Activation of the Wnt Pathway Interferes with Serum Response Element-driven Transcription of Immediate Early Genes*

Mutational activation of the Wnt signaling pathway is a common early event in colorectal tumorigenesis, and the identification of target genes regulated by this pathway will provide a better understanding of tumor progression. Gene expression profiling on oligonucleotide microarrays revealed reduced expression of the immediate early genes fos and fosB following stimulation of cells by Wnt-1. Further analysis demonstrated that serum or 12- O -tetradecanoylphorbol-13-acetate activation of several immediate early genes including fos , fosB , junB , and egr1 was inhibited by Wnt signaling. Wnt signaling inhibited transcriptional activation driven by the serum response element without altering the activation of the extracellular signal-regulated kinase cascade or ternary complex formation at the fos serum response element promoter. The Wnt-mediated repression of c-Fos, FosB, and JunB expression was consistent with a decrease in their binding to an AP-1 promoter element and decreased target gene transcription. The expression of fos , fosB , junB , and egr1 was also repressed in human colon tumors relative to patient matched normal tissue. By contrast, the fos family member fra-1 was up-regulated in the human colon tumors, suggesting a compensatory mechanism for the reduction in fos and fosB expression. The results indicate that Wnt signaling can repress the expression of certain immediate early genes, and that this effect is consistent with changes in gene expression observed in human colorectal tumors.

Mutational activation of the Wnt signaling pathway is a common early event in colorectal tumorigenesis, and the identification of target genes regulated by this pathway will provide a better understanding of tumor progression. Gene expression profiling on oligonucleotide microarrays revealed reduced expression of the immediate early genes fos and fosB following stimulation of cells by Wnt-1. Further analysis demonstrated that serum or 12-O-tetradecanoylphorbol-13-acetate activation of several immediate early genes including fos, fosB, junB, and egr1 was inhibited by Wnt signaling. Wnt signaling inhibited transcriptional activation driven by the serum response element without altering the activation of the extracellular signal-regulated kinase cascade or ternary complex formation at the fos serum response element promoter. The Wnt-mediated repression of c-Fos, FosB, and JunB expression was consistent with a decrease in their binding to an AP-1 promoter element and decreased target gene transcription. The expression of fos, fosB, junB, and egr1 was also repressed in human colon tumors relative to patient matched normal tissue. By contrast, the fos family member fra-1 was up-regulated in the human colon tumors, suggesting a compensatory mechanism for the reduction in fos and fosB expression. The results indicate that Wnt signaling can repress the expression of certain immediate early genes, and that this effect is consistent with changes in gene expression observed in human colorectal tumors.
Components of the Wnt signaling pathway are frequently mutated in human colon cancer where the most common genetic defect results in inactivation of the tumor suppressor adenomatous polyposis coli (reviewed in Ref. 1). Recently, mutations in axin, another member of the Wnt signaling pathway, were detected in human colon and hepatocellular cancers (2,3). The inactivation of either adenomatous polyposis coli or axin results in the deregulation of ␤-catenin, a signaling molecule that activates the T cell factor/lymphoid enhancer factor (TCF/ LEF) 1 family of transcription factors (4 -7). Oncogenic mutations in the ␤-catenin gene that affect the amino-terminal re-gion of the protein have also been identified in a wide range of human tumors including those derived from colonic and hepatic tissues (reviewed in Ref. 8). The result of all of these mutational events is the stabilization of ␤-catenin, an effect that is also elicited by the treatment of cells with members of the Wnt family of secreted ligands (9).
