Archs oral Bid. Vol. 36, No. 10, pp. 715-725,
0003-9969/91
1991
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SUBCELLULAR CO-LOCALIZATION AND CO-VARIATIONS OF TWO VITAMIN D-DEPENDENT PROTEINS IN RAT AMELOBLASTS ARIANE BERDAL,“~DOMINIQUEHOTTON,’ SHADI KAMYAB,’ PAULETTECUISINIER-GLEIZES’and HENRI MATHIEU’ ‘LJ120 INSERM, Hopital Robert Debre, 48 Boulevard Serurier, 75019 Paris and *Laboratoire d’histologie et d’embryologie, Faculti de chirurgie dentaire, Universitt Paris V, I rue Maurice Amoux, 92120 Montrouge, France (Accepred I8 April 1991) Summary-The immunocytochemical patterns of calbindin-D,, (CaBP 9 k) and calbindin-D,,, (CaBP 28 k) were compared by light and electron microscopy throughout amelogenesis. Labelling on serial sections and co-localization of CaBPs confirmed that the two proteins were restricted to a single cell type, the ameloblasts. Their quantity increased during presecretion, was stable during secretion and alternately high and low during the cyclic modulation of ameloblasts which occurs during maturation. Ruffle-ended ameloblasts contained the highest apparent concentration. Investigations with several fixatives indicated that the CaBPs were present in the cytosol and the nucleus, although there were slight differences with various fixatives by light microscopy. Their concentrations in these compartments varied in parallel throughout arnelogenesis. However, mitochondria contained only immunoreactive CaBP 9 k. While the distribution of CaBP 9 k in zones containing Golgi apparatus and rough endoplasmic reticulum was similar, CaBP 28 k concentration has, in another paper, been shown to be higher near the rough endoplasmic reticulum. Key words: ameloblasts, enamel, calcium, vitamin D-dependent calcium-binding proteins.
INTRODUCTION Calbindin-D,, (Cal3P 9 k) calbindin-D,,, (CaPB 28 k) are members of a superfamily of proteins that contain structurally similar calcium-binding sites (Perret, Lomri and ‘Thomasset, 1988). They are distributed in distinct ceil types of several mammalian organs (Christakos, Gabrieledes and Rhoten, 1989; Norman, Roth and Orci, 1982; Riad et al., 1988; Thomasset, Parkes and Cuisinier-Gleizes, 1982): mainly intestine, uterus, yolk sac and placenta for CaBP 9 k, kidney and cerebellum for CaBP 28 k. They have also been described in mineralized tissues, including growth-plate cartilage (Balmain ef al., 1986a, b; Zhou et ill., 1986), bone (Balmain et al., 1989; Celio, Norman and Heizmann, 1984; Christakos and Norman, 1978; Thomasset et al., 1982) and teeth (Berdal et al., 1989a; Celio et al., 1984; Elms and Taylor, 1987; Magloire et al., 1988; Taylor, 1984; Taylor, Gleason and Lankford, 1984). The synthesis of CaBPs is vitamin D-dependent, differently depending on the tissue type (for review see Christakos et al., 1989). In the rat enamel organ, the concentration of CaBP 28 k reaches _ 1% of that of the cytosolic proteins (Berdal et al., 19891,); it is restricted to ameloblasts and its immunolabelling density appears to vary with the stages of amelogenesis (Berdal et al., 1991). This pattern strongly suggests that CaBP 28 k could be Abbreviations: CaBP, calcium-binding protein; PBS, phosphate-buffered saline.
involved in amelogenesis, as this process is affected by vitamin D-deficiency (Berdal et al., 1987), which also causes CaBP depletion (Berdal et al., 1989b). Other studies indicate that CaBP 9 k is also present in the enamel organ (Taylor et al., 1984) in a lower concentration (Berdal er al., 1989~). We have now further investigated the relationships between the different stages of amelogenesis, CaBP 9 k and CaBP 28 k in the enamel organ. This tissue constitutes a unique example of an epithelium involved in calcium transport and extracellular mineralization in mammals (Bawden, 1989), where CaBP 28 k reaches the levels observed in kidney and intestine (Berdal et al., 1991) and is calcitriol dependent (Berdal et al., 1989a). The subcellular distribution of CaBPs was compared by light and electron microscopy, including co-localization. MATERIALS AND
METHODS
Antisera Antibodies to purified rat duodenum CaBP 9 k [gift from M. Thomasset, INSERM, U 120 (Thomasset et al., 1982)] were raised in New Zealand White rabbits as were antibodies to rat kidney CaPB 28 k [gift from A. Brehier, INSERM, U 120 (Intrator et al., 1985)]. Controls included replacement of the antisera with: non-immune serum (Nordic, Tilburg, the Netherlands), anti-sucrase serum (gift from J. P. Broyart, INSERM, U 120), preadsorbed anti-serum (1.5 pg CaBP 9 k/p1 anti-CaBP 9 k; 100 ng CaBP 28 k/p1 anti-CaBP 28 k). The rest of the immunolabelling 715
716
ARIANE BERDAL et al.
procedure was identical in controls and experimental samples as described above. Tissue processing localization
of
light -microscopic
immuno -
Preparation of samples. A total of 60 SpragueDawley rats (2-12 days old) were killed with ether. Various fixatives (acetic acid : ethanol, 4% paraformaldehyde in phosphate buffer, Carnoy’s mixture) were used. Some samples were fixed by immersion for 16 h. Other animals were anaesthetized and fixed by a 15min intracardiac perfusion through the left ventricle using a monostaltic pump (Touzart and Matignon, Vitry, France) followed by immersion for 90 min. The first molars were dissected out, rinsed in 0.1 M PBS (Eurobio, Paris, France) for 2 h and impregnated with 30% sucrose (Sigma, la Verpilliere,
Plate Figs l-5.
Localization
Fig. 1. Preameloblasts Fig. 2. Presecretory
of CaBP
(PA) in molars to contain
France) for 2 h in PBS. Sections were cut at -20°C in a cryostat (Wild Leitz, Rueil-Malmaison, France), placed on slides coated with poly+lysine (Sigma) and kept overnight at -4°C. Some fixed mandibles were dehydrated and embedded in paraffin. Fivemicron sections were cut, the paraffin removed, rehydrated and sections used for immunocytochemistry. Immunocytochemical study. All incubations and rinsings were done in PBS, pH 7.3, containing 0.5% bovine serum albumin (Institut Pasteur, Paris, France). Secondary antibodies and the streptavidinbiotin system (Amersham, les Ulis, France) were used to link peroxidase to the primary antibodies. The staining process was as follows: (1) non-specific binding sites were blocked with non-immune goat serum (1: 30; Nordic) for 30 min; (2) the sections were incubated with the primary antiserum at 27°C diluted 1:200 to 1: 1600 for CaBP 9 k and
1
9 k during
the presecretion
and secretion
of a 2-day-old rat; fixation with Carnoy’s no immunoreactive CaBP 9 k. x 600
stages.
solution.
ameloblasts (A) in molar of 2-day-old rat; fixation with Carnoy’s contain a light staining in the distal cytoplasm (arrow). x 600
Fig. 3. CaBP 9 k: stratum
intermedium
in incisor
from 30-day-old x 42,000
rat; fixation
with
They appear solution.
They
1% glutaraldehyde.
Fig. 4. Nucleus and neighbouring area of rough endoplasmic reticulum in secretory ameloblasts of incisors from 30-day-old rat; fixation with 3% paraformaldehyde and 0.5% glutaraldehyde. (a) CaBP 9 k: gold particles are scattered inside the nucleus (N) and in cytosolic area between the cisternae of rough endoplasmic reticulum (RER). There is some labelhng along the RER membranes on their cytosolic side (arrow). (b) Control: the cell appears almost devoid of labelling with non-immune serum. x 42,000 Fig. 5. CaBP 9 k: Golgi area in secretory ameloblasts of incisors from 30-day-old rat; fixation with 1% GA. Gold particles are distributed through the cytosohc zones surrounding either rough endoplasmic reticulum (RER) or Golgi (G) apparatus. x42,000 Plate 2 Figs 6 and 7. Localization Fig. 9 k: are and
of CaBP 9 k and CaBP 28 k in secretory
ameloblasts.
