Endocytosis has been referred to as being either receptor-mediated or fluid phase.1, 2 Receptor-mediated endocytosis is usually defined as clathrin-dependent.1 This is in part because the endocytotic cellular uptake of extracellular proteins frequently involves clathrin assemblies and clathrin adaptor protein (AP) complexes that are generally activated and assembled by a membrane-bound receptor-mediated event such as ligand binding. The most commonly studied AP complex is AP-2, found at the plasma membrane.3–9 AP-1 (Golgi-endosome trafficking) is also known to be dependent upon clathrin, but is associated with the trans-Golgi network (TGN) and endosomes.6, 10 Following the completion of the human genome project, which has in part included the discoveries of the adaptor protein AP-311 and AP-412, 13 complexes, it is accepted that AP-mediated, clathrin-independent trafficking can also occur,6, 10, 14–17 as well as clathrin-dependent but AP-2–independent endocytosis.18 Our previous understandings of “nonspecific” cellular uptake mechanisms are now being critically reevaluated based on these discoveries,19, 20 as well as the recent characterization of the elaborate molecular machinery required for macropinocytosis.21–23 Currently, there is no widely accepted model for the removal of the enamel matrix protein (EMP) debris (degraded EMPs) during enamel development (amelogenesis).
Mammalian enamel is a product of ameloblast cells that secrete a protein matrix into the extracellular enamel space. Ameloblasts orchestrate molecular events such that the protein matrix is removed and replaced by hydroxyapatite (Hap)-based mineral, eventually comprising ∼95% (weight %) of matured enamel.2 The formation of enamel involves two distinct stages recognized primarily on the activities and morphology of the enamel-producing cells (ameloblasts). The initial secretory function is followed by activities that control enamel maturation.2 During enamel maturation, ameloblasts must perform both a resorptive function, and act to control the movement of large quantities of ions required for the vast increase in volume of Hap crystallites that occurs at this stage. The cellular and molecular events of ameloblasts driving secretory function are well appreciated2, 24–29; however, the events regulating enamel maturation are only recently becoming better understood.30 As ameloblasts transition from the secretory stage to the maturation stage of amelogenesis their gene expression profiles dramatically change.30 Ameloblast function during enamel maturation can be broadly classified into functions that encompass the regulation of water31 and ion flux (eg, calcium, phosphate, bicarbonate, chloride),2 the maintenance of acid-base balance,26 and the removal of EMP debris.32
Some of our understanding of the removal of the organic enamel matrix during the events of amelogenesis is derived from rodent studies using a radiolabel amino acid “pulse” administered to the animal and studied at various time intervals following administration.2 Earlier studies that focused on the removal of the enamel organic matrix looked at either a passive diffusion of degraded matrix proteins past the dentin enamel junction (DEJ) and toward the dentin,33, 34 or nonspecific internalization (ie, macropinocytosis involving fluid-phase cellular uptake) by ameloblast cells,2, 24, 35–37 but have generally excluded receptor-mediated endocytosis. However, several studies have described coated pits and/or vesicles on the cytoplasmic surface of the apical pole of ameloblasts in the secretory stage38–40 and in the maturation stage, both during the smooth-ended41 and ruffle-ended36 phases. Note that during the maturation stage, ameloblasts change morphology, modulating from a ruffle-ended morphology (characterized by infolded plasma membranes at the apical end) to a smooth-ended morphology,42–45 with the ruffle-ended phase predominating during maturation.2, 46 Clathrin was first discovered in 1976 as being associated with coated vesicles,47 thus the coated pits/vesicles alluded to in these earlier studies38–40 likely represent clathrin-coated vesicles. Indeed, Franklin and colleagues40 conclude their study by stating that the “Tomes' processes of secretory ameloblasts are highly active in endocytosis” and that “some of this endocytosis is receptor-mediated.” Sasaki36 states that there are “two distinct endocytotic pathways in ruffle-ended ameloblasts: one involving direct pinosome formation and another involving coated vesicles.” Additional studies by Sasaki38, 39 and Sasaki and colleagues41 describe these endocytotic capabilities of the apical membranes of both the secretory and smooth-ended ameloblasts.
Additional insight into the resorptive abilities of ameloblasts came from intravenously (IV)-delivered horseradish peroxidase (HRP) experiments, or immunocytochemistry (IHC) of plasma proteins (albumin [ALB] and alpha 2HS-glycoprotein [AHSG]).37, 48–52 These plasma-derived large molecules were capable of intercellular movements from the circulation to the enamel matrix, and these molecules could also enter ameloblasts at the apical50, 51 and the basolateral membranes.37 These works collectively demonstrated that: (1) the intercellular movement of proteins from the papillary layer cells to the enamel matrix is possible, and thus the reverse direction intercellular movement of EMPs is also feasible; and (2) both the papillary layer cells and the ameloblasts absorb extracellular matrix proteins. During the secretory stage of amelogenesis the enamel organ is composed of four distinct cell layers (ameloblasts, stratum intermedium, stellate reticulum, and outer dental epithelium), and during the transition to maturation stage the stratum intermedium, stellate reticulum, and outer dental epithelium reorganize to form a structure with a rich vasculature known as the papillary layer.28 If attention is focused on maturation stage ameloblasts, the HRP uptake experiments by Skobe and Garant51 state that on the rare occasion they observed HRP in “reduced” (mature) ameloblasts it was at the apical membrane of ruffle-ended ameloblasts; this is in keeping with earlier work noting that one of the likely functions of the mature ameloblasts is the removal (resorption) of the organic material from the developing enamel.53 Similar experiments with HRP by Sasaki50 concur that it was the “distal” (apical) surface of ruffle-ended ameloblasts which was responsible for the absorption of HRP. Although these studies clearly show ameloblasts and papillary layer cells absorb extracellular proteins, they do not help answer the relative resorptive and absorptive contributions (uptake of ameloblast-derived enamel proteins) of ruffle-ended ameloblasts versus papillary layer cells, respectively. An additional challenge to the study of the movements of serum-derived molecules into enamel organ cells came with the more recent discoveries of a large presence of the ameloblast-derived secreted proteinases; most notably, matrix metallopeptidase 20 (Mmp20) expressed during secretory stage amelogenesis, and kallikrein-related peptidase 4 (Klk4) expressed during enamel maturation.54, 55 In addition, matrix metallopeptidases 2 and 9 (Mmp2 and Mmp9)56 and chymotrypsin-C (Ctrc)57 have been discussed as being present in the enamel organ at various stages of amelogenesis, although the significance and spatiotemporal distribution of these additional proteinases (Mmp2, Mmp9, and Ctrc) in the enamel organ is not yet as clearly defined as they have been for Mmp20 or Klk4.54–57 With the presence of these proteinases in the enamel matrix, the longevity of any protein that enters the enamel matrix is limited, including the enzymatic activity of HRP, making quantitative cell uptake observations difficult.
