15- Expression Requires ERK1/2 MAPK Activity Ashish Bhattacharjee, Anny Mulya, Srabani Pal, Biswajit Roy, Gerald M. Feldman and Martha K. Cathcart This information is current as of October 2, 2021. J Immunol 2010; 185:5211-5224; Prepublished online 22 September 2010; doi: 10.4049/jimmunol.1000514 http://www.jimmunol.org/content/185/9/5211 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Monocyte 15-Lipoxygenase Requires ERK1/2 MAPK Activity

Ashish Bhattacharjee,*,† Anny Mulya,*,† Srabani Pal,*,† Biswajit Roy,*,† Gerald M. Feldman,‡ and Martha K. Cathcart*,†

IL-13 induces profound expression of 15-lipoxygenase (15-LO) in primary human . Our studies have defined the func- tional IL-13R complex, association of Jaks with the receptor components, and the tyrosine phosphorylation of several Stat molecules in response to IL-13. Furthermore, we identified both p38MAPK and protein kinase Cd as critical regulators of 15-LO expression. In this study, we report an ERK1/2-dependent signaling cascade that regulates IL-13–mediated 15-LO gene expression. We show the rapid phosphorylation/activation of ERK1/2 upon IL-13 exposure. Our results indicate that Tyk2 kinase is required for the activation of ERK1/2, which is independent of the Jak2, p38MAPK, and protein kinase Cd pathways, suggesting bifurcating parallel regulatory pathways downstream of the receptor. To investigate the signaling mechanisms associated with the ERK1/2- Downloaded from dependent expression of 15-LO, we explored the involvement of transcription factors, with predicted binding sites in the 15-LO promoter, in this process including Elk1, early growth response-1 (Egr-1), and CREB. Our findings indicate that IL-13 induces Egr-1 nuclear accumulation and CREB serine phosphorylation and that both are markedly attenuated by inhibition of ERK1/2 activity. We further show that ERK1/2 activity is required for both Egr-1 and CREB DNA binding to their cognate sequences identified within the 15-LO promoter. Furthermore, by transfecting monocytes with the decoy oligodeoxyribonucleotides specific for Egr-1 and CREB, we discovered that Egr-1 and CREB are directly involved in regulating 15-LO gene expression. These studies http://www.jimmunol.org/ characterize an important regulatory role for ERK1/2 in mediating IL-13–induced monocyte 15-LO expression via the transcrip- tion factors Egr-1 and CREB. The Journal of Immunology, 2010, 185: 5211–5224.

he Th2 lymphocyte-derived cytokine IL-13 is a potent diverse clinical implications, we have pursued studies to under- activator of monocyte and function. IL-13 stand the regulation of IL-13–mediated 15-LO expression in pri- T induces the expression of the lipid-oxidizing 15- mary human monocytes. lipoxygenase (15-LO) in peripheral blood monocytes (1, 2). Lip- Our previous studies have characterized the functional monocyte are a family of lipid-peroxidating that cata- IL-13R complex and the downstream signaling events in primary by guest on October 2, 2021 lyzes the oxygenation of free and esterified polyunsaturated fatty monocytes (20). We have demonstrated the IL-13–induced het- acids to form the corresponding hydroperoxy fatty acid derivatives erodimerization of IL-13Ra1– and IL-4Ra–chains leading to the (3, 4). 15-LO catalyzes the conversion of 15(S) H(p)ETE from activation/phosphorylation of Jak2 and Tyk2 and tyrosine phos- arachidonate and 13(S) H(p)ODE from linoleate (5, 6). These phorylation of the receptor components (2, 20). We have also products are potent mediators of inflammation (7) and are found in reported the activation/tyrosine phosphorylation of specific Stat human atherosclerotic lesions (8–10). Several studies have sug- molecules Stat1, Stat3, Stat5, and Stat6 in response to IL-13 gested the involvement of 15-LO in the development of athero- stimulation (20). These studies have defined a regulatory signal sclerosis, , , cancer, renal injury, osteoporosis, and transduction pathway from the receptor to the nucleus in human neurodegenerative disorders (3, 11–18). Recently, it was also monocytes. shown that expression of the lipoxygenase gene Alox 15, which Our recent data indicated that IL-13 induces Stat1 and Stat3 Ser727 encodes 12/15-LO, induces cardiac inflammation and plays a cru- phosphorylation in addition to tyrosine phosphorylation (21, 22). cial role in the development of heart failure (19). Because of its Our studies also demonstrated the activation of p38MAPK and subsequent phosphorylation of Ser727 residues on Stat1 and Stat3 molecules as critical regulators in IL-13–induced 15-LO expres- *Department of Cell Biology, Lerner Research Institute, Cleveland Clinic and sion in primary human monocytes (21). We further observed the † Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of involvement of protein kinase C (PKC) d in forming a mole- Case Western Reserve University, Cleveland, OH 44195; and ‡Division of Monoclonal Antibodies, Office of Therapeutics, Research and Review, Center for Biologics Evalu- cular complex with tyrosine-phosphorylated Stat3 and subsequent ation and Research, Food and Drug Administration, Bethesda, MD 20892 PKCd-dependent Stat3 Ser727 phosphorylation for optimal ex- Received for publication February 16, 2010. Accepted for publication August 25, pression of 15-LO in response to IL-13 stimulation (22). 2010. One major mechanism involved in the regulation of inflam- This work was supported by National Institutes of Health Grants HL051068 and matory processes is the activation of ERK1/2 (23–25). They are HL087018 (to M.K.C.). members of the MAPK superfamily (p44MAPK/p42MAPK) and Address correspondence and reprint requests to Dr. Martha K. Cathcart, Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, play a major role in cell proliferation and differentiation (26, 27). Cleveland, OH 44195. E-mail address: [email protected] In response to a variety of stimuli, they are activated/phospho- Abbreviations used in this paper: BCS, bovine calf serum; CRE, cAMP response rylated (at specific Thr and Tyr residues) and can either act as element; Egr-1, early growth response-1; EGR1 CP, nonlabeled EGR1 Cold Probe; an upstream kinase for several cytosolic proteins that participate 15-LO, 15-lipoxygenase; MT, mutated; ODN, oligodeoxyribonucleotide; PBM, pe- ripheral blood monocyte; PCNA, proliferating cell nuclear Ag; PKC, protein kinase in different signal transduction pathways (28) or translocate to the C; WT, wild-type. nucleus, where they can directly or indirectly activate several www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000514 5212 ERK1/2 REGULATES 15-LO EXPRESSION transcription factors (28, 29), to regulate the expression of specific transferred to a polyvinylidene difluoride membrane, blocked with 5% (30, 31). BSA in PBS with 0.1% Tween 20 and subjected to immunoblotting with p- 133 505 d In the current study, we investigated the contribution of the Ser CREB, Egr-1, p-p38MAPK, p-ERK1/2, and p-Thr PKC Ab overnight. The hybridization signal was detected using SuperSignal West MEK–ERK1/2-mediated signaling pathway in IL-13 induction Pico Chemiluminescent substrate (Pierce, Rockford, IL). 15-LO protein of 15-LO expression through the activation of transcription factors was detected on Western blots following a previously described protocol early growth response-1 (Egr-1) and CREB in primary human mono- (2). For immunoprecipitation experiments, the lysates were incubated with d cytes. Our results indicate that MEK–ERK1/2-regulated stimulation anti-PKC Abs for 2 h at 4˚C with constant rotation and precipitated with prewashed protein A-Sepharose beads (Sigma-Aldrich, St. Louis, MO) of Egr-1 nuclear accumulation and CREB activation are directly at 4˚C overnight. Immunoprecipitates were thoroughly washed with a lysis linked to IL-13–induced 15-LO expression in human monocytes. buffer containing 1% Triton X-100, 150 mM NaCl, 50 mM NaF, 30 mM We demonstrate that activation of ERK1/2 is dependent on Tyk2 b-glycerophosphate, 0.5 mM phosphoserine, 0.5 mM phosphotyrosine, and independent of the Jak2, p38MAPK, and PKCd pathways. 1.0 mM phosphothreonine, 1.5 mM p-nitrophenylphosphate, 50 mM Tris m Thus, the MEK–ERK1/2-mediated signaling pathway represents (pH 7.4), 1 mM sodium orthovanadate, 500 M PMSF, and protease in- hibitor mixture (Sigma-Aldrich), and the immune complexes were released a bifurcation of signaling downstream of the monocytes IL-13R that by boiling the beads in SDS sample buffer and were then subjected to West- works in parallel with the previously identified Jak2, p38MAPK, and ern blot analysis as described earlier (20). Immunoblots were stripped and PKCd signaling pathways to regulate IL-13–stimulated 15-LO ex- reprobed to assess equal loading according to our previously published pression. Hence, this pathway is required but not sufficient for IL-13– protocol (20). induced 15-LO expression. This work is also the first report, to our Treatment of monocytes with sense/scrambled and antisense knowledge, on the involvement of transcription factors like CREB oligodeoxyribonucleotides for Jak2, Tyk2, and ERK1/2 and Egr-1 in regulating the expression of 15-LO gene in IL-13– The antisense oligodeoxyribonucleotide (ODN) sequences for human Jak2 Downloaded from stimulated primary human monocytes. and Tyk2 were selected based on our previously published literature (2). Control ODNs for Jak2 and Tyk2 consisted of complementary sense ODNs. The antisense ODN sequence for human ERK1/2 was also selected Materials and Methods based on the previous studies by Lee et al. (37). A scrambled ODN (for Reagents ERK antisense) was used as a control. All ODNs were end-modified (phosphorothioated, three bases of 59 and 39) oligodeoxyribonucleotides Recombinant human IL-13 was purchased from BioSource International to limit DNA degradation, and all were HPLC-purified preuse (Invitrogen,

