Canonical Notch Signaling is Required for Bone Morphogenetic Protein-mediated Human Osteoblast Differentiation,STEM CELLS

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Canonical Notch Signaling is Required for Bone Morphogenetic Protein-mediated Human Osteoblast Differentiation
STEM CELLS ( IF 4.0 ) Pub Date : 2020-06-24 , DOI: 10.1002/stem.3245
Yadav Wagley ₁ , Alessandra Chesi _{2,

3} , Parker K Acevedo ₁ , Sumei Lu ₂ , Andrew D Wells _{2,

4} , Matthew E Johnson _{2,

3} , Struan F A Grant _{2,

3,

5,

6,

7,

8} , Kurt D Hankenson ₁

Affiliation

Osteoblast differentiation of bone marrow‐derived human mesenchymal stem cells (hMSC) can be induced by stimulation with canonical Notch ligand, Jagged1, or bone morphogenetic proteins (BMPs). However, it remains elusive how these two pathways lead to the same phenotypic outcome. Since Runx2 is regarded as a master regulator of osteoblastic differentiation, we targeted Runx2 with siRNA in hMSC. This abrogated both Jagged1 and BMP2 mediated osteoblastic differentiation, confirming the fundamental role for Runx2. However, while BMP stimulation increased Runx2 and downstream Osterix protein expression, Jagged1 treatment failed to upregulate either, suggesting that canonical Notch signals require basal Runx2 expression. To fully understand the transcriptomic profile of differentiating osteoblasts, RNA sequencing was performed in cells stimulated with BMP2 or Jagged1. There was common upregulation of ALPL and extracellular matrix genes, such as ACAN, HAS3, MCAM, and OLFML2B. Intriguingly, genes encoding components of Notch signaling (JAG1, HEY2, and HES4) were among the top 10 genes upregulated by both stimuli. Indeed, ALPL expression occurred concurrently with Notch activation and inhibiting Notch activity for up to 24 hours after BMP administration with DAPT (a gamma secretase inhibitor) completely abrogated hMSC osteoblastogenesis. Concordantly, RBPJ (recombination signal binding protein for immunoglobulin kappa J region, a critical downstream modulator of Notch signals) binding could be demonstrated within the ALPL and SP7 promoters. As such, siRNA‐mediated ablation of RBPJ decreased BMP‐mediated osteoblastogenesis. Finally, systemic Notch inhibition using diabenzazepine (DBZ) reduced BMP2‐induced calvarial bone healing in mice supporting the critical regulatory role of Notch signaling in BMP‐induced osteoblastogenesis.

中文翻译：

骨形态发生蛋白介导的人类成骨细胞分化需要经典 Notch 信号传导

骨髓来源的人类间充质干细胞 (hMSC) 的成骨细胞分化可以通过经典 Notch 配体、Jagged1 或骨形态发生蛋白 (BMP) 的刺激来诱导。然而，这两种途径如何导致相同的表型结果仍然难以捉摸。由于 Runx2 被认为是成骨细胞分化的主要调节因子，我们在 hMSC 中用 siRNA 靶向 Runx2。这废除了 Jagged1 和 BMP2 介导的成骨细胞分化，证实了 Runx2 的基本作用。然而，虽然 BMP 刺激增加了 Runx2 和下游 Osterix 蛋白的表达，但 Jagged1 治疗也未能上调，这表明规范的 Notch 信号需要基础 Runx2 表达。为了充分了解分化成骨细胞的转录组学特征，在用 BMP2 或 Jagged1 刺激的细胞中进行 RNA 测序。ALPL 和细胞外基质基因（如 ACAN、HAS3、MCAM 和 OLFML2B）普遍上调。有趣的是，编码 Notch 信号（JAG1、HEY2 和 HES4）成分的基因是被这两种刺激上调的前 10 个基因之一。事实上，ALPL 表达与 Notch 激活同时发生，并在 BMP 与 DAPT（一种 γ 分泌酶抑制剂）一起给药后抑制 Notch 活性长达 24 小时，完全消除了 hMSC 成骨细胞生成。一致地，可以在 ALPL 和 SP7 启动子内证明 RBPJ（免疫球蛋白 kappa J 区的重组信号结合蛋白，Notch 信号的关键下游调节剂）结合。因此，siRNA 介导的 RBPJ 消融降低了 BMP 介导的成骨细胞生成。最后，

更新日期：2020-06-24

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