To identify the gene activities involved in enamel mineralization we previously conducted a genomewide transcriptome (DNA microarray) analysis of the enamel organ from rat incisors, comparing secretory and early-mid maturation and mid-late maturation stages.30, 58 Here we further characterize endocytotic-related activities in the enamel organ. We report that AP-2–mediated endocytosis and associated cellular activities are prominent features during enamel maturation. Our findings suggest that whereas all four AP complexes are expressed in the enamel organ, AP-2 is significantly upregulated during enamel maturation along with clathrin and clathrin-associated proteins.
Subjects and Methods
All vertebrate animal manipulation complied with Institutional and Federal guidelines. We have used both the rat and mouse incisor and molar models for our investigations. The rat and mouse incisors are similar in anatomy and show the same developmental sequences related to amelogenesis. The larger size of the rat incisors allowed us to more easily isolate secretory and maturation enamel organ cells for array and real-time PCR studies. For molar quantitative PCR (qPCR) this size distinction does not represent such a technical challenge. Likewise, immunolocalization studies are performed on rats and mice with appropriate species-specific antibodies.
Total RNA isolation and qPCR
Using the same dissection protocol described previously,30 10 male Wistar Hannover rats weighing between 100 and 110 g were used for tissue collection. For qPCR analysis secretory stage and maturation stage enamel organs were isolated.30 Cell homogenates were processed for RNA extraction by first filtering lysates through the QIAshredder (Qiagen, Valencia, CA, USA) column and then isolating RNA using the Qiagen RNeasy Mini kit. Reverse-transcribed PCR (RT-PCR) was obtained using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA), or the C-03 RT2 First Strand Kit from SABiosciences (Qiagen). Real-time PCR reactions were performed with either iQ SYBR Green Supermix (Bio-Rad) using rat-specific primers pairs designed and prepared by RealTimePrimers (Elkins Park, PA, USA; http://www.realtimeprimers.com/info.html; Supplemental Table S1A), or using RT2 Real-Time SYBR Green/Rox PCR master mix from SABiosciences (Qiagen) using primers prepared by SABiosciences following their respective recommended conditions. Note that the primers purchased from SABiosciences are proprietary, thus sequence information was not disclosed to us. Relative expression of mRNA was calculated using the delta-delta CT method.59, 60 The least variable housekeeping gene in the SA Biosciences custom-made array, which included hexokinase 2 (Hk2), beta-2 microglobulin (B2m), and ubiquitin C (Ubc), was β-actin (Actb), and hence we normalized all data to the expression of this gene. To assess potential DNA contamination, RGDC (Rattus norvegicus clone CA14 satellite DNA, GenBank accession# U26919) was included in the analysis.
The first mandibular molars from 4-day-old and 8-day-old Wistar Hannover rats were isolated and manually homogenized, and total RNA was extracted as described in the previous paragraph to be used for real-time PCR analysis. Molar dissections were undertaken to compare transcript levels of iron-transport–associated genes with their expression in incisors.
Wistar Hannover rats ∼200 g were perfused through the left ventricle with 4% paraformaldehyde (PFA). Mandibles were dissected out, soft tissues were removed, and hemimandibles were kept overnight in the same PFA solution at 4°C. Samples were decalcified in 4.13% EDTA (pH 7.3) for 4 weeks, washed, and embedded in paraffin for sectioning. Swiss Webster mice at day 10 postnatal were euthanized, mandibles were extracted, soft tissues were removed, and hemimandibles were immersed in 4% PFA overnight at 4°C. Samples were decalcified in 4.13% EDTA (pH 7.3) for 7 days, and tissues were embedded in paraffin and sectioned. Tissue sections were dewaxed and rehydrated and endogenous peroxidase was blocked with 0.3% H2O2 in methanol. Sections were blocked with 1% bovine serum albumin (BSA) and incubated overnight with primary antibodies to clathrin light chain (CLTA; ProteinTech, Chicago, IL, USA; Cat# 10852-1-AP at a dilution of 1:600), clathrin heavy chain (CLTC; AbCam, Cambridge, MA, USA; #ab21679 at a dilution of 1:1000), and adaptor-related protein complex 2, alpha 2 subnit (AP2A2; Sigma-Aldrich, St Louis, MO, USA; Cat #A9983 at a dilution of 1:200), all produced in a rabbit host. Additional rabbit-generated antibodies against transferrin receptor (TFRC; Protein Tech #10084-2-AP; diluted 1:50); ATPase, H+ transporting, lysosomal V0 subunit D2 (ATP6V0D2; AbCam #ab87059; diluted 1:20); mucolipin 1 (MCOLN1; AbCam #ab74857; diluted 1:500); Rab10 (ProteinTech #11808-AP; diluted 1:20); RAB24 (AbCam #Ab65058; diluted 1:300); and chloride channel, voltage-sensitive 7 (CLCN7; AbCam #ab86196; diluted 1:300) were also used. After washing, sections were incubated with biotinylated anti-rabbit secondary antibody (Vector Laboratories, Inc., Burlingame, CA, USA). Sections were then incubated with the R.T.U. Vectastain Elite ABC reagent (Vector Laboratories' catalogue# PK-7100) prior to incubation with either the AEC peroxidase substrate kit (Vector Laboratories; catalogue# SK-4200), or the DAB peroxidase substrate kit (Vector Laboratories; catalogue# SK-4100), as described in the protocol provided by the manufacturer. Sections were counter-stained with Mayer's hematoxylin, dehydrated through an ethanol series and a cover slip with mounting medium added.
