THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2003 by The … · 2003-10-29 · Phorbol Ester-dependent Activation of Peroxiredoxin I Gene Expression via a Protein Kinase C, Ras, p38 Mitogen-activated

Phorbol Ester-dependent Activation of Peroxiredoxin I GeneExpression via a Protein Kinase C, Ras, p38 Mitogen-activatedProtein Kinase Signaling Pathway*

Received for publication, July 21, 2003, and in revised form, September 4, 2003Published, JBC Papers in Press, September 5, 2003, DOI 10.1074/jbc.M307871200

Alexander Hess‡, Nastiti Wijayanti‡, Andrea Pathe Neuschafer-Rube§, Norbert Katz‡,Thomas Kietzmann§, and Stephan Immenschuh‡¶

From the ‡Institut fur Klinische Chemie und Pathobiochemie, Justus-Liebig-Universitat Giessen, Giessen D-35392,Germany and §Institut fur Biochemie und Molekulare Zellbiologie, Georg-August-Universitat Gottingen,Gottingen D-37073, Germany

The antioxidant protein peroxiredoxin (Prx) I is a thi-oredoxin peroxidase that is involved in the regulation ofproliferation and differentiation of mammalian cells.Here, it is shown that Prx I gene expression was inducedtranscriptionally by the phorbol ester 12-O-tetradecano-ylphorbol-13-acetate (TPA) in cultured rat liver tissuemacrophages and RAW264.7 monocytic cells. TPA-de-pendent induction of Prx I gene expression was medi-ated by two proximal activator protein-1 sites of the ratPrx I promoter region that were nuclear targets of c-Junas determined by transfection studies with luciferasereporter gene constructs and electrophoretic mobilityshift assays. The transcription factor Nrf2, however, wasnot involved in the regulation of Prx I promoter activity.Prx I gene induction by TPA was decreased by proteinkinase C inhibitors and overexpressed dominant nega-tive forms of Ras and MEKK1, but not Raf-1. The p38MAPK inhibitor SB202190 and overexpression of domi-nant negative mutants of MAPK kinase 4 (MKK4), MKK6,and p38 inhibited the TPA-dependent induction of Prx Igene transcription. In contrast, inhibitors of the JNK,SP600125, and the NF-�B signaling pathway, caffeic acidphenethyl ester, respectively, as well as overexpresseddominant negative MKK7 and I�B, had no effect on theup-regulation of Prx I reporter gene activity by TPA.Cotransfection of wild-type p38� and p38�, but not thatof p38� and p38�, increased Prx I promoter activity. Thedata indicate that a protein kinase C, Ras, MEKK1, p38MAPK signaling pathway plays a major role for the tran-scriptional up-regulation of Prx I gene expression.

Peroxiredoxin (Prx)1 I is an intracellular antioxidant proteinwith thioredoxin peroxidase activity that has initially been

purified from human HeLa cells (proliferation-associated gene;see Ref. 1), mouse peritoneal macrophages (mouse stress pro-tein 23; see Ref. 2), and rat liver (heme-binding protein 23; seeRef. 3). Prx I belongs to the Prx protein family and is charac-terized by conserved cysteine residues (4–7). Prxs have beenidentified in numerous organisms ranging from bacteria andplants up to vertebrates and mammals and are divided intothree classes: typical two-cysteine Prxs (Prx I-IV), atypicaltwo-cysteine Prxs (Prx V), and one-cysteine Prxs (Prx VI) (8, 9).The functions of these proteins comprise modulation of signaltransduction and cell proliferation, as well as protectionagainst oxidative stress (10–15). Therefore, Prxs may comple-ment other enzymes with antioxidant potential such as super-oxide dismutase, catalase, or glutathione peroxidase that havebeen ascribed protective roles in inflammation, cancer, or neu-rodegenerative disease (16–18).

The enzyme activity of Prxs is regulated by post-transla-tional mechanisms such as protein phosphorylation, proteoly-sis, and oligomerization (9, 19, 20). More recently, a redox-de-pendent mechanism for the reversible peroxidase activation ofPrx I involving the active site cysteine has been demonstratedby Woo et al. (21). However, little is known on the mechanismsthat regulate gene expression of Prxs in mammalian cells. Weand others (22–25) have shown previously that Prx I geneexpression is induced by a number of oxidative stress stimuliincluding heme, heavy metals, and oxidized low density li-poproteins in various cell culture models. Because Prx I ap-pears to play an important role in carcinogenesis as a tumorsuppressor (14, 15) the goal of the present study was to inves-tigate the regulation of Prx I gene expression by the phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA is atumor promoter that activates protein kinase C (PKC) (26, 27),and here it is demonstrated that TPA induces Prx I geneexpression in cultured monocytic cells. By transient transfec-tion of reporter gene constructs with the newly cloned 5�-flank-ing promoter region of the Prx I gene it is shown that activatorprotein-1 (AP-1) sites of the rat Prx I promoter region play acrucial role for the TPA-dependent gene induction via interac-tion with c-Jun. The induction of Prx I promoter activity ismediated by a signaling pathway involving PKC, Ras, mitogen-activated protein kinase (MAPK)/extracellular signal-regu-lated kinase (ERK) kinase kinase (MEKK) 1, and p38 MAPKisoforms.

* This work was supported in part by Grants SFB 402 A8 (to S. I.) andSFB 402 A1 (to T. K.) from the Deutsche Forschungsgemeinschaft. Thecosts of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “adver-tisement” in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s) AJ457059.

¶ To whom correspondence should be addressed: Institut fur KlinischeChemie und Pathobiochemie, Justus-Liebig-Universitat Giessen, Gaffky-strasse 11, Giessen 35392, Germany. Tel.: 49-641-99-41578; Fax: 49-641-99-41559; E-mail: [email protected].

1 The abbreviations used are: Prx, peroxiredoxin; AP-1, activatorprotein-1; ARE, antioxidant response element; BIM, bisindolylmaleim-ide; CAPE, caffeic acid phenethyl ester; EMSA, electrophoretic mobilityshift assay; ERK, extracellular signal-regulated kinase; JNK, c-JunN-terminal kinase; MAPK, mitogen-activated protein kinase; MEKK,MAPK/ERK kinase kinase; MKK, MAPK kinase; NF-�B, nuclear factor-

�B; PKC, protein kinase C; RE, regulatory element; rRNA, ribosomalRNA; stauro, staurosporine; TF, transcription factor; TPA, 12-O-tetradecanoylphorbol-13-acetate.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 46, Issue of November 14, pp. 45419–45434, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 45419

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EXPERIMENTAL PROCEDURES

Materials—Media M199 and Dulbecco’s modified Eagle’s mediumwere obtained from Invitrogen, nitrocellulose filters were fromSchleicher & Schuell, and radioisotopes and the ECL chemiluminescentdetection system for Western blot were from Amersham Biosciences.The nucleotide removal kit was from Qiagen, and the multiprime la-beling kit, restriction endonucleases, and terminal deoxynucleotidyltransferase were from New England Biolabs (Cambridge, MA). Falcontissue culture dishes were from BD Biosciences. All other chemicalswere purchased from Sigma and Roche Applied Science unless other-wise indicated.

Cell Isolation and Culture—Liver tissue macrophages (Kupffer cells)were isolated from livers of male Wistar rats (2 months old, body weight170 to 200 g) according to Knook and Sleyster (28) as described previ-ously (23). In brief, the liver was digested with Pronase/collagenase

solutions, and non-parenchymal cells were separated by density gradi-ent centrifugation. Liver tissue macrophages were purified by counter-flow elutriation (J2–21, JE-B6 rotor; Beckman Instruments, Fullerton,CA), and the obtained liver tissue macrophages were resuspended inmedium M199 containing 15% fetal calf serum, 100 units penicillin/mland 100 �g streptomycin/ml. Cell viability was assessed by trypan bluestaining. Cells were plated on 6-well plates (3 � 106 cells/well) forpreparation of RNA or protein. After 2 h cells were washed for elimi-nation of non-adherent cells. RAW264.7 cells were from American TypeCulture Collection (Manassas, VA) and were cultured in Dulbecco’smodified Eagle’s medium supplemented with 10% fetal calf serum, 100units penicillin/ml and 100 �g streptomycin/ml. Cell cultures were keptunder air/CO2 (19:1) at 100% humidity. Treatment of cells with TPAwas performed with serum-free medium.

