Adaptor protein complex 2–mediated, clathrin-dependent endocytosis, and related gene activities, are a prominent feature during maturation stage amelogenesis

Abstract

Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real time PCR, we show that the expression of clathrin and adaptor protein subunits are up-regulated in maturation stage rodent enamel organ cells. AP-2 is the most up-regulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin dependent endocytosis, thus implying the likelihood of a specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also up-regulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1), cluster of differentiation 63 and 68 (Cd63 and Cd68), ATPase, H+ transporting, lysosomal V0 subunit D2 (Atp6v0d2), ATPase, H+ transporting, lysosomal V1 subunit B2 (Atp6v1b2), chloride channel, voltage-sensitive 7 (Clcn7) and cathepsin K (Ctsk). Immunohistological data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain® showed up-regulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor regulated pathway for the endocytosis of enamel matrix proteins. These data together define an endocytotic pathway likely used by ameloblasts to remove the enamel matrix during enamel maturation.

Function and repair of dental enamel – Potential role of epithelial transport processes of ameloblasts

“…A systematic characterization of the whole-cell currents in isolated rat ameloblasts and ameloblast originated cell lines should provide vital information about the identity of such channels. Particularly, CFTR is hypothesized to play a key role in vectorial Cl À and HCO 3 -transport in ameloblasts [1,37] but gene array complex expressional studies have also revealed the presence of calciumactivated Cl À channels in these cells [1,31,38]. Their functional significance has not yet been investigated in ameloblasts.…”

“…The presently available data on the acidification process are contradictory [38,44,50], but it is clear that ameloblasts have the molecular machinery to secrete bicarbonate ions into the enamel space to buffer the eight moles of protons liberating during one mol of hydroxyapatite formation (Fig. 3).…”

“…3). Maturation ameloblasts are thought to express carbonic anhydrase 2 and 6 (CA2, CA6), anion exchanger 2 (AE2), Na þ -HCO 3 -cotransporter (NBCe1), Na þ -H þ exchanger 1 (NHE1), cystic fibrosis transmembrane conductance regulator (CFTR) and pendrin (SLC26A4) [7,31,38,44]. Studies on loss of function of several of these proteins have indicated their involvement in mineralization (Fig.…”

Dental and Cranial Pathologies in Mice Lacking the Cl−/H+‐Exchanger ClC‐7

“…In this study we examined whether ClC-7 is also directly involved in amelogenesis, based on the fact that ameloblasts, like osteoclasts, express V-ATPase (Lin et al, 1994; Josephsen et al, 2010; Damkier et al, 2014) and ClC-7 (Lacruz et al, 2013). In addition, the acidic pH of the enamel matrix bordering ruffle-ended ameloblasts (RA) has long been observed (Smith, 1998), although it remains unclear whether the acidic pH is the result of active acid secretion by ameloblasts, or is the by-product of hydroxyapatite crystal synthesis (Damkier et al, 2014).…”

High-fluoride promoted phagocytosis-induced apoptosis in a matured ameloblast-like cell line

“…In our previous study, we found that millimolar concentrations of fluoride reduced chloride-channel-dependent receptor-mediated endocytosis in porcine ameloblast-like PABSo-E cells, and stimulated an acidic intracellular environment 6 . Multiple ways of vesicular endocytic trafficking can coexist simultaneously in the same cell type including ameloblasts 7,8 . Acidification is an important marker of phagosome maturation and the phagocytosed particles can be degraded only when the phagosome achieves a sufficiently low pH 9 .…”