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J. Biol. Chem., Vol. 282, Issue 6, 3766-3777, February 9, 2007
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mRNA*
1
From the
Division of Critical Care, Department of Pediatrics and the ¶Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109 and the Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, August 2, 2006 , and in revised form, December 12, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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is a major cytokine produced by alveolar macrophages in response to pathogen-associated molecular patterns such as lipopolysaccharide. TNF- secretion is regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation occurs by modulation of TNF- mRNA stability via the binding of tristetraprolin (TTP) to the adenosine/uridine-rich elements found in the 3'-untranslated region of the TNF- transcript. Phosphorylation plays important roles in modulating mRNA stability, because activation of p38 MAPK by lipopolysaccharide stabilizes TNF- mRNA. We hypothesized that the protein phosphatase 2A (PP2A) regulates this signaling pathway. Our results show that inhibition of PP2A by okadaic acid or small interference RNA significantly enhanced the stability of TNF- mRNA. This result was associated with increased phosphorylation of p38 MAPK and MAPK-activated kinase 2 (MK-2). PP2A inhibition increased TTP phosphorylation and enhanced complex formation with chaperone protein 14-3-3. TTP physically interacted with PP2A in transfected mammalian cells. A functional consequence of TTP-14-3-3 complex formation appeared to be protection of TTP from dephosphorylation by inhibition of the binding of PP2A to phosphorylated TTP. Mutation of the MK-2 phosphorylation sites of TTP did not influence TNF- adenosine/uridine-rich element binding and did not alter the increased TNF- 3'-untranslated region-dependent luciferase activity induced by PP2A-small interference RNA silencing. Our data indicate that, although phosphorylation stabilizes TNF- mRNA, PP2A regulates the mRNA stability by modulating the phosphorylation state of members of the p38/MK-2/TTP pathway. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(TNF-)2 is a pro-inflammatory cytokine that influences a broad range of immunological processes, including autoimmune diseases, rheumatoid arthritis, septic shock, and acute respiratory distress syndrome. In these disease states, macrophages are widely recognized as cells that play a central role in the regulation of immune and inflammatory activities. In response to a variety of stimuli, for example LPS, macrophages generate pro-inflammatory cytokines such as TNF-, IL-1
, IL-6, IL-12, and oxidants. TNF- is an important alveolar macrophage secretory product, and depletion of alveolar macrophages reduces TNF- production, thereby neutralizing and attenuating LPS-induced lung inflammation (1). Therefore, understanding all potential regulatory pathways of TNF- expression is an important goal.
LPS-induced production of TNF- by monocyte/macrophages is regulated at both transcriptional and post-transcriptional levels. Transcriptional activation of TNF- occurs primarily through the binding of NF-
B to the TNF- promoter (2). Post-transcriptional regulation of TNF- occurs by modulation of its mRNA stability. This mRNA stability is dependent on a class II AU (adenosine/uridine)-rich element (ARE) found in the 3'-untranslated region (3'-UTR) of many cytokines, growth factors, and inflammatory mediators such as TNF-, IL-1, IL-8, granulocyte macrophage-colony stimulating factor, MIP-1 (macrophage inflammatory protein-1), vascular endothelial growth factor, and granulocyte-macrophage metalloproteinase-13 (3–6).
ARE-associated RNA-binding proteins are classified as stabilizing or destabilizing proteins depending on their effect on mRNA stability. Destabilizing proteins include tristetraprolin (TTP) (7–9), BRF1 (10), and AUF1 (11–13), which can directly bind to the AREs to promote mRNA decay. In contrast, stabilizing proteins such as HuR (14–16) inhibit mRNA decay. The mitogen-induced immediate-early gene product, TTP, is a prototypical member of zinc finger proteins that contain two CCCH zinc fingers and three tetraproline (PPPP) motifs (17, 18). Involvement of TTP in the post-transcriptional regulation of TNF- mRNA was suggested by the increased TNF- secretion from TTP-deficient mouse macrophages (19, 20). Lai et al. (8) demonstrated that integrity of both zinc fingers was required for TTP to directly bind to the TNF- ARE to promote mRNA decay. However, zinc fingers alone were not sufficient to mediate TNF- mRNA degradation, because truncation of either the carboxyl or amino ends of TTP caused a loss TTP function (21). Both ends of the TTP zinc fingers contain a cluster of phosphorylation sites (22), yet the effects of phosphorylation on the mRNA binding and destabilizing activity of TTP remain controversial (22–25).
