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Originally published In Press as doi:10.1074/jbc.M609768200 on August 2, 2007

J. Biol. Chem., Vol. 282, Issue 40, 29394-29400, October 5, 2007
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Retinoic Acid Inhibits beta-Catenin through Suppression of Cox-2

A ROLE FOR TRUNCATED ADENOMATOUS POLYPOSIS COLI*

Annie L. Eisinger{ddagger}§, Lincoln D. Nadauld{ddagger}§, Dawne N. Shelton{ddagger}§, Stephen M. Prescott{ddagger}§1, Diana M. Stafforini§2, and David A. Jones{ddagger}3

From the Departments of {ddagger}Oncological Sciences and Medicinal Chemistry and the §Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112

Received for publication, October 17, 2006 , and in revised form, August 1, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations in adenomatous polyposis coli (APC) underlie the earliest stages of colorectal carcinogenesis. Consequences of APC mutation include stabilization of beta-catenin, dysregulation of cyclooxygenase-2 (COX-2) expression, and loss of retinoic acid production, events with poorly defined interactions. Here we showed that treatment of zebrafish expressing a truncated form of Apc with either retinoic acid or a selective COX-2 inhibitor decreased beta-catenin protein levels and downstream signaling events. Interestingly, the destruction of beta-catenin in apc mutant embryos following Cox-2 inhibition required the presence of truncated Apc. These findings support roles for retinoic acid and Cox-2 in regulating the stability of beta-catenin following Apc loss. Furthermore, truncated Apc appears to retain the ability to target beta-catenin for destruction, but only in the absence of Cox-2 activity. This novel function of truncated Apc may provide a molecular basis for the efficacy of COX-2 inhibitors in the treatment of colon cancer.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Retinoids are important regulators of differentiation and cell proliferation. Induction of differentiation by retinoic acid has been observed in endothelial, melanoma, neuroblastoma, and lung cancer cells (1). Retinoic acid has been shown to inhibit the growth of breast cancer cell lines and to reduce the average number and incidence of tumors in an animal model of breast cancer (2–4). The anti-tumor potential of retinoids has been demonstrated by their ability to inhibit the growth of several human cancers, including colon and prostate cancer and melanoma (5–7). Retinoic acid mediates its effects by binding to its receptors, retinoic acid receptor (RAR),4 or retinoid X receptor (RXR), followed by heterodimerization and recognition of RAR element-containing promoters. In cells that express the transcription factor AP-1, retinoic acid regulates cell growth by inhibiting the formation of AP-1 complexes capable of DNA binding (8–10). In SW480 colon cancer cells, retinoic acid associates with its receptor RAR, and the complex sequesters beta-catenin from the TCF transcription factor, thus preventing transcription of beta-catenin/TCF target genes (11). In addition, RXR agonists induce degradation of beta-catenin through a proteasome-dependent process (12).

beta-Catenin is a multifunctional protein that transduces Wnt signals, mediates cell-cell adherens junctions through its interaction with E-cadherin, and stimulates cell proliferation. beta-Catenin is regulated by, and forms part of, a multiprotein complex that also includes APC, axin, glycogen synthase kinase-3beta (GSK-3beta), and casein kinase 1 (13–19). Under basal conditions, beta-catenin associates with APC and axin, and it becomes phosphorylated by casein kinase 1 and GSK-3beta (20, 21). Phosphorylation of beta-catenin marks the protein for ubiquitination and subsequent degradation by the proteasome (22–25). Mutations in members of the beta-catenin complex can compromise its integrity and ability to destabilize beta-catenin. For example, impaired function of APC, which is observed in ~85% of colorectal cancers, is accompanied by the stabilization and accumulation of beta-catenin and the uncontrolled activation of cell proliferation programs. Interestingly, mutations in APC also result in decreased retinoic acid biosynthesis (26–28).

