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Beta-Catenin stabilizes cyclooxygenase-2 mRNA by interacting with AU-rich elements of 3'-UTR.

Lee HK, Jeong S - Nucleic Acids Res. (2006)

Bottom Line: Expression of COX-2 mRNA is regulated by various cytokines, growth factors and other signals. beta-Catenin, a key transcription factor in the Wnt signal pathway, activates transcription of COX-2.Here we found that COX-2 mRNA was also substantially stabilized by activating beta-catenin in NIH3T3 and 293T cells.Taken together, we provided evidences for beta-catenin as an RNA-binding factor and a regulator of stabilization of COX-2 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, BK21 Graduate Program for RNA Biology, Institute of Nanosensor and Biotechnology, Dankook University, Seoul 140-714, Republic of Korea.

ABSTRACT
Cyclooxygenase-2 (COX-2) mRNA is induced in the majority of human colorectal carcinomas. Transcriptional regulation plays a key role in COX-2 expression in human colon carcinoma cells, but post-transcriptional regulation of its mRNA is also critical for tumorigenesis. Expression of COX-2 mRNA is regulated by various cytokines, growth factors and other signals. beta-Catenin, a key transcription factor in the Wnt signal pathway, activates transcription of COX-2. Here we found that COX-2 mRNA was also substantially stabilized by activating beta-catenin in NIH3T3 and 293T cells. We identified the beta-catenin-responsive element in the proximal region of the COX-2 3'-untranslated region (3'-UTR) and showed that beta-catenin interacted with AU-rich elements (ARE) of 3'-UTR in vitro and in vivo. Interestingly, beta-catenin induced the cytoplasmic localization of the RNA stabilizing factor, HuR, which may bind to beta-catenin in an RNA-mediated complex and facilitate beta-catenin-dependent stabilization of COX-2 mRNA. Taken together, we provided evidences for beta-catenin as an RNA-binding factor and a regulator of stabilization of COX-2 mRNA.

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Requirement of the proximal region of COX-2 3′-UTR for β-catenin-dependent stabilization. (A) The reporter construct containing 1455 nt of the COX-2 3′-UTR (connecting line) together with the luciferase coding region (filled box). AU-rich elements are indicated as vertical lines. (B) Luciferase assay. Cells were transfected with various luciferase reporters. Luciferase activities were measured after treatment with LiCl or KCl for 24 h. Three independent experiments were performed. (C) Schematic representation of the locations of the F1, F2 and F3 regions. Vertical lines represent AREs. (D) In vitro RNA degradation assays to identify the β-catenin-responsive element in the COX-2 3′-UTR. [α-32P]-labeled RNA substrates were incubated with cytoplasmic extracts from either vector or β-catenin-expressing NIH3T3 cells, and the reactions were stopped by adding stop buffer at the indicated times. Processed RNA was resolved on a 7 M urea/5% acrylamide gel and visualized by autoradiography. (E) In vitro analysis of mRNA decay upon MG132 treatment. Labeled F1 mRNA was incubated with cytoplasmic extracts from either DMSO (Control) or MG132-treated NIH3T3 cells, and decay was analyzed as in (D). (F) Stabilization of the F1 UTR by LiCl treatment. Labeled F1 mRNA was incubated with cytoplasmic extracts from either KCl (Control) or LiCl-treated NIH3T3 cells, and degradation was examined as in (D).
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fig2: Requirement of the proximal region of COX-2 3′-UTR for β-catenin-dependent stabilization. (A) The reporter construct containing 1455 nt of the COX-2 3′-UTR (connecting line) together with the luciferase coding region (filled box). AU-rich elements are indicated as vertical lines. (B) Luciferase assay. Cells were transfected with various luciferase reporters. Luciferase activities were measured after treatment with LiCl or KCl for 24 h. Three independent experiments were performed. (C) Schematic representation of the locations of the F1, F2 and F3 regions. Vertical lines represent AREs. (D) In vitro RNA degradation assays to identify the β-catenin-responsive element in the COX-2 3′-UTR. [α-32P]-labeled RNA substrates were incubated with cytoplasmic extracts from either vector or β-catenin-expressing NIH3T3 cells, and the reactions were stopped by adding stop buffer at the indicated times. Processed RNA was resolved on a 7 M urea/5% acrylamide gel and visualized by autoradiography. (E) In vitro analysis of mRNA decay upon MG132 treatment. Labeled F1 mRNA was incubated with cytoplasmic extracts from either DMSO (Control) or MG132-treated NIH3T3 cells, and decay was analyzed as in (D). (F) Stabilization of the F1 UTR by LiCl treatment. Labeled F1 mRNA was incubated with cytoplasmic extracts from either KCl (Control) or LiCl-treated NIH3T3 cells, and degradation was examined as in (D).

Mentions: To map the regions of the COX-2 3′-UTR responsible for stabilization we utilized luciferase reporters containing the full-length COX-2 3′-UTR or various deletions (Figure 2A). LiCl treatment has no significant effect on luciferase activity in cells transfected with either the luciferase control (ΔUTR) or luciferase lacking the F1 region of the COX-2 3′-UTR (ΔF1). In contrast, it increased the luciferase activities derived from the full-length COX-2 3′-UTR (Full) or the F1 region (F1), suggesting that the F1 region is responsible for the β-catenin-mediated stabilization of COX-2 mRNA (Figure 2B).


