A JNK-Independent Signaling Pathway Regulates TNF α-Stimulated, c-Jun-Driven FRA-1 Protooncogene Transcription in Pulmonary Epithelial Cells This information is current as of September 27, 2021. Pavan Adiseshaiah, Dhananjaya V. Kalvakolanu and Sekhar P. Reddy J Immunol 2006; 177:7193-7202; ; doi: 10.4049/jimmunol.177.10.7193 http://www.jimmunol.org/content/177/10/7193 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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A JNK-Independent Signaling Pathway Regulates TNF␣-Stimulated, c-Jun-Driven FRA-1 Protooncogene Transcription in Pulmonary Epithelial Cells1

Pavan Adiseshaiah,* Dhananjaya V. Kalvakolanu,† and Sekhar P. Reddy2*

〈mong the several effectors that mediate TNF-␣ action is AP-1, which consists of transcription factors belonging to the JUN and FOS families. Although the effects of TNF-␣ in immune cells, such as the induction of NF-␬〉, are well known, the mechanisms by which it induces transcriptional activation of AP-1 in pulmonary epithelial cells are not well defined. In this study, we report that TNF-␣ stimulates the expression of the FRA-1 protooncogene in human pulmonary epithelial cells using c-Jun, acting via a ,12-O-tetradecanoylphorbol-13 acetate response element located at ؊318. Although TNF-␣ stimulates phosphorylation of c-Jun the inhibition of JNK activity had no significant effect on FRA-1 induction. Consistent with this result, ectopic expression of a c-Jun Downloaded from mutant lacking JNK phosphorylation sites had no effect on the TNF-␣-induced expression of the promoter. In contrast, inhibition of the ERK pathway or ectopic expression of an ERK1 mutant strikingly reduced FRA-1 transcription. ERK inhibition not only blocked phosphorylation of Elk1, CREB, and ATF1, which constitutively bind to the FRA-1 promoter, but also suppressed the recruitment of c-Jun to the promoter. We found that short interfering RNA-mediated silencing of FRA-1 enhances TNF-␣-induced IL-8 expression, whereas overexpression causes an opposite effect. Our findings collectively indicate that ERK signaling plays key

roles in both Elk1, CREB, and ATF-1 activation and the subsequent recruitment of c-Jun to the FRA-1 promoter in response to http://www.jimmunol.org/ TNF-␣ in pulmonary epithelial cells. The Journal of Immunology, 2006, 177: 7193–7202.

ronchial epithelium and its associated tissues act as a in other cell types in response to TNF-␣. AP-1 is a dimeric com- primary interface for the interaction of a plethora of en- plex composed mainly of Jun (c-Jun, JunB, and JunD), Fos (c-Fos, vironmental stressors in the vertebrates. Acute lung in- FosB, Fra-1, and Fra-2), and ATF family . Fos/Jun dimers B 3 jury caused by pathogenic and toxic products activates the synthe- bind to 12-O-tetradecanoylphorbol-13-acetate (TPA) response el- sis of proinflammatory cytokines. These cytokines not only act ements (TREs, also known as AP-1 sites) and regulate the expres- directly on the lung cells themselves during the early phase of the sion of involved in cell proliferation, inflammation, and pul- response but also help recruit the cells of the immune system to monary defenses (7, 8). A combinatorial interaction among the by guest on September 27, 2021 alleviate the effects of the injury. Human pulmonary epithelial Jun, Fos, and ATF families of proteins has been shown (9) to cells are known (1) to secrete many proinflammatory cytokines. regulate expression in a signal, cell-type-, and stressor-spe- High-level expression of these cytokines has been (2) causally cific manner. The abundance and regulated autoinduction of cer- linked to the development of pulmonary diseases, such as chronic tain members of the AP-1 family in response to specific stimuli obstructive pulmonary disease and asthma. These proinflammatory control the duration and magnitude of a stress-related or mitogenic signals initiate intracellular signaling cascades, leading to an acti- response (10). Consistent with this observation, overexpression of vation of various immediate early transcription factors, which then some AP-1 proteins results in various diseases associated with in- bind to target sequences commonly found in the regulatory regions flammation. For example, targeted expression of JunB in T lym- of various cytokine and cytokine genes and activate their phocytes promotes high levels of Th2 cytokines (11). Abrogation transcription (3). of JunB in keratinocytes triggers chemokine/cytokine expression, One of the proinflammatory cytokines, TNF-␣, plays a critical leading to the development of psoriasis, whereas abrogation of role in diverse physiologic events and contributes to the develop- c-Jun has the opposite effect (12). A role for JunD in T lymphocyte ment of air pollutant-induced lung pathogenesis and airway re- proliferation and cell differentiation has been reported (13). Given modeling (4). Apart from NF-␬B, activation of immediate tran- that specific members of this family are rapidly induced and the scription factors such as AP-1 has been reported (3, 5, 6) to occur composition of AP-1 complex distinctly regulates gene ex- pression, an understanding of the mechanisms of activation of Jun

*Department of Environmental Health Sciences, Johns Hopkins University, Balti- and Fos members is critical to our understanding of the molecular more, MD 21205; and †Greenbaum Cancer Center and Department of Microbiology pathogenesis promoted by inflammatory stimuli. and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201 The mechanisms by which TNF-␣ induces effector functions in Received for publication April 17, 2006. Accepted for publication August 7, 2006. immune cells are well recognized. However, it is unclear how this The costs of publication of this article were defrayed in part by the payment of page cytokine stimulates the activation of immediate response genes, charges. This article must therefore be hereby marked advertisement in accordance such as transcription factors, that regulate subsequent expression with 18 U.S.C. Section 1734 solely to indicate this fact. of a variety of inflammatory mediators in pulmonary epithelial 1 This study was supported by National Institutes of Health Grants ES11863 and HL66109 and (to S.P.R.) and by National Cancer Institute Grants CA782282 and CA105005 (to D.V.K.). 2 Address correspondence and reprint requests to Dr. Sekhar P. Reddy, Department of 3 Abbreviations used in this paper: TPA, 12-O-tetradecanoylphorbol-13-acetate; TRE, Environmental Health Sciences, Johns Hopkins University, Bloomberg School of TPA response element; MMP, matrix metalloproteinases; WT, wild type; MEF, Public Health, 615 North Wolfe Street, Room E7610, Baltimore, MD 21205. E-mail mouse embryonic fibroblast; rRNA, rRNA-encoding DNA; ChIP, chromatin immu- address: [email protected] noprecipitation; siRNA, small interfering RNA; SRE, serum response element.