Western Blot Analysis—After washing of cell cultures twice with

FIG. 1. Induction of Prx I gene expression by TPA in cultures of rat liver tissue macrophages and RAW264.7 cells. Rat liver tissuemacrophages (Kupffer cells) and RAW264.7 cells were cultured as described under “Experimental Procedures.” Cell cultures were kept for 18 h inserum-free medium before treatment with TPA. A, rat liver tissue macrophages (left panel) or RAW264.7 cells (right panel) were cultured for 6 hunder control conditions or in the presence of TPA (0.5 �M). Total RNA (15 �g) was subjected to Northern analysis, and blots were sequentiallyprobed with a 32P-labeled cDNA for Prx I and a 28 S rRNA oligonucleotide. The size marker was the 18 S rRNA band. B, cultures of rat liver tissuemacrophages (left panel) and RAW264.7 cells (right panel) were maintained in the absence or presence of TPA (0.5 �M) for 16 h. Total protein (50�g) was subjected to Western blot analysis and probed with a polyclonal antibody against rat Prx I. Autoradiograms from representativeexperiments are shown in A and B, respectively. C–E, RAW 264.7 cells were cultured in the presence of TPA (0.5 �M) for the times indicated (C),for 6 h in the presence of increasing concentrations of TPA (D), or for 30 min with actinomycin D (1 �g/ml) before TPA (0.5 �M) was added foranother 6 h (E). Total RNA (15 �g) was subjected to Northern blot analysis as described in A. Autoradiograms were quantitated with aphosphorimager, and the signal of the 28 S rRNA band served as an internal standard. Values � S.E. represent the -fold induction of Prx I mRNAnormalized to 28 S rRNA from at least three independent experiments. Statistics, Student’s t test for paired values: *, significant difference TPAversus control; **, actinomycin D � TPA versus TPA, p � 0.05. AD, actinomycin D; con, control; LTM, liver tissue macrophages; RAW, RAW264.7cells.

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FIG. 2. Nucleotide sequence of the rat Prx I gene promoter and potential AP-1 and NF-�B sites. Nucleotides are numbered relative tothe transcription initiation site (�1), which is demonstrated by an arrow. The translational start site is indicated by ATG in bold. The TATA boxand DNA sequences with homology to the AP-1 and NF-�B consensus motifs are in bold. AP-1-A, AP-1-B, and AP-1-C sites that were examinedin detail are underlined.

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0.9% NaCl, total protein was prepared essentially as described (23).After addition of 1 ml of lysis buffer (0.1% SDS, 10 mM Tris, pH 7.4),cells were boiled for 5 min and homogenized by passing through a25-gauge needle. The homogenate was centrifuged for 5 min at 4 °C,

and the protein content was determined in the supernatant using theBradford method. 50 �g of total protein was loaded onto a 12% SDS-polyacrylamide gel and was blotted onto nitrocellulose membranes byelectroblotting. Membranes were blocked with Tris-buffered saline con-

FIG. 3. Identification of functional AP-1 sites in the rat Prx I promoter that mediate TPA-dependent induction. The indicated rat PrxI promoter fragments were cloned either into pGL3-Basic (A) or as enhancers in 5�-3� or 3�-5� orientation in front of the SV40 promoter intopGL3-Prom (B). The localization of AP-1-A, AP-1-B, and AP-1-C sites is indicated. The various reporter gene constructs were transientlytransfected into cultured RAW264.7 cells. 24 h after transfection cells were treated for 16 h with control medium or medium supplemented withTPA (0.5 �M) as indicated. Cell extracts were assayed for luciferase activity, and the -fold induction in each experiment relative to the control wasdetermined. The values are S.E. from four to six independent experiments with duplicates of each point. Statistics, Student’s t test for pairedvalues: *, significant difference TPA versus control, p � 0.05. C, Prx I promoter luciferase gene constructs with targeted mutations in AP-1-A andAP-1-B sites of pPrx558 were generated as described under “Experimental Procedures,” and point mutations in AP-1-A and AP-1-B sites areunderlined and in italics. These Prx I reporter gene constructs and a luciferase construct with six copies of the AP-1 consensus motif (pAP-1luc;see Ref. 29) were transiently transfected into cultured RAW264.7 cells. 24 h after transfection cells were treated for 16 h with control medium ormedium supplemented with TPA (0.5 �M) as indicated. Cell extracts were assayed for luciferase activity, and the -fold induction in each experimentrelative to the control was determined. The values are S.E. from four to six independent experiments with duplicates of each point. Statistics,Student’s t test for paired values: *, significant differences TPA versus control; **, pPrx558mutA � TPA versus pPrx558 � TPA, pPrx558mutAB � TPAversus pPrx558 � TPA, p � 0.05. HSV, herpes simplex virus promoter; Luc, luciferase; SV40, simian virus 40 promoter.



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taining 1% bovine serum albumin, 10 mM Tris/HCl, pH 7.5, and 0.1%Tween for 1 h at room temperature. The primary polyclonal antibody forPrx I (3) was added at 1:1000 dilution, and the blot was incubated for12 h at 4 °C. The secondary anti-rabbit IgG was diluted 1:8000, and theECL chemiluminescent detection system was used for detection accord-ing to the manufacturer’s instructions. The primary antibodies for thedetection of phosphorylated and total MAPKs were from Cell Signaling(Beverly, MA) and were applied at the concentrations recommended bythe manufacturer.

RNA Isolation, Northern Blot Analysis, and Hybridization—TotalRNA for Northern blotting was isolated as described previously (23).Equal quantities of RNA were separated on 1.2% agarose/2.2 M form-aldehyde gels. After electrophoresis, RNA was blotted onto nitrocellu-lose membranes and baked at 80 °C for 4 h. After prehybridization for4 h at 42 °C, blots were hybridized overnight with � [32P]dCTP-radio-labeled cDNA probes at 42 °C essentially as described (23). The hybrid-ization solution contained 6� SSC, 5� Denhardt’s solution (0.2% Ficoll400, 0.2% polyvinylpyrrolidone, and 0.2% bovine serum albumin), 0.5%SDS, 50% formamide, and 100 �g/ml denatured salmon sperm DNA.Blots were washed subsequently with 2� SSC/0.1% SDS (once) and0.1� SSC/0.1% SDS (twice) at 65 °C. Filters were autoradiographedwith x-ray films (X-OMAT RP; Eastman Kodak Co., Rochester, NY) at�70 °C for up to 48 h or were displayed to a phosphorimager screen for4–8 h, and radioactivity was measured with Imagequant software(Molecular Dynamics, Sunnyvale, CA). When nitrocellulose filters weresequentially hybridized with different probes the 32P-labeled cDNA wasremoved after autoradiography by two washing steps with boiling0.05� SSC/0.1% SDS for 15 min before rehybridization.

cDNA Probe—As a probe for hybridization the cDNA of rat Prx I wasapplied (22). The cDNA fragment was labeled by the oligomer methodwith � [32P]dCTP using the multiprime DNA labeling kit according tothe manufacturer’s instructions. To correct for differences in RNA load-ing filters were rehybridized with a 28 S ribosomal RNA (rRNA) oligo-nucleotide, which was labeled with [�-32P]dATP at the 5�-end with T4polynucleotide kinase.

