内质网应激反应通过 PERK-eIF2α-ATF4 通路介导，参与人牙髓细胞的成牙本质分化。
Endoplasmic reticulum stress response mediated by the PERK-eIF2α-ATF4 pathway is involved in odontoblastic differentiation of human dental pulp cells.
机构信息
Department of General Dentistry, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; College of Stomatology, Shanghai Jiao Tong University, Shanghai, China; National Center for Stomatology, National Clinical Research Center for Oral Diseases, Shanghai Key Laboratory of Stomatology, Shanghai, China.
Department of Stomatology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
出版信息
Arch Oral Biol. 2022 Jan;133:105312. doi: 10.1016/j.archoralbio.2021.105312. Epub 2021 Nov 16.
OBJECTIVE
RNA-activated protein kinase-like ER-resident kinase (PERK) was a major transducer of Endoplasmic reticulum (ER) stress response and it directly phosphorylated α-subunit of eukaryotic initiation factor 2 (eIF2α), which specifically promoted the translation of activating transcription factor 4 (ATF4), an important transcription factor in cells' differentiation. The purpose of this study was to establish whether ER stress mediated by PERK-eIF2α-ATF4 pathway was involved in odontoblastic differentiation of human dental pulp cells (DPCs).
METHODS
DPCs were isolated from extracted teeth and cultured in odontogenic medium. A recombinant lentiviral vector was constructed to transfect DPCs for PERK knockdown. Alkaline phosphatase (ALP) and Alizarin red S staining were used to characterize the odontoblastic differentiation. Real-time polymerase chain reactions (RT-PCR) were performed to analyze the genes' expressions in DPCs' odontoblastic differentiation. The mRNA and protein levels of ER stress markers were examined by RT-PCR and western blot.
RESULTS
DPCs cultured in odontogenic media showed increased ALP activity and mineralized nodule formation. Notably, treatment with differentiation medium resulted in the up-regulation of genes, such as osteocalcin (OCN), bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP), splicing x-box binding protein-1 (sXBP1), ATF4 and glucose-regulated protein 78 (GRP78). Meanwhile, the expressions of PERK-eIF2α-ATF4 pathway proteins, phosphorylated PERK, phosphorylated eIF2α and ATF4, increased in odontoblastic induction cells compared with controls. Furthermore, inhibition of PERK (PERK knockdown) decreased ALP activity and matrix mineralization in DPCs accompanied by the decrease expression of phosphorylated eIF2α and ATF4.
CONCLUSION
These results suggested that PERK-eIF2α-ATF4 pathway was involved in the odontoblastic differentiation of DPCs.
目的
RNA 激活蛋白激酶样内质网驻留激酶（PERK）是内质网（ER）应激反应的主要传感器，它直接磷酸化真核起始因子 2（eIF2α）的α亚基，特异性促进激活转录因子 4（ATF4）的翻译，ATF4 是细胞分化中的重要转录因子。本研究旨在确定 PERK-eIF2α-ATF4 通路介导的 ER 应激是否参与人牙髓细胞（DPC）的成牙本质分化。
方法
从提取的牙齿中分离 DPCs 并在成牙本质培养基中培养。构建重组慢病毒载体转染 DPCs 以敲低 PERK。碱性磷酸酶（ALP）和茜素红 S 染色用于鉴定 DPCs 的成牙本质分化。实时聚合酶链反应（RT-PCR）用于分析 DPCs 成牙本质分化中的基因表达。通过 RT-PCR 和 Western blot 检测 ER 应激标志物的 mRNA 和蛋白水平。
结果
在成牙本质培养基中培养的 DPCs 显示 ALP 活性增加和矿化结节形成。值得注意的是，用分化培养基处理会导致骨钙素（OCN）、骨涎蛋白（BSP）、牙本质涎磷蛋白（DSPP）、剪接 X 盒结合蛋白-1（sXBP1）、ATF4 和葡萄糖调节蛋白 78（GRP78）等基因上调。同时，与对照组相比，成牙本质诱导细胞中 PERK-eIF2α-ATF4 通路蛋白、磷酸化 PERK、磷酸化 eIF2α 和 ATF4 的表达增加。此外，PERK 抑制（PERK 敲低）降低了 DPCs 的 ALP 活性和基质矿化，同时降低了磷酸化 eIF2α 和 ATF4 的表达。
结论
这些结果表明 PERK-eIF2α-ATF4 通路参与了 DPC 的成牙本质分化。
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