Solute carrier family 9 member A5 regulated by TGF-β is necessary for dental enamel formation

In this study, we aimed to elucidate the functional significance of transforming growth factor-β (TGF-β)-regulated SLC9A5 (NHE5) in enamel formation.

Mouse ameloblast-derived mHAT9d cells were treated with TGF-β1, TGF-β2, or TGF-β3, and the expression of Slc9a family members was analyzed via RNA sequencing (RNA-seq) and quantitative PCR (qPCR). Slc9a5-deficient mice were generated to examine enamel morphology. Enamel volume, mineral density, and structure were assessed using micro-computed tomography (μCT), scanning electron microscopy (SEM), and electron probe microanalysis (EPMA). Protein expression was analyzed via SDS-PAGE, western blotting, and immunohistochemistry.

RNA-seq and qPCR analyses revealed that TGF-β1 and TGF-β3 strongly induced Slc9a5 in a dose-dependent manner; however, TGF-β2 had a minimal effect. In Slc9a5-deficient mice, incomplete crown formation and pit-like defects were evident on postnatal day 5; however, enamel protein profiles and thickness formation at day 11 were comparable to those in wild-type mice. In contrast, by day 70, μCT revealed marked thinning of enamel and reduced mineral density, SEM showed cracks and surface defects, and EPMA demonstrated significantly lower calcium-to-phosphorus (Ca/P) molar ratios than in wild-type mice. These findings indicate that loss of Slc9a5 slightly affects protein secretion but causes defective mineral maturation and enamel fragility.

SLC9A5 is a downstream target of TGF-β signaling, which is indispensable for enamel maturation because it maintains ion transport and extracellular pH homeostasis in ameloblasts. Its deficiency leads to reduced mineral density, altered Ca/P composition, and progressive enamel loss. These findings underscore the essential role of SLC9A5 in the long-term structural stability of enamel.