Cloning of the 5�-Flanking Region of the Prx I Gene Promoter—Theproximal promoter 5�-flanking region of the rat Prx I gene was isolated

using the Genome WalkerTM kit from Clontech (Palo Alto, CA), whichcontains separate pools (libraries) of uncloned, genomic DNA that havebeen predigested with EcoRV, ScaI, DraI, PvuII, or SspI and ligated toan oligonucleotide anchor (adapter primer 1). A nested PCR approachwas employed. The first PCR was performed with the adapter primer 1(as described in the kit) and a 25-bp primer from position 55 to 31 of thePrx I cDNA (3) (5�-CTTGCTATCAGCAGAGTCGAGCTAC-3�) on a DNAcycler (PerkinElmer Life Sciences) using a program of 94 °C � 25 s,72 °C � 3 min for seven cycles, 94 °C � 25 s, 67 °C � 3 min for 32 cycles,and 67 °C � 4 min for one cycle. After analysis of an aliquot of the PCRproducts on a 1% agarose gel, the PCR products were diluted 1:50 insterile deionized H2O and subjected to a second round of PCR. Thesecond PCR was performed with the adapter primer 2 and the nested26-bp primer from position 31 to 9 of the Prx I cDNA (5�-CAGGTCT-CACAAACAGAACCAACCGT-3�) using a program of 94 °C � 25 s,72 °C � 3 min for five cycles, 94 °C � 25 s, 67 °C � 3 min for 20 cycles,and 67 °C � 4 min for one cycle. The amplified PCR products wereseparated by electrophoresis, subcloned into the pCR2.1TM vector (In-vitrogen) to generate plasmid pCRPrxProm, and sequenced in bothdirections.

Plasmid Constructs—A MluI/XhoI fragment from plasmid pCRPrx-Prom was cloned into promoterless pGL3-Basic plasmid to generatepPrx1750. Deletion mutants pPrx1209, pPrx897, pPrx558, pPrx283,and pPrx127 were generated by PCR using oligonucleotides Prx1209(5�-CATAGCAAAAAGCAAACTTTC-3�), Prx897 (5�-GTTCTGTCTCAC-TTCCTCG-3�), Prx558 (5�-AAGAGACTTTTGGGGGACATC-3�), Prx283(5�-GCAGGGCCAGGAGAC-3�), and Prx127 (5�-GACCAGTGAAGCTC-TTTTC-3�) as forward primers, respectively, and oligonucleotide Prxba-sic (5�-TCTGGCACCTGCACTTG-3�) as a reverse primer. PCR productswere religated by standard procedures. To construct plasmidpPrx897/668 and pPrx668/897 specific primers with adjacent SacI orBglII restriction sites were generated. For pPrx897/668 the forwardprimer Prx897SacIF (5�-AATCGGAGCTCGTTCTGTCTCACTTCCCT-CGC-3�) with a SacI restriction site and the reverse primer Prx668Bg-lIIR (5�-GACTGAGATCTTGTTTGCTGAACACCATGAC-3�) with aBglII restriction site were used for PCR with pPrx1750 as template.After digestion with SacI and BglII the PCR fragment was cloned into

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the SacI/BglII sites of pGL3-Prom. Plasmid pPrx668/897 was con-structed accordingly with the forward primer Prx897BglIIF (5�-GACT-GAGATCTGTTCTGTCTCACTTCCTCGC-3�) and the reverse primerPrx668SacIR (5�-AATCGGAGCTCTGTTTGCTGAACACCATGAC-3�).In a similar fashion pPrx667/283 and pPrx283/667 were generated byusing the primer pairs Prx667SacIF (5�-AATCGGAGCTCCCATGGG-GTCGGGTCCCAG-3�) and Prx283BglIIR (5�-GACTGAGA TCTCCA-CTGTCATCAACAACCAAAG-3�) or Prx667BglIIF (5�-GACTGAGATC-TCCATG GGGTCGGGTCCCAG-3�) and Prx283SacIR (5�-AATCGGAG-CTCCCACTGTCATCAA CAACCAAAG-3�), respectively. pPrx282/108and pPrx108/282 were generated by using the primer pairsPrx282SacIF (5�-AATCGGAGCTCCAGGGCCAGGAGACCTAATG-3�)and Prx108BglIIR (5�-GACTGAGATCTGGAAAAGAGCTTCACTG-GTC-3�) and Prx282BglIIF (5�-GACTGAGATCTCAGGGCCAGGAGAC-CTAATG-3�) and Prx108SacIR (5�-AATCGGAGCTCGGAAAAGAGCT-TCACTGGTC-3�), respectively.

Plasmid pPrx558mutA was generated with the template pPrx558and the oligonucleotide MutAfor (5�-GTCAGACAGGAAGACTGTGTG-TATGGGC-3�) as forward primer and the oligonucleotide MutArev (5�-AACCCCTGTCTCTGTCATCTCTGTTCC-3�) as reverse primer. Plas-mid pPrx558mutB was generated with the oligonucleotide MutB2for(5�-AGTCAAAAGCTTATTCTAAAGTGCAGGGTC-3�) as forwardprimer and MutB2rev (5�-ACGGAGTCATAGTCTCTGTGCTACAGC-3�) and pPrx558 as template. To construct plasmid pPrx558mutABoligonucleotides MutAfor and MutArev were applied for PCR withplasmid pPrx558mutB as template. All PCR fragments were religatedby standard procedures, and the constructs were verified by DNAsequencing in both directions. The luciferase reporter gene constructpAP-1luc with six copies of the AP-1 enhancer was a gift from Dr. BrentCarter (University of Iowa College of Medicine, Iowa City, IA) (29), andpNF-�Bluc was purchased from Stratagene.

Expression vectors for constitutively active H-Ras (Q61L mutant),dominant negative H-Ras (S17N mutant), constitutively active Raf-1(Y340D mutant), and dominant negative Raf-1 (K375M mutant) havebeen described (30). Expression plasmids for MAPK signaling pathwaycomponents were generous gifts: MEKK1 and dominant negativeMEKK1 were from Dr. Melanie Cobb (Southwestern Medical Center,Houston, TX), dominant negative MAPK kinase (MKK)6 were from Dr.Sylvio Gutkind (National Institutes of Health, Bethesda, MD), domi-nant negative MKK4 were from Dr. Ulf Rapp (University of Wurzburg,Wurzburg, Germany), dominant negative MKK7 were from Dr. EisukeNishida (University of Kyoto, Kyoto, Japan), wild-type and dominantnegative mutants (AF) of p38�, p38�, p38�, and p38� were from Dr. J.Han (Scripps Research Institute, La Jolla, CA), and dominant negativeI�B were from Dr. Richard Gaynor (Southwestern Medical Center,Houston, TX). Expression vectors for c-Jun, dominant negative c-Jun,Nrf2 (nuclear factor E2-related factor 2), dominant negative Nrf2, andthe p3xStREluc reporter plasmid with three copies of the heme oxygen-ase-1 antioxidant response element (ARE) were provided by Dr. JawedAlam (Alton Ochsner Foundation, New Orleans, LA).

Transfection and Luciferase Assay—After growth for 16 h transfec-tion of plasmid DNA into RAW264.7 cells was performed by the lipo-some method using FuGENE (Roche Applied Science) according to themanufacturer’s instructions. Unless otherwise stated cells were trans-fected with 1 �g of reporter plasmid and 0.1 to 0.5 �g of cotransfectedexpression vectors. Equal amounts of DNA were adjusted with therespective empty expression vector. Cells were lysed with 1� luciferaselysis buffer, and luciferase activity was determined with a luciferaseassay system (Promega, Madison, WI) according to the manufacturer’sinstructions. Cells were either harvested 24 h after transfection or

FIG. 4. Effect of overexpressed c-Jun on TPA-dependent in-duction of Prx promoter activity. A, RAW264.7 cells were cotrans-fected with the indicated Prx I promoter luciferase gene constructs andempty control expression vector or an expression vector for dominantnegative c-Jun. 24 h after transfection cells were treated for 16 h withcontrol medium or medium supplemented with TPA (0.5 �M). Cellextracts were assayed for luciferase activity, and the rate of induction ineach experiment relative to the control was determined. The values areS.E. from three independent experiments with duplicates of each point.Statistics, Student’s t test for paired values: *, significant differencesTPA versus control; **, TPA � c-Jundn versus TPA control, p � 0.05. B,RAW264.7 cells were cotransfected with pPrx558 and empty expressionvector or an expression vector for wild-type c-Jun. 24 h after transfec-

tion cells were treated for 16 h with control medium or medium sup-plemented with a submaximal concentration of TPA (0.2 �M). Cellextracts were assayed for luciferase activity, and the rate of induction ineach experiment relative to the control was determined. The values areS.E. from three independent experiments with duplicates of each point.Statistics, Student’s t test for paired values: *, significant differencesTPA versus control, c-Jun versus control, TPA � c-Jun versus control;**, TPA � c-Jun versus c-Jun, p � 0.05. C, pPrx558 and pPrx558mutABwere cotransfected in parallel with empty expression vector or an ex-pression vector for wild-type c-Jun. Cell extracts were assayed forluciferase activity, and the rate of induction in each experiment relativeto the control was determined. The values are S.E. from three inde-pendent experiments with duplicates of each point. Statistics, Student’st test for paired values: *, significant differences pPrx558 � TPA versuspPrx558 control; **, pPrx558mutAB � c-Jun versus pPrx558 � c-Jun,p � 0.05. c-Jundn, dominant negative c-Jun.



