Effect of parathyroid hormone-related protein on odontogenic differentiation in human dental pulp cells

This study was approved by the Institutional Review Board of Chonnam National University Dental Hospital, Gwangju, Korea (IRB No. CNUDH-2016-009). Written informed consent was obtained from each patient included in this study. Extracted human third molars with pulp tissues were obtained from the Department of Oral Maxillofacial Surgery, Chonnam National University Dental Hospital. Tooth samples were removed aseptically, rinsed with Dulbecco’s phosphate-buffered saline solution (DPBS, Welgene, Daegu, South Korea), and placed in 60 mm dishes. The cells were cultured in growth media (GM) consisting of α-minimum essential medium (α-MEM, Gibco Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco Invitrogen) and 1% antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin, Gibco Invitrogen) in a humidified atmosphere of 5% CO₂ at 37 °C. For mineralization experiments, the cells were cultured in odontogenic induction medium (OM) containing 50 mg/mL ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA) and 10 mmol/L β-glycerophosphate (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). When the cells reached confluence, subcultures were performed and cells with passage numbers 3 to 4 were used for this study and rinsed with DPBS. The cells were detached with an appropriate quantity of trypsin solution (Gibco Invitrogen) for three minutes in a CO₂ incubator in a humidified atmosphere of 5% CO₂ at 37 °C. The cell suspension was transferred to a tube and gently centrifuged at 800 RPM for five minutes. After removing the supernatant, the cell pellet was gently resuspended in GM consisting of 10% α-MEM. The density of the viable cells was determined by counting in a hemocytometer and the cells were seeded in wells or dishes in the following experiments.

Recombinant human PTHrP (Sigma-Aldrich) was directly added at 1 nM or 10 nM to the OM. The cells were treated with PTHrP the next day after seeding and incubated with PTHrP for 3, 5, 7, or 14 days. The medium was changed with fresh medium containing PTHrP every two days.

HDPCs were seeded at a density of 1 × 10⁴cells per well in 96-well culture plates. After 24 h of culture, the cells were treated with PTHrP at different concentrations (1, 10, and 100 nM) for 24 h. After 24 h of culture, cell viability was analyzed by the WST-1 assay using an EZ-Cytox Enhanced Cell Viability Assay Kit (Daeil Lab Service, Seoul, Korea). Briefly, 10 μL of EZ-Cytox reagent was added to each well in the 96-well plate and incubated at 37 °C for four hours. The absorbance was measured at a wavelength of 420 nm using a spectrophotometer (Thermo Scientific Multiskan GO, Waltham, MA, USA).

HDPCs were seeded at a density of 2 × 10⁵ cells per well in 6-well culture plates with GM. After 24 h of culture, the cells were treated in OM with or without 1 and 10 nM PTHrP for three and five days. Total RNA was then extracted from the cells using Trizol reagent (Gibco Invitrogen) according to the manufacturer’s recommended protocol. Complementary DNA (cDNA) was synthesized using a random primer (Promega Biotech, Piscataway, NJ, USA) and an AccessQuick™ real-time polymerase chain reaction (RT-PCR) system (Promega, Madison, WI, USA). Quantitative RT-PCR was performed using a QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, CA, USA) on a 72-well Rotor-Gene 6000 (Corbett Research, Sydney, Australia). The primer sequences used for PCR are listed in Table 1. The relative gene expression levels were analyzed using the 2^-∆∆Ct method. To examine the effect of PTHrP, the same assay described above was performed for cells cultured in OM with or without PTHrP at 1 or 10 nM.

HDPCs were seeded at a density of 3 × 10⁵ cells per dish on 60 mm cell culture dishes and cultured in OM with or without PTHrP at 1 or 10 nM. The medium was replaced with fresh medium every two days. The PTHrP-induced odontogenic protein expression and activation of protein kinase B (PKB/AKT), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinases (JNK), and p38 mitogen-activated protein kinase (MAPK) were analyzed by Western blots. HDPCs were treated with or without PTHrP at 1 or 10 nM. The cells were washed twice with PBS and extracted with cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA). Then cell lysates were then centrifuged at 13,000 rpm for 10 min. The supernatants were collected and protein concentrations were determined using a Lowry Protein Assay Reagent Kit (Bio-Rad Laboratories, Hercules, CA, USA). The proteins were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis at 80 V for two hours and transferred to polyvinylidene difluoride membranes at 10 V overnight. After blocking with 5% non-fat dried skim milk in PBS containing 0.1% Tween 20 (PBST) at room temperature for one hour, the membranes were incubated with anti–dentin sialophosphoprotein (DSPP) (1:2000; Thermo Fisher Scientific), anti–dentin matrix protein (DMP)-1 (1:2000; Abcam, Cambridge, UK), anti-ERK (1:2000; Cell Signaling Technology), and anti-phospho-ERK (1:2000; Cell Signaling Technology) at 4 °C overnight. After washing three times with PBST, the membranes were then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibodies (1:10,000 Sigma-Aldrich) at room temperature for one hour. After washing with PBS five times, chemiluminescent HRP substrate (Millipore, Billerica, MA, USA) was used to visualize the protein signals with a Chemiluminescence Imaging System (Ez-capture; Atto, Tokyo, Japan).

