During craniofacial development, cranial neural crest (CNC) cells migrate into the developing face and form bone through intramembranous ossification. Loss of JAGGED1 (JAG1) signaling in the CNC cells is associated with maxillary hypoplasia or maxillary bone deficiency (MBD) in mice and recapitulates the MBD seen in humans with Alagille syndrome. JAGGED1, a membrane-bound NOTCH ligand, is required for normal craniofacial development, and Jagged1 mutations in humans are known to cause Alagille Syndrome, which is associated with cardiac, biliary, and bone phenotypes and these children experience increased bony fractures. Previously, we demonstrated deficient maxillary osteogenesis in Wnt1-cre;Jagged1f/f (Jag1CKO) mice by conditional deletion of Jagged1 in maxillary CNC cells. In this study, we investigated the JAG1 signaling pathways in a CNC cell line. Treatment with JAG1 induced osteoblast differentiation and maturation markers, Runx2 and Ocn, respectively, Alkaline Phosphatase (ALP) production, as well as classic NOTCH1 targets, Hes1 and Hey1. While JAG1-induced Hes1 and Hey1 expression levels were predictably decreased after DAPT (NOTCH inhibitor) treatment, JAG1-induced Runx2 and Ocn levels were surprisingly constant in the presence of DAPT, indicating that JAG1 effects in the CNC cells are independent of the canonical NOTCH pathway. JAG1 treatment of CNC cells increased Janus Kinase 2 (JAK2) phosphorylation, which was refractory to DAPT treatment, highlighting the importance of the non-canonical NOTCH pathway during CNC cells osteoblast commitment. Pharmacologic inhibition of JAK2 phosphorylation, with and without DAPT treatment, upon JAG1 induction reduced ALP production and, Runx2 and Ocn gene expression. Collectively, these data suggest that JAK2 is an essential component downstream of a non-canonical JAG1-NOTCH1 pathway through which JAG1 stimulates expression of osteoblast-specific gene targets in CNC cells that contribute to osteoblast differentiation and bone mineralization.

中文翻译：

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JAK2的非经典JAGGED1信号介导颅神经c细胞中的成骨细胞定型。

在颅面发育过程中，颅神经rest（CNC）细胞迁移到发育中的面部并通过膜内骨化形成骨骼。CNC细胞中JAGGED1（JAG1）信号的缺失与小鼠上颌骨发育不全或上颌骨缺乏症（MBD）有关，并概括了Alagille综合征患者所见的MBD。JAGGED1是膜结合的NOTCH配体，是正常颅面发育所必需的，已知人类中的Jagged1突变会导致Alagille综合征，该综合征与心脏，胆道和骨骼的表型有关，这些儿童的骨骨折增加。以前，我们通过有条件地删除上颌CNC细胞中的Jagged1来证明Wnt1-cre; Jagged1f / f（Jag1CKO）小鼠的上颌骨成骨不足。在这项研究中，我们研究了CNC细胞系中JAG1信号通路。用JAG1处理分别诱导成骨细胞分化和成熟标志物Runx2和Ocn，碱性磷酸酶（ALP）产生以及经典的NOTCH1靶标Hes1和Hey1。虽然在DAPT（NOTCH抑制剂）治疗后，JAG1诱导的Hes1和Hey1表达水平可预测地降低，但是在存在DAPT的情况下，JAG1诱导的Runx2和Ocn水平出乎意料地恒定，这表明CNC细胞中JAG1的作用独立于典型的NOTCH途径。JAG1处理CNC细胞增加了Janus Kinase 2（JAK2）磷酸化，这对DAPT治疗是难治的，突显了非经典NOTCH途径在CNC细胞成骨细胞定植过程中的重要性。JAK2磷酸化的药理抑制作用 不论是否使用DAPT，JAG1诱导后均会降低ALP的产生以及Runx2和Ocn基因的表达。总体而言，这些数据表明，JAK2是非规范JAG1-NOTCH1途径下游的重要组成部分，通过该途径，JAG1刺激CNC细胞中成骨细胞特异性基因靶标的表达，从而促进成骨细胞分化和骨矿化。