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. 2015 Nov 15;7(11):2379-96.
eCollection 2015.

Dspp mutations disrupt mineralization homeostasis during odontoblast differentiation

Jie Jia  1 , Zhuan Bian  1 , Yaling Song  1
Affiliations

Dspp mutations disrupt mineralization homeostasis during odontoblast differentiation

Jie Jia et al. Am J Transl Res. .

Abstract

The main pathological feature in isolated hereditary dentin disorders is the abnormality of dentin mineralization. Dentin sialophosphoprotein (DSPP) gene is the only identified causative gene for the disorders. The present study aims to explore the molecular association between Dspp mutations and the disrupted mineralization homeostasis during odontoblast differentiation. We generated lentivirus constructs with the mouse full-length wild type Dspp cDNA and 3 Dspp mutants and transfected them into mouse odontoblast-lineage cells (OLCs) which were then performed 21-day mineralization inducing differentiation. The formation of mineralized nodules was obviously fewer in mutants. 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, Col1 and Runx2, and proliferative markers for mineralization like Alp and Ocn, as well as mineral homeostasis feedback regulators Mgp and Htra1. 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. We are the first to use full-length mouse Dspp gene expression system to explore the mineralization mechanism by which inductors and inhibitors adjust each other during odontoblast differentiation. Our findings shed new light on association between Dspp and the dynamic homeostasis of mineralization inductors and inhibitors, and indicate the disruption of mineralization homeostasis might be a crucial reason for Dspp mutations resulting in dentin disorders.

Keywords: Dentin sialophosphoprotein; dentin; mineralization; mutation; odontoblast-lineage cells (OLCs).

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Figures

Figure 1
Figure 1
Sketch for 3 Dspp mutations and the results of sequencing the mutants (NM_010080). A. Sketch for 3 Dspp mutations (SPP cleavage site: signal peptide peptidase cleavage site). B-D. The results of sequencing the mutants. The upper pictures present the normal sequences, and the arrows indicate the location of mutations.
Figure 2
Figure 2
Formation of mineralization nodules and ALP activity assay. A. Alizarin red staining of OLCs transfected with LV-Dspp/M2/M4/M5 and LV-GFP on day 7 and 21. B. ALP activity in each group during the differentiation of transfected OLCs. *Indicates statistical difference between the test group and the control, P<0.05.
Figure 3
Figure 3
Heat map of selected identified genes P≤0.05. Hierarchical clustering of differentially expressed genes in LV-Dspp (A), LV-M5 (B) and LV-GFP (C) group. Each column represents an experimental condition, each row represents a gene, red indicates up-regulation, and green indicates down-regulation.
Figure 4
Figure 4
Categories of DEGs based on GO function in LV-GFP-VS-LV-Dspp (A) and LV-GFP-VS-LV-M5 (B). The results are summarized in three major categories: biological process, cellular component and molecular function. P≤0.05 as a threshold.
Figure 5
Figure 5
The scatter plot for KEGG enrichment result. Top 20 Statistics of KEGG pathway enrichment for LV-GFP-VS-LV-Dspp (A) and LV-GFP-VS-LV-M5 (B). Rich Factor is the ratio of differentially expressed gene numbers annoted in this pathway terms to all gene numbers annoted in this pathway term. Q≤0.05 as significantly enriched.
Figure 6
Figure 6
Expression of Dsp in each group during the differentiation of transfected OLCs. A. mRNA expression of Dsp was investigated by Real-time PCR analysis. B, C. Protein expression of Dsp was detected by Western-Blot. D. β-actin served as an internal control. *Indicates statistical difference between the test group and the control P < 0.05. It is worth mentioning that the levels of Dsp were thousands times higher in the test groups than in the control, resulting in the large margin of error bars, so the error bars in the graph were not indicated.
Figure 7
Figure 7
Expression of Bmp2 and Col1 in each group during the differentiation of transfected OLCs. mRNA expression of Bmp2 (A) and Col1 (C) were investigated by Real-time PCR analysis. Protein expression of Bmp2 (B, B’) and Col1 (D, D’) were detected by Western-Blot. (E) β-actin served as an internal control. *Indicates statistical difference between the test group and the control P < 0.05.
Figure 8
Figure 8
Expression of Runx2 and Ocn in each group during the differentiation of transfected OLCs. mRNA expression of Runx2 (A) and Ocn (C) were investigated by Real-time PCR analysis. Protein expression of Runx2 (B, B’) and Ocn (D, D’) were detected by Western-Blot. (E) β-actin served as an internal control. *indicates statistical difference between the test group and the control P < 0.05.
Figure 9
Figure 9
Expression of Mgp and Htra1 in each group during the differentiation of transfected OLCs. mRNA expression of Mgp (A) and Htra1 (C) were investigated by Real-time PCR analysis. Protein expression of Mgp (B, B’) and Htra1 (D, D’) were detected by Western-Blot. (E) β-actin served as an internal control. *indicates statistical difference between the test group and the control P < 0.05.
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