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Molecular Biology, Pathobiology and Genetics |
1 Fourth Department of Internal Medicine, School of Medicine, Kitasato University; 2 Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Kanagawa; 3 Department of Pathology, School of Medicine, Toho University; 4 Laboratory of Tumor Cell Biology, Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan; 5 Department of Pathology, Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, Massachusetts; and 6 Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences, University of Western Australia, Crawley, Western Australia, Australia
Requests for reprints: Ryouichi Horie, Fourth Department of Internal Medicine, School of Medicine, Kitasato University, 1-15-1 Sagamihara, Kanagawa 228-8555, Japan. Phone: 81-42-778-8111; Fax: 81-42-778-8441; E-mail: rhorie{at}med.kitasato-u.ac.jp.
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Abstract |
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B in ALCL and HL. These results show a common mechanism of CD30 overexpression in ALCL and HL, although the outcome of CD30 signaling differs between NPM-ALK-positive ALCL and NPM-ALK-negative ALCL, cutaneous ALCL, and HL as we recently reported. |
Introduction |
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Overexpression of CD30 and JunB is recognized as a common feature of Hodgkin-Reed-Sternberg (H-RS) cells and anaplastic large cell lymphoma (ALCL) cells (9–12). Recent cloning and characterization of the promoter region of the CD30 gene revealed that the CD30 promoter is composed of a microsatellite region containing CCAT repeats and a core promoter with Sp-1 binding sites. The Sp-1 site at –43 to –38 within the core promoter is responsible for maximum promoter activity (13, 14). Repeats of the CCAT motif within the CD30 microsatellite repress core CD30 promoter activity. Thus, it was hypothesized that the CD30 microsatellite may be truncated in CD30-overexpressing cells, possibly because of microsatellite instability (14, 15). We recently characterized the structure and function of the CD30 microsatellite from H-RS cell lines. The results showed that the CD30 microsatellite of H-RS cell lines are not truncated, and the AP-1 site (–377 to –371) located within the microsatellite relieves suppression by the microsatellite through interaction with JunB (10). This JunB-mediated induction of CD30 promoter seems to be one mechanism underlying high levels of CD30 promoter activity in H-RS cells (10).
In this report, we studied mechanisms underlying high levels of CD30 and JunB expression in ALCL and H-RS cells. We first examined interactions between overexpressed CD30 and JunB. Next, we investigated whether JunB-mediated induction of CD30 promoter also operates in ALCL cells. We found that CD30 signals regulate JunB expression dependent on the extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK) pathway, which may act to stabilize CD30 induction in ALCL and H-RS cells. The results show that tumor cells of ALCL and Hodgkin lymphoma (HL) share a common mechanism of CD30 and JunB induction.
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Materials and Methods |
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Inhibitors. DHMEQ is a nuclear factor B (NF-B) inhibitor that acts at the level of nuclear translocation of NF-B (18). Inhibitors for MEK1/2 (UO126), c-Jun-NH2-kinase (JNK; SP600125) and p38 MAPK (SB203580), were purchased from Cell Signaling Technology (Beverly, MA), BioMol (Plymouth Meeting, PA), and Sigma (Tokyo, Japan), respectively. Compounds were dissolved with DMSO and used for experiments at specified concentrations.
Northern blotting. Northern blot analysis was done essentially as described (10). Briefly, polyadenylic acid–selected RNA was size-fractionated on 1% formalin agarose gel electrophoresis and subsequently blotted onto Hybond-C extra-nitrocellulose membranes (Amersham Bioscience, Piscataway, NJ). Filters were hybridized in 4x SSC, 1x Denhardt's, 0.5% SDS, 0.1 mol/L NaPO4 (pH 7.0), 10% dextran Na at 65°C with 1.0 x 106 cpm/mL of random prime-labeled probes. After washings to a final stringency of 0.2x SSC and 0.1% SDS at 65°C, filters were exposed to XAR-5 films (Eastman Kodak, Rochester, NY) at –80°C. RT-PCR amplified cDNA fragments of human JunB and human glyceraldehyde-3-phosphate dehydrogenase were used as probes.
