Aberrantly expressed c-Jun and JunB are a hallmark of Hodgkin lymphoma cells, stimulate proliferation and synergize with NF-kappaB
2002; Springer Nature; Volume: 21; Issue: 15 Linguagem: Inglês
10.1093/emboj/cdf389
ISSN1460-2075
Autores Tópico(s)T-cell and Retrovirus Studies
ResumoArticle1 August 2002free access Aberrantly expressed c-Jun and JunB are a hallmark of Hodgkin lymphoma cells, stimulate proliferation and synergize with NF-κB Stephan Mathas Stephan Mathas Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, D-13125 Berlin, Germany Universitätsklinikum Charité, Robert-Rössle-Klinik, Humboldt University, Lindenberger Weg 80, D-13125 Berlin, Germany Search for more papers by this author Michael Hinz Michael Hinz Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, D-13125 Berlin, Germany Search for more papers by this author Ioannis Anagnostopoulos Ioannis Anagnostopoulos Institute for Pathology, Universitätsklinikum Benjamin Franklin, Free University, Hindenburgdamm 30, 12200 Berlin, Germany Search for more papers by this author Daniel Krappmann Daniel Krappmann Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, D-13125 Berlin, Germany Search for more papers by this author Andreas Lietz Andreas Lietz Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, D-13125 Berlin, Germany Search for more papers by this author Franziska Jundt Franziska Jundt Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, D-13125 Berlin, Germany Universitätsklinikum Charité, Robert-Rössle-Klinik, Humboldt University, Lindenberger Weg 80, D-13125 Berlin, Germany Search for more papers by this author Kurt Bommert Kurt Bommert Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, D-13125 Berlin, Germany Search for more papers by this author Fatima Mechta-Grigoriou Fatima Mechta-Grigoriou Unité des Virus Oncogènes, URA CNRS 1644, Institut Pasteur, 28 Rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Harald Stein Harald Stein Institute for Pathology, Universitätsklinikum Benjamin Franklin, Free University, Hindenburgdamm 30, 12200 Berlin, Germany Search for more papers by this author Bernd Dörken Bernd Dörken Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, D-13125 Berlin, Germany Universitätsklinikum Charité, Robert-Rössle-Klinik, Humboldt University, Lindenberger Weg 80, D-13125 Berlin, Germany Search for more papers by this author Claus Scheidereit Corresponding Author Claus Scheidereit Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, D-13125 Berlin, Germany Search for more papers by this author Stephan Mathas Stephan Mathas Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, D-13125 Berlin, Germany Universitätsklinikum Charité, Robert-Rössle-Klinik, Humboldt University, Lindenberger Weg 80, D-13125 Berlin, Germany Search for more papers by this author Michael Hinz Michael Hinz Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, D-13125 Berlin, Germany Search for more papers by this author Ioannis Anagnostopoulos Ioannis Anagnostopoulos Institute for Pathology, Universitätsklinikum Benjamin Franklin, Free University, Hindenburgdamm 30, 12200 Berlin, Germany Search for more papers by this author Daniel Krappmann Daniel Krappmann Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, D-13125 Berlin, Germany Search for more papers by this author Andreas Lietz Andreas Lietz Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, D-13125 Berlin, Germany Search for more papers by this author Franziska Jundt Franziska Jundt Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, D-13125 Berlin, Germany Universitätsklinikum Charité, Robert-Rössle-Klinik, Humboldt University, Lindenberger Weg 80, D-13125 Berlin, Germany Search for more papers by this author Kurt Bommert Kurt Bommert Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, D-13125 Berlin, Germany Search for more papers by this author Fatima Mechta-Grigoriou Fatima Mechta-Grigoriou Unité des Virus Oncogènes, URA CNRS 1644, Institut Pasteur, 28 Rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Harald Stein Harald Stein Institute for Pathology, Universitätsklinikum Benjamin Franklin, Free University, Hindenburgdamm 30, 12200 Berlin, Germany Search for more