Western blot analysis
Rab10, Rab24. and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) Western blot analyses were performed using protocols listed,30 and using the antibodies listed in the previous paragraph for IHC. In the case of the tubulin and clathrin Western blots, tissues were obtained from 200-g Wistar rats. Wistar rats were incapacitated by carbon dioxide inhalation and killed by cervical dislocation. Mandibles were removed and incisors were dissected free of the mandibular bone. One contralateral incisor was carefully wiped free of enamel organ and the apical border of the white opaque zone (marking the start of the maturation stage) and was marked by scoring the tooth with a scalpel blade. The remaining incisor, with the enamel organ in situ was snap frozen in liquid nitrogen. The frozen incisor was placed on a brass bar pre-cooled in liquid nitrogen and the previously marked/scored contralateral incisor was used to identify the boundary between the secretory stage and early maturation stage enamel organ. Approximately 4 mm of frozen enamel organ along with the underlying enamel were dissected apical to the boundary mark (giving a secretory stage enamel organ sample). Four millimeters (4 mm) of frozen enamel organ and underlying enamel were dissected incisal to the boundary mark (giving an early maturation stage sample). Finally, the next 4 mm of incisal frozen enamel organ were collected, the underlying enamel being too hard to cut at this stage (giving a late maturation stage sample). All samples were added immediately to a microcentrifuge tube containing 50 µL of nonreducing SDS-PAGE sample loading (containing a cocktail of protease inhibitors) and the tissue was homogenized using a pestle. Samples were heated at 90°C for 2 minutes, centrifuged at 20,000 g for 5 minutes, and 7.5-µL aliquots loaded on 10% minigels. Electrophoresis was carried out at 200 V and, once complete, gels were either stained with Coomassie brilliant blue or electrotransferred onto nitrocellulose membrane at 80 V for 1 hour. Blots were blocked overnight in 5% nonfat milk powder (Bio-Rad #170-6404) in Tris buffered saline containing 0.05% Tween (TTBS) at 4°C. Membranes were probed with the antibodies to CLTA and CLTC described in the previous paragraph for IHC (both diluted 1:1500 in TTBS) for 2 hours at room temperature. Blots were washed in TTBS and incubated in anti-rabbit immunoglobulin G (IgG) peroxidase conjugate (Sigma #A6154) for 1 hour at room temperature. Blots were washed and developed using a DAB staining kit (Sigma #D0426) in accordance with the manufacturer's instructions. Blots were then documented (Bio-Rad Chemidoc MP) and then incubated with anti-beta tubulin (TUBB) HRP conjugate (Abcam #ab21058) diluted 1:2000 at room temperature for 1 hour and developed using the DAB staining kit. Blots were documented and the tubulin bands used has a loading control to calculate relative amounts of clathrin present in each sample.
Micro–computed tomography, scanning electron microscopy, and microindentation hardness testing
To examine the enamel phenotype of Cd63-null61 and genetically-matched wild-type (WT) animals (siblings), micro–computed tomography (µCT) and scanning electron microscopy (SEM) were performed as described.62–66 Imaging was done on three WT and three Cd63-null mice in regions of fully matured enamel. Briefly, the samples were analyzed with Siemens MicroCAT II at the University of Southern California Molecular Imaging Center. The MicroCAT images were acquired with the X-ray source at 80 kVp and 250 µA. The data were collected at a high resolution of 9 µm. The reconstruction was done with Inveon Acquisition Workplace software (Siemens Medical Solutions, Knoxville, TN, USA) and COBRA reconstruction software (Visage Imaging Inc., San Diego, CA, USA). The acquisition proceeded for 28 to 29 minutes (750 rotation steps, 360-degree rotation). For each sample, 1024 × 1024 slices were taken for the area of interest.
Microindentation techniques were used to measure enamel hardness, also in fully matured enamel.63, 67, 68 For all mechanical testing, mature 2-month-old or older animals were studied. Twelve freshly extracted intact murine lower left incisor teeth, from 6 WT and 6 Cd63-null mice, arising from three litters, were studied. Teeth were kept moist at all times, mounted in slow-set epoxy resin, and sequentially ground in the sagittal plane to a 0.1-µm alumina finish using a semiautomatic polisher (Buehler, Lake Bluff, IL, USA). Loads of 100 g were used, with dwell times of 20 seconds using a customized manually operated Vickers microhardness tester. For each tooth, five indentations were made in the central parts of the enamel layers in the erupted incisal thirds of the teeth, but not including the incisal-most 1 mm, with the means being used to describe each tooth. Indentations were examined by light microscopy, using polarization, interference, light/dark field, and transillumination techniques. Measurements were made using a digital micrometer. Descriptive statistics were calculated and a Student's t test was performed (p < 0.05).
