von Willebrand 因子 D 和 EGF 结构域调节成釉细胞分化和牙釉质形成,Journal of Cellular Physiology

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von Willebrand 因子 D 和 EGF 结构域调节成釉细胞分化和牙釉质形成
Journal of Cellular Physiology ( IF 4.5 ) Pub Date : 2021-12-26 , DOI: 10.1002/jcp.30667
Kokoro Iwata ₁ , Keita Kawarabayashi ₂ , Keigo Yoshizaki ₃ , Tian Tian ₃ , Kan Saito ₄ , Asuna Sugimoto ₁ , Rika Kurogoushi ₁ , Aya Yamada ₄ , Akihito Yamamoto ₅ , Yasuei Kudo ₆ , Naozumi Ishimaru ₇ , Satoshi Fukumoto _{4,

8} , Tsutomu Iwamoto ₁

Affiliation

Department of Pediatric Dentistry/Special Needs Dentistry, Division of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
Department of Pediatric Dentistry, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan.
Orthodontics and Dentofacial Orthopedics Section, Division of Oral Health, Growth and Development, Kyushu University Faculty of Dental Science, Fukuoka, Japan.
Department of Oral Health and Development Sciences, Pediatric Dentistry Division, Tohoku University Graduate School of Dentistry, Sendai, Japan.
Department of Tissue Regeneration, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan.
Department of Oral Bioscience, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan.
Department of Oral Molecular Pathology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan.
Pediatric Dentistry Section, Division of Oral Health, Growth and Development, Kyushu University Faculty of Dental Science, Fukuoka, Japan.

细胞和组织特异性细胞外基质 (ECM) 成分通过调节细胞行为（例如细胞增殖和分化）在器官发育（包括牙齿）中发挥重要作用。在这里，我们首次证明冯维勒布兰德因子 D 和表皮生长因子 (EGF) 结构域 (Vwde)，一种以前未表征的 ECM 蛋白，在牙齿中特异性表达并调节内釉上皮细胞 (IEE) 中的细胞增殖和分化和牙釉质的形成。我们使用 NCBI 表达的小鼠序列标签数据库通过生物信息学将 Vwde 鉴定为一种新型 ECM 蛋白。Vwde 互补 DNA 编码 1773 个氨基酸，包含一个信号肽、一个 von Willebrand 因子 D 型结构域和串联的钙结合 EGF 样结构域。实时聚合酶链反应表明Vwde在牙齿组织中高度表达，但在其他组织中不表达，包括脑、肺、心脏、肝、肾和骨骼。原位杂交显示 IEE 在发育中的牙齿中表达Vwde信使 RNA。免疫染色显示 VWDE 位于 IEE 细胞周围区域的近端和远端。大众汽车在小鼠牙上皮衍生的 M3H1 细胞分化过程中被诱导。Vwde转染的M3H1细胞将VWDE蛋白分泌到培养基中并抑制细胞增殖，而促进成釉细胞分化。此外，Vwde 增加了细胞外信号调节激酶 1/2 和蛋白激酶 B 的磷酸化，并强烈诱导细胞间连接蛋白 N-钙粘蛋白 (Ncad) 的表达。有趣的是，内源性 Vwde 的抑制抑制了 Ncad 的表达。最后，我们使用 CRISPR-Cas9 系统创建了 Vwde 基因敲除小鼠。Vwde-null 小鼠显示出低矿物质密度、粗糙表面和牙釉质裂缝，表明牙釉质发育不全表型。

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von Willebrand factor D and EGF domains regulate ameloblast differentiation and enamel formation

Cell- and tissue-specific extracellular matrix (ECM) composition plays an important role in organ development, including teeth, by regulating cell behaviors, such as cell proliferation and differentiation. Here, we demonstrate for the first time that von Willebrand factor D and epidermal growth factor (EGF) domains (Vwde), a previously uncharacterized ECM protein, is specifically expressed in teeth and regulates cell proliferation and differentiation in inner enamel epithelial cells (IEEs) and enamel formation. We identified the Vwde as a novel ECM protein through bioinformatics using the NCBI expressed sequence tag database for mice. Vwde complementary DNA encodes 1773 amino acids containing a signal peptide, a von Willebrand factor type D domain, and tandem calcium-binding EGF-like domains. Real-time polymerase chain reaction demonstrated that Vwde is highly expressed in tooth tissue but not in other tissues including the brain, lung, heart, liver, kidney, and bone. In situ hybridization revealed that the IEEs expressed Vwde messenger RNA in developing teeth. Immunostaining showed that VWDE was localized at the proximal and the distal ends of the pericellular regions of the IEEs. Vwde was induced during the differentiation of mouse dental epithelium-derived M3H1 cells. Vwde-transfected M3H1 cells secreted VWDE protein into the culture medium and inhibited cell proliferation, whereas ameloblastic differentiation was promoted. Furthermore, Vwde increased the phosphorylation of extracellular signal-regulated kinase 1/2 and protein kinase B and strongly induced the expression of the intercellular junction protein, N-cadherin (Ncad). Interestingly, the suppression of endogenous Vwde inhibited the expression of Ncad. Finally, we created Vwde-knockout mice using the CRISPR-Cas9 system. Vwde-null mice showed low mineral density, rough surface, and cracks in the enamel, indicating the enamel hypoplasia phenotype. Our findings suggest that Vwde assembling the matrix underneath the IEEs is essential for Ncad expression and enamel formation.

更新日期：2021-12-26

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