papers by this author Bernd Dörken Bernd Dörken Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, D-13125 Berlin, Germany Universitätsklinikum Charité, Robert-Rössle-Klinik, Humboldt University, Lindenberger Weg 80, D-13125 Berlin, Germany Search for more papers by this author Claus Scheidereit Corresponding Author Claus Scheidereit Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, D-13125 Berlin, Germany Search for more papers by this author Author Information Stephan Mathas1,2, Michael Hinz1, Ioannis Anagnostopoulos3, Daniel Krappmann1, Andreas Lietz1, Franziska Jundt1,2, Kurt Bommert1, Fatima Mechta-Grigoriou4, Harald Stein3, Bernd Dörken1,2 and Claus Scheidereit 1 1Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Straße 10, D-13125 Berlin, Germany 2Universitätsklinikum Charité, Robert-Rössle-Klinik, Humboldt University, Lindenberger Weg 80, D-13125 Berlin, Germany 3Institute for Pathology, Universitätsklinikum Benjamin Franklin, Free University, Hindenburgdamm 30, 12200 Berlin, Germany 4Unité des Virus Oncogènes, URA CNRS 1644, Institut Pasteur, 28 Rue du Dr Roux, 75724 Paris, cedex 15, France *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:4104-4113https://doi.org/10.1093/emboj/cdf389 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info AP-1 family transcription factors have been implicated in the control of proliferation, apoptosis and malignant transformation. However, their role in oncogenesis is unclear and no recurrent alterations of AP-1 activities have been described in human cancers. Here, we show that constitutively activated AP-1 with robust c-Jun and JunB overexpression is found in all tumor cells of patients with classical Hodgkin's disease. A similar AP-1 activation is present in anaplastic large cell lymphoma (ALCL), but is absent in other lymphoma types. Whereas c-Jun is up-regulated by an autoregulatory process, JunB is under control of NF-κB. Activated AP-1 supports proliferation of Hodgkin cells, while it suppresses apoptosis of ALCL cells. Furthermore, AP-1 cooperates with NF-κB and stimulates expression of the cell-cycle regulator cyclin D2, proto-oncogene c-met and the lymphocyte homing receptor CCR7, which are all strongly expressed in primary HRS cells. Together, these data suggest an important role of AP-1 in lymphoma pathogenesis. Introduction In the multi-stage process of oncogenesis, tumor cells acquire a series of genetic alterations of regulatory genes that result in their escape from normal proliferation and cell-death control (Hanahan and Weinberg, 2000). An oncogenic potential is therefore inherent to components of mitogenic and apoptotic signaling pathways, including transcription factors such as activator protein-1 (AP-1) and nuclear factor κB (NF-κB). AP-1 is composed of homo- or heterodimers formed by related Jun (c-Jun, JunB, JunD), Fos (c-Fos, FosB, Fra-1, Fra-2) and ATF family proteins (Karin et al., 1997). Transcription of c-Jun and other AP-1 family members is rapidly and transiently stimulated by a number of extracellular signals, which trigger activation of the JNK, ERK1/2 or p38 MAP kinase pathways (Chang and Karin, 2001). AP-1 activity is regulated at the level of expression and post-translational modification of the different subunits (Karin et al., 1997; Leppä and Bohmann, 1999). This regulation requires a tight balance, as heterodimers composed of different Jun family members differ functionally and may even act antagonistically, as has been shown for c-Jun and JunB (Bakiri et al., 2000; Szabowski et al., 2000). AP-1 proteins are usually activated in response to stress signals, such as UV irradiation, but also by growth factor pathways, and promote mitogen-induced cell-cycle progression or regulate apoptosis by modulating cyclin D1 and p53 expression (Schreiber et al., 1999; Wisdom et al., 1999). The role of Jun/AP-1 in programmed cell death is complex and, depending on the cellular context and cell type, pro- or anti-apoptotic functions have been reported (Leppä and Bohmann, 1999). In line with the transcriptional activation in response to mitogenic signals, Jun proteins, alone or in cooperation with oncogenic proteins, can induce