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treated for another 18 h with TPA as indicated. Relative light unitswere correlated with sample protein.

Preparation of Nuclear Extracts and Electrophoretic Mobility ShiftAssay (EMSA)—Nuclear extracts were prepared essentially as de-scribed (30). The sequences of the oligonucleotides used for the EMSAare as follows: Prx I AP-1-A, 5�-GACAGGGTGACTAAGACAGGA-3�(spanning the Prx I AP-1-A site; see Fig. 2), Prx I AP-1-B, 5�-TGACTC-CTGACTCAAAAGCTT-3� (spanning the Prx I AP-1-B site; see Fig. 2),and Prx I AP-1-Amut, 5�-GACAGGGGTTCTCAGACAGGA-3�. TheAP-1 consensus oligonucleotide with the sequence 5�-CGCTTGAT-GAGTCAGCCGGAA-3� was from Promega. Equal amounts of comple-mentary oligonucleotides were annealed and labeled by 5�-end labelingwith [�-32P]dATP and T4 polynucleotide kinase. They were purifiedwith the nucleotide removal kit. Binding reactions were carried out ina total volume of 20 �l containing 50 mM KCl, 1 mM MgCl2, 1 mM EDTA,5% glycerol, 10 �g of nuclear extract, 250 ng poly(dI-dC), and 5 mM

dithioerythritol. For competition analyses an excess of unlabeled oligo-nucleotides was added. After preincubation for 5 min at room temper-ature, 1 �l of the labeled probe (104 cpm) was added, and the incubationwas continued for an additional 10 min. For supershift analysis 1 �l ofthe c-JunD (epitope corresponding to DNA binding domain), c-JunN(epitope mapping within the N-terminal domain), cAMP regulatoryelement (RE) binding protein-1 (24H4B), or SP-1 (PEP2-G) antibody (allobtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA), as wellas a rabbit preimmune serum, were added to the EMSA reaction, whichwas then incubated at 4 °C for 2 h. The electrophoresis was thenperformed with a 4.5% non-denaturing polyacrylamide gel in TBE

buffer (89 mM Tris, 89 mM boric acid, 5 mM EDTA) at 200 V. Afterelectrophoresis the gels were dried and exposed to x-ray films.

RESULTS

TPA-dependent Induction of Prx I Gene Expression in Cul-tured Rat Liver Tissue Macrophages and RAW264.7 Cells—Regulation of Prx I gene expression by TPA was examined incultures of rat liver tissue macrophages and the mouse mono-cytic cell line RAW264.7. As shown by Northern and Westernblot analysis TPA caused a pronounced induction of Prx ImRNA and protein expression, respectively (Fig. 1, A and B). InRAW264.7 cells Prx I mRNA up-regulation by TPA was time-dependent with a peak after 6 h (Fig. 1C). Moreover, a dose-dependent increase of Prx I mRNA expression was observedwith a maximum at a concentration of 0.5 �M TPA (Fig. 1D). Toprobe into the mechanism of TPA-dependent Prx I gene induc-tion RAW264.7 cells were treated with actinomycin D, whichinhibits cellular transcription. Induction of Prx I mRNA ex-pression by TPA was blocked by pretreatment with actino-mycin D suggesting that Prx I gene induction was regulatedon the transcriptional level (Fig. 1E). The data indicate thatPrx I gene expression is induced by TPA in cultured mono-cytic cells.

FIG. 5. Effect of overexpressed Nrf2on TPA-dependent induction of Prxpromoter activity. A, RAW264.7 cellswere cotransfected with the indicated PrxI promoter luciferase gene constructs andempty control expression vector or an ex-pression vector for Nrf2. 24 h after trans-fection cells were treated for 16 h withcontrol medium or medium supplementedwith TPA (0.5 �M) as indicated. Cell ex-tracts were assayed for luciferase activity,and the rate of induction in each experi-ment relative to the control was deter-mined. The values are S.E. from threeindependent experiments with duplicatesof each point. Statistics, Student’s t testfor paired values: *, significant differenceTPA versus control, p � 0.05. B,RAW264.7 cells were cotransfected withpPrx558 and empty expression vector oran expression vector for dominant nega-tive Nrf2. 24 h after transfection cellswere treated for 16 h with control mediumor medium supplemented with TPA (0.5�M) as indicated. Cell extracts were as-sayed for luciferase activity, and the rateof induction in each experiment relativeto the control was determined. The valuesare S.E. from three independent experi-ments with duplicates of each point. Sta-tistics, Student’s t test for paired values:*, significant difference TPA versus con-trol, p � 0.05. Nrf2dn, dominant negativeNrf2.



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Cloning of the Rat Prx I Gene Promoter and Identification ofPotential cis-Acting Elements—To analyze the molecular mech-anisms of TPA-dependent Prx I gene expression in more detailwe cloned the promoter and 5�-flanking region of the rat Prx Igene. For this purpose a nested PCR approach was employed asdetailed under “Experimental Procedures.” The obtained1750-bp DNA fragment was subcloned into pCR2.1, and thesequence was determined by standard sequencing methods anddeposited into GenBankTM (accession number AJ457059). Thetranscriptional start site was determined by primer extensionanalysis using total RNA from RAW264.7 cell cultures and ratliver (data not shown) and was localized 48 bp upstream of thetranslational start site (Fig. 2). Sequence comparison of thecloned rat Prx I promoter with the previously characterizedpromoter region of the human proliferation-associated geneshowed no sequence similarity (3, 31). A TATA box was iden-tified at position �62 relative to the transcription start site.Putative cis-acting elements were localized by sequence com-parison in the cloned promoter 5�-flanking region of the rat PrxI gene. Various candidate cis-acting sequences were identifiedincluding, but not limited to, AP-1 and NF-�B (Fig. 2). Inter-estingly, the extended sequence of the AP-1-A site exhibitedhigh homology to the consensus sequence of the ARE, GGTG-ACNNNGCA, which is a target of the transcription factor (TF)Nrf2. Nrf2 has been shown previously (32) to play an importantregulatory role for the stress-dependent induction of Prx I gene

expression (32). At this point the functional significance of theidentified DNA elements was unclear.

Functional Role of Proximal AP-1 Sites for the TPA-depend-ent Induction of Prx I Promoter Activity—The importance ofcis-acting elements in the regulation of Prx I gene expressionwas evaluated by ligating the cloned 5�-flanking promoter se-quence into the luciferase reporter vector pGL3-Basic to givepPrx1750. To identify REs that mediate TPA-dependent induc-tion of Prx I gene expression RAW264.7 cells were transfectedwith serially 5�-deleted Prx I promoter luciferase reporter geneconstructs. Luciferase activity of pPrx1750 was induced byTPA 9-fold, and the strongest TPA-dependent up-regulationof luciferase activity was observed for construct pPrx558(Fig. 3A).

