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Organic anion transport during rat enamel formation
Journal of Oral Biosciences 55 (2013) 40–46
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Original Article
Organic anion transport during rat enamel formation Ratnayake A.R.K. Ratnayake a, Dawud Abduweli a, Seong-Suk Jue b, Otto Baba a, Makoto J. Tabata a, Kaj Josephsen c,d, Ole Fejerskov c, Yoshiro Takano a,n a Section of Biostructural Science, Department of Bio-Matrix, Tokyo Medical and Dental University, Graduate School of Medical and Dental Sciences 1-5-45 Yushima, Bunkyo-ku 113-8549, Tokyo, Japan b School of Dentistry, Kyung Hee University, Seoul, South Korea c Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark d Department of Dentistry, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 November 2012 Received in revised form 2 December 2012 Accepted 3 December 2012 Available online 12 February 2013
Objective: The C-terminal end of nascent amelogenin is dissociated immediately after secretion and rapidly re-absorbed by ameloblasts, presumably by endocytosis. The purpose of this study was to test whether organic anion transporters (OATs) are also involved in the re-absorption process of enamel matrix proteins via non-endocytotic pathways. Materials and methods: Localization of OAT1, OAT2, and OAT3 in rat tooth germs was examined by immunohistochemistry using specific antibodies. Actual translocation of organic anions through the ameloblast layer was further tested by systemic tracer experiments in rats in which Lucifer Yellow (LY), a fluorescent organic anion, was used as a tracer. Results: In rat tooth germs, OAT2 was associated exclusively with the distal cell membranes of secretory ameloblasts where Tomes’ processes were developed and disappeared when matrix formation was terminated. On the other hand, OAT1 was absent in secretory ameloblasts and was colocalized with the ruffled border of ruffle-ended ameloblasts in the maturation stage. OAT3 was undetectable in ameloblasts and located instead only in the stratum intermedium cells. Systemic administration of LY resulted in intense labeling of immature enamel and also a transient labeling of the cytosol of secretory ameloblasts immunopositive for OAT2. In the maturation stage, cytosolic labeling of LY was negligible in all cells of the enamel organs, including ameloblasts. Conclusions: These data suggest the existence of OATs in rat tooth germs and their possible involvement in matrix re-absorption at least in the secretory stage of amelogenesis. & 2013 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Keywords: Organic anion transporter Ameloblast Enamel Absorption Lucifer Yellow
1. Introduction The organic anion transporter family is known to play an important role in the elimination of a variety of endogenous and exogenous harmful substances from the body [1]. Almost 2 decades ago, several groups cloned and identified a transport protein that is responsible for the complete excretion of para-aminohippuric acid (PAH) by the kidney [2,3]. This PAH transporter was found to interact with and excrete a variety of other negatively charged endogenous and exogenous protein molecules [4] and hence was renamed as organic anion transporter1 (OAT1) [5,6]. Thus far, 6 isoforms of OAT, all belonging to the solute carrier SLC 22 gene family, have been identified; in addition to the kidney, they are located in various organs, including liver, brain, and placenta,
n
Corresponding author. Tel.: þ81 3 5803 5439; fax: þ81 3 5803 5442. E-mail address: [email protected] (Y. Takano).
where they are involved in the excretion of a wide range of xenobiotics and endobiotics [4]. Tooth enamel formation or amelogenesis is roughly divided in 2 consecutive stages, the secretory stage and the maturation stage. In the secretory stage, tall columnar ameloblasts synthesize and secrete enamel matrix proteins. Once the full thickness of the enamel is laid down, the ameloblasts become typical transporting cells and regulate calcium influx and matrix removal in and out of the enamel throughout the process of enamel maturation [7–9]. For the highly mineralized enamel to form, extensive degradation and re-absorption of the organic matrix are essential [7]. To date, the absorption of degraded enamel matrix proteins is proposed to be mediated by ameloblasts via fluid phase endocytosis, upon which the proteins are processed in the lysosomal system [10–12]. Alternatively, the majority of the partially broken down matrix is removed by osmotic pressure created at stages of enamel maturation that involve smooth-ended ameloblasts [13]. In independent in vivo tracer experiments, we intravascularly injected rats with a fluorescent anionic dye as a tracer in order to
1349-0079/$ - see front matter & 2013 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.job.2012.12.002
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monitor its excretion from the kidneys. We noted intense fluorescence in the cytoplasm of secretory ameloblasts comparable to that in the transport epithelia of kidney tubules (unpublished data). We, therefore, hypothesized an involvement of the kidneytype non-endocytotic transcellular pathways in the elimination of degraded enamel matrix proteins by the enamel-forming cells. We envisaged this process might operate in addition to the classical endocytotic re-absorption pathways. To test this hypothesis, we performed an immunohistochemical study of the location of some organic anion transporters in the rat enamel organs. Moreover, we performed a systemic tracer experiment using a fluorescent tracer, Lucifer Yellow, a water-soluble organic anion, in order to monitor the actual movements of organic anions through the enamel organ in vivo.
2. Materials and methods All experimental protocols were approved by the Animal Welfare Committee of Tokyo Medical and Dental University (No. 0120176B) and carried out under the institutional guidelines for animal experimentation.
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2.2. Tracer experiment with Lucifer Yellow In order to trace the putative movement of organic anions in the growing tooth germs, we performed in vivo tracer experiments using Lucifer Yellow (dilithium salt, LYCH, MW, 457), a water soluble organic anion, as a fluorescent tracer. Under isoflurane anesthesia, 3-week-old male Wistar rats (n ¼12) were injected with 0.5% LY solution (1.5 mL/100 g body weight) into the external jugular vein within 30 s and euthanized by vascular perfusion as described elsewhere at 2–60 min after LY injection. Upper and lower jaws, including intact incisors and molars, were dissected and decalcified in 10% EDTA. Decalcified samples were dehydrated through an ascending ethanol series and embedded in Technovit 7100 (Heraeus, Wehrheim, Germany). All these processes were carried out in the dark to avoid loss of fluorescence in the tissue. Next, 3-mm Technovit sections were cut using a diamond knife and mounted on a glass slide with anti-fading mounting medium Vectashield (Vector Laboratories, Burlingame, CA, USA) and examined under an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan).