transformation, which is best characterized for c-Jun (Mechta-Grigoriou et al., 2001); however, JunB might also act as a tumor suppressor in certain cell types (Passegué et al., 2001). Although an oncogenic potential of Jun proteins was indicated in mouse models and mammalian cell lines, no recurrent involvement of these proteins, including chromosomal translocation or alterations of protein expression, have yet been identified in human diseases. Thus, their role for malignant transformation of human cells is not clear. A common lymphoma in humans is classical Hodgkin's disease (cHD). Characteristically, the malignant mononuclear Hodgkin-/multinuclear Reed–Sternberg (HRS) cells, which are considered to be derived from clonally expanded germinal center B cells, represent only a minor cellular fraction of the affected lymph nodes (Staudt, 2000). They are surrounded by reactive cells attracted by cytokines and chemokines, abundantly produced by HRS cells (Staudt, 2000). Despite great effort, the pathogenesis of HD is not understood. Molecules like CD30 are frequently expressed by HRS cells, but there is no hint of a link for these molecules to disease development (Dürkop et al., 1992). Furthermore, the pathogenic role of clonal copies of Epstein–Barr virus (EBV), present in cHD, has not yet been clarified (Chapman and Rickinson, 1998). As a characteristic for HRS cells, a constitutive activation of transcription factor NF-κB has been recognized (Bargou et al., 1996; Krappmann et al., 1999). In most normal cell types, NF-κB is only transiently activated, mainly by stress signals and in immune and inflammatory signaling events (Karin and Ben Neriah, 2000). As a major cause of constitutive NF-κB activation in HRS cells, the IκB kinase complex (IKK) is persistently activated, inducing high turnover of IκB proteins (Krappmann et al., 1999). Constitutive NF-κB accounts for the elevated expression of several genes typically associated with HRS cells, including cell cycle-regulating and anti-apoptotic genes, and contributes to proliferation, apoptosis resistance and tumorigenicity of HRS cells (Bargou et al., 1997; Hinz et al., 2001). Many physiological signaling pathways that activate IKK and NF-κB also stimulate MAP kinase signaling cascades and AP-1. The simultaneous activation of both groups of transcription factors allows a functional synergism, and relevant target genes, such as cyclin D1, GM-CSF and others, are regulated by both NF-κB and AP-1 (Stein et al., 1993; Herber et al., 1994; Thomas et al., 1997; Hinz et al., 1999). We therefore investigated the activity of AP-1 and MAP kinases in Hodgkin and non-Hodgkin lymphoma. We identified a constitutive, aberrant expression of the AP-1 complex, containing c-Jun and JunB, as a pathogenetic hallmark of Hodgkin lymphoma. With a specificity rarely documented for other oncogenes, this activity is found in the entire tumor cell population of all cHD patients tested. Surprisingly, in cultured cHD cells, AP-1 is up-regulated in a cell autonomous manner, independent of MAP kinases. AP-1 is required for cHD cell proliferation and stimulates expression of cyclin D2, proto-oncogene c-met and the CCR7 lymphocyte homing receptor. Results Strongly elevated AP-1 DNA-binding activity and Jun protein expression in HRS and ALCL cells, but not in other non-Hodgkin lymphoma cells A number of unstimulated Hodgkin and non-Hodgkin lymphoma cell lines were tested for AP-1 DNA-binding activity. A dramatically elevated constitutive activity was detected in all seven HRS (Figure 1A, lanes 1–7) and anaplastic large cell lymphoma (ALCL) cell lines (lanes 15–17), whereas all other non-Hodgkin cell lines lacked a comparable DNA-binding activity (lanes 8–14). Supershift analysis with c-Jun, JunB, JunD, c-Fos, Fra-1, Fra-2 or ATF-2 antibodies indicated that the AP-1 complex in all HRS cells predominantly contained c-Jun (Figure 1A). In addition, JunB was detectable in some HRS cell lines. In ALCL cells, c-Jun and JunB were also detectable, but Fra-2 was the main component (Figure 1A; data not shown). JunD was weakly present in all cell lines. In agreement with these results, highly elevated c-Jun mRNA