THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) IS EXPRESSED IN MATURATION STAGE AMELOBLASTS, ODONTOBLASTS AND BONE CELLS

Abstract

Patients with cystic fibrosis (CF) have mild defects in dental enamel. The gene mutated in these patients is CFTR, a Cl⁻ channel involved in transepithelial salt- and water transport and bicarbonate secretion. We tested the hypothesis that Cftr channels are present and operating in the plasma membranes of mouse ameloblasts.

Tissue sections of young mouse jaws and fetal human jaws were immunostained with various anti-Cftr antibodies. Specificity of the antibodies was validated in Cftr-deficient murine and human tissues. Immunostaining for Cftr was obtained in the apical plasma membranes of mouse maturation ameloblasts of both incisor and molar tooth germs. A granular intracellular immunostaining of variable intensity was also noted in bone cells and odontoblasts. In Cftr-deficient mice the incisors were chalky white and eroded much faster than in wild type mice. Histologically, only maturation ameloblasts of incisors were structurally affected in Cftr-deficient mice. Some antibody species gave also a positive cytosolic staining in Cftr-deficient cells. Transcripts of Cftr were found in maturation ameloblasts, odontoblasts and bone cells. Similar data were obtained in forming human dentin and bone.

We conclude that Cftr protein locates in the apical plasma membranes of mouse maturation ameloblasts. In mouse incisors Cftr is critical for completion of enamel mineralization and conceivably functions as a regulator of pH during rapid crystal growth. Osteopenia found in CF patients as well as in Cftr-deficient mice is likely associated with defective Cftr operating in bone cells.

Introduction

Cystic fibrosis (CF) is the most common lethal autosomal recessive disease in Caucasians, caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene [1]. CFTR is a chloride channel that is regulated by cyclic AMP-dependent protein phosphorylation and is required for transepithelial salt and water transport. The CFTR protein consists of two homologous repeats, each containing six membrane-spanning domains (MSD1&2, Fig. 1) followed by an intracellular nucleotide-binding domain (NBD1&NBD2). These two half-molecules are joined by an intracellular regulatory or R-domain. The MSD-domains form the central pore through which Cl⁻ ions cross the cell membrane. Recent evidence revealed that CFTR is also crucial for bicarbonate secretion [2–4]. Apart from its secretory function, CFTR controls the activity of other channels and transporters including the Na⁺/H ⁺ exchanger (NHE), HCO₃⁻/Cl⁻ exchanger and cotransporters, intermediate conductance outwardly rectifying (ICOR) Cl⁻ channels, and Ca⁺²- and volume-activated Cl⁻ channels (for review see [5, 6]).

Fig 1. Schematic map of the domains in the human CFTR gene.

MSD1 membrane spanning domain 1; NBD1 nucleotide binding domain 1; R regulatory domain; MSD2 membrane spanning domain 2; NBD2 nucleotide binding domain 2. The figures below the domains indicate amino acid numbering from 1-1480 of the hCFTR protein. To generate the Cftr knockout mouse strain a stop codon was inserted in the mouse gene after amino acid 487. The small black bars below the domains indicate approximate positions of the peptide sequences in human CFTR (or of corresponding sequences in mouse Cftr) used to generate antibody 3G11, CC24, MD1314 and 24-1. The grey bar indicates the position of the fragments for which the nucleotide sequence was detected by RT-PCR (Drawn and modified after: http://www.genet.sickkids.on.ca/cftr)

The tissues affected in CF patients include sweat glands, intestine, bile duct, pancreatic ducts, respiratory epithelia, submandibular glands, uterus and vas deferens [7–13]. Dental enamel, a product of epithelial cells is also (mildly) affected in some CF patients [14–17].

It was unclear for a long time whether enamel defects in CF patients were direct effects due to dysfunctional ameloblasts or secondary effects that follow medication, respiratory problems or malnutrition caused by intestinal obstruction, pancreatic insufficiency or poor intestinal absorption [14–17]. Enamel defects seen in Cftr knockout mice [18, 19], and detection of Cftr mRNA transcripts in developing teeth of wild type mice [20] suggested that this chloride channel operates also in ameloblasts.

