used to generate the antisense and mutated sequences constructs (see Figs. 5A and 8A for exact probe sequences). The total length of each probe produced by this method was 37 nt. Uncapped sense, antisense, and mutant probes were transcribed from the corresponding pGEM-3Z-hPGHS-1-3'UTR vector linearized with HindIII, using T7 RNA polymerase (PerkinElmer Life Sciences) in the presence of [-³²P]UTP (Amersham Biosciences, PB20383, 20 mCi/ml) and 0.5 mM each of ATP, CTP, and GTP and 0.05 mM UTP for 2 h at 37 °C. The radiolabeled probes were recovered by phenol extraction and ethanol precipitation with tRNA after digestion of DNA template with DNase/RNase Free. The radiolabeled transcripts were verified on acrylamide gels and used directly in RNA mobility-shift assays after dilution at 200,000 cpm/µl or at 1 pmol of probe per binding reaction, based on their respective specific activities.

Preparation of Cell Extracts-- The cells were incubated for 1 or 4 days with or without TPA, harvested by centrifugation, and washed twice in phosphate-buffered saline, and cellular extracts were obtained as described by Dignam (14). Protein concentrations were determined with the use of a BioRad protein assay kit, according to the manufacturer's instructions.

RNA Mobility-shift Assay-- Binding reactions were carried out with 10-20 µg of MEG-01 protein extract and 200,000 cpm of hPGHS-1-3'UTR ³²P-labeled transcript, and were incubated for 15 min at room temperature in 1× binding buffer (20 mM Hepes, pH 7.6, 1 mM EDTA, 10 mM (NH₄)₂SO₄, 1 mM dithiothreitol, Tween 20^® 0.2% (w/v), 30 mM KCl) in a total volume of 20 µl, to allow the formation of complexes. Heparin was added to different final concentrations and incubated for 10 min at room temperature. Samples were then placed on ice and irradiated for 10 min in a Stratalinker chamber (Stratagene 2400) at a distance of 6 cm from the light source, prior to electrophoresis on 5% native polyacrylamide (29:1 acrylamide:bis-acrylamide) gels in 0.5× TBE buffer. These binding reactions were analyzed by autoradiography. In some assays, cellular extracts were preincubated for 10 min at room temperature with different competitor RNAs or treatments, such as proteinase K, SDS, or heat denaturation before addition of the riboprobe.

SDS-PAGE Analysis of RNA-Protein Complex-- For complex C analysis, RNA-EMSA reactions submitted to UV irradiations as described above were resolved on 5% native gels. Complex C from 5 binding reactions was cut and pooled, and proteins were eluted in 50 mM (NH₄) ₂HCO₃ buffer containing 0.001% SDS and 0.02 M phenylmethylsulfonyl fluoride at 4 °C for 16 h with shaking. After elution, volume was concentrated on centrifugal filter devices (Centricon^® and Ultrafree^®-MC YM-10, Millipore) to a volume of 40 µl, and complex protein components were resolved on gradient (4-19%) polyacrylamide gels in denaturing conditions. Proteins were visualized with Sypro Tangerine protein gel stain (Molecular Probe, S12010). Dried gels were then exposed to PhosphorImaging screen for detection of proteins linked to the RNA probe.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Delayed PGHS-1 Protein Accumulation-- The temporal relationship between PGHS-1 mRNA, PGHS-1 protein, and PGHS-1 activity in TPA-treated MEG-01 cells was examined. The steady-state level of PGHS-1 mRNA was measured by Northern blot analysis and was normalized by comparison to -actin mRNA (Fig. 1A). In agreement with previous results we have reported, increase in mRNA was detected at maximum at 1 day after TPA treatment and remained high for at least 4 days (11). The steady-state level of PGHS-1 protein was measured by Western blot analysis (Fig. 1B). PGHS-1 enzyme increased disproportionately compared with PGHS-1 mRNA; there was no detectable PGHS-1 protein after 1 day of TPA stimulation and a strong signal after 4 days. Therefore, a significant delay in PGHS-1 protein accumulation occurred in TPA-treated MEG-01 cells. The activity of the PGHS-1 enzyme was measured by incubating the cells with the radioactive substrate arachidonic acid and analyzing the formation of TxB₂, the stable metabolite of TxA₂. The formation of TxB₂ increased proportionally to the levels of PGHS-1 enzyme, i.e. low at 1 day after TPA stimulation and high at 4 days (Fig. 1C), indicating that differences in PGHS-1 protein levels measured by Western blot are not an artifact. The possibility that thromboxane synthase protein and/or activity might limit the formation of TxB₂ (Fig. 1C) was excluded on the basis of a study reporting low formation of TxB₂ despite high levels of thromboxane synthase protein in unstimulated MEG-01 cells (24). Also, this study reports very little variation in thromboxane synthase levels after TPA stimulation.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Delay between PGHS-1 mRNA and PGHS-1 protein increases after TPA stimulation. A, representative autoradiograms of Northern blots from total RNA (15 µg) extracted from TPA-treated MEG-01 cells for 1 and 4 days. PGHS-1 2.8-kb and 4.5-kb transcripts were both detected after 1 day and 4 days of TPA treatment. Detection of -actin transcript (1.8 kb) was used as a loading control. B, representative Western blots of SDS-PAGE separated proteins (10 µg) from MEG-01 cells treated with TPA for 1 and 4 days. PGHS-1 protein (70 kDa) was only detected in the 4-day-TPA-treated lysate; actin protein (43 kDa) was detected to insure equal loading between lanes. C, PGHS-1 activity in TPA-treated MEG-01 cells for 1 and 4 days. Activity is a measure of [14C]thromboxane B2 (TxB2) formation (cpm/106 cells ± S.E.) from exogenously supplied [14C]arachidonic acid. Increased PGHS-1 protein levels are associated with an increase in TxB2 formation. All of the results are representative of three experiments.