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 7194 INTERPLAY BETWEEN c-Jun AND Elk1 AT THE FRA-1 PROMOTER cells. The FRA-1 was isolated as a TPA-inducible gene from Real-time RT-PCR monocytes, suggesting a role for this in cell TaqMan assays for mouse and human FRA-1, c-Jun, and differentiation (14). Human T cell leukemia virus type 1 Tax1 GAPDH were purchased from Applied Biosystems, and mRNA levels were activates the transcription of FRA-1 (15). Recently, we and others quantified in triplicate according to the supplier’s recommendations. The have shown that respiratory toxins that promote airway inflamma- absolute values for FRA-1 and c-Jun were normalized to that of GAPDH. tion, such as cigarette smoke (16), asbestos (17), and diesel ex- The relative value from the vehicle-treated control group was considered equal to one arbitrary unit. IL-8 and IL-6 expression was analyzed by a haust particles (18), strongly up-regulate the expression of FRA-1 LightCycler (Roche) using the SYBR Green QuantiTech RT-PCR kit in lung epithelial cells, suggesting a key role for this transcription (Qiagen).Primersequenceswere:IL-8sense,GTTTTTGAAGAGGGCTGA factor in airway inflammation, injury, and repair processes. FRA-1 GAATTC; IL-8 antisense, CATGAAGTGTTGAAGTAGATTTGC T; IL-6 up-regulates the expression of several matrix metalloproteinases sense, GGCAGAAAACAACCTGAACCT TC; IL-6 antisense, ACCTCAA ACTCCAAAAGACCAGTG; and 18S rRNA-encoding DNA (rRNA) sense, (MMPs), such as MMP-12 (19) and MMP-9 (18, 20–22), which GTAACCCGTTGAACCCCATT; 18S rRNA antisense, CCATCCAATC are known to promote airway inflammation. Although the activation GGTAGTAGCG. The reaction was performed in a 20-␮l final volume con- of c-Fos by cytokines has been investigated in great detail (23–25) in sisting of 25 ng of total RNA (IL-8 and IL-6) or 2.5 ng of total RNA (for 18S cells of the immune system, the induction of FRA-1 by cytokines and rRNA), 10 ␮l of QuantiTech SYBR Green PCR mastermix (Qiagen), and 1.5 its role in inflammatory responses in pulmonary epithelial cells are mM primers. Negative controls without template were included in all of the RT-PCR. Quantification of IL-8 and IL-6 mRNA in each sample was normal- poorly understood. In this study, we report that JNK activation is not ized to the abundance of its corresponding 18S rRNA in each sample. required for TNF-␣-induced, c-Jun-mediated FRA-1 transcription in Transient transfection assays pulmonary epithelial cells. The induction occurs instead via an ERK signaling pathway through the activation of Elk1, CREB, ATF, and Cells were transfected with 100 ng of IL-8-Luc or FRA-1 promoter reporter Downloaded from the subsequent recruitment of c-Jun to the promoter. construct, 1 ng of Renilla luciferase (pRL-TK) vector (Promega), and 25– 200 ng of empty or expression plasmids. At 18–24 h posttransfection, cells were serum-starved for 24 h and then treated with vehicle or TNF-␣. Cell Materials and Methods extracts were assayed for firefly and Renilla luciferase activities using a Reagents and plasmids commercially available kit (Promega). Luciferase activity of individual samples was normalized to that of Renilla luciferase activity (31).

Abs specific for c-Jun (SC-45X), FRA-1 (SC-605X), JNK1 (SC-474), EMSAs http://www.jimmunol.org/ ERK2 (SC-154), Elk1 (SC-355), and p-Elk1 (Ser383, SC-8406) were ob- tained from Santa Cruz Biotechnology. The ␤-actin Ab and phosphospe- Serum-starved cells were treated with TNF-␣ for 60 min, nuclear extracts cific Abs for JNK (T183/Y185), c-Jun (Ser73), and ERK (T202/Y204) were all were prepared, and the EMSAs were performed using 2–3 ␮g of nuclear obtained from Cell Signaling Technology. Phosphospecific-CREB (Ser133; extract and 32P-labeled double-stranded Ϫ318 TRE oligonucleotide as a catalog no. 05-667) and nonphosphorylated CREB (catalog no. 06-863) probe, as described previously (26). For supershift assays, nuclear extracts were purchased from Upstate Cell Signaling. Details concerning the vari- were incubated with 1–2 ␮g of specific Abs or nonimmune IgG on ice for ous deletion and mutant FRA-1 promoter reporter luciferase constructs 2 h before the addition of labeled probe. used in this study have been published elsewhere (26). Expression vectors coding for the c-Jun mutant (c-Jun TAM), Elk1 mutant (dn-Elk1), SRF Chromatin immunoprecipitation (ChIP) assays mutant (SRF-mt), ATF1 mutant (ATF1-mt), and CREB mutants (CREB- ChIP assays were conducted as described earlier (32): Cells (ϳ1 ϫ 107) mt) used in this study are detailed in our earlier publication (26). Plasmid were exposed to TNF-␣ for 60 min, and ChIP was performed using a by guest on September 27, 2021 constructs of wild-type (WT) c-Jun and mutant c-Jun lacking JNK phosphor- commercially available kit (Upstate Biotechnology). Chromatin was cross- ylation sites, serines 63 and 73 and threonines 91 and 93 (27), were gifts from Ϫ linked by adding formaldehyde (1%) to the tissue culture medium for 10 W. G. Kaelin, Jr. (Harvard Medical School, Boston, MA). The 165- to 19-bp min at 37°C. A fraction of the soluble chromatin (1%) was saved for promoter of human IL-8 (IL-8-Luc), which contains the functional motifs, ␬ measurement of total chromatin input. Precleared chromatin was incubated such as AP-1 and NF- B sites (21), fused to luciferase (Luc) gene was de- with specific Abs for 18 h at 4°C. DNA recovered from the immunopre- scribed elsewhere (28). cipitated products was used as a template for PCR with FRA-1 promoter- specific primers (32). After cross-linking and immunoprecipitation, puri- Cell culture fied DNA isolated from MEFs was subjected to PCR amplification for 40 cycles using primers specific for fra-1 promoter (GenBank accession no. A549, a human alveolar type II-like epithelial cell line, was maintained in AF017128): forward primer (Ϫ208/Ϫ185), 5Ј-GCGGAGCTCGGCCACA RPMI 1640 medium supplemented with 5% FBS and antibiotics (Invitrogen GGATTTTGTTTCGCCCT-3Ј and reverse primer (Ϫ44/Ϫ64), 5Ј-GGC Life Technologies). The primary human bronchial epithelial cells were cul- GCTAGCCCTCTGACGCAGCTGCCCAT-3Ј. PCR was performed at tured in MEM supplemented with growth factors according to the supplier’s 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 55°C for 45 s, and recommendation (Cambrex). Mouse embryonic fibroblasts (MEFs) lacking the Ϫ/Ϫ 72°C for 1 min, with a final extension at 72°C for 10 min. The amplified erk1 gene (erk1 ) and their isogenic WT cells (29) were cultured as previ- 165-bp DNA fragment was separated on gel electrophoresis. ously described (30). To generate stable cell lines that overexpress FRA-1, A549 cells were transfected with FRA-1 wild-type cDNA (gift from E. Small interfering RNA (siRNA) and gene expression analysis Tulchinsky, University of Leicester, U.K.) or an empty pCMV mammalian expression vector containing a selection marker neomycin. SMARTpool siRNA duplexes for c-Jun (catalog no. M-003268-01) and a Stable cell clones overexpressing FRA-1 (referred to as A549-F1 cells) scrambled siRNA (catalog no. D-001206-06-05) were purchased from Dhar- or a control empty vector (referred to as A549-C) were isolated following macon. To silence the endogenous FRA-1 expression, a plasmid-based ex- selection with 600 ␮g/ml G418 (Invitrogen Life Technologies), pooled, pression vector, pRNATin-H1.2/Neo (GenScript) containing FRA-1 siRNA and used for subsequent gene expression and functional studies. sequence, GGATCCCGCTGACTGCCACTCATGGTGCCACACCCACCA TGAGTGGCAGTCAGTTTTTTCCAAAAGCTT, was used. Empty vector was used as a control. A549 cells at 30–40% confluence were transfected with Northern and Western blot analyses siRNAs at 20 nM concentrations and were harvested at 48–72 h to determine For Northern blot analysis, cells were serum-starved for 24 h and subse- the effect of siRNA on the expression of endogenous c-Jun and FRA-1 using quently treated with TNF-␣ (10 ng/ml) for various times as indicated. Total Western blotting. For reporter assays, A549 cells were transfected with 100 ng RNA (15 ␮g/lane) was separated on a 1.2% agarose gel, blotted onto a of the 379-Luc promoter-reporter construct along with c-Jun or scrambled nylon membrane, and hybridized with 32P-labeled cDNAs of FRA-1 and siRNAs for 36 h. Cells were serum-starved overnight before stimulation with ␣ 18S RNA as previously described (31). For Western blot analysis, total TNF- , and luciferase activity was measured as described above. protein was extracted using a lysis buffer consisting of 20 mM Tris (pH Statistical analysis 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 ␤ Ϯ mM sodium pyrophosphate, 1 mM Na3VO4,5mM -glycerophosphate, Data are expressed as means SE. Statistical significance was determined and 1 ␮g/ml leupeptin. A comparable quantity of protein from each sample using t tests and accepted at p Ͻ 0.05. All assays were performed using two was separated on a 10% SDS-PAGE, and the membranes were probed with or three (n ϭ 2–3) independent samples, and each experiment was repeated specific Abs (Santa Cruz Biotechnology). at least two times. The Journal of Immunology 7195

