ERK and p38 Inhibit the Expression of 4E-BP1 Repressor of Translation through Induction of Egr-1*
Malvyne Rolli-Derkinderen
,
François Machavoine
,
Jay M. Baraban
,
Annabelle Grolleau ¶,
Laura Beretta ¶ and
Michel Dy
||
From the
CNRS FRE 2444, Université René Descartes Paris V, Hôpital Necker, Institut Federatif de Recherche Necker Enfants Malades, 75015 Paris, France,
Department of Neurosciences, Psychiatry, and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205,
¶ Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan 48109-0666
Received for publication, November 18, 2002
, and in revised form, February 27, 2003.
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ABSTRACT
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4E-BP1 plays a major role in translation by inhibiting cap-dependent translation initiation. Several reports have investigated the regulation of 4E-BP1 phosphorylation, which varies along with cell differentiation and upon various stimulations, but very little is known about the regulation of its expression. In a first part, we show that the expression of 4E-BP1 protein and transcript decreases in hematopoietic cell lines cultivated in the presence of phorbol 12-myristate 13-acetate (PMA). This decrease depends on the activation of the ERK/mitogen-activated protein kinases. 4E-BP1 expression also decreases when the p38/mitogen-activated protein kinase pathway is activated by granulocyte/macrophage colony-stimulating factor but to a lesser extent than with PMA. In a second part, we examine how 4e-bp1 promoter activity is regulated. PMA and granulocyte/macrophage colony-stimulating factor induce Egr-1 expression through ERK and p38 activation, respectively. Using a dominant negative mutant of Egr, ZnEgr, we show that this transcription factor is responsible for the inhibition of 4e-bp1 promoter activity. In a third part we show that histidine decarboxylase, whose activity and expression are inversely correlated with 4E-BP1 expression, is a potential target for the translational machinery. These data (i) are the first evidence of a new role of ERK and p38 on the translational machinery and (ii) demonstrate that 4E-BP1 is a new target for Egr-1.
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INTRODUCTION
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Control of mRNA translation plays a pivotal role in regulating gene expression under a variety of conditions in mammalian cells (1). The predominant step in translational regulation is the initiation phase, which consists of the recruitment of the 40 S ribosomal subunit to the mRNA (2). This occurs through recognition of the 5' cap structure (m7GpppX, where X is any nucleotide) by the cap-binding protein complex eIF4F (eukaryotic initiation factor 4F) which, in higher eukaryotes, consists of three subunits: eIF4A, eIF4E, and eIF4G. The initiation process is largely regulated through changes in the phosphorylation state of eIFs and other components involved in this process (3, 4). eIF4E activity is modulated by phosphorylation in response to mitogens, polypeptide hormones, tumor promoters, and growth factors in a mitogen-activated protein kinase (MAPK)1-MAPK signal-integrating kinase (MNK) pathway-dependent manner (5). In addition to the regulation of its phosphorylation, the activity of eIF4E is tightly controlled through reversible interaction with a family of inhibitory proteins termed 4E-BP (eIF4E-binding proteins). Of the three known proteins (4E-BP1, 4E-BP2, and 4E-BP3), 4E-BP1, also named PHAS-1, is the best characterized. 4E-BP1 specifically inhibits cap-dependent translation by competing with eIF4G for binding to the cap-binding factor eIF4E and consequently preventing the formation of the eIF4F complex (6). The affinity of the 4E-BPs to eIF4E depends on their phosphorylation state. Hypophosphorylated 4E-BPs interact with high affinity with eIF4E, whereas hyperphosphorylation of 4E-BPs, elicited by stimulation of cells with hormones, cytokines, or growth factors, results in an abrogation of eIF4E-binding activity. Activation of phosphatidylinositol 3-kinase or a downstream phosphatidylinositol 3-kinase effector, Akt/protein kinase B, and FRAP/mTOR (FKBP and rapamycin-associated protein), leads to 4E-BP1 hyperphosphorylation (7, 8, 9). Six phosphorylation sites have been identified in 4E-BP1: Thr37, Thr46, Ser65, Thr70, Ser83, and Ser112 (numbering according to human 4E-BP1). FRAP/mTOR phosphorylates 4E-BP1 on Thr37, Thr46, and Thr70 (9, 12), activation of p38-MSK1 (mitogen and stress kinase 1) pathway by UV light leads to phosphorylation on Thr37 and Ser65 (10), and the activation of the ERK pathway induces phosphorylation on Ser65, Thr37, Thr46, and Thr70 (11). A hierarchical phosphorylation of 4E-BP1 has been proposed: first on the Thr37 and Thr46 and then on Thr70 and Ser65 (12), showing that multiple phosphorylation events (most likely via different kinases) are required to release 4E-BP1 from eIF-4E. Recently it has been shown that 4E-BP1 cleavage by caspase is a new step in the regulation of translation in response to insulin (13).