6. Fixative-dependent immunostaining of CaBPs in molars from S-day-old rat. (a) Carnoy’s, CaBP the staining is restricted to the cytoplasm in ameloblasts (A). (b) Carnoy’s, control: ameloblasts (A) not stained when antigen-absorbed antibody is used. (c) Acid: alcohol, CaBP 28 k: both the nuclei cytoplasm of ameloblasts (A) appear strongly stained. (d) 4% paraformaldehyde, CaBP 28 k: the cytoplasm and nuclei of ameloblasts (A) are evenly stained. x 1000
Fig. 7. Co-localization of CaBPs in secretory-stage ameloblasts; CaBP 9 k localized by IS-nm gold particles and CaBP 28 k by 30-nm gold particles. (a) CaBPs: supranuclear area in the incisor of a 30-day-old rat; fixation with 3% paraformaldehyde and 0.2% glutaraldehyde. Mitochondria (M) appear to contain gold particles, while cisternae of rough endoplasmic reticulum (RER) do not. (b) CaBPs: Tomes’ process; fixation with 1% glutaraldehyde. Gold particles are located between secretion granules (sg). Sparse staining is present in enamel. (c) Control: Tomes’ process; fixation with I % glutaraldehyde. Particles were detected within enamel with anti-sucrase. x 42,000 Plate 3 Figs 8-10.
Co-variations
of the distribution
and co-localization
of CaBPs
during
the maturation
Figs 8 and 9. Immunostaining for CaBP 9 k and CaBP 28 k along the cusp side on serial sections molar from I I-day-old rat; fixation with Carnoy’s.
stage. of the
Fig. 8. (a) CaBP 9 k: immunonegative areas of ameloblasts (A) located in the cervical loop region. (b) CaBP 9 k: neighbouring immunopositive ameloblasts (A); oe: organ enamel. (c) CaBP 9 k: immunopositive ameloblasts (A) located by the cusp top. (d) CaBP 9 k: immunonegative ameloblasts (A) located at the cusp top. x 600 Fig. 9. (a) CaBP 28 k: area corresponding sac. (c) CaBP 28 k: area corresponding
to 8(a). (b) CaBP 28 k: area corresponding to 8(b); fs: follicular to 8(c). (d) CaBP 28 k: area corresponding to 8(d). x 600
Fig. 10. Co-localization of CaBP 9 k and CaBP 28 k in the incisor of a 30-day-old rat; fixation with 4% paraformaldehyde. (a) Many lS-nm (CaBP 9 k) and 30-nm (CaBP 28 k) particles are present in the ruffled border of ameloblasts (RE). (b) In contrast, there are few gold particles within the cytosol of smooth-ended ameloblasts (SE). x 42,000
CaBP 9 k and CaBP 28 k in ameloblasts
Plate
I
717
718
&mm
BERDALet al.
Plate 2
CaBP 9 k and CaBP 28 k in ameloblasts
Plate 3
719
120
ARIANEBERDALet al.
Plate 4
CaBP 9 k and CaBP 28 k in ameloblasts
1:200 to 1: 26800 for CaBP 28 k; (3) rinsed and incubated with biotinylated anti-rabbit IgG, diluted 1: 100 for 90min; (4.) rinsed and incubated with streptavidin-peroxidase, diluted 1: 300 for 30 min. Peroxidase was revealed by incubating the sections for 15 min at room temperature with 3,3’-diaminobenzidine (5 mg/lO ml; Sigma) plus 5 ~1 30% H,02 in 0.1 M tris-solution, pH 7.66. The sections were finally rinsed with Tris, mounted in neutral glycerine, and observed and photographed in a Zeiss Orthoplan microscope. Tissue processing for electron -microscopic immuno localization Preparation of samples. Twenty 30-40-day-old Sprague-Dawley rats (body weight 103 + 6 g) were anaesthetized with ether before perfusion. Fixation was with different solutions in 0.1 M PBS, pH 7.3, including: 4% paraformaldehyde (n = 5), 3% paraformaldehyde with either 0.2% (n = 5), or 0.5% glutaraldehyde (n = 5) and 1% glutaraldehyde (n = 5). After a 15-min perfusion, half-mandibles were immersed in the same fixative for 90 min, and rinsed in PBS at 4°C. Incisors were dissected out. Free aldehyde groups were blocked by immersion of the whole teeth in PBS-O.25 M NH,Cl (Merck, Paris, France) for 18 h at 4.“C. Dehydration was done in methanol in a graded series of temperatures (4 to - 30°C). Samples were progressively impregnated with Lowicryl K4M resin (Chemiche Werk Lowi, Walkraiburg, Fed. Rep. Germany) at -30°C. U.V. light polymerization was at -30°C for 48 h and continued at room temperature for 5 days. Gold-interference sections were cut on an Ultrotome 5 (LKB) and collected on 200.mesh nickel grids. Immunogold labelling of CaBP 9k. All incubations and rinsings were done in PBS, pH 7.3, at room temperature. Grids were placed on a droplet of non-immune goat serum (diluted 1: 30) for 30 min, to inhibit non-specific t,inding. Sections were directly incubated with the primary antibodies (diluted 1: 10 to 1: 1000) for 90 min. After rinsing, grids were incubated with biotinylated secondary antibodies (diluted 1:lOO) for 60min. They were again rinsed and incubated with streptavidin conjugated with 15-nm gold particles (Amersham; diluted 1: 20) for 30 min. The grids were finally rinsed with PBS and distilled water, coumerstained with uranyl acetate and lead citrate, and observed in a Philips 201 electron microsope. Co-localization of CaBP 9 k and CaBP 28k. CaBP 9 k was immunolabelled on one side of the grid, as described above, except that sections were not counterstained. CaBP 28 k was visualized on the other side as follows: (1) incubation with nonimmune goat serum (diluted 1: 30) for 30 min; (2) then directly with an+CaBP 28 k (diluted 1: 100 to 1: 1000) for 90 min; (.3) rinsing and incubation with
721
secondary antibodies linked to 30-nm gold particles (Biocell, Cardiff, U.K.; diluted 1: 20) for 60 min. The grids were rinsed in PBS and distilled water and stained with uranyl acetate and lead citrate. Nonrecognition of anti-CaBP 9 k by the anti-CaBP 28 k secondary antibodies was checked by using supplementary controls in which the antibodies raised to CaBP 28 k were omitted. Radioimmunoassay of CaBPs in SlOO cytosolic extracts of the enamel organ
Eight male and eight female Sprague-Dawley rats (Charles River) were used for radioimmunoassays. Other rats (140- and 300-day-old pooled males and females) were also used. They were anaesthetized with ether and decapitated. The enamel organs of both mandibular incisors were dissected out, as described by Berdal et al. (1991). Radioimmunoassays on SlOO extracts were as described previously (Thomasset et al., 1982). Enamel organ provided curves parallel to the one obtained with both standard CaBPs. RESULTS CaBP concentration in the enamel organ
In 30-day-old rats, the amounts of CaBP 9 k were different in males (69 & 16 ng/mg of total protein) and in females (154 f 13 ng/mg). The concentrations of CaBP 9 k and CaBP 28 k remained in the same proportion during the life-cycle of the rat ([CaBP 9 k] - 100 ng/mg; [CaBP 28 k] - 10 pg/mg). CaBP 9 k distribution in presecretion - and secretion stage ameloblasts (Figs l-5)
Young dividing preameloblasts appeared to be unstained (Fig. 1). Immunolabelling was first detected in presecretory ameloblasts adjoining the dentine (Fig. 2). The nuclei and cytosol in cells of the stratum intermedium contained a neglible number of gold particles, which corresponded to the background level (Fig. 3). Immunogold particles were present between cisternae of rough endoplasmic reticulum, and inside the nuclei of secretory ameloblasts [Fig. 4(a)]. Controls showed a low labelling [Fig. 4(b)]. The immunoreactivity appeared lower in areas containing Golgi apparatus versus the areas containing rough endoplasmic reticulum (Fig. 5). Non-ameloblastic epithelial cells in the enamel-free areas at the cusp tip of the molar, or in the Hertwig’s sheath where the root develops, never contained CaBP 9 k immunolabelling (not shown). Effects of fixation on intracellular distribution of CaBPs in secretion-stage ameloblasts (Figs 6 and 7)
The distribution of CaBPs was restricted to the cytoplasm in specimens fixed in Carnoy’s [Fig. 6(a), CaBP 9 k; Fig. 6(b), control]. In contrast, CaBPs
Plate 4 Fig. 11, Co-localization and co-variations of CaBPs inside nuclei during the maturation stage. CaBP 9 k and CaBP 28 k. in the incisor of a 30-day-old rat; fixation with 3% paraformaldehyde and 0.5% glutaraldehyde. (a) Ruffle-ended ameloblasts (RE) contain dense lahelling. (b) Smooth-ended ameloblasts (SE) contain little labelling.