The high rate of mineral acquisition during advanced stage enamel formation generates large quantities of protons and it has been proposed that ameloblasts buffer these protons by secreting bicarbonates into the enamel space [21–24]. Buffering these protons to near neutrality would enable further mineral growth [21, 23]. What molecular mechanisms are responsible for such vectorial bicarbonate secretion and at which developmental stage this happens is unknown. Ameloblasts form enamel in two steps: in secretory stage they secrete an enamel matrix that initiates and fosters formation of thin enamel crystals [reviewed by [21, 22]). This is followed by maturation stage. During first half of maturation stage the matrix is degraded and removed along with moderate increase in mineral. The second half of maturation stage when mineral acquisition is very high the (calculated) proton production is substantial [24]. In maturation phase two morphologically different types of cells have been recognized: ruffle-ended ameloblasts (the majority of the cells, with well developed membrane invaginations at their apical membranes), and smooth-ended ameloblasts with a smooth apical membrane [25]. During enamel maturation groups of these cells cyclically transform from ruffle-ended into smooth-ended cells and back multiple times, a process called modulation. Modulation is associated with alternation between narrow bands of enamel with neutral pH and much wider bands with slightly acidic pH, corresponding with smooth-ended cells and ruffle-ended cells, respectively [26–28]. The acidification is presumably is due to crystal growth. However in Cftr −/− mice maturation stage enamel is acidic, neutral pH bands are lacking [23] and matrix is retained [18]. The mutant ameloblasts do not modulate as assessed by failure to stain with glyoxal bis (2-hydroxyanil) (GHBA) [23] and enamel mineral accretion is severely reduced [17, 18, 19, 29]. These data suggest that a Cftr is critical for ameloblast modulation, pH regulation and for completion of enamel mineralization. Transcipts for Cftr mRNA have been detected in apical parts of mouse incisors and molars tooth germs [20]. The cells that express Cftr however have not been identified.

Given the relevance of Cftr for formation of fully matured enamel we tested the hypothesis that Cftr is expressed in the apical plasma membrane of maturation ameloblasts where it serves a critical role in pH regulation in developing enamel.

Materials and methods

Tissues

Jaws were collected from Cftr null mice (congenic FVB, three pups aged 13–17 days; five adult mice, aged 100–172 days), and littermate wild type controls (three pups and three adults). These ages covered all stages of enamel development in molar tooth germs and incisors. In addition, jaws from hamster (Mesocricetus auratus L) pups (total of 3, aged 3–7 days) and 16–20 week human fetal jaws (archival tissues) were used. Mice with targeted disruption in the Cftr gene (Cftr^tm2Cam knockout mice, backcrossed for more than 12 generations into the FVB background; Cftr −/−) were originally obtained from Dr. William Colledge (Cambridge, UK) [30]. The Cftr gene codes for a 1480 amino acid glycoprotein with molecular mass of 170 kD. The insertion of a HPRT minigene into the coding sequence of the murine cystic fibrosis gene (Cftr) at arg 487 introduced a termination codon in frame with the Cftr coding sequence to terminate prematurely the cftr protein within the first nucleotide binding domain (NBD1, Fig 1). The remaining portion consists of the N-terminal part and the first membrane-spanning domain (MSD1); this truncated Cftr-fragment is very labile and insoluble, and is degraded via proteasomes immediately after synthesis. The Cftr−/− mouse strain was kept on solid food in the animal facility at the Erasmus Medical Center in Rotterdam (The Netherlands) and genotyped using DNA from tail clippings. To reduce the incidence of intestinal obstruction, polyethylene glycol (17.6 mM; Klean Prep) was added to the drinking water. Human primary osteoblasts were obtained by outgrowth of cells from human morselized bone chips grown as cultures and provided by the Department of Orthopedics of the VU-University Hospital in Amsterdam. Freshly extracted human wisdom teeth were obtained from the Department of Oral and Maxillofacial Surgery of Academic Centre for Dentistry Amsterdam (ACTA). All animal procedures were approved by the Committee for Animal Care using national standards for treatment of animals. Collection of human material was approved by Medical Ethical Committees, and with informed consent of the patients.

Histological procedures

Mandibles and maxillae were excised, freed from skin and fixed by immersion in 5% paraformaldehyde in 0.1 M phosphate buffer and 2% sucrose (pH 7.3) overnight and rinsed in phosphate buffer. Some samples were decalcified in 5% EDTA containing 0.8 % formalin at 4°C for 3 weeks. Decalcified and non-decalcified tissues were embedded in paraffin wax and serial sections were made in a sagittal or cross-sectional plane 5–7 micrometer thick and mounted on polylysin-coated glass slides. Immediately before immunostaining sections were dewaxed in three changes of xylene and rehydrated using descending concentrations of ethanol. For orientation and selection of appropriate stages of differentation every tenth section was routinely stained with hematoxylin-eosin (HE). To compare staining of the different antibodies series of three adjacent sections from the same block were chosen and stained with each of the antibodies.

