RUNX2 is a master osteogenic transcription factor, and mutations in RUNX2 cause the inherited skeletal disorder cleidocranial dysplasia (CCD). Studies have revealed that RUNX2 is not only a downstream target of the bone morphogenetic protein (BMP) pathway but can also regulate the expression of BMPs. However, the underlying mechanism of the regulation of BMPs by RUNX2 remains unknown. In this project, we diagnosed a CCD patient with a 7.86-Mb heterozygous deletion on chromosome 6 containing all exons of RUNX2 by multiplex ligation-dependent probe amplification (MLPA) and whole-genome sequencing (WGS). Bone marrow mesenchymal stem cells (BMSCs) were further extracted from patient alveolar bone fragments (CCD-BMSCs), an excellent natural model to explore the possible mechanism. The osteogenic differentiation ability of CCD-BMSCs was severely affected by RUNX2 heterozygous deletion. Also, BMP4 decreased most in BMP ligands, and CHRDL1, a BMP antagonist, was abnormally elevated in CCD-BMSCs. Furthermore, BMP4 treatment essentially rescued the osteogenic capacity of CCD-BMSCs, and RUNX2 overexpression reversed the abnormal expression of BMP4 and CHRDL1. Notably, we constructed CRISPR/Cas9 Runx2^+/m MC3T3-E1 cells, which simulated a variant in CCD-BMSCs, to exclude the interference of other gene deletions and the heterogeneity of the genetic background of primary cells, and verified all findings from the CCD-BMSCs. Moreover, the luciferase reporter experiment showed that RUNX2 could inhibit the transcription of CHRDL1. Through immunofluorescence, the inhibitory effect of CHRDL1 on BMP4/Smad signaling was confirmed in MC3T3-E1 cells. These results revealed that RUNX2 regulated the BMP4 pathway by inhibiting CHRDL1 transcription. We collectively identified a novel RUNX2/CHRDL1/BMP4 axis to regulate osteogenic differentiation and noted that BMP4 might be a valuable therapeutic option for treating bone diseases.

RUNX2 是一种主要的成骨转录因子，RUNX2的突变导致遗传性骨骼疾病锁骨发育不良（CCD）。研究表明，RUNX2 不仅是骨形态发生蛋白 (BMP) 通路的下游靶点，还可以调节 BMPs 的表达。然而，RUNX2 调控 BMP 的潜在机制仍然未知。在这个项目中，我们诊断出一名 CCD 患者，其 6 号染色体上有 7.86-Mb 杂合缺失，包含RUNX2的所有外显子通过多重连接依赖性探针扩增（MLPA）和全基因组测序（WGS）。从患者牙槽骨碎片（CCD-BMSCs）中进一步提取骨髓间充质干细胞（BMSCs），这是探索可能机制的极好自然模型。RUNX2杂合缺失严重影响CCD-BMSCs的成骨分化能力。此外，BMP 配体中的 BMP4 下降最多，而 BMP 拮抗剂 CHRDL1 在 CCD-BMSCs 中异常升高。此外，BMP4 治疗基本上挽救了 CCD-BMSCs 的成骨能力，而 RUNX2 过表达逆转了 BMP4 和 CHRDL1 的异常表达。值得注意的是，我们构建了 CRISPR/Cas9 Runx2 ^+/mMC3T3-E1 细胞模拟了 CCD-BMSCs 中的一个变异体，排除了其他基因缺失的干扰和原代细胞遗传背景的异质性，并验证了 CCD-BMSCs 的所有发现。此外，荧光素酶报告实验表明RUNX2可以抑制CHRDL1的转录。通过免疫荧光，在 MC3T3-E1 细胞中证实了 CHRDL1 对 BMP4/Smad 信号传导的抑制作用。这些结果表明，RUNX2 通过抑制CHRDL1转录来调节 BMP4 通路。我们共同确定了一种新的 RUNX2/CHRDL1/BMP4 轴来调节成骨分化，并指出 BMP4 可能是治疗骨病的一种有价值的治疗选择。