N-terminal Dentin Sialoprotein fragment induces type I collagen production and upregulates dentinogenesis marker expression in osteoblasts

Abstract

Bone and dentin are mineralized extracellular matrices produced by osteoblasts and odontoblasts, respectively, and their major organic portion is type I collagen. Dentinogenesis Imperfecta (DGI) is one of the most common clinically- and genetically-based disturbances of dentin formation, causing irreversible dentin defects. Among several types of DGI, patients with DGI type II exhibit opalescent dentin with partial or complete pulp obliteration. It has been previously reported that the non-sense mutation (c.133C>T) in Dentin Sialophosphoprotein (DSPP) was identified in DGI type II patients at glutamine residue 45, resulting in the premature stop codon (p.Q45X). DSPP is known to be synthesized as a single gene product and further processed at Gly462-Asp463, resulting in the production of Dentin Sialoprotein (DSP) and Dentin Phosphoprotein (DPP). We hypothesized that the shorter form (Q45X) of N-terminal Dentin Sialoprotein (N-DSP) may cause over-production of type I collagen protein as obliterated pulp is occupied by dentin. To test this hypothesis, we generated mouse recombinant Glutathione-S-Transferase (GST)-N-DSP fusion protein, and the effect of GST-N-DSP was investigated in calvarial bone explant culture and MC3T3-E1 osteoblastic culture systems. Here we show that a significant increase in calvarial bone formation is observed by GST-N-DSP. GST-N-DSP accelerates MC3T3-E1 osteoblast cell growth and proliferation and subsequent osteoblast differentiation by inducing the expression of certain osteogenic markers such as type I collagen, Runx2, Osterix and ATF4. Interestingly, GST-N-DSP significantly enhances dentinogenesis marker gene expression including Dspp and Dmp1 gene expression in non-odontogenic MC3T3-E1 cells. To rule out any artificial effect of GST-tag, we also used the synthetic peptide of N-DSP and confirmed the results of N-DSP peptide were essentially similar to those of GST-N-DSP. Taken together, our data suggest that N-DSP promotes bone formation by accelerating osteoblast cell proliferation and subsequent osteoblast differentiation accompanied by marked up-regulation of the dentin matrix markers, such as Dspp and Dmp1 genes.

Cementogenic genes in human periodontal ligament stem cells are downregulated in response to osteogenic stimulation while upregulated by vitamin C treatment

“…1b) and the % was calculated against the total area. Due to the irregularity in the shape of the granules and tissues, they were outlined using the freehand selection method and the areas were measured by ImageJ (Alongi et al 2010; Jaha et al 2016). …”

Ex vivo Bone Models and Their Potential in Preclinical Evaluation

“…For testing the potency of growth factors and small molecules for bone-related diseases, explants obtained from mouse calvarial bones are regularly used because it is a relatively simple and inexpensive model [62,70,78,104,[112][113][114][115][116][117][118][119][120][121][122][123][124]. The addition of human prostate or breast cancer cells to mouse calvariae allowed investigating the effects of several potential cancer therapeutics [74,[125][126][127][128][129][130][131][132][133].…”

Manifestation and Mechanisms of Abnormal Mineralization in Teeth

“…This may be the explanation for pulp obliteration in DGI-II. 37 The dentinal tubules in DGI-II are discontinuous. 36 The irregular appearance of the dentinal tubules and low elastic modulus caused by mutation of DSPP account for reduced dentin mineral density and dentin hardness, which result in tooth fragility and deterioration 34,38 (Figure 3G−N).…”