Role of the NH2 -terminal fragment of dentin sialophosphoprotein in dentinogenesis.

Genetic studies have shown that dentin sialophosphoprotein (DSPP) plays an essential role in dentinogenesis (1–3). For example, the dentin in Dspp knockout mice is hypomineralized and the predentin is widened, creating a phenotype similar to that of dentinogenesis imperfecta type III in humans (3). While studies have demonstrated the critical role of DSPP in dentin formation, the mechanism(s) by which DSPP function(s) in biomineralization remain largely unclear. DSPP, first identified by cDNA cloning (4), is a large protein that undergoes proteolytic processing to form the NH2-terminal fragment known as dentin sialoprotein (DSP), proteoglycan form of the NH2-terminal fragment referred to as DSP-PG and a COOH-terminal fragment named dentin phosphoprotein (DPP) (5–7). The sequence of the NH2-terminal fragment (DSP or DSP-PG) lies in the 5′-portion, while the DPP sequence is located in the 3′-region of the DSPP transcript (4, 8–10). Studies done in our laboratory and others established the presence of a proteoglycan form of DSP (known as DSP-PG) (5–7). DSP/DSP-PG and DPP are abundant in the dentin extracellular matrix (ECM) whereas the protein representing the entire sequence (DSPP) is scarcely present, which led to the belief that the processed fragments may be the functional forms of DSPP. Recent work in our laboratory has shown that blocking the proteolytic fragmentation of DSPP inactivates the function(s) of this molecule during dentinogenesis (11). This observation provides strong evidence that the proteolytic processing of DSPP is an activation event which releases the functional fragments that exert distinct molecular role(s) during dentin formation (11). DPP, discovered in 1967, is the most abundant non collagenous protein (NCP) in the dentin ECM (12, 13). The unusual feature of DPP is the presence of large amounts of aspartic acid (Asp) and phosphoserine (Pse), mostly found in the repeating sequences of (Asp-Pse-Pse)n and (Asp-Pse)n (14,15). Several in vitro mineralization studies have indicated that DPP is an important initiator and modulator for the formation and growth of hydroxyapatite (HA) crystals (16–18). It is believed that the DPP secreted by the odontoblasts is transported to the mineralization front where it binds to collagen fibrils promoting the formation of initial HA. As this mineralization process proceeds, and more predentin is converted to dentin, these mineral crystals grow in an oriented fashion under the modulation of DPP and other non-collagenous proteins (NCPs) that bind to the growing HA faces (19, 20). DSP, discovered in 1981, is much less abundant than DPP in dentin ECM (21). DSP [protein without the glycosaminoglycan (GAG) chains]does not have a significant effect on the in vitro mineral formation and growth (22). Since there is no in vivo data concerning the role of DSP in dentinogenesis, the functions of this fragment is presently undefined. The recently discovered proteoglycan form of DSP (DSP-PG) is a major component in the proteoglycan pool of the dentin extracellular matrix (ECM) and appears to be more abundant than DSP (5, 6). We believe that the NH2-terminal fragment of DSPP (DSP/DSP-PG) must play a role in biomineralization, distinct from that of the COOH-terminal fragment of DSPP (DPP). DSP-PG from the NH2-terminal sequence of DSPP was found in the dentin ECM at higher amounts than the core protein DSP, which led us to believe that DSP-PG may be the functional form of the NH2-terminal fragment. We hypothesize that the role of DSP-PG may be to prevent the predentin from being mineralized too rapidly and to serve as an antagonist of DPP in dentinogenesis. In order to examine the function of the NH2-terminal fragment of DSPP in dentinogenesis, we expressed the NH2-terminal fragment of DSPP in the Dspp-null background (referred as “Dspp KO/DSP Tg”) to analyze the in vivo effects of this fragment in a gain of function experimental approach. In this study, we systematically characterized the dentin of Dspp KO/DSP Tg mice and compared it to the dentin from Dspp-null (Dspp knockout or Dspp KO) mice and wild type (WT) mice. We observed that the dentin of the Dspp KO/DSP Tg mice appeared much worse compared to the Dspp-null mice. The results from this investigation support our hypothesis that the NH2-terminal fragment of DSPP has an inhibitory role in dentinogenesis.