TGF-β and Physiological Root Resorption of Deciduous Teeth

Abstract

The present study was performed to examine how transforming growth factor β (TGF-β) in root-surrounding tissues on deciduous teeth regulates the differentiation induction into odontoclasts during physiological root resorption. We prepared root-surrounding tissues with (R) or without (N) physiological root resorption scraped off at three regions (R1-R3 or N1-N3) from the cervical area to the apical area of the tooth and measured both TGF-β and the tartrate-resistant acid phosphatase (TRAP) activities. The TGF-β activity level was increased in N1-N3, whereas the TRAP activity was increased in R2 and R3. In vitro experiments for the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-mediated osteoclast differentiation revealed that proteins from N1-N3 and R1-R3 enhanced the TRAP activity in RAW264 cells. A genetic study indicated that the mRNA levels of TGF-β1 in N1 and N2 were significantly increased, and corresponded with levels of osteoprotegerin (OPG). In contrast, the expression level of RANKL was increased in R2 and R3. Our findings suggest that TGF-β is closely related to the regulation of OPG induction and RANKL-mediated odontoclast differentiation depending on the timing of RANKL and OPG mRNA expression in the root-surrounding tissues of deciduous teeth during physiological root resorption.

Keywords: cytokine; gene expression; osteoclast; pediatric dentistry; protein expression; root resorption.

Figure A1

Effect of OPG against RANKL stimulated RAW264 cells. (A) iTRAP activity (n = 6 culture wells for each group) and (B) TRAP staining of RAW264 cells exposed by RANKL only (200 ng/mL) without (Control) or with the addition of various concentration of OPG (0.1 to 100 ng).

Figure 1

Extraction of proteins in root-surrounding tissues on deciduous incisor teeth. (A) Deciduous incisor teeth with (R) or without (N) root resorption from five-month-old pigs. The root-surrounding tissues were scraped off by dividing them into three regions (R1–R3 and N1–N3) at 1-cm intervals from the cervical area to the apical area of the tooth; (B) Flow chart indicates the procedures used to produce extracts for proteins in root-surrounding tissues. Sup: supernatant; Ppt: precipitate; (C) SDS-PAGE (5% to 20% gradient gel) stained with Simply Blue Safe Stain (Simply Blue) and Stains-all indicates each fraction.

Figure 2

Detection of alkaline phosphatase (ALP)-inducing activities and endogenous TRAP (eTRAP) activities in root-surrounding tissues, and identification of TGF-β isoforms. (A) ALP-inducing activity of HPDL cells exposed by protein extracts in N1–N3 and R1–R3 extracted with Triton X, NP-40, and SDS buffers from root surrounding tissues (n = 6 culture wells per sample); (B) enzyme-linked immunosorbent assay (ELISA) for the detection of TGF-β1, TGF-β2, and TGF-β3 in N1–N3 and R1–R3 extracted with Triton X; (C) eTRAP activity in N1–N3 and R1–R3 extracted with Triton X, NP-40, and SDS buffers from root surrounding tissues (n = 3 tests per sample).

Figure 3

In vitro experiment for RANKL (receptor activator of NF-κB ligand)-mediated osteoclast differentiation. (A) Induced TRAP (iTRAP) activity of RAW264 cells exposed by TGF-β isoforms (1 ng/mL each) with (RANKL) or without (Control) the addition of RANKL (200 ng/mL) (n = 6 culture wells per group); (B) TRAP staining for RAW264 cells exposed by TGF-β isoforms without (left panel) or with (right panel) RANKL (100 ng/mL); (C) iTRAP activity of RAW264 cells exposed by protein extracts in N1–N3 and R1–R3 of root surrounding tissues with or without the addition of GST (glutathione S-transferase)-RANKL (200 ng/mL) and SB431542 (1 µM). In the RANKL only sample, the dagger (†) indicates a significant difference between with and without SB431542. The asterisk (*) on the bar graph without SB431542 indicates a significant difference between each sample and RANKL only. In the group with SB431542, there were no significant differences between each sample and RANKL only. Statistical significance (* or †) was determined with an unpaired Student’s t-tests. In all cases, p < 0.05 was regarded as statistically significant.

Figure 4

Gene expression in root surrounding tissues. mRNA expressions via qPCR analysis of (A) TGF-β1, (B) TGF-β2, (C) TGF-β3, (D) RANKL, (E) RANK, (F) TRAP, (G) CALCR, (H) NFATc1 and (I) OPG were shown. Each ratio was normalized to the relative quantification data of each mRNA in N1–N3 and R1–R3 of root-surrounding tissues compared with a reference gene (GAPDH), which was generated on the basis of a mathematical model for relative quantification in a qPCR system (n = 6 tissues). Statistical significance (*) was determined with an unpaired Student’s t-tests. In all cases, p < 0.05 was regarded as statistically significant.