Immunoblot analysis. Immunoblotting experiments were done as described previously (19, 20). Cell lysates were prepared from 1 x 106 cells in a lysis buffer [10 mmol/L Tris-HCl (pH 7.4), 1% SDS, 1 mmol/L sodium orthovanadate, 0.1 mmol/L sodium molybdate, and 1 mmol/L phenylmethylsulfonyl fluoride]. Antibodies used were as follows: JunB (C-11), c-Jun (H-79), Raf-1 (E-10), and 
-tubulin (TU-02; all from Santa Cruz Biotechnology, Santa Cruz, CA), total ERK1/2 MAPK, phosphorylated (p-)ERK1/2 MAPK (Thr202/Tyr204), JNK, p38 MAPK, and p-p38 MAPK (all from Cell Signaling Technology), and CD30 (Dako, Kyoto, Japan).
Immunohistochemistry. Fluorescence immunostaining was done on cultured cells and signals were detected using confocal microscopy (Radiance 2000, Bio-Rad, Richmond, CA) as described (20, 21). Primary antibodies used were as follows: JunB (Santa Cruz), p-ERK1/2 MAPK or total ERK1/2 MAPK (both from Cell Signaling Technology).
Immunostaining on paraffin-embedded specimens of primary ALCL and HL samples was done as described (10). Primary antibodies used were as follows: p-ERK1/2 MAPK, total ERK1/2 MAPK (both from Cell Signaling Technology) or ALK (Immunotech, Marseille, France).
DNA constructs. Construction of various CD30 promoter–driven luciferase plasmids was described previously (10). An AP-1 site-dependent luciferase vector p(AP-1)7 was purchased from Stratagene (Tokyo, Japan). A JunB expression vector pME-JunB was described previously (10). The structure of the expression vector for the dominant-negative CD30, CD30
has been described (17). A c-Jun expression vector was kindly provided by Dr. Y. Kasuya (Department of Biochemistry and Molecular Pharmacology, Chiba University Graduate School of Medicine). Expression vectors for Raf-1 and its constitutively active form Raf-1 were kindly provided by Dr. S. Hattori (Division of Cellular Proteomics, BML, Institute of Medical Science, University of Tokyo).
Reporter gene assays. Activities of the CD30 promoter were studied by transient reporter gene assays. Renilla luciferase expression vector driven by the herpes simplex virus thymidine kinase promoter, pRL-TK (Promega, Madison, WI), was cotransfected to standardize the transfection efficiency in each experiment. For each transfection, 0.5 µg of reporter construct, 0.1 µg of pRL-TK, and if indicated, 1 µg each of the expression vector was used. Cells (2 x 105) were transfected using LipofectAMINE 2000 reagent (Invitrogen, Groningen, Netherlands) according to the manufacturer's instructions. Cells were harvested after 16 hours and luciferase activities were measured by Dual Luciferase assay kit (Promega). Representative results of triplicate experiments with mean and SDs are shown in the figures.
Treatment of cells with actinomycin D. To measure the half-life of JunB mRNA, cells were treated with 10 µg/mL actinomycin D, an inhibitor of RNA synthesis (Sigma). Cells were incubated for 0, 0.5, 1, 2, and 5 hours, respectively, after the addition of actinomycin D, then harvested and subjected to Northern blotting.
Electrophoretic mobility shift analysis. Electrophoretic mobility shift analysis (EMSA) was done as described (10). Double-stranded oligonucleotides containing the AP-1 consensus sites (5'-CGCTTGATGAGTCAGCCGGAA-3') were purchased from Promega. The nucleotide sequence of the CD30 microsatellite AP-1 site probe is as follows: 5'-CACTCACTGATTCATTTTACA-3' (nucleotide position –384 to –364). Nuclear extracts were prepared basically according to the method reported by Andrews and Faller (22). Antibodies used for supershift assays are as follows: JunB (C-11), c-Fos (H-125), c-Jun (H-79), FosB (H-75), Fra-1 (R-20), Fra-2 (L-15), CREB-1 (C-21), ATF-2 (C-19), and JunD (329; all from Santa Cruz Biotechnology).