Wnt signaling is initiated through the binding to a class of seven transmembrane receptors encoded by the frizzled genes (reviewed in Ref. 10). The activation of the receptor prevents a pivotal serine/threonine kinase, glycogen synthase kinase 3␤, from phosphorylating its substrates including Axin, adenomatous polyposis coli, and ␤-catenin (11)(12)(13). Consequently, unphosphorylated ␤-catenin is no longer recognized by components of an E3 ubiquitin ligase and thus accumulates throughout the cytoplasm and nucleus (14 -16). In the nucleus, ␤-catenin is known to interact with the TCF/LEF family of transcription factors and thereby regulate gene transcription (17,18). Several target genes that are directly regulated by the binding of ␤-catenin to TCF/LEF such as cyclin D1, c-myc, and matrilysin have already been identified (19 -21).
In addition to binding to the TCF/LEF family, ␤-catenin can also interact with other transcription factors. Recent evidence has shown a biochemical and biological interaction between ␤-catenin and retinoic acid receptors that results in the alteration of the gene expression profile in response to both Wnt and retinoic acid (22)(23). 2 Several members of the Sox family of transcription factors have also been shown to interact directly with ␤-catenin and alter Wnt-responsive gene expression (25). Cross talk between ␤-catenin and other transcriptional complexes might also be achieved through the direct interaction of ␤-catenin with the transcriptional coactivator p300/CBP or a member of the chromatin-remodeling complex Brg-1 (26 -28). Whichever mechanism is employed, inappropriate gene expression mediated by stabilized ␤-catenin could contribute to the progression of tumors driven by the Wnt pathway.
In an effort to better understand some of the changes that take place in Wnt-mediated transformation, a screen was performed on mouse C57MG cells to identify target genes of the pathway. Unexpectedly, the expression of several immediate early genes including fos and fosB was repressed by Wnt-1. Additional experiments demonstrated that the activation of these immediate early genes by serum or 12-O-tetradecanoylphorbol-13-acetate (TPA) was also inhibited by Wnt signaling. The inhibition of these immediate early genes was observed in human colon tumors as was a marked increase in mRNA levels of the fos family member fra-1. The inhibition of certain immediate early genes by Wnt signaling might represent an early step in tumor progression that then leads to subsequent selection for increased expression of other family members. * 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.

EXPERIMENTAL PROCEDURES
Cell Culture-C57MG cells with tetracycline-repressible Wnt-1 expression were grown as described previously (23). Cells were cultured in the presence or absence of 200 ng/ml of tetracycline for 48 h prior to treatment with 15% fetal bovine serum (Invitrogen) or 100 ng/ml of TPA (Sigma) for the indicated times.
Electrophoretic Mobility Shift Assay-Nuclear extracts were analyzed by electrophoretic mobility shift assay using Geneka Biotechnology AP-1 family and SRE Nushift kits according to instructions by the manufacturer. Probes were generated by annealing forward and reverse oligonucleotides and performing a 5Ј overhang fill-in reaction in the presence of ␣-[ 32 P]ATP. Forward and reverse oligos are as follows: fos wild type SRE, 5Ј-ACACAGGATGTCCATATTAGGACATC-3Ј and 5Ј-ACGCAGATGTCCTAATATGGACATC-3Ј; fos mutant ternary complex factor, 5Ј-ACACAAAATGTCCATATTAGGACATC-3Ј; and collagenase AP-1, 5Ј-ATAAAGCATGAGTCAGAC-3Ј and 5Ј-AGGTGTCT-GACTCATGCTTT-3Ј. Reactions were separated on 4% Tris-glycine gels overnight at 4°C and subjected to analysis on a Molecular Dynamics Storm PhosphorImager.
Luciferase Assay-COS-7 cells were transfected with 1 g of the indicated expression plasmids, 0.2 g of Renilla luciferase (pRL-tk), and 1 g of 5ϫ SRE luciferase reporter construct (Stratagene) using Effectene (Qiagen) transfection reagent according to the instructions by the manufacturer. Expression plasmids for ␤-catenin and Wnt-1 have been described elsewhere (30,31). The amino-terminal truncation of ␤-catenin encodes codons 90 -781, and the carboxyl-terminal truncation encodes codons 1-710. Q61L mutant Ha-Ras expression vector was purchased from Upstate Biotechnology. Cells were harvested 48 h after transfection. Luciferase activity in 10 l of lysate was analyzed using the Promega dual-luciferase reporter assay system and a Tropix TR717 microplate luminometer.