3. Results 3.1. Localization of OATs in the enamel organ
2.1. Animals and tissue preparation for immunohistochemistry Two- to three-week-old normal male Wistar rats weighing 25–40 g (n¼10) were anesthetized by isoflurane inhalation and perfused through the ascending aorta with saline for 2 min, followed by perfusion with 4% paraformaldehyde (PFA) in 0.1 M cacodylate buffer (pH 7.4) for 15 min. The upper and lower incisors and molar tooth germs with surrounding bones were dissected and further immersed in the same fixative overnight at 4 1C. The specimens were then decalcified for 2 weeks in neutral 10% ethylenediaminetetraacetic acid (EDTA) at 4 1C and routinely embedded in paraffin. The kidneys were also embedded in paraffin. Next, 4-mm thick longitudinal sections of the incisors and molar teeth were deparaffinized in xylene, rehydrated through a descending ethanol series, and rinsed in distilled water. In most cases, the sections were treated with TEG buffer (pH 9.0) for 15–20 min at 85 1C for antigen retrieval. After a brief wash in phosphate buffered saline (PBS), nonspecific binding sites were blocked by pre-incubating the sections in PBS containing 1% normal goat serum and 2% bovine serum albumin for 30 min.Subsequently, the section were incubated overnight at 4 1C with affinity purified polyclonal rabbit antibodies raised against a synthetic C-terminal peptide of the rat renal organic anion transporter 1 (OAT1; Gene Accession ]NP058920.1) and N-terminal peptide of rat renal OAT2 (Gene Accession ] 035913; Cat ] OAT11-A and -] OAT21-A; Alpha Diagnostics, San Antonio, TX, USA) and C-terminal peptide of rat renal OAT3 (Cat ] KE035; Transgenic Inc. Kobe, Japan). The specificity of the antibodies had already been validated by western blot and absorption tests [14–17]. After the sections were rinsed in PBS, they were incubated either with fluorescein isothiocyanate (FITC)- or streptavidin-conjugated goat-anti-rabbit IgG and processed for immunofluorescence and immunoperoxidase staining (ABC method) for the optical visualization of the immunoreactive sites. Negative controls were run by excluding primary antibodies from the reaction. Before microscopic examination, the sections were counterstained with DAPI (40 ,6-diamidino-2-phenylindole) or hematoxylin. Kidney sections were similarly immunostained and served as a positive control for OATs.
3.1.1. OAT1 In rat incisors, no OAT1 immunoreaction was observed in the cells of the enamel organ throughout the presecretory, secretory, and transitional stages of amelogenesis (Figs. 1a and 2d). Immunoreaction for OAT1 first appeared along the distal membranes of ameloblasts at the beginning of the maturation stage and increased in intensity toward the incisal end (Fig. 2a). The OAT1 immunoreaction was associated with the ruffled distal membranes of ruffle-ended ameloblasts (RA; Fig. 2b) and fluctuated in intensity in accord with the cyclical modulation of ameloblast morphology from the ruffle-ended to the smooth-ended cell types (Fig. 2c and e). OAT1 expression was very weak or negative when the ameloblasts were in the smooth-ended mode (Fig. 2c and e). Cells of the papillary layer did not show detectable OAT1 immunoreactivity. 3.1.2. OAT2 In contrast, distinct immunoreactions for OAT2 were observed only during enamel matrix secretion (Fig. 1b and c) located along the distal membranes of the Tomes’ processes of secretory ameloblasts (Fig. 1i). The OAT2 appeared at the onset of enamel matrix deposition (Fig. 1b) and persisted throughout the secretory stage terminating at the transition stage (Fig. 1c and d). In the molar tooth germs, an intense OAT2 immunoreaction was also located entirely to the distal end of secretory ameloblasts (Fig. 1k and l). 3.1.3. OAT3 In both incisors and molars, OAT3 immunoreactivity was confined exclusively to the cells of the stratum intermedium (Fig. 1e, f, g, j, and m), which showed a reaction corresponding to the peripheral part of the cells. In these cells, the OAT3 immunoreaction started in the presecretory stage (Fig. 1e) and extended into early maturation (Fig. 1g and m). 3.2. Localization of LY in the enamel organ In rat incisors, 15 min after intravenous injection of 0.5% LY solution, intense fluorescence of LY appeared in the full thickness of the growing enamel matrix and in the cytoplasm of secretory
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Fig. 1. Organic anion transporters (OATs) in the enamel organ of rat incisors and molars during the secretory stage of amelogenesis. (a) OAT1: Longitudinal section of the upper incisor showing absence of OAT1 immunoreactivity in the enamel organ cells throughout the secretory and transitional (T) stages. (b, c, d) OAT2: Combined images of a longitudinal section of the enamel organ of upper incisor at early (b), mid (c), and late (d) secretory stages. Distinct OAT2 immunoreaction appears at the distal end of ameloblasts concomitant with the onset of enamel matrix deposition (arrowhead in b) and is continuously expressed along the Tomes’ processes of secretory ameloblasts (arrow in c) until it disappears at the end of the secretory stage (bold arrow in d). (e, f, g) OAT3: OAT3 is located exclusively in the stratum intermedium (arrow in f) of the enamel organ through presecretory (e) to early maturation (g) stages of amelogenesis. (h, i, j) Enlarged images of OAT reactions at mid-secretory stage. OAT1 is negative (h), whereas OAT2 and OAT3 are located in the Tomes’ process (arrow in i) and stratum intermedium cells (arrow in j), respectively. (k, l, m) OAT2 (k, l) and OAT3 (m) immunoreactivity in the enamel organ cells of the third (k) and second (l, m) molar tooth germs. OAT2 is located along the Tomes’ processes of secretory ameloblasts (K, arrow in l) but abolished by the end of secretory stage (bold arrow) and remains negative in the transitional (T) and maturation stages. OAT3 is associated only with the stratum intermedium cells (SI in m). Dotted line in (k) indicates the mineralization front of dentin. Am, ameloblasts; bold arrows, border between secretory and transitional stages; arrowheads, onset of enamel formation; upward arrows, Tomes’ processes; downward arrows, stratum intermedium (SI). Bars in (a, d, g)¼100 mm; k ¼200 mm; j, m ¼ 20 mm.
ameloblasts. Fluorescence in the enamel matrix extended into the transitional stage of amelogenesis and then disappeared gradually. Odontoblasts, predentin, and osteoid in the surrounding bone also showed some fluorescence (Fig. 3). A closer examination of the enamel organ revealed that the LY labeling in ameloblasts was confined to the cytosol and nuclei, which were intensely labeled. No fluorescence was detectable in the lysosomal structures in the labeled ameloblasts (Fig. 3c and f). As shown in Fig. 3b, the labeling of ameloblasts with LY appeared exactly at the point where the enamel matrix started to form and disappeared abruptly at the point where the enamel matrix formation stopped (Fig. 3d). The intensity of LY labeling in the ameloblasts and the enamel matrix layer was time-dependent. After 5 min of injection, moderate fluorescence of LY was identified in the cytoplasm and nuclei of both ameloblasts and the stratum intermedium cells (Fig. 3e). Some fluorescence was also noted
in the nuclei of other cell types in the enamel organ. By 15 min, fluorescence in SI cells and other cells of the enamel organ had disappeared, whereas ameloblasts maintained moderate fluorescence in the cytosol (Fig. 3f). By 60 min, a considerable decrease in the intensity of LY fluorescence was noted in both ameloblasts and enamel matrix layers (data not shown). In the maturation stage, none of the cells of the enamel organ showed significant labeling with LY. Only the intercellular spaces between the smooth-ended ameloblasts (SA) and the narrow interface between SA and the maturing enamel surface (Fig. 3g) showed LY fluorescence, representing paracellular diffusion of the dye. LY fluorescence was also detected in the maturing enamel layer in specimens where the SA zone was located in early maturation. A similar pattern of LY fluorescence was observed in the enamel organ of rat molar tooth germs (data not shown).
