QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 7 November 2006
doi: 10.1242/jcs.03257

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pan, W. Articles by Li, L. Search for Related Content PubMed PubMed Citation Articles by Pan, W. Articles by Li, L.

ß-catenin relieves I-mfa-mediated suppression of LEF-1 in mammalian cells

Weijun Pan^*, Yingying Jia^*, Tao Huang, Jiyong Wang, Donglei Tao, Xiaoqing Gan and Lin Li

State Key Laboratory of Molecular Biology and Center of Cell Signaling, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

View larger version (26K): [in this window] [in a new window]   Fig. 1. Stably expressing I-mfa siRNA in P19 cells initiate myogenesis. (A) Protein expression levels in different kinds of stable cell lines. The levels of endogenous I-mfa, ß-catenin and ß-tubulin and exogenous C-LEF-1-HA were determined by western-blot analysis. ß-tubulin was measured as an internal control. (B,C) P19 cell lines that stably express a control plasmid (Ctr), I-mfa siRNA (siRNA) or C-LEF-1, or coexpress C-LEF-1 and I-mfa siRNA were induced to differentiate after aggregation in the presence or absence of Wnt3a. At Day 9, cells were collected. MHC protein levels were analyzed via western blotting and quantified through the use of an Odyssey II quantification system as previously described (Pan et al., 2005) (B). Cells were also fixed and stained with DAPI (blue) and an anti-MHC antibody (green) (C). We established several individual clones for every kind of stable cell line. All the clones showed similar results, and we present only the results from one of them.

View larger version (36K): [in this window] [in a new window]   Fig. 2. I-mfa inhibits endogenous Wnt/ß-catenin signaling in P19 cells. (A) P19 cells, as shown in Fig. 1B,C, were collected for RNA isolation and semi-quantitative RT-PCR analysis as described in the Materials and Methods. (B) I-mfa suppresses LEF-1 reporter activity in P19 cells. Cells were first transfected with control siRNA (si-Ctr) or I-mfa siRNA (si-I-mfa) and two days later with the LEF-1 reporter system. Twenty-four hours later, cells were treated with control-conditioned medium (Ctr CM) or Wnt3a-conditioned medium (Wnt3a CM) for an additional 8 hours before the activity was determined. (C) I-mfa siRNA elevate the expression of the Wnt target gene. Individual clones for different kinds of P19 stable cell line were cultured as a monolayer, and treated with Ctr CM or Wnt3a CM for 6 hours before RNA isolation. Semi-quantitative RT-PCR was performed for cyclin D1 and GAPDH detection. Relative mRNA levels of cyclin D1 were quantified by Labwork 2.0 UVP software and normalized with the amount of GAPDH. (D) I-mfa does not affect Wnt-induced free ß-catenin accumulation. P19 cells were transfected with I-mfa, GSK3ß and Axin1 expression plasmids or LacZ as indicated in the figure. Eighteen hours post-transfection, cells were treated with Ctr CM or Wnt3a CM for a further 6 hours and then collected for cell-fraction preparation and ß-catenin ELISA assay.

View larger version (23K): [in this window] [in a new window]   Fig. 3. ß-catenin relieves I-mfa-mediated suppression of LEF-1. (A) ß-catenin competes with LEF-1 for binding to I-mfa. HEK 293T cells were transfected with plasmids as indicated. Immunoprecipitation was performed with an anti-GluGlu antibody and analyzed with an anti-HA, anti-GluGlu or anti-Myc antibody. (B) ß-catenin relieves I-mfa inhibition of the LEF-1 reporter gene in mammalian cells. For NIH3T3 cells, transfection was performed with the LEF-1 reporter gene system and expression plasmid as indicated in the figures. Luciferase activity was determined 24 hours after transfection.

View larger version (45K): [in this window] [in a new window]   Fig. 4. Effect of endogenous ß-catenin on LEF-1-I-mfa interactions. P19 cells were treated with or without Wnt3a for 3 hours. Cells were harvested for cell fractionation and immunoprecipitation with an anti-LEF-1 or anti-Myc antibody (control antibody). Endogenous LEF-1, ß-catenin and I-mfa were detected with their respective antibodies.

View larger version (20K): [in this window] [in a new window]   Fig. 5. A model for the regulation of MRFs and TCF/LEF-1 by ß-catenin and I-mfa in myogenesis. In the absence of a canonical Wnt signal, I-mfa suppresses transcriptional activities of MRF and TCF/LEF-1 by binding to them. In the presence of canonical Wnt proteins, canonical Wnt signaling is activated, and the levels of ß-catenin increase in the cytoplasm and nucleus are elevated. ß-catenin then binds to I-mfa and relieves I-mfa suppression of TCF/LEF-1 and MRF, which is required for myogenic specification and development.