Prostaglandin E2 (PGE2), a cyclooxygenase-2 (COX-2) product, was recently shown to promote the stabilization of beta-catenin in colon cancer cell lines by displacing GKS-3beta from the beta-catenin complex, thus preventing its phosphorylation and degradation (29, 30). We previously showed that retinoic acid suppresses COX-2 expression in human cancer cell lines and in zebrafish embryos (31). Based on these findings, we hypothesized that retinoic acid antagonizes beta-catenin signaling by regulating the levels of PGE2 generated by COX-2. In this study, we demonstrated that beta-catenin is stabilized in three retinoic acid-deficient mutant zebrafish. In addition, supplementation of apc mutant zebrafish embryos with retinoic acid decreased beta-catenin protein expression by a mechanism that required attenuation of Cox-2 expression and decreased accumulation of PGE2. Treatment of wild-type embryos with PGE2 led to stabilization of beta-catenin, whereas reducing PGE2 levels in apc mutant zebrafish results in the degradation of beta-catenin. These combined results establish a role for PGE2 as a key determinant of beta-catenin signaling in vivo.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Embryo Culture and Zebrafish Stocks—Wild-type, apc mutant, and nls mutant Danio rerio (zebrafish) were maintained on a 14:10-h light:dark cycle. Fertilized embryos were collected following natural spawnings and allowed to develop at 28.5 °C. Control and experimental embryos were raised in 0.003% phenylthiourea to inhibit pigment formation.

Whole Mount in Situ Hybridization—Zebrafish embryos were fixed in sucrose-buffered 4% paraformaldehyde, rinsed in phosphate-buffered saline, dehydrated in methanol, and stored at -20 °C. The riboprobe for green fluorescent protein (GFP) was generated by linearization of pCRII (Invitrogen) containing GFP cDNA followed by in vitro transcription with SP6 or T7 RNA polymerase (Roche Diagnostics). The embryos were cleared in 70% glycerol/phosphate-buffered saline and photographed using an Olympus DP12 digital camera.

Quantitative RT-PCR—We isolated RNA from embryos and cell lines using an RNeasy kit (Qiagen) and then synthesized single-stranded cDNA from 1 µg of total RNA using Superscript III (Invitrogen). PCR was performed using a Light Cycler instrument and software (version 3.5, Roche Diagnostics). The sequences of PCR primers used were as follows: GFP forward, 5'-CCAGATCCGCCACAACATCG-3'; reverse, 5'-GTCCATGCCGAGAGTGATCC-3'. We performed duplicate PCRs using the LightCycler FastStart DNA Master SYBR Green 1 kit (Roche Diagnostics). The amplification conditions for GFP included 10 s of denaturation at 95 °C, 5 s of annealing at 57 °C, and 10 s of extension at 72 °C, for a total of 35 cycles. A template-free negative control was included in each experiment. Quantitative RT-PCR primers and conditions for amplification of 28 S rRNA, cox-2, and mmp9 were described previously (31, 32).

Morpholino and RNA Microinjections—We obtained morpholino oligonucleotides from Gene Tools LLC. Sequences for the apc and cox-2 morpholinos were described previously (27, 33). The raldh2 morpholino (5'-GTTCAACTTCACTGGAGGTCATC-3') and the control morpholino (5'-CCTCTTACCTCAGTTACAATTTATA-3') were dissolved in 1x Danieau buffer and adjusted to a final concentration of 1.0 mM. For microinjections, 1.0 nl of 1.0 mM morpholino was injected into wild-type embryos at the one- to four-cell stage. Messenger RNA for Cox-2 and Apc-(1–1318) were synthesized using mMessage mMachine (Ambion) according to the manufacturer's instructions. Cox-2 mRNA and Apc-(1–1318) mRNA were injected into embryos at the one-cell stage.