ARIANE BERDALet
722
appeared to be located in both the nucleus and cytoplasm with acid: alcohol [Fig. 6(c)] and aldehyde fixatives [Fig. 6(d); 4% paraformaldehyde and Figs 4 and 5, Figs 7-11; solutions of paraformaldehydeglutaraldehyde]. CaBP 9 k and CaBP 28 k co -localization in secretion stage ameloblasts (Figs 6 and 7)
The ameloblastic zone adjoining the stratum intermedium [Fig. 7(a)] contained numerous mitochondria immunostained for CaBP 9 k, but none stained for CaBP 28 k. Labelhng for both CaBPs was present in the cytosol between the rough endoplasmic reticulum and the mitochondria. Immunogold particles were located between secretory granules in Tomes’ process [Fig. 7(b)]. All immunocontrols showed low labelling density [Fig. 7(c); non-immune serum]. Co-localization and labelling co-variations of CaBP 9k and CaBP 28k in maturation-stage ameloblasts (Figs 8-l 1)
The immunostaining for CaBP 9 k (Fig. 8) and for CaBP 28 k (Fig. 9) in serial sections indicated that the distribution of the discontinuous, light-microscopic negative [(a) and (d)] and positive [(b) and (c)] areas for the two CaBPs was similar. Immunopositive ameloblasts showed a gradient of staining with the maximum near the cervical area [Figs 8(b) and 9(b)] and a minimum near the cusp tip [Figs 8(c) and 9(c)]. By electron microscopy, co-localization of CaBPs showed that ruffle-ended ameloblasts, with an interdigitated border [Fig. 10(a)], contained large amounts of labelling in their cytoplasm, while there were few gold particles in smooth-ended ameloblasts [Fig. 10(b)]. Similar variations were seen in the nuclei [Fig. 8(a); ruffle-ended ameloblasts and Fig. 8(b); smooth-ended ameloblasts in Fig. 111. DISCUSSION
Both CaBP 9 k and CaBP 28 k were shown in situ in the same cells by co-localization. Previous investigations suggested this possibility by labelling of different samples (Berdal et al., 1989b; Celia et al., 1984; Taylor, 1984; Taylor et al., 1984). Crossreactivity between the two proteins has been previously excluded by radioimmunoassay procedures (Thomasset et al., 1982). Western blotting of rat enamel-organ proteins also indicates that this is unlikely. A single band was obtained with anti-CaBP 28 k, with no labelling present at lower molecular weights (Berdal et al., 1989b, 1991). Thus, our immunocytochemical study indicates that the two proteins coexist in ameloblasts. Subcellular distribution of CaBPs within ameloblasts
Our findings show that immunonegative cells at the light microscopic level may contain immunoreactive protein at the electron microscopic level (CaBP 9 k and CaBP 28 k within smooth-ended ameloblasts here). Our investigation illustrates that fixation methods may also influence the distribution of CaBPs seen by immunolabelling, as suggested by analysis of the patterns obtained in other tissues [CaBP 9 k (Arnold, Kovaks and Murray, 1976; Balmain et al., 1986b; Riad et al., 1988; Taylor, 1981) and CaBP 28 k
al.
(Balmain et al., 1986a; Jande, Tolnai and Lawson, 1981; Schreiner et al., 1983; Thorens et al., 1982). Both CaBPs appeared to be restricted to the cytoplasm in material fixed with Carnoy’s but present in the nucleus and the cytoplasm of tissues fixed in all other fixatives at the light microscopic level. As Carnoy’s fluid is the only fixative that contains chloroform, this reagent may solubilize nuclear components. Indeed, measurements of CaBP in cell fractions have shown that CaBP 28 k, at least, is present in both nuclear and cytosolic compartments (Thorens et al., 1982). Moreover, proteins weighing less than 50 kDa penetrate passively and freely into the nucleus (Hunt, 1989). Therefore, in this study, the two CaBPs appeared in both the cytosol and nucleus. Moreover, the CaBP 9 k was only observed in mitochondria in the ameloblasts, as described in the cells of the yolk sac (Riad et al., 1988). Coexistence of CaBPs in calcium-transporting tissues
Radioimmunoassay for CaBP 9 k, compared to data for CaBP 28 k (Berdal et al., 1991), provided their ratio: ([CaPB 9 k]/[CaBP 28 k] = l-2%). The two CaBPs are present in the same cells not just in enamel organ but also in kidney (Screiner et al., 1983), growth plate cartilage (Balmain et al., 1986a, b; Zhou et al., 1986) and bone (Balmain et al., 1989; Ceho et al., 1984; Thomasset et al., 1982). It might therefore be that the two proteins are jointly involved in the physiology of calcium-handling tissues, albeit in different concentrations and with different functions as their subcellular distribution is different. Indeed, tissues not involved in transporting calcium contain only one CaBP (Norman et al., 1982; Thomasset et al., 1982; Christakos et al., 1989) pancreas or parathyroid containing CaBP 28 k and thymus and lung CaBP 9 k. 45Ca autoradiographic CaBP labelling
pattern
and fluctuations
in
The relatively low CaBP 9 k immunoreactivity in secretion-stage ameloblasts compared to maturationstage cells indicates that the concentration of the protein varies in the enamel organ, as suggested previously (Taylor et al., 1984). However, in contrast to Taylor’s findings, CaBP 9 k was detected in secretory cells in our study. A comparison of the distributions of CaBP 9 k (our study) and CaBP 28 k (Berdal et al., 1991), the immunolabelling of CaBPs in serial sections and their co-localization indicate that the concentrations of the two CaBPs in ameloblasts change in parallel throughout enamel formation. Calcium incorporation throughout amelogenesis is also irregular (Bawden and Wennberg, 1977): calcium transport appears to be cellularly controlled and spatially continuous during the secretion stage. The CaBPs were evenly distributed throughout the secretory ameloblasts. In contrast, calcium penetration is more massive and varies cyclically during the maturation stage (Takano et al., 1987). These variations in uptake of radioactivity correspond to cyclical transformations of ameloblast morphology from ruffle-ended to smoothended (for review see Bawden, 1989) and may therefore correlate with variations in the apparent concentrations of the two CaBPs. CaBPs were mostly
123
CaBP 9 k and CaBP 28 k in ameloblasts
concentrated in ruffle-,ended ameloblasts. These cells may transport calcium actively, as suggested by the chemical inactivation IofCa2+-ATPase (Takano et al., 1987). First hypothesis: CaBPs as mediators for transcellular calcium transport
In the intestinal epithelium, there are gradients of CaBP 9 k concentratmn corresponding to the active transport gradient: from the duodenum to the jejunum (Perret, Desplan and Thomasset, 1985) and along the crypt-villus axis (Van Corven, Roche and Van OS, 1985). CaBP 28 k immunoreactivity in kidney is present in the principal cells of the distal convoluted tubule (Berdal et al., 1991; Thorens et al., 1982), where there is a Ca-ATPase activity (Borke et al., 1989). CaBPs are thought to contribute to the vitamin D-dependent transcellular transport of calcium by acting as intracellular shuttles or by activating Ca-ATPase (Bronner, 1988; Morgan et al., 1986; Walters, 1989). They may function as such in ameloblasts, as prop’osed previously for CaBP 9 k only (Bawden, 1989). However, in vitro data suggest that, in tooth germs, where CaBPs are vitamin D-dependent (Berdal et al., 1989a), calcitriol does not affect calcium incorporation (Bawden, Deaton and Crenshaw, 1983; Bawden et al., 1985). Bringas et al. (1987) obtained normal enamel mineralization in a chemically defined medium without any .vitamin D supplementation. As autoradiographic studies suggest that the paracellular transport is massive during enamel mineralization (Nagai and Frank, ‘.975), quantitative analysis of calcium pathways is needed to evaluate the importance of this ‘calcitriol-dependent, CaBPs-mediated, transcellular calcium I.ransport’. Indeed, it represents a small but physiologically regulated part of the calcium pathway in the intestine (Van OS, 1987) and kidney (Costanzo and Windhager, 1987).
