RhoA and Rac1 are important for Amelogenin and DSPP expression

Morphogenesis and cytodifferentiation are distinct processes in tooth development. Cell proliferation predominates in morphogenesis, while differentiation involves change in form and change in gene expression. The cytoskeleton is essential for both processes, being regulated by small Rho GTPases. Objectives: In the present study, the distribution, expression and role of the GTPases RhoA and Rac1 during early tooth development was evaluated. Methods: the distribution and expression of RhoA and Rac1 were analyzed by immunohistochemistry, immunofluorescence and real-time polymerase chain reaction (real time PCR). The role of these proteins was evaluated by using inhibitors of the Rho GTPases (Clostridium difficile toxin A and Y27632) during tooth germ development inside the anterior eye chamber, followed by real time PCR analyzes. Results: both GTPases were strongly expressed during morphogenesis phase. During cytodifferentiation, RhoA was present in ameloblasts and odontoblasts, while Rac1 and its effector p21-activated kinase (Pak3) were observed only in ameloblasts in a punctual manner. The expression of RhoA mRNA and its effectors Rho kinase I and II (RockI and II), Rac1 and Pak3, increased after ameloblasts and odontoblasts differentiation, according to the expression of amelogenin and dentin sialophosphoprotein (DSPP). The inhibition of all Rho GTPases, by Clostridium difficile toxin A, completely abolished amelogenin and DSPP gene expression, while the specific inhibition of Rocks, by Y27632, had only a partial effect. Conclusions: these results suggest that both GTPases are important during tooth morphogenesis. Rho proteins are essential for differentiation of ameloblasts and odontoblasts, by regulating the expression of amelogenin and DSPP. RhoA and its effector RockI (highly expressed) contribute for this role. A specific function for Rac1 in ameloblasts, however, remains to be elucidated. Due to its punctual distribution, it is possible that Rac1 has a role in exocytosis/endocytosis. Supported by FAPESP N° 01/09047-2.