Orthopaedic Research Laboratory, Department of Orthopedic Surgery, Boston University School of Medicine, Boston, Massachusetts, USA.
J Cell Physiol. 2013 Nov;228(11):2232-42. doi: 10.1002/jcp.24396.
Runx2 and Runx3 are known to be expressed in the growth plate during endochondral bone formation. Here we addressed the functional role of Runx3 as distinct from Runx2 by using two models of postnatal bone repair: fracture healing that proceeds by an endochondral process and marrow ablation that proceeds by only an intramembranous process. Both Runx2 and Runx3 mRNAs were differentially up regulated during fracture healing. In contrast, only Runx2 showed increased expression after marrow ablation. During fracture healing, Runx3 was expressed earlier than Runx2, was concurrent with the period of chondrogenesis, and coincident with maximal aggrecan expression a protein associated with proliferating and permanent cartilage. Immunohistological analysis showed Runx3 protein was also expressed by chondrocytes in vivo. In contrast, Runx2 was expressed later during chondrocyte hypertrophy, and primary bone formation. The functional activities of Runx3 during chondrocyte differentiation were assessed by examining its regulatory actions on aggrecan gene expression. Aggrecan mRNA levels and aggrecan promoter activity were enhanced in response to the over-expression of either Runx2 and Runx3 in ATDC5 chondrogenic cell line, while sh-RNA knocked down of each Runx protein showed that only Runx3 knock down specifically suppressed aggrecan mRNA expression and promoter activity. ChIP assay demonstrated that Runx3 interactions were selective to sites within the aggrecan promoter and were only observed during early periods of chondrogenesis before hypertrophy. Our studies suggest that Runx3 positively regulates aggrecan expression and suggest that its function is more limited to cartilage development than to bone. In aggregate these data further suggest that the various members of the Runx transcription factors are involved in the coordination of chondrocyte development, maturation, and hypertrophy during endochondral bone formation.
Runx2 和 Runx3 已知在软骨内骨形成过程中在生长板中表达。在这里，我们通过两种成骨后骨修复模型来解决 Runx3 与 Runx2 功能不同的问题：通过软骨内过程进行的骨折愈合和仅通过膜内过程进行的骨髓消融。在骨折愈合过程中，Runx2 和 Runx3mRNA 均呈差异上调。相比之下，骨髓消融后仅 Runx2 表达增加。在骨折愈合过程中，Runx3 的表达早于 Runx2，与软骨生成期同时发生，与最大的聚集蛋白表达一致，聚集蛋白与增殖和永久性软骨相关。免疫组织化学分析显示，Runx3 蛋白也在体内软骨细胞中表达。相比之下，Runx2 在软骨细胞肥大和原发性骨形成过程中表达较晚。通过检查其对聚集蛋白基因表达的调节作用来评估 Runx3 在软骨细胞分化过程中的功能活性。在 ATDC5 软骨细胞系中，Runx2 和 Runx3 的过表达均增强了聚集蛋白 mRNA 水平和聚集蛋白启动子活性，而每种 Runx 蛋白的 sh-RNA 敲低表明，只有 Runx3 敲低特异性抑制了聚集蛋白 mRNA 表达和启动子活性。ChIP 测定表明，Runx3 相互作用是聚集蛋白启动子内的选择性，仅在肥大前的软骨生成早期观察到。我们的研究表明，Runx3 正向调节聚集蛋白的表达，并表明其功能在软骨发育中比在骨骼发育中更为有限。总之，这些数据进一步表明，Runx 转录因子的各种成员参与了软骨细胞发育、成熟和肥大的协调，以进行软骨内骨形成。
