ARTICLE |
Correspondence to: Dominique Hotton, Laboratoire de BiologieOdontologie, EA 2380, UP7, Institut Biomédical des Cordeliers, 1521 rue de l'Ecole de Médecine, 75006 Paris, France.
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Summary |
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Among the four existing isoforms of alkaline phosphatase (AP), the present study is devoted to tissue-nonspecific alkaline phosphatase (TNAP) in mineralized dental tissues. Northern blot analysis and measurements of phosphohydrolase activity on microdissected epithelium and ectomesenchyme, in situ hybridization, and immunolabeling on incisors confirmed that the AP active in rodent teeth is TNAP. Whereas the developmental pattern of TNAP mRNA and protein and the previously described activity were similar in supra-ameloblastic and mesenchymal cells, they differed in enamel-secreting cells, the ameloblasts. As previously shown for other proteins involved in calcium and phosphate handling in ameloblasts, a biphasic pattern of steady-state TNAP mRNA levels was associated with additional variations in ameloblast TNAP protein levels during the cyclic modulation process. Although the association of TNAP upregulation and the initial phase of biomineralization appeared to be a basic feature of all mineralized tissues, ameloblasts (and to a lesser extent, odontoblasts) showed a second selectively prominent upregulation of TNAP mRNA/protein/activity during terminal growth of large enamel crystals only, i.e., the maturation stage. This differential expression/activity for TNAP in teeth vs bone may explain the striking dental phenotype vs bone reported in hypophosphatasia, a hereditary disorder related to TNAP mutation. (J Histochem Cytochem 47:15411552, 1999)
Key Words: alkaline phosphatase, bone, dentin, enamel, mineralization, vitamin D, hypophosphatasia, in situ hybridization, immunolocalization
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Introduction |
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Alkaline phosphatases (APs) are ubiquitous in many species, from bacteria to human (
Investigations of TNAP in the skeleton have been mostly performed in bone and cartilage (
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Materials and Methods |
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Ten-day-old (n = 12), 30-day-old (n = 18), and 56-day-old (n = 30) male SpragueDawley rats (Charles River; St Aubin les Elbeuf, France) were used for this study.
Tissue Preparation for RNA Investigation
The rats were decapitated after carbon dioxide asphyxiation, the mandibles were rapidly collected, and the alveolar bone was removed under a stereoscopic microscope. The incisor was extracted and the distinct epithelial and mesenchymal odontogenic cells were microdissected. The apical portion containing the odontogenic organ and the early mineralization zone of enamel and dentin was removed to ensure purity of epithelial and mesenchymal samples. Lateral cuts were made along the cementoenamel junction and the enamel organ was scraped off the labial part of the incisor. Finally, the dental mesenchyme was collected. Dental samples and kidneys were maintained under liquid nitrogen. Epithelial and mesenchymal dissection procedures were validated by: (a) identification of enamel protein mRNA, i.e., encoding amelogenins in epithelial cells and osteocalcin mRNA in mesenchymal cells (not shown) and (b) comparative measurements of purified proteins and alkaline phosphatase activity in right and left incisors of the same animals.
Northern Blotting
Total RNA isolation was performed on 100200 mg of microdissected tissue from 56-day-old rats with an RNA extraction kit (Euromedex; Souffelweyersheim, France). Total RNA was electrophoretically fractionated on a 1% agarose formaldehyde gel and transferred onto nylon membranes. Filters were prehybridized, hybridized with 32P-labeled rat TNAP cDNA (G.A. Rodan; Merck Research Laboratories, West Point, PA), washed, and autoradiographed, as previously described (
Measurement of AP Activity
The samples from 56-day-old rats were washed twice in 0.1 M PBS (Sigma; La Verpillière, France) and rinsed in 100 mM sodium carbonatebicarbonate solution at pH 10.2. They were homogenized at 6000 rpm with a polytron homogenizer (Ultraturax; Ika, Germany) in a solution containing 0.1% Nonidet P 40 (Sigma) 1 mM MgCl2, and sonicated at 4C. Lysates were removed by centrifugation at 3000 x g for 5 min. AP activity was determined in the supernatant. The enzymatic activity was expressed as nmol of p-nitrophenol (PNP) released per minute per mg of protein at 37C. AP phosphohydrolase activity was assessed by measuring PNP release from p-nitrophenolphosphate (PNPP) by absorbance spectrophotometry at 410 nm and compared with a PNP standard solution (Sigma). The reaction was carried out in 500 µl of buffer solution (1.5 M 2-amino-2-methyl-1 propanol, pH 10.3, with 15 mM PNPP). After 15 min at 37C with 50 µl of the tissue lysate supernatant, the reaction was stopped with 2 ml of 0.1 M NaOH. Protein concentrations were determined by Lowry's modified method (
Preparation of Incisor Samples for In Situ Hybridization and Immunolabeling
After barbital anesthesia, rats (10-, 30-, and 56-day-old) received an intracardiac infusion of 4% paraformaldehyde15% sucrose in PBS (Sigma), pH 7.4, for 15 min. Mandibles were dissected out, fixed by immersion in the same fixative for 1 hr at 4C, and rinsed overnight in 15% sucrosePBS at 4C. The mandibles were cut either without decalcification (left incisors) or after decalcification (right incisors). These latter were rinsed for 4 hr in PBS at 4C and decalcified for 4 weeks at 4C in PBS with 4.13% disodium ethylenediaminetetraacetic acid (Sigma) and 0.2% paraformaldehyde (Sigma) pH 7.4, dehydrated, and paraffin-embedded. Ten-µm sections of dissected left incisors were made with a cryostat at -25C (MGW Lauda Leitz; Rockleigh, NJ). Sections were deposited onto 50 mg/ml poly-L-lysine (Sigma)-coated slides and were then dehydrated in a graded ethanol series and stored at 4C. The other sections (decalcified right incisors) were made with a paraffin microtome (Leica; Rueil Malmaison, France) and were deposited on silanized slides, deparaffinized, and rehydrated before use.
