Enamel Defects in Kallikrein 4 Null Mice

Klk4 is a serine protease with broad target sequence specificity that is thought to degrade accumulated enamel matrix proteins, facilitating their removal and replacement by mineral, which is essential for hardening the enamel layer. In humans, KLK4 mutations cause autosomal recessive hypomaturation amelogenesis imperfecta. The enamel is less mineralized and is pigmented owing to failure of mineral to replace organic matrix. Objectives: To better understand Klk4 function we have used gene targeting to knock out normal Klk4 expression, while replacing the 5' Klk4 coding sequence with a ß-galactosidase (ß-gal) reporter. Methods: Heads from heterozygous (Klk4+/-) and null (Klk4-/-) mice at ages 4, 8, and 12 days were fixed in paraformaldehyde, processed for ß-galactosidase staining, counterstained with hematoxylin, and observed by light microscopy. Incisors and molars at 7 weeks were examined by scanning electron microscopy (SEM). Day 14 Klk4 null mouse molars and incisors were characterized by immunohistochemistry using amelogenin polyclonal antibodies. Results: X-gal staining of mouse sections shows intensive signal over maturation stage ameloblasts. No signal is observed in secretory stage ameloblasts, odontoblasts, or any other tissue in the head. A thick organic layer is observed within maturation stage enamel only in the Klk4 null mouse, and this material stains strongly with amelogenin antibodies, identifying the organic material as residual enamel proteins. SEM analyses of erupted teeth showed that the enamel prism pattern is normal, but the enamel layer fractures above the dentino-enamel junction (DEJ). The maturation stage enamel crystals appear to be thinner than normal and readily separate from one another. Conclusion: The Klk4 null mouse retains enamel proteins within the maturation stage enamel, preventing proper maturation (growth in width and thickness) of the enamel crystallites. We conclude that Klk4 is necessary for enamel maturation. This study is supported by NIDCR/NIH grant DE019775.