DMP1 Fragments Distribute Differently in Bone, Dentin and Cells

Gene alteration experiments indicate that Dentin matrix protein 1 (DMP1) is critical for the mineralization of bone and dentin. In the extracellular matrix of bone and dentin, DMP1 is present as NH2-terminal (37 kDa) and COOH-terminal (57 kDa) fragments resulting from proteolytic processing. While the COOH-terminal fragment has a very high level of phosphorylation, ~50% of the NH2-terminal fragment occurs as a proteoglycan. Objectives: of this investigation was to examine the distribution pattern of the NH2-terminal and COOH-terminal fragments of DMP1 in bone, dentin, and MC3T3-E1 and HEK293 cell lines. Methods: Paraffin sections of rat long bones and mandibles and MC3T3-E1 and HEK293 cells were labeled immunohistochemically and analyzed with a Leica SP2 confocal microscope. Western blots of nuclear and cytosolic fractions from extracts of cultured cells and bovine predentin and dentin were compared to morphology. Results: In developing long bones, a dramatic difference in the distribution of the two fragments was seen in the metaphyseal growth plate; there was a lack of co-localization for the two fragments in the resting, proliferation and prehypertrophic zone where only the NH2-terminal fragment was present while the COOH-terminal fragment accumulated at the mineralization front. In both cell lines the COOH-terminal fragment localized in the nuclear compartment, while the NH2-terminal fragment gathered in the cytoplasm . Clear difference in the localization of the NH2-terminal and COOH-terminal fragments of DMP1 was observed. Conclusions: The difference in the localization of NH2-terminal and COOH-terminal fragments suggests that they play different roles in biomineralization. This investigation was supported by NIH grant DE005092 (CQ).