Gene suppression by tristetraprolin and release by the p38 pathway

Wei Zhu1, Maria A. Brauchle2, Franco Di Padova2, Hermann Gram2, Liguo New1, Koh Ono1, Jocelyn S. Downey1, and Jiahuai Han1

1 Department of Immunology, The Scripps Research Institute, La Jolla, California 92037; and 2 Novartis Pharma, 4002 Basel, Switzerland


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Tristetraprolin (TTP) is a zinc finger protein that has been implicated in the control of tumor necrosis factor (TNF) mRNA stability. We show here that TTP protein has a suppressive effect on promoter elements from TNF-alpha and interleukin-8 and that lipopolysaccharide (LPS) stimulation can release this suppression. The release in LPS-stimulated cells was found to be primarily mediated by the p38 pathway because activation of p38 is sufficient to remove the suppressive effect of TTP. Indeed, TTP seems to be a direct substrate of p38 in vivo since it is an excellent substrate of p38 in vitro, and mutation of potential phosphorylation sites in TTP prevents release of the suppression imposed on TNF transcription. We found TTP protein to be present at low levels in the resting macrophage cell line RAW 264.7 and to be quickly induced after LPS stimulation. The kinetics of TTP induction suggests a potential role of TTP as an important player in switching off LPS-induced genes after induction. In conclusion, TTP plays an important role in maintaining gene quiescence, and this quenching effect on transcription can be released by p38 phosphorylation of TTP.

inflammation; mitogen-activated protein kinase; gene suppression; TIS11


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

THE FOUR CHARACTERIZED p38 kinases (alpha , beta , gamma , and delta ) represent a family of proteins within the mitogen-activated protein kinase (MAPK) superfamily that are of fundamental importance in cell signaling (18). They are distinguishable from the extracellular signal-regulated kinase (ERK) or Jun NH2-terminal kinase (JNK) family by the presence of a TGY motif located on a loop lying between kinase domains VII and VIII on the protein, and phosphorylation of the threonine and tyrosine residues in this motif is required for its activation (1, 17, 31). Exposing monocytic cells to stimuli that place them under stress, such as bacterial lipopolysaccharide (LPS), tumor necrosis factor (TNF), osmotic shock, or ultraviolet radiation, rapidly leads to an increase in dual phosphorylation and the activation of p38 via the activation of upstream activators MAP kinase kinase (MKK) 3 and MKK6 (10, 12, 31). Chemical inhibition of the p38 pathway has been shown to reduce the mortality of mice given LPS and to block the production of cytokines such as interleukin (IL)-1beta and TNF (24, 29).

Not surprisingly, therefore, these kinases have been linked to a number of inflammatory processes and have been associated with the activation of different cell types, including lymphocytes (4), neutrophils (8, 20, 28), endothelial cells (16, 32), and monocytes (5, 10, 25). TNF itself is an important mediator of inflammatory responses, and many of the severe symptoms of diseases such as septic shock, arthritis, hypotension, and disseminated intravascular coagulation have been attributed to the action of TNF (6, 30, 36). Studies have shown that p38 regulates TNF production on both a transcriptional and posttranscriptional level (15). Not only can the TNF promoter be modulated by the p38 pathway, p38 along with other MAPKs may also directly target the RNA polymerase II complex to bring about transcriptional activation of TNF (37). Investigations into the posttranscriptional control of TNF have focused on the importance of AU-rich elements (ARE) present in the 3'-untranslated region (UTR) of TNF RNA as a region involved in regulating the stability of the mRNA and translational efficiency of the protein (19, 21). Experiments with knock-in mice containing deletions in the ARE of TNF mRNA have shown that blocking the p38 pathway reduces the stability of mRNA in full-length TNF but does not affect the stability of TNF mRNA lacking the ARE sequence, indicating that p38alpha /beta may act via the ARE to modulate TNF mRNA stability (19). However, the mechanism by which p38 regulates the ARE is unknown.

Recently, the proline-rich protein tristetraprolin (TTP; also known as TIS11) has also been linked to the regulation of TNF-alpha production (13, 21, 33). As a prototype for a group of CCCH zinc finger proteins, TTP was originally observed as an immediate-early gene that was induced in insulin-stimulated cells and that is serine phosphorylated upon stimulation (23). Northern blot data have shown that TTP RNA can be detected within multiple tissues (23, 26), and although the protein was at first thought to be localized to the nucleus, cytosolic localization of the protein was recently observed (7, 35). TTP was proposed to have a role in TNF-alpha synthesis when TTP-null animals developed symptoms of dermatitis, wasting, alopecia associated with anti-nuclear antibodies, and myeloid hyperplasia (33). The clinical presentation of this syndrome resembled disease associated with TNF-alpha overproduction and could be ameliorated by treatment with anti-TNF antibodies, indicating a role for TTP in the control of TNF synthesis or degradation. Immunoprecipitation and gel-shift analyses have shown that TTP binds to the ARE in the 3'-TNF mRNA (21), and studies have demonstrated that the half-lives of TNF-alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNAs are prolonged in TTP knockout animals (3). Taken together, these data point to an important role for TTP in promoting the destabilization of mRNA.