Antibodies

Four different Cftr antibodies were used, two raised against peptide sequences from human CFTR and two against sequences from mouse Cftr, all located in the part of the Cftr gene that had been truncated in the Cftr-null mutant mice (Fig. 1). A mouse monoclonal antibody to human CFTR-C-terminus (clone 24–1) was purchased from R&D Systems Europe, Abingdon, UK (catalogue MAB25031). According to specifications of the manufacturer clone 24–1 was raised against human CFTR exon 23 and 24 (amino acids 1377-1480) conjugated to gluthathion-S-transferase. The rabbit polyclonal anti-mouse Cftr antibody MD1314 is a new version of antibody R3195 [31, 32] and was raised against the 13-amino acid COOH terminal peptide sequence of rodent Cftr (amino acids 1468-1480), conjugated to bovine thyroglobulin. The antibodies were affinity purified on a peptide-epoxide activated Sepharose column, eluted with 4.9 mol/L MgCl₂, dialyzed and concentrated. Rat monoclonal 3G11, raised against the purified first nucleotide binding domain (NBD1) of mouse Cftr [33], was a kind gift of Prof. William E. Balch, Dept of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. To specifically detect CFTR in human tissues rabbit antibody CC24 was raised against peptide sequence 693-716 of the human CFTR molecule and affinity purified (Fig. 1; [34, 35].

Immunostaining

Dewaxed and rehydrated paraffin sections were washed in phosphate saline buffer (PBS). After blocking endogenous peroxidase by incubation for 3 min in 3% hydrogen peroxide in PBS, antigen retrieval was performed by heating sections in 10 mM of citrate buffer pH 6.0 in a microwave for 20 min at 95°C. When mouse monoclonal antibodies (clone 24-1) were used to stain mouse tissues, endogenous mouse immunoglobulines were blocked with several rounds of blocking solutions from a Histomouse kit (Zymed/Invitrogen, CA, USA) prior to incubation with primary antibodies. All antibody species were stained with anti-mouse, anti-rabbit and anti-rat ABC-peroxidase Vectastain kits (Elite, Vector, Burlingame, CA, USA). Sections were incubated with the first antibodies overnight at 4°C. Dilutions of the primary antibodies were 1:100–1:200 for clone 24-1 (1–2 μg/ml final concentrations); 1:200 for MD1314 (final concentration 1 μg/ml), 1:50 to 1:100 for 3G11 (final concentration 10–20 μg/ml) and 1:100 for CC24 (final concentration 1.2 μg/ml). As (negative) controls primary antibodies were replaced by non-immune IgG from rat, mouse or rabbit in the same concentrations (Dakocytomation, Glostrup, Denmark). After washing in PBS biotinylated secondary antibodies were applied for 1 h at room temperature. Subsequently the sections were washed and incubated with the Avidin-Biotin peroxidase Complex for 1 hour. The immunostaining was developed with diamino benzidin (DAB kit, Vector) and counterstained with methyl green or hematoxylin.

Validation of specificity of antibodies

MD1314, 24-1 and 3G11 were tested on incisors from Cftr-deficient mice to investigate whether the immunostaining was negative. Sections were selected containing early stages of ameloblast maturation before the mutation caused severe histological changes in the ameloblast structure. To validate the CC24 anti-human CFTR antibody rectal biopsies obtained from healthy adult patients (positive controls) and from homozygous ΔF508 CF patients (functional knockout, negative controls) were used.

Real Time Reversed Transcription Polymerase Chain Reaction (RT-PCR)

Prior to RNA isolation, all tissue samples were processed with MagNALyser (Roche Diagnostics, Almere, The Netherlands) according to the manufacturer’s instructions. Total RNA was extracted by the Trizol extraction method (Invitrogen) from freshly isolated mouse kidney, mouse tongue and rat maturation ameloblasts (collected by microdissection from adult rat mandibular incisors according [28]). Total RNA from human dental pulp was isolated using the MagNA Pure LC isolation robot and a tissue RNA isolation kit (Roche Diagnostics). Total RNA from mouse calvarial osteoblast-like cell line MC3T3 and primary human osteoblasts (3^rd passage) from hip bone was obtained using the NucleoSpin RNA/protein kit (Macherey-Nagel, Düren, Germany) according to the manufacturers’ instructions. First strand cDNA synthesis was performed in a 25 μl reverse transcription reaction buffer containing 100 ng of total RNA; 1 × Transcriptor RT buffer; 1 mM of each dNTP; 0.08 U random primers; 10 U protector RNAse inhibitor and 5 U reverse transcriptase (Roche Diagnostics). The reactions were incubated at 42°C for 45 min, followed by 95°C for 5 min to inactivate the transcriptase. For each PCR reaction 10-fold diluted cDNA from mouse MC3T3, kidney (positive control) and tongue (negative control), rat ameloblasts and human Universal Reference RNA (Clontech Mountain View CA, USA; cat. No 636538) were used. Universal reference RNA is a pool of total human RNA from a collection of different tissues, a positive control for human CFTR). Real-time PCR analysis was performed using the LightCycler 480 system based on SYBR Green I dye (Roche Applied Science, Indianapolis, IN, USA). cDNA was amplified according to the following parameters: a 10 min activation of hot start enzyme at 95°C followed by 50 cycles of a two step amplification program consisting of 10 sec denaturation at 95°C and 30 sec annealing, elongation and extension at 63°C. For human CFTR the following primers were used: forward primer starting in exon 4: 5′caaggaggaacgctctatcg-3 and reverse primer: 5′gtgccaatgcaagtccttca-3′ (ending in exon 6, nucleotides 471-727; expected length 257 bp, GI: NM_000492). For mouse Cftr forward primers starting in exon 4: 5′gccatttaccttggcataggc-3′ and reverse primer: 5′gccaaggcaagtccttcatca-3′ (ending in exon 5; nucleotides 435-669, expected length 235, GI: NM_021050.2). Independent PCR fragments, derived from the amplified CFTR/Cftr fragments, were sequenced using the BigDye Terminator Cycle Sequencing V3.1 Ready Reaction kit (Applied Biosystems, Nieuwerkerk aan de Ijssel, The Netherlands) and analyzed on an ABI PRISM 3100 Genetic Analyzer.