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Fig. 2. OAT1 expression during postsecretory stages of amelogenesis. (a) Immunofluorescence of OAT1 first appears along the distal end of ameloblasts at the beginning of the maturation stage (arrow). The reaction increases toward the incisal direction (double arrows). (b) High magnification image of ruffle-ended ameloblasts (RA) and papillary layer (PL) of the upper incisor showing intense OAT1 immunoreactivity along the ruffled distal membrane (arrowheads) of RA (ABC method). (c) OAT1 immunoreactivity at the distal end of ameloblasts fluctuates in accord with cyclical modulation of ameloblasts. The smooth-ended ameloblasts (SA) only show trace amounts of OAT1 signals along the distal membrane (ABC method). (d, e) OAT1 immunoreactivity in the molar tooth germs. OAT1 is negative during secretory stage (d), but it is expressed along the ruffled membranes of ruffle-ended ameloblasts (RA) and fluctuates in accord with RA–SA modulation (e). S, secretory stage; T, transitional stage; Am, ameloblasts. Bars in (a and c) ¼ 50 mm; b, e¼20 mm.
4. Discussion The present study attempted to explore the possible routes of transport of organic anions during enamel formation. Two different approaches were used. First, we evaluated the presence and location of some OATs in the enamel organ. Next, we studied the dynamics of a water-soluble organic anion that acts as a fluorescent tracer in vivo. The results indicated the presence of nonendocytotic transcellular pathways, which we suggest may contribute to the various ways in which enamel matrix proteins are removed from the forming enamel during the different stages of enamel formation. To date, few data are available on how the organic matrix of tooth enamel, once produced and laid down by the secretory ameloblasts, is removed from the enamel and further processed. A recent breakthrough in our understanding is that enamel matrix proteins are cleaved by matrix metalloproteinase 20 (MMP20) immediately after secretion and further cleaved by kallikreinrelated peptidase 4 (KLK4)/enamel matrix serine proteinase 1 (EMSP1) during early maturation [18,19]. The peptides are then endocytosed by ameloblasts and further degraded in the lysosomal vesicles in these cells [19–21]. However, since ameloblasts apparently continue to synthesize and secrete enamel matrix proteins up to the early stages of enamel maturation [22], it is difficult to clearly distinguish lysosomal vesicles containing overproduced nascent enamel proteins from those containing extracellular enamel proteins internalized by endocytosis in both secretory and maturation stages [23,24].
4.1. Distribution of OATs and its relation to organic anion tracer in the enamel organ In this study, we suggest the presence of some OATs in the cells of the enamel organ. They seem to be localized particularly along the distal cell membranes of secretory ameloblasts and the ruffled border membrane of RA in both rat incisors and molars. The antibodies we used for immunohistochemistry were rabbit polyclonal antibodies raised against the synthetic peptides of rat kidney OAT1, OAT2, and OAT3; all showed the predicted localization patterns in the kidney samples that were used as positive controls. We are aware that while immunoreactivity in these experiments is a strong indication of OAT presence in tooth germs, it is not a conclusive evidence. This is because specific antibodies can also bind ‘‘specifically’’ to epitopes of unrelated proteins under certain conditions [25,26], and hence, better characterization of OATs in the enamel organ needs to be made in future studies. Despite this caveat, however, the exact temporal correlation between OAT2 expression in secretory ameloblasts and internalization of fluorescent organic anions, LY, in the cytosol of these cells appears to support an involvement of OAT2 as an organic anion transporter in secretory ameloblasts. LY is impermeable to biological membranes of most cell types, and hence, loading of cells with LY is only accomplished by microinjection, scrapeloading, or electrophoresis [27]. An exception is when appropriate transporters are present in the cell membrane, as is the case for kidney tubule cells [28]. In this regard, it is worthy to note that, in
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Fig. 3. Localization of Lucifer Yellow (LY) and hypothetical models for its translocation in the enamel organ of rat incisor. (a) Panoramic view of the localization pattern of LY in the labial aspect of upper incisor viewed in a sagittal section (15 min after intravenous injection of 0.5% LY solution). Note intense fluorescence of LY in the cytoplasm of secretory ameloblasts (Am) and also in the full thickness of growing enamel layers (E). Weaker fluorescence of LY extends into early maturation. Odontoblasts (Ob), predentin and osteoidin surrounding bones also show some fluorescence. Dentin (D) does not show significant labeling. Horizontal arrow indicates incisor direction. (b, c, d) Enlarged views of (a) at the initial (b), mid (c), and terminal (d) portions of the secretory stage. Note that cytosolic and nuclear fluorescence appears in ameloblasts concomitant with onset of enamel matrix formation (arrow in b) and disappears abruptly at the end of secretory stage (arrow in d). (e, f) Time-related changes of LY labeling in the enamel organ cells at the mid-secretory stage. At 5 min after injection (e), strong fluorescence of LY is in the cytosol and nuclei of both ameloblasts and stratum intermedium cells (SI). By 15 min (f), fluorescence in SI disappears, whereas secretory ameloblasts maintain fluorescence. Note that cytoplasmic lysosomal vesicles do not show fluorescence (arrows), suggesting the absence of endocytotic incorporation of LY by these cells (inset, enlarged view of lysosomal vesicles without fluorescent signals). (g) LY localization in the maturation stage at 15 min after injection. Both ruffle-ended (RA) and smooth-ended ameloblasts (SA) and cells of papillary layer (PL) do not show LY incorporation. LY is depicted only in the narrow intercellular spaces between SA and along the border between SA and maturing enamel surface (arrows). (h) Diagrammatic illustration of hypothetical transcellular translocation model of LY: LY enters into the cytosol of secretory ameloblasts (Am) via gap junctions (GJ) in the proximal membrane or other transporters, diffuses through the cytosol, and finally is transported to the enamel surface by OAT2 in the distal membrane. (i) Diagrammatic illustration of hypothetical non-endocytotic re-absorption model: LY first penetrates the ameloblast layer (Am) through paracellular channels into the forming enamel and is transported back into the cytosol of ameloblasts via OAT2. (j) Diagrammatic illustration of the area of ruffle-ended ameloblasts (RA) indicating blockade of paracellular diffusion of LY by tight junction (TJ): OAT1 in the ruffled border may contribute to non-endocytotic internalization of enamel matrix fragments, but due to absence of LY in enamel matrix, the cytosol of RA may not show LY fluorescence. Green arrows in each model (h, i, j) indicate the putative direction of transport/diffusion of LY through the ameloblast layer. Bars in (a)¼ 500 mm; b, g¼ 50 mm. Bar in inset (f) ¼5 mm.
