Chordin-Like 1 Improves Osteogenesis of Bone Marrow Mesenchymal Stem Cells Through Enhancing BMP4-SMAD Pathway

Chordin-like 1 (CHRDL1) is a secreted glycoprotein with repeated cysteine-rich domains, which can bind to BMPs family ligands. Although it has been reported to play important roles in several systems, the exact roles of CHRDL1 on human bone mesenchymal stem cells (hBMSCs) osteogenesis remain to be explored. The present study aimed to investigate the roles of CHRDL1 on the osteogenic differentiation of hBMSCs and the underlying molecular mechanisms. We found that CHRDL1 was upregulated during hBMSCs osteogenesis, and rhBMP-4 administration could enhance CHRDL1 mRNA expression in a dose and time dependent manner. Knockdown of CHRDL1 did not affect hBMSCs proliferation, but inhibited the BMP-4-dependent osteogenic differentiation, showing decreased mRNA expression levels of osteogenic markers and reduced mineralization. On the contrary, overexpression of CHRDL1 enhanced BMP-4 induced osteogenic differentiation of hBMSCs. Moreover, in vivo experiments by transplanting CHRDL1 gene modified hBMSCs into nude mice defective femur models displayed higher new bone formation in CHRDL1 overexpression groups, but lower new bone formation in CHRDL1 knockdown groups, compared with control groups. In consistent with the bone formation rate, there were increased CHRDL1 protein expression in new bone formation regions of defective femur in CHRDL1 overexpression groups, while reduced CHRDL1 protein expression in CHRDL1 knockdown groups compared with control groups. These indicate that CHRDL1 can promote osteoblast differentiation in vivo. Furthermore, the mechanisms study showed that CHRDL1 improved BMP-4 induced phosphorylation of SMAD1/5/9 during osteogenic differentiation of hBMSCs. Besides, promotion of osteogenic differentiation and the activation of SMAD phosphorylation by CHRDL1 can be blocked by BMP receptor type I inhibitor LDN-193189. In conclusion, our results suggested that CHRDL1 can promote hBMSCs osteogenic differentiation through enhancing the activation of BMP-4-SMAD1/5/9 pathway.

类腱蛋白1（CHRDL1）是一种分泌型糖蛋白，具有重复的富含半胱氨酸结构域，可与骨形态发生蛋白（BMPs）家族配体结合。尽管已有报道称其在多个系统中发挥重要作用，但CHRDL1在人骨髓间充质干细胞（hBMSCs）成骨过程中的确切作用仍有待探索。本研究旨在探讨CHRDL1在hBMSCs成骨分化中的作用及其潜在的分子机制。我们发现CHRDL1在hBMSCs成骨过程中表达上调，重组人骨形态发生蛋白 - 4（rhBMP - 4）给药可呈剂量和时间依赖性地增强CHRDL1 mRNA的表达。敲低CHRDL1不影响hBMSCs的增殖，但抑制了BMP - 4依赖的成骨分化，表现为成骨标志物mRNA表达水平降低以及矿化减少。相反，过表达CHRDL1增强了BMP - 4诱导的hBMSCs成骨分化。此外，通过将CHRDL1基因修饰的hBMSCs移植到裸鼠股骨缺损模型中进行的体内实验显示，与对照组相比，CHRDL1过表达组的新骨形成增加，而CHRDL1敲低组的新骨形成减少。与骨形成率一致的是，与对照组相比，CHRDL1过表达组股骨缺损处新骨形成区域的CHRDL1蛋白表达增加，而CHRDL1敲低组的CHRDL1蛋白表达减少。这些表明CHRDL1可在体内促进成骨细胞分化。此外，机制研究表明，在hBMSCs成骨分化过程中，CHRDL1可提高BMP - 4诱导的SMAD1/5/9磷酸化。此外，BMP受体I型抑制剂LDN - 193189可阻断CHRDL1对成骨分化的促进作用以及对SMAD磷酸化的激活作用。总之，我们的研究结果表明，CHRDL1可通过增强BMP - 4 - SMAD1/5/9通路的激活来促进hBMSCs的成骨分化。