Results

Immunolocalization of Cftr in wild type mouse ameloblasts

Preliminary staining of wild type and knockout tissues with a commercially available monoclonal antibody (24-1) suggested that this antibody was not monospecific. Several other antibodies were then obtained which are often used in Cftr studies (review reference [35]) and their staining patterns in developing mouse teeth compared. Antibodies 3G11 and MD1314 produced virtually identical patterns of immunostaining, 24-1 differed in several aspects (Table 1).

Table 1.

Cftr staining in mouse incisors and bone

Cell Type	24-1		MD1314		3G11
	WT	KO	WT	KO	WT	KO
Secretory ameloblasts	+/− to +	+/−	(+/−)	−	−	−
Transitional ameloblast	+/−	+/−	+/−	−	+/−	−
Maturation ameloblasts*	+++	+++	+++	(+/−)−(+)	+++	−
Stellate reticulum/papillary cells	++	++	+	−	+	−
Odontoblasts	+	+	(+/−)	+/−	+/−	−
Osteoblasts	+	+	(+/−)	−	+/−	−
Osteocytes	+	+	+/−	−	+/−	−
Osteoclasts	+	+	+/−	−	+/−	−

− negative, +/− weak, variable, + moderate, ++ strong, +++ very strong

^*apical plasma membrane, WT= Cftr wild type mice; KO= Cftr knock out mice

In incisors of wild type mice 3G11 and MD1314 revealed a prominent positive staining in apical (facing enamel) plasma membrane of maturation ameloblasts (Fig. 2a, b, e) but not in presecretory or fully secretory ameloblasts (Fig. 2c). First (weak) staining was noted in the cytoplasm of late secretory and transitional ameloblasts. In young mice a weak intracellular staining was also seen in the adjacent stratum intermedium and stellate reticulum. MD1314 very occasionally gave a weak staining over the nuclei in a few secretory ameloblasts, odontoblasts and bone cells (not shown). Sections in which primary antibodies had been replaced by non-immune IgG were negative (not shown).

Fig. 2. Developmental expression and location of Cftr-protein in young mandibular mouse incisors using different antibodies.

(a) Incisal part of incisor stained with antibody 3G11. At the bottom left of boxed area: late secretory stage ameloblasts deposit the last matrix into the enamel space on top of dentin (D). To the right of the boxed area they are maturation ameloblasts (MA) ameloblasts. B bone; DP dental pulp; E enamel; EO enamel organ; ES enamel space. The boxed area is enlarged in Fig. 2b.

(b) Magnification of boxed area of Fig 2a. Arrows indicate a brown-orange positive staining at the apical end of young maturation ameloblasts facing the enamel (E). Faint staining in cytoplasm. Staining with 3G11

(c) Secretory ameloblasts (SA) along forming enamel (E) do not stain with 3G11

(d) Secretory ameloblasts (SA) stain diffusely with 24-1

(e) Maturation stage ameloblasts (MA) stain with MD1314. Arrows indicate a very strong positive staining over apical membrane. ES enamel space

(f) Maturation stage ameloblasts (MA) stain substantially, arrows point to positive staining over apical membrane not as conspicuous as in Fig 2e.

Adjacent sections from the same tissue block were used. Large arrow in Fig. 2a points into incisal direction; also for Figs 2b–f. Counterstaining hematoxylin. Original magnifications: a. 250× ; b-f 400×. Calibration bars indicate length in μm.

