Fig 1: Proposed working model of TNAP-mediated phosphate metabolism during enamel formation. This model is based on the experimental findings of the present study and integrates known pathways of phosphate transport and metabolism. While the data provide functional and biochemical evidence supporting TNAP-dependent Pi generation within the enamel matrix, the pathways depicted here should be interpreted as a data-supported working model and have not been fully validated in vivo. Further studies, including genetic and intervention-based approaches, will be required to establish the essentiality and regulatory relationships of each component. The thickness of arrows indicates the relative contribution of each pathway: thick lines, major pathways; medium lines, moderate contributions; thin lines, minor contributions. Arrow weights were assigned based on an integrated assessment of gene expression levels by qPCR and localization patterns determined by immunohistochemistry. TNAP may be released from the ameloblast membrane by a hypothetical releasing mechanism (indicated by “?”). A, at the secretory stage, the ANK-dependent pathway and matrix TNAP-dependent pathways derived from PPi and phosphorylated enamel proteins (amelogenins and 32-kDa enamelin) are depicted as major routes. Low-molecular-weight amelogenins (TRAP/LRAP) generated by MMP-20 processing are highlighted to reflect their higher efficiency of TNAP-mediated Pi release compared with P148 amelogenin. B, at the maturation stage, the ATP→PPi pathway mediated by ENPP1 is depicted as the major route, whereas Pi transport via XPR1 and ATP release via PANX3 are shown as moderate contributors, reflecting stronger immunostaining. In contrast, the ANK-dependent pathway is depicted as less prominent than at the secretory stage. The ATP→TNAP→Pi pathway is shown as a minor route with a thin dashed line. TNAP, tissue-nonspecific alkaline phosphatase; ANK, progressive ankylosis protein; MMP-20, matrix metalloproteinase-20; PANX3, pannexin 3; XPR1, xenotropic and polytropic retrovirus receptor 1.
Fig 2: Protease-mediated enhancement of TNAP-dependent phosphate release from enamel proteins.A, phosphorylated amelogenins (P148 amelogenin, TRAP, and LRAP) and 32-kDa enamelin were incubated with (+) or without (−) rhTNAP under identical conditions (n = 5). Pi generation was calculated as the difference between 20 h and 0 h (t20 − t0). The resulting Pi values were normalized to the molar amount of each substrate and expressed as mol ΔPi/mol protein. Asterisks (∗) indicate significant differences compared with the corresponding rhTNAP-untreated (−) sample for each substrate, and daggers (†) indicate significant differences compared with rhTNAP-treated P148 (+). Statistical comparisons among P148 amelogenin, TRAP, and LRAP were performed using the Mann–Whitney U test (∗p < 0.05) and the Tukey–Kramer test (†p < 0.05). For 32-kDa enamelin, significance relative to the rhTNAP-untreated (−) condition was evaluated using the Mann–Whitney U test (∗∗p < 0.05). B, SDS-PAGE analysis (16% Tricine gel) of P148 amelogenin, TRAP, and LRAP and 32-kDa enamelin before (0 h) and after 20 h incubation with rhTNAP. TRAP, LRAP, and 32-kDa enamelin exhibited shifts toward lower apparent molecular weights following incubation (red dashed lines). Protein amounts were not strictly normalized across substrates, as this analysis was designed to evaluate qualitative changes in band mobility associated with TNAP-mediated dephosphorylation, rather than to provide a quantitative comparison of enzymatic activity. C, P148 amelogenin was incubated for 20 h with rhTNAP and/or ex vivo MMP-20 (n = 5). Pi generation was calculated as the difference between 20 h and 0 h (t20 − t0), normalized to the molar amount of P148 amelogenin, and expressed as mol ΔPi/mol P148 amelogenin. P148 only, P148 amelogenin alone; P148 + rhTNAP, with rhTNAP; P148 + MMP-20, with MMP-20; P148 + MMP-20 + rhTNAP, with both MMP-20 and rhTNAP. Asterisks (∗) indicate significant differences compared with the P148 only group (Steel–Dwass test, ∗p < 0.05). D, corresponding SDS-PAGE analysis (16% Tricine gel) of the reactions shown in (C). Left panel: Lane 1, P148 only; Lane 2, P148 + rhTNAP; Lane 3, P148 + MMP-20; Lane 4, P148 + MMP-20 + rhTNAP; Lane 5, MMP-20 only. The amount of MMP-20 present in the reaction mixtures was approximately 12.5 ng, and protein bands were barely detectable in lane 5 under the present staining conditions. Differences in the apparent molecular weight of TRAP were observed between P148 + MMP-20 (Lane 3) and coincubation with MMP-20 and rhTNAP (Lane 4) (red arrows). In addition, faint low-molecular-weight bands (∼13 kDa) observed in lanes 1 to 4 were consistent with previously reported amelogenin processing products generated from P148 amelogenin. Right panel: additional SDS-PAGE analysis of purified recombinant rhTNAP alone (5 and 50 ng). The amount of rhTNAP present in the reaction mixtures used in this experiment was approximately 5 ng, and at this loading amount, rhTNAP was barely detectable by SDS-PAGE, as shown in the right panel. However, when 50 ng of purified recombinant rhTNAP was intentionally loaded, the protein became detectable, suggesting that the 5 ng amount used in the present experiment was below or close to the practical detection limit of Simply Blue Safe Stain. E, effects of partially purified porcine MMP-20 or KLK4 on ex vivo Pi release from enamel matrix mediated by endogenous TNAP in secretory-stage (soft enamel; left) and maturation-stage (hard enamel; right) samples (n = 6). The left panel shows MMP-20, and the right panel shows KLK4. Cont, immediately after reaction start (0 h); Ori, after 20 h incubation without protease; MMP-20 or KLK4, after 20 h incubation with partially purified porcine MMP-20 or KLK4; MMP-20 + Lev or KLK4 + Lev, after 20 h incubation with each protease in the presence of levamisole. Pi content is expressed per gram of enamel. Asterisks (∗) indicate significant differences compared with the Cont group, and daggers (†) indicate significant differences compared with the Ori group (Holm test, ∗p < 0.05; †p < 0.05). TNAP, tissue-nonspecific alkaline phosphatase; rhTNAP, recombinant human tissue-nonspecific alkaline phosphatase; LRAP, leucine-rich amelogenin peptide; KLK4, kallikrein 4; MMP-20, matrix metalloproteinase-20.
