[Frontiers in Bioscience 18, 493-503, January 1, 2013]

WNT3A modulates chondrogenesis via canonical and non-canonical Wnt pathways in MSCs

Feng Qu 1,2, Junliang Wang2, Ningru Xu 1, Chang Liu 2, Shuyuan Li 2, Ning Wang 2, Wei Qi 2, Haifeng Li 2, Chunbao Li 2, Zhenying Geng 1, Yujie Liu 2

1Medical school of Nankai University, 94 Weijin Road, Tianjin, China, 2Department of orthopedics, Chinese PLA general hospital, 28 Fuxing Road, Beijing, China

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Generation of vectors 3.2 .Rat MSC culture 3.3 .Colorimetric tetrazolium salt analysis
3.4. Gene expression analysis
3.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting
3.6. Ca2+_ mobilization assay
3.7. Chondrocyte differentiation and toluidine blue staining
3.8. Lentiviral infection
3.9. Subcellular fractionation 3.10. Data analysis
4. Results
4.1. Overexpression of WNT3A increases MSC proliferation and inhibits chondrogenesis
4.2. WNT3A activates both the canonical and the non-canonical Wnt pathways in MSCs
4.3. Inhibition of the Wnt canonical pathway by Dickkopf-related protein 1 (DKK1) abrogates WNT3A-induced proliferation but does not rescue the loss of the chondrocyte phenotype
4.4. Inhibition of the Ca2+/CaMKII pathway rescues loss of the chondrocyte phenotype but has little effect on WNT3A-induced proliferation of MSCs
5. Discussion
6. Acknowledgments
7. References

1. ABSTRACT

The multilineage commitment of mesenchymal stem cells (MSCs) is controlled via unknown mechanisms. In this study, we investigated the regulation of the differentiation of MSCs into chondrocytes via the Wnt signaling pathway. Overexpression of WNT3A in MSCs activated both the canonical and non-canonical Wnt pathways, which were responsible for different WNT3A-induced outcomes. WNT3A promoted MSC proliferation via the β-catenin-mediated canonical Wnt pathway, and inhibited chondrogenesis of MSCs via the calcium/calmodulin-dependent kinase II (CaMKII)-mediated non-canonical Wnt pathway. Interestingly, blockade of the canonical Wnt pathway by Dickkopf-related protein 1 exerted a synergistic effect on the inhibition of chondrogenesis of MSCs, while blockade of the non-canonical Wnt pathway by KN93 also exerted a synergistic effect on MSCs proliferation. These results suggest that the WNT3A-activated canonical and non-canonical pathways counteract each other in the setting of MSCs. This study provides evidence for the delicate regulation of the Wnt signaling cascade during chondrogenesis of MSCs, and suggests that genetic manipulation of the Wnt pathway may offer a powerful vehicle for modulating MSC differentiation in stem-cell-based cartilage repair.