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. 1999 May 11;96(10):5522-7.
doi: 10.1073/pnas.96.10.5522.

The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway

M Shtutman  1 , J Zhurinsky, I Simcha, C Albanese, M D'Amico, R Pestell, A Ben-Ze'ev
Affiliations

The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway

M Shtutman et al. Proc Natl Acad Sci U S A. .

Abstract

beta-Catenin plays a dual role in the cell: one in linking the cytoplasmic side of cadherin-mediated cell-cell contacts to the actin cytoskeleton and an additional role in signaling that involves transactivation in complex with transcription factors of the lymphoid enhancing factor (LEF-1) family. Elevated beta-catenin levels in colorectal cancer caused by mutations in beta-catenin or by the adenomatous polyposis coli molecule, which regulates beta-catenin degradation, result in the binding of beta-catenin to LEF-1 and increased transcriptional activation of mostly unknown target genes. Here, we show that the cyclin D1 gene is a direct target for transactivation by the beta-catenin/LEF-1 pathway through a LEF-1 binding site in the cyclin D1 promoter. Inhibitors of beta-catenin activation, wild-type adenomatous polyposis coli, axin, and the cytoplasmic tail of cadherin suppressed cyclin D1 promoter activity in colon cancer cells. Cyclin D1 protein levels were induced by beta-catenin overexpression and reduced in cells overexpressing the cadherin cytoplasmic domain. Increased beta-catenin levels may thus promote neoplastic conversion by triggering cyclin D1 gene expression and, consequently, uncontrolled progression into the cell cycle.

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Figures

Figure 1
Figure 1
Induction of cyclin D1 and β-catenin-responsive transactivation of the cyclin D1 promoter. (A) Cells from the 293T line were transfected with 4 μg of GFP, GFP-linked ΔN-β-catenin (GFP-ΔN-β-cat), or mutant β-cat Y33. Cell lysates were analyzed by Western blotting for levels of cyclin D1, β-catenin (β-cat), and vinculin. (B) Cells from the 293T line were transfected with 0.8 μg of a reporter plasmid containing 1,745 bp of the cyclin D1 promoter (−1745CD1Luc) or with the empty pA3 plasmid, together with 4 μg of an HA-tagged ΔN-β-catenin-encoding plasmid (pHA-ΔN-β-cat), a GFP expression construct, or pCIneo. The bars represent luciferase activity in cells transfected with pHA-ΔN-β-catenin divided by the activity in cells transfected with control plasmid (pCIneo). Each transfection was carried out in duplicate plates. The means ± SD from three separate transfections are shown.
Figure 2
Figure 2
Identification of the LEF-1 binding sequence in the cyclin D1 promoter. (A) Schematic representation of reporter constructs from the cyclin D1 promoter, deletion constructs, and the LEF-1-binding sequence between nucleotides −81 and −73 of the promoter (Insert). (B) The promoter deletion constructs of the cyclin D1 promoter (0.8 μg) shown in A were transfected into 293T cells as described in Fig. 1B.
Figure 3
Figure 3
The LEF-1-binding sequence in the cyclin D1 promoter is required for β-catenin-mediated transactivation. (A) Cells from the 293T line were transfected with the indicated reporter plasmid (0.8 μg), together with 4 μg of ΔN-β-catenin or the control plasmid (pCIneo). The promoter activity is presented as in Fig. 1B. (B) Schematic representation of mutations in the −163 cyclin D1 promoter construct, including an AT to GC change at nucleotides −75 and −74 (−163mtLefCD1LUC) and deletion of the LEF-1 binding site (−163ΔLefCD1LUC) between nucleotides −81 to −73. (C) Neuro 2A cells were transfected with 0.8 μg of −1745CD1Luc, 2 μg of ΔN-β-catenin, or a control plasmid (pCIneo), along with increasing amounts of a LEF-1 expression plasmid. DNA concentrations were kept constant with empty vector DNA. Transfections were carried out in triplicate and the means ± SD are presented.
Figure 4
Figure 4
Electrophoretic mobility-shift assays of the cyclin D1 promoter. (A) Duplex oligonucleotides containing the LEF-1 binding sequences of the cyclin D1 promoter (CD1), a consensus LEF-1 binding sequences (CD1TOP) and a substitution of nucleotides −75 and −74 from AT to GC (CD1FOP) were “end labeled” with [32P]dATP and incubated with in vitro translated LEF-1 and/or β-catenin or with anti-LEF-1 antibody. The protein–DNA complexes were separated by electrophoresis and visualized by autoradiography. (B) In vitro translated LEF-1 was incubated with the 32P-labeled CD1 oligonucleotide, and increasing amounts of unlabeled CD1 or CD1FOP oligonucleotides were used as competitors. The protein–DNA complexes were analyzed as in Fig. 4A.
Figure 5
Figure 5
Electrophoretic mobility-shift assays of the cyclin D1 promoter with SW480 nuclear lysates and regulation of cyclin D1 promoter transcriptional activity by APC and axin. (A) Oligonucleotides labeled with [32P]dATP (CD1, lanes 1–5; CD1FOP, lanes 6–8) were incubated with in vitro translated LEF-1 (lanes 1 and 2) or with nuclear extracts from SW480 cells (lanes 3–8) in the presence of 100-fold excess of cold competitor oligonucleotides. (B) SW480 cells were transiently transfected with 1 μg of −163CD1LUC or −163ΔLefCD1LUC and 2 μg of either GFP or APC plus axin expression vectors. The bars represent luciferase activity in the transfected cells after normalizing for transfection efficiency with β-galactosidase. Note that, in A, an additional, faster-migrating band was obtained with CD1 and the SW480 nuclear extract (X). This band also was seen with the mutant CD1FOP and therefore was considered nonspecific.
Figure 6
Figure 6
Decreased transcription from the cyclin D1 promoter and cyclin D1 protein in SW480 cells expressing the N-cadherin cytoplasmic domain. (A) Individual SW480 cell clones stably expressing different levels of the N-cadherin cytoplasmic domain were transiently transfected with 1 μg of −1745CD1LUC. The bars represent luciferase activity in cells transfected with −1745CD1LUC divided by luciferase activity in cells transfected with the empty pCIneo vector. (B) Total cellular proteins from the cells described in A were separated by SDS/PAGE and analyzed by Western blotting with antibodies that recognize the N-cadherin tail, β-catenin, and cyclin D1.
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References

    1. Ben-Ze’ev A, Geiger B. Curr Opin Cell Biol. 1998;10:629–639. - PubMed
    1. Kemler R. Trends Genet. 1993;9:317–321. - PubMed
    1. Behrens J, von Kries J P, Kuhl M, Bruhn L, Wedlich D, Grosschedl R, Birchmeier W. Nature (London) 1996;382:638–642. - PubMed
    1. Molenaar M, van de Wetering M, Oosterwegel M, Peterson-Maduro J, Godsave S, Korinek V, Roose J, Destree O, Clevers H. Cell. 1996;86:391–399. - PubMed
    1. Huber O, Korn R, McLaughlin J, Ohsugi M, Herrmann B G, Kemler R. Mech Dev. 1996;59:3–11. - PubMed

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