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.

成牙细胞主要负责合成和分泌细胞外基质蛋白，这对牙本质生成至关重要。我们之前针对成牙细胞谱系的单细胞谱和 RNAscope 显示，环磷酸腺苷反应元件结合蛋白 3 like 1 （Creb3l1） 在末端分化的成牙细胞中特异性富集。在本研究中，Wnt1 + 谱系中 Creb3l1 的缺失导致牙根伸长和牙本质沉积不足。通过高通量测序 （ATAC-seq） 和 RNA 测序进行转座酶可及染色质检测，发现在 CREB3L1 缺陷小鼠牙细胞 （mDPC） 中，闭合染色质区域附近的基因主要与间充质发育相关，下调基因主要与细胞分化、蛋白质生物合成和转运等生物过程有关，所有这些都表现为成牙细胞分化，细胞内蛋白显着减少，细胞外上清液蛋白下降幅度更大。牙本质基质蛋白 1 （Dmp1） 、牙本质唾液磷蛋白 （Dspp） 和跨膜蛋白 30B （Tmem30b） 被确定为直接转录调控靶点。TMEM30B 在分化的成牙细胞中密集表达，并且在体内和体外CREB3L1缺陷成牙细胞中均表现出显著下降。Tmem30b 的缺失损害了 mDPCs 中成牙细胞分化、蛋白质合成和蛋白质分泌的能力。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。 总的来说，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并部分通过 TMEM30B间接促进蛋白质分泌，参与牙本质生成并促进齿母细胞分化。