Signal transduction via MAPK plays a key role in a variety of cellular responses, including early embryonic development, cell death, growth factor-induced proliferation, and cell differentiation (14, 15, 16, 17, 18, 19, 20, 21). Three main pathways have been defined in mammalian cells: the classical ERKs, the c-Jun N-terminal kinases (JNK, also known as SAPK1), and the p38 (also termed SAPK2). ERK are stimulated by growth factors and cytokines, whereas JNK and p38 are generally activated by pro-inflammatory cytokines (i.e. IL-1
and tumor necrosis factor
) (22, 23, 24) and stresses such as osmotic shock, sodium arsenite, anisomycin, or UV irradiation (25, 26). More recently, it has been reported that a variety of hemopoietic growth factors, such as colony-stimulating factor-1 (CSF-1), granulocyte/macrophage colony-stimulating factor (GM-CSF), and IL-3, also activate p38 (27). The MAPK activate several transcription factors, such as CREB, ATF-2, Elk-1, Sap1a, and Sap2/Net/Elk3 members of the ternary complex factor (28, 29, 30, 31, 32) and stimulate transcription of a set of immediate early genes such as c-fos, fosB, c-jun, junB, junD, egr-1, egr-2, pip92, or nur77 (15, 33, 34, 35). Thereby they control the expression of various genes including interferon-
(36), tumor necrosis factor
, IL-1, IL-8 (37), IL-6 (24), inducible nitric-oxide synthase (38), E-selectin (39), VCAM-1 (40), CHOP (41), MEF2C (42), and dad-1, myc, or grb-2 (43).
A differential regulation of the translational machinery during human myeloid differentiation has been reported. Induction of HL-60 and U-987 cell differentiation by PMA or interferon-
into monocytic/macrophages results in a dephosphorylation and consequent activation of 4E-BP1. In contrast induction of HL-60 into the granulocytic differentiation by Me2SO decreases 4E-BP1 expression level, whereas it increases 4E-BP2 expression level (44, 45). Expression of 4E-BP2 is down-regulated during thymocyte maturation (46), but nothing is known about the mechanisms by which 4E-BP expression is modified. We derived from the pluripotent UT7 cell line a subpopulation, the UT7D1 cell line, which in the presence of the growth factor GM-CSF spontaneously expresses the mRNA coding for histidine decarboxylase (HDC). Stimulation of these cells during 24 h with PMA induces a basophil differentiation characterized by an induction of IL-4, IL-6, and IL-13 expression, the presentation of the basophil Bsp-1 antigen, and an increase in histamine production (47). Whereas the induction of IL-4, IL-6, and IL-13 expression is regulated at the transcriptional level, the HDC expression seems to be regulated at a post-transcriptional level (47, 48). Thus we examined the activity of the translational initiation machinery in PMA-treated UT7D1 cells. Such a treatment induced a decrease in 4E-BP1 expression, but 4E-BP2 or eIF4E expressions did not change. We also have shown that 4E-BP1 expression is negatively regulated at the transcriptional level by the ERK and p38 via the induction of Egr-1 expression. Ultimately we have provided new insights into the regulation of HDC expression at the translational level.
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EXPERIMENTAL PROCEDURES
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Cell Culture and TreatmentsHuman UT7D1, 11OC1, HEL, K562, and HMC1 cells were maintained in RPMI 1640 (Invitrogen) supplemented with 1% 100x L-glutamine, 10% fetal calf serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin. 2 ng/ml of recombinant GM-CSF (R&D) was added to the UT7D1 cell line. Passages were performed every 3 or 4 days at a concentration of 2 x 105 cells/ml. 10 µg/ml cycloheximide (Sigma), 10 ng/ml rapamycin, 25 µM LY294002, 3 µM bisindolylmaleimide, 10 µM U0126, 20 µM PD98059, or 10 µM SB203580 (Calbiochem) were added to the medium 30 min before PMA (Sigma) treatment (20 ng/ml) for 30 min to 48 h as indicated.
Northern Blot AnalysisTotal RNA were extracted by a modified method of Chomczynski and Sacchi (49), using TRIzol Reagent (Invitrogen) according to the manufacturer's instruction. 20 µg of total RNA were separated on agarose formaldehyde gels and blotted onto nylon membranes (HybondTM-N+; Amersham Biosciences). The membrane filters were hybridized with [32P]dATP random primed cDNA probes prepared from a partial 4e-bp1 cDNA fragment amplified per PCR (sense primer, 5'-GGACTACAGCACGACCCCCG-3', and antisense primer, 5'-TGACTCTTCACCGCCCGCCC-3'), a partial 4e-bp2 cDNA fragment amplified per PCR (sense, primer 5'-GGGGGACGGTCTTCTCCACC-3', and antisense primer, 5'-GCATGTTTCCTGTCGTGATTGTTC-3'), a partial hdc cDNA fragment amplified per PCR (sense primer, 5'-GCCCGATGCTGATGAGTCCT-3', and antisense primer, 5'-CACCGTCTTCTTCTTAGTCT-3') or a partial cDNA fragment of egr-1 (GenBankTM accession number AA507023
[GenBank]
). Hybridized filters were washed under high stringency conditions (0.1x SSC, 70 °C), analyzed by autoradiography, and quantified using QuantityOne. Equal loading of RNA was confirmed by stripping and reprobing the blots with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe or staining of the ribosomal RNA with ethidium bromide.
