Expression of Phosphophoryn Is Sufficient for the Induction of Matrix Mineralization by Mammalian Cells

Abstract

Mineralized tissues such as dentin and bone assemble extracellular matrices uniquely rich in a variety of acidic phosphoproteins. Although these proteins are presumed to play a role in the process of biomineralization, key questions regarding the nature of their contributions remain unanswered. First, it is not known whether highly phosphorylated proteins alone can induce matrix mineralization, or whether this activity requires the involvement of other bone/dentin non-collagenous proteins. Second, it remains to be established whether the protein kinases that phosphorylate these acidic proteins are unique to cells responsible for producing mineralized tissues. To begin to address these questions, we consider the case of phosphophoryn (PP), due to its high content of phosphate, high affinity for Ca 2؉ , and its potential role in hydroxyapatite nucleation. We have created a model system of biomineralization in a cellular environment by expressing PP in NIH3T3 fibroblasts (which do not produce a mineralized matrix); as a positive control, PP was expressed in MC3T3-E1 osteoblastic cells, which normally mineralize their matrices. We show that expression of PP in NIH3T3 cells is sufficient for the induction of matrix mineralization. In addition, assessment of the phosphorylation status of PP in these cells reveals that the transfected NIH3T3 cells are able to phosphorylate PP. We suggest that the phosphorylation of PP is essential for mineral formation. The principle goal of this study is to enrich the current knowledge of mineralized tissue phosphorylation events by analyzing them in the context of a complete cellular environment.In biologically induced mineralization, crystals are generally produced by a heterogeneous nucleation mechanism. The deposition of mineral crystals in bone, dentin, and cartilage is orchestrated by cells, and the growth of these crystals is facilitated through mineral-matrix interactions. Current data indicate an important role for non-collagenous extracellular matrix (ECM) 2 proteins associated with collagen fibrils in the process of biomineralization. The high affinity of these non-collagenous proteins for divalent cations may facilitate control of the nucleation and growth of the initial mineral deposits. In addition, these proteins may subsequently regulate the size, composition, microstructure, morphology, and the orientation of the resulting crystals formed (crystal growth) (1). Mineralized tissues are unique in their production of extracellular acidic phosphoproteins. These phosphoproteins may be of structural and/or regulatory significance to the formation of mineralized tissues, potentially acting as a key interface between the nucleating mineral crystal and surrounding collagen fibrils (2, 3). These phosphoproteins include: osteopontin (OPN) (4), bone sialoprotein (5, 6), dentin matrix protein 1 (DMP1) (7), phosphophoryn (PP) (8), osteonectin (9), bone acidic glycoprotein 75 kDa (BAG 75) (10), and MEPE (11), each accumulating in the extracellular matrices of bone and dentin to diffe...

Biomineralization Mechanisms: A New Paradigm for Crystal Nucleation in Organic Matrices

“…The intensity of binding at a molar ratio of 1 to 1 rather than at electrostatic equivalence suggested that a non-electrostatic binding was involved at the primary specific interaction site, emphasizing the probable specificity in DPP sequence responsible for its primary collagen localization. Though polyAsp can do the in vitro job of nucleating cAp, the evidence is strong that, although the sequence of DPP contains approximately 40% Asp residues, the phosphorylation of the Ser residues of DPP ( 50% of the amino acids) is crucial for its function in mediating mineralization [42] and, most recently, it has been shown that the introduction of a DPP cDNA into a non-mineralizing fibroblast cell leads to expression and secretion of phosphorylated DPP and the induction of HAp mineralization in the fibroblast cultures [43]. …”

Multifunctional ECM proteins in bone and teeth

“…However, recently, DPP has been shown to possess signaling functions that can initiate lineage specific differentiation of mesenchymal stem cells (29). DPP in the ECM can mediate cell adhesion and initiate integrin mediated signaling (30,31).…”

Mechanism of Mineralization of Collagen‐Based Connective Tissues

“…Murshed et al also showed fibroblasts could mineralize when tissue non-specific alkaline phosphatase was over-expressed and -glycerol phosphate was added, but since alkaline phosphatase and -glycerol phosphate generate mineral in the absence of a matrix [63], the fibroblast data does not prove that physiologic mineralization was taking place. On the other hand, without alkaline phosphatase and without an organic phosphate source, Sfeir et al have induced physiologic HA formation in fibroblasts that do not mineralize under control conditions, by inserting the gene for a dentin-specific protein, phosphophoryn [64]. Thus, it appears that collagen-based tissues can support mineralization with the right additional factors, but what factors are they -and which are essential?…”