MAP kinase phosphatase-1 expression is regulated by 15-deoxy-Δ12,14-prostaglandin J2 via a HuR-dependent post-transcriptional mechanism

Biochimica et Biophysica Acta 1849 (2015) 612–625

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MAP kinase phosphatase-1 expression is regulated by 15-deoxy-Δ12,14-prostaglandin J2 via a HuR-dependent post-transcriptional mechanism Joo Hong Woo, Jee Hoon Lee, Hyunmi Kim, Yuree Choi, Sang Myun Park, Eun-hye Joe, Ilo Jou ⁎ Department of Pharmacology and Chronic Inflammatory Disease Research Center, Ajou University School of Medicine, Suwon 443-721, Republic of Korea

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Article history: Received 26 November 2014 Received in revised form 4 March 2015 Accepted 14 March 2015 Available online 22 March 2015 Keywords: 15d-PGJ2 HuR MKP-1 Post-transcriptional regulation Inflammation Astrocytes

a b s t r a c t In the present study, we demonstrate a mechanism through which 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) induces MKP-1 expression in rat primary astrocytes, leading to the regulation of inflammatory responses. We show that 15d-PGJ2 enhances the efficiency of MKP-1 pre-mRNA processing (constitutive splicing and 3′-end processing) and increases the stability of the mature mRNA. We further report that this occurs via the RNAbinding protein, Hu antigen R (HuR). Our experiments show that HuR knockdown abrogates the 15d-PGJ2induced increases in the pre-mRNA processing and mature mRNA stability of MKP-1, whereas HuR overexpression further enhances the 15d-PGJ2-induced increases in these parameters. Using cysteine (Cys)-mutated HuR proteins, we show that the Cys-245 residue of HuR (but not Cys-13 or Cys-284) is critical for the direct binding of HuR with 15d-PGJ2 and the effects downstream of this interaction. Collectively, our data show that HuR is a novel target of 15d-PGJ2 and reveal HuR-mediated pre-mRNA processing and mature mRNA stabilization as important regulatory steps in the 15d-PGJ2-induced expression of MKP-1. The potential to use a small molecule such as 15d-PGJ2 to regulate the induction of MKP-1 at multiple levels of gene expression could be exploited as a novel therapeutic strategy aimed at combating a diverse range of MKP-1-associated pathologies. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Inflammation is a complex process comprising diverse mediators and signaling pathways. Various inflammatory stimuli can activate important members of the Mitogen-activated protein kinase (MAPK) family, including extracellular signal-regulated kinase (Erk), p38 MAPK and c-Jun N-terminal kinase (JNK) [1,2]. The activation of MAPKs requires their phosphorylation at conserved threonine and tyrosine residues within the activation motif, and such phosphorylation critically regulates the magnitude and duration of their actions [3]. The MAPK phosphatases (MKPs) act as endogenous negative regulators of MAPKs by directly dephosphorylating these conserved threonine and tyrosine residues. Among the MKP family members, MKP-1 preferentially dephosphorylates p38 MAPK and JNK upon stimulation by stress, cytokines and growth factors [4]. Studies using knockout mice demonstrated Abbreviations: 15d-PGJ2, 15-deoxy-Δ12,14-prostaglandin J2; HuR, Hu antigen R; MKP-1, MAPK phosphatase-1; ARE, AU-rich elements; 3′-UTR, 3′-untranslated region; CHX, cycloheximide; Act. D, actinomycin D; Dex, dexamethasone; NAC, N-acetyl-L-cysteine; IP, immunoprecipitation; BT, biotinylated; Cys, cysteine ⁎ Corresponding author at: Department of Pharmacology and Chronic Inflammatory Disease Research Center, Ajou University School of Medicine, 164, World cup-ro, Yeongtong-gu, Suwon-si, Gyeonggi-do, 443-721 Rep. of Korea. Tel.: +82 31 219 5061; fax: +82 31 219 5069. E-mail address: [email protected] (I. Jou).

http://dx.doi.org/10.1016/j.bbagrm.2015.03.004 1874-9399/© 2015 Elsevier B.V. All rights reserved.

that MKP-1 plays crucial roles in infections and inflammation, and the balance between MAPK signaling and MKP signaling has been strongly implicated in diverse pathological conditions [2,5–7]. Hence, pharmacological trials have intensely targeted various signaling molecules involved in the MAPK pathways. The cyclopentenone prostaglandin, 15d-PGJ2, has well-known antiinflammatory and anti-cancer effects. The reactive electrophilic α, β-unsaturated carbonyl moiety in the cyclopentenone ring of 15dPGJ2 covalently binds to the free thiol group of Cys residues in its target proteins, which include PPARγ [8], AP-1 [9] and NF-κB [10], leading to regulation of their transcriptional activity. Although 15d-PGJ2 is mainly produced in macrophages and immune cells, it is also synthesized in the brain [11], where it inhibits the induction of pro-inflammatory cytokines and chemokines, resulting in the suppression of activated astrocytes and microglia [1,12]. We previously reported that 15d-PGJ2 suppresses IFN-γ-induced MCP-1 expression in cultured brain astrocytes via the induction of MKP-1 and subsequent inhibition of JNK phosphorylation [1]. However, the mechanism through which 15d-PGJ2 regulates the induction of MKP-1 remained unknown. The MKP-1 promoter is constitutively activated in the absence of any stimulus, but the mRNA expression level of MKP-1 is relatively low in resting cells, because the transcription process is delayed in exon 1 downstream of the transcription start site [13]. In addition to this transcriptional regulation, MKP-1 expression is also critically modulated at

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the levels of transcript stability and protein stability. The inhibition of proteasome activity leads to rapid accumulation of the MKP-1 protein, while the selective phosphorylations of serine residues in MKP-1 regulate its ubiquitin-dependent degradation [14]. Stimuli known to induce MKP-1 expression have been shown to stabilize its transcript [15,16], thereby increasing MKP-1 expression. In general, the expression levels of inflammation-associated genes, including MKP-1, are believed to be largely dependent on post-transcriptional regulation. However, the underlying mechanisms are not well understood. HuR is a well characterized, ubiquitously expressed RNA-binding protein that is known to alter the stability and translation of many mRNAs, including those encoding various inflammatory mediators, cell cycle regulators, cytokines, growth factors, apoptosis-related proteins, proto-oncogenes, and tumor suppressors [17,18]. HuR acts via the preferential and high-affinity binding of U-rich and AU-rich elements (AREs) in target mRNAs [19]. It is predominantly nuclear; in response to various stimuli, it is exported from the nucleus to the cytoplasm along with synthesized mature mRNAs [20]. In addition to its effects on the stabilization and translation of cytoplasmic mRNA, nuclear HuR also contributes to pre-mRNA processing - splicing and 3′-end processing [21–24]. The activity of HuR is modulated by several post-translational modifications, including phosphorylation [25], methylation [26] and ubiquitination [27]. A previous study showed that HuR dramatically stabilizes the MKP-1 mRNA in hydrogen peroxide-treated cells by binding to AREs in the 3′-untranslated region (3′-UTR) [15]. Here, we examined the mechanism through which 15d-PGJ 2 increases MKP-1 expression levels in brain astrocytes. We report for the first time that 15d-PGJ2-induced MKP-1 up-regulation results from enhanced processing of the pre-mRNA and stabilization of the mature mRNA. These post-transcriptional controls are mainly mediated by the direct binding of 15d-PGJ2 to the Cys-245 residue of HuR. These results collectively reveal a novel anti-inflammatory mechanism of 15d-PGJ2 that acts through the HuR-dependent post-transcriptional regulation of MKP-1. This action mechanism could potentially be exploited to combat various MKP-1-related pathologies. 2. Materials and methods 2.1. Materials The anti-MKP-1 (sc-370), anti-HuR (sc-71290) and anti-Actin (sc-1616) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-Myc antibody (#2278) was obtained from Cell Signaling Technology (Denver, CO, USA). The horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (G-21040), rabbit anti-goat IgG (81-1620), Alexa-546-conjugated goat anti-mouse IgG (A11003) and FITC-conjugated streptavidin (43-4311) were obtained from Invitrogen (Carlsbad, CA, USA). The HRP-conjugated goat antirabbit IgG (111-035-003) and HRP-conjugated streptavidin (016-030084) were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and Pierce (Rockford, IL, USA), respectively. The anti-Tubulin (T5168) and anti-FLAG (F7425) antibodies and all other chemicals, including the 15d-PGJ2 (D8440), dexamethasone (D2915) and actinomycin D (A9415), were obtained from Sigma Aldrich (St. Louis, MO, USA). All primers were designed using the Primer 3 design tool (www.genome. wi.mit.edu/genome_software/other/primer3.html) under the default parameters. The primers and ribooligonucleotides were purchased from Bioneer (Daejeon, Korea). 2.2. Cell culture and RNA interference Primary cultures of cortical astroglial cells were prepared as described previously [1], with slight modifications. In brief, primary cortical astroglial cells were cultured from the cerebral cortices of 1-day-old Sprague Dawley rats (Samtako, Osan, Korea). Cell suspensions were prepared by mechanical digestion of the cortices in MEM (Gibco/Life

