A Critical Role of TRPM7 As an Ion Channel Protein in Mediating the Mineralization of the Craniofacial Hard Tissues - PubMed (original) (raw)
A Critical Role of TRPM7 As an Ion Channel Protein in Mediating the Mineralization of the Craniofacial Hard Tissues
Yukiko Nakano et al. Front Physiol. 2016.
Abstract
Magnesium ion (Mg(2+)) is the fourth most common cation in the human body, and has a crucial role in many physiological functions. Mg(2+) homeostasis is an important contributor to bone development, however, its roles in the development of dental mineralized tissues have not yet been well known. We identified that transient receptor potential cation channel, subfamily M, member 7 (TRPM7), was significantly upregulated in the mature ameloblasts as compared to other ameloblasts through our whole transcript microarray analyses of the ameloblasts. TRPM7, an ion channel for divalent metal cations with an intrinsic serine/threonine protein kinase activity, has been characterized as a key regulator of whole body Mg(2+) homeostasis. Semi-quantitative PCR and immunostaining for TRMP7 confirmed its upregulation during the maturation stage of enamel formation, at which ameloblasts direct rapid mineralization of the enamel matrix. The significantly hypomineralized craniofacial structures, including incisors, molars, and cranial bones were demonstrated by microCT analysis, von Kossa and trichrome staining in Trpm7 (Δkinase∕+) mice. A previously generated heterozygous mouse model with the deletion of the TRPM7 kinase domain. Interestingly, the skeletal phenotype of Trpm7 (Δkinase∕+) mice resembled those found in the tissue-nonspecific alkaline phosphatase (Alpl) KO mice, thus we further examined whether ALPL protein content and alkaline phosphatase (ALPase) activity in ameloblasts, odontoblasts and osteoblasts were affected in those mice. While ALPL protein in Trpm7 (Δkinase∕+) mice remained at the similar level as that in wt mice, ALPase activities in the Trpm7 (Δkinase∕+) mice were almost nonexistent. Supplemented magnesium successfully rescued the activities of ALPase in ameloblasts, odontoblasts and osteoblasts of Trpm7 (Δkinase∕+) mice. These results suggested that TRPM7 is essential for mineralization of enamel as well as dentin and bone by providing sufficient Mg(2+) for the ALPL activity, underlining the key importance of ALPL for biomineralization.
Keywords: TRPM7; alkaline phosphatase; biomineralization; bone; dentin; enamel; ion transport; magnesium homeostasis.
Figures
Figure 1
The expression pattern of TRPM7 in craniofacial hard tissue forming cells. (A) Semi quantitive PCR analysis showed that the expression levels of TRPM7 mRNA in mouse molar enamel organ progressively increased from pre-secretoy stage (P0) to secretory stage (P5) and to early maturation stage (P10) (**P < 0.01). (B) Immunohistochemical staining for TRPM7 on incisors and surrounding alveolar bone showed an increased intensity of TRPM7 as ameloblasts progressed from precursor cells at cervical loop to maturation stage (Ba–e). TRPM7 was also immunolocalized in odontoblasts (Bb,f), and osteoblasts (Bg). No immunoreactions were seen in those cells immunostained with non-specific rabbit IgG (Bh). Scale bars: 25 μm
Figure 2
A hypomineralized phenotype is present in enamel, dentin and cranial bones of _Trpm7_Δkinase∕+ mice. (A) Transluscent enamel is found in P14 wt mice. (B) The transparent mandibular incisor enamel of P14 Trpm7_Δkinase∕+ mice allows the red colored pulp tissue could be seen through. (C) 3-D microCT images of craniofacial structure show well mineralized incisors, entire molars, and craniofacial bones in wt mice. (D) At the same intensity threshold, the mineralized tissues are only detected in the crowns of molars, a small segment of incisors near the incisal end in Trpm7_Δkinase∕+. (E) A representive 2-D microCT image of sagittal section from P14 wt mouse hemimandible show the well contrasted enamel, dentin and alveolar bone. (F) The 2-D microCT image of sagittal section from P14 Trpm7Δkinase∕+ hemimandibles show contrasting image intensity consistent with mineralization only seen in the molar crown and incisal end of the incisor. Mineralized enamel, dentin and alveolar bone are detected in P14 wt first molar (G), and second molar (H). Clear contrasts in these tissues is only seen in part of first molar enamel, dentin and alveolar bone (I) and coronal enamel and dentin in the second molar (J) of P14 Trpm7Δkinase∕+ hemimandible.