our current LY tracer experiments, the lysosomal vesicles of secretory ameloblasts did not contain detectable fluorescent material, whereas the entire cytosol of the same cells displayed strong fluorescence of LY. Thus, it is obvious that the fluorescence in the cytosol of secretory ameloblasts as shown in Fig. 3 is not a reflection of LY being internalized by endocytosis but a consequence of non-endocytotic translocation of LY through the ameloblast membrane. Accordingly, the heavily loaded cytosol of
secretory ameloblasts with LY is an indication of the presence of functional channels or transporters for LY, and OATs are the likely candidates for such receptors. LY is known to diffuse freely into nuclei through nuclear pores [29]. Intense fluorescence of nuclei in the labeled ameloblasts as shown in Fig. 3b–f is therefore likely a consequence of passive diffusion of LY from the cytosol to nuclei through nuclear pores. Nuclear translocation of LY is not specific to ameloblasts, but a
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common phenomenon among various cell types. LY is a nontoxic compound without notable adverse effects on cell activities [30]. Accordingly, the accumulation of LY in the cytosol and nuclei of secretory ameloblasts likely reflects physiological translocation of organic anions in the enamel organ in our in vivo experiments, rather than a perturbation of cellular function by the dye. Since the orientation of putative transporters in secretory ameloblasts is yet to be determined, we propose 2 hypothetical models to explain LY transport. We term these the transcellular translocation model and the non-endocytotic re-absorption model (Fig. 3h, i, and j). In the transcellular translocation model, LY enters the stratum intermedium cells through OAT3, passes through gap junctions to the secretory ameloblasts, and is subsequently transported into the forming enamel by OAT2. In the non-endocytotic re-absorption model, LY penetrates the enamel organ paracellularly into the forming enamel and is then taken back up by ameloblasts via the transporter back into the cytosol. In previous tracer experiments using rat incisors, Sasaki et al. (1983) [12] and SASAKI (1984) [11] confirmed paracellular penetration of systemically administered horseradish peroxidase (HRP, 4.4 kDa), a proteinaceous tracer, into the enamel of rat incisors through the secretory ameloblast layer. We also confirmed a rapid paracellular diffusion of vascularly administered microperoxidase (MP, MW 1,900) and calcein or fluorexin (MW 622) through the secretory ameloblast layer of rat incisors (unpublished data). It is, therefore, likely that the intense labeling of immature enamel by LY (MW 457) is a consequence of paracellular diffusion of the dye through the secretory ameloblast layer (Fig. 3i).
spaces between RA [34] (Fig. 3j). Absence of LY fluorescence in the cytosol of SA, the site of paracellular diffusion in and out of maturing enamel, may be due to the absence of OAT1 in these cells. We assume that when the ameloblasts are in the SA mode, degraded fragments of enamel matrix proteins may simply be flushed out through the open intercellular channels between SA cells, which function as a sluice in the ameloblast layer [13]. Accordingly, although we currently have no direct evidence to support the functional significance of OAT1 in enamel maturation, we suggest that it is involved in the re-absorption of enamel matrix proteins during the process of enamel maturation. This could act as an additional mechanism to the endocytotic resorption of enamel matrix proteins by ameloblasts, as suggested in previous studies [19,20]. In conclusion, we have shown immunohistochemical localization of OAT1, OAT2, andOAT3 in the ameloblasts and stratum intermedium cells of the enamel organ of rat teeth. Our data support the role of the ameloblast layer as a transporting epithelium and suggest involvement of some OAT family members in the transportation of endogenous organic anions during the secretory and maturation stages of amelogenesis.
4.2. In situ cleavage of amelogenin and fate of degraded fragments
This work was supported in part by a JSPS Grant-in-Aid for Scientific Research (B) (No. 24390408) and also a grant from Aarhus University Research Fund (No. F-2007-FLS 1–67) and the Bagger-Sorensen Foundation. We thank Mr. Hachiro Iseki for his invaluable technical assistance and advice in tissue preparation and histological staining.
The nascent 25 kDa amelogenin is degraded by the co-secreted proteinase MMP20 in the surface layers of growing enamel immediately after secretion [18,19] and, in this process, a hydrophilic (anionic) C-terminal peptide of amelogenin comprising 25 amino acids (5 kDa) is dissociated [31,32]. Interestingly, however, the C-terminal fragments of amelogenin are reportedly undetectable in biochemical analysis of surface enamel [33]. Sasaki (1989) [33] explained that it is an indication of rapid re-absorption of the C-terminal fragments from surface enamel, possibly via endocytosis carried out by secretory ameloblasts. On the basis of our current observations, we suggest that at least a portion of the C-terminal fragments of amelogenin is re-absorbed by a nonendocytotic pathway via OAT2 in the distal membrane of ameloblasts for final cytosolic processing at the secretory stage of amelogenesis. In the subsequent maturation stage, where massive re-absorption of the further degraded enamel matrix proteins occurs, OAT1, instead of OAT2 is the major player in the putative non-endocytotic re-absorption carried out by ameloblasts. OAT1 in the ruffled border of RA cells may continue the uptake of remaining C-terminal fragments. Whether OAT1 also handles degraded amelogenins by KLK4 or EMSP1 in the early stages of enamel maturation is not yet known [32]. In the LY tracer experiment, the ameloblasts in the maturation stage did not show significant fluorescence of LY in the cytoplasm, unlike secretory ameloblasts. As shown in Fig. 3a, absence of LY fluorescence in the maturing enamel layer (except at the very beginning of the maturation zone) indicates a tight distal junctional complex between RA cells. The fluorescence of enamel in early maturation may be attributed to lateral diffusion of LY within the enamel layers. In RA, since OAT1 is located in the distal ruffled membrane, absence of fluorescence in the cytosol of these cells (Fig. 3a and g) can be explained by the absence of LY in the maturing enamel due to the distal tight sealing of the intercellular
Conflicts of interest All authors declare that there are no competing interests.