Because of the potential role of p38 on ARE and direct interaction between TTP and ARE, it is possible that p38 may act through TTP to effect TNF mRNA stability. We studied the relationship between p38 and TTP in a murine macrophage line, RAW 264.7, and found that TTP can indeed be phosphorylated and regulated by p38. However, we were unable to detect an effect of TTP on TNF mRNA stability in our experimental system. Interestingly, we found that TTP suppressed TNF transcription and that this effect could be diminished by p38 phosphorylation.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

Reporter constructs. Luciferase reporter constructs driven by murine TNF, IL-8, and thymidine kinase (TK) promoters were generated as previously described (37). Briefly, a 1-kb fragment from the murine TNF promoter was removed from Pro-CAT using BamHI and HindIII and was inserted in BglII and HindIII sites of pGL2. A progressive series of deletions was generated in the murine 5'-TNF promoter using PCR. Activator protein-1 (AP-1), cAMP-responsive element (CRE), serum-responsive element (SRE), and nuclear factor-kappa B (NF-kappa B) reporters were generated by inserting heptameric promoter sequences 5' to the luciferase reporter.

Other constructs. Expression constructs for MKK1, -5, and -7 and TTP were cloned into pcDNA3 as previously described (11, 14, 27). Site-directed mutagenesis of TTP constructs was performed by PCR using the QuickChange site-directed mutagenesis system (Stratagene, La Jolla, CA), according to the manufacturer's instructions. Mouse TNF cDNA containing both the mouse TNF promoter and 3'-UTR regions was generated as follows: the coding region of mouse TNF-alpha and part of the 3'-UTR was amplified by PCR from plasmid pcDNA3-TNF-alpha and annealed to the amplified portion of the 3'-UTR from the reporter plasmid pBSKs(-)-TNFpro/utr (which contains the chloramphenicol acetyltransferase gene flanked by the TNF promoter and 3'-UTR). This annealed fragment was used as a template to amplify the full-length TNF sequence, which was then blunt-end cloned into EcoRV-digested pBSKS(-). The cloned construct was digested with HindIII and XbaI and ligated into pcDNA3.

Plasmid preparation. All plasmid DNA used in the transfection experiments was prepared using CsCl2 gradient ultracentrifugation. Possible LPS and bacterial sugar and/or lipid contamination was subsequently removed with the Endotoxin Removal Affinity Resin (Associates of Cape Cod, Falmouth, MA).

Transfection. RAW 264.7 macrophages and 293 kidney endothelial cells were maintained in DMEM supplemented with 10% FBS, 2 mM glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin, and 1% nonessential amino acids. Cells were transfected using 0.6 µg of each plasmid per well using calcium phosphate precipitation and glycerol shock. Empty pcDNA3 vector was used to normalize the amount of total DNA used in each transfection.

Preparation of recombinant proteins. Escherichia coli BL21(DE3) was transformed with the vector pET14b containing cDNAs for TTP, p38alpha , or p38beta . Transformed bacteria were grown at 37°C in Luria-Bertani broth until reaching an absorbance at 600 nm of 0.5, at which time isopropyl-beta -D-thiogalactopyranoside was added at a final concentration of 1 mM for 5 h. Cells were collected by centrifugation at 800 g for 10 min, and the bacterial pellet was resuspended in 10 ml of 30 mM NaCl, 10 mM EDTA, 20 mM Tris-Cl, and 2 mM phenylmethylsulfonyl fluoride (PMSF) for every 100 ml of original bacterial culture. The cell suspension was sonicated, and cellular debris was removed by centrifugation at 10,000 g for 30 min. Recombinant proteins were purified from the cleared lysate using a nickel-nitriloacetic acid purification system (QIAGEN, Valencia, CA) or glutathione-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ) following the manufacturer's instructions.

Protein kinase assays. In vitro kinase assays were conducted at 37°C for 30 min using purified immunoprecipitate as the kinase, 5 mg of kinase substrate, 250 µM ATP, and 10 µCi of [gamma -32P]ATP in 20 ml of kinase substrate as described previously. Reactions were terminated by the addition of Laemmli sample buffer. Reaction products were resolved on 12% SDS-PAGE. Phosphorylated proteins were visualized by autoradiography and were quantified by phosphorimaging.

Metabolic labeling and immunoprecipitation. RAW cells (5 × 107) were metabolically labeled as previously described. Briefly, the ATP pool of cells was labeled using [32P]orthophosphate (1 mCi/ml for 2 h), and the cells were stimulated with LPS (10 ng/ml) for 0, 15, 30, 60, 120, or 180 min. TTP was immunoprecipitated using anti-TTP antibody. SDS-PAGE was performed on the immunoprecipitates, and the dried gel was exposed on a phosphorimaging cassette for days.

Western blotting. Cells were rapidly chilled on ice, washed with ice-cold washing buffer [10 mM Tris · HCl (pH 7.5), 150 mM NaCl, and 1 mM Na3VO4], and then lysed in 250 µl lysis buffer/1 × 106 cells [20 mM Tris · HCl (pH 7.5), 120 mM NaCl, 10% glycerol, 1 mM Na3VO4, 1 mM EDTA, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, and 1 mM PMSF]. The proteins were separated by SDS-PAGE and were transferred to a nitrocellulose membrane. Anti-phospho-p38 antibody (New England Biolabs, Beverly, MA) was used to detect the phosphorylated p38 isoform. Anti-TTP antibody was produced by injecting rabbits with recombinant TTP protein and was used to detect TTP.

