Program of Cellular and Molecular Biology, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, 8380453 Santiago, Chile; Millennium Institute on Immunology and Immunotherapy, Faculty of Medicine, University of Chile, 8380453 Santiago, Chile.
Department of Orthopedics and Traumatology, Clínica Las Condes, 7591047 Santiago, Chile.
Gene. 2019 Mar 1;687:228-237. doi: 10.1016/j.gene.2018.11.037. Epub 2018 Nov 14.
The in vitro process of chondrogenic differentiation of mesenchymal stem cells (MSCs) induces a pre-apoptotic hypertrophic phenotype, guided by the active status of the WNT/β‑catenin pathway. To achieve a stable chondrocyte phenotype for cartilage tissue engineering, it is necessary to gain a better understanding of specific genes that regulate the cartilage tissue phenotype. RNA sequencing (RNA-seq) analysis of tissue samples from bone, cartilage, growth plate and muscle show that Dickkopf-1 (DKK1), a natural WNT canonical signaling inhibitor, is expressed in cartilage tissue. This observation reinforces the concept that inhibition of the WNT/β‑catenin pathway is critical for preventing avoid chondrocyte hypertrophy in vitro. We used two doses of DKK1 in a pellet cell culture system to inhibit the terminal differentiation of chondrocytes derived from bone marrow mesenchymal stem cells (MSCs). Bone marrow MSCs were cultured in chondrogenic induction medium with 50 and 200 ng/ml of DKK1 for 21 days. The highest doses of DKK1 reduce β‑catenin expression and nuclear localization at day 21, concomitant with reduced expression and activity of hypertrophy markers collagen type X (COL10A1) and alkaline phosphatase (ALPL), thus decreasing the pre-hypertrophic chondrocyte population. Furthermore, DKK1 stimulated expression of collagen type II (COL2A1) and glycosaminoglycans (GAGs), which represent healthy articular cartilage markers. We conclude that exogenous DKK1 impedes chondrocyte progression into a prehypertrophic stage and stimulates expression of healthy articular cartilage markers by blocking the WNT/β‑catenin pathway. Hence, DKK1 may promote a mature healthy articular cartilage phenotype and facilitate cartilage tissue engineering for joint repair.
骨髓间充质干细胞（MSCs）向软骨细胞分化的体外过程会诱导前凋亡肥大表型，这一过程受到 WNT/β-连环蛋白通路的活性状态的指导。为了实现软骨组织工程中稳定的软骨细胞表型，有必要更好地了解调节软骨组织表型的特定基因。对骨、软骨、生长板和肌肉组织样本的 RNA 测序（RNA-seq）分析表明，Dickkopf-1（DKK1）是一种天然的 WNT 经典信号抑制剂，在软骨组织中表达。这一观察结果强化了这样一种概念，即抑制 WNT/β-连环蛋白通路对于防止体外软骨细胞肥大至关重要。我们在微球细胞培养系统中使用两种剂量的 DKK1 来抑制骨髓间充质干细胞（MSCs）来源的软骨细胞的终末分化。将骨髓 MSCs 在含有 50 和 200ng/ml DKK1 的软骨诱导培养基中培养 21 天。在第 21 天，最高剂量的 DKK1 减少了 β-连环蛋白的表达和核定位，同时降低了肥大标志物胶原类型 X（COL10A1）和碱性磷酸酶（ALPL）的表达和活性，从而减少了前肥大的软骨细胞群体。此外，DKK1 刺激了胶原类型 II（COL2A1）和糖胺聚糖（GAGs）的表达，这些标志物代表了健康的关节软骨标志物。我们得出结论，外源性 DKK1 通过阻断 WNT/β-连环蛋白通路来阻碍软骨细胞向肥大前阶段的进展，并刺激健康关节软骨标志物的表达。因此，DKK1 可能促进成熟的健康关节软骨表型，并促进用于关节修复的软骨组织工程。