Drug Treatments—To investigate the role of retinoic acid in beta-catenin expression, we incubated embryos with 900 nM all-trans-retinoic acid (Sigma) in Me2SO at 50% epiboly for 1 h. After washing with embryo water, we treated the embryos with 20 nM all-trans-retinoic acid at 30 and 54 hpf for 1 h and then repeated the washing step. Inhibition of Cox-2 was accomplished by treating embryos at 50% epiboly, 30 and 54 hpf with the selective COX-2 inhibitor NS-398 (Cayman Chemical). Embryos were incubated with 5 µM PGE2 (Cayman Chemical) in Me2SO for the time periods described in the figure legends. To antagonize the EP2 receptor, embryos were treated with 3 µM AH-6809 (Cayman Chemical) in Me2SO at 50% epiboly, 30 and 54 hpf. Inhibition of the proteasome was accomplished by incubating 54-hpf embryos with 20 µM MG-132 for 18 h before harvesting.

Antibodies and Western Blotting—We obtained extracts from zebrafish embryos in 1x Reporter Lysis Buffer (Promega) with the aid of a Dounce homogenizer. Lysates were frozen at -80 °C and then centrifuged at 12,000 x g for 2 min to remove the insoluble components. We assessed protein concentration in the supernatants (using the BCA protein assay; Pierce) and then subjected 20–60 µg of protein to electrophoresis on 7.5% denaturing polyacrylamide gels. After transfer to polyvinylidene difluoride membranes (PolyScreen), we stained the proteins using Ponceau S solution for 2 min and then blocked the remaining protein-binding sites with 5% nonfat dry milk in TBST. The primary antibodies used throughout this study were obtained from Cayman Chemical (anti-COX-2), Cell Signaling (anti-beta-catenin), and ICN Biomedicals (anti-actin). We then probed the membranes using horseradish peroxidase-labeled secondary antibody (BIOSOURCE) and a chemiluminescence detection system (Western Lightning) to reveal the presence of immunoreactive proteins.

PGE2 Enzyme Immunoassay—Prostaglandin E2 levels were measured in wild-type and apc mutant zebrafish embryos using a PGE2 enzyme immunoassay kit (Cayman Chemical).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Retinoic Acid Negatively Regulates Expression of beta-Catenin— We reported previously that APC is also an essential regulator of retinoic acid biosynthesis (26–28) and that retinoic acid suppressed the expression COX-2 (31). Interestingly, parallel work has shown that PGE2, a COX-2 product, promotes the stabilization of beta-catenin in colon cancer cell lines by displacing GKS-3beta from the beta-catenin destruction complex (29, 30). Based on these findings, we hypothesized that APC control of retinoic acid and COX-2 may function to regulate the stability of beta-catenin and consequent downstream signaling. To examine this possibility, we supplemented developing apc mutant zebrafish embryos with retinoic acid and found that this reduced levels of beta-catenin protein (Fig. 1A). The functional consequences of reduced beta-catenin were reflected by remarkably reduced transcript levels of two beta-catenin target genes, mmp9 (Fig. 1B) and mmp13 (not shown) (34, 35).

To test the hypothesis that the higher levels of beta-catenin seen in apc mutant embryos resulted from a lack of retinoic acid production, we next examined the converse situation. We measured the levels of beta-catenin in zebrafish neckless (nls) mutants in which disruption of the retinal dehydrogenase2(raldh2) gene results in profound retinoic acid deficiency (36). We found that nls embryos expressed higher levels of beta-catenin compared with wild-type embryos, a result that is consistent with our observations in apc mutants. These findings indicated that retinoic acid deficiency alone increased beta-catenin levels (Fig. 1C). This conclusion was further substantiated when we utilized Top-dGFP zebrafish, a genetically engineered reporter line used as a measure of the state of activation of beta-catenin (37). We found that antisense morpholino knockdown of Raldh2 in TOPdGFP embryos robustly increased dGFP expression, indicating beta-catenin activation (Fig. 1D). Taken together, our data suggested that Apc controls beta-catenin levels by a mechanism that involves retinoic acid.


