Wnt16 is involved in intramembranous ossification and suppresses osteoblast differentiation through the Wnt/beta-catenin pathway

In the course of embryonic development skeletal elements form either through intramembranous or endochondral ossification. Wnt proteins play diverse roles during vertebrate skeletal development. Wnt16 is a key factor in developing long bones, but its exact role in craniofacial bone formation remains unclear. This study was initially undertaken to investigate the expression of Wnt16 during craniofacial bone development in mouse embryos. Wnt16 expression in the osteoid of calvaria, maxilla, and mandible started later than that of ALP and osteocalcin (OCN), but before mineralization of the craniofacial bones, suggesting that Wnt16 is involved in intramembranous ossification in the head. To confirm this, MC3T3-E1 cells were transfected with an adenovirus containing Wnt16 (Ad-Wnt16). Ad-Wnt16 cells showed decreased ALP activity and less mineralized nodule formations compared with control cells. In addition, the mRNA levels of osteogenic markers were reduced. Moreover, Wnt16 activated beta-catenin signaling in MC3T3-E1 cells at both transcription and protein levels as shown by a TOPflash luciferase reporter gene assay and western blot analysis. On the other hand, Wnt/beta-catenin pathway blockade by Dickkopf 1 abrogated the suppression of mineralization by Wnt16. Our findings suggest that Wnt16 is involved in intramembranous ossification and suppresses osteoblast differentiation through the Wnt/beta-catenin pathway