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Role of NBCe1 and AE2 in Secretory Ameloblasts
1 University of Southern California, School of Dentistry, Center for Craniofacial Molecular Biology, 2250 Alcazar Street, CSA Room 103, Los Angeles, CA 90033, USA; Correspondence: * corresponding author, UCLA Division of Nephrology, 10833 Le Conte Avenue, Room 7-155 Factor Building, Los Angeles, CA 90095-1689, USA; ikurtz{at}mednet.ucla.edu
The H+/base transport processes that control the pH of the microenvironment adjacent to ameloblasts are not currently well-understood. Mice null for the AE2 anion exchanger have abnormal enamel. In addition, persons with mutations in the electrogenic sodium bicarbonate co-transporter NBCe1 and mice lacking NBCe1 have enamel abnormalities. These observations suggest that AE2 and NBCe1 play important roles in amelogenesis. In the present study, we aimed to understand the roles of AE2 and NBCe1 in ameloblasts. Analysis of the data showed that NBCe1 is expressed at the basolateral membrane of secretory ameloblasts, whereas AE2 is expressed at the apical membrane. Transcripts for AE2a and NBCe1-B were detected in RNA isolated from cultured ameloblast-like LS8 cells. Our data are the first evidence that AE2 and NBCe1 are expressed in ameloblasts in vivo in a polarized fashion, thereby providing a mechanism for ameloblast transcellular bicarbonate secretion in the process of enamel formation and maturation.
Key Words: amelogenesis • anion exchanger • enamel • sodium bicarbonate co-transporter
Enamel formation by ameloblasts is a complex process that involves the coordinated formation of apatite crystals within an organic matrix composed primarily of amelogenins (Smith, 1998). The function of ameloblasts changes temporally during secretory, transition, and maturation stages (Robinson et al., 1978). Each of these stages is characterized not only by changes in ameloblast morphology, but also by unique biochemical and functional processes that ensure the normal development of the extracellular matrix and mineralization of enamel. The process of enamel formation requires the tight control of extracellular pH and bicarbonate concentration (Sasaki et al., 1991; Smith, 1998). Apatite crystal growth, proteinase activity in the extracellular space, and the triggering of ameloblast morphologic changes are thought to be pH-dependent phenomena requiring that cellular transport processes controlling the extracellular fluid pH be coordinated in a highly controlled fashion (Smith, 1998;Takagi et al., 1998). During apatite crystal growth in vitro, it has been estimated that, in the generation of 1 mole of apatite, approximately 14 moles of protons are released (Smith et al., 2005). Moreover, given that enamel is the only mammalian tissue wherein post-volumetric crystal expansion occurs after the appositional growth phase is completed, the potential for excess proton release is much greater than for dentin and bone, for example. Previous studies have suggested that the ameloblasts form a barrier that is relatively impermeable to protons during the secretory stage of crystal seeding and during the maturation stage, when there is a growth in the volume of apatite crystals associated with a morphologic appearance of a ruffled apical ameloblast membrane (Smith, 1998; Hubbard, 2000). Moreover, it has been hypothesized that ameloblasts secrete bicarbonate across their apical membrane to buffer the proton load generated by apatite formation. The proton production rate and the bicarbonate secretory rate likely change temporally, given the pH differences that exist, depending on the ameloblast stage. Specifically, the net pH during the secretory stage is near neutral and becomes acidic during the transition stage, and modulates between neutral and acidic during the maturation stage (Sasaki et al., 1991; Takagi et al., 1998). SLC4 bicarbonate transporters play an important role in pH regulation and bicarbonate transport in various cell types (Pushkin and Kurtz, 2006). Transgenic mouse models and patient disease states have significantly improved our understanding of the role these transporter proteins play in mammalian biology. With regard to the process of enamel formation, mice lacking SLC4A2 encoding the anion-exchanger AE2 are edentulous (Gawenis et al., 2004). In addition, persons with SLC4A4 mutations encoding the electrogenic sodium bicarbonate cotransporter NBCe1 have enamel abnormalities (Dinour et al., 2004; Inatomi et al., 2004). Similar findings have recently been documented in mice lacking SLC4A4 (Gawenis et al., 2007). These findings suggest that AE2 and NBCe1 play an important role in ameloblast bicarbonate transport and in regulating the pH in the microenvironment adjacent to enamel formation. In this study, we examined the expression of AE2 and NBCe1 in mouse ameloblasts in vivo, to understand the roles of these two proteins in enamel biomineralization.
