Differential expression of prostaglandin endoperoxide H synthase-2 and formation of activated ß-cateninLEF-1 transcription complex in mouse colonic epithelial cells contrasting in Apc
J.M. Mei*,
N.G. Hord2,*,
D.F. Winterstein1,
S.P. Donald and
J.M. Phang3
Laboratory of Nutritional and Molecular Regulation, Division of Basic Sciences, National Cancer Institute and
1 Intramural Research Support Program, SAICFrederick, NCIFCRDC, Frederick, MD 21702, USA
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Abstract
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Mutations in Apc underlie the intestinal lesions in familial adenomatous polyposis and are found in >85% of sporadic colon cancers. They are frequently associated with overexpression of prostaglandin endoperoxide H synthase-2 (PGHS-2) in colonic adenomas. It has been suggested that Apc mutations are linked mechanistically to increased PGHS-2 expression by elevated nuclear accumulation of ß-cateninTcf-LEF transcription complex. In the present study, we show that PGHS-2 is differentially expressed in mouse colonic epithelial cells with distinct Apc status. Cells with a mutated Apc expressed markedly higher levels of PGHS-2 mRNA and protein and produced significantly more prostaglandin E2 than cells with normal Apc. Using electrophoretic mobility shift assays, we demonstrate that DNAß-cateninLEF-1 complex formation is differentially induced in these two cell lines in an Apc-dependent manner. Our data indicate that the differential induction of ß-cateninLEF-1 complex correlates closely with differential expression of PGHS-2. These findings support the hypothesis that the differential expression of PGHS-2 is mediated through the proposed ß-catenin/Tcf-LEF signaling pathway.
Abbreviations: APC, adenomatous polyposis coli; IFN
, interferon-
; LPS, lipopolysaccharide; PGE2, prostaglandin E2; PGHS-2, prostaglandin endoperoxide H synthase-2.
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Introduction
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Epidemiological and intervention studies support the hypothesis that prostaglandin endoperoxide H synthase-2 (PGHS-2) is associated with colorectal tumorigenesis (1,2). Moreover, high PGHS-2 expression in human colon cancer cells is closely associated with increased metastatic potential (3). Mutations in the tumor suppressor protein adenomatous polyposis coli (APC), on the other hand, have been demonstrated to play a role in the human inherited colorectal cancer susceptibility syndrome, familial adenomatous polyposis, and are found in >85% of colon tumors (47). Apc heterozygote mice, including transgenic Apc mice and Min (multiple intestinal neoplasia) mice, which carry germline Apc mutations, are highly susceptible to intestinal polyposis and adenocarcinoma formation (8,9).
The biological consequences of the acquisition of Apc heterozygosity in carcinogenesis have been linked to ß-catenin localization and turnover (1012). Loss of APC protein function results in dysregulation of ß-catenin turnover (1315) and nuclear accumulation of transcriptionally active ß-cateninTcf-LEF complexes (1618). While target genes for this Apc genotype-dependent transcription complex have not been fully identified, the PGHS-2 gene may be responsive to this transcription complex (19). PGHS-2 catalyzes the production of a host of bioactive arachidonate metabolites, including prostaglandin E2 (PGE2). This prostanoid has been shown to have anti-apoptotic activities (20,21) which may enhance neoplastic progression. Although a link between APC and ß-catenin has been reported, their role in non-malignant, non-transformed colonic epithelial cells in response to external stimuli has not been demonstrated.
Given the suggested connection between Apc genotype and the expression of PGHS-2, we sought a cultured cell model system consisting of colonic epithelial cells isolated from mice with similar genetic backgrounds but with different Apc status (a generous gift of Dr Robert Whitehead, Ludwig Institute for Cancer Research, Melbourne, Australia). This pair of non-transformed murine colon epithelial cell lines includes one that carries the ApcMin/+ mutation (22). These conditionally immortal cells are designated IMCE (ApcMin/+), derived from F1 hybrids resulting from the mating of ApcMin/+ and SV40LT antigen transgenic mice; YAMC (Apc+/+) are derived from an SV40LT antigen parental mouse (23). Since both YAMC and IMCE express the heat-labile SV40LT antigen that allows them to proliferate at 33°C, they revert to a non-transformed phenotype at the restrictive temperature of 39°C, at which proliferation of these cells ceases (22,23). The epithelial nature of these cells was demonstrated by staining with anti-keratin antisera. The genotype and expression of APC protein of these cell types have been confirmed by allele-specific PCR and by western immunoblotting, respectively (2224). Furthermore, these cells have been used to demonstrate that the ApcMin/+ mutation in IMCE cells can cooperate with stably transfected oncogenic ras to produce the transformed, tumorigenic phenotype (e.g. growth in soft agar and tumor formation in athymic mice) (24).