Lack of PGHS-1 Protein Synthesis at Day 1 after TPA Treatment-- The low levels of PGHS-1 protein at day 1 after TPA treatment despite a high level of transcript is consistent with either a low rate of protein synthesis or a high rate of protein degradation. In order to evaluate the importance of PGHS-1 protein degradation, cells were incubated for 8 h before harvesting with one of the following proteolytic system inhibitors: 100 µM MG-132, 20 µM PD-150606, or 100 µM chloroquine. The steady-state levels of PGHS-1 protein in presence or absence of MG-132, a proteasome inhibitor, were nearly identical, being undetectable in control cells and at day 1 after TPA treatment but high at day 4 (Fig. 2). As a positive control for MG-132 treatment, c-Myc protein, known to be degraded by the ubiquitin-proteasome system, was monitored. In agreement to the literature, c-Myc levels increased when MG-132 is added to the cells (15). PGHS-1 protein was also not detected in 1-day-TPA-treated cells subjected to other drugs such as the calpain inhibitor, PD-150606, or the lysosomal inhibitor, chloroquine, thus excluding a high rate of degradation as an explanation for the lack of PGHS-1 protein after 1 day of TPA treatment (data not shown).

View larger version (34K): [in this window] [in a new window]   Fig. 2.   No effect of the proteasome inhibitor MG-132 on the levels of PGHS-1 protein in the 1-day-TPA-treated cells. MEG-01 cells were treated or not with TPA during 1 and 4 days and incubated with or without MG-132 (100 µM). PGHS-1, actin (internal control), and c-Myc (62 kDa) (drug treatment control) proteins were detected by Western blot. The Western blot presented is representative of three independent experiments.

To confirm the low rate of synthesis of PGHS-1 protein at 1 day after TPA treatment, de novo synthesis was measured by pulse-labeling with [³⁵S]methionine. Labeled PGHS-1 levels in the lysate were determined by immunoprecipitation with rabbit anti-human PGHS-1 IgG, SDS-PAGE, and autoradiography. The lysate of cells treated with TPA for 4 days contained newly synthesized PGHS-1, but essentially no new PGHS-1 protein was detected after treatment of the cells for 1 day (Fig. 3A). These results suggest that the low level of PGHS-1 enzyme at day 1 after TPA treatment is due to a relatively lower level of PGHS-1 protein synthesis. To determine whether there is an increase in overall protein synthesis at 4 days after TPA treatment, we also analyzed labeled proteins before performing the PGHS-1 immunoprecipitation. Starting with the same number of viable cells (2 × 10⁷) at 1 and 4 days after TPA stimulation, we observed less overall incorporation of [³⁵S]methionine when cells are treated for 4 days compared with 1 day (Fig. 3B, left panel). Ribosomal abundance was also reduced in the TPA 4-day samples (Fig. 3B, right panel). Despite a decrease in the overall protein synthesis and a reduction in ribosomal abundance after 4 days of TPA treatment, the synthesis and steady-state levels of PGHS-1 protein were increased.

