Sclerostin promotes human dental pulp cells senescence

Human dental pulp collection

Healthy and fresh human premolars were extracted from 20- to 80-year-old patients who were under orthodontic or periodontal treatment in the Hospital of Stomatology, Wuhan University. All donors gave their informed consent. The teeth were divided into two groups: the young group contained 30 teeth from 20- to 30-year-old patients and the old group contained 20 teeth from 45- to 80-year-old patients.

A total of 10 teeth from each group were used for immunohistochemical analysis, and 10 teeth from each group for qRT-PCR analysis. The remaining 10 teeth from the young group were used for HDPC culture.

Immunohistochemical analysis

Teeth were fixed with 4% paraformaldehyde at room temperature and decalcified in 10% EDTA solution for more than 6 months. Teeth were cut into five-μm-thick serial sections, which were collected on poly-l-lysine-coated slides. For immunohistochemical staining, the sections were dewaxed and incubated with rabbit anti-sclerostin (1:200; Abclonal, Boston, MA, USA) at 4 °C overnight. The staining was performed using a biotin-streptavidin kit (ZhongShan Biotech, Beijing, China) before the sections were counterstained with hematoxylin.

Real-time PCR analysis

Total RNA was isolated using the RNAiso kit (Takara, Kusatsu, Japan). First-strand cDNA syntheses were performed by using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Kusatsu, Japan). Real-time polymerase chain reaction for sclerostin, p16, p53, p21, alkaline phosphatase (ALP), osteocalcin (OCN), osteocalcin (OPN), dentin sialophosphoprotein (DSPP) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was performed with the SYBR Green Kit (Takara, Kusatsu, Japan) using an Applied BioSystems 7900HT thermocycler (Applied Biosystems, Foster City, CA, USA). The primers are listed in Table 1. The relative amount or fold change of the target gene was normalized relative to the level of human GAPDH and the control groups.

Cells and cell culture

Human dental pulp cells were isolated from the healthy dental pulp of 10 premolars from young group. The dental pulp tissues were digested in a solution containing four mg/mL dispase and three mg/mL collagenase type I for 1 h at 37 °C and then cultured in α-modified essential medium (α-MEM; HyClone Laboratories, Inc., Logan, UT, USA) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) at 37 °C under 5% CO₂. Confluent monolayers were dissociated with 0.5% (w/v) trypsin-EDTA for subculture. HDPCs were serially subcultured until the cells spontaneously arrested their replication. In brief, 2 × 10⁵ HDPCs were plated in 60 mm diameter dishes and the medium was changed every 3 days. When the cells reach 80% confluence, all cells were dissociated with 0.5% (w/v) trypsin-EDTA and counted by a cell counting device (Vi-CELL™ XR; Beckman Coulter, Pasadena, CA, USA). The number of population doublings (PDs) was calculated using the following formula: PD = log₂ (cells harvested/cells seeded) + previous PD (Huh et al., 2016; Kitajima et al., 2011). For odontoblastic induction, the medium was changed to the odontoblastic induction medium, containing α-MEM, 5% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mmol/L β-glycerophosphate, 50 μg/mL ascorbic acid and 10 nmol/L dexamethasone (Sigma-Aldrich Co., St. Louis, MO, USA). Cultures were maintained with a medium change every 3 days.

Lentivirus packaging and cell infection

Full human SOST cds sequence and a human SOST sh-RNA were cloned and inserted into the PCDH-CMV-MCS-EF1-copGFP vector (GenePharma, Shanghai, China) and the GV298 vector (Genechem, Shanghai, China), respectively. Lentiviral particles were produced using three-plasmid systems, including pMD2.G, psPAX2 and the individual vectors, with NEOFECT™ DNA transfection reagent (Neofect, Beijing, China) according to the manufacturer’s instruction. For infection, HDPCs were incubated with lentiviral particles and polybrene (four μg/mL) in complete medium for 12 h. Cells with successful infection by pCDH-human-SOST were designated SOST, cells infected by sh-SOST were designated sh-SOST, and control cells infected by empty vector were designated Control and sh-Ctrl, respectively. The expression of sclerostin was quantified by real-time PCR and Western blot analysis. The mRNA and protein expression levels were significantly upregulated in the SOST group (P < 0.001, Figs. 1E and 1F), while they were knocked down by over 85% in the sh-SOST group 48 h after infection (P < 0.0001, Figs. 1G and 1H).

Western blot

Cells were lysed in M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Rockford, IL, USA) combined with a cocktail of protease inhibitors (Roche Molecular Biochemicals, Rotkreuz, Switzerland). Total lysate was loaded and separated by SDS/PAGE. Primary antibodies specific for sclerostin (1:1,000; Abclonal, Boston, MA, USA), phosphor-β-catenin (p-β-catenin) (1:1,000; Cell Signaling Technology, Danvers, MA, USA) and β-actin (1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Senescence-associated β-galactosidase assay

To determine senescence of HDPCs, an SA-β-Gal kit (Beyotime, Shanghai, China) was used according to the manufacturer’s instructions. In brief, cells seeded on slips were fixed with paraformaldehyde and incubated with senescence-associated β-galactosidase (SA-β-Gal) overnight. Senescent HDPCs were identified by blue-staining under standard light microscopy.

