Wnt/b-catenin signaling changes C2C12 myoblast proliferation and differentiation by inducing Id3 expression Long Zhang 1 , Songting Shi 1 , Juan Zhang, Fangfang Zhou, Peter ten Dijke ⇑ Dept. of Molecular Cell Biology and Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands article info Article history: Received 5 January 2012 Available online 3 February 2012 Keywords: Wnt/b-catenin Id3 Cell proliferation Myoblast abstract Canonical Wnt signaling plays important roles in regulating cell proliferation and differentiation. In this study, we report that inhibitor of differentiation (Id)3 is a Wnt-inducible gene in mouse C2C12 myoblasts. Wnt3a induced Id3 expression in a b-catenin-dependent manner. Bone morphogenetic protein (BMP) also potently induced Id3 expression. However, Wnt-induced Id3 expression occurred independent of the BMP/ Smad pathway. Functional studies showed that Id3 depletion in C2C12 cells impaired Wnt3a-induced cell proliferation and alkaline phosphatase activity, an early marker of osteoblast cells. Id3 depletion elevated myogenin induction during myogenic differentiation and partially impaired Wnt3a suppressed myogenin expression in C2C12 cells. These results suggest that Id3 is an important Wnt/b-catenin induced gene in myoblast cell fate determination. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Wnts are a family of growth factors controlling multiple biolog- ical processes such as embryogenesis, organogenesis and tumori- genesis [1–4]. In the presence of Wnt ligand, Wnt receptor frizzled (Fz) and its co-receptor low-density lipoprotein receptor-related protein-5 or 6 (LRP-5/6) recruit Axin and GSK3b to the plasma mem- brane, together with the scaffold protein Disheveled (Dvl) [5,6]. The membrane association of Axin and GSK3b disrupts the b-catenin destruction complex, resulting in accumulation of b-catenin in the nucleus, where it triggers target gene activation by displacing tran- scriptional repressors from DNA-bound LEF/TCF [7,8]. In recent years, canonical Wnt signaling pathway is identified to be crucial for bone formation and bone homeostasis. The mutations in LRP-5 profoundly affect skeletal development and result in low bone mass [9,10]. The Dickkopf-1 (Dkk-1) resistant LRP5V171 mutation leads to high bone density [11]. Conditional deletion of the b-catenin gene in osteoblasts leads to reduced bone-mass in vivo [12]. Canonical Wnt signaling is also reported to be involved in myogenic differen- tiation of mouse myoblast cells [13]. Id (inhibitor of DNA binding) proteins are helix–loop– helix (HLH) proteins, which lack basic region adjacent to the HLH domain that is essential for specific DNA binding in other bHLH pro- teins [14]. Id proteins repress bHLH proteins by binding and inter- fering with DNA interaction of HLH proteins. As direct target genes of bone morphogenetic proteins (BMPs), Id proteins regulate cell fate [15,16]. In this study, we found canonical Wnt/b-catenin signaling could induce Id3 expression independent of BMP/Smad signaling activation. Loss of function studies in C2C12 cells demon- strated that Id3 plays a pivotal role in canonical Wnt3a-induced cell fate determination. 2. Materials and methods 2.1. Reagent and plasmids GAPDH antibody (Sigma), Id3 antibody (C-20, Santa Cruz), P-Smad1 (#9511 Cell signaling) and Smad1 (SC-7965 Santa Cruz), Myogenin (ab1835, Abcam); BMP response element (BRE)-Luc reporter, b-catenin WT/SY plasmids were previously described [17–19]. Dominant negative Lymphoid enhancer binding factor (LEF)-1 was cloned by polymerase chain reaction (PCR) into pcDNA3.1 vector. 2.2. Cell culture Mouse myoblast C2C12, NIH3T3, C3H10T1/2, control and Wnt3a expressing L cells and HEK293T cells were maintained in growing Dulbecco’s modified Eagle medium (DMEM) supple- mented with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator with 5% CO 2 . C2C12 myoblasts were seeded at a concen- tration of 0.35 Â 10 6 cells per well in 6-well plates and cultured for 24 h to reach 100% confluence (day 0). To induce myogenic differ- entiation, cells were washed in PBS and cultured in low glucose DMEM supplemented with antibiotics and 2% heat-inactivated horse serum, referred to as differentiation medium (DM). All of 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.01.132 ⇑ Corresponding author. E-mail address: p.ten_dijke@lumc.nl (P. ten Dijke). 1 These authors contribute equally to this work. Biochemical and Biophysical Research Communications 419 (2012) 83–88 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc