Effects of inositol hexasulphate, a casein kinase inhibitor, on dentine phosphorylated proteins in organ culture of mouse tooth germs

Abstract

To study the effects of impaired protein phosphorylation on dentine formation and mineralization, inositol hexasulphate, an intracellular type I and type II casein kinase inhibitor, was used in an in vitro organotypic culture system. Mandibular first molar tooth germs were dissected from 18-day-old mouse embryos and cultured for 11 days with and without inositol hexasulphate at different concentrations. At 0.04–0.08 mM inhibitor, cellular alterations were not detected. Dentine displayed the characteristic purple-blue colour when Stains all, a specific stain for extracellular phosphoproteins, was used. At 0.1 mM, dentine failed to stain and mineralization did not occur, as seen from the von Kossa method. The presence of numerous lysosome-like vesicles inside cells indicated that the experiment was at the limits of cytotoxicity; higher concentrations induced severe cellular alterations. Therefore, quantitative radioautography was carried out on germs treated or not with the inhibitor at 0.1 mM. [ 33P]-phosphate incorporation showed that grain density in inhibited germs compared with that in control germs was about double in odontoblasts and half in the predentine/dentine compartment. In the presence of inositol hexasulphate the incorporation of [ 3H]serine into odontoblast cell bodies was unchanged between 2 and 24 h while in predentine/dentine, grain density was higher between 1 and 4 h, and reduced at 24 h. Both with [ 33P]phosphate and [ 3H]serine, labelling was seen throughout the porous dentine formed in vitro and not as a band located at the predentine/dentine junction, as is the case in vivo. With [ 3H]proline, in the presence of the inhibitor, a small reduction of grain density occurred in cell bodies, no significant difference was seen between 1 and 4 h in predentine/dentine, and more silver grains were present after 24 h both in cells and in the matrix. The radioautographic data support the view that the inhibitor interacts mostly with post-transductional phosphorylation and does not alter significantly other cell synthetic pathways and functions. Finally, the experiments presented here confirm that phophorylated proteins have a key role in dentine mineralization.