Cell culture assay
Ameloblast-like LS8 cells and culture conditions have been described.69, 70 A stock solution of 15 mg/µL EMP in 0.1% acetic acid was prepared using lyophilized Emdogain.69 Two separate experiments were performed. In experiment 1, LS8 cells were plated in six-well plates at 9 × 105 cells per well, and in experiment 2 at 1.2 × 106 cells per well. In each experiment 3x wells were used as controls (no Emdogain) and 3x wells were exposed to Emdogain. All cells were incubated for a period of 6 hours, after which the medium was replaced with fresh media. Cells were incubated overnight in standard cell-culture conditions (37°C, 5% CO2). Emdogain stock solution was added after 24 hours of incubation to a final concentration of 250 µg/mL (both experimental groups), or an equimolar concentration of acetic acid was added in the corresponding control wells for each experiment. Cells were treated for 6 hours and total RNA was isolated using RNeasy Mini Kit (Qiagen). Total RNA was quantified and tested for quality by photometric measurement using the Nanodrop (Thermo Fisher Scientific Inc., Wilmington, DE, USA). The QuantiTect Reverse Transcription Kit (Qiagen) was used for cDNA synthesis. qPCR was carried out on the iCycler (Bio-Rad) with mouse gene-specific primers (Supplemental Table S1B) and SYBR Green (Bio-Rad). Values were normalized to Gapdh using the delta-delta CT method.60
Gene selection criteria for qPCR analysis and tissue quality control criteria
Previously, using DNA array analysis, we identified that endocytotic-related activities were associated with the maturation stage of amelogenesis.30, 58 The array data suggested that components AP-2 and clathrin were clearly present in enamel organ cells, and although individual AP-2 and clathrin subunits did not meet the strict selection criteria for those most highly upregulated or downregulated gene transcripts,30, 58 the expression levels of many of the individual subunits increased in the maturation stage, thus warranting further investigation. Forty-nine genes were identified for further investigation based on the literature as representing the various activities that define ubiquitous endocytotic events (Supplemental Table S2). Real-time PCR was used on secretory stage and maturation stage enamel organ cells to better define endocytotic events in amelogenesis. Klk4,71 odontogenic ameloblast associated protein (Odam),30 Mmp20,72 and enamelin (Enam)73 were used as controls to confirm the accuracy of dental tissue dissections, and Actb as well as Hk2 were included as housekeeping controls. All in vivo, rat-derived data are normalized to Actb as reported.30, 58 As expected,30, 58, 74 Mmp20 and Enam mRNAs were significantly downregulated during enamel maturation, whereas Klk4 and Odam gene transcripts were highly upregulated during enamel maturation (Supplemental Table S2).
AP-2 and clathrin are prominent components in maturation stage
Quantitative mRNA data for each of the AP and clathrin subunits in secretory and maturation enamel organ cells and corresponding fold changes are shown (Supplemental Table S2). A graphical representation of these results is shown in (Fig. 1A, B). These data suggest that AP complex and clathrin-associated activities increase significantly during enamel maturation (Fig. 1A, B; AP-2 and clathrin subunits represented in black). It can be appreciated that in general, expression of individual AP-2 subunits are greater than for AP-1, AP-3, and AP-4 subunits. The adaptor protein complex 2, alpha 2 subunit (Ap2a2) and the clathrin light chain A (Clta) were upregulated ∼3.6-fold and ∼2.6-fold, respectively, during maturation (Supplemental Table S2, Fig. 1B). Note that levels of both Clta and the clathrin heavy chain (Cltc) were also identified in the secretory stage as well as maturation stage (Supplemental Table S2, Fig. 1A).
Immunostaining of clathrin and AP-2 protein in enamel organ cells
Immunoperoxidase staining of Clta, Cltc, and Ap2a2 identified the expression of these proteins both in ameloblasts and the papillary layer of maturation stage of enamel organ cells (Fig. 2A–D). For all three proteins, staining was more apparent in the cytoplasm of ameloblasts with a higher concentration at the apical pole (Fig. 2A–C, identified by a double asterisk). For Cltc, staining is apparent in the papillary layer cells. However, expression in the ameloblasts is less clear as staining may be due to an artifact created when the enamel layer separates from the apical pole of the cell during tissue processing (Fig. 2B). The apical pole is the area of the plasma membrane in close proximity to the surface of the enamel. These data are consistent with the expected localization of clathrin complexes involved with enamel matrix endocytosis.
Western blot analysis of clathrin light and heavy chains in the enamel organ cells
In keeping with the quantitative mRNA data, the Western blots for Clta and Cltc showed the presence of both proteins in enamel organ at all stages of development (secretion early maturation and late maturation) (Fig. 2E). The relative molecular mass of the proteins observed correlates well with the expected relative molecular mass values provided by the manufacturers of the CLTA and CLTC antibodies (∼27 kDa and ∼192 kDa, respectively). The results indicate that the CLTA and CLTC antibodies are highly specific for their relevant target proteins. Beta-tubulin (Tubb) was identified in all samples migrating as predicted at ∼50 kDa and showed that lanes were loaded equally with respect to cellular protein (Fig. 2F).
Iron transport and storage is upregulated during maturation stage amelogenesis in the rodent incisor
Although AP-2–mediated endocytosis has not been studied in enamel organ cells, AP-2–mediated endocytosis is widely recognized as a receptor-mediated, clathrin-dependent activity.75–78 A classically described receptor initiating AP-2/clathrin endocytosis is the transferrin receptor (Tfrc).79–81 Here both transferrin (Trf/Tf) and Tfrc transcripts were examined by qPCR. Little or no change was noted for Tf mRNA transcripts during amelogenesis but a 60-fold increase was seen for Tfrc in maturation relative to the secretory stage (Supplemental Table S2, Fig. 1). In keeping with these data, by IHC there is low immunoreactivity for Tfrc in early secretory enamel organ cells (data not shown), and there is increasing immunoreactivity from the secretory stage through the maturation stage in both the ameloblasts and papillary layer cells, with the stronger signal being seen in papillary layer cells (Fig. 3).