Figure 1
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  		FIGURE 1.
The expression of beta-catenin is suppressed by retinoic acid. A, immunoblot analysis for beta-catenin in wild-type (WT) embryos and apc mutant embryos treated with vehicle (Veh) (Me2SO) or retinoic acid (RA). beta-Actin protein levels were determined for normalization purposes. Data shown are representative of at least three independent experiments. B, mmp9 mRNA levels were determined by quantitative RT-PCR using specific primers and RNA isolated from wild-type embryos, apc mutant embryos treated with vehicle (Me2SO), and apc mutant embryos treated with retinoic acid (RA). Each transcript measurement was first normalized to the levels of 28 S rRNA. Relative changes in expression were determined by comparing the normalized number of mmp9 transcripts in apc mutants treated with Me2SO or retinoic acid to the number of normalized mmp9 transcripts in wild-type embryos. The data are expressed as the mean ± S.D., n = 3. C, immunoblot analysis for beta-catenin in wild-type and raldh2 mutant embryos at 72 hpf. beta-Actin protein levels were determined for normalization purposes. Data shown are representative of at least three independent experiments. D, GFP mRNA levels were determined by quantitative RT-PCR using specific primers and RNA isolated from TOPdGFP embryos injected with either control or raldh2 morpholinos (MO). The data are expressed as the mean ± S.D., n = 3.

 
Down-regulation of Cox-2 Is Required for Apc-mediated Inhibition of beta-Catenin Expression—Because retinoic acid suppressed Cox-2 expression in apc mutant zebrafish and given that two groups recently demonstrated that PGE2 stabilized beta-catenin in human colon cancer cell lines (29, 30), we next tested whether Cox-2 and PGE2 served as intermediates between retinoic acid and beta-catenin. Consistent with this idea, we first found that antisense morpholino-mediated knockdown of Cox-2 in apc mutant embryos significantly reduced beta-catenin protein levels (Fig. 2A) (33). Similarly, inhibition of Cox-2 activity with the selective antagonist NS-398 (33) strongly suppressed beta-catenin expression compared with Me2SO-treated embryos (Fig. 2C). Decreased activation of the beta-catenin target genes mmp9 (Fig. 2, B and D) and mmp13 (not shown) paralleled the reduced levels of beta-catenin. These results demonstrated that the catalytic activity of Cox-2 was necessary for up-regulation and activation of beta-catenin when the function of Apc was impaired.

Overexpression of Cox-2 Increases Expression of beta-Catenin in Wild-type Zebrafish—To investigate if Cox-2 regulated the expression of beta-catenin when the function of Apc was intact, we overexpressed Cox-2 in wild-type zebrafish embryos by injection of cRNA and then assessed the expression of beta-catenin using immunoblot analysis (Fig. 3A). This treatment resulted in the up-regulation of beta-catenin compared with vehicle-injected embryos (Fig. 3A). Moreover, overexpression of Cox-2 increased activation of beta-catenin target genes, as demonstrated by elevated expression of mmp9 (Fig. 3B). Finally, overexpression of Cox-2 also increased expression of dGFP in reporter TOPdGFP zebrafish (Fig. 3C). These studies indicated that signaling through Cox-2 results in the functional up-regulation of beta-catenin and activation of target genes in wild-type zebrafish embryos.