Phosphorylation of TTP is regulated by p38 MAPK/MK-2 pathway (22, 26, 27). Activation by MK-2 results in a complex formation with chaperone protein, 14-3-3 (22, 28). This association subsequently effects nuclear export of TTP (28, 29) and prevents TTP-associated mRNA from being targeted to the degradative machinery (30). Regulation of dephosphorylation of TTP, and its upstream pathway, is incompletely understood. PP2A is a serine-threonine protein phosphatase involved in the regulation of signal transduction, cell growth, and apoptosis (31–34). The core heterodimer of PP2A consists of a 36-kDa catalytic subunit (PP2A-C) and a 65-kDa regulatory subunit (PR65 or PP2A-A). This heterodimer interacts with a variable third subunit (PP2A-B isoforms) that influences cellular substrate specificity (35). The role of PP2A in the cellular response to inflammatory stimuli that causes cytokine expression has been increasingly investigated. In monocytes, inhibition of PP2A resulted in increased JNK activity and subsequent enhanced IL-1 secretion (36). However, the role of PP2A in the regulation of LPS-induced TNF- production remains unknown.
In the present study, we tested the hypothesis that PP2A is involved in the post-transcriptional regulation of TNF-. Our data showed that inhibition of PP2A increased TNF- mRNA stability. This effect was accompanied by increased p38 and MK-2 activity as well as increased phosphorylation of TTP. Interestingly, binding of TTP to 14-3-3 appeared to protect phosphorylated TTP from access by PP2A, thereby preventing its dephosphorylation.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B inhibitor), and anisomycin were purchased from Calbiochem. U0126 (MEK1/2 inhibitor) was purchased from Cell Signaling. Anti-PP2A subunit C (clone 1D6) was purchased from Upstate. High concentration alkaline phosphatase (70 units/µl) was purchased from Stratagene. Anti-phospho-p38 (Thr-180/Tyr-182), immobilized phospho-p38 (Thr-180/Tyr-182) antibody, and anti-phospho-MK-2 (Thr-334) were purchased from Cell Signaling. Rabbit anti-phospho-TTP (Ser-178) antibody (C3769) was generated by immunization with synthetic peptide LRQSI(pS)FSGLPC coupled to a mixture of keyhole limpet hemocyanin and ovalbumin (BIOSOURCE/Quality Controlled Biochemicals). The serum was diluted 1/500 for Western blotting. Anti-14-3-3 (sc-629), anti-HuR (3A2, sc-5261), anti-PP1 (sc-6105), and anti-Myc (9E10)-agarose conjugate (sc-40AC) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). c-Myc antibody (9E10) was obtained from Clontech, anti-phospho(Ser-82)-Hsp27 was obtained from Cell Signaling, and recombinant PP2A was obtained from Upstate. Anti-TTP (CARP-3) antibody (37) was kindly provided by W. Rigby (Dartmouth Medical School, Lebanon, NH).
Constructs—A luciferase construct containing the mouse TNF- 3'-UTR (pMT2-luc-UTR), a T3 polymerase-driven in vitro transcription construct containing the 90-nt-long AU-rich element of mouse TNF- 3'-UTR (T3-Stem-ARE), -globin reporter gene containing the ARE of TNF- (pTet-7B-ARE (TNF)), and MycHis-tagged wild type (pcDNA3-TTP-wt-MycHis), and constructs containing serine-to-alanine mutations of TTP (pcDNA3-TTP-S52A-MycHis, pcDNA3-TTP-S178A-MycHis, and pcDNA3-TTP-AA-MycHis(S52A,S178A)) have been described previously (30). Altered TTP-m1,2 (38) in which both zinc fingers were disrupted was kindly provided by T. Keith Blackwell (Joslin Diabetes Center, Boston, MA). GST-14-3-3 fusion protein construct and Myc-tagged 14-3-3 (39) was kindly provided by R. W. Holz (University of Michigan, Ann Arbor). Full-length mouse TNF- cDNA was amplified from the total RNA of mouse lung using one-step reverse transcription-PCR (Invitrogen) and inserted into the pcDNA3.1/V5-His TOPO TA vector (Invitrogen) to obtain pcDNA3.1-TNF-V5His. Control siRNA (D-001210-02) and the PP2A-siRNA (M-040657-00), PP1-siRNA (M-040960-00) targeting the catalytic subunit of mouse PP2A and PP1 were purchased from Dharmacon.