The intracellular ferrous ion (Fe2+) storage proteins, ferritin light chain and ferritin heavy chain (Ft and Fth1, respectively), were also analyzed. Both gene transcripts were present in remarkably high levels during maturation (threefold and 19-fold greater than Actb, respectively), and both increased (approximately eightfold and 14-fold, respectively) in the transition from secretory to maturation stages (Supplemental Table S2, Fig. 1). These data confirm earlier reports82, 83 indicating that the rodent maturation stage incisor enamel organ recruits, stores, and releases large quantities of iron during the later stages of enamel mineralization.
Expression of Tfrc is relatively unaltered during maturation stage in the rodent molar
We also investigated if the very characteristic iron transport and storage phenomena of late-stage amelogenesis of the rodent incisor were also evident in rodent molar teeth. We used qPCR to quantitate Tfrc mRNA levels from the enamel organ in rat mandibular first molars at day 4 (when the first molar enamel organ is primarily involved with secretion) and from day 8 (when the first molar enamel organ is primarily involved with maturation), and found no statistical differences (ANOVA; p > 0.05) in Tfrc levels between mRNA populations (data not included). These would suggest that iron-related cellular activities of rodent incisor and molar teeth differ significantly.
Lysosome/endosome-associated activities are upregulated during enamel maturation
Several endocytosis-related genes: lysosomal-associated membrane protein 1 (Lamp1), cluster of differentiation 63 (Cd63), cluster of differentiation 68 (Cd68), and lysosomal protein transmembrane 5 (Laptm5), were significantly upregulated during maturation stage amelogenesis when compared to secretory stage amelogenesis (Supplemental Table S2, Fig. 1A, B). Lysosomal-associated membrane protein 2 (Lamp2) was also examined, but no significant change was noted in expression levels between maturation and secretory stages (Supplemental Table S2). Besides their primary association with lysosomal and endosomal membranes,84–86 these proteins are also located on the plasma membrane and are involved with trafficking between the plasma membrane and endosomal/lysosomal structures involving an association with one or more adaptor protein complexes.10, 17, 81, 87–89 Also upregulated were members of the tetraspanin family: tetraspanin 4 (Tspan4) and tetraspanin 8 (Tspan8), and also Cd63 as noted at the beginning of this paragraph (Supplemental Table S2). These tetraspanins network and bind with integrins and play a role in cell-matrix dynamics.89–92 Tspan4, Tspan8, and Cd63 all increased approximately twofold in the transition from secretory stage to maturation stage amelogenesis. Our earlier data demonstrated Lamp1 and Cd63 protein in ameloblast cells including the plasma membrane at the apical pole,69, 72 and Lamp1, Lamp2, and Cd63 have been shown to directly interact with known EMPs in vitro.93 Thus, in addition to Tfrc, Lamp1, Lamp2, and Cd63 should also be considered potential EMP receptor candidates.69
Two subunits of the lysosomal proton pumps, these being the ATPase, H+ transporting, lysosomal V0 subunit D2 (Atp6v0d2), and the ATPase, H+ transporting, lysosomal V1 subunit B2 (Atp6v1b2), were upregulated during maturation stage amelogenesis. In addition, a lysosomal membrane-bound transient receptor potential cation channel (Mcoln1), and the lysosomal-specific enzymes cathepsin K, cathepsin S, dipeptidyl-peptidase 7, and tripeptidyl peptidase 1 (Ctsk, Ctss, Dpp7, and Tpp1, respectively) were also upregulated during maturation stage amelogenesis. Most notable from this list of upregulated genes are Atp6v1b2, Ctsk, Ctss, Dpp7, and Tpp1, that increased 3.1-fold, 10.1-fold, 2.7-fold, 1.8-fold, and 1.9-fold, respectively (Supplemental Table S2, Fig. 1). These data, being specific features of endosomes and lysosomes, are consistent with a role of endocytosis in the removal of the enamel extracellular organic matrix. Another related gene included for analysis was the T-cell, immune regulator 1, ATPase, H+ transporting, lysosomal V0 subunit A3 (Tcirg1) mediating acidification of eukaryotic intracellular organelles required for receptor-mediated endocytosis.94 Our data suggest that the levels of Tcirg1 mRNA are low relative to many of the other gene transcripts studied, and contrary to our published array data showing a significant increase in Tcirg1 transcripts (more than fivefold),30 qPCR data presented here suggest little or no fold change. This new data for Tcirg1 is also supported by a recently published study95 that suggests that if “ameloblasts translate the Tcirg1 transcripts,” then the resulting protein levels are “likely very low.” Immunolocalization was used to confirm expression of Atp6v0d2 and Mcoln1 in maturation stage ameloblasts and the surrounding papillary layer (Fig. 4A, C). There was an additional and notable observation that Atp6v0d2 has a very characteristic expression profile in odontoblasts, being essentially restricted to the secretory or apical pole and extending partially into the odontoblastic processes (Fig. 4B), suggesting that lysosomal proton pumps play an important role in dentinogenesis (dentin formation) as well as amelogenesis.
Two members of the RAS superfamily of small GTPases (Rab10 and Rab24) were also included for qPCR analysis because each has been implicated as having a function in endocytotic activities. Rab10 and Rab24 belong to a family of proteins that have distinct spatiotemporal profiles and identity within specific endocytotic apparatus.96, 97 Rab10 mRNA increased ∼1.6 fold, and Rab24 mRNA increased ∼2.9-fold (Supplemental Table S2). Rab10 and Rab24 proteins were expressed in mouse molar maturation stage ameloblasts and the papillary layer cells as shown by IHC (Fig. 4D, E). Western blot analysis was also used to examine the levels of Rab10 and Rab24 in secretory stage and maturation stage enamel organ cells (Fig. 4G). For Rab10 and Rab24 the qPCR and Western blot data (Supplemental Table S2, Fig. 4G) are confirmatory of our earlier array data30 showing the expression of both proteins increases in maturation stage, when compared to secretory stage, enamel organs.