and protein expression was seen in all seven HRS cell lines and, though weaker, in ALCL cells (Figure 1B). JunB mRNA and protein up-regulation was found in the majority of HRS and ALCL cell lines (Figure 1B). In contrast, JunD mRNA was equally present in all cell lines tested, although JunD protein expression appeared to be elevated in some HRS cell lines (Figure 1B). Thus, all HRS cell lines reveal a striking accumulation and enhanced DNA-binding activity of c-Jun, and in addition JunB is selectively overexpressed in most cases. Figure 1.Abundant constitutive Jun/AP-1 DNA-binding activity in unstimulated HRS cell lines. (A) Top panel, nuclear extracts of Hodgkin cell lines, as indicated, pro-B lymphoblastic leukemia (Reh), Burkitt's lymphoma (Namalwa, Daudi, BL60), myeloma (L363, INA-6), T lymphocytic leukemia (Molt-4) or ALCL [K299, SU-DHL-1 (DHL-1), DEL] cells were assayed for AP-1 DNA-binding activity by EMSA using the TRE site of the human collagenase promoter. Free DNA is not shown. n.s., non-specific. Bottom panel, supershift analysis (ss) of AP-1 components with nuclear extracts of lymphoma cell lines, as indicated. (B) Top panel, expression of c-Jun, JunB and JunD mRNA in various lymphoma cell lines, as indicated. GAPDH expression is shown as a control. NB, northern blot. Bottom panel, protein expression of c-Jun, JunB and JunD in various lymphoma cell lines, as indicated. As a control, expression of α-tubulin is shown. WB, western blot. Download figure Download PowerPoint Patients with cHD reveal high level c-Jun and JunB expression in the entire tumor cell population The expression pattern of c-Jun and JunB in cell lines suggests that the strong c-Jun and JunB expression could serve as a marker to discriminate various lymphoma subtypes. Indeed, when lymph node sections of patients with cHD were analyzed by immunohistochemistry, all HRS cells in all cases examined revealed strong and selective nuclear staining for c-Jun and JunB using various poly- or monoclonal antibodies (Figure 2A and C; Table I; data not shown). No differences were detectable between the histological subtypes or EBV-positive and -negative cases of cHD (data not shown). In marked contrast to cHD, neither c-Jun nor JunB expression was detectable in the tumor cells of patients with lymphocyte predominance Hodgkin's disease (LPHD), a rare subtype distinct from cHD (Figure 2B and D; Table I). Among a number of precursor and peripheral B- and T-cell non-Hodgkin lymphomas, only t(2;5)-positive ALCL stained positive for c-Jun and JunB, although inconsistently and less intensely, when compared with cHD (Figure 2E and F; Table I). The characteristic pattern of c-Jun and JunB overexpression among lymphoid malignancies thus establishes these proteins as unique markers for cHD. Interestingly, c-Jun-positive, activated extrafollicular B cells with an expression level similar to HRS cells were found in tonsils from patients with acute EBV infection (EBV latency III). Some of these cells revealed multi nuclear Reed–Sternberg cell morphology (Figure 2G). The further analysis of 12 cases of lymphoproliferations associated with an EBV infection (latency I, Burkitt lymphoma; latency II and III, post-transplantation lymphoproliferative disorders) revealed only one case, which strongly labeled for c-Jun, whereas in the other cases, no staining or only weak staining of the minority of tumor cells was observed (data not shown). In order to identify a possible normal counterpart of c-Jun- and JunB-positive malignant cells, we further analyzed sections from activated tonsillar lymphoid tissue from an additional 20 patients. Apart from rare, weakly stained lymphoid cells in the germinal center of some tonsils, this lymphoid tissue was negative for both proteins (Figure 2H; data not shown). Figure 2.High level c-Jun and JunB expression is a hallmark of malignant cells in cHD. Immunohistochemistry of representative biopsy specimen. (A) All HRS cells (arrows) in cHD, but not surrounding benign cells, reveal strong nuclear c-Jun staining. (B) Malignant cells (arrows) in LPHD do not express c-Jun in detectable amounts. (C) In all HRS cells in cHD, JunB is