Lin J X, Niu J X, Fu J, Feng H, Liu Y, Yuan G H, Chen Z
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, Wuhan430079, China.
Zhonghua Kou Qiang Yi Xue Za Zhi. 2025 Jan 9;60(1):33-42. doi: 10.3760/cma.j.cn112144-20240916-00349.
To investigate the role of WW domain containing E3 ubiquitin protein ligase 1 (WWP1) in enamel development of mice. Single-cell RNA sequencing data of incisor tissues of postnatal day 7 (P7) mice and mandibular first molar tooth germs of P3.5 mice were used to analyze the expression of Wwp1 in dental epithelial cells. Immunohistochemistry was performed to observe the distribution and expression levels of WWP1 in the epithelium of mouse incisors and mandibular first molar tooth germs. Wwp1 knockout (Wwp1 KO) mice were generated and collected with their control littermates at P1, P7, three mice per group, as well as at P14, P28, 2 months (2M), and 3M, six mice per group. The enamel volumes of molars and incisors were analyzed using micro-CT. Scanning electron microscopy was employed to examine the enamel cross-sections of Wwp1 KO and control mice. Energy dispersive spectroscopy (EDS) was used to analyze the calcium and phosphorus content of the enamel rod of incisors. Immunofluorescence was performed to detect the expression of amelogenin (AMELX) in the ameloblasts of Wwp1 KO and control mice. Additionally, LS-8 ameloblast-like epithelial cells were cultured, and Wwp1 siRNA or overexpression plasmids were transfected to knock down or overexpress WWP1. The protein levels of AMELX were then assessed by Western blotting. Single-cell sequencing result showed a high Wwp1 mRNA expression level in the epithelial cells of mouse incisors and mandibular molar tooth germs. Immunohistochemistry revealed the expression of WWP1 in presecretory, secretory, transitional, and mature ameloblasts. Wwp1 KO mice exhibited enamel developmental defects. The enamel volumes of molars and incisors in Wwp1 KO mice [(0.155±0.016), (0.300±0.017) μm] were reduced by 23.95% (<0.001) and 28.31% (<0.001) compared with the control group [(0.203±0.062), (0.418±0.023) μm] respectively. Scanning electron microscopy showed disorganized enamel structures in Wwp1 KO incisors and molars. EDS results showed the weight percent of calcium in the enamel rod of incisors decreased in Wwp1 KO mice [(20.74±0.91)%] compared with the control group [(30.30±3.83)%] (<0.001), and the calcium-to-phosphorus ratio decreased in Wwp1 KO mice (1.93±0.01) compared with the control group (2.02±0.01) (<0.001). Immunofluorescence showed weaker AMELX expression in ameloblasts of mandibular first molar tooth germs from P1 and P7 Wwp1 KO mice compared with the control group (<0.001, <0.001). In LS-8 cells, Wwp1 knocked-down led to a decrease of AMELX protein expression, while WWP1 overexpression resulted in an increased AMELX protein level. WWP1 promotes ameloblast differentiation and enamel matrix mineralization, playing a critical role in enamel formation.
研究含WW结构域的E3泛素蛋白连接酶1（WWP1）在小鼠牙釉质发育中的作用。利用出生后第7天（P7）小鼠切牙组织和P3.5小鼠下颌第一磨牙牙胚的单细胞RNA测序数据，分析Wwp1在牙上皮细胞中的表达。进行免疫组织化学观察WWP1在小鼠切牙和下颌第一磨牙牙胚上皮中的分布及表达水平。构建Wwp1基因敲除（Wwp1 KO）小鼠，并在P1、P7时收集其与对照同窝小鼠，每组3只；以及在P14、P28、2个月（2M）和3个月（3M）时收集，每组6只。使用显微CT分析磨牙和切牙的牙釉质体积。采用扫描电子显微镜观察Wwp1 KO小鼠和对照小鼠的牙釉质横截面。利用能量色散光谱（EDS）分析切牙牙釉质棒的钙和磷含量。进行免疫荧光检测Wwp1 KO小鼠和对照小鼠成釉细胞中釉原蛋白（AMELX）的表达。此外，培养LS-8成釉细胞样上皮细胞，转染Wwp1小干扰RNA（siRNA）或过表达质粒以敲低或过表达WWP1。然后通过蛋白质印迹法评估AMELX的蛋白水平。单细胞测序结果显示，Wwp1 mRNA在小鼠切牙和下颌磨牙牙胚的上皮细胞中表达水平较高。免疫组织化学显示WWP1在前分泌、分泌、过渡和成熟成釉细胞中均有表达。Wwp1 KO小鼠表现出牙釉质发育缺陷。与对照组[（0.203±0.062），（0.418±0.023）μm]相比，Wwp1 KO小鼠磨牙和切牙的牙釉质体积[（0.155±0.016），（0.300±0.017）μm]分别减少了23.95%（<0.001）和28.31%（<0.001）。扫描电子显微镜显示Wwp1 KO小鼠切牙和磨牙的牙釉质结构紊乱。EDS结果显示，与对照组[（30.30±3.83）%]相比，Wwp1 KO小鼠切牙牙釉质棒中钙的重量百分比降低[（20.74±0.91）%]（<0.001），且Wwp1 KO小鼠的钙磷比（1.93±0.01）低于对照组（2.02±0.01）（<0.001）。免疫荧光显示，与对照组相比，P1和P7的Wwp1 KO小鼠下颌第一磨牙牙胚成釉细胞中AMELX的表达较弱（<0.001，<0.001）。在LS-8细胞中，敲低Wwp1导致AMELX蛋白表达降低，而过表达WWP1则导致AMELX蛋白水平升高。WWP1促进成釉细胞分化和釉质基质矿化，在牙釉质形成中起关键作用。
