Bmi1 regulate tooth and mandible development by inhibiting p16 signal pathway

Abstract To determine whether the deletion of p16 can correct tooth and mandible growth retardation caused by Bmi1 deficiency, we compared the tooth and mandible phenotypes of homozygous p16‐deficient (p16−/−) mice, homozygous Bmi1‐deficient (Bmi1−/−) mice, double homozygous Bmi1 and p16‐deficient (Bmi1−/−p16−/−) mice to those of their wild‐type littermates at 4 weeks of age by radiograph, histochemistry and immunohistochemistry. Results showed that compared to Bmi1−/− mice, the dental mineral density, dental volume and dentin sialoprotein immunopositive areas were increased, whereas the ratio of the predentin area to total dentin area and that of biglycan immunopositive area to dentin area were decreased in Bmi1−/−p16−/− mice. These results indicate that the deletion of p16 can improve tooth development in Bmi1 knockout mice. Compared to Bmi1−/− mice, the mandible mineral density, cortical thickness, alveolar bone volume, osteoblast number and activity, alkaline phosphatase positive area were all increased significantly in Bmi1−/−p16−/− mice. These results indicate that the deletion of p16 can improve mandible growth in Bmi1 knockout mice. Furthermore, the protein expression levels of cyclin D, CDK4 and p53 were increased significantly in p16−/− mice compared with those from wild‐type mice; the protein expression levels of cyclin D and CDK4 were decreased significantly, whereas those of p27 and p53 were increased significantly in Bmi1−/− mice; these parameters were partly rescued in Bmi1−/−p16−/− mice compared with those from Bmi1−/− mice. Therefore, our results indicate that Bmi1 plays roles in regulating tooth and mandible development by inhibiting p16 signal pathway which initiated entry into cell cycle.


| INTRODUC TI ON
Bmi1 is a member of the family of polycomb (PcG) proteins. The PcG proteins are a group of transcriptional inhibitors that regulate the target gene by chromatin modification, which are closely related to the determinants of stem cell fate, X chromosome inactivation, cell differentiation and cell carcinogenesis. PcG proteins can be divided into two core protein complexes: the first is the multi-comb inhibitory complex 2 (PRC2), which plays a role in the initial stage of transcription inhibition; the second is the multicomb inhibitory complex 1 (PRC1), which maintains the stability of inhibitory state chromatin. 1 size and appearance at birth; however, they exhibited progressive post-natal growth retardation and died by early adulthood with signs of hematopoietic failure and neurological abnormalities. 6,7,9 It was reported that Bmi1 −/− mice had abnormal axial bone morphogenesis and increased bone marrow adipocytes. 8,10 Bmi1 maintains cell self-renewal and cell cycle progression to prevent cell senescence by inhibiting the transcription of the ink4a-arf gene encoding the cell cycle-dependent kinase inhibitory factor (CDKI). 11 A recent study has demonstrated that Bmi1 can bind directly to the Bmi1-responding element of the p16 promoter to repress its expression. 12 The ink4a-arf gene overlaps two antitumor proteins: p16 ink4a (p16) and p19 arf (p19). p16 selectively inhibits cyclin-dependent kinase 4/6 (CDK4/CDK6), thereby inhibiting phosphorylation of retinoblastoma protein (Rb). Rb inhibits downstream gene expression required for translation from the G1 phase into the S phase by binding to the cell cycle-related gene transcription factor E2F, resulting in cell growth arrest. 13,14 Studies showed that p16-positive cell deletion could significantly increase the weight of mice, muscle fibre diameter and fat layer thickness, thus delaying the ageing process. 15 As one of the downstream targets of Bmi1, knockout of p16 gene partially corrected the ability of neural stem cells and hematopoietic stem cells to self-renew. 13,16,17 It was reported that knockout of p16 and p19 genes in Bmi1 −/− mice improved the number, proliferation and activity of stem cells in Bmi1-deficient incisors, indicating that knockout of p16 and p19 partially rescued Bmi1-deficient incisor stem cell dysfunction. 18 We previously reported that Bmi1 deficiency resulted in defects in dentin and alveolar bone formation, and p16 was increased in Bmi1-deficient mandible. 19,20 However, the role of p16 in dentin development and mandibular osteogenesis in Bmi1 −/− mouse was unclear.
In this study, we showed that Bmi1 gene expression was downregulated with age, while p16 gene only expressed in the old stage.
To investigate whether the function of Bmi1 was mediated through p16 in regulating tooth and mandible development, compound mutant mice with homozygous deletion of both Bmi1 and p16 (Bmi1 −/− p16 −/− ) were generated. Their mandible phenotype was then compared with p16 −/− , Bmi1 −/− and wild-type mice at 4 weeks of age.

