Mouse mutant ttw (tiptoe walking) is an excellent model for
ectopic ossification. This mutant exhibits ossification in various soft tissues, which is histologically similar to human OPLL (
ossification of posterior longitudinal ligament of the spine). We previously reported that ttw is caused by a
nonsense mutation of the
nucleotide pyrophosphatase (ENPP1) gene, and that a polymorphism of the human ENPP1 gene is associated with OPLL. These facts indicate that ENPP1 regulates
ectopic ossification in vivo; however, the mechanism is unclear. ENPP1 is an ectoenzyme that generates
phosphate (Pi) and
pyrophosphate (PPi). PPi is a strong inhibitor of ossification. Abnormal Pi metabolism is observed in patients with OPLL, and diseases with abnormal Pi metabolism such as
hypophosphatemic rickets are frequently complicated by
ectopic ossification. These lines of evidence suggest Pi-PPi metabolism associated with ENPP1 may play an important role in regulation of
ectopic ossification. To clarify the molecular mechanism of
ectopic ossification in ttw, we examined the effect of dietary
phosphate and
calcium on the ttw phenotype and found a high dietary
phosphate-accelerated
ectopic ossification. Then we examined genes associated with the enhanced ossification in ttw on a high
phosphate diet by a differential display method. We identified nine mouse genes; six genes were up-regulated by the high
phosphate diet, and three were down-regulated. Six of the nine genes were novel and we cloned and characterized them. Two of the genes were highly specific to cartilage, suggesting their specific role in enchondral ossification. Our identification of the novel genes would give novel insight into the mechanism of
ectopic ossification and etiology of OPLL.