Regulation of calbindin-D(28k) expression by Msx2 in the dental epithelium

Abstract

Amelogenesis involves the coordinated expression of a set of molecules that includes enamel matrix proteins and calcium-binding proteins. Msx2 is a member of the divergent homeobox gene family and is instrumental in dental morphogenesis and biomineralization. This study focused on an EF-hand calcium-binding protein, calbindin-D(28k), which is highly expressed in dental epithelium. In vivo data showed that calbindin-D(28k) levels were higher in ameloblasts from Msx2(+/-) mice than Msx2(+/+) mice. Consistent with this finding, calbindin-D(28k) distribution was affected in transgenic mice with ectopic expression in root epithelium in rests of Malassez in Msx2(+/-) and more clearly in Msx2(-/-) mice. In accordance with these in vivo data, calbindin-D(28k) protein and mRNA levels were decreased in LS8 ameloblast-like cells by exogenous Msx2 overexpression. Furthermore, calbindin-D(28k) promoter activity (nt-1075/+34) was specifically diminished in the presence of Msx2 overexpression, showing that Msx2 behave as a transcriptional repressor for calbindin-D(28k) gene expression. In conclusion, Msx2 may control the spatiotemporally restricted frame of calbindin-D(28k) production in the dental epithelium in relation to enamel mineralization, as previously shown for amelogenin.

Figure 1.

Calbindin-D_28k expression in mouse incisor. Calbindin-D_28k protein was comparatively immunodetected in the incisors of 14-day-old Msx2^+/+ (A, F), Msx2^+/− (B, G), and Msx2^−/− (C, H) mice. Tissue sections (in ameloblast secretion stage) were fixed in 4% paraformaldehyde, decalcified in ethylenediaminetetraacetate, and embedded in paraffin. Then, 8-µm sections were analyzed by immunohistochemistry with rabbit anti–calbindin-D_28k or rabbit anti–β-galactosidase polyclonal antibodies (for Msx2) (D). Control sections (E) were incubated without primary antibody. Arrows indicate calbindin-D_28k increased staining in epithelial rests of Malassez cells from Msx2^+/+ to Msx2^−/− incisors. Ab, ameloblasts; Od, odontoblasts; P, pulp; ERM, epithelial rests of Malassez. Top row, scale bar = 20 µm; bottom row, scale bar = 500 µm.

Figure 2.

Msx2-mediated dose-dependent inhibition of calbindin-D_28k expression. (A) Calbindin-D_28k transcript levels were quantified by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). RNAs were isolated from LS8 cells after transient transfection with the Msx2 expression vector. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level was used for normalization. Differences were considered significant at p<0.05 (*). Mean ± SEM from a representative set of three independent experiments are shown. (a.u. for arbitrary units). (B) Whole-cell lysates were prepared from LS8 cells transiently transfected with Msx2 vector. Comparable amounts of cell lysate proteins were loaded into each lane and immunoblotted with a goat anti-actin antibody (top) and a rabbit anti–calbindin-D_28k antibody (bottom). The numbers indicate the molecular masses of marker proteins (in kilodaltons). The left panel shows the graphical representation of calbindin signal quantification (related to actin) of three independent experiments.

Figure 3.

Calbindin-D_28k promoter activity. Varying amounts of Msx2 expression plasmid were used (A) with or without appropriate siRNAs (B) and were transiently co-transfected into LS8 cells with 0.5 µg reporter construct. The involvement of Msx2 binding element was shown with the loss of inhibition of the deleted promoter activity (C). Each bar represents the mean ± SEM of three independent experiments. **p<0.01, *p<0.05. CAT, chloramphenicol acetyl-transferase; WT, wild-type.