Northern blotting and mRNA stability. Total RNA was extracted from cultured cells using an RNeasy RNA extraction kit (QIAGEN) according to the manufacturer's instructions. Total RNA (10 µg) was resolved on a 1% denaturing agarose gel and transferred to a nylon membrane using a Turboblotter capillary transfer system (Schleicher & Schuell, Keene, NH). TNF, TTP, or green fluorescent protein cDNAs were transcribed from bacterial promoters in their cloning vectors, and the mRNA was radiolabeled with [32P]UTP.

Nuclear run-on analysis. Nuclear run-on was performed as previously described using immobilized TNF or glyceraldehyde-3-phosphate dehydrogenase (GADPH) cDNA (9).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TTP affects cytokine production at a transcriptional level. To evaluate the relationship between TTP and p38 activation in macrophages, we analyzed TTP protein and p38 activation in LPS-stimulated RAW 264.7 cells. RAW cells were stimulated with 10 ng/ml LPS for different lengths of time, and lysates from these samples were run on SDS-PAGE. Immunoblotting the gel revealed that levels of TTP significantly increased ~1 h after LPS stimulation and appeared as a diffuse, shifted band of ~40-45 kDa, coinciding with increased levels of phosphorylated p38 protein (Fig. 1, A and B). Fitting its description as an early response gene, TTP protein induction was clearly seen to precede the increase in TNF mRNA (Fig. 1, A and C). The diffuse smearing observed for the TTP-specific band is indicative of a protein that is present in multiple phosphorylation states, and this was reinforced by the ability of calf alkaline phosphatase treatment to reduce the smear (data not shown). The activation of p38 that occurred shortly before TTP phosphorylation suggested that p38 might be involved in phosphorylating TTP. Northern blotting RNA purified from the time course of LPS-stimulated RAW cells with TNF cDNA showed that levels of TNF mRNA began to increase after 30 min of LPS stimulation and reached a peak at around the 2-h time point before gradually declining (Fig. 1C). TNF transcription, measured by run-on analysis (Fig. 1D), reached a peak at 1 h and then declined.


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Fig. 1.   Kinetics of lipopolysaccharide (LPS)-induced tristetraprolin (TTP) production, p38 activity, tumor necrosis factor-alpha (TNF) mRNA accumulation, and TNF transcription. RAW cells were treated with 10 ng/ml LPS for different times as indicated. A: TTP protein was detected by Western blotting and immunostaining using anti-TTP polyclonal antibody. TTP was induced at 1 h and subsequently was shifted and sustained for up to 24 h. Equal loading of protein in each lane was assessed by staining the blot with Ponceau S (data not shown). B: p38 activity was determined by anti-phospho-p38 (*P-p38; top). p38 was probed with anti-p38 antibody (bottom). C: levels of TNF mRNA were determined by Northern blotting. Equal loading was determined by ethidium bromide staining of rRNA (data not shown) D: rate of TNF transcription was determined by nuclear run-on assay. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

To evaluate the role of TTP on TNF gene expression, we began by analyzing its influence on TNF transcription. A TNF promoter-driven luciferase reporter was transfected into RAW cells in the presence or absence of TTP cDNA, and reporter constructs containing promoter elements from either TK or IL-8 were used as controls. As shown in Fig. 2A, TTP depressed the basal synthesis of luciferase driven by the TNF and IL-8 constructs to ~10% of the level of synthesis observed in the absence of TTP. To a lesser degree, TTP also suppressed the synthesis of luciferase in the viral promoter constructs, reducing the amount of protein to ~60% of basal.


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Fig. 2.   Suppressive effect of TTP on the activity of various promoters and deactivation of TTP by LPS stimulation. A: RAW cells were transfected with 1) the beta -galactosidase expression vector pCMVbeta , 2) the TTP expression vector pcDNA3-TTP or empty pcDNA3 vector, and 3) luciferase reporters driven by TNF promoter, interleukin (IL)-8 promoter, or thymidine kinase (TK) promoter. Luciferase activity was measured 36 h posttransfection. beta -Galactosidase activity was used to normalize the transfection efficiency. B: RAW cells were transfected with expression vectors as in A. Posttransfection (36 h), the cells were stimulated LPS (10 ng/ml) for 12 h or left unstimulated. Luciferase activity was measured as described in A. We set the reporter expression level in LPS-stimulated cells without coexpressed TTP at 100% to compare the relative expression of reporter genes. Comparable results were obtained in 5 experiments.

To examine whether TTP affected LPS-induced TNF reporter gene expression, we treated the TNF, TK, and IL-8 reporter-transfected cells with LPS (10 ng/ml). To make it easier for comparison, we compared relative induction by setting the luciferase activity in LPS-stimulated samples that were transfected with reporter alone as 100. The relative induction of reporter gene expression in the presence or absence of TTP is shown in Fig. 2B. LPS was able to remove the suppressing effect of TTP on all reporter genes, and, although TTP suppressed basal expression of the unstimulated reporter genes, this effect was abolished after LPS stimulation.