Inhibition of JunB expression by small interfering RNA. Target sequences of small interfering RNA (siRNA) 5'-AATGGAACAGCCCTTCTACCA-3' for JunB were described previously (10). Twenty picomoles of siRNAs were transduced into 2 x 105 of Karpas299, SUDHL1, and K562 and 1 x 105 of HEK 293 cells by LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instruction. Cells were plated and maintained in appropriate conditions and used in the following experiments. The scrambled oligonucleotide 5'-CATGCTTAAATGGGCCCATGA-3' was served as a control.
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Results |
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inhibited JunB promoter induction by CD30 overexpression (Fig. 1B). These results suggest that signals triggered by overexpressed CD30 can induce JunB expression in 293 cells.
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expression vector into ALCL and H-RS cell lines and studied levels of promoter activity and JunB expression. Results of reporter gene assay and confocal immunofluorescence microscopy showed down-regulation of JunB promoter activity and expression in cells transduced with FLAG-tagged CD30 (Fig. 1C, a and b). Collectively, these results provide evidence for CD30 signal-mediated induction of JunB expression in ALCL and H-RS cells.
Induction of JunB expression is not nuclear factor-B–dependent in anaplastic large cell lymphoma and Hodgkin-Reed-Sternberg cell lines. We next tried to clarify the CD30 signal pathway leading to induction of JunB in ALCL and H-RS cells. A recent report suggested that NF-B is involved in the induction of JunB (11, 23, 24). We showed that high levels of CD30 expression could drive constitutive NF-B activation in H-RS cells (17). Therefore, we hypothesized that CD30-mediated JunB induction is NF-B dependent. To test this hypothesis, we compared NF-B activation and JunB expression in ALCL and H-RS cell lines. Activation of NF-B and expression of JunB were examined by EMSA and by immunoblot analysis, respectively. The results clearly showed abundant JunB expression in the absence of NF-B activation in ALCL cell lines (Fig. 2A). Abundant JunB expression in ALCL and H-RS cell lines was also detected by Northern blot analysis (data not shown). Blockade of NF-B activity by DHMEQ did not affect JunB protein and mRNA expression. (Fig. 2B). The very short half-life of JunB mRNA excludes the possibility that the above result is due to a long JunB half-life (Fig. 2C). These results indicate that high levels of JunB expression are independent of NF-B activation in ALCL and H-RS cells, and suggest the possibility that CD30 signals other than NF-B activation are involved in the induction of JunB expression in these cells.
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Raf-1) activated the JunB promoter and CD30 signals enhanced Raf-1-mediated induction of the JunB promoter (Fig. 3B). We next directly examined expression of p-ERK1/2 by immunohistochemistry in 293CD30 cells. The result shows strong nuclear localized staining of p-ERK1/2 in 293CD30 compared with control 293vec cells (Fig. 3C). Together, these results suggest that the ERK1/2 MAPK pathway is involved in the CD30-mediated induction of JunB in ALCL and H-RS cell lines. Because overexpression of CD30 and JunB is reported to be a hallmark of H-RS and ALCL cells in vivo as well, we examined expression of p-ERK1/2 MAPK and total ERK1/2 MAPK in primary samples of ALCL and HL. Five samples of ALCL (ALK+), ALCL (ALK–) and HL examined showed restricted expression of p-ERK1/2 MAPK in tumor cell components, which was largely localized within the nucleus. Total ERK1/2 MAPK was expressed ubiquitously in lymph node cells and was predominantly localized within the cytoplasm. A primary breast cancer sample served as a positive control, p-ERK and total ERK showed the same expression pattern as those in ALCL and HL samples. Representative results are shown in Fig. 3D. Collectively, these results suggest involvement of the CD30-ERK1/2 MAPK pathway in the induction of JunB in ALCL and H-RS cells in vivo.