Acknowledgments
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ameloblasts during the maturation stage of amelogenesis in rat incisors. Anat Rec 1987;217:107–23. Burry RW. Specificity controls for immunocytochemical methods. J Histochem Cytochem 2000;48:163–6. Josephsen K, Smith CE, Nanci A. Selective but nonspecific immunolabeling of enamel protein-associated compartments by a monoclonal antibody against vimentin. J Histochem Cytochem 1999;47:1237–45. Hanani M. Lucifer yellow—an angel rather than the devil. J Cell Mol Med 2012;16:22–31. Masereeuw R, Moons MM, Toomey BH, Russel FG, Miller DS. Active lucifer yellow secretion in renal proximal tubule: evidence for organic anion transport system crossover. J Pharmacol Exp Ther 1999;289:1104–11. Stehno-Bittel L, Perez-Terzic C, Clapham DE. Diffusion across the nuclear envelope inhibited by depletion of the nuclear Ca2 þ store. Science 1995;270: 1835–8. Mobbs P, Becker D, Williamson R, Bate M, Warner A. Techniques for dye injection and cell labelling. In: Ogden DC, editor. Microelectrode Techniques, The Plymouth Workshop Handbook. 2nd ed.. Cambridge, UK: Company of Biologists; 1994. p. 361–87. Fincham AG, Moradian-Oldak J, Simmer JP. The structural biology of the developing dental enamel matrix. J Struct Biol 1999;126:270–99. Fukae M, Yamamoto R, Karakida T, Shimoda S, Tanabe T. Micelle structure of amelogenin in porcine secretory enamel. J Dent Res 2007;86:758–63. Sasaki S. In: Fearnhead RW, editor. Discussion in Tooth Enamel V. Yokohama: Florence Publishers; 1989. p. 76. Inai T, Sengoku A, Hirose E, Iida H, Shibata Y. Differential expression of the tight junction proteins, claudin-1, claudin-4, occludin, ZO-1, and PAR3, in the ameloblasts of rat upper incisors. Anat Rec (Hoboken) 2008;291:577–85.
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Original Article
Organic anion transport during rat enamel formation Ratnayake A.R.K. Ratnayake a, Dawud Abduweli a, Seong-Suk Jue b, Otto Baba a, Makoto J. Tabata a, Kaj Josephsen c,d, Ole Fejerskov c, Yoshiro Takano a,n a Section of Biostructural Science, Department of Bio-Matrix, Tokyo Medical and Dental University, Graduate School of Medical and Dental Sciences 1-5-45 Yushima, Bunkyo-ku 113-8549, Tokyo, Japan b School of Dentistry, Kyung Hee University, Seoul, South Korea c Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark d Department of Dentistry, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 November 2012 Received in revised form 2 December 2012 Accepted 3 December 2012 Available online 12 February 2013
Objective: The C-terminal end of nascent amelogenin is dissociated immediately after secretion and rapidly re-absorbed by ameloblasts, presumably by endocytosis. The purpose of this study was to test whether organic anion transporters (OATs) are also involved in the re-absorption process of enamel matrix proteins via non-endocytotic pathways. Materials and methods: Localization of OAT1, OAT2, and OAT3 in rat tooth germs was examined by immunohistochemistry using specific antibodies. Actual translocation of organic anions through the ameloblast layer was further tested by systemic tracer experiments in rats in which Lucifer Yellow (LY), a fluorescent organic anion, was used as a tracer. Results: In rat tooth germs, OAT2 was associated exclusively with the distal cell membranes of secretory ameloblasts where Tomes’ processes were developed and disappeared when matrix formation was terminated. On the other hand, OAT1 was absent in secretory ameloblasts and was colocalized with the ruffled border of ruffle-ended ameloblasts in the maturation stage. OAT3 was undetectable in ameloblasts and located instead only in the stratum intermedium cells. Systemic administration of LY resulted in intense labeling of immature enamel and also a transient labeling of the cytosol of secretory ameloblasts immunopositive for OAT2. In the maturation stage, cytosolic labeling of LY was negligible in all cells of the enamel organs, including ameloblasts. Conclusions: These data suggest the existence of OATs in rat tooth germs and their possible involvement in matrix re-absorption at least in the secretory stage of amelogenesis. & 2013 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Keywords: Organic anion transporter Ameloblast Enamel Absorption Lucifer Yellow
1. Introduction The organic anion transporter family is known to play an important role in the elimination of a variety of endogenous and exogenous harmful substances from the body [1]. Almost 2 decades ago, several groups cloned and identified a transport protein that is responsible for the complete excretion of para-aminohippuric acid (PAH) by the kidney [2,3]. This PAH transporter was found to interact with and excrete a variety of other negatively charged endogenous and exogenous protein molecules [4] and hence was renamed as organic anion transporter1 (OAT1) [5,6]. Thus far, 6 isoforms of OAT, all belonging to the solute carrier SLC 22 gene family, have been identified; in addition to the kidney, they are located in various organs, including liver, brain, and placenta,
n
Corresponding author. Tel.: þ81 3 5803 5439; fax: þ81 3 5803 5442. E-mail address: [email protected] (Y. Takano).
where they are involved in the excretion of a wide range of xenobiotics and endobiotics [4]. Tooth enamel formation or amelogenesis is roughly divided in 2 consecutive stages, the secretory stage and the maturation stage. In the secretory stage, tall columnar ameloblasts synthesize and secrete enamel matrix proteins. Once the full thickness of the enamel is laid down, the ameloblasts become typical transporting cells and regulate calcium influx and matrix removal in and out of the enamel throughout the process of enamel maturation [7–9]. For the highly mineralized enamel to form, extensive degradation and re-absorption of the organic matrix are essential [7]. To date, the absorption of degraded enamel matrix proteins is proposed to be mediated by ameloblasts via fluid phase endocytosis, upon which the proteins are processed in the lysosomal system [10–12]. Alternatively, the majority of the partially broken down matrix is removed by osmotic pressure created at stages of enamel maturation that involve smooth-ended ameloblasts [13]. In independent in vivo tracer experiments, we intravascularly injected rats with a fluorescent anionic dye as a tracer in order to
1349-0079/$ - see front matter & 2013 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.job.2012.12.002
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monitor its excretion from the kidneys. We noted intense fluorescence in the cytoplasm of secretory ameloblasts comparable to that in the transport epithelia of kidney tubules (unpublished data). We, therefore, hypothesized an involvement of the kidneytype non-endocytotic transcellular pathways in the elimination of degraded enamel matrix proteins by the enamel-forming cells. We envisaged this process might operate in addition to the classical endocytotic re-absorption pathways. To test this hypothesis, we performed an immunohistochemical study of the location of some organic anion transporters in the rat enamel organs. Moreover, we performed a systemic tracer experiment using a fluorescent tracer, Lucifer Yellow, a water-soluble organic anion, in order to monitor the actual movements of organic anions through the enamel organ in vivo.
2. Materials and methods All experimental protocols were approved by the Animal Welfare Committee of Tokyo Medical and Dental University (No. 0120176B) and carried out under the institutional guidelines for animal experimentation.
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2.2. Tracer experiment with Lucifer Yellow In order to trace the putative movement of organic anions in the growing tooth germs, we performed in vivo tracer experiments using Lucifer Yellow (dilithium salt, LYCH, MW, 457), a water soluble organic anion, as a fluorescent tracer. Under isoflurane anesthesia, 3-week-old male Wistar rats (n ¼12) were injected with 0.5% LY solution (1.5 mL/100 g body weight) into the external jugular vein within 30 s and euthanized by vascular perfusion as described elsewhere at 2–60 min after LY injection. Upper and lower jaws, including intact incisors and molars, were dissected and decalcified in 10% EDTA. Decalcified samples were dehydrated through an ascending ethanol series and embedded in Technovit 7100 (Heraeus, Wehrheim, Germany). All these processes were carried out in the dark to avoid loss of fluorescence in the tissue. Next, 3-mm Technovit sections were cut using a diamond knife and mounted on a glass slide with anti-fading mounting medium Vectashield (Vector Laboratories, Burlingame, CA, USA) and examined under an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan).
