Koo Y et al. (2023), RNF2 regulates Wnt/ß-catenin signaling via TCF7...

XB-ART-60534

Sci Rep 2023 Nov 13;131:19750. doi: 10.1038/s41598-023-47111-x.

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RNF2 regulates Wnt/ß-catenin signaling via TCF7L1 destabilization.

Koo Y , Han W , Keum BR , Lutz L , Yun SH , Kim GH , Han JK .

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The Wnt signaling pathway is a crucial regulator of various biological processes, such as development and cancer. The downstream transcription factors in this pathway play a vital role in determining the threshold for signaling induction and the length of the response, which vary depending on the biological context. Among the four transcription factors involved in canonical Wnt/ß-catenin signaling, TCF7L1 is known to possess an inhibitory function; however, the underlying regulatory mechanism remains unclear. In this study, we identified the E3 ligase, RNF2, as a novel positive regulator of the Wnt pathway. Here, we demonstrate that RNF2 promotes the degradation of TCF7L1 through its ubiquitination upon activation of Wnt signaling. Loss-of-function studies have shown that RNF2 consistently destabilizes nuclear TCF7L1 and is required for proper Wnt target gene transcription in response to Wnt activation. Furthermore, our results revealed that RNF2 controls the threshold, persistence, and termination of Wnt signaling by regulating TCF7L1. Overall, our study sheds light on the previously unknown degradation mechanism of TCF7L1 by a specific E3 ligase, RNF2, and provides new insights into the variability in cellular responses to Wnt activation.

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Species referenced: Xenopus laevis
Genes referenced: abcb6 axin1 ctnnb1 hipk2 lef1 myc rnf2 tcf7l1 tcf7l2
GO keywords: Wnt signaling pathway