These results suggest that TTP may act as a broad-spectrum promoter-suppressor, although the degree of promoter quenching may differ significantly between genes. Because TTP is expressed at low levels in resting macrophages, it may be preoccupied, interacting with endogenous genes and preventing transiently transfected genes from interacting with endogenous TTP. This may account to some extent for the higher basal activity observed from transfected genes compared with their endogenous counterparts. Our results showed a significant increase in the degree of induction of luciferase synthesis as a consequence of cotransfecting TNF reporter constructs with TTP because of the reduction in its basal expression.

A number of TNF luciferase reporter genes were produced with 5'-deletions in the TNF promoter (see Fig. 3A). These constructs were transfected into RAW cells in the presence or absence of TTP, and the degree of suppression that TTP exerted on luciferase expression was assessed. In this study, the suppressing effect of TTP gradually reduced with progressive deletions in the TNF promoter. However, suppression of luciferase synthesis by TTP was still observed even when the TNF promoter was deleted to position -36, suggesting that the minimal promoter of TNF is important in mediating the effect of TTP (Fig. 3A). A further experiment was then performed to determine the dependence of the suppression effect of TTP on minimal promoters. To achieve this, luciferase reporter constructs were assembled that contained identical minimal promoters but different consensus cis elements (i.e., AP-1, CRE, SRE, and NF-kappa B sites). As shown in Fig. 3B, the suppressive effect of TTP varied according to the promoter elements used in the construct. TTP suppressed the AP-1 promoter to the greatest extent, followed by the CRE and SRE promoters, whereas the NF-kappa B promoter was least affected. Collectively, these data suggest that TTP itself may not specifically recognize a consensus sequence but does preferentially regulate certain structures located in the whole promoter region. This is reminiscent of the interaction of multiple transcription factors that converge to exert their function.


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Fig. 3.   Comparison of the effect of TTP on the deletion mutants of the TNF promoter and its effect on different promoter elements. A: RAW cells were cotransfected with 1) the beta -galactosidase expression vector pCMVbeta , 2) the TTP expression vector pcDNA3-TTP or empty pcDNA3 vector, and 3) luciferase (Luc) reporters driven by TNF promoter 5'-deletion series. Cell extracts were prepared 48 h after transfection, and beta -galactosidase activity was used to normalize the transfection efficiency. The relative suppression was calculated by dividing the luciferase activity of the sample with coexpressed pcDNA3 by that with coexpressed TTP. B: RAW cells were transfected with 1) pCMVbeta , 2) pcDNA3 or pcDNA3-TTP, and 3) luciferase reporters driven by a basal promoter joined to tandem repeats of activator protein (AP)-1, cAMP-responsive element (CRE), serum-responsive element (SRE), or nuclear factor-kappa B (NF-kappa B). Cell extracts were prepared 48 h after transfection, and beta -galactosidase activity was used to normalize transfection efficiency. The relative suppression was calculated by dividing the luciferase activity of the pcDNA3 sample by that of the TTP sample.

Although it has been reported that TTP can mediate mRNA stability (21), this is unlikely to explain the results in Figs. 2 and 3, since the mRNA species transcribed from each reporter construct were identical and lacked the ARE associated with TTP-mediated mRNA destabilization. Nevertheless, to thoroughly exclude this possibility, we determined the half-life of RNA under the influence of TTP. To accomplish this, RAW cells were transfected with luciferase reporters driven by the TNF promoter with or without TTP cDNA and were stimulated with LPS 36 h after transfection. Cells were treated with actinomycin D (10 µg/ml) 2 h post-LPS stimulation and were harvested at regular intervals from this point. Luciferase RNA extracted from these samples was quantified using quantitative RT-PCR. As shown in Fig. 4A, TTP reduced the level of luciferase RNA transcribed from the TNF promoter in resting cells, and LPS stimulation led to an induction of luciferase mRNA. Similar to observations on luciferase activity, LPS stimulation eliminated the suppressive effect of TTP. As expected, TTP coexpression did not influence the half-life of luciferase mRNA (Fig. 4B). Therefore, we have established that TTP regulates TNF production at the level of transcription.


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Fig. 4.   Reporter mRNA stability is not altered by TTP. A: RAW cells were cotransfected with 1) the beta -galactosidase expression vector pCMVbeta , 2) the pcDNA3-TTP or pcDNA3 vector, and 3) luciferase reporters driven by the TNF promoter. LPS stimulation was applied 36 h after transfection, and cells were harvested 2 h later. B: another group of cells was treated with actinomycin D (10 µg/ml) 2 h after LPS stimulation (or in cells left unstimulated). Cells were harvested for analysis at 0, 30, 60, 90, or 120 min after addition of actinomycin D. Total RNA was extracted from these cells, and luciferase reporter mRNA was quantified using the quantitative RT-PCR instrument Taqman (PE Applied Biosystems, Foster City, CA). The level of beta -galactosidase mRNA was used as internal control to normalize transfection efficiency. Relative mRNA level in A was calculated by dividing the mRNA number of each sample by the number of the sample without coexpressed TTP and LPS stimulation.