View larger version (49K): [in this window] [in a new window]   Fig. 3.   Increased PGHS-1 de novo synthesis in the 4-day-TPA-treated cells but not in the 1-day-TPA-treated cells. A, MEG-01 cells treated with TPA during 1 and 4 days were pulse-labeled with [35S]methionine and PGHS-1 was immunoprecipitated. The autoradiograph represents one of three representative experiments. B, aliquots of pulse-labeled MEG-01 lysates not submitted to the immunoprecipitation step were resolved on SDS-PAGE (Left panel). rRNA 28S and 18S from either 1-day-TPA-treated or 4-day-TPA-treated MEG-01 cells were resolved on denaturing formaldehyde agarose gel and visualized by ethidium bromide staining (Right panel). These results showed a reduction in overall protein synthesis after 4 days of TPA treatment and are representative of three independent experiments.

Time- and TPA-dependent Protein Interaction with a Conserved 20-nt Segment in the PGHS-1 3'UTR-- The 3'-UTR of translationally regulated transcripts is a predominant site of regulation. In most cases of translational control, a cellular RNA-binding protein binds to a cis-acting element of the targeted message. Seeking for a potential site for regulation, we aligned the PGHS-1 3'-UTR from four different species and identified a 20-nt sequence perfectly conserved. For the human PGHS-1 transcript, the 20-nt sequence is located at the 491st nt after the stop codon (Fig. 4A). Interacting proteins were detected by an RNA electrophoretic mobility-shift assay (EMSA) by using the [-³²P]UTP-labeled PGHS-1 20-nt conserved sequence as a probe. In the presence of increasing concentrations of heparin (1-5 µg/µl), the probe was retarded by extracts from control MEG-01 cells or cells treated with TPA for 1 day; four RNA-protein complexes, termed A, B, C, and D, were formed (Fig. 4B). None of the four complexes was detected in extracts from cells treated with TPA for 4 days. Thus, RNA-EMSA demonstrated that the formation of complexes A-D with the 20-nt conserved sequence is both time- and TPA-dependent. Extracts from the 4-day-treated cells produced a single complex, termed T, the intensity of which increased with the duration of the TPA exposure (Fig. 4B). To exclude the possibility of protein degradation or of an underestimation of protein amount used in the 4-day-TPA extract, the four extracts were run on SDS-PAGE stained with silver and have shown similar profiles (Fig. 4C). The absence of complexes A-D with the 4-day-treated MEG-01 extract correlated with a high level of PGHS-1 protein in this extract as monitored by Western blot (Fig. 4D). These results suggest that the complexes A-D could comprise PGHS-1 mRNA-binding proteins regulating PGHS-1 translational activity negatively.

View larger version (55K): [in this window] [in a new window]   Fig. 4.   Time- and TPA-dependent complexes formation with a 20-nt conserved segment in the PGHS-1 3'-UTR. A, computer analysis (ClustalW 1.8 and Boxshade 3.21 programs) of the first 1000 nucleotides after the stop codon in the 3'-UTR of PGHS-1 from murine, rat, human, and ovine species allowed the identification of a 20-nt conserved segment (black box); asterisks represents conserved nucleotides. B, representative autoradiogram from RNA-EMSA in which the 32P-labeled PGHS-1 conserved 20-nt RNA probe was incubated with cellular protein extracts (10 µg) from MEG-01 cells treated or not with TPA, for 1 and 4 days, in presence of increasing amount of heparin. The arrows indicate the major complexes observed (A-D) with every MEG-01 extract, except the 4-day-TPA-treated extract. T indicates the band observed only in TPA-treated extracts. As a reference, the migration of xylene cyanol (XC) and bromphenol blue (BB) dyes is indicated. FP (free probe) shows the migration of the RNA probe in absence of protein extract. C, the quality of MEG-01 cellular extracts (10 µg) resolved on a SDS-PAGE was verified by silver staining. D, Western blot analysis of PGHS-1 protein levels in the cellular extracts (10 µg) used in EMSA. PGHS-1 protein is detected only in 4-day-TPA extract; actin was monitored as an internal control. All results are representative of three independent experiments.

