10.6084/M9.FIGSHARE.5918548
Qi Wu
Qi
Wu
Jing Li
Jing
Li
Pingping Song
Pingping
Song
Jing Chen
Jing
Chen
Yuanzhi Xu
Yuanzhi
Xu
Shengcai Qi
Shengcai
Qi
Ji Ma
Ji
Ma
Qiuhui Pan
Qiuhui
Pan
Knockdown of NRAGE induces odontogenic differentiation by activating NF-κB signaling in mouse odontoblast-like cells
<p><b>Purpose</b>: Neurotrophin receptor-interacting MAGE homologue (Nrage) plays an important role in bone development and the metabolism of normal skeletal structures. Our previous study showed that Nrage inhibited the odontogenic differentiation of mouse dental pulp cells. However, the potential roles and mechanism of Nrage in regulating odontogenic differentiation are unknown. The aim of this study was to investigate the molecular mechanism of Nrage in odontogenic differentiation of mouse odontoblast-like cells.</p> <p><b>Materials and methods</b>: Endogenous expression of Nrage was stably downregulated by lentivirus-mediated shRNA. Mineralized nodules formation was detected by alizarin red S staining. Dmp-1, Dspp, and ALP mRNA and protein levels were detected by qRT-PCR and western blotting, respectively. In addition, ALPase activity was detected. Confocal microscopy and co-immunoprecipitation (co-IP) were used to analyze the interactions between NRAGE and NF-κB signaling molecules. An IKK inhibitor was also used in the study.</p> <p><b>Results</b>: NRAGE expression in odontoblasts was downregulated during mouse first maxillary molar development. Moreover, NRAGE expression was downregulated during odontogenic differentiation of odontoblast-like cells. NRAGE knockdown significantly upregulated DMP1 and DSP expression, increased ALPase activity, and promoted mineralized nodule formation. In addition, NRAGE knockdown increased the translocation of NF-κB1 to the nucleus and phosphorylation levels of p65. Co-IP results showed that NRAGE bound to IKKβ. Most importantly, the promoting effect of Nrage knockdown on odontoblastic differentiation was reduced after treatment with an IKK inhibitor.</p> <p><b>Conclusions</b>: Our data confirmed that NRAGE is an important regulator of odontogenic differentiation of odontoblasts by inhibiting the NF-κB signaling pathway through binding to IKKβ.</p> <p><b>Abbreviations</b>: Nrage: neurotrophin receptor-interacting MAGE homologue; DSP: dentin sialophospho protein; DMP-1: dentin matrix protein-1; BMP: bone morphogenetic protein; Wnt: wingless; NF-κB: nuclear factor of activated B cells; DAPI: 4′,6-diamidino-2-phenylindole; KO: knockout; DPCs: dental pulp cells; AA: ascorbic acid; β-Gly: β-glycerophosphate; Dex: dexamethasone; co-IP: co-immunoprecipitation; IκB: inhibitor of NF-κB; IKK: IκB kinase</p>
Biochemistry
Cell Biology
Genetics
Molecular Biology
Physiology
Immunology
Developmental Biology
Cancer
Hematology
Taylor & Francis
2018
2018-02-23
2023-06-02
Journal contribution
748544 Bytes
10.1080/03008207.2018.1439484
CC BY 4.0