FIGURE 1. FRA-1 induction by TNF-␣ in pulmonary epithelial cells. A, A549 cells were serum-starved for 24 h and then treated with TNF-␣ (10 ng/ml) for various times as indicated. Northern blot analysis was conducted using a 32P-labeled FRA-1 cDNA probe. Arrows, Positions of the 3.3- and 1.7-kb transcripts of FRA-1. The membrane was stripped and probed with 28S RNA cDNA to monitor equal RNA loading of all samples. B, Cells were treated with actinomycin D (Act-D, 10 ␮g/ml) or vehicle (DMSO) for 30 min before stimulation without (Con) or with TNF-␣ for 90 min. FRA-1 and 28S RNA expression was analyzed as detailed above. A representative blot of two independent experiments is shown for A and B. C, Cells were transfected with various FRA-1 promoter reporter constructs as indicated along with a reference plasmid, pRL-TK. After overnight incubation, cells were serum-starved for 24 h and then stimulated without (Ⅺ) or with TNF-␣ (f) for 5 h. The promoter activity of the constructs was expressed using the basal activity of the 861-Luc as one unit. The fold activation of the individual reporters was calculated with the basal values of the respective construct set to one. The data represent the values of three independent samples of a representative experiment, which was repeated at least twice to obtain reproducible results. Downloaded from

Results 2B). Furthermore, the knockdown of c-Jun expression by siRNA TNF-␣-induced FRA-1 expression in pulmonary epithelial cells strongly reduced TNF-␣-stimulated luciferase activity (Fig. 2C, bar 2), when compared with control scrambled siRNA (Fig. 2C,

To understand the regulation of FRA-1 expression by cytokines, http://www.jimmunol.org/ A549 cells were treated with TNF-␣ for 0–360 min, RNA was isolated, and Northern blot analysis was performed using a 32P- labeled human FRA-1 cDNA as probe. As shown in Fig. 1A, TNF-␣ significantly stimulated FRA-1 mRNA expression as early as 30 min; the levels reached a maximum by 90 min and remained elevated through 360 min. The induction of FRA-1 mRNA expres- sion by TNF-␣ was also correlated with a corresponding increase in its protein levels and was determined by Western blotting (data not shown). The two alternatively spliced mRNA transcripts of by guest on September 27, 2021 FRA-1 were induced similarly, as previously reported (14). How- ever, pretreatment of cells with actinomycin D, an inhibitor of transcription, blocked TNF-␣-stimulated FRA-1 expression (Fig. 1B), indicating that the induction was mainly regulated at the tran- scriptional level. To map the promoter region required for TNF- ␣-inducible transcription, promoter-reporter constructs bearing various lengths of the 5Ј-flanking region of FRA-1 were trans- fected into A549 cells, and reporter gene expression was moni- tored (Fig. 1C). Consistent with our previous results, the 283-Luc yielded an ϳ4-fold higher basal activity when compared with the 105-Luc and 68-Luc constructs. However, the levels of reporter expression following TNF-␣ stimulation were unchanged, sug- FIGURE 2. TRE mediates TNF-␣-stimulated FRA-1 transcription. A, gesting these constructs lack the cis-elements required for the in- The position of the Ϫ318 TRE of the 379-Luc construct is shown. Ϫ duction. In contrast, the 328-Luc construct bearing the serum re- Mutations were introduced into the 318 TRE of the 379-Luc as pre- sponse element (SRE) had a 2-fold higher luciferase activity in viously described (32). Cells were transfected with 100 ng of 379-Luc and Ϫ318 TRE mutant construct (379-TRE mt) in the presence of the response to TNF-␣ (Fig. 2B). However, nearly a 5- to 7-fold rise pRL-TK plasmid. The TNF-␣-inducible promoter activity was ex- in promoter activity was noticed with the 379-Luc, 570-Luc, and pressed as fold change over the activity of the respective constructs in 861-Luc constructs, suggesting that the DNA sequences spanning unstimulated cells. B, Cells were transfected with 100 ng of empty or Ϫ379 and Ϫ283 regulate the induction by TNF-␣. c-Jun mutant expression vectors to determine the role of c-Jun in TNF- ␣-stimulated FRA-1 transcription. Fold induction was calculated with Ϫ ␣ The 318 TRE mediates c-Jun-dependent, TNF- -inducible the values for empty parental vector-transfected cells set to one. C, FRA-1 promoter activity Cells were transfected with the 379-Luc construct and pRL-TK plasmid The Ϫ379/Ϫ283 region harbors functional elements such as Ϫ318 in the presence of scrambled (Scr) or c-Jun siRNA (20 nM) sequences TRE (26) and SRE (32). Because AP-1 acts as a major downstream as previously described (32). After 36 h of transfection, cells were serum-starved and then treated without or with TNF-␣ for 5 h. The effector of TNF-␣-induced signaling, we first examined the role of Ϫ promoter activity (-fold activation) was calculated with the value for 318 TRE in mediating cytokine-inducible FRA-1 transcription. unstimulated cells set to one unit. Values are mean Ϯ SE from two Ϫ ␣ Disruption of 318 TRE site markedly reduced TNF- -inducible independent experiments conducted in duplicate (n ϭ 4). D, To confirm promoter activity (Fig. 2A). Consistent with this result, ectopic the knockdown of endogenous c-Jun expression, siRNA transfected expression of a c-Jun mutant lacking the transactivation domain cells were lysed in parallel experiments and immunoblotted with anti- greatly reduced (ϳ80%) TNF-␣-stimulated promoter activity (Fig. c-Jun and tubulin Abs. Lane 1, Scr-SiRNA and lane 2, c-Jun siRNA. 7196 INTERPLAY BETWEEN c-Jun AND Elk1 AT THE FRA-1 PROMOTER