TTP is regulated by the p38 MAPK pathway. Having observed that the suppressive effect of TTP is removed upon stimulation of RAW cells with LPS, we sought to address the mechanism leading to the deactivation of TTP. We tested several MAPK pathways known to be activated by LPS stimulation for their effects on TTP-mediated suppression of the TNF promoter. Dominant active mutants of different MAP MKKs were employed to selectively activate each MAPK pathway. Cotransfection of a TNF promoter-driven luciferase reporter in the presence or absence of TTP cDNA and different MKK constructs revealed that activation of the p38 pathway resulted in the highest production of luciferase. The gene suppression by TTP was disabled by p38 activation, whereas TTP was able to retard the induction of the TNF-driven luciferase reporters by ERK, JNK, and big MAP pathways (see Fig. 5A). These data indicated that, of the MAPKs, the p38 pathway predominantly regulates the role of TTP in LPS-stimulated TNF promoter activation. To more precisely address which isoforms of p38 may be responsible for nullifying TTP-mediated suppression, transfected RAW cells were treated with SB-203580, a chemical inhibitor of p38alpha and p38beta but not of the gamma - and delta -isoforms. SB-203580 inhibited LPS-induced release of TTP-mediated suppression in a dose-dependent manner, with an IC50 identical to that required to inhibit p38alpha in this cell line (Fig. 5B).


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Fig. 5.   TTP deactivation is specifically mediated by p38. A: p38 mediates TTP deactivation. RAW cells were cotransfected with 1) the beta -galactosidase expression vector pCMVbeta , 2) the pcDNA3-TTP or pcDNA3 vector, 3) luciferase reporters driven by the TNF promoter, and 4) expression plasmids for mitogen/extracellular signal-regulated kinase (MEK) 1(E), MEK5(D), MAP kinase kinase (MKK) 6(E), MKK7(D), or empty pcDNA3 vector. Cell extracts were prepared 48 h after transfection. beta -Galactosidase activity was used to normalize the transfection efficiency. The relative induction was calculated by dividing the luciferase activity of each sample by the activity without coexpressed TTP and MKK (or MEK). Filled and hatched bars represent the samples transfected with TTP or without TTP, respectively. B: SB-203580 inhibits LPS-induced deactivation of TTP. RAW cells were cotransfected with 1) the beta -galactosidase expression vector pCMVbeta , 2) the pcDNA3-TTP or pcDNA3 vector, and 3) luciferase reporters driven by the TNF promoter in the presence or absence of SB-203580. Posttransfection (36 h), the cells were stimulated with or without LPS (10 ng/ml) for 12 h. Cell extracts were prepared, and beta -galactosidase activity was used to normalize transfection efficiency. The degree of induction was calculated by dividing the luciferase activity of the samples treated with LPS by the activity of the corresponding sample without LPS. Filled and hatched bars represent the samples transfected with TTP or without TTP, respectively.

Phosphorylation of TTP. Although phosphorylation of TTP has been observed in cultured cells treated with serum and phosphorylation on serine-220 by ERK has been reported (34), no biological function of this phosphorylation has been demonstrated. Having demonstrated that TTP can be regulated by p38 in macrophages, the potential role of the p38 pathway in TTP phosphorylation was then examined. To determine this, RAW cells were metabolically labeled with 32P and stimulated with LPS. Cells were then harvested over a time course (0-180 min), and TTP was immunoprecipitated and resolved by SDS-PAGE. Autoradiography was used to detect phosphorylated TTP and produced a protein smear on the gel characteristic of a multiphosphorylated protein (Fig. 6A). These data confirmed the results shown in Fig. 1A. To determine whether the phosphorylation of TTP is p38 dependent, we thought to examine the phosphorylation of TTP in the presence of p38 inhibition. Pretreatment of cells with the p38 inhibitor SB-203580 blocked TTP induction (data not shown), which prevented us from determining the relationship between TTP phosphorylation and p38. To avoid this problem, we added SB-203580 30 min after LPS stimulation when TTP was induced (Fig. 1A). Adding SB-203580 at this time point not only inhibited further induction of TTP protein expression but also prevented TTP phosphorylation as judged by the absence of band smearing in the presence of SB-203580 (Fig. 6B). Because our data implied a role for p38 in regulating TTP, we used an in vitro kinase assay to determine whether TTP was a potential substrate for p38alpha (p38) and p38beta . Recombinant TTP was incubated with p38alpha or p38beta in the presence of [gamma -32P]ATP, and the reactions were stopped in SDS sample buffer, resolved on SDS-PAGE, and then finally exposed to X-ray film. TTP was an excellent substrate for both p38 isoforms (Fig. 6C). The efficacy of TTP phosphorylation by the two enzymes was about the same when the activities of p38alpha and p38beta were normalized using myelin basic protein and was much higher than other p38 substrates we have tested, including activating transcription factor-2, myocyte-specific enhancer factor 2C, and phosphorylated heat- and acid-stable protein-1 (data not shown). Because multiple bands were produced after phosphorylation, we analyzed the kinetics of TTP phosphorylation by p38. TTP was incubated with p38alpha in the presence of [gamma -32P]ATP over time before the reactions were stopped in SDS sample running buffer, resolved on SDS-PAGE, and then finally exposed to X-ray film and stained with Coomassie blue. The results shown in Fig. 6D clearly demonstrate that p38alpha promotes the incorporation of radiolabeled phosphate into TTP, producing a gradual increase in the phosphorylated protein with time [similar to that seen in LPS-stimulated RAW cells (Figs. 1A and 6A)]. Multistep phosphorylation was confirmed by the observed graded shift in TTP protein revealed with Coomassie blue staining of the gel (Fig. 6D, bottom). As seen in the stained gel, 25 min of incubating p38alpha and TTP caused such dissociation of the protein bands that almost no protein was visible. In contrast, the autoradiograph from this time showed a strongly phosphorylated band, indicating that hyperphosphorylated TTP produces the strongest shift in SDS-PAGE. p38alpha can therefore catalyze phosphorylation of TTP at multiple sites.