3. Results 3.1. Localization of OATs in the enamel organ
2.1. Animals and tissue preparation for immunohistochemistry Two- to three-week-old normal male Wistar rats weighing 25–40 g (n¼10) were anesthetized by isoflurane inhalation and perfused through the ascending aorta with saline for 2 min, followed by perfusion with 4% paraformaldehyde (PFA) in 0.1 M cacodylate buffer (pH 7.4) for 15 min. The upper and lower incisors and molar tooth germs with surrounding bones were dissected and further immersed in the same fixative overnight at 4 1C. The specimens were then decalcified for 2 weeks in neutral 10% ethylenediaminetetraacetic acid (EDTA) at 4 1C and routinely embedded in paraffin. The kidneys were also embedded in paraffin. Next, 4-mm thick longitudinal sections of the incisors and molar teeth were deparaffinized in xylene, rehydrated through a descending ethanol series, and rinsed in distilled water. In most cases, the sections were treated with TEG buffer (pH 9.0) for 15–20 min at 85 1C for antigen retrieval. After a brief wash in phosphate buffered saline (PBS), nonspecific binding sites were blocked by pre-incubating the sections in PBS containing 1% normal goat serum and 2% bovine serum albumin for 30 min.Subsequently, the section were incubated overnight at 4 1C with affinity purified polyclonal rabbit antibodies raised against a synthetic C-terminal peptide of the rat renal organic anion transporter 1 (OAT1; Gene Accession ]NP058920.1) and N-terminal peptide of rat renal OAT2 (Gene Accession ] 035913; Cat ] OAT11-A and -] OAT21-A; Alpha Diagnostics, San Antonio, TX, USA) and C-terminal peptide of rat renal OAT3 (Cat ] KE035; Transgenic Inc. Kobe, Japan). The specificity of the antibodies had already been validated by western blot and absorption tests [14–17]. After the sections were rinsed in PBS, they were incubated either with fluorescein isothiocyanate (FITC)- or streptavidin-conjugated goat-anti-rabbit IgG and processed for immunofluorescence and immunoperoxidase staining (ABC method) for the optical visualization of the immunoreactive sites. Negative controls were run by excluding primary antibodies from the reaction. Before microscopic examination, the sections were counterstained with DAPI (40 ,6-diamidino-2-phenylindole) or hematoxylin. Kidney sections were similarly immunostained and served as a positive control for OATs.
3.1.1. OAT1 In rat incisors, no OAT1 immunoreaction was observed in the cells of the enamel organ throughout the presecretory, secretory, and transitional stages of amelogenesis (Figs. 1a and 2d). Immunoreaction for OAT1 first appeared along the distal membranes of ameloblasts at the beginning of the maturation stage and increased in intensity toward the incisal end (Fig. 2a). The OAT1 immunoreaction was associated with the ruffled distal membranes of ruffle-ended ameloblasts (RA; Fig. 2b) and fluctuated in intensity in accord with the cyclical modulation of ameloblast morphology from the ruffle-ended to the smooth-ended cell types (Fig. 2c and e). OAT1 expression was very weak or negative when the ameloblasts were in the smooth-ended mode (Fig. 2c and e). Cells of the papillary layer did not show detectable OAT1 immunoreactivity. 3.1.2. OAT2 In contrast, distinct immunoreactions for OAT2 were observed only during enamel matrix secretion (Fig. 1b and c) located along the distal membranes of the Tomes’ processes of secretory ameloblasts (Fig. 1i). The OAT2 appeared at the onset of enamel matrix deposition (Fig. 1b) and persisted throughout the secretory stage terminating at the transition stage (Fig. 1c and d). In the molar tooth germs, an intense OAT2 immunoreaction was also located entirely to the distal end of secretory ameloblasts (Fig. 1k and l). 3.1.3. OAT3 In both incisors and molars, OAT3 immunoreactivity was confined exclusively to the cells of the stratum intermedium (Fig. 1e, f, g, j, and m), which showed a reaction corresponding to the peripheral part of the cells. In these cells, the OAT3 immunoreaction started in the presecretory stage (Fig. 1e) and extended into early maturation (Fig. 1g and m). 3.2. Localization of LY in the enamel organ In rat incisors, 15 min after intravenous injection of 0.5% LY solution, intense fluorescence of LY appeared in the full thickness of the growing enamel matrix and in the cytoplasm of secretory
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Fig. 1. Organic anion transporters (OATs) in the enamel organ of rat incisors and molars during the secretory stage of amelogenesis. (a) OAT1: Longitudinal section of the upper incisor showing absence of OAT1 immunoreactivity in the enamel organ cells throughout the secretory and transitional (T) stages. (b, c, d) OAT2: Combined images of a longitudinal section of the enamel organ of upper incisor at early (b), mid (c), and late (d) secretory stages. Distinct OAT2 immunoreaction appears at the distal end of ameloblasts concomitant with the onset of enamel matrix deposition (arrowhead in b) and is continuously expressed along the Tomes’ processes of secretory ameloblasts (arrow in c) until it disappears at the end of the secretory stage (bold arrow in d). (e, f, g) OAT3: OAT3 is located exclusively in the stratum intermedium (arrow in f) of the enamel organ through presecretory (e) to early maturation (g) stages of amelogenesis. (h, i, j) Enlarged images of OAT reactions at mid-secretory stage. OAT1 is negative (h), whereas OAT2 and OAT3 are located in the Tomes’ process (arrow in i) and stratum intermedium cells (arrow in j), respectively. (k, l, m) OAT2 (k, l) and OAT3 (m) immunoreactivity in the enamel organ cells of the third (k) and second (l, m) molar tooth germs. OAT2 is located along the Tomes’ processes of secretory ameloblasts (K, arrow in l) but abolished by the end of secretory stage (bold arrow) and remains negative in the transitional (T) and maturation stages. OAT3 is associated only with the stratum intermedium cells (SI in m). Dotted line in (k) indicates the mineralization front of dentin. Am, ameloblasts; bold arrows, border between secretory and transitional stages; arrowheads, onset of enamel formation; upward arrows, Tomes’ processes; downward arrows, stratum intermedium (SI). Bars in (a, d, g)¼100 mm; k ¼200 mm; j, m ¼ 20 mm.
