Functional model for amelogenesis: polarization and pH sensitivity of calcium uptake in ameloblast-derived HAT-7 cells.

Abstract

Enamel mineralization requires calcium (Ca²⁺) entering the matrix through the epithelium. The exact transepithelial Ca²⁺ transport process remains unclear. We have previously shown that rat-derived HAT-7 ameloblast cells, cultured in a polarized condition, achieve transepithelial bicarbonate transport. Our current aim was to determine the functional polarization of known amelogenesis-related Ca²⁺ transporters and channels in polarized HAT-7 cells. HAT-7 cells were cultured on Transwell membranes. The formation of a confluent monolayer was monitored by measuring transepithelial resistance. Relative mRNA expression of calcium channels was compared using TaqMan qPCR assays. Intracellular Ca²⁺ responses were followed by ratiometric Ca²⁺-imaging (fura-2 AM). Store-operated Ca²⁺ entry (SOCE) was activated by store depletion, using the SERCA inhibitor thapsigargin, while TRPM7 function was evaluated using the agonists naltriben and mibefradil. Functional polarization was determined by separate perfusion of the apical and basolateral membrane domains. The effects of both apical and basolateral extracellular acidification on calcium transport were also assessed. HAT-7 cells exhibited high Trpm7 mRNA expression, although much of the TRPM7 protein was localised to intracellular organelles. mRNAs for the SOCE channels Orai1, Orai2 and Orai3 were also detected. Both SOCE and TRPM7 activity showed functional polarization: SOCE being more prominent on the basolateral side while the TRPM7 activity was predominantly observed apically. Basolateral but not apical extracellular acidification reduced the basolateral SOCE-related Ca²⁺ entry. In summary, Ca²⁺ uptake mechanisms showed significant functional polarization in confluent HAT-7 monolayers, suggesting that HAT-7 cells may provide a model for transepithelial Ca²⁺ transport during enamel maturation.