To assess the regulatory capacity of putative AP-1 sites inthe Prx I promoter 5�-flanking region various reporter geneconstructs were generated by cloning fragments of the Prx Ipromoter into plasmid pGL3-Prom in front of the SV40 pro-moter. DNA sequences with the AP-1-A (�211 to �205; TGAC-TAA), AP-1-B (�505 to �499; TGACTCA), or AP-1-C (�829 to�823; TGAGTCA) sites were ligated in either 5�-3� or 3�-5�orientation into this luciferase reporter plasmid, respectively.Regulation of these constructs by TPA was compared with thatof pPrx887, which contains the promoter with the AP-1-A-Csites in the wild-type context (Fig. 3B). The strongest TPA-de-pendent induction of luciferase activity was observed for con-

FIG. 6. Binding of nuclear proteins to Prx I AP-1-A and AP-1-B sites. A, the AP-1 consensus sequence and the AP-1-A and AP-1-B sitesof the Prx I promoter are shown. Bases matching the AP-1 consensus sequence are in bold. B, EMSA. 32P-Labeled oligonucleotides containing eitherthe Prx I AP-1-A element or the AP-1 consensus sequence (Promega) were incubated with 4 �g of protein of nuclear extracts from eitherTPA-treated or control RAW264.7 cells as indicated. C, for competition analyses the radiolabeled AP-1-A oligonucleotide was incubated with 4 �gof nuclear extracts from control cells and a 20- or 50-fold molar excess of unlabeled AP-1-A or AP-1-Amut oligonucleotides as indicated (left panel).In addition, nuclear extracts from control cells were preincubated with 1 �l of antibodies against the N-terminal domain of c-Jun or the DNAbinding domain of c-Jun (Santa Cruz Biotechnology, Inc.) for 1 h at 4 °C before adding the labeled AP-1-A probe (right panel). D, radiolabeledAP-1-A or AP-1-B probes were incubated with either 4 or 8 �g of nuclear extracts from control cells and a 10- or 20-fold molar excess of unlabeledAP-1-A or AP-1-B oligonucleotides as indicated. DNA-protein complexes were analyzed by electrophoresis on 4.5% native polyacrylamide gels. AP-1cons, AP-1 consensus sequence; con, control; D, antibody against DNA binding domain of c-Jun; N, antibody against N-terminal domain of c-Jun;NE, nuclear extract; S, supershift.



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struct pPrx282/108, which contained the AP-1-A site (Fig. 3B).TPA-dependent induction of pPrx282/108, however, was mark-edly lower as compared with that of pPrx887 suggesting thatboth the AP-1-A and the AP-1-B sites may be functional in acooperative manner.

The functional role of the AP-1-A and AP-1-B sites was alsodetermined by mutation studies. Induction of luciferase activ-ity of pPrx558mutB by TPA was slightly reduced in comparisonwith that of wild-type pPrx558 (Fig. 3C), and a markedly stron-ger reduction of the TPA-mediated Prx I transcriptional activ-ity was observed for pPrx558mutA and pPrx558mutAB (Fig.3C). TPA-dependent regulation of pPrx558 was also comparedwith that of a luciferase reporter gene construct with six copiesof the prototypical AP-1 consensus sequence (pAP-1luc; see Ref.29). As demonstrated in Fig. 3C the induction of pPrx558 byTPA was markedly higher in comparison with that of pAP-1luc.Thus, the AP-1-A, and to a minor extent the AP-1-B, sitesmediate the induction of Prx I promoter activity by TPA.

Up-regulation of Prx I Promoter Activity by Overexpressedc-Jun but Not Nrf2—AP-1 exists as a dimer of Jun and Fosproteins both belonging to the basic leucine zipper family of

TFs (33). To investigate the regulatory role of c-Jun for Prx Ipromoter activity RAW264.7 cells were cotransfected with var-ious Prx I promoter luciferase gene constructs and an expres-sion vector encoding a dominant negative mutant of c-Jun.TPA-dependent induction of Prx I promoter activity was down-regulated by dominant negative c-Jun (Fig. 4A). Moreover, thebasal and the TPA-dependent induction of Prx I promoter ac-tivity was enhanced by overexpressed wild-type c-Jun (Fig.4B). In contrast, the pPrx558mutAB construct with targetedmutations in the AP-1-A and AP-1-B sites was only slightlyaffected by overexpressed c-Jun (Fig. 4C)

The extended DNA sequence of the AP-1-A site exhibits highhomology to the ARE consensus motif, which may serve as anuclear target for the TF Nrf2 (34, 35). To determine theregulatory potential of Nrf2 for this putative ARE we examinedthe regulation of Prx I reporter gene constructs by cotrans-fected expression vectors with either wild-type or dominantnegative forms of Nrf2. Overexpression of wild-type Nrf2 hadno effect on the basal or the TPA-regulated reporter geneactivity of pPrx1750 and pPrx558 (Fig. 5A) but markedly up-regulated luciferase activity of control plasmid p3xStRE (34)

FIG. 7. Inhibition of TPA-dependentinduction of Prx I gene expression bythe PKC inhibitors BIM and stauro.A, the indicated Prx I promoter luciferasegene constructs were transiently trans-fected into cultured RAW264.7 cells. 24 hafter transfection cells were treated for16 h with control medium or medium sup-plemented with TPA (0.5 �M), BIM (0.5�M), stauro (0.02 �M), TPA plus BIM, orTPA plus stauro as indicated. Cell ex-tracts were assayed for luciferase activity,and the rate of induction in each experi-ment relative to the control was deter-mined. The values are S.E. from threeindependent experiments with duplicatesof each point. Statistics, Student’s t testfor paired values: *, significant differ-ences pPrx1750 � TPA versus pPrx1750control, pPrx558 � TPA versus pPrx558control; **, pPrx1750 � TPA � BIMversus pPrx1750 � TPA; pPrx1750 �TPA � stauro versus pPrx1750 � TPA;pPrx558 � TPA � BIM versus pPrx558 �TPA, pPrx558 � TPA � stauro versuspPrx558 � TPA, p � 0.05. B, after 18 h inserum-free medium RAW264.7 weretreated for 6 h with control medium ormedium supplemented with TPA (0.5�M), BIM (0.5 �M), stauro (0.02 �M), TPAplus BIM, or TPA plus stauro as indi-cated. Total RNA was isolated and sub-jected to Northern blot analysis. The blotswere probed sequentially with a 32P-labeled cDNA of rat Prx I and a 28 SrRNA oligonucleotide. Values � S.E.given represent the -fold induction of PrxI mRNA normalized to 28 S rRNA levelsfrom three independent experiments. Sta-tistics, Student’s t test for paired values:*, significant differences TPA versus con-trol; **, TPA � BIM versus TPA, TPA �stauro versus TPA, p � 0.05.



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FIG. 8. Regulation of Prx I promoteractivity by overexpressed Ras, Raf-1,and MEKK-1. A, RAW264.7 cells werecotransfected with an expression vectorfor dominant negative Ras or empty ex-pression vector and luciferase gene con-struct pPrx558 in the presence or absenceof TPA (0.5 �M) as indicated. Cell extractswere assayed for luciferase activity, andthe rate of induction in each experimentrelative to the control was determined.The values are S.E. from three independ-ent experiments with duplicates of eachpoint. Statistics, Student’s t test forpaired values: *, significant differenceTPA versus control; **, TPA � Rasdn ver-sus TPA, p � 0.05. B, the effects of over-expressed dominant negative Raf-1 ordominant negative MEKK1 were deter-mined in RAW264.7 cells when luciferasereporter gene activity of pPrx558 plasmidwas activated by a cotransfected expres-sion vector for constitutively activatedRas as indicated. Cell extracts were as-sayed for luciferase activity, and the -foldinduction in each experiment relative tothe control was determined. Values areS.E. from three independent experimentswith duplicates of each point. Statistics,Student’s t test for paired values: *, sig-nificant differences Ras ca versus control,Ras ca � Raf-1dn versus Raf-1dn control;**, Ras ca � MEKK1dn versus Ras cacontrol, p � 0.05. C, RAW264.7 cells werecotransfected with expression vectors forwild-type MEKK1 and constitutively acti-vated Raf-1 or empty expression vectorsalong with luciferase gene constructpPrx558 as indicated. Cell extracts wereassayed for luciferase activity, and the-fold induction in each experiment rela-tive to the control was determined. Thevalues are S.E. from three independentexperiments with duplicates of eachpoint. Statistics, Student’s t test forpaired values: *, significant differencesMEKK1 versus control, p � 0.05.MEKK1dn, dominant negative MEKK1;Raf-1ca, constitutive activated Raf-1; Raf-1dn, dominant negative Raf-1; Ras ca,constitutive activated Ras; Rasdn, domi-nant negative Ras.