Strong and constitutive activator protein binding activity in anaplastic large cell lymphoma cell lines consists of JunB. We recently showed that strong and constitutive AP-1 binding activities consisting of JunB contributed to the high CD30 promoter activity through the AP-1 site in H-RS cells. Thus, we examined if this mechanism also operates in ALCL cells. We first investigated the binding activity for AP-1 in ALCL cell lines. EMSA analysis revealed that ALCL cell lines, like H-RS cell lines, show constitutively strong binding activity of AP-1, whereas other cell lines unrelated to H-RS or ALCL cells do not show significant AP-1 binding activity (Fig. 4A, left). AP-1 binding activity was very weak in K562 and absent in HEK293. Competition assays using SUDHL1 nuclear extracts and an unlabeled consensus probe clearly inhibited binding activity of AP-1, and an unlabeled oligomer spanning the AP-1 binding sequence in the CD30 promoter also inhibited binding of AP-1 (Fig. 4A, right).
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JunB activates CD30 promoter through the activator protein site in anaplastic large cell lymphoma cell lines. We next studied the role of JunB in driving CD30 promoter activity in ALCL cell lines. Introduction of a mutation in the AP-1 site significantly decreased the promoter activities in SUDHL1, Karpas299, and Mac1 cells, whereas it did not do so in K562 and HEK293 cell lines unrelated to ALCL (Fig. 5A). The decreased CD30 promoter activity caused by the introduction of a mutation in the AP-1 site was also observed when we used another ALCL cell line SR786 (data not shown).
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Luciferase activities driven by the CD30 promoter were suppressed in ALCL cell lines by repression of endogenous JunB with siRNA as we recently reported in H-RS cell lines (Fig. 5C). Modest reduction of CD30 promoter activity by JunB siRNA in K562 cells but not in 293 cells seems to be consistent with the previous observation that K562 cells show weak JunB expression and AP-1 activity compared with control 293 cells (10).
Collectively, these results provide evidence that the AP-1 site is functionally responsive to JunB, but not to c-Jun, and JunB expression contributes to high CD30 promoter activities not only in H-RS cell lines but also in ALCL cell lines.
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Discussion |
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Since we found that ligand-independent signaling from overexpressed CD30 triggers constitutive activation of NF-B in H-RS cells and others reported that multiple NF-B sites in the promoter regulate expression of JunB (11, 23, 24), we tested the possibility that an autonomous CD30-NF-B-JunB loop maintains CD30 overexpression in ALCL and H-RS cells through an NF-B–dependent mechanism. Surprisingly, the results showed that irrespective of the presence or absence of NF-B activation, JunB expression is shown both in ALCL and H-RS cell lines (Fig. 2A). Moreover, blockade of NF-B activation by a NF-B inhibitor did not significantly reduce JunB transcripts or CD30 promoter activity in H-RS cell lines (Fig. 2B). Furthermore, it was shown that an atypical H-RS cell line, HDMYZ, does not express CD30 and lacks JunB expression in the presence of NF-B activation (11). Collectively, these results indicate that JunB expression is dependent on CD30 signaling but not primarily on NF-B activation.
Our results present direct evidence for the existence of a constitutively active CD30-ERK1/2 MAPK-JunB pathway in ALCL and H-RS cell lines and show that this mechanism operates in vivo (Fig. 3). Our study further shows that the ERK1/2 MAPK pathway is involved in CD30 promoter regulation in these cells. Recent reports indicate the importance of the ERK1/2 MAPK pathway in the growth of H-RS and ALCL cells and suggest that inhibition of this pathway may have therapeutic value in HL. Our study confirms the importance of this pathway as a possible therapeutic target in HL and extends it to ALCL (27, 28).
