Kamalakar Archana, McKinney Jay M, Salinas Duron Daniel, Amanso Angelica M, Ballestas Samir A, Drissi Hicham, Willett Nick J, Bhattaram Pallavi, García Andrés J, Wood Levi B, Goudy Steven L
Department of Otolaryngology, USA.
Wallace H. Coulter Department of Biomedical Engineering, USA; George W. Woodruff School of Mechanical Engineering, Georgia Tech College of Engineering, Atlanta, GA, USA; The Atlanta Veterans Affairs Medical Center Atlanta, GA, USA.
Bone. 2021 Feb;143:115657. doi: 10.1016/j.bone.2020.115657. Epub 2020 Sep 25.
Craniofacial bone loss is a complex clinical problem with limited regenerative solutions. Currently, BMP2 is used as a bone-regenerative therapy in adults, but in pediatric cases of bone loss, it is not FDA-approved due to concerns of life-threatening inflammation and cancer. Development of a bone-regenerative therapy for children will transform our ability to reduce the morbidity associated with current autologous bone grafting techniques. We discovered that JAGGED1 (JAG1) induces cranial neural crest (CNC) cell osteoblast commitment during craniofacial intramembranous ossification, suggesting that exogenous JAG1 delivery is a potential craniofacial bone-regenerative approach. In this study, we found that JAG1 delivery using synthetic hydrogels containing O9-1 cells, a CNC cell line, into critical-sized calvarial defects in C57BL/6 mice provided robust bone-regeneration. Since JAG1 signals through canonical (Hes1/Hey1) and non-canonical (JAK2) NOTCH pathways in CNC cells, we used RNAseq to analyze transcriptional pathways activated in CNC cells treated with JAG1 ± DAPT, a NOTCH-canonical pathway inhibitor. JAG1 upregulated expression of multiple NOTCH canonical pathway genes (Hes1), which were downregulated in the presence of DAPT. JAG1 also induced bone chemokines (Cxcl1), regulators of cytoskeletal organization and cell migration (Rhou), signaling targets (STAT5), promoters of early osteoblast cell proliferation (Prl2c2, Smurf1 and Esrra), and, inhibitors of osteoclasts (Id1). In the presence of DAPT, expression levels of Hes1 and Cxcl1 were decreased, whereas, Prl2c2, Smurf1, Esrra, Rhou and Id1 remain elevated, suggesting that JAG1 induces osteoblast proliferation through these non-canonical genes. Pathway analysis of JAG1 + DAPT-treated CNC cells revealed significant upregulation of multiple non-canonical pathways, including the cell cycle, tubulin pathway, regulators of Runx2 initiation and phosphorylation of STAT5 pathway. In total, our data show that JAG1 upregulates multiple pathways involved in osteogenesis, independent of the NOTCH canonical pathway. Moreover, our findings suggest that JAG1 delivery using a synthetic hydrogel, is a bone-regenerative approach with powerful translational potential.
颅面骨丢失是一个复杂的临床问题，再生解决方案有限。目前，骨形态发生蛋白2（BMP2）在成人中用作骨再生疗法，但在儿童骨丢失病例中，由于担心危及生命的炎症和癌症，它未获得美国食品药品监督管理局（FDA）的批准。开发一种针对儿童的骨再生疗法将改变我们降低当前自体骨移植技术相关发病率的能力。我们发现，在颅面膜内成骨过程中，锯齿状蛋白1（JAG1）可诱导颅神经嵴（CNC）细胞向成骨细胞分化，这表明外源性递送JAG1是一种潜在的颅面骨再生方法。在本研究中，我们发现，将含有O9 - 1细胞（一种CNC细胞系）的合成水凝胶递送至C57BL/6小鼠的临界尺寸颅骨缺损处，JAG1可实现强大的骨再生。由于JAG1在CNC细胞中通过经典（Hes1/Hey1）和非经典（JAK2）NOTCH信号通路发挥作用，我们使用RNA测序来分析在用JAG1 ± DAPT（一种NOTCH经典信号通路抑制剂）处理的CNC细胞中激活的转录信号通路。JAG1上调了多个NOTCH经典信号通路基因（Hes1）的表达，而在存在DAPT的情况下这些基因的表达下调。JAG1还诱导了骨趋化因子（Cxcl1）、细胞骨架组织和细胞迁移调节剂（Rhou）、信号靶点（STAT5）、早期成骨细胞增殖促进因子（Prl2c2、Smurf1和Esrra）以及破骨细胞抑制剂（Id1）。在存在DAPT的情况下，Hes1和Cxcl1的表达水平降低，而Prl2c2、Smurf1、Esrra、Rhou和Id1的表达仍保持升高，这表明JAG1通过这些非经典基因诱导成骨细胞增殖。对用JAG1 + DAPT处理的CNC细胞进行信号通路分析发现，多个非经典信号通路显著上调，包括细胞周期、微管蛋白信号通路、Runx2起始调节因子和STAT5信号通路的磷酸化。总体而言，我们的数据表明，JAG1上调了多个参与骨生成的信号通路，且不依赖于NOTCH经典信号通路。此外，我们的研究结果表明，使用合成水凝胶递送JAG1是一种具有强大转化潜力的骨再生方法。