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Technologies, Grand Island, NY, USA) containing 10% FBS (Gemini, West Sacramento, CA, USA) and 0.1% antibiotic-antimycotic solution (Gibco/Life Technologies). The cells were dispensed to 75-cm2 T-flasks and cultured for 2 weeks. For astrocyte cultures, the flasks were shaken at 250 rpm for 18 hours at 37 °C to separate the microglia and oligodendrocytes from the astrocytes, as previously described [28]. The cells were washed five times with warm PBS, detached by trypsinization, and cultured in MEM containing 5% FBS. The purity of astrocytes was confirmed to be about 99% by flow cytometry (Supplementary Fig. S1A). Prior to the 15d-PGJ2 treatments, the concentration of FBS in the medium was adjusted from 5% to 1%, because 15d-PGJ2 has the potential to bind serum proteins via the Michael addition reaction [29]. For silencing of HuR, cells were transfected with an HuR-targeting siRNA [20 nM; 5′-GAUGCCAACUUGUACAUCA(dTdT)-3′] [30], an HuR 3'-UTR-targeting siRNA [50 nM; 5'-CCAGUAUAUUCCAGAGUCAAGCCU G(dTdT)-3'] or negative control siRNA [20 nM; 5′-CCUACGCCACCAAU UUCGU(dTdT)-3′] (Bioneer). Transfections were performed with Lipofectamine 2000 (Invitrogen), and the cells were further incubated for 48 h. 2.3. Plasmids The plasmid, pSG5 MKP-1-Myc, which harbors the MKP-1 coding region tagged with one copy of the c-Myc epitope at the C-terminus under control of the SV40 promoter, was the kind gift of Prof. K. T. Kim (Dept. of Life Science, Pohang University of Science and Technology, Korea). The full-length cDNA of rat wild-type (WT) HuR was amplified from a rat astrocytes cDNA library using Phusion High Fidelity DNA polymerase (Finnzymes, Keilaranta, Finland) and specific primers (forward 5′-AATTGAATTCTAATGGTTATGAAGACCACATG-3′ and reverse 5′-GATCAGATCTTATTTGTGGGACTTGTTGG-3′). The obtained cDNA was cloned into a p3XFLAG-CMV-7.1 vector (Sigma Aldrich) digested with EcoRI and BglII. Three mutant plasmids (p3XFLAG-C13S HuR, -C245S HuR and -C284S HuR), in which the indicated cysteines (TGC)of HuR were substituted with serines (TCC), were produced using a QuikChange Lightning Site-directed mutagenesis kit (Agilent Technologies, Wilmington, DE, USA), p3XFLAG-WT HuR (as the template) and specific primers (C13S, forward 5′-ACATGGCGGAAGACTC CAGGGATGACATTGG-3′ and reverse 5′-CCAATGTCATCCCTGGAGTCTT CCGCCATGT-3′; C245S, forward 5′-CTTCCTCGGGCTGGTCCATCTTCATC TACAAC-3′ and reverse 5′-GTTGTAGATGAAGATGGACCAGCCCGAGGA AG-3′; and C284S, forward 5′-GATTTCAACACCAACAAGTCCAAAGGGT TTGGTTTTGTG-3′ and reverse 5′-CACAAAACCAAACCCTTTGGACTTGT TGGTGTTGAAATC-3′). We substituted cysteine with serine instead of alanine because the former has a hydroxyl side chain that is structurally similar to the sulfhydryl side chain of cysteine. To add an N-terminal (His)6 to WT HuR or C245S HuR, DNA (p3XFLAG-WT HuR and -C245S HuR, respectively) was PCR amplified with primers containing an EcoRI restriction site (forward, 5′-GCGCGAATTCATGTCTAATGGTTATG AAGA-3′) and an XhoI restriction site (reverse, 5′-GGCCCTCGAGTTAT TTGTGGGACTTGTTGG-3′), and the obtained fragments were cloned into pET21a + (Novagen, Darmstadt, Germany) pre-digested with EcoRI and XhoI. 2.4. Cell lysis and Western blot analysis To prepare whole-cell lysates, cultured cells were washed twice with cold PBS, harvested in lysis buffer [50 mM Tris, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1% (v/v) Nonidet P-40, and 0.25% (v/v) protease inhibitor cocktail III (Calbiochem, Darmstadt, Germany)], incubated for 1 h on ice, and lysed by ultrasonication. Lysates were centrifuged at 12,000 × g for 10 min at 4 °C for removal of cell debris, and equal amounts of protein were separated by SDS-PAGE and electro-transferred onto a nitrocellulose membrane (Whatman, Dassel, Germany). The membrane was blocked for 60 min at room temperature with TBST (50 mM Tris-HCl, pH 7.4, 250 mM NaCl, and 0.1% Tween 20) containing 5% non-fat dry

