Abstract
BackgroundPluripotent 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 FindingsIn 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 14-3-3σ isoform. Knockdown of 14-3-3σ with siRNA reduced embryonic stem cell proliferation, while only 14-3-3σ transfection increased cell growth and partially rescued retinoid acid-induced growth arrest. Since the growth-enhancing action of 14-3-3σ was abrogated by β-catenin knockdown, we investigated the influence of 14-3-3σ overexpression on β-catenin/GSK-3β. 14-3-3σ bound GSK-3β and increased GSK-3β phosphorylation in a PI-3K/Akt-dependent manner. It disrupted β-catenin binding by the multiprotein destruction complex. 14-3-3σ overexpression attenuated β-catenin phosphorylation and rescued the decline of β-catenin induced by retinoid acid. Furthermore, 14-3-3σ enhanced Wnt3a-induced β-catenin level and GSK-3β phosphorylation. DKK, an inhibitor of Wnt signaling, abolished Wnt3a-induced effect but did not interfere GSK-3β/14-3-3σ binding.SignificanceOur findings show for the first time that 14-3-3σ plays an important role in regulating mouse embryonic stem cell proliferation by binding and sequestering phosphorylated GSK-3β and enhancing Wnt-signaled GSK-3β inactivation. 14-3-3σ is a novel target for embryonic stem cell expansion.
Highlights
Embryonic stem (ES) cells are pluripotent cells that possess selfrenewal properties and retain the capacity for differentiation into all 3 germ layer cells [1,2]
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 glycogen synthase kinase 3b (GSK-3b) and enhancing Wnt-signaled GSK-3b inactivation. 143-3s is a novel target for embryonic stem cell expansion
Our results demonstrate that 14-3-3s overexpression enhances GSK-3b phosphorylation and inactivation as well as increases interaction between 14-3-3s and GSK-3b
Summary
Embryonic stem (ES) cells are pluripotent cells that possess selfrenewal properties and retain the capacity for differentiation into all 3 germ layer cells [1,2]. Upon Wnt activation through binding to frizzled and/or LRP5/6 receptors, disheveled (Dvl) displaces GSK-3b from the APC complex resulting in reduced bcatenin 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. 14-3-3f was reported to interact with Chibby protein to export b-catenin from nucleus and attenuate the b-catenin transcriptional activity [22]. These results indicate that 14-3-3 proteins are functionally complex. We investigated the involvement of 14-3-3 proteins in regulating mouse ES cell (mESC) proliferation. We provide evidence that 143-3s isoform regulates mESC proliferation by binding and sequestering GSK-3b and enhancing Wnt3a-induced GSK-3b phosphorylation and inactivation. We provide evidence that 143-3s isoform regulates mESC proliferation by binding and sequestering GSK-3b and enhancing Wnt3a-induced GSK-3b phosphorylation and inactivation. 14-3-3s overexpression rescues retinoid acid (RA)-induced growth arrest by increasing GSK-3b phosphorylation and b-catenin level
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