(Camarillo, CA). Ab against rabbit 15-LO, cross-reacting with http://www.jimmunol.org/ Carlsbad, CA). human 15-LO, was raised in sheep and was kindly provided by Dr. Joseph The sequences of the ODNs are as follows: Jak2 antisense, 59-TCT TAA Cornicelli, Parke-Davis (New York, NY). Anti–phospho-p38MAPK, anti– CTC TGT TCT CGT TC-39; Jak2 sense, 59-GAA CGA GAA CAG AGT phospho-ERK1/2, anti–phospho-(Ser133)–CREB, anti-phosphothreonine 505 TAA GA-39; Tyk2 antisense, 59-CCA ACT TTA TGT GCA ATG TG-39; PKCd, anti-CREB, and anti-PCNA (mouse monoclonal) Abs were purchased Tyk2 sense, 59-CAC ATT GCA CATAAA GTT GG-39; ERK1/2 antisense, from Cell Signaling Technology (Beverly, MA) and diluted 1:1000 according 59-AGC AGA TAT GGT CAT TGC-39; and ERK1/2 scrambled, 59-TCG to the manufacturer’s protocol. The other primary Abs used in this study were: TCT ATA CCA GTA ACG-39. rabbit anti-human Egr-1 (C-19) and rabbit anti-human PKCd (C-20) from Primary human monocytes (5 3 106 cells/well) were plated in six-well Santa Cruz Biotechnology (Santa Cruz, CA) and anti-ERK1/2 from Millipore culture plates overnight. Cells were then transfected with Jak2 and Tyk2 (Billerica, MA). Pharmacological inhibitors such as PD98059 and SB202190 sense and antisense ODNs or with ERK1/2 scrambled and antisense ODNs were purchased from Calbiochem (La Jolla, CA). U0126 and Rottlerin were m at 2 M concentration using Mirus TransIt-Oligo Transfection Reagent by guest on October 2, 2021 purchased from Biomol (Butler Pike, PA). The inhibitors were dissolved in (Mirus Bio, Madison, WI) according to the manufacturer’s protocols, and dimethyl sulfoxide and stored at 220˚C as concentrated stock solutions. the incubation was continued for 48 h. For the transfection control, Isolation of human monocytes monocytes were incubated with the transfection reagent alone for 48 h. After this treatment, monocytes were exposed to IL-13 for another 30 min Human peripheral blood monocytes (PBMs) were isolated either by sep- or 24 h to study either the activation/phosphorylation of p44/p42 MAPK aration of mononuclear cells followed by adherence to bovine calf serum (ERK1/2) or 15-LO gene expression. (BCS)-coated flasks as described earlier (32) or by Ficoll-Hypaque sedi- mentation followed by countercurrent centrifugal elutriation (33, 34). Decoy oligonucleotide transfection PBMs purified by these two methods were identical in response to IL-13 and consistently .95% CD14+. These studies complied with all relevant Double-stranded decoy ODN containing the conserved promoter binding federal guidelines and institutional policies regarding the use of human site of the CREB, early growth response element-1 (Egr-1), and a scram- subjects. bled sequence were prepared from complementary single-stranded phos- phorothioate-modified oligonucleotides (ordered from Invitrogen) by melt- Immunoprecipitation and immunoblotting ing at 95˚C for 5 min, followed by a cooldown phase overnight. Human monocytes were plated in six-well culture plates overnight. Cells were then 3 6 PBMs (5 10 cells/well in 2 ml 10% BCS/DMEM) were either directly transfected with decoy ODNs using Superfect Transfection Reagent (Qia- treated with IL-13 (1 or 2 nM) or pretreated with pharmacological in- gen, Valencia, CA) according to the manufacturer’s instructions for 24 h. hibitors (30 min) followed by IL-13 treatment for different time intervals Monocytes were then incubated in the absence or presence of IL-13 for as indicated. Total, cytosolic, and nuclear extracts were prepared by pre- another 24 h for 15-LO mRNA quantification or 48 h for 15-LO protein viously published protocols (20, 35, 36). For the preparation of cytosolic detection. The single-stranded sequences of the decoy ODNs were as fol- and nuclear extracts, we washed primary human monocytes with 10 ml lows: CREB (consensus): sense, 59-AGA GAT TGC CTG ACG TCA GAG PBS (two times) to remove the traces of BCS/DMEM and pelleted by AGC TA-39; Egr-1 (consensus): sense, 59-CCC GGC GCG GGG GCG ATT m centrifugation. The cell pellet was resuspended in 200 l cold buffer A (10 TCG AGT C-39; and scrambled: sense, 59-AAC AGA AGC CAG GAA CCC mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 1mM TCC TCT-39, adapted from Grote et al. (38). PMSF, 0.1% Nonidet P-40, and protease inhibitor mixture [1:50 dilution]), kept on ice for 5–10 min, and centrifuged for 1 min in a microfuge. The RNA extraction and quantitative real-time PCR analysis supernatant (cytosolic extract) was transferred to a fresh tube. The nuclear pellet was resuspended in 100 ml ice-cold buffer C (20 mM HEPES [pH Monocytes (5 3 106 in 2 ml 10% BCS/DMEM) were plated in six-well 7.9], 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, 1mM PMSF, and protease culture plates. Two hours after plating, cells were treated with IL-13 for 24 inhibitor mixture [1:50 dilution]), vortexed for 10–15 s, kept on ice for 15 h. In some experiments, monocytes were pretreated with MEK inhibitors min, and centrifuged at 10,000 3 g for 10 min. The supernatant (nuclear (PD98059 and U0126) for 30 min or transfected with decoy ODNs as extract) was saved at 280˚C. Using our method of fractionation, we found described above followed by IL-13 treatment for 24 h. Total cellular RNA that proliferating cell nuclear Ag (PCNA) or cyclin appeared in the nuclear was extracted using the RNeasy mini kit from Qiagen. The cDNA was fraction but not in the cytosolic fraction, whereas b-tubulin appeared in the prepared by reverse transcription of 1 mg total RNA using random hex- cytosolic fraction but not in the nuclear fraction (22). After determining the amers as primers (Roche, Branchburg, NJ). One hundred nanograms protein concentration using the Bio-Rad protein assay reagent (Hercu- cDNA was used for quantification by RT-PCR using specific primers listed les, CA), lysate proteins (50 mg/lane) were resolved by 8% SDS-PAGE, in Table I. Human GAPDH gene was used as an internal standard for The Journal of Immunology 5213 sample normalization. RT-PCR reactions were performed in duplicate on sequence of the sense strand of this control competitor (CRE BS3C-15LO an ABI PRISM 7000 sequence detection system (Applied Biosystems, sense) was 59-TCT CCC TGA CGT CAA GAT AG-39. As a non- Foster City, CA) using SYBR Green PCR core reagent according to competitor, a mutated sequence of the cognate CRE- in the established protocols (21). The relative levels of 15-LO mRNA were cal- context of the same flanking sequences present in CRE BS3-15LO (CRE culated using the comparative cycle threshold method (DDCt) (39). BS3 MT-15LO) was used in this assay. The sequence of the sense strand of this noncompetitor (CRE BS3 MT-15LO) was 59-TCT CCC GAC CAT TransMax EGR activation assay AGA GAT AG-39. The detection of activated CREB was carried out by A TransMax EGR transcription factor assay kit from Genlantis (San Diego, monitoring the colorimetric readout at 450 nm with a reference wave- CA) was used to allow the rapid analysis of EGR-1 activation (DNA length of 655 nm using a Synergy HT Multi-Mode Microplate Reader from BioTek (Winooski, VT). binding activity) from nuclear extracts using the manufacturer’s protocol. This kit offers a chemiluminescence-based assay that specifically recog- Data analysis nizes EGR-1 DNA binding to its consensus DNA binding sequence (59- CGCCCCCGC-39) using a streptavidin-alkaline phosphatase conjugate The number of experiments analyzed is indicated in each figure. Band and a chemiluminescent substrate. The amount of signal (light intensity) intensities were quantified by densitometric analyses using a laser densi- generated is proportional to the amount of EGR-1 in the nuclear extract tometer (Microtek ScanMaker 8700, Microtek Lab, Cerritos, CA) and and was detected using a microplate luminometer (Multilabel Counter National Institutes of Health ImageQuant software (Bethesda, MD). Dif- Victor3, PerkinElmer, Wellesley, MA). Two reagent controls were included ferences among experiment groups were analyzed using one-way ANOVA, in every assay. The positive reagent control provides the maximum signal followed by Tukey’s multiple comparison tests to identify each group for each site, and the negative reagent control provides the background differences. Comparisons between two groups were done with Student t signal for each site. The results are expressed as relative light units after test analysis. All statistical analyses were performed with a GraphPad background was subtracted. Prism4 program (GraphPad, San Diego, CA), and p , 0.05 was considered statistically significant.