Fig 3: TNAP activity and protein levels in the enamel matrix.A, gross appearance of secretory-stage (soft enamel; cheesy appearance) and maturation-stage (hard enamel; chalky appearance) collected from the crown portion of porcine incisors from approximately 5-month-old animals. B, time-dependent changes in TNAP activity normalized to enamel sample weight (IU/L/mg enamel) in the Tris-soluble fractions (Soft-T, Hard-T) and carbonate-bicarbonate-soluble fractions (Soft-C and Hard-C) prepared from soft and hard enamel (n = 6). A marked increase in activity was observed in the soft-C and hard-C fractions. C, comparison of TNAP activity at 20 min (n = 6). Although high activity was detected in the C fractions, no significant difference was observed between Soft-C and Hard-C by Student’s t test (n.s.). D, quantification of TNAP protein levels by ELISA, normalized to enamel sample weight (n = 6). In both the T and C fractions, soft enamel showed significantly higher TNAP protein levels than hard enamel (∗p < 0.05, Welch’s t test). TNAP, tissue-nonspecific alkaline phosphatase.
Fig 4: Gene expression and immunohistochemical localization of cell-derived factors involved in PPi supply.A, relative expression levels of Panx3, Enpp1, Ank, and Xpr1 in secretory-stage (Sec) and maturation-stage (Mat) EOE, analyzed by qPCR (n = 6). Gene expression levels were normalized to Gapdh. Panx3 and Ank were significantly higher at the Sec, whereas Enpp1 was significantly higher at the maturation stage (∗p < 0.05, Welch’s t test). B, representative immunohistochemical images showing the localization of PANX3, ENPP1, ANK, and XPR1 in sections from the secretory and maturation stages. HE indicates hematoxylin and eosin staining, and NC indicates the negative control in which the primary antibody was omitted. E, enamel; AM, ameloblast; SI, stratum intermedium; PL, papillary layer. Scale bar = 50 μm. The HE and NC panels shown in Figure 5 Bare identical to those shown in Figure 2C, as the immunohistochemical analyses were performed simultaneously using the same tissue sections and experimental controls. C, subcellular distribution of alkaline phosphatase (ALP) activity in EOE. Cytosolic and membrane fractions were prepared from EOE, and TNAP activity was measured over time. TNAP activity was predominantly detected in the membrane fraction, whereas only minimal activity was observed in the cytosolic fraction, consistent with the membrane-associated nature of TNAP. Data are presented as mean ± SD (n = 6). TNAP, tissue-nonspecific alkaline phosphatase; EOE, enamel organ epithelium; ANK, progressive ankylosis protein; PANX3, pannexin 3; XPR1, xenotropic and polytropic retrovirus receptor 1.
Fig 5: Histochemical localization of in situ ALP (TNAP) activity in porcine incisors. Porcine incisors from approximately 5-month-old animals were mechanically cleared of soft tissues, including the dental pulp and enamel organ epithelium (EOE), and extensively washed with PBS before ALP in situ activity analysis. Because the predominant ALP isozyme in these specimens is TNAP, the detected ALP activity was considered to represent TNAP activity. Arrows indicate the axial direction from the crown side to the root side. A, external surface view. B, fractured surface view. C, negative control (no ALP in situ activity) corresponding to (A). D, negative control (no ALP in situ activity) corresponding to (B). On the external surface, broad TNAP activity is observed predominantly on the root side, whereas staining on the crown side is relatively weak. On the fractured surface, continuous TNAP activity is detected along the exposed dentin and enamel. In contrast, reddish soft tissue-like areas within the same field show no detectable TNAP activity. No purple-blue TNAP activity following the tooth contour is observed in the control specimens, in which ALP in situ activity was omitted, shown in (C and D). Scale bar = 5 mm. ALP, alkaline phosphatase; TNAP, tissue-nonspecific alkaline phosphatase.
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