Expression Patterns and Subcellular Localization of Carbonic Anhydrases Are Developmentally Regulated during Tooth Formation

Abstract

Carbonic anhydrases (CAs) play fundamental roles in several physiological events, and emerging evidence points at their involvement in an array of disorders, including cancer. The expression of CAs in the different cells of teeth is unknown, let alone their expression patterns during odontogenesis. As a first step towards understanding the role of CAs during odontogenesis, we used immunohistochemistry, histochemistry and in situ hybridization to reveal hitherto unknown dynamic distribution patterns of eight CAs in mice. The most salient findings include expression of CAII/Car2 not only in maturation-stage ameloblasts (MA) but also in the papillary layer, dental papilla mesenchyme, odontoblasts and the epithelial rests of Malassez. We uncovered that the latter form lace-like networks around incisors; hitherto these have been known to occur only in molars. All CAs studied were produced by MA, however CAIV, CAIX and CARPXI proteins were distinctly enriched in the ruffled membrane of the ruffled MA but exhibited a homogeneous distribution in smooth-ended MA. While CAIV, CAVI/Car6, CAIX, CARPXI and CAXIV were produced by all odontoblasts, CAIII distribution displayed a striking asymmetry, in that it was virtually confined to odontoblasts in the root of molars and root analog of incisors. Remarkably, from initiation until near completion of odontogenesis and in several other tissues, CAXIII localized mainly in intracellular punctae/vesicles that we show to overlap with LAMP-1- and LAMP-2-positive vesicles, suggesting that CAXIII localizes within lysosomes. We showed that expression of CAs in developing teeth is not confined to cells involved in biomineralization, pointing at their participation in other biological events. Finally, we uncovered novel sites of CA expression, including the developing brain and eye, the olfactory epithelium, melanoblasts, tongue, notochord, nucleus pulposus and sebaceous glands. Our study provides important information for future single or multiple gene targeting strategies aiming at deciphering the function of CAs during odontogenesis.

Importance of bicarbonate transport in pH control during amelogenesis — need for functional studies

“…The data are also in accordance with previous studies (Amasheh et al, 2009;Colegio, Van Itallie, McCrea, Rahner, & Anderson, 2002;Lal-Nag & Morin, 2009) showing the expression of Cldn1, Cldn4, and Cldn8 in maturation-stage ameloblasts (Hata, Kawamoto, Kawai, & Yamamoto, 2010;Inai, Sengoku, Hirose, Iida, & Shibata, 2008). As previous studies show, ion transporters necessary for intracellular and extracellular pH regulation such as Slc9a1/Nhe1, Slc4a2/Ae2, Slc4a4/Nbce1, Slc26a4/pendrin, and CFTR (Bronckers et al, 2011;Jalali et al, 2014; and the cytoplasmic Car2 isoform of carbonic anhydrases (Lacruz, Smith, Moffatt, et al, 2012;Reibring et al, 2014) are expressed by ameloblasts. We identified all of them in HAT-7 cells suggesting that this cell line is suitable as an experimental model for studying ameloblast acid/base transport.…”

“…We also found that the application of the membrane permeable carbonic anhydrase inhibitor acetazolamide partially inhibited the basolateral base flux in HAT‐7 cells suggesting that this mechanism is present, and coupled to H + extrusion via a basolateral Na + ‐H + exchanger such as NHE1 (Bori et al., ). Among the large number of different isoenzymes in the carbonic anhydrase gene family, the dominant isoform in ameloblasts is Car2 (Lacruz, Smith, Moffatt, et al., ; Reibring et al., ), the isoform that we found to be expressed in HAT‐7 cells (Bori et al., ).…”

Understanding Nucleus Pulposus Cell Phenotype: A Prerequisite for Stem Cell Based Therapies to Treat Intervertebral Disc Degeneration

“…Therefore, it is possible that other members of the CA family can serve as functional NP markers. Noteworthy, expression of CAII, CAIII, and CAVI has been shown in embryonic mouse notochord and/or NP [105]. In addition, robust expression of CAIII transcripts in early notochordal and NP has been shown [106].…”

The evolution of metazoan α-carbonic anhydrases and their roles in calcium carbonate biomineralization

“…In some metazoan groups, α-CAs have been described as a calcium-dissolving enzyme, as in the bones of vertebrates and the spicules of calcareous sponges such as Sycon raphanus [45]. Recently, eight CAs in murine, produced by ameloblasts, were identified to be involved in biomineralization during teeth development [46]. In corals, α-CA isoforms interact in the carbon cycle of the endosymbiotic zooxanthellae playing an indirect role in skeletal formation [19].…”