Transcription factor ELISA assay to quantitate the binding Downloaded from activity of EGR1 Results A TF ELISA EGR1 kit from Panomics (Panomics, Santa Clara, CA) was IL-13 stimulates the phosphorylation of ERK1/2 in primary used to assess the binding activity of EGR1 to the cognate sequence human monocytes identified within the 15-LO promoter according to the manufacturer’s instructions. In brief, activated EGR1 molecules from nuclear extracts Our previous studies demonstrated the involvement of different bound to an EGR1 consensus binding site (EGR1 probe, 59-GCGG- Ser/Thr kinases, including p38MAPK and PKCd in regulating 15- GGGCG-39) on a biotinylated oligonucleotide were immobilized on a LO expression in response to IL-13 stimulation (21, 22, 40). We http://www.jimmunol.org/ streptavidin-coated 96-well assay plate. The EGR1, bound to the oligo- were also interested in exploring the role of ERK1/2 (p44/p42 nucleotide, was detected by an Ab against EGR1, followed by an HRP- conjugated secondary Ab reaction with the tetramethylbenzidine substrate MAPK) signaling on IL-13 induction of 15-LO expression in to provide a colorimetric readout, which was taken at 450 nm. Nonlabeled primary human monocytes. To investigate the phosphorylation/ EGR1 Cold Probe (EGR1 CP) was used in this assay as a negative control activation status of ERK1/2 poststimulation of primary mono- to measure competition of the labeled probe. cytes with recombinant human IL-13 for 5, 15, 30, 60, and 120 For competitive binding experiments, oligodeoxyribonucleotides cor- min, we used a phospho-ERK1/2 Ab that specifically recognized responding to the cognate EGR1-binding site (as predicted by Genomatix, 202 204 http://www.genomatix.de) in the context of 15-LO promoter flanking the phosphorylated (Thr and Tyr ) form of ERK1/2. Our sequences (EGR1 BS-15LO) were used in this assay. The single-stranded Western analysis data of phospho-ERK1/2 and total-ERK1/2 are by guest on October 2, 2021 sequence of this competitor was as follows: EGR1 BS-15LO sense, 59- shown in Fig. 1A. We have also provided the densitometric 9 GAG AAC AGC AGG GGC GGC GGG GGA-3 . As a control competitor, analysis data for the ratio of phospho-ERK1/2 to total-ERK1/2 in we used the consensus EGR1-binding site in the context of flanking sequences present in the 15-LO promoter (EGR1 BSC-15LO). The se- a time-dependent manner (Fig. 1B). Our time-course experiment quence of the sense strand of this control competitor (EGR1 BSC-15LO for IL-13 induction of ERK1/2 phosphorylation (Fig. 1A) dem- sense) was 59-GAG AAC AGC GGG GGC GGC GGG GGA-39.As onstrated that the level of active ERK1/2 increased rapidly after 5 a noncompetitor, a mutated (MT) sequence of the cognate EGR1-binding min of IL-13 stimulation, declined after 5 min, and a second peak site in the context of 15-LO promoter flanking sequences (EGR1 BS MT-15LO) was used. The sequence of the sense strand of this non- of activation appeared after 60 min of IL-13 stimulation. We ob- competitor (EGR1 BS MT-15LO sense) was 59-GAG AAC AAA TTG served no significant difference in total ERK1/2 expression levels GGC GGC GGG GGA-39. All of these ODNs were purchased from In- upon IL-13 stimulation across the time points. These data suggest tegrated DNA Technologies (Coralville, IA). The biotinylated EGR1 probe that in addition to p38MAPK and PKCd (21, 40), a rapid biphasic concentration was 250 nM in the final reaction, whereas the final con- activation of ERK1/2 (another important member of the MAPK centration for the competitors and noncompetitors was 10 mM. family) is also mediated by IL-13 stimulation of primary human TransAM pCREB activity assay monocytes. ATransAM pCREB kit from Active Motif (Carlsbad, CA) was used to evalu- ERK1/2 regulates IL-13–stimulated 15-LO gene expression in ate the cAMP-responsive element (CRE) promoter-binding activity of ac- monocytes tivated (phosphorylated) CREB accordingto the manufacturer’s instructions. Nuclear extracts were incubated in a 96-well assay plate precoated with im- Because IL-13 stimulated ERK1/2 activation in primary human mobilized oligonucleotides containing the CREB consensus sequence (59- monocytes, we next examined whether ERK1/2 activity was re- TGACGTCA-39). The wild-type (WT) consensus oligonucleotide was pro- vided with the kit, which prevents CREB binding to the probe immobilized quired for IL-13–induced expression of 15-LO mRNA and protein. on the plate. Conversely, the MT oligonucleotide was provided as a control To investigate the role of ERK1/2 activity in regulating IL-13 and expected to have a limited effect on CREB binding. induction of 15-LO expression at the mRNA level, we used two For competitive binding experiments, different oligodeoxyribonucleo- inhibitors of the upstream kinase that regulates ERK1/2 phos- tides corresponding to the cognate CRE-binding sites (as predicted by phorylation and activation. Monocytes were pretreated with either Genomatix, http://www.genomatix.de) in the context of 15-LO promoter flanking sequences (CRE BS1-15LO, CRE BS2-15LO, and CRE BS3- MEK inhibitor PD98059 (Fig. 2A) or U0126 (Fig. 2B) at several 15LO) were used in this assay. The single-stranded sequences of these indicated doses for 30 min followed by incubation with IL-13 for competitor ODNs were as follows: CRE BS1-15LO sense, 59-TCC ATG an additional 24 h. Total RNA was extracted and subjected to CCA TGA AGT TTA TGT TAG TAT TC-39; CRE BS2-15LO sense, 59- quantitative real-time RT-PCR using the primers listed in Table I ACA CGT GCA TAA CTC CTA CCC CCA-39; and CRE BS3-15LO sense, 59-TCT CCC TCC CGT CAA GAT AG-39. As a control competi- to quantify the expression of 15-LO mRNA. As expected, the tor, we used the consensus CRE-binding site in the context of the same quantitative real-time RT-PCR experiments showed that incu- flanking sequences present in CRE BS3-15LO (CRE BS3C-15LO). The bation of monocytes with IL-13 for 24 h substantially induced 5214 ERK1/2 REGULATES 15-LO EXPRESSION

