Senescence: novel insight into DLX3 mutations leading to enhanced bone formation in Tricho-Dento-Osseous syndrome
The homeodomain transcription factor distal-less homeobox 3 gene (DLX3) is required for hair, tooth and skeletal development. DLX3 mutations have been found to be responsible for Tricho-Dento-Osseous (TDO) syndrome, characterized by kinky hair, thin-pitted enamel and increased bone density. Here we show that the DLX3 mutation (c.533 A>G; Q178R) attenuates osteogenic potential and senescence of bone mesenchymal stem cells (BMSCs) isolated from a TDO patient, providing a molecular explanation for abnormal increased bone density. Both DLX3 mutations (c.533 A>G and c.571_574delGGGG) delayed cellular senescence when they were introduced into pre-osteoblastic cells MC3T3-E1. Furthermore, the attenuated skeletal aging and bone loss in DLX3 (Q178R) transgenic mice not only reconfirmed that DLX3 mutation (Q178R) delayed cellular senescence, but also prevented aging-mediated bone loss. Taken together, these results indicate that DLX3 mutations act as a loss of function in senescence. The delayed senescence of BMSCs leads to increased bone formation by compensating decreased osteogenic potentials with more generations and extended functional lifespan. Our findings in the rare human genetic disease unravel a novel mechanism of DLX3 involving the senescence regulation of bone formation.The 27-yr-old female patient with typical TDO features was enrolled in this study (Supplementary Fig. S2A,B and C)11. Specially, thickened cortical and abnormally increased trabecular bone density of mandibular was clearly observed in the patient’s panoramic radiograph. The cephalometric radiograph of the patient showed a bossed frontal bone, a square jaw, increased bone density and thickness in parietal and calvaria bones, with a lack of pneumatization of the mastoids. Mandibular bone specimens of the patient were collected during implant surgery (Supplementary Fig. S3A). Bone marrow derived stem cells were isolated from the specimen (named as TDO-BMSCs) and reached 80% confluence after 14 days culture (Supplementary Fig. S3B). The missense mutation of DLX3 (c.533 A>G; Q178R) was confirmed by Sanger sequencing in the blood sample as well as in the BMSCs of the patient (Supplementary Fig. S2D). Further, BMSCs isolated from three gender- and age-matched normal donors as controls (named as CON-BMSCs). The flow cytometry assay, which showed that both TDO- and CON-BMSCs were negative for the hematopoietic markers CD34 and CD45, and positive for the mesenchymal stem cell-associated markers CD73, CD90, and CD106, indicated that DLX3 mutation did not affect expression of stem cell markers (Supplementary Fig. S3C).A 26-year-old female with typical TDO features11 and gender- and age-matched normal donors (n = 3) participated in this study with informed consent. The study was approved by the Ethics Committee of Peking University School and Hospital of Stomatology. And the whole methods were carried out in accordance with the relevant guidelines, including any relevant details. BMSCs were isolated from alveolar bone of the involved patient (TDO-BMSCs) and healthy donors (CON-BMSCs) during implantation surgery as described below. Briefly, the bone tissue was rinsed in sterile phosphate-buffered saline (PBS), cut into 1 mm3 cubes, and digested in collagenase I (Invitrogen Life Technologies, Grand Island, NY, USA) and dispase II (Roche, Indianapolis, IN) for 1 h at 37 °C. Cells were collected by centrifugation at 200 × g and seeded in a 100-mm culture dish in alpha minimum essential medium (Alpha-MEM, Gibco, Paisley, Scotland, UK) supplemented with 10% fetal bovine serum (FBS) (Gibco) and penicillin-streptomycin and L-glutamine. Following overnight culture at 37 °C in a humidified atmosphere of 5% CO2 and 95% air, unattached cells were discarded when the medium was changed. Cells at passage 3 were analyzed for stem cell-associated markers by flow cytometry. Details were described in supplementary.This work was supported by grants from the National Natural Science Foundation of China (81570961), the Natural Science Foundation of Beijing Municipality (7092113), the National Key Health Research Project Foundation of China during the 11th Five-Year Plan Period (2006BAI05A07), the Capital Medical Developing Foundation (2007-1005), the Beijing Natural Science Foundation Grant (5162013) and National Institute of Dental and Craniofacial Research (R90DE022527). We are grateful to the TDO patient and the normal healthy donors and for participating in this study.Author Contributions Na Zhao performed the measurement of all biomarkers, analyzed the data, created figures and drafted the manuscript. Dong Han and Yue Li carried out sample preparation and were involved in the analysis and interpretation of the data. Haochen Liu and Singwai Wong carried out plasmid construction. Zhengyi Cao carried out the EMSA experiments. Jian Xu assisted in figures creating. Xiaowei Zhang and Tao Cai provided a lot of technical guidance and assisted in manuscript revising. Yixiang Wang and Hailan Feng designed the study, supervised the experiments, supervised all data acquisition and supervised writing of article.