calcium during the secretion stage. Their nuclear and cytoplasmic fluctuations during the maturation stage could represent a key element of the molecular requirement for amelobast modulation. This process involves: (1) increases in CaBP concentration, which may be controlled by epidermal growth factor as shown in the intestine for CaBP 9 k (Bruns et al., 1989), as the optimal binding of this growth factor was detected in ruffle-ended ameloblasts (MartineauDoizC et al., 1987) where CaBPs were much concentrated; (2) an increase in the total calcium content of the ameloblast layer (Eisenmann, Ashrafi and Zaki, 1989) probably related to the increased number of intracellular calcium ligands in ruffle-ended cells as shown here; (3) activation of Ca-ATPase with the same gradient (Takano et al., 1987; Takano, 1989) as those observed here for CaBPs, which would participate in the decreasing calcium intracellular concentration achieved in smooth-ended ameloblasts. All methods presently used to quantify calcium in ameloblasts cannot discriminate between Ca2+ and bound calcium (for review see Bawden, 1989). Studies on CaBPs may provide indirect evidence. Sasaki and Garant (1987) reported that Ca2+-ATPase may be calmodulin dependent and regulate calcium-exchange activity during amelogenesis. Investigations on the distribution of CaBPs and their variations under metabolic stress could thus contribute to a better understanding of some subcellular mechanisms of the undissociable trans- . port and intracellular regulation of calcium within ameloblasts. study was supported by the Laboratoires CRINEX. We thank Mireille Eb, Michelle George and Patricia Motron for their technical assistance
Second hypothesis: CaBPs acting in the regulation of intracellular calcium
Acknowledgements-This
Some autoradiographic studies appear to negate even the existence of transcellular calcium transport in ameloblasts (Munhoz and Leblond, 1984). Our data may conversely suggest that CaBPs contribute to the regulation of intracellular calcium, specifically in cells that are surrounded by calcium movements. This hypothesis corresponds with (1) the known, non-negligible concentration of CaBPs in tissues that are not involved in calcium exportation such as brain, cerebellum or uterus (for review see Christakos et al., 1989); (2) the intranuclear presence of CaPBs; and (3) the temporal differences between the calcitriolinduced increase in active calcium transport and in cytosolic CaBP shown in the intestine (for review see Christakos et al., 1989). Ionic calcium acts as an intracellular messenger and its concentration is maintained within narrow physiological limits (Carafoli, 1987). The secretion of enamel matrix appears calcium-dependent in vitro (Woltgens et al., 1987). Vitamin D-deficiency not only induces depletion of CaBPs but also disturbs enamel elaboration I:Berdal et al., 1989~). CaBPs could thus be essential for enamel matrix deposition, by acting on the intracellular concentration of ionic
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Burke J. L., Caride A., Verma A. K., Penniston T. and Kumar R. (1989) Plasma membrane protein calcium pump and 28kDa calcium binding protein in cells of rat kidnev distal tubules. Am. J. Phvs. 257. F842-F849. Bringasb., Nakamura E., Evans J. and Slavkin H. C. (1987) Ultrastructural analysis of enamel formation during in vifro development using chemically-defined medium. Scanning Microsopy 1, 1103-l 108. Bronner F. (1988) Vitamin D-dependent active calcium transport: the role of CaBP. Calc. Tiss. Int. 43, 133-137. Bruns D. E., Krishnan A. V., Feldman D., Gray R. W., Christakos S., Hirsch G. N. and Bruns M. E. (1989) Epidermal growth factor increases intestinal calbindin-D 9K and 1,25-dihydroxyvitamin D receptors in neonatal rats. Endocrinology 125, 478485. Carafoli E. (1987) Intracellular calcium homeostasis. A. Rev. Biochem. 56, 395433.
Celio M. R., Norman A. W. and Heizmann H. W. (1984) Vitamin D-dependent calcium-binding protein and parvalbumin occur in bones and teeth. Calc. Tiss. Int. 36, 129-130.
Christakos S. and Norman A. W. (1978) Vitamin D-induced calcium-binding protein in bone tissue. Science 202, 70-71. Christakos S., Gabrieledes C. and Rhoten W. B. (1989) Vitamin D-dependent calcium-binding proteins: chemistry, distribution, functional considerations, and molecular biology. Endocr. Rev. 10, 3-36. Costanzo L. and Windhager E. (1987) Calcium and sodium transport by the distal-convoluted tubule of rat. Am. J. Phvsiol. 235. F492-F506. Eisenmann D.’ R., Ashrafi S. H. and Zaki A. E. (1989) Calcium levels in ruffle-ended and smooth-ended maturation ameloblasts. Connect Tiss. Res. 22, 838. Elms T. N. and Taylor A. N. (1987) Calbindin-D 28K localization in rat molars during odontogenesis. J. dent. Res. 66, 1431-1434. Hunt T. (1989) Cytoplasmic anchoring proteins and the control of nuclear localization. Cell 59, 949-951. Intrator S., Elion J., Thomasset M. and Brehier A. (1985) Purification, immunological and biochemical characteriz-
ation of rat 28 kDa cholecalcin (cholecalciferol-induced calcium-binding protein). Biochem. J. 231, 89-95. Jande S. S., Tolnai S. and Lawson D. E. M. (1981) Immunohistochemical localization of vitamin D-dependent calcium-binding protein in duodenum, kidney, uterus and cerebellum of chicken. Hisfochemistry 71, 99-l 16.
Magloire H., Joffre A., Azerad J. and Lawson D. E. M. (1988) Localization of 28 kDa calbindin in human odontoblasts. Cell Tiss. Res. 254, 341-348. Martineau-Doize P., Lai W. H., Warshawsky H. and Bergeron J. J. M. (1987) Specific binding sites for growth factor in bone and incisor enamel organ of the rat. In Development and Diseases of Cartilage and Bone Matrix,
pp. 389-399. Alan R. Liss,.Philadelphia, PA. Morean D. W.. Welton A. F.. Heick A. E. and Christakos S.71986) Specific in vitro activation of Ca, Mg-ATPase by vitamin D-dependent rat renal calcium binding protein (calbindin-D,,,). Biochem. biophys. Res. Commun. 138, 547-553. Munhoz C. 0. G. and Leblond C. P. (1974) Deposition of calcium phosphate into dentin and enamel as shown by radio autography of sections of incisor teeth following injection of 4sCa into rats. Calif: Tiss. Res. 15, 221-235. Nagai N. and Frank R. M. (1985) Transfort du 45Ca par autoradiographie en microscopic llectronique au tours de l’amelogendse. Calc. Tiss. Res. 19, 21 l-221. Norman A. W., Roth J. and Orci L. (1982) The vitamin D endocrine system: steroid metabolism, hormone receptors, and biological response (calcium-binding protein). Endocr. Rev. 3, 331-366.