ERK1/2 protein expression level by the antisense ODNs was also verified by reprobing the same blot with an Ab against total ERK1/2 (Fig. 2E, middle panel). Our results further show that IL-13 induction of 15-LO mRNA expression was significantly inhibited (pp , 0.025) by the treatment of monocytes with ERK1/ 2-specific antisense ODNs (Fig. 2F). The transfection control as well as the scrambled ODNs to ERK1/2 showed no inhibition of IL-13–induced 15-LO mRNA expression (Fig. 2F). These data confirm the results presented in Fig. 2A–D, in which pharmaco- logical inhibitors were employed and indicate that ERK1/2 expression/activity is required for IL-13–stimulated 15-LO gene expression. Tyk2 is required for IL-13–mediated ERK1/2 activation Results from our previously published studies clearly established the requirement of both Jak2 and Tyk2 kinases for the IL-13– mediated signaling, leading to the expression of 15-LO in primary human monocytes (2). In the heterodimeric IL-13R, Tyk2 is as- sociated with IL-13Ra1 and Jak2 is associated with IL-4Ra. Be- cause we demonstrated that IL-13 stimulated ERK1/2 activation in Downloaded from FIGURE 1. IL-13 induces phosphorylation of ERK1/2 in primary hu- human monocytes and activation of ERK1/2 was required for IL- man PBMs. To determine the role of IL-13 incubation time on ERK1/2 13–stimulated 15-LO gene expression, we next investigated the phosphorylation, human PBMs were incubated with 2 nM IL-13 for 5, 15, requirement for Jak2 and Tyk2 kinases for the IL-13–induced 30, 60, and 120 min. The monocyte cell lysates were immunoblotted with signaling pathways leading to the activation of ERK1/2 in primary 202 204 Abs that recognize the phosphorylated Thr and Tyr of ERK1/2 fol- monocytes. For these studies, monocytes were treated with anti- lowed by stripping and reprobing the same blot with an Ab recognizing sense or sense ODN against Jak2 or Tyk2 kinases for 48 h in http://www.jimmunol.org/ total ERK1/2. A, Western blot image of phosphorylated ERK1/2 and total ERK1/2 (representative experiment). B, The densitometric quantification presence of Mirus TransIt-oligo transfection reagent (Mirus Bio). of phospho-ERK1/2 to total ERK1/2 in IL-13–treated monocytes relative Posttreatment, the cells were exposed to IL-13 for another 30 min. to untreated monocytes. The data represent the mean 6 data range from Total cell lysates were extracted, and ERK1/2 phosphorylation/ two independent experiments. activation was evaluated. The results in Fig. 3A show that the antisense ODN (2 mM) against Tyk2 kinase inhibited the IL- 15-LO mRNA expression (.4000-fold [212]). Treatment with 13–mediated phosphorylation/activation of ERK1/2, whereas the PD98059 and U0126 both had profound, dose-dependent in- sense ODN and the vehicle controls had no effect on the activation hibitory effects on 15-LO mRNA levels. Cell viability was not of ERK1/2. Antisense ODN inhibition of Tyk2 protein expression by guest on October 2, 2021 affected by these inhibitors at the specified doses and times of level was also verified by reprobing the same blot with an Ab treatment. The highest indicated doses of those inhibitors down- against Tyk2 kinase (Fig. 3A, middle panel). These data thus in- regulated 15-LO mRNA expression almost completely after 24 h dicate that Tyk2 is the upstream receptor-associated tyrosine ki- of IL-13 treatment (Fig. 2A,2B). The mRNA levels of GAPDH nase of ERK1/2 phosphorylation/activation in human monocytes (used as control) were nearly identical in all the samples, in- in response to IL-13 stimulation. dicating specificity of the response. We next evaluated the effect of MEK/ERK1/2 inhibitors Jak2 is not involved in regulating the IL-13–stimulated PD98059 and U0126 on IL-13–induced 15-LO protein expression. activation of ERK1/2 Primary monocytes were incubated for 24 h, with or without IL-13, Next, we examined whether inhibition of Jak2 by the antisense in the presence or absence of these pharmacological inhibitors. ODN against Jak2 kinase also altered the IL-13–induced activation Postincubation, the monocytes were harvested and lysed, and 15- of ERK1/2. Although our results indicated substantial inhibition LO protein expression was detected on Western blots. The results, of Jak2 expression in monocytes treated with antisense to Jak2 (2 shown in Fig. 2C and 2D, indicate that both MEK inhibitors sup- mM) (Fig. 3B, middle panel), the reduced expression level of Jak2 pressed expression of 15-LO protein in a dose-dependent manner. had no inhibitory effect on the IL-13–induced activation of ERK1/ PD98059 treatment at 25–50 mM concentration and U0126 treat- 2 (Fig. 3B, upper panel). The bottom panel in Fig. 3B indicates ment at 20 mM concentration caused almost total inhibition of IL- nearly equal level of total ERK1/2 in all the lanes. These results 13–stimulated 15-LO protein expression. These findings present thus indicate that Jak2 is not required for IL-13–mediated acti- evidence that ERK1/2 activation is required for 15-LO gene ex- vation of ERK1/2 and ERK1/2-mediated regulation of 15-LO ex- pression in IL-13–treated human PBMs. pression in IL-13–treated primary human monocytes. To further test the direct involvement of ERK1/2 in regulating d IL-13–induced 15-LO gene expression, we blocked ERK1/2 ex- ERK1/2 does not regulate p38MAPK or PKC activation/ pression by treating monocytes with ERK1/2-specific antisense phosphorylation in human monocytes ODNs before IL-13 addition. As a control, scrambled ODNs to Previously, we demonstrated that activation of PKCd and p38MAPK ERK1/2 were used. Total cellular RNA or proteins were extracted pathways are independent, but they work in parallel to regulate IL- for real time RT-PCR or Western blot analysis, respectively. Our 13–induced 15-LO expression in human monocytes (40). To in- results, presented in Fig. 2E, indicate that the antisense ODN (2 vestigate whether ERK1/2 activity has any role in controlling the mM) against ERK1/2 profoundly inhibited the IL-13–stimulated activation of p38MAPK and/or PKCd and vice versa in IL-13– 15-LO protein expression (∼60%), whereas the scrambled ODNs stimulated monocytes, we performed a series of experiments using and the vehicle control had no effect on IL-13–induced 15-LO the selective pharmacological inhibitors of ERK1/2, p38MAPK, and protein expression (Fig. 2E, upper panel). Downregulation of PKCd followed by IL-13 stimulation in primary human monocytes. The Journal of Immunology 5215 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. ERK1/2 activity/expression is required for IL-13–induced 15-LO gene expression in primary human monocytes. Primary human monocytes (5 3 106/group) were pretreated with MEK inhibitors PD98059 (A, C, D) or U0126 (B, D) at various indicated doses for 30 min, followed by stimulation with 1 nM IL-13 for 24 h. In A and B, total cellular RNA was extracted, and RNA (1 mg) from each sample was subjected to quantitative real-time RT-PCR analysis. Amplification plots of real-time RT-PCR analysis showed the regulatory effect of ERK1/2 activity on IL-13 induction of 15-LO mRNA ex- pression. Postnormalization with GAPDH amplification, the fold induction of 15-LO mRNA expression for different groups was plotted. Data were collected from two independent experiments and shown as the mean 6 data range. In C and D, monocytes were harvested and lysed; 50 mg lysates were resolved by 8% SDS-PAGE, and 15-LO protein expression was detected on Western blots with a 15-LO–specific Ab. The blots were then stripped and reprobed with b-tubulin (C, D, lower panels) to assess equal loading. The arrows indicate the positions of 15-LO and b-tubulin based on the migration of 15-LO positive control and m.w. markers in adjacent lanes. Data in C and D are from representative experiments of three identical experiments that were performed. To show the direct involvement of ERK1/2 on IL-13–induced 15-LO mRNA and protein expression, human blood monocytes were transfected with or without 2 mM antisense or scrambled ODNs to ERK1/2 (E, F) according to protocols described in Materials and Methods prior to the addition of IL-13 (2 nM) for 24 h. In E, Total of 50 mg monocyte lysates were separated by SDS-PAGE and immunoblotted with a 15-LO–specific Ab (upper panel). The blot was reprobed with a total ERK1/2 Ab to examine the effect of antisense ODN on total ERK1/2 expression (middle panel). The same blot was then stripped and reprobed with b-tubulin Ab (lower panel) as a loading control. Data are from a representative experiment of three repeat experiments that showed similar results. In F, 15-LO mRNA expression of monocytes was detected. The fold induction of 15-LO mRNA expression for different groups was shown after correcting for the GAPDH mRNA levels. 15-LO mRNA and GAPDH mRNA (internal control) were quantified by real-time quantitative PCR analysis. Results are from a representative experiment of three performed in which data are the mean 6 SD (n = 3). Significant differences were determined by comparing the antisense or scrambled ODN (to ERK1/2)-treated groups to the transfection control (IL-13 treated). pp , 0.025.

Cells were preincubated with various doses of MEK/ERK1/2 in- was evaluated. As presented in Fig. 4A, pretreatment with U0126 or hibitors PD98059 and U0126 prior to IL-13 stimulation. Total cell PD98059 at a dose (10–20 mM and 50 mM, respectively) that sub- lysates were extracted, and p38MAPK phosphorylation/activation stantially inhibited ERK1/2 activation (41, 42) and 15-LO expres-

Table I. Primer list

Gene Forward Primer Reverse Primer 15LOX 59-GCTGGAAGGATCTAGATGACT-39 59-TGGCTACAGAGAATGACGTTG-39 GAPDH 59-CACCAACTGCTTAGCACCCC-39 59-TGGTCATGAGTCCTTCCACG-39 5216 ERK1/2 REGULATES 15-LO EXPRESSION

IL-13 induces Egr-1 expression and nuclear accumulation in human monocytes Because ERK1/2 activation by IL-13 stimulation was required for expression of 15-LO in primary monocytes, we investigated the effect of ERK1/2 in regulating transcription factors that might control 15-LO expression. In silico analysis of the 15-LO promoter (as predicted by Genomatix; http://www.genomatix.de) demon- strated that Elk1, Egr-1, and CREB transcription factor binding sites are present in the 15-LO promoter. We therefore investigated the role of IL-13 and ERK1/2 in stimulating these transcription factors in primary human PBMs. We observed that Elk1 is poorly expressed in primary mono- cytes. It was difficult to detect by Western analysis (data not shown). Next, we analyzed whether Egr-1 is expressed in human blood monocytes and whether IL-13 induced Egr-1 expression in monocytes. Our results showed a significant level of Egr-1 ex- pression in total cell lysates from monocytes (data not shown). To investigate the IL-13 induction of Egr-1 expression, we evaluated

both cytosolic and nuclear fractions. Although IL-13 induced Egr-1 Downloaded from protein expression in the cytosol (Fig. 5A, upper panel), the level of expression was modest. In contrast, Egr-1 nuclear accumulation was substantially induced by IL-13 (Fig. 5A, upper panel). Time- course experiments demonstrated that IL-13 significantly stimu- lated Egr-1 nuclear accumulation in a time-dependent manner. The maximal level of accumulation of Egr-1 in the nucleus was FIGURE 3. Tyk2 but not Jak2 antisense treatment inhibits phosphory- http://www.jimmunol.org/ lation/activation of ERK1/2 poststimulation with IL-13 in human mono- reached at 1 h and then declined (Fig. 5B, upper panel). We cytes. Monocytes were pretreated with antisense or sense ODNs to either verified nuclear localization of Egr-1 by evaluating the purity of Tyk2 (A) or Jak2 (B) according to protocols described in Materials and our nuclear preparation as described previously (22). The same Methods prior to the addition of IL-13 (2 nM) for 30 min. The cells were blot was reprobed with PCNA Ab (used as a nuclear marker) to lysed, and 50 mg cell extracts (from each sample group) were separated by assess equal loading (Fig. 5B, lower panel). SDS-PAGE and immunoblotted with anti–phospho-ERK1/2 Ab (A, B, upper panels). Blots were then reprobed with either Tyk2 or Jak2 Abs to CREB activation rather than expression is stimulated by IL-13 examine the effect of antisense ODN on Tyk2 and Jak2 expression (A, B, treatment in primary human monocytes middle panels, respectively). The same blots were stripped and reprobed

