GTPases RhoA and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts

Abstract

Morphogenesis and cytodifferentiation are distinct processes in tooth development. Cell proliferation predominates in morphogenesis; differentiation involves changes in form and gene expression. The cytoskeleton is essential for both processes, being regulated by Rho GTPases. The aim of this study was to verify the expression, distribution, and role of Rho GTPases in ameloblasts and odontoblasts during tooth development in correlation with actin and tubulin arrangements and amelogenin and dentin sialophosphoprotein (DSPP) expression. RhoA, Rac1, and Cdc42 were strongly expressed during morphogenesis; during cytodifferentiation, RhoA was present in ameloblasts and odontoblasts, Rac1 and its effector Pak3 were observed in ameloblasts; and Cdc42 was present in all cells of the tooth germ and mesenchyme. The expression of RhoA mRNA and its effectors RockI and RockII, Rac1 and Pak3, as analyzed by real-time polymerase chain reaction, increased after ameloblast and odontoblast differentiation, according to the mRNA expression of amelogenin and DSPP. The inhibition of all Rho GTPases by Clostridium difficile toxin A completely abolished amelogenin and DSPP expression in tooth germs cultured in anterior eye chamber, whereas the specific inhibition of the Rocks showed only a partial effect. Thus, both GTPases are important during tooth morphogenesis. During cytodifferentiation, Rho proteins are essential for the complete differentiation of ameloblasts and odontoblasts by regulating the expression of amelogenin and DSPP. RhoA and its effector RockI contribute to this role. A specific function for Rac1 in ameloblasts remains to be elucidated; its punctate distribution indicates its possible role in exocytosis/endocytosis.

Dental Enamel Structure Is Altered by Expression of Dominant Negative RhoA in Ameloblasts

“…RhoA and ROCK mRNA levels were recently reported to be elevated in WT rat first molars at PN5 [Biz et al, 2010], and we have observed a similar elevation in murine molars (not shown). Because the Rho dissociation inhibitor RhoGDI, which sequesters Rho to control its status, decreases shortly after birth [Hatakeyama et al, 2009], this pathway fluctuates exactly as the ameloblast cells undergo changes in structural/functional relationships.…”

Sp6 regulation of <i>Rock1 </i>promoter activity in dental epithelial cells

“…On the other hand, the Rho family inhibitor RhoGDI was downregulated during ameloblast secretory stage (24). Interestingly, expression of RhoA, the direct upstream activator of ROCK, and ROCK1 were also detected strongly in highly polarized ameloblasts, but weakly in non-polarized cells of inner epithelium and dental papilla during tooth development (9,10).…”

“…RhoA also regulates the expression of amelogenin and DSPP during cytodifferentiation of ameloblasts and odontoblasts, respectively (10). However, the regulation of Rock1 expression has not been investigated, although the activators of Rock1 have been widely discussed.…”

Rho GTPases in ameloblast differentiation

“…Further, they are involved in osteoclastogenesis as well as in hematopoiesis and hemopathies [12], [13]. Recently, evidence has emerged showing the involvement of Rho signaling in tooth development [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24]. In this article, we summarize some interesting recent findings that provide molecular insights into how signaling by Rho GTPases results in tooth development, focusing on ameloblast differentiation in particular.…”