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milk, and then incubated overnight at 4 °C with the indicated antibodies. The immunoreactive bands were visualized by incubation with HRP-conjugated anti-rabbit IgG, anti-mouse IgG or anti-goat IgG, followed by incubation with an enhanced chemiluminescence detection reagent (WESTSAVE gold; AbFrontier, Seoul, Korea). Band density was quantified using the ImageJ software, version 1.42q (National Institutes of Health, Bethesda, MD, USA). 2.5. Isolation of RNA For RNA analysis, cells were grown to 80–90% confluence and frozen at −20 °C until use (storage was sustained for no more than 2 weeks). The frozen cells were re-suspended with the RNAiso Plus extraction reagent (Takara, Kyoto, Japan) and lysed with gentle shaking for 5 min. Total RNA was extracted according to the manufacturer's instructions and precipitated using isopropanol in the presence of glycoblue (Ambion, Foster City, CA, USA). The concentration of total RNA was determined using a micro-volume nucleic acid spectrophotometer (ASP-2680; ACTGene, Piscataway, NJ, USA). Only samples having an OD260/OD280 ratio in the range of 1.7 ~ 2.1 were used. 2.6. Synthesis of cDNA Total RNA (1 μg) was reverse-transcribed at 42 °C for 40 min in 20-μl reaction volumes containing avian myeloblastosis virus reversetranscriptase (Finnzymes) and either random primers (for analysis of pre-mRNA and splicing intermediates; Takara) or oligo(dT) primers (for analysis of mature mRNAs; Promega, Madison, WI, USA). 2.7. PCR and quantitative real-time PCR (qPCR) PCR was performed in 20-μl reaction volumes containing 0.5 μM of each primer, 1/20 dilution of cDNA template reverse-transcribed from total RNA (1 μg) and Taq DNA polymerase (GenDEPOT, Barker, TX, USA). To amplify splicing intermediates of the MKP-1 pre-mRNA, we used specific primers (forward 5′-GATCAGCGAGCACTTGAGGA-3′ and reverse 5′-TGTGCAAAGGATAGCAAGGG-3′) and 29 cycles of 30 s at 94 °C, 30 s at 55 °C and 150 s at 72 °C. To amplify GAPDH (as a reference gene), we used specific primers (forward 5′-GGCCAAAAGGGTCATC ATC-3′ and reverse 5′-GTGATGGCATGGACTGTGG-3′) and 20 cycles of 30 s at 94 °C, 30 s at 55 °C and 30 s at 72 °C. To amplify MKP-1-Myc, we used specific primers (forward 5′-AGATCCTCTTCAGAGATGAGTT TGC-3′ and reverse 5′-GCGGAGTATTATCTCCCCCCAAC-3′) and

22 cycles of 30 s at 94 °C, 30 s at 55 °C and 30 s at 72 °C. The PCR products were analyzed by 1% agarose gel electrophoresis and stained with GelRed (Biotium, Hayward, CA, USA), and the band densities were quantified using the ImageJ software, version 1.42q. All RT-qPCR assays were performed according to the MIQE guidelines [31], using a Corbett Rotor-Gene 6000 (Qiagen, Valencia, CA, USA) and a SYBR green qPCR kit (Kapa Biosystems, Woburn, MA, USA). Each 15-μl qPCR reaction mixture contained 1/20 dilution of cDNA template reverse-transcribed from total RNA (1 μg), 250 nM of each primer, and the SYBR FAST qPCR master mix. The amplification profile consisted of an enzyme activation step at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C for 3 s and annealing/extension at 60 °C for 30 s. Fluorescence was measured once at the end of an annealing and extension step. A final dissociation stage was run to generate a melting curve for verification of amplification product specificity. The following primers were used for qPCR: MKP-1 pre-mRNA, forward 5′-ACTGAT GGACGAAGCCAGTG-3′ and reverse 5′-ACTTGCTCAAGAGTGCGGTC-3′; MKP-1 mature mRNA, forward 5′-TAGACTCCATCAAGGATGCTGG-3′ and reverse 5′-GCAGCTCGGAGAGGTTGTGAT-3′; and COX-2, forward 5′-TGGGAAGCTTTCTCCAACCT-3′ and reverse 5′-GTGAAGTGCTGGGC AAAGAA-3′. The specificity of qPCR was confirmed by the generation of a single product at the end of the 35 PCR cycles and observation of a single peak on the melting curve analysis. The mRNA levels were determined by the 2-ΔΔCt comparative method [32], with GAPDH used as a reference gene. 2.8. Analysis of mRNA decay To halt de novo mRNA synthesis, cells were treated with actinomycin D (final concentration, 2 μg/ml). Total RNA was purified using RNAiso Plus, RT-qPCR was performed, and the mRNA decay rate was calculated as a percentage of the transcript level in untreated cells. The mRNA decay rate was plotted as a function of the actinomycin D treatment time. The equation obtained from the best-fit linear solution was used to calculate the half-life of the mRNA. 2.9. Immunofluorescence/RNA-fluorescence in situ hybridization (IF/RNAFISH) IF/RNA-FISH was performed as described by Grünwald et al. [33]. Briefly, primary astrocytes were cultured on coverslips, fixed with 3% paraformaldehyde in PBS, quenched with PBS containing glycine, and permeabilized in 80% methanol. For immunofluorescence, the fixed

Fig. 1. 15d-PGJ2 induces MKP-1 expression in rat primary astrocytes. Primary astrocytes were treated with 10 μM 15d-PGJ2 for the indicated periods (A and C) or with various concentrations of 15d-PGJ2 for 6 h (B and D). MG132 (5 μM, 2 h) was used to confirm that the antibody was specific for MKP-1 (A and B). The protein levels of MKP-1 and Actin (loading control) were measured by Western blot analysis. Transcript levels of MKP-1 were detected by RT-qPCR.

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Fig. 2. 15d-PGJ2 does not regulate MKP-1 expression at the post-translational level. (A) (a) Primary astrocytes were transfected with pSG5 MKP-1-Myc, incubated for 48 h, and then exposed to 10 μM of 15d-PGJ2, 1 μM of dexamethasone (Dex) or 5 μM of MG132 for 3 h. Whole-cell extracts were subjected to Western blot analysis (WB). (b) The transcript levels of MKP-1Myc and GAPDH were assayed by RT-PCR. (B) Astrocytes were transfected with pSG5 MKP-1-Myc and incubated with 10 μg/ml of cycloheximide (CHX) for the indicated time periods in the presence of 15d-PGJ2 (10 μM) or Dex (1 μM). (C) Astrocytes were treated with MG132 (5 μM) for 2 h, the cells were washed, and protein synthesis was blocked by incubation with CHX (10 μg/ml) for the indicated time periods in the presence 15d-PGJ2 (10 μM) or Dex (1 μM). Whole-cell extracts were subjected to Western blot analysis. Data are given as the mean ± S.D. Average values of three independent experiments are shown; *p b 0.05, **p b 0.01.

cells were incubated with the anti-HuR antibody, washed, and fixed a second time with 3% paraformaldehyde in PBS. For FISH, cells were incubated with a denatured biotinylated oligo DNA probe (5′-TTCGTG GGGTGAACAGGGATGGAGACAGGGAAGTTGAAGA-3′), washed, and incubated with Alexa-546-conjugated anti-mouse IgG and FITC-labeled streptavidin. Finally, each coverslip was mounted using Vectashield containing DAPI (Vector Laboratories, Burlingame, CA, USA) and observed using a confocal microscope (Zeiss, Germany).