Chloride channels are identified in ameloblast lysosomes
Mammals express nine members of a class of voltage-sensitive chloride channels (Clcn1–7, plus Clcnka and Clcnkb) characterized by inwardly rectifying selectivity.98 Figure 5A shows real-time PCR for all nine members of the voltage-sensitive chloride channel family (also known as the CLC family of genes) in the secretory stage and maturation stage enamel organ, indicating that Clcn7 is the most upregulated of these nine CLC genes. One-way ANOVA indicates that Clnc7 expression significantly varies between secretory and maturation (p < 0.05) with post hoc analysis revealing a significant increase in maturation (p < 0.05). For Clcn3, significant differences were noted between stages (p < 0.05) with a marked decrease in maturation as shown in post hoc analysis (p < 0.05). All real-time PCR data are normalized to Actb. Clcn7 is mainly found in lysosomes.99, 100 Lysosomal bodies degrade internalized molecules within a very acidic luminal pH (pH 4.5) generated by electrogenic proton pumps.101 Vacuolar-type ATPase (V-ATPase) pumps generate proton influx into the lumen of lysosomes using energy derived from ATP hydrolysis.102 In addition to proton influx, maintenance of acidic luminal pH requires a parallel anion pathway.103–105 Clcn7 has been recognized as the main Cl– permeation pathway in lysosomes, perhaps functioning as a Cl–/H+ antiporter106 resulting in H+ efflux but facilitating large accumulations of Cl– in the lumen.99, 100 The identification of Clcn7 in maturation stage ruffled-ended ameloblasts, and its discrete localization to intracellular organelles (Fig. 5B) suggests that Clcn7 in ameloblasts functions primarily within lysosomes to acidify luminal pH, thus forming part of the endocytotic apparatus of these cells.
The enamel of Cd63-null mice is apparently unaltered
Earlier data show that Cd63 is present in the ameloblast cytoplasm and is also localized to Tomes' processes of polarized ameloblasts.72 Data here shows that there is an approximately twofold increase in Cd63 mRNA expression in maturation stage enamel organ cells when compared to secretory stage cells (Supplemental Table S2, Fig. 1B). With this knowledge we asked if Cd63-null animals61 had an associated enamel pathology. Matured enamel from three Cd63-null mice (mutant animals numbered #4, #7, and #8 and were 14 months, 2 months, and 2 months old, respectively)61 and three age-matched littermate controls (WT animals numbered #3, #5, and #6 and were 14 months, 2 months, and 2 months old, respectively) were examined by µCT (Fig. 6A–D), SEM (Fig. 6E–H), and microindentation hardness testing using methodologies as described.62–66 All animals tested were 2 months old or older. All mutant and WT teeth, eg, animal #8 mutant and animal #6 WT, showed normal characteristics in their gross and microanatomical features (Fig. 6A–H). From µCT the enamel thickness was measured at a region at the point of eruption, perpendicular to the enamel surface (as shown in Fig. 6A, C), and at the point of greatest width as judged from the µCT images (as shown in Fig. 6B, D). For all samples tested the enamel width ranged from 160 to 180 µm with no statistically significant difference between the mutant and WT teeth (as assessed by the Student's t test with p > 0.05). SEM images of cross-sections of teeth prepared from approximately the same point that the µCT data was collected (at the point of eruption and perpendicular to the enamel surface) and the enamel architecture for the mature tooth region of the mutant animal, when compared to WT, appears normal throughout the entire thickness (Fig. 6G compared to Fig. 6E), and the rod/interrod architecture of the mutant also appears normal (Fig. 6H compared to Fig. 6F). Microindentation and light microscopy did not identify any structural or hardness differences between the mutant and age-matched WT control mice (Fig. 6I). WT and mutant mice had mean enamel microhardness values of 2.54 (SD 0.19) and 2.49 (SD 0.22) GPa, respectively (p = 0.7). In summary, the matured enamel of Cd63-null and WT age-matched littermate controls are similar in linear thickness, structural architecture, and in microhardness, a surrogate for degree of mineralization, wear resistance, and acid resistance.
LS8 cells respond at a transcriptional level when treated with enamel matrix proteins
Using confocal microscopy it has been shown that ameloblast-like LS8 cells rapidly take up Alexa Fluor 594-labeled Emdogain.69 Here we investigate if such an uptake/endocytotic activity involves changes in gene transcription for selected genes intimately tied to AP-2–mediated and clathrin-dependent endocytosis. When Emdogain is added to plated LS8 cells at a concentration of 250 µg/mL, after 6 hours of exposure, significant change in mRNA levels, as determined by the Student's t test (p < 0.05), are noted for Ap2a2, Ap2b1, Ap2m1, Ap2s1, Cltc, Lamp1, Lamp2, and Tpp1 ranging from ∼1.4-fold (for Ap2s1) to ∼3.1-fold (for Cltc) (Fig. 7). Additional exposure times were not studied, but a more comprehensive study may be of value for individual gene transcripts to establish kinetic data for transcriptional upregulation. Changes in gene expression for Clta, Cltb, Cd63, and Dpp1 were less marked (Fig. 7). These data demonstrate that the uptake of EMPs (Emdogain) by LS8 cells results in changes in transcriptional activities of a number of genes involved in endocytosis, and provides an approach by which endocytosis by enamel cells can be further investigated.