Figure 3
Hypomineralization defects of enamel, dentin and alveolar bone in Trpm7Δkinase∕+ mice are characterized by histological analyses. Matrix mineralization was assessed by Von Kossa staining (A,B,E,F,I,J,M,N) and Goldner's Trichrome staining (C,D,G,H,K,L,O,P). In the wt incisor (A,C), secretory stage enamel matrix (En) is lighty positive (black) for Von Kossa staining (A), and is stained in red by Trichrome staining (C). Dentin matrix (Dn) is stained in black with Von Kossa (A), and is stained in blue with Trichrome staining (C). Non-mineralized/Von Kossa negative pre-dentin (pDn) is stained in light pink by Trichrome staining (C). In Trpm7Δkinase∕+ mice, both incisal enamel and dentin are negative for Von Kossa staining (B), and Trichrome stains the dentin matrix in light pink, similar to the color of non-mineralized pre-dentin (D). The alveolar bone matrix of wt mice is stained in black by Von Kossa (E), and light blue by Trichrome staining (G). The alveolar bone matrix of Trpm7Δkinase∕+ mice is negative for Von Kossa staining (F), and Trichrome staining on alveolar bone showed bone in Trpm7Δkinase∕+ mice is light pink (H), similar to osteoid. Unlike Von Kossa positive stained wt molar dentin (I,M), the entire dentin of the Trpm7Δkinase∕+ first molar is negative for Von Kossa staining (J) and shows pink/pre-dentin status by Trichrome staining (L). Interestingly, in the second molar of Trpm7Δkinase∕+ mice, both Von Kossa (N) and Trichrome (P) staining shows the coronal dentin as partially mineralized, but the root dentin is not mineralized. Enamel matrix of molars likely chipped off during the sectioning (seen as an enamel space/EnS), and the remaining matrix is positive for Von Kossa staining (N). Scale bars: 100 μm
Figure 4
There are no obvious morphological change in ameloblasts of Trpm7Δkinase∕+ mice. Morphodifferentiation of ameloblasts (Am) from pre-secretory stage to secretory stage in Trpm7Δkinase∕+ mice (F–J) is similar to that of wt mice (A–E). Am, ameloblast; SI, stratum intermedium; PL, papillary layer; scale bars: 25 μm
Figure 5
Acellular cementum is found on the root analog of wt mouse incisor as well as on the molar root. Acellular cementum, ususally seen as a blue line following toluidine blue staining, is visualized between periodontal ligment (PDL) and dentin (Dn) on wt molar (A,B). A similar staining pattern corresponding to acellular cementum can not be detected in Trpm7Δkinase∕+ mice (C,D). Acellular cementum, seen as a blue line by Toluidine Blue staining, is visualized along the incisors from the apical to incisal end in P14 wt mice (E–H, indicated by black arrows). However, this line (indicated by black arrow) only appears on the surface of the incisor root analog dentin near the incisal end in Trpm7Δkinase∕+ mice (I–L). Scale bars: 25 μm
Figure 6
Trpm7 gene deficiency does not affect the abundance of ALPL protein. In both wt and Trpm7Δkinase∕+ mice, ALPL was immunolocalized on the basal and lateral plasma membrane of osteoblasts (Os) (A,B) and odontoblasts (Od) (C,D) at the similar levels. In enamel organs of both wt and Trpm7Δkinase∕+ mice, ALPL was immunolocalized on plasma membrane of stratum intermedium cells (SI) and the cytoplasm of ameloblasts (Am) at secretory stage (E,F), and on the plasma membrane and cytoplasm of papillary layer cells (PL) and maturation ameloblasts (G,H). There was no notable difference in immunoreactive activities between wt and Trpm7Δkinase∕+ mice. Scale bars: 25 μm
Figure 7
Impaired alkaline phosphatase (ALPase) activity of osteoblasts, odotoblasts and ameloblasts in Trpm7Δkinase∕+ mice is partially retrieved by Mg2+ pre-treatment. (A) At the bone forming site, ALPase activity was detected (in red) on the plasma membrane of osteoblasts (Os) and its precursor cells (preOs) in wt mice. **(B)**There was no ALPase activity presented by osteoblasts in Trpm7Δkinase∕+ mice. (C) However, Mg2+ pre-treatment retrieved the ALPase activity on osteoblasts. (D) ALPase activity was detected at the basolateral surface of odontoblasts (Od) in wt mice. (E) While in Trpm7Δkinase∕+ mice, no ALPase activity was seen in the odontoblasts. (F) Mg2+ pre-treatment retrieved ALPase activity on odontoblasts. (J) ALPase activity was detected in stratum intermedium (SI), secretory ameloblasts (Am), and (G) papillary layer cells (P) at maturation stage in wt mice. In Trpm7Δkinase∕+ mice, ALPase activities were restricted only on the stratum intermedium at secretory stage, not in ameloblasts (H,K). Mg2+ pre-treatment retrieved the ALPase activity on ameloblasts and papillary layer cells (I,L). Dn, dentin; pre-Dn, pre-dentin; En, enamel; Scale bars: 25 μm.
## References
1. 1. Addison W. N., Azari F., Sorensen E. S., Kaartinen M. T., McKee M. D. (2007). Pyrophosphate inhibits mineralization of osteoblast cultures by binding to mineral, up-regulating osteopontin, and inhibiting alkaline phosphatase activity. J. Biol. Chem. 282, 15872–15883. 10.1074/jbc.M701116200 -DOI -PubMed 2. 1. Baker A. R., Silva N. F., Quinn D. W., Harte A. L., Pagano D., Bonser R. S., et al. (2006). Human epicardial adipose tissue expresses a pathogenic profile of adipocytokines in patients with cardiovascular disease. Cardiovasc. Diabetol. 5:1. 10.1186/1475-2840-5-1 -DOI -PMC -PubMed 3. 1. Barciszewska M. Z., Rapp G., Betzel C., Erdmann V. A., Barciszewski J. (2001). Structural changes of tRNA and 5S rRNA induced with magnesium and visualized with synchrotron mediated hydroxyl radical cleavage. Mol. Biol. Rep. 28, 103–110. 10.1023/A:1017951120531 -DOI -PubMed 4. 1. Beertsen W., VandenBos T., Everts V. (1999). Root development in mice lacking functional tissue non-specific alkaline phosphatase gene: inhibition of acellular cementum formation. J. Dent. Res. 78, 1221–1229. 10.1177/00220345990780060501 -DOI -PubMed 5. 1. Bernick S., Hungerford G. F. (1965). Effect of dietary magnesium deficiency on the bones and teeth of rats. J. Dent. Res. 44, 1317–1324. 10.1177/00220345650440063401 -DOI
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