Role of MSX1 in Osteogenic Differentiation of Dental Pulp Cells

Msh homeobox 1 (MSX1) is a transcriptional factor regulating embryonic development of limbs and craniofacial tissues including bone and teeth. The purpose of this study was to investigate contribution of MSX1 to the osteogenic potential and calcification-related phenotypic expression of dental pulp stromal/mesenchymal cells isolated from human teeth. Immunohistochemisitry of a 3 week-old mouse molar showed that MSX1 protein was localized to odontoblasts and pulpal mesenchymal cells at different levels and in different manners depending upon the position of the cells in pulp tissue. When dental pulp stromal/mesenchymal cells were exposed to osteogenesis-induction medium, runt-related transcription factor-2 (RUNX2), bone morphogenetic protein-2 (BMP2), alkaline phosphatase (ALPL) and osteocalcin (OCN) mRNA levels, as well as alkaline phosphatase activity, increased on days 4-12, and, thereafter, the matrix was calcified on day 14. However, knockdown of MSX1 with small interfering RNA abolished this induction of the osteoblast-related gene expression, alkaline phosphatase activity and calcification. Interestingly, DNA microarray and quantative PCR analyses revealed that the MSX1 knockdown induced the sterol regulatory element-binding protein 2 (SREBP2) transcriptional factor and its downstream target genes in cholesterol-synthesis pathway. Inhibition of cholesterol synthesis enhances osteoblast differentiation of various mesenchymal cells. Thus, MSX1 may down-regulate the cholesterol synthesis-related genes to ensure osteoblast differentiation of dental pulp stromal/mesenchymal cells. Extracted healthy deciduous teeth were collected from children with informed consent under the ethics codes of Hiroshima University. The pulp tissue specimens from deciduous teeth were minced, and digested with collagenase and dispase to isolate dental pulp stromal/mesenchymal cells (DPSCs). Small interfering RNA oligonucleotides were used to knockdown MSX1 expression in DPSCs. DPSCs were incubated with growth medium or osteogenesis-induction medium. Four days after the onset of differentiation, total RNA was extracted. DNA microarray analysis was performed using the Agilent SurePrint G3 Human GE v2 8x60K Microarray. Raw data were standardized by the global median normalization method using GeneSpring.