PP2A Phosphatase Assay—A non-radioactive, malachite green-based immunoprecipitation assay kit (Upstate%20Biotechnology">Upstate Biotechnology) was used to measure PP2A activity. MHS cells were either pretreated with OA or siRNA as described. Total cellular proteins were then extracted in Triton X-100 lysis buffer containing 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1% Triton X-100, and 0.5% Nonidet P-40 with no phosphatase inhibitors. Subunit C (catalytic) of PP2A was immunoprecipitated by anti-PP2A antibody. The precipitates were washed twice with lysis buffer and once with phosphatase assay buffer (50 mM Tris-HCl, pH 7.0, 0.1 mM CaCl2). The pellets were resuspended in assay buffer and incubated with 750 µM phosphopeptide for 15 min at 30 °C. Reactions (25 µl) were then transferred to a microtiter plate and incubated with 100 µl of malachite green reagent. Color was developed for 5 min, and changes in absorbance were measured at 650 nm in a Spectra-MAX 250 (Molecular Devices plate reader).
Northern Blot Analysis—Total RNA was extracted from subconfluent 6-well plate using TRIzol reagent (Invitrogen). A total of 10 µg of RNA was resolved by 1% agarose/6% formaldehyde phosphate-buffered gel electrophoresis, blotted onto Nytran membranes using 10 x SSC. Full-length 32P-labeled TNF- antisense RNA was synthesized in vitro using plasmid pcDNA3.1-TNF-V5His linearized with XhoI, T7 polymerase and Maxiscript reagents and purified with Megaclear columns (Ambion). The 32P-labeled -actin probe was synthesized by the same method using the linearized plasmid provided by the Maxiscript kit. Hybridization was conducted overnight at 65 °C in hybridization solution containing 50% formamide, 5x SSC, 50 mM Tris-HCl, pH 7.5, 0.1% sodium pyrophosphate, 1% SDS, 0.2% polyvinypyrolidone, 0.2% Ficoll, 5 mM EDTA, 0.2% bovine serum albumin, and 100 µg/ml yeast RNA. After washing twice at room temperature and twice at 65 °C with 2x SSC/1% SDS, the membranes were apposed to Kodak BioMax MR film and exposed at -80 °C for 3 h.
Luciferase Assay—MHS cells were transiently transfected with pMT2-luc-UTR using Lipofectamine 2000 (Invitrogen). Two days after transfection, cells were either pretreated with or without 1 µM OA for 1 h, washed once with cell culture medium, and then stimulated with 200 ng/ml LPS. For PP2A-siRNA silencing experiments, 0.3 x 106 cells plated in 12-well plates were first transfected with control siRNA or mouse PP2A-siRNA for 48 h and then cotransfected with pMT2-luc-UTR and siRNA for another 24 h. Cells harvested at different LPS treatment times were lysed in Reporter lysis buffer (Promega, Madison, WI), and luciferase activity was measured using a Berthold Autolumat Plus LB 953 system with the protocol of a 2-s measurement delay followed by a 10-s measurement read.
p38 MAPK and MAPKAPK2 Kinase Assay—4 x 106 MHS cells were lysed in 500 µl of lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM -glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). 500 µg of cell lysates was incubated with 20 µl of immobilized phospho-p38 antibody overnight at 4 °C. Beads were washed twice with lysis buffer and once with 1x kinase buffer and then incubated with 8 µg of recombinant ATF-2 (Cell Signaling) in 40 µl of kinase reaction buffer containing 25 mM Tris-HCl, pH 7.5, 5 mM -glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 200 µM ATP, and 8 µCi of [
-32P]ATP for 30 min at 30 °C. The reaction mixture was then boiled in an equal volume of 2x Laemmli buffer and subjected to 10% SDS-PAGE. Gels were dried and exposed at -80 °C for 5 h. MK-2 kinase assay was performed using a non-radioactive immunoprecipitation kinase assay kit from Upstate. Briefly, 1 mg of cell lysates was incubated with 10 µl of anti-MK-2-agarose conjugate at 4 °C for 1.5 h. Immunoprecipitated MK-2 was then incubated with 0.5 µg of recombinant Hsp27 in 50 µl of kinase buffer containing 100 µM ATP for 45 min at 30 °C. One-fifth of the sample was subjected to SDS-PAGE followed by immunoblotting with the phospho-Hsp27 antibody and a secondary antibody for enhanced chemiluminescence (Amersham Biosciences).
