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.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Research Project (R01)
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Special Emphasis Panel (ZRG1-MOSS-K (09))
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Wan, Jason
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University of Michigan Ann Arbor
Schools of Dentistry
Ann Arbor
United States
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Núñez, Stephanie M; Chun, Yong-Hee P; Ganss, Bernhard et al. (2016) Maturation stage enamel malformations in Amtn and Klk4 null mice. Matrix Biol 52-54:219-33
Bartlett, J D; Simmer, J P (2015) New perspectives on amelotin and amelogenesis. J Dent Res 94:642-4
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
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
Suzuki, M; Shin, M; Simmer, J P et al. (2014) Fluoride affects enamel protein content via TGF-β1-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
Hu, Jan C-C; Chan, Hui-Chen; Simmer, Stephen G et al. (2012) Amelogenesis imperfecta in two families with defined AMELX deletions in ARHGAP6. PLoS One 7:e52052

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