Protein Kinase Assays1 x 106 UT7D1 cells were washed three times with ice-cold phosphate-buffered saline and harvested in lysis buffer (20 mM Tris acetate, pH 7.0, 0.1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 10 mM
-glycerophosphate, 50 mM NaF, 5 mM pyrophosphate, 1% Triton X-100, 1 mM benzamidine, 2 µg/ml leupeptin, 0.1% (v/v)
-mercaptoethanol, 0.27 M sucrose, 0.2 mM phenylmethylsulfonyl fluoride). The lysates were clarified by centrifugation (5 min, 10,000 x g, 4 °C). Kinase assay was performed at 30 °C for 20 min using 20 µl of lysate in 25 µl of kinase buffer (50 mM sodium
-glycerophosphate pH 7.4, 10 mM magnesium acetate, 0.1 mM EDTA, 50 µM ATP) containing 1.5 µCi of [
-32P]ATP and 3 µg of Hsp25 (nicely provided by Professor Matthias Gaestel) as substrate. The proteins were resolved by electrophoresis in 7.520% gradient SDS-polyacrylamide gel. The gels were dried, and kinase activity was analyzed by autoradiography.
Western BlotThe cell lysates were electrophoresed on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (HybondTM; Amersham Biosciences) in 25 mM Tris, 40 mM
-aminocaproic acid, and 20% methanol at 1 mA/cm2 for 90 min. The membranes were blocked for 2 h at 4 °C and incubated overnight at 4 °C with 1:500 4E-BP1 rabbit antibodies (Santa Cruz Biotechnologies), 1:1000 phospho-ERK/MAPK (p44/p42), phospho-JNK/SAPK1 (p54/p46), phospho-p38/SAPK2, ERK/MAPK, JNK/SAPK1, or p38/SAPK2 antibodies (New England Biolabs). Immunoreactive proteins were detected according to the enhanced chemiluminescent protocol (ECL Plus; Amersham Biosciences) using 1:5000 horseradish peroxidase-linked anti-rabbit or anti-mouse secondary antibody. The blots were exposed to film (Hyper-film; Amersham Biosciences) for 110 min. The signals were quantified using QuantityOne.
Cloning of 4e-bp1 PromoterA sequence of 1020 bp upstream of the ATG of 4e-bp1 gene was cloned in pCDRIVE vector (Qiagen) by PCR using the 5'-CGGGGGTACCCCGCCTCAAACCCCTGGGCTC-3' sense primer, the 5'-CCGCTCGAGCGGGTCTCCTGTGCGCTGCAC-3' antisense primer, and the BAC clone RPCI-11701H6 (CHORI-BACPAC Resources) as matrix. This sequence was fused to the luciferase gene in the pGL2-Basic vector (Promega) using XhoI and KpnI restriction sites. Entire sequence was verified by sequencing the insert with pGL1 and pGL2 primers (Promega).
Transfection and Reporter AssaysHeLa cells were plated 24 h before transfection in 24-well plates so that they are 6070% confluent the day of transfection. Transfection was realized with LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's instructions. pIRES-EGFP was always cotransfected to estimate the level of transfection (fluorescence measure from lysates with the Victor2) and normalize the luciferase activity. This control level varied from 0.73 to 1.22 times. Luciferase reporter reagent (Promega) was used to measure the luciferase activity according to the manufacturer's instructions in a LB96V luminometer (Berthold Technologies).
Histamine and HDC AssaysHistamine concentrations in cell lysates were routinely determined by an automated continuous flow fluorometric technique (lower limit of sensitivity, 0.5 ng/ml), as previously described (49). Its specificity has been confirmed by radioimmunoassay (Immunotech, Marseille, France). HDC activity was measured by a radiochromatographic technique, as described before (49). Briefly, the cell pellets were resuspended in 50 mM ice-cold phosphate buffer and gently sonicated. Aliquots of the HDC-containing suspensions were then incubated in 50 mM phosphate buffer at a final concentration of 10 µM pyridoxal 5'-phosphate and 0.1 µM L-[3H]histidine (specific activity, 50 Ci/mmol). The incubations were stopped by the addition of perchloric acid (final concentration, 0.4 N) containing 0.3 M unlabeled histidine to minimize possible nonspecific decarboxylation of the remaining L-[3H]histidine. To assess the specificity of the assay, each reaction was performed with or without 105 M
-fluoromethylhistidine, the specific inhibitor of HDC. After centrifugation, the synthesized [3H]histamine was separated from the [3H]histidine by ion exchange chromatography on Amberlite CG-50 columns.
