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1 Laboratory of Molecular and Genetic Information, Institute for Molecular and Cellular Biosciences and 2 Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; 3 Second Department of Surgery, Gunma University School of Medicine, Gunma, Japan; 4 OTSUKA GEN Research Institute, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan; and 5 Department of Molecular Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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, and Axin or the closely related factor conductin/Axil and subjected to proteasome-mediated degradation. Although intact APC normally induces the degradation of ß-catenin, the mutant APCs found in most colon cancers are defective in this activity. Furthermore, of those tumors that contain intact APC, some have stabilizing mutations in ß-catenin itself. Mutations in ß-catenin and Axin have also been identified in many other types of tumors, including hepatocellular carcinoma, ovarian cancer, and endometrial cancer. Thus, constitutive activation of ß-catenin-TCF–mediated transcription is believed to be a critical step in the tumorigenesis of various types of tumors. It has been reported that ß-catenin-TCF–mediated transcription is regulated by various factors. Members of the Groucho family of proteins interact with TCF and act as a transcriptional repressor of Wnt target genes (4 , 5) . ICAT interferes with the interaction between ß-catenin and TCF and antagonizes Wnt signaling (6 , 7) . On the other hand, p300 activates ß-catenin-TCF–mediated transcription and is critical for ß-catenin–mediated neoplastic transformation (8 , 9) . Furthermore, it has been shown that the Drosophila gene Lgs acts as the functional homologue of BCL9, which was identified previously as a gene overexpressed in a precursor B-cell acute lymphoblastic leukemia cell line carrying the translocation t(1;14)(q21;q32), t(1;22)(q21;q11) (10 , 11) . Lgs activates Wnt signaling by physically linking Pygopus (Pygo) to ß-catenin (10 , 12, 13, 14) . In the present study, we show that a BCL9-like protein, B9L, interacts with ß-catenin and enhances ß-catenin-TCF–mediated transcription. Furthermore, we show that B9L is required for ß-catenin–induced transformation of RK3E cells and that expression of B9L is aberrantly elevated in colorectal tumors.
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Materials and Methods |
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Plasmids.
Hemagglutinin (HA)-tagged ß-catenin-S33Y, B9L, B9L
Cter, B9LPygo, BCL9, NLS-BCL9, and mouse Pygo1 were subcloned into pCDNA3.1 (Invitrogen, Carlsbad, CA), pEGFP (BD Biosciences Clontech), or pMX, respectively. We constructed B9LCter (B9L lacking amino acids 886-1384) by deleting the Bpu1102I fragment from the full-length B9L. We also constructed B9LPygo, a B9L mutant lacking amino acids 184–326, using PCR-based methods. NLS-BCL9 was constructed by adding the nuclear localization signal of the SV40 large T-antigen to the NH2 terminus of BCL9.
In vitro-Binding Assays.
We synthesized the 35S-labeled fragment of B9L (amino acids 245–564) using the coupled transcription-translation TNT system (Promega, Madison, WI). Glutathione S-transferase (GST) fusion proteins immobilized to glutathione-Sepharose were mixed with in vitro-translated proteins in buffer A [50 mmol/L Tris-HCl (at pH 8), 140 mmol/L NaCl, 1 mmol/L EDTA, 10 µg/mL leupeptin, and 2 µg/mL aprotinin] containing 1% Tripton X-100 for 1 hour at 4°C and washed three times with buffer A. Bound proteins were fractionated by SDS-PAGE followed by autoradiography.
Antibodies.
Antibody to B9L was prepared by immunizing rabbits with recombinant GST-B9L (amino acids 245–564). Antibodies to ß-catenin (C19220) and HA-tag (12CA5) were purchased from Transduction Laboratories (San Jose, CA) and Roche (Indianapolis, IN), respectively. Antibodies to Flag-tag (M2) and GFP (3E6) were from Sigma (St. Louis, MO) and Quantum Biotechnologies (Irvine, CA), respectively.
Cell Culture and Transfection.
293, RK3E and Plat-E cells were cultured in DMEM supplemented with 10% fetal bovine serum. SW480 cells were cultured in Leibovitzs L-15 medium supplemented with 10% fetal bovine serum. We transfected plasmids into these cells using Lipofectamine 2000 (Invitrogen) or FuGENE6 (Roche).
Immunostaining.