Perret C., Desplan C. and Thomasset M. (1985) Cholecalcin (a 9 kDa cholecalciferol-induced calcium-binding protein) messenger RNA. Distribution and induction by calcitriol in the rat digestive tract. Eur. J. Biochem. 150, 21 l-217. Perret C., Lomri N. and Thomasset M. (1988) Evolution of the E-F hand calcium-binding protein family: evidence for exon shuffling and intron insertion. J. Molec. Evol. 27, 351-364. Riad N. H., Bruns E. H., Fares N. H., Bruns D. E. and Herr J. C. (1988) Ultrastructural localization of the 9-kilodalton vitamin D-dependent calcium-binding protein in the murine intraplacental yolk sac. Anat. Rec. 222, 252-259.
Sasaki T. and Garant P. R. (1987) Calmodulin in rat incisor ameloblasts as revealed by protein A-gold immunocytochemistry. Calc. Tiss. Int. 40, 294-297. Schreiner D. S., Jande S. S., Parkes C. O., Lawson D. E. M. and Thomasset M. (1983) Immunocytochemical demonstration of two vitamin D-dependent calcium-binding proteins in mammalian kidney. Acta anaf. 117, l-44. Takano Y. (1989) Tooth Enamel (Ed. Fearnhead R. W.), Vol. 5. Florence Publishers, Yokohama, Japan. Takano Y., Matsuo S., Wasikawa S., Ichikawa H., Nishitawa S. and Akai M. (1987) The influence of vanadate on calcium uptake in maturing enamel of the rat incisor. J. dent. Res. 66, 1702-1707.
Taylor A. N. (1981) Immunocytochemical localization of the vitamin D-induced calcium-binding protein: relocation of antigen during frozen section processing. J. Histochem. Cytochem. 29, 65-73.
Taylor A. N. (1984) Tooth formation and 28,000 Dalton vitamin D-dependent calcium-binding protein. J. Hisfothem. Cytochem. 32, 159-164. Taylor A. N., Gleason W. A. and Lankford G. L. (1984) Rat intestinal calcium-binding protein: immunocytochemical localization in incisor ameloblasts. J. dent. Res. 63, 94-97.
Thomasset M., Parkes C. 0. and Cuisinier-Gleizes P. (1982) Rat calcium-binding proteins: distribution, development and vitamin D-dependence. Am. J. Physiol. 243, 483-488.
CaBP 9 k and CaBP 28 k in ameloblasts Thorens B., Roth J., Norman A. W., Perrelet A. and Orci L. (1982) Immunocytoclhemical localization of the vitamin D-dependent calcium-binding protein in chick duoden& J. Ceil Biol. 94, 115-122. Van Corven E. J. J. M.. Roche C. and Van OS C. H. (1985) Distribution of Ca++-ATPase, ATP-dependent Ca++: transport, calmodulin and vitamin D-dependent Ca+ +bindina nrotein along the villus-crvnt axis in rat duode&n. Biochim. b&hys. Acta 826,-274-282. Van OS C. H. (1987) Transcellular calcium transport in intestinal and renal epithelia cells. Biochim. biophys. Acta 206, 195-222.
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Walters J. R. F. (1989) Calbindin-D 9 kDa stimulates the calcium pump in rat enterocyte basolateral membranes. Am. J. Physiol. 2S6G, 124128. Woltgens J.. H. M., Lyaruu D. M., Bervoets Th. and Bronckers A. L. J. J. (1987) Effects of calcium and phosphate on secretion of enamel matrix and its subsequent mineralization in vitro. Adv. dent. Res. 1.196201. Zhou X. Y.. Demnster D. W.. Marion S. L.. Pike J. W.. Haussler M. R. and Clemens T. L. (1986) Bone vitamin D-dependent calcium-binding protein is localized in chondrocytes of growth plate cartilage. Calc. 7’iss. Znt. 38, 244-247.
0003-9969/91
1991
$3.00 + 0.00
Copyright 0 1991Pergamon Press plc
Printed in Great Britain. All rights reserved
SUBCELLULAR CO-LOCALIZATION AND CO-VARIATIONS OF TWO VITAMIN D-DEPENDENT PROTEINS IN RAT AMELOBLASTS ARIANE BERDAL,“~DOMINIQUEHOTTON,’ SHADI KAMYAB,’ PAULETTECUISINIER-GLEIZES’and HENRI MATHIEU’ ‘LJ120 INSERM, Hopital Robert Debre, 48 Boulevard Serurier, 75019 Paris and *Laboratoire d’histologie et d’embryologie, Faculti de chirurgie dentaire, Universitt Paris V, I rue Maurice Amoux, 92120 Montrouge, France (Accepred I8 April 1991) Summary-The immunocytochemical patterns of calbindin-D,, (CaBP 9 k) and calbindin-D,,, (CaBP 28 k) were compared by light and electron microscopy throughout amelogenesis. Labelling on serial sections and co-localization of CaBPs confirmed that the two proteins were restricted to a single cell type, the ameloblasts. Their quantity increased during presecretion, was stable during secretion and alternately high and low during the cyclic modulation of ameloblasts which occurs during maturation. Ruffle-ended ameloblasts contained the highest apparent concentration. Investigations with several fixatives indicated that the CaBPs were present in the cytosol and the nucleus, although there were slight differences with various fixatives by light microscopy. Their concentrations in these compartments varied in parallel throughout arnelogenesis. However, mitochondria contained only immunoreactive CaBP 9 k. While the distribution of CaBP 9 k in zones containing Golgi apparatus and rough endoplasmic reticulum was similar, CaBP 28 k concentration has, in another paper, been shown to be higher near the rough endoplasmic reticulum. Key words: ameloblasts, enamel, calcium, vitamin D-dependent calcium-binding proteins.
INTRODUCTION Calbindin-D,, (Cal3P 9 k) calbindin-D,,, (CaPB 28 k) are members of a superfamily of proteins that contain structurally similar calcium-binding sites (Perret, Lomri and ‘Thomasset, 1988). They are distributed in distinct ceil types of several mammalian organs (Christakos, Gabrieledes and Rhoten, 1989; Norman, Roth and Orci, 1982; Riad et al., 1988; Thomasset, Parkes and Cuisinier-Gleizes, 1982): mainly intestine, uterus, yolk sac and placenta for CaBP 9 k, kidney and cerebellum for CaBP 28 k. They have also been described in mineralized tissues, including growth-plate cartilage (Balmain ef al., 1986a, b; Zhou et ill., 1986), bone (Balmain et al., 1989; Celio, Norman and Heizmann, 1984; Christakos and Norman, 1978; Thomasset et al., 1982) and teeth (Berdal et al., 1989a; Celio et al., 1984; Elms and Taylor, 1987; Magloire et al., 1988; Taylor, 1984; Taylor, Gleason and Lankford, 1984). The synthesis of CaBPs is vitamin D-dependent, differently depending on the tissue type (for review see Christakos et al., 1989). In the rat enamel organ, the concentration of CaBP 28 k reaches _ 1% of that of the cytosolic proteins (Berdal et al., 19891,); it is restricted to ameloblasts and its immunolabelling density appears to vary with the stages of amelogenesis (Berdal et al., 1991). This pattern strongly suggests that CaBP 28 k could be Abbreviations: CaBP, calcium-binding protein; PBS, phosphate-buffered saline.