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	Figure 1 TCF7L1 undergoes degradation through ubiquitination following activation of the Wnt signaling pathway. (a) Western blot analysis was performed using HeLa cells. Cells were treated with 50 mM LiCl or mWnt3a conditioned media for 16 h. The cytosolic fraction was lysed with CB buffer, while the nuclear fraction was lysed with RIPA buffer following initial lysis with CB buffer. (b) HeLa cells treated with LiCl (25 mM and 50 mM), CHIR99021 (5 μM and 10 μM) and BIO (5 μM and 10 μM) conditioned media for 16 h and lysed with RIPA buffer. (c) Western blot analysis was performed using HeLa cells. Cells were treated with LiCl-conditioned media or transfected with the pt-β-catenin plasmid, followed by treatment with 10 μM MG132 for 12 h prior to harvesting. (d) In vitro ubiquitination assay was performed using control HeLa cells. TCF7L1-HA (6 μg) was transfected into HeLa cells. Then, the cells were treated with 50 mM LiCl-conditioned media for 16 h, followed by treatment with 10 μM MG132 for 12 h prior to harvesting with IP buffer. The cells were lysed with the IP buffer and subjected to immunoprecipitation with an HA antibody. Ubiquitination levels were determined by anti-UB-K48.
	Figure 2. Candidate gene screening through mass spectrometry and luciferase assay. (a) Schematic diagram depicting the process of screening candidate genes that interact with TCF7L1. (b) TOPflash luciferase assay was performed using control HeLa cells and stable RNF2 knockdown cell lines. Cells were transfected with the indicated plasmids (50 ng TK-Renilla reporter; 200 ng TOPflash) and then treated with 50 mM NaCl and LiCl conditioned media for 16 h. N = 3 independent experiments. (c) TOPflash luciferase assay was performed using RNF2 knockdown stable HeLa cells. Cells were transfected with the indicated plasmids (50 ng TK-Renilla reporter; 200 ng TOPflash; 1 μg; 2 μg pCS2 + RNF2) and then treated with 50 mM NaCl and LiCl conditioned media for 16 h. The negative control (Co) was treated with 50 mM NaCl conditioned media. The positive control (PC) is performed using control HeLa cells with 50 mM LiCl conditioned media. N = 3 independent experiments. (d) TOPflash luciferase assay was performed using control HeLa cells. Cells were transfected with the indicated plasmids (50 ng TK-Renilla reporter; 200 ng TOPflash; 0.5 μg pCS2 + TCF7L1; 2.5 μg pCS2 + RNF2) and then treated with 50 mM LiCl conditioned media for 16 h. N = 3 independent experiments.
	Figure 3 RNF2 interacts with TCF7L1 and destabilizes TCF7L1. (a) Coimmunoprecipitation assay was performed using control HeLa cells. The indicated plasmids (6 μg TCF7L1-HA and 6 μg myc-RNF2) were transfected into cells, which were then treated with MG132 (10 μM, 12 h) and subjected to immunoprecipitation using anti-myc and anti-HA antibodies. (b) Immunocytochemistry assay was performed using control HeLa cells. Cells were fixed and stained with anti-TCF7L1 (green) and anti-RNF2 (red) after treatment with 10 μM MG132 for 12 h. Scale bar represents 10 μm. (c) Western blot analysis was performed using control HeLa cells. TCF7L1-HA (6 μg) and myc-RNF2 plasmids (3 μg and 6 μg) were introduced into cells, which were then lysed with RIPA buffer. (d) Western blot analysis was performed using control HeLa cells. TCF7L1-HA (6 μg) and myc-RNF2 plasmids (3 μg and 6 μg) were introduced into cells, followed by treatment with 10 μM MG132 for 12 h before harvest and lysed with RIPA buffer. (e) In vitro ubiquitination assay was performed using control HeLa cells. TCF7L1-HA (6 μg) and myc-RNF2 plasmids (6 μg) were introduced into cells, followed by treatment with 10 μM MG132 for 12 h before harvesting. The cells were lysed using the IP buffer and subjected to immunoprecipitation with anti-HA. Ubiquitination levels were determined by anti-UB-K48. (f) Cycloheximide chase assay was performed using control 4T1 cells. myc-RNF2 plasmids (6 μg) were introduced into cells, which were then treated with cycloheximide (10 μM) for 2, 4, and 8 h. Cells were lysed with RIPA buffer.