Cells were transfected with expression plasmids, cultured for 24 hours, fixed with 3.7% formaldehyde in PBS. Fixed cells were stained with polyclonal antibody to B9L (1:200 dilution), monoclonal antibody to ß-catenin (1:200), or monoclonal antibody to Flag (1:400) for 60 minutes at room temperature. Staining patterns obtained with these antibodies were visualized by incubating with FITC-labeled antirabbit or rhodamine-B isothiocyanate-labeled antimouse IgG for 60 minutes at room temperature. The cells were photographed with a Carl Zeiss LSM510 laser scanning microscope (Zeiss, Thornwood, NY). Tumor samples were dissected, embedded in paraffin and sectioned at 10 µm. Sections were retrieved with microwave (600W) for 30 minutes in 10 mmol/L Citrate buffer (pH 6.0) and were stained with polyclonal antibodies overnight at 4°C followed by staining with secondary antibodies for 1 hour at room temperature. The nucleus was counterstained with To-Pro-3 (Molecular Probe, Eugene, OR).
Immunoprecipitation and Immunoblotting.
Nuclei were prepared from SW480 cells as described previously (15)
and lysed in 1.5 mL of lysis buffer [50 mmol/L Tris-HCl (pH 8.0), 140 mmol/L NaCl, 1% TritonX-100, 1 mmol/L dithiothreitol, 0.1 mmol/L sodium orthovanadate, 10 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, and 10 µg/mL aprotinin] on ice for 20 minutes. For experiments that used 293 cells, cells were transfected with expression plasmids, cultured for 24 hours, and lysed in lysis buffer. The lysates (500 µL) were incubated with antibodies (2 µg) for 2 hours at 4°C in the presence of protein G-Sepharose (Amersham Pharmacia, Piscataway, NJ). Blocking of antibodies was done by preincubating the antibodies for 2 hours at 4°C with an excess of the antigens used for immunization. After washing extensively with lysis buffer, samples were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane filter (Immobilon P, Millipore, Bedford, MA). We subjected the blot to immunoblotting analysis using alkaline phosphatase-conjugated mouse antirabbit or goat antimouse IgG (Promega) as a second antibody.
Luciferase Assay.
Cells (2 x 105 cells/12 well dish) were transfected with a total of 1.25 µg of the various combinations of plasmids: 0.2 µg of reporter plasmid (TOPtkLuciferase or FOPtkLuciferase; ref. 8
); 0.05 µg of internal control pRL-TK (Promega), 0.5 µg of pCS2+Wnt1, and indicated amount of wild-type and/or mutant B9L or BCL9 expression vectors. Empty pCS2+ and pEGFP-C2 vectors were used as filler DNA. Luciferase activities were measured 24 hours after transfection using the Dual-Luciferase Reporter Assay System (Promega).
Small Interfering RNAs.
Double-strand RNA used for RNA interference (RNAi) experiments were generated using the Silencer small interfering RNA Construction kit (Ambion, Austin, TX). The sequences of the region targeted for RNAi in human B9L cDNA was 5'-AACCAGATCTCGCCTAGCAAC-3'. The sequence of B9L-mut RNAi was 5'-AACCAGATATCGCCTAACAAC-3'. Cells were transfected with 80 pmol of double-stranded RNA and cultured for 96 hours.
Reverse Transcription-PCR Analysis of Gene Expression.
Total RNA was extracted from cancerous and corresponding noncancerous tissues, using RNeasy (Qiagen, Valencia, CA). We synthesized first-strand cDNA using random hexamers and Superscript II reverse transcriptase (Invitrogen). PCR reactions were done in 25 µL volumes and amplified for 1 minute at 94°C for initial denaturation, followed by 20 to 30 cycles at 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 2 minutes. The primer sequences used were as follows: rat AXIN2 forward primer, 5'-AGGTACATTGAGAACAACAGT-3' and reverse primer, 5'-AGAGACTTGCCATTGGC-3'; human B9L forward primer, 5'-ATGTTCAGCCCTGATCAGAG-3' and reverse primer, 5'-ATGGCGTACTTGGACATCTG-3'; and ß-actin forward primer, 5'-ACACTGTGCCCATCTACGAG-3' and reverse primer, 5'-ACTCCTGCTTGCTGATCCAC-3'. Flag tag forward primer, 5'-ATGGACTACAAGGACGATGAT-3' and ß-catenin reverse primer, 5’-GAGCAGGAGATTATGCAGTG-3’. The PCR products were electrophoresed on a 1% agarose gel and visualized by EtBr staining.
