Study of SIBLING Proteins in the Dentin of Hyp Mice

Objectives: X-linked hypophosphatemic rickets caused by mutations in the PHEX gene, is characterized by hypomineralization of bone and dentin, widening of osteoid seam and predentin, growth retardation and impaired renal re-absorption of phosphates. Based on the phenotypic similarity among Dmp1-null, Dspp-nul and PHEX-deficient (Hyp) mice along with the observation that PHEX protein preferentially cleaves peptide bonds at the NH2-termini of aspartic acid residues, we previously postulated that PHEX protein might be responsible for the proteolytic cleavage of dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP). To test this hypothesis and to analyze SIBLING proteins in the dentin of Hyp mice, we compared the expression of DMP1, DSPP, bone sialoprotein (BSP) and osteopontin (OPN) between Hyp and wild type mice. Methods: First, non-collagenous extracellular matrix proteins were extracted from dentin of Hyp and wild type mice. The extracts were separated by ion-exchange chromatography and protein-containing fractions were analyzed by SDS-PAGE with Stains-All staining and Western immunoblotting using antibodies specific to individual SIBLING proteins. Second, Immunohistochemistry was performed to analyze the difference in the expression and distribution of these SIBLING proteins between Hyp and wild type mice. Results: The NH2-terminal and COOH-terminal fragments of DMP1 and DSPP were present in the extracts of dentin extracellular matrix of Hyp mice, with a quantity similar to that observed for the wild type mice. BSP appeared more abundant in the dentin extracts of Hyp mice than in the wild type mice, while the amount of OPN was similar between the two groups. Conclusion: we concluded that PHEX protein is not the enzyme responsible for proteolytic processing of DMP1 and DSPP. The elevation of BSP may be a reflection of the histopathological changes in the Hyp mice (widening of predentin). Supported by NIH grant DE005092 (CQ).