HDPCs were seeded at a density of 2 × 10⁴cells per well in 24-well culture plates and cultured in GM and OM with or without PTHrP at 1 or 10 nM for seven days. Fresh GM and OM were replaced every two days. After seven days of exposure, the medium was removed. The cells were washed with PBS and fixed in 70% ethanol for one hour, followed by rinsing with distilled water three times. The fixed cells were treated with 300 μL alkaline phosphatase (ALP) staining reagent (1-step NBT/BCIP solution; Thermo Fisher Scientific) per well. After removing the staining reagent, a photograph was taken using an Officejet Pro L7580 scanner (Hewlett-Packard, Palo Alto, CA, USA). To quantitatively evaluate the staining results, the stain was treated with 10% cetylpyridinium chloride (pH = 7.0) for 30 min at room temperature, followed by absorbance measurement at a wavelength of 562 nm using a spectrophotometer (Thermo Scientific Multiskan GO).

HDPCs were seeded at a density of 2 × 10⁴cells per well in 24-well cell culture plates and cultured in GM and OM with or without PTHrP at 1 or 10 nM for 14 days. After 14 days, the cells were fixed in 70% ethanol and stained with 2% alizarin red S staining reagent (LIFELINE Cell Tech, Frederick, MD, USA). The results were recorded using an Officejet Pro L7580 scanner. For quantitative analysis, 10% cetylpyridinium chloride (pH = 7.0) was added to each sample and the absorbance value was measured at a wavelength of 540 nm using a spectrophotometer (Thermo Scientific Multiskan GO).

A WST-1 assay was performed to evaluate the cell viability of hDPCs treated with PTHrP at 1, 10, and 100 nM. The cell viability was not significantly different between the untreated group and the groups treated with different concentrations of PTHrP (Fig. 1a).

Quantitative RT-PCR analyses were performed to investigate the effect of PTHrP on odontogenic differentiation in hDPCs. The DSPP and DMP-1 mRNA expression levels were gradually increased with increasing concentrations of PTHrP three and five days after treatment. DSPP mRNA levels were significantly increased in the group treated with 10 nM PTHrP compared to the group not treated with PTHrP (^**P < 0.01) (Fig. 1b). DMP-1 mRNA levels were also significantly increased after treatment with PTHrP at concentration of 1 or 10 nM compared to the group not treated with PTHrP (^*P < .05 and ^***P < .001) (Fig. 1c).

Western blot analyses were performed to investigate the effect of PTHrP on the odontogenic differentiation in hDPCs. As shown in Fig. 1d, the protein levels of DSPP in the PTHrP-treated cells were higher than those in the no PTHrP group on days 3 and 5, although the difference was not statistically significant. The protein levels of DMP-1 were increased significantly in the group treated with 10 nM PTHrP on day 5 and slightly increased on day 3.

To verify the mineralization effect of PTHrP on hDPSCs, ALP staining and Alizarin red staining with or without 1 or 10 nM PTHrP treatment were performed. After seven days, ALP staining was significantly (^***P < 0.001) increased in all groups compared to that in the GM group. There was also a significant difference in ALP staining in the 1 nM or 10 nM PTHrP groups compared to the no PTHrP group (^##P < 0.01 and ^###P < 0.001). After 14 days, treatment with 1 and 10 nM PTHrP significantly enhanced calcium nodule deposition based on Alizarin red staining compared to the GM or no PTHrP groups (^#P < 0.05, ^**P < 0.01 and ^***P < 0.001) (Fig. 2).

To investigate the signaling pathways involved in the PTHrP-induced odontogenic differentiation of hDPCs, the phosphorylation of AKT, ERK, JNK, and p38 in hDPCs was assessed by Western blot analysis. The results showed that treatment with 10 nM PTHrP increased the levels of p-AKT, p-ERK1/2, p-JNK, and p-p38 five minutes after treatment. These increases were sustained 30 min or 60 min after treatment (Fig. 3a).

To further investigate the role of AKT, ERK, JNK, and p38 signaling in the odontogenic differentiation induced by PTHrP, cells were pretreated with or without 10 μM LY294002 (AKT inhibitor; Cell Signaling Technology, #9901), 10 μM U0126 (ERK inhibitor; Cell Signaling Technology, #9903), 10 μM SP600125 (JNK inhibitor; Cell Signaling Technology, #8177), or 10 μM SB203580 (p38 inhibitor; Cell Signaling Technology, #5633) for one hour, followed by treatment with 1 nM PTHrP for seven or 14 days. LY294002, U0126, SP600125, and SB203580 also significantly (^***P < 0.001) decreased the ALP staining and mineralized nodule formation induced by PTHrP (Fig. 3)b-e.