The Role of Amelogenin for pH Buffering During Amelogenesis

Objectives: Amelogenin (AMELX) and KLK4 are critical for proper enamel formation. AMELX is known to regulate mineral phase and crystal alignment in vitro, yet whether it buffers extracellular matrix pH is unclear. To investigate mechanisms for pH regulation without amelogenin during early amelogenesis and during maturation stage with excess amelogenin retention, in the Klk4-null model, compared to wildtype.
Methods: To compare secretory, early and late maturation stages, RNA and protein in wildtype, Amelx-KO and Klk4-KO first molars were extracted at postnatal days 5, 9, and 12, respectively. RNA expression was quantified by qPCR (n=6), protein expression by Western blot (n=3) and immunohistochemistry (n=3) for: AMELX (FL-191, Santa Cruz), ameloblastin (AMBN) (M300, Santa Cruz), enamelin (ENAM) (C-18, Santa Cruz), carbonic anhydrase 6 (CA6) (139560, US Biologics) and anion exchanger 2 (AE2) (ab42687, Abcam). Significance was tested with ANOVA.
Results: Amelx-KO mice: Upregulation was seen in secretory stage for AMBN protein (4-fold) and RNA expression of AMBN (2-fold, p=0.02), ENAM (2-fold, p=0.10), KLK4 (7-fold, p=0.08), AE2 (2-fold, p=0.06), and CA6 (9-fold, p=0.12). In early maturation stage, upregulation was 4-fold (p=0.06) for ENAM RNA and 12-fold for AE2 protein, while in late maturation stage AE2 protein was 100-fold higher, but RNA expression was reduced 2-fold (p<0.001) and 10-fold (p<0.001) for CA6. Klk4-KO mice showed in early maturation stage upregulation for AE2 protein expression (4-fold) and for CA6 RNA levels (15-fold, p=0.1) and for late maturation stage (10-fold) for AE2 protein expression, yet, CA6 RNA levels were downregulated (10-fold, p=0.08).
Conclusions: Upregulation of AE2 in secretory stage Amelx-KO as well as in maturation stage Klk4-KO enamel supports a role of amelogenin as well as its cleavage products for regulation of extracellular pH either directly through buffering capacity or through the regulation of the rate of apatite and related proton formation.
Acknowledgements. NIH/NIDCR R01DE025865 (FB)