TNF-␣-stimulated c-Jun expression precedes and is essential for subsequent FRA-1 induction We next examined the role of c-Jun in this process. We measured the message levels of c-Jun and FRA-1 by real-time PCR follow- ing TNF-␣ stimulation at 30 and 90 min. TNF-␣ treatment in- creased the expression levels of c-Jun after as little as 30 min, and the levels remained elevated up to 90 min (Fig. 4A, left panel). In contrast, FRA-1 induction by TNF-␣ peaked between 30 and 90 min (Fig. 4A, right panel). The increase in c-Jun and FRA-1 FIGURE 3. Analysis of c-Jun binding at the FRA-1 promoter. A, ChIP mRNA expression was confirmed at the protein level by Western analysis of c-Jun binding to the endogenous FRA-1 promoter. The arrows blot analysis using ␤-actin as a loading control (Fig. 4B). We next indicate the positions of the primers flanking Ϫ318 TRE that were used in examined the role of c-Jun in controlling FRA-1 expression using the ChIP assays. Cells were treated with TNF-␣ for 0, 30, or 60 min, and siRNAs. Cell cultures were transfected with c-Jun or a control- then chromatin protein-DNA complexes were cross-linked using formal- scrambled siRNA and then stimulated with TNF-␣. Total RNA dehyde. The purified nucleoprotein complexes were immunoprecipitated was isolated, and FRA-1 expression was measured by real-time with c-Jun Abs or nonimmune IgG and amplified by PCR as detailed in PCR (Fig. 4C). Transfection of c-Jun siRNA significantly dimin- Materials and Methods. Experiments were repeated at least twice to obtain ished TNF-␣-stimulated FRA-1 expression (Fig. 4C, bar 4), when B reproducible results. , The quantification of the amplified band with c-Jun compared with the scrambled siRNA control (Fig. 4C, bar 2). These Ab normalized against the input reference band. Downloaded from results (Figs. 3 and 4) demonstrate a requirement for c-Jun for TNF- ␣-stimulated FRA-1 induction in pulmonary epithelial cells. bar 1). The c-Jun-specific siRNA markedly suppressed endoge- nous c-Jun protein levels by Ͼ80% (Fig. 2D, lane 2), when com- ␣ pared with scrambled siRNA (Fig. 2D, lane 1). The expression JNK signaling is not essential for TNF- -inducible FRA-1 level of tubulin was comparable between these two samples, con- expression firming a specific inhibitory effect of c-Jun siRNA. The level of TNF-␣ stimulates the activation of the JNK pathway, and c-Jun http://www.jimmunol.org/ expression of c-Jun was similar for a scrambled siRNA and re- acts as a major downstream effector of JNK kinases in other cell agent control (data not shown). These results collectively indicate types. We therefore asked whether the JNK pathway was neces- a requirement for c-Jun in TNF-␣-stimulated FRA-1 expression in sary for TNF-␣-induced expression of FRA-1. Cells were serum- pulmonary epithelial cells. starved for 24 h, then treated with TNF-␣, and JNK1/2 activation was assessed using phosphospecific Abs (Fig. 5A). As anticipated, ␣ c-Jun is recruited to the FRA-1 promoter following TNF- TNF-␣ strongly stimulated the phosphorylation of JNK1/2 (Fig. stimulation 5A). However, pretreatment of cells with the JNK inhibitor ␣ We performed ChIP assays to examine the binding of c-Jun to the SP600125 suppressed TNF- -stimulated JNK1/2 activation (Fig. by guest on September 27, 2021 Ϫ318 TRE of the FRA-1 promoter in vivo following TNF-␣ stim- 5B, compare lane 4 and lane 1). In contrast, SP600125 did not ulation (Fig. 3A). In the unstimulated state, c-Jun bound only min- inhibit ERK1/2 phosphorylation. In contrast, treatment of cells with imally to the FRA-1 promoter (Fig. 3A, lane 1). However, TNF-␣ the ERK1/2 and p38 MAPK pathway inhibitors PD98059 (Fig. 5B, induced the binding of c-Jun to the promoter as early as 30 min lane 2) and SB202190 (Fig. 5B, lane 3), respectively, had no effect on (Fig. 3A, lane 2), and it remained high through 60 min (Fig. 3A, TNF-␣-stimulated JNK1/2 activation (Fig. 5B, lane 4). To examine lane 3). We chose these time points because FRA-1 message levels the role of JNK signaling in TNF-␣-stimulated FRA-1 expression, were maximal at 60–90 min after TNF-␣ stimulation (Fig. 1A). In RNA was isolated from cells stimulated with TNF-␣ in the presence contrast, ChIP assays of nonimmune IgG showed no amplification or absence of SP600125, and a Northern blot analysis was performed. of the FRA-1 promoter. Quantification of c-Jun binding revealed a As shown in Fig. 5C, JNK inhibition had no effect on the TNF-␣- nearly 8- to 12-fold increase in the induced binding of c-Jun to the induced expression of FRA-1. Similar results were obtained with pri- Ϫ318 TRE after TNF-␣ stimulation (Fig. 3B). Collectively, these mary cultures of human bronchial epithelial cells (Fig. 5D). results support a critical role for c-Jun in controlling TNF-␣- Above results suggest that the activation of the JNK pathway is induced FRA-1 transcription. not essential for TNF-␣-stimulated FRA-1 expression. To further

FIGURE 4. c-Jun is required for TNF-␣-stimulated FRA-1 expression. A, Cells were stimulated with TNF-␣ for 0–90 min, total RNA was isolated, and c-Jun and FRA-1 mRNA expression was analyzed by real-time PCR. Bars, Mean Ϯ SE of triplicates. B, Cell extracts (40 ␮g) isolated from cells treated with TNF-␣ as detailed above were immunoblotted using the c-Jun, FRA-1, and tubulin Abs. A representative blot of two independent experiments is shown. C, Cells were transfected with scrambled (Scr) or c-Jun (20 nM) siRNA sequences as detailed in Fig. 2C, and then stimulated without (Ⅺ) or with TNF-␣ (f) for 90 min. Total RNA was isolated and FRA-1 mRNA expression quantified by real-time PCR. Bars, Mean Ϯ SE (n ϭ 4). *, p Ͻ 0.05. The Journal of Immunology 7197 Downloaded from FIGURE 5. Effect of JNK pathway inhibition on TNF-␣-induced FRA-1 expression. A, Cells were stimulated with TNF-␣ for 0–60 min, cell ex- tracts were isolated, and JNK1/2 activation was analyzed using phos- phospecific Abs. Membranes were stripped and probed with JNK2 Abs. B, Cells were incubated with ERK inhibitor PD98059 (PD, 30 ␮M), p38 FIGURE 6. Effect of a c-Jun mutant lacking JNK phosphorylation sites inhibitor SB202190 (SB, 10 ␮M), or JNK inhibitor SP600125 (SP6, 10 on TNF-␣-induced FRA-1 promoter activity. A, The extracts isolated from