	Figure 4 Loss of RNF2 stabilizes nuclear TCF7L1. (a) Western blot analysis of control HeLa and HEK293T cells and RNF2 knockdown stable HeLa and HEK293T cells. Endogenous TCF7L1, β-catenin, and RNF2 levels were determined using anti-TCF7L1, anti-β-catenin, and anti-RNF2 antibodies, respectively. (b) Rescue assay: Western blot analysis of control HeLa cells and stable RNF2 knockdown HeLa cells. TCF7L1-HA (6 μg) and myc-RNF2 plasmids (3 μg and 6 μg) were introduced into cells, which were then lysed with RIPA buffer. (c) HeLa cells were fixed and stained with anti-TCF7L1 (green) and anti-RNF2 (red) antibodies. Scale bar represents 10 μm. (d) Quantification of nuclear intensity using ImageJ after fixing the contrast value. Quantification was performed for control HeLa cells and RNF2 knockdown stable HeLa cells (n = 7 and 11). (e) Cycloheximide chase assay of control HeLa cells and RNF2 knockdown stable HeLa cells. The cells were treated with 10 μM cycloheximide (CHX) for 2, 4, and 8 h and lysed with RIPA buffer. (f) Cycloheximide chase assay of RNF2 knockdown stable HeLa cells. myc-RNF2 plasmids (6 μg) were introduced into cells, which were then treated with 10 μM cycloheximide (CHX) for 2, 4, and 8 h. Cells were lysed with RIPA buffer. (g) Cycloheximide chase assay of control HEK293T cells and RNF2 knockdown stable HEK293T cells. TCF7L1-HA plasmids (6 μg) were introduced into cells, which were then treated with 10 μM cycloheximide (CHX) for 2, 4, and 8 h. Cells were lysed with RIPA buffer. (h) In vitro ubiquitination assay was performed using RNF2 knockdown stable HeLa cells and control HeLa cells. TCF7L1-HA (6 μg) and myc-RNF2 plasmids (6 μg) were introduced into cells, followed by treatment with 10 μM MG132 for 12 h, prior to harvesting. The cells were lysed with IP buffer and subjected to immunoprecipitation with the HA antibody. Ubiquitination levels were determined by anti-UB-K48.
	Figure 5 RNF2 control threshold and persistence of Wnt activation by regulating TCF7L1. (a) Western blot analysis of control HeLa cells and RNF2 knockdown stable HeLa cells. TCF7L1-HA (6 μg) plasmid was introduced into cells, which were then treated with LiCl (25 mM, 50 mM) conditioned media for 16 h. Cells were lysed with RIPA buffer. (b) Western blot analysis of RNF2 knockdown stable HeLa cells. Cells were treated with LiCl (25 mM, 50 mM, and 100 mM) conditioned media for 16 h. Cells were lysed with RIPA buffer. ABC refers to active β-catenin. (c) Western blot analysis of control HeLa cells and RNF2 knockdown stable HeLa cells. TCF7L1-HA (6 μg) plasmid was introduced into cells, which were then treated with LiCl (25 mM, 50 mM, and 100 mM) conditioned media for 16 h. Cells were lysed with RIPA buffer. (d) RT-PCR analysis of total RNAs extracted from control HeLa cells and RNF2 knockdown stable HeLa cells after treatment with LiCl (50 mM) conditioned media for 8, 16, and 24 h. Samples were subjected to cDNA synthesis and RT-PCR analysis. (e) Western blot analysis of control HeLa cells and RNF2 knockdown stable HeLa cells treated with LiCl (25 mM and 50 mM) and CHIR99021 (5 μM and 10 μM) conditioned media for 16 h. TCF7L1-HA (6 μg) plasmid was introduced into control HeLa cells. (f) Western blot analysis of control 4T1 cells treated with LiCl (25 mM and 50 mM) and CHIR99021 (5 μM and 10 μM) conditioned media for 16 h. (g) Western blot analysis of control HEK239T cells and 4T1 cells treated with LiCl (25 mM, 50 mM, and 100 mM) and conditioned media for 16 h. (h) Western blot analysis of control HeLa cells and RNF2 knockdown stable HeLa cells treated with 50 mM LiCl conditioned media for varying durations (30 min, 1 h, 2 h, 4 h, 8 h, 16 h, and 24 h).
	Figure 6. Predicted mechanism for RNF2 regulation of Wnt signaling through TCF7L1 ubiquitination. (a) Diagram of TCF7L1 deletion mutant used for the domain study. The image was generated using Adobe Illustrator software (version 27.3). (b) Western blot analysis was performed using control HeLa cells. A total of 6 μg of myc-TCF7L1 partial mutants and myc-RNF2 plasmids were introduced into cells, which were then lysed with RIPA buffer. (c) Proposed mechanism of the RNF2 regulation of Wnt signaling. RNF2 continuously degrades TCF7L1 through ubiquitination-mediated degradation to regulate TCF7L1 at low levels. TCF7L1 binding to a promoter region may avoid RNF2 ubiquitination. When Wnt is activated, TCF7L1 detaches from DNA and is exposed followed by the binding of ß-catenin and HIPK2 phosphorylation. Then, the TCF7L1 is degraded through RNF2-mediated ubiquitination. The image was generated using Adobe Illustrator software (version 27.3).

References [+] :

Amado, Effects of natural compounds on Xenopus embryogenesis: a potential read out for functional drug discovery targeting Wnt/β-catenin signaling. 2012, Pubmed, Xenbase