Autophagy Plays a Crucial Role in Ameloblast Differentiation

Abstract

Tooth enamel is generated by ameloblasts. Any failure in amelogenesis results in defects in the enamel, a condition known as amelogenesis imperfecta. Here, we report that mice with deficient autophagy in epithelial-derived tissues (K14-Cre;Atg7^F/F and K14-Cre;Atg3^F/F conditional knockout mice) exhibit amelogenesis imperfecta. Micro-computed tomography imaging confirmed that enamel density and thickness were significantly reduced in the teeth of these mice. At the molecular level, ameloblast differentiation was compromised through ectopic accumulation and activation of NRF2, a specific substrate of autophagy. Through bioinformatic analyses, we identified Bcl11b, Dlx3, Klk4, Ltbp3, Nectin1, and Pax9 as candidate genes related to amelogenesis imperfecta and the NRF2-mediated pathway. To investigate the effects of the ectopic NRF2 pathway activation caused by the autophagy deficiency, we analyzed target gene expression and NRF2 binding to the promoter region of candidate target genes and found suppressed gene expression of Bcl11b, Dlx3, Klk4, and Nectin1 but not of Ltbp3 and Pax9. Taken together, our findings indicate that autophagy plays a crucial role in ameloblast differentiation and that its failure results in amelogenesis imperfecta through ectopic NRF2 activation.

Keywords: Atg3; Atg7; ameloblasts; amelogenesis imperfecta; enamel formation; tooth development.

Figure 1.

Gross appearance of the teeth of wild-type and Atg7 mutant mice. (A) Incisors of wild-type (WT) and Atg7 conditional knockout (cKO) mice at 6 mo (6 M) of age. (B) Molars of WT and Atg7 cKO mice at 6 mo of age. (C) The region containing enamel in the lower incisor was divided into eight 1-mm-long cross sections (ES1–ES8) from near the apical loop to the gingival margin. Upper panel: a representation of a model WT tooth with sagittal micro–computed tomography (CT) sections (ES4–ES8) of the lower incisor at 6 mo of age. Lower panels: transverse micro-CT sections, taken at the indicated time points, of lower incisors from WT and Atg7 cKO mice. Scale bar: 1 mm. Color bar, density in Hounsfield units, range 516 to 3,100. (D) Quantification of mineral density and area of the enamel in incisors from WT and Atg7 cKO mice at 6 mo of age. **P < 0.01. ***P < 0.001. n = 6 per group. (E) Micro-CT images of molars from WT and Atg7 cKO mice at 6 mo of age. Color bar, density in Hounsfield units, range 60 to 350. (F) Quantification of enamel thickness in molars from WT and Atg7 cKO mice at 6 mo of age. ***P < 0.001. n = 6 per group.

Figure 2.

Histological appearance of the incisors of wild-type and Atg7 mutant mice. (A) Hematoxylin and eosin staining of sagittal sections of upper incisors from wild-type (WT) and Atg7 cKO mice. Scale bar: 10 μm. (B) Immunoblotting for the indicated molecules. (C) Immunohistochemical staining for the indicated molecules of upper incisors from WT and Atg7 cKO mice. Scale bars: 100 μm (left panels) and 10 μm (right panels). (D) Transmission electron microscopy in ameloblasts in incisors from WT and Atg7 cKO mice. Yellow arrow indicates the autophagosome. Red arrow indicates vesicle-like abnormal structures. (E) Immunohistochemical staining for the indicated molecules of upper incisors from WT and Atg7 cKO mice. Scale bar: 25 μm. (F) Berlin blue staining of sagittal sections of upper incisors from WT and Atg7 cKO mice. Scale bar: 10 μm.

Figure 3.

Identification of genes with compromised expression in the teeth of autophagy-deficient mice. (A) Schematic diagram of binding sites (BSs) for NRF2 in the promoter region (10 kb upstream from transcription start site) of each gene related to amelogenesis imperfecta. The conserved NRF2 BSs among 8 species were selected for experimental validation. (B) Quantitative reverse transcription polymerase chain reaction analyses for the indicated genes of upper incisors from wild-type (blue bars) and Atg7 cKO (green bars) mice. ***P < 0.001. n = 6 per group. (C) Chromatin immunoprecipitation analyses for each BS in Dlx3, Klk4, Nectin1, Bcl11c, Pax9, and Ltbp3. ***P < 0.001; ns, not significant. n = 6 per group.

Figure 4.

Expression of molecules related to amelogenesis in the upper incisors of autophagy-deficient mice. (A) In situ hybridization for the indicated genes in sagittal sections of upper incisors from wild-type (WT) and Atg7 conditional knockout (cKO) mice. Scale bar: 100 μm. (B) Immunohistochemical staining for the indicated molecules of upper incisors from WT and Atg7 cKO mice at 6 mo of age. Scale bar: 25 μm. (C) In situ hybridization for the indicated genes in sagittal sections of upper incisors from WT and Atg3 cKO mice. Scale bar: 100 μm. (D) Immunohistochemical staining for the indicated molecules of upper incisors from WT and Atg3 cKO mice at 6 mo of age. Scale bar: 25 μm.

Figure 5.

NRF2-dependent regulation of genes in autophagy-deficient ameloblasts. (A) Quantitative reverse transcription polymerase chain reaction (RT-PCR) analyses for the indicated genes in mHat9d cells treated with a control (blue bars) and Nrf2 overexpression (red bars) vector. ***P < 0.001. n = 6 per group. (B) Immunoblotting for the indicated molecules in wild-type (WT), Atg7 knockout (KO), and Atg3 KO mHAT9d cells. (C) Transmission electron microscopy in WT, Atg7 KO, and Atg3 KO mHAT9d cells. Yellow arrows indicate the autophagosomes. Red arrow indicates vesicle-like abnormal structures. Scale bars: 2 μm. (D) Quantitative RT-PCR analyses for the indicated genes in WT and Atg7 KO mHAT9d cells (upper panel) and WT and Atg3 KO mHAT9d cells (lower panel) with and without Nrf2 knockdown (KD). **P < 0.005. ***P < 0.001. n = 6 per group.