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RESULTS
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4E-BP1 Expression Decreases in Response to PMAPhosphorylation of 4E-BP1 occurs in response to mitogens or growth factors by activation of the phosphatidylinositol 3-kinase and/or MAPK signal transduction pathways and is rapamycin-sensitive (10, 11, 50). To assess to what extend PMA modifies 4E-BP1 phosphorylation and/or expression, we analyzed by Western blot the expression and phosphorylation of 4E-BP1 in UT7D1 cells treated with or without PMA for 24 h. It should be mentioned that in all experiments UT7D1 cells were placed in fresh culture medium and GM-CSF-starved for 120 min before the addition of the inhibitors. After 30 min, we added fresh GM-CSF alone or GM-CSF and PMA. 4E-BP1 migrates in 12% SDS-polyacrylamide gels as four bands (
,
,
, and
; Fig. 1A). The
band is the less phosphorylated form, and the
band is the highly phosphorylated isoform. According to our personal observations and in line with the literature, the
band represents the 4E-BP1 isoform phosphorylated on Thr37 and Thr46; the
isoform is phosphorylated on Thr37, Thr46, and Thr70; and the
isoform is phosphorylated on these three threonines and on Ser65 (11, 12). When the cells were cultivated with GM-CSF, the four phosphorylated forms of 4E-BP1 were present. In contrast, when the cells were treated with PMA, only the
,
, and
forms of 4E-BP1 were barely detected. These results not only reflect a change in the phosphorylation of 4E-BP1 but also a decrease in 4E-BP1 protein amount (Fig. 1A). In the presence of rapamycin, an inhibitor of FRAP/mTOR, or LY294002, an inhibitor of phosphatidylinositol 3-kinase, we observed an accumulation of the
less phosphorylated form of 4E-BP1 in the absence or in the presence of PMA (Fig. 1A). These results are in line with the data from literature that show that the Thr37, Thr46, and Thr70 are phosphorylated by the FRAP/mTOR. p38 and ERK can phosphorylate several sites of 4E-BP1, notably the Ser65 (10, 11). This regulation did not occur in our system because treatment of the cells with SB203580 or U0126 did not have any effect on the phosphorylation pattern of 4EBP1.2 Nevertheless we could not rule out changes that could have occurred at shorter times of treatment. Northern blot analysis showed that neither rapamycin nor LY294002 affected the transcript level of 4E-BP1, whereas the PMA application resulted in a loss of 4E-BP1 transcript (Fig. 1B). We also looked at the effect of PMA on 4E-BP1 in three different cell lines: 11OC1, HEL, and K562. Twenty-four hours of PMA treatment led to an accumulation of the less phosphorylated isoforms of 4E-BP1 in 11OC1 and K562 cells, whereas in the HEL cell line only the most highly phosphorylated form of 4E-BP1 was detectable (Fig. 1C). In all three cell lines PMA decreased 4E-BP1 expression (Fig. 1C).

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FIG. 1. Analysis of 4E-BP1 expression and phosphorylation in UT7D1 cells cultivated in the presence of PMA. GM-CSF-starved (2 h) UT7D1 cells were cultivated in the presence or absence of PMA for 24 h. Where shown, the cells were preincubated (30 min) with rapamycin (Rapa; 20 ng/ml) or LY294002 (LY; 25 µM) prior to the addition of GM-CSF (2 ng/ml) or GM-CSF and PMA (10 nM). Samples of cell lysates were subjected to SDS-PAGE followed by Western blotting using either anti-4E-BP1 or anti-actin antibodies (loading control) as indicated (A). 20 µg of total RNA was analyzed by Northern blotting using 4e-bp1 or 18 S cDNA probes (loading control) as indicated (B). 11OC1, HEL, or K562 cells were also cultivated 24h in the presence or absence of PMA (10 nM). Samples of cell lysates were analyzed as described for A (C).
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4e-bp1 Transcript Level Decreases, Whereas the Expression of 4e-bp2 Is Not Sensitive to PMATime course analysis showed that 4E-BP1 expression transiently decreased after 4h of GM-CSF (47 ± 17%; Fig. 2, A and C) and that the
phosphorylated form of 4E-BP1 disappeared after 4 h of GM-CSF treatment. The
less phosphorylated form was mainly detected after 24 h of GM-CSF treatment (Fig. 2A). The expression of 4E-BP1 protein decreased after 4 h of PMA and was undetectable after 24 h (32.5 ± 13% and 4 ± 1%; Fig. 2, A and C). Northern blot analysis showed that the transcript level was strictly correlated with the protein expression level, that PMA negatively regulated 4E-BP1 expression at the transcript level (Fig. 2, B and C), and that this effect lasted 48 h (Fig. 2, D and E). Because 4E-BP2 expression was also known to vary during cell differentiation, we analyzed its expression by Northern blot. The transcript of 4e-bp2 was only present at 24 h of culture in the presence or absence of PMA (Fig. 2, D and E). Taken together, these results showed that the two repressors of translation have different ways of regulating.

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FIG. 2. Kinetics of 4E-BP1 and 4e-bp2 expression. GM-CSF-starved (2 h) UT7D1 cells were cultivated in the presence or absence of PMA for different times as indicated. Samples of cell lysates were subjected to SDS-PAGE followed by Western blotting using either anti-4E-BP1 or anti-actin antibodies (loading control) as indicated (A). The means of quantification of two independent Western blotting analysis ± S.D. and a representative Northern blotting analysis are represented in C. The results are expressed as percentages from the quantification of the bands at time 0, which is considered to be 100%. 20 µg of total RNA was analyzed by Northern blotting using 4e-bp1, 4e-bp2, or GAPDH DNA probes (loading control) as indicated (B and D). The means of quantification of two independent Northern blotting analysis ± S.D. is represented in E. The results are expressed as percentages from the quantification of the bands from GM-CSF, 1 day, which is considered to be 100%.