| Radiography
Mandibles were fixed in PLP fixative (2% paraformaldehyde containing 0.075 mol/L lysine and 0.01 mol/L sodium periodate) overnight at 4°C. A Faxitron model 805 radiographic inspection system (Faxitron, München, Germany) took using contact radiographs, at 22 kV voltage and with a 4-minute exposure time. X-Omat TL film (Eastman Kodak, Rochester, NY, USA) was used and processed routinely.

| Micro-computed tomography (micro-CT)
Mandibles were fixed in PLP fixative (2% paraformaldehyde containing 0.075 mol/L lysine and 0.01 mol/L sodium periodate) overnight at 4°C. Then the samples were analysed by micro-CT with a SkyScan 1072 scanner and associated analysis software (SkyScan, Antwerp, Belgium) as described. 19 Briefly, the samples were enclosed in tightly fitting plastic wrap to prevent movement and dehydration.
Thresholding was applied to the images to segment the bone from the background. Two-dimensional images were used to generate three-dimensional renderings using the 3D Creator software supplied with the instrument. The resolution of the micro-CT images is 18.2 μm.

| Histology
Mandibles were fixed in PLP fixative overnight at 4°C and processed histologically as described. 19 Mandibles were decalcified in EDTAglycerol solution for 7-10 days at 4°C. Decalcified right mandibles were dehydrated and embedded in paraffin, and 5 μm sections were cut on a rotary microtome. The sections were stained with haematoxylin and eosin (HE), or histochemically for total collagen and ALP, or immunohistochemically as described below.

| Immunohistochemical staining
Immunohistochemical staining was carried out for biglycan and dentin sialoprotein (DSP), using the avidin-biotin-peroxidase complex technique with affinity-purified rabbit anti-mouse biglycan antibody (1:400, ab58562, Abcam, Cambridge, UK) and dentin sialoprotein (1:200, sc-33587, Santa Cruz, CA, USA) following previously described methods. 19 Briefly, deparaffinized and rehydrated sections were brought to a biol in 10 mmol/L sodium citrate buffer pH6.0 for antigen retrieval. And then the sections were incubated with 3% hydrogen peroxide to block endogenous peroxidase activity and then washed in Tris-buffered saline (pH 7.6). The slides were incubated with the primary antibodies overnight at 4°C. After rinsing with Tris-buffered saline for 5 minutes for 3 times, slides were incubated with secondary antibody. Then Vectastain Elite ABC reagent (Vector Laboratories, Burlingame, CA, USA) was used to incubated for 45 minutes. Staining was developed using 3, 3-diaminobenzidine (2.5 mg/mL) followed by counterstaining with Mayer's haematoxylin.

| Western blot analysis
Proteins were extracted from mandibles and quantitated using a protein assay kit (Bio-Rad, Mississauga, Ontario, Canada). Protein samples (20 μg) were fractionated by SDS-PAGE and transferred to nitrocellulose membranes. Immunoblotting was carried out as de-

| Computer-assisted image analysis
After HE staining or histochemical or immunohistochemical staining of sections from five mice of each genotype, images of fields were photographed with a Sony digital camera. Images of micrographs from single sections were digitally recorded using a rectangular template, and recordings were processed and analysed using Northern Eclipse image analysis software as described. 19

| Statistical analysis
Data from image analysis are presented as mean ± SEM. Statistical comparisons were made using a two-way ANOVA, with P < .05 considered significant.