Figure 2
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  		FIGURE 2.
Down-regulation of Cox-2 is required for Apc-mediated suppression of beta-catenin. A, immunoblot analysis for beta-catenin in wild-type (WT) embryos, and in apc mutant embryos injected with control (Con) or cox-2-specific morpholinos (MO). beta-Actin protein levels were determined for normalization purposes. Data shown are representative of at least three independent experiments. B, mmp9 mRNA levels were determined by quantitative RT-PCR using RNA isolated from wild-type embryos and apc mutant embryos injected with control (CO) or cox-2-specific morpholinos. Each transcript measurement was first normalized to the levels of 28 S rRNA. Relative changes in expression were determined by comparing the number of normalized mmp9 transcripts in apc mutants injected with control morpholino or cox-2-specific morpholino to the number of normalized mmp9 transcripts in wild-type embryos. The data are expressed as the mean ± S.D., n = 3. C, immunoblot analysis forbeta-catenin in wild-type embryos, apc mutant embryos treated with vehicle (Veh) (Me2SO), and apc mutant embryos treated with the COX-2-selective inhibitor NS-398. beta-Actin protein levels were determined for normalization purposes. Data shown are representative of at least three independent experiments. D, mmp9 mRNA levels were determined by quantitative RT-PCR using specific primers and RNA isolated from wild-type embryos and from apc mutant embryos treated with either vehicle (Me2SO) or NS-398. Each transcript measurement was first normalized to the levels of 28 S rRNA. Relative changes in expression were determined by comparing the normalized number of mmp9 transcripts in apc mutants treated with Me2SO or NS-398 to the number of normalized mmp9 transcripts in wild-type embryos. The data are expressed as the mean ± S.D., n = 3.

 
PGE2 Increases beta-Catenin Levels in Wild-type Zebrafish Embryos—Castellone et al. (29) recently demonstrated that PGE2 alters the stability of the beta-catenin degradation complex by promoting the dissociation of GSK-3beta from axin in human cell lines. To determine whether PGE2 played a similar role in vivo and following apc mutation, we investigated the role for PGE2 on the stabilization and activation of beta-catenin in apc mutant zebrafish embryos. We first determined the levels of PGE2 in apc mutant zebrafish embryos and found that the levels of PGE2 were severalfold higher than those present in wild-type controls (Fig. 4A). In addition, a specific EP2 receptor antagonist (AH-6809) blocked accumulation of beta-catenin in apc mutant zebrafish (Fig. 4B), thereby indicating that the high endogenous levels of PGE2 mediated the up-regulation of beta-catenin in vivo. To determine whether up-regulation of PGE2 was sufficient to increase beta-catenin levels, we treated wild-type zebrafish embryos with 5 µM PGE2 and saw increased beta-catenin protein levels in comparison to vehicle (Me2SO)-treated wild-type embryos (Fig. 4C). The PGE2-mediated stabilization of beta-catenin was accompanied by activation of mmp9 (Fig. 4D) and mmp13 (not shown). Additionally, treatment of TOPdGFP embryos with PGE2 increased expression of dGFP compared with Me2SO-treated controls reflecting functional activation of beta-catenin (Fig. 4E). To confirm that the observed increase in PGE2 and subsequent increase in beta-catenin depended on Apc loss, we injected apc mutant embryos with a region of Apc (Apc-(955–2075)), previously shown to down-regulate beta-catenin (38). As expected, expression of Apc-(955–2075) robustly reduced beta-catenin levels (Fig. 2F), an effect that was overcome by treatment with PGE2 (Fig. 4F). These results indicated that the effect of PGE2 on beta-catenin expression occurred downstream of Apc and supported a model wherein PGE2 mediates stabilization and activation of beta-catenin following Apc loss.


Figure 3
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  		FIGURE 3.
Overexpression of Cox-2 increases beta-catenin levels in wild-type zebrafish. A, immunoblot analysis for Cox-2 and beta-catenin in wild-type embryos injected with water or with cox-2 cRNA. beta-Actin protein levels were determined for normalization purposes. Data shown are representative of at least three independent experiments. Con, control. B, mmp9 mRNA levels were determined by quantitative RT-PCR using specific primers and RNA isolated from wild-type embryos injected with water or with cox-2 cRNA. Each transcript measurement was first normalized to the levels of 28 S rRNA. Relative changes in expression were determined by comparing the normalized number of mmp9 transcripts in wild-type embryos injected with cox-2 cRNA to the normalized number of transcripts in wild-type embryos. The data are expressed as the mean ± S.D., n = 3. C, whole mount in situ hybridization with a GFP-specific probe following injection of TOPdGFP embryos with COX-2 cRNA. Embryos shown are typical of ~50 examined of each type.