2.10. RNP-immunoprecipitation (RNP-IP) RNP-IP in cytosol and nucleus fraction was performed as described by Kuwano et al. [15]. Briefly, primary astrocytes were treated with 15d-PGJ2 (10 μM) and incubated for 6 h. For cell fractionation, cultured cells were washed twice with cold PBS, harvested in lysis buffer [50 mM Tris, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 0.5% (v/v) Nonidet P-40, 0.25% (v/v) protease inhibitor cocktail III (Calbiochem, Darmstadt, Germany),

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and 100 U/ml RNaseOUT (Invitrogen)] and incubated for 10 min on ice. The cells were then passed 20 times through a 26-gauge needle and the resulting lysate was centrifuged at 1,000 × g for 10 min at 4 °C. The

supernatant was transferred to a fresh tube and used as the cytosolic fraction. The precipitate was dissolved in lysis buffer and washed twice by being passed 10 times through a 26-gauge needle and centri-

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fuged at 1,000 × g for 10 min at 4 °C. The washed nuclei were dissolved in lysis buffer containing 0.5% TX-100 instead of 0.5% Nonidet P-40, and then disrupted by ultrasonication. For immunoprecipitation of endogenous mRNA-HuR complexes, each extract was incubated with control mouse IgG (ab81216, Abcam) or the anti-HuR antibody. Immunocomplexes were collected by pull-down using protein A/Gagarose and washed five times. The isolated RNA was reversetranscribed using oligo(dT) primers, and mature MKP-1 and GAPDH mRNAs were PCR amplified using the above-described primers and 29 cycles of 30 s at 94 °C, 30 s at 55 °C and 30 s at 72 °C. For whole cell lysates, RNP-IP was performed using a protocol from Niranjanakumari et al. [34] with slight modification. Briefly, harvested cells were dissolved in Buffer A (20 mM HEPES, pH7.6, 10 mM KCl, and 1.5 mM MgCl2) and formaldehyde was added to a final concentration of 0.1% (v/v). The mixture was incubated for 5 min on ice. Crosslinking was quenched by the addition of glycine (pH 7.0) at a final concentration of 0.25 M, and the mixture was incubated for further 10 min. After centrifugation, cells were washed and resuspended in 0.5 ml of Buffer A supplemented with 0.5% (v/v) Nonidet P-40, 0.25% (v/v) protease inhibitor cocktail III, and 100 U/ml RNaseOUT. Cell lysis was accomplished using TissueLyzer-II machine (Qiagen). Extract pre-cleared using protein A/G-agarose was incubated with control mouse IgG or the anti-HuR antibody. Immunocomplexes were collected by pull-down using protein A/G-agarose and washed five times. The beads are collected and digested with DNase I (AM2222, Ambion), followed by removal of DNase I using proteinase K (F202S, Finnzymes). RNA was extracted from the immunoprecipitates using the RNAiso Plus extraction reagent. The isolated RNA was reverse-transcribed using random primers. MKP-1, COX-2 and GAPDH mRNAs were determined by qPCR. 2.11. Pull-down assay To pull down biotinylated 15d-PGJ2 (15d-PGJ2-BT) adducts, cells were treated with 15d-PGJ2-BT (Cayman, Ann Arbor, MI, USA). Lysates (100 μg of protein) were prepared, mixed with 20 μl of a streptavidinagarose bead slurry (Pierce), and incubated at 4 °C for 2 h. The 15dPGJ2-BT adduct-streptavidin agarose complexes were collected by centrifugation at 1000 × g for 3 min at 4 °C and washed six times with mild rotation for 3 min at 4 °C in lysis buffer. Following centrifugation at 1000 × g for 3 min, each sample was mixed with 30 μl of 1X Laemmli sample buffer. Incorporation of biotin was assessed by Western blot analysis with HRP-conjugated streptavidin. 2.12. In vitro incorporation of biotinylated 15d-PGJ2 The incorporation of 15d-PGJ2-BT into (His)6-WT HuR and (His)6– C245S HuR was examined as described by Gayarre et al. [35]. Briefly, recombinant (His)6-WT HuR and (His)6-C245S HuR proteins were purified using a Protino protein purification system (Macherey-Nagel, Duren, Germany) according to the manufacturer's instructions. The obtained proteins were then incubated with DMSO (0.1%, v/v) or 15dPGJ2-BT (100 μM) for 30 min at room temperature in reaction buffer [50 mM Tris pH 7.4, 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1% (v/v)

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glycerol, 0.01% (v/v) NP-40]. The reaction was stopped by addition of 1X Laemmli sample buffer, the samples were separated by SDS-PAGE, and incorporated biotin was detected by Western blot analysis with HRP-conjugated streptavidin. 2.13. Statistical analysis Statistical significance was determined using the Origin 8.0 software (MicroCal, Northampton, MA, USA). All results are depicted as the mean and standard deviation (S.D.) of at least three independent experiments. The data were compared using the Student's t test, and statistical significance was set at p b 0.05. 3. Results 3.1. Treatment with 15d-PGJ2 time- and concentration-dependently induces MKP-1 mRNA and protein expression To examine the kinetics of the 15d-PGJ2-triggered induction of MKP1, rat brain astrocytes were incubated with 10 μM 15d-PGJ2 for the indicated periods (Fig. 1A) or with various concentrations of 15d-PGJ2 for 6 h (Fig. 1B). The 15d-PGJ2-induced expression of the MKP-1 protein became prominent within 3 h, peaked at 12 h, and decreased thereafter (Fig. 1A). When cells were treated with increasing concentrations of 15d-PGJ2 (starting from 2.5 μM) for 6 h, the induction of MKP-1 protein expression was first observed following treatment with 7.5 μM, increased up to 12.5 μM, and decreased thereafter (Fig. 1B). Our RTqPCR analysis revealed similar time- and dose-dependent expression patterns of MKP-1 transcripts (Fig. 1C and D), suggesting that the induction of MKP-1 by 15d-PGJ2 in astrocytes is dependent on transcriptional activation. Two representative inflammation-related molecules induced by 15d-PGJ2 in other cell types, COX-2 and HO-1 [36,37], showed similar time- and dose-dependent patterns, with a slight left-shift observed in the pattern of HO-1 expression (Supplementary Fig. S1B-E). MKP-1 and COX-2 proteins rapidly accumulated in the presence of the proteasome inhibitor, MG132, whereas HO-1 proteins did not (Fig. 1 and Supplementary Fig. S1B and C). 3.2. Post-translational regulation does not contribute to the 15d-PGJ2triggered induction of MKP-1 Our finding that inhibition of the ubiquitin-proteasome system triggered the rapid accumulation of MKP-1 proteins (Fig. 1) prompted us to examine whether 15d-PGJ2 post-translationally regulates MKP-1 expression. We utilized a vector construct (pSG5 MKP-1-Myc) that contained a 3′-UTR-free MKP-1 coding region under the control of the SV40 promoter, in order to exclude the effects of transcriptional and post-transcriptional regulation as much as possible. Whereas the exogenous MKP-1 protein (MKP-1-Myc) accumulated in cells treated with MG132 or dexamethasone (Dex; known to attenuate the proteasomal degradation of MKP-1 [38]), this effect was not observed in 15d-PGJ2treated cells (Fig. 2A, a). However, the transcript levels of exogenous MKP-1 did not significantly differ among the three groups (Fig. 2A, b). To further exclude the involvement of a post-translational mechanism,

Fig. 3. 15d-PGJ2 increases the pre-mRNA processing and mature mRNA stability of MKP-1. (A) Schematic representation of the rat DUSP1/MKP-1 gene showing the qPCR or PCR primer sets (arrows) used to analyze the pre-mRNA, the mature mRNA and the splicing intermediates of the pre-mRNA. Random primers and oligo(dT) primer were used to synthesize cDNA to analyze the pre-mRNA and mature mRNA of MKP-1 respectively. Primer set for MKP-1 pre-mRNA was designed to contain polyadenylation site (polyA site) within PCR product. The boxes represent exons (1, 2, 3 and 4) and the lines connecting the boxes represent introns (α, β and γ). (B) Astrocytes were treated with 15d-PGJ2 (10 μM) for 3 h. (a) The 3′-end processing efficiency of the MKP-1 pre-mRNA is represented as the ratio of the mature mRNA to the pre-mRNA. (b) The pre-mRNA and mature mRNA levels of MKP-1 were determined by RT-qPCR. (C) Splicing intermediates were detected by RT-PCR. (a) Four different PCR products, representing the unspliced form (1α2β3γ) and three splicing intermediates (12β3γ, 1α23γ and 123γ), are indicated. (b) The bar chart shows the relative band intensities of the PCR products. (D and E) Astrocytes were exposed to 15d-PGJ2 (10 μM) for 3 h (D) or the indicated periods (E), and then incubated in the presence of 2 μg/ml of actinomycin D (Act. D) for 30 min (D) or the indicated periods (E). The decay rate of transcripts is expressed as a percentage of the level of pre-mRNA or mature mRNA in cells that did not receive actinomycin D. Data are given as the mean ± S.D. Average values of three independent experiments are shown; *p b 0.05, **p b 0.01.