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ERK Is Activated by PMA and p38 Is Activated by GM-CSF in UT7D1 Cell LinePMA and GM-CSF are known to activate ERK and p38 (27, 51, 52, 53), and the balance between these two pathways may be critical in determining cell function (54). To determine the activity of ERK and p38 in our system, we analyzed their phosphorylation by Western blot using phosphospecific antibodies. As previously mentioned, UT7D1 cells were placed in fresh culture medium and GM-CSF-starved for 120 min before addition of the inhibitors. After 30 min, we added fresh GM-CSF alone or GM-CSF and PMA. Activated ERK2/p42 was detected after 30 min of PMA treatment (Fig. 3A). This activation was maximal at 2 h of treatment and decreased thereafter to become undetectable after 48 h. It should be noted that although ERK1/p44 and ERK2/p42 were found at similar levels in UT7D1 cells, the signal was always weaker for phospho-ERK1/p44 than for phospho-ERK2/p42, and the former could not be detected in all experiments (Figs. 3A and 5A). The SB203580 inhibitor of p38/SAPK2
and
did not affect ERK phosphorylation (Fig. 3A). The PD98059, an inhibitor of MEKs, almost completely prevented the effect of PMA on ERK phosphorylation (Fig. 3A). Phosphorylated p38 was detected after 2 h of GM-CSF treatment. We tested the activation of the p38 pathway in UT7D1 lysates by performing kinase assays using Hsp25 (56). p38 activation was maximal between 4 and 24 h and decreased thereafter (Fig. 3, B and C). The total amount of p38 did not vary during our experiments. PMA did not affect p38 phosphorylation, but the application of PD98059 increased its phosphorylation at 24 h and extended it at 48 h (Fig. 3, B and C). The SB203580 completely abolished p38 pathway activation (Fig. 3C) without affecting its phosphorylation (Fig. 3B).

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FIG. 3. Analysis of MAPK phosphorylation. GM-CSF-starved (2 h) UT7D1 cells were cultivated in the presence or absence of PMA for different times as indicated. Where shown, the cells were preincubated (30 min) with PD98059 (20 µM) or SB203580 (10 µM) prior to the addition of GM-CSF (2 ng/ml) or GM-CSF and PMA (10 nM). Samples of cell lysates were subjected to SDS-PAGE followed by Western blotting using either anti-phospho-p44/p42, anti-phospho-p38, or anti-p44/p42 and anti-p38 antibodies (loading control) as indicated (A and B). MAPK-activated protein kinase 2 kinase assays were performed (as described under "Experimental Procedures") with samples of cell lysates using Hsp-25 as substrate (C).
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FIG. 5. Indirect activation of ERK and p38 by OA mimics the effect of PMA, whereas hyperosmolarity activation of JNK does not. GM-CSF-starved (2 h) UT7D1 cells were cultivated in the presence or absence of fetal calf serum (10%), NaCl (0.25 M), OA (40 nM), or PMA for 24 h. Samples of cell lysates were subjected to SDS-PAGE followed by Western blotting using either anti-phospho-p44/-p42, anti-phospho-p54/p46, anti-phospho-p38, or anti-actin antibodies (loading control) as indicated (A). 20 µg of total RNA was analyzed by Northern blotting using 4e-bp1 or GAPDH cDNA probes (loading control) as indicated (B).
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Inhibition of ERK Abolished the Effect of PMA on 4E-BP1 Expression, and Inhibition of p38/SAPK2
and
Increased 4E-BP1 ExpressionTo analyze the role of ERK or p38 in the regulation of 4E-BP1 expression, we treated UT7D1 (Fig. 4A) or HMC1 (Fig. 4B) cells with different specific inhibitors prior to the addition of GM-CSF or PMA. Northern blot analysis showed that the application of PMA for 24 h entirely abolished 4E-BP1 transcript expression (Fig. 4A). This effect was blocked by the U0126 inhibitor of MEK (71.5% ± 19) or by the bisindolylmaleimide 1 inhibitor of protein kinase C (79% ± 33), a well known kinase that transduces signal from PMA to ERK cascade (Fig. 4A). The SB203580 inhibitor of p38/SAPKs2
and
had no effect on 4E-BP1 expression in the presence of PMA, but in the presence of GM-CSF alone, the pretreatment with SB203580 increased the transcript level of 4E-BP1 (165 ± 33%; Fig. 4A). On another cell line, the HMC1 cell line that grows independently of GM-CSF, the SB203580 had no effect at all, whereas the bisindolylmaleimide 1 and the PD98059 still reversed the PMA effect on 4E-BP1 expression (110.5 ± 18% and 59 ± 18%, respectively; Fig. 4B). This showed that 4E-BP1 expression is inhibited by both PMA-activated ERK and GM-CSF-activated p38.

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FIG. 4. PMA-activated ERK and GM-CSF-activated p38 inhibit 4E-BP1 expression. GM-CSF-starved (2 h) UT7D1 cells (A) or HMC1 (B) cells were cultivated in the presence or absence of PMA for 18 h. Where shown, the cells were preincubated (30 min) with bisindolylmaleimide (Bis; 3 µM), PD98059 (20 µM), U0126 (10 µM), or SB203580 (10 µM) prior to the addition of GM-CSF (2 ng/ml) or GM-CSF and PMA (10 nM). 20 µg of total RNA was analyzed by Northern blotting using 4e-bp1 cDNA probe as indicated. Equal loading of RNA was controlled by ethidium bromide staining of the ribosomal RNA (28 and 18 S). The columns represent the means of quantification of two to four independent Northern blotting analyses of 4e-bp1 mRNA expression ± S.D. The results are expressed as percentages of the quantification of the bands from GM-CSF or 0, which are considered as 100% for A and B, respectively. The columns labeled with asterisks show significant differences (p < 0.01) compared with the same treatment without inhibitor.
