Effect of PPAR-g Agonist and Antagonist on Amelogenin Promoter Activity

Amelogenin has an essential role in tooth development during enamel mineralization and is expressed selectively by ameloblast at different developmental stages. Regulation of amelogenin expression involves the preservation of pre-ameloblast precursor cells while providing abundant amelogenin expression in mature ameloblasts compared to preameloblasts. CCAAT/enhancer-binding protein alpha (C/EBP-a) is a mouse amelogenin gene transactivator and binds to its cognate sites to promote amelogenin transcription. Peroxisome proliferator-activated receptor gamma (PPAR-g), a nuclear hormone receptor, has been proposed to regulate C/EBP-a expression in adipogenesis; however, its role in amelogenesis remains unknown. Objectives: To explore the upstream regulation of C/EBP-a by using drugs to modulate PPAR-g signaling pathways. Methods: A stable cell line (LS8/p83) based on mouse ameloblast-like LS8 cells transfected with amelogenin reporter construct p83 was maintained in vitro in DMEM (Sigma)+FBS (10%)+penicillin (100 units/ml). 1) PPAR-g agonist (Rosiglitazone)(American Radiolabeled Chemicals), and PPAR-g antagonist (GW9662)(Tocris Cookson) (0.1uM, 1uM, 10uM) were added to LS8/p83. Controls received no drugs. 2) C/EBP-a (10 ng, 100ng, 1000ng) was transfected into LS8/p83 in the presence of GW9662 (10uM). pCMV-lacZ was co-transfected as an internal control. 3) C/EBP-a (100ng) was transfected into LS8/p83 in the presence of GW9662 (10 uM, 20 uM). Controls included C/EBP-a(+)/GW9662(-) and C/EBP-a(-)/ GW9662(-). After incubation for 24h in 37^oC, 5% CO₂, luciferase activities were measured. Results: GW9662 decreased luciferase activity, dose dependently, by 54% when compared to control. Rosiglitazone had little effect on luciferase activity. GW9662 (10uM) decreased activity of transfected C/EBP-a by 10% when compared to control. Increasing the GW9662 dose to 20uM improved the antagonistic activity by 18%. Conclusions: PPAR-g antagonist exerts an inhibitory effect on amelogenin promoter activity, suggesting that PPAR-g can modulate amelogenin promoter activity. However, PPAR-g might not be a direct upstream regulator of C/EBP-a. Future directions will explore the responsible signaling pathways.

NIH/NIDCR DE06988, Thai government CRN scholarship