abundantly expressed, with mostly nuclear localization (arrows). (D) Malignant cells (arrows) in LPHD do not show JunB expression. (E) c-Jun and (F) JunB expression in ALCL. In contrast to cHD, all cells shown are tumor cells. (G) Tonsil of a patient with infectious mononucleosis analyzed by double staining for c-Jun and CD20, indicating B-cell origin. The membrane staining of CD20 appears red, nuclear staining of c-Jun, brown. B cells with HRS cell-like morphology stain positive for c-Jun (arrows). (H) Normal tonsil. No c-Jun-positive cells are detectable. Download figure Download PowerPoint Table 1. c-Jun and JunB expression analysis in sections of B- and T-cell lymphoma Differentiation stage Lymphoma entity No. of cases Immunohistochemistry staining intensity ABSENTa WEAK Proportion of tumor cellsb STRONG Majority of tumor cellsc c-Jun expression Pre-GC B cells Mantle cell lymphoma 12 12 GC B cells (early) Burkitt lymphoma 13 11 2 Follicular lymphoma 10 10 LPHD 16 16 GC B cells (late) cHD 26 26 Post-GC B cells Diffuse large B-cell lymphoma 11 7 4 Extranodal marginal zone lymphoma 10 8 2 Plasmacytoma 13 6d 7 Precursor T cells T lymphoblastic 5 5 Peripheral T cells Not otherwise specified 5 2 3 ALCL t(2;5) 7 3 4 JunB expression Classical HD 18 18 LPHD 10 10 ALCL t(2;5) 16 2 14 Results of immunohistochemical analysis of diverse lymphoma entities, ordered for the cellular differentiation stages, are indicated. a In some cases, occasional neoplastic cells exhibited weak immunostaining (<5% of the neoplastic cell population). b 10–70% of neoplastic cells exhibited weak immunostaining. c 70–100% of neoplastic cells were mostly labeled intensively, with nearly 100% of HRS cells labeled strongly in all cases of cHD. d In four plasmacytoma cases, only cytoplasmic staining was observed. GC, germinal center. The constitutive AP-1 complex is distinct from the mitogen-induced complex and is formed in the absence of MAP kinase activity Since c-Jun and JunB complexes are usually activated by extracellular stimuli, we analyzed the effect of phorbolester treatment on AP-1 DNA-binding activity in HRS cells compared with non-Hodgkin cell lines (Figure 3A). AP-1 complexes with distinct electrophoretic migration compared with the constitutive complexes in HRS cells were rapidly induced by PMA in all cell types. These complexes were predominantly composed of c-Fos. Induction of expression and phosphorylation of c-Fos protein was confirmed by western blot analysis (Figure 3B). These data indicate that constitutive and induced AP-1 complexes in HRS cells are distinct. As MAP kinases are strong AP-1 inducers, we next analyzed MAPK expression and activity in the same cell lines (Figure 3C). No significant differences of JNK1, ERK1/2 or p38 protein expression were observed. All three groups of MAPKs were activated by either PMA or UV irradiation, as detected with phosphospecific antibodies, but HRS cells revealed no increased basal MAPK activity. Stimulation of JNK activity was verified by JNK1 in in vitro kinase assays (data not shown). UV light strongly induced c-Jun Ser63 phosphorylation in HRS and ALCL cells, leading to reduced protein mobility. However, c-Jun in unstimulated HRS cells was not Ser63 phosphorylated, confirming the absence of MAPK activity. Compared with the PMA or UV induction of c-Jun in Reh and Namalwa cells, its abundancy in unstimulated HRS cells was remarkably high. Furthermore, MAPK independence of the AP-1 activity in HRS cells was supported by treatment with the ERK1/2 inhibitor PD98059 and the p38 inhibitor SB203580, which did not alter the constitutive AP-1 activity (data not shown). Figure 3.Modulation of AP-1 and MAPK activity in Hodgkin and non-Hodgkin cell lines. (A) Phorbolester-induction of a distinct AP-1 DNA-binding activity in HRS and non-Hodgkin cells. Cells were left untreated or stimulated with PMA, as indicated. Extracts were assayed by EMSA with or without preincubation with antibodies against c-Fos or c-Jun. Supershifted protein–DNA complexes are indicated (ss). The PMA-induced AP-1 complex (**) migrates slower than the constitutive complex in HRS cells (*). n.s., non-specific. (B) Extracts of