Other studies showing that CD30 signals activate the ERK1/2 MAPK pathway and that ERK1/2 MAPK activates the JunB promoter support our conclusion that JunB expression is regulated by the CD30-ERK1/2 MAPK pathway (29, 30). Previous reports suggest that CD30 signals can activate ERK1/2 MAPK, JNK, and p38MAPK pathways (25, 26). We reported that NPM-ALK oncoprotein abrogates CD30-mediated activation of NF-B by direct interaction with TRAF proteins in ALCL cells. JNK and p38MAPK transduce signals depending on TRAF proteins and these pathways also seem to be interrupted by NPM-ALK oncoprotein in ALCL cells (21). These observations support the notion that the CD30-ERK1/2 MAPK-JunB pathway functions similarly in ALCL and H-RS cells.
We reported that it is not the truncation of the microsatellite but JunB-mediated relief of suppression by the microsatellite which is a mechanism underlying high levels of CD30 promoter activity in H-RS cells (10). Our observations also revealed the absence of truncation of the microsatellite in the CD30 promoter in ALCL cell lines Karpas299, SUDHL1, and SR786.7 Because the repressive activity of the microsatellite is thought to be mediated by binding of protein(s) to CCAT repeats within the microsatellite, this finding suggests that high levels of CD30 expression in ALCL cells cannot be ascribed only to alleviation of the repressive activity of the microsatellite by reduction of the CCAT repeat number. The results here show that JunB can activate the CD30 promoter through the AP-1 site in ALCL cells. These findings indicate that the same regulatory mechanisms of CD30 promoter induction operate in both ALCL and H-RS cells.
Our study shows that JunB is the major AP-1 binding component in ALCL cells, and JunB, but not c-Jun, can activate the CD30 promoter. These results substantiate the regulatory function of JunB in ALCL and H-RS cells. Although JunB has been thought to be less active and antagonize c-Jun (2, 31–34), recent reports suggest that JunB also regulates distinct target genes in a c-Jun-independent manner (5–8). Thus, in spite of constitutive expression of JunB and c-Jun in ALCL and H-RS cells, JunB seems to be the principal molecule that drives the CD30 promoter through the AP-1 site. However, our results do not exclude the possibility that molecule(s) other than JunB are also involved in CD30 promoter induction in ALCL and H-RS cells.
Lymphomas with CD30 overexpression can be divided into four major categories, HL, systemic ALCL NPM-ALK(+), systemic ALCL NPM-ALK(–), and cutaneous ALCL NPM-ALK(–). We recently reported that blockade of the constitutive CD30-TRAF-NF-B pathway by oncoprotein NPM-ALK is responsible for absence of NF-B activation in systemic ALCL, NPM-ALK(+) as further evidence that this lymphoma represents a unique entity among CD30-overexpressing lymphomas (21). The results obtained in this study suggest an autoregulatory mechanism by which levels of CD30 promoter activity are controlled by CD30-ERK1/2 MAPK signaling. CD30-ERK1/2 MAPK signals induce JunB expression that maintains high levels of the CD30 promoter in ALCL and H-RS cells. This autoregulatory mechanism of CD30 expression is common to ALCL and HL, although the outcome of CD30 signaling between ALCL NPM-ALK(+) and other lymphomas—HL, ALCL NPM-ALK(–), and cutaneous ALCL is different, as we recently reported (Fig. 6). However, this amplifying loop does not seem to exist in normal cells because the expression level of CD30 on activated lymphocytes is too weak to transduce self-activating signals and effects of CD30 cross-linking are transient (data not shown). Some negative feedback systems may prevent unregulated CD30 activation in nonmalignant lymphocytes.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Footnotes |
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Received 3/21/05. Revised 5/28/05. Accepted 6/28/05.
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References |
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B site downstream of the junB gene that mediates responsiveness to interleukin-6 in a murine plasmacytoma. J Biol Chem 1995;270:31129–35.This article has been cited by other articles:
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, 50- to 150-fold molar excess of the microsatellite oligomer. B, supershift assays of AP-1 and CREB/ATF transcription factors. Antibodies used are indicated above the lanes. Left, cell lines used. C, immunoblot analysis of c-Jun and JunB expression in ALCL and H-RS cell lines. Twenty micrograms of whole cell lysates were subjected to analysis. Left, antibodies used. Cell lines used are indicated above the lanes.