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Hyperosmolarity-induced Activation of JNK/SAPK1 Does Not Affect 4E-BP1 ExpressionWe next wanted to determine the effect of different stimuli able to activate the MAPK pathways on 4e-bp1 expression. Using Western blot analysis with phosphospecific antibodies, we analyzed the phosphorylation and thereby the activation level of the three main MAPK pathways after 8 h stimuli. Whereas the NaCl barely activated the ERK, it activated the p38 and strongly activated the JNK/SAPK1. The okadaic acid (OA) inhibitor of serine/threonine phosphatases indirectly activated the ERK and strongly activated the p38 but not the JNK (Fig. 5A). As shown by Northern blot analysis, the 4E-BP1 mRNA expression was not affected by 10% fetal calf serum or NaCl but decreased when UT7D1 cells were treated with OA to the same level as when cells were treated with PMA (Fig. 5B).
Cycloheximide Treatment Abolished PMA Effect on 4E-BP1 ExpressionThe MAPKs are known to regulate gene expression directly by phosphorylation of transcription factors or indirectly by controlling immediate early genes, which are themselves transcription factors. To distinguish which of these two kinds of transcription factors, ERK and p38, inhibit 4e-bp1 transcription, we blocked the protein synthesis with cycloheximide (CHX). When cells were cultivated in GM-CSF, CHX had no effect on the 4E-BP1 transcript level, neither at 4 h nor at 8 h (Fig. 6). In contrast, the PMA-induced inhibition of 4E-BP1 mRNA expression was completely reversed by the presence of CHX, demonstrating the necessity of protein neosynthesis for 4e-bp1 repression. We addressed the question of 4e-bp1 mRNA stability by performing a time course analysis of 4e-bp1 mRNA expression in the presence of actinomycin D (10 µg/ml from 0 to 270 min). The decrease of its expression was the same in the presence or the absence of PMA, showing that GM-CSF or PMA did not stabilize or destabilize 4e-bp1 mRNA.2

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FIG. 6. Cycloheximide treatment abolishes the PMA effect on 4E-BP1 expression, and egr-1 expression is induced by PMA. GM-CSF-starved (2 h) UT7D1 cells were cultivated in the presence or absence of PMA for different times as indicated. Where shown, the cells were preincubated (30 min) with cycloheximide (10 µg/ml). 20 µg of total RNA was analyzed by Northern blotting using 4e-bp1, egr-1, or GAPDH cDNA probes (loading control) as indicated.
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Egr-1 Expression Is Induced by PMA or GM-CSF in an ERK- or p38-dependent Manner, RespectivelyBecause Egr-1 expression is known to be under the control of MAPK activities under different conditions (55, 56), we analyzed its expression in UT7D1 cells. Northern blot analysis of egr-1 expression showed an induction of its transcript by PMA (Fig. 6). CHX pretreatment increased this effect (Fig. 6), suggesting that in UT7D1 cells Egr-1 exerts a negative feedback on its expression as was demonstrated (57). Western and Northern blot analysis showed that GM-CSF induced the expressions of Egr-1 protein and transcript, which were maximal at 1 h and decreased thereafter to reach the control level at 24 h (Fig. 7, A and B). The addition of PMA prolonged the Egr-1 induction until 24 h. Pretreatment of the UT7DI cells with U0126 or SB203580 abolished egr-1 induction by PMA or GM-CSF, respectively (Fig. 7C). This demonstrated that PMA-activated ERK or GM-CSF activated p38 induce Egr-1 expression.

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FIG. 7. Egr-1 expression is induced by GM-CSF-activated p38 or PMA-activated ERK. GM-CSF-starved (2 h) UT7D1 cells were cultivated in the presence or absence of PMA for different times as indicated. Samples of cell lysates were subjected to SDS-PAGE followed by Western blotting using anti-egr-1 or anti-actin antibodies (loading control) as indicated (A). 20 µg of total RNA was analyzed by Northern blotting using egr-1 or GAPDH cDNA probes (loading control) as indicated (B). In C cells are GM-CSF starved for 2 h and pretreated with U0126 (10 µM) or SB203580 (10 µM) before 1 h of treatment with GM-CSF alone or GM-CSF and PMA. 20 µg of total RNA was analyzed by Northern blotting as described in B.
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4e-bp1 Promoter ActivityTo examine whether Egr-1 is involved in ERK and/or p38 inhibition of 4e-bp1 transcription, we have cloned 1020 bp upstream of the ATG of 4e-bp1 human gene sequence as described under "Experimental Procedures." This sequence contains some potential Egr response element, Elk1, Sp1, AP4, or NF
B regulatory elements (Fig. 8A). We have fused them to the luciferase gene to measure whether this potential promoter responds to ERK and p38 activities and whether this activity depends on Egr-1. HeLa cells were transfected, and luciferase assays were performed as described under "Experimental Procedures." 4e-bp1 promoter activity was almost entirely abolished when the cells were stimulated with PMA, and the U0126 inhibitor reversed this effect, whereas the SB203580 did not (Fig. 8B). These inhibitors applied together did not have more effect than the U0126 alone. The OA diminished 4e-bp1 promoter activity too, and this effect was partially reversed by U0126 and significantly by SB203580. The two MAPK inhibitors had an additional effect (Fig. 8B). Coexpression of the ZnEgr dominant negative mutant of Egr did not have any effect on 4e-bp1 promoter activity in our control condition. In the presence of PMA or OA it not only reversed the PMA- or OA-induced inhibition of 4e-bp1 promoter but also increased it approximately 2-fold (Fig. 8B). Coexpression of the ZnEgr did not change the pGL2B nor the pGL2C activities, showing that the effect observed is specific to the 4e-bp1 promoter.