involved in amelogenesis, as this process is affected by vitamin D-deficiency (Berdal et al., 1987), which also causes CaBP depletion (Berdal et al., 1989b). Other studies indicate that CaBP 9 k is also present in the enamel organ (Taylor et al., 1984) in a lower concentration (Berdal er al., 1989~). We have now further investigated the relationships between the different stages of amelogenesis, CaBP 9 k and CaBP 28 k in the enamel organ. This tissue constitutes a unique example of an epithelium involved in calcium transport and extracellular mineralization in mammals (Bawden, 1989), where CaBP 28 k reaches the levels observed in kidney and intestine (Berdal et al., 1991) and is calcitriol dependent (Berdal et al., 1989a). The subcellular distribution of CaBPs was compared by light and electron microscopy, including co-localization. MATERIALS AND
METHODS
Antisera Antibodies to purified rat duodenum CaBP 9 k [gift from M. Thomasset, INSERM, U 120 (Thomasset et al., 1982)] were raised in New Zealand White rabbits as were antibodies to rat kidney CaPB 28 k [gift from A. Brehier, INSERM, U 120 (Intrator et al., 1985)]. Controls included replacement of the antisera with: non-immune serum (Nordic, Tilburg, the Netherlands), anti-sucrase serum (gift from J. P. Broyart, INSERM, U 120), preadsorbed anti-serum (1.5 pg CaBP 9 k/p1 anti-CaBP 9 k; 100 ng CaBP 28 k/p1 anti-CaBP 28 k). The rest of the immunolabelling 715
716
ARIANE BERDAL et al.
procedure was identical in controls and experimental samples as described above. Tissue processing localization
of
light -microscopic
immuno -
Preparation of samples. A total of 60 SpragueDawley rats (2-12 days old) were killed with ether. Various fixatives (acetic acid : ethanol, 4% paraformaldehyde in phosphate buffer, Carnoy’s mixture) were used. Some samples were fixed by immersion for 16 h. Other animals were anaesthetized and fixed by a 15min intracardiac perfusion through the left ventricle using a monostaltic pump (Touzart and Matignon, Vitry, France) followed by immersion for 90 min. The first molars were dissected out, rinsed in 0.1 M PBS (Eurobio, Paris, France) for 2 h and impregnated with 30% sucrose (Sigma, la Verpilliere,
Plate Figs l-5.
Localization
Fig. 1. Preameloblasts Fig. 2. Presecretory
of CaBP
(PA) in molars to contain
France) for 2 h in PBS. Sections were cut at -20°C in a cryostat (Wild Leitz, Rueil-Malmaison, France), placed on slides coated with poly+lysine (Sigma) and kept overnight at -4°C. Some fixed mandibles were dehydrated and embedded in paraffin. Fivemicron sections were cut, the paraffin removed, rehydrated and sections used for immunocytochemistry. Immunocytochemical study. All incubations and rinsings were done in PBS, pH 7.3, containing 0.5% bovine serum albumin (Institut Pasteur, Paris, France). Secondary antibodies and the streptavidinbiotin system (Amersham, les Ulis, France) were used to link peroxidase to the primary antibodies. The staining process was as follows: (1) non-specific binding sites were blocked with non-immune goat serum (1: 30; Nordic) for 30 min; (2) the sections were incubated with the primary antiserum at 27°C diluted 1:200 to 1: 1600 for CaBP 9 k and
1
9 k during
the presecretion
and secretion
of a 2-day-old rat; fixation with Carnoy’s no immunoreactive CaBP 9 k. x 600
stages.
solution.
ameloblasts (A) in molar of 2-day-old rat; fixation with Carnoy’s contain a light staining in the distal cytoplasm (arrow). x 600
Fig. 3. CaBP 9 k: stratum
intermedium
in incisor
from 30-day-old x 42,000
rat; fixation
with
They appear solution.
They
1% glutaraldehyde.
Fig. 4. Nucleus and neighbouring area of rough endoplasmic reticulum in secretory ameloblasts of incisors from 30-day-old rat; fixation with 3% paraformaldehyde and 0.5% glutaraldehyde. (a) CaBP 9 k: gold particles are scattered inside the nucleus (N) and in cytosolic area between the cisternae of rough endoplasmic reticulum (RER). There is some labelhng along the RER membranes on their cytosolic side (arrow). (b) Control: the cell appears almost devoid of labelling with non-immune serum. x 42,000 Fig. 5. CaBP 9 k: Golgi area in secretory ameloblasts of incisors from 30-day-old rat; fixation with 1% GA. Gold particles are distributed through the cytosohc zones surrounding either rough endoplasmic reticulum (RER) or Golgi (G) apparatus. x42,000 Plate 2 Figs 6 and 7. Localization Fig. 9 k: are and
of CaBP 9 k and CaBP 28 k in secretory
ameloblasts.
6. Fixative-dependent immunostaining of CaBPs in molars from S-day-old rat. (a) Carnoy’s, CaBP the staining is restricted to the cytoplasm in ameloblasts (A). (b) Carnoy’s, control: ameloblasts (A) not stained when antigen-absorbed antibody is used. (c) Acid: alcohol, CaBP 28 k: both the nuclei cytoplasm of ameloblasts (A) appear strongly stained. (d) 4% paraformaldehyde, CaBP 28 k: the cytoplasm and nuclei of ameloblasts (A) are evenly stained. x 1000
Fig. 7. Co-localization of CaBPs in secretory-stage ameloblasts; CaBP 9 k localized by IS-nm gold particles and CaBP 28 k by 30-nm gold particles. (a) CaBPs: supranuclear area in the incisor of a 30-day-old rat; fixation with 3% paraformaldehyde and 0.2% glutaraldehyde. Mitochondria (M) appear to contain gold particles, while cisternae of rough endoplasmic reticulum (RER) do not. (b) CaBPs: Tomes’ process; fixation with 1% glutaraldehyde. Gold particles are located between secretion granules (sg). Sparse staining is present in enamel. (c) Control: Tomes’ process; fixation with I % glutaraldehyde. Particles were detected within enamel with anti-sucrase. x 42,000 Plate 3 Figs 8-10.
Co-variations
of the distribution
and co-localization
of CaBPs
during
the maturation
Figs 8 and 9. Immunostaining for CaBP 9 k and CaBP 28 k along the cusp side on serial sections molar from I I-day-old rat; fixation with Carnoy’s.
stage. of the
Fig. 8. (a) CaBP 9 k: immunonegative areas of ameloblasts (A) located in the cervical loop region. (b) CaBP 9 k: neighbouring immunopositive ameloblasts (A); oe: organ enamel. (c) CaBP 9 k: immunopositive ameloblasts (A) located by the cusp top. (d) CaBP 9 k: immunonegative ameloblasts (A) located at the cusp top. x 600 Fig. 9. (a) CaBP 28 k: area corresponding sac. (c) CaBP 28 k: area corresponding
to 8(a). (b) CaBP 28 k: area corresponding to 8(b); fs: follicular to 8(c). (d) CaBP 28 k: area corresponding to 8(d). x 600
Fig. 10. Co-localization of CaBP 9 k and CaBP 28 k in the incisor of a 30-day-old rat; fixation with 4% paraformaldehyde. (a) Many lS-nm (CaBP 9 k) and 30-nm (CaBP 28 k) particles are present in the ruffled border of ameloblasts (RE). (b) In contrast, there are few gold particles within the cytosol of smooth-ended ameloblasts (SE). x 42,000
CaBP 9 k and CaBP 28 k in ameloblasts
Plate
I
717
718
&mm
BERDALet al.
Plate 2
CaBP 9 k and CaBP 28 k in ameloblasts
Plate 3
719
120
ARIANEBERDALet al.
