Studies of the DMP1 57-kDa functional domain both in vivo and in vitro

Dmp1-null mice and patients with mutations in dentin matrix protein 1 (DMP1) resulting in autosomal recessive hypophosphatemic rickets display similar skeletal defects. As mutations were observed in the last 18 amino acids of DMP1 in 1 subset of patients and as fragments of intact DMP1, a 37-kDa N-terminal and a 57-kDa C-terminal fragment, have been purified from bone and dentin, we hypothesized that the cleaved 57-kDa C-terminal fragment is the essential functional domain of DMP1. To test this hypothesis, different forms of recombinant DMP1 were expressed in 293EBNA, CHO and 2T3 cells. The results showed that DMP1 was processed into a 37-kDa N-terminal and a 57-kDa C-terminal fragment in vitro in all cell lines examined. DMP1 processing in CHO cells was blocked by a furin protease inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone, in a dose-dependent manner. Coexpression of PHEX, a potential upstream protease, had no apparent effect on DMP1 cleavage in 293EBNA cells, suggesting that PHEX may not be required for DMP1 processing. To test the in vivo role of the C-terminal fragment, transgenic mice overexpressing full-length DMP1 or the 57-kDa fragment controlled by the 3.6-kb Col1 promoter were generated. Overexpression of these transgenes had no effect on the wild-type skeleton, but on the Dmp1-null background showed expression in the osteoblast layer and throughout the bone matrix leading to the rescue of the null bone phenotype. This suggests that the 57-kDa C-terminal fragment may be able to recapitulate the function of intact DMP1 in vivo.