Dspp Mutations Disrupt Mineralization Homeostasis During Odontoblast Differentiation

Dentin defects associated with genetic disorders are classified as Dentinogenesis Imperfecta (DGI) and Dentin Dysplasis (DD), and both of them are divided into different subtypes. DGI has type ?, type ?, and type ? and DD has type ?, type ?. DGI-? and DGI-? and DD-? are isolated autosomal dominant dentin disorders which affect both the primary and the permanent dentition. The main pathological feature in isolated hereditary dentin disorders is the abnormality of dentin mineralization, including:1. Pulpal spaces are either smaller than normal or completely obliterated.2. Dentin is hypomineralized.3. The structural morphology of dentin tubules is abnormal.4. Amount of type ? collagen fiber bundles around the tubules.5. The feature of DEJ is abnormal, and the adjacent enamel arrayed irregulary. Dentin sialophosphoprotein gene (DSPP) is the only identified causative gene for these disorders. The gene encodes a single transcript which cleavages into two protein products:dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). DSP is a glycoprotein with a relatively high sialic-acid content and accounts for 5-8% of the dentin extracellular matrix (DECM) excluding collagen. Found as the major noncollagenous DECM protein, DPP is rich in aspartic acid and phosphorylated serine, and might regulate biomineralization processes by binding to the matrix of structural proteins, nucleating mineralization, and controlling crystal growth. Mineralization, occurring not only in bone but also in dentin, is a homeostasis between various nucleators and inhibitors of hydroxyapatite crystal formation, which involves both the enhancement of mineralization-inducing molecules and the induction of mineralization inhibitors. Although many mutations in DSPP gene had been identified in families with the above disorders, however, the molecular mechanisms involving in bridging the mutations and the resulting abnormal dentin mineralization have not been completely elucidated nowadays.Objective:To explore the molecular association between Dspp mutations and the disrupted mineralization homeostasis during dentin formation.Methods:we generated the wild type full-length Dspp cDNA lentivirus and 3 Dspp mutants which were consistent with the previous identified DSPP mutations in human dentin disorders, and these wild type lentivirus and mutants were transfected into mouse Odontoblast-lineage cells (OLC). After 2 weeks screening, we got the OLCs stably expressing the wild-type and mutant Dspp. Throughout 21-day mineralization inducing culture, we examined the Alp activity and mineralized nodules to test the differences among the groups of wild-type and mutant Dspp. We also performed Digital Gene Expression (DGE) to find factors that associated with mineralization. Last but not least, relative mRNA and protein expressions of specific mineralization inductors and inhibitors were further examined.Results:The results showed Dspp mutants affected the Alp activity as well as significantly reduced the number of mineralized nodules. Digital Gene Expression (DGE) showed that Dspp mutation affected the OLC differentiation in a degree. Further examination validated that Dspp (LV-Dspp) overexpressing OLCs possessed the ability to strictly orchestrate framework for mineralization inductors like Bmp2, Coll and Runx2, and proliferative markers for mineralization like Alp and Ocn, as well as mineral homeostasis feedback regulators Mgp and Htral. However, the missense mutation in Dspp signal peptide region (LV-M2) and the nonsense mutation (LV-M5) broke this orchestration. The results suggested that the mutant Dspp disrupt the dynamic homeostasis of mineralization during OLC differentiation.Conclusions:Our findings provide the evidence for association between Dspp and the dynamic homeostasis of mineralization inductors and inhibitors, and indicate the disruption of the homeostasis might be the crucial reason for Dspp mutation resulting in the dentin disorders.