Department of Endodontics, School and Hospital of Stomatology, Liaoning Provincial Key Laboratory of Oral Diseases, China Medical University, 117 Nanjing North Street, Heping District, Shenyang, Liaoning, 110002, People's Republic of China.
Cell Mol Biol Lett. 2024 Nov 27;29(1):145. doi: 10.1186/s11658-024-00664-9.
Differentiating dental pulp stem cells (DPSCs) into odontoblasts is a critical process for tooth self-repair and dentine‒pulp engineering strategies in the clinic. However, the mechanism underlying the regulation of DPSC odontoblastic differentiation remains largely unknown. Here, we demonstrated that BCL-2 interacting protein 3 (BNIP3)-dependent mitophagy is associated with importin subunit beta-1 (KPNB1)-activating transcription factor 4 (ATF4), which promotes DPSC odontoblastic differentiation.
The key genes involved in DPSC odontogenic differentiation were identified via bioinformatics. Stable silencing or overexpression of BNIP3 was performed to investigate its impact on DPSC differentiation in vitro (n ≥ 3). To explore the role of BNIP3 in vivo, tooth root fragments loaded with the hydrogel-transfected DPSC complex were implanted into nude mice (n ≥ 6). Dual-luciferase reporter assays and chromatin immunoprecipitation (ChIP) polymerase chain reaction (PCR) were conducted to explore the binding site of ATF4 to the BNIP3 promoter (n ≥ 3). Mitochondrial function experiments were performed to investigate the impact of ATF4-BNIP3 on mitochondria (n ≥ 3). Immunoprecipitation (IP) mass spectrometry (MS) was used to investigate the interaction between ATF4 and its binding protein, KPNB1. Plasmids containing wild-type (WT)/mutant (MUT)-nuclear localization signal (NLS) forms of ATF4 were constructed to determine the specific amino acid residues recognized by KPNB1 and their effects on DPSC odontoblastic differentiation (n ≥ 3).
Compared with those in the control group, the levels of autophagy and mitophagy, especially BNIP3-dependent mitophagy, were greater in the DPSC odontoblastic differentiation group (P < 0.05). Genetic silencing or overexpression of BNIP3 demonstrated that BNIP3 expression was positively correlated with the transition of DPSCs into odontoblasts both in vitro and in vivo (P < 0.05). ATF4 regulates the expression of BNIP3 by directly binding to approximately -1292 to -1279 bp and approximately -1185 to -1172 bp within the BNIP3 promoter region, which is associated with mitophagy and mitochondrial reactive oxygen species (mtROS) levels (P < 0.05). Moreover, ATF4 increased mitophagy, mitochondrial function, and cell differentiation potential via BNIP3 (P < 0.05). Mechanistically, KPNB1 is a novel interacting protein of ATF4 that specifically recognizes amino acids (aa) 280-299 within ATF4 to control its translocation into the nucleus and subsequent transcription and differentiation processes (P < 0.05).
We reported that the critical role of KPNB1/ATF4/BNIP3 axis-dependent mitophagy could provide new cues for the regeneration of the dental pulp‒dentin complex in DPSCs.
牙髓干细胞（DPSCs）向成牙本质细胞分化是牙齿自我修复和临床牙本质-牙髓工程策略的关键过程。然而，DPSC 成牙本质细胞分化调控的机制在很大程度上仍然未知。在这里，我们证明了 BCL-2 相互作用蛋白 3（BNIP3）依赖性自噬与输入蛋白亚基β-1（KPNB1）激活转录因子 4（ATF4）有关，后者促进 DPSC 成牙本质细胞分化。
通过生物信息学鉴定了与 DPSC 牙向分化相关的关键基因。通过稳定沉默或过表达 BNIP3 来研究其对体外 DPSC 分化的影响（n≥3）。为了探讨 BNIP3 在体内的作用，将负载水凝胶转染的 DPSC 复合物的牙根片段植入裸鼠（n≥6）。通过双荧光素酶报告基因检测和染色质免疫沉淀聚合酶链反应（ChIP-PCR）来研究 ATF4 与 BNIP3 启动子结合的结合位点（n≥3）。通过线粒体功能实验来研究 ATF4-BNIP3 对线粒体的影响（n≥3）。免疫沉淀（IP）质谱（MS）用于研究 ATF4 与其结合蛋白 KPNB1 之间的相互作用。构建含有野生型（WT）/突变（MUT）核定位信号（NLS）形式的 ATF4 的质粒，以确定 KPNB1 识别的特定氨基酸残基及其对 DPSC 成牙本质细胞分化的影响（n≥3）。
与对照组相比，成牙本质细胞分化组的自噬和噬线粒体，特别是 BNIP3 依赖性噬线粒体水平更高（P<0.05）。BNIP3 的遗传沉默或过表达表明，BNIP3 的表达与体外和体内 DPSCs 向成牙本质细胞的转化呈正相关（P<0.05）。ATF4 通过直接结合 BNIP3 启动子区域的大约-1292 至-1279 bp 和大约-1185 至-1172 bp 来调节 BNIP3 的表达，这与噬线粒体和线粒体活性氧（mtROS）水平有关（P<0.05）。此外，ATF4 通过 BNIP3 增加噬线粒体、线粒体功能和细胞分化潜能（P<0.05）。机制上，KPNB1 是 ATF4 的一种新的相互作用蛋白，它特异性识别 ATF4 内的 280-299 个氨基酸残基，以控制其向核内的转位及其随后的转录和分化过程（P<0.05）。
我们报道了 KPNB1/ATF4/BNIP3 轴依赖性噬线粒体的关键作用可以为 DPSCs 中牙髓-牙本质复合体的再生提供新的线索。
