Fang JingXian, Yamaza Haruyoshi, Uchiumi Takeshi, Hoshino Yoshihiro, Masuda Keiji, Hirofuji Yuta, Wagener Frank A D T G, Kang Dongchon, Nonaka Kazuaki
Department of Pediatric Dentistry, Graduate School of Dental Science, Kyushu University, Fukuoka, Japan.
Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Eur J Oral Sci. 2016 Jun;124(3):241-5. doi: 10.1111/eos.12270. Epub 2016 Apr 17.
Mutation of the dihydroorotate dehydrogenase (DHODH) gene is responsible for Miller syndrome, which is characterized by craniofacial malformations with limb abnormalities. We previously demonstrated that DHODH was involved in forming a mitochondrial supercomplex and that mutated DHODH led to protein instability, loss of enzyme activity, and increased levels of reactive oxygen species in HeLa cells. To explore the etiology of Miller syndrome in more detail, we investigated the effects of DHODH inhibition in the cells involved in skeletal structure. Dihydroorotate dehydrogenase in MC3T3-E1 cells derived from mouse calvaria osteoblast precursor cells was knocked down by specific small interfering RNAs (siRNAs), and cell proliferation, ATP production, and expression of bone-related genes were investigated in these cells. After depletion of DHODH using specific siRNAs, inhibition of cell proliferation and cell cycle arrest occurred in MC3T3-E1 cells. In addition, ATP production was reduced in whole cells, especially in mitochondria. Furthermore, the levels of runt-related transcription factor 2 (Runx2) and osteocalcin (Ocn) mRNAs were lower in DHODH siRNA-treated cells compared with controls. These data suggest that depletion of DHODH affects the differentiation and maturation of osteoblasts. This study shows that mitochondrial dysfunction by DHODH depletion in osteoblasts can be directly linked to the abnormal bone formation in Miller syndrome.
二氢乳清酸脱氢酶（DHODH）基因突变是米勒综合征的病因，其特征为颅面畸形合并肢体异常。我们之前证明，DHODH参与形成线粒体超复合物，且突变的DHODH会导致蛋白质不稳定、酶活性丧失以及HeLa细胞中活性氧水平升高。为了更详细地探究米勒综合征的病因，我们研究了抑制DHODH对参与骨骼结构的细胞的影响。利用特异性小干扰RNA（siRNAs）敲低源自小鼠颅盖成骨细胞前体细胞的MC3T3-E1细胞中的二氢乳清酸脱氢酶，并研究这些细胞中的细胞增殖、ATP生成以及骨相关基因的表达。使用特异性siRNAs耗尽DHODH后，MC3T3-E1细胞中出现细胞增殖抑制和细胞周期停滞。此外，全细胞尤其是线粒体中的ATP生成减少。此外，与对照组相比，经DHODH siRNA处理的细胞中与矮小相关转录因子2（Runx2）和骨钙素（Ocn）mRNA的水平较低。这些数据表明，耗尽DHODH会影响成骨细胞的分化和成熟。本研究表明，成骨细胞中DHODH耗尽导致的线粒体功能障碍可能与米勒综合征中异常的骨形成直接相关。