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FIG. 8. Egr-1 is responsible for PMA- or OA-induced inhibition of 4e-bp1 promoter activity. Egr response element (shaded), Elk1 (underlined), Sp1(boxed), AP4 (underlined), or NF B (underlined) potential regulatory elements were found in the first 1020 bp of the sequence of the 5' upstream region of the human 4e-bp1 gene (A). This promoter fused to the luciferase gene (bp1-p) was transfected (as described under "Experimental Procedures") in HeLa cells and after 24 h stimulated with OA or PMA for additional 12 h. Where indicated, the cells were preincubated with U0126 (U;10 µM) or SB203580 (SB;10 µM) inhibitors. The dominant negative mutant ZnEgr was cotransfected as indicated, and the basal luciferase expression was estimated by transfection of the pGL2B vector. Luciferase activity was measured as described under "Experimental Procedures" and normalized by measuring the fluorescence of EGFP expressed by the pIRES-EGFP vector, which was always cotransfected. The data are the means ± S.D. of three independent experiments performed in duplicate (B). The columns labeled with asterisks show significant differences (p < 0.01) compared with the same treatment without the different inhibitors or without the ZnEgr.
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U0126 and SB203580 Inhibit Histamine Production and HDC Activity without Modifying hdc mRNA ExpressionStimulation of UT7D1 cells for 24 h with PMA induces a basophil differentiation characterized by an induction of IL-4, IL-6, and IL-13 expression, the presentation of the basophil Bsp-1 antigen, and an increase in histamine production (47). Whereas the induction of IL-4, IL-6, and IL-13 expression is regulated at the transcriptional level, the HDC expression seems to be regulated at a post-transcriptional level (47, 48). To determine whether the post-transcriptional regulation of HDC could be translational, we examined histamine production and HDC activity and expression in UT7D1 cells cultivated with GM-CSF or GM-CSF and PMA in the presence or absence of U0126 and SB203580. As we have previously shown (47), PMA increased the intracellular histamine concentration about 10 times at 24 and 48 h (Fig. 9A). This production resulted from de novo synthesis and was the result of an increased HDC activity (47). As expected the HDC activity was increased
6 and 16 times at 24 and 48 h, respectively (Fig. 9A). Whereas U0126 had no effect, the presence of SB203580 significantly decreased both the histamine production and the HDC activity when cells were cultivated in GM-CSF (Fig. 9A). When UT7D1 cells were stimulated with PMA, the histamine production and the HDC activity were significantly inhibited in the presence of U0126 (Fig. 9A). This effect seemed to be amplified when U0126 and SB203580 inhibitors were both present in the culture medium (Fig. 9A). Northern blot analysis showed that the hdc mRNAs expression did not change in the presence of PMA nor in the presence of U0126 or SB203580 inhibitors (Fig. 9B). Taken together these data suggest that the post-transcriptional regulation of HDC could be done at the translational level, depending on 4E-BP1.

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FIG. 9. Histamine production and HDC activity are inhibited by U0126 and SB203580 inhibitors. GM-CSF-starved (2 h) UT7D1 cells were cultivated in the presence or absence of PMA for 24 h (open bars and B) or 48 h (black bars). Where shown, the cells were preincubated (30 min) with U0126 (U) or SB203580 (SB) prior to the addition of GM-CSF (2 ng/ml; left panel) or GM-CSF and PMA (10 nM; right panel). Intracellular histamine concentration and HDC activity were measured as described under "Experimental Procedures." The data are the means ± S.D. of two to five independent experiments. The columns labeled with asterisks show significant differences (p < 0.01) compared with the same treatment without inhibitor (A). 20 µg of total RNA was analyzed by Northern blotting using hdc or GAPDH cDNA probes (loading control) as indicated (B).
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DISCUSSION
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In UT7D1 cells, PMA quickly and transiently activates the ERK through a protein kinase C-dependent pathway but does not affect p38, which is activated by the GM-CSF. Activation of one of these two pathways represses 4E-BP1 transcription but at different levels. In contrast the JNK pathway, activated by hyperosmolarity, does not affect 4E-BP1 expression. This work is the first evidence for a role of the ERK and p38 on translational machinery member expression. As determined by our CHX experiments, inhibition of 4E-BP1 expression by the MAPK depends on protein neosynthesis. ERK and p38 have common targets that could play a role in this regulation: the immediate early genes egr-1, c-jun, and c-fos, for example. Egr-1 can be the activator of transcription as well as the inhibitor of transcription and, in UT7D1 cells, we have shown that Egr-1 expression is strictly inversely correlated with 4E-BP1 expression. On the 1020-bp sequence of 4e-bp1 promoter some putative Egr response element, AP4, SRF, SP1, Elk-1, or NF
B-binding sites can be identified. Performing reporter assays in HeLa cells with the ZnEgr dominant negative mutant, we have shown that 4e-bp1 is a new gene, the expression of which is inhibited by Egr transcription factors.