ameloblasts. Fluorescence in the enamel matrix extended into the transitional stage of amelogenesis and then disappeared gradually. Odontoblasts, predentin, and osteoid in the surrounding bone also showed some fluorescence (Fig. 3). A closer examination of the enamel organ revealed that the LY labeling in ameloblasts was confined to the cytosol and nuclei, which were intensely labeled. No fluorescence was detectable in the lysosomal structures in the labeled ameloblasts (Fig. 3c and f). As shown in Fig. 3b, the labeling of ameloblasts with LY appeared exactly at the point where the enamel matrix started to form and disappeared abruptly at the point where the enamel matrix formation stopped (Fig. 3d). The intensity of LY labeling in the ameloblasts and the enamel matrix layer was time-dependent. After 5 min of injection, moderate fluorescence of LY was identified in the cytoplasm and nuclei of both ameloblasts and the stratum intermedium cells (Fig. 3e). Some fluorescence was also noted
in the nuclei of other cell types in the enamel organ. By 15 min, fluorescence in SI cells and other cells of the enamel organ had disappeared, whereas ameloblasts maintained moderate fluorescence in the cytosol (Fig. 3f). By 60 min, a considerable decrease in the intensity of LY fluorescence was noted in both ameloblasts and enamel matrix layers (data not shown). In the maturation stage, none of the cells of the enamel organ showed significant labeling with LY. Only the intercellular spaces between the smooth-ended ameloblasts (SA) and the narrow interface between SA and the maturing enamel surface (Fig. 3g) showed LY fluorescence, representing paracellular diffusion of the dye. LY fluorescence was also detected in the maturing enamel layer in specimens where the SA zone was located in early maturation. A similar pattern of LY fluorescence was observed in the enamel organ of rat molar tooth germs (data not shown).
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Fig. 2. OAT1 expression during postsecretory stages of amelogenesis. (a) Immunofluorescence of OAT1 first appears along the distal end of ameloblasts at the beginning of the maturation stage (arrow). The reaction increases toward the incisal direction (double arrows). (b) High magnification image of ruffle-ended ameloblasts (RA) and papillary layer (PL) of the upper incisor showing intense OAT1 immunoreactivity along the ruffled distal membrane (arrowheads) of RA (ABC method). (c) OAT1 immunoreactivity at the distal end of ameloblasts fluctuates in accord with cyclical modulation of ameloblasts. The smooth-ended ameloblasts (SA) only show trace amounts of OAT1 signals along the distal membrane (ABC method). (d, e) OAT1 immunoreactivity in the molar tooth germs. OAT1 is negative during secretory stage (d), but it is expressed along the ruffled membranes of ruffle-ended ameloblasts (RA) and fluctuates in accord with RA–SA modulation (e). S, secretory stage; T, transitional stage; Am, ameloblasts. Bars in (a and c) ¼ 50 mm; b, e¼20 mm.
4. Discussion The present study attempted to explore the possible routes of transport of organic anions during enamel formation. Two different approaches were used. First, we evaluated the presence and location of some OATs in the enamel organ. Next, we studied the dynamics of a water-soluble organic anion that acts as a fluorescent tracer in vivo. The results indicated the presence of nonendocytotic transcellular pathways, which we suggest may contribute to the various ways in which enamel matrix proteins are removed from the forming enamel during the different stages of enamel formation. To date, few data are available on how the organic matrix of tooth enamel, once produced and laid down by the secretory ameloblasts, is removed from the enamel and further processed. A recent breakthrough in our understanding is that enamel matrix proteins are cleaved by matrix metalloproteinase 20 (MMP20) immediately after secretion and further cleaved by kallikreinrelated peptidase 4 (KLK4)/enamel matrix serine proteinase 1 (EMSP1) during early maturation [18,19]. The peptides are then endocytosed by ameloblasts and further degraded in the lysosomal vesicles in these cells [19–21]. However, since ameloblasts apparently continue to synthesize and secrete enamel matrix proteins up to the early stages of enamel maturation [22], it is difficult to clearly distinguish lysosomal vesicles containing overproduced nascent enamel proteins from those containing extracellular enamel proteins internalized by endocytosis in both secretory and maturation stages [23,24].
4.1. Distribution of OATs and its relation to organic anion tracer in the enamel organ In this study, we suggest the presence of some OATs in the cells of the enamel organ. They seem to be localized particularly along the distal cell membranes of secretory ameloblasts and the ruffled border membrane of RA in both rat incisors and molars. The antibodies we used for immunohistochemistry were rabbit polyclonal antibodies raised against the synthetic peptides of rat kidney OAT1, OAT2, and OAT3; all showed the predicted localization patterns in the kidney samples that were used as positive controls. We are aware that while immunoreactivity in these experiments is a strong indication of OAT presence in tooth germs, it is not a conclusive evidence. This is because specific antibodies can also bind ‘‘specifically’’ to epitopes of unrelated proteins under certain conditions [25,26], and hence, better characterization of OATs in the enamel organ needs to be made in future studies. Despite this caveat, however, the exact temporal correlation between OAT2 expression in secretory ameloblasts and internalization of fluorescent organic anions, LY, in the cytosol of these cells appears to support an involvement of OAT2 as an organic anion transporter in secretory ameloblasts. LY is impermeable to biological membranes of most cell types, and hence, loading of cells with LY is only accomplished by microinjection, scrapeloading, or electrophoresis [27]. An exception is when appropriate transporters are present in the cell membrane, as is the case for kidney tubule cells [28]. In this regard, it is worthy to note that, in
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Fig. 3. Localization of Lucifer Yellow (LY) and hypothetical models for its translocation in the enamel organ of rat incisor. (a) Panoramic view of the localization pattern of LY in the labial aspect of upper incisor viewed in a sagittal section (15 min after intravenous injection of 0.5% LY solution). Note intense fluorescence of LY in the cytoplasm of secretory ameloblasts (Am) and also in the full thickness of growing enamel layers (E). Weaker fluorescence of LY extends into early maturation. Odontoblasts (Ob), predentin and osteoidin surrounding bones also show some fluorescence. Dentin (D) does not show significant labeling. Horizontal arrow indicates incisor direction. (b, c, d) Enlarged views of (a) at the initial (b), mid (c), and terminal (d) portions of the secretory stage. Note that cytosolic and nuclear fluorescence appears in ameloblasts concomitant with onset of enamel matrix formation (arrow in b) and disappears abruptly at the end of secretory stage (arrow in d). (e, f) Time-related changes of LY labeling in the enamel organ cells at the mid-secretory stage. At 5 min after injection (e), strong fluorescence of LY is in the cytosol and nuclei of both ameloblasts and stratum intermedium cells (SI). By 15 min (f), fluorescence in SI disappears, whereas secretory ameloblasts maintain fluorescence. Note that cytoplasmic lysosomal vesicles do not show fluorescence (arrows), suggesting the absence of endocytotic incorporation of LY by these cells (inset, enlarged view of lysosomal vesicles without fluorescent signals). (g) LY localization in the maturation stage at 15 min after injection. Both ruffle-ended (RA) and smooth-ended ameloblasts (SA) and cells of papillary layer (PL) do not show LY incorporation. LY is depicted only in the narrow intercellular spaces between SA and along the border between SA and maturing enamel surface (arrows). (h) Diagrammatic illustration of hypothetical transcellular translocation model of LY: LY enters into the cytosol of secretory ameloblasts (Am) via gap junctions (GJ) in the proximal membrane or other transporters, diffuses through the cytosol, and finally is transported to the enamel surface by OAT2 in the distal membrane. (i) Diagrammatic illustration of hypothetical non-endocytotic re-absorption model: LY first penetrates the ameloblast layer (Am) through paracellular channels into the forming enamel and is transported back into the cytosol of ameloblasts via OAT2. (j) Diagrammatic illustration of the area of ruffle-ended ameloblasts (RA) indicating blockade of paracellular diffusion of LY by tight junction (TJ): OAT1 in the ruffled border may contribute to non-endocytotic internalization of enamel matrix fragments, but due to absence of LY in enamel matrix, the cytosol of RA may not show LY fluorescence. Green arrows in each model (h, i, j) indicate the putative direction of transport/diffusion of LY through the ameloblast layer. Bars in (a)¼ 500 mm; b, g¼ 50 mm. Bar in inset (f) ¼5 mm.