␮M) for 30 min and then treated with TNF-␣ for 30 min. DMSO was used http://www.jimmunol.org/ cells stimulated with TNF-␣ at various points were probed with phos- as vehicle control. Cell extracts (40 ␮g) were analyzed by Western blotting phospecific c-Jun (Ser73) Abs. Membranes were stripped and probed with using the phosphospecific JNK1/2 Abs. Membranes were stripped and sub- c-Jun Abs. B, The extracts isolated from cells stimulated with TNF-␣ in the sequently probed with phosphospecific ERK1/2 and total ERK2 Abs. A presence of MAP kinase inhibitors, PD98059, SB202190, and SP600125, representative blot of two independent experiments is shown. C, Cells were were analyzed by Western blotting using the phosphospecific c-Jun (Ser73) incubated with SP600125 (SP6, 10 ␮M) for 30 min and then treated with or JNK1/2 Abs. Membranes were stripped and probed with native c-Jun TNF-␣ for 90 min, and FRA-1 mRNA expression was analyzed by real- and tubulin Abs. A representative blot of two independent experiments is time PCR. Bars, Mean Ϯ SE (n ϭ 6). D, Primary cultured human bronchial shown. C and D, Cells were transfected with the 379-Luc promoter reporter epithelial (PHBE) cells were serum-starved for 2 h and then treated with construct (0.1 ␮g) in the presence of the parental empty vector (vector), SP600125 (SP6) before stimulation with TNF-␣ for 90 min. FRA-1 mRNA

c-Jun WT vector (c-Jun), or a mutant form of c-Jun lacking JNK phos- by guest on September 27, 2021 expression was analyzed by real-time PCR. Bars, Mean Ϯ SE (n ϭ 4). phorylation sites (⌬JNK-c-Jun). Both basal (C) and inducible (D) promoter activities were determined after normalization to the value of the empty parental vector-transfected cells, which was set to 100%. Bars, Mean Ϯ SE confirm this hypothesis, we examined the JNK-mediated phos- of triplicates of a representative experiment. phorylation of c-Jun at Ser73 in response to TNF-␣ under our ex- perimental conditions. As anticipated, TNF-␣ markedly stimulated c-Jun expression (Fig. 6A) and its phosphorylation (Fig. 6B). Pre- treatment of cells with SP600125 inhibited TNF-␣-stimulated JNK (Fig. 7A, lane 2) but returned to basal levels thereafter. As shown MAPK activation and the subsequent c-Jun phosphorylation. To in Fig. 7B, treatment of cells with the ERK inhibitor PD98059 rule out a role for JNK phosphorylation in c-Jun-dependent FRA-1 completely blocked TNF-␣-stimulated ERK1/2 activation. transcription, we transiently transfected cells with a c-Jun mutant Next, we analyzed the effect of the ERK inhibition on TNF-␣- (⌬JNK c-Jun) lacking JNK phosphorylation sites, Ser63 and Ser73 and Thr91 and Thr93 (27) (Fig. 6C, bar 2), then compared FRA-1 enhanced FRA-1 transcription. PD98059 markedly blocked TNF- ␣ promoter activation to that of the WT construct (Fig. 6C, bar 3). -stimulated FRA-1 mRNA expression (Fig. 7C). A similar result Ectopic expression of the c-Jun mutant robustly stimulated FRA-1 was obtained with another MEK-ERK pathway-specific inhibitor, promoter activity to a level equivalent to that of the WT protein. U0126. These results were further confirmed at the transcription Moreover, the c-Jun mutant had no effect on TNF-␣-induced re- level using FRA-1 reporter constructs in transient transfection as- ␣ porter expression (Fig. 6D, bar 3). Collectively, these results in- says (Fig. 7D). TNF- strongly stimulated FRA-1 promoter activ- dicate that JNK1/2 signaling and c-Jun phosphorylation do not ity (Fig. 7D, bar 1), but this stimulation did not occur in the pres- contribute to TNF-␣-stimulated FRA-1 expression. ence of ERK inhibitor PD98059 (Fig. 7D, bar 2), supporting a role for ERK signaling in controlling FRA-1 induction by TNF-␣.To The ERK1/2 pathway is essential for TNF-␣-induced FRA-1 further assess the importance of ERK1 signaling in the regulation expression of FRA-1 induction in lung epithelial cells, A549 cells were trans- A critical role for ERK1/2-dependent control of toxin- and mito- fected with the dominant-negative ERK1 (dn-ERK1) plasmid, and gen-stimulated FRA-1 expression has been demonstrated (16) in TNF-␣-stimulated FRA-1 promoter activity was analyzed. A control lung epithelial cells. We, therefore, examined the role of this path- transfection with empty expression vector was used for comparison. way in TNF-␣-stimulated FRA-1 expression. Cells were treated Overexpression of dn-ERK1 significantly inhibited both basal and with TNF-␣ for various times, and ERK1/2 kinase activation was TNF-␣-stimulated FRA-1 promoter-driven reporter expression, as determined by Western blot analysis with Abs specific for phos- compared with empty vector-transfected, TNF-␣-treated cells (Fig. phorylated (active) forms of ERK1/2 (Fig. 7A). The TNF-␣-stim- 7E). Taken together, these results strongly support a critical role for ulated phosphorylation of ERK1/2 kinases was robust at 15 min ERK signaling in controlling TNF-␣-induced FRA-1 transcription. 7198 INTERPLAY BETWEEN c-Jun AND Elk1 AT THE FRA-1 PROMOTER

studies on the Elk1 and CREB transcription factors that are targets of ERK signaling and are known (32) to regulate the induction of FRA-1 in response to tumor promoters and mitogens. As antici- pated, TNF-␣ stimulated the phosphorylation of Elk1, CREB, and ATF-1 after as little as 15 min (Fig. 8A, lane 2), and this stimu- lation was decreased in the presence of PD98059 (Fig. 8A, lanes 6 and 7). To examine the role of Elk1 and ATF/CREB proteins in the transcriptional up-regulation of FRA-1 by TNF-␣, cells were trans- fected with the reporter constructs bearing a mutation in the Elk1 binding site TCF and the ATF/CREB binding site of the FRA-1 promoter (Fig. 8B). The CArG element, flanking these sites, has been shown (33) to be critical for efficient binding of Elk1 to the SRE. We, therefore, examined the impact of mutations in the CArG element on TNF-␣-induced FRA-1 promoter activity. Mu- tation of the individual TCF site, the CArG box, or the ATF site significantly diminished (Ͼ50%) TNF-␣-induced reporter expres- sion, when compared with the results for the WT construct that FIGURE 7. The ERK pathway regulates TNF-␣-induced FRA-1 pro- lack these mutations (Fig. 8B). To further confirm the role of these moter activity. A, Cells were stimulated with TNF-␣ for various times, and transcription factors, we transfected cells with plasmids coding for Downloaded from extracts were analyzed by Western blotting with ERK1/2 Abs. B, Western dominant-negative mutants of the SRF, Elk1, ATF1, and CREB pro- ␮ ␣ blot showing the effect of PD98059 (20 M) on TNF- -stimulated ERK1/2 teins. Coexpression of SRF mutant or an Elk1 mutant significantly activation. C, Cells were incubated with the ERK inhibitors PD98059 (30 repressed TNF-␣-induced FRA-1 promoter activity (Fig. 8C). Ectopic ␮M) and UO126 (10 ␮M) for 30 min and then stimulated without (Ⅺ)or expression of the ATF1 and CREB mutants had a similar effect on with TNF-␣ (f) for 90 min. FRA-1 mRNA expression was analyzed by real-time PCR. Bars, Mean Ϯ SE (n ϭ 6). D, Effect of PD98059 on TNF- reporter gene expression (Fig. 8D). These results collectively suggest