As far as we know, the only repressor effect of Egr-1 was described on egr-1 gene itself to realize a negative feedback (57), and how Egr-1 is inhibitor rather than activator of transcription is not fully understood. It was first shown that ZnEgr inhibits Egr-1-dependent transcription by binding to the Egr response element of a promoter, but a recent work has demonstrated that ZnEgr disrupts the formation of a Egr-1/c-Jun complex (58). In our system c-Jun expression is induced after 4 h of PMA treatment of UT7D1.2 We cannot exclude the possibility that a Egr-1/c-Jun complex is formed on 4e-bp1 promoter to inhibit its activity, but at that time Egr-1/c-Jun has been shown to activate the transcription of the MAO-B gene (59). A detailed study of 4e-bp1 promoter regulation must be done to identify the mechanism by which retinoic acid or Me2SO represses 4E-BP1 expression. Nevertheless Egr-1 has a critical role in a variety of processes that include proliferation, apoptosis, and cell differentiation (and thereby oncogenesis) neuronal plasticity and ischemia. The new link that our work makes between Egr-1 and 4E-BP1 allows us to reconsider the role that 4E-BP1 could play in this process by being regulated through Egr-1. The induction of Egr-1 expression by PMA has been implicated in the megakaryocytic differentiation process of K562 cell line (60). According to our data, we can suppose that 4E-BP1 could be responsible for one or more of the characteristic changes that appear, like variations of cell morphology, adhesive properties, endomitosis, and expression of markers associated with megakaryocytes.
This work is the first evidence for a role of the ERK and p38 on translational machinery member expression, but a lot of works have been done demonstrating the role of the MAPK in the regulation of phosphorylation of eukaryotic initiation factors. MAPK regulate the eIF4E phosphorylation state through MNK1 and MNK2, which can integrate signals emanating from both types of MAPK pathway in response to mitogens, polypeptide hormones, tumor promoters, and growth factors (61, 62, 63, 64). This phosphorylation was first correlated with an increase rate in protein synthesis, and on the other hand, dephosphorylation coincides with a reduction of protein synthesis at metaphase, upon heat shock, and during adenovirus infection. However, a correlation between eIF4E phosphorylation and the overall translation rate is not observed in every situation (5), and the effects of phosphorylation on eIF4E are not completely understood. Whereas Minich et al. (66) have described an increased affinity of phosphorylated eIF4E for the cap, Scheper et al. (65) have shown that phosphorylation of eIF4E on Ser209 diminishes its ability to bind capped mRNA. A recent work of Knauf et al. (68) demonstrates that the phosphorylation of eIF4E by MNK1 and MNK2 inhibits the cap-dependent translation. Two recent studies have shown that ERK and p38, through p90/RSK1, can influence the translational machinery at another level. The eukaryotic elongation factor 2 kinase phosphorylates and inactivates eEF2. Insulin induces dephosphorylation of eEF2 and inactivation of eukaryotic elongation factor 2 kinase, and these effects are blocked by rapamycin. In contrast, regulation of eEF2 by stimuli that activate ERK or p38 is insensitive to rapamycin but blocked by inhibitors of MEKs or p38, respectively, consistent with the involvement of p90/RSK (69, 70). Taken together, these results put on to the fore the translational level of MAPK regulation of protein expression. In UT7D1 cell line, neither the 4E-BP2 nor the eIF4E expressions vary along with PMA stimulation.2 Nevertheless, we have measured an increase in protein neosynthesis when cells are cultivated with PMA, and the concentration of protein/cell is two times higher in PMA-stimulated cells than in GM-CSF-cultivated cells.2 Of course PMA induces a great increase in transcription level, and we cannot assign this effect to a 4E-BP1 decrease of expression. Nevertheless we have demonstrated that concerning the HDC, its PMA-induced increased activity does not depend on transcriptional regulation but seems to be translational. We have now to determine other transcripts, the translation of which depends on 4E-BP1. A recent work from Grolleau et al. (67) shows an interesting application of microarrays and proteomics that could be used to identify such transcripts.
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FOOTNOTES
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* This work was supported by an Association pour la Recherche sur le Cancer Fellowship (to M. R.-D.) and by Association pour la Recherche sur le Cancer Grants 5477 and 4422. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
|| To whom correspondence should be addressed: 161 rue de Sevres, 75743 Paris, Cedex 15, France. E-mail: dy{at}necker.fr.
1 The abbreviations used are: MAPK, mitogen-activated protein kinase; MNK, MAPK signal-integrating kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; IL, interleukin; CSF, colony-stimulating factor; GM, granulocyte/macrophage; PMA, phorbol 12-myristate 13-acetate; HDC, histidine decarboxylase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGFP, enhanced green fluorescent protein; MEK, MAPK/ERK kinase; OA, okadaic acid; CHX, cycloheximide. 
2 M. Rolli-Derkinderen, F. Machavoine, and M. Dy, unpublished results. 
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ACKNOWLEDGMENTS
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We are grateful to Prof. Matthias Gaestel for the generous gift of Hsp25 and for critical reading of the paper.
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