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Fig. 6.   TTP phosphorylation in vivo and by p38. A: LPS stimulates TTP phosphorylation (*P) in vivo. RAW cells were metabolically labeled with 32P (1 mCi/ml for 2 h) and then stimulated with LPS (or left unstimulated) for different periods of time. Cells were harvested, and TTP was immunoprecipitated from the cell lysates and analyzed on SDS-PAGE. B: p38 inhibitor inhibits LPS-induced shift of TTP protein. RAW cells were stimulated with LPS for periods of time as indicated. SB-203580 was added 30 min after LPS addition in samples marked. TTP protein in the cells was analyzed by Western blotting using anti-TTP antibody. C: bacterially expressed p38alpha or p38beta was incubated with recombinant TTP in a kinase reaction buffer for 30 min. Myelin basic protein (MBP) was used as a common substrate to normalize the activity of the two p38 isoforms. D: time course of TTP phosphorylation by p38alpha . Bacterially expressed TTP was incubated with p38alpha in kinase buffer containing [32P]ATP for different periods of time. Kinase reactions were stopped in SDS-sample buffer and analyzed on SDS-PAGE. The gel was exposed to X-ray film (C and D, top) or stained with Coomassie blue (D, bottom).

Effect of phosphorylation on TTP. We have shown that LPS stimulation of RAW cells results in both TTP phosphorylation (Fig. 6) and the removal of the quenching effect of TTP on the TNF promoter (Fig. 2). It is therefore conceivable that TTP is inactivated by phosphorylation. If this is true, then mutating the phosphorylated residues to alanine should produce TTP mutants resistant to deactivation. Because of the diffuse smear of phosphorylated TTP in vivo and in vitro, we were unable to precisely map the phosphorylation sites using biochemical methods (data not shown). Upon analysis of the TTP sequence, 12 proline-derived serine and threonine residues were found to be probable targets of p38alpha /beta phosphorylation. These 12 single-site mutations were generated and cotransfected into RAW cells along with the TNF reporter gene. However, no significant differences in promoter quenching or the release of that quenching were found between these mutants and the wild-type TTP gene (data not shown). To study this more comprehensively, further mutations were introduced into TTP (summarized in Table 1) for transfection experiments. These mutants maintained the ability to quench the basal level of background transcription from the reporter, but as the number of mutations increased, there was a corresponding decrease in the ability of LPS to release the suppression (Fig. 7A). Mutation of proline-derived serine-197, -214, -218, and -228 had no effect on LPS stimulation of the TNF promoter (mutant III in Fig. 7A). Serine-228 corresponds to serine-220 in murine TTP, previously identified as the residue primarily targeted by the MAPK ERK, indicating that ERK phosphorylation alone is insufficient to activate TTP. When threonine-271 was mutated along with the serines (mutant VI in Fig. 7A), the TTP became significantly more resistant to LPS stimulation. However, mutating threonine-271 alone had no effect on LPS stimulation, strongly arguing that multiple phosphorylation sites are involved in controlling TTP activity, and mutation of serine-90 and -93 also decreased the reactivity of the TNF promoter to TTP. Mutating 7 or 8 of the 12 phosphorylation sites (mutants VII and VIII in Table 1) on TTP caused markedly reduced phosphorylation by p38alpha compared with the wild-type gene (Fig. 7B) and abolished the band shift seen on SDS-PAGE of TTP (Fig. 7C). Mutating all 12 proline-derived sites completely prevented TTP phosphorylation by p38alpha . Together, these results strongly argue that phosphorylation by p38alpha is involved in deactivating TTP through multiple-site phosphorylation.

                              
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Table 1.   Sites mutated in TTP mutants



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Fig. 7.   Multiple mutations of the potential p38 phosphorylation sites in TTP led to a gradual resistance to LPS stimulation. A: RAW cells were cotransfected with 1) the beta -galactosidase expression vector pCMVbeta , 2) TTP or TTP mutants, and 3) luciferase reporters driven by the TNF promoter. Posttransfection (36 h), the cells were stimulated with or without LPS (1 ng/ml) for 12 h. Cell extracts were then prepared. Luciferase activity and beta -galactosidase activity were measured. beta -Galactosidase activity was used to normalize the transfection efficiency. Relative LPS response was calculated by dividing the luciferase activity of each sample by the activity of the sample that coexpressed wild-type TTP and was stimulated with LPS. Experiments were performed 3 times, with each experiment giving comparable results. B: recombinant proteins of TTP (TTP mutant VII, VIII, or XVI) were incubated with recombinant p38 in kinase buffer with [32P]ATP for 30 min. Kinase reactions were stopped in SDS sample buffer and analyzed on SDS-PAGE. The gel was exposed to X-ray film (top) and stained with Coomassie blue (bottom).