Animals All vertebrate animal manipulation complied with Institutional and Federal guidelines. The genetic background for the animals used to prepare formalin-fixed sections was a cross among strains CBA/J, DBA/2J, and C57BL/6J. These were "wild-type" animals with no genetic manipulation, but that had been used as control animals in previous studies (Paine et al., 2005; White et al., 2007). The genetic background of the animals for the frozen, unfixed sections was CD1 (ICR) (Harlan Labs, Indianapolis, IN, USA), and the sections were prepared and purchased from (Zyagen, San Diego, CA, USA).
Tissue Preparation and Immunolocalization
Ameloblast-like LS8 Cells
Expression of NBCe1 was Localized to the Basolateral Membrane of Secretory Ameloblasts The expression profile of NBCe1 in ameloblast cells was examined in mandibular sections prepared in a sagittal plane to include both molar and incisor teeth. The distribution of NBCe1 was clearly demonstrated (Fig. 1  ). Within the dental tissues, NBCe1 was highly expressed in secretory ameloblast cells in both the incisor (Figs. 1A, 1B) and molar teeth (Figs. 1C, 1D). As shown in a higher-power view (Figs. 1B, 1D), NBCe1 was expressed in a polarized fashion and was localized to the basolateral membrane in ameloblasts. Peptide-blocked antisera and pre-immune sera were used as controls and were negative (not shown).
Expression of AE2 was Localized to the Apical Membrane of Secretory Ameloblasts The expression profile of AE2 in ameloblast cells was initially examined in frozen, non-fixed sections because of the sensitivity of the AE2 antibody used in this study for fixation. The tissue was cut in a coronal (cross-sectional plane). Polarized, pre-secretory ameloblasts showed little immunoreactivity (data not shown). Secretory ameloblasts in incisor and molar teeth stained positively for AE2 (Fig. 2 ). In contrast to NBCe1 expression, the expression of AE2 was localized specifically to the apical membrane of secretory ameloblast cells (Figs. 2B, 2D). Unlike NBCe1, there was no expression of AE2 at the ameloblast basal membrane. Peptide-blocked antisera and pre-immune sera were used as controls and were negative (not shown).
RT-PCR Analysis Identified the Expression of NBCe1 and AE2 in Ameloblast-like LS8 Cells Unique variants of NBCe1 and AE2 are expressed in cultured ameloblast (LS8) cells (Fig. 3A ). To the extent that this cultured cell model is representative of secretory ameloblasts in vivo, AE2a and NBCe1-B appeared to be the predominant variant of each transporter that was expressed.
Proposed Cell Model of Bicarbonate Transport in Secretory Ameloblasts The polarized distribution of AE2 and NBCe1 suggests that these transporters play an important role in the transcellular vectorial transport of bicarbonate from the basolateral to the apical side of secretory ameloblasts. A cell model of bicarbonate transport in secretory ameloblasts, based on the expression profile of AE2, NBCe1 (this study), and CFTR (Arquitt et al., 2002; Sui et al., 2003), is presented (Fig. 3B  ). Basolateral bicarbonate uptake was mediated by NBCe1-B. In the absence of functional NBCe1-B, basolateral bicarbonate uptake and subsequent secretion was impaired, as suggested by the abnormal enamel phenotype in persons with NBCe1-B mutations and a mouse model phenocopying the loss of NBCe1 function (Dinour et al., 2004; Inatomi et al., 2004; Gawenis et al., 2007). Following basolateral bicarbonate uptake, apical bicarbonate secretion was mediated by AE2a coupled to CFTR-mediated chloride transport (Arquitt et al., 2002; Sui et al., 2003). Evidence for the essential role of AE2a-mediated bicarbonate transport in ameloblasts was indicated by the abnormal enamel phenotype detected in mice with loss of AE2 function (Gawenis et al., 2004).
