Phosphorylated and Non-phosphorylated Leucine Rich Amelogenin Peptide Differentially Affect Ameloblast Mineralization

Abstract

The Leucine Rich Amelogenin Peptide (LRAP) is a product of alternative splicing of the amelogenin gene. As full length amelogenin, LRAP has been shown, in precipitation experiments, to regulate hydroxyapatite (HAP) crystal formation depending on its phosphorylation status. However, very few studies have questioned the impact of its phosphorylation status on enamel mineralization in biological models. Therefore, we have analyzed the effect of phosphorylated (+P) or non-phosphorylated (-P) LRAP on enamel formation in ameloblast-like cell lines and ex vivo cultures of murine postnatal day 1 molar germs. To this end, the mineral formed was analyzed by micro-computed tomography, Field Emission Scanning Electron Microscopy, Transmission Electron Microscopy, Selected Area Electon Diffraction imaging. Amelogenin gene transcription was evaluated by qPCR analysis. Our data show that, in both cells and germ cultures, LRAP is able to induce an up-regulation of amelogenin transcription independently of its phosphorylation status. Mineral formation is promoted by LRAP(+P) in all models, while LRAP(-P) essentially affects HAP crystal formation through an increase in crystal length and organization in ameloblast-like cells. Altogether, these data suggest a differential effect of LRAP depending on its phosphorylation status and on the ameloblast stage at the time of treatment. Therefore, LRAP isoforms can be envisioned as potential candidates for treatment of enamel lesions or defects and their action should be further evaluated in pathological models.

Keywords: LRAP; ameloblastic cell line; ameloblasts; amelogenin; hydroxyapatite; leucine-rich amelogenin peptide; phosphorylation; tooth germ.

Figure 1

TEM and SAED analyses of mineral phases formed under mineralizing conditions by the LS8 cells in the absence and presence of added peptide. (A,B) CONTROL, no added peptide; (C,D) LRAP(+P); and (E,F) LRAP(–P). In (A,C,E), general crystal distribution can be observed, crystal characterization is presented in (B,D,F). As shown, ordered needle like HAP (based on the observed selected area diffraction patterns) crystals were formed in the control (A,B) and in the presence of LRAP(+P) (C,D) in the LS8 cells. In the presence of LRAP(–P), the HAP crystals (SAED pattern in inset) appeared thinner, longer, and grouped in bundles (E,F).

Figure 2

TEM and SAED analyses of mineral phases formed under mineralizing conditions by the ALC cells in the absence and presence of added peptide. (A,B) CONTROL, no added peptide; (C,D) LRAP(+P); and (E,F) LRAP(–P). In (A,C,E), general crystal distribution can be observed, crystal characterization is presented in (B–D,F). Very large round and elongated HAP crystals are present in the control (A,B). Upon addition of LRAP(+P) (C,D) and LRAP(–P) (E,F), needle-like HAP crystals were formed with LRAP(–P) potentiating, bundle formation.

Figure 3

Kinetic of expression of amelogenin gene in LS8 and ALC cells and in the cultured first molar germs. (A,B) Amelx quantitative PCR analyses for the LS8 (A) and ALC (B) cell lines at selected time-points. Amelx expression was normalized to GAPDH and Actin for each time-point and cell line. Average expression levels and standard deviation error were calculated from 3 different qPCR experiments (each run in triplicate, n = 3). At D2, both peptides induced a statistically significant increase in amelogenin transcripts relative to the control in the LS8 [*p < 0.05 for LRAP(+P) and ^***p < 10⁻⁴ for LRAP(–P)]. At D7, both peptides induced a similar increase for the ALC, statistically significant for LRAP(–P) (*p < 0.05) relative to the control. (C) Amelx transcripts levels were compared between D0 (PND1 germ) and cultured D9 germs. Inhibition of Amelx expression was observed in all conditions relative to the D0 germ. LRAP(+P) and LRAP(–P) treatment induced a statistically significant (*p < 0.05) increase in Amelx expression relative to the control.

Figure 4

Macroscopic views and mineral density and enamel volume of first molar germs cultured in the absence and presence of added peptide. (A) Photographs of first molar germs at D0 and after 9 days of culture in the absence (CONTROL) or presence of LRAP(+P) and LRAP(–P). Scale bar 500 μm. Mean mineral density (B) and enamel volume (C) were calculated from Micro-CT scans of first molar germs at D0 and after 9 days of culture. Addition of LRAP(+P) peptide lead to a statistically significant increase (^*p < 0.05) in enamel volume relative to the D9 control, no difference could be observed between LRAP(–P) treated and control samples. All samples presented an increased mineral density (B) and enamel volume (C) at D9 relative to D0 confirming tooth germ growth and maturation.

Figure 5

FE-SEM analyses of D9 first molar germs cultured in the absence and presence of added peptide (A–C) CONTROL, (D–F) LRAP(+P), and (G–I) LRAP(–P). Ameloblast pits and mineral organization were clearly observed for all three samples, confirming enamel growth in culture. Ameloblast pits appeared smaller and more spaced in molar germs treated with LRAP(+P) (E,F) relative to the control (B,C) and slightly wider in molar germs treated with LRAP(–P) (H,I) relative to the control.