RNA Gel Shift—Gel-shift analysis was performed as described previously (30). ARE-RNA (32P-labeled) was synthesized in vitro using the same method as described in Northern blot analysis. 1 x 108 MHS cells were lysed in 1 ml of lysis buffer containing 10 mM HEPES, pH 7.6, 3 mM MgCl2, 40 mM KCl, 5% glycerol, 0.5% Nonidet P-40, 2 mM dithiothreitol, and Complete protease inhibitors (Roche Applied Science). 10 µg of cell lysate was incubated with 5 x 105 cpm of ARE-RNA in 20 µl of binding buffer (20 mM HEPES, pH 7.6, 3 mM MgCl2, 40 mM KCl, 5% glycerol, 2 mM dithiothreitol) at room temperature for 15 min. RNase T1 (Ambion) was then added at a concentration of 10 units/µl, and the reaction was incubated for another 15 min. Prior to non-denaturing 6% polyacrylamide gel electrophoresis, the following antibodies were added for 15 min: 4 µg of goat anti-TTP (sc-8458, Santa Cruz Biotechnology), 1 µg of rabbit anti-14-3-3 (sc-629, Santa Cruz Biotechnology). Gels were then fixed in 12% MetOH/10% acetic acid, dried for 1 h at 60 °C, and exposed to x-ray film at -80 °C for 3–5 h.
RNA Immunoprecipitation—COS7 cells were transfected with pTet-Off, pTet-7B-ARE (TNF), and either pcDNA3 vector, pcDNA3-TTP-wt-MycHis, pcDNA3-TTP-m1,2-MycHis, or pcDNA3-TTP-AA-MycHis. After 24 h, cytoplasmic lysates were prepared in 1% Nonidet P-40, 50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM MgCl2, 10% glycerol, 1 mM dithiothreitol, 1 mM sodium vanadate, 50 mM NaF, 20 nM OA, 1 mg/ml heparin, and Complete protease inhibitors (Roche Applied Science). RNA was extracted from the cytoplasmic lysate using RNAqueous (Ambion). MycHis-tagged TTP was immunoprecipitated using anti-Myc (9E10) antibody and protein A/G beads (Ultralink, Pierce). After eight washes with lysis buffer, RNA was isolated from the immunoprecipitated material by phenol/chloroform extraction and analyzed by Northern blot analysis as described earlier (30).
HEK293 Cell Transfection and Immunoprecipitation Assays— HEK293 cells in 35-mm dishes (0.5 x 106 cells/dish) were transfected with MycHis-tagged TTP constructs using Lipofectamine2000 (Invitrogen). Transfected cells were washed twice with ice-cold phosphate-buffered saline and lysed for 30 min at 4 °C in 600 µl of Nonidet P-40 lysis buffer containing 20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor mixture (Roche Applied Science). Co-immunoprecipitation assays were performed by incubating cell lysates with Myc-probe-agarose beads for 3 h at 4 °C. Beads were then washed twice in lysis buffer before SDS-PAGE and immunoblotting analysis.
siRNA Transfection—COS7 cells were transfected using Lipofectamine 2000 (Invitrogen) with 100 nM of either a control siRNA duplex (D0) or an siRNA duplex targeting MK-2 (M2). The sequences (sense strand) were: D0, 5'-GCAUUCACUUGGAUAGUAA-3'; M2, 5'-UCACCGAGUUUAUGAACCA-3' (from Ambion). After 48 h, cells were re-seeded and transfected again with the same siRNA duplexes together with either pcDNA3 (vector) or pcDNA3-TTP-wt-MycHis. Cells were cultured for an additional 48 h in serum-free medium. Where indicated, cells were treated for 30 min with 10 µg/ml anisomycin (Sigma) prior to lysis in SDS-sample buffer. HEK293 cells were transfected with control siRNA or human PP2A-siRNA using the same method as COS7 cell transfection. Control siRNA (D-001210-02) and PP2A-siRNA (L-003598-00), targeting the catalytic subunit of human PP2A, were purchased from Dharmacon.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Secretion in MHS Cells—To investigate the role of PP2A in regulating LPS-induced TNF- secretion from alveolar macrophages OA was used to examine the effect of phosphatase inhibition on TNF- production. MHS cells representative of mouse alveolar macrophages were treated with varying concentrations of OA. The catalytic unit of PP2A was immunoprecipitated from MHS cell lysates and subjected to an in vitro phosphatase assay. OA inhibited PP2A specific activity in a dose-dependent manner with nearly 90% inhibition of phosphatase activity achieved at 1 µM OA (Fig. 1A). As a result, subsequent studies used a standard protocol of 1-h pretreatment with 1 µM OA followed by culture medium change prior to LPS stimulation. This OA dose achieved PP2A inhibition with only modest affects on PP1-specific activity (data not shown).