Retrovirus Production and Infection.
The packaging cell line Plat-E was transfected with the retroviral expression constructs following the manufacturer’s instructions. Forty-eight hours after transfection, supernatants containing retroviruses were harvested, diluted 1:1 with fresh medium, and added to RK3E cells (1 x 105 cells/60-mm dishes). Virus-infected cells were selected for 24 hours in the presence of 3 µg/µl of puromycin.
Colony Formation in Soft Agar.
Colony formation assays in soft agar were done essentially as described previously (16)
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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. It has been shown that the Drosophila gene Lgs acts as the functional homologue of BCL9, despite a low degree of sequence homology (10)
. Lgs was found to activate Wnt signaling by physically linking Pygo (10
, 12, 13, 14)
to ß-catenin, and the Pygo-interacting domain (HD1) as well as HD2 and HD3 in Lgs is conserved in both B9L and BCL9 (Fig. 1A)
. However, Lgs does not contain the region corresponding to the COOH-terminal regions of B9L and BCL9 containing HD4–6. Northern blot analysis detected an mRNA of 7.5 kb, which is expressed at moderate levels in mouse kidney, liver, lung, and at low levels in testis, brain, spleen, heart, and skeletal muscle (data not shown). Expression of B9L mRNA increases during development of the mouse embryo (data not shown).
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. Two-hybrid assays that use various deletion fragments of B9L revealed that HD2 (amino acids 302–530) of B9L is involved in its interaction with ß-catenin (Fig. 1A)
. Also, HD2 of BCL9 was found to interact with ß-catenin. These results are consistent with a previous finding that the Lgs HD2 interacts with ß-catenin. On the other hand, armadillo repeats 1–6 of ß-catenin are sufficient for binding to B9L.
We next examined whether B9L is associated with ß-catenin in living cells. We generated antibody to a fragment of B9L and confirmed that the antibody reacts specifically with a B9L fragment-GST fusion (data not shown). When lysates from SW480 cells were subjected to immunoprecipitation and subsequent immunoblotting with anti-B9L antibody, we detected an Mr 200,000 protein, and precipitation of this protein was inhibited by preincubation of the antibody with antigen (Fig. 1C)
. These results suggest that B9L is an Mr 200,000 protein. In addition to SW480 cells, B9L was expressed at high levels in various cell lines including COS-7, HeLa, and RK3E cells, but at barely detectable levels in 293 cells. We then subjected a lysate from SW480 cells to immunoprecipitation with anti-B9L antibody, and immunoblotted the precipitates with anti-ß-catenin antibody. B9L was found to coprecipitate with ß-catenin (Fig. 1C)
, and coprecipitation was inhibited by preincubation of anti-B9L antibody with antigen (Fig. 1C)
. In addition, we confirmed the interaction between B9L and ß-catenin by a pull-down assay with antitag antibodies using lysates from 293 cells transfected with green fluorescent protein (GFP)-tagged B9L along with HA-tagged ß-catenin-S33Y (Fig. 1D)
. These results suggest that ß-catenin is associated with B9L in living cells. In these experiments, we used a mutant form of ß-catenin, ß-catenin-S33Y, which was initially identified in human colorectal tumors and which contains a tyrosine (Y) instead of the normal serine at residue 33 (S33). This change renders the protein resistant to APC-mediated degradation.
Immunohistochemical analysis that uses anti-B9L antibody showed that B9L is localized in the nucleus in SW480 cells (Fig. 2A)
. When 293 cells were transfected with GFP-tagged full-length B9L (GFP-B9L), B9L distributed homogenously throughout the nucleus, and a certain amount of B9L was localized to punctate nuclear bodies (Fig. 2B)
. A truncated mutant B9L that lacks the COOH-terminal region including HD4–6 (GFP-B9LCter) showed similar staining patterns. When 293 cells were transfected with ß-catenin-S33Y, it was found to distribute diffusely throughout the whole cell. However, when 293 cells were transfected with ß-catenin along with GFP-B9L or GFP-B9LCter, ß-catenin was found to colocalize with GFP-B9L or GFP-B9LCter in the punctate nuclear bodies as well as in the nucleoplasm (Fig. 2C)
. In these cells, TCF was also found to colocalize with ß-catenin and GFP-B9L (data not shown). These results are consistent with our finding that ß-catenin interacts with B9L. Furthermore, our results raise the possibility that B9L may have the ability to translocate ß-catenin to the nucleus.
