State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, China.
Department of Cariology and Endodontics, School of Stomatology, Wuhan University, Wuhan, China.
Int J Oral Sci. 2024 Oct 10;16(1):59. doi: 10.1038/s41368-024-00322-y.
Odontoblasts are primarily responsible for synthesizing and secreting extracellular matrix proteins, which are crucial for dentinogenesis. Our previous single-cell profile and RNAscope for odontoblast lineage revealed that cyclic adenosine monophosphate responsive element-binding protein 3 like 1 (Creb3l1) was specifically enriched in the terminal differentiated odontoblasts. In this study, deletion of Creb3l1 in the Wnt1+ lineage led to insufficient root elongation and dentin deposition. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing were performed to revealed that in CREB3L1-deficient mouse dental papilla cells (mDPCs), the genes near the closed chromatin regions were mainly associated with mesenchymal development and the downregulated genes were primarily related to biological processes including cell differentiation, protein biosynthesis and transport, all of which were evidenced by a diminished ability of odontoblastic differentiation, a significant reduction in intracellular proteins, and an even greater decline in extracellular supernatant proteins. Dentin matrix protein 1 (Dmp1), dentin sialophosphoprotein (Dspp), and transmembrane protein 30B (Tmem30b) were identified as direct transcriptional regulatory targets. TMEM30B was intensively expressed in the differentiated odontoblasts, and exhibited a significant decline in both CREB3L1-deficient odontoblasts in vivo and in vitro. Deletion of Tmem30b impaired the ability of odontoblastic differentiation, protein synthesis, and protein secretion in mDPCs. Moreover, overexpressing TMEM30B in CREB3L1-deficient mDPCs partially rescued the extracellular proteins secretion. Collectively, our findings suggest that CREB3L1 participates in dentinogenesis and facilitates odontoblastic differentiation by directly enhancing the transcription of Dmp1, Dspp, and other differentiation-related genes and indirectly promoting protein secretion partially via TMEM30B.
成牙本质细胞主要负责合成和分泌细胞外基质蛋白，这些蛋白对牙本质的形成至关重要。我们之前的单细胞图谱和 RNAscope 研究牙本质细胞谱系表明，环腺苷酸反应元件结合蛋白 3 样 1（Creb3l1）在终末分化的成牙本质细胞中特异性富集。在这项研究中，Wnt1+谱系中 Creb3l1 的缺失导致根伸长和牙本质沉积不足。进行了转座酶可及染色质的高通量测序（ATAC-seq）和 RNA 测序分析，结果表明在 CREB3L1 缺陷型小鼠牙乳头细胞（mDPC）中，靠近封闭染色质区域的基因主要与间充质发育有关，下调的基因主要与包括细胞分化、蛋白质生物合成和运输在内的生物学过程有关，这些都表现为成牙本质细胞分化能力减弱、细胞内蛋白质含量显著减少以及细胞外上清液蛋白质含量下降更为明显。牙本质基质蛋白 1（Dmp1）、牙本质涎磷蛋白（Dspp）和跨膜蛋白 30B（Tmem30b）被鉴定为直接转录调控靶标。TMEM30B 在分化的成牙本质细胞中强烈表达，在体内和体外 CREB3L1 缺陷型成牙本质细胞中均显著下降。Tmem30b 的缺失损害了 mDPC 中成牙本质细胞的分化、蛋白质合成和蛋白质分泌能力。此外，在 CREB3L1 缺陷型 mDPC 中过表达 TMEM30B 部分挽救了细胞外蛋白的分泌。总之，我们的研究结果表明，CREB3L1 通过直接增强 Dmp1、Dspp 和其他分化相关基因的转录以及间接部分通过 TMEM30B 促进蛋白分泌，参与牙本质的形成并促进成牙本质细胞的分化。