Staining with mouse 24-1 antibody showed a different pattern. The apical plasma membrane of maturation ameloblasts was immunopositive as well but less conspicuous than with the other two antibodies due to a consistent and robust cytoplasmic staining (Fig. 2f). A weak diffuse staining with 24-1 was already noted in secretory ameloblasts (Fig. 2d) in contrast to 3G11 (Fig. 2c) and MD1314 that were negative. Staining with 24-1 was also more widely distributed over other cells including non-epithelial cells (see below). This pattern did not change after several rounds of blocking of endogenous mouse IgG.

Staining of sagittal sections of lower incisors with 3G11 or MD1314 persisted in all maturation ameloblasts from early to late stages without overt interruption by non-stained cells (Fig. 3a). Only occasionally a small gap of some unstained maturation ameloblasts was seen with their apical end either damaged or not present in the plane of the section. In early maturation phase staining formed a broad fuzzy but intense band over the apical membrane and later became thinner and sharper in mid- and late-maturation cells.

Fig. 3. Low power view of immunostaining in maturation stage of an adult wild type mouse incisor (a), structural changes in knockout ameloblasts (b, c) and validation of antibodies tested on sections of Cftr-null mutants (d–i).

(a.) Two consecutive parts of a lower incisor of an adult wild type mouse sectioned sagittally and stained with MD1314. At the top left the first immunostaining becomes apparent (arrowhead) near transitional ameloblasts (TA). More incisally (in the direction of the large arrow) maturation ameloblasts (MA) stain strongly over their apical membranes. The bottom end of the incisor part (small arrow) continues with the second part of the incisor at the top right (small arrow). Staining at the apical end of the ameloblasts is uninterrupted except for a small group of cells (bracket) that did not include their apical part in the plane of sectioning. The most incisal part of the incisor was lost during the histological procedures.

(b, c) Early maturation ameloblasts of an incisor of a wild type (WT) and knockout (KO) adult mouse. EO enamel organ. B bone (HE staining)

(d, e) Wild type and knockout maturation ameloblasts stained with 3G11.

(f, g) Wild type and knockout maturation ameloblasts stained with MD1314.

(h, i) Wild type and knockout maturation ameloblasts stained with 24-1.

Counterstaining hematoxylin. Original magnifications: a. 100×; b, c, 400×; d-i; 1000×. Calibration bars indicate length in μm.

Basically similar data as in incisors were found in mouse molar tooth germs: apical ends of maturation ameloblasts including ameloblasts on the enamel-free area at the tip of the cusps stained strongly with MD1314 and 3G11 (Fig 4a, b, d), whereas 24-1 gave a more widely distributed cytoplasmic staining (Fig. 4c).

Fig. 4. Immunostaining for Cftr in developing molar tooth germs and bones.

(a) Wild type mouse molar tooth germ stained with 3G11. Apical membranes of maturation ameloblasts (MA) are immunopositive, odontoblasts (O) and cells of dental pulp (DP) are immunonegative. Undecalcified section, * preparation artefacts

(b) Wild type mouse molar tooth germ stained with MD1314. Strong staining is present in maturation ameloblasts (MA). Bottom right: staining in ameloblasts along enamel free area (EFA). D dentin, E enamel, Decalcified, * shrink artefact

(c) Hamster first molar tooth germ stained with 24-1. Secretory ameloblasts (SA) are positive with discrete intracellular staining, also seen in other cells of the enamel organ. Very prominent distinct cytoplasmic staining is seen in odontoblast layer (O) and in bone cells (B). Preameloblasts (PA) of second molar tooth germ are immunonegative. DP dental papilla, E enamel D dentin

(d) Mouse molar tooth germ stained with 3-fold normal working concentration MD1314. Maturation ameloblasts (MA) show strong apical staining and varying staining in cytoplasm. Note a weak staining in the odontoblasts (O) layer. ES enamel space, D dentin. The staining in odontoblast (O) layer varies from very weak to moderate positive.

(e) Odontoblasts (o) of a human fetal incisor stained with human specific CC24.

(f) Developing alveolar bone of human fetal jaw stained with CC24.

(g) Positive osteoclasts in hamster alveolar bone stained with 24-1

(h, i)Positive and negative human osteoclasts from human alveolar bone stained with CC24 and counterstained with hematoxylin (b, e, f, h i, blue nuclei) and methyl green (a, c, d, green nuclei only weakly).

Original magnifications a, b, c 250×; c, d 100×; g 400×; inset f, e, h, i 1000× Calibration bars indicate length in μm

Changes in ameloblast structure of incisors in Cftr-null mice

Cftr-null mice were viable but generally smaller than wild type littermates, and all their teeth erupted. Unlike wild type incisors that were orange-pigmented incisors of mutant mice were chalky white and showed substantial abrasion in agreement with previous reports [18, 19]. Molar teeth of Cftr knockout mice did not show overt changes.