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containing three copies of the ARE (data not shown). In addi-tion, basal or TPA-regulated Prx I promoter activity was notaffected by overexpressed dominant negative Nrf2 (Fig. 5B).From the data it is concluded that Prx I promoter gene activityis up-regulated by c-Jun but not by Nrf2.

Binding of c-Jun to the AP-1-A Site of the Prx I Promoter—In EMSA studies we examined the binding affinity of nuclearproteins from TPA-treated or untreated RAW264.7 cells toradiolabeled oligonucleotides with the Prx I AP-1-A site or theAP-1 consensus motif (Fig. 6A). No difference in DNA bindingaffinity from control or TPA-treated cells was observed for thePrx I AP-1-A oligonucleotide (Fig. 6B). By contrast, nuclearextracts from TPA-treated cells formed a stronger DNA-proteincomplex with the AP-1 consensus oligonucleotide as comparedwith extracts from control cells. The DNA-protein band formedwith AP-1-A was completely abolished by a 20-fold molar ex-cess of unlabeled AP-1-A oligonucleotide. By contrast, an excessof an oligonucleotide with a targeted mutation in the AP-1-Asite (AP-1-Amut) did not affect the intensity of this band (Fig.6C, left panel). Incubation of the binding reaction of the AP-1-Aoligonucleotide with an antibody against the N-terminal do-main of c-Jun produced a supershifted band. Moreover, addi-tion of an antibody raised against the DNA binding domain ofc-Jun attenuated the intensity of the DNA-protein complex(Fig. 6C, right panel). No obvious alteration of the AP-1-ADNA-protein band was observed with antibodies for the TFscAMP RE-binding protein or SP-1 (data not shown) suggestingthat the AP-1-A site is a nuclear target sequence of c-Jun.

The AP-1-B site conferred a relatively minor TPA responsewhen compared with AP-1-A (Fig. 3). To compare the DNAbinding affinities of these two elements for nuclear proteins,equal amounts of nuclear extracts from RAW264.7 cells wereincubated with radiolabeled AP-1-A and AP-1-B oligonucleo-tides. A markedly stronger DNA-protein complex formed withthe AP-1-A oligonucleotide as compared with that of AP-1-B.Moreover, a 10-fold molar excess of cold AP-1-A completelyabolished the DNA-protein complex of radiolabeled AP-1-B,whereas a 10-fold molar excess of AP-1-B did not abolish theAP-1-A DNA-protein band (Fig. 6D).

Attenuation of TPA-dependent Induction of Prx I Gene Ex-pression by PKC Inhibitors—TPA activates PKC by interactionwith the regulatory PKC domain (26, 27). To investigate thesignaling pathways that are involved in TPA-dependent induc-tion of Prx I promoter activity we tested the effects of the PKCinhibitors bisindolylmaleimide (BIM) and staurosporine(stauro). Both BIM and stauro strongly reduced TPA-depend-ent induction of pPrx1750 and pPrx558 luciferase activity (Fig.7A). The effect of BIM and stauro was also examined for TPA-dependent regulation of the endogenous Prx I gene expressionby Northern blot analysis. Both agents inhibited the inductionof Prx I mRNA levels by TPA (Fig. 7B) suggesting that the PKCpathway is involved in TPA-dependent regulation of Prx I geneexpression.

Role of Ras and MEKK1 for the TPA-dependent Induction ofPrx I Promoter Activity—A potential target of PKC signaling isthe low molecular weight GTP-binding protein Ras (36). Toinvestigate the role of Ras for the induction of Prx I geneexpression by TPA we examined the regulation of Prx I pro-moter activity by cotransfected expression vectors for dominantnegative and constitutively activated Ras. TPA-dependent in-duction of pPrx558 reporter gene activity was inhibited byoverexpressed dominant negative Ras (Fig. 8A). Overexpres-sion of constitutively activated Ras increased the reporter geneactivity of pPrx558 (Fig. 8B). We also determined the regula-tory role of the Ras effector molecules Raf-1 and MEKK1 on PrxI promoter activity. Overexpression of dominant negative Raf-1

did not inhibit Ras-dependent Prx I induction (Fig. 8, A and B),whereas overexpression of a dominant negative mutantMEKK1 markedly inhibited Ras-dependent induction of Prx Ipromoter activity (Fig. 8B).

Next, we examined the effect of overexpressed wild-typeMEKK1, which is highly active when overexpressed. Cotrans-fected wild-type MEKK1 had a marked stimulatory effect onreporter gene activity of pPrx558, whereas overexpression ofconstitutive activated Raf-1 only had a minor effect (Fig. 8C).The data suggest that activation of Ras and MEKK1 is involvedin the TPA-dependent induction of Prx I gene expression.

Activation of MAPKs by TPA in RAW264.7 Cells—MEKK1activates downstream MAPK signaling cascades, specificallythe JNK and p38 pathways. To determine the potential role ofMAPKs for TPA-mediated Prx I gene activation RAW264.7cells were treated with 0.5 �M TPA for various lengths of time,and cell extracts were analyzed for phosphorylated and totalMAPKs by Western blotting. Phosphorylated ERK1 and ERK2were detected only to a minor degree in untreated cells, and anincrease in the levels of these species was readily observed witha maximum after 15 min (Fig. 9). Phosphorylation of JNK andp38 was also observed for up to 4 h (Fig. 9). The data indicatethat ERK, JNK, and p38 MAPKs are activated by TPA inRAW264.7 cells.

Role of p38 in the Regulation of TPA-dependent Prx I Pro-moter Induction—MEKK1 activates the JNK signaling path-way through phosphorylation of MKK4 and MKK7. In addition,MEKK1 can stimulate the p38 MAPK through phosphorylationof MKK3 and MKK6 (37). Activation of MKK4, however, canalso stimulate the p38 MAPK activity (37). To further investi-gate the signaling pathways downstream of MEKK1 that maybe involved in the transcriptional Prx I gene regulation wedetermined the effects of pharmacological MAPK inhibitors, aswell as that of overexpressed dominant negative MKKs, onTPA-dependent Prx I promoter induction. The up-regulation ofPrx I promoter activity by TPA was inhibited by pretreatmentwith the p38 inhibitor SB202190 but not by the JNK inhibitorSP600125 (Fig. 10A). Overexpressed dominant negative MKK4inhibited the TPA-dependent induction of Prx I promoter ac-tivity, whereas overexpressed dominant negative MKK7,which is a specific activator of JNK, had no effect on Prx I gene

FIG. 9. Activation of MAPKs by TPA in RAW264.7 cells.RAW264.7 cells were cultured as described under “Experimental Pro-cedures.” Cell cultures were kept for 18 h in serum-free medium beforetreatment with TPA (0.5 �M) for 0.25, 0.5, 1, 2, and 4 h as indicated.Total protein (50 �g) was subjected to Western blot analysis and probedwith polyclonal antibodies against various MAPKs. Filters were ini-tially used to detect phosphorylated (phospho) MAPKs and thenstripped and probed with antibodies that detect total MAPKs. Similarresults were obtained in two-three independent experiments. Autora-diograms from representative experiments are shown, respectively.



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reporter gene activity (Fig. 10B). Overexpression of dominantnegative MKK6 markedly inhibited TPA-dependent inductionof Prx I promoter activity (Fig. 10B).

To investigate the role of p38 in the regulation of Prx I geneexpression, we examined the effect of overexpressed dominantnegative p38�, �, �, and � isoforms (AF) on TPA- and MEKK1-dependent induction of Prx I promoter activity. TPA-dependentinduction of pPrx558 was inhibited to a different extent byvarious overexpressed dominant negative p38 isoforms (Fig.11A). Similarly, the induction of Prx I promoter activity byMEKK1 was decreased by overexpressed dominant negativep38 isoforms. We also determined the effect of cotransfectedwild-type p38 isoforms on the reporter gene activity of pPrx558.Overexpression of p38� and p38�, but not that of p38� andp38�, up-regulated the basal activity of Prx I promoter activity(Fig. 11B). In contrast, none of these p38 isoforms affected thepromoter activity of the pPrx558mutAB reporter gene con-struct with targeted mutations in the AP-1-A and AP-1-B sites(data not shown). Taken together, the data demonstrate thatactivation of the p38 MAPK pathway plays a major role for theTPA-dependent activation of Prx I gene expression.