our current LY tracer experiments, the lysosomal vesicles of secretory ameloblasts did not contain detectable fluorescent material, whereas the entire cytosol of the same cells displayed strong fluorescence of LY. Thus, it is obvious that the fluorescence in the cytosol of secretory ameloblasts as shown in Fig. 3 is not a reflection of LY being internalized by endocytosis but a consequence of non-endocytotic translocation of LY through the ameloblast membrane. Accordingly, the heavily loaded cytosol of
secretory ameloblasts with LY is an indication of the presence of functional channels or transporters for LY, and OATs are the likely candidates for such receptors. LY is known to diffuse freely into nuclei through nuclear pores [29]. Intense fluorescence of nuclei in the labeled ameloblasts as shown in Fig. 3b–f is therefore likely a consequence of passive diffusion of LY from the cytosol to nuclei through nuclear pores. Nuclear translocation of LY is not specific to ameloblasts, but a
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common phenomenon among various cell types. LY is a nontoxic compound without notable adverse effects on cell activities [30]. Accordingly, the accumulation of LY in the cytosol and nuclei of secretory ameloblasts likely reflects physiological translocation of organic anions in the enamel organ in our in vivo experiments, rather than a perturbation of cellular function by the dye. Since the orientation of putative transporters in secretory ameloblasts is yet to be determined, we propose 2 hypothetical models to explain LY transport. We term these the transcellular translocation model and the non-endocytotic re-absorption model (Fig. 3h, i, and j). In the transcellular translocation model, LY enters the stratum intermedium cells through OAT3, passes through gap junctions to the secretory ameloblasts, and is subsequently transported into the forming enamel by OAT2. In the non-endocytotic re-absorption model, LY penetrates the enamel organ paracellularly into the forming enamel and is then taken back up by ameloblasts via the transporter back into the cytosol. In previous tracer experiments using rat incisors, Sasaki et al. (1983) [12] and SASAKI (1984) [11] confirmed paracellular penetration of systemically administered horseradish peroxidase (HRP, 4.4 kDa), a proteinaceous tracer, into the enamel of rat incisors through the secretory ameloblast layer. We also confirmed a rapid paracellular diffusion of vascularly administered microperoxidase (MP, MW 1,900) and calcein or fluorexin (MW 622) through the secretory ameloblast layer of rat incisors (unpublished data). It is, therefore, likely that the intense labeling of immature enamel by LY (MW 457) is a consequence of paracellular diffusion of the dye through the secretory ameloblast layer (Fig. 3i).
spaces between RA [34] (Fig. 3j). Absence of LY fluorescence in the cytosol of SA, the site of paracellular diffusion in and out of maturing enamel, may be due to the absence of OAT1 in these cells. We assume that when the ameloblasts are in the SA mode, degraded fragments of enamel matrix proteins may simply be flushed out through the open intercellular channels between SA cells, which function as a sluice in the ameloblast layer [13]. Accordingly, although we currently have no direct evidence to support the functional significance of OAT1 in enamel maturation, we suggest that it is involved in the re-absorption of enamel matrix proteins during the process of enamel maturation. This could act as an additional mechanism to the endocytotic resorption of enamel matrix proteins by ameloblasts, as suggested in previous studies [19,20]. In conclusion, we have shown immunohistochemical localization of OAT1, OAT2, andOAT3 in the ameloblasts and stratum intermedium cells of the enamel organ of rat teeth. Our data support the role of the ameloblast layer as a transporting epithelium and suggest involvement of some OAT family members in the transportation of endogenous organic anions during the secretory and maturation stages of amelogenesis.
4.2. In situ cleavage of amelogenin and fate of degraded fragments
This work was supported in part by a JSPS Grant-in-Aid for Scientific Research (B) (No. 24390408) and also a grant from Aarhus University Research Fund (No. F-2007-FLS 1–67) and the Bagger-Sorensen Foundation. We thank Mr. Hachiro Iseki for his invaluable technical assistance and advice in tissue preparation and histological staining.
The nascent 25 kDa amelogenin is degraded by the co-secreted proteinase MMP20 in the surface layers of growing enamel immediately after secretion [18,19] and, in this process, a hydrophilic (anionic) C-terminal peptide of amelogenin comprising 25 amino acids (5 kDa) is dissociated [31,32]. Interestingly, however, the C-terminal fragments of amelogenin are reportedly undetectable in biochemical analysis of surface enamel [33]. Sasaki (1989) [33] explained that it is an indication of rapid re-absorption of the C-terminal fragments from surface enamel, possibly via endocytosis carried out by secretory ameloblasts. On the basis of our current observations, we suggest that at least a portion of the C-terminal fragments of amelogenin is re-absorbed by a nonendocytotic pathway via OAT2 in the distal membrane of ameloblasts for final cytosolic processing at the secretory stage of amelogenesis. In the subsequent maturation stage, where massive re-absorption of the further degraded enamel matrix proteins occurs, OAT1, instead of OAT2 is the major player in the putative non-endocytotic re-absorption carried out by ameloblasts. OAT1 in the ruffled border of RA cells may continue the uptake of remaining C-terminal fragments. Whether OAT1 also handles degraded amelogenins by KLK4 or EMSP1 in the early stages of enamel maturation is not yet known [32]. In the LY tracer experiment, the ameloblasts in the maturation stage did not show significant fluorescence of LY in the cytoplasm, unlike secretory ameloblasts. As shown in Fig. 3a, absence of LY fluorescence in the maturing enamel layer (except at the very beginning of the maturation zone) indicates a tight distal junctional complex between RA cells. The fluorescence of enamel in early maturation may be attributed to lateral diffusion of LY within the enamel layers. In RA, since OAT1 is located in the distal ruffled membrane, absence of fluorescence in the cytosol of these cells (Fig. 3a and g) can be explained by the absence of LY in the maturing enamel due to the distal tight sealing of the intercellular
Conflicts of interest All authors declare that there are no competing interests.
Acknowledgments
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