Fig 1.
PKP1 expressed in developing tooth germs.
A, Differentially expressed genes identified by microarray analysis of teeth as compared to whole embryos at E14. Highlighted plot indicates Pkp1. Red and blue plots represent up- and down-regulated genes, respectively. B, qRT-PCR analysis of Pkp1 expression in teeth, skin, lungs, livers, kidneys, hearts, eyes, and brains of E14.5 embryos after normalization to Gapdh mRNA expression. C, qRT-PCR analysis of Pkp1 expression in teeth obtained from E11 to P7 a normalization to Gapdh 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.
Fig 2.
Pkp1 knockdown inhibits tooth germ growth and dental epithelial cell proliferation.
A, CLDE cell proliferation 3 days after treatment with control or Pkp1 siRNA, as determined by CCK-8 assay. B, BrdU incorporation by CLDE cells with or without Pkp1 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 Pkp1 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 Pkp1 expression in cultured tooth germ after normalization to Gapdh mRNA expression. F, CLDE cell proliferation in the presence of Wnt3a was evaluated using a CCK-8 assay after transfection with control or Pkp1 siRNA. G, TCF/LEF promoter activity after stimulation with Wnt3a was examined using a luciferase assay after transfection with control or Pkp1 siRNA. *P <0.01, Error bars represent the mean ± S.D.
Fig 3.
Extracellular Ca2+ induces PKP1 translocation from nucleus to plasma membrane.
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).
Fig 4.
Wnt signaling induces translocation of PKP1 from membrane to nucleus.
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.
Fig 5.
N terminus of PKP1 is required for nuclear translocation.
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.
Fig 6.
Expression patterns of PKP1 and ZO-1 in P1 incisors.
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 Pkp1 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 Pkp1 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.
Fig 7.
PKP1 regulates ameloblast differentiation and ZO-1 localization.
A, PKP1, ZO-1, Desmoplakin, and E-cadherin expressions in CLDE cells transfected with control or Pkp1 siRNA, as detected by immunocytochemistry. Nuclei were counterstained with DAPI. B, Enlarged image of disturbed ZO-1 expression following Pkp1 knockdown. Nuclei were counterstained with DAPI. C, CLDE cells were cultured for 1 week in the presence of Ca2+. Immunoprecipitation was performed with control IgG and anti-PKP1 antibodies, then ZO-1 and PKP1 binding was assessed by western blotting. Input lysates were confirmed by probing for ZO-1 (bottom). D, Immunoprecipitation was performed with control IgG and anti-ZO-1 antibodies, then PKP1 and ZO-1 binding was assessed by western blotting. Input lysates were confirmed by probing for PKP1 (bottom).