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secretion. At 2- and 4-h time points (Fig. 1B), the amount of TNF- secreted into culture supernatants approximately doubled in the OA-treated group. By 6 h of stimulation, the augmentation of TNF- produced in the presence of OA was less but remained significant (Fig. 1B). Although stimulation with 500 ng/ml LPS increased the absolute value of TNF-, the relative amount of OA-mediated augmentation, while significant, was not further enhanced. Therefore, 200 ng/ml LPS were used in all subsequent experiments. Moreover, OA treatment alone did not change the basal level of TNF- secretion, so that the enhancing effects were only observed following LPS stimulation.
To demonstrate that the augmented TNF- secretion by OA was specifically attributable to PP2A inhibition instead of PP1, silencing of PP2A and PP1 was achieved by transfecting MHS cells with either PP2A- or PP1-siRNA (Fig. 2). Western blot showed that the endogenous expression of PP2A and PP1 in MHS cells were both reduced by siRNA transfection (Fig. 2A). Similar to OA treatment, PP2A-siRNA silencing significantly increased TNF- secretion after LPS stimulation (Fig. 2B). Compared with control siRNA-transfected cells, the amount of secreted TNF- was nearly doubled in the PP2A-silencing group at all time points (Fig. 2B). Of note, no increase in TNF- secretion was observed following PP1 silencing at any time (Fig. 2B). As expected on the basis of transfecting less confluent cell cultures with siRNA, the overall level of TNF- secretion was slightly less with control siRNA transfection (Fig. 2B) as compared with non-transfected cells (see Fig. 1). Thus, attributing the OA-induced augmentation of TNF- secretion to PP2A inhibition was corroborated by PP2A-siRNA silencing experiments. When feasible, siRNA was used as a complimentary strategy to confirm results from OA treatment studies.
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K) and subsequently activated the NF-B pathway (40), we surmised the effect of OA was largely independent of transcription. Instead we hypothesized that post-transcriptional regulation was being affected.
PP2A Inhibition Prevents Decay of TNF- mRNA—Post-transcriptional regulation of TNF- involves the mRNA stability of its transcript. To examine the role of PP2A in the regulation of the TNF- mRNA stability, MHS cells pretreated with or without OA were stimulated with LPS for 2 h and then treated with 5 µg/ml actinomycin D to block further transcription. Total RNA was extracted at the indicated time intervals. Northern blot analysis revealed that the decay of TNF- mRNA was significantly prevented by OA pretreatment (Fig. 3). This result suggested that the effect of OA treatment on TNF- secretion was due to the stabilization of mRNA transcript.
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3'-UTR-dependent Luciferase—To further characterize the effects of PP2A inhibition on TNF- mRNA stability, a luciferase reporter construct that incorporated the 3'-UTR of TNF- (pMT2-luc-UTR) was transiently transfected into MHS cells and used to assess mRNA stability of transcribed luciferase. Transfected cells were pretreated with or without OA followed by LPS stimulation, and luciferase activity was measured at different time points. In response to OA, 2-, 15-, and 11-fold increases in luciferase activity were observed at the 2-, 4-, and 6-h time points, respectively (p < 0.01 for all time points, Fig. 4A, left panel).
Similar to OA treatment, knock-down of endogenous PP2A by siRNA also substantially increased (
10-fold) TNF-3'-UTR-dependent luciferase activity at all time points tested (Fig. 4A, right panel). Because siRNA transfections were performed on less confluent cell cultures than used in OA studies, luciferase activity was normalized to micrograms of protein. As a result, no effect was seen using control siRNA as compared with non-transfected cells. Taken together, these results indicate that the unstable elements conferred by the 3'-UTR of TNF- mRNA were suppressed by the inhibition of PP2A in MHS cells.
It has been demonstrated that activation of p38 MAPK stabilizes TNF- mRNA (26, 27). To determine whether the effect of PP2A on TNF- mRNA stability was mediated via the p38 pathway, inhibitors of p38, JNK, MEK1/2, and NF-B were added 30 min after stimulation with LPS to transfected cells that had been pretreated with OA. Results showed that luciferase activity was significantly inhibited only by the p38-specific inhibitor SB 203580 (5 µM) (Fig. 4B). These data provided additional support to the hypothesis that PP2A regulated TNF- mRNA stability via the p38 pathway.