Plate 4
CaBP 9 k and CaBP 28 k in ameloblasts
1:200 to 1: 26800 for CaBP 28 k; (3) rinsed and incubated with biotinylated anti-rabbit IgG, diluted 1: 100 for 90min; (4.) rinsed and incubated with streptavidin-peroxidase, diluted 1: 300 for 30 min. Peroxidase was revealed by incubating the sections for 15 min at room temperature with 3,3’-diaminobenzidine (5 mg/lO ml; Sigma) plus 5 ~1 30% H,02 in 0.1 M tris-solution, pH 7.66. The sections were finally rinsed with Tris, mounted in neutral glycerine, and observed and photographed in a Zeiss Orthoplan microscope. Tissue processing for electron -microscopic immuno localization Preparation of samples. Twenty 30-40-day-old Sprague-Dawley rats (body weight 103 + 6 g) were anaesthetized with ether before perfusion. Fixation was with different solutions in 0.1 M PBS, pH 7.3, including: 4% paraformaldehyde (n = 5), 3% paraformaldehyde with either 0.2% (n = 5), or 0.5% glutaraldehyde (n = 5) and 1% glutaraldehyde (n = 5). After a 15-min perfusion, half-mandibles were immersed in the same fixative for 90 min, and rinsed in PBS at 4°C. Incisors were dissected out. Free aldehyde groups were blocked by immersion of the whole teeth in PBS-O.25 M NH,Cl (Merck, Paris, France) for 18 h at 4.“C. Dehydration was done in methanol in a graded series of temperatures (4 to - 30°C). Samples were progressively impregnated with Lowicryl K4M resin (Chemiche Werk Lowi, Walkraiburg, Fed. Rep. Germany) at -30°C. U.V. light polymerization was at -30°C for 48 h and continued at room temperature for 5 days. Gold-interference sections were cut on an Ultrotome 5 (LKB) and collected on 200.mesh nickel grids. Immunogold labelling of CaBP 9k. All incubations and rinsings were done in PBS, pH 7.3, at room temperature. Grids were placed on a droplet of non-immune goat serum (diluted 1: 30) for 30 min, to inhibit non-specific t,inding. Sections were directly incubated with the primary antibodies (diluted 1: 10 to 1: 1000) for 90 min. After rinsing, grids were incubated with biotinylated secondary antibodies (diluted 1:lOO) for 60min. They were again rinsed and incubated with streptavidin conjugated with 15-nm gold particles (Amersham; diluted 1: 20) for 30 min. The grids were finally rinsed with PBS and distilled water, coumerstained with uranyl acetate and lead citrate, and observed in a Philips 201 electron microsope. Co-localization of CaBP 9 k and CaBP 28k. CaBP 9 k was immunolabelled on one side of the grid, as described above, except that sections were not counterstained. CaBP 28 k was visualized on the other side as follows: (1) incubation with nonimmune goat serum (diluted 1: 30) for 30 min; (2) then directly with an+CaBP 28 k (diluted 1: 100 to 1: 1000) for 90 min; (.3) rinsing and incubation with
721
secondary antibodies linked to 30-nm gold particles (Biocell, Cardiff, U.K.; diluted 1: 20) for 60 min. The grids were rinsed in PBS and distilled water and stained with uranyl acetate and lead citrate. Nonrecognition of anti-CaBP 9 k by the anti-CaBP 28 k secondary antibodies was checked by using supplementary controls in which the antibodies raised to CaBP 28 k were omitted. Radioimmunoassay of CaBPs in SlOO cytosolic extracts of the enamel organ
Eight male and eight female Sprague-Dawley rats (Charles River) were used for radioimmunoassays. Other rats (140- and 300-day-old pooled males and females) were also used. They were anaesthetized with ether and decapitated. The enamel organs of both mandibular incisors were dissected out, as described by Berdal et al. (1991). Radioimmunoassays on SlOO extracts were as described previously (Thomasset et al., 1982). Enamel organ provided curves parallel to the one obtained with both standard CaBPs. RESULTS CaBP concentration in the enamel organ
In 30-day-old rats, the amounts of CaBP 9 k were different in males (69 & 16 ng/mg of total protein) and in females (154 f 13 ng/mg). The concentrations of CaBP 9 k and CaBP 28 k remained in the same proportion during the life-cycle of the rat ([CaBP 9 k] - 100 ng/mg; [CaBP 28 k] - 10 pg/mg). CaBP 9 k distribution in presecretion - and secretion stage ameloblasts (Figs l-5)
Young dividing preameloblasts appeared to be unstained (Fig. 1). Immunolabelling was first detected in presecretory ameloblasts adjoining the dentine (Fig. 2). The nuclei and cytosol in cells of the stratum intermedium contained a neglible number of gold particles, which corresponded to the background level (Fig. 3). Immunogold particles were present between cisternae of rough endoplasmic reticulum, and inside the nuclei of secretory ameloblasts [Fig. 4(a)]. Controls showed a low labelling [Fig. 4(b)]. The immunoreactivity appeared lower in areas containing Golgi apparatus versus the areas containing rough endoplasmic reticulum (Fig. 5). Non-ameloblastic epithelial cells in the enamel-free areas at the cusp tip of the molar, or in the Hertwig’s sheath where the root develops, never contained CaBP 9 k immunolabelling (not shown). Effects of fixation on intracellular distribution of CaBPs in secretion-stage ameloblasts (Figs 6 and 7)
The distribution of CaBPs was restricted to the cytoplasm in specimens fixed in Carnoy’s [Fig. 6(a), CaBP 9 k; Fig. 6(b), control]. In contrast, CaBPs
Plate 4 Fig. 11, Co-localization and co-variations of CaBPs inside nuclei during the maturation stage. CaBP 9 k and CaBP 28 k. in the incisor of a 30-day-old rat; fixation with 3% paraformaldehyde and 0.5% glutaraldehyde. (a) Ruffle-ended ameloblasts (RE) contain dense lahelling. (b) Smooth-ended ameloblasts (SE) contain little labelling.
ARIANE BERDALet
722
appeared to be located in both the nucleus and cytoplasm with acid: alcohol [Fig. 6(c)] and aldehyde fixatives [Fig. 6(d); 4% paraformaldehyde and Figs 4 and 5, Figs 7-11; solutions of paraformaldehydeglutaraldehyde]. CaBP 9 k and CaBP 28 k co -localization in secretion stage ameloblasts (Figs 6 and 7)
The ameloblastic zone adjoining the stratum intermedium [Fig. 7(a)] contained numerous mitochondria immunostained for CaBP 9 k, but none stained for CaBP 28 k. Labelhng for both CaBPs was present in the cytosol between the rough endoplasmic reticulum and the mitochondria. Immunogold particles were located between secretory granules in Tomes’ process [Fig. 7(b)]. All immunocontrols showed low labelling density [Fig. 7(c); non-immune serum]. Co-localization and labelling co-variations of CaBP 9k and CaBP 28k in maturation-stage ameloblasts (Figs 8-l 1)
The immunostaining for CaBP 9 k (Fig. 8) and for CaBP 28 k (Fig. 9) in serial sections indicated that the distribution of the discontinuous, light-microscopic negative [(a) and (d)] and positive [(b) and (c)] areas for the two CaBPs was similar. Immunopositive ameloblasts showed a gradient of staining with the maximum near the cervical area [Figs 8(b) and 9(b)] and a minimum near the cusp tip [Figs 8(c) and 9(c)]. By electron microscopy, co-localization of CaBPs showed that ruffle-ended ameloblasts, with an interdigitated border [Fig. 10(a)], contained large amounts of labelling in their cytoplasm, while there were few gold particles in smooth-ended ameloblasts [Fig. 10(b)]. Similar variations were seen in the nuclei [Fig. 8(a); ruffle-ended ameloblasts and Fig. 8(b); smooth-ended ameloblasts in Fig. 111. DISCUSSION
Both CaBP 9 k and CaBP 28 k were shown in situ in the same cells by co-localization. Previous investigations suggested this possibility by labelling of different samples (Berdal et al., 1989b; Celia et al., 1984; Taylor, 1984; Taylor et al., 1984). Crossreactivity between the two proteins has been previously excluded by radioimmunoassay procedures (Thomasset et al., 1982). Western blotting of rat enamel-organ proteins also indicates that this is unlikely. A single band was obtained with anti-CaBP 28 k, with no labelling present at lower molecular weights (Berdal et al., 1989b, 1991). Thus, our immunocytochemical study indicates that the two proteins coexist in ameloblasts. Subcellular distribution of CaBPs within ameloblasts
Our findings show that immunonegative cells at the light microscopic level may contain immunoreactive protein at the electron microscopic level (CaBP 9 k and CaBP 28 k within smooth-ended ameloblasts here). Our investigation illustrates that fixation methods may also influence the distribution of CaBPs seen by immunolabelling, as suggested by analysis of the patterns obtained in other tissues [CaBP 9 k (Arnold, Kovaks and Murray, 1976; Balmain et al., 1986b; Riad et al., 1988; Taylor, 1981) and CaBP 28 k
al.
