Role of Phosphophoryn in mineralizing and non-mineralizing mammalian cells

Phosphophoryn (PP) is one of the major dentin non-collagenous proteins. It is rich in serine and aspartic acid, and is highly phosphorylated (~ 200 phosphate per molecule). Due to its high phosphate content, net negative charge, and high Ca²⁺ binding affinity, it is recognized as one of the key molecules driving dentin mineralization.

Objectives: To study the role of PP in the Biomineralization process and characterize the mineral induced by PP. This will provide us a model to study PP induced mineralization and the consequent interaction of non-collagenous extra-cellular matrix proteins from two different cell types (mineralizing and non-mineralizing cells). We focus on how these different environments control the various forms of calcium phosphate (CaP) phases.

Methods: We studied the effect of PP on the crystallographic structure and morphologies of the resultant CaPs using XRD, FTIR, and HRTEM. The matrix mineralization was induced by over-expressing PP (exon 5 of DSPP) in fibroblastic (NIH-3T3) and osteoblastic (MC-3T3) mammalian cells.

Results: Alizarin red and von kossa positive staining confirmed the presence of mineral phases. XRD and FTIR analyses following the overexpression of PP in NIH3T3 cells further corroborated the presence of CaP structures. FTIR confirmed the presence of phosphate, carbonate linkages, and hydroxyapatite type phases. These results will be correlated with the biomineralization process in MC3T3 over-expressing PP and the interaction of PP with Ca²⁺ and PO₄^3- ions when immobilized in various substrates using a single and double diffusion high pressure cell apparatus. These unique models will allow us to unequivocally study the biomineralization process in the presence and absence of cells.

Conclusions: The data indicate that even the extracellular matrix of fibroblast cells expressing PP would mineralize. The mineral analysis data will be combined with our proteomics data to better understand the biomineralization process.

The research was supported by NIH-NIDCR DE016123