Research Article JunB Induced by Constitutive CD30–Extracellular Signal-Regulated Kinase 1/2 Mitogen-Activated Protein Kinase Signaling Activates the CD30 Promoter in Anaplastic Large Cell Lymphoma and Reed-Sternberg Cells of Hodgkin Lymphoma Mariko Watanabe,1 Masataka Sasaki,1 Kinji Itoh,3 Masaaki Higashihara,1 Kazuo Umezawa,2 Marshall E. Kadin,5 Lawrence J. Abraham,6 Toshiki Watanabe,4 and Ryouichi Horie1,4 1Fourth Department of Internal Medicine, School of Medicine, Kitasato University; 2Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Kanagawa; 3Department of Pathology, School of Medicine, Toho University; 4Laboratory of Tumor Cell Biology, Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan; 5Department of Pathology, Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, Massachusetts; and 6Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences, University of Western Australia, Crawley, Western Australia, Australia Abstract characterization of the promoter region of the CD30 gene revealed High expression of CD30 and JunB is characteristic of tumor that the CD30 promoter is composed of a microsatellite region cells in anaplastic large cell lymphoma (ALCL) and Hodgkin containing CCAT repeats and a core promoter with Sp-1 binding lymphoma (HL). Possible interactions of CD30 and JunB were sites. The Sp-1 site at À43 to À38 within the core promoter is examined in this study. We found that the CD30 promoter in responsible for maximum promoter activity (13, 14). Repeats of tumor cells of both nucleophosmin (NPM)-anaplastic lympho- the CCAT motif within the CD30 microsatellite repress core ma kinase (ALK)–positive and NPM-ALK-negative ALCL and CD30 promoter activity. Thus, it was hypothesized that the CD30 HL is regulated by a constitutively active CD30–extracellular microsatellite may be truncated in CD30-overexpressing cells, signal-regulated kinase (ERK) 1/2 mitogen-activated protein possibly because of microsatellite instability (14, 15). We recently kinase (MAPK). Phosphorylation of ERK1/2 MAPK was characterized the structure and function of the CD30 microsatellite confirmed in nuclei of tumor cells in both ALCL and HL. from H-RS cell lines. The results showed that the CD30 micro- satellite of H-RS cell lines are not truncated, and the AP-1 site CD30-ERK1/2 MAPK signals induce JunB expression, which maintains high activity of the CD30 promoter. JunB induction (À377 to À371) located within the microsatellite relieves seems to be largely independent of nuclear factor KB in ALCL suppression by the microsatellite through interaction with JunB and HL. These results show a common mechanism of CD30 (10). This JunB-mediated induction of CD30 promoter seems to be overexpression in ALCL and HL, although the outcome of one mechanism underlying high levels of CD30 promoter activity in CD30 signaling differs between NPM-ALK-positive ALCL and H-RS cells (10). NPM-ALK-negative ALCL, cutaneous ALCL, and HL as we In this report, we studied mechanisms underlying high levels of CD30 and JunB expression in ALCL and H-RS cells. We first recently reported. (Cancer Res 2005; 65(17): 7628-34) examined interactions between overexpressed CD30 and JunB. Next, we investigated whether JunB-mediated induction of CD30 Introduction promoter also operates in ALCL cells. We found that CD30 signals JunB is a member of the activator protein (AP-1) transcription regulate JunB expression dependent on the extracellular signal- family. AP-1 is composed of homo- or heterodimers of the regulated kinase (ERK) 1/2 mitogen-activated protein kinase related Jun (c-Jun, JunB, JunD), Fos (Fos, FosB, Fra1, Fra2) and (MAPK) pathway, which may act to stabilize CD30 induction in ATF, CREB families (1). AP-1 is involved in different biological ALCL and H-RS cells. The results show that tumor cells of ALCL processes including cell differentiation, proliferation, and apo- and Hodgkin lymphoma (HL) share a common mechanism of CD30 ptosis (2, 3). Transcription of AP-1 family members is rapidly and JunB induction. and transiently stimulated by multiple extracellular signals (4). c-Jun and JunB have antagonistic functions in biological processes such as oncogenic transformation and cell prolifera- Materials and Methods tion. However, JunB also regulates distinct target genes in a Cell lines and cell cultures. K562 and HEK293 cell lines were obtained c-Jun-independent manner and exerts specific functions (5). from the Japanese Cancer Research Resources Bank (Tokyo, Japan) and Overexpression of JunB has been reported to be associated with Fujisaki Cell Biology Center (Okayama, Japan). Nucleophosmin (NPM)- neoplastic transformation (2, 6–8). anaplastic lymphoma kinase (ALK)+ ALCL cell lines (SUDHL1, Karpas299, Overexpression of CD30 and JunB is recognized as a common and SR786) and H-RS cell lines (HDLM2, L428, and L540) were purchased from the German Collection of Microorganisms and Cell Cultures feature of Hodgkin-Reed-Sternberg (H-RS) cells and anaplastic (Braunschweig, Germany). Mac1 is a