Iron Deposition and Ferritin Heavy Chain (Fth) Localization in Rodent Teeth

Wen and Paine BMC Research Notes 2013, 6:1 http://www.biomedcentral.com/1756-0500/6/1 RESEARCH ARTICLE Open Access Iron Deposition and Ferritin Heavy Chain (Fth) Localization in Rodent Teeth Xin Wen and Michael L Paine* Abstract Background: An iron rich layer on the labial surface is characteristic of the enamel of rodent incisors. In order to address a role for iron content in continuously growing incisors during odontogenesis, we studied iron deposition patterns in enamel and dentine using Perls’ blue staining and ferritin heavy chain (Fth) immunolocalization. Fth expression is regulated by iron level; therefore its localization can be used as a sensitive indicator for iron deposition. Results: Sagittal sections of 4-week old rat incisors showed a gradual increase in iron level in the enamel organ from secretory to maturation stages. In addition, iron was detected in ameloblasts of erupting third molars of 4-week old rats, suggesting iron plays a role in both incisor and molar development. In odontoblasts, the presence of iron was demonstrated, and this is consistent with iron’s role in collagen synthesis. Using postnatal 3-, 6-, 9-day old mice, the spatial and temporal expression of Fth in tooth development again indicated the presence of iron in mature ameloblasts and odontoblasts. Conclusions: While these data do not explain what functional role iron has in tooth formation, it does highlight a significant molecular activity associated with the formation of the rodent dentition. Keywords: Amelogenesis, Enamel, Endosomes, Ferritin, Immunohistochemistry, Iron Background Rodent incisors are characterized by yellowish pigmentation at labial side due to the presence of iron, with an iron content of about 0.030% in the whole upper incisors and 0.027% in the whole lower incisors [1]. Electron microscopy has shown that iron is found only in the region of the enamel organ associated with maturation [2]. The function of iron in enamel is not understood. A quantitative analysis on butterflyfish Chaetodon miliaris teeth found that those feed on harder prey have more iron than those that feed on softer-bodied prey, suggesting that iron serves as a strengthening agent to resist abrasion and cracking [3], and this is equally feasible in rodent incisor teeth. Furthermore, the iron concentration is inversely related to the level of calcium in the lingual edge of the tooth cap of butterflyfish [4], consistent with earlier observations that rats with a diet high in calcium showed decreased iron pigmentation in enamel [5] while incisors of iron deficient rats showed higher calcium * Correspondence: Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry, University of Southern California, Los Angeles, USA content in outer enamel [6]. This also suggests that iron and calcium may be able to reversibly substitute for each other in hydroxyapatite. It has also been proposed that iron can decrease the solubility of crystallized hydroxyapatite because iron density positively correlates with acid-resistance of outer enamel [7]. In addition, many knockout or transgenic animals targeting the silencing or overexpression of enamel gene products result in an enamel with a chalky white appearance and structural defects, suggesting the incorporation of iron into enamel is linked to the normal process of enamel formation [8,9]. Iron is essential to all living organisms. The most abundant iron-containing proteins are hemoproteins that are involved in oxygen transport and delivery. In addition, iron’s ability to shuttle between ferric iron (Fe3+) and ferrous iron (Fe2+) makes it especially useful in electron transport and enzyme catalysis. By the same token, unregulated iron can cause cellular damage by catalyzing reactions leading to the production of toxic oxygen radicals [10,11]. Excess iron that is not for immediate use is stored in ferritin, a shell-like structure with a central, Fe3+ containing, © 2013 Wen and Paine; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Wen and Paine BMC Research Notes 2013, 6:1 http://www.biomedcentral.com/1756-0500/6/1 cavity. Mammalian ferritins are 24-subunit heteropolymers made of two different subunit types, a heavy and light chain, coded by Fth and Ftl genes respectively. The early embryonic lethality in Fth knockout mice suggests an critical role for ferritin during organismal development [12]. The expression of Fth and Ftl is post-transcriptionally regulated by iron level [13]. When cellular iron levels are low, the iron regulatory proteins IRP1 and IRP2 bind to iron responsive elements, IREs, located in the 5’ untranslated region of the Fth and Ftl mRNA, and block the translation initiation of both genes. When iron levels are high, the iron-bound IRPs dissociate from the mRNA, thereby allowing translation of Fth and Ftl to proceed [14,15]. Given the high iron content in mature enamel, not surprisingly, Fth was identified as one of the genes most highly up-regulated in maturation ameloblasts when compared to secretory ameloblasts [16]. Earlier electron microscopic studies have also shown that ferritin is present only in maturation ameloblasts and papillary layer, but not in secretory ameloblasts [2,17]. Iron also functions as a cofactor of prolyl hydroxylase, which catalyzes formation of hydroxyl proline, a key step in collagen’s triple helix formation [18]. Since collagens comprise of 90% of dentin extracellular matrix molecules [19], iron is presumably present in odontoblasts for producing collagen. However, few studies have shown the presence of iron in odontoblasts, probably due to much lower iron level when compared to that in ameloblasts, and also the low sensitivity of iron staining method. Based on the knowledge that the amount of ferritin responds to iron levels [13], the presence of iron in odontoblasts was implied with immunolocalization of ferritin in this study. Published reports on the presence of iron and ferritin in teeth have mainly been limited to observations in ameloblasts and in the enamel of rodent incisors [2,20]. Iron uptake in developing rat molars has been observed with autoradiographic methods [21]. In the present study, the increasing iron deposit and ferritin expression in the enamel organ cells of rat incisors, throughout amelogenesis, is demonstrated. Additional data are also presented to illustrate the presence of iron in ameloblasts of molar teeth prior to eruption. The spatiotemporal expression profiles of Fth throughout incisor and molar tooth development are also shown using postnatal 3-, 6-, 9-day old mouse samples. Methods Page 2 of 11 with sodium pentobarbital at about 5 mg per 100 gram of rat body weight. After the lack of reflex was co (...truncated)