PGHS-2 expression is known to be responsive to inflammatory stimuli such as interferon-
(IFN
) and lipopolysaccharide (LPS). We first examined the expression of PGHS-2 induced by IFN
and LPS in YAMC and IMCE cells monitored at both the mRNA and protein levels (Figure 1
). After 12 h induction, PGHS-2 mRNA levels, assessed with a murine cDNA PGHS-2 probe (Oxford Biomedical Research, Oxford, MI), were markedly higher in IMCE cells than that in YAMC cells (Figure 1A
). Interestingly, although PGHS-2 mRNA was not detected in unstimulated YAMC cells, it was consistently observed in unstimulated IMCE cells, indicating basal expression of PGHS-2 in IMCE cells in the absence of inflammatory stimuli. Protein levels of PGHS-2 were also examined using an anti-PGHS-2 monoclonal antibody (Transduction Laboratories, Lexington, KY). Following 24 h treatment, PGHS-2 levels were markedly increased whereas both PGHS-1 and actin levels, used as controls, remained unchanged (Figure 1B
). Similar to the mRNA findings, PGHS-2 protein also showed basal expression in unstimulated IMCE cells. The induction of PGHS-2 was much higher in IMCE cells than in YAMC cells. Although Figure 1
only shows data on PGHS-2 mRNA and protein expression after 12 and 24 h induction, respectively, we have observed differential expression in YAMC and IMCE cells of PGHS-2 mRNA and protein with various durations of stimulation (data not shown).
In situ PGHS-2 enzymatic activity was assessed by measuring the amount of PGE2 (Cayman Chemical, Ann Arbor, MI) accumulated in the medium after cells were stimulated for 24 h with IFN
and LPS. Although basal PGE2 levels in YAMC were barely detectable, the levels of PGE2 in medium from unstimulated IMCE cells were twice those from YAMC cells (Figure 2
). More dramatically, the induced generation of PGE2 in either YAMC or IMCE cells was significantly higher than that of their unstimulated controls (P < 0.01). The difference in PGE2 generation between stimulated YAMC and IMCE cells was markedly widened, indicating that PGE2 generation in IMCE cells is more responsive to inflammatory stimulation. Sulindac sulfide, known to inhibit cyclooxygenase activity of both PGHS-2 and PGHS-1, blocked PGE2 production in both YAMC and IMCE cells (Figure 2
).
Significantly, neither cell type, under either transforming or non-transforming conditions, forms tumors in athymic nude mice or grows in soft agar. Only when transfected with the oncogenic form of the ras gene do these cells exhibit tumorigenic characteristics (24). Therefore, the present study using non-malignant cells differing in Apc status and inflammatory stimuli, i.e. IFN
and LPS, directly addresses the association between ApcMin/+ mutation(s), PGHS-2 expression and environmental factors. These data are consistent with the hypothesis that Apc mutation(s) affects PGHS-2 expression (19). Our results also show that PGHS-2 expression increased under the influence of proinflammatory stimuli. IMCE cells showed a strong propensity to overexpress PGHS-2 and overproduce PGE2 under environmental stress, i.e. stimulation with cytokine and endotoxin.