In Situ Hybridization
A 2.2-kb fragment of rat TNAP subcloned into Bluescript-SK+ plasmid (gift from M. Vogel and G.A Rodan; Merck Research Laboratories) was linearized with BamHI or PvuII endonucleases (Promega; Madison, WI). [35S]-UTP-labeled single-stranded antisense and sense probes were synthesized in vitro using T7 and T3 polymerases, respectively (Promega). In situ hybridization was performed as previously described (
Immunocytochemical Procedures
Monoclonal murine primary antibodies specific for rat TNAP (M. Vogel and G. Rodan) were used. Paraffin sections were treated with 0.3% hydrogen peroxide in 0.1 M Tris-HCl, pH 7.6, for 10 min to inhibit endogenous peroxidase activity. After rinsing in Tris-HCl solution, the sections were incubated overnight in Tris-HCl containing 1:30 nonimmune goat serum (Nordic; Tilburg, The Netherlands) to block nonspecific binding sites and were then incubated with serial dilutions of monoclonal rat TNAP antibodies (from 1:750 to 1:4000) for 2 hr at room temperature, rinsed in 1% Tris-HClbovine serum albumin (BSA), and incubated with biotinylated polyclonal rabbit anti-mouse secondary antibodies at a 1:100 dilution for 1 hr. After incubation in 1:300 diluted extravidinperoxidase (Sigma) for 30 min, the immunoreactive sites were visualized by 3-3'-tetrachloride diaminobenzidine oxidation (Sigma), 5 mg/10 ml in 0.1 M Tris-HCl, pH 7.6, with 0.03% hydrogen peroxide. Sections were rinsed in Tris-HCl, dehydrated, and mounted in Depex (Gurr; OSI, France). Sections were lightly counterstained with Harris hematoxylin solution (Sigma). Irrelevant murine immunoglobulins (1:7501:4000) were used as negative controls.
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Results |
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Biochemical Investigations of Rat TNAP
The measurement of phosphohydrolase-specific activity of AP was realized in 56-day-old rat samples. This activity was consistently and significantly higher (p= 0.006) in microdissected mesenchyme (15,184 ± 911 nmol PNP/min at 37C/mg proteins) than in epithelium (7145 ± 214 nmol PNP/min at 37C/mg proteins). The reproductibility of microdissection procedures was established by the low variability between measurements (<3%) on paired left and right incisors of the same rat (Table 1). The Km values for epithelial and mesenchymal samples were identical (2.1 mM).
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Northern blot analysis was serially performed with TNAP and GAPDH probes using mRNA from two different portions of the tooth containing epithelial (EO) and mesenchymal (M) differentiated cells. The kidney (K) was used as the reference organ for rat TNAP. The transcripts (2.5 kb) expressed in dental epithelium and mesenchyme corresponded to that of the TNAP enzyme observed in the kidney (Figure 1).
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Distribution of TNAP mRNA and Protein in the Mandible
The forming alveolar bone (Figure 2) and adjoining cells of the follicular sac surrounding the incisor contained immunoreactive alkaline phosphatase. Progenitor and differentiated bone cells of the mandible (Figure 3) also consistently showed AP immunostaining, with an apparent decrease from early stages of osteoblasts/recently embedded osteocytes to the stages of older osteocytes. TNAP protein was present in both the cell membrane and cytoplasmic compartments of bone cells. Immunocontrols showed no labeling (Figure 4).