Sequence Specificity of Time- and TPA-dependent Binding-- The sequence specificity of all observed complexes was determined using an antisense probe that included the same restriction site sequences present in the sense probe and the PGHS-1 20-nt sequence in the opposite orientation (Fig. 5A). As shown in Fig. 5B (left panel), only one complex (complex C, indicated by the arrow) was not detected with the antisense probe incubated with extracts from control MEG-01 cells. Complex T, observed with the 4-day TPA extract, was also formed with the antisense probe (Fig. 5B, right panel). Therefore the formation of complexes A, B, D, and T is both time- and TPA-dependent but not sequence specific. Addition of an excess amount of the unlabeled 20-nt sense probe to the binding reactions diminished the formation of complex C in a concentration-dependent manner (data not shown). The next experiments were designed to study complex C formation, which is time- and TPA-dependent and specific to the 20-nt conserved sequence in the PGHS-1 3'-UTR.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Specific binding activity to the PGHS-1 conserved 20-nt segment. A, sequence of the sense (S) and antisense (As) probes used in RNA-EMSA. The bold sequence corresponds to the PGHS-1 20-nt sequence, and the underlined sequences indicate the restriction sites used for the cloning in the pGEM-3Z plasmid; the italic characters correspond to the T7 RNA polymerase transcription promoter. B, RNA-EMSA performed with the radiolabeled S or As transcript in presence of cellular extracts (20 µg) from control MEG-01 cells (left panel) or 4-day-TPA-treated cells (right panel) and heparin (2.5 µg/µl). The sequence-specific complex, complex C, not formed with the antisense probe, is indicated by the arrow. Complex T was formed with both the sense and the antisense probes. These results are representative of three different experiments.

The protein content in the complex C was confirmed by digestion or denaturation of the control MEG-01 extract before its addition to the binding reaction. Incubation of cell extracts with proteinase K alone or in presence of SDS, or denaturing cell extracts with SDS or heat all prevented complex formation (Fig. 6, line 4 to 7). Heating the probe before incubation with untreated MEG-01 extracts did not abolish complex formation (Fig. 6, line 8). Incubation of the probe with RNase A/T1 before incubation with a cell extract from control MEG-01 (Fig. 6, line 9) did not lead to complex formation, indicating that complexes do not result from binding with free labeled nucleotides or aborted shorter probes. Thus, formed complexes include protein(s), and the nucleotide sequence rather than the conformation were critical for the binding of proteins.

View larger version (46K): [in this window] [in a new window]   Fig. 6.   Formed complexes include protein(s) and RNA. The radiolabeled 20-nt transcript (P) or the control MEG-01 extract (E) was treated as indicated below prior to binding reactions in presence of heparin (2.5 µg/µl). Lanes: 1, free probe; 2, positive controlformation of complexes A-D; 3, binding reaction with bovine serum albumin (10 µg)no complex formed; lanes 4-7: extract denaturation by proteinase K (0.5 mg/ml) (K), proteinase K (2.5 mg/ml) + SDS (0.1%) (K/S), SDS (1%) (S), or heat (85 °C, for 15 min) (H)no complex formation occurred; lanes 8-9: RNA probe denaturation by heat (70 °C, for 5 min) (H) or digestion with RNases A/T1 (1 U/µl) (D)complex formation occurred with the heated probe only. The autoradiogram is representative of three experiments.

Identification of Uridylate Residues Responsible for Binding-- To identify the nucleotides of the conserved sequence that are crucial for complex C formation, we performed competition experiments and design mutants lacking binding activity. Extracts prepared from control MEG-01 cells were preincubated with increasing amounts of ribonucleotides, either poly(U), poly(C), or poly(A), before the addition of the sense probe to the binding reactions. As shown in Fig. 7, the formation of complex C was partially competed by poly(U) additions as low as 10 ng and completely competed by an excess of 1000 ng. However, poly(C) or poly(A) did not significantly attenuate its formation at any concentration tested. The binding activity from MEG-01 extracts therefore binds preferentially U-rich sequence correlating with the fact that 50% of the nucleotides composing the 20-nt conserved sequence are U residues, of which four are found consecutively. Next, we designed mutated probes listed in figure 8A and used them in RNA-EMSA. First, we arbitrarily substituted 1 of 2 nucleotides (mutant 1) and observed that the formation of complex C was reduced but not abolished. Next, we targeted the four consecutive U residues in the conserved sequence and analyzed their binding activity. As shown in Fig. 8, substitution of the UUUU sequence to CUGA, UUGA, and CUUA (mutants 3-5 respectively) completely abolished the formation of complex C. Mutating only the second of the four consecutive U residues (UCUU, mutant 2) permitted formation of complex C. Therefore, the U residues of the 20-nt sequence and in particular the UUUU motif are important for the formation of complex C. 