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we examined the stability of exogenous and endogenous MKP-1 protein among cells in which de novo protein expression had been blocked by cycloheximide (CHX). We found that exogenous MKP-1 protein has a half-life of ~2.3 h in control cells. Dex-treatment effectively inhibited the degradation of exogenous MKP-1 protein. 15d-PGJ2-treatment rather decreased the half-life of exogenous MKP-1 protein (Fig. 2B). Consistent with the data from our MKP-1-overexpression experiments, endogenous MKP-1 protein accumulated by MG132 was stabilized in Dex-treated cells but not in 15d-PGJ2-treated cells (Fig. 2C). Collectively, 15d-PGJ2-induced MKP-1 induction did not result from the blockage of protein degradation, which is different from the action mechanism of Dex or MG132. 3.3. 15d-PGJ2 regulates MKP-1 expression primarily at the posttranscriptional level A previous report indicated that the gene expression of MKP-1 is arrested at the transcriptional elongation step under basal conditions, and progresses in response to specific stimuli [13]. Based on this finding, we hypothesized that the 15d-PGJ2-induced expression of MKP-1 is mainly controlled by a post-transcriptional mechanism. To test this hypothesis, we designed specific primer sets for qPCR and PCR experiments examining the 3′-end processing efficiency, constitutive splicing efficiency and mRNA decay rate of MKP-1 (Fig. 3A). The 3′-end processing efficiency of MKP-1 pre-mRNA was measured as the ratio of mature mRNA to pre-mRNA, largely as described by Decorsière et al. [39]. No PCR product was obtained in our negative control experiments, which included single-stranded cDNA transcribed with random primers, and single-stranded cDNA transcribed with oligo(dT) primers in the absence of reverse transcriptase. Furthermore, no MKP-1 pre-mRNA PCR product was obtained from cDNA transcribed with oligo(dT) primers (Supplementary Fig. S2A). In contrast, 15d-PGJ2 treatment significantly increased the 3′-end processing efficiency of the MKP-1 pre-mRNA (Fig. 3B, a), with ratio changes that reflected a more prominent increase in the level of the mature mRNA over that of the pre-mRNA (Fig. 3B, b). Based on this finding, we examined the effect of 15d-PGJ2 on the constitutive splicing efficiency of the MKP-1 pre-mRNA. Spliced products were detected by PCR using a forward primer located in exon 1 and a reverse primer located in intron C (thus excluding PCR products amplified from the mature mRNA). Four PCR products were generated: a long fragment corresponding to the unspliced pre-mRNA (1α2β3γ), and three smaller fragments corresponding to intermediates from which the first and/or second introns had been spliced (12β3γ, 1α23γ and 123γ). Treatment with 15d-PGJ2 significantly increased the levels of the splicing intermediates, 12β3γ and 123γ, compared to the unspliced forms (Fig. 3C). Relatively little product corresponded to the 1α23γ intermediate, from which only intron β had been removed. This suggests that the introns are sequentially spliced in their order of appearance during the constitutive splicing process. To assess the effect of 15d-PGJ2 on the mRNA stability of MKP-1, we used actinomycin D and examined the mRNA decay rates. We did not observe any difference in the decay of pre-mRNAs in 15dPGJ2-treated and control cells, but the stability of the mature mRNA was significantly higher in 15d-PGJ2-treated cells (Fig. 3D). To examine the effect of 15d-PGJ2 on the kinetics of MKP-1 mRNA metabolism, we treated cells with actinomycin D in the presence or absence of 15d-PGJ2 for the indicated periods, and used RT-qPCR to examine the relevant mRNA levels. The half-life of the mature MKP-1 mRNA was increased from 16.5 min in untreated astrocytes to 34.7 min in cells treated with 15d-PGJ 2 for 3 h and 46.4 min in cells treated with 15d-PGJ2 for 12 h (Fig. 3E). In contrast, the halflife of GAPDH was not affected by 15d-PGJ2 during the tested time period. Based on this result, a treatment time of 3 h was used for our assessments of mRNA decay, unless otherwise indicated.

Consistent with a previous finding in human osteosarcoma cells [36], 15d-PGJ2 treatment also stabilized the COX-2 mRNA in rat primary astrocytes (Supplementary Fig. S2B). Collectively, these results reveal that the 15d-PGJ2-triggered induction of MKP-1 expression is regulated by post-transcriptional mechanisms, including enhancement of pre-mRNA processing and stabilization of the mature mRNA. 3.4. HuR mediates the ability of 15d-PGJ2 to enhance the pre-mRNA processing and mature mRNA stability of MKP-1 The cooperative actions of RNA-binding proteins and cis-acting elements in mRNA determine the post-transcriptional regulation of gene expression. Among the RNA-binding proteins, HuR has been shown to play pleiotropic roles in splicing [22], polyadenylation [21], enhanced mRNA stability [15,17,18] and translation [15]. Thus, we herein used siRNA knockdown and exogenous HuR overexpression to examine whether HuR is involved in the 15d-PGJ2-triggered induction of MKP1 expression. We found that transfection of the HuR-targeting siRNA efficiently reduced both HuR protein expression and 15d-PGJ2induced MKP-1 expression, suggesting that HuR is involved in this process (Fig. 4A). Next, astrocytes transfected with control or HuR siRNA were incubated in the presence or absence of 15d-PGJ2, and assays were run to test the 3′-end processing efficiency, amounts of splicing intermediates, and stability of the MKP-1 mRNA. Our results revealed that HuR knockdown inhibited the 15d-PGJ2-induced 3′-end processing of the MKP-1 pre-mRNA (Fig. 4B, a), enhancement of mature MKP-1 mRNA levels (Fig. 4B, b), and induction of MKP-1 splicing intermediates (Fig. 4C). The decay rate of the MKP-1 pre-mRNA was not altered by 15d-PGJ2 treatment of HuR-knockdown cells (Fig. 4D), but the 15dPGJ2-induced stabilization of the mature MKP-1 mRNA was significantly reduced in these cells (Fig. 4E). Conversely, exogenous expression of FLAG-tagged WT HuR increased the stability (Fig. 4 F) and transcript levels (Fig. 4G) of the MKP-1 mRNA following its induction by 15dPGJ2, compared to mock-transfected cells. Collectively, these results indicate that the 15d-PGJ2-triggered induction of MKP-1 at the posttranscriptional level is regulated in a HuR-dependent manner. 3.5. 15d-PGJ2 increases the bind of HuR to MKP-1 mRNA To examine how 15d-PGJ2 induces HuR to mediate the mRNA processing and subsequent protein expression of MKP-1, we first test whether 15d-PGJ2 regulates the previously reported interaction between HuR and the MKP-1 mRNA [15,40]. To directly show the association of HuR and MKP-1 mRNA in vivo, we used IF/RNA-FISH. To verify the specificity of the oligo cDNA probe designed to detect the MKP-1 mRNA, we transfected astrocytes with control or MKP-1-targeting siRNA, treated the cells with 15d-PGJ2 for 3 h, and then subjected the cells to RNA-FISH. The MKP-1 mRNA was observed as diffuse, granulelike accumulations in the cytoplasm of 15d-PGJ2-treated astrocytes, but was not detected in MKP-1 siRNA-transfected cells subjected to the same treatment (Fig. 5A, a). RNA hybridization with a cDNA probe and co-staining with a specific anti-HuR antibody revealed that the co-localization between the mRNA transcripts for MKP-1 and HuR was greatly enhanced in 15d-PGJ2-treated cells (Fig. 5A, b). Kim et al. reported that an exposure of 15d-PGJ2 at a high dose or for a long period of time induced stress granule formation [41]. Thus, we also test the effect of 15d-PGJ2 on the stress granule formation under our experimental condition. Punctuated cytoplasmic staining of TIA-1, a marker of stress granule, was more frequently shown in control cells than in 15dPGJ2-treated cells (Supplemental Fig. S2C). In addition, cytosolic HuR is co-localized with TIA-1 in control cells. However it was diminished in 15d-PGJ2-treated cells. This result indicated that 15d-PGJ2 inhibited the formation of stress granule under our experimental condition. To quantify the interaction between HuR and the mature MKP-1 mRNA, we performed RNP-IP in control and 15d-PGJ2-treated cells. HuR-bound mature MKP-1 mRNAs were detected in immune