 
Truncated Apc Is Required for beta-Catenin Degradation Following Inhibition of COX-2—Mutations in APC are thought to impair formation of a functional beta-catenin degradation complex. Surprisingly, we found that retinoic acid treatment, or inhibition of Cox-2 activity, each caused loss of beta-catenin protein expression in apc mutant zebrafish despite the presence of a typical, truncated form of Apc. This suggested that truncated Apc may retain some ability to form a functional beta-catenin degradation complex. To address this issue we first asked whether beta-catenin degradation following Cox-2 inhibition required the action of the proteasome. To do so, we decreased PGE2 accumulation in apc mutants using NS-398 but also tested the effects of MG-132, an inhibitor of the proteasome. We found that MG-132 treatment attenuated the ability of NS-398 to decrease beta-catenin levels, indicating that apc mutant embryos degraded beta-catenin through a proteasome-dependent process (Fig. 5A).


Figure 4
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  		FIGURE 4.
PGE2 induction of beta-catenin. A, PGE2 levels in 72-hpf wild-type (WT) and apc mutant zebrafish embryos were analyzed by a PGE2 enzyme immunoassay. The data are expressed as the mean ± S.D., n = 3. B, immunoblot analysis for beta-catenin in wild-type and in apc mutant embryos treated with either vehicle (Veh) (Me2SO) or an EP2 receptor antagonist (AH-6809). beta-Actin protein levels were determined for normalization purposes. Data shown are representative of at least three independent experiments. C, treatment of wild-type embryos with 5 µM PGE2 at 50% epiboly and at 24 hpf for 0.5–2 h increased beta-catenin protein levels compared with treatment with vehicle (Me2SO). beta-Actin protein levels were determined for normalization purposes. D, mmp9 mRNA levels were determined by quantitative RT-PCR using specific primers and RNA isolated from wild-type embryos treated with vehicle (Me2SO) or with 5 µM PGE2 for 6 h. Each transcript measurement was first normalized to the levels of 28 S rRNA. Relative changes in expression were determined by comparing the number of normalized mmp9 transcripts in wild-type embryos treated with PGE2 to the number of normalized transcripts in wild-type embryos treated with Me2SO. The data are expressed as the mean ± S.D., n = 3. E, whole mount in situ hybridization with a GFP-specific probe following treatment of TOPdGFP embryos with vehicle (Me2SO) or with 5 µM PGE2 for 6 h. F, apc mutant zebrafish embryos were injected with a wild-type Apc construct (Apc-(955–2075)). The levels of beta-catenin were decreased as judged by immunoblot analyses. Treatment of Apc-(955–2075)-injected embryos with 5 µM PGE2 for 6 h inhibited the ability of wild-type Apc to suppress beta-catenin, resulting in increased expression of the protein. beta-Actin protein levels were determined for normalization purposes. Data shown are representative of at least three independent experiments.

 


Figure 5
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  		FIGURE 5.
Truncated Apc is required for degradation of beta-catenin in apc mutant embryos following Cox-2 inhibition. A, immunoblot analysis for beta-catenin in wild-type (WT) embryos, apc mutant embryos treated with vehicle (Veh) (Me2SO), and apc mutant embryos treated with the COX-2-selective inhibitor NS-398 or NS-398 and the proteasome inhibitor MG-132. beta-Actin protein levels were determined for normalization purposes. Data shown are representative of at least three independent experiments. B, immunoblot analysis for beta-catenin in untreated wild-type embryos, and in embryos injected with an apc splice-blocking morpholino and subsequently treated with vehicle (Me2SO) or with NS-398. Inhibition of Cox-2 failed to promote degradation of beta-catenin in APC morphant zebrafish embryos. However, co-injection of Apc-(1–1318) restored degradation of beta-catenin apc morphants but only after treatment with NS-398. Data shown are representative of at least three independent experiments. C, mmp9 mRNA levels were determined by quantitative RT-PCR using RNA isolated from wild-type embryos treated as described in B. Each transcript measurement was first normalized to the levels of 28 S rRNA. The data are expressed as the mean ± S.D., n = 3.