View larger version (71K): [in this window] [in a new window]   Fig. 7.   Importance of uridylate residues for the formation of the PGHS-1 3'-UTR 20-nt segment specific complex. Cellular extracts (20 µg) from control MEG-01 cells were incubated with the radiolabeled PGHS-1 20-nt sequence in absence or presence of the indicated amounts (ng) of poly(U) (pU), poly(C) (pC), or poly(A) (pA) homoribopolymers and analyzed by RNA-EMSA. Heparin (2.5 µg/µl) was added to the binding reactions. As shown by the arrow, complex C formation was prevented in presence of poly(U). A positive binding reaction with a control extract in absence of competition is shown on the left, and a binding reaction with a 4-day-TPA-extract is shown on the right. FP, free probe. These observations are representative of three independent experiments.

View larger version (59K): [in this window] [in a new window]   Fig. 8.   Mutations in the PGHS-1 3'-UTR 20-nt segment affecting the specific binding activity. A, sequences of the non-mutated probe (NM) and of 5 mutated probes (M1 to M5) used in RNA-EMSA. Italic characters indicate different point mutations into the conserved 20-nt sequence (bold characters), and more particularly into the UUUU tetranucleotide (framed sequence). For each mutated probe, formation of complex C is reported based on the RNA-EMSA results. B, RNA-EMSA representative of three independent experiments where 32P-labeled non-mutated probe or mutant probe was incubated in presence of control MEG-01 lysate (20 µg) and heparin (2.5 µg/µl). As shown by the arrow, complex C formation was observed using NM, M1, or M2 probe, whereas complex C formation was absent with probe M3, M4, or M5.

Characterization of the PGHS-1 mRNA-binding Protein(s)-- To further characterize the protein(s) that interact with the 20-nt conserved region of the PGHS-1 3'-UTR, we first resolved, on native gels, binding reactions submitted to UV irradiations. Complex C was cut, eluted from the gel, and resolved on a gradient SDS-PAGE. Gels were first stained with SYPRO Tangerine stain to visualize all the proteins. Dried gels were then exposed to a PhosphorImaging screen to identify proteins interacting with the RNA probe. The stained gel showed that complex C is composed of at least 5 proteins with apparent molecular mass varying from 20-100 kDa with two major bands at ~45 kDa and 100 kDa (Fig. 9, left panel). Strikingly, a doublet migrating at around 116 kDa and a band at 45 kDa were radioactive, indicating that these proteins are likely to bind the 20-nt sequence (Fig. 9, right panel).

View larger version (53K): [in this window] [in a new window]   Fig. 9.   Binding of 45- and 116-kDa proteins to the PGHS-1 3'-UTR 20-nt conserved segment. Eluted proteins from complex C were separated on a 4-19% gradient gel and visualized by SYPRO Tangerine dye staining (left panel) and then by PhosphorImaging exposure (right panel). Complex C was composed of proteins varying in mass from ~20 to ~100 kDa. A radioactive doublet in the 116-kDa region and a single band in the 45-kDa region were observed, suggesting that the corresponding proteins interact directly with the conserved sequence. Results are representative of three independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

As a first step to characterize PGHS-1 expression, we analyzed steady-state levels of PGHS-1 mRNA and protein in a megakaryocytic cell line induced to differentiate. The levels of PGHS-1 mRNA were maximal at 1 day and remained elevated for at least 4 days after treatment with TPA, while PGHS-1 protein was not detected at 1 day, but levels were high at day 4. We hypothesized that a delay at the translational step was responsible for the lack of PGHS-1 protein at day 1 after TPA treatment. To support this hypothesis, we measured de novo synthesis of PGHS-1 enzyme with metabolic labeling experiments and found no PGHS-1 synthesized at day 1, but synthesis did occur at day 4. As a potential mechanism that may mediate the lack of PGHS-1 translation in control MEG-01 cells or early after TPA stimulation, we report the binding of 45- and 116-kDa proteins to a 20-nt conserved sequence located in the 3'-UTR of the PGHS-1 transcript.