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Developmental Pattern of TNAP Protein and mRNA During Amelogenesis
The expression of TNAP mRNA and protein was analyzed in the incisor enamel organ (Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 and Figure 12). Similar data concerning the developmental patterns of AP mRNA and protein at the various ameloblast stages were obtained in 10-, 20-, and 56-day-old rat incisors. They are illustrated in the plates obtained from 56-day-old rats. The developmental pattern was followed from the presecretion stage (Figure 5) to the secretion (Figure 6) and maturation (Figure 7 Figure 8 Figure 9 and Figure 12) stages, ordered on the longitudinal axis of the samples. The apical loop that contains undifferentiated epithelial and mesenchymal cells was devoid of TNAP mRNA (not shown) and protein (Figure 2). At the end of the enamel presecretion stage, the TNAP transcripts were detected in presecretory ameloblasts and stratum intermedium cells (Figure 5A and Figure 5B). TNAP mRNA expression persisted in stratum intermedium cells during the secretion stage, whereas TNAP mRNA dramatically decreased in secretory ameloblasts (Figure 6A and Figure 6B). This distribution was observed throughout the secretion stage. Maturation stage ameloblasts showed progressively increasing apparent concentrations of TNAP transcripts from the transition phase (Figure 7A and Figure 7B) to the first ameloblast modulation (not shown). The highest and apparently stable steady-state mRNA levels were then observed throughout the maturation stage (Figure 8A and Figure 8B). The TNAP mRNA and protein distribution patterns were identical, except in the ameloblasts during the maturation stage. Ruffle-ended ameloblasts showed intense labeling during the modulation process, especially adjacent to the enamel matrix inside the ruffled border, in contrast with the smooth-ended ameloblasts, which were almost devoid of staining, at least on the smooth border (Figure 9). TNAP mRNA was detected in supra-ameloblastic cells with a decreasing gradient from the transition stage (Figure 7A and Figure 7B) throughout the successive ameloblast modulation cycles, resulting in almost complete absence of mRNA in supra-ameloblastic cells (Figure 8A and Figure 8B). The same pattern was observed for TNAP protein in supra-ameloblastic cells (Figure 9). Finally, immu-nostaining was also found in the extracellular enamel matrix bordering the apical pole of the ameloblasts in the maturation stage (Figure 10).
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Expression of TNAP mRNA and Protein in Dentinogenic Cells
Consistent with the data obtained with TNAP antibodies, TNAP mRNA appeared to be present in the odontoblasts and subodontoblastic cells (Figure 5A and Figure 5B), but the relative abundance of TNAP mRNA decreased in odontoblasts with the progressive deposition and biomineralization of mantle dentin followed by orthodentin. TNAP protein immunolabeling was present in the cell membrane of odontoblasts and subodontoblastic cells (Figure 11). Immunostaining was distributed in the cytoplasmic compartment and plasma membrane, from the supranuclear area to the secretory pole of odontoblasts. As observed in osteoblasts and epithelial cells, the nuclei of odontoblasts and subodontoblastic cells were devoid of labeling (Figure 11). The extracellular signal evidenced by serial dilutions of TNAP antibodies in the predentindentin border (1:1000 dilution, Figure 12 vs 1:750 dilution, Figure 11) became progressively more obvious, suggesting enrichment of TNAP epitopes in this extracellular zone compared to the intracellular compartments. Such an effect of TNAP antibody dilution was also observed for the extracellular labeling of enamel matrix (Figure 10).
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Discussion |
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This study was devoted to the comparative investigation of AP protein and mRNA in three different mineralized tissues: the best characterized bone, used as a reference system, as well as enamel and dentin, which have previously been mainly investigated separately by enzymatic methods, as many studies have been devoted to the fine analysis of AP activity by light and electron microscopy of bone and teeth (for reviews see
The developmental pattern of AP mRNA, protein (as shown here), and enzymatic activity (see review by
Odontogenic cells synthesize a set of molecules involved in the control of calcium and phosphate bioavailability, presumably useful for nucleation and growth of apatite crystals (see review by
The present cellular and tissue distribution of immunoreactive AP is consistent with the proposed pathways for AP synthesis and processing (
A striking feature of the comparison among bone, enamel, and dentin is that proteins important for the formation of mineralized tissues, e.g., TNAP, are expressed not only in the "main" cells, which secrete matrix proteins, osteoblasts, ameloblasts, and odontoblasts (for review, see
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Acknowledgments |
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Supported by EA 2380 funds, PHRC AOM 96067, and Laboratories Novartis, Santé Familiale.
Received for publication February 23, 1999; accepted July 20, 1999.
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