Like Egr-1, we also observed substantial levels of CREB ex- by guest on October 2, 2021 with an Ab recognizing total ERK1/2 to assess equal loading (A, B, lower pression in total cell lysates from monocytes (data not shown). To panels). The results are representative of three independent experiments. examine whether the expression/activation of CREB is modulated by IL-13, we first checked the effect of IL-13 treatment on CREB expression in monocytes. CREB was hardly expressed in the sion (Fig. 2) had no detectable effect on IL-13–induced p38 MAPK cytosol, whereas the expression level of CREB was significant in phosphorylation (Fig. 4A). These data suggest that p38MAPK is the nuclear extract. IL-13 treatment failed to induce CREB ex- not a downstream target of ERK1/2 in the IL-13–induced signaling pression in either the cytosolic or nuclear fraction (Fig. 5A, lower pathway in human monocytes. panel). We next evaluated the ability of IL-13 to induce the ac- To determine whether p38MAPK was upstream of ERK1/2, we tivation of CREB (phosphorylation at Ser133), which has been treated cells with the p38MAPK inhibitor SB202190 prior to IL-13 reported as one of the major downstream targets of ERK1/2 (25, activation and evaluated ERK1/2 activation/phosphorylation. Pre- 45–49). Our results clearly indicate that IL-13 stimulated Ser133 treatment with SB202190 at a dose that significantly inhibited phosphorylation of CREB in a time-dependent manner in the p38MAPK activity and IL-13–induced 15-LO expression (21, 43) nuclear fraction (Fig. 5C, upper panel). We observed a transient caused no inhibition of IL-13–induced activation/phosphorylation phosphorylation of CREB that peaked at 1 h after IL-13 treat- of ERK1/2 (Fig. 4B). ment, with the signal diminishing thereafter. Nuclear localization In a similar experiment, we preincubated cells with various indi- of CREB activation (Ser133 phosphorylation) was further con- cated doses of U0126 followed by IL-13 activation. Pretreatment firmed by evaluating our method of fractionation as described of monocytes with U0126 (even at 20 mM concentration) could earlier (22). Equal loading was also verified by reprobing the blot 505 not block the IL-13–induced Thr phosphorylation of PKCd with anti-PCNA Ab (Fig. 5C, lower panel). (Fig. 4C). In a reciprocal experiment, we pretreated cells with the PKCd selective inhibitor rottlerin (different doses) for 30 min be- IL-13 induces nuclear translocation of ERK1/2 fore IL-13 stimulation for another 30 min. Total cell lysates were Our results revealed that IL-13 treatment had no effect on the ERK1/ extracted, and ERK1/2 phosphorylation/activation was evaluated. 2 protein expression levels in the cytosol (Fig. 5D, upper panel), As presented in Fig. 4D, pretreatment with rottlerin at a dose but it strongly facilitated the nuclear import of ERK1/2 (Fig. 5D, (5 mM) that profoundly inhibited PKCd activation (44) and 15-LO lower panel). This result suggests that upon IL-13 stimulation, expression (40) had no significant effect on IL-13–induced ERK1/2 ERK1/2 molecules are dual-phosphorylated by upstream kinase activation/phosphorylation. These results together with our prior (MEK1/2) and translocate from the cytoplasm to the nucleus. published work (40) indicate that IL-13–mediated activation of Our results demonstrated a significant increase of ERK1/2 protein ERK1/2 is independent of p38MAPK and PKCd activation, yet in the nuclear extract within 1 h and remained stationary thereafter both are required to act in parallel to regulate 15-LO expression. until the 4-h time point (Fig. 5D, lower panel). The maximal level The Journal of Immunology 5217 Downloaded from FIGURE 4. Activation of ERK1/2 by IL-13 is independent of both the p38MAPK and PKCd pathways. Human monocytes 5 3 106/group (A, B, D) and 10 3 106/group (C) were pretreated with MEK inhibitors U0126 (A, C) and PD98059 (A, lower panel), p38MAPK inhibitor SB202190 (B), and the PKCd selective inhibitor rottlerin (D) at different indicated doses for 30 min, pre-exposure to IL-13 (1 nM) for 15 min (A, C) or 30 min (B, D). A total of 50 mg cell lysate was subjected to Western blot analysis (A, B, D) using anti–phospho-p38MAPK Ab (A) and anti–phospho-ERK1/2 Ab (B, D). The membrane was stripped and reprobed with anti-ERK1/2 Ab to assess equal loading (B, D, lower panels). Whole-cell lysates were immunoprecipitated with an Ab against PKCd (C). The immunoprecipitates were then resolved by 8% SDS-PAGE for immunoblotting with an anti–phospho-Thr505 PKCd Ab (C, upper panel). The blot was stripped and reprobed with PKCd Ab (C, lower panel) to confirm equal immunoprecipitation. Data are from a representative ex- http://www.jimmunol.org/ periment of four repeat experiments that showed similar results. of IL-13–driven nuclear import of ERK1/2 (between 1 and 4 h) reduction of nuclear Egr-1 levels compared with cells treated with coincides with the peak expression level of Egr-1 (1 h) and peak IL-13 alone (∼72%; Fig. 6A, lower panel,6B, upper panel). IL-13– phosphorylation level of CREB (1 h) in the nuclear extract. induced nuclear accumulation of Egr-1 was almost completely abolished (∼97%) in the presence of 20 mM concentration of IL-13 induction of Egr-1 nuclear translocation requires ERK1/2 U0126 (Fig. 6A, lower panel,6B, upper panel). These results activity in primary human monocytes suggest that the MEK–ERK1/2 MAPK pathway is an important To determine whether the MEK–ERK1/2 MAPK pathway was regulator of IL-13–mediated induction of nuclear Egr-1 levels. In- by guest on October 2, 2021 linked to IL-13–mediated upregulation of Egr-1 nuclear translo- terestingly, pretreatment of monocytes with the same pharmaco- cation, monocytes were pretreated with MEK/ERK1/2 inhibitors logical inhibitors (with the same indicated doses) did not inhibit the PD98059 (12.5–25 mM) and U0126 (20 mM) prior to incubation IL-13–stimulated Egr-1 protein expression level (∼4-fold increased with IL-13. Our results, presented in Fig. 6A (lower panel)and6B level of expression compared with the untreated control) in the (upper panel), indicate that IL-13 substantially stimulated the level cytosolic extracts (Fig. 6A, upper panel). of accumulation of Egr-1 in the nucleus (∼35–40-fold enhanced To explore the specific role of ERK1/2 in regulating IL-13– level of nuclear accumulation of Egr-1 compared with the untreated stimulated Egr-1 nuclear accumulation, the effect of the p38MAPK control). Monocytes pre-exposed to PD98059 displayed significant specific inhibitor SB202190 (10 mM) was again investigated.

FIGURE 5. IL-13 stimulates Egr-1 expression/ nuclear accumulation, CREB serine phosphoryla- tion, and nuclear translocation of ERK1/2. Freshly isolated human blood monocytes (10 3 106/group) were left untreated or directly treated with IL-13 (1 nM) for either 1 h (A) or for different time inter- vals as indicated (B–D). Cytosolic (40 mg/lane) and nuclear extracts (10 mg/lane) were resolved by 8% SDS-PAGE and subjected to immunoblotting using Abs against Egr-1 (A, upper panel, B, upper panel), CREB (A, lower panel), phospho-CREB (Ser133) (C, upper panel), and ERK1/2 (D). In B and C, the same blots were stripped and reprobed using Ab against PCNA (lower panels) to assess equal load- ing. Results are from a representative experiment of three performed. 5218 ERK1/2 REGULATES 15-LO EXPRESSION