Beta-Catenin stabilizes cyclooxygenase-2 mRNA by interacting with AU-rich elements of 3'-UTR.

Lee HK, Jeong S - Nucleic Acids Res. (2006)

Requirement of the proximal region of COX-2 3′-UTR for β-catenin-dependent stabilization. (A) The reporter construct containing 1455 nt of the COX-2 3′-UTR (connecting line) together with the luciferase coding region (filled box). AU-rich elements are indicated as vertical lines. (B) Luciferase assay. Cells were transfected with various luciferase reporters. Luciferase activities were measured after treatment with LiCl or KCl for 24 h. Three independent experiments were performed. (C) Schematic representation of the locations of the F1, F2 and F3 regions. Vertical lines represent AREs. (D) In vitro RNA degradation assays to identify the β-catenin-responsive element in the COX-2 3′-UTR. [α-32P]-labeled RNA substrates were incubated with cytoplasmic extracts from either vector or β-catenin-expressing NIH3T3 cells, and the reactions were stopped by adding stop buffer at the indicated times. Processed RNA was resolved on a 7 M urea/5% acrylamide gel and visualized by autoradiography. (E) In vitro analysis of mRNA decay upon MG132 treatment. Labeled F1 mRNA was incubated with cytoplasmic extracts from either DMSO (Control) or MG132-treated NIH3T3 cells, and decay was analyzed as in (D). (F) Stabilization of the F1 UTR by LiCl treatment. Labeled F1 mRNA was incubated with cytoplasmic extracts from either KCl (Control) or LiCl-treated NIH3T3 cells, and degradation was examined as in (D).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636482&req=5

fig2: Requirement of the proximal region of COX-2 3′-UTR for β-catenin-dependent stabilization. (A) The reporter construct containing 1455 nt of the COX-2 3′-UTR (connecting line) together with the luciferase coding region (filled box). AU-rich elements are indicated as vertical lines. (B) Luciferase assay. Cells were transfected with various luciferase reporters. Luciferase activities were measured after treatment with LiCl or KCl for 24 h. Three independent experiments were performed. (C) Schematic representation of the locations of the F1, F2 and F3 regions. Vertical lines represent AREs. (D) In vitro RNA degradation assays to identify the β-catenin-responsive element in the COX-2 3′-UTR. [α-32P]-labeled RNA substrates were incubated with cytoplasmic extracts from either vector or β-catenin-expressing NIH3T3 cells, and the reactions were stopped by adding stop buffer at the indicated times. Processed RNA was resolved on a 7 M urea/5% acrylamide gel and visualized by autoradiography. (E) In vitro analysis of mRNA decay upon MG132 treatment. Labeled F1 mRNA was incubated with cytoplasmic extracts from either DMSO (Control) or MG132-treated NIH3T3 cells, and decay was analyzed as in (D). (F) Stabilization of the F1 UTR by LiCl treatment. Labeled F1 mRNA was incubated with cytoplasmic extracts from either KCl (Control) or LiCl-treated NIH3T3 cells, and degradation was examined as in (D).
Mentions: To map the regions of the COX-2 3′-UTR responsible for stabilization we utilized luciferase reporters containing the full-length COX-2 3′-UTR or various deletions (Figure 2A). LiCl treatment has no significant effect on luciferase activity in cells transfected with either the luciferase control (ΔUTR) or luciferase lacking the F1 region of the COX-2 3′-UTR (ΔF1). In contrast, it increased the luciferase activities derived from the full-length COX-2 3′-UTR (Full) or the F1 region (F1), suggesting that the F1 region is responsible for the β-catenin-mediated stabilization of COX-2 mRNA (Figure 2B).

Bottom Line: Expression of COX-2 mRNA is regulated by various cytokines, growth factors and other signals. beta-Catenin, a key transcription factor in the Wnt signal pathway, activates transcription of COX-2.Here we found that COX-2 mRNA was also substantially stabilized by activating beta-catenin in NIH3T3 and 293T cells.Taken together, we provided evidences for beta-catenin as an RNA-binding factor and a regulator of stabilization of COX-2 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, BK21 Graduate Program for RNA Biology, Institute of Nanosensor and Biotechnology, Dankook University, Seoul 140-714, Republic of Korea.

ABSTRACT
Cyclooxygenase-2 (COX-2) mRNA is induced in the majority of human colorectal carcinomas. Transcriptional regulation plays a key role in COX-2 expression in human colon carcinoma cells, but post-transcriptional regulation of its mRNA is also critical for tumorigenesis. Expression of COX-2 mRNA is regulated by various cytokines, growth factors and other signals. beta-Catenin, a key transcription factor in the Wnt signal pathway, activates transcription of COX-2. Here we found that COX-2 mRNA was also substantially stabilized by activating beta-catenin in NIH3T3 and 293T cells. We identified the beta-catenin-responsive element in the proximal region of the COX-2 3'-untranslated region (3'-UTR) and showed that beta-catenin interacted with AU-rich elements (ARE) of 3'-UTR in vitro and in vivo. Interestingly, beta-catenin induced the cytoplasmic localization of the RNA stabilizing factor, HuR, which may bind to beta-catenin in an RNA-mediated complex and facilitate beta-catenin-dependent stabilization of COX-2 mRNA. Taken together, we provided evidences for beta-catenin as an RNA-binding factor and a regulator of stabilization of COX-2 mRNA.

Show MeSH
Related in: MedlinePlus