Cell proliferation assay

Human dental pulp cells were seeded at a density of 2 × 10³ cells/well in a 96-well plate and cultured for 24 h, and then the medium was replaced with fresh medium. On days 1, 2, 3 and 4, the density of viable cells within each well was quantified with the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) according to the manufacturer’s protocols. The absorbance at 450 nm was measured to calculate the number of viable cells in each well. A well with medium and CCK-8 solution but without cells was used as the baseline.

Alkaline phosphatase staining and ALP activity assay

Alkaline phosphatase staining was performed according to the manufacturer’s instructions (Beyotime, Shanghai, China). For ALP activity, cells were lysed with 0.1% Triton X-100 and 50 μL lysate was mixed with 100 μL p-nitrophenyl phosphate (four mg/mL). The mixture was incubated at 37 °C for 15–20 min. The reaction was stopped by the addition of 0.5N NaOH (100 μL) and read spectrophotometrically at 405 nm. The protein concentration of the lysate was determined as described in the manufacturer’s instructions of the Pierce BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). The enzyme activity was quantified by a p-nitrophenol standard curve and normalized by protein concentration.

Alizarin red staining

Cells were cultured in 12-well cell culture dishes in the odontoblastic induction medium for 14 days. Then, cells were fixed with 4% paraformaldehyde and stained with 2% alizarin red. The stain was desorbed with 10% cetylpyridinium chloride for 1 h and the absorbance was examined at 562 nm.

Statistical analysis

All experiments were repeated at least three times. Quantitative results are expressed as the mean ± standard deviation. Data were analyzed by t-test and one-way analyses of variance using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). P-values < 0.05 were considered statistically significant.

Expression of sclerostin is increased in senescent human dental pulp and subculture-induced senescent HDPCs

The immunohistochemistry assay showed that the expression of sclerostin in young human pulp was very low, and positive sclerostin staining was only found in odontoblasts of young dental pulp. In contrast, strong staining of sclerostin was observed throughout the senescent human dental pulp, especially in the odontoblasts lining near the pre-dentin (Figs. 1A and 1B). Similarly, qRT-PCR analyses showed a higher expression level of sclerostin mRNA in senescent dental pulp, along with increased expression levels of p16, p53 and p21 (Fig. 1C).

Rapidly proliferating HDPCs were serially subcultured until the cells spontaneously arrested replication. HDPCs completing 16 and 54 PDs were defined as young and senescent HDPCs, respectively (Mehrazarin et al., 2011). In line with the higher expression levels of sclerostin, p16, p53 and p21 in senescent dental pulp, qRT-PCR analyses showed significantly higher expression levels of sclerostin, p16, p53 and p21 in senescent HDPCs (Fig. 1D).

Sclerostin induces HDPCs senescence

To access the role of sclerostin in senescence of HDPCs, sclerostin was overexpressed in early-passaged HDPCs. As shown in Fig. 2, sclerostin overexpression significantly increased the number of SA-β-Gal-positive cells. In addition, the CCK-8 assay showed significantly decreased proliferation and higher expression levels of p16, p53 and p21 in sclerostin-overexpressing HDPCs (Figs. 2C and 2D). In contrast, knockdown of sclerostin in late-passaged HDPCs showed fewer SA-β-Gal-positive cells, increased proliferation and decreased p16, p53 and p21 expressions (Figs. 2E–2H). In conclusion, these results suggest that sclerostin induces HDPCs senescence and inhibits HDPCs proliferation via the p16 and p53 signaling pathways.

Sclerostin inhibits odontoblastic differentiation of HDPCs

To determine the effect of sclerostin on HDPC differentiation, we compared the odontoblastic differentiation ability between early-passaged SOST and Control groups. As shown in Fig. 3, sclerostin overexpression significantly decreased the odontoblastic differentiation of early-passaged HDPCs as shown by the decreased mineralization nodules formation and a decline in ALP activity along with lower expression levels of odontoblastic differentiation markers such as ALP, OPN, CON and DSPP (Figs. 3A–3C). Moreover, knockdown of sclerostin rescued the decreased odontoblastic differentiation of senescent HDPCs (Figs. 3D–3E). These data imply that the increased expression of sclerostin contributed to the impaired odontoblastic differentiation potential of senescent HDPCs.

The Wnt/β-catenin pathway may be involved in the progress of HDPCs senescence related to higher expression of sclerostin

To clarify the mechanisms underlying sclerostin-related HDPCs senescence, we examined the Wnt/β-catenin pathway activity. Western blot analysis showed that overexpression of sclerostin suppressed the activity of the Wnt/β-catenin pathway by increasing the expression of p-β-catenin and sclerostin knockdown significantly increased the activity of the Wnt/β-catenin pathway (Fig. 4). These results suggest that sclerostin might accelerate senescence of HDPCs in a Wnt signaling pathway-dependent mechanism.