SB202190 did not inhibit IL-13–induced Egr-1 nuclear accumu- lation in primary monocytes (Fig. 6B, upper panel). ERK1/2- dependent accumulation of Egr-1 in the nuclear compartment was verified as described previously (22). Equal loading of the nuclear fraction was assessed by reprobing the same blot with the nuclear marker PCNA (Fig. 6B, lower panel). Tyk2 is involved in IL-13–induced nuclear accumulation of Egr-1 As Tyk2 is the upstream regulator of ERK1/2 phosphorylation/ activation in IL-13– stimulated human monocytes, we further in- vestigated whether Tyk2 is required for Egr-1 nuclear accumula- tion in response to IL-13 stimulation. Our results, presented in Fig. 6C (upper panel), indicate that antisense ODNs (2 mM) against Tyk2 almost completely inhibited the IL-13–induced Egr-1 nu- clear accumulation, whereas the Tyk2 sense ODNs and the trans- fection control had no inhibitory effect on the level of accumu- lation of Egr-1 in the nucleus. By using the antisense ODNs against Jak2 in the same experiment, we further confirmed that Jak2 is not involved in IL-13–stimulated nuclear accumulation of Downloaded from Egr-1 (Fig. 6C, upper panel). Equal loading of the nuclear fraction was further assessed by reprobing the same blot with PCNA (Fig. 6C, lower panel). The specificity and effectiveness of these anti- senses have been documented previously by our laboratory and are also shown in Fig. 3. http://www.jimmunol.org/ IL-13–induced DNA binding activity of Egr-1 transcription factor requires ERK1/2 kinase activity in human monocytes We next investigated the requirement of ERK1/2 kinase activity in mediating the IL-13–stimulated binding of Egr-1 to its consensus DNA binding sequence. We used a chemiluminescence-based TransMax Egr transcription factor assay method to evaluate the binding activity of Egr-1 in response to IL-13 stimulation. Our results demonstrated that Egr-1 has a basal level of binding ac- tivity to its consensus DNA binding sequence that was en- by guest on October 2, 2021 hanced (.2-fold) by IL-13 treatment (Fig. 6D). IL-13 induction of Egr-1 DNA binding activity was inhibited by the pretreatment of monocytes with MEK/ERK1/2 inhibitors PD98059 and U0126 in a dose-dependent manner. PD98059 at 25 mM and U0126 at 20 mM concentration brought back the IL-13–stimulated Egr-1 DNA binding activity almost to the basal level (Fig. 6D). In con- trast, the p38MAPK inhibitor SB202190 at 10 mM concentration showed no inhibition on IL-13–stimulated binding of activated Egr-1 to its nascent DNA binding sequence in primary monocytes (Fig. FIGURE 6. IL-13–induced Egr-1 nuclear accumulation and Egr-1 ac- 6D). These results implicate a specific role of ERK1/2 kinase acti- tivation (DNA binding activity) requires Erk1/2 activity in human mono- vity in regulating Egr-1 DNA binding activity in response to IL-13 6 6 cytes. Human monocytes 10 3 10 /group (A, B) and 5 3 10 /group (D) stimulation. were pretreated with MEK inhibitors PD98059 and U0126 (at different indicated doses) (A, B, D) and p38MAPK inhibitor SB202190 (10 mM) (B, ERK1/2 activity is required for CREB activation in D) for 30 min, followed by IL-13 stimulation (1 nM) for 1 h or directly IL-13–stimulated monocytes stimulated with IL-13 for the same period of time (A, B, D). Nuclear (20 To determine whether ERK1/2 was involved in the IL-13–stimu- mg/lane) (A, lower panel, B, upper panel) and cytosolic (40 mg/lane) (A, lated activation (Ser133 phosphorylation) of CREB, we also in- upper panel) extracts were separated by SDS-PAGE and immunoblotted with an Ab against Egr-1 (A, upper panel of B). The same blot was vestigated the effect of MEK/ERK1/2 inhibitors PD98059 and stripped and reprobed with anti-PCNA Ab (B, lower panel) as a loading U0126 on IL-13 induction of CREB serine phosphorylation. The control. In A, human rEgr-1 was used as a positive control. In C, mono- results of a representative experiment are shown in Fig. 7A (upper cytes (10 3 106/group) were pretreated with antisense or sense ODNs to panel). Monocytes were pretreated with these pharmacological Tyk2 and Jak2 according to protocols described in Materials and Methods inhibitors for 30 min prior to the addition of IL-13 and were either prior to the addition of IL-13 (2 nM) for 1 h. Nuclear extracts (20 mg/lane) treated or left untreated with IL-13 for 1 h and harvested. The were separated by 8% SDS-PAGE and immunoblotted with an Ab against serine phosphorylation status of CREB in the nuclear extract was Egr-1 (C, upper panel). The same blot was stripped and reprobed with anti- PCNA Ab (C, lower panel) as a loading control. The results are repre- sentative of three independent experiments. To detect Egr-1 activity (D), 1 Data are the mean 6 SD (n = 3). Significant differences were determined mg nuclear extracts/well were run in triplicate to measure the binding of by comparing each group to the IL-13–treated group as the control Egr-1 to its consensus DNA binding sequence. The positive and negative according to the Data analysis section under Materials and Methods. pp , x reagent control wells were also included in each assay and run in triplicate. 0.001; no significant difference. The Journal of Immunology 5219 determined by Western blots using a phosphoserine-CREB–spe- IL-13–induced monocytes. In contrast, the p38MAPK specific in- cific Ab. The results, presented in Fig. 7A (upper panel), indicate hibitor SB202190 (10 mM) demonstrated no inhibitory effect on that PD98059 inhibited IL-13–stimulated CREB Ser133 phos- IL-13 induction of CREB Ser133 phosphorylation in monocytes. phorylation in a dose-dependent manner. The degree of inhibition These results support a specific regulatory role of ERK1/2 in was significant (p , 0.0008) when we used 25 mM concentration mediating the activation (Ser133 phosphorylation) of CREB in the of PD98059. U0126 at 10 mM concentration also significantly nuclear extracts. Nuclear localization of IL-13–mediated CREB (p , 0.0006) downregulated Ser133 phosphorylation of CREB in Ser133 phosphorylation was verified by evaluating the purity of the nuclear extract as described previously (22). Equal loading of the nuclear fraction was further verified by reprobing the same blot with the nuclear marker PCNA (Fig. 7A, lower panel). Tyk2 is involved in IL-13–induced phosphorylation/activation of CREB As ERK1/2 activity regulates CREB phosphorylation/activation in IL-13–induced human monocytes, we further investigated whether Tyk2 is required for CREB serine phosphorylation/activation in the nucleus after IL-13 stimulation. Our results, shown in Fig. 7B (upper panel), demonstrate that downregulation of Tyk2 by using antisense ODNs (2 mM) against Tyk2 significantly reduced the IL- 13–induced CREB activation (Ser133 phosphorylation), whereas Downloaded from the Tyk2 sense ODNs and the transfection control had no in- hibitory effect on IL-13–mediated CREB serine phosphorylation/ activation in the nucleus. In contrast, downregulation of Jak2 by using the antisense ODNs directed to Jak2 showed no inhibitory effect on IL-13 induction of CREB Ser133 phosphorylation in

