Stress-Related Genes are Upregulated in Maturation Stage Ameloblasts

The enamel organ contains amelobasts that are responsible for enamel formation on the developing tooth. Ameloblasts undergo several stages of differentiation during enamel development (secretory, transition, maturation). It has been previously shown that TGF-b is expressed in most differentiating ameloblasts but is absent in pre-secretory ameloblasts. However, the relative expression level of TGF-b during the secretory, transition and maturation stages of enamel development is not yet known. Because overexpression of TGF-b1 in ameloblasts induces apoptosis and because approximately 50% of ameloblasts undergo apoptosis during transition through maturation stages, we hypothesized that TGF-b mediated signaling pathways may be responsible for inducing ameloblast cell death in vivo. Objective: Here, we ask if TGF-b expression is upregulated during the maturation stage and if stress-related genes are also induced during this stage. Methods: The ameloblast lineage cell line, ALC, was treated with several concentrations of TGF-b. Assays were performed to quantify cell proliferation and survival. Transcript levels of several stress-related genes were quantified by real-time RT-PCR. Further, stress-related transcripts present in enamel organs from first molars of postnatal 4 day-old mice (secretory stage) were compared to those from 11 day-old mice (maturation stage) using real-time RT-PCR. Immunohistochemistry results identified cells expressing TGF-b within the mouse enamel organ. Results: We show that mouse molar TGF-b expression was upregulated at the maturation stage and that several stress-related factors including ATF3, c-jun, c-fos, C/EBP-b, Egr-1 and Bax were also expressed at increased levels during this stage. Further, ALC cells also exhibit decreased Bcl2-Bax ratio as well as reduced cell survival on exposure to TGF-b. Conclusions: TGF-b expression positively correlates with increased expression of stress-related transcripts in vivo. Thus, TGF-b may play a role in initiating ameloblast apoptosis during transition through maturation stage of enamel development. Supported by NIDCR grant DE016276.