Structural changes were seen in maturation stage ameloblasts of adult Cftr-knockout mice, but presecretory and secretory cells looked normal, similar as reported [18]. Polarization of the maturation ameloblasts was gradually lost in mutant incisors, the cells shortened, and nuclei became round (Fig. 3b, 3c). The cells accumulated yellow-brown grains and later also fat-like droplets of various sizes not seen in wild type cells. All these changes were much more prominent in older adult mutant mice than in young mutant mice.

Validation of the specificity of the antibodies

In knockout tissues 3G11 and MD1314 failed to stain incisor maturation ameloblasts at normal antibody working dilutions that gave a strong staining in apical membrane (Fig. 3d–g). However, by increasing the concentration of primary antibodies 3 fold also MD1314 gave a weak cytoplasmic reaction in maturation ameloblasts in null mutants (not shown). Antibody 24-1 consistently showed a very strong cytoplasmic staining in null-mutant ameloblasts without a clear apical staining (Fig. 3h, 3i).

Immunostaining in mineralizing connective tissue cells

Surprisingly, some of the antibodies also stained mineralizing connective tissue cells (Fig. 4). Antibody 3G11 gave no in connective tissue cells including bone and pulp (Fig. 4a) but with 24-1 a fine granular intracellular staining was seen in odontoblasts, osteoblasts, osteocytes and some osteoclasts (Fig 4c, g; Table 1). This staining with 24-1 was moderate in developing mouse tissues but very strong in developing hamster jaws (Fig. 4c, g). Also human embryonic bone- and dentin-forming cells stained positive with 24-1 (not shown). With MD1314 staining in odontoblasts and bone cells of rodents was weak and variable (Fig. 4d).). In Cftr-null mice a strong (24-1) or variable but weak (MD1314) staining persisted in odontoblasts and bone cells.

A recent report showing 24-1 immunostaining of human bone cells [36] prompted us to investigate whether this staining was CFTR-specific. The human specific antibody CC-24 raised to a completely different portion of CFTR than 24-1 (Fig. 1) gave a similar granular cytoplasmic staining in odontoblasts (Fig. 4e) and bone cells of fetal human jaws, including preosteoblasts, osteoblasts, osteocytes and some of the osteoclasts (Fig. 4f, h, i). In control tissues CC24 stained luminal plasma membranes of rectum glands of healthy humans but failed to do so in biopsies from Δ F508 CF patients (not shown).

Transcipts for Cftr and CFTR

By RT-PCR transcripts for Cftr were detected in mouse osteoblast-like cells (MC3T3), human primary osteoblasts, rat maturation ameloblasts, and human dental pulp tissue including odontoblasts, but not in murine tongue (Fig. 5). Starting with an equal amount of RNA the positive controls (mouse kidney, human Universal reference mRNA) started to become visible after 30 cycles. The appearance of transcripts in cells from pulp and bone required at least 35 cycles indicating that expression in mineralizing connective tissue cells was much lower than in soft tissues with established Cftr expression. Sequencing of the amplified fragments confirmed that they originated from mouse Cftr and human CFTR-mRNA.

Fig. 5. Transcripts of Cftr in bone and dental cells of mouse/rat (A) and humans (B).

A: Lane 1: mouse MC3T3 osteoblast-like cells; lane 2: mouse kidney (positive control); lane 3: rat maturation stage ameloblasts; lane 4: mouse tongue (negative control). Mouse primers for exon 4-5, expected size 235 bp. Y-axis: nucleotide size (100 bp ladder)

B: lane 1: commercial human reference (positive control); lane 2: human dental pulp; lane 3: human osteoblasts (subject 1); lane 4: human osteoblasts (subject 2); lane 5: water (negative control). Human primers for exon 4-6, expected size 257 bp. Y-axis: nucleotide size (50 b)

Discussion

Cftr and enamel formation

Our data are consistent with the hypothesis that Cftr channels are present in mouse ameloblasts and functionally active in pH regulation in incisors as previously suggested [18, 19, 20, 22, 23]. Here we demonstrate that Cftr immunostaining is not present in secretory ameloblasts but highly prominent in the apical plasma membranes of maturation ameloblasts, the cells that are severely affected in Cftr-null mutant mice ([18], present data). All maturation ameloblasts were immunostained for Cftr. This staining pattern is different from that for anion exchanger isoform 2 (Ae2), in which small groups of maturation ameloblasts - presumably smooth-ended cells - did not stain or stained only patches of their cell bodies [37, 38]. The fact that after eruption incisor enamel rapidly erodes [18, present data) and contains less calcium than wild type incisors [19, 29] demonstrates that Cftr is critical for functioning of maturation ameloblasts, similar as Ae2 [37, 38].