Expression Levels of AE2a and NBCe1-B at Different pHs in LS8 Cells
The results of the present study demonstrate that ameloblasts possess specific cellular transport processes for the efficient secretion of bicarbonate during enamel formation, and provide the basis for a model of transcellular bicarbonate transport during enamel formation. Specifically, basolateral bicarbonate influx was mediated by NBCe1, and bicarbonate secretion across the apical membrane was mediated by AE2. If it is assumed that the ameloblast-like LS8 cell line’s gene expression profile is representative of ameloblasts in vivo, the variants of AE2 and NBCe1 expressed on the apical and basolateral membranes of secretory ameloblasts are AE2a and NBCe1-B, respectively. In the LS8 cell model system, the levels of AE2a and NBCe1-B mRNAs were pH-dependent, with both transcripts showing the highest level of expression below pH 7.0. The effect of the pH microenvironment on the erosion of enamel in adult teeth is well-known (Brown et al., 2007). The pH in the ameloblast microenvironment also plays a critical role in enamel maturation during tooth development. Mice with a targeted disruption of AE2 (Gawenis et al., 2004) have been reported to be edentulous. Whether these mice exhibit failure of tooth eruption vs. lack of tooth formation is unknown. Recently, it has been shown that mice lacking AE2a/b have an abnormality in incisor enamel maturation, whereas molars are less severely affected (Lyaruu et al., 2008). Our studies complement these findings and further suggest that it is the lack of the AE2a variant that plays a key role in preventing normal enamel maturation. In addition to AE2, mice lacking the CFTR Cl– channel also have hypomineralized incisor enamel and retention of enamel matrix protein (Wright et al., 1996a,b). These findings are in keeping with those from experiments that have localized CFTR mRNA expression to the apical tooth bud (Arquitt et al., 2002), and with the finding that the enamel matrix pH is generally lower and is modulated differently in CFTR-deficient mice (Sui et al., 2003). While CFTR is expressed in developing molars, the molar enamel formation in CFTR-deficient mice is normal. Similarly, incisor-molar differences have been detected in AE2a/b-deficient mice and may result from differences between these types of teeth in extracellular pH regulation and/or ameloblast function. The loss of AE2 function results in a more severe tooth phenotype than either the loss of NBCe1 (Dinour et al., 2004; Inatomi et al., 2004; Gawenis et al., 2007) or CFTR-mediated transport (Wright et al., 1996a,b). Specifically, the incisors of mice lacking NBCe1 have a chalky white appearance and are prone to enamel fracture (Gawenis et al., 2007). In persons with loss of NBCe1 function, enamel defects have been described that appear as raised white and chalk-like spots (Inatomi et al., 2004). The NBCe1 mutations in these individuals involved all 3 NBCe1 variants (NBCe1A-C). Based on the results of the present study, it appears that the NBCe1-B variant mediates basolateral bicarbonate transport in ameloblasts. Importantly, the tooth phenotype in persons and mice with loss of NBCe1-B function is not characteristic of those with comparable metabolic acidosis. We hypothesize that the enamel defects are due to abnormal ameloblast function (loss of NBCe1-B transport per se), rather than to systemic acidosis. The specific membrane expression of AE2a and NBCe1-B in ameloblasts indicates that these cells should be viewed as a polarized layer of cells analogous to other bicarbonate-transporting (secretory) epithelia. We currently do not possess a complete detailed model of ion transport in ameloblasts, and it is therefore possible that, in addition to AE2a and NBCe1-B, other transport proteins, such as CFTR (Wright et al., 1996a,b; Arquitt et al., 2002; Sui et al., 2003), are involved in maintaining and modulating bicarbonate secretion at the different phases of ameloblast development. The exact role of CFTR in overall transcellular bicarbonate transport in ameloblasts is currently unknown and could involve an alteration in apical Cl–/HCO3– exchange mediated by AE2a, independent of its Cl– channel activity, CFTR-mediated apical bicarbonate secretion per se, and the indirect effect of CFTR on NBCe1-B function via changes in basolateral membrane potential (Sui et al., 2003). In addition, recent studies of pancreatic duct bicarbonate transport have documented the role of SLC26 transporters (SLC26A6) in regulating CFTR activity and its relevance to cystic fibrosis (Wang et al., 2006). It would therefore be of interest in future studies to address the potential role of SLC26 transporters in ameloblast bicarbonate secretion. It is currently thought that proton release during hydroxyapatite formation is buffered by the subsequent secretion of bicarbonate by ameloblasts (Smith, 1998). The finding that ameloblasts possess transport machinery for secreting bicarbonate raises the possibility that bicarbonate secretion by ameloblasts occurs independent of proton release during hydroxyapatite formation, and increases the peri-apical pH to a value that favors hydroxyapatite formation. Factors that potentially signal ameloblasts to secrete bicarbonate are predicted to play a central role in the biology of enamel formation.
This work was supported by NIH Grants DE06988, DE013404, DE014867, DK58563, DK63125, DK07789, DK077162, and ES012935, by the Max Factor Family Foundation, the Richard and Hinda Rosenthal Foundation, and the Fredricka Taubitz Fund. Received for publication June 4, 2007. Revision received December 20, 2007. Accepted for publication January 10, 2008.
Journal of Dental Research, Vol. 87, No. 4,
391-395 (2008) This article has been cited by other articles:
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