Effect of TTP on the stability of transfected TNF mRNA. TTP-deficient mice develop a clinical syndrome closely resembling hyperexpression of TNF, and it has been demonstrated that the stability of TNF mRNA increases in bone marrow-derived macrophages from these animals (3). In vitro studies show that TTP can directly bind to the ARE sequence found in the TNF 3'-UTR (21, 22). Overexpressing TTP affected the level of an ectopically expressed TNF mRNA. Because TTP coexpression resulted in a shorter form of TNF mRNA, it was suggested that TTP promotes deadenylation of TNF mRNA (21). We therefore wished to evaluate whether phosphorylation of TTP by p38 has any effect on TNF mRNA stability. We adapted the same system described by Lai et al. (21), except that the construct used for expressing TNF mRNA was different. The construct we used contained the full-length TNF coding sequence and the complete 3'-UTR and TNF poly(A) signal, whereas that used by Lai et al. contained the full-length TNF coding sequence and the TNF 3'-UTR truncated after ARE sequence followed by 33 adenylate residues. TNF expression plasmid was cotransfected into 293 cells with varying amounts of TTP expression vector and was incubated for 40 h. Total RNA from these cells was then used to measure the amount of both TTP and TNF mRNA by Northern blot. As observed by Lai et al., TTP first negatively and then positively affected the amount of TNF mRNA in the cell (Fig. 8A, top) such that increasing the amount of TTP from 0 to 0.5 µg caused a gradual decrease in the amount of TNF. However, above this level, when 1.0 or 4.0 µg of TTP were used, the amount of TNF mRNA increased. The expression of TTP correlated with the amount of plasmid used in the experiments (Fig. 8A, bottom). In contrast to the data described by Lai et al., we did not see a short form of TNF mRNA on any occasion. To determine whether the alteration in the amount of TNF mRNA in the presence of TTP was due to the ability of TTP to modulate mRNA stability, we examined the effect of TTP on the decay of TNF mRNA. The 293 cells were cotransfected with expression constructs of TNF, TTP, and MKK6(E) in different combinations. Later (24 h), the cells were treated with actinomycin D (10 µg/ml) to inhibit further transcription and were incubated for 0, 1, 2, 3, or 4 h, and total RNA isolates were prepared from these cells. Northern blot analysis of these isolates showed that TTP decreased the total amount of TNF-alpha mRNA visible on the blot but did not alter the rate of decay of the RNA (Fig. 8B). MKK6(E) slightly extends TNF mRNA half-life, and TTP still had no effect when cotransfected with MKK6(E). TNF mRNA half-life was ~3 h in 293 cells. These data indicate that TTP has no effect on TNF mRNA stability in this experimental system.


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Fig. 8.   TTP does not affect TNF mRNA stability in 293 cells. A: 293 cells were cotransfected with pEGFPN1, pcDNA3-TNF, and varying amounts of pcDNA3 or pcDNA3-TTP and incubated for 40 h. Total RNA was extracted from these cells, and amounts of green fluorescent protein (GFP), TTP, and TNF-alpha mRNAs were determined by Northern blot. B: 293 cells were cotransfected with 1) pcDNA3-TNF, 2) pcDNA3-MKK6(E) or pcDNA3, and 3) pcDNA3-TTP or pcDNA3. Cells were incubated for 16 h and treated with actinomycin D (10 µg/ml) for different times. At the end of this time, RNA was isolated from the cells, equally loaded on agarose gel (data not shown), and Northern blotted with a TNF probe.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TTP knockout mice possess a pathology that not only closely resembles TNF overproduction but can also be prevented by administering the mice anti-TNF antibody (33). It is therefore clear that TNF is involved in the pathology of TTP-associated disease. What is less clear, however, is precisely how TTP regulates TNF production. Previous work has indicated that TTP destabilizes the mRNAs of TNF and GM-CSF in vivo (3, 21). Along with these data, our results assert that TTP can act as a transcriptional repressor for a broad range of promoters, including the TNF and IL-8 promoters, lowering the basal level of transcription. Stimulating macrophages with LPS may have the effect of removing TTP-imposed squelching. The p38 pathway activated by LPS stimulation appears to be a primary signaling pathway responsible for modulating TTP activity. Because TTP protein expression was quickly induced in macrophages after LPS stimulation, the regulation of TNF expression by TTP may be dynamic. The preexisting protein may prevent expression of TNF in the absence of stimulation, and the newly synthesized TTP may function in TNF gene downregulation.