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(19, 20). MK-2 phosphorylates TTP at two major serine sites, Ser-52 and Ser-178, both of which serve as 14-3-3 binding sites (22, 28, 30). To measure MK-2-induced phosphorylation of TTP, a phospho-specific antibody was raised against Ser-178. The antibody recognized endogenous TTP in lysates of LPS-activated Raw264.7 macrophages (Fig. 6A, lane 2), whereas treatment of the lysate with alkaline phosphatase abolished the signal (lane 3). To confirm specificity of this antibody, COS7 cells were transiently transfected with TTP constructs. The novel, phospho-specific antibody recognized both human and mouse TTP (Fig. 6B, lanes 2 and 3) but failed to recognize TTP that was mutated at Ser-178 (lanes 5 and 6). After knocking down MK-2 in COS7 cells by siRNA, the phospho-(Ser-178)-TTP signal was reduced by 50%, both in the absence and presence of the p38-MAPK activator anisomycin (Fig. 6C). Consistent with prior studies (22, 30), this result confirmed that MK-2 was required for phosphorylation of TTP at Ser-178.
To examine the effects of PP2A on TTP phosphorylation, MHS cells pretreated with or without OA were stimulated with LPS for 1 and 2 h. Phosphorylated TTP was pulled down by GST-14-3-3 beads in vitro and analyzed using the phospho-(Ser-178)-TTP antibody. The amount of total TTP in the cell lysates was detected with an affinity-purified TTP antibody (CARP-3). LPS induced TTP expression with its phosphorylation being apparent by 1 h (Fig. 6D, upper panel). At 2 h, a second, lower mobility band was induced, suggesting an enhanced phosphorylation state. OA strongly increased the phosphorylated bands, whereas incubation with recombinant PP2A significantly decreased phospho-(Ser-178)-TTP (Fig. 6D, upper panel). p38 activation is known to increase TTP expression by preventing its proteolytic degradation via the proteasome (41) or by activating its expression at the transcriptional level. Because OA treatment increased p38 activity (Fig. 5A), it was crucial to determine the total amount of TTP expression in these experiments. As expected, increased total TTP expression was seen in MHS cells after OA treatment (Fig. 6D, bottom panel). However, a substantial amount of OA-augmented TTP protein was the phosphorylated isoform, because densitometry analysis showed that total TTP expression increased 2.5-fold after OA treatment, whereas the phosphorylated isoform (Ser-178 of TTP) increased 4.2-fold.
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Binding of 14-3-3 and PP2A to TTP showed an inverse correlation when tested on non-phosphorylatable mutations of TTP (Fig. 7A). Thus, we hypothesized that 14-3-3 association with TTP may prevent its dephosphorylation by PP2A. To test this, HEK293 cells were co-transfected with wild-type TTP and gradually decreasing amounts of Myc-tagged 14-3-3 construct. Western blotting confirmed equal TTP expression but a decreasing amount of 14-3-3 in the cell lysates (Fig. 7B, upper panel). These same blots were stripped and examined for phospho-(Ser-178)-TTP. In this case, a decreasing amount of phospho-TTP was detected in the cells that had less expression of 14-3-3 (Fig. 7B, bottom panel). Finally, knocking down PP2A by siRNA in this model system increased the signal of phospho-(Ser-178)-TTP by 50%, whether or not 14-3-3 was overexpressed (Fig. 7C, lanes 2 and 4). Also, whether PP2A was knocked down or not, the absence of 14-3-3 overexpression correlated with a diminished phospho-TTP signal (lanes 3 and 4). These data provided further evidence that TTP-14-3-3 complex formation appeared to prevent TTP dephosphorylation by PP2A.
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Effects of TTP Phosphorylation on TNF- ARE Binding—To determine the effect of TTP phosphorylation on mRNA binding, RNA gel-shift assays were performed. Cytoplasmic extracts from LPS-stimulated MHS cells were incubated with a TNF- ARE probe without the addition of supershifting antibodies (Fig. 9A). Of the five major bands resolved by non-denaturing gel electrophoresis, LPS induced one band designated in the prior literature as C4 (lanes 2 and 3) (27). Pretreatment with OA strongly increased this C4 band signal (lanes 4 and 5) by 3.3-fold as compared with non-treated samples (lanes 2–3).
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We therefore used an RNA-immunoprecipitation method to examine the ability of wild-type TTP (mTTP-wt) and Ser to Ala double mutation TTP (TTP-S52A/S178A) to co-immunoprecipitate a reporter mRNA containing the TNF--ARE (Fig. 9C). Globin-ARE (TNF) mRNA was effectively precipitated by both constructs with nearly identical efficiency. The TTP-m1,2 expressed possesses an altered zinc finger section of TTP such that it cannot bind TNF- mRNA and was used as a negative control to demonstrate the specificity of this experiment (lane 3). These data suggested that TTP binding to the TNF- ARE may be independent of the two MK-2-dependent phosphorylation sites (22, 28, 30).