monocytes (Fig. 7B, upper panel). Equal loading of the nuclear http://www.jimmunol.org/ fraction was verified as before by reprobing the same blot with PCNA (Fig. 7B, lower panel). ERK1/2 activity is involved in IL-13–induced DNA binding activity of CREB in primary human monocytes After already demonstrating in Fig. 7A that IL-13 induction of CREB activation is regulated by the ERK1/2 kinase activity, we further examined the role of ERK1/2 in mediating the DNA binding activity of activated (phosphorylated) CREB in primary by guest on October 2, 2021 monocytes. Using an ELISA-based TransAM method and em- ploying specific phospho-CREB primary Ab, we showed that IL-13 treatment facilitated DNA binding activity of IL-13–stimulated CREB transcription factor (Fig. 7C). Furthermore, pretreatment of monocytes with MEK/ERK1/2 inhibitors PD98059 and U0126 for 30 min before IL-13 stimulation significantly attenuated the extent FIGURE 7. ERK1/2 regulates IL-13–stimulated CREB serine phos- of DNA binding activity of phosphorylated CREB in a dose- phorylation and CREB activation (CRE promoter site binding activity). dependent manner. The WT consensus oligonucleotides (used as Human monocytes (5 3 106/group) (A, C) were pretreated with MEK inhibitors PD98059 and U0126 (at different indicated doses) (A, C) and a competitor for CREB binding) also significantly reduced the p38MAPK inhibitor SB202190 (10 mM) (A, C) for 30 min, followed by CREB activation (Fig. 7C). Conversely, the p38MAPK inhibitor stimulation with 1 nM IL-13 for 1 h or directly exposed to IL-13 for the SB202190 at 10 mM concentration and the MT consensus oligo- same period of time (A, C). In case of A, nuclear extracts (10 mg/lane) were nucleotides showed negligible effects on IL-13–induced DNA resolved by 8% SDS-PAGE and subjected to immunoblotting using Abs binding activity of CREB in primary monocytes. These results against phospho-CREB (Ser133)(upper panel). The same blot was stripped indicate the involvement of ERK1/2 kinase activity in regulating and reprobed with anti-PCNA Ab (lower panel) to assess equal loading. In CREB activation and related DNA binding activity in response to 6 B, monocytes (10 3 10 /group) were pretreated with antisense or sense IL-13 stimulation. ODNs to Tyk2 and Jak2 as in Fig. 6C. Nuclear extracts (20 mg/lane) were separated by SDS-PAGE and immunoblotted with an Ab against phospho- In monocytes treated with IL-13, Egr-1 and CREB specifically CREB (Ser133)(B, upper panel). The same blot was stripped and reprobed with anti-PCNA Ab (B, lower panel) as a loading control. The results are bind to their cognate sequences derived from the 15-LO representative of three repeat experiments. In case of pCREB activation promoter (C), 5 mg nuclear extracts/well were run in duplicate to perform an To demonstrate that Egr-1 and CREB transcription factors can bind immunodetection of activated CREB. Nuclear extracts from forskolin- to their cognate DNA binding sites present in the 15-LO promoter stimulated WI-38 cells were used as a positive control for activated CREB. after IL-13 stimulation, we performed competitive binding exper- The WT and MT consensus oligonucleotides were used to monitor the iments using ELISA-based activation assays for Egr-1 and CREB. specificity of the assay. The results are representative of three independent experiments. Values are mean 6 SEM of three separate experiments. Our results indicated that the basal level of binding activity of Egr-1 ∼ Significant differences were determined by comparing each group to the to its consensus DNA binding sequence was enhanced ( 3-fold) by IL-13–treated monocytes as the control as described in the Data analysis IL-13 treatment (Fig. 8A). EGR1 CP completely abolished the section under Materials and Methods. pp , 0.05; ppp , 0.001; xno sig- elevated level of EGR1 activation after IL-13 stimulation. EGR1 nificant difference. BS-15LO, oligodeoxyribonucleotides corresponding to the cog- 5220 ERK1/2 REGULATES 15-LO EXPRESSION nate EGR1-binding site in the context of 15-LO promoter flanking 15-LO promoter flanking sequences, was used as a noncompetitor sequences, was used as a competitor in this assay and significantly and showed no inhibition of IL-13–induced DNA binding activity inhibited (pp , 0.002) the IL-13–induced EGR1 activation. EGR1 of EGR1 in primary human monocytes (Fig. 8A). BSC-15LO, oligodeoxyribonucleotides containing the consensus For CREB activation, our results demonstrated that IL-13 EGR1-binding site in the context of flanking sequences present stimulated the binding activity of CREB to its consensus DNA in the 15-LO promoter, used as a control competitor for EGR1 binding sequence (.3-fold compared with the untreated control) binding in this assay, also significantly downregulated the EGR1 (Fig. 8B). For competitive binding, CRE BS1-15LO, CRE BS2- activation (Fig. 8A). In contrast, EGR1 BS MT-15LO, a mutated 15LO, and CRE BS3-15LO (three different oligonucleotides sequence of the cognate EGR1-binding site in the context of corresponding to the cognate cAMP-responsive element [CRE]- binding sites in the context of flanking sequences present in the 15-LO promoter) were added prior to addition of the nuclear ex- tract. Although CRE BS1-15LO and CRE BS2-15LO (used as competitors for CREB binding) showed some inhibitory effects on IL-13–induced DNA binding activity of CREB in primary monocytes, CRE BS3-15LO significantly attenuated the IL-13– induced CREB DNA binding (pp , 0.001) (Fig. 8B). CRE BS3C- 15LO (oligodeoxyribonucleotides containing the consensus CRE- binding site in the context of the same flanking sequences present in CRE BS3-15LO) was also used as a competitor for comparison. Addition of CRE BS3C-15LO in this assay profoundly down- Downloaded from regulated IL-13–stimulated CREB DNA binding (pp , 0.001). The WT consensus oligodeoxyribonucleotides also significantly reduced CREB DNA binding (Fig. 8B). Conversely, CRE BS3 MT-15LO (an MT sequence of the cognate CRE-binding site in the context of the same flanking sequences present in CRE BS3- 15LO) and the MT consensus oligodeoxyribonucleotides were http://www.jimmunol.org/ used as noncompetitors in this assay and caused essentially no inhibition of IL-13–induced DNA binding activity of CREB (Fig. 8B). These data show the direct binding of Egr-1 and CREB transcription factors to their cognate DNA binding sites in the context of flanking sequences present in the 15-LO promoter after IL-13 stimulation in primary monocytes. Egr-1 and CREB regulate IL-13–induced 15-LO expression in primary human monocytes by guest on October 2, 2021 We have demonstrated that IL-13 induced Egr-1 protein expres- sion, CREB activation, and Egr-1 and CREB DNA binding activity in primary human monocytes (Figs. 5–7). Our results further reveal that inhibition of IL-13–induced ERK1/2 activation by MEK/ ERK1/2 specific pharmacological inhibitors downregulated these effects (Figs. 6, 7). To investigate whether Egr-1 and CREB are the downstream target genes of ERK1/2 signaling that play a sig- nificant role on IL-13–induced 15-LO expression in human monocytes, we transfected monocytes with decoy ODNs specific for Egr-1 and CREB consensus sequences and evaluated their impact on 15-LO expression. After 24 h of transfection with the FIGURE 8. In IL-13–stimulated monocytes, Egr-1 and CREB bind to decoy ODNs, we stimulated the cells with IL-13 for an additional their cognate DNA binding sequences located in the 15-LO promoter. 24 or 48 h and quantified the mRNA and protein expression level Human monocytes (5 3 106/group) (A, B) were either untreated or directly of 15-LO relative to monocytes transfected with scrambled decoy stimulated with IL-13 (2 nM) for 1 h. To measure the binding of Egr-1 to its cognate DNA binding sequence present in the 15-LO promoter, 5 mg using quantitative real-time PCR and Western blot analysis, re- nuclear extracts/well were run in triplicate using Panomics TF ELISA kits spectively. 15-LO mRNA and protein expression levels were de- (Panomics) for EGR1 (A). EGR1 BS-15LO and EGR1 BSC-15LO were termined from these experiments and the results are shown in Fig. used as competitors in this assay, whereas EGR1 BS MT-15LO was used 9A and 9B, respectively. as a noncompetitor. EGR1 CP was provided by the kit and used at a con- Fig. 9A illustrates that transfection of monocytes with Egr-1 and centration of 10 mM in the final reaction. In B,5mg nuclear extracts/well CREB decoy ODNs (2 mM each) significantly inhibited IL-13– were run in triplicate to detect activated CREB using a TransAM pCREB induced 15-LO mRNA expression levels as compared with the kit. CRE BS1-15LO, CRE BS2-15LO, CRE BS3-15LO, and CRE BS3C- levels in monocytes transfected with scrambled decoy ODN (50% 15LO were used as competitors, whereas CRE BS3 MT-15LO was used as for Egr-1 and 78% for CREB). Cotransfection with a combination a noncompetitor. All of the competitors and noncompetitors were used at of both Egr-1 and CREB decoy ODNs (2 mM each) inhibited 15- a concentration of 20 pmol/well. In both A and B, the results are repre- sentative of three independent experiments. Data are the mean 6 SD (n = LO mRNA levels even further (90% inhibition with the combi- 3). Significant differences in both the experiments were determined by nation) as compared with the scrambled decoy ODN transfected comparing each group to the IL-13–treated monocytes as the control as monocytes. Transfection of monocytes in the absence of the decoy described in the Data analysis section under Materials and Methods. pp , ODNs (transfection control) as well as in the presence of the 0.002 in A; pp , 0.001 in B. scrambled decoy ODNs followed by IL-13 stimulation showed no The Journal of Immunology 5221