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Fig. 4. HuR mediates 15d-PGJ2-induced MKP-1 expression. (A–E) Astrocytes were transfected with control siRNA (20 nM) or HuR-targeting siRNA (20 nM) for 48 h, and then treated with 15d-PGJ2 (10 μM) for 6 h. (A) HuR and MKP-1 protein levels were examined by Western blot analysis. The bar chart represents the relative quantitation of band density. (B) The 3′-end processing efficiency of the MKP-1 pre-mRNA (a) and the pre- and mature mRNA levels of MKP-1 (b) were determined as described in Fig. 3B. (C) The generation (a) and relative density (b) of splicing intermediates were assayed as described in Fig. 3C. (D and E) The decay rates of the pre-mRNA (D) and mature mRNA (E) of MKP-1 were determined by incubation in the presence of 2 μg/ml of actinomycin D (Act. D) for the indicated periods. The decay rate of transcripts is expressed as a percentage of the level of pre-mRNA or mature mRNA in cells that did not receive actinomycin D. (F and G) Cells were mock-transfected or transfected with p3XFLAG-WT HuR for 24 h, and then treated with 15d-PGJ2 for 6 h. (F) (a) MKP-1 mRNA decay rates were determined as described in E. (b) Exo- and endogenous HuR protein levels were examined by Western blot analysis. (G) The MKP-1 mRNA level was determined by RT-qPCR. Data are given as the mean ± S.D. Average values of three independent experiments are shown; *p b 0.05, **p b 0.01.

complexes collected from both the cytoplasmic and nuclear fractions, and the levels were higher in 15d-PGJ2-treated cells compared to controls (Fig. 5B, a). In addition, 15d-PGJ2 largely increased the interaction

of HuR with MKP-1 mRNA in immune complexes collected from whole cell lysates (Fig. 5B, b). These results indicate that 15d-PGJ2 increases the association between HuR and MKP-1 mRNA.

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3.6. 15d-PGJ2 directly binds to Cys-245 of HuR Next, we tested whether 15d-PGJ2 directly interacts with HuR. Previous studies showed that 15d-PGJ2 covalently binds to the free sulfhydryl groups of protein-incorporated Cys residues via a Michael addition

reaction, thereby modulating the functions of its target proteins [8–10]. Using a biotinylated analog of 15d-PGJ2 (15d-PGJ2-BT), we examined the formation of covalent protein adducts between HuR and 15d-PGJ2. We observed adduct formation as early as 30 min after the start of the reaction, thereafter, the adduct level peaked at 1 h, and

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Fig. 6. 15d-PGJ2 directly binds to Cys-245 of HuR. (A) Astrocytes were treated with 10 μM of biotinylated 15d-PGJ2 (15d-PGJ2-BT) for 6 h. (a) MKP-1 mRNA levels were measured by RTqPCR, and MKP-1 and tubulin were examined by Western blot (WB) analysis. (b) The decay rate of the MKP-1 mRNA was determined as described in Fig. 3D. (B and C) Astrocytes were pretreated with the indicated concentrations of NAC for 30 min, and then incubated with 15d-PGJ2-BT (10 μM) for 1 h (B) or 15d-PGJ2 (10 μM) for 6 h (C). Equal amounts of protein were examined by Western blot analysis. 15d-PGJ2 adducts were detected using HRP-conjugated streptavidin (B). (D) Astrocytes were mock transfected or transfected with p3XFLAG-WT HuR or -mutant HuRs (-C13S, -C245S and -C284S) for 24 h, and then incubated with 15d-PGJ2-BT (10 μM) for 1 h. Precipitates collected using streptavidin-agarose (pull-down) or whole-cell lysates (15 μg of protein; input), and 15d-PGJ2-BT adducts and the indicated proteins were examined by Western blot analysis using HRP-conjugated streptavidin (for the 15d-PGJ2-BT adducts) or the appropriate antibodies. (E) In vitro incorporation of 15d-PGJ2-BT was assessed using purified (His)6-WT HuR and (His)6-C245S HuR, as described in the Materials and Methods section, followed by Western blot analysis. Ponceau S staining was used to confirm that the proteins had successfully transferred onto the membrane. The 15d-PGJ2-BT adducts and HuR proteins were detected using HRP-conjugated streptavidin and the anti-HuR antibody, respectively.

was detected until 24 h (Supplementary Fig. S3A). Similar to the effect of 15d-PGJ2, 15d-PGJ2-BT increased MKP-1 expression (Fig. 6A, a) and delayed mRNA decay (Fig. 6A, b). When cells were pre-treated with N-acetyl-L-cysteine (NAC, a competitor for Cys binding), both adduct formation (Fig. 6B) and MKP-1 protein expression (Fig. 6C) decreased, suggesting that one or more of the Cys residues of HuR served as targets for 15d-PGJ2-BT. The expression levels of COX-2 and HO-1 were dose-dependently decreased by NAC (Fig. 6C), providing further ev-

idence suggesting that the 15d-PGJ2-induced up-regulations of these target proteins arise via direct interaction(s) with Cys residue(s) of the target protein(s). Since HuR has three candidate Cys residues for 15d-PGJ2 binding, we generated three mutants in which the individual Cys residues were substituted with Ser residues, as described in the Materials and methods section. Astrocytes were transfected with WT or mutant HuR in the presence of 15d-PGJ2-BT, and 15d-PGJ2 adducts were collected by

Fig. 5. 15d-PGJ2 increases the amount of HuR-bound mature MKP-1 mRNA. (A) Astrocytes were transfected with control siRNA (20 nM) or MKP-1-targeting siRNA (20 nM), incubated for 48 h, and treated with 15d-PGJ2 for 3 h. (a) The MKP-1 mRNA (green) was detected by RNA-FISH analysis, as described in the Materials and Methods section. Nuclei (blue) were stained with DAPI. (b) The colocalization of HuR proteins (red) and MKP-1 mRNAs (green) in control- and 15d-PGJ2-treated cells was detected by IF and RNA-FISH, respectively, as described in the Materials and Methods section. Bar = 10 μm. (B) (a) Cells were treated with 15d-PGJ2 for 3 h and cytosolic and nuclear fractions were prepared as described in the Materials and Methods section. left, The association of MKP-1 mRNAs with HuR proteins was analyzed by immunoprecipitation using an anti-HuR antibody or normal mouse IgG, followed by RT-PCR. (Input) Total RNA (1 μg) was subjected to RT-PCR. right, Western blot (WB) analysis was performed using anti-HuR, -Tubulin, and -Lamin B antibodies. HuR was used as a control for equal amounts of input protein for RNP-IP. Tubulin and Lamin B were used as markers for the cytosolic and nuclear fractions, respectively. (b) left, Cells were treated with 15d-PGJ2 for 6 h and whole cell lysates were prepared as described in the Materials and Methods section. MKP-1 and GAPDH mRNA in immune complex obtained using anti-HuR antibody or normal mouse IgG were measured by RT-qPCR. right, Western blot analysis was performed using anti-HuR and -Tubulin antibodies. HuR was used as a control for equal amounts of input protein for RNP-IP.