Our current results suggest that protein extracts from control or 1-day-TPA-treated MEG-01 cells bound to the 20-nt conserved sequence in the 3'-UTR. This binding referred to as complexes A-D, was occurring in the absence of PGHS-1 synthesis. To the opposite, the synthesis of PGHS-1 enzyme correlated with the formation of complex T. The conserved sequence in the PGHS-1 3'-UTR appears to be unique, as a BLAST search has revealed that it is not found in any other gene. Similar to previously reported mRNA cis element, there is only one copy of the 20-nt cis element within the PGHS-1 message. A literature search has allowed us to identify other known 20-nt cis elements in the 3'-UTRs of c-fos (16), ₂-adrenergic receptor (17), peptidylglycine -amidating monooxygenase (18), and ribonucleotide reductase R2 (19) that mediate translational regulation. These 20-nt sequences have no homology to the PGHS-1 3'-UTR conserved sequence but, like PGHS-1 conserved sequence, all contain stretches of uridylate residues important for the binding activity. The sequence-specific complex formed (complex C) with the 20-nt conserved sequence includes proteins of 116 and of 45 kDa that cross-linked to the RNA probe. Based on our observations that the U stretch is crucial for protein binding, we predict that proteins known to bind A + U-rich nucleotide regions, including the heterogeneous nuclear ribonucleoproteins, in particular heterogeneous nuclear ribonucleoproteins C, migrating at ~45 kDa, are potential candidates (20).

The importance of translational regulation for the expression of the PGHS-1 gene is only speculative at this stage. The overall reduction of ribosomal RNA after TPA treatment is consistent with previous reports of a suppression of ribosomal biogenesis that accompany terminal differentiation of myeloid cells (21). As suggested, a decrease in ribosomal biogenesis may be necessary to allow cells to redirect gene expression away from proteins involved in growth and toward those characterizing differentiated cells. For the differentiating MEG-01 cells, PGHS-1 protein synthesis would be favored despite a significant reduction of the levels of ribosomes, strengthening the importance of translational regulation for PGHS-1 gene expression. We have reported that PGHS-1 protein levels show a consistent increase over the entire course of differentiation of MEG-01 cells, that is, from blast cell to platelet-shedding megakaryocyte (10). It is well established that platelets represent one of richest source of PGHS-1 enzyme in the body, but the molecular mechanisms leading to this are unknown (22). The most likely role of the translational regulation we document here would be to prevent the accumulation of PGHS-1 protein early in megakaryocyte differentiation. In this context, when differentiation of the platelet precursor cells is triggered by the hematopoietic factors, the levels of PGHS-1 mRNA would rapidly increase, but protein synthesis would be delayed until differentiation of the cells has reached the stage of releasing platelets. In a model similar to megakaryocytic maturation, erythroid differentiation is associated with the translational silencing of the 15-lipoxygenase gene (23). 15-Lipoxygenase mRNA is synthesized in the erythroid precursors of the bone marrow but remained untranslated until the maturation of reticulocytes into red blood cells. Interestingly, PGHS-1 enzyme metabolizes the same substrate as 15-lipoxygenase (arachidonic acid), and both enzymes are found at high levels in enucleated blood cells (the platelets and the red blood cells, respectively).

In summary, in the setting of TPA-induced MEG-01 differentiation, the increase in the steady state levels of PGHS-1 protein could be accounted for by the increase in PGHS-1 synthesis, based on [³⁵S]methionine labeling, suggesting that translational regulation is a significant means of PGHS-1 regulation. We have identified a 20-nt conserved region in the 3'-UTR of the PGHS-1 transcript as a potential regulatory site. We report a correlation between the association of proteins to this cis element and the lack of PGHS-1 protein synthesis. We have identified a stretch of four U residues within the 20-nt conserved sequence that is essential for binding activity. Last, we have determined the size of the PGHS-1 mRNA-binding proteins, and their identity remains to be elucidated.

	ACKNOWLEDGEMENT

We thank Adele Boudreau for her technical contribution.

	FOOTNOTES

^* This work was supported in part by a grant from the Canadian Institutes of Health Research (to O. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a studentship from the "Formation de Chercheurs et l'Aide à la Recherche" (FCAR, Quebec).

^§ To whom correspondence should be addressed. Tel.: 613-562-5800 (ext. 8402); Fax: 613-562-5440; E-mail: olaneuvi@uottawa.ca.

Published, JBC Papers in Press, September 16, 2002, DOI 10.1074/jbc.M207007200

	ABBREVIATIONS

The abbreviations used are: PGHS, prostaglandin endoperoxide H synthase; TPA, 12-O-tetradecanoylphorbol-13-acetate; MEG-01, human megakaryocytic cell line, UTR, untranslated region; Tx, thromboxane; TLC, thin layer chromatography; EMSA, electrophoretic mobility shift assay; nt, nucleotide.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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