Tristetraprolin (TTP)-14-3-3 Complex Formation Protects TTP from Dephosphorylation by Protein Phosphatase 2a and Stabilizes Tumor Necrosis Factor-α mRNA
2006; Elsevier BV; Volume: 282; Issue: 6 Linguagem: Inglês
10.1074/jbc.m607347200
ISSN1083-351X
AutoresLei Sun, Georg Stoecklin, Susan Van Way, Vania Hinkovska‐Galcheva, Ren-Feng Guo, Paul Anderson, Thomas P. Shanley,
Tópico(s)Molecular Biology Techniques and Applications
ResumoTumor necrosis factor (TNF)-α is a major cytokine produced by alveolar macrophages in response to pathogen-associated molecular patterns such as lipopolysaccharide. TNF-α secretion is regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation occurs by modulation of TNF-α mRNA stability via the binding of tristetraprolin (TTP) to the adenosine/uridine-rich elements found in the 3′-untranslated region of the TNF-α transcript. Phosphorylation plays important roles in modulating mRNA stability, because activation of p38 MAPK by lipopolysaccharide stabilizes TNF-α 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-α 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-α adenosine/uridine-rich element binding and did not alter the increased TNF-α 3′-untranslated region-dependent luciferase activity induced by PP2A-small interference RNA silencing. Our data indicate that, although phosphorylation stabilizes TNF-α mRNA, PP2A regulates the mRNA stability by modulating the phosphorylation state of members of the p38/MK-2/TTP pathway. Tumor necrosis factor (TNF)-α is a major cytokine produced by alveolar macrophages in response to pathogen-associated molecular patterns such as lipopolysaccharide. TNF-α secretion is regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation occurs by modulation of TNF-α mRNA stability via the binding of tristetraprolin (TTP) to the adenosine/uridine-rich elements found in the 3′-untranslated region of the TNF-α transcript. Phosphorylation plays important roles in modulating mRNA stability, because activation of p38 MAPK by lipopolysaccharide stabilizes TNF-α 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-α 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-α adenosine/uridine-rich element binding and did not alter the increased TNF-α 3′-untranslated region-dependent luciferase activity induced by PP2A-small interference RNA silencing. Our data indicate that, although phosphorylation stabilizes TNF-α mRNA, PP2A regulates the mRNA stability by modulating the phosphorylation state of members of the p38/MK-2/TTP pathway. Tumor necrosis factor-α (TNF-α) 2The 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. 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-α, IL-1β, IL-6, IL-12, and oxidants. TNF-α is an important alveolar macrophage secretory product, and depletion of alveolar macrophages reduces TNF-α production, thereby neutralizing and attenuating LPS-induced lung inflammation (1Berg J.T. Lee S.T. Thepen T. Lee C.Y. Tsan M.F. J. Appl. Physiol. 1993; 74: 2812-2819Crossref PubMed Scopus (83) Google Scholar). Therefore, understanding all potential regulatory pathways of TNF-α expression is an important goal. LPS-induced production of TNF-α by monocyte/macrophages is regulated at both transcriptional and post-transcriptional levels. Transcriptional activation of TNF-α occurs primarily through the binding of NF-κB to the TNF-α promoter (2Shakhov A.N. Collart M.A. Vassalli P. Nedospasov S.A. Jongeneel C.V. J. Exp. Med. 1990; 171: 35-47Crossref PubMed Scopus (735) Google Scholar). 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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 (17Lai W.S. Stumpo D.J. Blackshear P.J. J. Biol. Chem. 1990; 265: 16556-16563Abstract Full Text PDF PubMed Google Scholar, 18Varnum B.C. Lim R.W. Sukhatme V.P. Herschman H.R. Oncogene. 1989; 4: 119-120PubMed Google Scholar). Involvement of TTP in the post-transcriptional regulation of TNF-α mRNA was suggested by the increased TNF-α secretion from TTP-deficient mouse macrophages (19Carballo E. Gilkeson G.S. Blackshear P.J. J. Clin. Invest. 1997; 100: 986-995Crossref PubMed Scopus (124) Google Scholar, 20Carballo E. Lai W.S. Blackshear P.J. Science. 1998; 281: 1001-1005Crossref PubMed Google Scholar). Lai et al. (8Lai W.S. Carballo E. Strum J.R. Kennington E.A. Phillips R.S. Blackshear P.J. Mol. Cell. Biol. 1999; 19: 4311-4323Crossref PubMed Scopus (638) Google Scholar) demonstrated that integrity of both zinc fingers was required for TTP to directly bind to the TNF-α ARE to promote mRNA decay. However, zinc fingers alone were not sufficient to mediate TNF-α mRNA degradation, because truncation of either the carboxyl or amino ends of TTP caused a loss TTP function (21Rigby W.F. Roy K. Collins J. Rigby S. Connolly J.E. Bloch D.B. Brooks S.A. J. Immunol. 2005; 174: 7883-7893Crossref PubMed Scopus (55) Google Scholar). Both ends of the TTP zinc fingers contain a cluster of phosphorylation sites (22Chrestensen C.A. Schroeder M.J. Shabanowitz J. Hunt D.F. Pelo J.W. Worthington M.T. Sturgill T.W. J. Biol. Chem. 2004; 279: 10176-10184Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar), yet the effects of phosphorylation on the mRNA binding and destabilizing activity of TTP remain controversial (22Chrestensen C.A. Schroeder M.J. Shabanowitz J. Hunt D.F. Pelo J.W. 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Immunol. 