Transcriptional regulation of gene expression has been studied intensively. Although transcriptional suppressors are equally important as activators, their functions remain very poorly defined. The induction of cytokine genes is well accepted to be important for inflammation and many other pathological changes, and alteration of their basal activities is also important, especially in controlling the progression of chronic inflammatory disorders. The production of TTP knockout mice has successfully demonstrated that increasing the basal activity of TNF leads to a serious chronic inflammatory disorder. Gene suppression by TTP plays a very important role in keeping the basal level of the TNF gene quiescent, which is essential for preventing the development of chronic inflammatory diseases. As with gene activation, gene suppression can occur at multiple levels. TTP has been shown to suppress gene expression by reducing mRNA stability, and our data suggest that TTP may also exert an effect on transcription to suppress promoter basal activity. The suppression of the basal expression of genes, an often neglected aspect of genetic control, is undoubtedly physiologically important. Another important role of gene suppressors is turning genes off after induction. As suggested by others, the induction of TTP constitutes a feedback mechanism (2). The upregulation of TTP may function at both transcriptional and posttranscriptional levels to downregulate cytokine genes after induction.

We have shown that TTP is phosphorylated and exists in multiple phosphorylation states in cells. Several lines of evidence suggest that p38 is a kinase for TTP. LPS stimulation of monocytes leads to the activation of p38 kinase, and this quickly precedes the phosphorylation of TTP. p38 directly phosphorylates TTP in vitro, producing a smear and shift of TTP bands when analyzed on SDS-PAGE, and this is also seen in LPS-stimulated cells. p38 inhibitor inhibited LPS-induced TTP phosphorylation. Although we were unable to map the in vivo and in vitro phosphorylation sites using biochemical methods because of the diffused multiple phosphorylation bands of TTP, mutation of potential phosphorylation sites of p38 gradually reduced TTP regulation, indicating that p38 is indeed directly regulating TTP activity. However, the involvement of other kinases in phosphorylating TTP cannot be excluded. In agreement with this, mutation of all potential p38 phosphorylation sites failed to render TTP totally unregulatable. It should be noted that p38 appears to have a dual role in TTP regulation. TTP can be deactivated by p38 phosphorylation, and TTP induction is p38 dependent. The latter can be considered as a feedback response from a former event, since there is a temporal difference between these two events. The effect of TTP in gene suppression may be determined by the overall effect of TTP protein level and its phosphorylation level.

The method by which phosphorylation alters the TTP-mediated promoter squelching is open to question. It is possible that phosphorylation alters the binding capacity of TTP, but since the phosphorylation sites we have mapped have a physiological effect outside the zinc finger domain (which is around amino acids 100-170 in our clone), we have to look to interactions that may occur in the carboxy-terminal domain of the protein. Sequence homology to different RNA polymerase II molecules in this region might suggest that TTP interacts with molecules of the basal transcriptional machinery or another transcription factor. It is also possible that phosphorylation alters the intracellular localization or the stability of the protein.

Lai et al. (21) have previously discussed the involvement of TTP in binding and destabilizing TNF mRNA. In an attempt to evaluate whether p38 acts via TTP to regulate TNF mRNA stability, we examined TNF mRNA stability in 293 cells, the system used by Lai et al. Although we confirmed that transfection of increasing amounts of TTP first reduces and then increases the amount of TNF mRNA, Northern blot analysis failed to reveal any significant change in the stability of TNF mRNA affected by TTP. When we carefully look at the data presented in the published report, 4 h of actinomycin D treatment in TTP-cotransfected cells produced no detectable change in TNF mRNA in their experiments (Fig. 2 in Ref. 21). The only difference between the results is that we did not observe the smaller band reportedly caused by deadenylation. The artificial poly(A) used in their construct may contribute to this unique phenomenon, which may provide a tool to facilitate the study of TNF mRNA stability. However, further evaluation is needed, since a clear band of deadenylated TNF mRNA was seen neither in macrophages nor in 293 cells. It is true that TTP-null mice show TNF and GM-CSF mRNA with an increased half-life, and this unarguably demonstrates a role for TTP in the stability of TNF, but indirect effects still potentially exist. TTP exerted no effect on TNF mRNA stability in 293 cells, and it is open to question whether TTP phosphorylation affects TNF mRNA stability, since this could not be assessed from our experiments. Several ARE-binding proteins have been identified and have been shown to regulate the stability of different ARE-bearing mRNAs. The relationship between these ARE-binding proteins and how they interact would be interesting to ascertain. It could be that these molecules act in a coordinated manner, such that transfection of a single molecule cannot reproduce the TTP-promoted destabilization of TNF mRNA observed in knockout mice.


    ACKNOWLEDGEMENTS

We thank Dr. Ivan Lindley for the interleukin-8 reporter construct and J. V. Kuhns for excellent secretarial assistance.


    FOOTNOTES

This work was supported by California Cancer Research Program Subcontract no. 99-00521V-10121 and National Institutes of Health (NIH) Grant AI-41637 (J. Han).

M. Brauchle was supported by a fellowship from the Max-Planck Society of Germany. L. New was supported by National Institutes of Health Grant HL-07195.

This is publication no. 13535-IMM from the Department of Immunology, The Scripps Research Institute, La Jolla, CA.

Address for reprint requests and other correspondence and present address of J. S. Downey: Division of Mycobacterial Research, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK (E-mail: jdowney{at}nimr.mrc.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6 February 2001; accepted in final form 3 April 2001.


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