Abstract
Ameloblasts are ectoderm-derived cells that produce an extracellular enamel matrix that mineralizes to form enamel. The development and use of immortalized cell lines, with a stable phenotype, is an important contribution to biological studies as it allows for the investigation of molecular activities without the continuous need for animals. In this study we compare the expression profiles of enamel-specific genes in two mouse derived ameloblast-like cell lines: LS8 and ALC cells. Quantitative PCR analysis indicates that, relative to each other, LS8 cells express greater mRNA levels for genes that define secretory-stage activities (Amelx, Ambn, Enam, and Mmp20), while ALC express greater mRNA levels for genes that define maturation-stage activities (Odam and Klk4). Western blot analyses show that Amelx, Ambn, and Odam proteins are detectable in ALC, but not LS8 cells. Unstimulated ALC cells form calcified nodules, while LS8 cells do not. These data provide greater insight as to the suitability of both cell lines to contribute to biological studies on enamel formation and biomineralization, and highlight some of the strengths and weaknesses when relying on enamel epithelial organ-derived cell lines to study molecular activities of amelogenesis.
Highlights
Dental enamel, the most highly mineralized and hardest tissue in vertebrates, is comprised of highly organized hydroxyapatite (Hap) crystallites formed by ameloblast cells (Smith, 1998)
Higher mRNA levels for Amelx, Ambn, Enam, and matrix metallopeptidase 20 (Mmp20) were noted in LS8 cells, while ALC cells expressed higher mRNA levels for odontogenin ameloblast associated (Odam) and kallikrein related-peptidase 4 (Klk4)
Expression of Amtn in both cell lines was negligible. These expression levels were compared to mRNA transcripts in mouse first molars isolated at PN3, 6, and 9 (Figure 1B). This in vivo derived molar data clearly demonstrated a shift from a more dominate secretory function, with high levels of expression for Amelx, Ambn, Enam, and Mmp20 at PN3, to a more dominant maturation-stage gene expression profile, with the highest levels of Amtn, Odam, and Klk4 seen at PN9
Summary
The most highly mineralized and hardest tissue in vertebrates, is comprised of highly organized hydroxyapatite (Hap) crystallites formed by ameloblast cells (Smith, 1998). Dental enamel formation is tightly controlled by ameloblast cells whose differential activities toward enamel formation can be broadly divided into the initial secretory-stage, followed by a maturation-stage (Smith, 1998; Lacruz et al, 2013b). The enamel organ transcriptome for secretory-stage vs maturationstage amelogenesis is remarkably different from one another and highlights the changing cellular events orchestrated for each stage of enamel formation (Lacruz et al, 2012c, 2013b). LS8 (Lacruz et al, 2012a) and HAT-7 cells (Zheng et al, 2013) have been used to investigate circadian rhythm gene activities related to enamel formation. PABSo-E cells have been used to study calcium-sensing receptor activities in enamel formation (Mathias et al, 2001). ALC cells are noted to form calcified nodules when cultured in conditioned Dulbecco’s modified Eagle’s medium (DMEM) (Nakata et al, 2003), suggesting that ALC cells may be a model more suitable to the study of maturation-stage events of amelogenesis, such as ion transport associated with mineral phase maturation
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