unstimulated or stimulated cells were analyzed in a western blot for c-Fos induction. (C) MAPK pathways are not constitutively activated in HRS cells, as revealed by mitogen and UV-light induction. The HRS cell lines L428 and L1236 or control cell lines were stimulated with PMA (P), UV-irradiated (UV) or left untreated. Whole-cell extracts were analyzed in western blots for MAPK activation and c-Jun N-terminal phosphorylation with phosphospecifc antibodies (pERK1/2, pp38, pJNK, pc-Jun), and for expression of the kinases and c-Jun with the respective antibodies, as indicated. Download figure Download PowerPoint In HRS cells, c-Jun is largely activated by an autoregulatory mechanism at the transcriptional level, while JunB is under NF-κB control One reason for highly elevated c-Jun protein levels in HRS cells in the absence of MAPK activity, which usually precedes induction of c-Jun, might be enhanced protein or RNA stability. We therefore determined c-Jun protein stability by pulse–chase analysis (Figure 4A). The half-life of c-Jun protein in L428, L1236 and Namalwa cells was between 120 and 170 min; in KMH-2 cells, it was slightly longer. This is in good agreement with the determined c-Jun half-life in other cell types (Lamph et al., 1988); thus prolonged protein stability cannot explain the drastic c-Jun accumulation in HRS cells. Furthermore, the c-Jun mRNA half-life in HRS cells was ∼40 min, similar to the value reported for other cell types (Figure 4B; Blattner et al., 2000). Consequently, elevated expression must be primarily due to increased transcription. Figure 4.High level c-Jun/AP-1 expression is not caused by increased protein or mRNA half-life. (A) For pulse–chase analysis, cells were labeled with 35[S]methionine and chased with medium containing unlabeled amino acids for the times indicated. Immunoprecipitated c-Jun was separated by SDS–PAGE and quantitated with a phosphoimager. Note that exposition time for KMH-2, L1236 and L428 samples was overnight and for Namalwa, 10 days. (B) c-Jun transcript stability was assessed by actinomycin D treatment of L1236 cells for the times indicated, followed by northern analysis (bottom panel). A quantitation of the c-Jun signal, corrected for GAPDH, is shown for a triplicate experiment (top panel). Error bars denote SDs. Download figure Download PowerPoint The c-Jun promoter activity is autoregulated by c-Jun/AP-1 (Angel et al., 1988). When comparing the activities of the wild-type promoter (WT) with a mutant (MT) lacking AP-1 sites, the latter conferred a several-fold activation in HRS but not in Namalwa cells (Figure 5A; data not shown). This indicates that AP-1 in HRS cells is transcriptionally active and that the c-Jun promoter is autoregulated. The finding of an autoregulation of c-Jun by Jun/AP-1 was supported by two further experiments. First, the AP-1 DNA-binding activity to both AP-1-like sites of the c-Jun promoter (URE1, −72 to −63; URE2, −191 to −182; Stein et al., 1992) was strongly enhanced in HRS cell lines compared with non-Hodgkin cell lines, as observed for the TRE site (Figures 5B and 1A). Supershift analysis revealed, especially at the URE2 site, a similar subunit composition as found for the TRE site (data not shown). Secondly, down-regulation of the AP-1 activity in L428 HRS cells by A-Fos, a dominant repressor of AP-1 (Olive et al., 1997), reduced the c-Jun protein expression level (Figure 5C). Figure 5.Constitutive c-Jun is positively autoregulated, whereas JunB is a target gene of NF-κB. (A) Autostimulatory regulation of the c-Jun promoter by Jun/AP-1. c-Jun promoter–luciferase constructs with wild-type (c-JunPWT) or mutated (c-JunPMT) AP-1 sites were transfected into Namalwa (left panel) or L1236 HRS (right panel) cells. As a control, both constructs were activated by PMA, which acts through AP-1 and non-AP-1 sites (Marinissen et al., 1999). Luciferase activity was determined for triplicate experiments. Error bars denote SDs. (B) Nuclear extracts of the HRS cell lines HD-MyZ, L428 and L1236, as well as the non-Hodgkin cell lines Reh and Namalwa, were analyzed by EMSA for AP-1 DNA-binding activity to the URE sites 1 and 2 of the c-Jun