␣-stimulated FRA-1 promoter activity. Cells were transfected with the 379- that SRF and TCF proteins, such as Elk1, ATF1, and CREB, regulate http://www.jimmunol.org/ Luc reporter (100 ng) and pRL-TK (1 ng). The fold-activation of was TNF-␣-stimulated FRA-1 expression through the SRE (TCF and calculated with the basal values of the respective DMSO or PD98059 CArG) and the ATF sites located in the enhancer region. treated samples as one unit. Bars, Mean Ϯ SE (n ϭ 4). E, Cells were transfected with the 379-Luc reporter and pRL-TK along with an equimo- ERK1 kinase regulates FRA-1 induction by TNF-␣ lar amount of empty vector or dominant negative ERK1 (dn-ERK1) plas- ␣ To examine the role of the ERK1 pathway in FRA-1 induction, we mid, and the TNF- -stimulated FRA-1 promoter activity was analyzed with Ϫ/Ϫ the value for empty vector-transfected cells set to one. *, p Ͻ 0.05. used MEFs lacking the erk1 gene (erk1 ) and compared the magnitude of fra-1 induction by TNF-␣ in these cells to that in isogenic WT cells. We examined the activation of the ERK1/2 ␣ by guest on September 27, 2021 Inhibition of the ERK pathway suppresses TNF- -induced Elk1 pathway in these two cell types by Western blot analysis using and CREB phosphorylation phosphospecific Abs. As shown in Fig. 9A, TNF-␣ strongly stim- To determine the downstream effector mechanisms by which ERK ulated both ERK1 and ERK2 phosphorylation in WT cells signaling controls FRA-1 induction by TNF-␣, we focused our (cf lanes 1 and 2). As expected, there was no ERK1 activation in

FIGURE 8. The SRE is essential for TNF-␣-induced FRA-1 promoter activity. A, Cells stimulated with TNF-␣ for 0, 15, and 30 min (lanes 1-3) and the extracts were analyzed by Western blotting using phosphospecific Elk1 (Ser383) and CREB (Ser133) Abs. Membranes were stripped and probed with total Elk1 Abs to monitor equal loading. A (right panel, lanes 4-7), cells were treated with PD98059 and then stimulated with TNF-␣ for 30 min, and Elk1 and CREB/ATF1 activation was analyzed. DMSO was used as a vehicle control (lanes 4 and 5). Note that CREB (Ser133) Abs also recognize the phosphospecific form of ATF1 (Upstate Cell Signaling). B, A549 cells were transfected with the FRA-1 promoter-reporter constructs bearing mutations within the TCF site, the CArG box, and the ATF site. pRL-TK plasmid was used as an internal control. C and D, Cells were transfected with the 379-Luc along with the pRL-TK plasmid in the presence of an equimolar amount of either empty vector, mutant SRF (dn-SRF), or Elk1 (dn-Elk1) plasmid (C). The effects of mutant ATF1 (dn-ATF-1) and CREB (dn-CREB) plasmids on TNF-␣-stimulated FRA-1 promoter is shown in D. TNF-␣-induced promoter activity (-fold activation) was calculated with the value for unstimulated cells set to one unit. Data shown are mean Ϯ SE of triplicates from a typical .Statistically significant difference at p Ͻ 0.05 ,ء .experiment The Journal of Immunology 7199

FIGURE 9. ERK1 is critical for TNF-␣-stimulated FRA-1 induction. A, The activation of ERK1/2 kinases by TNF-␣ in the WT and erk1Ϫ/Ϫ MEFs. The MEFs were serum-starved for 2 h and then stimulated with TNF-␣ or TPA (10 ng/ml) for 15 min, and ERK activation was analyzed using phosphospecific Abs. B, The effects of ERK1 deficiency on endogenous fra-1 mRNA expression. MEFs were stimulated without (Ⅺ) or with TNF-␣ (f) for 90 min, and fra-1 mRNA expression was analyzed by real-time PCR. Bars, Mean Ϯ SE (n ϭ 6). C, After transfection with the 379-Luc FRA-1 promoter construct, WT and erk1Ϫ/Ϫ MEFs were treated with TNF-␣, and luciferase activity was analyzed. erk1Ϫ/Ϫ MEFs, which lack this gene (Fig. 9A, lane 4). In transient Role of FRA-1 in TNF-␣-stimulated proinflammatory gene transfection assays, TNF-␣ strongly stimulated FRA-1 promoter transcription Downloaded from Ϫ/Ϫ activity in WT MEFs as compared with the erk1 MEFs (Fig. To examine a role for FRA-1 in regulating TNF-␣-induced pul- ␣ 9B). Furthermore, the ERK inhibitor PD98059 repressed TNF- - monary epithelial responses, we used two complimentary ap- stimulated FRA-1 promoter activity in both cell types. We further proaches: 1) an RNAi-mediated knockdown of gene expression validated these results at the level of endogenous fra-1 expression and 2) stable overexpression. To silence endogenous FRA-1 ex- ␣ levels by real-time PCR (Fig. 9C). Treatment of cells with TNF- pression, A549 cells were transfected with FRA-1 shRNA expres- stimulated fra-1 mRNA expression in WT MEFs. However, the sion vector or empty vector; after a 48-h incubation, cell lysates http://www.jimmunol.org/ magnitude of the induction was greatly diminished in the erk1Ϫ/Ϫ MEFs when compared to WT cells. To further validate these re- sults, we treated WT cells with the MEK-ERK pathway-specific inhibitors PD98059 and U0126 and examined the endogenous fra-1 expression. Treatment of WT MEFs with either PD98059 or U0126 obliterated the TNF-␣-stimulated response. These results collectively indicate a prominent role for ERK signaling, espe- cially ERK1, in regulating fra-1 induction by TNF-␣. by guest on September 27, 2021 ERK1/2 signaling is essential for the recruitment of c-Jun to the FRA-1 promoter We have previously shown (32) that mutations in the Ϫ318 TRE or TCF and CArG sites of SRE as well as the flanking ATF site ablate the mitogen-induced FRA-1 promoter activity. Because inhibition of the JNK pathway did not block c-Jun activation, and recruitment of c-Jun following TNF-␣ stimulation precedes FRA-1 induction, we wondered whether inhibition of the ERK pathway affects the recruit- ment of c-Jun at the promoter. For this purpose, we exposed cells to TNF-␣ for 60 min in the presence or absence of the ERK inhibitor UO126, which completely blocks FRA-1 induction, and analyzed the recruitment of c-Jun using ChIP assays as detailed in Materials and Methods. As shown in Fig. 10, TNF-␣ strongly enhanced the binding of c-Jun at the promoter (lanes 2 and 3). However, pretreatment of FIGURE 10. Effect of ERK inhibition on the recruitment of c-Jun to the cells with ERK inhibitor before TNF-␣ stimulation markedly reduced Ͼ FRA-1 promoter. A, A549 cells were serum-starved for 24 h and incubated ( 80%) the recruitment of c-Jun at the FRA-1 promoter (Fig. 10, with UO126 (10 ␮M) or DMSO for 30 min before treatment without (Ϫ) lanes 5 and 6). In a complementary experiment, we performed a ChIP or with TNF-␣ (ϩ). After a 60-min incubation, formaldehyde was added to Ϫ/Ϫ analysis to determine whether the lack of erk1 altered the recruit- the cells to cross-link chromatin. ChIP assays were performed using c-Jun ment of c-Jun at the endogenous fra-1 promoter in MEFs. The WT Abs and nonimmune IgG as detailed in Materials and Methods. Experi- Ϫ Ϫ and erk1 / MEFs were stimulated with or without TNF-␣, DNA- ments were repeated at least twice to obtain reproducible results. C, Quan- protein complexes were cross-linked, and ChIP assays were per- tification of amplified DNA band intensity normalized against input DNA formed using mouse fra-1 promoter-specific primers as detailed in from two independent experiments is shown (n ϭ 4). C, The position of Materials and Methods. As expected, ChIP assays with the nonim- forward (F) and reverse (R) primers used to amplify the fra-1 promoter Ϫ Ϫ mune IgG showed no amplification of the fra-1 promoter (data not encompassing the TRE site located at nucleotide position 119/ 113 is shown. D,WTanderk1Ϫ/Ϫ MEFs were serum-starved for 2 h and stim- shown). The binding of c-Jun at the promoter is very low under ulated without (Ϫ) or with TNF-␣ (ϩ). ChIP assays (bottom) were per- steady-state conditions (Fig. 10B, lanes 1 and 2). However, the re- formed using c-Jun Abs or nonimmune IgG (data not shown) using fra-1 ␣ cruitment of c-Jun was strongly enhanced following TNF- treatment promoter-specific primers as detailed in Materials and Methods. A repre- (Fig. 10B, lanes 3 and 4). In contrast, the binding of c-Jun to the fra-1 sentative blot of two independent experiments performed in duplicate is Ϫ Ϫ promoter was significantly diminished in MEFS lacking the erk1 / shown. E, Quantification of amplified DNA band intensity normalized signaling (cf lanes 7 and 8 with lanes 3 and 4). against input DNA from two independent experiments (n ϭ 4) is shown. 7200 INTERPLAY BETWEEN c-Jun AND Elk1 AT THE FRA-1 PROMOTER

