Endoplasmic Reticulum (ER) Stress Inducible Factor Cysteine-Rich with EGF-Like Domains 2 (Creld2) Is an Important Mediator of BMP9-Regulated Osteogenic Differentiation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are multipotent progenitors that can undergo osteogenic differentiation under proper stimuli. We demonstrated that BMP9 is one of the most osteogenic BMPs. However, the molecular mechanism underlying BMP9-initiated osteogenic signaling in MSCs remains unclear. Through gene expression profiling analysis we identified several candidate mediators of BMP9 osteogenic signaling. Here, we focus on one such signaling mediator and investigate the functional role of cysteine-rich with EGF-like domains 2 (Creld2) in BMP9-initiated osteogenic signaling. Creld2 was originally identified as an ER stress-inducible factor localized in the ER-Golgi apparatus. Our genomewide expression profiling analysis indicates that Creld2 is among the top up-regulated genes in BMP9-stimulated MSCs. We confirm that Creld2 is up-regulated by BMP9 in MSCs. ChIP analysis indicates that Smad1/5/8 directly binds to the Creld2 promoter in a BMP9-dependent fashion. Exogenous expression of Creld2 in MSCs potentiates BMP9-induced early and late osteogenic markers, and matrix mineralization. Conversely, silencing Creld2 expression inhibits BMP9-induced osteogenic differentiation. In vivo stem cell implantation assay reveals that exogenous Creld2 promotes BMP9-induced ectopic bone formation and matrix mineralization, whereas silencing Creld2 expression diminishes BMP9-induced bone formation and matrix mineralization. We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation. Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.

间充质干细胞（Mesenchymal stem cells, MSCs）是一类多能祖细胞，可在适宜刺激下发生成骨分化。本研究团队此前证实，骨形态发生蛋白9（Bone Morphogenetic Protein 9, BMP9）是骨形态发生蛋白（Bone Morphogenetic Protein, BMP）家族中最具成骨活性的成员之一。然而，BMP9启动的间充质干细胞成骨信号通路背后的分子机制仍未明确。通过基因表达谱分析（gene expression profiling），我们筛选得到数个BMP9成骨信号通路的候选介导因子。本研究聚焦于其中一类信号介导因子，深入探究富含半胱氨酸且含EGF样结构域2（cysteine-rich with EGF-like domains 2, Creld2）在BMP9启动的成骨信号通路中的功能角色。Creld2最初被鉴定为一种内质网应激（ER stress）诱导因子，定位于内质网-高尔基体（ER-Golgi apparatus）。我们的全基因组表达谱分析（genomewide expression profiling）结果显示，Creld2是BMP9刺激的间充质干细胞中上调幅度最为显著的基因之一。我们进一步验证了BMP9可诱导间充质干细胞中Creld2的表达上调。染色质免疫沉淀（Chromatin Immunoprecipitation, ChIP）分析表明，Smad1/5/8能够以BMP9依赖性方式直接结合至Creld2的启动子区域。在间充质干细胞中外源过表达Creld2，可增强BMP9诱导的早期及晚期成骨标志物的表达，并促进基质矿化过程。反之，沉默Creld2的表达则会抑制BMP9诱导的成骨分化。体内干细胞植入实验结果显示，外源过表达Creld2可促进BMP9诱导的异位骨形成与基质矿化，而沉默Creld2的表达则会削弱BMP9诱导的骨形成及基质矿化进程。我们进一步证实，Creld2定位于内质网，且内质网应激诱导剂可增强BMP9诱导的成骨分化。本研究结果强烈提示，Creld2可能直接受BMP9调控，且内质网应激应答或许在成骨分化的调控中发挥关键作用。