promoter. Free DNA is not shown. n.s., non specific. (C) c-Jun protein expression is down-regulated by inhibition of AP-1. Forty hours after transfection of L428 cells with A-Fos, cells were FACS-sorted and whole-cell extracts were analyzed for c-Jun and α-tubulin expression in western blots. (D) JunB but not c-Jun is activated by constitutive NF-κB in HRS cells. L428 cells were infected with an adenovirus expressing IκBαΔN (I) or with a control adenovirus (M). Whole-cell extracts of uninfected cells (C) and cells infected for 24 and 48 h with Adv–IκBαΔN (I 24h and I 48h, respectively) and control virus (M 24h and M 48h, respectively) were analyzed by EMSA for NF-κB and AP-1 DNA-binding activity (top panel) and western blot analysis for expression of c-Jun and JunB (bottom panel). (E) 70Z/3 and IKKγ-deficient 70Z/3–1.3E2 cells were stimulated with LPS. Whole-cell extracts were analyzed for JunB and, as a control, p65 protein expression by western blot analysis. Download figure Download PowerPoint To examine whether AP-1 activity was connected to the constitutive NF-κB activation in HRS cells, we infected L428 cells with an adenovirus encoding the NF-κB repressor IκBαΔN. NF-κB DNA-binding activity was strongly reduced after infection, as described previously (Figure 5D; Hinz et al., 2001). AP-1 activity decreased only moderately at 48 h. While c-Jun protein levels were not affected, JunB amounts dropped strongly at the latest time point. Thus, our data indicate that, in contrast to c-Jun, JunB is a cellular target gene of constitutive NF-κB in HRS cells. To confirm regulation of JunB by the IKK/NF-κB pathway, 70Z/3 pre-B cells and an IKKγ-deficient variant, 70Z/3–1.3E2 (Yamaoka et al., 1998), were stimulated with lipopolysaccharide (LPS; Figure 5E). In fact, JunB was induced only in IKKγ-expressing cells. To analyze a possible modulation of AP-1 transcriptional activity by NF-κB, we determined the effect of IκBαΔN on the pRTU14 AP-1 reporter in L428 cells. No alteration of AP-1 transcriptional activity was detectable (data not shown), indicating that c-Jun, not JunB, is the major transactivating component of the constitutive AP-1 complex in HRS cells. Constitutive c-Jun/AP-1 promotes proliferation and activates expression of cyclin D2, c-Met and CCR7 in HRS cells To investigate the functional consequences of constitutive AP-1 activity, we transiently transfected HRS cells with A-Fos. Due to low transfection efficiencies, green fluorescent protein (GFP)-cotransfected cells were sorted by FACS. As expected, AP-1 DNA-binding activity was diminished upon A-Fos expression, whereas NF-κB DNA binding and transcriptional activities remained unchanged (Figure 6A; data not shown). Down-modulation of AP-1 activity resulted in a marked reduction of cell growth, compared with mock-transfected cells (Figure 6A). No growth inhibition was observed after transfection of the Burkitt lymphoma cell line BL60, which lacks constitutive AP-1 activity (data not shown; see Figure 1A). G1–S-phase transition in HRS cells could be under the control of cyclin D2, which is expressed at elevated levels in HRS cells, is stimulated by constitutive NF-κB, and contains AP-1 sites in the promoter region (Brooks et al., 1996; Hinz et al., 2001). Indeed, A-Fos caused a noticeable decline of cyclin D2 expression (Figure 6B), indicating that constitutively activated AP-1 and NF-κB cooperate to induce cyclin D2 expression in HRS cells. Figure 6.Functional analysis of AP-1 activity in Hodgkin and ALCL cell lines. (A) Down-regulation of constitutive AP-1 activity by the c-Jun/AP-1 repressor A-Fos reduces proliferation in L428 HRS cells. Whole-cell lysates of FACS-sorted cells were analyzed by EMSA for AP-1 and NF-κB DNA-binding activity. Mock- (open diamonds) or A-Fos- (filled squares) transfected cells (2 × 105) were seeded after sorting and cell numbers were determined in the following 4 days. Results are the means of triplicate measurements. Error bars denote SDs. (B) Western blot analysis for A-Fos protein expression with an anti-FLAG tag antibody and for cyclin D2 and, as a control, cdk4 expressi
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