IADR Abstract Archives
NCKX4 Is Critical For Enamel Matrix Protein Amelogenin Enzymatic Hydrolysis
Objectives: Enamel is the hardest and most mineralized tissue in the human body. Maturation ameloblasts are the major cells responsible for removing the organic matrix proteins, while also transporting calcium and phosphate into the enamel space to orchestrate the formation of this tissue. NCKX4, a potassium (K+) dependent sodium (Na+)/ calcium (Ca2+) exchanger, is highly expressed in the maturation stage of ameloblasts. Mice with the loss-of-function of NCKX4 (Nckx4-/-) display poorly mineralized enamel and a delayed removal of enamel matrix proteins in the late stage enamel matrix. This goal of this study is to investigate the role of NCKX4 in the removal of amelogenins by enamel matrix serine protease 1 (KLK4).
Methods: Hemimandibles from Nckx4+/+ and Nckx4-/- mice were subjected to microCT analyses to determine the mineral density of craniofacial hard tissues. First molars from Nckx4+/+ and Nckx4-/- mice were dissected on postnatal day 5 and 12, and total RNA was extracted to synthesize cDNA libraries, which served as templates for RT-qPCR gene expression analyses. Maturation stage of enamel matrix proteins extracted from Nckx4+/+ and Nckx4-/- mouse first molars were separated on SDS-PAGE gels followed by Coomassie staining and Western Blotting. Sagittal sections of hemimandibles from Nckx4+/+ and Nckx4-/- mice on postnatal day 21-22 were paraffin-embedded and processed for RNAscope analyses.
Results: Nckx4-/- mice had significantly reduced enamel mineral density, but similar dentin and alveolar bone mineral density as compared to Nckx4+/+ mice. Protein analyses showed that large size of amelogenin hydrolyzed fragments were retained in the maturation stage Nckx4-/- mouse enamel matrix although Klk4 transcripts were upregulated in Nckx4-/- mouse transition and mature ameloblasts compared to that of the wild type cells.
Conclusions: KLK4 enzymatic efficiency and amelogenins extracted from enamel matrix will be analyzed by kinetic analysis and mass spectrometry to confirm the roles of NCKX4 in the proper removal of enamel matrix proteins from the extracellular matrix through regulating the enzymatic hydrolysis of amelogenins.
Methods: Hemimandibles from Nckx4+/+ and Nckx4-/- mice were subjected to microCT analyses to determine the mineral density of craniofacial hard tissues. First molars from Nckx4+/+ and Nckx4-/- mice were dissected on postnatal day 5 and 12, and total RNA was extracted to synthesize cDNA libraries, which served as templates for RT-qPCR gene expression analyses. Maturation stage of enamel matrix proteins extracted from Nckx4+/+ and Nckx4-/- mouse first molars were separated on SDS-PAGE gels followed by Coomassie staining and Western Blotting. Sagittal sections of hemimandibles from Nckx4+/+ and Nckx4-/- mice on postnatal day 21-22 were paraffin-embedded and processed for RNAscope analyses.
Results: Nckx4-/- mice had significantly reduced enamel mineral density, but similar dentin and alveolar bone mineral density as compared to Nckx4+/+ mice. Protein analyses showed that large size of amelogenin hydrolyzed fragments were retained in the maturation stage Nckx4-/- mouse enamel matrix although Klk4 transcripts were upregulated in Nckx4-/- mouse transition and mature ameloblasts compared to that of the wild type cells.
Conclusions: KLK4 enzymatic efficiency and amelogenins extracted from enamel matrix will be analyzed by kinetic analysis and mass spectrometry to confirm the roles of NCKX4 in the proper removal of enamel matrix proteins from the extracellular matrix through regulating the enzymatic hydrolysis of amelogenins.