Qianqian Gao1, Jianping Ge1, Yanqin Ju1, Changsheng Li1, Jian Gao1, Meisheng Wu1, Shouliang Zhao2. 1. Department of Conservative Dentistry, Laboratory of Oral Biomedical Science and Translational Medicine, School of Stomatology, Tongji University, Shanghai, China. 2. Hua Shan Hospital, Shanghai Fudan University, China. Electronic address: slzhao@fudan.edu.cn.
Abstract
OBJECTIVE: Voltage-gated inward Ca2+ currents (ICa) are triggered by cell depolarization and commonly produce transient increases in the cytoplasmic free Ca2+ concentration. The CaV1.2 distal C-terminus is susceptible to proteolytic cleavage, which yields a truncated CaV1.2 subunit and a cleaved C-terminal fragment (CCt or DCT). Stem cells from the apical papilla (SCAPs) has a capacity for differentiation into the odontoblastic-like cells in vitro and dentin forming in vivo, which makes SCAPs advantages in tissue engineering and regenerative endodontic. The aim of this study was to investigate the effect of CaV1.2 and its distal C-terminal fragment in the odontoblastic differentiation of rat SCAPs (stem cells from the apical papilla). DESIGN: In this study, we generated stable CaV1.2 knockdown and DCT over-expressed rSCAPs using short hairpin RNA and DCT gene containing Lentivirus vectors, respectively. The transfected apical papilla cells were induced to differentiate into the odontoblast-like cells, and the expression of markers for odontoblastic differentiation were analyzed by alizarin red staining, Real-time Polymerase chain reaction (RT-PCR), and Western blot analysis. RESULTS: The knockdown of CaV1.2 and excess expression of DCT both suppressed the expression of DSPP, ALP in mRNA level and the formation of calcium nodules. CONCLUSIONS: Our results suggest that CaV1.2 and DCT play important roles in the differentiation of rSCAPs, DCT might act as a transcription factor and regulate the differentiation of rSCAPs.
OBJECTIVE: Voltage-gated inward Ca2+ currents (ICa) are triggered by cell depolarization and commonly produce transient increases in the cytoplasmic free Ca2+ concentration. The CaV1.2 distal C-terminus is susceptible to proteolytic cleavage, which yields a truncated CaV1.2 subunit and a cleaved C-terminal fragment (CCt or DCT). Stem cells from the apical papilla (SCAPs) has a capacity for differentiation into the odontoblastic-like cells in vitro and dentin forming in vivo, which makes SCAPs advantages in tissue engineering and regenerative endodontic. The aim of this study was to investigate the effect of CaV1.2 and its distal C-terminal fragment in the odontoblastic differentiation of rat SCAPs (stem cells from the apical papilla). DESIGN: In this study, we generated stable CaV1.2 knockdown and DCT over-expressed rSCAPs using short hairpin RNA and DCT gene containing Lentivirus vectors, respectively. The transfected apical papilla cells were induced to differentiate into the odontoblast-like cells, and the expression of markers for odontoblastic differentiation were analyzed by alizarin red staining, Real-time Polymerase chain reaction (RT-PCR), and Western blot analysis. RESULTS: The knockdown of CaV1.2 and excess expression of DCT both suppressed the expression of DSPP, ALP in mRNA level and the formation of calcium nodules. CONCLUSIONS: Our results suggest that CaV1.2 and DCT play important roles in the differentiation of rSCAPs, DCT might act as a transcription factor and regulate the differentiation of rSCAPs.
Keywords:
L-type calcium channel; Odontoblastic differentiation; Overexpression; Short hairpin RNA; Stem cells from apical papilla; The distal C-terminus of Ca(V)1.2