11D). An equal number of viable cells were plated on a 6-well plate, serum-starved, and then stimulated with or without TNF-␣ for 6 h. Total RNA was isolated and IL-8 expression was analyzed. TNF-␣ markedly (9-fold) stimulated IL-8 mRNA expression (Fig. 11D, bar 2), as compared with untreated cells (Fig. 11D, bar 1). The basal level expression of IL-8 is significantly lower in A549-F1 as compared with A549-C cells. Moreover, FRA-1 over- expression completely suppressed the TNF-␣-stimulated expres- sion of IL-8 (Fig. 11D, bar 4). Consistent with this result, the magnitude of TNF-␣-stimulated IL-8 promoter-driven reporter ex- pression was remarkably lower in A549-F1 cells (Fig. 11E, bar 4) as compared with A549-C (Fig. 11E, bar 2). Collectively, these data indicate that the FRA-1 induction by TNF-␣ may play a role in dampening a sustained IL-8 induction by TNF-␣.

FIGURE 11. The effects of FRA-1 on TNF-␣-induced proinflammatory cytokine gene transcription. A, A549 cells were transfected with a plasmid Discussion construct containing a FRA-1 siRNA sequence (FRA1-sh) and incubated In this study, we have shown that c-Jun, a major effector of JNK1/2 for 48 h. Empty vector without siRNA sequence was used as control. signaling, is required for FRA-1 protooncogene induction by Downloaded from Whole-cell lysates were prepared and immunoblotted with anti-FRA-1 and TNF-␣ in pulmonary epithelial cells. Unexpectedly, inhibition of ␤ -actin Abs. A representative immunoblot of three independent experi- the JNK pathway, which is known to play a critical role in AP-1 ments is shown. B, A549 cells transfected with the empty vector or plas- activation in other cell types, failed to suppress TNF-␣-stimulated mid-bearing FRA1-siRNA as in A and then treated without (Ⅺ) or with TNF-␣ (f) for 6 h. IL-8 gene expression was analyzed by real-time RT- FRA-1 expression in both A549 and primary cultured human bron- -p Ͻ 0.02. C, An equal amount of whole-cell chial epithelial cells (Fig. 5, C and D). Furthermore, ectopic ex ,ءء p Ͻ 0.01 and ,ء .PCR lysates (40 ␮g) isolated from the stable FRA-1 overexpressing (A549-F1) pression of a c-Jun mutant lacking the N-terminal JNK phosphor- http://www.jimmunol.org/ or control empty vector (A549-C) cells were immunoblotted with anti- ylation sites did not suppress TNF-␣-inducible FRA-1 promoter FRA-1 and ␤-actin Abs. Results shown are from two independent exper- activation (Fig. 6D). A requirement of c-Jun, but not the JNK1/2 iments. D, A549-F1 and A549-C cells were treated without (Ⅺ) or with (f) pathway, indicates that JNK1/2-dependent c-Jun phosphorylation TNF-␣ for 6 h, and IL-8 mRNA expression was analyzed. Data are rep- may not be essential for TNF-␣-stimulated FRA-1 promoter trans- ء resentative of two independent experiments performed in duplicate. , activation in pulmonary epithelial cells. The JNK pathway, in con- Ͻ p 0.001. E, Stable A549-F1 and A549-C were cotransfected with 100 ng trast, has been implicated (34) in FRA-1 induction by TNF-␣ in of IL-8-Luc reporter along with 1 ng of a reference plasmid, pRL-TK. MEF cells. JNK-deficient MEFs, lacking both Jnk1 and Jnk2 TNF-␣-unstimulated (Ⅺ) and -stimulated (f) luciferase activity was de- termined as detailed in Fig. 1. The values obtained from the untreated (Ⅺ) genes, showed diminished levels of fra-1 expression in response to ␣ by guest on September 27, 2021 A549-C cells were set as 1.0. Data represent mean Ϯ SD from at least two TNF- (34). Thus, it seems that the JNK1/2 pathway regulates p Ͼ 0.001. FRA-1 transcription in a cell type-specific manner. Consistent with ,ء .three independent experiments done in triplicate this view and our results, Catani et al. (35) have shown that ascor- bate, which blocks the activation of the JNK pathway, strongly were prepared, and the knockdown of endogenous FRA-1 expres- up-regulates FRA-1 expression in the HACAT cell line of epithe- sion was analyzed by Western blot analysis (Fig. 11A). FRA-1 lial origin. On the contrary, we have shown (16) that JNK1/2 in- shRNA suppressed ϳ60% of the total level of FRA-1 protein (Fig. hibition blocks cigarette smoke-stimulated FRA-1 expression in 11A, lane 2), as compared with the empty vector-transfected con- human bronchial epithelial cells, underscoring the notion that the trol (Fig. 11A, lane 1). The siRNA exerted no effect on the ex- JNK1/2 pathway regulates FRA-1 transcription in a context-depen- pression of ␤-actin (Fig. 11A, bottom panel) or ERK2 (data not dent manner. shown) protein. Given a critical role of IL-8 in mediating TNF-␣- The c-Jun activity is primarily regulated at the level of its tran- induced phenotypic effects, we have analyzed the effects of FRA-1 scription and posttranslational modifications. c-Jun expression is silencing on IL-8 expression. FRA-1-silencing caused a significant rapidly induced within the first 30 min by a variety of mitogenic increase (3-fold) in the basal level expression of IL-8, as compared and stress stimuli, as well as in response to cytokines in multiple with vector-transfected control (Fig. 11B). As expected, TNF-␣ cell types (36). The induction of the c-Jun gene is controlled by markedly stimulated IL-8 expression (Fig. 11B, bar 2). However, multiple regulatory elements, including TRE, GC box, CAAT box, knockdown of FRA-1 further significantly enhanced the TNF-␣- ATF, and sites (37). Several transcription factors that bind enhanced IL-8 mRNA expression (Fig. 11B,cfbars 2 and 4). to these elements are targets of ERK1/2, ERK5, JNK1/2, and p38 These results indicate that FRA-1 induction by TNF-␣ may play a kinases, which have been shown (38) to regulate c-Jun transcrip- role in attenuating IL-8 induction by TNF-␣. To confirm this no- tion in response to stress, cytokine, and mitogenic stimuli. For tion, we have stably overexpressed FRA-1 in A549 cells (termed example, ATF and c-Jun, which bind to the ATF/Jun site, are as A549-F1) and its expression was confirmed by immunoblot targets of ERK1/2, JNK, and p38 kinases (39). In contrast, MEK5- analysis (Fig. 11C, lane 2). As anticipated, empty vector-bearing ERK5 signaling regulates c-Jun transcription via a phosphoryla- A549 cells (A549-C) showed a little expression of FRA-1 (Fig. tion of the MEF family of transcriptions factors that bind to the 11C, lane 1). The biological activity of ectopically expressed functional MEF site located next to the TATA box (40, 41). In FRA-1 in A549-F1 cells was significantly high compared with addition to transcriptional induction, posttranslational modifica- A549-C cells, as assessed by EMSA using a TRE as probe and by tions, such as phosphorylation of c-Jun play key roles in c-Jun- transfection assays using TRE-Luc as a reporter, and was markedly dependent gene transcription. The N-terminal phosphorylation of (4-fold) higher in A549-F1 cells, as compared with A549-C cells c-Jun protein by JNK kinases enhances both transactivation po- (data not shown). We next examined the effects of ectopically tential and the stability of c-Jun protein (42), which otherwise un- expressed FRA-1 on TNF-␣ stimulated IL-8 expression (Fig. dergoes ubiquitin-dependent proteolysis (43). The Journal of Immunology 7201