The NF-�B Signaling Pathway Is Not Involved in the Regu-lation of Prx I Promoter Activity—The NF-�B signaling path-

way has been demonstrated to play a regulatory role for TPA-dependent induction of various genes (38–40). Because theproximal Prx I promoter region contains a putative NF-�B site(Fig. 2) we investigated the potential regulatory role of NF-�Bsignaling by examining the effect of the NF-�B inhibitor caffeicacid phenethyl ester (CAPE), as well as that of overexpresseddominant negative I�B, on TPA-dependent induction of Prx Ipromoter activity. Pretreatment of cell cultures with CAPE hadno effect on TPA-dependent reporter gene induction ofpPrx1750. Moreover, TPA-dependent induction of this con-struct was not inhibited by overexpressed dominant negativeI�B (Fig. 12B). Both CAPE and overexpressed dominant neg-ative I�B markedly inhibited the TPA-dependent induction of acontrol reporter gene construct with five copies of the NF-�Bsite (Fig. 12B). The data suggest that activation of the NF-�Bsignaling pathway does not play a role for TPA-dependentinduction of Prx I promoter activity.

DISCUSSION

Prx I belongs to the antioxidant protein group of Prxs thatare characterized by thiol-specific peroxidase activity (4–6, 8,9). The major findings of the present study on the gene regu-lation of Prx I are as follows. 1) The tumor promoter TPA

FIG. 10. Effects of pharmacologicalMAPK inhibitors and overexpresseddominant negative mutants of MKKson TPA-dependent induction of Prx Ipromoter activity. A, RAW264.7 cellswere transiently transfected with the lu-ciferase gene construct pPrx558. Cellswere treated for 30 min with SP600125(10 �M) and SB202190 (5 �M) before add-ing TPA (0.5 �M) for 16 h. In each exper-iment the -fold induction of luciferase ac-tivity was determined relative to thepPrx558 control, which was set equal to 1.The values are S.E. from at least threeindependent experiments with duplicatesof each point. Statistics, Student’s t testfor paired values: *, significant differ-ences of TPA versus control, TPA �SP600125 versus control; **, TPA �SB202190 versus TPA, p � 0.05. B,RAW264.7 cells were cotransfected withthe luciferase reporter gene constructpPrx558 and empty control expressionvector or expression vectors with the dom-inant negative mutants of the indicatedMKKs. 24 h after transfection cells weretreated for 16 h with control medium ormedium supplemented with TPA (0.5�M). Cell extracts were assayed for lucif-erase activity, and the TPA-dependent in-duction in each experiment relative to therespective control was determined. Thevalues are S.E. from three independentexperiments with duplicates of eachpoint. Statistics, Student’s t test forpaired values: *, significant differences ofTPA versus control, MKK7dn � TPAversus MKK7dn control; **, significantdifferences of MKK4dn � TPA versus con-trol � TPA, MKK6dn � TPA versus con-trol � TPA, p � 0.05.



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induces Prx I in cultured rat liver tissue macrophages and inthe mouse monocytic cell line RAW264.7. 2) The transcrip-tional induction of Prx I by TPA is mainly regulated via twoproximal AP-1 sites localized in the proximal promoter 5�-flanking region that bind c-Jun but not Nrf2. 3) A PKC/Ras/MEKK1/p38 MAPK signaling pathway mediates the TPA-de-pendent induction of Prx I gene expression.

Transcriptional Induction of Prx I Gene Expression by theTumor Promoter TPA—Peroxidase activity of Prx I has beenshown to be regulated on the post-translational level by variousmechanisms (for a review see Ref. 9) including protein phos-phorylation by cyclophilin-dependent kinase-2 (19), interactionwith cyclophilin A (41), C-terminal proteolysis (20), and redox-dependent modification (21). Here, it is reported that geneexpression of Prx I is transcriptionally induced by the phorbolester TPA, which is known to be a tumor promoter. In culturedrat liver tissue macrophages and RAW264.7 cells expression ofthe endogenous Prx I gene was induced by TPA in a time- anddose-dependent manner (Fig. 1). To examine the molecularmechanisms of TPA-dependent Prx I induction we cloned the5�-flanking promoter region of the rat Prx I gene (Fig. 2), which

showed little similarity with the promoter sequence of thehuman homologue proliferation-associated gene (1, 31). By de-letion and mutation studies two proximal AP-1 sites, AP-1-Aand AP-1-B, were demonstrated to mediate the TPA-dependentinduction of Prx I promoter activity (Fig. 3). The AP-1-A sitethat matches the classical AP-1 consensus sequence (TGA(G/C)TCA) in 6 of 7 bp conferred a major TPA response to the PrxI promoter as compared with the AP-1-B site that is homolo-gous with the AP-1 consensus sequence (Fig. 6A). The strongerTPA responsiveness of the AP-1-A site coincided with a higherDNA binding affinity of this element for nuclear proteins fromRAW264.7 cells (Fig. 6D). This may suggest that DNA se-quences adjacent to the AP-1-A and AP-1-B sites are involvedin the regulation of the TPA-dependent induction of Prx Ipromoter activity. Such a conclusion is also supported by thefinding that a reporter gene construct with the Prx I promoterwas more strongly induced by TPA as compared with a lucif-erase gene construct containing six copies of the AP-1 consen-sus motif (see Fig. 3C and Ref. 29). TPA-dependent gene induc-tion via AP-1 sites has been demonstrated previously for othergenes such as the monoamine oxidase B gene in HepG2 hepa-

FIG. 11. Regulation of Prx I pro-moter activity by overexpressed p38isoforms. A, RAW264.7 cells were co-transfected with expression vectors fordominant negative mutants of p38�,p38�, p38�, and p38�, along with lucifer-ase reporter gene construct pPrx558. Incontrol experiments pPrx558 was co-transfected with empty expression vector.24 h after transfection cells were treatedfor 16 h with control medium or mediumsupplemented with TPA (0.5 �M). Cell ex-tracts were assayed for luciferase activity,and the -fold induction in each experi-ment relative to the control was deter-mined. The values are S.E. from threeindependent experiments with duplicatesof each point. Statistics, Student’s t testfor paired values: *, significant differenceof TPA versus control; **, significant dif-ferences of p38� dn � TPA versus control� TPA, p38� dn � TPA versus control �TPA, p38� dn�TPA versus control �TPA, p38� dn � TPA versus control �TPA, p � 0.05. B, RAW264.7 cells werecotransfected with expression vectors forwild-type p38�, p38�, p38�, and p38� andluciferase reporter gene constructpPrx558 or empty expression vector as acontrol. Cell extracts were assayed for lu-ciferase activity, and the rate of inductionin each experiment relative to the controlwas determined. The values are S.E. fromthree independent experiments with du-plicates of each point. Statistics, Stu-dent’s t test for paired values: *, signifi-cant differences of p38� versus control,p38� versus control; p � 0.05. p38dn,dominant negative p38.



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toma cells (42) or the involucrin A gene in keratinocyte cultures(43, 44).

AP-1 sites, also designated TPA-response elements, are nu-clear targets for homo- or heterodimers of Jun and Fos (33).The findings of an inhibition of TPA-dependent induction byoverexpressed dominant negative c-Jun (Fig. 5A) and inductionby wild-type c-Jun (Fig. 5B) strongly suggest that this proteinis involved in TPA-dependent induction of Prx I promoter ac-tivity. The up-regulation of Prx I promoter activity by c-Junalso correlated with the observation that AP-1-A was a nuclearbinding site for c-Jun (Fig. 6). The extended DNA sequence ofthe AP-1-A site was homologous in 10 of 12 bp with the con-sensus motif of the ARE that is a target of the TF Nrf2 (34, 35).Nrf2 has been shown previously (32) to play a major role forPrx I gene induction, because Prx I gene expression was notup-regulated by the stress stimuli sodium arsenite, cadmiumchloride, or menadione in cell cultures from Nrf2(�/�) mice.