RESULTS
We performed a screen on C57MG mouse breast epithelial cells to identify genes whose expression is altered by the activation of the Wnt pathway. 2 We identified two genes from the AP-1 family of transcription factors, fos and fosB, whose expression was decreased in the presence of Wnt-1. To further explore this finding, we examined the effect of Wnt signaling on the activation of AP-1 family transcription factors by serum stimulation of C57MG cells. Quantitative RT-PCR analysis showed a significant repression of serum-induced fos and fosB transcript levels in the presence of Wnt (Fig. 1A). The Wntmediated inhibition was also observed at the level of protein expression for both c-Fos and FosB but not for another c-Fos family member, Fra-1 (Fig. 1E). In multiple experiments, a consistent 50 -90% reduction in serum-induced fos and fosB transcript and protein levels was observed in Wnt-treated cells compared with untreated controls. To determine whether this effect was specific for serum stimulation, we treated C57MG cells with other stimuli that have been reported to activate fos expression. We tested dibutyrl cAMP, forskolin, TPA, tumor necrosis factor, and interleukin-6, but only TPA treatment resulted in the stimulation of fos expression in C57MG cells (data not shown). Similar to the results obtained with serum treatment, TPA-induced fos and fosB expression was again diminished in Wnt-treated cells (Fig. 1B).
Other AP-1 family transcription factors are activated following serum and TPA stimulation in different cell types. An analysis of transcript levels for all the family members following serum and TPA stimulation revealed that the expression of a third AP-1 family member, junB, was also reduced (Fig. 1C), whereas the expression of the other family members remained unchanged in the presence of Wnt (data not shown). The rapid and transient activation of junB paralleled those observed for fos and fosB, suggesting that they may have a similar mechanism of activation. In contrast, the time course of activation of c-jun and fra-1 was more delayed and sustained and not inhibited by Wnt treatment (data not shown). Both serum and TPA stimulation are known to activate transcription through SREs in the promoters of many target genes including fos, fosB, and junB (32). Another immediate early gene, early growth regulated gene-1 (egr1), is rapidly and transiently activated through an SRE in response to various stimuli (33,34), and its expression was also repressed by Wnt treatment of C57MG cells (Fig. 1C). These results suggest that Wnt interferes with the activation of a subset of genes whose expression is activated in response to SRE-mediated stimuli.
Serum and TPA both activate SRE-driven promoters via the Ras-mitogen-activated protein kinase pathway. However, Wnt treatment did not inhibit the activation of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase, ERK, or the ternary complex factor Elk-1 as visualized by Western blotting with phospho-specific antibodies (Fig. 2). Thus, the activation of the signaling pathway leading to the SRE promoter complex is intact, suggesting that the inhibition by Wnt occurs at the level of the promoter. Next, we examined the assembly of the SRE promoter complex. A ternary structure containing serum response factor (SRF) and ternary complex factor assembles at the SRE. Depending on the cell and promoter context, this complex can either assemble upon stimulation or is constitutively bound to the SRE (35)(36)(37). Electrophoretic mobility shift assays with the fos SRE oligonucleotide probe demonstrated that the complex is preformed prior to serum stimulation and remains unchanged following stimulation of C57MG cells (Fig. 3). Furthermore, Wnt treatment was unable to alter the formation of this complex on the fos SRE, suggesting that Wnt-mediated inhibition occurred downstream of transcription factor binding to the SRE.