AP-2–mediated endocytosis is a receptor-mediated, clathrin-dependent activity.75–78 The classically described receptors include the low-density lipoprotein receptor (Ldlr), transferrin receptor (Tfrc), and the epidermal growth factor receptor (Egfr).79–81 It is intriguing that Tfrc transcripts increase significantly (∼60-fold) during maturation stage amelogenesis in the rodent incisor. Also intriguing is the fact that, using the yeast two-hybrid system, our earlier data identified the iron transport protein transferrin (Tf) as a protein binding partner of Enam.107 Transferrin, being an extracellular ferric ion (Fe3+) transport protein synthesized primarily in the liver, testis, and central nervous system,108 would not be expected to have significant mRNA fluctuations in enamel cells. The levels of Tf mRNA were relatively unaltered in the enamel organ cells from secretory to maturation stages (Supplemental Table S2), thus Tf can be used as an additional control gene transcript. One possible scenario for the removal of the enamel matrix debris would be through a direct interaction between EMP debris and Tf, and a resulting EMP/Tf/Tfrc-initiated AP-2 endocytotic pathway. Other protein partners of Tf have been identified, and these include the GABA(A) receptor-associated protein (Gabarap),109 leukocyte cell-derived chemotaxin 2 (Lect2),110 insulin-like growth factor 1 and 2 (Igf1 and Igf-2),111 and insulin-like growth factor binding proteins 1–6 (Igfbp1–6).112 Alternatively, EMPs may bind directly to Tfrc, resulting in an EMP/Tfrc-initiated AP-2 endocytotic pathway. Tfrc does have at least one other protein-binding partner besides Tf. Tfrc binds hemochromatosis (Hfe), which is a membrane protein, and because of the competitive nature of Tf and Hfe, this is a mechanism used to modulate cellular Fe3+ uptake.113 Ferritin, transferrin, and transferrin-binding sites have been studied in the maturation stage ameloblasts,82, 83 and it is clear from these studies that Tfrc is likely involved in the movement of Fe3+ into maturation stage ameloblasts from the papillary layer for later incorporation into the enamel matrix. Real-time PCR data shows that the ferritin light polypeptide (Ftl) is upregulated approximately eightfold, and Fth1 is upregulated ∼14-fold, when comparing maturation stage to secretory stage amelogenesis.
Our data suggest that there may be multiple ligands for Tfrc in amelogenesis, including EMP protein debris. However there is a note of caution with this interpretation; in rodents, maturing incisor enamel incorporates significant concentrations of iron, giving enamel a characteristic pigmentation,114 yet these changes in iron activities during enamel maturation may be limited to rodent incisor teeth. For example, we have used qPCR to quantitate Tfrc mRNA levels from the enamel organ in rat first molars at day 4 (when the first molar enamel organ is primarily involved with secretion), and from day 8 (when the first molar enamel organ is primarily involved with maturation), and found no statistical differences (ANOVA at p > 0.05) in Tfrc levels between mRNA populations. Thus, Tfrc could be considered a potential EMP receptor candidate; however, data from rodent molar teeth or from non-rodent species may question or dismiss Tfrc as a universal EMP receptor. It should be highlighted that amelogenesis in rodent incisors and molars differ in some characteristics and gene expression profiles, such as iron handling and transport. Hence Tfrc expression in incisors warrants further investigation.
Another established major pathway for the uptake of Tf located at the apical pole of some polarized epithelia (eg, the small intestine, renal proximal tubule, the visceral yolk sac, and the placental cytotrophoblast) is through the megalin-dependent cubilin-mediated endocytotic pathway.115–119 From our array data30, 58 there is no suggestion that either megalin (Lrp2) or cubilin (Cubn) are upregulated significantly during maturation stage amelogenesis, or even expressed at any appreciable level in enamel epithelia. This would suggest that in ameloblasts the megalin-dependent cubilin-mediated endocytotic pathway is not significantly active during amelogenesis and thus we have not investigated this link further.
Individually and collectively, Lamp1, Lamp2, and Cd63 have also been described as membrane-bound protein receptors initiating AP endocytotic activities by direct interaction with all four AP complexes.10, 17, 81, 87–89 This initial LAMP-AP complex recognition activity, and the subsequent trafficking of Lamp1, Lamp2, and Cd63 from the cell membrane to the lysosome, is initiated by a direct protein-protein interaction between a lysosomal targeting motif (GYXX∅; where X is any amino acid and ∅ is a bulky hydrophobic amino acid) located at the cytoplasmically located C′-terminus of all three LAMPs, and the mu/µ subunit of either AP-1, AP-2, AP-3, or AP-4 (Ap1m1, Ap2m1, Ap3m1, or Ap4m1, respectively).75, 87, 88, 120–122 We have shown that amelogenin (Amelx), or more accurately in the alternatively spliced amelogenin isoform LRAP, through a proline-rich region (PLSPILPELPLEAW), interacts directly with Lamp1, Lamp2, and Cd63 through a well-defined, externalized, 20–amino acid domain/motif, with high but not absolute homology, common to all three LAMP proteins.93 In Cd63 this binding domain is contained within the externalized “EC2” domain.93 This proline-rich Amelx/LRAP binding region is hydrophobic, largely disordered, and accessible to the external environment.93 We have also demonstrated that the externalized EC2 domain of Cd63 also interacts with full-length Enam and ameloblastin (Ambn).93, 107
Although our published data69, 93 speculated on EMP-LAMP interactions initiating AP complex-mediated endocytosis, it was unclear which of the four AP complexes was involved. Data presented here suggest that AP-2–mediated endocytosis is a prominent feature of amelogenesis, thus we are suggesting that AP-2–mediated, clathrin-dependent endocytosis may be a major pathway responsible for the resorption of some, if not all, of the enamel matrix organic debris. Using an osteoclast-based cell culture, this same activity has been described for the uptake of EMPs in osteoclasts.123 Whereas the transcript levels of AP-2 individual subunits are most prominent during amelogenesis, both the basal levels and fold changes of AP-1 subunits are also significant and suggest that AP-2 (to a greater extent), as well as AP-1 and AP-3 (to a lesser extent), may play a role in amelogenesis. Our data indicate that AP-4 is likely not involved to any significant degree in amelogenesis; however, all AP complexes are ubiquitously expressed in all cell types studied to date so specific functional roles for AP-1, AP-3, and/or AP-4, during amelogenesis, are feasible. In addition to the upregulation of all AP-2 subunit transcripts during enamel maturation, many lysosome/endosome–specific transcripts are also upregulated, most notably Lamp1, Cd63, Cd68, Atp6v1b2, and Ctsk. In an earlier study by Tye and colleagues124 qPCR was used to demonstrate that many of the lysosomal proteases, including Ctsk, Ctss, Dpp7, and Tpp1, were expressed in mouse mature enamel organ cells; however, the changing expression levels at various stages of amelogenesis were not investigated.124 We have also investigated the expression of Clcn7, and other CLC family members, in the enamel organ of secretory stage and maturation stage enamel organ cells. From the CLC data we believe that Clcn7 is involved in the lysosomal pathway of ameloblasts, in a similar fashion as has been described for the role of Clcn7 in osteoclasts.125, 126 In addition, our cell culture–based real-time PCR data presented here, showing significant upregulation of AP-2 and clathrin subunits, along with other endocytotic gene transcripts in LS8 cells following exposure to enamel matrix proteins, further support an enamel matrix–initiated signaling activity of enamel cells resulting in endocytosis. There thus appears ample evidence that receptor-mediated endocytosis is a feature of maturation amelogenesis.
