Kallikrein 4 (Klk4) is a serine protease expressed during dental enamel formation. In humans, KLK4 defects cause autosomal recessive, pigmented, hypomaturation amelogenesis imperfecta, a poorly understood condition for which there is no cure. Through gene targeting we have developed a Klk4 knock-out/ss-galactosidase knock-in mouse exhibiting enamel manifestations homologous to the human condition. Unlike wild-type mice, the Klk4 null mice retain enamel proteins in the enamel layer, and the enamel delaminates at or near the dentino-enamel junction. The Klk4 knock-out/ss-galactosidase knock- in mouse offers unique opportunities to better define the temporal and spatial patterns of Klk4 expression and to gain valuable information concerning Klk4's role in dental enamel formation in vivo. Furthermore we have developed efficient procedures for the isolation of matrix metalloproteinase-20 and Klk4 for in vitro analyses. Our overriding hypothesis is that Klk4 is required for enamel maturation and functions as a part of the system for removing enamel proteins that were secreted and partially digested during the secretory stage of amelogenesis.
Five specific aims are posed: SA1: To determine the temporal and spatial expression of Klk4 in ameloblasts during the secretory, transition, and maturation stages and in the underlying odontoblasts. SA2: To characterize enamel formation in the absence of Klk4 expression. SA3: To characterize the enzymatic activity of Klk4 on amelogenin, ameloblastin and enamelin. SA4: To investigate the expression of protease activated receptors (PARs) by ameloblasts. SA5: To identify other organs and tissues that express Klk4. The expression of Klk4 in ameloblasts and odontoblasts is determined by histochemistry using the ss- galactosidase (lacZ) expression assay and by immunohistochemistry at the light and electron microscopy levels. The enamel layer of the Klk4 null mouse is characterized by SEM, TEM, radiography, microCT, Knoop microhardness testing, and by determining protein and mineral contents in regularly-spaced increments along the developing maxillary and mandibular incisors. The residual enamel protein in the Klk4 null mouse is extracted and characterized, and digested with Klk4 to learn how Klk4 degrades the organic portion of maturation stage enamel. We determine if PARs are expressed in developing teeth by RT-PCR and in situ hybridization, and determine which PARs can be cleaved by Klk4 using synthetic fluorescent peptides. We expect to prove that Klk4 aggressively cleaves enamel proteins during the maturation stage of amelogenesis and that this function is essential for the proper maturation of enamel crystals.

Public Health Relevance

Kallikrein 4 (Klk4) is a serine protease expressed during dental enamel formation. In humans, defects in the KLK4 gene cause inherited enamel malformations known as autosomal recessive hypomaturation amelogenesis imperfecta, a poorly understood condition for which there is no cure. Through gene targeting we have developed a Klk4 knock-out mouse that has this disease. We will characterize the Klk4 knock-out to better understand the pathology in human patients and hope, eventually, to discover a cure.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE019775-04
Application #
8272467
Study Section
Special Emphasis Panel (ZRG1-MOSS-K (09))
Program Officer
Wan, Jason
Project Start
2009-09-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
4
Fiscal Year
2012
Total Cost
$350,977
Indirect Cost
$116,713
Name
University of Michigan Ann Arbor
Department
Biology
Type
Schools of Dentistry
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Kawasaki, Kazuhiko; Hu, Jan C-C; Simmer, James P (2014) Evolution of Klk4 and enamel maturation in eutherians. Biol Chem 395:1003-13
Simmer, James P; Richardson, Amelia S; Wang, Shih-Kai et al. (2014) Ameloblast transcriptome changes from secretory to maturation stages. Connect Tissue Res 55 Suppl 1:29-32
Suzuki, M; Shin, M; Simmer, J P et al. (2014) Fluoride affects enamel protein content via TGF-?1-mediated KLK4 inhibition. J Dent Res 93:1022-7
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
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
Bartlett, John D; Yamakoshi, Yasuo; Simmer, James P et al. (2011) MMP20 cleaves E-cadherin and influences ameloblast development. Cells Tissues Organs 194:222-6
Smith, Charles E; Richardson, Amelia S; Hu, Yuanyuan et al. (2011) Effect of kallikrein 4 loss on enamel mineralization: comparison with mice lacking matrix metalloproteinase 20. J Biol Chem 286:18149-60
Simmer, James P; Hu, Yuanyuan; Richardson, Amelia S et al. (2011) Why does enamel in Klk4-null mice break above the dentino-enamel junction? Cells Tissues Organs 194:211-5
Simmer, J P; Papagerakis, P; Smith, C E et al. (2010) Regulation of dental enamel shape and hardness. J Dent Res 89:1024-38