(Balmain et al., 1986a; Jande, Tolnai and Lawson, 1981; Schreiner et al., 1983; Thorens et al., 1982). Both CaBPs appeared to be restricted to the cytoplasm in material fixed with Carnoy’s but present in the nucleus and the cytoplasm of tissues fixed in all other fixatives at the light microscopic level. As Carnoy’s fluid is the only fixative that contains chloroform, this reagent may solubilize nuclear components. Indeed, measurements of CaBP in cell fractions have shown that CaBP 28 k, at least, is present in both nuclear and cytosolic compartments (Thorens et al., 1982). Moreover, proteins weighing less than 50 kDa penetrate passively and freely into the nucleus (Hunt, 1989). Therefore, in this study, the two CaBPs appeared in both the cytosol and nucleus. Moreover, the CaBP 9 k was only observed in mitochondria in the ameloblasts, as described in the cells of the yolk sac (Riad et al., 1988). Coexistence of CaBPs in calcium-transporting tissues
Radioimmunoassay for CaBP 9 k, compared to data for CaBP 28 k (Berdal et al., 1991), provided their ratio: ([CaPB 9 k]/[CaBP 28 k] = l-2%). The two CaBPs are present in the same cells not just in enamel organ but also in kidney (Screiner et al., 1983), growth plate cartilage (Balmain et al., 1986a, b; Zhou et al., 1986) and bone (Balmain et al., 1989; Ceho et al., 1984; Thomasset et al., 1982). It might therefore be that the two proteins are jointly involved in the physiology of calcium-handling tissues, albeit in different concentrations and with different functions as their subcellular distribution is different. Indeed, tissues not involved in transporting calcium contain only one CaBP (Norman et al., 1982; Thomasset et al., 1982; Christakos et al., 1989) pancreas or parathyroid containing CaBP 28 k and thymus and lung CaBP 9 k. 45Ca autoradiographic CaBP labelling
pattern
and fluctuations
in
The relatively low CaBP 9 k immunoreactivity in secretion-stage ameloblasts compared to maturationstage cells indicates that the concentration of the protein varies in the enamel organ, as suggested previously (Taylor et al., 1984). However, in contrast to Taylor’s findings, CaBP 9 k was detected in secretory cells in our study. A comparison of the distributions of CaBP 9 k (our study) and CaBP 28 k (Berdal et al., 1991), the immunolabelling of CaBPs in serial sections and their co-localization indicate that the concentrations of the two CaBPs in ameloblasts change in parallel throughout enamel formation. Calcium incorporation throughout amelogenesis is also irregular (Bawden and Wennberg, 1977): calcium transport appears to be cellularly controlled and spatially continuous during the secretion stage. The CaBPs were evenly distributed throughout the secretory ameloblasts. In contrast, calcium penetration is more massive and varies cyclically during the maturation stage (Takano et al., 1987). These variations in uptake of radioactivity correspond to cyclical transformations of ameloblast morphology from ruffle-ended to smoothended (for review see Bawden, 1989) and may therefore correlate with variations in the apparent concentrations of the two CaBPs. CaBPs were mostly
123
CaBP 9 k and CaBP 28 k in ameloblasts
concentrated in ruffle-,ended ameloblasts. These cells may transport calcium actively, as suggested by the chemical inactivation IofCa2+-ATPase (Takano et al., 1987). First hypothesis: CaBPs as mediators for transcellular calcium transport
In the intestinal epithelium, there are gradients of CaBP 9 k concentratmn corresponding to the active transport gradient: from the duodenum to the jejunum (Perret, Desplan and Thomasset, 1985) and along the crypt-villus axis (Van Corven, Roche and Van OS, 1985). CaBP 28 k immunoreactivity in kidney is present in the principal cells of the distal convoluted tubule (Berdal et al., 1991; Thorens et al., 1982), where there is a Ca-ATPase activity (Borke et al., 1989). CaBPs are thought to contribute to the vitamin D-dependent transcellular transport of calcium by acting as intracellular shuttles or by activating Ca-ATPase (Bronner, 1988; Morgan et al., 1986; Walters, 1989). They may function as such in ameloblasts, as prop’osed previously for CaBP 9 k only (Bawden, 1989). However, in vitro data suggest that, in tooth germs, where CaBPs are vitamin D-dependent (Berdal et al., 1989a), calcitriol does not affect calcium incorporation (Bawden, Deaton and Crenshaw, 1983; Bawden et al., 1985). Bringas et al. (1987) obtained normal enamel mineralization in a chemically defined medium without any .vitamin D supplementation. As autoradiographic studies suggest that the paracellular transport is massive during enamel mineralization (Nagai and Frank, ‘.975), quantitative analysis of calcium pathways is needed to evaluate the importance of this ‘calcitriol-dependent, CaBPs-mediated, transcellular calcium I.ransport’. Indeed, it represents a small but physiologically regulated part of the calcium pathway in the intestine (Van OS, 1987) and kidney (Costanzo and Windhager, 1987).
calcium during the secretion stage. Their nuclear and cytoplasmic fluctuations during the maturation stage could represent a key element of the molecular requirement for amelobast modulation. This process involves: (1) increases in CaBP concentration, which may be controlled by epidermal growth factor as shown in the intestine for CaBP 9 k (Bruns et al., 1989), as the optimal binding of this growth factor was detected in ruffle-ended ameloblasts (MartineauDoizC et al., 1987) where CaBPs were much concentrated; (2) an increase in the total calcium content of the ameloblast layer (Eisenmann, Ashrafi and Zaki, 1989) probably related to the increased number of intracellular calcium ligands in ruffle-ended cells as shown here; (3) activation of Ca-ATPase with the same gradient (Takano et al., 1987; Takano, 1989) as those observed here for CaBPs, which would participate in the decreasing calcium intracellular concentration achieved in smooth-ended ameloblasts. All methods presently used to quantify calcium in ameloblasts cannot discriminate between Ca2+ and bound calcium (for review see Bawden, 1989). Studies on CaBPs may provide indirect evidence. Sasaki and Garant (1987) reported that Ca2+-ATPase may be calmodulin dependent and regulate calcium-exchange activity during amelogenesis. Investigations on the distribution of CaBPs and their variations under metabolic stress could thus contribute to a better understanding of some subcellular mechanisms of the undissociable trans- . port and intracellular regulation of calcium within ameloblasts. study was supported by the Laboratoires CRINEX. We thank Mireille Eb, Michelle George and Patricia Motron for their technical assistance
Second hypothesis: CaBPs acting in the regulation of intracellular calcium
Acknowledgements-This
Some autoradiographic studies appear to negate even the existence of transcellular calcium transport in ameloblasts (Munhoz and Leblond, 1984). Our data may conversely suggest that CaBPs contribute to the regulation of intracellular calcium, specifically in cells that are surrounded by calcium movements. This hypothesis corresponds with (1) the known, non-negligible concentration of CaBPs in tissues that are not involved in calcium exportation such as brain, cerebellum or uterus (for review see Christakos et al., 1989); (2) the intranuclear presence of CaPBs; and (3) the temporal differences between the calcitriolinduced increase in active calcium transport and in cytosolic CaBP shown in the intestine (for review see Christakos et al., 1989). Ionic calcium acts as an intracellular messenger and its concentration is maintained within narrow physiological limits (Carafoli, 1987). The secretion of enamel matrix appears calcium-dependent in vitro (Woltgens et al., 1987). Vitamin D-deficiency not only induces depletion of CaBPs but also disturbs enamel elaboration I:Berdal et al., 1989~). CaBPs could thus be essential for enamel matrix deposition, by acting on the intracellular concentration of ionic
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