To ascertain whether the induced expression of PGHS-2 in either YAMC or IMCE cells could be associated with the proposed ß-catenin/Tcf-LEF pathway, we performed EMSA using a 32P-labeled murine LEF-1 consensus sequence oligonucleotide as probe (5'-CACCCTTTGAAGCTC-3') (Gibco BRL, Grand Island, NY) (Figure 3
). DNAß-cateninLEF-1 complex formation was increased in both YAMC and IMCE cells by inflammatory stimuli, but more so in IMCE cells (Figure 3A
). The difference between these two cell lines in the amount of DNAß-cateninLEF-1 complex is striking, demonstrating its differential regulation in YAMC and IMCE cells. The specificity of the complex was shown by competition with unlabeled LEF-1 probe (Figure 3A
) but not with non-homologous probe (data not shown). The addition of anti-ß-catenin polyclonal antibody (Sigma, St Louis, MO) to the reaction mixture caused an antibody-specific supershifting of the complex in both YAMC and IMCE cells (Figure 3B and C
). This is direct evidence showing the participation of ß-catenin in this DNA-binding transcription complex with LEF-1. We have repeatedly observed that this supershift increased with increasing anti-ß-catenin antibody concentration (Figure 3B
). Furthermore, failure of rabbit serum IgG (Sigma) to cause a supershift in either cell line demonstrates the specificity of this finding (Figure 3C
). Anti-E-cadherin antibody was also used as a control and failed to cause any supershift (data not shown). Based on the data from Figure 3A
, the DNAß-cateninLEF-1 complex in IMCE cells in response to IFN
/LPS is ~5-fold of that in YAMC cells. In order to achieve optimal separation and supershifting in IMCE cells, the amount of nuclear protein loaded onto the gel was adjusted to a 5:1 ratio between YAMC and IMCE cells (5 and 1 µg, respectively) (Figure 3C
). In the face of similar amounts of complexes formed, the same amount of anti-ß-catenin antibody caused a similar amount of supershift in both YAMC and IMCE cells (Figure 3C
).
Overall, these data clearly associate a ß-catenin-containing DNA-binding protein or protein complex residing in the nuclear extracts of IFN
/LPS-treated cells with the conditions known to produce PGHS-2 overexpression. This finding suggests that Apc mutations play a permissive and perhaps amplifying role for inducers of PGHS-2, such as IFN
and LPS, frequently produced under inflammatory conditions. Furthermore, chronic infection and inflammation in diseases affecting the colon, such as chronic ulcerative colitis, predispose the subjects to higher cancer risk and increase the frequency of colon malignancy (25). This supports the notion that ß-cateninLEF-1 complex acts as a crucial regulator of the differential expression of PGHS-2 mRNA, protein and PGE2 generation in response to inflammatory stimuli in non-malignant, non-transformed colonic epithelial cells contrasting in Apc status, such as YAMC and IMCE cells.
The identification of ß-catenin, often found to be bound with cadherins at the adherens junctions, as part of an important transcription complex with the DNA-binding proteins Tcf-LEF indicates an essential role for this protein in colon carcinogenesis (12,1618). Although the target genes controlled by this transcription pathway have not been fully identified, evidence supports PGHS-2, frequently overexpressed in colon cancer cells, as being one of these genes (19). We found that the PGHS-2 5' regulatory region does, in fact, contain consensus LEF-1-binding motifs (data not shown). The data presented in this study support the hypothesis that ApcMin/+ mutation(s) causes PGHS-2 overexpression by increasing the level of ß-catenin available to form DNA-binding complexes capable of increasing the expression of PGHS-2 mRNA. However, the regulatory mechanism(s) through which ß-catenin plays a central role has not been completely elucidated.
It has been suggested recently that superstabilization of ß-catenin, an E-cadherin-binding protein involved in cellcell adhesion and communication, by Apc mutation(s) is among the main factors causing enhanced PGHS-2 expression mediated through the ß-cateninTcf-LEF heterodimeric transcription complex (16,17,19). It is now known that ß-catenin is constantly degraded by GSK-3ß and APC protein in the presence of other important mediators, such as axin, in normal mammalian cells (26). Therefore, nuclear ß-catenin levels remain relatively low and ß-catenin is normally concentrated and evenly distributed in the cytoplasm in healthy cells (1013). Mutations of Apc, GSK-3ß and/or ß-catenin itself would enable ß-catenin to elude the destruction process (16,17). Thus, up-regulation of ß-catenin levels occurs, resulting in increased transcriptional activation, in concert with the DNA-binding proteins Tcf-LEF, of certain genes, such as PGHS-2. Our findings presented in this study provide experimental evidence for the connection between Apc mutation(s) and PGHS-2 inducibility in non-malignant and non-transformed cells in response to inflammatory stimuli.
These data support the hypothesis that Apc is associated with PGHS-2 induction, through affecting either the nuclear ß-catenin levels and/or the amount of ß-catenin-containing DNA-binding complex formation.
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Acknowledgments
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The authors acknowledge the National Cancer Institute for allocation of computing time and staff support at the Frederick Biomedical Supercomputing Center of the Frederick Cancer Research and Development Center.
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Notes
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2 Present address: Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824, USA 
3 To whom correspondence should be addressedEmail: phang{at}mail.ncifcrf.gov 
* These two authors contributed equally to this study 
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Received August 25, 1998;
revised November 3, 1998;
accepted December 1, 1998.