Integrin b 3 in Iron Transportation during Enamel Formation 1155

Rodent incisors exhibit pigmentation on their labial surfaces. Although previous studies have shown that this pigment is composed of iron, the existence of other elements has not been investigated. This study found that the lower incisors of CD61, also known as integrin b3, null mice (CD61) lacked pigmentation. Although ameloblasts differentiated and formed enamel normally, no ferric ion accumulation was observed in maturation-stage ameloblasts in CD61 mice. Surface elements of control and CD61 lower incisors were compared by x-ray photoelectron spectroscopy (XPS). XPS analysis detected C, Ca, N, O, and P on the labial surfaces of lower incisors of both mice, whereas Fe was detected only in control samples. No peak of non-ferrous metal or other element was detected in either group. Quantitative RT-PCR analysis of 18 iron-transportation-related genes with mRNA from maturation-stage ameloblasts and ALC, a pre-ameloblastic cell line, was performed. The results suggested that CD61 regulates the expressions of Slc11a2 and Slc40a1, both of which are involved in iron transportation in epithelial tissues. These results suggested that the pigment on the labial surface of mouse incisors is composed of Fe and that both anemia and reduction of irontransporting proteins may cause the loss of pigmentation in CD61 mice. KEY WOrDs: odontogenesis, incisor, mice, pigmentation, x-ray photoelectron spectroscopy, iron transporter. IntrODuctIOn As a continuously growing tooth, the rodent incisor is recognized as a good model for the study of tooth development and dental stem cells. Only the labial surfaces of incisors are covered with enamel, which exhibits brown pigmentation in rodent species including rats and mice. Deposition of the pigment is found to start after enamel maturation, just prior to eruption, and is thought to increase the hardness and acid resistance of enamel (Yanagawa et al., 2004). Previous observations have shown that ferric ion accumulates in the supranuclear region of ameloblasts and is deposited during the maturation stage (Warshawsky and Smith, 1974; Takano and Ozawa, 1981; Hu et al., 2011). Rats fed with iron-deficient food exhibit a loss of pigmentation, and electron microprobe analysis shows a decrease in Fe (Halse, 1973; Yanagawa et al., 2004). Thus, the pigment is thought to be composed of iron. Chalky white enamel that lacks pigmentation on incisors has been observed in many mouse strains (reviewed in Hu et al., 2007; Urzua et al., 2011, unless noted) that are deficient in enamel proteins, their degradation enzymes, fibroblast growth factor signaling (Lin et al., 2009), ion transporters, extracellular matrix proteins (Kii et al., 2006), transcription factors (Yanagawa et al., 2004), or cell adhesion molecules (Barron et al., 2008; Yoshida et al., 2010; Huang et al., 2011). Among these mutants, iron transportation defects have been reported in mice mutant for a transcription factor Nrf2 (Nrf2) and a cell adhesion molecule Nectin-1 (Nectin-1). Nrf2 mice exhibited a lack of or diffused iron deposition in ameloblasts and the stratum intermedium layer (Yanagawa et al., 2004). In Nectin-1 mice, diffused localization of ferric ion and iron-laden cells has been observed (Barron et al., 2008). These results suggest that iron transportation plays an important role during pigmentation. CD61, also known as integrin β3, is a cell-surface molecule forming a heterodimer with integrin αIIb or αv, which bind to various extracellular matrices, including vitronectin and fibronectin (Nieswandt and Watson, 2003). A signal from CD61 plays an important role in various physiological processes, including platelet function and stem-cell maintenance (Nieswandt and Watson, 2003; Asselin-Labat et al., 2007; Umemoto et al., 2012). In humans, mutations in CD61 cause Glanzmann thrombasthenia (GT), a condition associated with impaired platelet function (Chen et al., 1992). Mice lacking CD61 are used as animal models for GT (Hodivala-Dilke et al., 1999). This study found that CD61 mice lacked pigmentation on lower incisors, despite the absence of obvious defects in enamel formation. Although it has been reported that the pigment is composed of iron, the other elements have not been well-studied. The role of integrin signaling for iron transporters in requirement of Integrin β3 for Iron transportation during Enamel Formation at PENNSYLVANIA STATE UNIV on May 22, 2015 For personal use only. No other uses without permission. jdr.sagepub. com Downloaded from © 2012 International & American Associations for Dental Research J Dent Res 91(12) 2012 Integrin b3 in Iron Transportation during Enamel Formation 1155 ameloblasts is not well-understood. The aim of this study was to investigate the detailed elemental composition of the pigment and the effect of integrin deficiency on iron transporters in mouse ameloblasts.