Enamel Without Enamelin: Our long-term objective is to understand the molecular mechanisms of normal and diseased dental enamel formation. Mutations in the human enamelin gene cause autosomal dominant amelogenesis imperfecta. In this investigation, we study enamelin's functions during amelogenesis. The two Specific Aims propose to investigate enamelin function by: SA 1: characterizing enamel formation in the absence of mouse enamelin expression. SA 2: characterizing enamelin protein structural and functional properties.
In Specific Aim 1 we generate a knock-in mouse that replaces the enamelin gene with the coding region for bacterial 13-galactosidase fused to a mouse nuclear localization signal (NLS-lacZ). We then compare enamel formed in the absence of enamelin expression to enamel formed in the wild-type mice. Expression of the enamelin-NLS-lacZ reporter gene will be characterized by X-gal staining in frozen sections and whole mounts. Biochemical analyses will include SDS-PAGE, Western blotting, and amino acid analysis. Mineral analyses will include Light Transmission Microscopy (LTM), Scanning Electron Microscopy (SEM) to look for pitting, chipping or other surface defects, Transmission Electron Microscopy (TEM) to determine crystal morphology, X-ray microanalysis to determine elemental composition, Selected-Area Electron Diffraction (SAED) to determine mineral type, Dual-Energy X-ray Absorption (DEXA) to measure mineral density, histomorphometric measurement and Faxitron radiology for high-resolution X-ray imaging to determine enamel thickness, and Micro-computed tomography for 3-D reconstruction for mineral area and volume.
In Specific Aim 2 enamelin proteolytic cleavage products are isolated from the pig enamel matrix and characterized to reveal their posttranslational modifications. How these enamelins influence crystal growth in vitro is analyzed using pH stat and constant composition assays of hydroxyapatite formation, seeded growth assays for inhibition of hydroxyapatite growth, and by analysis of hydroxyapatite growth habit by X-ray diffractometry This study will determine if enamelin catalyzes enamel crystal nucleation and crystal elongation, or controls crystal habit. The findings will establish an appropriate knowledge base for framing future hypotheses concerning the molecular mechanisms by which enamelin controls enamel biomineralization.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE011301-08
Application #
6891388
Study Section
Special Emphasis Panel (ZRG1-OBM-2 (07))
Program Officer
Small, Rochelle K
Project Start
1997-01-01
Project End
2008-06-30
Budget Start
2005-07-01
Budget End
2006-06-30
Support Year
8
Fiscal Year
2005
Total Cost
$314,636
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Dentistry
Type
Schools of Dentistry
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Chan, Hsun-Liang; Giannobile, William V; Eber, Robert M et al. (2014) Characterization of periodontal structures of enamelin-null mice. J Periodontol 85:195-203
Hu, Jan C-C; Hu, Yuanyuan; Lu, Yuhe et al. (2014) Enamelin is critical for ameloblast integrity and enamel ultrastructure formation. PLoS One 9:e89303
Chan, Albert H-L; Lertlam, Rangsiyakorn; Simmer, James P et al. (2013) Bodyweight assessment of enamelin null mice. Biomed Res Int 2013:246861
Simmer, S G; Estrella, N M R P; Milkovich, R N et al. (2013) Autosomal dominant amelogenesis imperfecta associated with ENAM frameshift mutation p.Asn36Ilefs56. Clin Genet 83:195-7
Chen, Chiung-Fen; Hu, Jan Cc; Bresciani, Eduardo et al. (2013) Treatment considerations for patient with Amelogenesis Imperfecta: a review. Braz Dent Sci 16:7-18
Chen, Chiung-Fen; Hu, Jan Ching Chun; Estrella, Maria Regina Padilla et al. (2013) Assessment of restorative treatment of patients with amelogenesis imperfecta. Pediatr Dent 35:337-42
Simmer, James P; Richardson, Amelia S; Hu, Yuan-Yuan et al. (2012) A post-classical theory of enamel biomineralizationýýý and why we need one. Int J Oral Sci 4:129-34
Hu, Jan C-C; Lertlam, Rangsiyakorn; Richardson, Amelia S et al. (2011) Cell proliferation and apoptosis in enamelin null mice. Eur J Oral Sci 119 Suppl 1:329-37
Smith, Charles E; Hu, Yuanyuan; Richardson, Amelia S et al. (2011) Relationships between protein and mineral during enamel development in normal and genetically altered mice. Eur J Oral Sci 119 Suppl 1:125-35
Chan, H-C; Mai, L; Oikonomopoulou, A et al. (2010) Altered enamelin phosphorylation site causes amelogenesis imperfecta. J Dent Res 89:695-9

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