To determine whether Wnt is able to directly inhibit SREactivated transcription, we transfected COS-7 cells with a con-struct that expresses luciferase under the control of a multimerized fos SRE promoter. The luciferase reporter was activated when co-transfected with a construct expressing an activated mutant of Ha-Ras compared with a construct expressing green fluorescent protein (Fig. 4). The addition of a plasmid encoding Wnt-1 was able to reduce Ha-Ras-activated SRE transcription by 50%. The SRE reporter construct contains only the core fos SRE sequence, suggesting that Wnt mediates its inhibitory effect on transactivation from the SRE itself and does not require additional promoter elements. The expression of a stabilized form of ␤-catenin, a key downstream mediator of Wnt signaling, also inhibited the Ha-Ras-activated SRE transactivation. The amino-and carboxyl-terminal sequences of ␤-catenin, both of which contribute to ␤-catenin-mediated transactivation (38,39), were required for the inhibition of Ha-Rasmediated fos SRE activation.
To assess the cell type specificity of the Wnt-mediated inhibition of immediate early gene activation, we examined five cell lines that are known to respond to Wnt stimulation. Whereas Wnt-3A inhibited the activation of fos in the cell lines COS-7, NIH3T3, and C57MG, inhibition was not observed in C3H10T1/2 or 293 cells (data not shown). This finding suggests that there might be a cell type-specific component necessary to mediate the inhibition by Wnt. To determine whether this inhibition of AP-1 family members occurs in human cancers, we examined human colorectal tumors, a cancer in which the Wnt pathway is activated in the vast majority of cases. We compared human colon tumors to matched normal colon samples from 12 different patients for immediate early gene transcript levels. For fos, 5 of 12 patients had a 50% or greater reduction in expression in tumors relative to normal tissue, whereas at least 9 of 12 sample pairs exhibited a 50% or greater reduction in the expression of fosB, junB and egr1 (Fig. 5A).
A previous report describing increased expression of c-jun and fra-1 transcripts in response to ectopic overexpression of ␤-catenin in colorectal cancer cells and immortalized colonocytes prompted us to examine the expression levels of other AP-1 family members in human colon tumors (40). Transcript levels of c-jun, junD, fra-2, and several members of the atf family were unchanged in tumor versus normal colorectal tissue samples (data not shown). However, the expression of fra-1 was increased more than 2-fold in 10 of 12 of the colon tumor C57MG cells were untreated or induced to express Wnt-1 for 48 h prior to serum stimulation for the indicated times. Nuclear extracts were incubated with labeled probes representing fos wild type SRE (wt SRE) or an SRE containing a mutated ternary complex factor (TCF) binding site (mTCF). Reactions were separated on 4% Tris-glycine gels and analyzed on a PhosphorImager. The mobilities of SRF complexes bound to an SRE probe containing or lacking a TCF binding site are indicated at right. The specificity of the SRF containing complexes were confirmed by mutation of the SRF binding site (data not shown). samples (Fig. 5B). Although stimulation by Wnt-1 did not result in an increase in fra-1 in any of the Wnt-responsive cultured cell lines, Fra-1 transcript and protein levels were already relatively high as observed with the C57MG cells ( Fig.  1A and data not shown). Therefore, it was possible that Fra-1 might be able to compensate for the loss of c-Fos and FosB in the formation of the AP-1 heterodimer. Mobility shift assays using the collagenase AP-1 promoter element demonstrates that there is indeed a diminished c-Fos supershift in nuclear extracts of Wnt-treated C57MG cells (Fig. 6A). Furthermore, upon quantitation of supershifts performed on all the AP-1 family members, the relative contribution of FosB and JunB to the AP-1 complex was diminished, whereas that of Fra-1 and JunD was elevated in the extracts from Wnt-treated cells (Fig.  6B and data not shown). These results suggest that although Fra-1 and JunD protein levels are unchanged, they might compensate for the loss of c-Fos and JunB, respectively, in the formation of the AP-1 complex in cells with an activated Wnt pathway.