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. Furthermore, in cells transfected with ß-catenin along with GFP-BCL9, both proteins colocalized to the punctate structures in the cytoplasm (Fig. 2E)
. Addition of the nuclear localization signal of the SV40 large T-antigen (18)
to the NH2 terminus of BCL9 (NLS-BCL9) resulted in nuclear translocalization and accumulation of BCL9 in the punctate nuclear bodies. We also examined whether Pygo has any effect on the subcellular localization of BCL9. When 293 cells were transfected with BCL9 along with Pygo, BCL9 was found to localize to the nucleus and to accumulate in the punctate nuclear bodies (Fig. 2F)
. ß-Catenin was also found to localize in the nucleus in the presence of exogenously expressed BCL9 and Pygo (data not shown). By contrast, subcellular localization patterns of B9L were not altered by the presence of Pygo (data not shown). Thus, although the structure of BCL9 is very similar to B9L, BCL9 presumably needs to interact with Pygo to localize in the nucleus.
The fact that B9L and ß-catenin colocalize in the nucleus raises the possibility that B9L is involved in ß-catenin-TCF–regulated transcription. We therefore examined the effect of B9L on Wnt-1–induced transactivation of a reporter plasmid that contains optimal TCF-binding sites upstream of a luciferase reporter gene. Expression of Wnt-1 in 293 cells greatly enhanced the activity of the reporter gene, and coexpression of B9L with Wnt-1 further enhanced its activity (Fig. 3B)
. In contrast, B9L did not show any effect in the absence of Wnt-1. When a reporter plasmid containing mutated TCF-binding sites was used, little change in reporter activity was observed, confirming the specificity of this finding. Similar results were obtained when ß-catenin-S33Y was used instead of Wnt-1 (data not shown). The mutant GFP-B9LCter, which contains both ß-catenin- and Pygo-binding domains but lacks the COOH-terminus (Fig. 3A)
, failed to enhance the effect of Wnt-1 (Fig. 3B)
. In contrast, the mutant B9LPygo, which lacks only the Pygo-binding domain HD1, could enhance Wnt-1–induced transactivation as efficiently as wild-type B9L (Fig. 3C)
. However, Pygo itself could enhance Wnt-1–induced transactivation in the presence of B9L. Consistent with the fact that B9L is expressed at barely detectable levels in 293 cells, Pygo did not show any effect in the absence of exogenously expressed B9L. These results suggest that the COOH-terminal region of B9L is crucial for its transactivation activity whereas its interaction with Pygo is dispensable for this activity.
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, whereas NLS-BCL9 exhibited activity comparable with that of B9L (Fig. 3C)
. Furthermore, BCL9 could enhance Wnt-1–induced transactivation when cotransfected with Pygo. Thus, Pygo-mediated nuclear translocation of BCL9 is presumably important for its transactivation activity. In addition, overexpression of Pygo further enhanced the effect of NLS-BCL9 on Wnt-1–induced transactivation, whereas Pygo showed no effect in the absence of exogenously expressed BCL9. These results suggest that Pygo has the potential to stimulate the effect of BCL9 on Wnt-regulated transcription, in addition to its ability to translocate BCL9 to the nucleus.
In colorectal tumor cells, ß-catenin-TCF–regulated transcription is constitutively activated because of inactivation of APC or activation of ß-catenin (1, 2, 3)
. B9L was found to activate ß-catenin-TCF–regulated transcription when transfected into SW480 cells, which contains mutated APC (Fig. 3D)
. To further assess the significance of B9L in SW480 cells, we tried to generate a dominant-negative mutant of B9L. We found that GFP-B9LCter abrogates the effect of B9L in a dose-dependent manner when coexpressed with B9L in 293 cells (Fig. 3E)
. Consistent with the fact that B9L is expressed at barely detectable levels in 293 cells, GFP-B9LCter did not inhibit transcription activated by Wnt-1 alone in the absence of exogenously expressed B9L (Fig. 3B)
. However, when GFP-B9LCter was overexpressed in SW480 cells, which express B9L endogenously, it was found to inhibit transcription of the reporter gene (Fig. 3D)
. To examine the specificity of B9L action in SW480 cells, we designed double-stranded RNA interference oligonucleotides (19)
against B9L (B9L-RNAi) and a mutant control (B9L-mut RNAi). Immunoblotting experiments revealed that introduction of B9L-RNAi but not B9L-mut RNAi into SW480 cells decreased the amount of endogenous B9L (Fig. 3F)
. When we tested the effect of these oligonucleotides on ß-catenin-TCF–regulated transcription, we found that B9L-RNAi but not B9L-mut RNAi inhibited transcription of the reporter gene (Fig. 3D)
. As expected, this B9L-RNAi–mediated inhibition was abrogated by overexpression of GFP-B9L but not GFP-B9LCter. In these experiments, GFP-B9L and GFP-B9LCter were expressed at similar levels (data not shown). These results suggest that endogenous B9L plays an important role in enhancing ß-catenin-TCF–mediated transcription in colorectal tumor SW480 cells.