| p16 knockout improved teeth and mandible growth and mineralization of Bmi1-deficient mice
To clarify the effect of p16 knockout on the growth and minerali-

| p16 knockout promoted dentin maturation and formation in Bmi1-deficient mice
In order to clarify the effect of p16 knockout on dentin maturation and formation in Bmi1 −/− mice, HE staining was performed on the paraffin section of the first molar. The ratio of predentin to total dentin was significantly increased in the Bmi1 −/− mice com-

| p16 knockout accelerated formation of alveolar bone in Bmi1-deficient mice
In order to clarify whether the effect of p16 gene knockout on osteo-

| p16 knockout regulated cell cycle-related factor protein expression in Bmi1-deficient mandible
To clarify whether p16 gene knockout was associated with changes in cell cycle-related factors, the expression of cell cycle-related factors of dental and mandible proteins were analysed by Western blot.
Protein expressions of the Cyclin D and CDK4 in the Bmi1 −/− mice were significantly downregulated, while p27 and p53 were upregulated compared with that of WT mice; these four indicators in the F I G U R E 2 p16 knockout increased the tooth volume and alveolar bone volume in Bmi1-deficient mice. Paraffin-embedded sections through the first molars from 4-wk-old wild-type (WT), p16 −/− , Bmi1 −/− and Bmi1 −/− p16 −/− mice stained with (A) HE, and with (B) serious red for total collagen. Scale bars represent 400 μm. (C) Dental volume of incisors (mm 2 ), (D) dental volume of the first molars (mm 2 ), (E) dental alveolar bone volume (mm 2 ) and (F) cortical thickness (μm) were measured. Each value is the mean ± SEM of determinations in five animals of each group. *P < .05 relative to the wild-type mice

| D ISCUSS I ON
In this study, p16 was a cell cycle-dependent kinase inhibitor, knockout of p16 gene could partially correct the abnormal expression of Previous studies have shown that the number of Bmi1-deficient apical stem cells was reduced, the ability of the lingual cervical ring cells to enamel differentiation was weakened, and the deposition of enamel was reduced. Ink4a/Arf deletion can restore the capacity and the number of lumbar neck rings, but was unable to improve enamel developmental barrier. 18 It has been reported that the expression level of p16 gene was increased by 5 to 21 times in the central nervous system and peripheral nervous system of Bmi1 deficiency in all ages, and the gene expression level of p19 was only increased by 1.4-3 times. 22 Therefore, we believed that p16 played an important role in the nervous system development and maintenance with Bmi1 deletion. Teeth are derived from the differentiation of neural crest cells. p16 deletion can significantly improve by dentin development disorders caused by Bmi1 deficiency, which may be related to the ability that p16 deletion can restore the expression of DSP. DSP is a specific protein produced by odontoblast cells during odontoblast differentiation. 23 The expression of DSP in Bmi1 −/− teeth was significantly reduced, indicating that the function of odontoblast cells Previous studies have shown that the deficiency of Ink4a/Arf failed to correct the phenotype of osteoblast formation and proliferation caused by deletion of Bmi1. 16 However, the lack of Ink4a/ Arf was able to partially correct the neurological developmental disorder resulted from Bmi1 deletion. 13,17 We doubted whether p16 gene knockout can improve the neural crest cells developed from the mandible phenotype in Bmi1-deficient mice. Our results showed that knockout p16 can increase the ratio of alveolar bone, osteoblast number, osteoblast surface and ALP activity in Bmi1 −/− mice, and suggested that p16 gene knockout can increase the ability to bone formation in Bmi1-deficient osteoblasts. In summary, results of this study showed that Bmi1 can inhibit p16 and promote cell cycle progression, thus playing a role in promoting the growth of teeth and mandible.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest. Project administration (lead); supervision (equal); writing-original draft (lead); writing-review & editing (equal).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.