Altering the composition of the AP-1 heterodimer is thought to alter the specificity of gene activation. Through knockout and transgenic mice studies, collagenase and stromelysin have been identified as specific targets of c-Fos (41,42). Consequently, in cells where the Wnt pathway is activated and fos expression is diminished, fos target gene activation should also be reduced. Indeed collagenase and stromelysin transcript levels were significantly reduced in the presence of Wnt treatment (Fig. 7). This observation suggests that although Fra-1 might substitute for c-Fos in binding to the collagenase AP-1 promoter, it cannot compensate for fos target gene activation. DISCUSSION The activation of the Wnt pathway is a common event in human cancer and occurs with high frequency at an early stage in the progression of colorectal cancer. Gene expression profiles of Wnt-treated C57MG cells demonstrated a reduction in the expression of several immediate early genes including the AP-1 family members fos, fosB, and junB. The expression of these genes as well as another transcription factor, egr1, was significantly reduced in human colon tumors relative to normal tissue, whereas fra-1 expression was highly up-regulated. It is not clear whether the reduced expression of certain AP-1 family members actually offers a selective advantage in tumor progression, or whether this represents an unavoidable outcome of positive selection for hyperactive Wnt signaling. In the latter scenario, the counter selection for increased fra-1 expression might be required to maintain the transformed phenotype in the face of reduced expression of other AP-1 family members. In either case, alterations in the level and composition of these transcription factors by activation of the Wnt pathway probably modify the array of genes that are normally regulated by these factors. This observation was evident from the reduced expression of the fos target genes collagenase and stromelysin in C57MG cells stimulated by Wnt-1. That these genes were not down-regulated in human colorectal cancers (data not shown) suggests that the cancer cells have engaged alternative mechanisms for their activation.
Serum and TPA stimulation result in a rapid and transient activation of fos, fosB, junB, and egr1, which was blocked by the pretreatment of cells with Wnt. The SRE in the promoters of these genes represents a convergence point for many different stimuli including serum and TPA (32,43). Serum invokes both a Rho-dependent and ERK-dependent signaling component, whereas TPA is solely dependent on the Raf-mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-ERK signaling pathway for full activation of fos and egr1 promoters (44). Wnt inhibits the activation of fos by TPA and serum equally well, suggesting that the point at which Wnt signals interfere with activation is downstream of Raf. However, TPA stimulation of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase, ERK, and Elk-1 phosphorylation were unchanged in the presence of Wnt, suggesting that repression occurs at the level of the promoter and transcriptional complex. Serum response factor is regulated by signal transduction pathways and by its interaction with additional factors (45). At the fos SRE, for example, SRF forms a ternary complex with members of the ternary complex factor family such as Elk-1. Ternary complex factors potentiate signals emanating from mitogen-activated protein kinases into transcriptional activation, and binding at the SRE is required to link the fos and egr1 promoters to ERK signaling (46 -48). In the C57MG cells, the ternary complex was constitutively formed and unchanged by Wnt treatment as determined by its association with the fos SRE oligonucleotide probe. Even though transcription factor binding and phosphorylation of the upstream signaling components were unchanged, the activation of the Wnt pathway was still able to inhibit fos SRE transactivation using the minimal SRE sequence. This finding suggests that additional promoter elements are not required to mediate the inhibition, and that Wnt is able to interfere with the ability of the ternary complex to activate transcription.