The molecular mechanisms responsible for buffering protons during amelogenesis are largely unknown. During secretory phase amelogenins are abundantly present in the enamel space and will likely buffer a major part of the protons [21, 22]. However, when amelogenins have been removed in maturation stage, it is very conceivable that bicarbonates are secreted into the enamel space to buffer protons [21, 24,]. Several reports speculated how ameloblasts could regulate pH changes in enamel by producing [39,40] and secreting bicarbonates using carbonic anhydrase and a set of (largely unknown) ion exchangers/cotransporters and chloride channels in the plasma membranes including Cftr [22–24]. In a recent model it was proposed that secretory ameloblasts import and secrete bicarbonates through activity of apical Ae2a and basolateral sodium bicarbonate cotransporter 1 (Nbc1) [41]. Previous [18, 23] and present data strongly suggest that in maturation stage Cftr is a critical factor in pH regulation and essential for completion of enamel mineralization. In figure 6 we present a more detailed working model for the function of Cftr in (ruffle-ended) ameloblasts.

Fig 6. Working model depicting the putative role of Cftr in extracellular pH regulation by ruffle-ended ameloblasts.

Apatite forms in the enamel space from ionic calcium, phosphate and water; this generates 8 protons per unit of cell hydroxyapatite [21]. These protons are buffered by bicarbonates secreted into the enamel space by maturation ameloblasts [21, 23]. Bicarbonate is generated by activity of cytosolic carbonic anhydrase-type 2 (Car2, [39, 40]). Bicarbonates are also imported through the basal plasma membrane plausibly by Na⁺-2HCO₃⁻ cotransporter isotype 1 (Nbc1 or Slc4a4, [42, 43]). Intracellular bicarbonate is exchanged for Cl⁻ basolaterally by sodium-independent anion exchanger-2 (Ae2 or Slc4a2 [37, 38]). At the apical plasma membrane the imported Cl⁻ is conduced into the enamel space through the Cftr channel ([23], present study). The extracellular Cl⁻ is then exchanged for one or more bicarbonates by (a) hypothetic member(s) of the anion transporter Slc26a family that colocalize(s) with Cftr [4, 44]. Several Slc26a members may be present in the same apical membrane simultaneously. Cftr interacts physically with Slc26a members, which strongly stimulates (+) the exchange activity of the Slc26a [44, 45]. The Cftr can also be permeable for bicarbonates [2, 4]. The electrogenic Nbc1 cotransporter at the basal membrane induces plasma membrane hyperpolarization; such sustained hyperpolarization stimulates HCO₃⁻ – efflux across the apical membrane. The bicarbonates secreted into the enamel space react with protons to form carbonic acid that can form CO₂ and water, catalyzed by a putative carbonic anhydrase 6 (Car6, [46]). Water is used partly to form new apatites, partly reabsorbed by ameloblasts. The CO₂ can pass the apical membrane and be reused intracellularly to form bicarbonate by Car2. To prevent intracellular acidification the protons generated by Car2 are pumped out by basolateral H⁺/K⁺ ATPase activity (H⁺/K⁺, [47]) or by (a) hypothetic sodium hydrogen exchanger(s) (NEH, [5]). Any excess of intracellular sodium is exchanged for potassium by a Na⁺/K⁺-ATPase (not drawn). Question marks: hypothetic; ZO: zonula occludens

The model is principally based on pH regulation by cells of pancreatic duct that generate and secrete large quantities of bicarbonates into the lumen of the ducts [for review see 3, 5, 42]. The model builds on recent reports of enamel defects in knockout mice deficient in Ae2 [38, 39], Nbc1 [43] and Cftr [18, present study]. In the model Cftr in ameloblasts has three major functions: (i) As classical chloride channel Cftr transduces intracellular Cl⁻ into the enamel space (Fig. 6). Anion-transporters in the apical plasma membranes that colocalize with Cftr exchange the extracellular Cl⁻ for intracellular bicarbonates. These anion-transporters are likely member(s) of the solute carrier family 26a (Slc26a) [5, 44]. Slc26a is a group of 10 different members of multifunctional anion transporters, some of which exchange one Cl⁻ for one bicarbonate (electroneutral exchange, e.g., Slc26a4), while others exchange with different stoichiometry (electrogenic exchange, e.g, one Cl− for two bicarbonates as for Slc26a6 or two Cl⁻ for one bicarbonate as for Slc26a3;[44]). Electrogenic exchange influences membrane potential of the plasma membranes and thus the rate of efflux or influx of anions. (ii) Cftr also controls bicarbonate efflux by directly stimulating the transport activity of Slca26 members and vice versa. Coexpression studies in cell lines showed that the Cftr molecule physically interacts through its R- (regulatory-) domain with the STAS-domain (Sulphate Transporter and Anti-Sigma factor antagonist) of Slc26a3 and Slc26a6 [44, 45]. This interaction results in 5–6 fold enhanced transport activity by Slca26 [44, 45]. (iii) Cftr on its own is also permeable for bicarbonate [2, 4]. Though earlier studies showed that Cftr is 3–4 fold less permeable for bicarbonate than for Cl⁻ more recent studies in pancreatic duct cells show that Cftr can contribute up to 60% of the total amount of bicarbonate efflux [4].