 
To investigate whether some form of Apc was essential for degradation of beta-catenin following Cox-2 inhibition, we suppressed the expression of full-length Apc in wild-type embryos using an Apc-specific splice-blocking antisense morpholino. As expected, Apc morphants expressed higher total levels of beta-catenin, reflecting functional down-regulation of Apc (Fig. 5B) and higher levels of Cox-2 (data not shown and see Ref. 31). However, in contrast to our observations in apc mutant embryos, blockade of PGE2 synthesis in Apc morphants failed to reduce levels of beta-catenin (Fig. 5B). We could rescue this defect by co-injection of a cRNA encoding amino acids 1–1318 of Apc, the region precisely representing the truncated form expressed in apc mutant zebrafish. Injection of Apc morphants with Apc-(1–1318) cRNA alone had no effect on beta-catenin protein levels. In contrast, injection of Apc morphants with Apc-(1–1318) cRNA along with treatment by NS-398 robustly decreased beta-catenin protein levels (Fig. 5B) and expression of mmp9 (Fig. 5C). These observations indicated that Apc-(1–1318) is required for the degradation of beta-catenin seen in the presence of Cox-2 inhibition. This conclusion is consistent with results shown in Fig. 2, C and D, demonstrating that inhibition of PGE2 synthesis with a Cox-2 inhibitor restored beta-catenin to nearly basal levels in the presence of truncated Apc.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We previously reported that the tumor suppressor APC is an essential regulator of retinoic acid biosynthesis (27). Additional studies indicated that APC control of retinoic acid biosynthesis occurs through the transcriptional regulator CtBP-1 and that this control is independent of beta-catenin (39, 40). Although the regulation of retinoic acid biosynthesis appears independent of the actions of beta-catenin, the converse may not hold true. Indeed, a number of studies suggest that retinoic acid may antagonize the actions of beta-catenin. First, studies in SW480 cells indicated that activated RARs sequestered beta-catenin from the TCF transcription factor, thus preventing transcription of beta-catenin/TCF target genes (11). Similarly, RXR agonists appear to accelerate the destruction of beta-catenin through a proteasome-dependent process (12). The mechanism leading to the destruction of beta-catenin following RXR activation is currently unclear.

Recent evidence implicates retinoic acid in suppressing the expression of Cox-2 downstream of Apc, a process that presumably leads to decreased PGE2 accumulation (31). Given the recent demonstrations that the COX-2 product, PGE2, stabilized beta-catenin (29, 30), we reasoned that retinoic acid may antagonize beta-catenin by a mechanism that required attenuation of Cox-2 expression and decreased accumulation of PGE2. We tested this possibility using three independent approaches as follows: down-regulation of Cox-2 expression, inhibition of Cox-2 enzymatic activity, and Cox-2 overexpression. The results of our studies consistently supported a role for Cox-2 as a positive regulator of beta-catenin signaling. These findings extend previous studies and support a model wherein retinoic acid suppresses beta-catenin signaling by inhibiting the expression of Cox-2.