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pull-down assays using streptavidin-agarose. Both endogenous HuR and exogenous FLAG-WT HuR were detected in the collected 15d-PGJ2 adducts (Fig. 6D, pull-down). In contrast, we did not observe precipitation of other RNA-binding proteins, including TIA-1, TIAR, hnRNP A/B/ C/D, TTP and CUGBP1/2 (Supplementary Fig. S3B). When cells were transfected with increasing amounts of the FLAG-WT HuR construct, the proportions of FLAG-WT HuR in the 15d-PGJ2 adducts increased accordingly (Supplementary Fig. S3C). HuR was not detected in 15d-PGJ2 adducts pulled down from lysates of cells pre-treated with NAC (Supplementary Fig. S3D), confirming that the interaction of 15d-PGJ2 with HuR occurs via a Cys residue. Moreover, the detection of exogenous FLAG-WT HuR was greatly reduced by mutation of Cys-245, but not Cys-13 or Cys-284 (Fig. 6D, pull-down). However, traces of FLAGC245S HuR were still detected in 15d-PGJ2 adducts, and endogenous HuR was also decreased in 15d-PGJ2 adducts obtained from cells expressing FLAG-C245S HuR. To further explore whether 15d-PGJ2 directly binds to Cys-245 of HuR, we performed in vitro incorporation assays with 15d-PGJ2-BT and purified recombinant (His)6-WT HuR and (His)6-C245S HuR. Our results indicated that 15d-PGJ2-BT bounds to (His)6-WT HuR but not (His)6-C245S HuR (Fig. 6E). Together, these results indicate that 15d-PGJ2 regulates the expression of MKP-1 by directly binding to Cys-245 of HuR. 3.7. Cys-245-mutant HuR inhibits the 15d-PGJ2-induced increases in the pre-mRNA processing and mature mRNA stability of MKP-1 To examine the events downstream of the Cys-245 mutation of HuR, astrocytes expressing FLAG-WT HuR or FLAG-C245S HuR were exposed to 15d-PGJ2. Our experiments revealed that the 3′-end processing efficiency of the MKP-1 pre-mRNA (Fig. 7A, a) and the level of mature MKP-1 mRNA (Fig. 7A, b) were significantly higher in 15d-PGJ 2 treated WT HuR-expressing cells compared to similarly treated C245S HuR-expressing cells. Under basal conditions, there was no difference in the splicing intermediate levels of the WT and C245S HuR-overexpressing cells (Fig. 7B). 15D-PGJ2 treatment conspicuously increased the level of the 123γ form (from which introns α and β had been spliced) in WT HuR-overexpressing cells, whereas C245S HuRoverexpressing cells treated with 15d-PGJ2 showed general decreases in both unspliced and spliced intermediates compared to the basal levels (Fig. 7B). Furthermore, 15d-PGJ2 stabilized the mature MKP-1 mRNA more highly in WT HuR-expressing cells than in C245S HuRexpressing cells (Fig. 7C). Similarly, COX-2 mRNA levels (Fig. 7D, a) and stability (Fig. 7D, b) were increased by 15d-PGJ2 in WT HuRexpressing cells, but these effects were inhibited in C245S HuRexpressing cells. To prove the functional relevance of C245 of HuR in 15d-PGJ2-induced HuR binding to MKP-1 mRNA, RNP-IP assay was performed by using cells expressing either WT or C245S HuR protein. To achieve the maximum effect of over-expression of WT or C245S HuR, endogenous HuR was silenced using siRNA targeting the HuR 3'-UTR. Both MKP-1 and COX-2 mRNA enriched in RNP-IP from WT HuRexpressing cells were decreased in C245S HuR-expressing cells (Fig. 7E). This result indicated that the binding of 15d-PGJ2 to C245 of HuR has an important role in the interaction between HuR and MKP-1 mRNA. Our observations collectively indicate that the binding of 15dPGJ2 to the C245 residue of HuR is critical for the 15d-PGJ2-induced post-transcriptional regulation of MKP-1 expression. 4. Discussion We previously demonstrated that 15d-PGJ2 suppresses IFN-γstimulated MCP-1 expression in astrocytes by inducing the expression of MKP-1 [1]. Here, we further show that 15d-PGJ2 induces MKP-1 expression by increasing the efficiency of its pre-mRNA processing and the stability of its mature mRNA, and that this is achieved by the direct binding of 15d-PGJ2 to Cys-245 of HuR.