NPM-ALK-negative cutaneous ALCL large cell lymphoma (ALCL) cells (9–12). Recent cloning and cell line (16). Establishment of an HEK293 transformant stably over- expressing CD30 was described previously (17). Nonadherent cell lines were cultured in RPMI 1640 and adherent cells in DMEM with supplementation Requests for reprints: Ryouichi Horie, Fourth Department of Internal Medicine, of recommended concentrations of FCS and antibiotics. School of Medicine, Kitasato University, 1-15-1 Sagamihara, Kanagawa 228-8555, Japan. n n Phone: 81-42-778-8111; Fax: 81-42-778-8441; E-mail: [email protected]. Inhibitors. DHMEQ is a nuclear factor B (NF- B) inhibitor that acts at I2005 American Association for Cancer Research. the level of nuclear translocation of NF-nB (18). Inhibitors for MEK1/2 doi:10.1158/0008-5472.CAN-05-0925 (UO126), c-Jun-NH2-kinase (JNK; SP600125) and p38 MAPK (SB203580), Cancer Res 2005; 65: (17). September 1, 2005 7628 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. CD30 Promoter Induction by CD30-ERK1/2-JunB were purchased from Cell Signaling Technology (Beverly, MA), BioMol JunB expression vector pME-JunB was described previously (10). The (Plymouth Meeting, PA), and Sigma (Tokyo, Japan), respectively. Com- structure of the expression vector for the dominant-negative CD30, CD30D pounds were dissolved with DMSO and used for experiments at specified has been described (17). A c-Jun expression vector was kindly provided by concentrations. Dr. Y. Kasuya (Department of Biochemistry and Molecular Pharmacology, Northern blotting. Northern blot analysis was done essentially as Chiba University Graduate School of Medicine). Expression vectors for described (10). Briefly, polyadenylic acid–selected RNA was size-fractionated Raf-1 and its constitutively active form DRaf-1 were kindly provided by on 1% formalin agarose gel electrophoresis and subsequently blotted onto Dr. S. Hattori (Division of Cellular Proteomics, BML, Institute of Medical Hybond-C extra-nitrocellulose membranes (Amersham Bioscience, Piscat- Science, University of Tokyo). away, NJ). Filters were hybridized in 4Â SSC, 1Â Denhardt’s, 0.5% SDS, Reporter gene assays. Activities of the CD30 promoter were studied by j 6 0.1 mol/L NaPO4 (pH 7.0), 10% dextran Na at 65 C with 1.0 Â 10 cpm/mL transient reporter gene assays. Renilla luciferase expression vector driven by of random prime-labeled probes. After washings to a final stringency of the herpes simplex virus thymidine kinase promoter, pRL-TK (Promega, 0.2Â SSC and 0.1% SDS at 65jC, filters were exposed to XAR-5 films Madison, WI), was cotransfected to standardize the transfection efficiency (Eastman Kodak, Rochester, NY) at À80jC. RT-PCR amplified cDNA in each experiment. For each transfection, 0.5 Ag of reporter construct, fragments of human JunB and human glyceraldehyde-3-phosphate dehydro- 0.1 Ag of pRL-TK, and if indicated, 1 Ag each of the expression vector was genase were used as probes. used. Cells (2 Â 105) were transfected using LipofectAMINE 2000 reagent Immunoblot analysis. Immunoblotting experiments were done as (Invitrogen, Groningen, Netherlands) according to the manufacturer’s described previously (19, 20). Cell lysates were prepared from 1 Â 106 cells instructions. Cells were harvested after 16 hours and luciferase activities in a lysis buffer [10 mmol/L Tris-HCl (pH 7.4), 1% SDS, 1 mmol/L sodium were measured by Dual Luciferase assay kit (Promega). Representative orthovanadate, 0.1 mmol/L sodium molybdate, and 1 mmol/L phenyl- results of triplicate experiments with mean and SDs are shown in the methylsulfonyl fluoride]. Antibodies used were as follows: JunB (C-11), c-Jun figures. (H-79), Raf-1 (E-10), and a-tubulin (TU-02; all from Santa Cruz Bio- Treatment of cells with actinomycin D. To measure the half-life of technology, Santa Cruz, CA), total ERK1/2 MAPK, phosphorylated JunB mRNA, cells were treated with 10 Ag/mL actinomycin D, an inhibitor (p-)ERK1/2 MAPK (Thr202/Tyr204), JNK, p38 MAPK, and p-p38 MAPK of RNA synthesis (Sigma). Cells were incubated for 0, 0.5, 1, 2, and 5 hours, (all from Cell Signaling Technology), and CD30 (Dako, Kyoto, Japan). respectively, after the addition of actinomycin D, then harvested and Immunohistochemistry. Fluorescence immunostaining was done on subjected to Northern blotting. cultured cells and signals were detected using confocal microscopy Electrophoretic mobility shift analysis. Electrophoretic mobility shift (Radiance 2000, Bio-Rad, Richmond, CA) as described (20, 21). Primary analysis (EMSA) was done as described (10). Double-stranded oligonucleo- antibodies used were as follows: JunB (Santa Cruz), p-ERK1/2 MAPK or total tides containing the AP-1 consensus sites (5V-CGCTTGATGAGTCAGCCG- ERK1/2 MAPK (both from Cell Signaling Technology). GAA-3V) were purchased from Promega. The nucleotide sequence of the Immunostaining on paraffin-embedded specimens of primary ALCL and CD30 microsatellite AP-1 site probe is as follows: 5V-CACTCACTGATT- HL samples was done as described (10).