c-Jun has been shown to mediate various cellular responses in a the respective Ϫ318 TRE, Ϫ274 TCF and the Ϫ248 ATF sites JNK phosphorylation-dependent and -independent manner. Al- (detailed in Fig. 2 of Ref. 32) of the FRA-1 enhancer. Consistent though phosphorylation of c-Jun by JNK on Ser63 and Ser73 is with this notion, mutational inactivation of the Ϫ318 TRE, Ϫ274 required to protect cells from UV-induced cell death, it is not re- TCF, or Ϫ248 ATF sites crippled TNF-␣ inducibility of the FRA-1 quired for cell growth (41). Consistent with this, although c-Jun promoter (Fig. 8B). Conversely, coexpression of mutant forms of deletion leads to embryonic lethality (44), the c-Jun mice lacking c-Jun, Elk1, SRF, ATF1, or CREB repressed FRA-1 induction the JNK phosphorylation sites, Ser63 and Ser73, are viable, fertile, (Fig. 8, C and D). The inability of c-Jun to bind to the FRA-1 and displayed no phenotypic defects (45). However, these mutant promoter in the absence of ERK signaling and the fact that Elk1, mice are less susceptible to kinate-induced neuronal apoptosis than SRF, and CREB are bound to the promoter in the steady state the WT mice (42, 45). c-Jun-regulated cell cycle progression (46) suggest that the activation of Elk1, SRF, and CREB proteins by and Ras-induced cellular transformation (47, 48) do not require the ERK signaling may facilitate, in some way, the recruitment of JNK-induced phosphorylation at Ser63 and Ser73 of c-Jun. Several c-Jun at the FRA-1 promoter in response to TNF-␣. This effect recent studies have shown (49, 50) that JNK signaling is dispens- seems to occur independently of JNK signaling. able for transactivation of c-Jun. For example, interaction of c-Jun Our findings indicate that FRA-1 may play a key role in regu- with CBP coactivator or RNA helicase requires the N-terminal lating TNF-␣-induced proinflammatory cytokine gene expression region but not the JNK phosphorylation sites. Consistent with (Fig. 11). Silencing of FRA-1 enhanced both basal and TNF-␣- these findings, it has been shown (51) that JNK phosphorylation of stimulated IL-8 expression. In contrast, FRA-1 overexpression c-Jun, which is essential for disassociation of c-Jun from the caused a repression of IL-8 gene expression. The suppressive ef-

HDAC3 repressor, is not essential for subsequent transcriptional fect of FRA-1 on IL-8 gene expression appears to be regulated at Downloaded from activation of c-Jun. In contrast, JNK phosphorylation sites are re- the level of transcription (Fig. 11E). Our results are consistent with quired for an efficient interaction of c-Jun with TCF4 and the sub- a recent report (63) that demonstrated a negative role for FRA-1 in sequent recruitment of ␤-catenin on the c-Jun promoter, thereby attenuating or limiting the IL-1-induced IL-8 gene expression in resulting in an enhanced c-Jun transcription (52). Thus, it appears non-pulmonary epithelial cells. In that study, the authors have that the status of c-Jun JNK phosphorylation has a distinct effect on shown that a delayed recruitment of FRA-1 to the IL-8 promoter

gene transcription. counteracts c-FOS and NF-␬B-mediated IL-1-induced IL-8 ex- http://www.jimmunol.org/ Our findings show a prominent role for ERK1/2 in controlling pression. Similarly, we have noticed that TNF-␣-stimulated c-FOS TNF-␣-induced FRA-1 transcription, despite the transient nature of expression precedes FRA-1 induction in pulmonary epithelial cells the activation of this MAPK pathway by TNF-␣ (Figs. 7 and 9). (data not shown). Thus, a repression of IL-8 induction by FRA-1 Our analysis revealed that the ERK pathway is required for TNF- may probably be mediated by the displacement of c-FOS from the ␣-stimulated Elk1, CREB, and ATF1 phosphorylation (Fig. 8A) IL-8 promoter. and is consistent with the previous suggestion (53) that these pro- In summary, induction of the FRA-1 by TNF-␣ occurs indepen- teins are putative substrates for ERKs. We have previously shown dently of the JNKs. Instead, ERKs seem to play a critical role in (26, 32, 54) using ChIP assays that these proteins are constitutively this process. Our findings also suggest that FRA-1 may attenuate bound to a critical SRE of the FRA-1 promoter in pulmonary ep- the magnitude of the TNF-␣-induced activation of IL-8 expression by guest on September 27, 2021 ithelial cells. Similarly, a variety of external stimuli, such as epi- in pulmonary epithelial cells. dermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, and cigarette smoke, also did not enhance their binding to the pro- Acknowledgments moter. A similar scenario exists for c-Fos, whose promoter is oc- We thank all of the scientists for providing us with the various expression cupied by these factors in vivo in the unstimulated state (55, 56). vectors used in this study. We also thank Suneetha Peddakama and Won Based on these observations, we speculate that ERK inhibition Kyung Lee for technical assistance on real-time PCR and Bill Spannhake likely affects the activation of the DNA-bound Elk1, CREB, and for his help in the analysis of IL-6 and IL-8 expression. ATF proteins. Consistent with this notion, the translocation of ERKs from the cytoplasm to the nucleus following external stimuli Disclosures has been firmly established (57). Finally, the phosphorylation of The authors have no financial conflict of interest. Elk1 by MAPK has been shown (26, 32, 54) to enhance its inter- action with the coactivator p300, leading to gene transcription. References 133 Phosphorylation of CREB at Ser is critical for CBP recruitment 1. Bals, R., and P. S. Hiemstra. 2004. Innate immunity in the lung: how epithelial to the promoter in response to mitogenic and stress signals (60). cells fight against respiratory pathogens. Eur. Respir. J. 23: 327–333. 2. 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