The therapeutic potentials of 15d-PGJ2 are closely related to its ability to modulate gene expression, as many of the reported targets of 15d-PGJ2 are transcription factors or their regulators (e.g., PPARγ [8], AP-1 [9], NF-κB [10] and Keap1 [42]). However, recent results have suggested that 15d-PGJ2 can also regulate gene expression via a post-transcriptional mechanism. For example, 15d-PGJ 2 was found to up-regulate death receptor 5 by increasing its mRNA stability rather than affecting its transcriptional activity [43]. Similarly, 15d-PGJ2 was shown to increase the stability of the COX-2 mRNA in human osteosarcoma [36]. It is possible that these effects could reflect the involvement of 15d-PGJ2 target proteins in post-transcriptional steps. However, the detailed molecular mechanism(s) underlying these post-transcriptional changes were previously unknown. Here, we demonstrate for the first time that HuR is a target of 15d-PGJ2, and that their interaction modulates MKP-1 expression at the posttranscriptional level. Using biotinylated 15d-PGJ2 and site-directed mutants of HuR, we reveal that the binding of 15d-PGJ2 to Cys-245 of HuR contributes to enhancing the pre-mRNA processing and mature mRNA stability of MKP-1 by promoting the binding of HuR to MKP-1 mRNA (Figs. 6 and 7). Previous studies showed that the stabilization of the mature mRNA is a mechanism through which MKP-1 expression is induced in response to various stimuli [15,16]. Several recent reports have revealed that both pre-mRNA processing and mature mRNA stabilization play important roles in post-transcriptional gene regulation [44]. Indeed, our present data show that 15d-PGJ2 increases not only the stability of the mature MKP-1 mRNA but also the processing of the MKP-1 pre-mRNA (e.g., constitutive splicing and 3′-end processing) (Fig. 3). HuR knockdown and mutation at Cys245 of HuR were found to inhibit these 15d-PGJ2-induced post-transcriptional regulations (Figs. 4 and 7). Here, we report for the first time that pre-mRNA processing is another target for 15d-PGJ2-induced MKP-1 expression. Transcriptomewide analysis using high-throughput techniques (e.g., PAR-CLIP and RIP-chip) indicated that HuR is coupled with both pre-mRNA processing and mature mRNA stability of many genes [23,24]. These reports showed that HuR binds only to 3'-UTR of human MKP-1 mRNA. However, HuR-binding motifs [45] are located within intron as well as 3'-UTR of rat MKP-1 mRNA. Although published data did not show the interaction of HuR with MKP-1 mRNA intron, we think more research is needed. We next questioned how the binding of 15d-PGJ2 to HuR could posttranscriptionally regulate gene expression. Various stimuli are known to stabilize transcripts by increasing the association between HuR and its target mRNA [15,30]. Furthermore, we found that 15d-PGJ2 stabilized the COX-2 mRNA more strongly than the MKP-1 mRNA (Fig. 3E and Supplementary Fig. S2B). This result is paralleled by the numbers of AREs in the 3′-UTRs of these two mRNAs [40,46]. Accordingly, we hypothesized that 15d-PGJ2 might increase the association between HuR and the MKP-1 mRNA. Our experiments revealed that the direct binding of 15d-PGJ2 to C245 of HuR increases the interaction between HuR and the MKP-1 mRNA (Figs. 5 and 7). And we found that 15d-PGJ2 selectively changes the binding activity of HuR to its putative binding motifs scattered over the 3′-UTR of the MKP-1 mRNA (Supplementary Fig. S4). These results suggest that 15d-PGJ2 might increase the interaction of HuR and MKP-1 mRNA via selective use of AREs in 3'-UTR of MKP-1 mRNA. Given that HuR plays pleiotropic roles in post-transcriptional regulation, we speculate that the binding of 15d-PGJ2 to HuR may trigger alterations in the extensive network of RNA-protein- and/or proteinprotein-interaction-based machineries (e.g. spliceosomes, pre-mRNA 3′-end processing complexes and mRNA decay complexes). For example, studies have shown that HuR regulates 3′-end processing by blocking the interaction between polyadenylation cleavage stimulation factor and its target RNA [21], and that a physical interaction between RNPC1 and HuR is required to stabilize the p21 mRNA [47]. Notably, the third RNA recognition motif (RRM3) near the C terminus of HuR

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Fig. 7. The binding of 15d-PGJ2 to Cys245 of HuR is required for the 15d-PGJ2-induced enhancements of MKP-1 pre-mRNA processing and mature mRNA stability. Primary astrocytes transfected with p3XFLAG-WT HuR or p3XFLAG-C245S HuR were exposed to 15d-PGJ2 (10 μM) for 3 h. (A) The 3′-end processing efficiency of the MKP-1 pre-mRNA (a) and the preand mature mRNA levels of MKP-1 (b) were examined as described in Fig. 3B. (B) The generation (a) and relative density (b) of the splicing intermediates were analyzed as described in Fig. 3C. (C) The decay rate of the mature MKP-1 mRNA was determined as described in Fig. 3D. (D) (a) COX-2 mRNA levels were determined by RT-qPCR. (b) To determine the decay rate of the COX-2 mRNA, de novo mRNA synthesis was halted by treatment with actinomycin D for 60 min. The decay rate of transcripts is expressed as a percentage of the COX-2 mRNA level in cells that did not receive actinomycin D. (E) left, MKP-1, COX-2 and GAPDH mRNA in immune complex obtained using anti-HuR antibody were measured as described in Fig. 5B, b. right, Western blot analysis was performed using anti-HuR and -Tubulin antibodies. HuR was used as a control for equal amounts of input protein for RNP-IP. Data are presented as the mean ± S.D. Average values of at least three independent experiments are shown; *p b 0.05, **p b 0.01.

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has been shown to mediate HuR oligomerization [48,49], exon skipping in alternative RNA splicing [22], and the interaction with RNPC1 and subsequent effects on the mRNA stability of p21 [47]. Cys-245 of HuR (i.e., the binding target of 15d-PGJ2) is located within RRM3 [48,49]. This may suggest that the binding of 15d-PGJ2 to HuR could change the conformational structure of RRM3, promoting HuR oligomerization and/or modulating the interaction between HuR and other proteins. Consistent with this concept, traces of C245S HuR were detected and endogenous HuR was decreased in 15d-PGJ2 adducts obtained from C245S HuR-expressing cells (Fig. 6D). As HuR was recently shown to homodimerize through Cys-13 [50], we hypothesize that our finding might reflect the formation of endogenous/exogenous HuR homodimers via Cys-13. We did not observe any decrease in endogenous or exogenous HuR in 15d-PGJ2 adducts collected from C13S HuRexpressing cells (Fig. 6D, pull-down), suggesting that mutation of Cys245 in HuR may block 15d-PGJ2-induced HuR oligomerization. Future studies are warranted to examine this possibility. 15d-PGJ2 regulates gene expression at multiple levels depending on the applied concentration and cellular circumstances, triggering alterations in diverse cellular functions. Under our experimental conditions, 15d-PGJ2 appeared to sequentially regulate gene expression in a concentration-dependent fashion, with the induction of HO-1 commencing at a lower concentration of 15d-PGJ2 than the responses of MKP-1 and COX-2 (Fig. 1 and Supplementary Fig. S1B-E). 15d-PGJ2 induces HO-1 expression by up-regulating the transcriptional activity of Nrf-2 [42], whereas the 15d-PGJ2-triggered up-regulations of MKP1 and COX-2 are mainly regulated post-transcriptionally via HuR (Fig. 7). At high concentrations, 15d-PGJ2 blocks the translation of many genes by directly binding to eukaryotic initiation factor 4A (eIF4A) [41]. Collectively, the present and previous findings suggest that 15d-PGJ2 can differentially regulate the expression of diverse genes through the coordinated interplay of regulatory mechanisms at the transcriptional, post-transcriptional and translational levels. In summary, we herein outline the mechanism through which 15dPGJ2 regulates MKP-1 expression. The covalent binding of 15d-PGJ2 to Cys-245 of HuR appears to enhance the pre-mRNA processing and mature mRNA stability of MKP-1, leading to increases in MKP-1 expression. Given the importance of MKP-1 and its differential regulation by small molecules such as 15d-PGJ2, a better understanding of the contextspecific regulation of MKP-1 could give rise to novel targets for the future treatment of inflammatory brain diseases. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbagrm.2015.03.004. Transparency Document The Transparency document associated with this article can be found, in the online version. Acknowledgement The authors thank Prof. K. T. Kim (Dept. of Life Science, Pohang University of Science and Technology, Korea) for providing the plasmid, pSG5 MKP-1-Myc. This work was supported by a National Research Foundation of Korea Grant funded by the Korean Government (MSIP: NRF2012R1A5A2048183). References [1] J.H. Lee, J.H. Woo, S.U. Woo, K.S. Kim, S.M. Park, E.H. Joe, I. Jou, The 15-deoxy-delta 12,14-prostaglandin J2 suppresses monocyte chemoattractant protein-1 expression in IFN-gamma-stimulated astrocytes through induction of MAPK phosphatase-1, J. Immunol. 181 (2008) 8642–8649. [2] M. Hammer, J. Mages, H. Dietrich, A. Servatius, N. Howells, A.C. Cato, R. Lang, Dual specificity phosphatase 1 (DUSP1) regulates a subset of LPS-induced genes and protects mice from lethal endotoxin shock, J. Exp. Med. 203 (2006) 15–20.

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