2001; 166: 966-972Crossref PubMed Scopus (133) Google Scholar). However, the role of PP2A in the regulation of LPS-induced TNF-α production remains unknown. In the present study, we tested the hypothesis that PP2A is involved in the post-transcriptional regulation of TNF-α. Our data showed that inhibition of PP2A increased TNF-α 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. Cells and Reagents—The mouse alveolar macrophage cell line (MHS cell line) was purchased from the ATCC and was cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum, 2 mm l-glutamine, 4.5 g/liter glucose, 10 mm HEPES, 1.0 mm sodium pyruvate, penicillin (100 units/ml), streptomycin (100 μg/ml), and 0.05 mm 2-mercaptoethanol. The HEK293 cell line was cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum. COS7 and Raw264.7 cells were cultured as described previously (30Stoecklin G. Stubbs T. Kedersha N. Wax S. Rigby W.F. Blackwell T.K. Anderson P. EMBO J. 2004; 23: 1313-1324Crossref PubMed Scopus (423) Google Scholar). LPS (Escherichia coli, serotype O55:B5) was purchased from Sigma. Okadaic acid (OA) was purchased from Biomol and dissolved in Me2SO. SB 203580 (p38 inhibitor), JNK inhibitor I, JNK inhibitor II, BAY 11–7082 (NF-κB 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 β (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 (37Brooks S.A. Connolly J.E. Diegel R.J. Fava R.A. Rigby W.F. Arthritis Rheum. 2002; 46: 1362-1370Crossref PubMed Scopus (57) Google Scholar) was kindly provided by W. Rigby (Dartmouth Medical School, Lebanon, NH). Constructs—A luciferase construct containing the mouse TNF-α 3′-UTR (pMT2-luc-UTR), a T3 polymerase-driven in vitro transcription construct containing the 90-nt-long AU-rich element of mouse TNF-α 3′-UTR (T3-Stem-ARE), β-globin reporter gene containing the ARE of TNF-α (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 (30Stoecklin G. Stubbs T. Kedersha N. Wax S. Rigby W.F. Blackwell T.K. Anderson P. EMBO J. 2004; 23: 1313-1324Crossref PubMed Scopus (423) Google Scholar). Altered TTP-m1,2 (38Johnson B.A. Geha M. Blackwell T.K. Oncogene. 2000; 19: 1657-1664Crossref PubMed Scopus (68) Google Scholar) 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 14-3-3β (39Sun L. Bittner M.A. Holz R.W. J. Biol. Chem. 2003; 278: 38301-38309Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) was kindly provided by R. W. Holz (University of Michigan, Ann Arbor). Full-length mouse TNF-α 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-TNFα-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 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 × SSC. Full-length 32P-labeled TNF-α antisense RNA was synthesized in vitro using plasmid pcDNA3.1-TNFα-V5His linearized with XhoI, T7 polymerase and Maxiscript reagents and purified with Megaclear columns (Ambion). The 32P-labeled β-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, 5× 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 2× 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 × 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 × 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 β-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 1× 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 β-glycerophosphate, 2 mm dithiothreitol, 0.1 mm Na3VO4, 200 μm ATP, and 8 μCi of [γ-32P]ATP for 30 min at 30 °C. The reaction mixture was then boiled in an equal volume of 2× 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 (30Stoecklin G. Stubbs T. Kedersha N. Wax S. Rigby W.F. Blackwell T.K. Anderson P. EMBO J. 2004; 23: 1313-1324Crossref PubMed Scopus (423) Google Scholar). ARE-RNA (32P-labeled) was synthesized in vitro using the same method as described in Northern blot analysis. 1 × 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 × 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 (30Stoecklin G. Stubbs T. Kedersha N. Wax S. Rigby W.F. Blackwell T.K. Anderson P. EMBO J. 2004; 23: 1313-1324Crossref PubMed Scopus (423) Google Scholar). HEK293 Cell Transfection and Immunoprecipitation Assays— HEK293 cells in 35-mm dishes (0.5 × 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. PP2A Inhibition by OA or siRNA Increased LPS-induced TNF-α Secretion in MHS Cells—To investigate the role of PP2A in regulating LPS-induced TNF-α secretion from alveolar macrophages OA was used to examine the effect of phosphatase inhibition on TNF-α 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). MHS cells were pretreated with OA and then stimulated with 200 or 500 ng/ml LPS for 2, 4, and 6 h. OA treatment significantly augmented TNF-α secretion. At 2- and 4-h time points (Fig. 1B), the amount of TNF-α secreted into culture supernatants approximately doubled in the OA-treated group. By 6 h of stimulation, the augmentation of TNF-α 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-α, 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-α secretion, so that the enhancing effects were only observed following LPS stimulation. To demonstrate that the augmented TNF-α 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-α secretion after LPS stimulation (Fig. 2B). Compared with control siRNA-transfected cells, the amount of secreted TNF-α was nearly doubled in the PP2A-silencing group at all time points (Fig. 2B). Of note, no increase in TNF-α 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-α 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-α 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. Because recent studies suggested that PP2A positively regulated I kappa kinase (IκK) and subsequently activated the NF-κB pathway (40Kray A.E. Carter R.S. Pennington K.N. Gomez R.J. Sanders L.E. Llanes J.M. Khan W.N. Ballard D.W. Wadzinski B.E. J. Biol. Chem. 2005; 280: 35974-35982Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), 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-α mRNA—Post-transcriptional regulation of TNF-α involves the mRNA stability of its transcript. To examine the role of PP2A in the regulation of the TNF-α 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-α mRNA was significantly prevented by OA pretreatment (Fig. 3). This re
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