Gene knockout animal models are often used to explain genotype-phenotype relationships. Cd63 knockout mice (Cd63-null) have recently been described,61 and we have examined the enamel phenotype and mechanical properties of these animals by µCT, SEM, and microindentation using methods previously published.64, 127 Here we found that in this Cd63 knockout mice model61 there is no clearly apparent phenotype that distinguishes the enamel from the knockout animal when compared to age-matched non-mutated littermates. What then remains an enigma is that, to the authors' knowledge, no dental disease states have been ascribed to mutations to any of the AP-2 subunits, or other genes highlighted in this study. There is, however, every reason to suggest that there is a functional redundancy between the four AP complexes, and also between Lamp1, Lamp2, and Cd63 in amelogenesis. For example, in Lamp1-null mice, Lamp2 is upregulated and appears to compensate for the loss of Lamp1 function,84, 128 whereas Lamp2-null mice are more critically impacted.128, 129 In the model for receptor-mediated endocytosis of EMPs, it is possible that Lamp1, Lamp2, and Cd63 all share a common activity; thus, mutating or eliminating the function of one or two of these proteins may have little impact on endocytosis during enamel maturation. This type of functional redundancy is noted among the AP complex subunits. For example, there are three unique genes coding for the sigma subunit of AP-1 (Ap1s1, Ap1s2, and Ap1s3) and mutations to one can be compensated for by one of the others.130 There are two mu subunits of AP-1 (Ap1m1 and Ap1m2) and these serve distinct functions with AP-1 clathrin-associated protein complexes, although each can compensate for the other if required.131 From knockout animals, it also appears that disruptions to the AP-3 complex, by targeting the delta subunit (Ap3d1), may be compensated for in some, but not all, cellular activities by an upregulation of AP-1 activity.132–134 Another example of functional redundancy among the groups of genes studied is seen in the subunits of the vacuolar proton-pumping ATPase, where Atp6v1b2 subunit upregulation can compensate for the loss of the Atp6v1b1 subunit in Atp6v1b1-null animals.135
Data presented indicate that AP-2–mediated, clathrin-dependent endocytosis is a prominent feature of amelogenesis, and endocytotic activity in enamel cells increases during enamel maturation. We also identified that Lamp1, Cd63, and Tfrc increase during enamel maturation. Membrane-bound Lamp1, Cd63, and/or Tfrc, through direct or indirect interaction with EMP debris, may initiate endocytotic activities during amelogenesis; however, such associations between the EMPs and membrane-bound receptors are only speculative at this stage and require further investigation. Finally, we present an ameloblast-like cell model system (LS8 cells) that endocytose EMPs, resulting in the upregulation of gene transcription for selected genes related to AP-2 and clathrin-associated endocytosis. These animal- and cell-derived in vivo data strongly suggest that the endocytosis of EMP debris involves ligand-receptor signaling activities where the ligands are the EMP. Taken together, these data advances our understanding of endocytotic-related events during the final stages of enamel mineralization.
Note Added in Proof
We also want to acknowledge that during the final data analysis and the writing of this manuscript, a fifth AP complex (AP-5) was identified.136 AP-5 associates with the late endosome compartment and does not associate with clathrin,136 thus the authors do not believe any of the data, or conclusions made in our study, are invalid.
All authors state that they have no conflicts of interest.
This work was supported by grants DE013404 and DE019629 from the National Institutes of Health. The heads of the Cd63 knockout mice were a kind gift from Drs. Paul Saftig and Renate Lüllmann-Rauch (University of Kiel, Germany). We thank Dr. Malcolm Snead for his continued support throughout this project, Ms. Susan Smith for assistance with Western blot data and analyses, Ms. Angelica Frausto and Mr. Pablo Bringas Jr. for their assistance with the tissue preparation for immunohistochemistry, and Ryan Park and Grant Dagliyan of the USC Molecular Imaging Center for their assistance in the collection and analysis of the µCT data. We also thank the two anonymous reviewers of this manuscript for their supportive comments and critiques that were of great value in crafting the final version of this article.
Authors' roles: RSL, SJB, SPL, CTO, CES, and MLP conceived and designed the experiments; RSL, SJB, XW, JMJ, SV, PH, and SNW performed the experiments; RSL, SJB, XW, JMJ, SV, PH, SNW, SPL, CTO, CES, and MLP analyzed the data; and RSL, SJB, SNW, SPL, CTO, CES, and MLP wrote the manuscript. All listed authors critically read, edited, and approved the final manuscript. MLP accepts full responsibility for the integrity of the data analysis.