on understanding the mechanistic impact of the ERK1/2-dependent signaling cascade in IL-13–stimulated primary human monocytes. Earlier, our group showed that IL-13 induced the tyrosine phosphorylation of Jak2 and Tyk2 (IL-13R–associated tyrosine kinases) in human monocytes and that activation of both of these Jak kinases was essential for downstream signaling by IL-13, leading to the induction of 15-LO expression (2). We further demonstrated that Jak2 and Tyk2 are associated with IL-4Ra and IL-13Ra1, respectively, in the heterodimeric IL-13R complex (20). The results of our Jak2/Tyk2 antisense experiment thus clearly indicate that an IL-13Ra1–Tyk2–ERK1/2-dependent pathway ex- ists in human monocytes, which acts in parallel with the IL-4Ra– Jak2-dependent signaling cascade to regulate the 15-LO gene ex- pression in monocytes in response to IL-13 stimulation. Although our work focused on the involvement of IL-13Ra1- associated protein tyrosine kinase Tyk2 in mediating IL-13– dependent activation of ERK1/2 in primary monocytes, the sig- naling machinery triggered downstream of Tyk2 is still unknown. In the context of Tyk2-mediated signal transduction, it has been shown that Ras and Raf-1 (a critical downstream effector of Ras) Downloaded from FIGURE 9. The effect of Egr-1 and CREB decoy ODNs on IL-13– activity is essential for Tyk2-dependent mitogenesis and supports induced 15-LO mRNA and protein expression. To determine the role of the general importance of the protein–tyrosine kinase/Ras effector Egr-1 and CREB on IL-13–induced 15-LO mRNA and protein expression, pathways in mitogenic signal transduction (58). Taken together, human blood monocytes (5 3 106/group) were transfected with or without a highly probable scenario is that the Tyk2-dependent ERK1/2 2 mM scrambled, Egr-1, or CREB decoy ODNs or a combination of both activation by IL-13 utilizes the Ras–Raf–MEK1/2–ERK1/2 path- Egr-1 and CREB decoy ODNs at 1 or 2 mM for 24 h. Twenty-four hours way. This is the topic of future studies. http://www.jimmunol.org/ posttransfection, cells were stimulated with 2 nM IL-13 for 24 h (mRNA) We showed that IL-13 activation of the MEK–ERK1/2 pathway or 48 h (protein). A, 15-LO mRNA expression of monocytes was detected increased Egr-1 levels in the nucleus as well as DNA binding posttransfection with decoy ODNs as indicated. Data represent the percent activity and that both are required for maximal expression of 15- expression of 15-LO relative to monocytes transfected with the scrambled LO in primary monocytes. Egr-1 is a zinc finger transcription decoy ODNs after correcting for the GAPDH mRNA levels. 15-LO mRNA and GAPDH mRNA (internal control) were quantified by real-time quan- factor that plays a key master regulatory role in multiple cardio- titative PCR analysis. B, 15-LO and b-tubulin (control) protein expression vascular pathological processes including , cardiac of monocytes are shown posttransfection with decoy ODNs as indicated. hypertrophy, ischemia, and angiogenesis (59) and recently was Data shown are representative of three independent experiments and are described as a major link between infection and atherosclerosis by guest on October 2, 2021 presented as the mean 6 SEM. Significant differences were detected by (60, 61). Recent studies also demonstrated Egr-1 to be a key comparing the groups either to the scrambled decoy treated group or to the regulator in the pathogenesis of IL-13–induced inflammatory and Egr-1 decoy-treated (2 mM) group as described in the Data analysis sec- remodeling responses (62). Egr-1 activation via the MEK–ERK1/2 tion under Materials and Methods. pp , 0.001; ppp , 0.05. pathway has been reported to be involved in tissue factor ex- pression and TNF-a gene expression in human monocytes and THP-1 monocytic cells (63). Our results not only provide the first inhibition of 15-LO protein expression as compared with the evidence, to our knowledge, that IL-13 induces Egr-1 expression/ nontransfected monocytes induced by IL-13 (Fig. 9B); however, activity in primary human monocytes, but also demonstrate the similar to the 15-LO mRNA results shown in Fig. 9A, both Egr-1 detailed mechanism by which IL-13 stimulates Egr-1 nuclear and CREB decoy ODNs markedly attenuated IL-13–induced 15- accumulation and DNA binding activity. Our data clearly establish LO protein expression (Fig. 9B). Cotransfection of Egr-1 and a signaling pathway involving IL-13R, receptor-associated tyro- CREB decoy ODNs (1 mM each) significantly inhibited the IL- sine kinase Tyk2, and the activated MEK–ERK1/2 cascade in 13–stimulated 15-LO protein expression as compared with scram- regulating IL-13 induction of Egr-1 accumulation in the nuclear bled decoy ODN transfected monocytes (Fig. 9B). These data compartment and binding of Egr-1 to its cognate DNA binding provide the first direct evidence, to our knowledge, that the tran- sequence located within the 15-LO promoter. scription factors Egr-1 and CREB are important regulators of IL- In this study, we also provide evidence that IL-13 induced 15- 13–induced 15-LO gene expression in primary human monocytes LO transcription by the activation of CREB transcription factor (Fig. 10). through Ser133 phosphorylation and its regulation by the ERK1/2 MAPKs. CREB belongs to the basic leucine zipper family of Discussion transcription factors that binds to CREs within target gene pro- In this study, we demonstrated a crucial role for ERK1/2 in IL-13– moters with activation initiated by phosphorylation at Ser133. This mediated 15-LO gene expression in primary human monocytes. activation of CREB facilitates the DNA binding activity of the ERK1/2 are members of the MAPK family that has been impli- protein in many cell types (25, 45, 49, 64, 65). Upon phosphor- cated in IL-13R regulation (50), IL-13–induced eotaxin release ylation of CREB at Ser133, the transcriptional coactivator CREB- (51–53), and type I collagen gene regulation (54). ERK1/2 MAPK binding protein, a histone acetyl , is recruited to CREB. activation has also been reported to play a critical role in medi- This, in turn, promotes the assembly of the basal transcriptional ating IL-13–induced inflammation and alveolar remodeling (23). machinery (66–70). It is already reported that CREB plays a Expression of 15-LO has been implicated in the biosynthesis of pivotal role in the progression of inflammation by regulating the anti-inflammatory lipid mediators (55, 56) and in the pathogenesis expression of inflammatory genes, such as mucin and cyclo- of inflammation and atherosclerosis (3, 57). We, therefore, focused 2 (25, 71, 72). Our results in the current study clearly 5222 ERK1/2 REGULATES 15-LO EXPRESSION

FIGURE 10. Model of the bifurcating pa- rallel signal transduction pathways down- stream of the IL-13R that regulate 15-LO gene expression in primary human mono- cytes. The IL-13Ra1–Tyk2-mediated sig- naling pathway that is required for IL-13– inducible Egr-1 and CREB activation via MEK–ERK1/2 in primary human mono- cytes is shown in the dotted-line box. Downloaded from http://www.jimmunol.org/ show that IL-13 activation of CREB requires ERK1/2 activity; the containing PKCd, p38MAPK, and tyrosine-phosphorylated Stat3 pharmacological inactivation of ERK1/2 MAPKs abolished (22) (A. Bhattacharjee and M.K. Cathcart, unpublished observa- the IL-13–induced Ser133 phosphorylation and CRE promoter- tions) that is required for the serine phosphorylation of Stat3, binding activity of CREB (Fig. 7A,7C). In addition, our results probably by p38MAPK-mediated interaction between PKCd and also demonstrated that IL-13 induces the translocation of ERK1/2 tyrosine-phosphorylated Stat3. from the cytosol to the nucleus, implying that the translocated In this study, we investigated whether IL-13–mediated activation ERK1/2 MAPKs are involved in regulating CREB serine phos- of ERK1/2 MAPKs were related to p38MAPK and PKCd activa- by guest on October 2, 2021 phorylation/activation and binding of CREB to its cognate se- tion in the IL-13–induced signaling pathways in monocytes. By quences identified within the 15-LO promoter. using selective pharmacological inhibitors of ERK1/2, p38MAPK, Several transcription factors have been reported to regulate 15- and PKCd, we demonstrated that IL-13 activation of ERK1/2 LO expression. Although many of these studies showed evidence MAPKs is independent of p38MAPK- and PKCd-mediated sig- for Stat6-dependent transcriptional regulation of 15-LO expres- naling cascades in primary human monocytes. The existence of sion and activity (73–76), our previous studies demonstrated the these parallel signal transduction pathways, that although are in- involvement of Stat1 and Stat3 as well as Stat6 as transcrip- dividually not sufficient for IL-13 induction of 15-LO expression, tional regulators of 15-LO in IL-13–stimulated monocytes (21) are all required for 15-LO expression, suggests the importance of (A. Bhattacharjee and M.K. Cathcart, unpublished observa- tight regulation of 15-LO gene expression. As 15-LO is a very tions). Earlier GATA6-mediated transcriptional regulation of 15- important enzyme for monocyte function and required in many LO expression was reported in human colorectal cancer cells cellular processes, cells probably operate their own machinery in (77, 78). Requirement of the transcription factor KU 70/80 lupus such a way that several independent but parallel pathways must autoantigen (DNA helicase) was also demonstrated for IL-13 in- orchestrate simultaneously to stimulate the upregulation of 15-LO duction of 15-LO gene expression in human epithelial A549 cells gene expression in an IL-13–dependent manner. (79). Recent studies further identified the transcription factor In summary, we report in this study the transcriptional regulation SPI1 (a member of the Ets family of transcription factors), which of 15-LO by IL-13–mediated activation of two different tran- was responsible for increased transcriptional activation of the scription factors Egr-1 and CREB and the involvement of a MEK– ALOX15 (reticulocyte-type 15-LO) gene in (80). The ERK1/2-dependent signaling pathway in regulating this process. results of our decoy experiments in this study strongly implicate Our results schematically presented in Fig. 10 indicate the IL-13 both Egr-1 and CREB as potent transcriptional regulators of the signaling in monocytes through the membrane-bound hetero- 15-LO gene in IL-13–stimulated primary human monocytes and dimeric IL-13R complex (comprised of IL-13Ra1 and IL-4Ra) provide insights into the regulation of 15-LO transcription in these and the requirement of receptor-associated tyrosine kinases Jak2 important inflammatory cells. and Tyk2, which are activated in response to IL-13 stimulation and We have previously reported that IL-13–mediated induction of influence the 15-LO expression in monocytes (2, 20). We suggest 15-LO requires Jak2- and Tyk2-mediated tyrosine phosphoryla- the existence of two distinct bifurcating parallel regulatory path- tion of several Stat family members in primary human monocytes ways downstream of the IL-13R. One is the IL-4Ra–Jak2- (2, 20). Our previous studies also demonstrated the involvement of dependent signaling cascade that is required for IL-13–stimulated the serine-threonine kinases p38MAPK and PKCd in regulating Stat3 tyrosine phosphorylation (A. Bhattacharjee and M.K. Cath- IL-13–driven 15-LO expression (21, 22, 40). Our recent work cart, unpublished observations) followed by the formation of an identified the formation of an IL-13–stimulated signaling complex IL-13–stimulated cytosolic signaling complex containing PKCd, The Journal of Immunology 5223 p38MAPK, and tyrosine-phosphorylated Stat3 (22) (A. Bhatta- attenuates atherogenesis in LDL receptor-deficient mice. Circulation 104: 1646– 1650. charjee and M.K. Cathcart, unpublished observations). 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