The present data clearly indicate that the activity of the rat PrxI promoter is not regulated by Nrf2 (Fig. 5), which could beexplained by the fact that other functional AREs may be local-ized outside of the cloned Prx I promoter sequence.

Signaling Pathways of TPA-dependent Prx I Gene Activa-tion—Attenuation of the TPA-dependent induction of Prx Igene expression by PKC inhibitors (Fig. 7) indicated that thiskinase is involved in Prx I gene regulation. This finding issimilar to a previous report (25) in which Prx I gene activationby sodium arsenite was mediated by a PKC-dependent mech-anism in a murine osteoblast cell line. PKC can either bedirectly activated by interaction of TPA with the regulatoryPKC domain, or, alternatively, TPA can stimulate PKC indi-rectly via up-regulation of NADPH oxidase-mediated produc-tion of reactive oxygen species (26, 27). An indirect mechanismof TPA-dependent induction of gene expression has been shownpreviously (45) for the human manganese superoxide dis-

FIG. 12. Effects of CAPE and overexpressed dominant negative I�B on TPA-dependent induction of Prx I promoter activity. A,RAW264.7 cells were transfected with luciferase gene constructs either driven by wild-type 1750 bp of the rat Prx I promoter (pPrx1750) or fivecopies of NF-�B sites (pNF�Bluc). 24 h after transfection cells were preincubated with CAPE for 30 min (4 �g/ml) before adding TPA (0.5 �M) for16 h as indicated. In each experiment the -fold induction of luciferase activity by TPA was determined relative to the control, which was set equalto 1. The values are S.E. from at least three independent experiments with duplicates of each point. Statistics, Student’s t test for paired values:*, significant difference of pPrx1750 � TPA versus pPrx1750 control, pPrx1750 � CAPE � TPA versus pPrx1750 � CAPE, pNF�Bluc � TPA versuspNF�Bluc control; **, pNF�Bluc � CAPE � TPA versus pNF�Bluc TPA; p � 0.05. B, cells were cotransfected with either dominant negative I�Bexpression vector or empty expression vector and luciferase gene construct pPrx1750 or pNF�Bluc as indicated. 24 h after transfection cells wereincubated for 16 h with control medium or medium supplemented with TPA (0.5 �M). Cell extracts were assayed for luciferase activity, and therate of induction in each experiment relative to the control was determined. The values are S.E. from three independent experiments withduplicates of each point. Statistics, Student’s t test for paired values: *, significant difference of pPrx1750 � TPA versus pPrx1750 control,pPrx1750 � I�Bdn � TPA versus pPrx1750 � I�Bdn, pNF�Bluc � TPA versus pNF�Bluc control; **, pNF�Bluc � I�Bdn � TPA versus pNF�BlucTPA; p � 0.05. I�Bdn, dominant negative I�B.



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mutase gene in lung carcinoma cells. An important effectormolecule of PKC is the low molecular weight GTP-bindingprotein Ras (36), which is involved in the induction of Prx Igene expression by TPA as indicated by experiments with dom-inant negative and constitutively activated Ras mutants (Fig.8). Activation of the NF-�B pathway has been shown previously(46, 47) to be involved in TPA- and Ras-dependent signalingbut does not seem to play a role for the induction of Prx Ipromoter activity by TPA in our cell culture model (Fig. 12).Stimulation of Ras, however, can also activate the Raf-1/MEK/ERK, MEKK1/MEK4,7/JNK, or MEKK1/MEK3,6/p38 signal-ing cascades (for a review see Ref. 37). The three MAPKs, ERK,JNK, and p38, are phosphorylated by treatment with TPA inRAW264.7 cells (Fig. 9). Because overexpression of dominantnegative Raf-1 had no effect on Ras-dependent induction of PrxI promoter activity, the Raf-1/MEK/ERK signaling pathwayappears not to be involved in Prx I gene regulation. Ras-de-pendent induction of Prx I gene transcription, however, wasinhibited by overexpressed dominant negative MEKK1, and, inaddition, overexpression of wild-type MEKK1 markedly in-duced Prx I promoter activity (Fig. 8). Two major downstreamtargets of MEKK1 are the JNK and p38 MAPK pathways (37).Studies with pharmacological inhibitors of MAPKs and withdominant negative forms of MKKs suggested that activation ofp38 rather than that of JNK is involved in the induction of PrxI gene expression by TPA (Fig. 10). This conclusion was sup-ported by the inhibitory effect of overexpressed dominant neg-ative mutants of various p38 isoforms on TPA-dependent Prx Ipromoter induction (Fig. 11). Similar to a recent report (48) inwhich AP-1-mediated gene regulation has been shown to bemodulated in a highly specific manner by various p38 isoformsin human breast cancer cells, overexpressed p38 isoforms haddifferent regulatory effects on Prx I promoter regulation inRAW264.7 cells (Fig. 11). The findings of the present study alsoagree with a previous report (49) demonstrating that TPA-de-pendent activation of the matrix metalloprotease-9 is mediatedvia p38 activation in UMSCC and NIH3T3 cells. Interaction ofAP-1 with the TF TATA-binding protein may be involved inTPA-dependent gene activation via the p38 signaling pathwayas demonstrated recently by Carter et al. (29).

Physiological Functions of Prx I—Prx I belongs to the typicaltwo-cysteine Prxs (Prx I-IV), which are distinct from the atyp-ical two-cysteine Prx, Prx V, and the one-cysteine Prx, Prx VI(8, 9). Prx I has thioredoxin-dependent peroxidase activity andadds to the cellular defense mechanisms against oxidativestress (9). Prxs are involved in the cellular regulation of signaltransduction, apoptosis, and proliferation (9). It has been re-ported previously that Prxs regulate hydrogen peroxide-medi-ated signal transduction by growth factors and tumor necrosisfactor � in NIH3T3 and A431 cells (11) and that of thyrotropinin FRTL-5 thyroid cells (50). A developmental difference ofbacterial and eukaryotic two-cysteine Prxs may have evolvedas to their regulatory role for hydrogen-dependent signaling(21). Because Prxs are both major target molecules of peroxide,as well as peroxide-degrading enzymes in primary cultures ofLeydig cells, Rabilloud et al. (51) have suggested that the ratioof active to inactive Prx enzyme may protect against stress-mediated apoptosis. Prx I has also been suggested to be a tumorsuppressor by regulating cell proliferation and transformationvia direct interaction with the oncogenes c-Abl and c-Myc. Wenand Van Etten (14) have shown that Prx I can directly bind toc-Abl and inhibit its cytostatic properties. Moreover, it has beendemonstrated recently (15) that Prx I can affect the regulatoryproperties of c-Myc by interaction with the Myc Box II domain.

The results of the present study provide novel information onthe regulatory mechanisms of Prx I gene expression. Further

investigations along this line will help to understand the phys-iology and pathophysiology of Prx I gene expression in healthand disease and may result in specific therapeutic interven-tions in various diseases such as cancer and inflammation.

Acknowledgments—We thank E. Welzel and D. Brusius for excellenttechnical assistance and Dr. J. Alam, Dr. B. Carter, Dr. M. Cobb, Dr. R.Gaynor, Dr. S. Gutkind, Dr. J. Han, Dr. M. Nishida, and Dr. U. Rapp forthe generous supply of plasmids.

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Thomas Kietzmann and Stephan ImmenschuhAlexander Hess, Nastiti Wijayanti, Andrea Pathe Neuschäfer-Rube, Norbert Katz,

Protein Kinase C, Ras, p38 Mitogen-activated Protein Kinase Signaling PathwayPhorbol Ester-dependent Activation of Peroxiredoxin I Gene Expression via a

doi: 10.1074/jbc.M307871200 originally published online September 5, 20032003, 278:45419-45434.J. Biol. Chem.

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