Date of Award

7-2011

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Carolyn Gibson, PhD

Second Advisor

Li Peng, DDS, PhD

Third Advisor

Marcus Bell, PhD

Abstract

Exposure to high levels of fluoride during tooth development will lead to mineralization defects and a decrease in enamel strength, known as enamel fluorosis. It has been proposed that fluoride may alter the cytoskeleton of ameloblasts, cells that secrete enamel during tooth development. This change is carried out through the Rho pathway, more specifically through RhoA. RhoA is stimulated by Wnt3a, a part of the Wnt pathway involved in regulation of cell proliferation and differentiation. The main component of the Wnt pathway, β-catenin, is expressed in dental epithelium and acts by linking cadherins to the actin cytoskeleton in cell adhesion. This study examined the effect of sodium fluoride on the Wnt and Rho pathways in ameloblast-lineage cells (ALC) from a mouse animal model. ALC were treated with Dkk-1 and sFRP-2, extracellular receptor inhibitors of the Wnt pathway. Treatment with β-cateninsiRNA was used as an intracellular inhibitor of Wnt, while RhoADN plasmid and Y-27632 were added to inhibit RhoA and Rho-associated protein kinase (ROCK) pathway components, respectively. Following treatment with inhibitors and 1.5 mM NaF, cells were analyzed via Rho pulldown assay, immunoprecipitation, Western blot, Topflash luciferase assay, and alkaline phosphatase assay. Western blot and Topflash luciferase assay results verified that both the Wnt and Rho pathways were up-regulated by NaF. Wnt was discovered to be located upstream from the Rho pathway as confirmed by treatment with Wnt pathway cell receptor inhibitors, Dkk-1 and sFRP-2, leading to a decrease in RhoA and ROCK activity. Inhibition of the Rho pathway with RhoADN plasmid and Y-27632 caused up-regulation of Wnt pathway activity, which was found to negatively regulate ALC differentiation. These data suggest that the crosstalk between these pathways could lead to the development of enamel fluorosis.

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