The mechanism of transcriptional activation via the SRE remains to be fully resolved. Many factors, such as ATF-6, YY-1, C/EBP␤, TFII-I, ASC-2, SRC-1, and CBP/p300 have been reported to either activate or repress SRE-dependent transcrip-tional activity in transient transfection assays (49 -56). ␤-Catenin signaling might function to repress a cell type-dependent co-activator or recruit a co-repressor that associates with the ternary complex. This hypothesis is supported by the observation that Wnt treatment does not repress fos activation in all cell types, including some cell lines that are known to respond to Wnt by activating ␤-catenin/LEF-mediated transcription. In addition, ␤-catenin has been reported to bind to transcriptional cofactors directly through sequences contained in its aminoand carboxyl-terminal regions (26,57,58), domains that we observed to be essential for the inhibition of SRE transactivation. We were unable to detect ␤-catenin in the ternary complex by antibody supershift (data not shown), which suggests that ␤-catenin itself might not directly repress SRE-induced transcription and instead may alter the function or expression of a secondary effector. It is not clear whether the repression of SRE by ␤-catenin is dependent upon the activation of the LEF/TCF transcription factors defined by the canonical Wnt signaling pathway. The carboxyl-terminal segment of ␤-catenin, which is required for the activation of LEF/TCF transcription factors (38), was also needed for the repression of SRE-induced transcription. Interestingly, the amino-terminal segment of ␤-catenin was also required for the repression of SRE. However, the contribution of this region of ␤-catenin to the activation of the LEF/TCF factors remains controversial. Although considered dispensable in some studies (38), others have reported severely diminished activity with amino-terminal deletion mutants (59). Finally, the reporter constructs employed for structure function studies of ␤-catenin probably do not fully recapitulate endogenous gene induction by this signaling pathway.
Immediate early genes, particularly members of the AP-1 family, are involved in a number of cellular processes such as proliferation, differentiation, apoptosis, and oncogenic transformation (reviewed in Ref. 60). Despite the striking induction of immediate early gene expression in response to mitogenic stimulation, the knockouts of the mouse fos or fosB genes demonstrate that neither gene product is essential for the viability, proliferation, and differentiation of most cell types (61)(62)(63). However, the fos/fosB double mutant fibroblasts proliferate slower, suggesting that they may compensate for each other in the single knockouts (64). Despite the reduction in both fos and fosB following Wnt pathway activation, treated C57MG cells progressed through the cell cycle at a rate compared with untreated cells (data not shown). Therefore, it is possible that other genes could compensate for the reduction in both fos and fosB levels. Here we have entertained fra-1 as a candidate gene that could compensate for the inhibition of fos and fosB expression by Wnt. This hypothesis is supported by several observations. Both c-Fos and Fra-1 show high homology in their leucine zipper and DNA-binding domains and bind DNA with indistinguishable specificity in c-Jun heterodimers (65). Fra-1 substituted for c-Fos and FosB in the dimer formation at an AP-1 promoter in Wnt-treated C57MG cells. Also, fra-1 rescued fos-dependent functions in the development of knock-in mice (66). Finally, fra-1 transcripts were highly induced in colon tumors compared with normal controls that exhibited a striking reduction in fos and fosB levels.
If Fra-1 can simply compensate for the reduction in c-Fos and FosB levels, why would activation of the Wnt pathway result in a substitution of these transcription factors? The answer may lie in the structural difference between c-Fos and Fra-1. Fra-1 lacks the transactivation domain of c-Fos and is unable to compensate for the activation of c-Fos target genes despite compensating for the normal development of a mouse (66). Wnt treatment resulted in a similar reduction in c-Fos target genes, collagenase and stromelysin, even though Fra-1 substituted for FIG. 7. Effect of Wnt-1 on fos target gene transcription. RNA from C57MG cells treated as described in Fig. 1 was subjected to quantitative RT-PCR analysis for mouse collagenase and stromelysin mRNA transcripts. Each graph is a representative of at least five independent experiments. c-Fos in binding to the collagenase AP-1 promoter. In several models of oncogenic transformation, fos appeared to be dispensable (42). Instead, Fra-1 was up-regulated, and c-Fos and FosB serum inducibility was lost in Ha-Ras-transformed fibroblasts, suggesting that Fra-1 is a preferred AP-1 component in Ha-Ras transformation (67). A distinct subset of genes may be regulated through the activation of the Wnt pathway by the formation of various functional AP-1 dimers or by the regulation of other transcription factors such as Egr-1. This "reprogramming" of gene expression profiles may be an important part of cancer progression resulting from activation of the Wnt/␤-catenin pathway.