Ser-52/178 
Ala-52/178 Mutation of TTP Did Not Disrupt the Increased Luciferase Activity Induced by PP2A-siRNA Silencing—Because Ser-52/178 to Ala-52/178 mutation did not change the binding of TTP to TNF- ARE, we tested the effects of this mutation on luciferase reporter gene expression and whether knocking down PP2A altered this expression. Compared with vector control, co-transfection of reporter gene (pMT2-luc-UTR) with either wild-type TTP (mTTP-wt) or TTP-S52A/S178A strongly inhibited the luciferase gene expression (data not shown). However, higher luciferase activity was observed for wild-type TTP than the mutated TTP (Fig. 10, left panel compared with right panel). This finding is consistent with prior reports of MK-2 phosphorylating TTP to stabilize TNF- mRNA (21, 30, 42). Knock-down of PP2A increased the luciferase activity of both constructs, although less dramatically with the mutated TTP (Fig. 10). Nevertheless, it was surprising that PP2A silencing increased the luciferase activity of the mutated TTP to any degree as the two MK-2 phosphorylation sites in this construct have been mutated. It may be that the increased p38/MK-2 activity caused by PP2A silencing was responsible for the increased luciferase activity of the mutated TTP. Alternatively, other phosphorylation sites on TTP, independent of MK-2, that regulate TNF- mRNA stability could be additional targets by PP2A.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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), chemokines, and adhesion molecules that mediate pathological responses in inflammatory disease states (43, 44). As a result, a number of studies have examined the transcriptional and post-transcriptional mechanisms regulating LPS-induced TNF- expression.
Post-transcriptional regulation of TNF- is mediated by binding of TTP to the AU-rich elements in the 3'-untranslated region of TNF- mRNA. The p38 MAPK has been shown to increase TNF- expression via ARE-mediated mRNA stability (26, 27). This action of p38 is mediated through MK-2, which phosphorylates TTP in vitro and in vivo (22, 30, 45, 46). It has been established that regulation of phosphorylated proteins results from a balance between the activity of specific kinases and phosphatases. The current studies aimed to elucidate the role of PP2A in the post-transcriptional regulation of TNF-. We observed that modulating PP2A activity greatly enhanced LPS-induced TNF- mRNA stability. These data provide the first evidence for the involvement of PP2A in the post-transcriptional regulation of TNF- production. Although previous results have shown that TTP phosphorylation modulates TNF- expression (21, 30), our understanding of the molecular mechanism by which phospho-TTP controls inflammatory gene expression remains incomplete.
Previous studies have established that binding of phosphorylated TTP to 14-3-3 (22, 28) mediates a variety of cellular functions. For example, the TTP-14-3-3 interaction was reported to be responsible for the nuclear exportation of TTP (29, 47). This observation suggests subcellular localization of the complex was an important contributor to function. This concept of localization-dependent function was supported by the additional observation that TTP-14-3-3 interaction reduced the entry of TTP into the stress granule after arsenite treatment in COS7 cells (30). In the current study, we have identified a potential additional and novel function of TTP-14-3-3 association. Our results demonstrate that interaction with 14-3-3 protects phosphorylated TTP from access by the catalytic unit of PP2A thus preventing its dephosphorylation.
We hypothesize that this protective role of TTP-14-3-3 complex formation against PP2A dephosphorylation is crucial for stabilizing cytokine messenger RNA after stimulus exposure. Our experiments suggest that LPS-induced p38 MAPK/MK-2 phosphorylation is quick and transient. Phosphorylation peaks at 30 min and is followed by subsequent, rapid dephosphorylation. In contrast, TTP phosphorylation usually persists for several hours (27, 30). Thus, it appears that 14-3-3 binding may be critical for maintaining a prolonged phosphorylation state of TTP once upstream kinases have been deactivated. Subsequently, via an unknown mechanism, 14-3-3 is dissociated, leaving the unbound TTP to be accessed by PP2A for dephosphorylation, thereby promoting mRNA decay. A recent study reported that phosphorylation of 14-3-3 by IK and protein kinase C
resulted in the dissociation of 14-3-3 from the 4GalT1 mRNA thereby enhancing mRNA stability (48). These reports support our hypothesis that dissociation of 14-3-3 from its binding to TTP might be dependent on the phosphorylation state of 14-3-3. Such a function could reflect another layer to the complexity of TTP-mediated mRNA stability.
