14-3-3s Regulates b-Catenin-Mediated Mouse Embryonic Stem Cell Proliferation by Sequestering GSK-3b Tzu-Ching Chang1., Chia-Chia Liu1,2., En-Wei Hsing1, Shu-Man Liang1, Ya-Hui Chi1, Li-Ying Sung2, Shau- Ping Lin2, Tang-Long Shen3, Bor-Sheng Ko1,4, B. Linju Yen1, Shaw-Fang Yet1, Kenneth K. Wu1*, Jun- 1 Yang Liou * 1 Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan, 2 Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, 3 Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan, 4 Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan Abstract Background: Pluripotent embryonic stem cells are considered to be an unlimited cell source for tissue regeneration and cell-based therapy. Investigating the molecular mechanism underlying the regulation of embryonic stem cell expansion is thus important. 14-3-3 proteins are implicated in controlling cell division, signaling transduction and survival by interacting with various regulatory proteins. However, the function of 14-3-3 in embryonic stem cell proliferation remains unclear. Methodology and Principal Findings: In this study, we show that all seven 14-3-3 isoforms were detected in mouse embryonic stem cells. Retinoid acid suppressed selectively the expression of 14-3-3s isoform. Knockdown of 14-3-3s with siRNA reduced embryonic stem cell proliferation, while only 14-3-3s transfection increased cell growth and partially rescued retinoid acid-induced growth arrest. Since the growth-enhancing action of 14-3-3s was abrogated by b-catenin knockdown, we investigated the influence of 14-3-3s overexpression on b-catenin/GSK-3b. 14-3-3s bound GSK-3b and increased GSK-3b phosphorylation in a PI-3K/Akt-dependent manner. It disrupted b-catenin binding by the multiprotein destruction complex. 14-3-3s overexpression attenuated b-catenin phosphorylation and rescued the decline of b-catenin induced by retinoid acid. Furthermore, 14-3-3s enhanced Wnt3a-induced b-catenin level and GSK-3b phosphorylation. DKK, an inhibitor of Wnt signaling, abolished Wnt3a-induced effect but did not interfere GSK-3b/14-3-3s binding. Significance: Our findings show for the first time that 14-3-3s plays an important role in regulating mouse embryonic stem cell proliferation by binding and sequestering phosphorylated GSK-3b and enhancing Wnt-signaled GSK-3b inactivation. 14- 3-3s is a novel target for embryonic stem cell expansion. Citation: Chang T-C, Liu C-C, Hsing E-W, Liang S-M, Chi Y-H, et al. (2012) 14-3-3s Regulates b-Catenin-Mediated Mouse Embryonic Stem Cell Proliferation by Sequestering GSK-3b. PLoS ONE 7(6): e40193. doi:10.1371/journal.pone.0040193 Editor: Qingzhong Xiao, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, United Kingdom Received February 8, 2012; Accepted June 2, 2012; Published June 29, 2012 Copyright: 2012 Chang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by The National Science Council (99-3111-B-400-001, 100-2321-B-400-021, 99-3111-B-400-005 and 100-2321-B-400-020) and The National Health Research Institutes (00A1-CSPP08-014) of Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jliou@nhri.org.tw (JYL); kkgo@nhri.org.tw (KKW) . These authors contributed equally to this work. Introduction Embryonic stem (ES) cells are pluripotent cells that possess self- renewal properties and retain the capacity for differentiation into all 3 germ layer cells [1,2]. Because of their high proliferation capability, pluripotency and low immunogenicity, ES cells are considered to be a valuable source for cell therapy, tissue regeneration, drug testing and developmental biology [3,4]. ES cell proliferation and renewal are maintained by diverse factors that activate the renewal genetic program via selective signaling pathways [5,6] among which the b-catenin pathway plays a pivotal role [7,8]. At the basal state, b-catenin is associated with a multiprotein destruction complex composed of APC (adenoma- tous polyposis coli), axin, casein kinase 2 and glycogen synthase kinase 3b (GSK-3b) where it is phosphorylated and degraded via ubiquitin/proteasome [9–11]. Upon Wnt activation through binding to frizzled and/or LRP5/6 receptors, disheveled (Dvl) displaces GSK-3b from the APC complex resulting in reduced b- catenin degradation, and increased cytosolic b-catenin which is translocated to nucleus where it is associated with Tcf/Lef transcription factor to drive the expression of renewal and proliferative genes. Experimental data have provided convincing evidence for the crucial role of GSK-3b/b-catenin in ES cell renewal [12–14]. GSK-3b is a serine/threonine protein kinase which was originally discovered as an enzyme that phosphorylates and inactivates glycogen synthase in response to insulin, and was subsequently reported to phosphorylate b-catenin and facilitate b- catenin ubiquitination and degradation [15]. GSK-3b inhibition was shown to maintain ES cells in the renewal state [14]. Thus, GSK-3b occupies a central position in controlling b-catenin and PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e40193