Plakophilin-1, a Novel Wnt Signaling Regulator, Is Critical for Tooth Development and Ameloblast Differentiation

<div><p>Tooth morphogenesis is initiated by reciprocal interactions between the ectoderm and neural crest-derived mesenchyme, and the Wnt signaling pathway is involved in this process. We found that Plakophilin (PKP)1, which is associated with diseases such as ectodermal dysplasia/skin fragility syndrome, was highly expressed in teeth and skin, and was upregulated during tooth development. We hypothesized that PKP1 regulates Wnt signaling via its armadillo repeat domain in a manner similar to β-catenin. To determine its role in tooth development, we performed <i>Pkp1</i> knockdown experiments using ex vivo organ cultures and cell cultures. Loss of <i>Pkp1</i> reduced the size of tooth germs and inhibited dental epithelial cell proliferation, which was stimulated by Wnt3a. Furthermore, transfected PKP1-emerald green fluorescent protein was translocated from the plasma membrane to the nucleus upon stimulation with Wnt3a and LiCl, which required the PKP1 N terminus (amino acids 161 to 270). Localization of PKP1, which is known as an adhesion-related desmosome component, shifted to the plasma membrane during ameloblast differentiation. In addition, <i>Pkp1</i> knockdown disrupted the localization of Zona occludens 1 in tight junctions and inhibited ameloblast differentiation; the two proteins were shown to directly interact by immunoprecipitation. These results implicate the participation of PKP1 in early tooth morphogenesis as an effector of canonical Wnt signaling that controls ameloblast differentiation via regulation of the cell adhesion complex.</p></div

Wnt signaling induces translocation of PKP1 from membrane to nucleus.

<p>A, CLDE cells were transfected with PKP1-EmGFP, then treated with or without Wnt3a for 24 h. Nuclear translocation was detected by confocal microscopy. Nuclei were stained with DAPI. B, Enlarged image of PKP1-EmGFP nuclear translocation following stimulation with Wnt3a. C, Nuclear translocation ratio of CLDE cells treated with Wnt3a (*P <0.01). Error bars represent the mean ± S.D. D, CLDE cells were transfected with PKP1-EmGFP, then treated with 40 mM LiCl. Nuclear translocation was detected by confocal microscopy. E, Nuclear translocation ratios with different doses of LiCl in CLDE cells.</p

Pkp1 knockdown inhibits tooth germ growth and dental epithelial cell proliferation.

<p>A, CLDE cell proliferation 3 days after treatment with control or <i>Pkp1</i> siRNA, as determined by CCK-8 assay. B, BrdU incorporation by CLDE cells with or without <i>Pkp1</i> knockdown. The ratio was calculated as BrdU-positive cell number/DAPI-stained nuclei. C, Seven-day organ cultures of E13 tooth germs transfected with control or <i>Pkp1</i> siRNA. D, Relative tooth size plot (n = 12), with the average tooth germ size in the control siRNA group set at 1.0. E, qRT-PCR analysis of <i>Pkp1</i> expression in cultured tooth germ after normalization to <i>Gapdh</i> mRNA expression. F, CLDE cell proliferation in the presence of Wnt3a was evaluated using a CCK-8 assay after transfection with control or <i>Pkp1</i> siRNA. G, TCF/LEF promoter activity after stimulation with Wnt3a was examined using a luciferase assay after transfection with control or <i>Pkp1</i> siRNA. *P <0.01, Error bars represent the mean ± S.D.</p

N terminus of PKP1 is required for nuclear translocation.

<p>A, Schematic representation of p120-catenin and PKP1-EmGFP constructs. PKP1-FL, full-length PKP1; PKP1-delN1, with deletion of N-terminal a.a. 1–160; PKP1-delN2, with deletion of N-terminal a.a. 1–270. All constructs contained the armadillo repeat domain. B, Nuclear translocation of PKP1-FL, -delN1, and -delN2 expressed in CLDE cells with or without LiCl treatment, as detected by confocal microscopy. C, Nuclear translocation ratios of CLDE cells transfected with PKP1-FL, -delN1, and -delN2 with or without LiCl treatment. D, Cell proliferation was determined after 72 h of culture by counting relative numbers of CLDE cells transfected with PKP1-FL and -delN2 with or without Wnt3a (*P <0.01). Error bars represent the mean ± S.D.</p

Extracellular Ca²⁺ induces PKP1 translocation from nucleus to plasma membrane.

<p>A, Cell proliferation was analyzed using a CCK-8 assay after stimulation with 100 ng/ml NT-4 or 1.5 mM Ca²⁺. B, AMBN expression was analyzed by qRT-PCR after stimulation with 100 ng/ml NT-4 or 1.5 mM Ca²⁺ (*P <0.01). Error bars represent the mean ± S.D. C, CLDE cells were cultured with 1.5 mM Ca²⁺ for 1 week, then the expressions of PKP1, ZO-1, E-cadherin, β-catenin, AMBN, and Sox2 were assessed by western blotting. GAPDH was used as a loading control. D. CLDE cells were cultured with or without Ca²⁺ for 48 h. Expressions of PKP1 (green, center panel) and transfected PKP1-EmGFP were detected by immunocytochemistry, and visualized by confocal microscopy. Nuclei were stained with DAPI (blue).</p

PKP1 expressed in developing tooth germs.

<p>A, Differentially expressed genes identified by microarray analysis of teeth as compared to whole embryos at E14. Highlighted plot indicates <i>Pkp1</i>. Red and blue plots represent up- and down-regulated genes, respectively. B, qRT-PCR analysis of <i>Pkp1</i> expression in teeth, skin, lungs, livers, kidneys, hearts, eyes, and brains of E14.5 embryos after normalization to <i>Gapdh</i> mRNA expression. C, qRT-PCR analysis of <i>Pkp1</i> expression in teeth obtained from E11 to P7 a normalization to <i>Gapdh</i> mRNA expression. D, PKP1 (green) expression in E13, E14, E16, and P1 mice, as detected by immunocytochemistry. Broken lines represent basement membrane of teeth. Enlarged images are shown below each panel. de, dental epithelium; dm, dental mesenchyme; iee, inner enamel epithelium; ek, enamel knot; sr, stellate reticulum; si, stratum intermedium; dp, dental papilla.</p

Expression patterns of PKP1 and ZO-1 in P1 incisors.

<p>A, PKP1 (green), ZO-1 (green), and AMBN (red) expression in cervical loop of P1 incisors at pre-secretory and secretory stages, as determined by immunocytochemistry. Nuclei were stained with DAPI (blue). iee, inner enamel epithelium; oee, outer enamel epithelium; sr, stellate reticulum; si, stratum intermedium; dp, dental papilla. B, CLDE cells were cultured after transfection with control or <i>Pkp1</i> siRNA in the presence of Ca²⁺ for 48 h, then the expressions of AMBN, ZO-1, Desmoplakin, PKP1, and β-catenin were assessed by western blotting. GAPDH was used as a loading control. C, CLDE cells were cultured after transfection with control or <i>Pkp1</i> siRNA in the presence of Ca²⁺ for 48 h. Differential interference contrast image (left) and immunolabeling of keratin 14 (green; center panel and enlarged in right panel) are shown.</p