Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
Biomedical Research Center and Key Laboratory of Biotherapy of Zhejiang Province, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, PR China; Department of Mechanical and Automation Engineering, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
Biomaterials. 2017 Nov;145:33-43. doi: 10.1016/j.biomaterials.2017.08.031. Epub 2017 Aug 16.
N-cadherin, a transmembrane protein and major component of adherens junction, mediates cell-cell interactions and intracellular signaling that are important to the regulation of cell behaviors and organ development. Previous studies have identified mimetic peptides that possess similar bioactivity as that of N-cadherin, which promotes chondrogenesis of human mesenchymal stem cells (hMSCs); however, the molecular mechanism remains unknown. In this study, we combined the N-cadherin mimetic peptide (HAVDI) with the self-assembling KLD-12 peptide: the resultant peptide is capable of self-assembling into hydrogels functionalized with N-cadherin peptide in phosphate-buffered saline (PBS) at 37 °C. Encapsulation of hMSCs in these hydrogels showed enhanced expression of chondrogenic marker genes and deposition of cartilage specific extracellular matrix rich in proteoglycan and Type II Collagen compared to control hydrogels, with a scrambled-sequence peptide after 14 days of chondrogenic culture. Furthermore, western blot showed a significantly higher expression of active glycogen synthase kinase-3β (GSK-3β), which phosphorylates β-catenin and facilitates ubiquitin-mediated degradation, as well as a lower expression of β-catenin and LEF1 in the N-cadherin peptide hydrogels versus controls. Immunofluorescence staining revealed significantly less nuclear localization of β-catenin in N-cadherin mimetic peptide hydrogels. Our findings suggest that N-cadherin peptide hydrogels suppress canonical Wnt signaling in hMSCs by reducing β-catenin nuclear translocation and the associated transcriptional activity of β-catenin/LEF-1/TCF complex, thereby enhancing the chondrogenesis of hMSCs. Our biomimetic self-assembled peptide hydrogels can serve as a tailorable and versatile three-dimensional culture platform to investigate the effect of biofunctionalization on stem cell behavior.
N-钙黏蛋白是一种跨膜蛋白，也是黏着连接的主要成分，它介导细胞-细胞相互作用和细胞内信号转导，对细胞行为和器官发育的调节至关重要。先前的研究已经鉴定出具有与 N-钙黏蛋白相似生物活性的模拟肽，这些模拟肽促进了人间充质干细胞（hMSCs）的软骨生成；然而，其分子机制尚不清楚。在这项研究中，我们将 N-钙黏蛋白模拟肽（HAVDI）与自组装的 KLD-12 肽结合：所得的肽能够在 37°C 的磷酸盐缓冲盐水中（PBS）自组装成具有 N-钙黏蛋白肽功能化的水凝胶。与对照水凝胶相比，hMSC 包封在这些水凝胶中显示出软骨形成标志物基因的表达增强和富含蛋白聚糖和 II 型胶原的软骨特异性细胞外基质的沉积，在软骨形成培养 14 天后，具有随机序列的肽。此外，Western blot 显示，在 N-钙黏蛋白肽水凝胶中，活性糖原合酶激酶-3β（GSK-3β）的表达显著升高，GSK-3β 磷酸化β-连环蛋白并促进其泛素介导的降解，而β-连环蛋白和 LEF1 的表达则降低。免疫荧光染色显示，N-钙黏蛋白模拟肽水凝胶中β-连环蛋白的核定位明显减少。我们的研究结果表明，N-钙黏蛋白肽水凝胶通过减少β-连环蛋白的核易位及其与β-连环蛋白/LEF-1/TCF 复合物相关的转录活性，从而抑制 hMSC 中的经典 Wnt 信号通路，增强 hMSC 的软骨生成。我们的仿生自组装肽水凝胶可以作为一种可定制和多功能的三维培养平台，用于研究生物功能化对干细胞行为的影响。