It has been established that binding of TTP to 14-3-3 stabilizes TNF- mRNA. This is at least in part p38/MK-2-dependent, because mutation of two key serine residues affected luciferase expression mediated by the TNF- 3'-UTR. Importantly, in the current studies, this mutated TTP had reduced binding to 14-3-3 and showed increased association with PP2A. Thus, the enhanced mRNA decay observed with the Ser-mutated TTP appears consistent with our hypothesis. However, these results cannot exclude other phosphorylation sites as being critically involved in the stability of mRNA transcript. Consistent with our observations, recent MK-2/TTP function studies (21) as well as additional studies employing the MK-2 knock-out mice (42) have suggested that alternative pathways other than p38/MK-2 could modify TTP to affect its function. An indirect effect of p38/MK-2 activation caused by PP2A inhibition could play a role in the increased stability of TNF- mRNA; however, direct dephosphorylation of TTP by PP2A was substantiated by the present data. These two regulatory mechanisms (p38/MK-2-dependent versus independent pathways) may not be mutually exclusive. Instead the regulatory mechanisms may act at different stages of TTP activation/phosphorylation. For an example, p38 and MK-2 act immediately upon cellular activation to phosphorylate TTP and stabilize mRNA. In contrast, PP2A may function at a later stage to dephosphorylate TTP thereby affecting mRNA stability.
The current studies also examined the effect of TTP phosphorylation on its binding to mRNA. Other studies exploring this question have reported varying results (22–25). Carballo et al. reported that phosphorylated TTP bound less to a granulocyte macrophage-colony stimulating factor ARE probe than dephosphorylated TTP. This finding suggested that the increased stability of granulocyte macrophage-colony stimulating factor mRNA after LPS stimulation was caused by the release of TTP from the transcript (23). In a similar manner, Hitti et al. (42) reported that a lower phosphorylation state of TTP correlated to increased binding to a TNF- ARE probe. In contrast to these observations, we did not detect a difference between wild-type TTP and the Ser-mutated TTP (S52A/S178A) in their ability to bind to a globin-TNF-ARE reporter mRNA. This was corroborated by RNA gel-shift studies that showed an increased C4 band after OA treatment that was supershifted by both TTP and 14-3-3 antibodies. Thus, phosphorylation of TTP, at least at the MK-2-dependent sites, did not appear to disrupt mRNA binding. These RNA-binding data are consistent with reports showing the effect of phosphorylation of other RNA-binding proteins to their target AU-rich elements such as the binding of BRF1 to an IL-3 ARE (49).
Because TTP is considered a destabilizing RNA-binding protein, it would have been predicted that increased mRNA stability should have been associated with less RNA binding by TTP. Our current data suggest there may be additional effects mediated by PP2A regulation of the phosphorylation state if TTP. We speculate that TTP may regulate mRNA stability via the interaction with additional regulatory proteins. The presence of such putative regulatory proteins that could comprise a particular RNA-binding complex may influence the ultimate state of a mature mRNA transcript. In support of this notion, 14-3-3 binding appears to prevent TTP-bound RNA from moving to the degradation machinery, thereby increasing mRNA stability (30). One possible explanation is that 14-3-3 binding is antagonistic to the interaction of TTP with RNA degradation machinery. TTP-14-3-3 complex formation could impair the localization of the transcript to either the exosome, a multiprotein complex of 3'-to-5' exoribonucleases (50, 51), or the decapping complex possessing the 5'-to-3' exoribonuclease Xrn1 (52, 53). Therefore, TTP-RNA binding alone may not be the only indicator of mRNA processing. Identification of additional putative proteins involved in the ARE-binding complex requires further study. Further insight into the role of these proteins in modifying mRNA stability will be necessary. Finally, understanding how this function is influenced by their phosphorylation state and regulated by phosphatases such as PP2A is necessary to increase our understanding of the molecular regulation of cytokine gene expression.
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FOOTNOTES |
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1 To whom correspondence should be addressed: Division of Critical Care, Dept. of Pediatrics, University of Michigan Medical School, 109 Zina Pitcher Place, 4460 Basic Science Research Building, Ann Arbor, MI 48109. Tel.: 734-764-5302; Fax: 734-647-5624; E-mail: tshanley{at}med.umich.edu.
2 The abbreviations used are: TNF, tumor necrosis factor; ARE, (adenosine/uridine)-rich element; 3'-UTR, 3'-untranslated regions; TTP, tristetraprolin; MAPK, mitogen-activated kinase; MK-2, mitogen activate protein kinase-activated kinase 2; PP2A, protein phosphatase 2A; OA, okadaic acid; LPS, lipopolysaccharide; IL-1, interleukin-1; JNK, c-Jun NH2-terminal kinase; MEK, MAPK/extracellular signal-regulated kinase kinase; GST, glutathione S-transferase; siRNA, small interference RNA.
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