In addition to cytosolic carbonic anhydrase type 2 maturation ameloblasts also express mRNA transcripts of carbonic anhydrase type 6 (Car6), a secreted type of carbonic anhydrase that may operate in maturation enamel, where it could catalyse the formation of CO₂ and H₂O from carbonic acid formed by bicarbonate buffering of the protons in the enamel space ([46], Fig. 6). However, the translation into protein and the localization of Car6 protein have not been assessed yet.

How pH is regulated in smooth-ended ameloblasts and how Cftr functions in this cell type is presently unknown. The substantial morphological changes that occur when ruffle-ended cells transform into smooth-ended cells including the temporary disappearance of lateral H⁺K⁺-ATPase activity [47] and possibly also Ae2 [37, 38] suggest that smooth-ended cells function fundamentally different from ruffle-ended cells.

Collectively, these data confirm previous reports that Cftr is critical for enamel mineralization in mouse incisors. Cftr potentially functions as regulator of pH in the enamel fluid by conducing Cl⁻ and bicarbonates into enamel and by stimulating the activity of several (hypothetic) types of anion transporters to secrete bicarbonates.

Antibody specificity and cftr in bone cells

A striking finding was that also bone cells express Cftr. Using antibody 24-1 Shead et al. [36] reported cytoplasmic immunostaining in normal human fetal bone cells and showed the presence of the expected mature (fully glycosylated) 180 kD protein in bone tissue extracts. However, considering the well-established apical localization of human CFTR in T84 cells [48], used as a positive control in the Shead study, it is remarkable that their immunostaining of T84 cells with 24-1 was cytoplasmic, with strong perinuclear staining [36]. We report here that antibody 24-1 does not react exclusively with Cftr but also detects epitopes different from Cftr; this was demonstrated by a very strong immunostaining of maturation ameloblasts in Cftr-deficient ameloblasts. In support of this it is also pertinent to report that by Western blotting with 24-1 but not with 3G11 and MD1314 we can detect immunopositive bands in extracts from intestine of Cftr-null mice showing a similar molecular weight as authentic Cftr (de Jonge et al. unpublished data). This also indicates that 24-1 is not monospecifc and thus the positive staining in bone cells [36] may not merely reflect the presence of Cftr protein. In a more extensive study we here present evidence that mouse and human bone cells contain low levels of Cftr mRNA and are positively stained by at least two monospecific antibody species in addition to 24-1. These findings strongly support the concept that osteoblasts and odontoblast do express low amounts of Cftr protein.

Both CF patients and Cftr-deficient mice are known to develop osteopenia [49–52], even in the absence of overt respiratory and gastrointestinal diseases or steroid treatment [50, 51]. Three weeks old Cftr-deficient mice had reduced bone mineral density and increased bone resorption relative to bone formation [50]. In skeletally mature (28-weeks old) mutant mice some of these bone defects had improved compared with 12-weeks old mutants but defects persisted in cortical and trabecular bone structure [51]. Based on the increase in the number of osteoblasts and osteoclasts it was suggested that peak bone mass in Cftr-deficient mice may have been delayed and bone turnover increased compared with age-matched wild type mice [51]. Our data show that Cftr protein is present in murine and human bone cells. Expression of Cftr in bone cells and odontoblasts was not seen in plasma membranes but intracellular. There is evidence that Cftr is expressed at a low level in membranes of organelles in other non-epithelial cell types, e.g. lymphocytes, phagocytes, alveolar macrophages and muscle tissue [53–59]. In some of these cell types Cftr has been implied to act as Cl⁻ channel for phagosomal acidification in alveolar macrophages [55, 56], but other studies question such a function [57, 58]. Skeletal muscle has been shown to express low levels of Cftr in the sarcoplasmic reticulum, and calcium homeostasis was dysregulated in Cftr-deficient muscles [59]. In analogy with these cell types Cftr in bone cells may likewise function as a Cl⁻ channel or glutathione transporter [59] in intracellular organelles. Poor functioning of Cftr in organelle membranes of bone cells may underlie the changes in bone metabolism in Cftr-deficient mice or in CF patients, resulting in osteopenia.

In conclusion, we report here that maturation ameloblasts in rodents express Cftr in their apical plasma membrane. The data suggest that Cftr may functionally be involved in pH regulation. Bone- and dentin-forming cells express Cftr mainly intracellularly but the function of Cftr in this compartment remains to be elucidated. Poor functioning of Cftr in Cftr-deficient bone cells may be responsible for development of osteopenia.