The goal of this application is to characterize the role of matrix metalloproteinase-20 (MMP20) and N- cadherin in ameloblast movement and cell-cell attachment during dental enamel development. MMP20 is essential for dental enamel formation. People and mice lacking functional MMP20 have strikingly malformed dental enamel that is thin, soft, and easily abrades from the underlying dentin. In Mmp20 null mice, the secretory stage ameloblasts do not enter the maturation stage of development properly and, once there, the ameloblasts overlap and grow atop one another. This suggests that ameloblast cell-cell attachment and signaling is altered in Mmp20 null mice. Cadherins are a family of proteins that span the cell membrane mediating attachment to identical cadherins present on adjacent cells. p120-catenin (p120) stabilizes cadherins to the cell surface and absence of p120 significantly reduces the presence cell surface cadherins. Previously, we showed that ablation of p120 in mice also results in malformed enamel that abrades from the teeth. Therefore, both MMP20 and cadherins are required for enamel formation. We will determine (AIM 1) how loss of MMP20 affects ameloblast cell-cell interaction. We hypothesize that MMP20 cleaves the extracellular domain of cadherins, which releases intracellular signaling molecules from the disrupted cadherin complex, such as ?-catenin, that are essential for enamel formation. Importantly, our preliminary data demonstrate that MMP20 cleaves the extracellular domain of E-cadherin and we propose to test our hypothesis by use of a stably transfected ameloblast derived cell line (ameloblast-lineage cells, ALC) that can be induced to express high levels of activated MMP20. Normal enamel has a decussating (interlacing) rod pattern. Each rod is formed by one ameloblast and each rod preserves a complete record of the migratory path of the ameloblast that formed it. Mmp20 null mouse enamel has either a highly dysplastic rod pattern or no rod pattern at all. We will determine (AIM 2) if MMP20 enhances ameloblast movement. We hypothesize that MMP20 cleaves the extracellular domains of cadherins and that this is required for ameloblasts to move synchronously in rows to form the complex decussating enamel rod patterns. Intriguingly, it is at precisely the initiation of movement that the ameloblasts switch from expressing predominantly E-cadherin to predominantly N-cadherin. N-cadherin expression in epithelial cells promotes cell movement. This opens exciting possibilities wherein MMP20 may facilitate the cadherin switch and facilitate cell movement via cadherin hydrolysis. We will determine (AIM 3) if N-cadherin ablation in ameloblasts disrupts the normal decussating enamel rod pattern. We hypothesize that the E-, to N-cadherin switch is essential for ameloblast movement and, therefore, for establishing the decussating enamel rods. Our overall hypothesis is that the E- to N-cadherin switch allows ameloblasts to move laterally in rows to form the decussating enamel rod pattern and that MMP20 facilitates this process by releasing these extracellular cadherin contacts and associated intracellular signaling factors.

Public Health Relevance

Ameloblast cells of the enamel organ are responsible for enamel development and these cells move in rows and signal to one another. When matrix metalloproteinase-20 (MMP20) is deleted from the mouse genome, the dental enamel becomes severely malformed. Importantly, four different MMP20 mutations are currently known to cause severely malformed dental enamel in humans. Therefore, MMP20 function is clinically important and is essential for human health. We will determine if MMP20 facilitates ameloblast movement and cell signaling by cleaving extracellular ameloblast cell-cell attachments that then release intracellular signaling molecules.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE016276-08
Application #
8711075
Study Section
Skeletal Biology Development and Disease Study Section (SBDD)
Program Officer
Wan, Jason
Project Start
2004-12-01
Project End
2015-08-31
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
8
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Forsyth Institute
Department
Type
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02142
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Bartlett, John D; Simmer, James P (2014) Kallikrein-related peptidase-4 (KLK4): role in enamel formation and revelations from ablated mice. Front Physiol 5:240
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Shin, Masashi; Hu, Yuanyuan; Tye, Coralee E et al. (2014) Matrix metalloproteinase-20 over-expression is detrimental to enamel development: a Mus musculus model. PLoS One 9:e86774
Guan, Xiaomu; Bidlack, Felicitas B; Stokes, Nicole et al. (2014) E-cadherin can replace N-cadherin during secretory-stage enamel development. PLoS One 9:e102153
Yamakoshi, Yasuo; Simmer, James P; Bartlett, John D et al. (2013) MMP20 and KLK4 activation and inactivation interactions in vitro. Arch Oral Biol 58:1569-77
Wang, S-K; Hu, Y; Simmer, J P et al. (2013) Novel KLK4 and MMP20 mutations discovered by whole-exome sequencing. J Dent Res 92:266-71
Pugach, M K; Suggs, C; Li, Y et al. (2013) M180 amelogenin processed by MMP20 is sufficient for decussating murine enamel. J Dent Res 92:1118-22

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