Castellone et al. (29) recently elucidated the mechanism by which PGE2 stabilizes beta-catenin in colorectal carcinoma cells. They showed that PGE2 promoted the dissociation of GSK-3beta from axin within the beta-catenin destruction complex. This dissociation prevented GSK-3beta-mediated phosphorylation and degradation of beta-catenin. Details concerning the interplay between other complex members were not addressed. Our findings showed that the enzymatic activity of Cox-2 was necessary for stabilization of beta-catenin in vivo. We found that treatment of wild-type zebrafish embryos with PGE2 increased beta-catenin protein levels and activated beta-catenin target genes in vivo. Conversely, reducing PGE2 levels in apc mutant zebrafish by inhibiting Cox-2 resulted in degradation of beta-catenin. These findings provide solid evidence demonstrating that PGE2 plays a critical role in the stabilization of beta-catenin in vivo and places Cox-2 activity upstream of beta-catenin stabilization following apc mutation.

Our studies also revealed an interesting feature about APC and the nature of the beta-catenin destruction complex. Previous studies have demonstrated that most APC mutations, including the one in apc mutant zebrafish, result in a truncated protein that no longer binds axin and that binding of APC and axin is necessary for degradation of beta-catenin (16, 19). However, a number of studies have suggested that truncated forms of APC retain beta-catenin binding activity (19, 41).

apc mutant zebrafish recapitulate a typical apc mutation that is seen in human polyposis and colorectal cancers (42). Therefore, they provide a unique in vivo experimental system for examining the downstream responses to wild-type or truncated Apc (42). In this case, inhibition of PGE2 accumulation in embryos expressing this truncated form of Apc resulted in the degradation of beta-catenin. In contrast, elimination of the entire Apc molecule using an antisense morpholino approach prevented the destruction of beta-catenin following inhibition of Cox-2. Our observations are consistent with a model in which mutant Apc retains the ability to form a complex with beta-catenin, but the nature of this complex is such that degradation is severely impaired by high levels of PGE2. It is possible that this truncated form of Apc retains enough beta-catenin binding to aid in its degradation. Indeed, while this manuscript was under review, Schneikert et al. (43) reported that truncated APC regulates beta-catenin transcriptional activity in a cell cycle-dependent manner. This regulation, however, did not appear to result from changes in beta-catenin stability.

In summary, we have shown that retinoic acid antagonizes beta-catenin by regulating levels of Cox-2 and PGE2. These in vivo data concur with previous findings in colorectal carcinoma cells and provide further evidence to support a role for PGE2 in the dissociation of the beta-catenin destruction complex, accumulation of beta-catenin, migration to the nucleus, and activation of target genes. Moreover, our studies show that a typical truncated form of Apc retains the ability to target beta-catenin for destruction by the proteasome, but only in the absence of Cox-2 activity. These findings place retinoic acid and Cox-2 as intermediates in the Apc/Wnt signaling pathway and have important implications regarding the consequences of APC mutation in colorectal cancer. Apc-mediated destruction of beta-catenin following inhibition of PGE2 biosynthesis may explain, in part, the molecular basis for the efficacy of COX-2 inhibitors in the treatment of colon cancer.


   FOOTNOTES
 
* This work was supported by the American Cancer Society, the NCI, National Institutes of Health, and Huntsman Cancer Foundation. 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. Back

1 Present address: Oklahoma Medical Research Foundation, Oklahoma City, OK 73104. Back

2 To whom correspondence may be addressed: Huntsman Cancer Institute, University of Utah, 2000 Circle of Hope, Salt Lake City, UT 84112. Tel.: 801-585-3402; E-mail: diana.stafforini{at}hci.utah.edu. 3 To whom correspondence may be addressed. Tel.: 801-585-6107; E-mail: david.jones{at}hci.utah.edu.

4 The abbreviations used are: RAR, retinoic acid receptor; APC, adenomatous polyposis coli; COX-2, cyclooxygenase-2; TCF, T-cell factor; PGE2, prostaglandin E2; hpf, hours post-fertilization; RT, reverse transcription; RXR, retinoid X receptor; GSK-3beta, glycogen synthase kinase-3beta; GFP, green fluorescent protein. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Richard Dorsky (University of Utah) for providing the TOPdGFP zebrafish.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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