Because activation of ß-catenin-TCF–mediated transcription is known to be crucial for tumorigenesis (1, 2, 3)
, we speculated that B9L may enhance the transforming activity of ß-catenin. It has been reported that ß-catenin-S33Y induces neoplastic transformation of the RK3E cell line (16)
. We therefore examined the effect of B9L on ß-catenin-S33Y–induced transformation of RK3E cells. For this purpose, we generated retroviruses encoding ß-catenin-S33Y (Ret-ß-catenin-S33Y), B9L (Ret-B9L), or B9LCter (Ret-B9LCter). As reported previously, infection of RK3E cells with Ret-ß-catenin-S33Y induced dense foci of morphologically transformed cells after 3 weeks (Fig. 4A)
. When RK3E cells were infected with Ret-B9L before infection with Ret-ß-catenin-S33Y, the number of transformed foci was increased significantly. By contrast, infection of RK3E cells with Ret-B9LCter before infection with Ret-ß-catenin-S33Y resulted in fewer transformed foci. Ret-B9L and Ret-B9LCter did not show any effect on cells not infected with Ret-ß-catenin-S33Y. Consistent with these results, reverse transcription (RT)-PCR analysis revealed that expression levels of the known ß-catenin target gene, AXIN2 (20)
, were increased by expression of B9L but decreased by B9LCter (Fig. 4B)
. In addition, B9L, B9LCter, and ß-catenin-S33Y were expressed at the expected levels. These results suggest that B9L plays an important role in ß-catenin–induced transformation of RK3E cells.
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. In the other eight cases, B9L expression was almost equal to that of the noncancerous tissues. On the other hand, AXIN2 expression was enhanced in 12 of 14 cases, suggesting that Wnt signaling is aberrantly activated in at least 86% of tumors examined. Immunostaining experiments also showed that B9L is expressed at high levels in colorectal tumors but at low levels in noncancerous epithelium (Fig. 4D)
. We have shown here that B9L interacts with ß-catenin and enhances ß-catenin-TCF–mediated transcription. Lgs was shown previously to function as an adapter to physically link Pygo to ß-catenin, and this recruitment of Pygo was suggested to be sufficient to activate ß-catenin-TCF–mediated transcription (10) . However, we found that the COOH-terminal region of B9L, which is not conserved in Lgs, is crucial for its transactivation activity, whereas its interaction with Pygo is dispensable for this activity. Possibly, the COOH-terminal region may mediate the recruitment of, for example, a coactivator or a chromatin-related factor. On the other hand, BCL9 seems to require both Pygo and the COOH-terminal region, despite its close sequence similarity to B9L. Consistent with the importance of ß-catenin-TCF–mediated transactivation in tumorigenesis (1, 2, 3) , B9L was found to enhance the transforming activity of ß-catenin. Furthermore, B9L was found to be overexpressed in about 43% of colorectal tumors. These findings suggest that B9L plays an important role in tumorigenesis induced by aberrant activation of Wnt signaling. In addition, it would be interesting to examine the relationship between B9L overexpression and the clinical behavior of colorectal tumors. Further elucidation of the function of B9L may give insights into the mechanism of tumorigenesis as well as the development of antitumor reagents and methods for diagnosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
Requests for reprints: Tetsu Akiyama, Laboratory of Molecular and Genetic Information, Institute for Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan, akiyama{at}iam.u-tokyo.ac.jp
Received 6/25/04. Revised 9/10/04. Accepted 10/13/04.
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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